Difco Manual

11th Edition
Important Notice
Product labels/package inserts take precedence over the formula or instructions listed in the manual.
Generic names may be substituted for trade names in the ingredient list, providing more information
regarding animal origin, i.e. pancreatic digest of casein instead of Casitone.
During 1999, catalog numbers will be changed to meet new UCC/EAN128 labeling requirements.
Please visit us at www.bd.com/microbiology for more information.
Table of Contents
Foreword ................................................................................................... vii
Introduction.................................................................................................ix
Monographs .................................................................................................1
Culture Media and Ingredients, Dehydrated ..............................................19
Culture Media, Prepared ..........................................................................585
Stains and Indicators ................................................................................595
Serology and Immunology.......................................................................607
Reference Guides .....................................................................................811
Indices ...................................................................................................... 843
Alphabetical Index .............................................................................845
Numerical Index .................................................................................855
First Edition
1927
Second Edition
1929
Third Edition
1931
Fourth Edition
1933
Fifth Edition
1935
Sixth Edition
1939
Seventh Edition
1943
Eighth Edition
1948
Ninth Edition
Reprinted
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Tenth Edition
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Eleventh Edition
1953
1953
1956
1958
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1962
1963
1964
1965
1966
1967
1969
1971
1972
1974
1977
1984
1985
1994
1996
1998
Copyright 1998 by
Difco Laboratories,
Division of Becton Dickinson and Company
Sparks, Maryland 21152 USA
vi
The Difco Manual
Foreword
Foreword
This edition of the DIFCO MANUAL, the eleventh published since 1927, has been extensively revised and
rewritten. The purpose of the Manual is to provide information about products used in microbiology. The
Manual has never been intended to replace any official compendium or the many excellent standard text
books of scientific organizations or individual authors.
Difco is perhaps best known as the pioneer in bacteriological culture media. Numerous times one will find
the trademarks Difco or Bacto preceding the names of materials used by scientists in their published
papers. Because Difco products have been readily available worldwide longer than any others, Difco
products have become the common-language reagents of the microbiological community. Standardized
products readily available worldwide are essential for corroborative studies demanded by rigorous science.
Recommendation and approval have been extended to our products by the authors of many standard text
books and by the committees on methods and procedures of scientific societies throughout the world. Difco
products continue to be prepared according to applicable standards or accepted formulae. It is expected that
they will be used only by or under the supervision of microbiologists or other professionals qualified by
training and experience to handle pathogenic microorganisms. Further, it is expected that the user will be
throughly familiar with the intended uses of the formulations and will follow the test procedures outlined in
the applicable official compendia and standard text books or procedures manual of the using laboratory.
Grateful acknowledgment is made of the support we have received from microbiologists throughout the
world. It is our desire to continue and extend our services to the advancement of microbiology and related
sciences.
Difco Laboratories
Division of Becton Dickinson and Company
The Difco Manual
vii
Introduction
Introduction
Microbiology, through the study of bacteria, emerged as a defined
branch of modern science as the result of the monumental and immortal
research of Pasteur and Koch. In 1876, Robert Koch, for the first time
in history, propagated a pathogenic bacterium in pure culture outside
the hosts body. He not only established Bacillus anthracis as the
etiological agent for anthrax in cattle, but he inaugurated a method of
investigating disease which ushered in the golden age of medical
bacteriology.
Early mycologists, A. de Bary and O. Brefeld, and bacteriologist,
R. Koch and J. Schroeter, pioneered investigations of pure culture
techniques for the colonial isolation of fungi and bacteria on solid
media. Koch, utilizing state-of-the-art clear liquid media which he
solidified with gelatin, developed both streak and pour plate methods
for isolating bacteria. Gelatin was soon replaced with agar, a solidifying
agent from red algae. It was far superior to gelatin in that it was
resistant to microbial digestion and liquefaction.
The capability of Koch to isolate disease-producing bacteria on solidified culture media was further advanced by manipulating the cultural
environment using meat extracts and infusions so as to reproduce, as
closely as possible, the infected hosts tissue. The decade immediately
following Kochs epoch-making introduction of solid culture media
for the isolation and growth of bacteria ranks as one of the brightest in
the history of medicine because of the number, variety, and brilliance of
the discoveries made in that period. These discoveries, which, as Koch
himself expressed it, came as easily as ripe apples fall from a tree,
were all dependent upon and resulted from the evolution of correct
methods for the in vitro cultivation of bacteria.
The fundamental principles of pure culture isolation and propagation
still constitute the foundation of microbiological practice and research.
Nevertheless, it has become more and more apparent that a successful
attack upon problems unsolved is closely related to, if not dependent
upon, a thorough understanding of the subtle factors influencing
bacterial metabolism. With a suitable culture medium, properly
used, advances in microbiology are more readily made than when
either the medium or method of use is inadequate. The microbiologist
of today is, therefore, largely concerned with the evolution of methods
for the development and maintenance of microbial growth upon which
an understanding of their unique and diversified biological and
biochemical characteristics can be investigated. To this end, microbiologists have developed innumerable enrichment culture techniques
for the isolation and cloning of microorganisms with specific nutritional requirements. These organisms and their unique characteristics
have been essential to progress in basic biological research and modern
applied microbiology.
The study of microorganisms is not easy using microscopic single cells.
It is general practice to study pure cultures of a single cell type. In the
laboratory, microbiological culture media are utilized which contain
various nutrients that favor the growth of particular microorganisms in
pure cultures. These media may be of simple and defined chemical
composition or may contain complex ingredients such as digests of
plant and animal tissue. In particular, the cultivation of bacteria is
The Difco Manual
dependent upon nutritional requirements which are known to vary

widely. Autotrophic bacteria are cultivated on chemically defined or
synthetic media while heterotrophic bacteria, for optimal growth, may
require more complex nutrients such as peptones, meat or yeast
extracts. These complex mixtures of nutrients readily supply fastidious
heterotrophic bacteria with vitamins and other growth-promoting
substances necessary for desired cultivation. The scientific literature
abounds with descriptions of enriched, selective and differential
culture media necessary for the proper isolation, recognition and
enumeration of various bacterial types.
Almost without exception whenever bacteria occur in nature, and this
is particularly true of the pathogenic forms, nitrogenous compounds
and carbohydrates are present. These are utilized in the maintenance
of growth and for the furtherance of bacterial activities. So complex is
the structure of many of these substances, however, that before they
can be utilized by bacteria they must be dissimilated into simpler
compounds then assimilated into cellular material. Such metabolic
alterations are affected by enzymatic processes of hydrolysis, oxidation,
reduction, deamination, etc., and are the result of bacterial activities of
primary and essential importance. These changes are ascribed to the
activity of bacterial enzymes which are both numerous and varied. The
processes involved, as well as their end-products, are exceedingly
complex; those of fermentation, for example, result in the production
of such end-products as acids, alcohols, ketones, and gases including
hydrogen, carbon dioxide, methane, etc. The study of bacterial
metabolism, which defines the organized chemical activities of a cell,
has led to the understanding of both catabolic or degradative activities
and anabolic or synthetic activities. From these studies has come a
better understanding of the nutritional requirements of bacteria, and in
turn, the development of culture media capable of producing rapid and
luxuriant growth, both essential requisites for the isolation and study
of specific organisms.
Studies to determine the forms of carbon, hydrogen, and nitrogen which
could most easily be utilized by bacteria for their development were
originally carried on by Naegeli1 between 1868 and 1880, and were
published by him in the latter year. Naegelis report covered the use of
a large variety of substances including carbohydrates, alcohols, amino
acids, organic nitrogen compounds, and inorganic nitrogen salts.
The first reference to the use of peptone for the cultivation of microorganisms is that made by Naegeli in the report referred to above, when
in 1879, he compared peptone and ammonium tartrate. Because of its
content amino acids and other nitrogenous compounds which are
readily utilized by bacteria, peptone soon became one of the most
important constituents of culture media, as it still remains. In the light
of our present knowledge, proteins are known to be complex compounds
composed of amino acids joined together by means of the covalent
peptide bond linkage. When subjected to hydrolysis, proteins yield
polypeptides of various molecular sizes, metapeptones, proteoses,
peptones and peptides, down to the level of simple amino acids. The
intermediate products should be considered as classes of compounds,
rather than individual substances, for there exists no sharp lines of
demarcation between the various classes. One group shades by
imperceptible degrees into the next. All bacteriological peptones, thus,
are mixtures of various products of protein hydrolysis. Not all the
ix
Introduction
products of protein decomposition are equally utilizable by all

bacteria. In their relation to proteins, bacteria may be divided into two
classes; those which decompose naturally occurring proteins, and those
which require simpler nitrogenous compounds such as peptones and
amino acids.
The relation of amino acids to bacterial metabolism, and the ability of
bacteria to use these compounds, have been studied by many workers.
Duval,2,3 for example, reports that cysteine and leucine are essential in
the cultivation of Mycobacterium leprae. Kendall, Walker and Day4
and Long5 reported that the growth of M. tuberculosis is dependent
upon the presence of amino acids. Many other workers have studied the
relation of amino acids to the growth of other organisms, as for example,
Hall, Campbell, and Hiles6 to the meningococcus and Streptococcus;
Cole and Lloyd7 and Cole and Onslow8 to the gonococcus; and Jacoby
and Frankenthal9 to the influenza bacillus. More recently Feeley, et
al. 34 demonstrated that the nonsporeforming aerobe, Legionella
pneumophila requires L-cysteine . HCI for growth on laboratory media.
Indispensable as amino acids are to the growth of many organisms,
certain of them in sufficient concentration may exert an inhibitory
effect upon bacterial development.
From the data thus far summarized, it is apparent that the problem
of bacterial metabolism is indeed complicated, and that the phase
concerned with bacterial growth and nutrition is of the utmost practical
importance. It is not improbable that bacteriological discoveries such
as those with Legionella pneumophila await merely the evolution of
suitable culture media and methods of utilizing them, just as in the past
important discoveries were long delayed because of a lack of similar
requirements. Bacteriologists are therefore continuing to expend much
energy on the elucidation of the variations in bacterial metabolism,
and are continuing to seek methods of applying, in a practical way, the
results of their studies.
While the importance of nitrogenous substances for bacterial growth
was recognized early in the development of bacteriological technique,
it was also realized, as has been indicated, that bacteria could not
always obtain their nitrogen requirements directly from protein. It
is highly desirable, in fact essential, to supply nitrogen in readily
assimilable form, or in other words to incorporate in media proteins
which have already been partially broken down into their simpler and
more readily utilizable components. Many laboratory methods, such
as hydrolysis with alkali,10 acid,11,12,13 enzymatic digestion,8,14,15,16,17,18
and partial digestion of plasma10 have been described for the preparation
of protein hydrolysates.
The use of protein hydrolysates, particularly gelatln and casein, has
led to especially important studies related to bacterial toxins by
Mueller, et al.20-25 on the production of diphtheria toxin; that of Tamura,
et al.25 of toxin of Clostridium welchii; that of Bunney and Loerber27,28
on scarlet fever toxin, and of Favorite and Hammon29 on Staphylococcus
enterotoxin. In addition, the work of Snell and Wright30 on the
microbiological assay of vitamins and amino acids was shown to
be dependent upon the type of protein hydrolysate utilized. Closely
associated with research on this nature are such studies as those of
Mueller31,32 on pimelic acid as a growth factor for Corynebacterium
diphtheriae, and those of OKane33 on synthesis of riboflavin by
staphylococci. More recently, the standardization of antibiotic susceptibility testing has been shown to be influenced by peptones of culture
media. Bushby and Hitchings35 have shown that the antimicrobial activities of trimethoprim and sulfamethoxazole are influenced considerably by the thymine and thymidine found in peptones of culture media.
In this brief discussion of certain phases of bacterial nutrition, we have
attempted to indicate the complexity of the subject and to emphasize
the importance of continued study of bacterial nutrition. Difco Laboratories has been engaged in research closely allied to this problem in
its broader aspects since 1914 when Bacto Peptone was first introduced.
Difco dehydrated culture media, and ingredients of such media, have
won universal acceptance as useful and dependable laboratory adjuncts
in all fields of microbiology.
References
1. Sitzber, math-physik. Klasse Akad. Wiss. Muenchen, 10:277,
1880.
2. J. Exp. Med., 12:46, 1910.
3. J. Exp. Med., 13:365, 1911.
4. J. Infectious Diseases, 15:455, 1914.
5. Am. Rev. Tuberculosis, 3:86, 1919.
6. Brit. Med. J., 2:398, 1918.
7. J. Path. Bact., 21:267, 1917.
8. Lancet, II:9, 1916.
9. Biochem, Zelt, 122:100, 1921.
10. Centr. Bakt., 1:29:617, 1901.
11. Indian J. Med. Research, 5:408, 1917-18.
12. Compt. rend. soc. biol., 78:261, 1915.
13. J. Bact., 25:209, 1933.
14. Ann. de LInst., Pasteur, 12:26, 1898.
15. Indian J. Med. Research, 7:536, 1920.
16. Sperimentale, 72:291, 1918.
17. J. Med. Research, 43:61, 1922.
18. Can. J. Pub. Health, 32:468, 1941.
19. Centr. Bakt., 1:77:108, 1916.
20. J. Bact., 29:515, 1935.
21. Brit. J. Exp. Path., 27:335, 1936.
22. Brit. J. Exp. Path., 27:342, 1936.
23. J. Bact., 36:499, 1938.
24. J. Immunol., 37:103, 1939.
25. J. Immunol., 40:21, 1941.
26. Proc. Soc. Expl. Biol. Med., 47:284, 1941.
27. J. Immunol., 40:449, 1941.
28. J. Immunol., 40:459, 1941.
29. J. Bact., 41:305, 1941.
30. J. Biol. Chem., 139:675, 1941.
31. J. Biol. Chem., 119:121, 1937.
32. J. Bact., 34:163, 1940.
33. J. Bact., 41:441, 1941.
34. J. Clin. Microbiol., 8:320, 1978.
35. Brit. J. Pharmacol., 33:742, 1968.
The Difco Manual
Section !I
Monographs
History of Difco Laboratories

Difco Laboratories, originally
known as Ray Chemical, was
founded in 1895. This company
produced high quality enzymes,
dehydrated tissues and glandular
products to aid in the digestion
process. Ray Chemical acquired
Digestive Ferments Company,
a company that specialized in
producing digestive enzymes
for use as bacterial culture media
ingredients. The experience of
processing animal tissues, purifying enzymes and performing
dehydration procedures created
Original Difco Laboratories
a smooth transition to the
Manufacturing facility.
preparation of dehydrated
culture media. In 1913, the Digestive Ferments Company moved to
Detroit, Michigan, and dropped the name, Ray Chemical.
Difco pursued the challenging task of producing bacterial antisera

and antigens. Lee Laboratories, a subsidiary, remains one of the
largest manufacturers of bacterial antisera. Additional firsts for Difco
Laboratories came in the 1950s with the development of C Reactive
Protein Antiserum, Treponemal Antigen and Antistreptolysin Reagents.
After 1895, meat and other protein digests were developed to stimulate
growth of bacteria and fungi. The extensive research performed on
the analysis of pepsin, pancreatin and trypsin (and their digestive
processes) led to the development of Bacto Peptone. Bacto Peptone,
first introduced in 1914, was used in the bacteriological examination
of water and milk as a readily available nitrogen source. Bacto Peptone
has long been recognized as the standard peptone for the preparation
of bacteriological culture media.
Bactrol Disks were introduced by Difco Laboratories in 1972. Bactrol

Disks are water-soluble disks containing viable microorganisms of
known cultural, biochemical and serological characteristics used for
quality control testing. Bactrol Disks became the first of many
products manufactured by Difco for use in quality control.
The development of Proteose Peptone, Proteose Peptone No. 2 and

Proteose Peptone No. 3 was the result of accumulated information that
no single peptone is the most suitable nitrogen source for growing
fastidious bacteria. Proteose Peptone was developed for use in the
preparation of diphtheria toxin of high and uniform potency. Bacto
Tryptose was originally formulated to provide the growth requirements
of Brucella. Bacto Tryptose was also the first peptone prepared that
did not require the addition of infusions or other enrichments for the
isolation and cultivation of fastidious bacteria.
The Digestive Ferments Company began the preparation of diagnostic
reagents in 1923. Throughout the development of products used in the
diagnosis of syphilis and other diseases, Difco worked closely with
and relied on the direct involvement of expert scientists in the field.
Bacto Thromboplastin, the first manufactured reagent used in
coagulation studies, was developed in the early 1930s. This product
was another in a long line of many firsts for Difco Laboratories.
In 1934, the Digestive Ferments Company chose an acronym, Difco,
to rename the company. The focus of Difco Laboratories was to
develop new and improved culture media formulations.
After World War II, the microbiology and health care fields expanded
rapidly. Difco focused on the development of microbiological and
immunological products to meet this growing demand. In the 1940s,
The Difco Manual
Throughout the 1950s and 1960s, Difco continued to add products

for clinical applications. Bacto Blood Cultures Bottles were developed
to aid in the diagnosis and treatment of sepsis. Difco Laboratories
pioneered in the preparation of reagents for in vitro propagation and
maintenance of tissue cells and viruses.
With the discovery of penicillin, a brand new branch of microbiology
was born. Difco initiated developmental research by preparing
antibiotic disks for use in a theorized disk diffusion procedure.
The result was Bacto Sensitivity Disks in 1946, followed by DispensO-Discs in 1965.
In the 1960s, Difco Laboratories became the largest manufacturer of
microbiological culture media by acquiring the ability to produce agar.
Difco offers the same premier gold standard, Bacto Agar, today.
In 1983, Difco purchased the Paul A. Smith Company, later to be known

as Pasco. A semi-automated instrument, the Pasco MIC/ID System, is
used for bacterial identification and sensitivity testing. The Pasco Data
Management System can be used in industrial and clinical laboratories,
either alone or as a back up to automated systems.
In 1992, ESP, an automated continuous monitoring blood culture
system, was introduced. ESP was the first blood culture system to
detect both gas production and consumption by organism growth. The
technology continued with ESP Myco, an adaptation to the system that
allowed for growth, detection and susceptibility testing of mycobacteria
species. The ESP clinical system was sold to AccuMed International
in 1997.
In 1995, Difco Laboratories celebrated 100 years in business. In 1995,
Difco was the first U.S. microbiology company to receive ISO 9001
certification. The International Organization for Standardization (ISO)
verifies that Difco Laboratories maintains quality standards for the
worldwide microbiology industry.
In 1997, Difco Laboratories, the industrial microbiology leader, was
purchased by the clinical microbiology leader, Becton Dickinson
Microbiology Systems, to form the largest microbiology company in
the world. Together, Becton Dickinson Microbiology Systems and
Difco Laboratories look forward to an even stronger future with our
combined commitment to serving microbiologists worldwide.
Monographs
Section I
History of Microbiology and Culture Media

The science of microbiology evolved from a series of significant
discoveries. The Dutch microscopist, Anton van Leeuwenhoek, was
the first to observe bacteria while examining different water sources.
This observation was published in 1676 by the Royal Society in
London. Anton van Leeuwenhoek was also the first to describe the
parasite known today as Giardia lamblia. In 1667, the discovery of
filamentous fungi was described by Robert Hooke.
After microorganisms were visually observed, their growth or
reproduction created a major controversy. The conflict was over the
spontaneous generation theory, the idea that microorganisms will grow
spontaneously. This controversy continued for years until Louis
Pasteurs renowned research. Pasteur realized that the theory
of spontaneous generation must be refuted for the science of
microbiology to advance. The controversy remained even after
Pasteurs successful experiment using heat-sterilized infusions.
Two important developments were required for the science of
microbiology to evolve. The first was a sophisticated microscope; the
second was a method for culturing microorganisms. Compound
microscopes were developed in Germany at the end of the sixteenth
century but it was not until the early nineteenth century that
achromatic lenses were developed, allowing the light in the microscope
to be focused.
In 1719, Leeuwenhoek was the first to attempt differentiation of
bacteria by using naturally colored agents such as beet juice. In 1877,
Robert Koch used methylene blue to stain bacteria. By 1882,
Robert Koch succeeded in staining the tubercle bacillus with
methylene blue. This landmark discovery was performed by using
heat to penetrate the stain into the organism. Two years later Hans
Christian Gram, a Danish pathologist, developed the Gram stain. The
Gram stain is still widely used in the differentiation of gram-positive
and gram-negative bacteria.
In 1860, Pasteur was the first to use a culture medium for growing
bacteria in the laboratory. This medium consisted of yeast ash, sugar
and ammonium salts. In 1881, W. Hesse used his wifes agar
(considered an exotic food) as a solidifying agent for bacterial growth.
The study of fungi and parasites lagged behind other microorganisms.
In 1839, ringworm was the first human disease found to be caused by
fungi, followed closely by the recognition of Candida albicans as
the cause of thrush. It was not until 1910 that Sabouraud introduced
a medium that would support the growth of pathogenic fungi. The
interest of scientists in studying fungi was often related to crop
protection. There continues to be a close connection between
mycology and botany today.
By 1887, a simple device called the Petri dish revolutionized
microbiology. With the invention of the Petri dish, the focus turned to
culture media formulations. With all the research being performed,
scientists began to replace gelatin with agar because it was resistant to
microbial digestion and liquefaction.
Early years at Difco Laboratories.
The study of immunity began after the discovery of the tubercle

bacillus by Robert Koch. With this acclaimed discovery, the
involvement of bacteria as agents of disease became evident. The first
rational attempts to produce artificial active immunity were by
Pasteur in 1880 during his work with cholera.
Antibiotics had a dramatic beginning with the famous discovery of
penicillin by Alexander Fleming in 1928. Fleming found a mold spore
that accidentally landed on a culture of staphylococci. It was not until
the late 1930s that scientists could purify penicillin and demonstrate
its antibacterial effects. Commercial production of penicillin began
as a combined wartime project between the United States and
England. This project was the beginning of the fermentation industry
and biotechnology.
Around 1930, certain growth factors, including factor X and V, were
shown to be important in bacterial nutrition. In the early 1950s, most
of the vitamins were also characterized as co-enzymes. This detailed
information lead scientists to develop an understanding of
biochemical pathways.
A booming development of microbiology began after World War II.
Molecular biology, biotechnology and the study of genetics were fields
of extraordinary growth. By 1941, the study of microbiology and
genetics came together when Neurospora crassa, a red bread mold,
was used to study microbial physiology. The study of bacterial
genetics moved dramatically forward during the 1940s following the
discovery of antibiotic resistance. The birth of molecular biology
began in 1953 after the publication by Watson and Crick of the
structure of DNA.
In 1953, viruses were defined by Luria as submicroscopic entities,
capable of being introduced into specific living cells and of
reproducing inside such cells only. The work of John Enders on
culturing viruses lead to the development of vaccines. Enders
The Difco Manual
Section I
Monographs
demonstrated that a virus could be grown in chick embryos and would

lose its ability to cause disease after successive generations. Using
this technique, Salk developed the polio vaccine.
With rapid advances in technologies and instrumentation, the basic

culture media and ingredients listed in this Manual remain some of the
most reliable and cost effective tools in microbiology today.
One organism that has made a great contribution to molecular

biology is Escherichia coli. In 1973, Herbert Boyer and Stanley Cohen
produced recombinant DNA through plasmid transformation.
The researchers found that the foreign gene not only survived, but
copied the genetic material. This study and similar others started a
biotechnology revolution that has gained momentum over the years.
References
In the 1980s, instrumentation entered the microbiology laboratory.

Manual procedures could be replaced by fully automated instruments
for bacterial identification, susceptibility testing and blood culture
procedures. Immunoassays and probe technologies are broadening the
capabilities of the microbiologist.
1. Marti-Ibanez, F. 1962. Baroque medicine, p. 185-195. In

F. Marti-Ibanez (ed.). The epic of medicine. Clarkson N. Potter,
Inc., New York, N.Y.
2. Wainwright, M., and J. Lederberg. 1992. History of
microbiology, p. 419-437. In J. Lederberg (ed.), Encyclopedia of
microbiology, vol 2. Academic Press Inc., New York, N.Y.
Microorganism Growth Requirements

Microorganism growth on culture media depends on a number of
important factors:
Proper nutrients must be available.
Oxygen or other gases must be available, as required.
Moisture is necessary.
The medium must have an appropriate pH.
Proper temperature relations must prevail.
The medium must be free of interfering bioburden.
Contamination must be prevented.
A satisfactory microbiological culture medium must contain available
sources of:
Carbon,
Nitrogen,
Inorganic phosphate and sulfur,
Trace metals,
Water,
Vitamins.
These were originally supplied in the form of meat infusion. Beef or
yeast extracts frequently replace meat infusion in culture media. The
addition of peptones, which are digests of proteins, provides readily
available sources of nitrogen and carbon.
The pH of the culture medium is important for microorganism growth.
Temperature is another important parameter: mesophilic bacteria and
fungi have optimal growth at temperatures of 25-40C; thermophilic
(heat loving) organisms grow only at temperatures greater than 45C;
psychrophilic (cold loving) organisms require temperatures below
20C. Human pathogenic organisms are generally mesophiles.
The Difco Manual
Common Media Constituents

Media formulations are developed on the ability of bacteria to use
media components.
CONSTITUENTS
SOURCE
Amino-Nitrogen
Peptone, protein hydrolysate,

infusions and extracts
Growth Factors
Blood, serum, yeast extract or

vitamins, NAD
Energy Sources
Sugar, alcohols and carbohydrates
Buffer Salts
Phosphates, acetates and citrates
Mineral Salts and Metals
Phosphate, sulfate, magnesium,

calcium, iron
Selective Agents
Chemicals, antimicrobials and dyes
Indicator Dyes
Phenol red, neutral red
Gelling agents
Agar, gelatin, alginate, silica gel
Media Ingredients
Peptone, protein hydrolysates, infusions and extracts are the
major sources of nitrogen and vitamins in culture media. Peptones are
water-soluble ingredients derived from proteins by hydrolysis or
digestion of the source material, e.g. meat, milk.
Carbohydrates are employed in culture media as energy sources and
may be used for differentiating genera and identifying species.
Buffers maintain the pH of culture media.
Monographs
Selective Agents include Bile Salts, dyes and antimicrobial agents.

Bile Salts and desoxycholate are selective for the isolation of
gram-negative microorganisms, inhibiting gram-positive cocci.
Dyes and indicators are essential in the preparation of differential and
selective culture media. In these formulations, dyes act as bacteriostatic
agents, inhibitors of growth or indicators of changes in acidity or
alkalinity of the substrate.
Antimicrobial agents are used in media to inhibit the growth of bacteria,
yeasts and fungi.
Solidifying agents, including agar, gelatin and albumin, can be added
to a liquid medium in order to change the consistency to a solid
or semisolid state.
Environmental Factors in Culture Media

Atmosphere
Most bacteria are capable of growth under ordinary conditions of oxygen
tension. Obligate aerobes require the free admission of oxygen, while
anaerobes grow only in the absence of atmospheric oxygen. Between
these two groups are the microaerophiles, which develop best under
partial anaerobic conditions, and the facultative anaerobes, which are
capable of growing in the presence or absence of oxygen. Anaerobic
conditions for growth of microorganisms are obtained in a number of ways:
Addition of small amounts of agar to liquid media;
Addition of fresh tissue to the medium;
Addition of a reducing substance to the medium; e.g., sodium
thioglycollate, thioglycollic acid and L-cystine;
Displacement of the air by carbon dioxide;
Section I
Absorption of the oxygen by chemicals;

Inoculation into the deep layers of solid media or under a layer
of oil in liquid media.
Many microorganisms require an environment of 5-10% CO2. Levels
greater than 10% are often inhibitory due to a decrease in pH as
carbonic acid forms. Culture media vary in their susceptibility to form
toxic oxidation products if exposed to light and air.
Water Activity
Proper moisture conditions are necessary for continued luxuriant
growth of microorganisms. Organisms require an aqueous environment
and must have free water. Free water is not bound in complex
structure and is necessary for transfer of nutrients and toxic waste
products. Evaporation during incubation or storage results in loss of
free water and reduction of colony size or total inhibition of
organism growth.
Protective Agents and Growth Factors

Calcium carbonate, soluble starch and charcoal are examples of
protective agents used in culture media to neutralize and absorb toxic
metabolites produced by bacterial growth.
NAD (V factor) and hemin (X factor) are growth factors required by
certain bacteria; e.g., Haemophilus species, and for enhanced growth
of Neisseria species.
Surfactants, including Tween 80, lower the interfacial tension around
bacteria suspended in the medium. This activity permits more rapid
entry of desired compounds into the bacterial cell and can increase
bacterial growth.
Culture Media Ingredients Agars

History
Agar was discovered in 1658 by Minora Tarazaemon in Japan. 1
According to legend, this Japanese innkeeper threw surplus seaweed
soup into the winter night and noticed it later transformed into a gel by
the nights freezing and the days warmth.2 In 1882, Koch was the first
to use agar in microbiology.3,4 Walter Hesse, a country doctor from
Saxony, introduced Koch to this powerful gelling agent.5 Hesse
had learned about agar from his wife, Fanny Hesse, whose family had
contact with the Dutch East Indies where agar was being used for
jellies and jams.3,5,6 The term agar-agar is a Malaysian word that
initially referred to extracts from Eucheuma, which yields carrageenan,
not agar.5
By the early 1900s, agar became the gelling agent of choice instead
of gelatin. Agar was found more suitable because it remained solid at
the temperatures required for growth of human pathogens and was

resistant to breakdown by bacterial enzymes.
Production of agar in the United States was started just before the
beginning of World War II as a strategic material.5 In the 1940s,
bacteriological-grade agar manufactured by the American Agar
Company of San Diego, California, served as reference agar for the
evaluation of the characteristics of other culture media components,
such as peptones.5
Characteristics
Agar is a phycocolloid, a water-soluble polysaccharide, extracted from
a group of red-purple marine algae (Class Rhodophyceae) including
Gelidium, Pterocladia and Gracilaria. These red-purple marine algae
are widely distributed throughout the world in temperate zones.
The Difco Manual
Section I
Monographs
trapped in the frozen water. The ice is then washed from the agar,
eliminating the contaminants. The Ice Agar process results in greater
consistency and freedom from interposing contaminants when used
in microbiological procedures.
Product Applications
Bacto Agar is optimized for beneficial calcium and magnesium
content. Detrimental ions such as iron and copper are reduced. Bacto
Agar is recommended for clinical applications, auxotrophic studies,
bacterial and yeast transformation studies and bacterial molecular
genetics applications.7,8
Agar Flake is recommended for use in general bacteriological
purposes. The quality is similar to Bacto Agar. The flakes are more
wettable than the granules found in Bacto Agar.
Agar is derived from a group of red-purple marine algae as pictured above.
For Difco Agars, Gelidium is the preferred source of agar. The most
important properties of agar are:5
Good transparency in solid and gel forms to allow identification
of colony type;
Consistent lot-to-lot gel strength that is sufficient to withstand
the rigors of streaking but not so stiff that it affects diffusion
characteristics;
Consistent gelling (32-40C) and melting (approximately
85C) temperatures, a property known as hysteresis;
Essential freedom from metabolically useful chemicals such as
peptides, proteins and fermentable hydrocarbons;
Low and regular content of electronegative groups that could
cause differences in diffusion of electropositive molecules
(e.g., antibiotics, nutrients);
Freedom from toxic substances (bacterial inhibitors);
Freedom from hemolytic substances that might interfere with
normal hemolytic reactions in culture media;
Freedom from contamination by thermophilic spores.
Agars are normally used in final concentrations of 1-2% for
solidifying culture media. Smaller quantities of agar (0.05-0.5%) are
used in culture media for motility studies (0.5% w/v) and growth
of anaerobes (0.1%) and microaerophiles.2
The Manufacturing Process

Difco Laboratories selects the finest Gelidium marine algae from world
sources and requires algae harvested from water where the temperature is both constant and temperate. Bacto Agar and Agar Granulated
are produced from an Ice Agar purification process. Agar is insoluble
in cold water but is colloidally dispersible in water above 90C.2 When
an agar gel is frozen, the agar skeleton contracts toward the center of
the mass as a membrane, leaving ice as a separate phase.2
Through a variety of processes, the agar is extracted from the Gelidium,
resulting in a liquid agar that is purified. The liquid agar is first gelled
and then frozen, causing the soluble and suspended contaminants to be
The Difco Manual
Agar Granulated is qualified to grow recombinant strains of

Escherichia coli (HB101) and Saccharomyces cerevisiae. Agar
Granulated may be used for general bacteriological purposes where
clarity is not a strict requirement. This agar was developed to
address the special needs of the Biotechnology Industry for large
scale applications.
Noble Agar is the purest form of Difco agar. It is washed extensively
and bleached to remove extraneous material. The result is a white
powder in dry form, clear and colorless in solution and when solidified
in plates. This agar is suitable for immunodiffusion studies, for use in
some electrophoretic applications and as a substrate for mammalian
and plant tissue culture.
Agar Technical is suitable for many general bacteriological
applications. This agar is not as highly processed as other Difco agars
and has lower technical specifications. This agar is not recommended
for growth of fastidious organisms.
References
1. C. K. Tsend. 1946. In J. Alexander (ed.). 6:630. Colloid
Chemistry. Reinhold Publishing Corp., New York, N. Y.
2. Selby, H. H., and T. A. Selby. 1959. Agar. In Whister (ed.).,
Industrial gums. Academic Press Inc., New York, NY.
3. Hitchens, A. P., and M. C. Leikind. 1939. The introduction
of agar-agar into bacteriology. J. Bacteriol. 37:485-493.
4. Koch, R. 1882. Die Atiologie der Turberkulose. Berl. Klin.
Wochenschr. 19:221- 230.
5. Armisen, R. 1991. Agar and agarose biotechnological applications.
Hydrobiol. 221:157-166.
6. Hesse, W. 1894. Uber die quantitative Bestimmung der in der
Luft enthaltenen Mikroorganismen. Mitt. a. d. Kaiserl. Gesh.
Berlin 2:182-207.
7. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular
cloning, a laboratory manual, 2nd ed. Cold Spring Harbor
Laboratory Press, New York, N.Y.
8. Schiestl, R. H., and R. D. Geitz. 1989. High efficiency
transformation of intact yeast cells using single stranded nucleic
acids as a carrier. Current Genetics 16:339-346.
Monographs
Section I
Culture Media Ingredients Peptones and Hydrolysates

Preparation of Peptones
The composition of peptones varies with the origin and the method of
preparation. Some common sources of peptone include:
Typical fermentation process.
History
Peptones were originally described by Naegeli in 1879.3 In this report,
Naegeli compared peptone and ammonium tartrate. With the rich amino
acid and nitrogen compounds readily utilized by bacteria, peptone
soon became one of the most important constituents of culture media.
The importance of peptone as a nutritive source was demonstrated
by Klinger.4
Bacto Peptone was introduced commercially in 1914, and became the
standard peptone for the preparation of bacteriological culture media.
The development of Bacto Proteose Peptone, Bacto Proteose Peptone
No. 2 and Bacto Proteose Peptone No. 3 resulted from accumulated
information that no single peptone is the most suitable nitrogen source
for culturing fastidious bacteria. Extensive investigations were
undertaken at Difco Laboratories using peptic digests of animal tissue
prepared under varying digestion parameters. Bacto Tryptone was
developed by Difco Laboratories while investigating a peptone
particularly suitable for the elaboration of indole by bacteria.
Meat (fresh, frozen or dried)

Fish (fresh, dried)
Casein
Gelatin
Keratin (horn, hair, feathers)
Ground Nuts
Soybean Meal
Cotton Seed
Sunflower Seeds
Microorganisms (yeasts, algae, bacteria)
Guar Protein
Blood
Corn Gluten
Egg Albumin
Demineralized water is added to these protein sources to form a thick
suspension. The digestion process follows with an acid or enzyme. Acid
and alkaline hydrolyses are performed by boiling the protein with
mineral acids or strong alkalis at increased pressure to raise the
temperature of the reaction. This procedure can decrease the vitamin
content of the protein and a portion of the amino acid content.
Digestion with proteolytic enzymes is performed at lower
temperatures and normal atmospheric pressure. This process is often
less harmful to the protein and amino acids. Microbial Proteoses,
Papain, Pancreatin and Pepsin are used most often by Difco
Laboratories in the manufacture of peptones.
The peptone suspension is then centrifuged and filtered. The
suspension is concentrated to approximately 67% total solids and the
product now appears as a syrup. This peptone syrup is spray dried
and packaged.
Infusions and Extracts
Other non-chemically defined ingredients, including Bacto Liver, Bacto

Beef Heart for Infusion and Bacto Yeast Extract can serve
as nitrogen or carbon sources. Infusions of meat were first employed
as nutrients in culture media. It was discovered that for many routine
procedures in the preparation of culture media, extracts have the
advantage of greater ease in preparation, uniformity and economy
than infusions.
The water-soluble fractions of materials such as muscle, liver, yeast

cells and malt are usually low in peptides but contain valuable
extractives such as vitamins, trace metals and complex carbohydrates.5
It is common practice to combine infusions and peptones to obtain the
best of both products.5 Bacto Yeast Extract, Bacto Malt Extract, Bacto
Beef Heart for Infusion and Bacto Beef Extract are examples of
extracts and infusions manufactured by Difco Laboratories for use in
the preparation of culture media.
Protein Biochemistry
Peptone Performance
Proteins consist of amino acids joined together by means of the

covalent peptide bond linkage. When the bonds are hydrolyzed,
proteins yield polypeptides of various molecular sizes, proteoses,
peptones and peptides down to the level of simple amino acids.
Bacteriological peptones are mixtures of various products of protein
hydrolysis, organic nitrogen bases, inorganic salts and trace elements.
The quality and performance of peptones, infusions and extracts are

very dependent on the freshness or preservation of the raw materials.5
Extensive quality control testing is performed on all peptones and other
culture media ingredients during the manufacturing process and on the
final product. Certificates of Analysis supply information from the
manufacturer on lot specific final testing of a product.
The Difco Manual
Section I
A typical analysis was performed on Difco peptones and hydrolysates

to aid in the selection of products for research or production
needs when specific nutritional characteristics are required. The
specifications for the typical analysis include:
Physical characteristics
Nitrogen content
Amino acids
Inorganics
Vitamins
Biological testing
The quality of peptones and culture media ingredients is truly assessed
by their ability to support adequate growth of various microorganisms
when incorporated into the medium. 6 The nature of peptones,
infusions and extracts will then play a major role in the growth
performance properties of the medium and, in turn, advance the
science of microbiology.6
Media Ingredients
Autolyzed Yeast
Autolyzed Yeast is a desiccated product containing both the soluble
and insoluble portions of autolyzed bakers yeast. Autolyzed Yeast is
recommended for the preparation of yeast supplements used in the
microbiological assay of riboflavin and pantothenic acid.7,8, Autolyzed
Yeast provides vitamins, nitrogen, amino acids and carbon in
microbiological culture media.
Beef
Beef Heart for Infusion
Beef and Beef Heart for Infusion provide nitrogen, amino acids and
vitamins in microbiological culture media. Beef is desiccated,
powdered, fresh lean beef, prepared especially for use in beef infusion
media. Large quantities of beef are processed at one time to secure a
uniform and homogenous product. Beef Heart for Infusion is prepared
from fresh beef heart tissue and is recommended for preparing heart
infusion media. Beef Heart for Infusion is processed from large
volumes of raw material, retaining all the nutritive and growth
stimulating properties of fresh tissues.
Beef Extract
Beef Extract, Desiccated
Beef Extract and Beef Extract, Desiccated are replacements for
infusion of meat. Beef Extract and Beef Extract, Desiccated provide
nitrogen, vitamins, amino acids and carbon in microbiological culture
media. Beef Extract is standard in composition and reaction and
generally used to replace infusion of meat. In culture media, Beef
Extract is usually employed in concentration of 0.3%. Beef Extract,
Desiccated, the dried form of Beef Extract, was developed to provide a
product for ease of use in handling. Beef Extract is in the paste form.
The products are to be used in a 1 for 1 substitution.
Bile Salts
Bile Salts No. 3
Bile Salts and Bile Salts No. 3 are used as selective agents for the
isolation of gram-negative microorganisms, inhibiting gram-positive
The Difco Manual
Monographs
cocci. Bile is derived from the liver. The liver detoxifies bile salts by
conjugating them to glycine or taurine. A bile salt is the sodium salt of
a conjugated bile acid. Bile Salts and Bile Salts No. 3 contain bile
extract standardized to provide inhibitory properties for selective
media. Bile Salts No. 3 is a modified fraction of bile acid salts,
providing a refined bile salt. Bile Salts No. 3 is effective at less than
one-third concentration of Bile Salts.
Casamino Acids
Casamino Acids, Technical
Casamino Acids, Vitamin Assay
Casamino Acids, Casamino Acids, Technical and Casamino Acids,
Vitamin Assay are derived from acid hydrolyzed casein. Casein is a
milk protein and a rich source of amino acid nitrogen. Casamino
Acids, Casamino Acids, Technical and Casamino Acids, Vitamin
Assay are added to media primarily because of their organic nitrogen
and growth factor components; their inorganic components also play
a vital role.9 Casamino Acids is recommended for use with microbiological cultures that require a completely hydrolyzed protein as a
nitrogen source. In Casamino Acids, hydrolysis is carried on until all
the nitrogen in the casein is converted to amino acids or other compounds of relative chemical simplicity. The hydrolysis of Casamino
Acids, Technical is carried out as in the preparation of Casamino
Acids, but the sodium chloride and iron content have not been decreased
to the same extent. Casamino Acids, Vitamin Assay is an acid digest of
casein specially treated to markedly reduce or eliminate certain
vitamins. It is recommended for use in microbiological assay media
and in growth promotion studies.
Casein Digest
Casein Digest is an enzymatic digest of casein, providing a distinct
source of amino acids for molecular genetics media. Casein Digest is
used as a nitrogen and amino acid source for microbiological culture
media. Casein Digest is similar to N-Z Amine A. This product is
digested under conditions different from other enzymatic digests
of casein, including Tryptone and Casitone.
Casitone
Casitone is a pancreatic digest of casein. Casitone is recommended
for preparing media where an enzymatic hydrolyzed casein is
desired. Casein is a rich source of amino acid nitrogen. This product
is used to support the growth of fastidious microorganisms and its
high tryptophan content makes it valuable for detecting indole
production.
Fish Peptone No. 1
Fish Peptone No. 1 is a non-mammalian, non-animal peptone used
as a nitrogen source in microbiological culture media. Fish Peptone
No. 1 is a non-bovine origin peptone, to reduce Bovine Spongiform
Encephalopathy (BSE) risk. This peptone was developed by Difco
Laboratories for pharmaceutical and vaccine production and can
replace any peptone, depending on the organism and production
application.
Monographs
Section I
Gelatin
Gelatin is a protein of uniform molecular constitution derived chiefly
by the hydrolysis of collagen.10 Collagens are a class of albuminoids
found abundantly in bones, skin, tendon, cartilage and similar tissues
of animals. 10 Gelatin is used in culture media to detect gelatin
liquifaction by bacteria and as a nitrogen and amino acid source.
Peptamin
Peptamin, referred to as Peptic Digest of Animal Tissue, complies
with the US Pharmacopeia XXIII (USP). 11 Peptamin provides
nitrogen, amino acids, vitamins and carbon in microbiological culture
media. Diluting and rinsing solutions, Fluid A and Fluid D, contain
0.1% Peptamin.
Gelatone
Gelatone is a pancreatic digest of gelatin, deficient in carbohydrates.
Gelatone is used as a media ingredient for fermentation studies and,
alone, to support the growth of non-fastidious microorganisms.
Gelatone is in granular form for convenience in handling and is
distinguished by a low cystine and tryptophan content.
Peptone, Bacto
Peptone Bacteriological, Technical
Bacto Peptone and Peptone Bacteriological, Technical are enzymatic
digests of protein and rich nitrogen sources. Bacto Peptone was
introduced in 1914 and became the standard peptone for the
preparation of culture media. Peptone Bacteriological, Technical can
be used as the nitrogen source in microbiological culture media when
a standardized peptone is not essential. Both peptones have a high
peptone and amino acid content and only a negligible quantity of
proteoses and more complex nitrogenous constituents.
Liver
Liver is prepared from large quantities of carefully trimmed fresh beef
liver. Liver is a desiccated powder of beef liver. The nutritive factors of
fresh liver tissue are retained in infusion prepared from Liver.
Liver is used as a source of nitrogen, amino acids and vitamins in
microbiological culture media. The reducing substances contained in
liver create an anaerobic environment, necessary to support the growth
of anaerobes. One hundred thirty-five (135) grams of desiccated Liver
are equivalent to 500 grams of fresh liver.
Malt Extract
Malt Extract is obtained from barley, designed for the propagation of
yeasts and molds. Malt Extract is particularly suitable for yeasts and
molds because it contains a high concentration of carbohydrates,
particularly maltose. This product is generally employed in
concentrations of 1-10%. Malt Extract provides carbon, protein and
nutrients for the isolation and cultivation of yeasts and molds in
bacterial culture media.
Neopeptone, Difco
Neopeptone is an enzymatic digest of protein. Neopeptone contains
many peptide sizes in combination with vitamins, nucleotides,
minerals and other carbon sources. Neopeptone is particularly well
suited in supplying the growth requirements of fastidious bacteria. This
peptone is extremely valuable in media for the cultivation of
pathogenic fungi. Growth of these microorganisms is rapid and colony
formation is uniform and typical.
Oxgall
Oxgall is manufactured from large quantities of fresh bile by rapid
evaporation of the water content. Bile is composed of fatty acids, bile
acids, inorganic salts, sulphates, bile pigments, cholesterol, mucin,
lecithin, glycuronic acids, porphyrins and urea. The use of Oxgall
ensures a regular supply of bile and assures a degree of uniformity
impossible to obtain with fresh materials. It is prepared for use in
selective media for differentiating groups of bile tolerant bacteria.
Oxgall is used as a selective agent for the isolation of gram-negative
microorganisms, inhibiting gram-positive bacteria. The major
components of Oxgall are taurocholic and glycocholic acids.
10
Proteose Peptone
Proteose Peptone No. 2
The development of Proteose Peptone, Proteose Peptone No. 2 and
Proteose Peptone No. 3 is the result of accumulated information
demonstrating that no single peptone is the most suitable nitrogen
source for culturing fastidious bacteria. Proteose Peptone is an
enzymatic digest of protein high in proteoses. Many factors account
for the suitability of Proteose Peptone for the culture of fastidious
pathogens, including the nitrogen components, buffering range and the
high content of proteoses. Proteose Peptone No. 2 and Proteose
Peptone No. 3 are enzymatic digests of protein. Proteose Peptone
No. 2 is used for producing bacterial toxins and is suitable for media
of nutritionally less-demanding bacteria. Proteose Peptone No. 3 is
a modification of Proteose Peptone, adapted for use in the preparation
of chocolate agar for propagation of Neisseria species and chocolate
tellurite agar for Corynebacterium diphtheriae.
Sodium Deoxycholate
Sodium Taurocholate
Sodium Desoxycholate is the sodium salt of desoxycholic acid. Since
Sodium Desoxycholate is a salt of a highly purified bile acid, it is used
in culture media in lower concentrations than in naturally occurring
bile. Sodium Taurocholate is the sodium salt of a conjugated bile acid.
Sodium Taurocholate contains about 75% sodium taurocholate in
addition to other naturally occurring salts of bile acids. Sodium
Desoxycholate and Sodium Taurocholate, like other bile salts, are used
as selective agents in microbiological culture media. They are used
to aid in the isolation of gram- negative microorganisms, inhibiting
gram-positive organisms and spore forming bacteria.
The Difco Manual
Section I
Soytone
Soytone No. 2
Soytone is an enzymatic digest of soybean meal. Soytone No. 2 is a
papaic digestion of soybean meal. The nitrogen source in Soytone and
Soytone No. 2 contains the naturally occurring high concentrations of
vitamins and carbohydrates of soybean. Soytone No. 2 minimizes the
risk of Bovine Spongiform Encephalopathy (BSE) in vaccine
production because the origin of this product is plant.
TC Lactalbumin Hydrolysate
TC Yeastolate
TC Lactalbumin Hydrolysate is an enzymatic digest of lactalbumin for
use as an enrichment in tissue culture media. Lactalbumin is a protein
derived after removal of casein from milk. TC Yeastolate is a
desiccated, clarified, water soluble portion of autolyzed fresh yeast prepared and certified for use in tissue culture procedures.
TC Yeastolate is a source of vitamin B complex.
Tryptone
Tryptone is a pancreatic digest of casein used as a nitrogen source in
culture media. Casein is the main protein of milk and is a rich source
of amino acid nitrogen. Tryptone is rich in tryptophan, making it
valuable for use in detecting indole production.12 The absence of
detectable levels of carbohydrates in Tryptone makes it a suitable
peptone in differentiating bacteria on the basis of their ability to
ferment various carbohydrates.
Tryptose
Tryptose is a mixed enzymatic hydrolysate with distinctive nutritional
properties. The digestive process of Tryptose results in assorted
peptides, including those of higher molecular weight. Tryptose was
originally developed as a peptone particularly adapted to the growth
requirements of Brucella.
Monographs
References
1. Nash, P., and M. M. Krenz. 1991. Culture Media, p. 1226-1288.
In A. Balows, W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg,
and H. J. Shadomy (ed.), Manual of clinical microbiology, 5th ed.
American Society for Microbiology, Washington, D.C.
2. De Feo, J. 1986. Properties and applications of hydrolyzed
proteins. ABL. July/August, 44-47.
3. Naegeli. 1880. Sitzber, math-physik. Klasse Akad. Wiss.
Muenchen. 10:277.
4. Klinger, I. J. 1917. The effect of hydrogen ion concentration on
the production of precipitates in a solution of peptone and its
relation to the nutritive value of media. J. Bacteriol. 2:351-353.
5. Bridson, E. Y. 1990. Media in microbiology. Rev. Med. Microbiol.
1:1-9.
6. Alvarez, R. J., and M. Nichols. 1982. Formulating microbiological culture media-a careful balance between science and art.
Dairy Food Sanitation 2:356- 359.
7. J. Ind. Eng. Chem., Anal. Ed. 1941. 13:567.
9. Nolan, R. A., and W. G. Nolan. 1972. Elemental analysis of
vitamin-free casamino acids. Appl. Microbiol. 24:290-291.
10. Gershenfeld, L., and L. F. Tice. 1941. Gelatin for bacteriological
use. J. Bacteriol. 41:645-652.
11. United States Pharmacopeial Convention. 1995. The United
States pharmacopeia, 23rd ed. The United States Pharmacopeial
Convention. Rockville, MD.
12. J. Bacteriol. 1933. 25:623.
Yeast Extract
Yeast Extract, Technical
Yeast Extract and Yeast Extract, Technical are water soluble portions
of autolyzed yeast containing vitamin B complex. Yeast Extract is
an excellent stimulator of bacterial growth and used in culture media.
The autolysis is carefully controlled to preserve the naturally
occurring B-complex vitamins. Yeast Extract is generally employed
in the concentration of 0.3-0.5%, with improved filterability at
20%. Yeast Extract, Technical is used in bacterial culture media when
a standardized yeast extract is not essential. Yeast Extract, Technical
was developed to demonstrate acceptable clarity and growth
promoting characteristics. Yeast Extract and Yeast Extract, Technical
also provide vitamins, nitrogen, amino acids and carbon in
The Difco Manual
11
Monographs
Section I
Media Preparation
The preparation of culture media from dehydrated media requires
accuracy and attention to preparation. The following points are
included to aid the user in successful and reproducible preparation
of culture media.
Dehydrated Media and Ingredients
Store in a cool (15-30C), dark and dry area unless otherwise
specified.
Note date opened.
Check expiry (applied to intact container).
Verify that the physical characteristics of the powder are typical.
Glassware / Plasticware
Use high quality, low alkali borosilicate glass.
Avoid detergent residue.
Check for alkali or acid residue with a few drops of brom thymol
blue pH indicator (yellow is acidic; blue is alkaline).
Quantities of media in excess of two liters may require an

extended autoclave time to achieve sterilization. Longer
sterilization cycles can cause nutrient concentration changes
and generation of inhibitory substances.
Adding Enrichments and Supplements
Enrichments and supplements tend to be heat sensitive.
Cool medium to 45-55C in a waterbath prior to adding
enrichments or supplements.
Ensure adequate mixing of the basal medium with enrichments
or supplements by swirling to mix thoroughly.
Sterile broths may be cooled to room temperature before adding
enrichment.
pH
Commercial dehydrated media are designed to fall within the
specified pH range after steam sterilization. The pH tends to fall
approximately 0.2 units during steam sterilization.
Use vessels at least 2-3 times the volume of medium.
For filter sterilization, adjust the pH, if necessary, prior to filtering.
Discard (recycle) etched or chipped glassware.
Avoid excessive pH adjustments.
Do not used etched glassware.

Equipment
Use measuring devices, scales, pH meters, autoclaves and other
equipment that are frequently and accurately calibrated.
Water
Use distilled or deionized water.
pH 5.5-7.5.
Dissolving the Medium
Accurately weigh the appropriate amount of dehydrated medium.
Dissolve the medium completely.
Agitate the medium while dissolving.
Take care to not overheat. Note media that are very sensitive to
overheating. Overheated media will frequently appear darker. Do
not heat in a microwave.
Sterilization
The autoclave set-temperature should be 121C.
Routine autoclave maintenance is important. Ask manufacturer
to check for hot and cold spots.
The recommended 15 minute sterilization assumes a volume
of 1 liter or less. Larger volumes may require longer
cycles. Check with your autoclave manufacturer for
recommended load configurations.
12
Dispensing Media
Ensure gentle mixing during dispensing.
Cool the medium to 50-55C prior to dispensing to reduce
water evaporation.
Dispense quickly.
If using an automatic plate dispenser, dispense general purpose
media before dispensing selective media.
Immediately recover or recap tubes to reduce the chance of
contamination. Leave Petri dish covers slightly open for 1-2 hours
to obtain a dry surface.
Storage and Expiry
In general, store steam-sterilized plated media inverted in a
plastic bag or other container in a dark refrigerator for up to
1-2 weeks.
Quality Control
For media prepared in-house, each lot of every medium must
be tested.
Maintain Quality Control Organisms appropriately.
Maintain appropriate records.
Report deficiencies to the manufacturer.
The following table is a troubleshooting guide to assist in the
preparation of reliable culture media.
The Difco Manual
Section I
PROBLEM
Monographs
Abnormal color of medium

Incorrect pH
Nontypical precipitate
Incomplete solubility
Darkening or carmelization
Toxicity
Tract substances (Vitamins)
OTHER CAUSES
Storage at high temperature

Hydrolysis of ingredients
pH determined at wrong temperature
Inadequate heating
Inadequate convection in a too small flask
Loss of gelation property

Loss of nutritive value or
selective or differential
properties
Contamination
Burning or scorching
Airborne or environmental sources
of vitamins
Hydrolysis of agar due to pH shift
Not boiling medium
Burning or scorching
Presence of strong electrolytes, sugar
solutions, detergents, antiseptics, metallic
poisons, protein materials or other
substances that may inhibit the inoculum
Improper sterilization
Poor technique in adding enrichments and
pouring plates
Not boiling agar containing medium
Key
A
Deteriorated Dehydrated Medium
Incorrect Weighing
Repeated Remelting
Improperly Washed Glassware
Incomplete Mixing
Dilution by a Too Large Inoculum
Impure Water
Overheating
Media Sterilization
Sterilization is any process or procedure designed to entirely eliminate
viable microorganisms from a material or medium. Sterilization should
not be confused with disinfection, sanitization, pasteurization or
antisepsis which are intended to inactivate microorganisms, but may
not kill all microorganisms present. Sterilization can be accomplished
by the use of heat, chemicals, radiation or filtration.1
Sterilization with Heat1

The principal methods of thermal sterilization include 1) moist heat
(saturated steam) and 2) dry heat (hot air) sterilization. Heat kills
microorganisms by protein denaturation and coagulation. Moist heat
has the advantage of being more rapid and requiring lower temperatures
than dry heat. Moist heat is the most popular method of culture media
sterilization. When used correctly, it is the most economical, safe and
reliable sterilization method.
The Difco Manual
Moist Heat Sterilization

Water boils at 100C, but a higher temperature is required to kill
resistant bacterial spores in a reasonable length of time. A temperature
range of 121-124C for 15 minutes is an accepted standard condition
for sterilizing up to one liter of culture medium. The definition of
autoclave at 121C for 15 minutes refers to the temperature of the
contents of the container being held at 121C for 15 minutes, not to the
temperature and time at which the autoclave has been set.2 The steam
pressure of 15 pounds per square inch at this temperature aids in the
penetration of the heat into the material being sterilized. If a larger
volume is to be sterilized in one container, a longer period should be
employed. Many factors can affect sterility assurance, including size
and contents of the load and the drying and cooling time. Certain
products may decompose at higher temperature and longer cycles. For
this reason, it is important that all loads be properly validated.
The basic principles for validation and certification of a sterilizing
process are enumerated as follows:3
1. Establish that the processing equipment has the capability of
operating within the required parameters.
13
Monographs
Section I
2. Demonstrate that the critical control equipment and

instrumentation are capable of operating within the prescribed
parameters for the process equipment.
3. Perform replicate cycles representing the required operational
range of the equipment and employing actual or simulated
product. Demonstrate that the processes have been carried out
within the prescribed protocol limits and, finally, that the
probability of microbial survival in the replicate processes
completed is not greater than the prescribed limits.
4. Monitor the validated process during routine operation.
Periodically as needed, requalify and recertify the equipment.
5. Complete the protocols and document steps 1-4, above.
For a complete discussion of process validation, refer to appropriate
references.
Ensuring that the temperature is recorded correctly is vital. The
temperature must reach all parts of the load and be maintained for the
desired length of time. Recording thermometers are employed for the
chamber and thermocouples may be buried inside the load.
Carmelization or darkening of the medium;

Loss of nutritive value;
Loss of selective or differential properties.
There are certain media (e.g., Hektoen Enteric Agar and Violet Red
Bile Agar) that should not be autoclaved. To dissolve these media
formulation, heat to boiling to dissolve completely. It is important
to follow all label directions for each medium. Media supplements
should be sterile and added aseptically to the sterilized medium,
usually at 45-55C.
Dry Heat Sterilization1

Dry heat is employed for materials such as metal instruments that could
be corroded by moist heat, powders, ointments and dense materials
that are not readily penetrated by steam. Because dry heat is effective
only at considerably higher temperatures and longer times than moist
heat, dry heat sterilization is restricted to those items that will
withstand higher temperatures. The dry heat time for sterilization is
120 minutes at 160C.
For best results when sterilizing culture media, plug tubes or flasks
of liquids with nonabsorbent cotton or cap loosely. Tubes should be
placed in racks or packed loosely in baskets. Flasks should never be
more than two-thirds full. It is important to not overload the autoclave
chamber and to place contents so that there is a free flow of steam
around the contents. After sterilizing liquids, the chamber pressure must
be reduced slowly to atmospheric pressure. This allows the liquid to
cool below the boiling point at atmospheric pressure before opening
the door to prevent the solution from boiling over.
Chemical Sterilization1
In autoclave operation, all of the air in the chamber must be expelled

and replaced by steam; otherwise, hot spots and cold spots will
occur. Pressure-temperature relations of a properly operated autoclave
are shown in the table below.
Radiation Sterilization1
Pressure-Temperature Relations in Autoclave

(Figures based on complete replacement of air by steam)
PRESSURE IN POUNDS
5
10
15
20
25
30
TEMPERATURE (C)
TEMPERATURE (F)
109
115
121
126
130
135
228
240
250
259
267
275
Over-sterilization or prolonged heating will change the composition of

the medium. For example, carbohydrates are known to break down in
composition upon overheating. Over-sterilizing media can cause a
number of problems, including:
Incorrect pH;
A decrease in the gelling properties of agar;
The development of a nontypical precipitate;
14
Chemical sterilization employs gaseous and liquid sterilants for

certain medical and industrial instruments. The gases include ethylene
oxide, formaldehyde and beta-propiolactone. The liquid sterilants
include glutaraldehyde, hydrogen peroxide, peracetic acid, chlorine
dioxide and formaldehyde. Chemical sterilization is not employed in
the preparation of culture media. For a complete discussion of this
topic, consult appropriate references.
Radiation sterilization is an optional treatment for heat-sensitive

materials. This includes ultraviolet light and ionizing radiation.
Ultraviolet light is chemically active and causes excitation of atoms
within the microbial cell, particularly the nucleic acids, producing
lethal mutations. This action stops the organism from reproducing. The
range of the ultraviolet spectrum that is microbiocidal is 240-280 nm.
There is a great difference in the susceptibility of organisms to
ultraviolet radiation; Aspergillus niger spores are 10 times more
resistant than Bacillus subtilis spores, 50 times more resistant than
Staphylococcus aureus and Escherichia coli, and 150 times more
resistant than influenza virus.
Because most materials strongly absorb ultraviolet light, it lacks
penetrating power and its applications are limited to surface treatments.
Much higher energy, 100 to millions of times greater, is generated by
ionizing radiations. These include gamma-rays, high energy X-rays and
high energy electrons.
Ionizing radiation, unlike ultraviolet rays, penetrates deeply into
atoms, causing ionization of the electrons. Ionizing radiation may
directly target the DNA in cells or produce active ions and free radicals
that react indirectly with DNA.
Gamma radiation is used more often than x-rays or high-energy
electrons for purposes of sterilization. Gamma rays are generated by
The Difco Manual
Section I
radioactive isotopes, cobalt-60 being the usual source. Gamma

radiation requires many hours of exposure for sterilization. Validation
of a gamma irradiation procedure includes:4
Establishment of article materials compatibility;
Establishment of product loading pattern and completion of dose
mapping in the sterilization container;
Establishment of timer setting;
Demonstration of the delivery of the required sterilization dose.
The advantages of sterilization by irradiation include low chemical
reactivity, low measurable residues, and few variables to control.3
Gamma irradiation is used for treating many heat-sensitive products
that can also be treated by gaseous sterilization, including medical
materials and equipment, pharmaceuticals, biologicals, certain
prepared media and laboratory equipment.
Sterilization by Filtration1,3
Filtration is a useful method for sterilizing liquids and gases. Filtration
excludes microorganisms rather than destroying them. Two major types
of filters may be used, depth filters and membrane filters.
The membrane filter screens out particles, while the depth filter
entraps them. Membrane filters depend largely on the size of the pores
to determine their screening effectiveness. Electrostatic forces are also
important. A membrane filter with an average pore size of 0.8 m will
retain particulate matter as small as 0.05 m. For removing bacteria, a
pore size of 0.2 m is commonly used. For retention of viruses and
mycoplasmas, pore sizes of 0.01-0.1 m are recommended. Cocci and
bacilli range in size from about 0.3 to 1 m in diameter. Most viruses
are 0.02-0.1 m, with some as large as 0.25 m.
Rating the pore size of filter membranes is by a nominal rating that
reflects the capability of the filter membrane to retain microorganisms
of size represented by specified strains. Sterilizing filter membranes
are membranes capable of retaining 100% of a culture of 10 7
microorganisms of a strain of Pseudomonas diminuta (ATCC 19146)
per square centimeter of membrane surface under a pressure of
not less than 30 psi. These filter membranes are nominally rated 0.22
m or 0.2 m. Bacterial filter membranes (also known as analytical
filter membranes), which are capable of retaining only larger
microorganisms, are labeled with a nominal rating of 0.45 m.
Membrane filters are used for the commercial production of a number
of pharmaceutical solutions and heat-sensitive injectables. Serum
for use in bacterial and viral culture media are often sterilized by
filtration, as well as some sugars that are unstable when heated.
Membrane filtration is useful in testing pharmaceutical and medical
products for sterility.
Sterility Assurance1
Sterility Assurance is the calculated probability that a microorganism
will survive sterilization. It is measured as the SAL, Sterility
Assurance Level, or degree of sterility. For sterility assurance,
Bacillus stearothermophilus which contains steam heat-resistant spores
is employed with steam sterilization at 121C.
The Difco Manual
Monographs
Testing Sterilizing Agents1,5

Sterilization by moist heat (steam), dry heat, ethylene oxide and ionizing radiation is validated using biological indicators. The methods of
sterilization and their corresponding indicators are listed below:
STERILIZATION METHOD
BIOLOGICAL INDICATOR
Steam
Dry heat
Ethylene oxide
Ionizing radiation
Filtration
Bacillus stearothermophilus
Bacillus subtilis var. niger
Bacillus subtilis var. globigii
Bacillus pumilus
Pseudomonas diminuta
For moist heat sterilization, paper strips treated with chemicals that
change color at the required temperature may be used.
The heat-resistant spores of B. stearothermophilus are dried on paper
treated with nutrient medium and chemicals. After sterilization, the
strips are incubated for germination and growth, and a color change
indicates whether they have or have not been activated. Spore strips
should be used in every sterilization cycle.
Glossary1,6
Bioburden is the initial population of living microorganisms in the
product or system being considered.
Biocide is a chemical or physical agent intended to produce the death
of microorganisms.
Calibration is the demonstration that a measuring device produces
results within specified limits of those produced by a reference
standard device over an appropriate range of measurements.
Death rate is the rate at which a biocidal agent reduces the number
of cells in a microbial population that are capable of reproduction.
This is determined by sampling the population initially, during
and following the treatment, followed by plate counts of the surviving
microorganisms on growth media.
D value stands for decimal reduction time and is the time required in
minutes at a specified temperature to produce a 90% reduction in the
number of organisms.
Microbial death is the inability of microbial cells to metabolize and
reproduce when given favorable conditions for reproduction.
Process validation is establishing documented evidence that a
process does what it purports to do.
Sterility Assurance Level is generally accepted when materials
are processed in the autoclave and attain a 10-6 microbial survivor
probability; i.e., assurance of less than one chance in one million that
viable microorganisms are present in the sterilized article.3
Sterilization process is a treatment process from which the probability
of microorganism survival is less than 10-6, or one in a million.
15
Monographs
Thermal Death Time and Thermal-Chemical Death Time are terms

referring to the time required to kill a specified microbial population
upon exposure to a thermal or thermal-chemical sterilizing agent
under specified conditions. A typical thermal death time value with
highly resistant spores is 15 minutes at 121C for steam sterilization.
References
1. Block, S. 1992. Sterilization, p. 87-103. Encyclopedia of
microbiology, vol. 4. Academic Press, Inc., San Diego, CA.
2. Cote, R. J., and R. L. Gherna. 1994. Nutrition and media,
p. 155-178. In P. Gerhardt, R. G. E. Murray, W. A. Wood, and N. R.
Krieg (ed.), Methods for general and molecular bacteriology.
Section I
3. The United States Pharmacopeia (USP XXIII) and The

National Formulary (NF 18). 1995. Sterilization and
sterility assurance of compendial articles, p. 1976-1980.
United States Pharmacopeial Convention Inc., Rockville, MD.
4. Perkins, J. J. 1969. Principles and methods of sterilization
in health sciences, 2nd ed. Charles C. Thomas, Springfield, IL.
5. Leahy, T. J. 1986. Microbiology of sterilization processes. In F. J.
Carleton and J. P. Agalloco (ed.), Validation of aseptic
pharmaceutical processes. Marcel Dekker, Inc. New York, N.Y.
6. Simko, R. J. 1986. Organizing for validation. In F. J. Carleton and
J. P. Agalloco (ed.), Validation of aseptic pharmaceutical processes.
Marcel Dekker, Inc., New York, N.Y.
Quality Control Organisms

Bacteria Control Strain Source
An integral part of quality control testing includes quality control
organisms. Microorganisms should be obtained from reputable sources,
for example, the American Type Culture Collection (ATCC) or
other commercial sources.
9. Store vials at or below -50C (freezer) for one year. Organisms

will keep longer (indefinitely) if stored in an ultra low temperature
freezer or in a liquid nitrogen tank.
To use a frozen culture:

1. Thaw the vial quickly.
Maintenance / Frozen Stock Cultures
2. Use the culture directly or subculture.
If using commercial stock cultures, follow the manufacturers

recommendations for growth and maintenance.
3. Discard any unused cell suspension.
To prepare frozen stock cultures of Staphylococcus species,

Streptococcus species, Enterobacteriaceae and Pseudomonas
aeruginosa:
1. Reconstitute the stock culture, if necessary.
2. Inoculate multiple plates of a general purpose medium (e.g., TSA
or blood agar).
3. Incubate plates for 18-24 hours in an appropriate atmosphere and
at the recommended temperature.
4. Check for purity and correct colony morphology.
5. If necessary, verify biochemical tests.
6. Remove sufficient growth from a confluent area to prepare a 0.5
McFarland standard (1-2 x 108 CFU/ml). For fastidious organisms,
adjust to a 1 McFarland.
7. Suspend the growth in 50-100 ml of cryoprotective medium, e.g.,
Tryptic Soy Broth with 10-15% Glycerol, Skim Milk or sterile
defibrinated sheep blood.
8. Dispense 0.5-1.0 ml into sterile glass or plastic freezing vials.
Prepare enough vials for one year of storage. Assume only
one freeze/thaw cycle per vial. Assume at least one fresh culture
every four weeks.
16
Working Cultures
Prepare no more than three serial subcultures from a frozen stock
culture.
1. Inoculate an agar slant or plate with the frozen stock culture and
incubate overnight.
2. Store the working culture at 2-8C or at room temperature for up
to four weeks.
3. Check for purity and appropriate colony morphology.
OR
1. Use the frozen stock culture directly as a working culture.
Maintain anaerobic cultures in Cooked Meat Medium or another suitable anaerobic medium. Alternatively, use frozen anaerobic cultures.
Test Procedure
1. Inoculate an agar plate from the working culture.
2. Incubate overnight.
3. Suspend 3-5 isolated colonies with typical appearance in a small
volume (0.5-1.0 ml) of TSB. Incubate 4-5 hours in an appropriate
atmosphere and temperature.
4. Adjust the turbidity to 0.5 McFarland and 0.08-0.1 absorbance units
at 625 nm.
The Difco Manual
Section I
OR
1. Adjust an overnight culture to a 0.5 McFarland.
2. Plate 0.01 ml of the specimen to confirm a colony count of
1-2 x 108 CFU/ml. If using a frozen culture, confirm the appropriate
density.
To Test Cultural Response

Non-Selective Media
Dilute the cell suspension 1:100 in normal saline or purified water.
Inoculate each plate with 0.01 ml to give 1-2 x 104 CFU/plate. Reduce
the inoculum ten fold, if necessary, to obtain isolated colonies.
Monographs
Results
For general-purpose media, sufficient, characteristic growth and
typical colony morphology should be obtained with all test strains.
For selective media, growth of designated organisms is inhibited and
adequate growth of desired organisms is obtained. Color and hemolytic
reaction criteria must be met.
Reference
National Committee for Clinical Laboratory Standards. 1996.
Quality assurance for commercially prepared microbiological culture
media, 2nd ed. Approved standard. M22-A2, vol. 16, no. 16. National
Committee for Clinical Laboratory Standards, Wayne, PA.
Selective Media and Tubed Media

Dilute the cell suspension 1:10 in normal saline or purified water. Streak
each plate with 10.01 ml of the suspension to provide 1-2 x 105 CFU/
plate. Reduce the inoculum ten fold, if necessary, to avoid
overwhelming some selective media.
Typical Analysis
Typical chemical compositions have been determined on media
ingredients. The typical analysis is used to select products for research
or production needs when specific nutritional characteristics are
required. The specifications for the typical analysis include:
Physical characteristics,
Nitrogen content,
Amino acids,
Inorganics,
Vitamins, and
Biological testing.
All values are presented as weight/weight; % = g/100 g.
Glossary
Nitrogen
Total Nitrogen: Total nitrogen is usually measured by the
Kjeldhal digestion or titration method. Not all organic nitrogen is
nutritive. Percent (%) nitrogen x 6.25 % proteins, peptides or amino
acids present.
Amino Nitrogen: The amino nitrogen value shows the extent of
protein hydrolysis by measuring the increase in free amino groups.
This is a nutritionally meaningful value.
pH
Changes in pH from specified values, either after storage or processing,
indicate deterioration. These changes are usually accompanied by
darkening of the end product. Hydrolysates vary in their pH resistance
according to their inherent buffering (phosphate) capacity.
Ash
Phosphates
The higher the ash content, the lower the clarity of the prepared
ingredient. The ash content includes sodium chloride, sulfate, phosphates, silicates and metal oxides. Acid-insoluble ash is typically
from silicates found in animal fodder.
High-phosphate ingredients may be unsuitable for pH indicator media

due to the inherent buffering of phosphates. However, phosphates do
aid in gas production, which can be enhanced by deliberate addition of
sodium phosphate.
Moisture
Sodium Chloride
Lower moisture levels (<5%) are preferred. Higher moisture levels in

dehydrated ingredients may reduce stability. In the presence of high
moisture and high ambient temperatures, chemical interactions will
cause darkening of the product and falling pH. These characteristics
indicate product deterioration.
The NaCl content may reflect significant pH adjustments during

processing, e.g., acid hydrolysates. (See Ash).
The Difco Manual
Trace Metals
Trace metals can directly antagonize antimicrobial activity in vitro
or impact toxin production (e.g., C. diphtheriae toxin production is
17
Monographs
maximal at low concentrations, 0.1- 0.2 mg/l, and inhibited at high

concentrations). Chelating agents (e.g., citrate) may be added to
culture media to sequester trace metals and clarify the media.
Antigenic Schema for Salmonella

Update of the Kauffmann-White Schema1
The Centers for Disease Control has modified the Kauffmann-White
antigenic schema originally proposed by Ewing.1-3 The updated schema
are used with Difco Salmonella Antisera as an aid in the serological
identification of Salmonella.
All of the Salmonella serovars belong to two species, S. bongori
containing 18 serovars and S. enterica containing the remaining
2300-plus serovars which are divided among six subspecies.1 The six
subspecies of S. enterica are:
S. enterica subsp. enterica (I or 1)
S. enterica subsp. salamae (II or 2)
S. enterica subsp. arizonae (IIIa or 3a)
S. enterica subsp. diarizonae (IIIb or 3b)
S. enterica subsp. houtenae (IV or 4)
S. enterica subsp. indica (VI or 6)
The legitimate species name for the above strains is S. choleraesuis.
However, this name may be confused with the serotype named
choleraesuis. At the International Congress for Microbiology in 1986,
the International Subcommittee for Enterobacteriaceae agreed to adopt
the species name S. enterica.4 LeMinor and Popoff5 published a
request to the Judicial Commission to use S. enterica as the official
species name. The Judicial Commission ruled that S. choleraesuis is
the legitimate name.6,7 S. enterica is used in many countries and is
favorably accepted as the species name.3,8 The Centers for Disease
Control has adopted this designation until the problem of naming this
species is resolved.1
Nomenclature and classification of these bacteria are ever changing.9
Salmonella and the former Arizona should be considered a single
genus, Salmonella.10 All serovars in subspecies enterica are named.
Serovars in other subspecies (except some in subspecies salamae and
houtenae) are not named. It is recommended that laboratories report
named Salmonella serovars by name and unnamed serovars by
antigenic formula and subspecies. For the most recent information on
nomenclature, consult appropriate references.1,3,9,10,12
Serotypes of Salmonella are defined based on the antigenic structure
of both somatic or cell wall (O) antigens and flagellar (H) antigens.
The antigenic formula gives the O antigen(s) first followed by the
H antigen(s). The major antigens are separated by colons and the
components of the antigens separated by commas. For example, the
antigenic formula for Salmonella typhimurium is Salmonella
1,4,5,12:i:1,2. This means that the strain has O antigen factors 1,4,5
and 12, the flagella phase 1 antigen I, and flagella phase 2 antigens
1 and 2.
Complete identification of Salmonella requires cultural isolation,
biochemical characterization and serotyping. Any serological results
obtained before biochemical identification must be considered as
18
Section I
presumptive identification only. Consult Reference 1 and other appropriate references for complete identification of Salmonella.1,3,9,11-14
References
1. McWhorter-Murlin, A. C., and F. W. Hickman-Brenner. 1994.
Identification and serotyping of Salmonella and an update of the
Kauffmann-White Scheme. Centers for Disease Control and
Prevention, Atlanta, GA.
2. Kauffmann, F. 1969. Enterobacteriaceae, 2nd ed. Munksgaard,
Copenhagen.
3. Ewing, W. H. 1986. Edwards and Ewings identification of
Enterobacteriaceae, 4th ed. Elsevier Science Publishing Co. Inc.,
New York, NY.
4. Penner, J. L. 1988. International committee on systematic
bacteriology taxonomic subcommittee on Enterobacteriaceae. Int.
J. Syst. Bacteriol. 38:223-224.
5. LeMinor, L., and M. Y. Popoff. 1987. Request for an opinion.
Designation of Salmonella enterica sp. nov., nom. rev., as the type
and only species of the genus Salmonella. Int. J. Syst. Bacteriol.
37:465-468.
6. Wayne, L. G. 1991. Judicial Commission of the International
Committee on Systematic Bacteriology. Int. J. Syst. Bacteriol.
41:185-187.
7. Wayne, L. G. 1994. Actions of the Judicial Commission of the
International Committee on Systematic Bacteriology on requests
for opinions published between January 1985 and July 1993. Int.
J. Syst. Bacteriol. 44:177.
8. Old, D. C. 1992. Nomenclature of Salmonella. J. Med. Microbiol.
37:361-363.
9. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and
R. H. Yolken. 1995. Manual of clinical microbiology, 6th ed.
10. Farmer, J. J., III, A. C. McWhorter, D. J. Brenner, and
G. D. Morris. 1984. The Salmonella-Arizona group of
Enterobacteriaceae: nomenclature, classification and reporting.
Clin. Microbiol. Newsl. 6:63-66.
11. Isenberg, H. D. (ed.) 1992. Clinical microbiology procedures handbook, vol. 2. American Society for Microbiology, Washington, D.C.
12. Holt, J. G., N. R. Krieg, P. H. Sneath, J. T. Staley,
S. T. Williams. 1994. Bergeys manual of determinative
bacteriology, 9th ed. Williams & Wilkins, Baltimore, MD.
13. Andrews, W. H., G. A. June, P. Sherrod, T. S. Hammack,
and R. M. Amaguana. 1995. Food and drug administration
bacteriological analytical manual, 8th edition. AOAC International,
Gaithersburg, MD.
14. Russell, S. F., J. DAoust, W. H. Andrews, and J. S. Bailey. 1992.
Salmonella. In C. Vanderzant and D. F. Splittstoesser (eds.),
Compendium of methods for the microbiological examination
of foods, 3rd ed. American Public Health Association,
Washington, D.C.
The Difco Manual
Section II
Agar
Agar
Bacto Agar . Agar Flake . Agar, Granulated . Agar Noble
Agar Bacteriological Technical Agar Bacteriological Technical is a solidifying agent used in pre
paring microbiological culture media. Although Agar Bacteriological

Technical has wider quality control parameters than other
bacteriological agars, solubility, gelation temperature and solidity are
carefully monitored to permit its use.
Intended Use
Bacto Agar is a solidifying agent in which extraneous matter,
pigmented portions and salts have been reduced to a minimum. Bacto
Agar is used in preparing microbiological culture media.
Summary and Explanation
Agar Flake is a solidifying agent used in preparing microbiological

culture media.
Agar is a phycocolloid extracted from a group of red-purple marine

algae (Class Rhodophyceae) including Gelidium, Pterocladia and
Gracilaria. Gelidium is the preferred source for Difco agars. Impurities,
debris, minerals and pigment are reduced to specified levels
during manufacture.
Agar, Granulated is a solidifying agent used in preparing microbiological culture media.

Agar Noble is a solidifying agent that is essentially free of impurities.
It is used in electrophoretic and nutritional procedures and in preparing
microbiological culture media when increased purity is required.
Agar was first suggested for microbiological purposes in 1881 by

Fannie Hesse.1, 2 By the early 1900s, agar became the gelling agent of
choice over gelatin because agar remains firm at growth temperatures
User Quality Control

Identity Specifications
BACTO AGAR
Dehydrated
Appearance:
AGAR FLAKE
Very light beige, free

flowing, homogeneous,
granules.
Solution is very light amber;
very slightly to slightly
opalescent. Clarity is less
than 10 Nephelometric
turbidity units.
16-20%
Less than or equal to 6.5%
300-3,000 ppm
Solution
1.5% solution
soluble in distilled
or deionized water
upon boiling
Loss on Drying (LOD)
Ash6
Calcium
g/g (ppm)
Magnesium
g/g (ppm)
Melting Point
83-89C
Gelation Point
32-39C
50-1,000 ppm
AGAR, GRANULATED
AGAR NOBLE
AGAR
BACTERIOLOGICAL TECHNICAL
Off-white to light
beige, free flowing,
flakes.
Solution is very
light to light amber,
very slightly to
slightly opalescent.
Very light beige to

light tan, free flowing
homogeneous, granules.
Solution is very light
to medium amber,
very slightly opalescent
to opalescent.
White to off-white, free

flowing,homogeneous,
fine granules.
Solution is colorless,
clear to very
Less than or equal to 20%

2-5.2%
Less than or equal
to 3,400 ppm
Less than or equal
to 1,850 ppm
Greater than or
equal to 85C
32-39C
Less than or equal to 20%

Less than or equal to 6.5%
Less than or equal
to 3,000 ppm
Less than or equal
to 1,000 ppm
83-89C
Less than or equal to 20% Less than or equal to 20%

Less than or equal to 2% Less than or equal to 6.5%
100-2,600 ppm
Less than or equal
to 3,000 ppm
0-750 ppm
Less than or equal
to 1,300 ppm
Greater than or
Greater than or
equal to 85C
equal to 85C
32-39C
32-39C
32-39C
Very light to medium

beige, free flowing,
homogeneous.
Solution is very light
to medium amber,
opalescent.
Cultural Response
Prepare the agar formulation of Nutrient Broth (0003) or LB Broth, Miller (0446) by adding 1.5% agar.
Sterilize and pour plates. Inoculate with 100-1,000 CFU of the indicated test organisms and incubate
at 35 2C for 18-24 hours. Record recovery.
Nutrient Broth with:

Escherichia coli ATCC 25922*
Staphylococcus aureus ATCC 25923*
LB Broth, Miller with:
Escherichia coli ATCC 33694 (HB101)
Saccharomyces cerevisiae ATCC 9763
BACTO
AGAR
AGAR
FLAKE
Good
Good
Good
Good
*These cultures are available as Bactrol Disks and should be

used as directed in the Bactrol Disks Technical Information.
The Difco Manual
AGAR,
GRANULATED
AGAR
NOBLE
AGAR BACTERIOLOGICAL
TECHNICAL
Good
Good
Good
Good
Good
Good
Can of
Bacto Agar
21
Agar
Section II
for many pathogens. Agar is also generally resistant to a breakdown

by bacterial enzymes. The use of agar in microbiological media
significantly contributed to the advance of microbiology, paving the
way for pure culture isolation and study.
Agar is a gel at room temperature, remaining firm at temperatures
as high as 65C.3 Agar melts at approximately 85C, a different
temperature from that at which it solidifies, 32-40C. This property is
known as hysteresis. Agar is generally resistant to shear forces; however,
different agars may have different gel strengths or degrees of stiffness.
Agar is typically used in a final concentration of 1-2% for solidifying
culture media. Smaller quantities (0.05-0.5%) are used in media for
motility studies (0.5% w/v) and for growth of anaerobes (0.1%) and
microaerophiles.3
Specifications for bacteriological grade agar include good clarity,
controlled gelation temperature, controlled melting temperature, good
diffusion characteristics, absence of toxic bacterial inhibitors, and
relative absence of metabolically useful minerals and compounds.
Product Applications
Bacto Agar is optimized for beneficial calcium and magnesium
content. Detrimental ions such as iron and copper are reduced. Bacto
Agar is recommended for clinical applications, auxotrophic studies,
bacterial and yeast transformation studies, and bacterial molecular
genetics applications.4, 5
Agar Flake is recommended for general bacteriological purposes. The
quality is similar to Bacto Agar. However, the flakes are more easily
wetted than the granules found in Bacto Agar.
Agar, Granulated is qualified for culturing recombinant strains of
Escherichia coli (HB101) and Saccharomyces cerevisiae. Agar,
Granulated may be used for general bacteriological purposes where
clarity is not a strict requirement.
Noble Agar is extensively washed and bleached. This agar should be
used for applications where extreme clarity and high purity are required.
Noble Agar is suitable for immunodiffusion, some electrophoretic
applications, and as a substrate for mammalian or plant tissue culture.
Agar Bacteriological Technical is suitable for many bacteriological
applications. This agar is not highly processed, has broader technical
specifications than other Difco agars, and is not recommended for
growth of fastidious organisms.
Typical Analysis
BACTO
AGAR
AGAR,
GRANULATED
AGAR
NOBLE
AGAR
BACTERIOLOGICAL
TECHNICAL
Physical Characteristics
Ash (%)
Color
Texture
3.6
3.4
1.3
4.1
lt. beige
lt. beige
off white
lt beige
granular
granular
fine
granular
free-flowing free-flowing granular free-flowing
free flowing
Clarity, 1.5% Soln (NTU) 4.3
5.3
3.7
26.2
Loss on Drying (%)
17.3
12.2
16.0
18.2
pH, 1.5% Soln
6.5
6.6
5.7
6.9
600
560
700
613
Gel Strength (g/cm2)
Gelation Point(C)
35C
35C
35C
36C
Melting Point (C)
88C
88C
87C
88C
22
AGAR,
GRANULATED
AGAR
NOBLE
AGAR
BACTERIOLOGICAL
TECHNICAL
<1,000
<1,000
<1,000
<1,000
<1,000
<1,000
4,300
2,725
0.179
0.021
<0.001
<0.001
0.002
<0.001
0.068
<0.001
<0.005
<0.005
0.121
0.837
1.778
0.841
<0.001
<0.001
0.133
<0.005
<0.001
<0.001
0.003
<0.001
0.041
<0.001
<0.005
0.010
0.079
0.776
1.710
0.868
<0.001
<0.001
0.015
<0.050
<0.001
<0.001
<0.001
<0.001
0.002
<0.001
<0.050
<0.050
0.022
0.335
0.663
0.333
<0.001
<0.001
0.110
0.172
<0.001
<0.001
0.002
<0.001
0.093
<0.001
<0.005
0.015
0.124
0.932
0.367
0.646
<0.001
<0.001
BACTO
AGAR
Biological Testing (CFU/g)

Spore Count
Standard Plate Count
Inorganics (%)
Calcium
Chloride
Cobalt
Copper
Iron
Lead
Magnesium
Manganese
Nitrate
Phosphate
Potassium
Sodium
Sulfate
Sulfur
Tin
Zinc
Precautions
1. For Laboratory and Manufacturing Use.
2. Follow proper established laboratory procedures in handling and
disposing of infectious materials.
Storage
Store dehydrated agar below 30C. Dehydrated agar is very hygroscopic.
Keep container tightly closed.
Expiration Date
The expiration date applies to the product in its intact container when
stored as directed. Do not use the product if it fails to meet specifications
for identity and performance.
Procedure
Materials Provided
Bacto Agar
Agar Flake
Agar, Granulated
Agar Noble
Agar Bacteriological Technical
Materials Required But Not Provided

Materials vary depending on the application.
Method of Preparation
Method of preparation varies depending on the application.
Specimen Collection and Preparation

Obtain and process specimens according to the techniques and
procedures established by laboratory policy.
Test Procedure
See appropriate references for specific procedures using Bacto Agar,
Agar Flake, Agar, Granulated, Agar Noble or Agar Bacteriological
Technical.
The Difco Manual
Section II
2xYT
Results

Refer to appropriate references and procedures for results.
References
1. Hesse, W. 1894. ber die quantitative Bestimmung der in der Luft
enthaltenen Mikroorganismen. Mitt. a.d. Kaiserl. Gesh. Berlin
2:182-207.
2. Hitchens, A. P., and M. C. Leikind. 1939. The introduction of
agar-agar into bacteriology. J. Bacteriology 37:485-493.
3. Selby, H. H., and T. A. Selby. 1959. Agar. In Whister (ed.),
Industrial gums. Academic Press Inc., New York, NY.
cloning, a laboratory manual, 2nd ed. Cold Spring Harbor
Laboratory Press, NY, NY.
5. Schiestl, R. H., and R. Daniel Geitz. 1989. High efficiency
transformation of intact yeast cells using single stranded nucleic
acids as a carrier. Current Genetics 16:339-346.
Bacto 2xYT
Intended Use
Bacto 2xYT is used for cultivating recombinant strains of Escherichia coli.

2xYT is a nutritionally rich growth medium designed for growth of
recombinant strains of Escherichia coli. This medium is also used for
propagation of M13 bacteriophage for sequencing and phage display
Packaging
Bacto Agar
100
1
2
10
Agar Flake
500 g
0970-17
Agar, Granulated
100 g
2 kg
10 kg
0145-17
0145-07
0145-08
Agar Noble
100 g
500 g
0142-15
0142-17
Agar Bacteriological Technical
500 g
2 kg
10 kg
0812-17
0812-07
0812-08
Principles of the Procedure

Tryptone and Yeast Extract provide the necessary nutrients and cofactors
required for excellent growth of E. coli. Sodium Chloride is included to
provide a suitable osmotic environment.
Formula
2xYT
Formula Per Liter
Cultural Response
Prepare 2xYT per label directions. Inoculate and incubate at
35 2C for 18-24 hours.
ORGANISM
Escherichia coli (C600)

Escherichia coli (JM103)
Escherichia coli (HB101)
Escherichia coli (DH-1)
ATCC
INOCULUM
CFU
GROWTH
23724
39403
47014
33694
33849
53868
100-300
100-300
100-300
100-300
100-300
100-300
Good
Good
Good
Good
Good
Good
The cultures listed are the minimum that should be used for
performance testing.
The Difco Manual
0140-15
0140-01
0140-07
0140-08
research.1-3 The components of 2xYT provide nitrogen and growth

factors that allow bacteriophage to reproduce in large quantities
without exhausting the host. E. coli grows more rapidly in this rich
medium because it provides amino acids, nucleotide precursors,
vitamins and other metabolites that the cell would otherwise have to
synthesize.2

Dehydrated Appearance: Light beige, free-flowing, homogeneous.
Solution:
3.1% solution, soluble in distilled or
deionized water. Solution is light to
medium amber, clear.
Prepared Medium:
Light to medium amber, clear.
Reaction of 3.1%
Solution 25C:
pH 7.0 0.2
g
lb
kg
kg
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 g
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Final pH 7.0 0.2 at 25C
Precautions
1. For Laboratory Use.
2. Follow proper established laboratory procedure in handling and
Storage
Store the dehydrated medium below 30C. The powder is very hygroscopic. Keep container tightly closed.
Store the prepared medium at 2-8C.
Expiration Date
stored as directed. Do not use a product if it fails to meet specifications
23
A-1 Medium
Section II
Procedure
Test Procedure
Materials Provided
Please consult appropriate references for recommended test procedures.1-3
2xYT
Results
Growth is evident in the form of turbidity.
Flasks with closures

Distilled or deionized water
Autoclave
Incubator (35C)
References

cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y.
2. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G.
Seidman, J. A. Smith, and K. Struhl. 1994. Current protocols in
molecular biology, vol 1. Current Protocols, New York, N.Y.
3. Davis, L. G., M. D. Dibner, and J. F. Battey. 1986. Basic
methods in molecular biology. Elsevier, New York, N.Y.
Refer to appropriate references for specimen collection and preparation.
Packaging
1. Dissolve 31 grams in 1 liter of distilled or deionized water.
2. Autoclave at 121C for 15 minutes.
2xYT
Bacto A-1 Medium
500 g
0440-17-0
Intended Use
Bacto A-1 Medium is used for detecting fecal coliforms in water.
Also Known As
A-1 Medium is also referred to as A-1 Broth.
Since the early 1900s enumeration of coliform organisms, specifically

E. coli, has been used to determine water purity. Elevated-temperature,
most-probable-number (MPN) methods are routinely used for the
analysis of water and food samples for the presence of fecal coliforms.
One limiting factor in using E. coli is the length of time required for
complete identification.1 A-1 Medium was formulated to hasten the
recovery of E. coli and reduce the incidence of false positive cultures.

Dehydrated Appearance: Light beige, lumpy.
Solution:
3.15% solution, soluble in distilled or deionized water
on boiling. Solution is light amber, opalescent immediately
after sterilization. Solution is light amber, clear, may have
flocculent precipitate upon cooling.
Prepared Medium:
(When cooled to room temperature) - Light amber, clear,
flocculent precipitate may be present.
Reaction of 3.15%
Solution at 25C:
pH 6.9 0.1
Cultural Response
Prepare A-1 Medium per label directions. Prepare tubes by placing fermentation vials
and 10 ml amounts of medium into tubes. Inoculate and incubate at
35 2C for 3 hours. Transfer tubes to a 44.5C waterbath for 21 2 hours.
ORGANISM
Bacillus subtilis
Enterobacter aerogenes
Enterococcus faecalis
Escherichia coli
Escherichia coli
ATCC
INOCULUM
CFU(APPROX.)
GROWTH
6633
13048*
19433*
25922*
13762
100
100
100
100
100
none
poor to good/may produce gas
none to poor
good/with gas production
good/with gas production
The cultures listed are the minimum that should be used for performance testing.
Bacillus subtilis is available as Subtilis Spore Suspension.
24
Uninoculated
tube
Escherichia coli
ATCC 25922
with fermentation vial

used as directed in Bactrol Disk Technical Information.
The Difco Manual
Section II
A-1 Medium
In 1972 Andrews and Presnell developed A-1 Medium. A-1 Medium

recovers E. coli from estuarine water in 24 hours instead of 72 hours,
and in greater numbers without the preenrichment step.2 Using a
3-hour preincubation step for the enumeration of coliforms in
chlorinated wastewater gave results that were statistically comparable
to those obtained in the two-step MPN technique.3
A-1 Medium can be used in a single-step procedure for the detection

of fecal coliforms in source water, seawater, treated wastewater and
foods. Prior enrichment in a presumptive medium is not required.4
A-1 Medium conforms to standard methods for the isolation of fecal
coliforms in water and foods.4,5,6
NOTE: For 10 ml water samples, prepare double-strength medium to

ensure the ingredient concentrations are not reduced below those of
the standard medium.4
Tryptone provides the nitrogen, vitamins, minerals and amino acids in

A-1 Medium. Lactose is the carbon source and, in combination with
Salicin, provides energy for organism growth. Sodium Chloride
maintains the osmotic balance of the medium. Triton X-100 is a surfactant.
Formula
A-1 Medium
Formula Per Liter
g
g
g
g
ml
Precautions
Storage
Store the dehydrated medium below 30C. The dehydrated medium is
very hygroscopic. Keep container tightly closed.
Store prepared medium in the dark at room temperature for no longer
than 7 days.4
Expiration Date
stored as directed. Do not use a product if it fails to meet the specifications
Procedure
Materials Provided
A-1 Medium

Glassware
Fermentation vials
Autoclave
Incubator (35C)
Waterbath (44.5C)
Test tubes
The Difco Manual
Suspend 31.5 grams in 1 liter distilled or deionized water.

Heat to boiling to dissolve completely.
Dispense into tubes containing inverted fermentation vials.
Autoclave at 121C for 10 minutes.

Obtain and process specimens according to the procedures established
by laboratory policy or standard methods.4,5,6
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Salicin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Triton X-100 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.
2.
3.
4.
Test Procedure
1. Inoculate tubes of A-1 Medium as directed in standard methods.4,5,6
2. Incubate at 35 0.5C for 3 hours.
3. Transfer tubes to a water bath at 44.5 0.2C and incubate for an
additional 21 2 hours.
4. Maintain water level in bath above level of liquid in inoculated tubes.
Results5
Gas production in the inverted vial, or dissolved gas that forms fine
bubbles when slightly agitated, is a positive reaction indicating the
presence of fecal coliforms. Calculate fecal coliform densities using
MPN tables from standard methods.
Limitations of the Procedure

1. Since the nutritional requirements of organisms vary, some strains may
be encountered that fail to grow or grow poorly on this medium.
2. Fecal coliform counts are usually greater than E. coli counts.5
3. Interpretation of test procedure using A-1 Medium requires
understanding of the microflora of the specimen.5
References
1. Andrews, W. H., C. D. Diggs, and C. R. Wilson. 1975.
Evaluation of a medium for the rapid recovery of Escherichia coli
from shellfish. Appl. Microbiol. 29:130- 131.
2. Andrews, W. H., and M. W. Presnell. 1972. Rapid recovery of
Escherichia coli from estuarine water. Appl. Microbiol.
23:521-523.
3. Standridge, and Delfino. 1981. Appl. Environ. Microbiol. 42:918.
4. Eaton, A. D., L. S. Clesceri, and A. E. Greenberg (ed.). 1995.
Standard methods for the examination of water and wastewater,
19th ed. American Public Health Association, Washington, D.C.
5. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992.
Compendium of methods for the microbiological examination of
food, 3rd ed. American Public Health Association, Washington, D.C.
6. Association of Official Analytical Chemists. 1995. Bacteriological
analytical manual, 8th ed. AOAC International, Gaithersburg, MD.
Packaging
A-1 Medium
500 g
1823-17
25
AC Broth & AC Broth w/o Dextrose
Section II
Bacto AC Broth
Bacto AC Broth w/o Dextrose
Intended Use
Bacto AC Broth is used for cultivating a wide variety of microorganisms
and for the sterility testing of turbid or viscous solutions and other
materials not containing mercurial preservatives.
Bacto AC Broth w/o Dextrose is used, with the addition of a carbohydrate,
for cultivating a wide variety of microorganisms.

AC Broth and AC Broth w/o Dextrose possess growth-promoting
properties for voluminous growth of a wide variety of microorganisms.
Christensen1 and Malin and Finn2 reported that AC Medium does not
exhibit the toxicity shown by media containing sodium thioglycollate.

AC Broth
Dehydrated Appearance: Light tan, free-flowing, homogeneous.
Solution:
deionized water. Solution is medium
to dark amber, clear to very slightly
opalescent.
Prepared Tubes:
Light to medium amber, clear to very
Reaction of 3.4%
Solution at 25C:
pH 7.2 0.2
AC Broth w/o Dextrose
Solution:
deionized water. Solution is medium
to dark amber, clear to very slightly
opalescent.
Prepared Tubes:
Medium to dark amber, clear to very
Reaction of 2.92%
Solution at 25C:
pH 7.2 0.2
Cultural Response
Prepare AC Broth or AC Broth w/o Dextrose per label
directions. Inoculate and incubate at 35 2C for 18-48 hours.
Corynebacterium diphtheriae
Type mitis
Streptococcus pneumoniae
Streptococcus pyogenes
ATCC
INOCULUM
CFU
8024 100-1,000
6305 100-1,000
19615* 100-1,000
GROWTH
good
good
good
*This culture is available as Bactrol Disks and should be used
as directed in Bactrol Disks Technical Information.
26
AC Broth w/o Dextrose has the same formula as AC Broth except that
the dextrose is omitted, allowing for the addition of other carbohydrates
if desired.

Proteose Peptone No. 3, Beef Extract, and Malt Extract provide
the carbon and nitrogen sources required for good growth of a wide
variety of organisms. Vitamins and cofactors required for growth as
well as additional sources of nitrogen and carbon are provided by Yeast
Extract. Dextrose is included in AC Broth as a carbon energy source.
Ascorbic Acid is added to clarify the solution.
Formula
ORGANISM
Several early studies reported on the wide variety of organisms able to

grow on AC Medium.3,4,5 AC Broth is suitable for use in the detection
of obligately aerobic contaminants in biologicals and other products.
AC Broth and AC Broth w/o Dextrose are also useful in the isolation
and cultivation of many common pathogenic and saprophytic aerobes.6
The media can be used to test the sterility of biologicals and solutions
that do not contain mercurial preservatives. Fluid Thioglycollate
Medium should be employed for the sterility testing of solutions
containing mercurial preservatives.
AC Broth
Formula Per Liter
Bacto Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Bacto Malt Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
g
g
g
g
g
g

Formula Per Liter
Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
g
g
g
g
g
Precautions
Storage
Store the dehydrated media below 30C. The dehydrated media are
AC Broth
Store prepared medium at 15-30C. After prolonged storage, reheat in
flowing steam or a boiling water bath for a few minutes to drive off
dissolved gases. Cool without agitation.
Store prepared medium at 15-30C.
The Difco Manual
Section II
APT Agar & APT Broth
Expiration Date
Results
The expiration date applies to the products in their intact containers

when stored as directed. Do not use a product if it fails to meet
specifications for identity and performance.
Procedure
Materials Provided
AC Broth

Glassware
Autoclave
Incubator (35C)
1. Suspend appropriate amount of medium in 1 liter distilled or
deionized water:
AC Broth - 34 grams;
AC Broth w/o Dextrose - 29.2 grams.
2. If necessary, warm slightly to dissolve completely.
3. Dispense as desired. Autoclave at 121 C for 15 minutes.
If the medium is not used the same day it is sterilized, place in
flowing steam or a boiling water bath for a few minutes to drive off
dissolved gases. Allow to cool without agitation.
Test Procedure

1. When reheating prepared media to drive off dissolved gases do not
overheat because this may result in decreased growth.
References
1. Paper read at New York Meeting. Am. Pub. Health Assoc., 1944.
2. Malin, B., and R. K. Finn. 1951. The use of a synthetic resin in
anaerobic media. J. Bacteriol. 62:349-350.
3. Reed, G. B., and J. H. Orr. 1943. Cultivation of anaerobes and
oxidative-reduction potentials. J. Bacteriol. 45:309-320.
4. Schneiter, R., J. E. Dunn, and B. H. Caminita. 1945. Studies
in connection with the selection of a satisfactory culture medium
for bacterial air sampling. Pub. Health Reports 60:789-806.
5. Kolb, R. W., and R. Schneiter. 1950. The germicidal and
sporicidal efficacy of methyl bromide for Bacillus anthracis.
J. Bacteriol. 59:401-412.
6. MacFaddin, J. D. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, vol. 1, p. 13-14.
Williams & Wilkins, Baltimore, MD.
Packaging
AC Broth
500 g
10 kg
0317-17
0599-08
See appropriate references for specific procedures.
Bacto APT Agar

Bacto APT Broth
Intended Use
Bacto APT Agar is used for cultivating heterofermentative lactobacilli
and other organisms requiring high thiamine content. It is also used for
maintaining stock cultures of Lactobacillus viridescens ATCC 12706
used in the assay of thiamine.
requiring bacteria, specifically Lactobacillus viridescens. Their

formulations led to the development of APT Agar and APT Broth.
The lactic acid bacteria, a group of acid producing bacteria, include
the genera Streptococcus, Leuconostoc, Pediococcus and Lactobacillus.3
These organisms are widespread in nature and are associated with
bacterial spoilage of foods such as dairy, meat and vegetable
products.3 One use of APT Agar and APT Broth is for cultivating
these heterofermentative lactic acid bacteria from food products.3
Bacto APT Broth is used for culturing Lactobacillus viridescens

ATCC 12706 used in the assay of thiamine. It is also used for cultivating
heterofermentative lactobacilli and other organisms requiring high
thiamine content.
APT Agar and APT Broth are also used in the microbiological assay of
thiamine. In the assay, APT Agar is the maintenance medium that
preserves the viability and sensitivity of Lactobacillus viridescens
ATCC 12706. APT Broth is used for growing Lactobacillus viridescens
ATCC 12706 and preparing the inoculum.
Also Known As
All Purpose Tween
APT Agar and APT Broth contain Tryptone as a source of carbon,

nitrogen, vitamins and minerals. Yeast Extract supplies B-complex
vitamins which stimulate bacterial growth. Dextrose is the carbohydrate. The Manganese Chloride, Magnesium Sulfate and Ferrous
Sulfate provide ions used in replication by lactobacilli. Sorbitan
Monooleate Complex is a source of fatty acids required by lactobacilli.
Bacto Agar is the solidifying agent in APT Agar.

Evans and Niven1 investigated cultivating the heterofermentative
lactobacilli that cause the faded or greenish discoloration of cured meat
products, while Deibel, Evans and Niven2 investigated thiamine
The Difco Manual
27
APT Agar & APT Broth
Section II
Formula
APT Broth
Formula Per Liter
APT Agar
Formula Per Liter
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Thiamine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . 0.001
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Manganese Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.14
Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.04
Sorbitan Monooleate Complex . . . . . . . . . . . . . . . . . . . . . . 0.2
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
Precautions
Storage
APT Agar
Dehydrated Appearance: Light beige, free-flowing,
homogeneous.
Solution:
6.12%, soluble in distilled or
deionized water on boiling.
Solution, upon cooling, is medium
amber, clear to slightly opalescent,
may have a slight precipitate.
Prepared Medium:
Medium amber, clear to slightly
opalescent, may have a slight
precipitate.
Reaction of 6.12%
Solution at 25C:
pH 6.7 0.2

APT Broth
Solution:
4.62%, soluble in distilled or
deionized water with slight heating.
Solution, upon cooling, is light to
medium amber, clear to very slightly
precipitate.
Prepared Medium:
Light to medium amber, clear to
very slightly opalescent without
significant precipitate.
Reaction of 4.62%
Solution at 25C:
pH 6.7 0.2
Materials Required but not Provided
Cultural Response
Prepare APT Agar and APT Broth per label directions.
Inoculate and incubate at 35 2C for 24-48 hours.
ORGANISM
ATCC
Lactobacillus fermentum
Lactobacillus viridescens
9338
12706
INOCULUM
CFU
GROWTH
100-1,000
100-1,000
good
good
28

Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Thiamine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . 0.001
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.04
Expiration Date
Procedure
Materials Provided
APT Agar
APT Broth
Glassware
Autoclave
Incubator (35C)
1. Suspend the medium in 1 liter distilled or deionized water:
APT Agar:
61.2 grams;
APT Broth:
46.2 grams.
2. Heat to boiling to dissolve completely.
4. Avoid overheating.

Test Procedure
For maintaining stock cultures of Lactobacillus viridescens
ATCC 12706 prepare a stab inoculation. Prepare stock cultures in
triplicate at monthly intervals. One of the transfers is saved for the
The Difco Manual
Section II
Acetate Differential Agar
preparation of stock cultures. The others are used to prepare inoculum

in APT Broth for assay as needed. Following incubation at 35-37C for
24-48 hours, store stock cultures at 2-8C.
Results
3. Vedamuthu, E. R., M. Raccach, B. A. Glatz, E. W. Seitz, and M.

S. Reddy. 1992. Acid-producing microorganisms, p. 225-238. In
C. Vanderzant, and D. F. Splittstoesser (ed.), Compendium of
methods for the microbiological examination of foods, 3rd ed.
American Public Health Association, Washington, D.C.
Packaging
References
1. Evans, J. B., and C. F. Niven, Jr. 1951. Nutrition of the
heterofermentative lactobacilli that cause greening of cured meat
products. J. Bact. 62:599-603.
2. Deibel, R. H., J. B. Evans, and C. F. Niven, Jr. 1957.
Microbiological assay for thiamine using Lactobacillus
viridescens. J. Bact. 74:818-821.
APT Agar
500 g
2 kg
10 k
0654-17
0654-07
0654-08
APT Broth
500 g
0655-17
Bacto Acetate Differential Agar
Intended Use
Bacto Acetate Differential Agar is used for differentiating microorganisms
of the Shigella genus from those of the Escherichia genus.
Also Known As
Acetate Differential Agar is also known as Sodium Acetate Agar.

Although classified taxonomically as different species for clinical
reasons, Shigella species and E. coli are essentially the same genus and
species. Their DNA relatedness is high, they are difficult to differentiate
biochemically, and they cross-react serologically.1 One way they can
be differentiated is by using a medium containing sodium acetate as a
sole source of carbon. Many strains of E. coli are able to use acetate as
a carbon source, whereas typical cultures of Shigella are unable to grow.
Trabulsi and Ewing 2 developed Acetate Differential Agar by
substituting sodium acetate for sodium citrate in their basal medium,
Simmons Citrate Agar. They demonstrated that none of the Shigella
tested grew on the Acetate Differential Agar. A large percentage of
E. coli strains, belonging to various O antigen groups, did use the
acetate within 2 to 7 days of incubation.
The majority of Salmonella, Citrobacter, Klebsiella, Enterobacter and
Serratia groups use acetate and grow on Acetate Differential Agar
within 1 to 7 days. Proteus and Providencia groups, however, fail
to grow on the medium. Several standard methods list Acetate

Dehydrated Appearance: Medium yellowish-tan to light green, free-flowing,
homogeneous.
Solution:
on boiling. Solution is emerald green, slightly opalescent.
Prepared Medium:
Emerald green to green, slightly opalescent.
Reaction of 2.92%
Solution at 25C:
pH 6.7 0.1
Cultural Response
Prepare Acetate Differential Agar per label directions. Inoculate the medium
and incubate at 35 2C for 2-7 days. Acetate utilization is indicated by a color
change of the slant from green to blue.
ORGANISMS
Escherichia coli
Shigella sonnei
ATCC
GROWTH
25922*
good
25931* poor to good
APPEARANCE
blue
green
The organisms listed are the minimum that should be used for performance testing.
The Difco Manual
Uninoculated
tube
Escherichia coli
ATCC 25922
29
Section II
Differential Agar as a possible medium for the differentiation of

Enterobacteriaceae.2,3,4
Incubator (35C)
0.85% NaCl solution
Acetate Differential Agar consists of a mixture of salts and sodium

acetate as a sole source of carbon. Brom Thymol Blue is added to
detect the alkaline products resulting from acetate utilization. Mono
Ammonium Phosphate and Dipotassium Phosphate provide buffering
capability. Bacto Agar is a solidifying agent.
1.
2.
3.
4.
5.
Formula
Formula Per Liter
Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Mono Ammonium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . 1
Bacto Brom Thymol Blue . . . . . . . . . . . . . . . . . . . . . . . . 0.08
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Test Procedure
g
g
g
g
g
g
g
Precautions
2. IRRITANT. IRRITATING TO EYES, RESPIRATORY SYSTEM
AND SKIN. Avoid contact with skin and eyes. Do not breathe dust.
Wear suitable protective clothing. Keep container tightly closed.
FIRST AID: In case of contact with eyes, rinse immediately with
plenty of water and seek medical advice. After contact with skin,
wash immediately with plenty of water. If inhaled, remove to fresh
air. If not breathing, give artificial respiration. If breathing is
difficult, give oxygen. Seek medical advice. If swallowed seek
medical advice immediately and show this container or label.
Storage
Expiration Date
Procedure
Materials Provided

Boil to dissolve completely.
Dispense into tubes to allow a 10 mm butt and a 30 mm slant.
Allow tubes to cool in a slanted position to give the recommended
butt and slant size.
1. Inoculate agar slant surfaces with 16-18 hour cultures emulsified

in 1 ml of 0.85% sodium chloride solution.
2. Incubate aerobically at 35 2C for at least 7 days; read daily,
examining for a change in the color of the medium from green
to blue.
Results
Positive: Blue
Negative: Green

1. Some strains of E. coli and nonmotile, anaerogenic E. coli
(Alkalescens-Dispar) grow slowly or not at all and, thus, may give
a false-negative reaction.
2. Further biochemical, physiological and serological tests are required
to differentiate species.
3. False-positive results may occur from a too heavy inoculum.
4. MacFaddin5 suggests that correct results occur only when some
syneresis fluid is present in the bottom of the tube (junction of the
slant and butt).
References
1. Gray, L. D. 1995. Escherichia, Salmonella, Shigella, and Yersinia,
pp. 450-456. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C.
Tenover, and R. H. Yolken (eds.). Manual of clinical microbiology,
6th ed. American Society for Microbiology, Washington, D.C.
2. Vanderzant, C., and D. F. Splittstoesser (eds.). 1992. Compendium of methods for the microbiological examination of food,
3rd ed. American Public Health Association, Washington, D.C.
3. Andrews, W. H., G. A. June, and P. S. Sherrod. 1995. Shigella,
p. 6.01-6.06. In Bacteriological analytical manual, 8th ed. AOAC
International, Gaithersburg, MD.
4. MacFaddin, J. F. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, vol. 1, p. 17-20.
Packaging
500 g
0742-17
Glassware
Autoclave
30
The Difco Manual
Section II
Actinomycete Isolation Agar & Glycerol
Bacto Actinomycete Isolation Agar

Bacto Glycerol
Intended Use
Bacto Actinomycete Isolation Agar is used with added glycerol for
isolating and cultivating actinomycetes from soil and water.
Bacto Glycerol is used in preparing microbiological culture media.
Formula

Although some genera are important to human medicine, most of the
actinomycetes are part of the indigenous flora of soil, water, and
vegetation. Actinomycetes may impart a musty odor to water or a
muddy flavor to fish.2 Actinomycetes can cause massive growths which
will form a thick foam in the activated sludge process, causing a
disruption in wastewater treatment. 3,4 Actinomycetes are gram
positive, acid-fast cells, growing as filaments that may branch and may
form irregularly shaped rods and cocci.
Olsen1 formulated Actinomycete Isolation Agar for isolating and
cultivating actinomycetes from soil and water. The formula,
supplemented with Glycerol, is a highly purified fermentable alcohol
used occasionally for differentiating certain bacteria and in media for
isolating and culturing fastidious bacteria.

Actinomycete Isolation Agar contains Sodium Caseinate which is a
source of nitrogen. Asparagine is an amino acid and a source of

Actinomycete Isolation Agar
Solution:
deionized water on boiling. Solution
is light to medium amber, opalescent
to opaque with precipitation.
Prepared Medium:
Medium amber, opalescent.
Reaction of 2.2%
Solution with 0.5%
Glycerol at 25C:
pH 8.1 0.2

Formula Per Liter
Sodium Caseinate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Asparagine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
Sodium Propionate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.001
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g

Glycerol
Not applicable
Precautions
2. MAY BE IRRITATING TO EYES, RESPIRATORY SYSTEM
AND SKIN. (US) Avoid contact with skin and eyes. Do not breathe
dust. Wear suitable protective clothing. Keep container tightly closed.
Storage
Store Glycerol at 15-30C.
Expiration Date
Cultural Response
Prepare Actinomycete Isolation Agar per label directions
with the addition of 0.5% Glycerol. Inoculate and incubate at
30 2C for up to 72 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Streptomyces achromogenes
Streptomyces albus
Streptomyces lavendulae
12767
3004
8664
100-1,000
100-1,000
100-1,000
good
good
good
The Difco Manual
organic nitrogen. Sodium Propionate is a substrate used in anaerobic

fermentation. Dipotassium Phosphate provides buffering capability to
maintain pH balance. Magnesium Sulfate and Ferrous Sulfate provide
sources of sulfates and metallic ions. Bacto Agar is the solidifying
agent. The added Glycerol is a source of carbon.
Procedure
Materials Provided
Glycerol
31
Agar Medium No. F
Section II
Results
Glassware
Petri dishes
Autoclave
Incubator (30C)
References
1.
2.
3.
4.
Suspend 22 grams in 1 liter distilled or deionized water.

Add 5 grams Glycerol.

1. Collect specimens in sterile containers or with sterile swabs.
Transport immediately to the laboratory, in accordance with
recommended guidelines.
2. Process each specimen as appropriate for that specimen.
Test Procedure
Inoculate medium and incubate at 30C for up to 72 hours.
Bacto Agar Medium No. F
Intended Use
Bacto Agar Medium No. F is a selective medium used for detecting
Enterobacteriaceae and other gram-negative bacteria in pharmaceutical
products.
1. Olsen, E. H. 1960. Personal Communication.

2. Eaton, A. D., L. S. Clesceri, and A. E. Greenberg. 1995.
3. Lechevalier, H. A. 1975. Actinomycetes of sewage-treatment
plants. Environ. Protection Technol. Ser., EPA-600/2-75-031,
U. S. Environmental Protection Agency, Cincinnati, OH.
4. Lechevalier, M. P., and H. A. Lechevalier. 1974. Nocardia
amarae, sp. nov., an actinomycete common in foaming activated
sludge. Int. J. Syst. Bacteriol. 24:278.
Packaging
Glycerol
100
500
100
500
g
g
g
g
0957-15
0957-17
0282-15
0282-17

Agar Medium No. F is based on the formula for Agar Medium F (Agar
Medium with Bile, Crystal Violet, Neutral Red and Glucose) described
in DAB, 10th Edition. Agar Medium No. F is recommended for use in
the detection of Enterobacteriaceae and other gram-negative bacteria
in pharmaceuticals.1

Dehydrated Appearance: Beige, free-flowing, homogeneous.
Solution:
5.15% solution, soluble in distilled
or deionized water on boiling. Solution
is reddish-purple, slightly opalescent.
Prepared Medium:
Reddish-purple, slightly opalescent
without a precipitate.
Reaction of 5.15%
Solution at 25C:
pH 7.4 0.2
Cultural Response
Prepare Agar Medium No. F per label directions. Inoculate
and incubate at 35 2C for 18-24 hours.
INOCULUM
CFU
ORGANISM
ATCC
Escherichia
11775 100-1,000
RECOVERY
COLONY
DESCRIPTION
good
reddish-purple, may
have a slight precipitate
around the colonies
Salmonella
9184 100-1,000
good
reddish-purple, may
gallinarum
have a slight precipitate
around the colonies
Staphylococcus 6538 1,000-2,000 inhibited
aureus
32
Agar Medium No. F, based on Violet Red Bile Agar and Violet Red Bile
Glucose Agar, uses Sodium Cholate instead of the Bile Salts
No. 3 used in Violet Red Bile Agar and Violet Red Bile Glucose Agar.
Carbon and nitrogen sources required for growth of a variety of
organisms are provided by Bacto Peptone and Yeast Extract. Selectivity
is due to the presence of Crystal Violet and Sodium Cholate which
markedly to completely inhibit growth of gram-positive microorganisms.
Bacto Agar is the solidifying agent.
Differentiation is based on the fermentation of Dextrose and Lactose.
Organisms growing in this medium that can ferment dextrose, such as
members of the family Enterobacteriaceae, produce a localized pH
drop which, followed by absorption of the Neutral Red, imparts a
reddish-purple color to the colony. A zone of precipitated Sodium
Cholate may also be present due to this drop in pH. These reactions are
further intensified in those organisms that can ferment both lactose
and dextrose.
Formula
Agar Medium No. F
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
g
g
g
g
The Difco Manual
Section II
Amino Acid Assay Media
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Cholate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.03
Crystal Violet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.002
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
Precautions
2. Follow proper, established laboratory procedures in handling and
Storage
very hygroscopic. Keep container tightly closed. Store the prepared
medium at 2-8C.
Expiration Date
Procedure
Materials Provided
Agar Medium No. F

Lactose Broth
Enterobacteriaceae Enrichment Broth Mossel (EE Broth Mossel)
Incubator (35C)
Polysorbate 20 or Polysorbate 80
1. Suspend 51.5 grams in 1 liter distilled or deionized water.
3. Sterilize by steaming for 30 minutes. Do Not Autoclave.

1. Collect samples in sterile containers and transport immediately to
the laboratory following recommended guidelines.1,2
2. Process each sample using procedures appropriate for that

sample.1,2
Test Procedure1,2
1. Pre-enrich the sample in Lactose Broth. If the sample is insoluble
in water, add 0.1 ml of polysorbate 20 or polysorbate 80 to the
Lactose Broth.
2. Homogenize the mixture and incubate at 35 2C for 2-5 hours.
3. Transfer 1 ml of enriched Lactose Broth to 100 ml of EE Broth
Mossel (Enterobacteriaceae Enrichment Broth-Mossel).
4. Incubate at 35 2C for 24-48 hours.
5. Subculture all enrichment broth cultures showing growth onto
Agar Medium No. F.
7. Examine plates for the presence of presumptive Enterobacteriaceae
colonies.
Results
Colonies of the family Enterobacteriaceae are reddish-purple in color
and are generally surrounded by a zone of precipitated bile salt. Growth
of gram-positive organisms is markedly to completely suppressed.
Further biochemical testing is necessary to confirm the presence and
identification of Enterobacteriaceae. Consult appropriate references
for further information on identification of Enterobacteriaceae.3,4
References
1. DAB, 10th Edition. 1991. V.2 Biology, V.2.1.8 Proving Certain
Microorganisms, VIII.10 Media (Microbiological Pollution),
Frankfurt/Main.
2. British Pharmacopoeia, Volume II, Appendix XVI. 1988.
HMSO, London.
3. Farmer, J. J. 1995. Enterobacteriaceae: introduction and
identification. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C.
Tenover, and R. H. Yolken (ed.). Manual of clinical microbiology,
4. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey &
Scotts diagnostic microbiology, 9th ed. Mosby-Year Book, Inc.,
St. Louis, MO.
Packaging
Agar Medium No. F
500 g
0666-17

Bacto Lysine Assay Medium . Bacto Methionine Assay Medium
Bacto Cystine Assay Medium
Intended Use
Bacto Lysine Assay Medium is used for determining lysine concentration
by the microbiological assay technique.
Bacto Methionine Assay Medium is used for determining methionine
concentration by the microbiological assay technique.
The Difco Manual
Bacto Cystine Assay Medium is used for determining L-cystine

Also Known As
Lysine Assay Medium, Methionine Assay Medium and Cystine Assay
Medium are also referred to as Amino Acid Assay Media.
33

Amino Acid Assay Media are prepared for use in the microbiological
assay of amino acids. Three types of media are used for this purpose:
1. Maintenance Media: For carrying the stock culture to preserve the
viability and sensitivity of the test organism for its intended purpose.
2. Inoculum Media: To condition the test culture for immediate use.
3. Assay Media: To permit quantitation of the amino acid under test.
They contain all the factors necessary for optimal growth of the test
organism except the single essential amino acid to be determined.
Amino Acid Assay Media are prepared according to the formulations
of Steel et al.1 They are used in the microbiological assay of amino
acids using Pediococcus acidilactici ATCC 8042 as the test organism.

Lysine Assay Medium, Methionine Assay Medium and Cystine Assay
Medium contain all the factors essential for the growth of Pediococcus
acidilactici ATCC 8042, except the amino acid under assay. The
addition of the amino acid in specified increasing concentrations gives
a growth response by the test organism.
Formula
Lysine Assay Medium, Methionine Assay Medium, or
Cystine Assay Medium

Lysine Assay Medium, Methionine Assay Medium,
or Cystine Assay Medium
Dehydrated Appearance: White to off-white, homogeneous,
may have a tendency to clump.
Solution:
5.25% (single strength) and 10.5%
(double strength) solution, soluble
in distilled or deionized water upon
boiling. Solution (single strength) is
light to medium amber, clear to
slightly opalescent, may have a
slight precipitate.
Prepared Medium:
Single strength-light to medium
amber, clear.
Reaction of 5.25%
Solution at 25C:
pH 6.7 0.2
Cultural Response
Prepare Lysine Assay Medium, Methionine Assay Medium and
Cystine Assay Medium per label directions. These media will
support the growth of Pediococcus acidilactici ATCC 8042
when supplemented with the appropriate amino acid. Test
Lysine Assay Medium by creating a standard curve using
L-Lysine at 0 to 300 g per 10 ml. Test Methionine Assay
Medium by creating a standard curve using DL-Methionine at
0 to 60 g per 10 ml. Test Cystine Assay Medium by creating
a standard curve using L-Cystine at 0 to 50 g per 10 ml.
The test organism listed is the minimum used for performance testing.
34
Section II
All amino acid assay media contain the following formula. Omit the
particular amino acid to be assayed from the medium.
Formula Per Liter
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Ammonium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . 1.2
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Manganese Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Adenine Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Guanine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Uracil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Xanthine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Thiamine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Pyrodoxine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Pyridoxamine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . 600
Pyridoxal Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . 600
Calcium Pantothenate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Nicotinic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
p-Aminobenzoic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Folic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Glycine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
DL-Alanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4
Bacto Asparagine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8
L-Aspartic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
L-Proline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
DL-Serine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
DL-Tryptophane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
L-Glutamic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6
L-Histidine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . 0.124
DL-Phenylalanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
DL-Threonine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4
L-Tyrosine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
DL-Valine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
L-Lysine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
DL-Methionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
DL-Isoleucine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
DL-Leucine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
L-Arginine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . 0.484
g
g
g
g
g
g
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
g
g
g
g
g
g
g
g
g
mg
g
g
g
g
g
g
g
g
g
g
g
g
Precautions
2. Great care to avoid contamination of media or glassware must be
taken in microbiological assay procedures. Extremely small
amounts of foreign material may be sufficient to give erroneous
results. Scrupulously clean glassware free from detergents and
other chemicals must be used. Glassware is heated to 250C for at
least 1 hour to burn off any organic residues that might be present.
3. Methionine Assay Medium and Cystine Assay Medium
IRRITANT. IRRITATING TO EYES, RESPIRATORY SYSTEM
The Difco Manual
Section II

TARGET ORGAN(S): Kidney, Bladder.
air. If not breathing, give artificial respiration. If breathing is difficult, give oxygen. Seek medical advice. If swallowed seek medical
advice immediately and show this container or label.
4. Take precautions to keep sterilizing and cooling conditions uniform
throughout the assay.
Storage
Store the dehydrated media at 2-8C. The dehydrated medium is very
hygroscopic and may be stored in a container with calcium chloride
or other desiccant. Keep container tightly closed.
Expiration Date
Procedure
Materials Provided
Lysine Assay Medium or
Methionine Assay Medium or
3.
4.
5.
6.
Dispense 5 ml amounts into tubes, evenly dispersing the precipitate.

Add standard or test samples.
Adjust tube volume to 10 ml with distilled or deionized water.

Assay samples are prepared according to references given in the specific
assay procedure. The samples should be diluted to approximately the
same concentration as the standard solution.
Test Procedure
Stock Culture and Inoculum
Stock cultures of Pediococcus acidilactici ATCC 8042 are prepared by
stab inoculation into tubes of Lactobacilli Agar AOAC or Micro Assay
Culture Agar. Incubate cultures at 35-37C for 24 hours. Store stock
cultures at 2-8C. Make transfers at monthly intervals in triplicate.
The inoculum for assay is prepared by subculturing the test organism
into 10 ml Lactobacilli Broth AOAC or Micro Inoculum Broth. Incubate
at 35-37C for 16-24 hours. After incubation, centrifuge the cells under
aseptic conditions and decant the liquid supernatant. Wash the cells
3 times with 10 ml sterile 0.85% NaCl solution. After the third wash,
resuspend the cells in 10 ml sterile 0.85% NaCl solution. Dilute the
10 ml cell suspension with the appropriate amount of sterile 0.85%
NaCl solution. (See Table 1 below.) One drop of the diluted inoculum
suspension is used to inoculate each of the assay tubes.
Amino Acid Solution
Prepare stock solutions of each amino acid as described in Table 1. If
the DL form is used, twice the concentration of the amino acid is required.
Prepare the stock solutions fresh daily.
Increasing amounts of the standard or the unknown and sufficient distilled
or deionized water to give a total volume of 10 ml per tube, are added
to the tubes containing 5 ml of the rehydrated medium. The appropriate
volumes of the standards and their final concentrations are listed in the table.
Measure the growth response turbidimetrically or titrimetrically.
Turbidimetric readings are made after incubation at 35-37C for 16-20
hours. Titrimetric readings are made after incubation at 35-37C for 72 hours.
It is essential that a standard curve be constructed each time an assay is
run. Conditions of autoclaving and temperature of incubation that
influence the standard curve readings cannot always be duplicated.

Glassware
Autoclave
Stock culture of Pediococcus acidilactici ATCC 8042
Sterile tubes, optically standardized
Centrifuge
Spectrophotometer (660 nm)
L-Lysine HCl
DL-Methionine
L-Cystine
Sterile 0.85% NaCl
Lysine Assay Medium, Methionine Assay Medium, and Cystine
Assay Medium
1. Suspend 10.5 grams in 100 ml distilled or deionized water.
2. Boil for 2-3 minutes to dissolve completely.
Results
1. Prepare a standard concentration response curve by plotting the
response readings against the amount of standard in each tube, disk
or cup.
Table 1. Preparation of inoculum dilution, amino acid stock and working solution.
ASSAY MEDIUM
TEST CULTURE
PREPARATION OF
INOCULUM DILUTION
(CELL SUSPENSION +
(STERILE 0.85% NaCI)
PREPARATION OF
AMINO ACID STOCK SOLUTION
(AMINO ACID) + (DISTILLED H2O)
Cystine Assay
Medium
Pediococcus acidilactici
ATCC 8042
1 ml + 19 ml
L-cystine
Lysine Assay
Medium
Methionine Assay
Medium
ATCC 8042
ATCC 8042
1 ml + 19 ml
L-lysine
1 ml + 19 ml
DL-methionine 1.2 g + 1,000 ml
The Difco Manual
1 g + 100 ml + 1 ml
HCl heated, then cooled,
add up to 1,000 ml
6 g + 1,000 ml
STANDARD
VOLUME OF STANDARD
WORKING SOLUTION
WORKING
(STOCK SOLUTION) +
SOLUTION
(DISTILLED H2O)
(ml/10 ml TUBE)
FINAL
AMINO ACID
CONCENTRATION
g/10 ml
1 ml + 99 ml
0, 0.5, 1, 1.5,
2, 2.5, 3, 4, 5
0.0, 5, 10, 15, 20,

25, 30, 40, 50
1 ml + 99 ml
0, 0.5, 1, 1.5,
2, 2.5, 3, 4, 5
0, 0.5, 1, 1.5,
2, 2.5, 3, 4, 5
0.0, 30, 60, 90, 120,

150, 180, 240, 300
0.0, 6, 12, 18, 24,
30, 36, 48, 60
1 ml + 99 ml
35
Anaerobic Agar
Section II
2. Determine the amount of amino acid at each level of assay solution

by interpolation from the standard curve.
3. Calculate the concentration of amino acid in the sample from the
average of these volumes. Use only those values that do not vary
more than 10% from the average. Use the results only if two thirds
of the values do not vary more than 10%.
References
Packaging
1. The test organism used for inoculating an assay medium must be

cultured and maintained on media recommended for this purpose.
2. Aseptic technique should be used throughout the assay procedure.
3. The use of altered or deficient media may cause mutants having
different nutritional requirements that will not give a satisfactory
response.
Lysine Assay Medium
100 g
0422-15*
Methionine Assay Medium
100 g
0423-15*
100 g
0467-15*
Bacto Anaerobic Agar
4. For successful results of these procedures, all conditions of the

assay must be followed precisely.
1. Steel, Sauberlich, Reynolds, and Baumann. 1949. J. Biol.

Chem. 177:533.
*Store at 2-8C
Intended Use
Bacto Anaerobic Agar is used for cultivating anaerobic microorganisms.

Brewer1 described a special Petri dish cover that allowed surface growth
of anaerobes and microaerophiles without anaerobic equipment. The
microorganisms were grown on an agar-based medium having a low
oxidation-reduction potential. Anaerobic Agar is a modification of
Brewers original formula. This medium is suitable for standard
plating procedures used in cultivating anaerobic bacteria.2,3,4

Solution:
deionized water on boiling. Light
amber, slightly opalescent. As the
medium cools, it becomes green
due to aeration.
Prepared Medium:
Light green, slightly opalescent.
Reaction of 5.8%
Solution at 25C:
pH 7.2 0.2
Cultural Response
ATCC
INOCULUM
CFU
GROWTH
25285*
13124*
100-1,000
100-1,000
good
good
*These cultures are available as Bactrol Disks and should be used
36
Casitone provides the nitrogen, vitamins and amino acids in Anaerobic

Agar. Dextrose is a carbon source. Sodium Chloride maintains the
osmotic equilibrium. Sodium Thioglycollate and Sodium Formaldehyde
Sulfoxylate are reducing agents. Methylene Blue serves as an indicator
of anaerobiosis with a blue color indicating the presence of oxygen.
Anaerobic Agar
Formula Per Liter
Bacto Casitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Sodium Thioglycollate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Sodium Formaldehyde Sulfoxylate . . . . . . . . . . . . . . . . . . . 1
Methylene Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.002
g
g
g
g
g
g
g
Prepare Anaerobic Agar per label directions. Inoculate the

medium and incubate at 35 2C under anaerobic conditions
for 18-48 hours.
Bacteroides fragilis
Clostridium perfringens
Formula
ORGANISM
Anaerobic bacteria cause a variety of infections in humans, including

otitis media, oral infections, endocarditis, meningitis, wound infections
following bowel surgery or trauma, and bacteremia.5,6 Anaerobic
bacteria are the predominant flora colonizing the skin and mucous
membranes of the body.3 Anaerobes vary in their sensitivity to oxygen
and nutritional requirements.2 Anaerobic bacteria lack cytochromes and
thus are unable to use oxygen as a terminal electron acceptor.3
Precautions
disposing of infectious material.
Storage
The Difco Manual
Section II
Expiration Date
Procedure
Materials Provided
Anaerobic Agar

Glassware
Autoclave
Incubator (35C)
Waterbath (45-50C) (optional)
Sterile Petri dishes
Brewer Anaerobic Petri dish covers (optional)
1.
2.
3.
4.

Autoclave at 121C for 15 minutes. Cool to 45-50C.
Dispense as desired.

Anaerobic bacteria are overlooked or missed unless the specimen is
properly collected and transported to the laboratory.2 Obtain and
process specimens according to the techniques and procedures
established by institutional policy.
Test Procedure
Standard Petri Dishes:2
1. Inoculate a properly obtained specimen onto the medium and streak
to obtain isolated colonies.
2. Immediately incubate anaerobically at 35C.
3. Examine at 24 hours if incubating plates in an anaerobic chamber.
Examine at 48 hours if incubating plates in an anaerobic jar or
anaerobic pouch.
4. Extended incubation may be necessary to recover some anaerobes.
Brewer Anaerobic Agar Plates:
1. Dispense 50-60 ml of Anaerobic Agar into a standard Petri dish.
For best results use porous tops to obtain a dry surface.
2. Inoculate the surface of the medium by streaking; avoid the edges
of the plates.
3. Replace the standard Petri dish lid with a sterile Brewer anaerobic
Petri dish cover. The cover should not rest on the Petri dish bottom.
The inner glass ridge should seal against the uninoculated periphery of the agar. It is essential that the sealing ring inside the cover
is in contact with the medium. This seal must not be broken before
the end of the incubation period. A small amount of air is
caught over the surface of the medium; however, the oxygen in this
space reacts with reducing agents in the medium to form an
anaerobic environment.
The Difco Manual
Anaerobic Agar
4. Incubate aerobically as desired.

For a complete discussion on anaerobic and microaerophilic
bacteria from clinical specimens, refer to the appropriate
procedures outlined in the references.2,3,4 For the examination of
anaerobic bacteria in food, refer to Standard Methods.7,8,9
Results

1. Since the nutritional requirements of organisms vary, some
strains may be encountered that fail to grow or grow poorly on
this medium.
2. Clinical specimens must be obtained properly and transported to
the laboratory in a suitable anaerobic transport container.2
3. The microbiologist must be able to verify quality control of the
medium and determine whether the environment is anaerobic.2
4. The microbiologist must perform aerotolerance testing on each
isolate recovered to ensure that the organism is an anaerobe.2
5. Methylene blue is toxic to some anaerobic bacteria.
References
1. Brewer, J. H. 1942. A new Petri dish and technique for use in the
cultivation of anaerobes and microaerophiles. Science 95:587.
2. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures
handbook, American Society for Microbiology, Washington, D.C.
3. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Etiological
agents recovered from clinical material, p. 474-503. Bailey
& Scotts diagnostic microbiology, 9th ed. Mosby-Year Book, Inc.
St. Louis, MO.
R. H. Yolken (ed.). 1995. Manual of clinical microbiology, 6th ed.
5. Balows, A., W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg,
and H. J. Shadomy (ed.). 1991. Manual of clinical microbiology,
6. Smith, L. D. S. 1975. The pathogenic anaerobic bacteria, 2nd ed.
Charles C. Thomas, Springfield, Il.
analytical manual, 8th ed. AOAC International, Gaithesburg, MD.
8. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compendium of methods for the microbiological examination of food,
9. Marshall, R. T. (ed.). 1992. Standard methods for the microbiological examination of dairy products, 16th ed. American Public
Health Association, Washington, D.C.
Packaging
Anaerobic Agar
500 g
0536-17
37
Antibiotic Assay Media
Section II
Bacto Antibiotic Assay Media

Bacto Antibiotic Medium 1 . Bacto Antibiotic Medium 2
Bacto Antibiotic Medium 19
Intended Use
Also Known As
Bacto Antibiotic Assay Media are used for determining antibiotic

potency by the microbiological assay technique.1,6,7
DIFCO
PRODUCT NAME

Antibiotic Medium 1
Dehydrated Appearance: Beige, homogeneous, free-flowing.
Solution:
or deionized water upon boiling;
light to medium amber, very slightly
to slightly opalescent.
Prepared Medium:
Light to medium amber, slightly
opalescent.
Reaction of 3.05%
Solution at 25C:
pH 6.55 0.05
Antibiotic Medium 2
Dehydrated Appearance: Light tan, homogeneous, free-flowing.
Solution:
lightmedium amber, very slightly
Prepared Medium:
Light-medium amber, slightly
opalescent.
Reaction of 2.55%
Solution at 25C:
pH 6.55 0.05
Antibiotic Medium 3
Dehydrated Appearance: Tan, free-flowing, homogeneous.
Solution:
or deionized water; light to medium
amber, clear.
Prepared Medium:
Reaction of 1.75%
Solution at 25C:
pH 7.0 0.05
continued on following page
38
GROVE AND
RANDALL8
USP1
Antibiotic
Penassay
Medium 1
Medium 1
Seed Agar
Antibiotic
Penassay
Medium 2
Medium 2
Base Agar
Antibiotic
Penassay
Medium 3
Medium 3
Broth
Antibiotic
Yeast
Medium 4
Beef Agar
Antibiotic Streptomycin Medium 5
Medium 5
Assay Agar
Antibiotic
Medium 8
Medium 8
Antibiotic
Polymyxin
Medium 9
Medium 9
Base Agar
Antibiotic
Polymyxin Medium 10
Medium 10 Seed Agar
Antibiotic
Neomycin
Medium 11 Assay Agar

Antibiotic
Medium 12
Antibiotic
Medium 19
Medium 19
21 CFR6
AOAC7
Medium 1
Agar Medium A
Medium 2
Agar Medium C
Medium 3 Broth Medium A

Medium 4
Agar Medium B
Medium 5
Agar Medium E
Medium 8
Agar Medium D
Medium 9
Medium 10
Medium 11
Agar Medium J
Medium 19

The activity (potency) of an antibiotic can be demonstrated under
suitable conditions by its inhibitory effect on microorganisms.1 Reduction
in antimicrobial activity may reveal changes not demonstrated by
chemical methods.1 Antibiotic assays are performed by the cylinder plate
method and the turbidimetric tube assay. The cylinder plate method,
first described by Abraham et al.2 for the assay of penicillin, was later
modified by Foster and Woodruff3 and by Schmidt and Moyer4 et al.
Antibiotic Assay Media are prepared according to the specifications
of the U.S. Pharmacopeia (USP) XXIII1, European Pharmacopeia,
Code of Federal Regulations (21CFR6) and the Association of Official
Analytical Chemists (AOAC)7. The Antibiotic Media are identified
numerically and also, where applicable, with names assigned by Grove
and Randall in Assay Methods of Antibiotics.8 Antibiotic Medium 19
corresponds to the use described in Outline of Details for Official
Microbiological Assays of Antibiotics.9
The Difco Manual
Section II
The use of standardized culture media and careful control of all test
conditions are fundamental requisites in the microbiological assay of
antibiotics in order to achieve satisfactory test results.

Cylinder Plate Assay
This method is based on the diffusion of an antibiotic solution from a
cylinder placed on the surface of an inoculated agar medium. The
diameter of a zone of inhibition after incubation depends, in part, on
the concentration or activity of the antibiotic. This method is used in
the assay of commercial preparations of antibiotics, as well as in the
User Quality Control cont.
Antibiotic Medium 4
Solution:
deionized water on boiling; light
amber, very slightly opalescent.
Prepared Medium:
Light amber, very slightly to slightly
opalescent.
Reaction of 2.65%
Solution at 25C:
pH 6.55 0.05
Antibiotic Medium 5
Solution:
deionized water on boiling; light to
medium amber, very slightly to
Prepared Medium:
opalescent.
Reaction of 2.55%
Solution at 25C:
pH 7.9 0.1
Antibiotic Medium 8
Solution:
Prepared Medium:
opalescent.
Reaction of 2.55%
Solution at 25C:
pH 5.85 0.05
Antibiotic Medium 9
Solution:
deionized water upon boiling; light to
medium amber, slightly opalescent,
may have a slight flocculent precipitate.
Prepared Medium:
opalescent with slight flocculent
precipitate.
Reaction of 5.0%
Solution at 25C:
pH 7.25 0.05
The Difco Manual
quantitative determination of antibiotics in body fluids, animal feeds

and other materials.
Turbidimetric Assay
The turbidimetric method is based on the inhibition of growth of a
microbial culture in a fluid medium containing a uniform solution of an
antibiotic.1 Turbidimetric determinations have the advantage of requiring a short incubation period, providing test results after 3 or 4 hours.
However, the presence of solvents or other inhibitory materials may
influence turbidimetric assays more markedly than cylinder plate assays.
Use of this method is appropriate only when test samples are clear.
Antibiotic Medium 10
Dehydrated Appearance: Beige, homogeneous, moist with a
tendency to clump.
Solution:
medium amber, very slightly to slightly
opalescent.
Prepared Medium:
opalescent.
Reaction of 5.2%
Solution at 25C:
pH 7.25 0.05
Solution:
opalescent.
Prepared Medium:
opalescent.
Reaction of 3.05%
Solution at 25C:
pH 8.0 0.1
Dehydrated Appearance: Tan, homogeneous, free-flowing.
Solution:
opalescent.
Prepared Medium:
opalescent.
Reaction of 6.25%
Solution at 25C:
pH 6.1 0.1
Solution:
deionized water upon boiling; medium
amber, very slightly to slightly
opalescent.
Prepared Medium:
Medium amber, slightly opalescent.
Reaction of 6.0%
Solution at 25C:
pH 6.1 0.1
39
Section II
Formula
Cultural Response
Antibiotic Medium 1
Antibiotic Medium 2
Prepare Antibiotic Medium 1 or Antibiotic Medium 2 per label
ORGANISM
Staphylococcus aureus
ATCC
INOCULUM
CFU
GROWTH*
6538P
30-300
good
Antibiotic Medium 3
Prepare Antibiotic Medium 3 per label directions. Inoculate and
incubate at 35 2C for up to 24 hours.
ORGANISM
Enterococcus faecium
Escherichia coli
Klebsiella pneumoniae
ATCC
INOCULUM
CFU
GROWTH*
10541
10536
10031
6538P
approx. 107
approx. 107
approx. 107
approx. 107
good
good
good
good
Antibiotic Medium 4
Prepare Antibiotic Medium 4 per label directions. Inoculate and
incubate at 35 2C for 40-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH*
Micrococcus luteus
9341
30-300
good
Antibiotic Medium 5
Antibiotic Medium 8
ORGANISM
ATCC
INOCULUM
CFU
GROWTH*
Bacillus subtilis
6633
30-300
good
Antibiotic Medium 9
ORGANISM
ATCC
INOCULUM
CFU
GROWTH*
Bordetella bronchiseptica
4617
30-500
good
Prepare Antibiotic Medium 11 per label directions. Inoculate
ORGANISM
ATCC
INOCULUM
CFU
GROWTH*
Micrococcus luteus
Staphylococcus epidermidis
9341
12228
30-300
30-300
good
good
ORGANISM
ATCC
INOCULUM
CFU
GROWTH*
Saccharomyces cerevisiae
2601
30-300
good
*When tested in an appropriate antibiotic assay procedure in parallel
with a previously approved lot of material, inhibition of growth
should produce the specified zones and be comparable to the
previously approved lot.6
40
Antibiotic Medium 1 (Penassay Seed Agar)

Formula Per Liter
Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5
Bacto Casitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g

Antibiotic Medium 2 (Penassay Base Agar)
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g

Antibiotic Medium 3 (Penassay Broth)
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5
Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . 3.68
Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . 1.32
g
g
g
g
g
g
g

Antibiotic Medium 4 (Yeast Beef Agar)
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g

Antibiotic Medium 5 (Streptomycin Assay Agar)
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g

Antibiotic Medium 8
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g

Antibiotic Medium 9 (Polymyxin Base Agar)
Formula Per Liter
Bacto Casitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 g
Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g
The Difco Manual
Section II
Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g

Antibiotic Medium 10 (Polymyxin Seed Agar)
Formula Per Liter
Precautions
Storage
Bacto Casitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Polysorbate 80 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
g
g
g
g
g
g
g

Antibiotic Medium 11 (Neomycin Assay Agar)
Formula Per Liter
Store dehydrated Antibiotic Media (except Antibiotic Medium 10)

below 30C. Store dehydrated Antibiotic Medium 10 at 2-8C. The
dehydrated medium is very hygroscopic. Keep container tightly closed.
Expiration Date
stored directed. Do not use a product if it fails to meet specifications
Procedure

Bacto Casitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g

Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
g
g
g
g
g
g

Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23.5
g
g
g
g
g
g
Materials Provided
Antibiotic Medium 1
Antibiotic Medium 2
Antibiotic Medium 3
Antibiotic Medium 4
Antibiotic Medium 5
Antibiotic Medium 8
Antibiotic Medium 9

Glassware
Autoclave
Incubator
Sterile tubes
Waterbath
Test organisms
Maintenance medium for test organisms
Cylinder Plate Assay: Petri dishes 20 x 100 mm with suitable covers
Stainless steel or porcelain cylinders
Turbidimetric Assay: Glass or plastic tubes
Selection of Media for the Microbiological Assay of Antibiotics1,6
Antibiotic
Assay Method
Organism
ATCC
Amikacin
Amoxicillin
Amphotericin B
Ampicillin
Bacitracin
Bacitracin
Capreomycin
Turbidimetric
Cylinder Plate
Cylinder Plate
Cylinder Plate
Cylinder Plate
Cylinder Plate
Turbidimetric
Micrococcus luteus
Micrococcus luteus
Micrococcus luteus
Micrococcus luteus
6538P*
9341
9763
9341
7468
10240**
10031
Maintenance Inoculum
Medium
Medium
1
1
19
1
1
1
1
1
1
19
1
1
1
1
Cylinder Plate
Base
Seed
Layer Layer
Turbidimetric
Assay Medium
3
11
11
2
1
11
19
11
1
1
3
The Difco Manual
41
Section II
Selection of Media for the Microbiological Assay of Antibiotics1,6 cont.

Antibiotic
Assay Method
Organism
ATCC
Carbenicillin
Cefaclor
Cefadroxil
Cefamandole
Cefazolin
Cefotaxime
Cefoxitin
Cephalexin
Cephaloglycin
Cephaloridine
Cephalothin
Cephapirin
Cephradine
Chloramphenicol
Chlortetracycline
Chlortetracycline
Chlortetracycline
Clindamycin
Cloxacillin
Colistimethate, sodium
Colistin
Cyclacillin
Cycloserine
Dactinocycin
Demeclyocycline
Dicloxacillin
Dihydrostreptomycin
Doxycycline
Erythromycin
Erythromycin
Gentamicin
Gramicidin
Hygromycin B
Kanamycin
Kanamycin B
Lincomycin
Lincomycin
Meclocycline
Methacycline
Methicillin
Cylinder Plate
Cylinder Plate
Cylinder Plate
Cylinder Plate
Cylinder Plate
Cylinder Plate
Cylinder Plate
Cylinder Plate
Cylinder Plate
Cylinder Plate
Cylinder Plate
Cylinder Plate
Cylinder Plate
Turbidimetric
Cylinder Plate
Turbidimetric
Turbidimetric
Cylinder Plate
Cylinder Plate
Cylinder Plate
Cylinder Plate
Cylinder Plate
Turbidimetric
Cylinder Plate
Turbidimetric
Cylinder Plate
Cylinder Plate
Turbidimetric
Turbidimetric
Cylinder Plate
Cylinder Plate
Cylinder Plate
Turbidimetric
Cylinder Plate
Turbidimetric
Cylinder Plate
Cylinder Plate
Turbidimetric
Turbidimetric
Turbidimetric
Cylinder Plate
Pseudomonas aeruginosa
Escherichia coli
Bacillus cereus
Micrococcus luteus
Micrococcus luteus
Bacillus subtilis
Bacillus subtilis
Micrococcus luteus
Micrococcus luteus
Bacillus subtilis
Bacillus subtilis
Micrococcus luteus
25619
6538P
6538P
6538P
6538P
6538P
6538P
6538P
6538P
6538P
6538P*
6538P
6538P
10536
11778**
6538P*
9144**
9341
6538P*
4617
4617
9341
6538P*
6633
6538P*
6538P
6633
10031
6538P*
9341
9341**
12228
10541
6633**
6538P
6633
9341**
6538P
6538P
6538P*
6538P
Medium
Medium
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1 or 3
1
3
1
1
1 or 3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Cylinder Plate
Base
Seed
Layer Layer
9
2
2
2
2
2
2
2
2
2
2
2
2
1
1
1 or 3
1
1
1
1
10
1
1
1
1
1
1
1
1
1
1
1
1
3
8
1
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1 or 3
1
3
Turbidimetric
Assay Medium
8
3
3
11
2
9
9
11
11
1
10
10
11
3
3
2
5
1
5
3
3
11
11
11
11
11
3
5
5
5
11
3
3
3
2
1
42
The Difco Manual
Section II
Selection of Media for the Microbiological Assay of Antibiotics1,6 cont.

Medium
Medium
Antibiotic
Assay Method
Organism
ATCC
Mitomycin
Nafcillin
Natamycin
Neomycin
Neomycin
Netilmicin
Novobiocin
Novobiocin
Nystatin
Oleandomycin
Oleandomycin
Oxacillin
Oxytetracyline
Oxytetracycline
Paromomycin
Penicillin G
Penicillin V
Plicomycin
Polymyxin B
Procaine Penicillin
Rifampin
Rolitetracycline
Sisomicin
Spectinomycin
Streptomycin
Streptomycin
Streptomycin
Tetracycline
Tobramycin
Troleandomycin
Tyrothricin
Vancomycin
Cylinder Plate
Cylinder Plate
Cylinder Plate
Cylinder Plate
Turbidimetric
Cylinder Plate
Cylinder Plate
Cylinder Plate
Cylinder Plate
Cylinder Plate
Cylinder Plate
Cylinder Plate
Cylinder Plate
Turbidimetric
Cylinder Plate
Cylinder Plate
Cylinder Plate
Cylinder Plate
Cylinder Plate
Cylinder Plate
Cylinder Plate
Turbidimetric
Cylinder Plate
Turbidimetric
Cylinder Plate
Cylinder Plate
Turbidimetric
Turbidimetric
Turbidimetric
Turbidimetric
Turbidimetric
Cylinder Plate
Bacillus subtilis
Micrococcus luteus
Micrococcus luteus
Bacillus cereus
Micrococcus luteus
Bacillus subtilis
Escherichia coli
Bacillus subtilis
Bacillus subtilis
Bacillus subtilis
6633
1
6538P
1
9763
19
6538P***
1
10031
1
12228
1
9341**
1 or 3
12228
1
2601
19
9341**
1 or 3
12228
1
6538P
1
11778**
1
6538P*
1
12228
1
6538P*
1
6538P
1
6538P
1
4617
1
9341**
1 or 3
6633
1
6538P*
1
12228
1
10536
1
6633
1
6633**
32
10031
1
6538P*
1
6538P*
1
10031
1
10541
3
6633
1
1
1
19
1
1
1
1 or 3
1
19
1 or 3
1
1
1
1
1
1
1
1
1 or 3
1
1
1
1
1
1
1
1
1
3
1
Cylinder Plate
Base
Seed
Layer Layer
8
2
11
Turbidimetric
Assay Medium
8
1
19
11
3
11
2
2
11
2
11
2
1
19
11
11
1
8
3
11
2
2
8
9
1
2
11
1
1
8
10
4
2
3
11
11
3
5
5
5
5
3
3
3
3
3
* For USP methods, use Staphylococcus aureus ATCC 29737.

** Specified by AOAC for Drugs in Feeds.
*** For USP methods, use Staphylococcus epidermidis ATCC 12228.
1. Suspend the appropriate amount of medium in 1 liter distilled or
deionized water:
Antibiotic Medium 1 - 30.5 grams;
The Difco Manual

Antibiotic Medium 9 - 50 grams;
Antibiotic Medium 10 - 52 grams;
Antibiotic Medium 19 - 60 grams.
2. Boil to dissolve completely (except Antibiotic Medium 3, which
dissolves without boiling).
43

4. Cool medium to 45-50C.
5. Antibiotic Medium 11, only: To alter the pH, add 1N HCl or 1N
NaOH to the medium at 45-50C.
6. Dispense as appropriate.

Test Organism Preparation

Maintain stock cultures on agar slants and make transfers at 1- or
2-week intervals. Prepare the inoculum for assay by washing growth
from a fresh 24-48 hour agar slant using sterile distilled water, saline
or Antibiotic Medium 3 and further dilute the culture to obtain the
desired organism concentration. In some turbidimetric assays, a 18- to
24-hour culture of the test organism in Antibiotic Medium 3, diluted to
obtain the optimal number of organisms, is used.
When Bacillus subtilis is used as the test organism, inoculate it on
Antibiotic Medium 1 and incubate at 37C for 1 week, wash spores
from the agar surface, and heat the spores at 56C for 30 minutes. Wash
the spores 3 times in distilled water, heat again at 65C for 30 minutes,
and then dilute to the optimal concentration. This inoculum preparation
should produce a sharp zone in the assay.
Antibiotic Medium modified by the addition of 300 mg manganese
sulfate (MnSO4.H2O) per liter often aids the sporulation of B. subtilis
and may be used in preparing the spore suspension. A standardized
spore suspension prepared from B. subtilis ATCC 6633 is available as
Bacto Subtilis Spore Suspension.
When B. cereus var. mycoides is required, inoculate the organism on
Antibiotic Medium 1 and incubate at 30C for 1 week. Wash and
prepare the spores as for B. subtilis, above. A standardized spore
suspension of B. cereus var. mycoides is available as Bacto Cereus
Spore Suspension.
Cylinder Plate Assay

Use 20 x 100 mm Petri dishes with sufficient depth so that cylinders
used in the assay will not be pushed into the medium by the cover.
Porcelain covers glazed on the outside, only, are recommended.
Use stainless steel or porcelain assay cylinders having the following
dimensions ( 0.1 mm): 8 mm outside diameter, 6 mm inside diameter
and 10 mm long.1 Carefully clean the cylinders to remove all residues,
using an occasional acid bath, i.e., with approximately 2N nitric acid
or with chromic acid.1 Four or six cylinders are generally used per
plate, evenly spaced on a 2.8 cm radius.
To assure accurate assays, work on a level surface to obtain uniformly
thick base and seed layers in the Petri dish. Allow the base layer to
solidify and then overlay the seed layer containing a proper concentration of the test organism. The amount of medium in the layers varies
for different antibiotics, with most assays specifying a 21 ml base layer
and a 4 ml seed layer. In any case, dishes with flat bottoms are required
to assure complete coverage of the bottom of the dish when small
amounts of base medium are used. Tilt the plate to obtain even coverage
of the base layer by the seed layer and allow it to solidify in a level
position. Plates should be used the same day as prepared.
44
Section II
Turbidimetric Assay
Use glass or plastic test tubes (i.e., 16 x 125 mm or 18 x 150 mm)
that are relatively uniform in length, diameter and thickness and
substantially free from surface blemishes.1 Tubes that will be placed in
the spectrophotometer should be matched and free of scratches or
blemishes. 1 Clean the tubes thoroughly to remove all antibiotic
residues and traces of cleaning solution and, prior to subsequent use,
sterilize tubes that have been previously used.1
Prepare working dilutions of the antibiotic reference standards in
specific concentrations. To a 1 ml quantity of each solution in a
suitable tube, add 9 ml of inoculated broth, as required. Prepare similar
solutions of the assay materials containing approximately the same
amounts of antibiotic activity and place in tubes. Incubate the tubes for
3-4 hours at the required temperature, generally in a water bath. At
the end of the incubation period, stop growth by adding 0.5 ml of
1:3 formalin. Determine the amount of growth by measuring light
transmittance with a suitable spectrophotometer. Determine the
concentration of the antibiotic by comparing the growth obtained with
that given by reference standard solutions.
For a complete discussion of antibiotic assay methods, refer to
appropriate procedures outlined in the references.1,5,6,7
Results
Refer to appropriate procedures for results.1,5,6,7

1. Since the nutritional requirements of organisms vary, some strains
may be encountered that fail to grow or grow poorly on this
medium.
References
States pharmacopeia, 23rd ed. Biological Tests and Assays,
p. 1690-1696. The United States Pharmacopeial Convention,
Rockville, MD.
2. Abraham. 1941. Lancet. 2:177.
3. Foster and Woodruff. 1943. J. Bacteriol. 46:187.
4. Schmidt, W. H., and A. J. Moyer. 1944. Penicillin. I. Methods of
assay. J Bacteriol. 47:199.
5. European Pharmacopoeia. 1994. Council of Europe, 2nd ed.
Maisonneuve S. A. Sainte-Ruffine, FR.
6. Federal Register. 1992. Tests and methods of assay of
Antibiotics and Antibiotic-Containing Drugs. Fed. Regist.
21:436.100-436.106.
7. Association of Official Analytical Chemists. 1995. Official
methods of analysis of AOAC International, 16th ed. AOAC
International, Arlington, VA.
8. Grove, D. C., and W. A. Randall. 1955. Assay methods of
antibiotics. Medical Encyclopedia Inc., New York, NY.
9. Kirshbaum, A., and B. Arret. 1967. Outline of details for
official microbiological assays of antibiotics. J. Pharm.
Sci. 56:512.
The Difco Manual
Section II
Aseptic Commissioning Medium
Packaging
Antibiotic Medium 5
500 g
0277-17
500 g
2 kg
10 kg
0263-17
0263-07
0263-08
Antibiotic Medium 8
500 g
0667-17
Antibiotic Medium 9
500 g
0462-17
Antibiotic Medium 2
500 g
10 kg
0270-17
0270-08
500 g
0463-17
500 g
0593-17
Antibiotic Medium 3
500 g
2 kg
0243-17
0243-07
500 g
0669-17
Antibiotic Medium 4
500 g
0244-17
500 g
0043-17
Antibiotic Medium 1
Bacto Aseptic Commissioning Medium
Intended Use
Bacto Aseptic Commissioning Medium is a fluid medium used in
validating aseptic packing lines.

Aseptic Commissioning Medium is a basic medium in which growth
can be demonstrated by either acid or gas production. It is ideally suited
for validating and commissioning aseptic packing and filling lines.
Principles of Procedure
Peptone and Yeast Extract provide basic nutrients. Sucrose is a
Dehydrated Appearance: Beige to pink, free-flowing,
homogeneous.
Solution:
or deionized water on warming,
orange-red, clear.
Prepared Medium:
Orange-red, clear.
Reaction of 1.75%
Solution at 25C:
pH 7.2 0.2
Cultural Response
Prepare the medium per label directions. Inoculate test
organisms into tubes with fermentation vials and incubate at
Bacillus cereus
Escherichia coli
ATCC
INOCULUM
CFU

Formula Per Liter
Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
Sucrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
g
g
g
mg
g
14579
13048*
25922*
25923*
100-1,000
100-1,000
100-1,000
100-1,000

Storage
Expiration Date
Procedure
Materials Provided
GROWTH ACID
good
good
good
good
GAS
+
+
+** + or
+
**May revert to alkaline after prolonged incubation.
The Difco Manual
Formula
Precautions
ORGANISM
carbohydrate source. Phenol Red is a pH indicator. Sodium Chloride

maintains the osmotic balance.

2. Heat gently to dissolve completely.
45
Azide Blood Agar Base
Section II
Test Procedure
1. Dispense reconstituted medium into the packing line upstream of
the sterilization process.
2. Incubate final packs at 30C, as appropriate, for up to 7 days.
production by a color change of the medium to yellow. Growth is

indicated by turbidity in the medium.
Packaging
Results
500 g
5 kg
1862-17
1862-03
Gas production is demonstrated by swelling of the pack and acid
Bacto Azide Blood Agar Base
Intended Use
Bacto Azide Blood Agar Base is used for isolating streptococci and
staphylococci; for use with blood in determining hemolytic reactions.
Also Known As
Blood Agar Base may be abbreviated as BAB.

Solution:
deionized water upon boiling. Light
to medium amber, very slightly to
slightly opalescent without
Prepared Medium:
opalescent without precipitate.
With 5% blood, cherry red, opaque.
Reaction of 3.3%
Solution at 25C:
pH 7.2 0.2
Cultural Response
Prepare Azide Blood Agar Base per label directions, enrich
with 5% sterile defibrinated blood. Inoculate prepared medium
and incubate at 35 2C. Read plates for growth, hemolysis,
colony size at 18-24 and 40-48 hours.
ORGANISM
Enterococcus
faecalis
Escherichia coli
Staphylococcus
aureus
Staphylococcus
epidermidis
Streptococcus
pneumoniae
Streptococcus
pyogenes
ATCC
INOCULUM
CFU
GROWTH
HEMOLYSIS
19433*
100-1,000
good
25922* 1,000-2,000 inhibited

25923* 100-1,000
good
alpha/
gamma
beta
12228*
100-1,000
good
gamma
6305
100-1,000
good
alpha
19615*
100-1,000
good
beta
46

In 1933, Edwards1 used a liquid medium containing Crystal Violet and
Sodium Azide as a selective broth in the isolation of mastitis
streptococci. Snyder and Lichstein2,3 reported that 0.01% Sodium Azide
in blood agar prevented the swarming of Proteus species,
and permitted the isolation of streptococci from mixed bacterial
populations. Packer 4 modified Edwards medium and prepared
Infusion Blood Agar containing 1:15,000 Sodium Azide and
1:500,000 Crystal Violet for the study of bovine mastitis. Mallmann,
Botwright and Churchill5 reported that Sodium Azide exerted a
bacteriostatic effect on gram negative bacteria. The Azide Blood
Agar Base formulation was based on the work of these researchers.
Azide Blood Agar Base is used in the isolation of gram positive
organisms from clinical and non-clinical specimens. Azide Blood Agar
Base can be supplemented with 5-10% sheep, rabbit or horse blood
for isolating, cultivating and determining hemolytic reactions of
fastidious pathogens.

Tryptose and Beef Extract provide nitrogen, vitamins, carbon and
amino acids. Sodium Chloride maintains osmotic balance. Sodium
Azide is the selective agent, suppressing the growth of gram negative
bacteria. Bacto Agar is the solidifying agent.
Supplementation with 5-10% blood provides additional growth factors
for fastidious microorganisms, and is used to determine hemolytic
patterns of bacteria.
Formula
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Azide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
Precautions
2. HARMFUL. HARMFUL BY INHALATION AND IF
SWALLOWED. IRRITATING TO EYES, RESPIRATORY
SYSTEM AND SKIN. Avoid contact with skin and eyes. Do
not breathe dust. Wear suitable protective clothing. Keep
The Difco Manual
Section II
container tightly closed. TARGET ORGAN(S): Cardiovascular,

Lungs, Nerves.
plenty of water and seek medical advice. After contact with
skin, wash immediately with plenty of water. If inhaled, remove
to fresh air. If not breathing, give artificial respiration. If breathing
is difficult, give oxygen. Seek medical advice. If swallowed
seek medical advice immediately and show this container or label.
Storage
Expiration Date
Procedure
Materials Provided

Glassware
Autoclave
Incubator (35C)
Waterbath (45-50C)
Sterile defibrinated blood (optional)
1.
2.
3.
4.

Autoclave at 121C for 15 minutes. Cool to 50C.
To prepare blood agar, aseptically add 5% sterile defibrinated blood
to the medium at 45-50C. Mix well.
5. Dispense into sterile Petri dishes.

Collect specimens in sterile containers or with sterile swabs. Transport
immediately to the laboratory in accordance with recommended
guidelines outlined in the references.
Test Procedure
1. Process each specimen as appropriate, and inoculate directly
onto the surface of the medium. Streak for isolation with an
inoculating loop, then stab the agar several times to deposit
beta-hemolytic streptococci beneath the agar surface. Subsurface
growth will display the most reliable hemolytic reactions
demonstrating both oxygen-stable and oxygen-labile streptolysins.6
2. Incubate plates aerobically, anaerobically or under conditions
of increased CO 2 (5-10%) in accordance with established
laboratory procedures.
The Difco Manual
Results
Examine plates for growth and hemolytic reactions after 18-24 and
40-48 hours of incubation. Four different types of hemolysis on blood
agar media can be described:7
a. Alpha ()-hemolysis is the reduction of hemoglobin to
methemoglobin in the medium surrounding the colony, causing
a greenish discolorization of the medium.
b. Beta()-hemolysis is the lysis of red blood cells, resulting in a
clear zone surrounding the colony.
c. Gamma()-hemolysis indicates no hemolysis. No destruction of
red blood cells occurs, and there is no change in the medium.
d. Alpha-prime (` )-hemolysis is a small zone of complete
hemolysis that is surrounded by area of partial lysis.

1. Nutritional requirements of organisms vary. Strains may be
encountered that fail to grow or grow poorly on this medium.
2. Azide Blood Agar Base is intended for selective use and should be
inoculated in parallel with nonselective media.
3. Hemolytic patterns of streptococci grown on Azide Blood Agar
Base are somewhat different than those observed on ordinary blood
agar. Sodium azide enhances hemolysis. Alpha and beta zones may
be extended.4
4. Hemolytic patterns may vary with the source of animal blood or
base medium used.6
References
1. Edwards, S. J. 1933. The diagnosis of Streptococcus mastitis by
cultural methods. J. Comp. Pathol. Ther. 46:211.
2. Snyder, M. L., and H. C. Lichstein. 1940. Sodium azide as an
inhibition substance of gram-negative bacteria. J. Infect. Dis.
67:113.
3. Lichstein, H. C., and M. L. Snyder. 1941. The inhibition of the
spreading growth of Proteus and other bacteria to permit the
isolation of associated streptococci. J. Bacteriol. 42:653.
4. Packer, R. A. 1943. The use of sodium azide (NaN3) as an
inhibition substance of gram-negative bacteria. J. Infect. Dis.
67:113.
5. Mallmann, Botwright, and Churchill. 1943. J. Bacteriol. 46:343.
6. Ruoff, K. L. 1995. Streptococcus, p. 299-305. In P. R. Murray,
E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.),
Manual of clinical microbiology, 6th ed. American Society for
Microbiology, Washington, D.C.
handbook, vol. 1. American Society for Microbiology,
Washington, D.C.
Packaging
500 g
10 kg
0409-17
0409-08
47
Azide Dextrose Broth
Section II
Bacto Azide Dextrose Broth
Intended Use
Formula
Formula Per Liter
Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sodium Azide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Azide Dextrose Broth is used for cultivating streptococci in

water and wastewater.

The formula for Azide Dextrose Broth originated with Rothe at the
Illinois State Health Department.1 In a comparative study, Mallmann
and Seligmann2 investigated the detection of streptococci in water and
wastewater using Azide Dextrose Broth. Their work supported use
of the medium in determining the presence of streptococci in water,
wastewater, shellfish and other materials. Azide Dextrose Broth has
also been used for primary isolation of streptococci from foodstuffs3,4
and other specimens of sanitary significance as an indication of fecal
contamination.
Azide Dextrose Broth is specified for use in the presumptive test
of water and wastewater for fecal streptococci by the Multiple-Tube
Technique.5

Azide Dextrose Broth contains Beef Extract and Tryptose as sources
of carbon, nitrogen, vitamins and minerals. Dextrose is a fermentable
carbohydrate. Sodium Chloride maintains the osmotic balance of the
medium. Sodium Azide inhibits cytochrome oxidase in gram-negative
bacteria.
Group D streptococci grow in the presence of azide, ferment glucose,
and cause turbidity.
4.5
15
7.5
7.5
0.2
g
g
g
g
g
Precautions
2. IRRITANT. HARMFUL BY INHALATION AND IF SWALLOWED. IRRITATING TO EYES, RESPIRATORY SYSTEM
TARGET ORGAN(S): Cardiovascular, Lungs, Nerves.
Storage

Solution:
3.47% (single strength) and 6.94% (double strength)
solution, soluble in distilled or deionized water.
Single-strength solution is light to medium amber,
clear to very slightly opalescent; double-strength
solution is medium to dark amber, clear.
Prepared Medium:
Light to medium amber, clear (single strength).
Reaction of 3.47%
Solution at 25C:
pH 7.2 0.2
Cultural Response
Prepare Azide Dextrose Broth per label directions. Inoculate and incubate
at 35 2C for 18-48 hours.
ORGANISM
Escherichia coli
ATCC
INOCULUM
CFU
GROWTH
19433*
25922*
100-1,000
1,000-2,000
good
inhibited
Uninoculated
tube
ATCC 29212
*These cultures are available as Bactrol Disks and should be used as directed in Bactrol Disks Technical Information.
48
The Difco Manual
Section II
B12 Assay Medium USP
Expiration Date
Results
A positive test is indicated by turbidity (cloudiness) in the broth. A

negative test remains clear.
Procedure
Materials Provided
All Azide Dextrose Broth tubes showing turbidity after 24- or 48-hours
incubation must be subjected to the Confirmed Test Procedure.
Consult appropriate references for details of the Confirmed Test
Procedure5 and further identification of Enterococcus.5,6
1. Azide Dextrose Broth is used to detect presumptive evidence of

fecal contamination. Further biochemical testing must be done for
confirmation.
2. For inoculum sizes of 10 ml or larger, use double strength medium
to prevent dilution of ingredients.5,6
Glassware
Tubes with closures
Autoclave
Incubator (35C)
Rehydrate with proportionally less water when liquid inocula will
exceed 1 ml.

Presumptive Test Procedure5

1. Inoculate a series of Azide Dextrose Broth tubes with appropriately
graduated quantities of sample. Use sample quantities of 10 ml or
less. Use double-strength broth for 10 ml inocula. Consult an
appropriate reference for suggested sample sizes.5
2. Incubate inoculated tubes at 35 2C for 20-48 hours.
3. Examine each tube for turbidity at the end of 24 2 hours. If
no turbidity is evident, reincubate and read again at the end of
48 3 hours.
References
1. Rothe. 1948. Illinois State Health Department.
2. Mallmann, W. L., and E. B. Seligmann. 1950. A comparative
study of media for the detection of streptococci in water and
sewage. Am. J. Public Health 40:286.
3. Larkin, E. P., W. Litsky, and J. E. Fuller. 1955. Fecal
streptococci in frozen foods. I. A bacteriological survey of some
commercially frozen foods. Appl. Microbiol. 3:98.
4. Splittstoesser, D. F., R. Wright, and G. J. Hucker. 1961. Studies
on media for enumerating enterococci in frozen vegetables. Appl.
Microbiol. 9:303.
5. Eaton, A. D., L. S. Clesceri, and A. E. Greenberg (eds.). 1995.
6. MacFaddin, J. F. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, vol. 1. Williams
& Wilkins, Baltimore, MD.
Packaging
500 g
0387-17
Bacto B12 Assay Medium USP
Intended Use
Bacto B12 Assay Medium USP is used for determining vitamin B12
Also Known As
USP is an abbreviation for United States Pharmacopeia.

Vitamin Assay Media are used in the microbiological assay of vitamins.
Three types of media are used for this purpose:
viability and sensitivity of the test organism for its intended purpose;
2. Inoculum Media: To condition the test culture for immediate use;
3. Assay Media: To permit quantitation of the vitamin under test.
The Difco Manual
B12 Assay Medium USP is used in the microbiological assay of vitamin

B12 according to the procedures of the Vitamin B12 Activity Assay in
USP1 and the Cobalamin (Vitamin B12 Activity) Assay in AOAC.2
Lactobacillus delbrueckii subsp. lactis ATCC 7830 (Lactobacillus
leichmannii) is the test organism used in this procedure.

B12 Assay Medium USP is a vitamin B12-free dehydrated medium
containing all other nutrients and vitamins essential for the cultivation
of L. delbrueckii subsp. lactis ATCC 7830. To obtain a standard curve,
USP Cyanocobalamin Reference is added in specified increasing
concentrations giving a growth response that can be measured
titrimetrically or turbidimetrically.
49
Section II
Formula
Precautions

Formula Per Liter

2. Great care must be taken to avoid contamination of media or glassware in microbiological assay procedures. Extremely small
other chemicals must be used. Glassware must be heated to 250C
for at least 1 hour to burn off any organic residues that might be present.
3. Take precautions to keep sterilization and cooling conditions uniform
Bacto Vitamin Assay Casamino Acids . . . . . . . . . . . . . . . . 15

Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4
DL-Tryptophane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4
Adenine Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Uracil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Xanthine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
p-Aminobenzoic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Pyridoxine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Pyridoxal Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Pyridoxamine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . 800
Folic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Sorbitan Monooleate Complex . . . . . . . . . . . . . . . . . . . . . . . 2
g
g
g
g
g
g
g
mg
mg
mg
mg
mg
mg
g
mg
mg
mg
mg
mg
g
g
g
g
g
mg
mg
mg
g

Dehydrated Appearance: Very light to light beige, homogeneous,
with a tendency to clump.
Solution:
4.25% (single strength) or 8.5%
in distilled or deionized water on
boiling for 2-3 minutes. Light amber,
clear, may have a slight precipitate
(single strength).
Prepared Medium:
(Single strength) very light to light
amber, clear, may have a slight
precipitate.
Reaction of 4.25%
Solution at 25C:
pH 6.0 0.1
Cultural Response
Prepare B12 Assay Medium USP per label directions. Prepare
a standard curve using USP Cyanocobalamin Reference
Standard at levels of 0.0 to 0.25 ng per 10 ml. The medium
supports the growth of L. delbrueckii subsp. lactis ATCC 7830
when supplemented with cyanocobalamin (vitamin B12).
50
Storage
Store the dehydrated medium at 2-8C. The dehydrated medium is very
hygroscopic. Keep container tightly closed.
Expiration Date
Procedure
Materials Provided

Glassware
Autoclave
Stock culture of Lactobacillus delbrueckii subsp. lactis ATCC 7830
Lactobacilli Agar AOAC or B12 Culture Agar USP
Lactobacilli Broth AOAC or B12 Inoculum Broth USP
Sterile 0.85% saline
Spectophotometer or nephelometer
B12 Culture Agar USP
B12 Inoculum Broth USP
Cyanocobalamin USP (vitamin B12)
1.
2.
3.
4.
5.
6.
Suspend 8.5 grams in 100 ml distilled or deionized water.

Heat to boiling for 2-3 minutes to dissolve completely.
Distribute 5 ml amounts into tubes, evenly dispersing the precipitate.

Assay samples are prepared according to references given in the
specific assay procedures. For assay, the samples should be diluted to
approximately the same concentration as the standard solution.
Test Procedure
Follow assay procedures as outlined in USP1 or AOAC.2 Use levels
of B12 in the preparation of the standard curve according to these
The Difco Manual
Section II
B12 Culture Agar USP & B12 Inoculum Broth USP
references. It is essential that a standard curve be constructed each time

an assay is run. Autoclave and incubation conditions can influence the
standard curve reading and cannot always be duplicated.
Generally satisfactory results are obtained with B12 at the following
levels: 0.0, 0.025, 0.05, 0.075, 0.1, 0.125, 0.15, 0.2 and 0.25 ng per
assay tube (10 ml).
Stock cultures of L. delbrueckii subsp. lactis ATCC 7830 are
prepared by stab inoculation into 10 ml of B12 Culture Agar USP or
Lactobacilli Agar AOAC. After 16-24 hours incubation at 35-37C, the
cultures are kept refrigerated. The inoculum for assay is prepared by
subculturing a stock culture of L. delbrueckii subsp. lactis into
10 ml of B12 Inoculum Broth USP. For a complete discussion on B12
Culture Agar USP and B12 Inoculum Broth USP, refer to USP.1
Results
or cup.
2. Determine the amount of vitamin at each level of assay solution by
interpolation from the standard curve.
3. Calculate the concentration of vitamin in the sample from the
Bacto B12 Culture Agar USP

Bacto B12 Inoculum Broth USP
more than 10% from the average and use the results only if two
thirds of the values do not vary more than 10%.

2. For successful results to these procedures, all conditions of the
different nutritional requirements and will not give a satisfactory
response.
References
1. The United States Pharmacopeial Convention. 1995. The United
Convention Inc., Rockville, MD.
Packaging
100 g
0457-15
Intended Use
Bacto B12 Inoculum Broth USP is used for preparing the inoculum
of Lactobacillus delbrueckii subsp. lactis ATCC 7830 used in the
Vitamin B12 Activity Assay.
Bacto B12 Culture Agar USP is used for cultivating L. delbrueckii subsp.
lactis ATCC 7830 used in the Vitamin B12 Activity Assay.
Also Known As
USP is an abbreviation for United States Pharmacopeia.

Vitamin Assay Media are prepared for use in the microbiological assay
of vitamins. Three types of media are used for this purpose:
They contain all the factors necessary for optimal growth of the
test organism except the single essential vitamin to be determined.
Lactobacillus species grow poorly on non-selective culture media and
require special nutrients. Mickle and Breed2 reported the use of
tomato juice in culture media for lactobacilli. Kulp,3 while investigating
the use of tomato juice on bacterial development, found that growth of
Lactobacillus acidophilus was enhanced.
The Difco Manual
B 12 Culture Agar USP is recommended for maintaining stock

cultures of L. delbrueckii subsp. lactis ATCC 7830 (Lactobacillus
leichmannii) for use in the Vitamin B12 Activity Assay according to
US Pharmacopeia (USP).1
B 12 Inoculum Broth USP is used for preparing the inoculum of
L. delbrueckii subsp. lactis ATCC 7830 in the microbiological assay
of vitamin B12 according to USP.1

Proteose Peptone No. 3 provides the nitrogen and amino acids in B12
Culture Agar USP and B12 Inoculum Broth USP. Yeast Extract is the
vitamin source in the formulas. Tomato Juice is added to create the
proper acidic environment. Dextrose is the carbon source, and Sorbitan
Monooleate Complex acts an emulsifier. Potassium Phosphate Dibasic
acts as the buffering agent in B 12 Inoculum Broth USP, and
Monopotassium Phosphate is the buffering agent in B12 Culture Agar
USP. Bacto Agar is the solidifying agent in B12 Culture Agar USP.
Formula
Formula Per Liter
Tomato Juice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Bacto Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . . 7.5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
ml
g
g
g
g
g
g
51
B12 Culture Agar USP & B12 Inoculum Broth USP
Section II

Formula Per Liter
Tomato Juice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Potassium Phosphate Dibasic . . . . . . . . . . . . . . . . . . . . . . . . 2
ml
g
g
g
g
g
other chemicals must be used.
Storage
Store the dehydrated media at 2-8C. The dehydrated media are very
hygroscopic. Keep containers tightly closed.
Expiration Date
Precautions
Procedure
Materials Provided

Solution:
or deionized water upon boiling.
Solution is light to medium amber,
opalescent when hot, slightly
opalescent with flocculent precipitate
when cooled.
Prepared Medium:
flocculent precipitate.
Reaction of 4.7%
Solution at 25C:
pH 6.8 0.1
Dehydrated Appearance: Tan, homogeneous, tendency to
clump.
Solution:
is medium to dark amber, opalescent
when hot, clear when cooled to room
temperature.
Prepared Medium:
Medium amber, clear.
Reaction of 3.2%
Solution at 25C:
pH 6.8 0.1
Cultural Response
B12 Culture Agar USP or B12 Inoculum Broth USP
Prepare B12 Culture Agar USP or B12 Inoculum Broth USP per
label directions. Inoculate medium with test organism and
ORGANISM
ATCC
INOCULUM
CFU
Lactobacillus delbrueckii
subsp. lactis
7830
300-1,000
The culture listed is the minimum that should be used for

52
GROWTH
good

Glassware
Autoclave
Incubator
Inoculating needle
deionized water:
47 grams
B12 Inoculum Broth USP 32 grams
2. Boil to dissolve completely. (B12 Culture Agar)
3. Dispense 10 ml amounts into tubes.
5. Allow tubes of B12 Culture Agar to cool in an upright position.
Stock Culture
1. Prepare stock cultures in triplicate in sterile B12 Culture Agar USP.
2. Inoculate the tubes using a straight wire inoculating needle.
3. Incubate cultures for 16-24 hours at any temperature between
30-40C, but held constant within 0.5C.
4. Store at 2-8C.
5. Before using a fresh culture for assay, make no fewer than 10
successive transfers of the culture in a 2 week period.
6. Prepare stab cultures at least three times each week and do not use
a culture for preparing assay inoculum if over 4 days old.
Inoculum
Prepare inoculum as described in USP.1

The Difco Manual
Section II
BAGG Broth
Test Procedure
References
For a complete discussion of vitamin assay methodology, refer to

appropriate procedures outlined in USP.1

Convention Inc. Rockville, MD.
2. Mickle, and Breed. 1925. Technical Bulletin 110, NY State
Agriculture Ex. Station.
3. Kulp, J. W. L., and V. White. 1932. Modified medium for plating
Lactobacillus acidophilus. Science 76:17.
Results
For test results of vitamin assay procedures refer to USP.1

response.
Packaging
100 g
0541-15*
100 g
0542-15*
*Store at 2-8C
Bacto BAGG Broth

In developing Buffered Azide Glucose Glycerol (BAGG) Medium,
Hajna1 modified the formula of SF Broth as specified by Hajna and
Perry.2 Hajna found that adding glycerol to SF Medium enhanced
dextrose fermentation by Enterococcus faecalis. Decreasing the
concentration of brom cresol purple allowed for easier detection of a
color change within 24 hours. The BAGG Broth formulation made the
original SF Medium more useful in testing for fecal contamination of
water and other materials.
Intended Use
Bacto BAGG Broth is used for presumptively identifying and
confirming fecal streptococci.
Also Known As
Buffered Azide Glucose Glycerol Medium

Dehydrated Appearance: Light beige with a slight green tint, free-flowing,
homogeneous.
Solution:
containing 0.5% glycerol. Solution is purple, clear.
Prepared Tubes:
Purple, clear.
Reaction of 3.6%
Solution at 25C:
pH 6.9 0.2
Cultural Response
Prepare BAGG Broth per label directions. Inoculate tubes in duplicate
and incubate at 35 2C and 45 1.0C for 18-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
Escherichia coli
19433* 100-1,000
27270
100-1,000
25922* 1,000-2,000
19615* 1,000-2,000
GROWTH
good
good
markedly to
completely inhibited
markedly to
ACID
PRODUCTION
+ (yellow)
+ (yellow)
Uninoculated
tube
ATCC 29212
The Difco Manual
53
BAGG Broth
Section II

BAGG Broth contains Tryptose as a source for carbon, nitrogen,
vitamins and minerals. Dextrose is a fermentable carbohydrate.
Sodium Chloride maintains the osmotic balance of the medium.
Sodium Azide inhibits gram-negative bacteria. Brom Cresol Purple is
a pH indicator.
Enterococci grow in the presence of azide and ferment glucose,
producing an acid pH that changes the color of the medium.
Formula
BAGG Broth
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Azide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Bacto Brom Cresol Purple . . . . . . . . . . . . . . . . . . . . . . . 0.015
Glycerol
Tubes with closures
Autoclave
Incubators (35 2C, 45 1C)
1. Dissolve 36 grams in 1 liter distilled or deionized water containing
5 ml glycerol. Rehydrate with proportionally less water when
liquid inocula will exceed 1 ml.
2. Dispense into tubes with closures.
3. Autoclave at 114 -118C for 15 minutes.

g
g
g
g
g
g
g
Precautions
Test Procedure1-3
1. Inoculate duplicate tubes with sample. Use single-strength medium
for inocula of 1 ml or less. Use double-strength medium for
inocula of 10 ml.
2. Incubate one set of tubes at 35 2C for 18-48 hours. Incubate the
second set at 45 1C for 18-48 hours.
3. Read tubes for growth and acid production.

2. HARMFUL. HARMFUL BY INHALATION AND IF SWALLOWED. IRRITATING TO EYES, RESPIRATORY SYSTEM
Results1-3
Storage
1. Hajna, A. A. 1951. A buffered azide glucose-glycerol broth for

presumptive and confirmative tests for fecal streptococci. Pub.
Health Lab. 9:80-81.
2. Hajna, A. A., and C. A. Perry. 1943. A comparative study of
presumptive and confirmative media for bacteria of the
coliform group and for fecal streptococci. Am. J. Pub. Health
33:550-556.

Expiration Date
Procedure
1. A positive test is indicated by the production of a yellow color

(acid) throughout the medium. This result is presumptive evidence
of the presence of fecal streptococci. Further testing must be
performed to confirm this result. Consult appropriate references
for further identification of Enterococcus.3
2. A negative result is indicated by no change in the medium (purple
color).

1. The concentration of the medium must be adjusted to the inoculum
size. Refer to discussion in Test Procedure.
References
Materials Provided
Packaging
BAGG Broth
BAGG Broth
500 g
0442-17

Glassware
54
The Difco Manual
Section II
BG Sulfa Agar, SBG Enrichment & SBG Sulfa Enrichment
Bacto BG Sulfa Agar . Bacto SBG Enrichment

Bacto SBG Sulfa Enrichment
Intended Use
Bacto BG Sulfa Agar is used for isolating Salmonella.
Bacto SBG Enrichment and Bacto SBG Sulfa Enrichment is used for
enriching Salmonella prior to isolation procedures.
Also Known As
BG is an abbreviation for Brilliant Green and SBG is an abbreviation
for Selenite Brilliant Green.

Salmonellosis continues to be an important public health problem
worldwide, despite efforts to control the prevalence of Salmonella in

BG Sulfa Agar
Dehydrated Appearance: Pink, free flowing, homogeneous.
Solution:
is very dark amber, very slightly to
Prepared Plates:
Dark reddish-amber, slightly
opalescent.
Reaction of 5.9%
Solution at 25C:
pH 6.9 0.2
SBG Enrichment
homogeneous.
Solution:
or deionized water; green,
opalescent with slight precipitate.
Prepared Medium:
Green, opalescent without
significant precipitation.
Reaction of 2.37%
Solution at 25C:
pH 7.2 0.2
SBG Sulfa Enrichment
homogeneous.
Solution:
or deionized water; green,
opalescent without significant
precipitation.
Prepared Medium:
Green, opalescent without
Reaction of 2.42%
Solution at 25C:
pH 7.2 0.2
domesticated animals. Infection with non-typhi Salmonella often causes

mild, self-limiting illness.1 The illness results from consumption of
raw, undercooked or improperly processed foods contaminated with
Salmonella. Many of these cases of Salmonella-related gastroenteritis
are due to improper handling of poultry products. Various poultry
products are routinely monitored for Salmonella before their distribution for human consumption, but in many instances, contaminated
food samples elude detection.
BG Sulfa Agar is a highly selective medium. Osborne and Stokes2
added 0.1% sodium sulfapyridine to Brilliant Green Agar to enhance
the selective properties of this medium for Salmonella. This formula is
recommended as a selective isolation medium for Salmonella following
enrichment. It is also recommended for direct inoculation with primary
specimens for Salmonella isolation.
For food testing, BG Sulfa Agar has been used for detection of Salmonella
in low and high moisture foods.3,4 It has also been used for detecting
Salmonella in feeds and feed ingredients.5 This medium is recommended
when testing foods for Salmonella following USDA guidelines.6,7
SBG Enrichment and SBG Sulfa Enrichment are prepared according
to the formulas described by Stokes and Osborne.8 The researchers
found that whole egg and egg yolk reduced the selective properties of
selenite brilliant green enrichment.2 They also found that the addition
of sulfapyridine restored these selective properties.2
SBG Enrichment and SBG Sulfa Enrichment are selective enrichments
for the isolation of Salmonella species, especially from egg products.
The shell and the contents of the egg at the time of oviposition are
generally sterile or harbor very few microorganisms.9,10,11 Contamination of the shell occurs afterwards from nesting material, floor
litter, and avian fecal matter.12 Salmonellae are of most concern in
egg products.12

In BG Sulfa Agar, Proteose Peptone and Yeast Extract provide nitrogen,
vitamins and minerals. Lactose and Sucrose are the sources of
carbohydrates in the medium. Brilliant Green and Sodium Pyridine
are complementary in inhibiting gram-positive bacteria and most
gram-negative bacilli other than Salmonella spp. Phenol Red is the
pH indicator that turns the medium a yellow color with the formation
of acid when lactose and/or sucrose is fermented. Bacto Agar is a
solidifying agent.
Bacto Peptone provides the nitrogen, minerals and amino acids in
SBG Enrichment and SBG Sulfa Enrichment. Yeast Extract is the vitamin
source. D-Mannitol is the carbon source to stimulate organism growth.
The phosphates acts as buffers in the enrichments. Sodium Taurocholate,
Sodium Selenite and Brilliant Green are the selective agents. The
selective agents are used to inhibit gram positive organisms and
enteric bacteria other than Salmonella. Sodium Sulfapyridine is added
in SBG Sulfa Enrichment to increase selectivity.
The Difco Manual
55
Section II
Formula
Sodium Sulfapyridine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5

Sodium Selenite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Bacto Brilliant Green . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.005
BG Sulfa Agar
Formula Per Liter
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Saccharose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Sulfapyridine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Brilliant Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0125
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.08
g
g
g
g
g
g
g
g
g

SBG Enrichment
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto D-Mannitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Taurocholate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Selenite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Bacto Brilliant Green . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.005
g
g
g
g
g
g
g
g

Formula Per Liter
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto D-Mannitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sodium Taurocholate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
5
5
1
g
g
g
g
g
Precautions
1. BG Sulfa Agar
For Laboratory Use.
SBG Enrichment, SBG Sulfa Enrichment
For Laboratory Use.
2. SBG Enrichment
VERY TOXIC. VERY TOXIC BY INHALATION AND IF
SWALLOWED. DANGER OF CUMULATIVE EFFECTS. (EC)
IRRITATING TO EYES, RESPIRATORY SYSTEM AND SKIN.
Avoid contact with skin and eyes. Do not breathe dust. Wear suitable protective clothing. Keep container tightly closed. TARGET
ORGAN(S): Kidney, Liver, Spleen.
air. If not breathing, give artificial respiration. If breathing is difficult,
give oxygen. Seek medical advice. If swallowed, induce vomiting;
g
g
g
g

VERY TOXIC. VERY TOXIC BY INHALATION AND IF
SWALLOWED. DANGER OF CUMULATIVE EFFECTS. (EC)
Uninoculated
plate
Salmonella typhimurium
ATCC 14028
Cultural Response
BG Sulfa Agar
Prepare BG Sulfa Agar per label directions. Inoculate and incubate
the plates at 35 2C for 18-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
COLOR OF
COLONIES/MEDIUM
Enterococcus faecalis 29212* 1,000-2,000

none
/no change
Escherichia coli
25922* 100-1,000 none to poor yellow-green
Salmonella
14028* 100-1,000
good
pink-white/red
typhimurium
SBG Enrichment and SBG Sulfa Enrichment

Prepare SBG Enrichment and SBG Sulfa Enrichment per label
directions. Inoculate tubes with the test organisms. Incubate
inoculated medium at 35 2C for 18-24 hours. After incubation,
subculture onto prepared plates of MacConkey Agar.
ORGANISM
Escherichia coli
Salmonella
typhimurium
Shigella sonnei
ATCC
INOCULUM
CFU
GROWTH
25922* 100-1,000 none to poor

14028* 100-1,000
good
9290
100-1,000
poor to fair
COLONY COLOR
ON MACCONKEY
pink, if any
colorless
colorless
56
The Difco Manual
Section II

Avoid contact with skin and eyes. Do not breathe dust. Wear
suitable protective clothing. Keep container tightly closed.
TARGET ORGAN(S): Kidney, Liver, Spleen.
difficult, give oxygen. Seek medical advice. If swallowed, induce
vomiting; seek medical advice immediately and show this container
or label.
Storage
Store the dehydrated medium and enrichments below 30C. The dehydrated
products are very hygroscopic. Keep container tightly closed.

Examine prepared media for growth. Positive tubes should be subcultured
onto prepared media for isolation and identification of bacteria.
Store prepared BG Sulfa Agar plates at 2-8C.
Expiration Date
1. On BG Sulfa Agar colonies of Salmonella sp. vary from red to

pink to white depending on length of incubation and strain.13
2. BG Sulfa Agar is normally orange-brown in color; however, on
incubation, it turns bright red and returns to normal color at room
temperature.13
3. S. typhi does not grow adequately on BG Sulfa Agar. Shigella sp.
do not grow on BG Sulfa Agar.13
4. Do not sterilize BG Sulfa Agar longer than 15 minutes; longer
periods decrease the selectivity of the medium.
5. Since BG Sulfa Agar is highly selective, it is recommended
that less selective media, such as MacConkey Agar, be used
simultaneously.
6. SBG Enrichment and SBG Sulfa Enrichment should be used
in conjunction with selective prepared medium for bacterial
identification.
Procedure
Materials Provided
BG Sulfa Agar
SBG Enrichment

Bunsen burner or magnetic hot plate
Autoclave
Waterbath (45-50C)
Petri dishes
Incubator (35C)
Sterile test tubes
BG Sulfa Agar
1. Suspend 59 grams in 1 liter distilled or deionized water.
3. Autoclave at 121C for 15 minutes. Avoid overheating which will
decrease selectivity.
4. Cool to 45-50C in a waterbath.
1. Dissolve the appropriate amount of medium in 1 liter distilled or
deionized water:
SBG Enrichment
23.7grams/liter
SBG Sulfa Enrichment 24.2 grams/liter
2. Boil gently for 5-10 minutes.
3. Avoid overheating. DO NOT AUTOCLAVE.
The Difco Manual
For information about specimen preparation and inoculation of food

samples, consult appropriate references.7,12
Results
BG Sulfa Agar
The typical Salmonella colonies appear as pink-white to red opaque
colonies surrounded by a brilliant red medium. The few lactose and/or
sucrose fermenting organisms that grow are readily differentiated due
to the formation of a yellow-green colony surrounded by an intense
yellow-green zone. BG Sulfa Agar is not suitable for the isolation of
S. typhi or Shigella; however, some strains of S. typhi may grow forming
red colonies.
References
1. Flowers, R. S., W. Andrews, C. W. Donnelly, and E. Koenig.
1993. Pathogens in milk and milk products, p. 103-212. In
R. T. Marshall (ed.), Standard methods for the examination of
dairy products, 16th ed. American Public Health Association,
Washington, D.C.
2. Osborn, W. W., and J. L. Stokes. 1955. A modified selenite
brilliant green medium for the isolation of Salmonella from egg
products. Appl. Microbiol. 3:295-299.
3. DAoust, J. Y., C. Maishment, D. M. Burgener, D. R. Conley,
A. Loit, M. Milling, and U. Purvis. 1980. Detection of
Salmonella in refrigerated preenrichment and enrichment broth
cultures. J. Food Prot. 43:343-345.
4. DAoust, J. Y. 1984. Effective enrichment-plating conditions for
detection of Salmonella in foods. J. Food Prot. 47:588-590.
5. DAoust, J. Y., A. Sewell, and A. Boville. 1983. Rapid cultural
methods for detection of Salmonella in feeds and feed ingredients.
J. Food Prot. 46:851-855.
6. Moats, W. A. 1981. Update on Salmonella in foods: selective plating
media and other diagnostic media. J. Food Prot. 44:375-380.
57
Baird-Parker Agar Base & EY Tellurite Enrichment
Section II
7. Federal Register. 1996. Pathogen reduction; hazard analysis

and critical point (HACCP) systems; final rule. Fed. Regis.
61:38917-38925.
8. Osborn, W. W., and J. L. Stokes. 1955. Appl. Microbiol. 3:217.
9. Brooks, J. and D. J. Taylor. 1955.Rep. Rd. Invest., Bd. 60, H. M.
S. O. London, England.
10. Forsythe, R. H., J. C. Ayres, and J. L. Radlo. 1953. Factors
affecting the microbiological populations of shell eggs. Food
Technol. 7:49.
11. Stadelman, W. J., A. I. Ikeme, R. A. Roop, and S. E. Simmons.
1982. Thermally processed hard-cooked eggs. Poultry Science
61:388.
Bacto Baird-Parker Agar Base

Bacto EY Tellurite Enrichment
13. MacFaddin, J. F. 1985. Media for isolation-cultivation-identificationmaintenance of medical bacteria, Vol. 1. Williams & Wilkins,
Baltimore, MD.
Packaging
BG Sulfa Agar
500 g
0717-17
SBG Enrichment
500 g
0661-17
500 g
0715-17
Intended Use
Bacto Baird-Parker Agar Base is used with Bacto EY Tellurite
Enrichment in isolating and enumerating staphylococci in foods and
other materials.
EY Tellurite Enrichment is also known as Egg Yolk Tellurite

Enrichment.
Summary And Explanation

The formulation of Baird-Parker Agar was published in 1962.1 It is a
selective medium for isolation and presumptive identification of
coagulase-positive staphylococci.
Also Known As
Baird-Parker is also known as Egg Tellurite Glycine Pyruvate Agar
(ETGPA) based on its composition.
Uninoculated
plate
ATCC 25923

Baird Parker Agar Base
Solution:
medium amber, slightly opalescent.
Prepared Medium (Final):Yellow, opalescent.
Reaction of 6.3%
Solution at 25C:
pH 6.9 + 0.1
EY Tellurite Enrichment
Appearance:
Canary yellow, opaque suspension
with a resuspendable precipitate.
Cultural Response
Baird-Parker Agar Base, EY Tellurite Enrichment
Prepare Baird-Parker Agar per label directions. Inoculate and
ORGANISM
Bacillus subtilis
Escherichia coli
Proteus mirabilis
58
ATCC
INOCULUM
CFU
GROWTH
APPEARANCE
LECITHINASE
HALOS
6633
25922*
25933
25923*
6538
14990
1,000
1,000
1,000
100
100
100
poor to fair
none
good
good
good
poor to good
brown
brown
black
black
black
N/A
+
+
The cultures listed are the minimum that should

be used for performance testing.
*These cultures are available as Bactrol Disks
and should be used as directed in Bactrol Disks
Technical Information.
The Difco Manual
Section II
Baird-Parker Agar Base & EY Tellurite Enrichment
Baird-Parker Agar is widely used and is included in many Standard

Methods procedures for testing foods, dairy products and other
materials. 2,3,4,5,6 Coagulase-positive staphylococci can grow and
reproduce in cosmetic products and these should be tested using
standard microbiological methods.4 In 1995. the American Public
Health Association (APHA) published proposed procedures for
testing swimming pools for coagulase-positive staphylococci.7

Baird-Parker Agar Base contains Tryptone and Beef Extract as
carbon and nitrogen sources for general growth. Yeast Extract supplies
B-complex vitamins which stimulate bacterial growth. Glycine and
Sodium Pyruvate stimulate growth of staphylococci. The selectivity of
the medium is due to Lithium Chloride and Potassium Tellurite
(provided in EY Tellurite Enrichment) which suppress growth of
organisms other than staphylococci. The differentiation of coagulasepositive staphylococci depends on the Potassium Tellurite and Egg Yolk
(provided in the EY Tellurite Enrichment). Staphylococci that contain
lecithinase break down the Egg Yolk and cause clear zones around the
colonies. An opaque zone of precipitation may form due to lipase
activity. Reduction of Potassium Tellurite, also a characteristic of
coagulase-positive staphylococci, causes blackening of the colonies.
Baird-Parker Agar Base

Formula Per Liter
g
g
g
g
g
g
g

Egg yolk emulsion containing Potassium Tellurite.
Precautions
2. Baird Parker Agar Base
HARMFUL. IRRITATING TO EYES, RESPIRATORY SYSTEM
AND SKIN. MAY CAUSE HARM TO THE UNBORN CHILD.
ORGAN(S): Blood, Kidneys, Nerves.
The Difco Manual
Store Baird Parker Agar Base below 30C. The powder is very
Store EY Tellurite Enrichment at 2-8C.
Expiration Date
Procedure
Materials Provided

Flask with closure
Autoclave
Petri dishes
Waterbath (45-50C)
Incubator (35C)
Formula
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Glycine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Sodium Pyruvate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Lithium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Storage
1. Suspend 63 grams Baird-Parker Agar Base in 950 ml distilled or

deionized water.
5. Warm EY Tellurite Enrichment to 45-50C and mix thoroughly to
resuspend the precipitate.
6. Aseptically add 50 ml of prewarmed enrichment to the medium.
Mix thoroughly.

Certain foods and other materials may require repair-selective
enrichment if injured cells are suspected or selective enrichment if raw
food materials or nonprocessed foods containing large numbers of
competing microorganisms are being tested.2 Consult standard references
for specific instructions for the type of material being tested.2,3,4,5
Test Procedure
1. Prepare dilutions of test samples if indicated by standard
procedure.2,3,4,5
2. Transfer 1 ml of sample to each of 3 Baird-Parker Agar plates and
distribute over the surface using a sterile, bent glass rod.
3. Allow the inoculum to be absorbed by the medium (about 10 minutes)
before inverting the plates.
4. Incubate at 35-37C for 45-48 hours.
5. Examine plates having 20-200 colonies, counting colonies typical
of S. aureus.
Results
Coagulase-positive staphylococci produce black, shiny, convex colonies
with entire margins and clear zones, with or without an opaque zone,
around the colonies.
59
Beef Extract & Beef Extract, Desiccated
Coagulase-negative staphylococci produce poor or no growth. If growth

occurs, colonies are black; clear or opaque zones are rare.
Most other organisms are inhibited or grow poorly. If growth occurs,
colonies are light to brown-black with neither clear nor opaque zones.
Section II
4
5.
Baird-Parker Agar is selective for coagulase-positive staphylococci but

other bacteria may grow. Microscopic examination and biochemical
tests will differentiate coagulase-positive staphylococci from other
microorganisms.
6.
References
7.
1. Baird-Parker, A. C. 1962. An improved diagnostic and selective

medium for isolating coagulase-positive staphylococci. J. Appl.
Bacteriol. 25:12-19.
2. Lancette, G. A., and S. R. Tatini. 1992. Staphylococcus aureus,
p. 533-550. In C. Vanderzant, and D. F. Splittstoesser (ed.), Compendium of methods for the microbiological examination of foods,
R. T. Marshall, (ed.), Standard methods for the microbiological
Bacto Beef Extract

Bacto Beef Extract, Desiccated
examination of dairy products, 16th ed. American Public Health

Association, Washington, D.C.
Association of Official Analytical Chemists. 1995. Bacteriological
Andrews, W. H. 1995. Microbial Methods, p. 1-119. Official
International. Arlington, VA.
United States Pharmacopeial Convention. 1995. The United
States Pharmacopeia, 23rd ed. The United States Pharmacopeial
Convention, Rockville, MD.
Eaton, A. D., L. S. Clesceri, and A. E. Greenberg (ed.). 1995.
Recreational waters, p. 9.26-9.27. In Standard methods for the
examination of water and wastewater, 19th ed. American Public
Packaging
100
500
2
10
g
g
kg
kg
0768-15
0768-17
0768-07
0768-08
6 x 100 ml
0779-73
Intended Use
Bacto Beef Extract and Bacto Beef Extract, Desiccated are used in
preparing microbiological culture media.

Beef Extract is prepared and standardized for use in microbiological
culture media, where it is generally used to replace infusions of meat.
Culture media containing Beef Extract have been recommended for
use in the bacteriological examination of water, milk and other materials
where having media of uniform composition is important.
Beef Extract has been employed by many investigators. Bedell and
Lewis1 used it in their medium for the study of non-sporulating
anaerobes of the intestinal tract. Hutner2 used a medium containing
Beef Extract as a stock broth in the study of nutritional needs of
streptococci. Beef Extract is the formula of Potato Infusion Agar for
the cultivation of Brucella. Fletcher Medium Base, Starch Agar,
Dextrose Agar, Dextrose Broth and CLED Agar all contain Beef
Extract to enhance the growth of bacteria. Antibiotic Assay media
specified by US Pharmacopeia3 includes Beef Extract in the formula.
Several media containing Beef Extract are recommended in standard
methods for multiple applications.4,5,6
In culture media, Beef Extract is usually employed in concentrations
of 0.3%. Concentrations may vary slightly according to the requirements
of individual formulas, but do not often exceed 0.5%. Beef Extract
60
may be relied upon for biochemical studies, particularly fermentation

reactions, because of its independence from fermentable substances
that would interfere with the accuracy of such determinations.
Beef Extract, Desiccated, the dried form of Beef Extract, was developed
to provide a product for ease of use in handling. Beef Extract is in the
paste form. The products are to be used in a one for one substitution,
however variations tend to be formulation specific and require actual

Beef Extract and Beef Extract, Desiccated are replacements for infusion
of meat. Beef Extract and Beef Extract, Desiccated provide nitrogen,
vitamins, amino acids and carbon in several formulations of microbiological culture media.
Typical Analysis
BEEF EXTRACT
BEEF EXTRACT, DESICCATED
Ash (%)
Clarity, 1% Soln (NTU)
Filterability (g/cm2)
Loss on Drying (%)
pH, 1% Soln
24.1
116.8
0.1
77.2
5.4
10.2
1.7
0.6
2.5
6.9
0.2
<0.1
11.2
3.8
33.8
14.0
2.2
15.7
Carbohydrate (%)
Total
Nitrogen Content (%)

Total Nitrogen
Amino Nitrogen
AN/TN
The Difco Manual
Section II
Beef Extract & Beef Extract, Desiccated

BEEF EXTRACT
Amino Acids (%)

Alanine
Arginine
Aspartic Acid
Cystine
Glutamic Acid
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine
2.54
1.39
1.67
0.18
6.01
4.14
4.94
0.53
1.00
1.45
0.30
<0.01
2.16
0.90
0.67
0.05
1.99
0.86
8.96
5.66
4.30
0.17
12.55
16.25
2.50
1.45
3.63
3.27
1.08
2.00
9.58
2.10
1.42
0.32
1.03
2.62

Beef Extract
Dehydrated Appearance: Medium to dark brown paste.
Solution:
0.3% solution - soluble in distilled
or deionized water upon warming.
Light to medium amber in color,
clear, no precipitate.
Reaction of 0.3%
Solution at 25C:
pH 6.9 0.2
Dehydrated Appearance: Medium to dark brown, free-flowing,
homogeneous powder.
Solution:
0.3% solution - soluble in distilled
or deionized water at a 0.3%
concentration. 0.3% solution is light
to medium amber in color, clear
Reaction of 0.3%
Solution at 25C:
pH 6.6-7.4
Cultural Response
Beef Extract
Prepare a sterile solution of 0.3% Beef Extract or Beef Extract,
Desiccated, and 0.5% Bacto Peptone. Adjust the pH to 6.9-7.1.
Inoculate tubes with the test organisms, incubate at 35 2C
for 18-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
14028*
25923*
100-1,000
100-1,000
good
good
used as directed in Bactrol Disks Technical Information.
The Difco Manual
BEEF EXTRACT
Inorganics (%)
Calcium
Chloride
Cobalt
Copper
Iron
Lead
Magnesium
Manganese
Phosphate
Potassium
Sodium
Sulfate
Sulfur
Tin
Zinc
0.068
1.284
<0.001
<0.001
<0.001
<0.001
0.239
<0.001
5.458
5.477
2.315
0.629
0.707
<0.001
<0.001
0.018
1.576
<0.001
0.001
0.001
<0.001
0.022
<0.001
0.345
1.994
2.774
0.829
0.661
<0.001
0.002
Vitamins (g/g)
Biotin
Choline (as Choline Chloride)
Cyanocobalamin
Folic Acid
Inositol
Nicotinic Acid
PABA
Pantothenic Acid
Pyridoxine
Riboflavin
Thiamine
Thymidine
0.1
1171.5
0.5
3.3
4113.2
774.7
20.0
91.0
7.3
0.4
<0.1
1093.4
0.1
1300.0
<0.1
0.6
2100.0
138.1
40.5
8.7
2.8
<0.1
<0.1
111.3

Coliform
Salmonella
Spore Count
Thermophile Count
negative
negative
299
117
33
negative
negative
585
690
28
Precautions
Storage
Store the dehydrated product below 30C. The dehydrated ingredient
is very hygroscopic. Keep container tightly closed.
Expiration Date
Procedure
Materials Provided
Beef Extract
61
BiGGY Agar
Section II

Materials vary depending on the medium being prepared.
2. Formula allowances may be required due to the lower sodium

chloride concentration of Beef Extract, Desiccated.
References
Refer to the final concentration of Beef Extract or Beef Extract,

Desiccated in the formula of the medium being prepared. Add Beef
Extract or Beef Extract, Desiccated as required.
1. Bedell and Lewis. 1938. J. Bacteriol. 36:567.

2. Hutner. 1938. J. Bacteriol. 35:429.
States pharmacopeia, 23rd. Ed. The United States Pharmacopeial
5. Association of Official Analytical Chemists. 1995. Bacteriological analytical manual, 8th ed. AOAC International,
Gaithersburg, MD.

Test Procedure
See appropriate references for specific procedures using Beef Extract
or Beef Extract, Desiccated.
Results

may be encountered that fail to grow or grow poorly on prepared
medium.
Bacto BiGGY Agar
Packaging
Beef Extract
500 g
0126-17
500 g
10 kg
0115-17
0115-08
Intended Use
Bacto BiGGY Agar is used for isolating and differentiating
Candida spp.
Also Known As
BiGGY Agar is an abbreviation for Bismuth Glucose Glycine Yeast Agar.
BiGGY Agar is also referred to as Nickerson Agar and Nickerson
Candida Elective Agar.

BiGGY Agar is a modification of the formula described by Nickerson.1,2
This medium was developed while studying sulfite reduction of
Candida species. Nickerson described BiGGY Agar as a selective
and differential medium for the isolation of Candida albicans.
C. albicans can be differentiated from other Candida species based on
colony morphology.
Candidiasis is the most frequently encountered opportunistic fungal
infection.3 It is caused by a variety of species of Candida, with
Candida albicans being the most frequent etiological agent, followed
by Candida tropicalis and Candida (Torulopsis) glabrata.3 Candida
species can be present in clinical specimens as a result of environmental
contamination, colonization or actual disease process.4

Yeast Extract provides the nitrogen, vitamins and amino acids in
BiGGY Agar. Glycine is used to stimulate growth. Dextrose is the carbon
62
source. Candida species reduce bismuth sulfite, and colonies become

brown to black in color. Bismuth Sulfite Indicator is also used as a
selective agent against bacteria, often present as normal flora. Bacto
Agar is used as the solidifying agent.
Formula
BiGGY Agar
Formula Per Liter
Glycine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bismuth Sulfite Indicator . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
g
g
g
g
g
Precautions
Storage
Expiration Date
The Difco Manual
Section II
BiGGY Agar
Procedure
C. tropicalis
Materials Provided
BiGGY Agar
Discrete dark brown colonies with black centers

and sheen, medium sized, diffuse blackening
of the surrounding medium after 72 hours of
incubation.
C. pseudotropicalis Large, dark reddish-brown colonies, flat with

slight mycelial fringe.
Glassware
Incubator (30C)
Waterbath (optional)
C. krusei
Large flat wrinkled colonies with silvery black

top, brown edge and yellow halo.
C. parakrusei
Medium sized flat wrinkled colonies with red

dish-brown color and yellow mycelial fringe.
C. stellatoidea
Medium size, flat, dark brown colonies; very

light mycelial fringe.
2. Heat to boiling to dissolve completely. Avoid overheating. DO
NOT AUTOCLAVE.
3. Evenly disperse the flocculent precipitate when dispensing.

Test Procedure
For a complete discussion on the isolation and identification of yeast
species refer to the procedures described in appropriate references.3,4
Results
Colony morphology according to Nickerson2 after 48 hours of incubation
on BiGGY Agar:
C. albicans
Intensely brown-black colonies with slight
mycelial fringe, medium sized, no diffusion.

this medium.
2. Pigmented bacterial and yeast-like fungi are usually inhibited on
BiGGY Agar. They can be differentiated by microscopic examination, if necessary. Dermatophytes and molds seldom appear and
are easily recognized by development of aerial mycelia.5
3. Further growth characteristic and biochemical tests are needed
to differentiate yeasts, particularly identification of Candida
species.5
Uninoculated
plate
Candida albicans
ATCC 10231

Solution:
4.9% solution, soluble upon boiling in
distilled or deionized water. Solution is
very light to light amber, opalescent
with a flocculent dispersable precipitate.
Prepared Medium:
Very light to light amber, opalescent
with a flocculent precipitate.
Reaction of 4.9%
Solution at 25C:
pH 6.8 0.2
Cultural Response
Prepare BiGGY Agar per label directions. Inoculate and
ORGANISM
ATCC
INOCULUM
CFU
COLONY
DESCRIPTION
GROWTH
Candida albicans 10231
100-1,000 brown to black, no diffusion

good
into medium, no sheen
Candida kefyr
4135 100-1,000
reddish brown, flat
good
colonies, no diffusion
Candida tropicalis 750
100-1,000
brown to black, sheen,
good
black diffusion into medium
Escherichia coli 25922* 1,000-2,000
markedly inhibited
The Difco Manual
63
Bile Esculin Agar Base & Bile Esculin Agar
Section II
4. It is recommended that BiGGY Agar be prepared fresh, just prior

to use.1,2
5. Do not use slants because the reactions are unsatisfactory.1,2
References
1. Nickerson, W. J. 1947. Biology of pathogenic fungi. The Chronica
Botanica Co., Waltham, MA.
2. Nickerson, W. J. 1953. Reduction of inorganic substances by
yeasts. I. Extracellular reduction of sulfite by species of Candida.
J. Infect. Dis. 93:43.
St. Louis, MO.
4. Warren, N. G., and K. C. Hazen. 1995. Candida, Cryptococcus,

and other yeasts of medical importance, p. 723-737. In P. R. Murray,
E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken (ed.).,
Packaging
BiGGY Agar
Bacto Bile Esculin Agar Base

Bacto Bile Esculin Agar
Also Known As
Intended Use
100 g
500 g
0635-15
0635-17
Bacto Bile Esculin Agar Base (with added esculin) and Bacto Bile
Esculin Agar are differential media used for isolating and presumptively
identifying group D streptococci.
Bile Esculin Agar is also known as Bile Esculin Medium (BEM). The
spelling, aesculin, is often seen in literature.
Bile Esculin Agar Base and Bile Esculin Agar are prepared according
to the formulation described by Swan1 and further evaluated by
Uninoculated
plate
ATCC 29212

Dehydrated Appearance: Greenish, light to medium beige,
homogeneous, free-flowing.
Solution:
6.3% solution Bile Esculin Agar Base;
6.4% solution Bile Esculin Agar: soluble
in distilled or deionized water on boiling.
Solutions are medium to dark amber,
slightly opalescent; media with
esculin have a bluish cast.
Prepared Plates:
Greenish to medium amber, slightly
opalescent; media with esculin have a
bluish cast.
Reaction of
Solution at 25C:
6.3% solution Bile Esculin Agar Base;
6.4% solution Bile Esculin Agar:
pH 6.6 0.2
Cultural Response
Prepare Bile Esculin Agar Base or Bile Esculin Agar per label directions. Add
0.1% esculin to Bile Esculin Agar Base. Inoculate and incubate at 35 2C for
18-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
ESCULIN
HYDROLYSIS
29212*
19615*
100-1,000
2,000-10,000
good
inhibited
+ = positive, blackening of medium
= negative, no change
64
The Difco Manual
Section II
Facklam and Moody.2 Rochaix3 first noted the value of esculin hydrolysis
in the identification of enterococci. Meyer and Schnfeld4 added bile
to the esculin medium and demonstrated that 61 of 62 enterococci
strains were able to grow and hydrolyze esculin, while the other
streptococci could not.
Molecular taxonomic studies of the genus Streptococcus have placed
enterococci, previously considered group D streptococci, in the distinct
genus Enterococcus.6 Streptococci with Lancefield group D antigen
include the nonhemolytic species Streptococcus bovis.7 The ability to
hydrolyze esculin in the presence of bile is a characteristic of enterococci
and group D streptococci.
Swan1 compared the use of an esculin medium containing 40% bile
salts with the Lancefield serological method of grouping. He reported
that a positive reaction on the bile esculin medium correlated with a
serological group D precipitin reaction. Facklam and Moody,2 in
a comparative study of tests used to presumptively identify group D
streptococci, found that the bile esculin test provided a reliable means
of identifying group D streptococci and differentiating them from
non-group D streptococci. Facklam5 further confirmed the usefulness
of Bile Esculin Agar in another study differentiating enterococci/group
D streptococci from non-group D streptococci.
Lindell and Quinn8 showed that the medium is also useful in the
differentiation of the Klebsiella-Enterobacter-Serratia group from other
Enterobacteriaceae. Edberg et al.9 recommended the medium for routine
testing of the Enterobacteriaceae in order to differentiate
Klebsiella-Enterobacter-Serratia spp. Bile Esculin Agar is listed in standard
procedures for the microbiological examination of food products.10-13
Precautions
2. Bile Esculin Agar Base:
Wear suitable gloves and eye/face protection. Use only in well
ventilated areas. Keep container tightly closed.
Bacto Bile Esculin Agar:
TARGET ORGAN(S): Lungs.
Storage

Organisms positive for esculin hydrolysis hydrolyze the glycoside
esculin to esculetin and dextrose. The esculetin reacts with the ferric citrate
to form a dark brown or black complex. Oxgall (bile) is used to inhibit
gram-positive bacteria other than enterococci. Beef Extract and Bacto
Peptone provide the carbon and nitrogen sources required for growth of a
wide variety of organisms. Bacto Agar is the solidifying agent.

Expiration Date
Formula
Procedure
Bile Esculin Agar

Formula Per Liter
Materials Provided

Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Esculin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Bacto Oxgall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Ferric Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g

Bile Esculin Agar Base
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Oxgall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Ferric Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
The Difco Manual
g
g
g
g
g

Bile Esculin Agar

Glassware
Autoclave
Incubator (35C)
Esculin (to be added to Bile Esculin Agar Base)
Filter-sterilized horse serum (optional)
Petri dishes
Tubes with closures
1. Suspend the specidied amount of medium in 1 liter distilled or
deionized water:
Bile Esculin Agar Base - 63 grams
Bile Esculin Agar - 64 grams
65

3. Bile Esculin Agar Base, only: Add 1 gram (or another desired
amount) of Esculin and mix thoroughly.
4. Autoclave at 121C for 15 minutes. Overheating may cause
darkening of the media.
5. Cool to 50-55C.
6. If desired, aseptically add 50 ml of filter-sterilized horse serum.
Mix thoroughly.
7. Dispense as desired.

Test Procedure
Results

1. The bile esculin test was originally formulated to identify
enterococci. However, the properties of growth on 40% bile media
and esculin hydrolysis are characteristics shared by most strains of
Group D streptococci.14 The bile esculin test should be used in
combination with other tests to make a positive identification.
Facklam14 and Facklam et al.15 recommend a combination of the bile
esculin test and salt tolerance (growth in 6.5% NaCl). Streptococcus
bovis will give a positive reaction on Bile Esculin Agar, but unlike
Enterococcus spp., it cannot grow on 6.5% NaCl or at 10C.16
2. Bile Esculin Agar should be considered a differential medium, but
with the addition of sodium azide (which inhibits gram-negative
bacteria) the medium can be made more selective (see Bile Esculin
Azide Agar).
3. Occasional viridans strains will be positive on Bile Esculin Agar
or will display reactions that are difficult to interpret.17 Of the
viridans group, 5 to 10% may be able to hydrolyze esculin in the
presence of bile.16
4. Use a light inoculum when testing Escherichia coli on Bile Esculin
Agar. Wasilauskas18 suggests that the time required for an isolate
to hydrolyze esculin is directly proportional to the size of the
inoculum. For a tabulation of those Enterobacteriaceae that can
hydrolyze esculin, refer to Farmer.19
References
1. Swan, A. 1954. The use of bile-esculin medium and of Maxteds
technique of Lancefield grouping in the identification of enterococci
(group D streptococci). J. Clin. Pathol. 7:160.
2. Facklam, R. R., and M. D. Moody. 1970. Presumptive identification of group D streptococci: The bile-esculin test. Appl.
Microbiol. 20:245.
3. Rochaix, A. 1924. Milieux a leculine pour le diagnostid
differentieldes bacteries du groups strepto-entero-pneumocoque.
Comt. Rend. Soc. Biol. 90: 771-772.
4. Meyer, K., and H. Schnfeld. 1926. ber die Untersheidung des
Enterococcus vom Streptococcus viridans und die Beziehunger
66
Section II
beider zum Streptococcus lactis. Zentralbl. Bakteriol. Parasitenkd.

Infektionskr. Hyg. Abt. I Orig. 99:402-416.
5. Facklam, R. R. 1973. Comparison of several laboratory media for
presumptive identification of enterococci and group D streptococci.
Appl. Microbiol. 26:138.
6. Schleifer, K. H., and R. Kilpper-Balz. 1987. Molecular and
chemotaxonomic approaches to the classification of streptococci,
enterococci and lactococci: a review. Syst. Appl. Microbiol. 10:1-19.
7. Ruoff, K. L. 1995. Streptococcus. P. R. Murray, E. J. Baron, M. A.
Pfaller, F. C. Tenover and R. H. Yolken (eds.), Manual of clinical
microbiology, 6th ed. American Society for Microbiology,
Washington, D.C.
8. Lindell, S. S., and P. Quinn. 1975. Use of bile-esculin agar for rapid
differentiation of Enterobacteriaceae. J. Clin. Microbiol. 1:440.
9. Edberg, S. C., S. Pittman, and J. M. Singer. 1977. Esculin
hydrolysis by Enterobacteriaceae. J. Clin. Microbiol. 6:111.
10. Bacteriological Analytical Manual. 1995. 8th ed. AOAC
11. Vanderzant, C., and D. F. Splittstoesser (eds.). 1992. Compendium of methods for the microbiological examination of foods,
12. Marshall, R. T. (ed.) 1992. Standard methods for the examination
of dairy products, 16th ed. American Public Health Association,
Washington, D.C.
13. Atlas, R. M. 1995. Handbook of microbiological media for the
examination of food. CRC Press, Boca Raton, FL.
14. Facklam, R. 1972. Recognition of group D streptococcal species
of human origin by biochemical and physiological tests. Appl.
Microbiol. 23:1131.
15. Facklam, R. R., J. F. Padula, L. G. Thacker, E. C. Wortham,
and B. J. Sconyers. 1974. Presumptive identification of group A,
B, and D streptococci. Appl. Microbiol. 27:107.
16. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey
& Scotts diagnostic microbiology, 9th ed. Mosby-Year Book,
Inc. St. Louis, MO.
17. Ruoff, K. L., S. I. Miller, C. V. Garner, M. J. Ferraro, and S. B.
Calderwood. 1989. Bacteremia with Streptococcus bovis and
Streptococcus salivarius: clinical correlates of more accurate
identification of isolates. J. Clin. Microbiol. 27:305-308.
18. Wasilauskas, B. L. 1971. Preliminary observations on the rapid
differentiation of the Klebsiella-Enterobacter-Serratia group on
bile-esculin agar. Appl. Microbiol. 21:162.
19. Farmer, J. J., III. 1995. Enterobacteriaceae. P. R. Murray, E. J.
Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken (eds.), Manual
of clinical microbiology, 6th ed. American Society for Microbiology,
Washington, D.C.
Packaging
500 g
0878-17
Bile Esculin Agar
100 g
500 g
0879-15
0879-17
10 g
0158-12
Esculin
The Difco Manual
Section II
Bile Esculin Azide Agar
Bacto Bile Esculin Azide Agar
Intended Use
Bacto Bile Esculin Azide Agar is used for isolating, differentiating and
presumptively identifying group D streptococci.
Also Known As
Bile Esculin Azide (BEA) Agar conforms with Selective Enterococcus
Medium (SEM) and Pfizer Selective Enterococcus Medium (PSE).

Bile Esculin Azide Agar is a modification of the medium reported by
Isenberg1 and Isenberg, Goldberg and Sampson.2 The formula modifies
Bile Esculin Agar by adding sodium azide and reducing the concentration
of bile. The resulting medium is more selective but still provides
for rapid growth and efficient recovery of group D streptococci.
Enterococcal streptococci were previously grouped in the genus
Streptococcus with the Lancefield group D antigen. Molecular taxonomic
studies have shown that enterococci were sufficiently different from
other members of the genus Streptococcus to warrant the separate
genus Enterococcus.6 Other streptococci with the group D antigen
exist in the genus Streptococcus, such as the non-hemolytic species
Streptococcus bovis.9
The ability to hydrolyze esculin in the presence of bile is a characteristic
of enterococci and group D streptococci. Esculin hydrolysis and bile
tolerance, as shown by Swan3 and by Facklam and Moody4, permit the
isolation and identification of group D streptococci in 24 hours. Sabbaj,

Dehydrated Appearance: Light beige to medium beige,
free-flowing, homogeneous.
Solution:
is medium to dark amber with bluish
cast, very slightly to slightly opalescent
without significant precipitate.
Prepared Medium:
Medium to dark amber with bluish
cast, slightly opalescent.
Reaction of 5.7%
Solution at 25C:
pH 7.1 0.2
Cultural Response
Prepare Bile Esculin Azide Agar per label directions. Inoculate
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
ESCULIN
HYDROLYSIS
Enterococcus 29212* 100-1,000

good
positive, blackening
faecalis
of the medium
Escherichia 25922* 1,000-2,000 marked to
negative, no color
coli
complete inhibition
change
The Difco Manual
Sutter and Finegold5 evaluated selective media for selectivity, sensitivity,

detection, and enumeration of presumptive group D streptococci from
human feces. Bile Esculin Azide Agar selected for S. bovis, displayed
earlier distinctive reactions, and eliminated the requirement for
special incubation temperatures.
Brodsky and Schiemann6 evaluated Pfizer Selective Enterococcus
Medium (Bile Esculin Azide Agar) in the recovery of fecal streptococci
from sewage effluent on membrane filters and found the medium to be
highly selective for enterococci. Jensen7 found that Bile Esculin Azide
Agar supplemented with vancomycin combines differential and
selective properties to rapidly isolate vancomycin-resistant enterococci
from heavily contaminated specimens.

Organisms positive for esculin hydrolysis hydrolyze the glycoside
esculin to esculetin and dextrose. Esculetin reacts with ferric ammonium
citrate to form a dark brown or black complex. Oxgall (bile) inhibits
gram-positive bacteria other than enterococci, while sodium azide
inhibits gram-negative bacteria. Tryptone and Proteose Peptone No. 3
provide nitrogen, vitamins and minerals. Yeast Extract provides
vitamins and cofactors required for growth, as well as additional
sources of nitrogen and carbon. Sodium chloride maintains the
osmotic balance of the medium. Bacto Agar is the solidifying agent.
Formula
Formula Per Liter
Bacto Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . . . 3
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Bacto Oxgall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Esculin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Ferric Ammonium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Azide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.15
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g
g
g
Precautions
2. HARMFUL. IRRITATING TO EYES, RESPIRATORY SYSTEM
AND SKIN. HARMFUL BY INHALATION AND IF SWALLOWED. Avoid contact with skin and eyes. Do not breathe dust.
give oxygen. Seek medical advice. If swallowed seek medical
Storage
67
Biotin Assay Medium
Expiration Date
Procedure
Materials Provided

Glassware
Autoclave
Incubator (35C)
Petri dishes
Horse Serum, filter sterilized (optional)
2. Boil to dissolve completely.
3. Autoclave at 121C for 15 minutes. Overheating may cause
darkening of the medium.
4. If desired, aseptically add 50 ml of filter-sterilized horse serum.
Mix thoroughly.

Test Procedure
For isolation of group D streptococci, inoculate the sample onto a small
area of one quadrant of a Bile Esculin Azide Agar plate and streak for
isolation. This will permit development of discrete colonies. Incubate
at 35C for 18-24 hours. Examine for colonies having the characteristic
morphology of group D streptococci.
Results
Group D streptococci grow readily on this medium and hydrolyze
esculin, resulting in a dark brown color around the colonies after
18-24 hours incubation.
Section II
exhibit growth on the medium (less than 1 mm, white-gray

colonies), but they will show no action on the esculin.2
2. Other than the enterococci, Listeria monocytogenes consistently
blackens the medium around colonies. After 18-24 hours, there may
be a reddish to black-brown zone of hydrolysis surrounding pinpoint
Listeria colonies. After 48 hours, white-gray pigmented colonies will
be seen. Listeria do not attain the same degree of esculin hydrolysis
displayed by enterococci in this short incubation period.2
References
1. Isenberg, H. D. 1970. Clin. Lab. Forum. July.
2. Isenberg, H. D., D. Goldberg, and J. Sampson. 1970. Laboratory
studies with a selective enterococcus medium. Appl. Microbiol.
20:433.
3. Swan, A. 1954. The use of bile-esculin medium and of Maxteds
technique of Lancefield grouping in the identification of
enterococci (group D streptococci). J. Clin. Pathol. 7:160.
4. Facklam, R. R., and M. D. Moody. 1970. Presumptive
identification of group D streptococci: The bile-esculin test.
5. Sabbaj, J., V. L. Sutter, and S. M. Finegold. 1971. Comparison
of selective media for isolation of presumptive group D
streptococci from human feces. Appl. Microbiol. 22:1008.
6. Brodsky, M. H., and D. A. Schiemann. 1976. Evaluation of Pfizer
selective enterococcus and KF media for recovery of fecal
streptococci from water by membrane filtration. Appl. Environ.
Microbiol. 31:695-699.
7. Jensen, B. J. 1996. Screening specimens for vancomycin-resistant
Enterococcus. Laboratory Medicine 27:53-55.
8. Schleifer, K. H., and R. Kilpper-Balz. 1987. Molecular and
chemotaxonomic approaches to the classification of streptococci,
enterococci and lactococci: a review. Syst. Appl. Microbiol. 10:1-19.
9. Ruoff, K. L. 1995. Streptococcus. In P. R. Murray, E. J. Baron,
M. A. Pfaller, F. C. Tenover, and R. H. Yolken (eds.), Manual of
clinical microbiology, 6th ed. American Society for Microbiology,
Washington, D.C.
Packaging
1. Staphylococcus aureus and Staphylococcus epidermidis may
Bacto Biotin Assay Medium
100 g
500 g
2 kg
0525-15
0525-17
0525-07
Bacto Biotin Assay Medium is used for determining biotin concentration

by the microbiological assay technique.

Assay media contain all the factors necessary for optimal growth of
the test organism except the single essential vitamin to be determined.
Biotin Assay Medium is prepared for use in the microbiological assay of
biotin using Lactobacillus plantarum ATCC 8014 as the test organism.
Vitamin Assay Media are used in the microbiological assay of

vitamins. Three types of media are used for this purpose:
Biotin Assay Medium is a biotin-free dehydrated medium containing

all other nutrients and vitamins essential for the cultivation of
L. plantarum ATCC 8014. The addition of biotin standard in specified
increasing concentrations gives a growth response by this organism
that can be measured titrimetrically or turbidimetrically.
Intended Use
68
The Difco Manual
Section II
Biotin Assay Medium
Formula

Biotin Assay Medium

Formula Per Liter
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
DL-Tryptophane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
Adenine Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Uracil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Storage
g
g
g
g
g
mg
mg
mg
mg
mg
mg
mg
mg
g
g
g
g
mg
mg
mg
Precautions
2. Take great care to avoid contamination of media or glassware for
microbiological assay procedures. Extremely small amounts of
foreign material may be sufficient to give erroneous results.
Scrupulously clean glassware, free from detergents and other
chemicals, must be used. Glassware must be heated to 250C for at
3. Take precautions to keep sterilization and cooling conditions
uniform throughout the assay.

Expiration Date
Procedure
Materials Provided
Biotin Assay Medium

Lactobacilli Agar AOAC
Centrifuge
Spectrophotometer
Biotin
Glassware
Autoclave
Sterile tubes
Stock culture of Lactobacillus plantarum ATCC 8014
1.
2.
3.
4.
5.
6.

Boil 2-3 minutes to dissolve completely.

Dehydrated Appearance: Light beige, homogeneous with a
tendency to clump.
Solution:
3.75% (single strength) solution,
soluble in distilled or deionized water
on boiling 2-3 minutes. Light amber,
clear, may have a slight precipitate.
Prepared Medium:
(Single strength) light amber, clear,
may have slight precipitate.
Reaction of 3.75%
Solution at 25C:
pH 6.8 0.2
Cultural Response
Prepare Biotin Assay Medium per label directions. Prepare a
standard curve using biotin at levels of 0.0, 0.1, 0.2, 0.3, 0.4,
0.5, 0.6, 0.8 and 1 ng per 10 ml. The medium supports the
growth of L. plantarum ATCC 8014 when prepared in single
strength and supplemented with biotin.
The Difco Manual

Test Procedure
Stock Cultures
Stock cultures of the test organism, L. plantarum ATCC 8014, are
prepared by stab inoculation of Lactobacilli Agar AOAC. After
16-24 hours incubation at 35-37C, the tubes are stored in the
refrigerator. Transfers are made weekly.
Inoculum
Inoculum for assay is prepared by subculturing from a stock culture
of L. plantarum ATCC 8014 to 10 ml of single-strength Biotin
Assay Medium supplemented with 0.5 ng biotin. After 16-24 hours
incubation at 35-37C, the cells are centrifuged under aseptic conditions
and the supernatant liquid decanted. The cells are washed three times
with 10 ml sterile 0.85% saline. After the third wash, the cells are
resuspended in 10 ml sterile 0.85% saline and finally diluted 1:100
with sterile 0.85% saline. One drop of this suspension is used to inoculate
each 10 ml assay tube.
69
Bismuth Sulfite Agar
Standard Curve
run. Autoclave and incubation conditions can influence the standard
curve reading and cannot always be duplicated. The standard curve is
obtained by using biotin at levels of 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8
and 1 ng per assay tube (10 ml).
The concentration of biotin required for the preparation of the
standard curve may be prepared by dissolving 0.1 gram of d-Biotin
or equivalent in 1,000 ml of 25% alcohol solution (100 g per ml).
Dilute the stock solution by adding 2 ml to 98 ml of distilled water.
This solution is diluted by adding 1 ml to 999 ml distilled water, giving
a solution of 2 ng of biotin per ml. This solution is further diluted by
adding 10 ml to 90 ml distilled water, giving a final solution of 0.2 ng
of biotin per ml. Use 0.0, 0.5, 1, 1.5, 2, 2.5, 3, 4 and 5 ml of this final
solution. Prepare the stock solution fresh daily.
Biotin Assay Medium may be used for both turbidimetric and titrimetric
analysis. Before reading, the tubes are refrigerated for 15-30 minutes
to stop growth. Turbidimetric readings should be made after 16-20
hours at 35-37C. Titrimetric determinations are made after 72 hours
incubation at 35-37C. The most effective assay range, using Biotin
Assay Medium, has been found to be between 0.1 ng and 1 ng biotin.
For a complete discussion of antibiotic assay methodology, refer to
appropriate procedures outlined in the references.1,2
Results
Calculations
response readings against the amount of standard in each tube,
disk or cup.
Bacto Bismuth Sulfite Agar
Section II

more than 10% from the average. Use the results only if two
thirds of the values do not vary by more than 10%.

cultured and maintained on media recommended for this
purpose.
response.
4. For successful results to these procedures, all conditions of the assay must be followed precisely.
References
1. Federal Register. 1992. Tests and methods of assay of antibiotics
and antibiotic-containing drugs. Fed. Regist. 21:436.100-436.106.
States pharmacopeia, 23rd ed. Biological tests and assay,
p. 1690-1696. The United States Pharmacopeial Convention,
Rockville, MD.
Packaging
Biotin Assay Medium
100 g
0419-15
Intended Use
Bacto Bismuth Sulfite Agar is used for isolating Salmonella spp,
particularly Salmonella typhi, from food and clinical specimens.

domesticated animals. Infection with nontyphi Salmonella often causes
mild, self-limiting illness.1 Typhoid fever, caused by S. typhi, is
characterized by fever, headache, diarrhea, and abdominal pain, and
can produce fatal respiratory, hepatic, splenic, and/or neurological
damage. These illnesses result from consumption of raw, undercooked
or improperly processed foods contaminated with Salmonella. Many
cases of Salmonella-related gastroenteritis are due to improper
handling of poultry products. United States federal guidelines require
various poultry products to be routinely monitored before distribution
for human consumption but contaminated food samples often
elude monitoring.
Bismuth Sulfite Agar is a modification of the Wilson and Blair2-4
formula. Wilson5,6 and Wilson and Blair2-4 clearly showed the superiority of Bismuth Sulfite medium for isolation of S. typhi. Cope and
Kasper7 increased their positive findings of typhoid from 1.2 to 16.8%
70
among food handlers and from 8.4 to 17.5% among contacts with
Bismuth Sulfite Agar. Employing this medium in the routine laboratory
examination of fecal and urine specimens, these same authors 8
obtained 40% more positive isolations of S. typhi than were obtained
on Endo medium. Gunther and Tuft,9 employing various media in a
comparative way for the isolation of typhoid from stool and urine
specimens, found Bismuth Sulfite Agar most productive. On Bismuth
Sulfite Agar, they obtained 38.4% more positives than on Endo Agar,
33% more positives than on Eosin Methylene Blue Agar, and 80%
more positives on Bismuth Sulfite Agar than on the Desoxycholate
media. These workers found Bismuth Sulfite Agar to be superior to
Wilsons original medium. Bismuth Sulfite Agar was stable, sensitive
and easier to prepare. Green and Beard,10 using Bismuth Sulfite Agar,
claimed that this medium successfully inhibited sewage organisms.
The value of Bismuth Sulfite Agar as a plating medium after
enrichment has been demonstrated by Hajna and Perry.11
Since these earlier references to the use of Bismuth Sulfite Agar, this
medium has been generally accepted as routine for the detection of
most Salmonella. The value of the medium is demonstrated by the many
references to the use of Bismuth Sulfite Agar in scientific publications,
laboratory manuals and texts. Bismuth Sulfite Agar is used in microbial limits testing as recommended by the United States Pharmacopeia.
In this testing, pharmaceutical articles of all kinds, from raw materials
to the finished forms, are evaluated for freedom from Salmonella spp.12
The Difco Manual
Section II
For food testing, the use of Bismuth Sulfite Agar is specified for the
isolation of pathogenic bacteria from raw and pasteurized milk, cheese
products, dry dairy products, cultured milks, and butter.1,13-15 The use
of Bismuth Sulfite Agar is also recommended for use in testing clinical
specimens.16,17 In addition, Bismuth Sulfite Agar is valuable when
investigating outbreaks of Salmonella spp., especially S. typhi.18-20
Bismuth Sulfite Agar is used for the isolation of S. typhi and other
Salmonella from food, feces, urine, sewage and other infectious
materials. The typhoid organism grows luxuriantly on the medium,
forming characteristic black colonies, while gram-positive bacteria
and members of the coliform group are inhibited. This inhibitory
action of Bismuth Sulfite Agar toward gram-positive and coliform
organisms permits the use of a much larger inoculum than possible
with other media employed for similar purposes in the past. The use
of larger inocula greatly increases the possibility of recovering the
pathogens, especially when they are present in relatively small
numbers. Small numbers of organisms may be encountered in the early
course of the disease or in the checking of carriers and releases.

In Bismuth Sulfite Agar, Beef Extract and Bacto Peptone provide
nitrogen, vitamins and minerals. Dextrose is an energy source.
Disodium phosphate is a buffering agent. Bismuth sulfite indicator and
brilliant green are complementary in inhibiting gram-positive bacteria
and members of the coliform group, while allowing Salmonella to
grow luxuriantly. Ferrous sulfate is for H2S production. When H2S is
present, the iron in the formula is precipitated, giving positive cultures
the characteristic brown to black color with metallic sheen. Agar is a
solidifying agent.
Formula
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3
Bismuth Sulfite Indicator . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Brilliant Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.025
g
g
g
g
g
g
g
g
Precautions
2. HARMFUL. MAY CAUSE SENSITIZATION BY INHALATION. IRRITATING TO EYES, RESPIRATORY SYSTEM AND
SKIN. Avoid contact with skin and eyes. Do not breathe dust. Wear
Salmonella typhi
ATCC 19430
Uninoculated
plate

Dehydrated Appearance: Light beige to light green, free-flowing,
homogeneous.
Solution:
is light green, opaque with a flocculent
precipitate that must be dispersed by
swirling contents of flask.
Prepared Plates:
Light grey-green to medium green,
opaque with a flocculent precipitate.
Reaction of 5.2%
solution at 25C:
7.7 0.2
Cultural Response
Prepare Bismuth Sulfite Agar per label directions.
Inoculate and incubate the plates at 35 2C for 24-48 hours.
ORGANISM
ATCC
CFU
GROWTH
COLONY COLOR
Escherichia coli
25922* 1,000-2,000 partial inhibition brown to green
Salmonella typhi
19430 100-1,000
good
black w/metallic sheen
Salmonella typhimurium 14028* 100-1,000
good
black or greenish-grey,
may have sheen
Enterococcus faecalis 29212* 1,000-2,000 markedly inhibited
ATCC 14028
The Difco Manual
71
Section II
Storage
Results
Store the dehydrated medium below 30C. The dehydrated medium

is very hygroscopic. Keep container tightly closed. Store prepared
plates at 2-8C.
The typical discrete S. typhi surface colony is black and surrounded by

a black or brownish-black zone which may be several times the size of
the colony. By reflected light, preferably daylight, this zone exhibits a
distinctly characteristic metallic sheen. Plates heavily seeded with
S. typhi may not show this reaction except near the margin of the mass
inoculation. In these heavy growth areas, this organism frequently
appears as small light green colonies. This fact emphasizes the
importance of inoculating plates so that some areas are sparsely populated
with discrete S. typhi colonies. Other strains of Salmonella produce black
to green colonies with little or no darkening of the surrounding medium.
Generally, Shigella spp. other than S. flexneri and S. sonnei are
inhibited. Shigella flexneri and Shigella sonnei strains that do grow on
this medium produce brown to green, raised colonies with depressed
centers and exhibit a crater-like appearance.
Expiration Date
Procedure
Materials Provided

Waterbath (45-50C)
Petri dishes
Incubator (35C)
2. Heat to boiling no longer than 1-2 minutes to dissolve. Avoid
overheating. DO NOT AUTOCLAVE.
4. Gently swirl flask to evenly disperse the flocculent precipitate.
Dispense into sterile Petri dishes.
NOTE: Best results are obtained when the medium is dissolved
and used immediately. The melted medium should not be allowed
to solidify in flasks and remelted. Current references suggest
that the prepared plated medium should be aged for one day
before use.13,21

1. Collect specimens or food samples in sterile containers or with
sterile swabs and transport immediately to the laboratory following
recommended guidelines.1,13-20
2. Process each specimen, using procedures appropriate for that
specimen or sample.1,13-20
Test Procedure
For isolation of Salmonella spp. from food, samples are enriched and
selectively enriched. Streak 10 l of selective enrichment broth onto
Bismuth Sulfite Agar. Incubate plates for 24-48 hours at 35C.
Examine plates for the presence of Salmonella spp. Refer to appropriate
references for the complete procedure when testing food samples.1,13-15
For isolation of Salmonella spp. from clinical specimens, inoculate
fecal specimens and rectal swabs onto a small area of one quadrant of
the Bismuth Sulfite Agar plate and streak for isolation. This will
permit the development of discrete colonies. Incubate plates at 35C.
Examine at 24 hours and again at 48 hours for colonies resembling
Salmonella spp.
For additional information about specimen preparation and inoculation
of clinical specimens, consult appropriate references.16-20
72
E. coli is partially inhibited. Occasionally a strain will be encountered

that will grow as small brown or greenish glistening colonies. This
color is confined entirely to the colony itself and shows no metallic
sheen. A few strains of Enterobacter aerogenes may develop on this
medium, forming raised, mucoid colonies. Enterobacter colonies
may exhibit a silvery sheen, appreciably lighter in color than that
produced by S. typhi. Some members of the coliform group that
produce hydrogen sulfide may grow on the medium, giving colonies
similar in appearance to S. typhi. These coliforms may be readily
differentiated because they produce gas from lactose in differential
media, for example, Kligler Iron Agar or Triple Sugar Iron Agar. The
hydrolysis of urea, demonstrated in Urea Broth or on Urea Agar Base,
may be used to identify Proteus sp.
To isolate S. typhi for agglutination or fermentation studies, pick
characteristic black colonies from Bismuth Sulfite Agar and subculture
them on MacConkey Agar. The purified colonies from MacConkey
Agar may then be picked to differential tube media such as
Kligler Iron Agar, Triple Sugar Iron Agar or other satisfactory
differential media for partial identification. All cultures that give
reactions consistent with Salmonella spp. on these media should be
confirmed biochemically as Salmonella spp. before any serological
testing is performed. Agglutination tests may be performed from the
fresh growth on the differential tube media or from the growth on
nutrient agar slants inoculated from the differential media. The growth
on the differential tube media may also be used for inoculating
carbohydrate media for fermentation studies.

1. It is important to streak for well isolated colonies. In heavy growth
areas, S. typhi appears light green and may be misinterpreted as
negative growth for S. typhi.22
2. S. typhi and S. arizonae are the only enteric organisms to exhibit
typical brown zones on the medium. Brown zones are not produced
by other members of the Enterobacteriaceae. However, S. arizonae
is usually inhibited.22
3. Colonies on Bismuth Sulfite Agar may be contaminated with
other viable organisms; therefore, isolated colonies should be
subcultured to a less selective medium (e.g., MacConkey Agar).22
4. Typical S. typhi colonies usually develop within 24 hours;
however, all plates should be incubated for a total of 48 hours to
allow growth of all typhoid strains.22
The Difco Manual
Section II
Blood Agar Base & Blood Agar Base No. 2
5. DO NOT AUTOCLAVE. Heating this medium for a period longer

than necessary to just dissolve the ingredients destroys its selectivity.
References
Marshall, R. T. (ed.), Standard methods for the examination of
Washington, D.C.
2. Wilson, W. J., and E. M. Blair. 1926. A combination of bismuth
and sodium sulphite affording an enrichment and selective
medium for the typhoid-paratyphoid groups of bacteria. J. Pathol.
Bacteriol. 29:310.
3. Wilson, W. J., and E. M. Blair. 1927. Use of a glucose bismuth
sulphite iron medium for the isolation of B. typhosus and
B. proteus. J. Hyg. 26:374-391.
4. Wilson, W. J., and E. M. Blair. 1931. Further experience of the
bismuth sulphite media in the isolation of Bacillus typhosus and
Bacillus paratyphosus from faeces, sewage and water. J. Hyg.
31:138-161.
5. Wilson, W. J. 1923. Reduction of sulphites by certain bacteria in
media containing a fermentable carbohydrate and metallic salts.
J. Hyg. 21:392.
6. Wilson, W. J. 1928. Isolation of B. typhosus from sewage and
shellfish. Brit. Med. J. 1:1061.
7. Cope, E., and J. Kasper. 1937. A comparative study of methods
for the isolation of typhoid bacilli from the stool of suspected
carriers. Proceedings of local branches of the Society of American
Bacteriologists. J. Bacteriol. 34:565.
8. Cope, E. J., and J. A. Kasper. 1938. Cultural methods for the
detection of typhoid carriers. Am. J. Public Health 28:1065-1068.
9. Gunther, M. S., and L. Tuft. 1939. A comparative study of media
employed in the isolation of typhoid bacilli from feces and urine.
J. Lab. Clin. Med. 24:461-471.
10. Green, C. E., and P. J. Beard. 1938. Survival of E. typhi in sewage
treatment plant processes. Am. J. Public Health 28:762-770.
11. Hajna, A. A., and C. A. Perry. 1938. A comparative study of
selective media for the isolation of typhoid bacilli from stool
specimens. J. Lab. Clin. Med. 23:1185-1193.
Bacto Blood Agar Base

Bacto Blood Agar Base No. 2
13. Andrews, W. H., G. A. June, P. S. Sherrod, T. S. Hammack, and

R. M. Amaguana. 1995. Salmonella, p. 5.01-5.20. In FDA
bacteriological analytical manual, 8th ed. AOAC International,
Gaithersburg, MD.
14. Flowers, R. S., J. DAoust, W. H. Andrews, and J. S. Bailey.
1992. Salmonella, p. 371-422. In Vanderzant, C. and D. F.
Splittstoesser (ed.), Compendium of methods for the microbiological
examination of foods, 3rd ed. American Public Health Association,
Washington, D.C.
15. Andrews, W. H. (ed). 1995. Microbiological Methods, p. 17.1-17.119.
In Cunniff, P. (ed.), Official methods of analysis of AOAC
International, 16th ed. AOAC International, Arlington, VA.
16. Washington, J. A. 1981. Initial processing for culture of
specimens, p. 91-126. Laboratory procedures in clinical microbiology, p. 749. Springer-Verlag New York Inc. New York, NY.
17. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994.
Microorganisms encountered in the gastrointestinal tract,
p. 234-248. Bailey & Scotts diagnostic microbiology, 9th ed.
Mosby-Year Book, Inc. St. Louis, MO.
18. Gray, L. D. 1995. Escherichia, Salmonella, Shigella and Yersinia,
p. 450-456. In Murray, P. R., E. J. Baron, M. A. Pfaller, F. C.
Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology,
19. Citron, F. 1992. Initial processing, inoculation, and incubation of
aerobic bacteriology specimens, p. 1.4.1-1.4.19. In Isenberg, H. D.
(ed.), Clinical microbiology procedures handbook, vol. 1. American
Society for Microbiology, Washington, D.C.
20. Grasnick, A. 1992. Processing and interpretation of bacterial
fecal cultures, p. 1.10.1-1.10.25. In Isenberg, H. D. (ed.), Clinical
microbiology procedures handbook, vol. 1. American Society for
21. DAoust, J. Y. 1977. Effect of storage conditions on the
performance of bismuth sulfite agar. J. Clin. Microbiol. 5:122-124.
22. MacFaddin, J. F. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, Vol. 1. Williams
Packaging
100 g
500 g
10 kg
0073-15
0073-17
0073-08
Intended Use
Bacto Blood Agar Base is used for isolating and cultivating a wide
variety of microorganisms and, with added blood, for cultivating
fastidious microorganisms.
Bacto Blood Agar Base No. 2 is used for isolating and cultivating
fastidious microorganisms with or without added blood.
The Difco Manual
Also Known As
Blood Agar Base is abbreviated as BAB, and may be referred to as
Infusion Agar.

Blood agar bases are typically supplemented with 5-10% sheep,
rabbit or horse blood for use in isolating, cultivating and determining
hemolytic reactions of fastidious pathogenic microorganisms. Without
enrichment, blood agar bases can be used as general purpose media.
73
In 1919, Brown1 experimented with blood agar formulations for the

effects of colony formation and hemolysis; the growth of pneumococci
was noticeably influenced when the medium contained peptone
manufactured by Difco.
Blood Agar Base is a modification of Huntoons2 Hormone Medium
with a slight acidic composition. Norton3 found the pH of 6.8 to be
advantageous in culturing streptococci and pneumococci. Blood Agar
Base No. 2 is a nutritionally rich medium for maximum recovery of
Blood Agar Base media are specified in Standard Methods4,5,6 for
food testing.

Blood Agar Base formulations have been prepared using specially
selected raw materials to support good growth of a wide variety of
Infusion from Beef Heart and Tryptose provide nitrogen, carbon,
amino acids and vitamins in Blood Agar Base. Proteose Peptone No. 3
Section II
is the nitrogen source for Blood Agar Base No. 2 while Yeast Extract
and Liver Digest provide essential carbon, vitamin, nitrogen and
amino acids sources. Both media contain Sodium Chloride to maintain
osmotic balance and Bacto Agar as a solidifying agent. Blood
Agar Bases are relatively free of reducing sugars, which have been
reported to adversely influence the hemolytic reactions of
beta-hemolytic streptococci.7
Supplementation with blood (5-10%) provides additional growth
factors for fastidious microorganisms and is the basis for
determining hemolytic reactions. Hemolytic patterns may vary
with the source of animal blood or type of base medium used.8
Chocolate agar for isolating Haemophilus and Neisseria species can
be prepared from Blood Agar Base No. 2 by supplementing the
medium with 10% sterile defibrinated blood (chocolatized).
ATCC 25923
ATCC 19615

Blood Agar Base
Solution:
Prepared Medium:
Without blood -light to medium
amber, slightly opalescent.
With 5% sheep blood - cherry red,
opaque.
Reaction of 4.0%
Solution at 25C:
pH 6.8 0.2
Blood Agar Base No. 2
Solution:
3.95% solution, soluble in distilled or deionized
water upon boiling; medium to dark amber very slightly
to slightly opalescent, without significant precipitate.
Prepared Medium:
Without blood-medium to dark amber, slightly
opalescent, without significant precipitate.
With 5% sheep blood-cherry red, opaque.
Reaction of 3.95%
Solution at 25C:
pH 7.4 0.2
Cultural Response
Prepare Blood Agar Base or Blood Agar Base No. 2 per
label directions with and without 5% sterile defibrinated
sheep blood. Inoculate and incubate at 35 2C under
approximately 10% CO2 for 18-24 hours.
ORGANISM
ATCC 6305
ATCC
Escherichia coli
25922
25923*
Streptococcus pneumoniae 6305
19615*
INOCULUM
CFU
GROWTH
HEMOLYSIS
100-1,000
100-1,000
100-1,000
100-1,000
good
good
good
good
beta
alpha
beta
74
The Difco Manual
Section II
Formula
Blood Agar Base

Formula Per Liter
Collect specimens in sterile containers or with sterile swabs and transport

Beef Heart, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . 500

Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g

Formula Per Liter
Liver Digest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
g
g
g
g
g
Precautions
Storage
Expiration Date
Procedure
Materials Provided
Blood Agar Base

Glassware
Autoclave
Incubator (35C)
Sterile defibrinated blood
Blood Agar Base - 40 grams;
Blood Agar Base No. 2 - 39.5 grams.
3. Autoclave at 121C for 15 minutes. Cool to 45-50C.
4. To prepare blood agar, aseptically add 5% sterile defibrinated
blood to the medium at 45-50C. Mix well.
5. To prepare chocolate agar, add 10% sterile defibrinated blood
to Blood Agar Base No. 2 at 80C. Mix well.
The Difco Manual
Test Procedure
growth will display the most reliable hemolytic reactions owing to
the activity of both oxygen-stable and oxygen-labile streptolysins.8
2. Incubate plates aerobically, anaerobically or under conditions of
increased CO2 (5-10%) in accordance with established laboratory
procedures.
Results
Examine the medium for growth and hemolytic reactions after 18-24
and 48 hours incubation. Four types of hemolysis on blood agar media
can be described:9
a. Alpha hemolysis () is the reduction of hemoglobin to
methemoglobin in the medium surrounding the colony. This causes
a greenish discoloration of the medium.
b. Beta hemolysis () is the lysis of red blood cells, producing a
c. Gamma hemolysis () indicates no hemolysis. No destruction of
red blood cells occurs and there is no change in the medium.
d. Alpha-prime hemolysis (` ) is a small zone of complete hemolysis
that is surrounded by an area of partial lysis.

1. Blood Agar Base media are intended for use with blood
supplementation. Although certain diagnostic tests may be
performed directly on this medium, biochemical and, if indicated,
immunological testing using pure cultures are recommended
for complete identification. Consult appropriate references for
further information.
this medium.
3. Hemolytic reactions of some strains of group D streptococci
have been shown to be affected by differences in animal blood.
Such strains are beta-hemolytic on horse, human and rabbit
blood agar and alpha-hemolytic on sheep blood agar.8
4. Colonies of Haemophilus haemolyticus are beta-hemolytic on
horse and rabbit blood agar and must be distinguished from
colonies of beta-hemolytic streptococci using other criteria. The
use of sheep blood has been suggested to obviate this problem since
sheep blood is deficient in pyridine nucleotides and does not
support growth of H. haemolyticus.10
5. Atmosphere of incubation has been shown to influence hemolytic
reactions of beta-hemolytic streptococci.8 For optimal performance,
incubate blood agar base media under increased CO2 or anaerobic
conditions.
75
Bordet Gengou Agar Base
Section II
References
1. Brown, J. H. 1919. The use of blood agar for the study of
streptococci, NY Monograph No. 9. The Rockefeller Institute for
Medical Research.
2. Huntoon, F. M. 1918. Hormone Medium. A simple medium
employable as a substitute for serum medium. J. of Infect.
Dis. 23:169-172.
3. Norton, J. F. 1932. Bacteriology of pus. J. Lab Clin. Med. p. 558-564.
4. Association of Official Analytical Chemists. 1995.
Bacteriological analytical manual, 8th ed., App. 3.08-3.09. AOAC
5. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compendium
of methods for the microbiological examination of food, 3rd ed.,
p. 1113. American Public Health Association, Washington, D.C.
6. Atlas, R. 1993. Handbook of microbiological media, p.136-138.
CRC Press, Boca Raton, FL.
7. Casman, E. 1947. A noninfusion blood agar base for neisseriae,
pneumococci and streptococci. Am. J. Clin. Path. 17:281-289.

E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken
(ed.). Manual of clinical microbiology, 6th ed. American Society
for Microbiology, Washington, D.C.
9. Isenberg, H. D. (ed.). 1992. Interpretation of aerobic bacterial
growth on primary culture media, Clinical microbiology
procedures handbook, vol.1, p. 1.6.1-1.6.7. American Society for
10. Baron, E. J, L. R. Peterson, and S. M. Finegold. 1994. Bailey
& Scotts diagnostic microbiology, 9th ed., p. 415. Mosby-Year
Book, Inc. St. Louis, MO.
Packaging
Blood Agar Base
100 g
500 g
2 kg
0045-15
0045-17
0045-07
500 g
0696-17
Bacto Bordet Gengou Agar Base
Intended Use
Bacto Bordet Gengou Agar Base is used with added blood for isolating
Bordetella pertussis and other Bordetella species.
Bordet Gengou Agar Base is a modification of the medium originally

described by Bordet and Gengou2 in 1906 for the cultivation of
Haemophilus pertussis, now Bordetella pertussis. The original formula
used a base medium consisting of 1% glycerol and potato extract with
Also Known As
Bordet Gengou Agar Base is also referred to as B-G Agar Base and
Bordet-Gengou Potato-Glycerol Agar.1
Uninoculated
plate
Bordetella pertussis
ATCC 8467

Solution:
3.0% solution, soluble upon boiling in
distilled or deionized water containing
1% glycerol; light to medium amber,
opalescent, may have a slight precipitate.
Prepared Medium:
Plain - Light to medium amber,
opalescent, may have a precipitate.
With 15% blood - Cherry red, opaque.
Reaction of 3.0%
Solution at 25C:
pH 6.7 0.2
Cultural Response
Prepare Bordet Gengou Agar Base enriched with 15% sterile
defibrinated blood per label directions. Inoculate and incubate
ORGANISM
ATCC
INOCULUM
CFU
Bordetella bronchiseptica 4617

30-300
Bordetella parapertussis MDH 32472 30-300
8467
30-300
GROWTH
GROWTH W/15%
W/O BLOOD
RABBIT BLOOD
good
poor to good
poor to good
good
good
good
Bordetella parapertussis
MDH 32472
76
The Difco Manual
Section II
an equal volume of human or rabbit blood. The modified medium is

prepared according to the formula recommended by the American
Public Health Association.3 Eldering and Kendrick4 reported that the
addition of 1% proteose peptone or neopeptone increased growth of
B. pertussis, thereby increasing the yield of vaccine.
The genus Bordetella consists of four species: Bordetella pertussis,
B. parapertussis, B. bronchiseptica and B. avium.5 All Bordetella are
respiratory pathogens, residing on the mucous membranes of the
respiratory tract. B. pertussis and B. parapertussis are uniquely human
pathogens. B. pertussis is the major cause of whooping cough or
pertussis. B. parapertussis is associated with a milder form of the
disease. 6 B. bronchiseptica is an opportunistic human pathogen
associated with both respiratory and non-respiratory infections, often
occurring in patients having close contact with animals. 5
B. bronchiseptica has not been reported to cause pertussis. There have
been no reports of recovery of B. avium from humans.5
The cough plate method for the diagnosis of whooping cough was
originally reported by Chievitz and Meyer.7 This technique is no longer
recommended. Nasopharyngeal washings or a nasopharyngeal swab
(calcium alginate on a wire handle) should be collected within the first
week of paroxysmal coughing.8

Infusion from Potato provides nitrogen, vitamins and amino acids.
Glycerol is a carbon source. Sodium Chloride maintains the osmotic
balance of the medium. Bacto Agar is a solidifying agent. The addition
of blood provides essential growth requirements for Bordetella species.
Many factors will inhibit growth of B. pertussis, including fatty acids
present in nasal secretions or cotton from the collection swab. Starch,
present from the Potato Infusion, absorbs fatty acids.
Modified Bordet Gengou medium, enriched with 15-20% blood, yields
typical B. pertussis growth. The colonies appear small, white, opaque
and surrounded by a characteristic zone of hemolysis that is not sharply
defined but merges diffusely into the medium. The zone of hemolysis
is usually absent if 30% or more blood is added to the medium and
cannot be seen on charcoal-containing media.9 Sterile, defibrinated
sheep or rabbit blood can be used in preparing the medium.
Formula
Formula Per Liter
Potato, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 g
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Precautions
Storage
The Difco Manual
Expiration Date
Procedure
Materials Provided

Glassware
Autoclave
Incubator (35C)
Waterbath (45-50C)
1. Suspend 30 grams in 1 liter distilled or deionized water containing
10 grams of glycerol.
4. Cool to 45-50C. Aseptically add 15% sterile defibrinated sheep
or rabbit blood. Mix well.
Specimen Collection and Preparation9

Specimens should be obtained during the early phases of the disease
and prior to the convalescent stage and antimicrobial therapy. The
specimen of choice is duplicate nasopharyngeal swabs. Direct plating
of the specimen at bedside is recommended; when this is not possible,
submerge both swabs into Regan-Lowe transport medium.
Test Procedure9
1. Roll one of the swabs over the primary inoculation area of the
Bordet Gengou plate and streak for isolation. Return the swab to
the transport medium. Incubate the transport medium for 48 hours.
Plate the swabs onto a duplicate set of media.
2. Incubate the culture plates at 35C for 5-7 days in a moist chamber.
Increased CO 2 is not recommended. Growth of B. pertussis
appears in 3-5 days. Other bordetellae species can appear in 1-3 days.
3. Nasopharyngeal specimens may contain staphylococci that produce
a diffusible substance inhibitory to B. pertussis growth. For these
specimens, use a plating medium with methicillin (2.5 g/ml) or
cephalexin (40 g/ml) and a medium without antimicrobics.
4. Isolates suspected of being B. pertussis should be confirmed by
using a specific antiserum in either the slide agglutination or
fluorescent antibody staining techniques.10
Results
For a complete discussion on the isolation and identification of
Bordetella species refer to the appropriate procedures outlined in
the references.

77
Bovine Albumin 5%
Section II
2. Some Haemophilus species will grow on Bordetella isolation

media and may cross-react with B. pertussis antisera. It may be
prudent to rule out X and V factor dependence.
References
1. MacFaddin, J. F. 1985. Media for isolation-cultivationidentification- maintenance of medical bacteria, vol. 1, p. 86-92.
2. Bordet, J., and D. Gengou. 1906. Le microbe de la coqueluche.
Ann. Inst. Pasteur 20:731.
3. Kendrick, P. L., E. Eldering, and W. L. Bradford. 1970.
Whooping cough, p.106-117. In H. L. Bodily, E. L. Updyke, and
J. O. Mason (ed.), Diagnostic procedures for bacterial, mycotic and
parasitic infections, 5th ed. American Public Health Association,
New York, NY.
4. Eldering, E., and P. L. Kendrick. 1936. Some practical
considerations in B. pertussis vaccine preparation. Am. J. Public
Health 24:309.
5. Marcon, M . J. 1995. Bordetella, p. 566-573. In P. R. Murray, E. J.

Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual
Washington, D.C.
6. Linneman, C. C., and E. B. Pery. 1977. Bordetella parapertussis:
recent experience and a review of the literature. Am. J. Dis. Child.
131:560-563.
7. Chievitz, J., and A. H. Meyer. 1916. Recherches sur la
coqueluche. Ann. Inst. Pasteur 30:503.0
Bailey & Scotts diagnostic microbiology, 9th ed. Mosby-Year
Book, Inc., St. Louis, MO.
9. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures handbook, vol. 1. American Society for Microbiology, Washington, D.C.
10. Koneman, E. W. 1988. Color atlas and textbook of diagnostic
microbiology, 3rd ed. J. B. Lippincott Company, Washington, D.C.
Packaging
Bacto Bovine Albumin 5%
Intended Use
Bacto Bovine Albumin 5% is used to enrich media for cultivating a
large variety of microorganisms and tissue cells.
Also Known As
Bovine Albumin can be abbreviated as BSA.1

Bovine Albumin 5%
Appearance:
Light amber, clear to very slightly
opalescent.
Sterility Test:
Negative.
Reaction of
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepare Dubos Broth Base per label directions, substituting
Bovine Albumin 5% for Dubos Medium Albumin. Inoculate
and incubate at 35 2C under CO2 for up to three weeks.
ORGANISM
ATCC
INOCULUM
CFU
Mycobacterium
intracellulare
Mycobacterium
tuberculosis H37Ra
Mycobacterium
tuberculosis H37Ra
13950
100-1,000
good
25177
100-1,000
good
27294
100-1,000
good
GROWTH
100 g
500 g
0048-15
0048-17

Davis and Dubos2 recommended the use of bovine albumin at a final
concentration of 0.5% in liquid media for culturing Mycobacterium
tuberculosis. In this study, bovine albumin neutralized the toxicity of
fatty acids and permitted more luxuriant growth of M. tuberculosis.
Ellinghausen and McCullough3 used bovine albumin fraction V at a final
concentration of 1% in liquid, semisolid and solid media for culturing
leptospires. Morton et al.4 demonstrated that 1% bovine albumin
stimulated growth of Mycoplasma (PPLO).
Bovine Albumin can be added to normally sterile specimens, tissues
and body fluids for direct inoculation onto culture media used for
isolating mycobacteria. BSA is also used as an enrichment when
contaminated specimens are digested.
Bovine Albumin 5%, modified with added sodium chloride and
dextrose, is available as Dubos Medium Albumin.

Bovine Albumin 5% is a filter sterilized solution of Bovine Albumin
Fraction V. BSA is suggested as a culture media enrichment because its
buffering capacity and detoxifying effect on specimen sediment.1 Bovine
Albumin 5% also increases adhesion of the specimen to solid media.1
Precautions
3. Mycobacterial organisms are BioSafety Level 2 pathogens. The
handling of clinical specimen material that is potentially infected
with mycobacteria should be performed in a Class I or II biological
safety cabinet (BSC).1
Storage
Store Bovine Albumin 5% at 2-8C.
78
The Difco Manual
Section II
Brain Heart Infusion Media
Expiration Date
Procedure
Materials Provided
Bovine Albumin 5%

Materials vary depending on the specimen collected and the procedure
performed.
Refer to the final concentration of Bovine Albumin in the procedure
being used to inoculate specimens. A 0.2% solution of Bovine Albumin
5% is recommended for the inoculation of sterile and contaminated
specimens when isolating mycobacteria.1

Many different specimen types can be collected for mycobacterial
cultures but the majority will be from the respiratory tract.1 Tissues,
body fluids, urine, blood and gastric aspirates can also be tested for the
presence of mycobacteria. Refer to the procedures established by
laboratory policy or to appropriate references for specific guidelines
on specimen collection and processing.
Test Procedure
Sterile Specimens for the Isolation of Mycobacteria1
Normally sterile tissues may be ground in 0.2% BSA and inoculated
directly in culture media. Concentrate body fluids before inoculation
because they normally contain only a small number of mycobacteria.
Centrifuge fluids at 3,000 x g and inoculate the sediment onto liquid
or solid media. For a complete discussion of the inoculation of sterile
specimens, refer to appropriate references.
Contaminated Specimens for the Isolation of Mycobacteria1

A concentration of 0.2% Bovine Albumin fraction V can be added to
specimen sediment that has been digested and centrifuged by the
NALC-NaOH digestion method. Using a separate sterile pipette for
each tube, add 1-2 ml of 0.2% BSA, then resuspend the sediment with
the pipette or by shaking the tube gently by hand.
Several digestion procedures exist. Consult appropriate references for
a complete discussion on all digestion and decontamination methods
and other testing procedures.
Results
All media should be examined closely for evidence of growth. Refer to
the procedure established by laboratory policy or to appropriate
references on typical growth patterns and confirmation tests.

1. Bovine Albumin 5% is not recommended for use with Bactec
because BSA may delay detection times.1
References
1. Nolte, F. S., and B. Metchock. 1995. Mycobacterium, p. 400-437.
In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and
2. Davis and Dubos. 1945. J. Bacteriol. 55:11.
3. Ellinghausen and McCullough. 1962. Bacteriol. Proc. 62:54.
4. Morton, H. E., P. E. Smith, N. B. Williams, and C. F.
Eickenberg. 1951. Isolation of pleuropneumonia-like organisms
from human saliva: A newly detected member of the oral flora.
J. Dent. Res. 30:415-422.
Packaging
Bovine Albumin 5%
12 x 20 ml
0668-64

Bacto Brain Heart Infusion . Bacto Brain Heart Infusion Agar
Bacto Clostridium Difficile Antimicrobic Supplement CC
Bacto Brain Heart CC Agar . Bacto Brain Heart Infusion w/PAB
and Agar . Bacto Brain Heart Infusion w/o Dextrose
Intended Use
Bacto Brain Heart Infusion is used for cultivating fastidious microorganisms, including streptococci, pneumococci and meningococci.
Bacto Brain Heart Infusion Agar is used for cultivating fastidious
microorganisms, especially fungi and yeasts, and, with added
antibiotics, for isolating fungi.
Bacto Clostridium Difficile Antimicrobic Supplement CC is used with
The Difco Manual
Brain Heart Infusion Agar in preparing Clostridium Difficile Agar.

Bacto Brain Heart CC Agar is used for isolating and cultivating
fastidious fungi.
Bacto Brain Heart Infusion w/PAB and Agar is used for cultivating
fastidious organisms, particularly from blood containing sulfonamides.
Bacto Brain Heart Infusion w/o Dextrose is used for cultivating
fastidious organisms.
79
Also Known As
Brain Heart Infusion is abbreviated as BHI.

In 1919, Rosenow 1 devised an excellent medium for culturing
streptococci by supplementing dextrose broth with brain tissue.
Hayden2 revised Rosenows procedure by adding crushed marble to
the medium and reported favorable growth of organisms from dental
pathogens. Brain Heart Infusion is a modification of the media
described by Rosenow1 and Hayden2 in which infusion from calf
brains has replaced the brain tissue and disodium phosphate has
replaced the calcium carbonate buffer.
Brain Heart Infusion Agar is used for cultivating a variety of fastidious
microorganisms, fungi and yeasts. This medium is used in combination
with penicillin and streptomycin. Roseburg, Epps and Clark3 reported
that the isolation and cultivation of Actinomyces israelii was enhanced
on Brain Heart Infusion with 2% agar compared with 1% dextrose
infusion agar. Howell4 used Brain Heart Infusion with the addition
of 2% Bacto Agar and 10% sterile defibrinated horse blood for the
cultivation of Histoplasma capsulatum.
Brain Heart Infusion Agar can be used with Clostridium Difficile
Antimicrobic Supplement CC, a selective supplement containing
lyophilized cycloserine and cefoxitin, for the preparation of
Section II
Clostridium Difficile Agar. The complete medium is based on the

formula of Willey and Bartlett5 and recommended for use in the isolation
of Clostridium difficile from fecal specimens. C. difficile is the major
cause of antibiotic-associated diarrhea and pseudomembranous colitis.6
Brain Heart CC Agar is prepared with chloramphenicol and cycloheximide (Actidione) according to the formulation of Ajello et al.7 and
McDonough et al.8 These selective agents restrict growth of bacteria
and saprophytic fungi. Brain Heart CC Agar is used in the isolation of
fungi that cause systemic disease, such as Histoplasma capsulatum
and Blastomyces dermatiditis.
Brain Heart Infusion media are specified in several standard methods
references for food testing.9,10,11 Standard Methods for the Examination
of Water and Wastewater recommends Brain Heart Infusion media in
tests for the verification of fecal streptococci.12
Brain Heart Infusion is recommended by the National Committee for
Clinical Laboratory Standards (NCCLS) for the preparation of inocula
used in antimicrobial susceptibility tests.13
Brain Heart Infusion w/o Dextrose is a basal medium used with added
carbohydrates for fermentation studies.
Modifications of BHI media include:14
Brain Heart Infusion Agar with penicillin (20,000 U) and
streptomycin (40 mg) for the selective isolation of pathogenic
Prepared Medium:
Reaction of 5.2%
Solution at 25C:
Brain Heart Infusion

Solution:
deionized water; light to medium amber,
clear without significant precipitate.
Prepared Medium:
Light to medium amber, clear without
Reaction of 3.7%
Solution at 25C
pH 7.4 0.2
Brain Heart Infusion Agar
Solution:
medium amber, slightly opalescent to
opalescent with a flocculent precipitate.
Prepared Medium:
Plain - light to medium amber, slightly
opalescent with a precipitate.
With 5% sheep blood-cherry
red, opaque.
Reaction of 5.2%
Solution at 25C:
pH 7.4 0.2
Brain Heart CC Agar
Solution:
deionized water on boiling; medium
amber, slightly opalescent without
80
Medium amber, slightly opalescent

pH 7.4 0.2
Brain Heart Infusion w/PAB and Agar

Solution:
medium amber, slightly opalescent.
Prepared Medium:
Light to medium amber, slightly opalescent.
Reaction of 3.8%
Solution at 25C:
pH 7.4 0.2
Brain Heart Infusion w/o Dextrose
Solution:
deionized water; light to medium
amber, clear.
Prepared Medium:
Reaction of 3.5%
Solution at 25C:
pH 7.4 0.2
Clostridium Difficile Antimicrobic Supplement CC
Lyophilized Appearance: White, homogeneous cake.
Solution:
Soluble in 5 ml sterile distilled or
deionized water. Colorless, clear.
Microbial Limits Test: Satisfactory (negative).
Reaction of Rehydrated
Vial at 25C:
pH 5.9-6.3
The Difco Manual
Section II
fungi from specimens heavily contaminated with bacteria and

saprophytic fungi;
Brain Heart Infusion with 3% sodium chloride for the isolation of
Vibrio parahaemolyticus;
Brain Heart Infusion with agar, yeast extract, sodium chloride,
inactivated horse serum and penicillin for the cultivation of
fastidious fungi;
Brain Heart Infusion with casein to support the growth of
Serratia marcescens;
Brain Heart Infusion with 0.7% agar to support the growth of
staphylococcal species for the production of enterotoxin; and,
Brain Heart Infusion with rabbit serum and yeast extract for the
cultivation of Mycoplasma equirhinis.

Infusion from Beef Heart, Calf Brains and Proteose Peptone provide
nitrogen, carbon, sulfur and vitamins in Brain Heart Infusion media.
Dextrose is a carbon energy source that facilitates organism growth.

Sodium Chloride maintains the osmotic balance of the medium. Disodium Phosphate is a buffering agent. Bacto Agar is a solidifying agent.
The nutritionally rich broth formulation of Brain Heart Infusion
supports growth of a variety of microorganisms, as does the medium
when supplemented with agar and/or blood. BHI (broth) is often used
as a blood culture medium and as a basal medium for metabolic tests,
particularly for identifying streptococci.15 BHI with 0.5% Polysorbate 80 can be used for detecting Mycobacterium avium-intracellulare
complex organisms and M. tuberculosis from blood cultures.15
Brain Heart Infusion Agar is used in the aminoglycoside and
vancomycin screen test for resistant enterococci.16 BHI Agar with
5-10% sheep blood and chloramphenicol (16 g/ml) and gentamicin
(5 g/ml) will inhibit the growth of bacteria while allowing growth of
dimorphic fungi.15 This agar can be used as a primary plating medium
Cultural Response
Brain Heart Infusion (0037)
Brain Heart Infusion w/o Dextrose (0502)
Prepare the selected medium per label directions. Inoculate
ORGANISM
Neisseria meningitidis
ATCC
INOCULUM
CFU
GROWTH
13090*
6305
19615*
100-1,000
100-1,000
100-1,000
good
good
good
Brain Heart Infusion Agar (0418)

Prepare medium with and without 5% sheep blood per label
directions. Inoculate and incubate Aspergillus aerobically at
30 2C for 18-72 hours; incubate all other organisms
aerobically at 35 2C with 5-10% CO2 for 18-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
Aspergillus niger
16404
6305
19615
25923*
100-1,000
100-1,000
100-1,000
100-1,000
GROWTH GROWTH w/5%

PLAIN
SHEEP BLOOD
good
good
good
good
good
good
good
good
Brain Heart Infusion w/PAB and Agar (0499)

Prepare medium per label directions. Inoculate and incubate
at 35 2C for 18-48 hours under appropriate atmospheric
conditions.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
25285
13090*
6305
19615*
100-1,000
100-1,000
100-1,000
100-1,000
good
good
good
good
Brain Heart CC Agar (0483)

Prepare medium per label directions. Inoculate and incubate at
25 2C for up to 7 days
ORGANISM
Aspergillus niger
Candida albicans
Escherichia coli
Trichophyton mentagrophytes
The Difco Manual
ATCC
16404
10231
25922*
9533
INOCULUM
CFU
GROWTH
100-1,000 inhibited
100-1,000 fair to good
100-1,000 inhibited
good
1 mm2
Uninoculated
tube
ATCC 13090
ATCC 19615

Prepare 500 ml Brain Heart Infusion Agar supplemented with 5%
sterile sheep blood and 5 ml Clostridium Difficile Antimicrobic
Supplement CC. Inoculate and incubate at 35 2C under
anaerobic conditions for 24-72 hours.
ORGANISM
ATCC
INOCULUM
CFU
Clostridium difficile
Clostridium difficile
Clostridium
perfringenes
17858 100-1,000
9689
100-1,000
13124* 1,000-2,000
Escherichia coli
25922* 1,000-2,000
33186
1,000-2,000
GROWTH
good
good
markedly to
markedly to
markedly to
as directed in Bactrol Disk Technical Information.
81
Section II
for the growth of fungi since it has been shown to yield better recovery
than the previously recommended Sabouraud Dextrose Agar.15 In
Brain Heart CC Agar, chloramphenicol is used as a broad-spectrum
antibiotic to inhibit a wide range of bacteria; cycloheximide inhibits
saprophytic fungi. Sheep blood provides essential growth factors for
fastidious fungi.
Clostridium Difficile Agar (Brain Heart Infusion Agar supplemented
with 5% sheep blood or 7% horse blood and Clostridium Difficile
Antimicrobic Supplement CC) improves the growth and recovery of
C. difficile. Clostridium Difficile Agar markedly to completely inhibits
most aerobic and anaerobic enteric organisms other than C. difficile.5
The final concentration of cycloserine and cefoxitin in Clostridium
Difficile Agar is 250 mcg/ml and 10 mcg/ml, respectively.
Brain Heart CC Agar can be supplemented with sheep blood (5-10%)
for enrichment and gentamicin (5 mg/l) for additional selectivity.14
McDonough et al17 demonstrated that the temperature of incubation
affects the sensitivity of some pathogenic fungi to antibiotics. Incubate
the medium containing antibiotics at room temperature. The specimen
source and the type of fungus suspected will indicate the isolation
medium to be used. Both an antimicrobic-containing medium and
a non-selective medium should be used on primary isolates with
incubation at both 25C and 37C.
Brain Heart Infusion w/PAB and Agar contains p-aminobenzoic acid
(0.05 g/l) to neutralize sulfonamides in the blood of patients receiving
this therapy. This formulation will also inactivate streptomycin in the
ratio of 10 ml of medium to 100 units of streptomycin. The addition of
0.1% agar to Brain Heart Infusion w/PAB and Agar provides optimum
conditions for aerobic organisms, microaerophiles and obligate anaerobes.
Brain Heart Infusion W/PAB and Agar

Formula Per Liter
Formula

2. Brain Heart CC Agar: HARMFUL. HARMFUL BY INHALATION
AND IF SWALLOWED. POSSIBLE RISK OF IRREVERSIBLE
EFFECTS. POSSIBLE RISK OF HARM TO THE UNBORN
CHILD. Do not breathe dust. In case of accident or if you feel
unwell, seek medical advice immediately (show the label where
possible). Wear suitable protective clothing. Keep container tightly
closed. TARGET ORGAN(S): Eyes/Ears, Cardiovascular, Muscles,
Blood, Lymph Glands, Nerves, Urogenital.
difficult, give oxygen. Seek medical advice. If swallowed, induce
or label.
3. Brain Heart CC Agar: Avoid overheating or holding the medium
in the melted state. Doing so tends to reduce the selective properties
of the medium.
4. When testing human serum, treat all specimens as infectious agents.

Formula Per Liter
Calf Brains, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . 200
Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
g
g
g
g
g
g

Formula Per Liter
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g

Formula Per Liter
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
82
g
g
g
g
g

Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
p-Aminobenzoic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05
g
g
g
g
g
g
g
g

Brain Heart CC Agar
Formula Per Liter
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Chloramphenicol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Cycloheximide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500
g
g
g
g
g
g
g
mg
mg

Formula per 5 ml
Cycloserine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 mg
Cefoxitin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 mg
Precautions
Storage
Store Clostridium Difficile Antimicrobic Supplement CC at 2-8C.
The Difco Manual
Section II
Expiration Date
Procedure
Materials Provided
Brain Heart CC Agar

Glassware
Autoclave
Incubator
Sterile tubes
Anaerobic system for Clostridium Difficile Agar
1. Suspend an appropriate amount of the selected medium in 1 liter
distilled or deionized water:
Brain Heart Infusion - 37 grams;
Brain Heart Infusion Agar - 52 grams;
Brain Heart CC Agar - 52 grams;
Brain Heart Infusion w/PAB and Agar - 38 grams;
Brain Heart Infusion w/o Dextrose - 35 grams.
2. If the medium contains agar (Brain Heart Infusion Agar, Brain
Heart CC Agar and Brain Heart Infusion w/PAB and Agar), heat it
to boiling to dissolve completely. Avoid overheating.
3. Autoclave at 121C for 15 minutes. Cool to room temperature.
Clostridium Difficile Agar
1. Rehydrate and sterilize 500 ml of Brain Heart Infusion Agar per
label directions. Cool to 45-50C.
2. Aseptically rehydrate Clostridium Difficile Antimicrobic
Supplement CC with 5 ml sterile distilled or deionized water.
Invert the vial gently several times to dissolve the contents. Use
immediately.
3. Aseptically add 5% sterile defibrinated sheep blood or 7%
defibrinated horse blood and 5 ml of Clostridium Difficile
Antimicrobic Supplement CC to the rehydrated medium.
4. Mix thoroughly, avoiding the formation of bubbles, and dispense
into sterile Petri dishes.

Test Procedure
See appropriate references for specific procedures using Brain Heart
Infusion Media.
The Difco Manual

1. Inoculate a representative portion of the specimen directly onto
the surface of a freshly prepared or previously reduced Clostridium
Difficile Agar plate and streak for isolation. The inoculum should
include mucous, blood or membranous material, if present.
2. Incubate at 35C under anaerobic conditions.
3. Examine for growth after 24-48 hours incubation.
For a complete discussion on the isolation and identification
of Clostridium difficile refer to appropriate procedures in the
references.15,18,20
Results
After 24 hours incubation, colonies of C. difficile appear
non-hemolytic, 1-3 mm in diameter, off-white to gray, flat and circular
with an undulated edge. Colonies become larger (3-5 mm) after
48 hours incubation.

may be encountered that fail to grow or grow poorly on this medium.
2. Certain pathogenic fungi may be inhibited by the antibiotics in
Brain Heart CC Agar.19
3. Clostridium Difficile Antimicrobic Supplement CC is intended for
use in the preparation of Clostridium Difficile Agar. Although
this medium is selective for C. difficile, additional testing using
pure cultures is necessary for complete identification. Consult
appropriate references for further information.15,18,20
4. Suspected colonies of C. difficile should be Gram stained and
subcultured anaerobically and aerobically on blood agar for
complete identification.
5. Demonstration of the C. difficile toxin in feces in the presence
of clinically evident pseudomembranous colitis is required for
definitive diagnosis.20
References
1. Rosenow, E. C. 1919. Studies on elective localization. J. Dent.
Research 1:205- 249.
2. Hayden, R. L. 1923. Elective localization in the eye of bacteria
from infected teeth. Arch. Int. Med. 32:828-849.
3. Roseburg, T., L. J. Epps, and A. R. Clark. 1944. A study of the
isolation, cultivation and pathogenicity of Actinomyces israeli
recovered from the human mouth and from actinomycosis in man.
J. Infect. Dis. 29:390.
4. Howell, A. 1948. The efficiency of methods for the isolation of
Histoplasma capsulatum. Public Health Reports 63:173-178.
5. Willey, S. H., and J. G. Bartlett. 1979. Cultures for Clostridium
difficile in stools containing a cytotoxin neutralized by Clostridium
sordellii antitoxin. J. Clin. Microbiol. 6:880-884.
6. Lyerly, D. M., D. E. Lockwood, S. H. Richardson, and T. D.
Wilkins. 1982. Biological activities of toxins A and B of
Clostridium difficile. Infect. Immum. 35:1147- 1150.
7. Ajello, L., L. K. Georg, W. Kaplan, and L. Kaufman. 1966.
Laboratory manual for medical mycology (CDC), U.S. DHEW,
Center for Disease Control, Atlanta, GA.
83
Brain Heart Infusion, Porcine
Section II
8. McDonough, E. S., L. K. Georg, L. Ajello, and S. Brinkman.

1960. Growth of dimorphic human pathogenic fungi on media
containing cycloheximide and chloramphenicol. Mycopathol.
Mycol. Appl. 13:113.
9. Cunnif, P. (ed). 1995. Official Methods of Analysis AOAC
11. Vanderzant, C., and D. F. Splittstoesser (ed). 1992. Compendium of methods for the microbiological examination of food,
12. Greenberg, A. E., L. S. Clesceri, and A. D. Eaton (ed.). 1995.
Membrane filter techniques, 9,72-74. Standard methods for the
13. National Committee for Clinical Laboratory Standards. 1994.
M11-A3, Vol. 13, No. 26, Methods for antimicrobial susceptibility
testing of anaerobic bacteria. National Committee for Clinical
Laboratory Standards, Villanova, PA.
14. Atlas, R. M. 1993. Handbook of microbiological media, p. 147-153,
& Scotts diagnostic microbiology, 9th ed. Mosby-Year Book, Inc.
St. Louis, MO.
16. Swenson, J. M., N. C. Clark, M. J. Ferraro, D. F. Sahm,
G. Doern, M. A. Pfaller, L. B. Reller, M. P. Weinstein,
R. J. Zabransky, and F. C. Tenover. 1994. Development of a
standardized screening method for detection of vancomycinresistant enterococci. J. Clin. Microbiol. 32:1700-1704.
17. McDonough, E. S., L. Ajello, L. K. Georg, and S. Brinkman.

1960. In vitro effects of antibiotics on yeast phase of Blastomyces
dermatitidis and other fungi. J. Lab. Clin. Med. 55:116-119.
handbook, vol. 1. American Society for Microbiology, 6th ed.
19. Georg, L. K., L. Ajello, and C. Papageorge. 1954. Use of
cycloheximide in the selective isolation of fungi pathogenic to man.
J. Lab Clin. Med. 44:422-428.
20. Onderdonk, A. B., and S. D. Allen. 1995. Clostridium, p. 574 -586.
In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H.
Yolken (ed.). Manual of clinical microbiology, 6th ed. American
Packaging
100
500
2
10
g
g
kg
kg
0037-15
0037-17
0037-07
0037-08
100 g
500 g
2 kg
0418-15
0418-17
0418-07
Brain Heart CC Agar
500 g
0483-17
500 g
0499-17
10 kg
Clostridium Difficile Antimicrobic

Supplement CC
6 x 5 ml
0502-08
3194-57*
*Store at 2-8C
Bacto Brain Heart Infusion, Porcine
Intended Use
Bacto Brain Heart Infusion, Porcine is used for cultivating a wide

variety of microorganisms.
Solution:
or deionized water. Light to medium
amber, clear.
Reaction of 3.7%
Solution at 25C:
pH 7.4 0.2
Cultural Response
Prepare medium per label directions. Inoculate tubes with test
organisms and incubate at 35 2C for 18-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
13090*
6305
19615*
100-1,000
100-1,000
100-1,000
fair
good
good
84
Also Known As
Brain Heart Infusion, Porcine is abbreviated as BHI, Porcine.

Rosenow1 devised an excellent medium for culturing streptococci by
supplementing Dextrose Broth with brain tissue. Hayden,2 revising
Rosenows procedure by adding crushed marble to the medium,
reported favorable growth of organisms from dental pathogens. Brain
Heart Infusion (0037) is a modification of the media described
by Rosenow1 and Hayden.2 Infusion from calf brains has replaced the
brain tissue and Disodium Phosphate has replaced the Calcium
Carbonate buffer.
Brain Heart Infusion, Porcine was developed as an alternative to Brain
Heart Infusion formula, and replaces calf brains and beef heart with
porcine brains and heart. Brain Heart Infusion, Porcine was developed
for pharmaceutical and vaccine production and can replace the
traditional BHI depending on organism and production application.
The Difco Manual
Section II
Brewer Anaerobic Agar
BHI, Porcine was formulated with no bovine components to minimize

Bovine Spongiform Encephalopathy (BSE) risk.
The nutritionally rich formula of BHI is used to grow a variety of
microorganisms The original Brain Heart Infusion media are specified
in standard methods for multiple applications.3,4,5,6
Infusion from pork brains, infusion from pork heart and Pork Peptone
No. 2 provides nitrogen, carbon, sulfur and vitamins in Brain Heart
Infusion, Porcine. Dextrose is the carbon energy source to facilitate
organism growth. Sodium Chloride maintains the osmotic balance of
the medium. Disodium Phosphate is the buffering agent.
1. Dissolve 37 grams in 1 liter distilled or deionized water.

Glassware
Autoclave
Incubator

Formula
Formula Per Liter
Pork Brains, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . 200
Pork Heart, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . 250
Bacto Pork Peptone No. 2 . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Test Procedure
g
g
g
g
g
g
Precautions
Storage
Expiration Date
Procedure
Materials Provided
Bacto Brewer Anaerobic Agar
Intended Use
Bacto Brewer Anaerobic Agar is used for cultivating anaerobic and
microaerophilic bacteria.

Brewer1 described a special Petri dish cover that allowed surface growth
of anaerobes and microaerophiles without anaerobic equipment. The
microorganisms were grown on agar with a low oxidation-reduction potential. Brewer Anaerobic Agar was originally formulated and
modified for the procedure described by Brewer.1 This medium is
The Difco Manual
See appropriate references for specific procedures using Brain

Heart Infusion.
Results
References
1. Rosenow, E. C. 1919. Studies on elective localization. J. Dent.
Res. 1:205-249.
2. Hayden, R. L. 1923. Elective localization in the eye of bacteria
from infected teeth. Arch. Int. Med. 32:828-849.
of methods for the microbiological examination of food, 3rd ed.
6. Cunnif, P. (ed).1995. Official methods of analysis, AOAC
Packaging
500 g
0561-17
suitable for standard plating procedures used in cultivating

anaerobic bacteria.2,3,4
Anaerobic bacteria cause a variety of infections in humans, including
otitis media, oral infections, endocarditis, meningitis, wound infections
following bowel surgery or trauma and bacteremia. 5,6 Anaerobic
bacteria are the predominant flora colonizing the skin and mucous
membranes of the body.3 Anaerobes vary in their sensitivity to oxygen
and nutritional requirements.2 Anaerobic bacteria lack cytochromes
and thus are unable to use oxygen as a terminal electron acceptor.3

Tryptone, Proteose Peptone No. 3 and Yeast Extract provide the nitrogen,
vitamins and amino acids in Brewer Anaerobic Agar. Dextrose
85
Section II
is the carbon source, and Sodium Chloride maintains osmotic

equilibrium. Sodium Thioglycollate and Sodium Formaldehyde
Sulfoxylate are the reducing agents. Resazurin serves as an
indicator of anaerobiosis with a pink color indicating the presence of
oxygen. Bacto Agar is the solidifying agent.
Formula
Expiration Date
Procedure
Materials Provided

Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Sodium Formaldehyde Sulfoxylate . . . . . . . . . . . . . . . . . . . 1
Resazurin, Certified . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.002
g
g
g
g
g
g
g
g
g
Precautions
Storage

Glassware
Autoclave
Incubator (35C)
Brewer Anaerobic Petri dish covers (optional)
1.
2.
3.
4.


Test Procedure

Solution:
amber, slightly opalescent while hot,
turning red on aeration and cooling.
Prepared Medium:
Light pink ring at outer edge, light
amber in center, slightly opalescent.
Reaction of 5.8%
Solution at 25C
pH 7.2 0.2
Cultural Response
Prepare Brewer Anaerobic Agar per label directions. Inoculate
the plates using the streak method. Incubate plates at 35 2C
anaerobically for 40-48 hours.
ORGANISM
Clostridium beijerinckii
ATCC
INOCULUM
CFU
GROWTH
25285*
17795
12924
100-1,000
100-1,000
100-1,000
good
good
good
*This culture is available as Bactrol Disks and should be used as
directed in Bactrol Disks Technical Information.
86
Standard Petri Dishes:2

1. Inoculate a properly obtained specimen onto the medium, and
streak to obtain isolated colonies.
2. Immediately incubate anaerobically at 35 2C.
3. Examine at 24 hours if incubating plates in an anaerobic chamber.
Examine at 48 hours if incubating plates in an anaerobic jar or
pouch, or if using Brewer anaerobic dish cover.
4. Extended incubation may be necessary to recover some anaerobes.
Brewer Anaerobic Agar Plates:
1. Dispense 50-60 ml of Brewer Anaerobic Agar into a standard
Petri dish. For best results use porous tops to obtain a dry surface.
2. Inoculate the surface of the medium by streaking; avoid the edges
of the plates.
3. Replace the standard Petri dish lid with a sterile Brewer anaerobic
dish cover. The cover should not rest on the Petri dish bottom.
The inner glass ridge should seal against the uninoculated
periphery of the agar. It is essential that the sealing ring inside the
cover is in contact with the medium. This seal must not be broken
before the end of the incubation period. A small amount of air is
caught over the surface of the medium, and the oxygen in this space
reacts with the reducing agents to form an anaerobic environment.
4. Incubate aerobically as desired.
For a complete discussion on anaerobic and microaerophilic
bacteria from clinical specimens, refer to the appropriate
procedures outlined in the references.2,3,4 For the examination of
anaerobic bacteria in food refer to standard methods.7,8,9
The Difco Manual
Section II
Brilliant Green Agar
Results

Charles C. Thomas, Springfield, IL.
Bacteriological analytical manual, 8th ed. AOAC International,
Gaithersburg, MD.
9. Marshall, R. T. (ed.). 1993. Standard methods for the
microbiological examination of dairy products, 16th ed. American
Public Health Association, Washington, D.C.

isolate recovered to ensure the organism is an anaerobe.2
References
1. Brewer, J. H. 1942. A new Petri dish and technique for use in the
cultivation of anaerobes and microaerophiles. Science 95:587.
Etiological agents recovered from clinical material, p. 474-503.
Packaging
500 g
10 kg
0279-17
0279-08
Bacto Brilliant Green Agar
Intended Use

worldwide, despite efforts to control the prevalence of Salmonella
Bacto Brilliant Green Agar is used for isolating Salmonella other than
Salmonella typhi.
Uninoculated
plate
ATCC 14028

Solution:
water on boiling. Solution is brownish-green,
clear to very slightly opalescent.
Prepared Plates:
Orangish-brown, very slightly to
Reaction of 3.6%
Solution at 25C:
pH 6.9 0.2
Cultural Response
Prepare Brilliant Green Agar per label directions. Inoculate and
incubate the plates at 35 2C for 18-24 hours.
ORGANISM
Escherichia coli
Salmonella enteritidis
ATCC
INOCULUM
CFU
25922* 2,000-10,000
13076 100-1,000
GROWTH
COLONY
MORPHOLOGY
none to poor
good
yellow-green
pink-white
w/red medium
Salmonella typhi
19430 100-1,000
none to poor
red
Salmonella typhimurium 14028*
30-300
good
pink-white
w/red medium
Staphylococcus aureus 25923* 2,000-10,000 markedly inhibited
The Difco Manual
*These cultures are available as Bactrol Disks and should

be used as directed in Bactrol Disks Technical Information.
87
Section II
in domesticated animals. Infection with non-typhi Salmonella

often causes mild, self-limiting illness.1 The illness results from
consumption of raw, undercooked or improperly processed foods
contaminated with Salmonella. Many of these cases of Salmonellarelated gastroenteritis are due to improper handling of poultry
products. Various poultry products are routinely monitored for
Salmonella before their distribution for human consumption, but
in many instances, contaminated food samples elude monitoring.
The use of Brilliant Green Agar as a primary plating medium for
the isolation of Salmonella was first described by Kristensen, Lester
and Jurgens 2 who reported it useful for the differentiation of
paratyphoid B from other intestinal gram-negative bacilli. Later,
Kauffmann3 modified their formula and used Brilliant Green Agar
in addition to Tetrathionate Broth for the isolation of Salmonella
from stool specimens. Gallon and Quan4 increased their positive
Salmonella findings by using Tetrathionate Broth and plating
on Brilliant Green Agar. Broh-Kahn5 showed that the Kauffmann
modification of Brilliant Green Agar permitted the use of heavy
inocula to obtain maximum recovery of Salmonella from fecal
specimens. Miller and Tate6 found that the addition of 20 mg per liter of
sodium novobiocin to Brilliant Green Agar reduced or completely
inhibited nuisance organisms commonly seen on agar media used for
isolating salmonellae. Brilliant Green Agar with Novobiocin is also
recommended for use when testing food for Salmonella.7
Brilliant Green Agar is recommended for use in testing clinical
specimens.8,9 The outstanding selectivity of this medium permits
the use of moderately heavy inocula, which should be evenly
distributed over the surface. Brilliant Green Agar is valuable
when investigating outbreaks of Salmonella spp., other than S. typhi
and S. paratyphi.8,9 In addition, Brilliant Green Agar is used in the
microbial limits test as recommended in the United States
Pharmacopeia. The microbial limits test is performed to ensure
that pharmaceutical articles are free of Salmonella spp.10
Precautions
Storage
plates at 2-8C.
Expiration Date
The expiration date applies to the product in its intact container
Procedure
Materials Provided

Autoclave
Waterbath (45-50C)
Petri dishes
Incubator (35C)
1.
2.
3.
4.
5.

Autoclave at 121C for 15 minutes. Avoid overheating.
Cool to 45-50C in a waterbath.
In Brilliant Green Agar, Proteose Peptone No. 3 and Yeast Extract

provide nitrogen, vitamins and minerals. Lactose and Saccharose
are the carbohydrates in the medium. Phenol Red is the pH indicator
that turns the medium a yellow color with the formation of acid when
lactose and/or sucrose is fermented. Sodium Chloride maintains the
osmotic balance in the medium. Brilliant Green inhibits gram-positive
bacteria and most gram-negative bacilli other than Salmonella spp.
Lactose/sucrose fermenters are usually inhibited.11 Bacto Agar is the
solidifying agent.
Formula
For isolation of Salmonella from clinical specimens, inoculate fecal

specimens and rectal swabs onto a small area of one quadrant of the
Brilliant Green Agar plate and streak for isolation. This will permit
the development of discrete colonies. Incubate plates at 35C.
Examine plates after 18-24 hours for colonies with characteristic
morphologies associated with Salmonella spp.

Formula Per Liter
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Brilliant Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0125
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.08
88
g
g
g
g
g
g
g
g
1. Collect specimens in sterile containers or with sterile swabs and

transport immediately to the laboratory following recommended
guidelines.7.8.9
2. For specific information about specimen preparation and
inoculation of clinical specimens, consult the appropriate
references.7,8,9
Test Procedure
Results
The typical Salmonella colonies appear as pink-white opaque colonies
surrounded by a brilliant red medium. The few lactose and/or sucrose
fermenting organisms that grow are readily differentiated due to the
formation of a yellow-green colony surrounded by an intense
The Difco Manual
Section II
Brilliant Green Agar Modified
yellow-green zone. Brilliant Green Agar is not suitable for the

isolation of S. typhi or Shigella; however, some strains of S. typhi may
grow forming red colonies.

1. Colonies of Salmonella spp. vary from red-pink-white depending
on length of incubation and strain.11
2. Medium is normally orangish-brown in color; however, on
incubation, it turns bright red but returns to normal color at
room temperature.11
3. Studies by Taylor12 showed that slow lactose fermenters, Proteus,
Citrobacter, and Pseudomonas may grow on Brilliant Green
Agar as red colonies.
4. In routine examination of clinical specimens or other materials for
the gram-negative intestinal pathogens, other primary plating
media such as MacConkey Agar, and fluid enrichments such as
Tetrathionate Broth and Selenite Broth, should be used with
Brilliant Green Agar.
5. S. typhi does not grow adequately on this medium. Shigella spp. do
not grow.11
References
1993. Pathogens in milk and milk products, p. 103-212. In R. T.
Marshall, (ed.). Standard methods for the examination of
Washington, D.C.
2. Kristensen, M., V. Lester, and A. Jurgens. 1925. On the use of
trypsinized casein, brom thymol blue, brom cresol purple,
phenol red and brilliant green for bacteriological nutrient media.
Br. J. Exp. Pathol. 6:291.
3. Kauffmann, F. 1935. Weitere Erfahrungen mit den kombinierten
Anreicherungsverfahren fr Salmonellabacillen. Z. Hyg.
Infektionskr. 117:26.
4. Galton, M. M., and M. S. Quan. 1944. Salmonella isolated in

Florida during 1943 with the combined enrichment method of
Kauffmann. Am. J. Public Health 34:1071.
5. Broh-Kahn, R. H. 1946. The laboratory diagnosis of enteric
infections caused by the Salmonella-Shigella group. Military
Surgeon 99:770-776.
6. Tate, C. R., and R. G. Miller. 1990. Modification of brilliant
green agar by adding sodium novobiocin to increase selectivity for
Salmonella. The Maryland Poultryman 4:7-11.
7. Federal Register. 1993. Chicken Disease Caused by Salmonella
enteritidis; proposed rule. Fed. Regis. 58:41048-41061.
8. Pezzlo, M. (ed.). 1992. Aerobic bacteriology, p. 1.0.1-1.20.47. In
H. D.Isenberg, (ed.), Clinical microbiology procedures handbook,
vol. 1. American Society for Microbiology, Washington, D.C.
p. 450-456. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C.
10 United States Pharmacopeial Convention. 1995. The United
11. MacFaddin, J. F. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, vol. 1. Williams &
Wilkins, Baltimore, MD.
12. Taylor, W. I. 1965. Isolation of shigellae. I. Xylose lysine
agars: New media for isolation of enteric pathogens. Am. J. Clin.
Pathol. 44:471.
Packaging
100 g
500 g
0285-15
0285-17
Bacto Brilliant Green Agar Modified
Intended Use
Bacto Brilliant Green Agar Modified is used for isolating Salmonella
from water, sewage and foodstuffs.

Kampelmacher1 proposed the formula for a selective medium to isolate
Salmonella from pig feces and minced meat. Brilliant Green Agar
Modified is more selective than Desoxycholate Citrate Agar and other
brilliant green media, and inhibits the growth of Pseudomonas
aeruginosa and Proteus sp. which may resemble Salmonella. Salmonella
cholerasuis grows well on Brilliant Green Agar Modified, but poorly
on Desoxycholate Citrate Agar.2
Brilliant Green Agar Modified is recommended for the isolation of
Salmonella, other than Salmonella typhi, from water and associated
The Difco Manual
materials3 and meat and meat products.4 It is recommended by the British

Poultry Meat Society5 for the examination of poultry and poultry products.
The recommended procedures include using complementary selective
culture media and techniques to increase the likelihood of isolating
multiple serotypes of Salmonella from samples.6

Brilliant Green Agar Modified contains Beef Extract and Bacto Peptone
as sources of carbon, nitrogen, vitamins and minerals. Yeast Extract
supplies B-complex vitamins which stimulate bacterial growth. Lactose
and Sucrose are carbohydrate sources. In the presence of Phenol Red, a
pH indicator, nonlactose and/or nonsucrose-fermenting Salmonella will
produce red colonies. Brilliant Green inhibits gram positive organisms
and many gram negative bacteria, except Salmonella. Bacto Agar is a
solidifying agent.
89
Section II
Formula

Formula Per Liter
Glassware
Petri dishes
Autoclave
Incubator (42C)
Sterile Blender Jar
Buffered Peptone Water
Muller Kauffmann Tetrathionate Broth
Selenite Brilliant Green Medium

Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Disodium Hydrogen Phosphate . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Dihydrogen Phosphate . . . . . . . . . . . . . . . . . . . . . 0.6
Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sucrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.09
Brilliant Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0047
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.0
g
g
g
g
g
g
g
g
g
g
2. Heat to boiling to dissolve completely. DO NOT AUTOCLAVE.
Precautions

Meat and Meat Products

1. Weigh 25 g of the sample into a sterile blender jar and add 225 ml
of Buffered Peptone Water. Macerate for a sufficient time to give
15,000-20,000 revolutions.
2. Aseptically transfer the contents of the blender jar to a 500 ml flask.
Incubate at 37 0.1C for 16-20 hours.
3. Transfer 10 ml samples to 100 ml Muller Kauffmann Tetrathionate
Broth and to 100 ml of Selenite Brilliant Green Medium.
4. Incubate the Muller Kauffmann Tetrathionate Broth at 42-43C
and the Selenite Brilliant Green Enrichment at 37C.
Sewage Polluted Natural Water
This procedure is applicable to the isolation of Salmonella spp. other
than S. typhi.
Storage
Expiration Date
Procedure
Materials Provided
Uninoculated
plate
ATCC 14028

Dehydrated Appearance: Pink, free-flowing, homogeneous.
Solution:
deionized water on boiling. Solution is
orange-brown, clear to slightly opalescent.
Prepared Medium:
Orange-brown, clear to slightly
opalescent.
Reaction of 5.2%
Solution at 25C:
pH 6.9 0.1
Cultural Response
Prepare Brilliant Green Agar Modified per label directions.
ORGANISM
Escherichia coli
ATCC
INOCULUM
CFU
25922* 1,000-2,000
GROWTH
COLONY COLOR
completely to
partially inhibited
Proteus mirabilis NCTC 11938 1,000-2,000 completely to
partially inhibited
good
green
red
red
90
The Difco Manual
Section II
Brilliant Green Bile Agar
1. Inoculate 25 ml aliquots of the sample into 25 ml of double strength

Buffered Peptone Water (1810) and incubate at 37C for 18 hours.
2. Transfer 1 ml samples into 10 ml of Muller Kauffmann
Tetrathionate Broth.
3. Incubate at 43C for 48 hours.
Test Procedure
1. Subculture the broths at 18-24 hours and at 48 hours onto Brilliant
Green Agar (Modified).
2. Examine for typical colonies of Salmonella after overnight
incubation at 37C.
Results
Salmonella will produce red colonies.

1. Due to the nutritional requirements and inhibitory characteristics
of the organisms themselves, organisms other than Salmonella spp.,
such as Morganella morgani and some Enterobacteriaceae may
grow on the medium.
2. Confirmatory tests, such as fermentation reactions and
seroagglutination, should be carried out on all presumptive
Salmonella spp.
References
1. Guinee, P. A., and E. H. Kampelmacher. 1962. Antonie van
Leeuwenhoek. 28:417-427.
2. Heard, T. W., N. E. Jennet, and A. H. Linton. 1969. British
Veterinary Journal 125:635-644.
3. H. M. S. O. 1982. Methods for the isolation and identification of
salmonellae (other than Salmonella typhi) from water and
associated materials.
4. International Organisation for Standardisation. 1974. Draft
International Standard ISO/DIS 3565. Geneva.
5. British Poultry Meat Society. 1982. A manual of recommended
methods for the microbiological examination of poultry and
poultry products.
6. Harvey, R. W. S., and T. H. Price. 1976. J. Hygiene Camb.
77:333-339.
Packaging
500 g
1880-17
Bacto Brilliant Green Bile Agar
Uninoculated
plate
ATCC 13048

Dehydrated Appearance: Light purple, free-flowing, homogeneous.
Solution:
is bluish purple, very slightly to
Prepared Medium:
Bluish purple, slightly opalescent.
Reaction of 2.06%
Solution at 25C:
pH 6.9 0.2
Cultural Response
Prepare Brilliant Green Bile Agar per label directions. Inoculate
using the pour plate technique and incubate the plates at 35 2C
for 18-24 hours.
ORGANISM
ATCC
INOCULUM
CFU
Enterobacter aerogenes 13048* 100-1,000

Escherichia coli
25922* 100-1,000
GROWTH
COLONY COLOR
good
good
pink
deep red with
bile precipitate
14028 100-1,000
good
colorless to
light pink
25923* 1,000-2,000 marked to
complete inhibition
Escherichia coli
ATCC 25922
ATCC 13076
The Difco Manual
91
Section II
Intended Use
Also Known As

Brilliant Green Bile Agar (BGBA) is also known as Brilliant Green

Agar2 (BGA).
Storage

Bacto Brilliant Green Bile Agar is used for isolating, differentiating

and enumerating coliform bacteria.
Noble and Tonney1 described Brilliant Green Bile Agar for determining
the relative density of coliform bacteria in water and sewage. The
medium is particularly useful in selectively isolating Salmonella spp.
from other coliform bacteria. The American Public Health Association
(APHA) specifies a qualitative procedure to isolate and identify
Salmonella spp. from water and wastewater using concentration,
enrichment and selective growth.2
Brilliant Green Bile Agar contains Bacto Peptone as a source of carbon,
nitrogen, vitamins and minerals. Lactose is a fermentable carbohydrate.
Oxgall (bile) and brilliant green inhibit gram-positive bacteria and most
gram-negative bacteria except coliforms. Basic Fuchsin is a pH
indicator. Monopotassium phosphate is a buffering agent. Agar Noble is
a solidifying agent.
Differentiation of the coliforms is based on fermentation of lactose.
Bacteria that ferment lactose produce acid and, in the presence of basic
fuchsin, form deep red colonies with a pink halo. Bacteria that do
not ferment lactose form colorless to faint pink colonies. Coliform
bacteria typically ferment lactose, producing deep red colonies, while
Salmonella spp., which do not ferment lactose, produce colorless to
faint pink colonies.
Materials Provided

Glassware
Autoclave
Incubator (35)
Formula

Formula Per Liter
Test Procedure
g
g
g
g
g
g
g
g
g
g
Precautions
2. POSSIBLE RISK OF IRREVERSIBLE EFFECTS. (US) Avoid
contact with skin and eyes. Do not breathe dust. Wear suitable
protective clothing. Keep container tightly closed. TARGET
ORGAN(S): Liver, Thyroid.
92
Expiration Date
Procedure
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.25

Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.9
Bacto Oxgall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.00295
Sodium Sulfite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.205
Ferric Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0295
Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . 0.0153
Agar Noble . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.15
Erioglaucine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0649
Bacto Basic Fuchsin . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0776
Brilliant Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0000295
This medium is light sensitive. Protect from exposure to direct sunlight.
Results

1. The medium is sensitive to light, particularly direct sunlight, which
produces a decrease in the productivity of the medium and a change
in color from deep blue to purple or red. The medium should be
prepared just prior to use and, when necessary to store the
medium, it should be kept in the dark.
References
1. Nobel and Tonney. 1935. J. Am. Water Works Assoc. 27:108.
Packaging
500 g
0014-17
The Difco Manual
Section II
Brilliant Green Bile 2%
Bacto Brilliant Green Bile 2%
Intended Use
Formula
Bacto Brilliant Green Bile 2% is used for confirming the presence of

coliform organisms in water and foods.

Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Oxgall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Brilliant Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0133
Also Known As
Brilliant Green Bile Broth
Brilliant Green Lactose Bile Broth, 2%
Brilliant Green Lactose Bile Broth
Brilliant Green Bile Lactose Broth
g
g
g
g
Precautions

The coliform group of bacteria includes aerobic and facultatively
anaerobic gram-negative non-sporeforming bacilli that ferment lactose
and form acid and gas at 35C within 48 hours. Members of the
Enterobacteriaceae comprise the majority of this group but organisms
such as Aeromonas species may also be included.
Procedures to detect and confirm coliforms are used in testing water,
foods, dairy products and other materials.1,2,3,4,5 The procedures begin
with a presumptive test that, when positive, is confirmed by using
Brilliant Green Bile 2%.

Bacto Peptone is a source of carbon and nitrogen for general growth
requirements. Oxgall (bile) and Brilliant Green inhibit gram-positive
bacteria and many gram-negative bacteria other than coliforms.
Lactose is a carbohydrate source. Bacteria that ferment lactose and
produce gas are detected.

AND SKIN. Avoid contact with skin and eyes. Do not breathe
dust. Wear suitable gloves and eye/face protection. Use only in
well ventilated areas. Keep container tightly closed. TARGET
ORGAN(S): Lungs.

Dehydrated Appearance: Greenish-beige, free-flowing, homogeneous.
Solution:
4.0% solution soluble in distilled or deionized water
on warming, if necessary; emerald green, clear
Prepared Medium:
Emerald green, clear.
Reaction of 4.0%
Solution at 25C:
pH 7.2 0.2
Cultural Response
Prepare Brilliant Green Bile 2% per label directions. Inoculate medium and
incubate at 35 2C for 48 hours.
ATCC
INOCULUM
CFU
Escherichia coli
13048*
25922*
25923*
100-1,000
100-1,000
1,000-2,000
19433
1,000-2,000
ORGANISM
GROWTH
good
good
marked to
complete inhibition
marked to
complete inhibition
GAS
PRODUCTION
+
+
Uninoculated
tube
Escherichia coli
ATCC 25922
The Difco Manual
93
mBrilliant Green Broth
Storage
Store the dehydrated medium below 30C. The powder is very
Expiration Date
Procedure
Materials Provided

Flask with closure
Test tubes with caps
Fermentation tubes
Autoclave
Incubator
2. Warm slightly to dissolve completely.
3. Dispense required amount in tubes containing inverted fermentation vials.

Process specimens according to established procedures for the type
of material being tested.1,2,3,4,5
Test Procedure
Section II
1. Subculture from a positive presumptive coliform specimen in

Lauryl Tryptose Broth (LST) or from typical coliform-type colonies
on Violet Red Bile Agar (VRBA) to tubes of Brilliant Green Bile 2%.
2. Incubate at 35C for 48 2 hours.
3. Examine for bubbles (gas) in the fermentation tube.
Results
Positive: Bubbles (gas) in fermentation tube.
Negative: No bubbles (gas) in fermentation tube.
References
2. Christen, G. L., P. M. Davidson, J. S. McAllister, and L. A. Roth.
1993. Coliform and other indicator bacteria, p. 247-269. In
R. T. Marshall (ed.). Standard methods for the microbiological
3. Hitchins, A. D., P. A. Hartman, and E. C. D. Todd. 1992.
Coliforms - Escherichia coli and its toxins, p. 325-369. In
C. Vanderzant and D. F. Splittstoesser (ed.). Compendium of
4 Hitchins, A. D., P. Feng, W. D. Watkins, S. R. Rippey, and L. A.
Chandler. 1995. Escherichia coli and the coliform bacteria,
p. 4.01-4.29. In Bacteriological analytical manual, 8th ed.
AOAC International, Gaithersburg, MD.
5. Andrews, W. H. 1995. Microbial methods, p. 1-119. In Official
Packaging
Consult standard references for specific instructions for the type of

material being tested.1,2,3,4,5
Bacto mBrilliant Green Broth
100
500
2
10
g
g
kg
kg
0007-15-6
0007-17-4
0007-07-6
0007-08-5
Intended Use
Bacto mBrilliant Green Broth is used for recovering and differentiating
Salmonella from primary water samples by membrane filtration.

mBrilliant Green Broth is primarily used as a selective-differential
medium for Salmonella species. Salmonella species cause many types
of infections from mild, self-limiting gastroenteritis to life-threatening
typhoid fever.4 The most common form of Salmonella disease is
self-limiting gastroenteritis with fever lasting less than two days and
diarrhea lasting less than 7 days.4
mBrilliant Green Broth is a modification of Kauffmanns1 Brilliant
Green Agar in which the agar has been omitted and all other ingredients
are at double strength.
94
Kabler and Clark2 used mBrilliant Green Broth in a membrane filtration

procedure originally developed by Geldreich and Jeter.3 In this technique,
an appropriate volume of water is filtered through the membrane filter.
The filter is placed on an absorbent pad saturated with m Tetrathionate
Broth Base. After incubation, the membrane is transferred to another
absorbent pad saturated with mBrilliant Green Broth and incubated.
Following incubation, the membrane is transferred to a fresh pad
saturated with urease test reagent.

Proteose Peptone No. 3 provides the nitrogen, minerals and amino
acids in mBrilliant Green Broth. Yeast Extract is the vitamin source.
Lactose and Saccharose are the carbohydrates for bacterial growth.
Sodium Chloride maintains the osmotic balance of the medium and
Phenol Red is the dye used as an indicator of carbohydrate fermentation.
Brilliant Green is the selective agent.
The Difco Manual
Section II
Formula

Formula Per Liter
Glassware
Sterile absorbent pad
Membrane filtration equipment
Incubator (35C)
Sterile Petri dishes, 50 x 9 mm

Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.16
Brilliant Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.025
g
g
g
g
g
g
g
Precautions
Storage
Use the rehydrated medium within 24 hours.
Expiration Date
1.
2.
3.
4.
5.
Suspend 7.6 grams in 100 ml of distilled or deionized water.

Heat to boiling to dissolve completely. Do not autoclave.
Cool to room temperature.
Dispense 2 ml amounts onto sterile absorbent pads.
Use the rehydrated medium within 24 hours.

Obtain and process water samples according to the techniques and
Test Procedure
1.
2.
3.
4.
Inoculate a water sample using the membrane filtration procedure.

Place the filter on a pad saturated with mBrilliant Green Broth.
Incubate at 35 2C in a humid atmosphere for 18-24 hours.
After incubation, examine for growth and the color of the colonies.
Escherichia coli
ATCC 25922
Procedure
Materials Provided

Solution:
deionized water; greenish-red, slightly
opalescent.
Prepared Medium
Greenish-red, slightly opalescent.
Reaction of 7.6%
Solution at 25C:
pH 6.9 0.2
ATCC 14028
Cultural Response
Prepare mBrilliant Green Broth per label directions. Inoculate using the
membrane filter technique and incubate at 35 2C in a humid atmosphere
for 18-24 hours.
ORGANISM
Escherichia coli
ATCC
INOCULUM
CFU
GROWTH
COLOR OF COLONY
25922*
13076
14028*
20-80
20-80
20-80
good
good
good
yellow
pink to red
pink to red
*These cultures are available as Bactrol Disks and should be used as directed in
Bactrol Disks Technical Information.
The Difco Manual
95
Brucella Agar & Brucella Broth
Section II
Results
Salmonella species form pink to red colonies.

2. Kabler and Clark. 1952. Am. J. Public Health 42:390.

3. Geldreich and Jeter. 1952. Bacteriol. Proc. SAB., Boston, MA.
Packaging
References
Anreicherungsverfahren fur Salmonellabacillen. Z. Hyg.
Bacto Brucella Agar

Bacto Brucella Broth
Intended Use
Bacto Brucella Agar is used for isolating and cultivating Brucella.
Bacto Brucella Broth is used for cultivating Brucella and other

Brucella Agar
Solution:
or deionized water with frequent
agitation on boiling. Light amber,
very slightly to slightly opalescent.
Prepared Medium:
Light amber, slightly opalescent.
Reaction of 4.3%
Solution at 25C:
pH 7.0 0.2
Brucella Broth
Solution:
or deionized water. Light amber,
clear to very slightly opalescent,
no precipitate.
Prepared Medium:
opalescent, no precipitate.
Reaction of 2.8%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepare Brucella Agar or Brucella Broth per label directions.
Inoculate and incubate at 35 2C under 5-10% CO2 for
24-72 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Brucella abortus
Brucella melitensis
Brucella suis
4315
4309
4314
100-1,000
100-1,000
100-1,000
good
good
good
500 g
0494-17
Brucella Agar and Brucella Broth are prepared according to the APHA
formula for Albimi Broth, which is used for the isolation of Brucella
species.1 Brucellosis is a zoonotic disease with a domestic-animal
reservoir.2 Transmission by milk, milk products, meat and direct
contact with infected animals is the usual route of exposure.2
Brucella Agar is used as a general medium for the cultivation of
fastidious microorganisms, e.g., Streptococcus pneumoniae,
Streptococcus viridans and Neisseria meningitidis.3 With the addition
of blood, Brucella Agar can be used to determine hemolytic reactions
of pathogenic bacteria.3 Brucella Agar can be used as a base for the
isolation of Campylobacter species.3
Brucella Broth is recommended for the isolation of Brucella species
from blood cultures.4,5 Brucella Broth is specified in the Compendium
of Methods for the Microbiological Examination of Food.6

Peptamin provides nitrogen and amino acids. Tryptone provides nitrogen.
Yeast Extract adds essential vitamins. Dextrose is a carbon source;
Sodium Bisulfite enhances growth. Sodium Chloride maintains the
osmotic balance. Bacto Agar is the solidifying agent in Brucella Agar.
Supplemental blood (5-10%) provides additional growth factors for
fastidious microorganisms and is used to determine hemolytic reactions.
Formula
Brucella Agar
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Peptamin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Bisulfite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g

Brucella Broth
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Peptamin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
g
g
g
g
g
g
96
The Difco Manual
Section II
Bryant and Burkey Medium
Precautions
Test Procedure

2. Brucella species are classified as Biosafety Level 3 pathogens. All
manipulations with live cultures and antigens must be confined to
a Class II biological safety cabinet (BSC).2
For a complete discussion of the isolation and identification of

Brucella, refer to appropriate procedures outlined in the references.2,4,5
Storage

2. Hemolytic reactions of many microorganisms are different on
horse blood from those on sheep blood agar; e.g., some Group D
streptococci exhibit beta hemolysis on horse blood but not on sheep
blood and are mistaken for Group A.3

Expiration Date
Procedure
Materials Provided
Brucella Agar
Brucella Broth

Glassware
Autoclave
Incubator (35C)
Sterile Petri dishes or tubes
1. Brucella Agar: Suspend 43 grams in 1 liter distilled or deionized
water and boil to dissolve completely.
Brucella Broth: Dissolve 28 grams in 1 liter distilled or deionized
water.
3. OPTIONAL: To prepare Brucella Blood Agar, aseptically add
5-10% sterile defibrinated blood at 45-50C. Mix well.
Results
Refer to apropriate references and procedures for results.
References
1. Hausler, W. J. (ed.). 1976. Standard methods for the examination
Washington, D.C.
2. Moyer, N. P., and L. A. Holcomb. 1995. Brucella, p. 549-555. In
P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H.
3. MacFaddin, J. D. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, vol. 1, p.110-114.
handbook. American Society for Microbiology, Washington, D.C.
St. Louis, MO.
Packaging
Brucella Agar
500 g
2 kg
10 kg
0964-17
0964-07
0964-08
Brucella Broth
500 g
10 kg
0495-17
0495-08

procedures established by institutional policy.
Bacto Bryant and Burkey Medium
Intended Use
Bacto Bryant and Burkey Medium is used for detecting and
enumerating spores of lactate-fermenting Clostridium in milk and
dairy products.

Bryant and Burkey Medium is based on the lactate fermentation media
described by Rosenberger1 and Bryant and Burkey2, as modified by
The Difco Manual
Bergre et al,3 who reported that their medium could be used for detecting
and enumerating C. tyrobutyricum spores in milk and dairy products.3-5

Tryptone, Yeast Extract, Beef Extract Desiccated and L-Cysteine
Hydrochloride provide nutrients and cofactors required for good
growth of clostridia. Selectivity of this medium is achieved through
the addition of Sodium Acetate, which is also the principal promoter of
97
Section II
germination by C. tyrobutyricum spores. 6 Sodium Lactate is

fermented under anaerobic conditions by C. tyrobutyricum and other
lactate-fermenting clostridia, producing hydrogen and carbon dioxide.
Gas production is demonstrated by an upward movement of a paraffin
plug which is overlaid on the medium. Resazurin is included in the
medium to show anaerobiosis, turning from pink (aerobic) to colorless
under anaerobic conditions.
Storage
1. Store the dehydrated medium below 30C. The powder is very
2. Store prepared medium at 2-8C.
Expiration Date
During processing, the sample is heated at 75C for 15 minutes to kill

vegetative cells and activate germination of spores.
Formula
Procedure

Formula Per Liter
Materials Provided
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Bacto Beef Extract, Desiccated . . . . . . . . . . . . . . . . . . . . . 7.5
Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
L-Cysteine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Sodium Lactate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Resazurin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0025
g
g
g
g
g
g
g

Autoclave
Incubator (35 2C)
Precautions
NOTE: This product contains sodium lactate; it is not necessary to add

sodium lactate during preparation.


2. Dispense 10 ml amounts into tubes.

Solution:
medium amber, clear when hot,
becoming red upon cooling.
Reaction of 3.8%
Solution at 25C:
pH 5.9 0.2
Cultural Response
Prepare Bryant and Burkey Medium per label directions.
Inoculate using Most Probable Number (MPN) method and
incubate at 35 2C for 6 days.
ORGANISM
Clostridium
tyrobutyricum
Clostridium
tyrobutyricum
Clostridium
tyrobutyricum
Clostridium
tyrobutyricum
ATCC OR
STRAIN
GAS
INOCULUM
GROWTH
PRODUCTION
CNRZ 500 MPN method
good
>1 cm of gas
CNRZ 510 MPN method
good
>1 cm of gas
CNRZ 608 MPN method
good
>1 cm of gas
good
>1 cm of gas
25755
MPN method
98
1. Collect food samples in sterile containers and transport immediately

to the laboratory following recommended guidelines.
2. Process each food sample using procedures appropriate for that
sample.
Test Procedure
Three-tube Most Probable Number (MPN) Method
1. Before use, heat tubes to boiling for 10 minutes to regenerate
anaerobic conditions. Note: This step is required only with tubes
stored under aerobic conditions. Tubes stored under anaerobic
conditions or freshly sterilized tubes do not need additional heating.
2. Prepare 10-fold dilutions of the sample and inoculate triplicate
tubes of Bryant and Burkey Medium with 1 ml of each sample
dilution.
3. Pour approximately 2 ml of melted paraffin (60-65C), previously
autoclaved at 121C for 20 minutes, into each tube.
4. Heat the tubes at 75C for 15 minutes to kill vegetative cells and
activate spores; allow to cool to room temperature.
5. Incubate tubes at 35C for 6 days.
6. Examine tubes for growth and gas production after 48 hours of
incubation and daily for up to 6 days.
Results
Tubes showing both growth and production of gas (indicated by upward
movement of the paraffin more than 1 cm) are considered positive for
the presence of lactate-fermenting clostridial spores. Determine the
spore count using the Most Probable Number (MPN) method.
The Difco Manual
Section II
Buffered Peptone Water & Modified Buffered Peptone Water
References
1. Rosenberger, K. F. 1951. The development of methods for the
study of obligate anaerobes in silage. Proc. Soc. Appl. Bacteriol.
14:161-164.
2. Bryant, M. P., and L. A. Burkey. 1956. The characteristics of
lactate-fermenting sporeforming anaerobes from silage.
3. Bergre, J. L., P. Gouet, J. Hermier, and G. Mocquot. 1968. Les
Clostridium du groupe butyrique dans les produits laitiers. Ann.
Inst. Pasteur Lille. 19:41-54.
4. Cerf, O., and J. L. Bergre. 1968. La numration des spores de
Clostridium et son application au lait et aux produits laitiers.
Numration des diffrents groupes de Clostridium. Le Lait

48:501-519.
5. Bergre, J. L. 1979. Developpement de lensilage. Ses
consquences sur la qualit du lait et des produits laiters. Revue
laitre franaise.
6. Touraille, C., and J. L. Bergre. 1974. La germination de la spore
de Clostridium tyrobutyricum. Biochimie. 56:404-422.
Packaging
500 g
2 kg
0645-17
0645-07
Bacto Buffered Peptone Water

Bacto Modified Buffered Peptone Water
Intended Use
Dehydrated Appearance: Cream-white to light tan, free-flowing,
homogeneous.
Solution:
deionized water. Solution is light
amber, clear.
Prepared Medium:
Light amber, clear.
Reaction of 2.0%
Solution at 25C:
pH 7.2 0.2
Modified Buffered Peptone Water
Dehydrated Appearance: Light beige, free-flowing, homogenous.
Solution:
amber, clear.
Prepared Medium:
Light amber, clear.
Reaction of 2.5%
Solution at 25C:
pH 7.2 0.2
Prepare Buffered Peptone Water or Modified Buffered Peptone
Water per label directions. Inoculate and incubate at 35 2C
for 18-24 hours.
Salmonella typhi

Edel and Kampelmacher1 noted that food preservation techniques
involving heat, desiccation, preservatives, high osmotic pressure or pH
changes cause sublethal injury to salmonellae. Preenrichment in a
nonselective medium allows for repair of cell damage and facilitates
the recovery of salmonellae. Lactose Broth is frequently used for this
purpose but it may be detrimental to recovering salmonellae.2 Buffered
Peptone Water maintains a high pH over the preenrichment period and
results in repair of injured cells that may be sensitive to low pH.3 This
is particularly important for vegetable specimens which have a low
buffering capacity. These media can be used for testing dry poultry
feed.4 Buffered Peptone Water is a standard methods medium.5
Modified Buffered Peptone Water provides additional buffering capacity
when organisms have been enriched in a pre-enrichment medium
containing a high carbohydrate concentration.
Cultural Response
ORGANISM
Bacto Buffered Peptone Water is used for preenriching damaged

Salmonella species from food specimens to increase recovery.
Bacto Modified Buffered Peptone Water is used for preenriching
Salmonella species from food specimens to increase recovery.
ATCC
INOCULUM
CFU
GROWTH
13076
19430
14028*
10-100
10-100
10-100
good
good
good
Buffered Peptone Water and Modified Buffered Peptone Water contain

Peptone as a source of carbon, nitrogen, vitamins and minerals.
Sodium Chloride maintains the osmotic balance. Phosphates buffer the
medium.
Formula
Formula Per Liter
Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Phosphate, Dibasic . . . . . . . . . . . . . . . . . . . . . . . . 3.5
Potassium Phosphate, Monobasic . . . . . . . . . . . . . . . . . . . 1.5
g
g
g
g

The Difco Manual
99
Bushnell-Haas Broth
Section II

Formula Per Liter
Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Phosphate, Dibasic . . . . . . . . . . . . . . . . . . . . . . . . . 7
Potassium Phosphate, Monobasic . . . . . . . . . . . . . . . . . . . . 3
g
g
g
g
Precautions
2. Buffered Peptone Water
MAY BE IRRITATING TO EYES, RESPIRATORY SYSTEM
IRRITANT. MAY BE IRRITATING TO EYES, RESPIRATORY
SYSTEM AND SKIN. (US) Avoid contact with skin and eyes. Do
not breathe dust. Wear suitable protective clothing. Keep container
tightly closed.

Autoclave
Incubator (35C)
1. Dissolve the medium in 1 liter distilled or deionized water:
Buffered Peptone Water - 20 grams;
Modified Buffered Peptone Water - 25 grams.

Collect specimens according to recommended guidelines.
Test Procedure
Test specimens according to recommended guidelines.
Results
Growth is indicated by turbidity.

1. The types and numbers of competing flora in the test sample can
affect recovery and may overgrow salmonellae.
References
Procedure
1. Edel, W., and E. H. Kampelmacher. 1973. Bull. World Hlth.

Org. 48:167-174.
2. Angelotti, R. 1963. Microbiological quality of foods. Academic
Press, New York.
3. Sadovski, A. Y. 1977. J. Food Technol. 12:85-91.
4. Juven, B. J., N. A. Cox, J. S. Bailey, J. E. Thomson, O. W.
Charles, and J. V. Schutze. 1984. Recovery of Salmonella from
artificially contaminated poultry feeds in non-selective and
selective broth media. Jour. of Food Prot. 47:299-302.
5. Flowers, R. S., J-Y. DAoust, W. H. Andrews, and J. S. Bailey.
1992. Salmonella, p. 371-422. In C. Vanderzant, and D. F.
Splittstoesser (ed.). Compendium of methods for the microbiological
Washington, D.C.
Materials Provided
Packaging
Storage
Expiration Date

500 g
2 kg
10 kg
1810-17
1810-07
1810-08
500 g
1833-17

Glassware
Bacto Bushnell-Haas Broth
Intended Use
Bacto Bushnell-Haas Broth is used for studying microbial utilization
of hydrocarbons.
Also Known As
Bushnell-Haas Broth is also referred to as Bushnell-Haas marine
salts broth.
100

Bushnell-Haas Broth, prepared according to the formula described by
Bushnell and Haas1, is used to evaluate the ability of microorganisms
to decompose hydrocarbons. It is formulated without a carbon source
which allows for the addition of alternate hydrocarbons such as kerosene,
light and heavy mineral oils, paraffin wax, and gasoline.
Bushnell-Haas was recommended for the microbiological examination
of fuels by the Society for Industrial Microbiology (SIM) Committee
on Microbiological Deterioration of Fuels.2 The medium was used to
The Difco Manual
Section II
Bushnell-Haas Broth
enumerate total heterotrophs and hydrocarbon degradation

by microorganisms during bioremediation of Prince William Sound
following the Exxon Valdez oil spill.3,4

Magnesium Sulfate, Calcium Chloride, and Ferric Chloride provide
trace elements necessary for bacterial growth. Potassium Nitrate is a
nitrogen source, while Monopotassium Phosphate and Ammonium
Phosphate Dibasic provide buffering capability.
Formula

TARGET ORGAN(S): Blood, Liver, Nerves.
Storage
Bushnell-Haas Broth
Formula Per Liter
Calcium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.02
Ammonium Phosphate Dibasic . . . . . . . . . . . . . . . . . . . . . . 1
Potassium Nitrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Ferric Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05
g
g
g
g
g
g

medium at 2-8C.
Expiration Date
Precautions
Procedure

Materials Provided
Dehydrated Appearance: Beige with pink tint, free-flowing,
homogeneous.
Solution:
0.327% solution not soluble in
distilled or deionized water, white
precipitate remains. Solution, after
autoclaving, is colorless to very
light amber, clear supernatant over
yellow-orange precipitate.
Prepared Medium:
Colorless to very light amber, clear
supernatant over yellow-orange
precipitate.
Reaction of 0.327%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepare Bushnell-Haas Broth per label directions. Inoculate in
duplicate with the test organisms. Add sterile mineral oil (the
hydrocarbon source) to one set. Incubate at 25-30C
for up to 1 week.
RECOVERY
w/Hydrocarbon
ATCC
INOCULUM
PLAIN
9027
10145
14207
27853*
100-1,000
100-1,000
100-1,000
100-1,000
none to poor
none to poor
none to poor
none to poor
good
good
good
good
*The cultures are available as Bactrol Disks and should be used
The Difco Manual

Glassware
Autoclave
Incubator (25-30C)
ORGANISM
Bushnell-Haas Broth
1. Dissolve 3.27 grams in 1 liter distilled or deionized water.
2. Dispense as desired and autoclave at 121C for 15 minutes.
3. Cool to 45-50C.
NOTE: A precipitate that is white prior to sterilization and turns yellow
to orange after sterilization is normal.

1. Collect samples in sterile containers or with sterile swabs and
transport immediately to the laboratory.
Test Procedure
1.
2.
3.
4.
Inoculate the collected sample directly into the broth.

Overlay the broth with a sterile hydrocarbon source.
Incubate aerobically at 25-30C.
Examine tubes daily for growth for up to one week.
Results
Organisms capable of degrading hydrocarbons should show growth in
the Bushnell-Haas Broth supplemented with a hydrocarbon source.

1. Because the nutritional requirements of organisms vary, some strains
may be encountered that fail to grow or grow poorly in this medium.
References
1. Bushnell, L. D., and H. F. Haas. 1941. The utilization of certain
hydrocarbons by microorganisms. J. Bacteriol. 41:653-673.
101
CLED Agar
Section II
2. Allred, R. C., R. J. DeGray, R. W. Edwards, H. G. Hedrick, D.

E. Klemme, M. Rogers, M. Wulf, and H. Hodge. 1963. Proposed
procedures for microbiological examination of fuels. SIM Special
Publications, Number 1. Merck, Sharp & Dohme Research
Laboratories, Rahway, NJ.
3. Bragg, J. R., J. C. Roffall, and S. McMillen. 1990. Column flow
studies of bioremediation in Prince William Sound. Exxon
Production Research Co., Houston, TX.
Bacto CLED Agar
4. Brown, E. J., and J. F. Braddock. 1990. Sheen Screen,

a miniaturized most-probable-number method for enumeration
of oil-degrading microorganisms. Appl. Environ. Microbiol.
56:3895-3896.
Packaging
Bushnell-Haas Broth
500 g
10 kg
0578-17
0578-08
Intended Use
Bacto CLED Agar is used for cultivating, differentiating and enumerating
bacteria in urine.
Also Known As
CLED Agar is an abbreviation for Cystine Lactose-ElectrolyteDeficient Agar.

Sandys1 developed an electrolyte-deficient medium that prevented
Proteus from swarming. Mackey and Sandys2 modified the formula by
substituting lactose and sucrose for mannitol, and increasing the
amount of indicator and agar. While investigating this medium for a
dip slide technique for urine cultures, the researchers further modified
the formula. The revised formula omitted sucrose and added cysteine
and was called Cystine Lactose-Electrolyte-Deficient medium.3
CLED Agar is recommended in the spread plate technique or as a dip

slide for the detection of bacteria in urine. This medium supports the
growth of urinary pathogens and provides distinct colony morphology.
CLED medium lacks an electrolyte (salt) which is necessary for growth
or other characteristics of certain bacteria.4 Many European laboratories
use Cystine Lactose-Electrolyte-Deficient (CLED) Agar.5

Beef Extract, Bacto Peptone and Tryptone provide the nitrogen, vitamins
and amino acids in CLED Agar. L-Cystine is added as a growth supplement
for cystine-dependent coliforms. Lactose is included as a carbon source.
Organisms capable of fermenting lactose will lower the pH and change
the color of the medium from green to yellow. Brom Thymol Blue is
used as a pH indicator. Bacto Agar is used as a solidifying agent.
Uninoculated
plate
Escherichia coli
ATCC 25922

Dehydrated Appearance: Beige with slight green tint, free-flowing,
homogeneous.
Solution:
deionized water upon boiling. Solution
is bluish-green, very slightly opalescent
without precipitate.
Prepared Medium:
Bluish-green, very slightly opalescent
Reaction of 3.6%
Solution:
pH 7.3 0.2
Cultural Response
Prepare CLED Agar per label directions. Inoculate by spread
plate technique and incubate at 35 2C for 18-24 hours.
ORGANISM
Escherichia coli
Proteus vulgaris
ATCC
INOCULUM
CFU
GROWTH
COLONY
COLOR
25922* 100-1,000
good
yellow
8427 100-1,000 good, swarming blue to
inhibited
blue-green
Staphylococcus aureus 25923* 100-1,000
good
yellow
Proteus vulgaris
ATCC 8427
ATCC 25923
102
The Difco Manual
Section II
Campylobacter Agar Base, Campylobacter Agar Kit Blaser & Campylobacter Agar Kit Skirrow
Formula
CLED Agar
Formula Per Liter
Obtain and process specimens according to the techniques and procedures

established by laboratory policy. For best results, inoculate medium
with specimen as soon as possible.

Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.128
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g
Test Procedure
For a complete discussion on collection and processing of urine cultures
refer to appropriate references.5,6,7
Results
Precautions


2. CLED Agar is basically non-selective. However, due to electrolyte
exclusion, the growth of Shigella species is usually inhibited.4
Storage
Expiration Date
Procedure
Materials Provided
CLED Agar
Glassware
Autoclave
Incubator (35C)
Waterbath (45-50)
Sterile Petri dishes (optional)
Sterile dip slides (optional)
1.
2.
3.
4.

References
1. Sandys, G. H. 1960. A new method of preventing swarming of
Proteus spp. with a description of a new medium suitable for use
in routine laboratory practice. J. Med. Lab. Technol. 17:224.
2. Mackey, J. P., and G. H. Sandys. 1965. Laboratory diagnosis of
infections of the urinary tract in general practice by means of a
dip-inoculum transport medium. Br. Med. J. 2:1286.
3. Mackey, J. P., and G. H. Sandys. 1966. Diagnosis of urinary tract
infections. Br. Med. J. 1:1173.
4. MacFaddin, J. D. 1985. Media for isolation-cultivationidentification- maintenance of medical bacteria, vol. 1. Williams
St. Louis, MO.
7. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and R.
H. Yolken (ed.). 1995. Manual of clinical microbiology, 6th ed.
Packaging
CLED Agar
500 g
10 kg
0971-17
0971-08
Bacto Campylobacter Agar Base . Bacto Campylobacter Agar

Kit Blaser . Bacto Campylobacter Agar Kit Skirrow
Intended Use
Also Known As
Bacto Campylobacter Agar Base is used with blood and Bacto

Campylobacter Antimicrobic Supplement B (Blaser), Bacto
Campylobacter Antimicrobic Supplement S (Skirrow) or other
antibiotics in isolating and cultivating Campylobacter.
Campylobacter Agar Kit Skirrow is used to prepare Campylobacter

Agar, Skirrows or Skirrows Campylobacter Agar.
The Difco Manual
Campylobacter Agar Kit Blaser is used to prepare Campylobacter Agar,

Blasers or Blasers Campylobacter Agar.
103

The genus Campylobacter was proposed in 1963 for Vibrio fetus, a
species not exhibiting true characteristics of Vibrionaceae.1 In 1977,
Skirrow succeeded in isolating C. jejuni from fecal samples. Skirrow
used a selective medium, incubated at 42C in an atmosphere of
5% oxygen, 10% carbon dioxide and 85% nitrogen. Skirrow
confirmed this organism as a major etiologic agent of human enteritis,1
an infection acquired through ingestion of water or food contaminated
with the microorganism.
The Skirrow formulation includes blood agar supplemented with
vancomycin, polymyxin B and trimethoprim for the selective isolation
of C. fetus subsp. jejuni.2 Blaser et al. further Incorporated cephalothin
and amphotericin B to improve inhibition of normal enteric flora.
In 1983, spiral-shaped organisms resembling campylobacteria were
isolated from the human stomach. The discovery sparked renewed
interest in the etiology of human type B gastritis.1 After genetic analysis,
the genus Helicobacter was created and most attention focused on
H. pylori. Specimens of gastric biopsies, brushings, or aspirates are
used for the detection of H. pylori. Chocolate agar and brain heart
Section II
infusion or brucella agar, enriched with 5 to 7% horse or rabbit blood,

will support the growth of H. pylori.1
The Skirrow formulation is recommended for clinical specimens.1
Campylobacter Agar Base is specified for food testing in Standard
Methods.3,4

Campylobacter Agar Base is a nutritionally rich medium based on
Blood Agar Base No. 2, rather than on Brucella Agar, to support more
luxuriant Campylobacter growth because Trimethoprim is more active
in Blood Agar Base No. 2. Supplementation of the base with antimicrobial agents as described by Skirrow2 and Blaser et al.5,6 provides for
markedly reduced growth of normal enteric bacteria and improved
recovery of C. fetus supsp. jejuni from fecal specimens. Growth of
fungi is markedly to completely inhibited with Campylobacter
Antimicrobic Supplement B due to the presence of amphotericin B.
Campylobacter fetus
subsp. jejuni
ATCC 33291
Uninoculated
plate

Campylobacter Agar Base
Solution:
deionized water upon boiling; medium to
dark amber, clear to slightly opalescent.
Prepared Medium:
Without blood: medium to dark amber,
very slightly to slightly opalescent
With 10% sheep blood: cherry red,
opaque.
Reaction of 3.95%
Solution at 25C:
pH 7.4 0.2
Campylobacter Antimicrobic Supplement B
Lyophilized Appearance: Bright medium yellow cake or powder.
Rehydrated Appearance: Yellow suspension.
Prepared Medium:
Blaser formulation: opaque, medium cherry red.
Campylobacter Antimicrobic Supplement S
Lyophilized Appearance: White cake or powder.
Rehydrated Appearance: Colorless, clear.
Prepared Medium:
Skirrow formulation: translucent, dark red.
Cultural Response
Prepare Campylobacter Agar Blaser or Skirrow per label directions. Inoculate and incubate at 42C for 40-48 hours.
ORGANISM
Campylobacter fetus subsp. jejuni

Candida albicans
Escherichia coli
ATCC
CFU
GROWTH
APPEARANCE
33291
10231
33186
25922*
100-1,000
2,000-10,000
2,000-10,000
2,000-10,000
good
marked to complete inhibition
non-hemolytic, mucoid, gray colonies
This organism is tested on Campylobacter Agar Blaser, only.

*This culture is available as Bactrol Disks and should be used as directed in Bactrol Disks Technical Information.
104
The Difco Manual
Section II
Formula
Do not use a product if it fails to meet specifications for identity and

performance.

Formula Per Liter
Liver Digest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Ingredients per vial
10 ml vial
Vancomycin . . . . . . . . . . . . . . . . . . . . . . . 10
Polymyxin B . . . . . . . . . . . . . . . . . . . . 2,500
Trimethoprim . . . . . . . . . . . . . . . . . . . . . . 5
Cephalothin . . . . . . . . . . . . . . . . . . . . . . . 15
Amphotericin B . . . . . . . . . . . . . . . . . . . . 3
mg
units
mg
mg
mg

Ingredients per vial
10 ml vial
Vancomycin . . . . . . . . . . . . . . . . . . . . . . . 10 mg
Polymyxin B . . . . . . . . . . . . . . . . . . . . 2,500 units
Trimethoprim . . . . . . . . . . . . . . . . . . . . . . 5 mg
g
g
g
g
g
Materials Provided

5 ml vial
5
1,250
2.5
7.5
1
mg
units
mg
mg
mg
5 ml vial
5 mg
1,250 units
2.5 mg
Precautions
2. Campylobacter Antimicrobic Supplement B
AND SKIN. MAY CAUSE SENSITIZATION BY INHALATION
AND SKIN CONTACT. POSSIBLE RISK OF IRREVERSIBLE
EFFECTS. MAY CAUSE HARM TO THE UNBORN CHILD.
Avoid contact with skin and eyes. Do not breathe dust. Wear suitable
protective clothing. Keep container tightly closed. Target Organs:
Blood, Kidneys, Ears, Bone Marrow.
AND SKIN. MAY CAUSE SENSITIZATION BY INHALATION
AND SKIN CONTACT. MAY CAUSE HARM TO THE UNBORN
CHILD. Avoid contact with skin and eyes. Do not breathe dust.
Target Organs: Kidneys, Ears, Bone Marrow.
Storage
Store lyophilized and rehydrated Campylobacter Antimicrobic Supplements B and S at 2-8C. Use the rehydrated supplement within 24 hours.
Expiration Date
stored as directed.
The Difco Manual
Procedure
Specimen collection containers or sterile rectal swabs

Microaerophilic environment system
Bunsen burner or incinerator
Sterile defibrinated blood or sterile lysed horse blood
Inoculating loops
Incubator (42C)
Campylobacter Agar Base:
1. Suspend 39.5 grams of Campylobacter Agar Base in 1 liter of
distilled or deionized water.
4. Cool to 45-50C. Aseptically add 5-7% sterile lysed horse blood
(final concentration) or 10% sterile defibrinated sheep blood
(final concentration).
5. Aseptically add 1% rehydrated Campylobacter Antimicrobic
Supplement B or Campylobacter Antimicrobic Supplement S
(10 ml per liter or 5 ml per 500 ml of basal medium). Mix well.
6. Dispense 20 ml amounts into 90 mm Petri dishes.
1. Aseptically rehydrate the lyophilized supplement with 5 or 10 ml
of sterile distilled or deionized water, depending on label directions.
2. Invert the vial gently several times to dissolve the powder. Use
within 24 hours of rehydration.

Fecal specimens should be collected in sterile containers or with a
sterile rectal swab and transported immediately to the laboratory for
processing. If the specimen cannot be inoculated onto appropriate
media within four hours after collection, the specimen should be
maintained or transported in Cary-Blair Transport Medium.1
Test Procedure
1. Inoculate the specimen directly onto the surface of the prepared
Campylobacter Agar plate and streak for isolation.
2. Incubate at 42C under a microaerophilic atmosphere containing
5-6% oxygen and 3-10% carbon dioxide. Consult appropriate
references for specific information on establishing a microaerophilic
environment.1,3,7
Results
The colonies of Campylobacter species appear as non-hemolytic, flat
and gray with an irregular edge or raised and round with a mucoid
appearance. Some strains may appear tan or slightly pink. Swarming
105
Candida BCG Agar Base
or spreading may be observed on moist surfaces. Growth of normal

enteric bacteria is markedly to completely inhibited. Growth of fungi
is markedly to completely inhibited on Campylobacter Agar Blaser.
Colonies are selected for further biochemical characterization.
Identification is based on a positive oxidase reaction and characteristic
darting motility in a wet mount.1 For further differentiation into
species and biotypes, test for catalase activity, urease, hydrogen
sulfide production, nitrate reduction, hippurate, indoxyl acetate, DNA
hydrolysis and susceptibility to cephalothin and nalidixic acid.1

1. Campylobacter Agar prepared with either Campylobacter Antimicrobic Supplement S or Campylobacter Antimicrobic Supplement
B is selective primarily for Campylobacter species. Biochemical
testing using a pure culture is necessary for complete identification.
Consult appropriate references for further information.1,3,7
2. Growth of Campylobacter fetus subsp. intestinalis may be
dramatically inhibited on Campylobacter Agar Blaser due to the
presence of cephalothin. The use of Campylobacter Agar Skirrow
and incubation at 35C is suggested when isolating this organism
from mixed populations.
3. Some strains of C. fetus subsp. jejuni may be encountered that fail
to grow or grow poorly on prepared Campylobacter Agar.
4. Some strains of normal enteric organisms may be encountered that
are not inhibited or only partially inhibited on Campylobacter Agar.
References
R. H. Yolken (ed.). Manual of clinical microbiology, 6th ed.
2. Skirrow, M. D. 1977. Campylobacter enteritis: A new disease.
Br. Med. J. 2:9-11.
3. Vanderzant, C., and D. F. Splittstoesser (ed). 1992. Compendium of methods for the microbiological examination of food,
Bacto Candida BCG Agar Base
Section II

5. Blaser, M. J., V. Berkowitz, F. M. LaForce, J. Cravens, L. B.
Reller, and W. L. Wang. 1979. Campylobacter enteritis: clinical
and epidemiologic features. Ann. Intern. Med. 91:179-185.
6. Blaser, M. J., J. Cravens, B. W. Powers, and W. L. Wang. 1978.
Campylobacter enteritis associated with canine infection. Lancet
(ii):979-980.
7. Koneman E. W., S. D. Allen, W. M. Janda, P. C.
Schreckenberger, W. C. Winn. 1983. Color atlas and textbook
of diagnostic microbiology, 5th ed. J. B. Lippencott-Raven
Publishers. Washington, D.C.
Packaging
Campylobacter Agar Kit Blaser
To prepare: 6 x 1 liter
Campylobacter
Antimicrobic Supplement B
Campylobacter Agar Kit Blaser
To prepare: 6 x 500 ml
Campylobacter
Antimicrobic Supplement B
Campylobacter Agar Kit Skirrow
To prepare: 6 x 1 liter
Campylobacter
Antimicrobic Supplement S
To prepare: 6 x 500 ml
Campylobacter
Antimicrobic Supplement S
2 kg
1820-07
3279-32
6 x 39.5 grams
6 x 10 ml
3279-40
6 x 19.75 grams
6 x 5 ml
3280-32
6 x 39.5 grams
6 x 10 ml
3280-40
6 x 19.75 grams
6 x 5 ml
Intended Use
Bacto Candida BCG Agar Base is used with added neomycin in isolating
and differentiating Candida from primary specimens.
Also Known As
Candida BCG Agar Base is an abbreviation for Candida Brom Cresol
Green Agar Base.

Candida BCG Agar Base is prepared according to the formulation of
Harold and Snyder.1 Candida BCG Agar Base was developed after a
study demonstrated triphenyltetrazolium chloride (TTC) employed in
Pagano Levin medium retarded the growth of some Candida species.
Harold and Snyder1 used brom cresol green as the indicator, which is
nontoxic to Candida species. This medium is primarily used for
106
demonstrating morphological and biochemical reactions characterizing

the different Candida species for clinical diagnosis.
Candidiasis is the most frequently encountered opportunistic fungal
infection. 2 It is caused by a variety of species of Candida, with
Candida albicans being the most frequent etiological agent, followed
by Candida tropicalis and Candida (Torulopsis) glabrata.2 Candida
species can be present in clinical specimens as a result of environmental
contamination, colonization, or actual disease process.3
Principle of the Procedure

Bacto Peptone provides the nitrogen and amino acids in Candida BCG
Agar Base. Yeast Extract is the vitamin source. The high concentration
of Dextrose provides carbon as an energy source in this formula. Bacto
Agar is the solidifying agent. Brom cresol green is the pH indicator,
and acid production changes the medium from blue-green to yellow.
Due to pH changes, specific color patterns appear in the base and
surface of colonies for differentiation of Candida species.
The Difco Manual
Section II
Neomycin is added to the medium in a concentration of 500 g/ml.

Neomycin and brom cresol green act as selective agents to inhibit
bacteria in Candida BCG Agar Base.4
Formula
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Brom Cresol Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.02
g
g
g
g
g

Glassware
Autoclave
Incubator (30C)
Waterbath (45-55C)
Neomycin (500 g/ml)
1.
2.
3.
4.
Precautions

Cool the medium to 50-55C. Add sterile neomycin (500 g/ml).
Mix well.

Storage
Test Procedure

Refer to the scheme for yeast identification.3 For a complete discussion

on the isolation and identification of Candida species refer to the
procedures described in the appropriate references.2,3,5
Expiration Date
Procedure
Materials Provided

established by laboratory policy.
Results
Identification of Candida species on the basis of colony morphology
on Candida BCG Agar follows:
C. albicans: Colonies appear as blunt cones 4.5-5.5 mm diameter with
smooth edges and surfaces; coarse feathery growths may arise from
the center of the colony base to penetrate the medium. The color of
Uninoculated
plate
Candida albicans
ATCC 10231

Dehydrated Appearance: Beige to blue-green, free-flowing,
homogeneous.
Solution:
deionized water on boiling, blue-green,
slightly opalescent to opalescent,
may have a precipitate.
Prepared Medium:
Blue-green to greenish blue,
slightly opalescent to opalescent;
Reaction of 6.6%
Solution at 25C:
pH 6.1 0.1
Cultural Response
Prepare Candida BCG Agar Base per label directions. Inoculate medium
using the streak plate technique, and incubate at 30 2C for 24-48 hours.
ORGANISM
Candida albicans
Candida tropicalis
Escherichia coli
ATCC
INOCULUM
CFU
GROWTH
COLOR OF
MEDIUM
10231
3869
25922*
100-1,000
100-1,000
1,000-2,000
good
good
inhibited
yellow
yellow
green
Candida tropicalis
ATCC 3869
The Difco Manual
107
Candida Isolation Agar
the base and surface of the colonies is yellowish to bluish green

with the intensity diminishing from a gray green center spot to
paleness at the edge, although some strains may show a distinct
green outer ring.
C. stellatoidea: Colonies appear convex 4.0-5.0 mm in diameter, with
smooth edges and smooth to irregular surfaces; there is a fine central
basal feathery growth penetrating the medium. The color of both
base and surface of colonies is yellow to green, the intensity of which
may or may not diminish from center to border but is usually light.
C. tropicalis: Colonies appear convex or as low cones 4.5-5.0 mm in
diameter with smooth to undulate edges, and smooth to granular or
ridged surfaces; deeply stained feathery growth arises from several
points in the base of the colony to form an effusive cloud. The
color of the submerged growth is normally an intense blue green
compared with that of the base which is much lighter; the surface
is uniformly pale and may be yellowish green to green, reflecting a
lower pH than observed of the base.
C. pseudotropicalis: Colonies appear convex, 4.5-5.5 mm in diameter
with undulate to smooth edges, and smooth surfaces; occasionally
the surface is membranous but all colonies are shiny in appearance,
and there is feathering growth emerging from several points in the
base of the colony. The color of a large central area in the base of the
colony is a medium green, which diminishes in intensity toward the
edge; a similar distribution of color occurs on the surface, but this
green is bright in hue and is never grayed as it is with C. tropicalis.
C. krusei: Colonies appear as low cones 4.5-5.0 m in diameter with
pseudohyphal edges, which may be weakly contractile or spreading,
and have dull surfaces. There is abundant lightly colored growth
penetrating the medium from the base of the colony. The base of
the colony is a medium blue green in the center diminishing in
intensity to paleness at the edge; the surface is usually a light green
to yellow green without much concentration in any part.
C. parapsilosis: Colonies appear as convex to low cones 3.5-4.5 mm
in diameter with smooth or slightly spreading edges, but vary from
smooth to granular or rough surfaces; there is no submerged
growth. The color for both base and surface of the colony is blue
Bacto Candida Isolation Agar
Section II
green over much of the colony, being more intense in the base than
the surface which is modified by a thin grayish film of cells; the
intensity in color fades abruptly leaving a broad pale edge.
C. guilliermondii: Colonies appear as low cones 4.0-5.0 mm in
diameter with very smooth edges and highly glossy surfaces; there
maybe a weak, fine feathered submerged growth. Both base and
surface of the colony tend to have blue centers of medium intensity
fading into a pale edge; however the surface may be blue green
with the central third lightened with gray.
C. glabrata: Colonies are smooth and convex, 4.6-5.0 mm diameter;
the surface color pattern is pale green in the center which becomes
medium green at the edge, and the base has the same color pattern
but of less intensity.

1. Since the nutritional requirements of yeast vary, some strains may
References
1. Harold, W., and M. Snyder. 1968. Personal Communication.
St. Louis, MO.
and other yeasts of medical importance, p. 723-737. In P. R.
Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken
(ed.), Manual of clinical microbiology, 6th ed. American Society
Packaging
500 g
0835-17
Intended Use
Bacto Candida Isolation Agar is used for isolating and differentiating
Candida albicans.

Candida Isolation Agar is a nutritionally rich medium that supports
growth of many yeasts and molds and is differential for Candida
albicans. Candida Isolation Agar was developed using a modification
of YM Agar as described by Fung and Liang.1 Goldschmidt demonstrated
that YM Agar with Aniline Blue WS could be used to identify
C. albicans in clinical samples with high accuracy and predictability.2
Aniline Blue is metabolized by C. albicans to produce a fluorescent
moiety that can be detected under long wave UV light.2

Yeast Extract provides nitrogen, carbon, vitamins and cofactors. Malt
Extract provides carbon, protein and nutrients. Bacto Peptone provides
108
additional carbon and nitrogen. Dextrose is an energy source. Aniline

Blue is a fluorescent indicator. Bacto Agar is a solidifying agent.
Formula
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Aniline Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
g
g
g
g
g
g
Precautions
The Difco Manual
Section II
Storage
Store dehydrated medium below 30C. The dehydrated medium is very
Expiration Date
Procedure
Materials Provided
light following incubation at 30C for 18-24 hours. Non-C. albicans

isolates do not fluoresce.

1. Strains of Candida albicans have been reported that are false
negative for fluorescence on this medium.2
2. Strains of C. parapsilosis, C. krusei, and C. pulcherrima that fluoresce
on this medium may be encountered.2 These strains may be distinguished from C. albicans based on germ tube formation in serum.2,5
References

Glassware
Autoclave

1. Specimens should be collected in sterile containers or with sterile
swabs and transported immediately to the laboratory according to
recommended guidelines.3,4
Test Procedure
1. Process each specimen as appropriate for that specimen and inoculate directly onto the surface of the medium. Streak for isolation.
2. Incubate plates aerobically at 30C for 18-72 hours.
3. Examine plates for growth after 18-72 hours of incubation.
1. Fung, D. Y. C., and C. Liang. 1988. A new fluorescent agar for

the isolation of Candida albicans. Bull. Inf. Lab. Serv. Vet. (France)
29/30:1-2.
2. Goldschmidt, M. C., D. Y. C. Fung, R. Grant, J. White, and T.
Brown. 1991. New aniline blue dye medium for rapid identification
and isolation of Candida albicans. J. Clin. Micro. 29:1095-1099.
3. Miller, J. M., and H. T. Holmes. 1995. Specimen collection and
handling, p. 19- 32. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C.
Tenover, and R. H. Yolken, (ed.), Manual of clinical microbiology,
4. Splittstoesser, D. F., and C. Vanderzant (ed.). 1992. Compendium of methods for the microbiological examination of foods,
5. Murray, P. R, E. J. Baron, M. A. Pfaller, F. C. Tenover, and
American Society for microbiology, Washington, D.C.
Packaging
Results
Colonies of C. albicans fluoresce yellow-green under long wave UV
500 g
0507-17
Candida albicans
ATCC 10231

Dehydrated
Medium Appearance: Beige, free-flowing, homogeneous.
Solution:
water on boiling. Solution is medium blue,
very slightly opalescent.
Prepared Plates:
Medium blue, slightly opalescent.
Reaction of 4.1%
Solution at 25C:
pH 6.2 0.2
Cultural Response
Prepare Candida Isolation Agar per label instructions. Inoculate
and incubate plates aerobically at 30 2C for 18-72 hours.
ORGANISM
Bacillus subtilis
Candida albicans
Escherichia coli
ATCC
INOCULUM
CFU
GROWTH
FLUORESCENCE
6633
10231
25922*
100-1,000
100-1,000
100-1,000
good
good
good
negative
positive
negative
The Difco Manual
109
Casamino Acids, Casamino Acids, Technical & Vitamin Assay Casamino Acids
Section II
Bacto Casamino Acids . Bacto Casamino Acids, Technical

Bacto Vitamin Assay Casamino Acids
Intended Use

Casamino Acids
Dehydrated Appearance: Very light beige, free-flowing,
homogeneous.
Solution:
1% solution-very light amber, clear
solution.
2% solution-Light amber, clear,
upon slight heating.
Reaction of a 2%
Solution at 25C:
pH 5.8-6.65
homogeneous.
Solution:
1% solution, soluble in distilled or
deionized water. Solution is colorless
to very light amber and clear.
Reaction of 1%
Solution at 25C:
pH 5.0-7.5
Vitamin Assay Casamino Acids
Solution:
deionized water on boiling. Very
light to light amber, clear, may have
a slight precipitate.
Reaction of 3%
Solution at 25C:
pH 6.5-8.5
Cultural Response
Casamino Acids and Casamino Acids, Technical
Prepare a 1% solution and adjust the pH to 7.2 0.2. Inoculate
tubes with the test organisms, and incubate at 35 2C for
18-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Escherichia coli
Salmonella typhi
25922*
19430
100-1,000
100-1,000
good
good


Vitamin Assay Casamino Acids is prepared in various vitamin
assay media to determine the vitamin content. It should not
contain a vitamin content higher than 20% above the
following values:
Vitamin B12
0.2 nanograms/gram
Biotin
0.3 nanograms/gram
Folic Acid
3.3 nanograms/gram
Niacin
0.17 micrograms/gram
Pantothenate
0.04 micrograms/gram
Riboflavin
0.1 micrograms/gram
Thiamine
0.1 micrograms/gram
110
Bacto Casamino Acids is used in preparing microbiological

culture media.
Bacto Casamino Acids, Technical is used in the preparation of
Bacto Vitamin Assay Casamino Acids is used in vitamin assay
procedures.
Also Known As
Casamino Acids are also referred to as Casein Hydrolysate (Acid) or
Casein Peptone, Acid Hydrolysate.

Casamino Acids is acid hydrolyzed casein having low sodium chloride
and iron concentrations. Casamino Acids is recommended for use in
microbiological culture media that require a completely hydrolyzed
protein as a nitrogen source. Casamino Acids is prepared according to
the method described by Mueller and Miller1 and Mueller and Johnson.2
Mueller3 prepared diphtheria toxin in a medium containing a casein
hydrolysate as the source of nitrogen. It was shown that the high
sodium chloride content was the limiting factor in the amount of toxin
that could be produced in this medium. Mueller and Miller1 described
a method to reduce the sodium chloride and iron content of the
hydrolyzed casein. This hydrolyzed casein, supplemented with
inorganic salts, growth factors, cystine, maltose and an optimum
amount of iron, was used to prepare diphtheria toxin.1,3 Casamino
Acids duplicates this specially treated hydrolyzed casein.
In Casamino Acids, hydrolysis is carried out until all the nitrogen in
the casein is converted to amino acids or other compounds of relative
chemical simplicity. Casamino Acids is particularly well suited for
nutritional studies, microbiological assays, and in the semi-synthetic
medium for testing disinfectants.4 Casamino Acids is also used in the
preparation of tetanus toxins, and pertussis vaccines, and for sulfonamide
inhibitor studies.5
Casamino Acids, Technical is acid hydrolyzed casein. The hydrolysis
is carried out as in the preparation of Casamino Acids, but the sodium
chloride and iron content of this product have not been decreased to
the same extent. Casamino Acids, Technical is recommended for use
in culture media where amino acid mixtures are required for a nitrogen
source, and the sodium chloride content is slightly increased. It is
particularly valuable in studying the growth requirements of bacteria.
Casamino Acids, Technical is prepared according to the method
suggested by Mueller1 for use in the preparation of diphtheria toxin.
Mueller and Hinton6 used Casamino Acids, Technical in a medium for
primary isolation of gonococcus and meningococcus. Casamino Acids,
Technical was used in agar-free media for the isolation of Neisseria,
and in a tellurite medium for the isolation of Corynebacterium,
described by Levin.7 Wolf 8 used Casamino Acids, Technical in the
preparation of a medium for the testing of disinfectants.
Vitamin Assay Casamino Acids is an acid digest of casein specially
treated to markedly reduce or eliminate certain vitamins. It is
The Difco Manual
Section II
Casamino Acids, Casamino Acids, Technical & Vitamin Assay Casamino Acids
recommended for use in microbiological assay media and in studies of

the growth requirements of microorganisms. Vitamin Assay Casamino
Acids is commonly used as the amino acid source in early phases of
nutrition work.9 Sarett10 used Vitamin Assay Casamino Acids as the
acid hydrolyzed casein in his studies on p-aminobenzoic acid and
p-teroylglutamic acid as growth factors for Lactobacillus species.
Several media containing Casamino Acids are specified in standard
methods for multiple applications.11,12,13

Casamino Acids, Casamino Acids, Technical and Vitamin Assay
Casamino Acids are acid hydrolyzed casein. Casein is milk protein,
and a rich source of amino acid nitrogen. Casamino Acids, Casamino
Acids, Technical and Vitamin Assay Casamino Acids provide nitrogen,
vitamins, carbon and amino acids in microbiological culture media.
Although Casamino Acids, Casamino Acids, Technical, and Vitamin
Assay Casamino Acids are added to media primarily because of their
organic nitrogen and growth factor components, their inorganic
components also play a vital role.14
Formula
Casamino Acids is a dehydrated acid hydrolyzed casein in which
Sodium Chloride and Iron are present in low concentrations permitting
toxin production.
Casamino Acids, Technical is a dehydrated acid hydrolyzed casein.
The Sodium Chloride and Iron content have not been reduced to same
extent as Casamino Acids.
Vitamin Assay Casamino Acids is an acid hydrolyzed casein used to
prepare media for microbiological assay of vitamins.
Typical Analysis
Ash (%)
Loss on Drying (%)

pH, 1% Soln
4.5
6.4
10.5
8.8
AN/TN
83.8
3.26
2.20
4.76
0.16
15.30
1.31
1.66
3.34
5.47
Lysine
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine
5.71
1.28
2.11
6.17
2.19
2.41
<0.01
0.47
4.30
<0.001
7.400
<0.001
<0.001
<0.001
<0.001
0.002
<0.001
Phosphate
Potassium
Sodium
Sulfate
Sulfur
Tin
Zinc
3.325
0.410
8.710
0.045
0.420
<0.001
<0.001
The Difco Manual
<5.0
<0.1
<0.1
1.8
1.2
<30.0

Coliform
Salmonella
Spore Count
negative
negative
390

Thermophile Count
950
25
Precautions
Storage
Store the dehydrated product below 30C. The dehydrated product is
Expiration Date
Procedure
Materials Provided
Casamino Acids
Refer to the final concentration of Casamino Acids, Casamino Acids,
Technical or Vitamin Assay Casamino Acids in the formula of the
medium being prepared. Add Casamino Acids, Casamino Acids,
Technical or Vitamin Assay Casamino Acids as required.

Test Procedure
See appropriate references for specific procedures using Casamino
Acids, Casamino Acids, Technical or Vitamin Assay Casamino Acids.
Results
Inorganics (%)
Calcium
Chloride
Cobalt
Copper
Iron
Lead
Magnesium
Manganese
PABA
Pantothenic Acid
Pyridoxine
Riboflavin
Thiamine
Thymidine
Amino Acids (%)

Alanine
Arginine
Aspartic Acid
Cystine
Glutamic Acid
Glycine
Histidine
Isoleucine
Leucine
Biotin
<0.1
Choline (as Choline Chloride) 160.0
Cyanocobalamin
<0.1
Folic Acid
<0.1
Inositol
<100.0
Nicotinic Acid
<20.0

24.4
0.5
2.9

Total Nitrogen
Amino Nitrogen
Vitamins (g/g)
References
1. Mueller and Miller. 1941. J. Immunol. 40:21.
2. Mueller and Johnson. 1941. J. Immunol. 40:33.
3. Mueller. 1939. J. Immunol. 37:103.
111
Casein Digest
Section II
4.
5.
6.
7.
8.
9.
Klarman and Wright. 1945. Soap and San. Chem. 21:113.

Straus, Dingle and Finland. 1941. J. Immunol. 42:331.
Mueller and Hinton. 1941. Proc. Soc. Exp. Biol. Med. 48:330.
Levin. 1943. J. Bacteriol. 46:233.
Wolf. 1945. J. Bacteriol. 49:463.
Nolan, R. A. 1971. Amino acids and growth factors in vitamin-free
casamino acids. Mycol. 63:1231-1234.
10. Sarett. 1947. J. Biol. Chem. 171:265.
of methods for the microbiological examination of food, 3rd. ed.
Bacto Casein Digest
Packaging
Casamino Acids
100
500
2
10
g
g
kg
kg
0230-15
0230-17
0230-07
0230-08
500 g
10 kg
0231-17
0231-08
100 g
500 g
0288-15
0288-17
Intended Use
Bacto Casein Digest is used in preparing microbiological culture media.
Also Known As
Casein Digest is similar to N-Z-Amine A.

Solution:
1%, 2%, and 10% solutions, soluble
in distilled or deionized water:
1%-Light amber, clear;
2%-Medium amber, clear;
10%-Dark amber, clear, no significant
precipitate.
Reaction of 1%
Solution at 25C:
pH 7.2 0.2
Cultural Response
Prepare NZM Broth per formula. Inoculate and incubate at
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Bacillus subtilis
Escherichia coli (DH5)
Streptomyces avermitilis
6633
33694
47014
53868
9763
31267
100-1,000
100-1,000
100-1,000
100-1,000
100-1,000
100-1,000
good
good
good
good
good
good

14. Nolan, R. A., and W. G. Nolan. 1972. Elemental analysis of
vitamin-free casamino acids. Appl. Microbiol. 24:290-291.
Bacillus subtilis is available as Subtilis Spore Suspension.
Casein Digest, an enzymatic digest of casein, was developed for use in

molecular genetics media. This product is digested under conditions
different from other enzymatic digests of casein, including Tryptone
and Casitone.
Casein Digest is contained in the formulas of NZ media (NZCYM
Broth, NZYM Broth and NZM Broth), which are used for cultivating
recombinant strains of Escherichia coli. E. coli grows rapidly in these
rich media because they provide amino acids, nucleotide precursors,
vitamins and other metabolites that the cells would otherwise have to
synthesize.1 Consult appropriate references for recommended test
procedures using NZ media.1,2

Casein Digest is a nitrogen and amino acid source for microbiological
culture media. Casein is raw milk protein, a rich source of amino acid
nitrogen.
Precautions
Storage
Store Casein Digest below 30C. The product is very hygroscopic.
Expiration Date
Procedure
Materials Provided
Casein Digest

112
The Difco Manual
Section II
Casitone
References
Refer to the final concentration of Casein Digest in the formula of the

medium being prepared. Add Casein Digest as required.

Seidman, J. A. Smith, and K. Struhl (ed.). 1994. Current
protocols in molecular biology, vol.1. Current Protocols,
New York, NY.
cloning: a laboratory manual, 2nd ed. Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY.

Obtain and process specimens according to the techniques and procedures established by laboratory policy.
Test Procedure
See appropriate references for specific procedures using Casein Digest.
Packaging
Results
Casein Digest
500 g
0116-17
Bacto Casitone
Intended Use
Bacto Casitone is used in preparing microbiological culture media.

Dehydrated Appearance: Tan, free-flowing granules.
Solution:
1%, 2% and 10% solutions are
soluble in distilled or deionized water.
1%-Light amber, clear, no precipitate.
2%-Light to medium amber, clear,
10%-Medium to dark amber, clear
to very slightly opalescent, may
have a precipitate.
Reaction of 1%
Solution at 25C:
pH 6.8 - 7.4
Cultural Response
All solutions are prepared with the pH adjusted to 7.2 - 7.4.
TEST
SOLUTION
Fermentable
2%
Carbohydrates
Indole
0.1%
Production
Acetylmethylcarbinol 0.1%
Production
Hydrogen Sulfide
1%
Production
Toxicity
2%w/0.5%
NaCl &
1.5% Agar
Toxicity
2%w/0.5%
NaCl &
1.5% Agar
ORGANISM
Escherichia
coli
Escherichia
coli
Enterobacter
aerogenes
Salmonella
typhi
Escherichia
coli
ATCC
RESULT
INOCULUM
25922* negative
25922* positive
13048* positive
6539 positive
25922*
good 100-1,000
growth
Staphylococcus 25923* good 100-1,000

aureus
growth
Casitone, is recommended for preparing media where an enzymatic

hydrolyzed casein is desired. Casitone is used to support the growth of
fastidious microorganisms. The high tryptophan content of Casitone
makes it valuable for use in detecting indole production.
Dubos Broth and Dubos Oleic Agar media that support the growth of
Mycobacterium tuberculosis contain Casitone. Media used for the
enumeration of coliforms in water, m Endo Agar and m Endo Broth
MF, use Casitone as a nitrogen source. Several Thioglycollate media
used for detecting microorganisms in normally sterile materials,
include Casitone as a nitrogen and amino acid source.
Casitone is recommended for preparing media for sterility testing
according to US Pharmacopeia XXIII (USP).1 Several media containing
Casitone are specified in standard methods2,3,4,5 for multiple applications.

Casitone is a pancreatic digest of casein. Casein is the main protein of
milk, and a rich source of amino acid nitrogen.
Precautions
Storage
Expiration Date
Typical Analysis
Ash (%)
Loss on Drying (%)

pH, 1% Soln
3.7
7.2
Carbohydrate (%)
Total
The Difco Manual
7.0
0.6
1.7
0.2
113
Casman Medium Base
Section II
Total Nitrogen
Amino Nitrogen
13.3
4.7
AN/TN
35.3
Amino Acids (%)

Alanine
Arginine
Aspartic Acid
Cystine
Glutamic Acid
Glycine
Histidine
Isoleucine
Leucine
3.01
3.76
6.61
0.02
20.03
1.97
2.17
4.16
8.74
Lysine
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine
13.62
1.71
4.02
8.57
4.82
3.74
0.14
2.09
4.06
0.010
0.110
<0.001
<0.001
0.003
<0.001
0.019
<0.001
Phosphate
Potassium
Sodium
Sulfate
Sulfur
Tin
Zinc
2.604
0.162
3.073
0.339
0.676
<0.001
0.004
Inorganics (%)
Calcium
Chloride
Cobalt
Copper
Iron
Lead
Magnesium
Manganese
Vitamins (g/g)
Biotin
0.2
Cyanocobalamin
<0.1
Folic Acid
0.8
Inositol
980.0
Nicotinic Acid
20.3
PABA
Pantothenic Acid
Pyridoxine
Riboflavin
Thiamine
Thymidine
15.9
7.7
1.3
0.4
<0.1
342.9

Coliform
Salmonella
Spore Count
negative
negative
300

Thermophile Count
1850
100
Procedure
Materials Provided
Refer to the final concentration of Casitone in the formula of the

medium being prepared. Add Casitone as required.

Test Procedure
See appropriate references for specific procedures using Casitone.
Results
References
States Pharmacopeia, 23rd ed. Sterility test, p. 1686-1690. The
5. Marshall, R. T. (ed.). 1993. Standard methods for the examination
of dairy products, 16th ed., American Public Health Association,
Washington, D.C.
Packaging
Casitone
Casitone
100 g
500 g
10 kg
0259-15
0259-17
0259-08
Bacto Casman Medium Base
Intended Use
Bacto Casman Medium Base is used with blood in isolating fastidious
microorganisms under reduced oxygen tension.

In 1947, Casman1,2,3 described an infusion-free medium enriched with
5% blood for fastidious microorganisms incubated anaerobically. This
medium replaced labor intensive formulas containing fresh meat
infusion and unheated and heated blood.1 Casman adjusted the
medium after experiments revealed that nicotinamide disrupted the
action of a blood enzyme that inactivates V factor (NAD).2 Using
unheated human blood in the formula, Haemophilus influenzae grew
well and Neisseria was inhibited. The concentration of nicotinamide
was lowered to support growth of Neisseria species.2,3
114
Casman Agar Base with rabbit blood can be used for the cultivation
and maintenance of Gardnerella vaginalis.4

Proteose Peptone No.3, Tryptose and Beef Extract provide nitrogen,
vitamins and amino acids. Nicotinamide enhances growth of
N. gonorrhoeae and H. influenzae by impeding the removal of
coenzyme (V factor) by nucleotidase from the enriched blood. The
small amount of Dextrose is added to enhance growth of pathogenic
cocci. Sodium chloride maintains the osmotic balance of the medium.
Para-aminobenzoic acid is a preservative. Corn starch is added to
ensure that any toxic metabolites produced are absorbed, to neutralize
glucose inhibition of beta-hemolysis 4 and to enhance growth of
Neisseria species. Agar Noble is a solidifying agent.
The Difco Manual
Section II
Casman Medium Base
Formula
Procedure
Casman Medium Base

Formula Per Liter
Materials Provided
Casman Medium Base

Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Nicotinamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Corn Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Agar Noble . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
g
g
g
g
g
g
g
g
g

Glassware
Autoclave
Incubator (35C)
Sterile water-lysed blood
Precautions
Storage
Expiration Date
1.
2.
3.
4.

Add 5% sterile blood and 0.15% sterile water-lysed blood solution
(one part blood to three parts water). Omit water-lysed blood if
sterile blood is partially lysed.
5. Dispense into sterile Petri dishes or as desired.

Uninoculated plate
with enrichment
Neisseria gonorrhoeae
CDC 116
with enrichment

Solution:
deionized water with frequent agitation
on boiling. Light to medium amber
with a ground glass appearance.
Prepared Medium:
Without blood, light to medium
amber with a ground glass appearance.
With 5% blood, cherry red opaque.
Reaction of 4.3%
Solution at 25C:
pH 7.3 0.2
Cultural Response
Prepare Casman Medium Base per label directions, enrich
with 5% sterile blood and 0.15% sterile water-lysed blood
solution. Inoculateprepared medium and incubate at 35 2C
under increased CO2 for 18-48 hours.
ORGANISM
Haemophilus influenzae
ATCC
10211
CDC 116
6305
19615*
INOCULUM
CFU
GROWTH
w/BLOOD
100-1,000
100-1,000
100-1,000
100-1,000
good
good
good
good
The Difco Manual
115
Cetrimide Agar Base
Section II
Test Procedure
of fastidious microorganisms, refer to the procedures described in
appropriate references.5,6
Results
using pure cultures are recommended for complete identification.

Consult appropriate references for further information.5,6
6. Improper specimen collection, environment, temperature, CO2
level, moisture and pH can adversely affect the growth and
viability of the organism.
References

2. Nicotinamide in concentrations greater than 0.005% inhibits growth
of some N. gonorrhoeae strains; however, only slight stimulation
of growth of H. influenzae occurs with this amount.1
3. Hemolytic reactions of some strains have been shown to be
affected by differences in animal blood.6
reactions of beta-hemolytic streptococci.6
5. Casman Medium Base is intended for use with supplementation.
Although certain diagnostic tests may be performed directly on this
medium, biochemical and, if indicated, immunological testing
1. Casman, E. P. 1947. A noninfusion blood agar base for Neisseriae,

Pneumococci and Streptococci. Am. J. Clin. Pathol. 27:281.
2. Casman, E. P. 1942. J. Bacteriol. 43:33.
3. Casman, E. P. 1947. J. Bacteriol. 53:561.
4. MacFaddin, J. D. 1985. Media for isolation-cultivationidentification-maintenance medical bacteria, vol. 1, p. 141-143.
6. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures handbook, vol.1. American Society for Microbiology. Washington, D.C.
Packaging
Casman Medium Base
Bacto Cetrimide Agar Base
500 g
0290-17
Also Known As
Cetrimide Agar Base is also referred to as Pseudosel Agar,
Pseudomonas Selective Agar Base or Pseudomonas Selective Medium.
Intended Use
Bacto Cetrimide Agar Base is used for isolating and cultivating
Pseudomonas aeruginosa.
Uninoculated
plate
ATCC 27853

Solution:
4.53% solution with 1% glycerol,
soluble on boiling in distilled or
deionized water. Light amber,
opalescent, with precipitate.
Prepared Medium:
Light amber, opalescent, with
precipitate.
Reaction of 4.53%
Solution at 25C:
pH 7.2 0.2
Cultural Response
Prepare Cetrimide Agar Base per label directions with 1% glycerol.
Inoculate prepared medium and incubate at 35 2C for 18-48 hours.
ORGANISM
ATCC
Pseudomonas
27853*
aeruginosa
Escherichia coli
25922*
Staphylococcus aureus 25923*
INOCULUM
CFU(approx.)
COLONY
GROWTH
1,000
good
1,000-2,000
1,000-2,000
COLOR
yellow-green
to blue
inhibited
inhibited
116
The Difco Manual
Section II
Cetrimide Agar Base
Procedure
Pseudomonas aeruginosa has one of the broadest ranges of infectivity

among pathogens, and is the most frequently isolated nonfermentative
bacillus in clinical specimens.3 It is a significant cause of burn and
nosocomial infections.4 The ability of P. aeruginosa strains to destroy
tissue may be related to the production of various extracellular
enzymes.3 In addition, virulent strains produce an exotoxin A, which
inhibits protein synthesis.3
Materials Provided
Pseudomonas aeruginosa produces a number of water-soluble

pigments, including the yellow-green or yellow-brown fluorescent
pigment pyoverdin. 4 When pyoverdin combines with the blue
water-soluble pigment pyocyanin, the bright green color characteristic
of P. aeruginosa is created.4 Fluorescent pigment-producing strains
fluoresce under short-wave ultraviolet light, and are observed
at 254 nm using a standard Woods lamp.3 Agar containing cetrimide
has been used successfully to isolate P. aeruginosa from
contaminated specimens.5
King, Ward and Raney1 developed Medium A (Tech Agar) to enhance
the production of pyocyanin in Pseudomonas species. Cetrimide Agar
Base is prepared according to this formula with the addition
of cetrimide.1 Brown and Lowbury2 used cetrimide in the Medium B
formulation of King, Ward and Raney1 to demonstrate the production
of fluorescein in P. aeruginosa.
Cetrimide Agar Base

Glassware
Autoclave
Incubator (35C)
Waterbath (45-50C)
Glycerol
1. Suspend 45.3 grams in 1 liter distilled or deionized water
containing 10 ml of glycerol.
4. Dispense into sterile Petri dishes, or as desired.

Test Procedure
Cetrimide Agar Base is recommended in the examination of food

and in United States Pharmacopeia (USP XXIII) for use in Microbial
Limit Tests.7
For the isolation of P. aeruginosa plates of Cetrimide Agar Base may

be inoculated by the streak method from nonselective medium or
directly from the specimen. When plating directly from the specimen
the inoculum level should be sufficiently high.
Results
Bacto Peptone provides the nitrogen, vitamins and amino acids in

Cetrimide Agar Base. Magnesium Chloride and Potassium Sulfate
enhance the production of pyocyanin and fluorescein.8 Cetrimide
(cetyltrimethylammonium bromide) is the selective agent. Cetrimide
acts as a quaternary ammonium cationic detergent causing nitrogen
and phosphorous to be released from bacterial cells other than
P. aeruginosa. Bacto Agar is the solidifying agent. Cetrimide Agar
Base is supplemented with 1% Glycerol as a source of carbon.
Examine plates or tubes for the presence of characteristic blue,

blue-green, yellow-green pigment. Pseudomonas aeruginosa typically
produce both pyocyanin and fluorescein.


2. The type of peptone used in base may affect pigment production.1,9
3. No single medium can be depended upon to exhibit all pigment
producing P. aeruginosa strains.
4. Occasionally some enterics will exhibit a slight yellowing of the
medium; however, this coloration is easily distinguished from
fluorescein production since this yellowing does not fluoresce.1
5. Some nonfermenters and some aerobic spore formers may exhibit
a water-soluble tan to brown pigmentation on this medium.
Serratia strains may exhibit a pink pigmentation.1
6. Studies of Lowbury and Collins10 showed Ps. aeruginosa may
lose its fluorescence under UV if the cultures are left at room
temperature for a short time. Fluorescence reappears when plates
are reincubated.
7. Further tests are necessary for definitive identification of
P. aeruginosa.
Storage
References
Formula
Cetrimide Agar Base
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Magnesium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4
Potassium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Cetrimide (Cetyltrimethylammonium Bromide) . . . . . . . . 0.3
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.6
g
g
g
g
g
Precautions

The Difco Manual
1. King, E. O., M. K. Ward, and E. E. Raney. 1954. Two simple

media for the demonstration of pyocyanin and fluorescein. J. Lab.
Clin. Med. 44:301.
117
Chapman Stone Medium
Section II
2. Brown, V.I., and E. J. L. Lowbury. 1965. Use of an improved

Cetrimide Agar Medium and of culture methods for Pseudomonas
aeruginosa. J. Clin. Pathol. 18:752.
Nonfermentative gram-negative bacilli and coccobacilli,
p. 386-405. Bailey & Scotts diagnostic microbiology, 9th ed.
Mosby-Year Book, Inc., St. Louis, MO.
4. Gilligan, P. H. 1995. Pseudomonas and Burkholderia, p. 509-519.
R. H. Yolken (ed.)., Manual of clinical microbiology, 6th ed.
5. Robin, T., and J. M. Janda. 1984. Enhanced recovery of
Pseudomonas aeruginosa from diverse clinical specimens on a
new selective agar. Diag. Microbiol. Infect. Dis. 2:207.
Bacto Chapman Stone Medium
7. The United States Pharmacopeia. 1995. Microbiological Limits

Tests, The United States pharmacopeia, 23rd ed. United States
Pharmacopeial Convention, Rockville, MD.
9. Goto, S., and S. Enomoto. 1970. Nalidixic acid cetrimide agar:
A new selective plating medium for the selective isolation of
Pseudomonas aeruginosa. Jpn. J. Microbiol. 14:65.
10. Lowbury, E. J. L., and A. G. Collins. 1955. The use of a new
cetrimide product in a selective medium for Pseudomonas
aeruginosa. J. Clin. Pathol. 8:47.
Packaging
Cetrimide Agar Base
Glycerol
100
500
100
500
g
g
g
g
0854-15
0854-17
0282-15
0282-17
Also Known As
Chapman Stone Medium conforms with Chapman Stone Agar.
Intended Use
Bacto Chapman Stone Medium is used for isolating and differentiating
staphylococci based on mannitol fermentation and gelatinase activity.

Dehydrated Appearance: Light beige, free-flowing, homogeneous
with a tendency to cake.
Solution:
light amber, opalescent with precipitation.
Prepared medium:
Light to medium amber, opalescent
with a precipitate.
Reaction of 20.2%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepare Chapman Stone Medium per label directions.
Inoculate and incubate at 30 2C for 18-48 hours. Add Brom
Cresol Purple indicator to determine mannitol fermentation
(yellow = positive).
ORGANISM
ATCC
INOCULUM
CFU
HALO
MANNITOL
GROWTH (Gelatinase) FERMENTATION
Escherichia
25922* 100-1,000 inhibited
coli
Staphylococcus 25923* 100-1,000 good
aureus
epidermidis

Chapman Stone Medium is prepared according to the formula
described by Chapman.1 It is similar to Staphylococcus Medium 110,
previously described by Chapman,2 except that the sodium chloride
concentration is reduced to 5.5% and ammonium sulfate is included in
the formulation. The inclusion of ammonium sulfate in the medium
negates the need to add a reagent after growth has been obtained in
order to detect gelatinase activity by Stones method. Chapman Stone
Medium is especially recommended for suspected food poisoning
studies involving Staphylococcus.3 It is selective, due to the relatively
high salt content, and is differential due to pigmentation, mannitol
fermentation, and the presence or absence of gelatin liquefaction.

Yeast Extract and Tryptone provide nitrogen, carbon, sulfur, vitamins,
and trace nutrients essential for growth. Gelatin serves as a substrate
for gelatinase activity. Ammonium Sulfate allows detection of gelatin
hydrolysis. D-Mannitol is the fermentable carbohydrate. Sodium
Chloride acts as a selective agent because most bacterial species are
inhibited by the high salt content. Dipotassium Phosphate provides
buffering capability. Bacto Agar is the solidifying agent.
Formula
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Bacto D-Mannitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Ammonium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g
g
118
The Difco Manual
Section II
Charcoal Agar
Precautions
Test Procedure

2. HARMFUL. HARMFUL IF SWALLOWED. (EC) IRRITATING
TO EYES, RESPIRATORY SYSTEM AND SKIN. Avoid contact
with skin and eyes. Do not breathe dust. Wear suitable protective
clothing. Keep container tightly closed. TARGET ORGAN(S):
Lungs, Intestines.
1. Streak a sample of the specimen onto the surface of the agar. Make
several stabs into the medium along the streak.
2. Incubate, aerobically, at 30 2C for up to 48 hours.
3. Examine for growth and the presence or absence of clear zones
around colonies.
4. To determine mannitol fermentation, add a few drops of Brom
Cresol Purple to areas on the medium from which colonies have
been removed. Any change in color of the indicator, compared with
that of the uninoculated medium, indicates fermentation of mannitol.
Storage
very hygroscopic. Keep container tightly closed. Store prepared medium
at 2-8C.
Expiration Date
Results
Mannitol fermentation: Positive = change in color of the indicator to
yellow.
Gelatinase activity: Positive Stone reaction = formation of clear zones
around the colonies.
Any mannitol-positive, yellow or orange colonies surrounded by a clear
zone are presumptively identified as Staphylococcus aureus. White or
nonpigmented colonies, with or without a clear zone, are probably
S. epidermidis.
1. Confirm the presumptive identification of pathogenic staphylococci

with additional tests, such as coagulase activity.
2. Enterococci and/or Group D streptococci may exhibit growth on
the medium and show slight mannitol fermentation. The colonies,
however, are tiny and can easily be differentiated from staphylococci
by Gram stain and the catalase test.3
References
Glassware
Autoclave
Incubator (30C)
Brom Cresol Purple
1. Chapman, G. H. 1948. An improved Stone medium for the isolation

and testing of food-poisoning staphylococci. Food Res. 13:100-105.
2. Chapman, G. H. 1946. A single culture medium for selective
isolation of plasma-coagulating staphylococci and for improved
testing of chromogenesis, plasma coagulation, mannitol fermentation,
and the Stone reaction. J. Bacteriol. 51:409-410.
3. MacFaddin, J. F. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria. Williams &
Procedure
Materials Provided
3. Autoclave at 121C for 10 minutes. Omit sterilization if prepared
medium is to be used within 12 hours.
Packaging
500 g
10 kg
0313-17
0313-08
Bacto Charcoal Agar
Intended Use
Bacto Charcoal Agar is used for cultivating fastidious organisms,
especially Bordetella pertussis, for vaccine production and stock
culture maintenance.
The Difco Manual

Charcoal Agar is prepared according to the method of Mishulow,
Sharpe and Cohen. 1 The authors found this medium to be an
efficient substitute for Bordet-Gengou Agar in the production of
B. pertussis vaccines.
The genus Bordetella consists of four species: Bordetella pertussis, B.
parapertussis, B. bronchiseptica and B. avium.2 All Bordetella are
119
Charcoal Agar
Section II
respiratory pathogens, residing on the mucous membranes of the

respiratory tract. B. pertussis is the major cause of whooping cough
or pertussis. B. parapertussis is associated with a milder form of
the disease.3 B. bronchiseptica is an opportunistic human pathogen
associated with both respiratory and non-respiratory infections,
often occurring in patients having close contact with animals. 2
B. bronchiseptica has not been reported to cause pertussis. There
have been no reports of recovery of B. avium from humans.2

Charcoal Agar supplemented with Horse Blood is used for the

cultivation and isolation of Haemophilus influenzae.4

Infusion from Beef Heart and Bacto Peptone provide the nitrogen,
carbon and amino acids in Charcoal Agar. Yeast Extract is a vitamin
source. Sodium Chloride maintains osmotic balance. Bacto Agar is a
solidifying agent. Soluble Starch absorbs toxic metabolites. Norit SG,
charcoal, provides growth requirements and selective properties.
Formula
Charcoal Agar
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Soluble Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Norit SG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
g
g
g
g
g
g
g
Storage
Expiration Date
Procedure
Materials Provided
Charcoal Agar

Glassware
Autoclave
Incubator (35C)
Waterbath (45-50C)
1.
2.
3.
4.

Mix thoroughly during dispensing to uniformly distribute
the charcoal.
Precautions
Uninoculated
plate
ATCC 4617

Dehydrated Appearance: Gray, free-flowing, homogeneous.
Solution:
deionized water on boiling; black,
opaque with a precipitate.
Prepared Medium:
Black, opaque.
Reaction of 6.25%
Solution at 25C
pH 7.3 0.2
Cultural Response
Prepare Charcoal Agar per label directions. Inoculate and
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Bordetella parapertussis
4617
15237
8467
100-1,000
100-1,000
100-1,000
good
good
good
120
The Difco Manual
Section II
Choline Assay Medium

Test Procedure
For a complete discussion on the isolation and maintenance of
fastidious microorganisms refer to the procedures described in
appropriate references.2,4,5
Results

2. Charcoal has a tendency to settle out of the medium. Swirl the flask
gently when dispensing to obtain a uniform charcoal suspension.4
References
1. Mishulow, L., L. S. Sharpe, and L. L. Cohen. 1953. Beef-heart
charcoal agar for the preparation of pertussis vaccines. Am. J.
Public Health, 43:1466.
Bacto Choline Assay Medium
2. Marcon, M. J. 1995. Bordetella, p. 566-573. In P. R. Murray, E. J.

Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.).,
3. Linneman, C. C., and E. B. Pery. 1977. Bordetella parapertussis;
recent experience and a review of the literature. Am. J. Dis. Child.
131:560-563. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C.
Tenover, and R. H. Yolken (ed.)., Manual of clinical microbiology,
4. MacFaddin, J. F. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, vol 1, p. 154-159.
Washington, D.C.
Packaging
Charcoal Agar
500 g
0894-17
Bacto Choline Assay Medium is used for determining choline concentration by the microbiological assay technique.

Choline Assay Medium is a slight modification of the medium
described by Horowitz and Beadle.1 Neurospora crassa ATCC 9277
is the test organism used in this microbiological assay.

Choline Assay Medium is a choline-free dehydrated medium containing

all other nutrients and vitamins essential for the cultivation of N. crassa
ATCC 9277. The addition of choline standard in specified increasing
concentrations gives a growth response by this organism that can be
measured gravimetrically.
Intended Use

Dehydrated Appearance: White, free-flowing, homogeneous.
Solution:
(double strength) solution, soluble in
distilled or deionized water upon
boiling. Solution is colorless, clear,
Prepared Medium:
Colorless, clear, may have a slight
precipitate.
Reaction of 2.85%
Solution at 25C:
pH 5.5 0.2
Cultural Response
Prepare Choline Assay Medium per label directions. Prepare
a standard curve using choline at levels from 0 to 25 g per
10 ml. The medium supports the growth of Neurospora crassa
ATCC 9277 when supplemented with choline chloride.
The Difco Manual
Formula
Formula Per Liter
Bacto Sucrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Ammonium Nitrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Potassium Sodium Tartrate . . . . . . . . . . . . . . . . . . . . . . . . 11.4
Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
Calcium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
Sodium Borate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 700
Ammonium Molybdate . . . . . . . . . . . . . . . . . . . . . . . . . . . 500
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1
Cuprous Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
Manganese Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Zinc Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.6
g
g
g
g
g
g
g
g
g
g
mg
g
g
mg
121
Section II
Precautions
Test Procedure

3. Great care must be taken to avoid contamination of media or
glassware in microbiological assay procedures. Extremely small
for at least 1 hour to burn off any organic residues that might be
present.
Remove 1 loop of spores from a 48-hour culture of N. crassa

ATCC 9277 grown on Neurospora Culture Agar and suspend it in
100 ml sterile saline. Add 1 drop of this spore suspension to each
flask of medium. Incubate at 25-30C for 3 days. At the end of the
incubation period, steam the flask at 100C for 5 minutes. Remove
all the mycelium from the flask using a stiff wire needle or glass rod,
press dry between paper towels, and roll into a small pellet. Dry the
pellet at 100C in a vacuum oven for 2 hours. (A glazed porcelain
spot plate is convenient for handling the mycelium during drying and
weighing.) Weigh to the nearest 0.5 mg. A standard curve is then
constructed from the weights obtained, and the unknown determined
by interpolation. In the assay for choline, 50 ml Erlenmeyer flasks
containing a total volume of 10 ml each are used.
Storage

obtained by using choline at levels of 0.0, 2.5, 5, 10, 15, 20 and 25 g
per assay flask (10 ml). The most effective assay range using Choline
Assay Medium is between 2.5 and 30 g choline.

Expiration Date
The concentration of choline required for the preparation of the standard

curve may be prepared by dissolving 0.5 grams choline chloride in
1,000 ml distilled water. This is the stock solution (500 g per ml).
Dilute the stock solution by adding 1 ml to 99 ml distilled water. Use
0.0, 0.5, 1, 2, 3, 4 and 5 ml of this diluted solution per flask. Prepare
the stock solution fresh daily.
Results
Glassware
Autoclave
Stock culture of Neurospora crassa ATCC 9277
Inoculating loop
Neurospora Culture Agar
Wire needle or glass rod
Paper towels
Vacuum oven
Porcelain spot plate
Scale

or cup.
Procedure
Materials Provided
1.
2.
3.
4.
5.
6.

Dispense 5 ml amounts into flasks, evenly dispersing the precipitate.
Adjust flask volume to 10 ml with distilled or deionized water.

response.
References
1. Horowitz and Beadle. 1943. J. Biol. Chem. 150:325.

Packaging
122
100 g
0460-15
The Difco Manual
Section II
Columbia Blood Agar Base, Columbia Blood Agar Base EH & Columbia Blood Agar Base No. 2
Bacto Columbia Blood Agar Base . Bacto Columbia Blood

Agar Base EH . Bacto Columbia Blood Agar Base No. 2
Intended Use
Bacto Columbia Blood Agar Base is used for cultivating fastidious

microorganisms with or without the addition of blood.
Bacto Columbia Blood Agar Base EH is used with blood in isolating
and cultivating fastidious microorganisms.
Bacto Columbia Blood Agar Base No. 2 is used with blood in isolating
and cultivating fastidious microorganisms.
Columbia Blood Agar Base uses specially selected raw materials to

support good growth of fastidious microorganisms. Two peptones,
Pantone (a casein hydrolysate) and Bitone (an infusion peptone),
provide nitrogen, carbon, amino acids and vitamins. Tryptic Digest
of Beef Heart provides additional nitrogen and amino acids. Corn
Starch, originally proposed by the authors of this medium, increases
growth of Neisseria and enhances the hemolytic reactions of some
streptococci.1 Agar is a solidifying agent. Sodium Chloride maintains
the osmotic balance of the medium.
Columbia Blood Agar Base No. 2 and Columbia Blood Agar Base EH
are similar in composition to Columbia Blood Agar Base. However,
different peptones are used to improve and enhance hemolysin
production while minimizing antagonism or loss in activity of streptococcal hemolysins. Columbia Blood Agar Base No. 2 contains Bitone
H while Columbia Blood Agar Base EH contains Bitone H Plus. Both
formulations contain Pantone, Enzymatic Digest of Animal Tissue,
Starch, Sodium Chloride and Agar.
Blood agar bases are relatively free of reducing sugars, which have
been reported to adversely influence the hemolytic reactions of
-hemolytic streptococci.3 Supplementation with blood (5-10%)
provides additional growth factors for fastidious microorganisms and
aids in determining hemolytic reactions. Hemolytic patterns may vary
with the source of animal blood and the type of basal medium used.4
Also Known As

Columbia blood agar base media are typically supplemented with
5-10% sheep, rabbit or horse blood for use in isolating, cultivating
and determining the hemolytic reactions of fastidious pathogenic
microorganisms. Without enrichment, Columbia Blood Agar Base can
be used as a general purpose medium.
Columbia Blood Agar Base was patterned after the Columbia Agar
formulation described by Ellner et al. of Columbia University. 1
Columbia Blood Agar Base No. 2 and Columbia Blood Agar Base EH
(Enhanced Hemolysis) are modifications of Columbia Blood Agar
Base. Columbia Blood Agar Base No. 2 provides clearer hemolytic
reactions with Streptococcus group A while Columbia Blood Agar Base
EH provides dramatic, enhanced hemolysis.
Columbia Blood Agar Base is specified in the Compendium of Methods
for the Microbiological Examination of Foods.2
Escherichia coli
ATCC 25922
ATCC 25922

Columbia Blood Agar Base
Solution:
deionized water on boiling, light to
medium amber, opalescent with a
fine precipitate.
Prepared Medium:
Plain - light to medium amber, slightly
opalescent to opalescent with a fine
precipitate.
With 5% sheep blood - cherry red, opaque.
Reaction of 4.4%
Solution at 25C:
pH 7.3 0.2
Columbia Blood Agar Base No. 2
Solution:
medium amber, opalescent.
Prepared Medium:
Reaction of 3.9%
Solution at 25C:
pH 7.3 0.2
ATCC 6305
ATCC 19615
On Columbia Blood Agar Base
The Difco Manual
123
Columbia Blood Agar Base, Columbia Blood Agar Base EH & Columbia Blood Agar Base No. 2
Formula
Section II


Formula Per Liter
Bacto Pantone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Bitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Tryptic Digest of Beef Heart . . . . . . . . . . . . . . . . . . . . . . . . 3
Corn Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Storage
g
g
g
g
g
g

Expiration Date

Columbia Blood Agar Base EH
Formula Per Liter
Procedure
Bacto Pantone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Bacto Bitone H Plus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Enzymatic Digest of Animal Tissue . . . . . . . . . . . . . . . . . . . 3
Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Materials Provided
g
g
g
g
g
g

Glassware
Autoclave
Incubator (35C)

Formula Per Liter
Bacto Pantone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Bacto Bitone H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Enzymatic Digest of Animal Tissue . . . . . . . . . . . . . . . . . . . 3
Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
g
g
g
g
g
g
Columbia Blood Agar Base - 44 grams;
Columbia Blood Agar Base EH - 39 grams;
Columbia Blood Agar Base No. 2 - 39 grams.
Precautions
ATCC 25923

Solution:
deionized water on boiling, light to medium
amber, clear to slightly opalescent.
Prepared Medium:
With 5% sheep blood - medium to
bright cherry red, opaque.
Reaction of 3.9%
Solution at 25C:
pH 7.3 0.2
Cultural Response
Prepare the medium with and without 5% sterile defibrinated
sheep blood per label directions. Inoculate and incubate at
35 2C under 5-10% CO2 for 18-48 hours.
ORGANISM
Escherichia coli
ATCC
INOCULUM
CFU
GROWTH
HEMOLYSIS
25922*
25923*
6305
19615*
100-1,000
100-1,000
100-1,000
100-1,000
good
good
good
good
N/A
beta
alpha
beta
124
The Difco Manual
Section II
Columbia Broth

4. To prepare blood agar, aseptically add 5% sterile defibrinated blood

Collect specimens in sterile containers or with sterile swabs and
transport immediately to the laboratory in accordance with
recommended guidelines outlined in the references.
Test Procedure
1. Process each specimen as appropriate and inoculate directly onto
the surface of the medium. Streak for isolation with an inoculating
loop, then stab the agar several times to deposit -hemolytic
streptococci beneath the agar surface. Subsurface growth will
demonstrate the most reliable hemolytic reactions due to the
activity of both oxygen-stable and oxygen-labile streptolysins.4
Results
1. Examine plates for growth and hemolytic reactions after 18-24 and
48 hours of incubation. Four types of hemolysis on blood agar
media can be described:5
a. Alpha-hemolysis () is the reduction of hemoglobin to
methemoglobin in the medium surrounding the colony, causing
a greenish discolorization of the medium.
b. Beta-hemolysis () is the lysis of red blood cells, producing a
c. Gamma-hemolysis () indicates no hemolysis. No destruction
of red blood cells occurs and there is no change in the medium.
d. Alpha-prime-hemolysis ( ) is a small zone of complete hemolysis

1. Blood agar base media are intended for use with blood
supplementation. Although certain diagnostic tests may
be performed directly on these media, biochemical and, if
indicated, immunological testing using pure cultures is
recommended for complete identification. Consult appropriate
references for further information.
Bacto Columbia Broth
3. Hemolytic reactions of some strains of group D streptococci have

been shown to be affected by differences in animal blood. Such
strains are -hemolytic on horse, human and rabbit blood agar and
-hemolytic on sheep blood agar.4
4. Colonies of Haemophilus haemolyticus are -hemolytic on horse
and rabbit blood agar and must be distinguished from colonies
of -hemolytic streptococci using other criteria. The use of sheep
blood has been suggested to obviate this problem since sheep
blood is deficient in pyridine nucleotides and does not support
growth of H. haemolyticus.6
reactions of -hemolytic streptococci.4 For optimal performance,
incubate blood agar media under increased CO2 or anaerobic conditions.
References
1. Ellner, P. D., C. J. Stoessel, E. Drakeford, and F. Vasi. 1966.
A new culture medium for medical bacteriology. Am. J. Clin.
Pathol. 45:502-504.
2. Vanderzant, C. and D. F. Splittstoesser (ed.). 1992. Compendium of methods for the microbiological examination of foods,
3. Casman, E. P. 1947. A noninfusion blood agar base for neisseriae,
pneumococci and streptococci. Am. J. Clin Pathol. 17:281-289.
E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.).
5. Isenberg, H. D. (ed). 1992. Clinical microbiology procedures handbook, vol.1. American Society for Microbiology, Washington, D.C.
Scotts diagnostic microbiology, 9th ed. Mosby-Year Book, Inc.
St. Louis, MO.
Packaging
500 g
2 kg
10 kg
0792-17
0792-07
0792-08
500 g
2 kg
10 kg
0790-17
0790-07
0790-08
500 g
2 kg
10 kg
0793-17
0793-07
0793-08
Intended Use
Bacto Columbia Broth is used for cultivating fastidious microorganisms.

Columbia Broth is prepared according to the formulation described by
Morello and Ellner. 1 In their study Columbia Broth, a medium
developed for blood cultures, was superior to a commonly used
general purpose broth for faster growth of Staphylococcus aureus,
E. coli and streptococci (viridans and enterococcus groups). Columbia
The Difco Manual
Broth, in the presence of CO2 and supplemented with SPS, is an

excellent blood culture medium.2 In the study by Morello and Ellner,1
the addition of sodium polyanetholsulfonate (SPS) in Columbia Broth
was emphasized. SPS is an anticoagulant that inhibits serum
bactericidal activity against many bacteria, inhibits phagocytosis,
inactivates complement, and neutralizes lysozymes and the
aminoglycoside class of antibiotics.2

Columbia Broth was formulated from Pantone and Bitone. Dextrose is
added to the formula as a carbon energy source. The medium is
125
Columbia Broth
Section II
buffered with Tris. Corn Starch is omitted to reduce opalescence.1

Cysteine is the reducing agent. Magnesium and Iron are added to
facilitate organism growth.
Formula
Expiration Date
Procedure
Columbia Broth
Formula Per Liter
Bacto Pantone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Bitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
L-Cysteine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . 0.1
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Magnesium Sulfate Anhydrous . . . . . . . . . . . . . . . . . . . . 0.1
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.02
Sodium Carbonate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6
Tris (Hydroxymethyl) Aminomethane . . . . . . . . . . . . . . 0.83
Tris (Hydroxymethyl) Aminomethane HCl . . . . . . . . . . . 2.86
g
g
g
g
g
g
g
g
g
g
g
Materials Provided
Columbia Broth

Glassware
Autoclave
Incubator (35C)
Waterbath (45-50C)
Sterile tubes
Sodium polyanetholesulfonate (SPS)
Final pH 7.5 0.2 at 25oC
Precautions
Storage

2. Warm slightly if necessary to dissolve completely.
3. OPTIONAL: Sodium polyanetholesulfonate (SPS) may be added
at this time with agitation to ensure a uniform solution. The culture
medium should contain 0.025 to 0.05% SPS.
4. Distribute in suitable containers. Autoclave at 121C for 15 minutes.
5. Allow to cool to room temperature before using.

Test Procedure
Dehydrated Appearance: Light beige, free-flowing homogeneous.

Solution:
or deionized water on warming.
Solution is light amber, clear to
very slightly opalescent, may have
a slight amount of fine precipitate.
Prepared Medium:
amount of fine precipitate.
Reaction of 3.5%
Solution at 25C:
pH 7.5 0.2
Cultural Response
Prepare Columbia Broth per label directions. Inoculate and
incubate at 35 2C under appropriate conditions for
18-48 hours. Incubate Bacteroides fragilis anaerobically.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
13090
25923*
19615*
25285
27853
100-1,000
100-1,000
100-1,000
100-1,000
100-1,000
good
good
good
good
good
126
Process clinical specimens from different body sites as described in

Clinical Microbiology Procedures Handbook,2 Manual of Clinical
Microbiology3 or according to laboratory procedures.
Results

2. Neisseria spp. may be inhibited by SPS in Columbia Broth. The
addition of 1.2% gelatin may counteract the inhibitory effect, but
SPS may also inhibit other organisms.2
3. Opalescence in Columbia Broth cannot always be relied upon as
evidence of bacterial growth in the bottle.
4. It is possible for significant numbers of viable bacteria to be present
in an inoculated and incubated blood culture bottle without the
usual signs of bacterial growth.
References
1. Morello, J. A., and P. D. Ellner. 1969. New medium for blood
cultures. Appl. Microbiol. 17:68-70.
2. Isenberg, H. D. (ed). 1992. Clinical microbiology procedures handbook, vol.1. American Society for Microbiology, Washington, D.C.
The Difco Manual
Section II
Columbia CNA Agar

Bacto Columbia CNA Agar
Intended Use
Bacto Columbia CNA Agar is used with added blood in isolating
gram-positive cocci.
Also Known As
Packaging
Columbia Broth
Formula
Columbia CNA Agar

Formula Per Liter

Columbia CNA Agar is Columbia Blood Agar Base supplemented with
colistin (10 g/ml) and nalidixic acid (15 g/ml). The antimicrobial
agents suppress growth of Enterobacteriaceae and Pseudomonas species
0944-17
0944-07
while allowing yeasts, staphylococci, streptococci and enterococci

to grow.4 Certain gram-negative organisms, such as Gardnerella
vaginalis and some Bacteriodes species, can grow very well on
Columbia CNA Agar with blood.4 Colistin disrupts the cell membrane
of gram-negative organisms; it is particularly effective against
Pseudomonas species.2 Nalidixic acid blocks DNA replication in
susceptible bacteria and acts against many gram-negative bacteria.2
Columbia CNA Agar is also referred to as Colistin Nalidixic Acid Agar.

Ellner et al.1 described Columbia CNA Agar, a variation of Columbia
Blood Agar Base that is selective for gram-positive cocci. The
antimicrobics colistin and nalidixic acid select for gram-positive
organisms and fungi by suppressing gram-negative bacteria. 2
Columbia CNA Agar is recommended as a primary plating medium
when culturing urine specimens.3
500 g
2 kg
Bacto Pantone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Bitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Corn Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Colistin Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Nalidixic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
mg
mg
g

ATCC 25923
Uninoculated
plate

Solution:
deionized water upon boiling, light to
medium amber, slightly opalescent
to opalescent with a fine precipitate.
Prepared Medium:
Without blood: light to medium amber,
slightly opalescent to opalescent with
a fine precipitate.
With 5% sheep blood: cherry red,
opaque.
Reaction of 4.4%
Solution at 25C:
pH 7.3 0.2
Cultural Response
Prepare Columbia CNA Agar with and without 5% sheep blood
per label directions. Inoculate both media and incubate at
35 2C for 18-24 hours under 10% CO2.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
w/o BLOOD w/BLOOD HEMOLYSIS
Proteus
12453 1,000-2,000 markedly markedly
mirabilis
inhibited inhibited
Staphylococcus 25923* 100-1,000
good
good
beta
aureus
Streptococcus
6305 100-1,000
good
good
alpha
pneumoniae
Streptococcus 19615* 100-1,000
good
good
beta
pyogenes
The Difco Manual
ATCC 19615
127
Columbia CNA Agar
Precautions
Storage
Expiration Date
whenstored as directed. Do not use a product if it fails to meet
specificationfor identity and performance.
Procedure
Materials Provided
Columbia CNA Agar

Glassware
Autoclave
Incubator (35C)
Waterbath
1.
2.
3.
4.
5.

Autoclave at 121 C for 15 minutes. Avoid overheating.
Cool to 45-50C.
Aseptically add 5% sterile defibrinated blood to the medium at
45-50C. Mix well.
6. Dispense into sterile Petri dishes or tubes as desired.

Test Procedure
1. Inoculate specimens directly onto the surface of the medium. Streak
for isolation with an inoculating loop, then stab the agar several
times to deposit beta-hemolytic streptococci beneath the agar
surface. Subsurface growth will display the most reliable hemolytic
reactions due to the activity of both oxygen-stable and
oxygen-labile streptolysins.5
Results
Examine plates for growth and hemolytic reactions after 18-24 and
48 hours incubation. Four different types of hemolysis on blood agar
128
Section II

methemoglobin in the medium surrounding the colony. This
causes a greenish discolorization of the medium.
b. Beta ()-hemolysis is the lysis of red blood cells, resulting in a
c. Gamma ()-hemolysis indicates no hemolysis. No destruction of
red blood cells occurs and there is no change in the medium.
d. Alpha-prime (` )-hemolysis is a small zone of complete hemolysis

1. Because the nutritional requirements of organisms vary, some
this medium.
strains are beta-hemolytic on horse, human and rabbit blood agar
and alpha-hemolytic on sheep blood agar.5
3. Colonies of Haemophilus haemolyticus are beta-hemolytic on horse
and rabbit blood agar and must be distinguished from colonies
on beta-hemolytic streptococci using other criteria. The use of
sheep blood has been suggested to obviate this problem since sheep
blood is deficient in pyridine nucleotides and does not support
growth of H. haemolyticus.4
4. Atmosphere of incubation has been shown to influence
hemolytic reactions of beta-hemolytic streptococci.5 For optimal
performance, incubate blood agar base media under increased CO2
or anaerobic conditions.
5. Proteus species occasionally grow on CNA Agar and may initially
be confused with streptococci because of the small size of the
colonies.2
References
1. Ellner, P. D., C. J. Stoessel, E. Drakeford, and F. Vasi. 1966.
A new culture medium for medical bacteriology. Am. J. Clin.
Pathol. 45:502-504.
2. Estevez, E. G. 1984. Bacteriologic plate media: review of
mechanisms of action. Lab Med. 15:258-262.
3. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures handbook, vol.1. American Society for Microbiology, Washington, D.C.
4. Baron, E. J., L. R. Peterson, and S.M. Finegold. 1994. Bailey &
St. Louis, MO.
Packaging
Columbia CNA Agar
500 g
2 kg
0867-17
0867-07
The Difco Manual
Section II
Cooke Rose Bengal Agar & Antimicrobic Vial A
Bacto Cooke Rose Bengal Agar

Bacto Antimicrobic Vial A
Intended Use
Bacto Cooke Rose Bengal Agar is used with or without Bacto Antimicrobic Vial A in isolating fungi from environmental and food specimens.
Bacto Antimicrobic Vial A is used in preparing microbiological
culture media.

Cooke Rose Bengal Agar is a selective medium for the isolation of
fungi prepared according to the formula of Cooke.1 Selectivity of the
medium may be increased by the addition of antibiotics.
A variety of materials and methods have been used to inhibit bacteria
in an attempt to isolate fungi from mixed flora. Fungi are extremely
successful organisms, as evidenced by their ubiquity in nature.2
Waksman3 described an acid medium consisting of peptone, dextrose,
inorganic salts and agar for the isolation of fungi from soil. Cooke1
used the Waksman3 medium without adjustment to investigate the
isolation of fungi from sewage. It was discovered that Soytone was
particularly suitable for use in this medium and that the combination
of chlortetracycline, or oxytetracycline, with rose bengal increased the
selectivity of the medium.
Antimicrobic Vial A contains sterile, desiccated chlortetracycline

(Aureomycin). It was originally used in preparing DTM Agar
described by Taplin, Azias, Rebell and Blank4 for the isolation of
dermatophytes. Antimicrobic Vial A is applicable for use in various media
requiring this antibiotic. Cooke1 preferred chlortetracycline in Cooke
Rose Bengal Agar due to the increased stability of the antibiotic.

Soytone provides nitrogen, carbon and vitamins in Cooke Rose Bengal
Agar. Dextrose is an energy source. Rose Bengal and chlortetracycline
selectively inhibit bacterial growth and restrict the size and height of
colonies of more rapidly growing molds. Monopotassium Phosphate
provides buffering capability. Magnesium Sulfate is a source of divalent
cations. Bacto Agar is a solidifying agent.
Formula
Cooke Rose Bengal Agar
Formula Per Liter
Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Rose Bengal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.035
g
g
g
g
g
g

Aspergillus niger
ATCC 16404
Uninoculated
plate

Dehydrated Appearance: Pinkish tan, free-flowing, homogeneous.
Solution:
water on boiling. Solution is pinkish red,
without a significant precipitate.
Prepared Medium:
Deep pink, slightly opalescent
Reaction of 3.6%
Solution at 25C:
pH 6.0 0.2
Antimicrobic Vial A
Lyophilized Appearance:
Rehydrated Appearance:
Solution:
Microbial Limits Test:
Potency (Cup-Plate Assay):
Yellow cake or powder.

Yellow, clear solution.
Soluble in 10 ml distilled or deionized water.
Negative.
90-140% of labeled potency.
Cultural Response
Prepare Cooke Rose Bengal Agar with 35 g per ml chlortetracycline (Antimicrobic Vial A)
per label directions. Inoculate and incubate at 25-30C for up to 72 hours.
ORGANISM
Aspergillus niger
Candida albicans
Escherichia coli
Saccharomyces cerevisae
The Difco Manual
ATCC
INOCULUM CFU
GROWTH
16404
26790
25922*
9763
30-300
30-300
1,000-2,000
30-300
good
good
good
Candida albicans
ATCC 26790
The cultures listed are the minimum that should be used

for performance testing.
*This culture is available as Bactrol Disks and should be
129
Cooked Meat Medium
Section II
Antimicrobic Vial A
1. Aseptically add 10 ml sterile distilled or deionized water to

Antimicrobic Vial A.
2. Agitate gently to dissolve completely.
3. The resulting concentration of the rehydrated solution is 2.5 mg
chlortetrycycline per ml.
4. Cool to 45C.
5. OPTIONAL: To increase selectivity, aseptically add 14 ml of
rehydrated Antimicrobic Vial A to achieve a final concentration of
35 g of chlortetracycline per ml of medium or an appropriate
amount of another antibiotic.
Antimicrobic Vial A contains 25 mg sterile desiccated chlortetracycline

(Aureomycin) per 10 ml vial.
Precautions
2. Antimicrobic Vial A
HARMFUL. MAY CAUSE ALLERGIC EYE, RESPIRATORY
SYSTEM AND SKIN REACTION. (US) POSSIBLE RISK OF
HARM TO THE UNBORN CHILD. Avoid contact with skin and
eyes. Do not breathe dust. Wear suitable protective clothing. Keep
container tightly closed. Target Organs: Teeth, Bones.
Storage
Store Antimicrobic Vial A at 2-8C.
Expiration Date
Procedure
Materials Provided
Antimicrobic Vial A

Glassware
Autoclave
Incubator

Test Procedure
Refer to appropriate references for specific procedures on the isolation
and cultivation of fungi.
Results

References
1. Cooke. 1922. J. Bact. 7:339.
2. Dixon, D. M., and R. A. Fromtling. 1995. Morphology, taxonomy,
and classification of the fungi, p. 699-708. In P. R. Murray, E. J.
Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.). Manual
Washington, D.C.
3. Waksman. 1954. Antibiotics and Chemotherapy 4:657.
4. Taplin, Azias, Rebell, and Blank. 1969. Arch. Dermatol. 99:203.
Packaging
Antimicrobic Vial A
Antimicrobic Vial A
Bacto Cooked Meat Medium
Intended Use
Bacto Cooked Meat Medium is used for cultivating anaerobic
microorganisms and for maintaining stock cultures.
Also Known As
Cooked Meat Medium (CMM) is also called Chopped Meat
Medium.
130
500 g
0703-17
6 x 10 ml
3333-60
In 1890, Theobald Smith1 made use of fresh unheated animal tissue for
cultivating anaerobic organisms. Tarozzi 2 confirmed Smiths 1
findings and discovered the meat-broth could be heated to
104-105C for 15 minutes without destroying medium nutrients.
A steam sterilized emulsion of brain tissue in water was employed
by von Hibler 3.4 for cultivating anaerobic microorganisms.
Von Hibler 3,4 found organisms in cooked brain broth were less
susceptible to harmful effects of toxic metabolic products than in
The Difco Manual
Section II
Cooked Meat Medium
carbohydrate serum media. Robertson5 substituted beef heart for brain

tissue and found successful results. Cooked Meat Medium is prepared
according to the formulation of Robertson.5
The capacity of Cooked Meat Medium to detoxify metabolic products
of microorganisms makes it an excellent maintenance and
growth medium. A study of various formulations used to grow and
maintain clinical isolates of anaerobic bacteria found Chopped Meat
Broth superior.6
Cooked Meat Mediums ability to initiate growth in a small inoculum
makes it valuable for the primary culture of clinical specimens. Cooked
Meat Medium can be supplemented with vitamin K1 (1% alcohol
solution) and hemin (1% solution) for clinical isolates. 7 This
modification is used as a general enrichment for anaerobes, and as a
backup for anaerobic jar or chamber failure.7
Chopped Meat Carbohydrate Medium and Chopped Meat Glucose
Medium is used for cultivation and maintenance of anaerobic
bacteria. 7,8,9 Cooked Meat Medium is recommended in the
Bacteriological Analytical Manual10 for use in the examination of
Clostridium botulinum from food and in the Compendium of
Methods for the Microbiological Examination of Foods.11
Formula
Procedure
Beef Heart and Proteose Peptone provide the nitrogen, vitamins and
amino acids in Cooked Meat Medium. Sodium Chloride maintains
the osmotic balance of the medium. The low concentration of
Dextrose is sufficient as the energy source, but not high enough to
accumulate toxic metabolites. This formulation provides an effective
maintenance medium.
Solid meat particles provide favorable growth conditions for
anaerobes due to the reducing action of -SH (sulfhydryl) groups of
muscle protein.2,3,4 Sulfhydryl groups are more accessible in denatured
proteins, therefore the use of cooked meat particles is preferred.9
Cooked Meat Medium

Formula Per Liter
Beef Heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
g
g
g
g
Precautions
Storage
Expiration Date
Materials Provided
Cooked Meat Medium

Glassware
Autoclave
Incubator (35C)
Sterile tubes with closures

Dehydrated Appearance: Brown pellets.
Prepared Medium:
Medium amber, clear supernatant over insoluble pellets.
Reaction of 12.5%
Solution at 25C:
pH 7.2 0.2
Cultural Response
Prepare Cooked Meat Medium per label directions. Inoculate and incubate
medium at 35 2C for 40-48 hours.
ORGANISM
Bacteroides vulgatus
Clostridium novyi
Clostridium sporogenes
ATCC
INOCULUM
CFU
GROWTH
8482
7659
12924
11437
25923*
100-1,000
100-1,000
100-1,000
100-1,000
100-1,000
good
good
good
good
good
*This culture is available as Bactrol Disks and should be used as directed in
The Difco Manual
Uninoculated
tube
Clostridium
sporogenes
ATCC 11437
131
Corn Meal Agar
1. Suspend 12.5 grams in 100 ml distilled or deionized water (1.25 g
per 10 ml).
2. Let stand until all particles are thoroughly wetted and form an
even suspension.
3. Autoclave at 121C for 15 minutes. Reduce pressure slowly.
4. Cool without agitation.
5. If not used within 24 hours, reheat (100C) prior to use to drive off
dissolved oxygen.

Test Procedure
1. Inoculate specimen well into the meat particles (bottom of the
tube). Tissue specimens should be ground prior to inoculation.
2. Growth is indicated by turbidity and/or the presence of gas bubbles.
3. For a complete discussion on the isolation and identification
of aerobic and anaerobic bacteria, refer to appropriate procedures
outlined in the references.
Results
Limitations
this medium.
References
Section II
2. Tarozzi, G. 1905. Uber ein leicht in aerober Weise ausfuhrbares

Kulturmittel von einigen bis jetzt fuu strenge Anaeroben
gehlatenen Keimen. Zentralb. Bakteriol. 38:619.
3. von Hibler, E. 1899. Beitrage zur Kenntnis der durch anaerobe
Spaltpilze erzeugen Infektions-krankheiten der Tiere und des
Menschen etc. Centr. Bakteriol. 25: 513,594,631.
4. von Hibler, E. 1908. Untersuchungen uber die pathogenen
Anaerobier, Jena: Verlag Fischer.
5. Robertson, M. 1916. Notes upon certain anaerobes isolated from
wounds. J. Pathol. Bacteriol. 20:327.
6. Claros, M. C., D. M. Citron, and E. J. C. Goldstein. 1995.
Survival of anaerobic bacteria in various thioglycollate and chopped
meat broth formulations. J. Clin. Microbiol. 33:2505-2507.
7. Isenberg, H. E. (ed.). 1992. Clinical microbiology procedures
8. Atlas, R. M. 1993. Handbook of microbiological media,
p. 224-226. CRC Press, Boca Raton, FL.
9. MacFaddin, J. D. 1985. Media for isolation-cultivationidentification- maintenance of medical bacteria, vol. 1, p. 240-246.
Gaithersburg, MD.
Compendium of methods for the microbiological examination of food,
Packaging
Cooked Meat Medium
1. Smith, T. 1890. Centr. Bakteriol. 7:509.
Bacto Corn Meal Agar
100 g
500 g
10 kg
0267-15
0267-17
0267-15r
Bacto Corn Meal Agar is used for stimulating the production of

chlamydospores by most strains of Candida albicans and for
cultivating phytopathological fungi.
Corn Meal Agar has been used with varying degrees of success for
showing chlamydospore formation in C. albicans. Chlamydospore
production is the best diagnostic criterion for identification of the
pathogenic yeast C. albicans.2 Kelly and Funigeillo3 reported that the
addition of 1% Tween 80 enhanced chlamydospore formation by
C. albicans. With this improvement, Corn Meal Agar may be the most
accurate routine tool available for identification of C. albicans.4
Numerous culture media formulations have been described for the

detection, isolation, and identification of Candida albicans, the
etiological agent in candidiasis. The various media were designed
to bring out morphological or physiological characteristics in this
organism which would differentiate it from other members of the
genus as well as from other genera.
Infusion from corn meal is a source of carbon, protein and nutrients.

Bacto Agar is a solidifying agent.
Intended Use
One of the most important differential characteristics of C. albicans

in its ability to form chlamydospores on certain media. This property
is perhaps the best criterion for identification. Corn Meal Agar is
valuable for morphologic differentiation of many yeast-like organisms.
It suppresses vegetative growth of many fungi while stimulating
sporulation.1
132
Formula
Corn Meal Agar
Formula Per Liter
Corn Meal, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . . . 50 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
Precautions
The Difco Manual
Section II
Corn Meal Agar

Storage

recommended guidelines.5

Test Procedure6
Expiration Date
Corn Meal Agar
1. Using a sterile inoculating needle, lightly touch the yeast colony, then
make two streaks approximately 1.5 cm long each and 1.0 cm apart.
2. Flame the needle, and allow it to cool. Lightly make an S-shaped
streak back and forth across the two streak lines.
3. Flame sterilize a cover glass. Allow it to cool, then place it over the
streak marks.
4. Incubate at 22-26C for 72 hours.
Results
Glassware
Autoclave
Sterile Inoculating Needle
Cover Glass
1. Examine plates for the presence of chlamydospores.
Procedure
Materials Provided

1. Corn Meal Agar with the addition of 1% Tween 80 should not be
the only medium used for identification of C. albicans since
C. stellatoidea and C. tropicalis also produce chlamydospores on
this medium.7
2. Repeated subculture of some Candida strains will result in the
reduced ability to form chlamydospores.
References
1. Baron, E. J., and S. M. Finegold. 1990. Formulas and

preparation of culture media and reagents, p. A-10. Bailey & Scotts
Diagnostic Microbiology, 8th ed. The C. V. Mosby Company,
St. Louis, MO.
2. Duncan, J., and J. Floeder. 1963. A comparison of media for the
production of chlamydospores by Candida albicans. Am. J. Med.
Tech. 29:199-206.
3. Kelly, J. P., and F. Funigiello. 1959. Candida albicans: A study
of media designed to promote chlamydospore production. J. Lab.
& Clin. Med. 53:807- 809.
4. Gordon, M. A., and G. N. Little.1963. Effective dehydrated
media with surfactants for identification of Candida albicans.
J. of Int. Soc. for Human and Animal Mycol. 2:171-175.
handling, p. 19- 32. In P. R. Murray, E. J. Baron, M. A. Pfaller, F.
C. Tenover, and R. H. Yolken, (ed.), Manual of clinical microbiology, 6th ed. American Society for Microbiology, Washington, D.C.
Washington, D.C.
Dehydrated Medium
Appearance:
Solution:
Reaction of 1.7%
Solution at 25C:
Yellow, free-flowing, homogeneous.

is light amber, slightly opalescent to
opalescent, may have a slight, fine
precipitate.
pH 6.0 0.2
Cultural Response
Prepare Corn Meal Agar per label directions. Inoculate using
the spread plate method. Prepare a heavy suspension of
C. albicans, dip a sterile inoculating loop into the suspension,
and cut a 2 cm X through the medium. Place a cover slip over
the X. Incubate at 20-25C for 40-48 hours and up to four
days, if required. Examine plates for chlamydospores which,
when produced by some Candida species, appear as double
walled spheres on cover slip plates.
ORGANISM
ATCC
INOCULUM
CFU
Aspergillus niger
16404 100-1,000
Candida albicans
10231* 100-1,000
Saccharomyces cerevisiae 9763 100-1,000
RECOVERY CHLAMYDOSPORES
good
good
good
The Difco Manual
Packaging
Corn Meal Agar
500 g
0386-17
133
Cystine Heart Agar
Section II
Bacto Cystine Heart Agar
Enrichment with 2% hemoglobin provides additional growth factors.

Without enrichment, Cystine Heart Agar supports excellent growth of
gram-negative cocci and other pathogenic microorganisms.6 Rabbit
blood and antimicrobial agents can be added to this medium.5
Intended Use
Bacto Cystine Heart Agar is used with Bacto Hemoglobin for
cultivating Francisella tularensis and without enrichment for
cultivating gram-negative cocci and other microorganisms.
Formula
Cystine Heart Agar
Formula Per Liter
Also Known As

Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Cystine Heart Agar with added hemoglobin is also referred to as

Cystine Glucose Blood Agar.

Francisella tularensis was first described in humans in 1907.1 Several
media formulations were employed to isolate this microorganism.
Initial formulations contained egg or serum and were difficult
to prepare. Edward Francis,2 who dedicated his career to the study of
this organism, reported that blood dextrose cystine agar was a
satisfactory medium for cultivating this fastidious pathogen. Shaw3
added 0.05% cystine and 1% dextrose to Heart Infusion Agar for the
cultivation of F. tularensis.
While experimenting with Francis blood dextrose cystine agar, Rhamy4
added hemoglobin to Cystine Heart Agar to develop a satisfactory
medium for growth of F. tularensis.
Cystine Heart Agar is the medium of choice for isolating F. tularensis.1,5
g
g
g
g
g
g
Final pH 6.8 0.2 at 25%C
Precautions
2. Francisella tularensis is a Biosafety Level 2 pathogen that can be
transmitted by aerosols or by penetration of unbroken skin.5
Wearing of gowns, gloves and masks is advocated for laboratory
staff handling suspected infectious material.
Storage

Infusions from Beef Heart, Proteose Peptone and L-Cystine provide

nitrogen, vitamins and amino acids in Cystine Heart Agar. Dextrose is
a carbon source. Sodium chloride maintains the osmotic balance and
Francisella tularensis
with enrichment
ATCC 29684
with enrichment
ATCC 13090

Solution:
opalescent, may have fine precipitate.
Prepared Medium:
Plain - Light to medium amber, slightly
opalescent, may have fine precipitate.
With Hemoglobin - Chocolate, opaque.
Reaction of 5.1%
Solution at 25C:
pH 6.8 0.2
Cultural Response
Prepare Cystine Heart Agar per label directions. Incubate inoculated
medium at 35 2oC aerobically for 18-48 hours. Neisseria meningitidis
should be incubated under 5- 10% CO2.
ORGANISM
Francisella tularensis
ATCC
INOCULUM
CFU
29684
13090*
100-1,000
100-1,000
GROWTH
w/o HEMOGLOBIN
w/HEMOGLOBIN
fair
good
good
good
134
The Difco Manual
Section II
Cystine Tryptic Agar
Expiration Date
Test Procedure
1. Inoculate and streak specimens as soon as possible. For a complete

discussion on the inoculation and identification of Francisella,
consult appropriate references.
2. Overgrowth by contaminating organisms can be reduced by
incorporating 100- 500 units penicillin per ml into the medium.6
Procedure
Materials Provided
Cystine Heart Agar

Glassware
Autoclave
Incubator (35C)
Hemoglobin Solution 2% or Hemoglobin (optional)
Enriched Medium:
4. Add 100 ml sterile 2% hemoglobin solution and mix well. Use:
Hemoglobin Solution 2%; or,
Prepare a 2% hemoglobin solution as follows: Place 2 g r a m s
of Hemoglobin in a dry flask. Add 100 ml of cold
distilled or deionized water while agitating vigorously.
Continue intermittent agitation for 10-15 minutes until
solution is complete. Autoclave at 121C for 15 minutes.
Cool to 50-60C
5. Dispense into sterile Petri dishes or tubes.
Unenriched Medium:
Results

strains may be encountered that fail to grow or grow poorly
on this medium.
References
1. Stewart, S. J. 1995. In P. R. Murray., E. J. Baron, M. A.
Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical
Washington, D.C.
2. Francis, E. 1928. Symptoms, diagnosis and pathology of tularemia.
J. Am. Med. Assoc. 91:1155-1160.
3. Shaw, F. W. 1930. Culture medium for Bacterium tularense.
Zentr. Bakt. I. Abt. Orig. 118:216-217.
4. Rhamy, B. W. 1933. A new and simplified medium for Pasteurella
tularensis and other delicate organisms. Am. J. Clin. Pathol.
3:121-124.
6. Stewart, S. J. 1995. Francisella, p. 545-548. In P. R. Murray,
E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken (ed.),
Packaging
Cystine Heart Agar
500 g
0047-17
Hemoglobin
100
500
2
10
g
g
kg
kg
0136-15
0136-17
0136-07
0136-08
6 x 100 ml
3248-73

Collect specimens in sterile containers or with sterile swabs. Transport
Hemoglobin Solution 2%
Bacto Cystine Tryptic Agar
Also Known As
Cystine Tryptic Agar is a semi-solid basal medium prepared according

to the formula of Vera.1 Many tests used to differentiate among members
of the Enterobacteriaceae determine the organisms ability to utilize a
carbohydrate with the production of acid metabolic end products.2 CTA
is free of fermentable carbohydrates, and the carbohydrate content can
be adjusted for specific reactions. The carbohydrate concentration used
most frequently in fermentation reactions is 0.5 or 1%.
Cystine Tryptic Agar is abbreviated as CTA, and referred to as

CT Medium.
Some researchers prefer 1% to insure against reversion of the reaction

due to depletion of the carbohydrate by the microorganism.
Intended Use
Bacto Cystine Tryptic Agar is used with added carbohydrates in differentiating microorganisms based on fermentation reactions and motility.
The Difco Manual
135
Section II
The low agar content of Cystine Tryptic Agar provides a suitable

environment for motility studies. Motility determination aids in the
identification of bacteria. CTA can also be used as a maintenance medium
for stock cultures.3,4 This formula will support the growth of fastidious
organisms, e.g., Streptococcus pneumonia and Corynebacterium species.4

Tryptose provides the nitrogen, vitamins and amino acids in Cystine
Tryptic Agar. L-Cystine and Sodium Sulfite are added to this formula
to stimulate growth. Sodium Chloride maintains the osmotic balance
of the medium. Phenol Red is the pH indicator. Bacto Agar maintains
an Eh potential which facilitates anaerobic growth, and aids in dispersion of reducing substances and CO2 formed in the environment.5 The
agar is also used for the determination of motility.
Formula
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Sulfite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.017
g
g
g
g
g
g
Precautions
Storage
Expiration Date
Procedure
Materials Provided

Glassware
Autoclave
Incubator (35C)
Sterile 5-10% carbohydrate solution
Sterile tubes
1.
2.
3.
4.
Suspend 28.5 grams in 1 liter of distilled or deionized water.

To prepare fermentation medium, use one of the following methods:
A. Add 5-10 grams carbohydrate before sterilization.
or B. Dissolve 28.5 grams medium in 900 ml water, sterilize
and aseptically add 100 ml sterile of carbohydrate solution.

Solution:
deionized water upon boiling. Red,
very slightly opalescent without
Prepared Medium:
Red, very slightly opalescent without
precipitate.
Reaction of 2.85%
Solution at 25C:
pH 7.3 0.2
Cultural Response
Prepare Cystine Tryptic Agar per label directions with and
without 0.5% dextrose. Inoculate tubes by straight stab and
incubate at 35C for 18-48 hours.
ORGANISM
ATCC
MOTILITY
ACID PRODUCTION
w/ DEXTROSE
subsp. mitis
Escherichia coli
Neisseria gonorrhoeae (CDC 98)
8024
25922*
43070*
+
+
Uninoculated
tube
Corynebacterium
diptheriae
ATCC 8024
with dextrose
Escherichia coli
ATCC 25922
with dextrose
136
The Difco Manual
Section II
Czapek-Dox Broth & Czapek Solution Agar

Test Procedure
For a complete discussion on motility and carbohydrate fermentation
studies refer to procedures described in appropriate references.2,6,7
Results
1. Fermentation of the test carbohydrate is observed when acid is
formed and the medium turns from red to yellow.
2. Motility of an organism is evident as a haze of growth extending
into the agar from the stab line.2

2. CTA requires a heavy inoculum.5
3. Prolonged incubation may lead to changes in pH indicator or
abnormal lactose/sucrose reactions with Neisseria pathogens.8,9
4. Neisseria species usually produce acid only in the area of stabs
(upper third). If there is a strong acid (yellow color) throughout the
medium, a contaminating organism may be present. If in doubt
about a tube containing a Neisseria species, a Gram stain and
oxidase test should be performed on the growth.5
References
1. Vera, H. D. 1948. A simple medium for identification and maintenance of the gonococcus and other bacteria. J. Bacteriol. 55:531.

Scotts Diagnostic Microbiology, 9th ed. Mosby-Year Book, Inc.,
St. Louis, MO.
3. Myers, R. M., and G. Koshy. 1961. Beta-hemolytic streptococci
in survey throat cultures in an Indian population. Am. J. Public
Health 51:1872.
4. Alford, J. A., G. E. Wiese, and J. J. Gunter. 1955. Heat
resistance in Corynebacterium and the relationship of the genus to
Microbacterium. J. Bacteriol. 69:516.
5. MacFaddin, J. D. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, vol. 1, p. 254-259,
802-804. Williams & Wilkins, Baltimore, MD.
8. Faur, Y. C., M. H. Weisburd, and M. E. Wilson. 1975.
Carbohydrate fermentation plate medium for confirmation of
Neisseria species. J. Clin. Microbiol. 1:294.
9. Applebaum, P. C., and R. B. Lawrence. 1979. Comparison of
three methods for identification of pathogenic Neisseria species.
J. Clin. Microbiol. 9:598.
Packaging
500 g
0523-17
Bacto Czapek-Dox Broth

Bacto Czapek Solution Agar
Dipotassium Phosphate is the buffering agent, and Potassium Chloride

contains essential ions. Magnesium Sulfate and Ferrous Sulfate sources
of cations. Bacto Agar is the solidifying agent in Czapek Solution Agar.
Intended Use
Formula
Bacto Czapek-Dox Broth and Czapek Solution Agar are used for
cultivating fungi and bacteria capable of using inorganic nitrogen.
Czapek-Dox Broth
Formula Per Liter

Czapek-Dox Broth and Czapek Solution Agar are a modification of the
Czapek1 and Dox2 formula prepared according to Thom and Raper.3
The media are prepared with only inorganic sources of nitrogen and
chemically defined compounds sources of carbon. Czapek-Dox media
are useful in a variety of microbiological procedures, including soil
microbiology and fungi and mildew resistance tests. Thom and Raper3
reported Czapek-Dox Broth and Czapek Solution Agar will produce
moderately vigorous growth of most saprophytic aspergilli and yield
characteristic mycelia and conidia.
Czapek Solution Agar is recommended in Standard Methods for the
Examination of Water and Wastewater5 for the isolation of Aspergillus,
Penicillium and related fungi.

Saccharose is the sole carbon source, and Sodium Nitrate is the sole
nitrogen source in Czapek-Dox Broth and Czapek Solution Agar.
The Difco Manual
Sodium Nitrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Potassium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01
g
g
g
g
g
g

Czapek Solution Agar
Formula Per Liter
Sodium Nitrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g
137
Czapek-Dox Broth & Czapek Solution Agar
Section II
Precautions

Czapek-Dox Broth
Procedure
Materials Provided

Czapek-Dox Broth
Storage
Expiration Date
Glassware
Autoclave
Incubator
Czapek-Dox Broth

Czapek-Dox Broth
Solution:
deionized water. Solution is colorless,
clear to very slightly opalescent and
Prepared Medium:
Colorless, clear to very slightly
opalescent, may have slight precipitate.
Reaction of 3.5%
Solution at 25C:
pH 7.3 0.2
homogeneous.
Solution:
deionized water on boiling; light amber,
opalescent with a uniform flocculent
precipitate.
Prepared Medium:
Light amber, slightly opalescent;
may have slight precipitate.
Reaction of 4.9%
Solution at 25C:
pH 7.3 0.2
Cultural Response
Czapek-Dox Broth
Prepare Czapek-Dox Broth per label directions. Inoculate tubes
with the test organisms. Incubate inoculated medium at 30 2C
for 48-72 hours.
Prepare Czapek Solution Agar per label directions. Inoculate
prepared medium with the test organisms. Incubate at 30 2C
for 18-48 hours, or up to 72 hours if necessary.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Aspergillus niger
Candida albicans
9642
10231
100-1,000
100-1,000
good
good
138


Test Procedure
Refer to appropriate references for specific procedures for the cultivation
of fungi and bacteria capable of utilizing inorganic nitrogen.
Results
References
1. Czapek, F. 1902-1903. Untersuchungen uber die
stickstoffgewinnung und EiweiBbildung der Pflanze. Beitr.
Chem. Physiol. Pathol. 1:540.
2. Dox, A. W. 1910. The intracellular enzymes of Penicillium and
Aspergillus with special references to those of P. camenberti. U.S.
Dept. Agr. Bur. Anim. Ind. Bull. 120:70.
3. Thom, C., and K. B. Raper. 1945. Manual of the aspergilli, vol. 39.
4. Thom, C., and M. B. Church. 1926. The aspergilli. Williams and
Wilkins Co., Baltimore, MD.
5. Eaton, A. D., L. S. Cleseri, and A. E. Greenberg (ed.). 1995.
Packaging
Czapek-Dox Broth
500 g
0338-17
500 g
0339-17
The Difco Manual
Section II
DCLS Agar
Bacto DCLS Agar
Formula
DCLS Agar
Formula Per Liter
Intended Use

Bacto Proteose Peptone No.3 . . . . . . . . . . . . . . . . . . . . . . . . 7
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Saccharose (sucrose) . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Thiosulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Desoxycholate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.03
Bacto DCLS Agar is used for isolating gram-negative enteric bacilli.
Also Known As
DCLS is an abbreviation for Desoxycholate Citrate Lactose
Saccharose Agar.

DCLS Agar is a modification of SS Agar and the Desoxycholate
Citrate Agar described by Leifson1. Coliform organisms capable of
fermenting lactose or sucrose are generally inhibited. Gram positive
bacteria are suppressed.
While studying enteric pathogens on Endo medium, Holt-Harris and
Teague2 used lactose and sucrose in the development of a nutrient agar
containing methylene blue and eosin. Some coliforms ferment sucrose
more readily than lactose. The addition of sucrose (saccharose) allows
nonpathogenic sucrose-fermenting organisms to produce red colonies.
The red colonies are easily recognized, reducing the number of false
positive reactions.

Beef Extract and Proteose Peptone No. 3 provide nitrogen, vitamins
and amino acids. Lactose and Saccharose (sucrose) provide
fermentable carbohydrates. Sodium Citrate, Sodium Thiosulfate and
Sodium Desoxycholate are selective agents. Bacto Agar is the
solidifying agent. Neutral Red is the indicator.
g
g
g
g
g
g
g
g
g
Precautions
Uninoculated
plate
ATCC 14028

Dehydrated Appearance: Light beige to light pink, free-flowing,
homogeneous.
Solution:
deionized water upon boiling. After
boiling, orange-red, clear to very
slightly opalescent, without
Prepared Medium:
Orange-red, slightly opalescent.
Reaction of 4.95%
Solution at 25C:
pH 7.2 0.2
Cultural Response
Prepare DCLS Agar per label directions. Inoculate prepared
medium and incubate at 35 2C for 18-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
29212* 1,000-2,000
Escherichia coli
25922* 100-1,000

Shigella flexneri
12022* 100-1,000
COLOR OF
GROWTH
COLONY
marked to
complete inhibition
marked to
red,
complete inhibition if present
good
colorless to pink
fair to good
colorless to pink
The Difco Manual
139
D/E Neutralizing Agar & D/E Neutralizing Broth
Section II
Storage
Results

Typical coliforms that rapidly ferment sucrose and/or lactose will form
red, opaque colonies. Shigella and Salmonella species will produce
colorless to slightly pink, transparent colonies.
Expiration Date
Procedure
Materials Provided
DCLS Agar

Glassware
Incubator (35C)
1.
2.
3.
4.
5.

Heat to boiling to dissolve completely. DO NOT AUTOCLAVE.
Cool to 50-55C.
Dispense into sterile Petri dishes, or as desired.
Allow prepared medium to dry for about 2 hours with the covers
partially removed.

Test Procedure
For a complete discussion on the isolation and identification of enteric
pathogens from clinical specimens, refer to the procedures described
in appropriate references.3,4
Bacto D/E Neutralizing Agar

Bacto D/E Neutralizing Broth

this medium.
2. DO NOT AUTOCLAVE MEDIUM. DO NOT OVERHEAT.
3. DCLS Agar is intended for selective use and should be inoculated
in parallel with nonselective media.
4. Colonies suspected of being enteric pathogens must be confirmed
biochemically and, if required, serologically.
References
1. Leifson, E. 1935. New culture media based on sodium
desoxycholate for the isolation of intestinal pathogens and for
the enumeration of colon bacilli in milk and water. J. Pathol.
2. Holt-Harris, J. E., and O. Teague. 1916. A new culture medium
for the isolation of Bacillus typhosus from stools. J. Infect.
Dis. 18:596-601.
R. H. Yolken (ed.). 1995. Manual of clinical microbiology,
handbook, vol.1. American Society for Microbiology,
Washington, D.C.
Packaging
DCLS Agar
500 g
0759-17
Intended Use
chemicals. D/E Neutralizing media neutralize higher concentrations

of residual antimicrobials when compared with other standard
neutralizing formulations such as Letheen media, Thioglycollate
media, and Neutralizing Buffer.2,3
D/E Neutralizing Agar and Broth are also known as Dey-Engley

Neutralizing Agar and Broth.
Complete neutralization of disinfectants is important because

disinfectant carryover can cause a false no-growth test result. D/E
Neutralizing media effectively neutralize the inhibitory effects of
disinfectant carryover,4,5 allowing differentiation between bacteriostasis
and the true bactericidal action of disinfectant chemicals. This is a
critical characteristic to consider when evaluating a disinfectant. D/E
Neutralizing media are recommended for use in disinfectant evaluation,
environmental sampling (swab and contact plate methods) and the
testing of water-miscible cosmetics in accordance with Cosmetic,
Toiletry and Fragrance Association (CTFA) guidelines.6
D/E Neutralizing media, developed by Dey and Engley,1 neutralize

a broad spectrum of disinfectants and preservative antimicrobial
D/E Neutralizing Agar and Broth contain Tryptone which provides the
carbon and nitrogen sources required for growth of a wide variety of
Bacto D/E Neutralizing Agar is used for neutralizing and determining

the bactericidal activity of antiseptics and disinfectants.
Bacto D/E Neutralizing Broth is used for determining the bactericidal
activity of antiseptics and disinfectants based on neutralizing the
chemical and detecting organisms remaining after treatment.
Also Known as
140
The Difco Manual
Section II
organisms. Yeast Extract provides vitamins and cofactors required for

growth and additional nitrogen and carbon. Dextrose is a source of
fermentable carbohydrate. Sodium Thioglycollate neutralizes
mercurials. Sodium Thiosulfate neutralizes iodine and chlorine.
Sodium Bisulfite neutralizes formaldehyde and gluteraldehyde.
Lecithin neutralizes quaternary ammonium compounds and Polysorbate
80 neutralizes phenols, hexachlorophene, formalin and, with lecithin,
ethanol.11 Brom Cresol Purple is used as a colorimetric indicator to
demonstrate the production of acid from the fermentation of dextrose.
D/E Neutralizing Broth

Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Polysorbate 80 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Lecithin (Soy Bean) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Bacto Brom Cresol Purple . . . . . . . . . . . . . . . . . . . . . . . . 0.02
D/E Neutralizing Agar uses Bacto Agar as a solidifying agent.
Final pH 7.6 0.2 at 25 C
Formula
Precautions
D/E Neutralizing Agar

Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Polysorbate 80 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Lecithin (Soy Bean) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g

2. D/E Neutralizing Agar
HARMFUL. MAY CAUSE SENSITIZATION BY INHALATION. IRRITATING TO EYES, RESPIRATORY SYSTEM AND
Final pH 7.6 0.2 at 25 C

Dehydrated Medium: Bluish-grey, homogeneous, appears moist
and lumpy.
Solution:
water on boiling. Lavender, opaque with an
even suspension of fine particles.
Prepared Medium: Lavender, opaque with a fine precipitate.
Reaction of 5.4%
Solution at 25C:
7.6 0.2
Dehydrated Medium: Bluish-grey, homogeneous, appears moist
and lumpy.
Solution:
water on warming. Purple, opaque with an even
suspension of fine particles.
Prepared Medium: Purple, opaque with an even suspension of particles.
Reaction 3.9%
Solution at 25C:
7.6 0.2
ATCC 25923
Cultural Response
D/E Neutralizing Agar: Neutralization Test
Prepare medium per label directions. Inoculate 50 ml of D/E Neutralizing Agar with 0.1 ml of a heavy suspension of each test organism
and dispense into 150 x 15 mm Petri dishes of D/E Neutralizing Agar and Plate Count Agar. Place 1/2 inch sterile blank disks on each
plate. Dispense 0.1 ml of each disinfectant solution onto two disks per medium. Incubate at 35 2C for 40-48 hours. D/E Neutralizing
Agar should exhibit no zones of inhibition or zones significantly smaller than those found on Plate Count Agar.
The Difco Manual
141
Section II
Cultural Response
D/E Neutralizing Broth: Toxicity Test
Prepare medium per label directions with and without added
disinfectants. Inoculate with 100-1,000 CFU of test organism.
Incubate at 35 2C for 40-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
Bacillus subtilis
Escherichia coli
6633
25922
27853
14028
25923
100-1,000
100-1,000
100-1,000
100-1,000
100-1,000
GROWTH
good
good
good
good
good

Storage
Expiration Date
Procedure
Materials Provided

Glassware
Autoclave
1. Suspend 54 grams in 1 liter of distilled or deionized water.
2. Heat to boiling to dissolve.
Uninoculated
tube
Bacillus subtilis
ATCC 6633
Escherichia coli
ATCC 25922

1. Suspend 39 grams in 1 liter of distilled of deionized water.
2. Heat to dissolve.

Test Procedure
D/E Neutralizing Agar and D/E Neutralizing Broth are used in a
variety of procedures. Consult appropriate references for further
information.6
Results
References
ATCC 27853
142
ATCC 14028
ATCC 25923
1. Engley, F. B., Jr., and B. P. Dey. 1970. A universal neutralizing

medium for antimicrobial chemicals. Presented at the Chemical
Specialties Manufacturing Association (CSMA) Proceedings, 56th
Mid-Year Meeting.
2. Dey, B. P., and F. B. Engley, Jr. 1983. Methodology for recovery
of chemically treated Staphylococcus aureus with neutralizing
medium. Appl. Environ. Microbiol. 45:1533-1537.
The Difco Manual
Section II
DNase Test Agar & DNase Test Agar w/Methyl Green
3. Dey, B. P., and F. B. Engley, Jr. 1978. Environmental sampling

devices for neutralization of disinfectants. Presented at the 4th
International Symposium on Contamination Control.
4. Dey, B. P., and F. B. Engley, Jr. 1994. Neutralization of antimicrobial chemicals by recovery media. J. Microbiol. Methods 19:51-58.
5. Dey, B. P., and F. B. Engley, Jr. 1995. Comparison of Dey and
Engley (D/E) Neutralizing medium to Letheen Medium and
Standard Methods Medium for recovery of Staphylococcus aureus
from sanitized surfaces. J. Ind. Microbiol. 14:21-25.
6. Curry, A. S., J. G. Graf, and G. N. McEwen, Jr. (ed.). 1993.

CTFA Microbiology Guidelines. The Cosmetic, Toiletry and
Fragrance Association, Washington, D.C.
Packaging
500 g
10 kg
0686-17
0686-08
500 g
0819-17
Bacto DNase Test Agar

Bacto DNase Test Agar w/Methyl Green
Intended Use
Bacto DNase Test Agar and Bacto DNase Test Agar w/Methyl Green
are used for differentiating microorganisms based on deoxyribonuclease activity.

In 1956, Weckman and Catlin1 showed a correlation between increased
DNase activity of Staphylococcus aureus and positive coagulase activity. They suggested that DNase activity could be used to identify
potentially pathogenic staphylococci. DiSalvo2 confirmed their results
by obtaining excellent correlation between the coagulase and DNase
activity of staphylococci isolated from clinical specimens. Jeffries,
Holtman and Guse3 incorporated DNA in an agar medium to study

DNase production by bacteria and fungi. Polymerized DNA precipitates
in the presence of 1N HCl, making the medium opaque. Organisms
that degrade DNA produce a clear zone around an inoculum streak.
Fusillo and Weiss4 studied the calcium requirements of staphylococci
for DNase production and concluded that additional calcium was
unnecessary when a complete nutritive medium was used.
Kurnick5 showed that methyl green combines with highly polymerized
DNA at pH 7.5. When combination does not take place, the color fades,

DNase Test Agar
Solution:
is light to medium amber, very slightly
to slightly opalescent, may have a
slight precipitate.
Prepared Medium:
precipitate.
Reaction of 4.2%
Solution at 25C:
pH 7.3 0.2
DNase Test Agar w/Methyl Green
Dehydrated Appearance: Light beige with a slight green tint,
Solution:
green, very slightly to slightly opalescent,
Prepared Medium:
Green, slightly opalescent, may have a slight precipitate.
Reaction of 4.2%
Solution at 25C:
pH 7.3 0.2
ATCC 25923
DNase Test Agar
The Difco Manual
143
DNase Test Agar & DNase Test Agar w/Methyl Green
Section II
creating a clear zone around the growth. Applying this principle, Smith,
Hancock and Rhoden6 modified DNase Test Agar with added methyl green
to detect staphylococci, streptococci and Serratia. When using DNase
Test Agar w/Methyl Green, acid does not have to be added to the plate.
Mannitol fermentation can be determined simultaneously with DNase
production by adding 10 grams of mannitol and 0.025 grams of phenol
red to the DNase Test Agar prior to sterilization.7

Tryptose is a source of nitrogen, amino acids and carbon. Deoxyribonucleic Acid enables the detection of DNase that depolymerizes DNA.
Sodium Chloride provides essential ions while maintaining osmotic
balance. Methyl Green is a colorimetric indicator. Bacto Agar is a
solidifying agent.
Formula
Precautions
Storage
Expiration Date
Procedure
Materials Provided
DNase Test Agar

Formula Per Liter
DNase Test Agar

Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Deoxyribonucleic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g

Glassware
Autoclave
1N Hydrochloric acid (DNase Test Agar)

Formula Per Liter
1. Suspend 42 grams of either DNase Test Agar or DNase Test Agar
w/Methyl Green in 1 liter distilled or deionized water.
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Deoxyribonucleic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
Methyl Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05 g
ATCC 25923
Uninoculated
plate
User Control Quality cont.
Cultural Response
DNase Test Agar
Prepare DNase Test Agar or DNase Test Agar w/Methyl Green
per label directions. Inoculate and incubate at 35 2C for
18-24 and up to 48 hours. Flood DNase Test Agar (only) with
1N hydrochloric acid prior to observing DNase activity.
ORGANISM
Serratia marcescens
ATCC
INOCULUM
CFU
GROWTH
DNASE
TEST
8100*
25923*
12228*
19615*
100-1,000
100-1,000
100-1,000
100-1,000
good
good
good
good
+
+
*These cultures are available as Bactrol Disks and should be used as
ATCC 12228
144
The Difco Manual
Section II
DRBC Agar
References
1. Weckman, B. G., and B. W. Catlin. 1957. Deoxyribonuclease

activity of micrococci from clinical sources. J. Bacteriol. 73:747-753.
2. DiSalvo, J. W. 1958. Deoxyribonuclease and coagulase activity of
micrococci. Med. Tech. Bull. U. S. Armed Forces Med. J. 9:191.
3. Jeffries, C. D., D. F. Holtman, and D. G. Guse. 1957. Rapid
method for determining the activity of microorganisms on nucleic
acid. J. Bacteriol. 73:590- 591.
4. Fusillo, M. H., and D. L. Weiss. 1959. Qualitative estimation of
staphylococcal deoxyribonuclease. J. Bacteriol. 78:520.
5. Kurnick, N. B. 1950. The determination of deoxyribonuclease activity by methyl green: application to serum. Arch. Biochem. 29:41.
6. Smith, P. B., G. A. Hancock, and D. L. Rhoden. 1969. Improved
medium for detecting deoxyribonuclease-producing bacteria. Appl.
Microbiol. 18:991.
Test Procedure
1. Inoculate plates by spotting or streaking with a heavy inoculum
of the test organism. Use a spot approximately 5 mm in diameter
or a 1-2 cm streak approximately 5 mm wide.
2. Incubate plates at 35 2C for 18-24 hours and up to 48 hours.
3. DNase Test Agar: Flood the plates with 1N hydrochloric acid.
DNase Test Agar w/Methyl Green: Do NOT flood with 1N
hydrochloric acid.
4. Observe for clearing around the spot or streak. Record results.
Results
DNase Test Agar:
A zone of clearing around the spot

or streak indicates DNase activity.
DNase Test Agar

w/Methyl Green:
A decolorized zone (halo) around the spot or

streak indicates DNase activity.
Packaging
1. The composition of the culture medium, the degree of aeration,

pH, temperature and incubation period are important factors
influencing DNase activity in the culturing and testing the
micrococci.7
DNase Test Agar
100 g
500 g
0632-15
0632-17
100 g
500 g
0220-15
0220-17
Bacto DRBC Agar

Solution:
is reddish pink, very slightly to slightly
opalescent.
Prepared Medium:
Bright pink, very slightly to slightly
opalescent.
Reaction of 3.16%
Solution at 25C:
pH 5.6 0.2
Cultural Response
Prepare DRBC Agar per label directions. Inoculate and
incubate plates at 25C for 5 days.
ORGANISM
ATCC
INOCULUM
CFU
Aspergillus niger
1015
Stab
Candida albicans
Escherichia coli
Micrococcus luteus
10231
25922*
10240
GROWTH
good-colonies white to
salt and pepper to black
100-1,000 good-colonies pink,smooth, raised
1,000-2,000
none to poor
1,000-2,000
none to poor
Aspergillus niger
ATCC 1015
The Difco Manual
145
DRBC Agar
Section II
Intended Use
Bacto DRBC Agar is used for the enumeration of yeasts and molds.
Also Known As
Dichloran Rose Bengal Chloramphenicol Agar

DRBC Agar is based on the Dichloran Rose Bengal Chlortetracycline
(DRBC) Agar formula described by King, Hocking and Pitt.1 DRBC
Agar conforms with APHA guidelines for the mycological examination
of foods, containing chloramphenicol rather than chlortetracycline as
proposed by King, Hocking and Pitt.2 DRBC Agar is a selective
medium that supports good growth of yeasts and molds.
Storage
1. Store the dehydrated medium below 30C. The powder is very
2. Protect medium from light.
3. Store prepared medium in the dark at 2-8C.
Expiration Date
Proteose Peptone No. 3 provides nitrogen, vitamins and minerals.

Dextrose is a carbohydrate source. Phosphate is a buffering agent.
Magnesium Sulfate is a source of divalent cations and sulfate. The
antifungal agent, Dichloran, is added to the medium to reduce colony
diameters of spreading fungi. The pH of the medium is reduced from
7.2 to 5.6 for improved inhibition of the spreading fungi.1 The
presence of Rose Bengal in the medium suppresses the growth of
bacteria and restricts the size and height of colonies of the more
rapidly growing molds. The concentration of Rose Bengal is reduced
from 50 g/ml to 25 g/ml as found in Rose Bengal Chloramphenicol
Agar for optimal performance with Dichloran. Chloramphenicol is
included in this medium to inhibit the growth of bacteria present in
environmental and food samples. Inhibition of growth of bacteria and
restriction of spreading of more rapidly growing molds aids in the
isolation of slow-growing fungi by preventing their overgrowth by more
rapidly growing species. In addition, Rose Bengal is taken up by yeast
and mold colonies, which allows these colonies to be easily
recognized and enumerated. Reduced recovery of yeasts may be
encountered due to increased activity of Rose Bengal at pH 5.6.1 Bacto
Agar is a solidifying agent.
Formula
g
g
g
g
g
g
g
g
Precautions
2. TOXIC. MAY CAUSE CANCER. POSSIBLE RISK OF HARM
TO THE UNBORN CHILD. Avoid contact with skin and eyes. Do
tightly closed. TARGET ORGAN(S): Blood, Nerves, Lymph
Glands, Eyes.
146
Materials Provided
DRBC Agar

Peptone Water
Autoclave
Incubator (25C)
1. Suspend 31.6 grams in 1 liter of distilled or deionized water.

Prepare sample for surface inoculation following recommended
guidelines. 2,3 The use of 0.1% Peptone Water as the diluent is
recommended.
DRBC Agar
Formula Per Liter
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Potassium Phosphate Monobasic . . . . . . . . . . . . . . . . . . . . . 1
Dichloran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.002
Rose Bengal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.025
Chloramphenicol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Procedure2,3
Test Procedure
1. Inoculate 0.1 ml of appropriate decimal dilutions of the sample in
duplicate onto the surface of DRBC Agar plates. The plates should
be dried overnight at room temperature. Spread the inoculum over
the entire surface of the plate using a sterile, bent-glass rod.
2. Incubate plates upright at 22-25C. Examine for growth of yeasts
and molds after 3, 4 and 5 days incubation.
Results
Colonies of molds and yeasts should be apparent within 5 days of
incubation. Colonies of yeast appear pink due to the uptake of Rose
Bengal. Report the results as colony forming units per gram or milliliter
of sample.
References
1. King, A. D., A. D. Hocking, and J. I. Pitt. 1979. Dichloran-rose
bengal medium for the enumeration and isolation of molds from
foods. Appl. and Environ. Microbiol. 37:959-964.
The Difco Manual
Section II
Decarboxylase Differential Media
2. Mislivec, P. B., L. R. Beuchat, and M. A. Cousin. 1992. Yeasts

and molds, p. 239-249. In C. Vanderzant, and D. F. Splittstoesser,
(ed.). Compendium of methods for the microbiological examination
of foods, 3rd ed. American Public Health Association.
Washington, D.C.
3. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compendium of methods for the microbiological examination of foods,
3rd ed. American Public Health Association. Washington, D.C.
Packaging
DRBC Agar
500 g
0587-17

Bacto Decarboxylase Base Moeller . Bacto Decarboxylase
Medium Base . Bacto Lysine Decarboxylase Broth
Intended Use
Bacto Decarboxylase Base Moeller is a basal medium which, with
added lysine, arginine, ornithine, or another amino acid, is used
for differentiating bacteria based on their ability to decarboxylate
amino acids.
Bacto Decarboxylase Medium Base, with added lysine, arginine, or
ornithine, is used for differentiating bacteria based on amino acid
decarboxylation.
Bacto Lysine Decarboxylase Broth is used for differentiating microorganisms based on lysine decarboxylation.
Also Known As
Decarboxylase Base Moeller is also referred to as Moeller Decarboxylase
Broth Base. Decarboxylase Medium Base is also known as Decarboxylase
Medium Base, Falkow or Decarboxylase Basal Medium.

Moeller 1,2,3 described the amino acid decarboxylase test for distinguishing
between various microorganisms. He determined the usefulness of using
this enzyme system in the differentiation of the Enterobacteriaceae.4,5
The production of lysine, arginine, ornithine, and glutamic acid
decarboxylase by various members of this family provided a useful
adjunct to other biochemical tests used for the speciation and identification
of the Enterobacteriaceae.
Carlquist6 developed a medium using the lysine decarboxylase reaction
to differentiate Salmonella arizonae (Arizona) from Citrobacter
(Bethesda-Ballerup biotype). Falkow7 obtained valid and reliable results
with a lysine decarboxylase medium he developed to differentiate and
identify Salmonella and Shigella. Although his modification of the
Moeller formula was originally described as a lysine medium only,
further study by Falkow and then by Ewing, Davis and Edwards,8
substantiated the use of the medium for ornithine and arginine
decarboxylase reactions as well.
Ewing, Davis and Edwards8 compared the Falkow decarboxylase medium
base to the Moeller medium and reported that, although the two methods
compared favorably in most cases, the Moeller medium was found to
be more reliable for cultures of Klebsiella and Enterobacter. They concluded
that the Moeller method should be regarded as the standard or reference
The Difco Manual
method, although the Falkow formula is suitable for determining

decarboxylase reactions for most members of the Enterobacteriaceae
except for Klebsiella and Enterobacter. The Moeller medium is also
particularly useful in the identification of Aeromonas, Plesiomonas,
Vibrio spp., and nonfermentative gram- negative bacilli.9
Decarboxylase Base Moeller conforms with the Moeller formulation
while Decarboxylase Medium Base is prepared according to the formula
described by Falkow. Lysine Decarboxylase Broth is the Falkow
medium with L-Lysine added in 0.5% concentration.
Decarboxylase tests are important in the differentiation and identification
of a wide variety of microorganisms and are outlined in numerous
standard methods.10-13

Decarboxylase Base Moeller, Decarboxylase Medium Base and Lysine
Decarboxylase Broth consist of Bacto Peptone and Beef Extract which
supply carbon and nitrogen. Dextrose is a fermentable carbohydrate.
Yeast Extract provides vitamins and cofactors required for growth as
well as additional sources of nitrogen and carbon. As applicable, Brom
Cresol Purple and Cresol Red are pH indicators. The Pyridoxal is an
enzyme cofactor for the amino acid decarboxylase. The amino acids
lysine, ornithine, and arginine are added to the basal media to detect
the production of the enzymes specific for these substrates.
When the media are inoculated with bacteria that are able to ferment
the dextrose, acids are produced that lower the pH and change the
indicator from purple to yellow. If the bacteria produce the appropriate
decarboxylase, the production of amines raises the pH of the medium
causing the indicator to change from yellow to a light or deep purple.
Decarboxylation of lysine yields cadaverine, while decarboxylation
of ornithine yields putrescine. Arginine is first hydrolyzed to
ornithine and then decarboxylated to putrescine. If decarboxylation
does not occur the medium remains acidic (yellow). Control tubes of
basal media, that do not contain an amino acid, should be inoculated
to verify reactions.
To obtain proper reactions, inoculated tubes must be protected from
the air. This is done to avoid false alkalinization at the surface of the
medium, which could cause a decarboxylase negative bacteria to appear
to be positive. This can be done by overlaying a medium with sterile
mineral oil as suggested by Ewing, Davis and Edwards.8
147
Section II
Formula
Decarboxylase Medium Base

Formula Per Liter
Decarboxylase Base Moeller

Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Cresol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.005
Pyridoxal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.005
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
g
g
g
g
g
g
g
g
g
g
Dehydrated Appearance: Light to medium tan, homogeneous,
free-flowing.
Solution:
deionized water on warming.
Prepared Tubes:
Yellowish-red, slightly opalescent.
pH at 25C:
6.0 0.2
Dehydrated Appearance: Light beige, homogeneous, free-flowing.
Solution:
Prepared Tubes:
Purple, clear.
pH at 25C:
6.8 0.2
Lysine Decarboxylase Base
Solution:
Prepared Tubes:
Purple, clear w/o significant precipitate.
pH at 25C:
6.8 0.2
Escherichia coli
ATCC 25922
Escherichia coli
Shigella flexneri
ATCC 25922
ATCC 12022
w/ Lysine
Shigella flexneri
ATCC 12022
w/ Lysine
Cultural Response
Prepare media per label directions. Where necessary add
appropriate amounts of amino acids to be tested. Inoculate with
approx. 1,000 CFUs of test organisms and overlay test tubes with
sterile mineral oil. Incubate at 35 2C for 18-48 hours. Purple
color indicates a positive decarboxylase reaction. Yellow color
indicates a negative decarboxylase reaction.
ATCC
GROWTH
Escherichia coli
25922*
good
Shigella flexneri
12022*
good
ORGANISM
REACTION
w/o LYSINE
w/ LYSINE
yellow
()
yellow
()
purple
(+)
yellow
()

ORGANISM
Salmonella
typhimurium
Proteus
vulgaris
ATCC
GROWTH
14028* good
13315*
LYSINE
REACTION
ORNITHINE
ARGININE
purple
(+)
yellow
()
purple
(+)
yellow
()
purple
(+)
yellow
()
148
Uninoculated
tube
ATCC 14028
Proteus vulgaris
ATCC 13315
The Difco Manual
Section II
Procedure
Lysine Decarboxylase Broth

Formula Per Liter
Materials Provided
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
L-Lysine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
g
g
g
g
g
Precautions
Storage
Store the dehydrated media below 30C. The dehydrated media is very
Expiration Date
when stored as directed. Do not use a product if it fails to meet specifications
Lysine Decarboxylase Base

ORGANISM
Escherichia coli
Proteus vulgaris
ATCC
GROWTH
REACTION
25922*
13315*
good
good
purple (+)
yellow ()


Glassware
Autoclave
Incubator (37C)
Wire loops (bacteriological)
L-lysine, L-arginine, L-ornithine, or other L-amino acids (to be added
to either Decarboxylase Base Moeller or Decarboxylase Medium Base)
Sterile mineral oil
1N NaOH
Methods of Preparation
1. Suspend 10.5 grams in 1 liter distilled or deionized water and heat
to dissolve completely.
2. Add 10 grams L-amino acid (or 20 grams DL-amino acid) and
agitate to dissolve completely. When adding ornithine which is
highly acidic, adjust the pH with NaOH (approximately 4.6 ml 1 N
NaOH per liter) prior to sterilizing.
3. Dispense 5 ml amounts into screw capped test tubes.
1. Suspend 9 grams in 1 liter distilled or deionized water and warm to
dissolve completely.
2. Add 5 grams L-amino acid (or 10 grams DL-amino acid) and warm
to dissolve completely. Adjust the pH with NaOH (if necessary)
prior to sterilizing.

Only pure cultures of enteric bacteria taken from purification plates
or agar slants are to be used for biochemical tests. A presumptive
identification of the bacteria under investigation should be made
on the basis of morphological and cultural characteristics prior to
biochemical testing.
Test Procedure
Escherichia coli
ATCC 25922
Proteus vulgaris
ATCC 13315
The Difco Manual
1. Inoculate the prepared tubes with a 24 hour pure culture using a

bacteriological loop. A control tube should also be inoculated.
2. Aseptically overlay all inoculated tubes, including the control tube,
with 4-5 mm sterile mineral oil.
3. Incubate tubes at 35 2C for up to 4 days. Tubes must be read
daily after 24 hours incubation. Observe for a change in color from
purple to yellow to purple.
149
Demi-Fraser Broth Base & Fraser Broth Supplement
Results
See appropriate reference for the expected decarboxylase reactions of
the Enterobacteriaceae and other organisms.14

1. Biochemical characteristics of the Enterobacteriaceae serve to
confirm presumptive identification based on cultural, morphological,
and/or serological findings. Therefore, biochemical testing should
be attempted on pure culture isolates only and subsequent to
differential determinations.
2. The decarboxylase reactions are part of a total biochemical profile
for members of the Enterobacteriaceae and related organisms.
Results obtained from these reactions, therefore, can be considered indicative of a given genus or species. However, conclusive
and final identification of these organisms cannot be made solely
on the basis of the decarboxylase reactions.
3. If layers of yellow and purple appear after incubation, shake the
test tube gently before attempting to interpret results.
4. If a reaction is difficult to interpret, compare the tube in question
to an uninoculated control tube. Any trace of purple after 24 hours
of incubation is a positive test.
5. A gray color may indicate reduction of the indicator. Additional
indicator may be added before the results are interpreted.12
6. Salmonella gallinarum gives a delayed positive ornithine
decarboxylase reaction, requiring 5-6 days incubation.3 Many
strains of E. coli, including those that ferment adonitol, may
exhibit a delayed reaction.3
7. Decarboxylase Medium Base is not satisfactory for the determination
of lysine decarboxylase activity with the two genera Klebsiella and
Enterobacter.
8. The lysine decarboxylase activity in Salmonella is used to differentiate this group from Citrobacter freundii. Salmonella paratyphi A,
however, gives an atypical negative reaction (yellow color of
medium) in 24 hours when Decarboxylase Medium Base is used.15
References
1. Moeller, V. 1954. Activity determination of amino acid decarboxylases in Enterobacteriaceae. Acta Pathol. Microbiol. Scand. 34:
102-111.
2. Moeller, V. 1954. Distribution of amino acid decarboxylases in
Enterobacteriaceae. Acta Pathol. Microbiol. Scand. 34: 259-277.
3. Moeller, V. 1955. Simplified tests of some amino acid decarboxylases for arginine dihydrolase system. Acta Pathol. Microbiol.
Scand. 36: 158-172.
Bacto Demi-Fraser Broth Base

Bacto Fraser Broth Supplement
Section II
4. Gale, E. F. 1940. The production of amines by bacteria. Biochem.

J. 34: 392, 583, 846.
5. Gale, E. F. 1941. Production of amines by bacteria. 4. The
decarboxylation of amino-acids by organisms of the groups
Clostridium and Proteus. Biochem. J. 35: 66-79.
6. Carlquist, P. R. 1956. A biochemical test for separating paracolon
groups. J. Bacteriol. 71: 339-341.
7. Falkow, S. 1958. Activity of lysine decarboxylase as an aid in the
identification of Salmonella and Shigella. Am. J. Clin. Pathol. 29: 598.
8. Ewing, W. H., B. R. Davis, and P. R. Edwards. 1960. The
decarboxylase reaction of Enterobacteriaceae and their value in
taxonomy. Publ. Health Lab. 18: 77-83.
9. Baron, E J., L R. Peterson, and S. M. Finegold (eds.). 1994.
10. Harmon, S. M., D. A. Kautter, D. A. Golden, and E. J.
Rhodehamel. 1995. Bacteriological analytical manual, 8th ed.
AOAC International, Arlington, VA.
13. Greenberg, A. E., L. S. Clesceri, and A. D. Eaton (eds.). 1995.
Packaging
100 g
500 g
0890-15
0890-17
500 g
10 kg
0872-17
0872-08
100 g
500 g
10 kg
0215-15
0215-17
0215-08
Intended Use
Bacto Demi-Fraser Broth Base is used with Bacto Fraser Broth
Supplement in selectively and differentially enriching Listeria from
foods.
150

Fraser Broth Base and Fraser Broth Supplement are based on the Fraser
Broth formulation of Fraser and Sperber.1 The medium is used in the
rapid detection of Listeria from food and environmental samples.
Demi-Fraser Broth Base is a modification of Fraser Broth Base in
which the nalidixic acid and acriflavine concentrations have been
reduced to 10 mg/l and 12.5 mg/l respectively, in accordance with
AFNOR guidelines.2
The Difco Manual
Section II
Demi-Fraser Broth Base & Fraser Broth Supplement

Tryptose, Beef Extract and Yeast Extract provide carbon and nitrogen
sources and the cofactors required for good growth of Listeria. Sodium
Phosphate and Potassium Phosphate buffer the medium. Selectivity is
provided by Lithium Chloride, Nalidixic Acid and Acriflavine. The
high Sodium Chloride concentration of the medium inhibits growth of
enterococci.
All Listeria species hydrolyze esculin, as evidenced by a blackening
of the medium. This blackening results from the formation of
6,7-dihydroxycoumarin, which reacts with ferric ions.1 Ferric ions are
added to the final medium as Ferric Ammonium Citrate in Fraser
Broth Supplement.
Formula
Demi-Fraser Broth Base
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Potassium Phosphate, Monobasic . . . . . . . . . . . . . . . . . . 1.35
Esculin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Nalidixic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01
Acriflavine HCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0125
g
g
g
g
g
g
g
g
g
g
Fraser Broth Supplement

Ingredients per 10 ml vial
Ferric Ammonium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
One vial is added to one liter of basal medium
Precautions
2. Demi-Fraser Broth Base: HARMFUL. IRRITATING TO EYES,
RESPIRATORY SYSTEM AND SKIN. MAY CAUSE HARM TO
THE UNBORN CHILD. Avoid contact with skin and eyes. Do not
breathe dust. Wear suitable protective clothing. Keep container
tightly closed. TARGET ORGAN(S): Blood, Kidneys, Nerves.
Fraser Broth Supplement: IRRITANT. IRRITATING TO EYES,
RESPIRATORY SYSTEM AND SKIN. Avoid contact with skin
and eyes. Do not breathe mist. Wear suitable protective clothing.

Solution:
water. Solution is medium amber, clear to slightly
opalescent, may have a fine precipitate.
Prepared Medium:
Medium amber, very slightly to slightly opalescent,
Reaction of 5.5%
Solution at 25C:
pH 7.2 0.2
Solution Appearance: Dark brown solution.
Cultural Response
Prepare Demi-Fraser Broth per label directions. Inoculate and incubate
ORGANISM
ATCC
INOCULUM
CFU
GROWTH/APPEARANCE
Escherichia coli
25922* 1,000-2,000
inhibited
Listeria monocytogenes 19114 100-1,000 good growth/blackening of the medium
29212* 1,000-2,000
markedly to completely inhibited
Uninoculated
tube
Listeria monocytogenes
ATCC 19114
The cultures listed above are the minimum that should be used for performance testing.
The Difco Manual
151
Desoxycholate Agar
Section II

Sample Collection and Preparation
Storage
Test Procedure2

1. Pre-enrich the sample in Demi-Fraser Broth. Incubate for 18-24 hours

at 35 2C. Subculture onto Oxford Medium or PALCAM Medium.
2. Transfer 0.1 ml of the pre-enrichment culture into 10 ml of Fraser
Broth and incubate for 48 hours at 37C. Subculture onto Oxford
Medium or PALCAM Medium after 18-24 hours and again after
42-48 hours of incubation.
3. Examine Oxford Medium or PALCAM Medium plates for the
appearance of presumptive Listeria colonies.
4. Confirm the identity of all presumptive Listeria by biochemical
and/or serological testing.
Store Fraser Broth Supplement at 2-8C.

Expiration Date
Procedure
Materials Provided

Glassware
Autoclave
Fraser Broth
Oxford Medium
PALCAM Medium
1. Dissolve 55 grams of Demi-Fraser Broth Base in 1 liter distilled or
deionized water.
3. Aseptically add 10 ml Fraser Broth Supplement. Mix well.
Bacto Desoxycholate Agar
Results
The presence of Listeria is presumptively indicated by the blackening
of Demi-Fraser Broth after incubation for 24-48 hours at 35C.
Confirmation of the presence of Listeria is made following subculture
onto appropriate media and biochemical/serological identification.
References
1. Fraser, J., and W. Sperber. 1988. Rapid detection of Listeria in
food and environmental samples by esculin hydrolysis. Journal of
Food Protection 51:762- 765.
2. Lassociation franaise de normalisation (AFNOR). 1993. Food
Microbiology- Detection of Listeria monocytogenes-Routine
Method, V 08-055. AFNOR, Paris, France.
Packaging
500 g
10 kg
0653-17-0
0653-07-0
6 x 10 ml
0211-60-2
Intended Use
Bacto Desoxycholate Agar is used for isolating and differentiating
gram-negative enteric bacilli.
Also Known As
Deoxycholate Agar (Sodium Deoxycholate Agar)
NOTE: Alternate spelling1 - Deoxy-.

Desoxycholate Agar as formulated by Leifson2 demonstrated improved
recovery of intestinal pathogens from specimens containing normal
intestinal flora. The medium was an improvement over other
media of the time because the chemicals, citrates and sodium
desoxycholate, in specified amounts, worked well as inhibitors. This
medium has been used to screen for Salmonella sp. and Shigella sp.
from clinical specimens.3
152

Bacto Peptone provides nitrogen and carbon for general growth
requirements. Lactose is the fermentable carbohydrate. Sodium
chloride and dipotassium phosphate maintain the osmotic balance of
the medium. Sodium desoxycholate, ferric citrate and sodium citrate
inhibit growth of gram-positive bacteria. Neutral red is a pH indicator.
Differentiation of enteric bacilli is based on fermentation of lactose.
Bacteria that ferment lactose produce acid and, in the presence of
neutral red, form red colonies. Bacteria that do not ferment lactose
form colorless colonies. The majority of normal intestinal bacteria
ferment lactose (red colonies) while Salmonella and Shigella species
do not ferment lactose (colorless colonies).
Formula
Desoxycholate Agar
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
The Difco Manual
Section II
Desoxycholate Agar
Sodium Desoxycholate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Ferric Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.03
g
g
g
g
g
g
g
Bunsen burner or heating plate

Incubator (35C)
Petri dishes
2. Boil 1 minute with frequent, careful agitation to dissolve completely.
Avoid overheating.
3. DO NOT AUTOCLAVE.
Precautions
Storage
For a complete discussion on the isolation of enteric bacilli, refer to

appropriate procedures outlined in the references.

Test Procedure
Results
Expiration Date
References
Procedure
Materials Provided
Desoxycholate Agar
1. MacFaddin, J. F. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, p. 269-275, vol 1.

desoxycholate for the isolation of intestinal pathogens and for the
enumeration of colon bacilli in milk and water. J. Pathol. Bacteriol.
40:581-599.
Glassware

Dehydrated Appearance: Pinkish beige, free-flowing, homogeneous.
Solution:
is reddish orange, very slightly to
slightly opalescent with no
Prepared Medium:
Orange, slightly opalescent.
Reaction of 4.5%
Solution at 25C:
pH 7.3 0.2
Cultural Response
Prepare Desoxycholate Agar per label directions. Inoculate
using the pour plate method and incubate plates at 35 2C
for 18-24 hours.
ORGANISM
Escherichia coli
ATCC
INOCULUM
CFU
GROWTH
APPEARANCE
29212* 1,000-2,000 marked to
complete inhibition
25922* 30-300
good
pink w/bile precipitate
14028* 30-300
good
colorless
ATCC 13048
The Difco Manual
153
Desoxycholate Citrate Agar
Section II
Packaging
3. Balows, A., W. J. Mausler, K. L. Herrmann, H. D. Isenberg,

and H. J. Shadony (ed.) 1991. Manual of clinical microbiology.
Desoxycholate Agar
500 g
0273-17
Bacto Desoxycholate Citrate Agar
Intended Use
Bacto Desoxycholate Citrate Agar is used for isolating enteric bacilli,

particularly Salmonella and many Shigella species.
Infusion from Meat is a source of carbon and nitrogen. This ingredient

is used because the inhibition of coliforms produced is greater than
when an extract or simple peptone is used.2 Desoxycholate Citrate Agar
contains Proteose Peptone No. 3 as a source of carbon, nitrogen,
vitamins and minerals. Lactose is a carbohydrate. Sodium Citrate and
Sodium Desoxycholate inhibit gram positive bacteria, coliforms and
Proteus species. Ferric Ammonium Citrate aids in the detection of H2S
producing bacteria. Neutral Red is a pH indicator. Bacto Agar is a
solidifying agent.
Also Known As
NOTE: Deoxy-; alternate spelling.1

Desoxycholate Citrate Agar is a modification of Desoxycholate Agar
formulated by Leifson.2 His original medium demonstrated improved
recovery of intestinal pathogens from specimens containing normal
intestinal flora by using citrates and sodium desoxycholate in specified
amounts as inhibitors to gram positive bacteria.
In the presence of neutral red, bacteria that ferment lactose produce

acid and form red colonies. Bacteria that do not ferment lactose form
colorless colonies. If the bacteria produce H2S, the colonies will have
black centers. The majority of normal intestinal bacteria ferment
lactose and do not produce H2S (red colonies without black centers).
Salmonella and Shigella spp. do not ferment lactose but Salmonella
may produce H2S (colorless colonies with or without black centers).
Lactose-fermenting colonies may have a zone of precipitation around
them caused by the precipitation of desoxycholate in the presence of acid.
Leifson modified his original medium by increasing the concentration

of sodium citrate and sodium desoxycholate and found Desoxycholate
Citrate Agar reliable for isolating many Salmonella and Shigella
species.2
Desoxycholate Citrate Agar effectively isolates intestinal pathogens
(Salmonella and Shigella species) by inhibiting coliforms and many
Proteus species.1 This medium is widely used by clinical laboratories.3
Uninoculated
plate
ATCC 14028

Dehydrated Appearance: Pinkish-beige, free-flowing, homogeneous.
Solution:
water on boiling. Solution is orange-red,
Prepared Medium:
Orange-red, slightly opalescent.
Reaction of 7.0%
Solution at 25C:
pH 7.5 0.2
Cultural Response
Prepare Desoxycholate Citrate Agar per label directions.
ORGANISM
Enterococcus
faecalis
Escherichia
coli
Salmonella
typhimurium
ATCC
INOCULUM
CFU
29212* 1,000-2,000
GROWTH
APPEARANCE
marked to
complete inhibition
25922* 100-1,000
partial
pink with bile
inhibition
precipitate
14028* 100-1,000
fair to good
colorless
H 2S
154
The Difco Manual
Section II
Desoxycholate Lactose Agar
Formula
2. Heat and boil briefly with frequent, careful agitation to dissolve

completely. Avoid overheating.

Formula Per Liter
Meat, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Ferric Ammonium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Sodium Desoxycholate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.02
g
g
g
g
g
g
g
g
Precautions
Storage
Expiration Date

Test Procedure
1. Inoculate specimen directly onto surface of medium.
2. Incubate plates at 35 2C for 18-24 hours. Plates can be incubated
for an additional 24 hours if no lactose fermenters are observed.
Results
Non-lactose fermenters produce transparent, colorless to light pink or
tan colored colonies with or without black centers. Lactose fermenters
produce a red colony with or without a bile precipitate.

1. Coliform strains may be encountered that will grow on this
medium, making it difficult to detect pathogens.
2. Heavy inocula should be distributed over the entire surface of the
medium prevent complete masking of pathogens by coliform
organisms.
References
Glassware
Petri dishes
Incubator (35C)

40: 581-599.
3. Farmer III, J. J., and M. T. Kelly. 1991. Enterobacteriaceae. p.
360-383. In A. Balows, W. J. Hausler, Jr., K. L. Herrmann, H. D.
Isenberg and H. J. Shadomy (ed.), Manual of clinical microbiology,
Packaging
Procedure
Materials Provided
500 g
0274-17
Bacto Desoxycholate Lactose Agar
Intended Use
Bacto Desoxycholate Lactose Agar is used for isolating and
differentiating gram-negative enteric bacilli and for enumerating
coliforms from water, wastewater, milk and dairy products.
Also Known As
NOTE: Deoxy-; alternate spelling.1

Desoxycholate Lactose Agar is a modification of Desoxycholate
Agar formulated by Leifson.2 His original medium demonstrated
improved recovery of intestinal pathogens from specimens containing
The Difco Manual
normal intestinal flora by using citrates and sodium desoxycholate

in specified amounts as inhibitors to gram-positive bacteria.
Standard Methods manuals for dairy3 and water4 specified a modification
of Desoxycholate Agar to contain less sodium desoxycholate and,
accordingly, be less inhibitory to gram-positive bacteria. This
formulation, known as Desoxycholate Lactose Agar, was used in pour
plate procedures for isolation and enumeration of coliforms in milk,
water and other specimens.

Bacto Peptone provides nitrogen and carbon for general growth
requirements. Lactose is a fermentable carbohydrate. Sodium
155
Section II
Chloride maintains the osmotic balance of the medium. Sodium

Desoxycholate and Sodium Citrate inhibit growth of gram-positive
bacteria. Neutral Red is a pH indicator. Bacto Agar is a solidifying
agent.
Expiration Date
Differentiation of enteric bacilli is based on fermentation of lactose.

Bacteria that ferment lactose produce acid and, in the presence of
neutral red, form red colonies. Bacteria that do not ferment lactose
form colorless colonies. The majority of normal intestinal bacteria
ferment lactose (red colonies) while Salmonella and Shigella species
do not ferment lactose (colorless colonies).
Procedure
Materials Provided

Glassware
Bunsen burner or heating plate
Incubator(35C)
Petri dishes
Formula
Formula Per Liter
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.03
g
g
g
g
g
g
g
2. Boil 1 minute with frequent, careful agitation to dissolve
completely. Avoid overheating.
Precautions

Test Procedure
Storage
Results

Uninoculated
plate
Escherichia coli
ATCC 25922

Dehydrated Appearance: Pinkish beige, free-flowing, homogeneous.
Solution:
is pinkish-red, very slightly to slightly
opalescent.
Prepared Medium:
Pinkish-red, very slightly to slightly
opalescent.
Reaction of 4.25%
Solution at 25C:
pH 7.1 0.2
Cultural Response
Prepare Desoxycholate Lactose Agar per label directions. Inoculate
the medium and incubate at 35 2C for 18-24 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
APPEARANCE
Enterococcus faecalis 29212* 1000-2,000 markedly inhibited
Escherichia coli
25922* 30-300
good
pink w/bile precipitate
Salmonella typhimurium 14028* 30-300
good
colorless
156
The Difco Manual
Section II
Dextrose Agar & Dextrose Broth
References
1. MacFaddin, J. F. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, p. 269-275, vol 1.
40:581-599.
3. American Public Health Association. 1960. Standard methods
Bacto Dextrose Agar

Bacto Dextrose Broth
for the examination of dairy products microbiological and

chemical, 11th ed. American Public Health Association,
Washington, D.C.
for the examination of water and wastewater, 11th ed. American
Packaging
500 g
0420-17
Intended Use
Bacto Dextrose Agar is used for cultivating a wide variety of
microorganisms with or without added blood.
Bacto Dextrose Broth is used for cultivating fastidious microorganisms
and for detecting gas from enteric bacilli.

In 1932, Norton1 recommended a basal medium containing 0.5-1%
dextrose with approximately 5% defibrinated blood for the isolation of
many fastidious bacteria, including Haemophilus and Neisseria.
Dextrose is an energy source used by many organisms. The high
concentration of this ingredient makes Dextrose Agar a suitable
medium for the production of early, abundant organism growth and

Dextrose Agar
Dehydrated Appearance: Medium beige, homogeneous,
free-flowing.
Solution:
deionized water on boiling; medium
opalescent.
Prepared Medium:
Plain - Medium amber, slightly
precipitate.
With blood - Cherry-red, opaque.
Reaction of 4.3%
Solution at 25C
pH 7.3 0.2
Dextrose Broth
Solution:
deionized water; light to medium amber,
Prepared Medium:
Light to medium amber.
Reaction of 2.3%
Solution at 25C:
pH 7.2 0.2
The Difco Manual
shortening the lag periods of older cultures. Because of the increased

dextrose content, Dextrose Agar is not suitable for observation of
hemolysis when supplemented with 5% sheep, rabbit or horse blood.
Dextrose Broth is a highly nutritious broth suitable for the isolation of
fastidious organisms and specimens containing a low inoculum. The
addition of 0.1-0.2% agar to Dextrose Broth facilitates anaerobic
growth and aids in dispersion of reducing substances and CO2 formed
in the environment.2 The low agar concentration provides suitable
conditions for both aerobic growth in the clear upper zone and for
microaerophilic and anaerobic growth in the lower, flocculent agar zones.
Dextrose Agar and Dextrose Broth are specified in the Compendium
of Methods for the Microbiological Examination of Foods.3

Beef Extract and Tryptose provide nitrogen, amino acids and vitamins.
Dextrose is a carbon source, and the increased concentration is a distinguishing characteristic of this medium from other formulations used
Cultural Response
Dextrose Agar
Prepare Dextrose Agar per label directions with and without sterile
defibrinated sheep blood. Inoculate and incubate at 35 2C
under proper atmospheric conditions for 18-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
w/o BLOOD w/5% SHEEP BLOOD
Neisseria meningitidis 13090* 100-1,000

Streptococcus pyogenes 19615* 100-1,000
poor
good
good
good
good
good
Dextrose Broth
Prepare Dextrose Broth per label directions with and without
0.1% Bacto Agar; dispense into tubes containing fermentation
vials. Inoculate and incubate at 35 2C under proper
atmospheric conditions. Read growth and gas production at
15-24 and 40-48 hours.
ORGANISM
Escherichia coli
ATCC GROWTH
25922*
13090*
19615*
25923*
good
good
good
good
CFU
100-1,000
100-1,000
100-1,000
100-1,000
GAS
GROWTH
PRODUCTION w/1% AGAR
good
good
good
good
157
Dextrose Starch Agar
Section II
as blood agar bases. Bacto Agar is a solidifying agent.

Supplementation with 5% blood provides additional growth factors for

Formula
Dextrose Agar
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g

Dextrose Broth
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
g
g
g
g
Precautions
Dextrose Agar
4. OPTIONAL: To prepare blood agar, aseptically add 5% sterile
defibrinated blood to the medium at 45-50C. Mix well.
Dextrose Broth
2. Dissolve in distilled or deionized water.
4. Dispense into tubes.

Specimens are obtained and processed according to the techniques and
Test Procedure
For a complete discussion on microorganism isolation and identification, refer to appropriate references.
Results
Storage

Expiration Date

References
Procedure
Materials Provided
Dextrose Agar
Dextrose Broth

Glassware
Autoclave
Incubator (35C)
1. Norton. 1932. Bacteriology of pus. J. Lab. Clin. Med. 17:558-565.

3. Vanderzant, C. and D. F. Splittstoesser (ed.). 1992. Compendium
Packaging
Dextrose Agar
Dextrose Broth
500 g
500 g
0067-17
0063-17
Bacto Dextrose Starch Agar
Intended Use
Bacto Dextrose Starch Agar is used for cultivating pure cultures of
Neisseria gonorrhoeae and other fastidious microorganisms.

Dextrose Starch Agar is recommended as a complete solid medium for
158
the propagation of pure cultures of Neisseria gonorrhoeae. This

highly nutritious medium without additives will also support
excellent growth of N. meningitidis, Streptococcus pneumoniae and
S. pyogenes. Dextrose Starch Agar, in half concentration, is
recommended as a Stock Culture Agar for the maintenance of
N. gonorrhoeae, N. meningitidis and other organisms not capable
The Difco Manual
Section II
of hydrolyzing starch. This medium cannot be used to maintain

stock cultures of organisms capable of splitting starch; acid production
from starch will create an unsatisfactory environment.
Dextrose Starch Agar was used by Wilkins, Lewis and Barbiers1 in an
agar dilution procedure to test the activity of antibiotics against
Neisseria species.

Proteose Peptone No. 3 and Gelatin provide the nitrogen, vitamins and
amino acids in Dextrose Starch Agar. Soluble Starch improves growth
response. Dextrose is a carbon source. Sodium chloride maintains the
osmotic balance of the medium, and disodium phosphate is a buffering
agent. Bacto Agar is the solidifying agent.
Formula
Formula Per Liter
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
g
g
g
g
g
g
g
Precautions
Storage
Expiration Date
Procedure
Material Provided

Glassware
Autoclave
Incubator (35C)
1.
2.
3.
4.


Test Procedure
N. gonorrhoeae and other fastidious pathogens, refer to the procedures
described in Clinical Microbiology Procedures Handbook2 and Manual
of Clinical Microbiology.3

Solution:
6.5 % solution, soluble in distilled
or deionized water on boiling; light
amber, opalescent with a precipitate.
Prepared Medium:
Light amber, opalescent with a
precipitate.
Reaction of 6.5%
Solution at 25C
pH 7.3 0.2
Cultural Response
Prepared Dextrose Starch Agar per label directions. Incubate
inoculated medium at 35 2C for 18-48 hours under
5-10% CO2.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
CDC 98*
13090 98*
19615 98*
100-1,000
100-1,000
100-1,000
good
good
good
The Difco Manual
Results

2. This medium is not recommended for isolation of gonococci from
mixed cultures.
References
1. Wilkins, Lewis, and Barbiers. 1956. Antibiot. Chemother. 6:149.
2. Isenberg, H. D. (ed.) 1992. Clinical microbiology procedures handbook, vol.1. American Society for Microbiology, Washington, D.C.
3. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover and
Packaging
500 g
10 kg
0066-17
0066-08
159
Dextrose Tryptone Agar
Section II
Bacto Dextrose Tryptone Agar
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
Bacto Brom Cresol Purple . . . . . . . . . . . . . . . . . . . . . . . . 0.04 g
Intended Use
Bacto Dextrose Tryptone Agar is used for cultivating thermophilic
flat-sour microorganisms associated with food spoilage.

In the 1930s, the National Canners Association specified the use of
Dextrose Tryptone Agar for isolating flat sour organisms from food
products.1 Flat sour spoilage of canned foods is caused by Bacillus
coagulans (Bacillus thermoacidurans). Bacterial growth results in a
0.3-0.5 drop in pH, while the ends of the can remain flat. B. coagulans
is a soil microorganism that can be found in canned tomato products
and dairy products. Conditions favorable for multiplication of the
bacterium can result in spoilage of the food product.2
Dextrose Tryptone Agar can also be used to isolate other food spoilage
bacteria: mesophilic aerobic spore formers in the genera Bacillus and
Sporolactobacillus and thermophilic flat sour spore formers such as
B. stearothermophilus.2
Precautions
Storage
Expiration Date
Procedure
Materials Provided

Dextrose Tryptone Agar contains Tryptone to provide carbon and nitrogen
sources for general growth requirements. Dextrose is the carbohydrate
source. Brom Cresol Purple is the pH indicator. Bacto Agar is the
solidifying agent.
Formula
Formula Per Liter

Glassware
Autoclave
Petri dishes
Incubator
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Uninoculated
plate
Bacillus coagulans
ATCC 7050

Dehydrated Appearance: Light, greenish-beige, free-flowing,
homogeneous.
Solution:
deionized water on boiling; purple, very
slightly to slightly opalescent without
Prepared Medium:
Purple, slightly opalescent without
Reaction of 3.0%
Solution at 25C:
pH 6.7 0.2
Cultural Response
Prepare Dextrose Tryptone Agar per label directions. Inoculate
plates and incubate at 55C for 36-48 hours. Examine cultures for
growth. A change in the color of the medium from purple to yellow
indicates dextrose fermentation.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
APPEARANCE
Bacillus coagulans
7050
7953
100-1,000
100-1,000
good
good
yellow
yellow
160
The Difco Manual
Section II
Differential Reinforced Clostridial Agar
Results
1.
2.
3.
4.

Cool to room temperature.
References
1. National Canners Association. 1933. Bacterial standards
for sugar.

Test Procedure
Packaging
Dextrose Tryptone Agar
500 g
0080-17
Bacto Differential Reinforced Clostridial Agar
Intended Use
Differential Reinforced Clostridial Agar (DRCA) is based on

Differential Reinforced Clostridial Medium, but with the addition
of agar.
Bacto Differential Reinforced Clostridial Agar is used for enumerating

and cultivating sulfite-reducing clostridia.
The assay is performed using unheated and heat shocked tubes of

DRCA containing replicate dilutions of the test sample. Blackening of
the medium is presumptive evidence for the presence of sulfite-reducing
clostridia. In this method, heat shocked tubes showing blackening are
confirmatory for clostridia. Non-heat shocked tubes showing blackening
Also Known As
Differential Reinforced Clostridial Agar is also known as DRCA.

Differential Reinforced Clostridial Medium was developed by Gibbs
and Freame in 1965 1. The medium could be used to enumerate
clostridia in foods using the Most Probable Number (MPN) method.
Uninoculated
plate
Clostridium septicum
ATCC 12464

Solution:
is light to medium amber, clear to
slightly opalescent while hot; upon
cooling, solution becomes light red.
Prepared Medium:
Light pink, very slightly to slightly
opalescent.
Reaction of 4.25%
Solution at 25C:
pH 7.1 0.2
Cultural Response
Prepare Differential Reinforced Clostridial Agar per label
directions. Inoculate and incubate at 35C in an anaerobic
environment for 72 hours.
ORGANISM
ATCC
APPROXIMATE
INOCULUM CFU
RECOVERY
BLACK COLONIES
Clostridium bifermentans
Clostridium septicum
638
12924
12464
100
100
100
Good
Good
Good
+
+
+
The Difco Manual
161
Section II
must be heat shocked to kill off vegetative cells and subcultured into
DRCA to confirm the presence of sulfite-reducing clostridia.
Incubator (35C)
Ringers solution or 0.1% peptone water
Tryptone, Bacto Peptone, Beef Extract, Yeast Extract, Starch, and

L-Cysteine provide nutrients and co-factors required for good growth
of clostridia. Dextrose is included in the medium as an energy source.
Partial selectivity of the medium is achieved through the addition of
Sodium Acetate. Bacto Agar has been incorporated into this medium
as a solidifying agent. Anaerobiosis in the medium is detected by the
redox indicator Resazurin. The addition of Ferric Ammonium Citrate
to the medium is used to detect sulfite reduction. Blackening of the
medium is due to the formation of iron sulfide.
1.
2.
3.
4.
Formula
g
g
g
g
g
g
g
g
g
g
g
g
Precautions
Storage
hygroscopic. Keep container tightly closed. Store prepared medium
at 2-8C.
Expiration Date
Procedure
Materials Provided
Material Required But Not Provided

Anaerobic Jar Complete
Autoclave
162

the laboratory following recommended guidelines.4,5
2. Process each sample using procedures appropriate for that
sample.4,5
Test Procedure

Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Beef Extract, Desiccated . . . . . . . . . . . . . . . . . . . . . . 8
L-Cysteine HCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Resazurin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.002
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Suspend 42.5 grams in 1 liter of distilled or deionized water.

Dispense 10 ml portions into tubes.
1. Prepare serial 10-fold dilutions of the sample in 1/4 strength

Ringers solution or 0.1% peptone water.
2. Depending on the amount of the initial sample, transfer 1 ml or 0.1
ml of the appropriate dilution, prepared in step 1, to the bottom of
a molten (45-50C) DRCA tube. Prepare a duplicate tube using the
same procedure.
3. Tighten the caps on the tubes.
4. Heat one of the duplicate DRCA tubes prepared in step 2 to 80 1C
for 10 minutes to kill vegetative cells.
5. Incubate both tubes, heat-shocked and non-heat-shocked, at 35 1C
for 5 days; examine for sulfite reduction.
Results
The presence of clostridia is presumptively indicated by blackening in
the medium. Heat-shocked tubes showing blackening should be
considered confirmatory for the presence of sulfite-reducing clostridia.

1. Non-heat-shocked cultures showing blackening must be heat
shocked and subcultured to DRCA for confirmation.
References
1. Gibbs, B. M., and B. Freame. 1965. Methods for the recovery of
clostridia from foods. J. Appl. Microbiol. 28:95-143.
2. Miller, N. J., O. W. Gerrett, and T. S. Prickett. 1939. Anaerobic
technique, a modified deep agar shake. Food Research 4:447-51.
3. Mikrobiologische Untersuchungsverfahren gem Anlage 3 (zu
4 Abs. 3) der Mineral-und Tafelwasserverordnung vom 1.8. 1984,
Untersuchung auf sulfitreduzierende, sporenbildende Anaerobier.
5. MacFaddin, J. F. 1985. Media for isolation-cultivation-identificationmaintenance of medical bacteria, vol. 1. Williams & Wilkins,
Baltimore, MD.
Packaging
500 g
0641-17
The Difco Manual
Section II
Dubose Albumin Broth, Dubos Broth Base, Dubos Medium Albumin, Dubos Oleic Agar Base & Dubos Oleic Albumin Complex
Bacto Dubos Albumin Broth . Bacto Dubos Broth Base

Bacto Dubos Medium Albumin . Bacto Dubos Oleic Agar Base
Bacto Dubos Oleic Albumin Complex
Intended Use
Bacto Dubos Broth Base is used with Bacto Dubos Medium Albumin
for rapidly cultivating pure cultures of Mycobacterium tuberculosis.
tuberculosis each year.1 During the mid 1980s, the number of tuberculosis (TB) cases in the U.S. began increasing. Prior to this time, the
number of cases in the U.S. had been decreasing, reaching a low in
1984.2 Non-tuberculous mycobacteria infections have also increased
since the mid 1980s.3
Bacto Dubos Oleic Agar Base is used with Bacto Dubos Oleic
Albumin Complex and penicillin for isolating and determining the
susceptibility of Mycobacterium tuberculosis.
Dubos Broth is prepared according to the Dubos, Fenner and Pierce4

modification of the medium originally described by Dubos and Davis5
and Dubos and Middlebrook.6
Dubos and Middlebrook6 described Dubos Oleic Medium Albumin as

suitable for primary isolation and cultivation of the tubercle bacillus
and for studying colony morphology. In comparative studies, Dubos
Oleic Albumin Agar Medium was superior to other media studied for
primary isolation.7,8
Bacto Dubos Albumin Broth is used for rapidly cultivating

Mycobacterium tuberculosis.
Mycobacterial infections, particularly tuberculosis, are a worldwide

health problem. Almost three million people worldwide die of

Dubos Albumin Broth
Appearance:
Almost colorless, clear to very
Reaction of
Solution at 25C:
pH 7.0 0.2
Dubos Broth Base
Solution:
deionized water. Solution is very light
to light amber, clear, may have a
slight precipitate.
Reaction of 0.65%
Solution at 25C:
pH 6.6 0.2
Dubos Medium Albumin
Appearance:
Very light amber, clear liquid.
Reaction of
Solution at 25C:
pH 6.6 0.2
Dubos Oleic Agar Base
Solution:
opalescent with fine precipitate.
Reaction of 2%
Solution at 25C:
pH 6.6 0.2
Dubos Oleic Albumin Complex
Appearance:
Light amber, clear liquid without
precipitate.
Reaction of
Solution at 25C:
pH 6.8 0.2
There are two types of solid culture media for the primary isolation of
mycobacteria, those that have coagulated egg as a base and those that
have agar. Lowenstein formulations are examples of media that
contain egg; Middlebrook and Dubos formulations contain agar.
Agar based media are not liquified by contaminating proteolytic
organisms but overgrowth may occur. These media are recommended
for specimens from nonsterile sites.9 The medium is clear so colonies
of mycobacteria can be viewed through a stereo microscope even if
contaminating organisms are present. Colonies can be observed in 10
to 12 days.
Drugs may be added to Dubos media in exact concentrations because
the medium is solidified with agar rather than by inspissation. Also,
there is less drug inactivation when egg ingredients are not present.
Mycobacteria grow more rapidly in broth media. Primary culture of all
specimens in broth media is recommended.10 Tween 80 in the medium
acts as a surfactant, dispersing the bacilli, which increases growth.

Casitone and Asparagine are sources of nitrogen. Disodium Phosphate
and Monopotassium Phosphate are sources of phosphates and,
along with Calcium Chloride, help maintain the pH of the medium.
Magnesium Sulfate, Ferric Ammonium Sulfate, Zinc Sulfate and
Copper Sulfate are sources of trace metals and sulfates. Bacto Agar is
the solidifying agent.
Formula
Dubos Albumin Broth
Formula Per Liter
Bacto Dubos Broth Base . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5 g
Distilled or Deionized Water . . . . . . . . . . . . . . . . . . . . . . 900 ml
Bacto Dubos Medium Albumin . . . . . . . . . . . . . . . . . . . . 100 ml
The Difco Manual
163
Dubose Albumin Broth, Dubos Broth Base, Dubos Medium Albumin, Dubos Oleic Agar Base & Dubos Oleic Albumin Complex
Dubos Broth Base

Formula Per Liter
Bacto Casitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5

Bacto Asparagine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Tween 80 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
Disodium Phosphate (Anhyd.) . . . . . . . . . . . . . . . . . . . . . . 2.5
Ferric Ammonium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . 50
Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Zinc Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
Copper Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
g
g
g
g
g
mg
mg
mg
mg
mg

A 5% solution of albumin fraction V from bovine plasma and
7.5% dextrose in normal saline.

Formula Per Liter
Bacto Casitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Bacto Asparagine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Disodium Phosphate (Anhyd.) . . . . . . . . . . . . . . . . . . . . . . 2.5
Ferric Ammonium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . 50
Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Zinc Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
Copper Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
mg
mg
mg
mg
mg
g
Cultural Response
Dubos Albumin Broth
Prepare medium from Dubos Broth Base and Dubos Medium
Albumin per label directions or use prepared Dubos Albumin
Broth. Inoculate and incubate at 35 2C under 5-10% CO2
for up to 21 days.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Mycobacterium fortuitum
Mycobacterium intracellulare
Mycobacterium kansasii
Mycobacterium scrofulaceum
Mycobacterium tuberculosis H37 Ra
6841
13950
12478
19981
25177
300-1,000
300-1,000
300-1,000
300-1,000
300-1,000
good
good
good
good
good
Dubos Oleic Agar

Prepare medium from Dubos Oleic Agar Base and Dubos Oleic
Albumin Complex per label directions. Inoculate and incubate
at 35 2C under 5-10% CO2 for up to 21 days.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Escherichia coli
25922* 1,000-2,000 partial

inhibition
6841 300-1,000 good
13950 300-1,000 good
12478 300-1,000 good
19981 300-1,000 good
Mycobacterium tuberculosis H37 Ra 25177 300-1,000 good
164
Section II
A 0.05% solution of alkalinized oleic acid in a 5% solution of albumin

fraction V in normal saline (0.85%).
Precautions
3. Dubos Broth Base
IRRITATING TO EYES, RESPIRATORY SYSTEM AND
Storage
Store Dubos Broth Base and Dubos Oleic Agar Base dehydrated
below 30C. The dehydrated medium is very hygroscopic. Keep
container tightly closed.
Store Dubos Albumin Broth, Dubos Medium Albumin and Dubos
Oleic Albumin Complex at 2-8C.
Store prepared media at 2-8C.
Expiration Date
Procedure
Materials Provided
Dubos Albumin Broth
Dubos Broth Base

Glycerol
Penicillin (for preparing Dubos Oleic Agar Base)
Glassware
Autoclave
Incubator (CO2, 35C)
Dubos Broth
1. Dissolve 1.3 grams Dubos Broth Base in 180 ml distilled or
deionized water (or 170 ml water and 10 ml Glycerol).
The Difco Manual
Section II
2.
3.
4.
5.
m E Agar & Esculin Iron Agar

Cool below 50C.
Aseptically add 20 ml Dubos Medium Albumin and mix thoroughly.
Dispense into tubes.
Dubos Oleic Agar

1. Suspend 4 grams Dubos Oleic Agar Base in 180 ml distilled or
deionized water.
4. Cool to 50-55C.
5. Aseptically add 20 ml Dubos Oleic Albumin Complex and 5,000
-10,000 units penicillin (25-50 units per ml medium).
6. Mix thoroughly.
7. Dispense into sterile tubes or plates.
Gross contamination interfered with the growth of the

mycobacteria.
Proper aerobic conditions and increased CO2 tension were not
provided during incubation.
2. Mycobacteria are strict aerobes and growth is stimulated by
increased levels of CO2. Screw caps on tubes or bottles should
remain loose for a free exchange of CO2.
References
Mycobacteria grow on the medium or in the broth.
1. Musser, J. M. 1995. Antimicrobial agent resistance in Mycobacteria:

molecular genetic insights. Clin. Microbiol. Rev. 8:496-514.
2. Klietmann, W. 1995. Resistance and susceptibility testing for
Mycobacterium tuberculosis. Clin. Microbiol. Newsletter 17:65-69.
3. Nolte, F. S., and B. Methcock. 1995. Mycobacterium, p. 400-437.
Yolken (ed.), Manual of clinical microbiology, 6th ed. American
4. Am. Rev. Tuberculosis, 1950, 61:66.
5. J. Exp. Med., 1946, 83:409.
6. Am. Rev. Tuberc., 1947, 56:334.
7. A. Rev. Tuberculosis, 1950, 61:563.
8. Am. J. Clin. Path., 1950, 20:678.
9. Isenberg, H. D. (ed.). 1994. Clinical microbiology procedures handbook, suppl. 1. American Society for Microbiology, Washington, D.C.
10. Tenover, F. C., J. T. Crawford, R. E. Huebner, L. J. Geiter,
C. R. Horsburgh, Jr., and R. C. Good. 1993. The resurgence of
tuberculosis: is your laboratory ready? J. Clin. Microbiol. 31:767-770.
Packaging
1. Negative culture results do not rule out active infection by mycobacteria. Some factors that are responsible for unsuccessful cultures are:
The specimen was not representative of the infectious material,
i.e., saliva instead of sputum.
The mycobacteria were destroyed during digestion and
decontamination of the specimen.
Dubos Albumin Broth

1. Collect specimens in sterile containers and transport immediately
Test Procedure
1. Inoculate the specimen onto/into the medium and incubate tubes
for up to eight weeks.
2. Examine tubes for growth.
Results
Bacto m E Agar
Bacto Esculin Iron Agar
Intended Use
Bacto m E Agar is used with nalidixic acid and triphenyl tetrazolium
chloride in isolating and differentiating enterococci from water by
membrane filtration and in an in situ esculin test on Bacto Esculin Iron
Agar.
Bacto Esculin Iron Agar is used for enumerating enterococci from water
by membrane filtration based on esculin hydrolysis.
Also Known As
Esculin Iron Agar is abbreviated as EIA.
The Difco Manual
Dubos Broth Base

20 tubes
500 g
12 x 20 ml
500 g
12 x 20 ml
1022-39
0385-17
0309-64
0373-17
0375-64

Enterococcus species are a subgroup of fecal streptococci that
includes E. faecalis, E. faecium, E. gallinarum, and E. avium. 1
Enterococci are differentiated from other streptococci by their
ability to grow in 6.5% sodium chloride, at pH 9.6, and at 10C and
45C.1 The enterococci portion of the fecal streptococcus group
is a valuable bacterial indicator for determining the extent of fecal
contamination of recreational surface waters.1
Slanetz and Bartley2 first reported quantitating enterococci by the
membrane filter method in 1957. A wide range of levels of enterococci
in water can be enumerated and detected because small or large
volumes of water can be analyzed by the membrane filter technique.3
In 1961, Kenner et al. 4 described the KF method for detecting
and quantitating fecal streptococci. In 1966, Isenberg et al.5 reported
a plating procedure with differentiation based on esculin hydrolysis.
Levin, Fischer and Cabelli6 compared the KF method with Isenbergs
165
Section II
plating method, and found the latter method resulted in better

recovery of fecal streptococci. They developed m E Agar as a primary
isolation medium for enterococci, and Esculin Iron Agar as an
in situ substrate test medium for identifying organisms capable of
hydrolyzing esculin.6
Two research projects by the Environmental Protection Agency (EPA)
evaluated the relationships between swimming-associated illness
and the ambient densities of indicator bacteria. 7,8 The studies
demonstrated that enterococci have a better correlation with
swimming-associated illness for both marine and fresh waters than
fecal coliforms. Escherichia coli has a correlation in fresh water equal
to enterococci but does not correlate as well in marine waters.7,8 This
suggests that enterococci may be better indicator organisms for some
recreational waters.7,8
m E Agar and Esculin Iron Agar are prepared according to the
formulas specified in Standard Methods.1 These media are used in
the membrane filter technique for the isolation of fecal streptococcus
and enterococcus groups.1 This procedure can be used to test marine
and fresh water sources.
m E Agar with the addition of 0.075% indoxyl B-D glucoside
(m EI Agar) is recommended by the U.S. EPA as a one step procedure
for the isolation and identification of enterococci in recreational
water.9 This method is used in the EPA Beaches Environmental
Assessment Closure and Health (BEACH) Program. m EI Agar

eliminates the necessity of transferring the incubated membrane to
Esculin Iron Agar.

m E Agar is a highly selective and differential primary isolation
medium that supports good growth of enterococci. Bacto Peptone
and Yeast Extract provides carbon, nitrogen, minerals, vitamins
and other growth factors for organism growth. Sodium Chloride
maintains the osmotic balance of the medium. Nalidixic Acid and
Sodium Azide act as selective agents to inhibit gram negative
bacteria. Actidione inhibits fungi. At the concentration in the
formula, 2,3,5 triphenyl tetrazolium chloride (TTC) dyes enterococci
colonies. TTC slightly inhibits growth of other microorganisms.
In addition, the elevated incubation temperature of 41C inhibits
some indigenous microbial flora. Esculin is hydrolyzed by enterococci
to form esculetin and dextrose. The esculetin reacts with the iron
salt (ferric ammonium citrate) contained in the medium to produce
a black to reddish brown complex that appears in the medium
surrounding the colonies. The production of black to reddish brown
complex verifies the colonies as enteroccci and facilitates their
enumeration. Bacto Agar is the solidifying agent in the medium.
m E Agar
ATCC 29212

m E Agar
Solution:
upon boiling. Light to medium amber with bluish cast,
Prepared Medium:
Light to medium amber with blue cast, slightly opalescent.
Reaction of 7.12%
Solution at 25C:
pH 7.1 0.2
Esculin Iron Agar
Dehydrated Appearance:
Solution:
amber with
Prepared Medium:
Reaction of 1.65%
Solution at 25C:
Tan to dark tan, free-flowing, homogeneous.

1.65%, soluble in distilled or deionized water upon boiling. Medium
blue cast, very slightly opalescent without significant precipitate.
Medium amber with blue cast, slightly opalescent without precipitate.
Esculin Iron Agar

ATCC 29212
pH 7.1 0.2
Cultural Response
Prepare m E Agar per label directions and pour into 9 x 50 mm plates. Dilute the test organisms and filter through membrane filters.
Place the filters on m E Agar plates and incubate the plates in an upright position for 48 hours at 41 0.5C. Remove the filters and
place over prepared Esculin Iron Agar plates. After 20 minutes incubation at 41 0.5C, count colonies giving positive esculin
reaction (formation of black or reddish brown precipitate).
ORGANISM
Escherichia coli
ATCC
INOCULUM CFU/10 ml
GROWTH ON m E AGAR
REACTION ON ESCULIN IRON AGAR
29212*
33186
25922*
20-60
20-60
20-60
good/pink to red colonies

good/pink to red colonies
black or red/brown ppt

black or red/brown ppt
inhibited
*These cultures are available as BactrolTM Disks and should be used as directed in Bactrol Disks Technical Information.
166
The Difco Manual
Section II
Formula
m E Agar
Formula Per Liter
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Esculin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Actidione . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05
Sodium Azide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.15
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g

Esculin Iron Agar
Formula Per Liter
Esculin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
Precautions
2. m E Agar
HARMFUL BY INHALATION AND IF SWALLOWED.
(US) IRRITATING TO EYES, RESPIRATORY SYSTEM AND
SKIN. (US) Avoid contact with skin and eyes. Do not breathe dust.
TARGET ORGAN(S): Cardiovascular, Lungs, Nerves
FIRST AID: In case of contact with eyes, rinse immediately
with plenty of water and seek medical advice. After contact with
skin, wash immediately with plenty of water. If inhaled,
remove to fresh air. If not breathing, give artificial respiration.
If breathing is difficult, give oxygen. Seek medical advice. If
swallowed seek medical advice immediately and show this
container or label.
Storage
Expiration Date
Procedure
Materials Provided
m E Agar
Esculin Iron Agar

Bacto TTC Solution 1%
Nalidixic acid
Indoxyl -D glucoside (optional)
The Difco Manual

Membrane filter equipment
Sterile pipettes
Sterile 47 mm, 0.45 m, gridded membrane filters
Autoclave
Glassware
Dilution blanks
41C incubator or waterbath
Fluorescent lamp
Magnifying lens
m E Agar
3. Autoclave at 121C for 15 minutes. Cool to 45C.
4. Add 0.024 grams of nalidixic acid and 1.5 ml TTC Solution 1%
(0.015 grams triphenyl tetrazolium chloride).
5. Adjust to pH 7.1 if necessary.
6. Dispense 4-5 ml into 9 x 50 mm Petri dishes.
Note: Nalidixic acid is soluble in water with an alkaline pH.
Esculin Iron Agar
4. Dispense 4-5 ml into 9 x 50 mm Petri dishes.

Collect water samples as described in Standard Methods for the
Examination of Water and Wastewater.1
Test Procedure
1. Follow the membrane filter procedure described in Standard
Methods for the Examination of Water and Wastewater.1
2. Choose a sample size so that 20-60 colonies will result.
3. Place the filter on an m E Agar plate and incubate for 48 hours
at 41 0.5C.
4. After incubation, remove the filter from m E Agar and place
it on Esculin Iron Agar plate. Retain at room temperature for
approximately 20-30 minutes.
5. Incubate Esculin Iron Agar at 41 0.5C for 20 minutes.
Results
Pink to red enterococci develop a black or reddish-brown
precipitate on the underside of the filter.1 Count colonies using a
fluorescent lamp and a magnifying lens.1 Report results as estimated
number or organisms per 100 ml of water.

this medium.
2. m E Agar and Esculin Iron Agar should be used in sequence.
167
EC Medium
Section II
3. Incubation at 41C is recommended.

4. Approximately 10% false-positive esculin reactions may be expected.
When used as m EI Agar, U.S. EPA reports a 6.0% false positive
and 6.5% false negative rate with mE Agar.
References
2. Slanetz, L. W., and C. H. Bartley. 1957. Numbers of enterococci
in water, sewage, and feces determined by the membrane filter
technique with an improved medium. J. Bacteriol. 74:591-595.
3. ASTM. 1996. Annual book of ASTM standards. Section 11,
Water and environmental technology. PCN: 01-110296-16.
ASTM, West Conshohocken, PA.
4. Kenner, R. A., H. F. Clark, and P. W. Kabler. 1960. Fecal
streptococci I. Cultivation and enumeration of streptococci in
surface waters. Appl. Microbiol. 9:15-20.
Bacto EC Medium
5. Isenberg, H. D., D. Goldberg, and J. Sampson. 1970.

Laboratory studies with a selective enterococcus medium. Appl.
6. Levin, M. A., J. R. Fischer, and V. J. Cabelli. 1975. Membrane
filter technique for enumeration of enterococci in marine waters.
Appl. Microbiol. 30:66-70.
7. Cabelli, V. J. 1981. Health effects criteria for marine recreational
waters. U.S. Environmental Protection Agency. EPA-600/1-80-031.
Cincinnati, OH.
8. Dufour, A. P. 1983. Health effects criteria for fresh recreational
waters. U.S. Environmental Protection Agency. Cincinnati, OH.
9. U.S. Environmental Protection Agency. 1997. EPA method 1600:
Membrane filter test method for enterococci in water. U.S. Environmental Protection Agency. EPA-821-R-97-004. Washington, D.C.
Packaging
m E Agar
100 g
500 g
0333-15
0333-17
Esculin Iron Agar
100 g
0488-15
Intended Use
Bacto EC Medium is used for differentiating and enumerating
coliforms in water, wastewater, shellfish and foods.
Also Known As
EC Medium was developed by Hajna and Perry1 in an effort to

improve the methods for the detection of the coliform group and
E. coli. This medium consists of a buffered lactose broth with the
addition of 0.15% Bile Salts No. 3. Growth of spore forming
bacteria and fecal streptococci is inhibited by the bile salts, while
growth of E.coli is enhanced by its presence. The medium can be
EC Medium is also referred to as EC Broth. EC is an abbreviation for

Escherichia coli.

Dehydrated Appearance Light beige, free-flowing,
homogeneous.
Solution:
deionized water. Light amber, clear.
Prepared Medium:
Light amber, clear.
Reaction of 3.7%
Solution at 25C:
pH 6.9 0.2
Cultural Response
Prepare EC Medium per label directions. Inoculate tubes with
the test organisms, and incubate at 44.5 0.2C for 24 2
hours. Read tubes for growth and gas production.
ORGANISM
ATCC
Enterococcus faecalis 19433

Escherichia coli
25922*
Escherichia coli
8739
INOCULUM
CFU
GROWTH
GAS
PRODUCTION
1,000
1,000
1,000
inhibited
good
good
+
+
Uninoculated
tube
Escherichia coli
ATCC 25922
*This culture is available as Bactrol Disk and should be used as directed in Bactrol Disk Technical Information.
168
The Difco Manual
Section II
EC Medium
used at 37C for the detection of coliform organisms or at 45.5C

for the isolation of E. coli.
Procedure
In a further evaluation of EC Medium and Lauryl Tryptose Broth,

Perry and Hajna 2 reported the results obtained from eleven
different laboratories examining a variety of waters, milk and
shellfish. The results indicate that the media are highly specific
for coliform bacteria. Fishbein and Surkiewicz 3 used the EC
confirmation test for recovery of E. coli from frozen foods and nut
meats. This study3 showed that the test is optimal when conducted
at 45.5C, with incubation limited to 24 hours.
EC Medium
EC Medium is employed in elevated-temperature tests for

distinguishing organisms of the total coliform group that also
belong to the fecal coliform group.4 The fecal coliform test, using
EC Medium, is applicable to investigations of drinking water,
stream pollution, raw water sources, wastewater treatment systems,
bathing waters, seawaters and general water-quality monitoring.
Prior enrichment in presumptive media is required for optimum
recovery of fecal coliforms when using EC Medium.
4,5,6
EC Medium is used in standard methods for food and water testing.
Tryptose provides the nitrogen, vitamins and amino acids in

EC Medium. Lactose is the carbon source. Bile Salts No. 3 is the
selective agent against gram positive bacteria, particularly
bacilli and fecal streptococci. Dipotassium Phosphate and
Monopotassium Phosphate are the buffering agents. Sodium Chloride
maintains the osmotic balance of the medium.
Glassware
Fermentation vials
Autoclave
Incubator or waterbath
1.
2.
3.
4.

Warm slightly to dissolve completely.

by laboratory policy or standard methods.4,5,6
Follow the methods and procedures as stated in standard methods.4,5,6
Results
Gas production with growth in EC Medium within 24 hours or less is
considered a positive fecal coliform reaction. Failure to produce gas
with little or no growth, is a negative reaction.4
Formula
EC Medium
Formula Per Liter
g
g
g
g
g
g
Precautions
Storage
Expiration Date
when stored as directed. Do not use a product if it fails to meet the
The Difco Manual
Test Procedure
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Bile Salts No. 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Materials Provided

2. False-negative reactions in recovering coliforms from water supplies
can occur due to low pH, refrigeration and use of bactericidal or
bacteriostatic agents.7
References
1.
2.
3.
4.
Hajna and Perry. 1943. Am. J. Public Health 33:550.

Hajna and Perry. 1944. Am. J. Public Health 34:735.
Fishbein and Surkiewicz. 1964. Appl. Microbiol. 12:127.
Eaton, A. D., L. S. Clesceri, and A. E. Greenberg (ed.). 1995.
7. Ray, B. 1986. Impact of bacterial injury and repair in food
microbiology: Its past, present and future. J. Food Prot. 49:651.
Packaging
EC Medium
100 g
500 g
10 kg
0314-15
0314-17
0314-08
169
EC Medium with MUG
Section II
Bacto EC Medium with MUG
Intended Use
Bacto EC Medium with MUG is used for detecting Escherichia coli
in water, food and milk.
Also Known As
EC is an abbreviation for Escherichia coli.

EC Medium was developed by Hajna and Perry1 to improve the
methods for the detection of coliforms and E. coli. This medium
consists of a buffered lactose broth with the addition of 0.15% Bile
Salts No. 3. Growth of spores formers and fecal streptococci were
inhibited by the bile salts, while growth of E. coli is enhanced. EC
Medium with MUG is the same formula as EC Medium with the
addition of 4-methylumbelliferyl--D-glucuronide.
Feng and Hartman2 developed a rapid assay for E. coli by incorporating
4-methylumbelliferyl--D-glucuronide (MUG) into Lauryl Tryptose
Broth at a final concentration of 100 g/ml. Robison3 compared the
fluorogenic assay with present methodology and found that total
agreement between the two methods was 94.8%. Moburg4 determined
the amount of MUG could be reduced to a final concentration of
50 g/ml without adversely affecting results. Koburger and Miller5
recommended the incorporation of MUG into EC Broth for use in
testing shellfish.
EC Medium with MUG is prepared according to the formula specified
by US EPA6 and standard methods for water and food testing.7,8
Tryptose provides the nitrogen, vitamins and amino acids in EC

Medium with MUG. Lactose is the carbon source in this medium. Bile
Salts No. 3 is the selective agent against gram-positive bacteria,
particularly bacilli and fecal streptococci. Dipotassium Phosphate and
Monopotassium Phosphate are buffering agents. Sodium Chloride
maintains the osmotic balance of the medium.
E. coli produces the enzyme glucuronidase that hydrolyzes MUG to
yield a fluorogenic product that is detectable under long-wave (366 nm)
UV light. The addition of MUG to EC Medium provides another
criterion, in addition to growth response and gas production, to
determine the presence of E. coli in food and environmental samples.
Formula
EC Medium with MUG
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
MUG (4-methylumbelliferyl--D-glucuronide) . . . . . . . 0.05
g
g
g
g
g
g
g
Precautions

Dehydrated Appearance:
Solution:
Prepared Medium:
Reaction of 3.71%
Solution at 25C:
Light beige, free-flowing, homogeneous.

3.71% solution, soluble in distilled or deionized water; light amber, clear.
Light amber, clear.
pH 6.9 0.2
Cultural Response
Prepare EC Medium with MUG per label directions. Inoculate tubes in duplicate. Incubate the
first set at 35 2C for 24 hours and the second set at 44.5 0.2C. Read fluorescence under
a long-wave UV light.
ORGANISM
Escherichia coli
ATCC
INOCULUM
CFU
GROWTH AT
35C/GAS
GROWTH AT
44.5C/GAS
FLUORESCENCE
13048*
25922*
19433*
100-1,000
100-1,000
100-1,000
good/+
good/+
inhibited/
inhibited/
good/+
inhibited/
*These cultures are available as Bactrol Disks and should be used as directed in Bactrol Disks
170
Escherichia coli
ATCC 25922
The Difco Manual
Section II
EE Broth Mossel
Storage
Expiration Date
Procedure
Materials Provided
EC Medium with MUG

Test tubes
Fermentation vials
Sterile pipettes
Incubator 35C, 44.5C
Long-wave UV lamp
Autoclave
1.
2.
3.
4.
5.

Dispense into test tubes containing inverted fermentation vials.
Before opening the autoclave, allow the temperature to drop below
75C to avoid entrapping air bubbles in the fermentation vials.

Collect food, water or other environmental samples in accordance with
recommended procedures.6,7,8
Test Procedure
Follow the methods and procedures as stated in appropriate references.6,7,8
Results
Following incubation, observe tubes for growth, production of gas and
fluorescence. Positive gas production is demonstrated by displacement
of the medium from the fermentation vial. Positive MUG reactions
exhibit a bluish fluorescence under long-wave (approximately 366 nm)
UV light. Typical strains of E. coli are positive for both gas production
Bacto EE Broth Mossel
and fluorescence. Non-E. coli coliforms that grow may exhibit

fluorescence but will not produce gas.

2. Strains of E. coli that fail to grow in EC Medium with MUG, fail to
produce gas, or fail to produce glucuronidase may infrequently be
encountered.
3. Strains of Salmonella, Shigella and Yersinia that produce glucuronidase may be encountered. These strains must be distinguished
from E. coli on the basis of other parameters, i.e., gas production,
growth at 44.5C.
4. The presence of endogenous glucuronidase in shellfish samples
may cause false positive fluorescent reactions at the presumptive
stage. To prevent this problem, the use of EC Medium with MUG
in the confirmatory stage has been recommended.5
References
1. Hajna and Perry. 1943. Am. J. Public Health 33:550.
2. Feng, P. C. S., and P. A. Hartman. 1982. Fluorogenic assays for
immediate confirmation of Escherichia coli. Appl. Environ.
Microbiol. 43:1320-1329.
3. Robison, B. J. 1984. Evaluation of a fluorogenic assay for detection
of Escherichia coli in foods. App. Environ. Microbiol. 48:285-288.
4. Moberg, L. J. 1985. Fluorogenic assay for rapid detection of
Escherichia coli in food. Appl. Environ. Microbiol. 50:1383-1387.
5. Koburger, J. A., and M. L. Miller. 1985. Evaluation of a
fluorogenic MPN procedure for determining Escherichia coli in
oysters. J. Food Prot. 48:244-245.
6. Federal Register. 1991. National primary drinking water regulation;
analytical techniques; coliform bacteria. Fed. Regist. 56:636-643.
Packaging
EC Medium with MUG
100 g
500 g
0022-15
0022-17
Intended Use
Bacto EE Broth Mossel is used for selectively enriching and detecting
Enterobacteriaceae, particularly from foods.

EE Broth Mossel is prepared according to the formula of Mossel,
Visser and Cornelissen.1 The formula contains dextrose to facilitate
growth of most Enterobacteriaceae, thus insuring the detection of
Salmonella and other lactose- negative organisms. EE Broth Mossel
The Difco Manual
should be used as an enrichment broth, followed by a selective

medium, e.g., Violet Red Bile Agar.
The enumeration of Enterobacteriaceae is of great concern in monitoring
the sanitary condition of food. Enterobacteriaceae can be injured in
food-processing procedures, which include exposure to low temperatures,
sub-marginal heat, drying, radiation, preservatives or sanitizers.2
Recovery relies on proper resuscitation of damaged cells.

Tryptose provides nitrogen, vitamins and amino acids. Dextrose
is a carbon source. Disodium Phosphate and Monopotassium
171
EE Broth Mossel
Section II
Phosphate are buffering agents. Brilliant Green and Oxgall are

selective agents.
Formula
EE Broth Mossel
Formula Per Liter
Expiration Date
Procedure
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Brilliant Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0135
Bacto Oxgall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
g
g
g
g
g
g
Materials Provided
EE Broth Mossel

Glassware
Autoclave
Incubator (35C)
Waterbath
Precautions
Storage
2. Dispense 120 ml amounts into 250 ml flasks.
3. Heat at 100C (waterbath or flowing steam) for 30 minutes.

Test Procedure
1. Inoculate flasks of EE Broth Mossel with approximately 10 grams
of homogenized food or other material to be tested.
2. Shake the inoculated medium thoroughly for a few seconds to mix well.
3. Incubate for a total of 20-24 hours at 35-37C. Shake the flasks
after the first 3 hours of incubation.

Dehydrated Appearance: Light green, homogeneous, free-flowing.
Solution:
4.5% solution, soluble in distilled or deionized water;
emerald green and clear.
Prepared Medium:
Emerald green, clear.
Reaction of 4.5%
Solution at 25C
pH 7.2 0.2
Cultural Response
Prepare EE Broth Mossel per label directions. Inoculate the medium and incubate
at 35 2C for 18-24 hours and up to 48 hours if necessary.
ORGANISM
Escherichia coli
ATCC
INOCULUM
CFU
GROWTH
ACID
PRODUCTION
25922*
25923*
100-1,000
100-1,000
good
inhibited
+ (yellow)
172
Uninoculated
tube
Escherichia coli
ATCC 25922
The Difco Manual
Section II
EMB Agar
4. Prepare plates such as Violet Red Bile Agar for streaking. To ensure
recovery of dextrose fermenters, add 1% dextrose before boiling.
5. Streak a loopful of the enrichment culture onto the prepared plates.
6. Incubate the plates for 18-24 hours at 35-37C. Examine for the
presence of coliforms which appear pink to purplish-red on Violet
Red Bile Agar. The color of coliform colonies may vary if a different
medium is used.
For a complete discussion on Enterobacteriaceae in food testing, refer
to procedures in Standard Methods.3,4
References
Acid production causes the color of EE Broth Mossel to become

yellow. A negative reaction results in no color change and the medium
remains green.
1. Mossel, D. A. A., M. Vissar, and A. M. R. Cornellisen. 1963. The

examination of foods for Enterobacteriaceae using a test of the
type generally adopted for the detection of salmonellae. J. Appl.
2. Hartman, P. A., and S. A. Minnich. 1981. Automation for rapid
identification of Salmonella in foods. J. Food Prot. 44:385-386.
of food, 3rd ed. American Public Health Association,
Washington, D.C.
Packaging

EE Broth Mossel
Results
Bacto EMB Agar
500 g
10 kg
0566-17
0566-08
Intended Use
Bacto EMB Agar is used for isolating and differentiating gramnegative enteric bacilli.
Also Known As
EMB Agar is also known as Eosin Methylene Blue Agar
The original Eosin Methylene Blue Agar was the formulation of HoltHarris and Teague.1 The use of eosin and methylene blue as indicators
gave sharp and distinct differentiation between colonies of lactose
fermenting and nonfermenting organisms. Sucrose was included in the
medium to detect members of the coliform group that fermented
sucrose more readily than lactose. Lactose-positive colonies were black
Escherichia coli
ATCC 25922
Uninoculated
plate

Dehydrated Appearance: Pinkish purple, free flowing, homogeneous.
Solution:
is green with orange cast, opalescent
with a uniform flocculent precipitate.
Prepared Plates:
Purple with a greenish-orange cast,
opalescent, may have a fine
precipitate.
Reaction of 3.6%
Solution at 25C:
pH 7.2 0.2
Cultural Response
Prepare EMB Agar per label directions. Inoculate and incubate
ORGANISM
Enterococcus
faecalis
Escherichia
coli
Salmonella
typhimurium
ATCC
INOCULUM
CFU
29212*
1,000
GROWTH
APPEARANCE
colorless
25922* 100-1,000
partial
inhibition
good
14028* 100-1,000
good
blue-black w/dark centers

and green metallic sheen
colorless to amber
ATCC 14028
The Difco Manual
173
EMB Agar
Section II
or possessed dark centers with transparent, colorless peripheries.

Lactose- or sucrose-negative colonies were colorless. The Eosin
Methylene Blue Agar of Holt-Harris and Teague had definite
advantages over the Fuchsin Sulfite Agar of Endo. The EMB Agar
formulation was more sensitive, more accurate, more stable, and gave
an earlier differentiation between the lactose fermenters and lactose
and sucrose nonfermenters.
Two years after Holt-Harris and Teague had introduced their new
medium, Levine 2 described an Eosin Methylene Blue Agar for
differentiating fecal and nonfecal coliforms. Levines medium
differentiated salmonellae and other lactose nonfermenters from the
coliform organisms.
EMB Agar is a combination of the Levine and the Holt-Harris and
Teague formulae. EMB Agar is selective due to the presence of inhibitors
and differential based on the ability of some organisms to ferment
carbohydrates with the absorption of eosin and methylene blue.
EMB Agar is recommended for use in examining clinical specimens
for enteric pathogens.3,4,5 The medium enables the isolation and differentiation of gram-negative enteric bacilli.

Peptone is a source of nitrogen and other nutrients in the formulation.
Eosin and methylene blue are dyes which combine to form a precipitate
at an acid pH. The dyes act both as pH indicators and inhibitors.
Gram-positive bacteria are partially inhibited on the medium. Lactose
and Sucrose are fermentable carbohydrates. Phosphate acts as a buffer.
Formula
EMB Agar
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Sucrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5
Eosin Y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4
Methylene Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.065
g
g
g
g
g
g
g
Precautions
Storage
Store the dehydrated medium below 30C. The dehydrated medium is very
hygroscopic. Keep container tightly closed. Store prepared plates at 2-8C.
Expiration Date
Expiration date applies to the product in its intact container when stored
as directed. Do not use a product if it fails to meet specifications for
identity and performance.
Procedure
Materials Provided
EMB Agar
174

Autoclave
Waterbath (45-50)C
Petri dishes
Incubator (35C)
1.
2.
3.
4.
5.

Dispense into sterile Petri dishes. Evenly disperse the precipitate
when dispensing.

guidelines.3,4,5
2. For specific information about specimen preparation and inoculation
of clinical specimens, consult appropriate references.3,4,5
Test Procedure
For isolation of enteric pathogens from clinical specimens, inoculate
fecal specimens and rectal swabs onto a small area of one quadrant
of the EMB Agar plate and streak for isolation. This will permit
development of discrete colonies. Incubate plates at 35C. Examine
plates at 24 hours and again at 48 hours for colonies with characteristic
morphologies associated with potential pathogens.
Results
Salmonella and Shigella colonies are translucent and amber colored or
colorless. Coliforms that use lactose and/or sucrose produce blue-black
colonies with dark centers and greenish metallic sheen. Other coliforms
such as Enterobacter form mucoid, pink colonies. Strains of
Enterococcus faecalis are partially inhibited on this medium and
appear as colorless colonies.

1. EMB Agar is only moderately inhibitory. Some staphylococci,
streptococci and yeast may grow. They will appear as small,
pinpoint colonies. Gram-negative nonfermenting bacilli may grow
and appear as non-lactose fermenters. Biochemical tests are necessary
for further identification to genus or species.6
2. Some strains of Salmonella and Shigella may not grow on EMB
Agar.6 It is recommended that a nonselective, differential medium
(MacConkey Agar or Hektoen Enteric Agar) and a selective medium
(Bismuth Sulfite Agar, SS Agar or Desoxycholate Citrate Agar) be
run in parallel with EMB Agar.
3. Sterilization reduces the methylene blue, leaving the medium
orange in color. The normal purple color of the medium may be
restored by gentle mixing. If the reduced medium is not shaken to
oxidize the methylene blue, a dark zone beginning at the top and
extending downward through the medium will gradually appear.
The sterilized medium normally contains a flocculent precipitate
which should not be removed. By cooling to 50C and gently
The Difco Manual
Section II
EVA Broth
mixing the medium before pouring it into plates, the flocculation

will be finely dispersed.
4. Greenish metallic sheen is not always present. The presence of the
greenish metallic sheen is not diagnostic for E. coli.6
5. Store and incubate EMB Agar plates in the dark. Visible light can
alter the ability of the medium to support microbial growth,
especially of Proteus spp.7
References
for the isolation of Bacillus typhosa from stools. J. Infect. Dis.
18:596-600.
2. Levine, M. M. 1918. Differentiation of E. coli and B. aerogenes
on a simplified Eosin-Methylene Blue Agar. J. Infect. Dis. 23:43.
Bacto EVA Broth
Intended Use
Bacto EVA Broth is used for detecting and confirming enterococci in
water and other specimens as an indication of fecal contamination.
Also Known As
EVA Broth is also known as Ethyl Violet Azide Broth.

H. D. Isenberg, (ed.), Clinical microbiology procedures handbook,
St. Louis, MO.
7. Girolami, R. L., and J. M. Stamm. 1976. Inhibitory effect of light
on growth-supporting properties of Eosin Methylene Blue Agar.
Appl. Environ. Microbiol. 31:141.
Packaging
EMB Agar
100
500
2
10
g
g
kg
kg
0076-15
0076-17
0076-07
0076-08
Dextrose Broth presumptively identified the streptococci. However,

because gram-positive bacteria other than enterococci grow in the
medium, confirmation is necessary. Litsky et al.2 studied various dyes
and selective agents and formulated a medium using ethyl violet and
sodium azide as selective agents. The medium known as Ethyl
Violet Azide (EVA) Broth is specific for enterococci. In conjunction
with Azide Dextrose Broth, EVA Broth is used to confirm the presence
of enterococci.

The presence of enterococci in water and other specimens indicates
fecal contamination. Mallmann and Seligmann1 compared various
enrichment media for detecting fecal streptococci and found that Azide
EVA Broth contains Tryptose as a source of carbon, nitrogen, vitamins

and minerals. Dextrose is the carbohydrate. Sodium Azide and

Solution:
3.58% solution, soluble in distilled or deionized water.
Solution is light amber, clear to very slightly opalescent.
Reaction of 3.58%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepare EVA Broth per label directions. Inoculate and incubate the tubes at
ORGANISM
Escherichia coli
ATCC
INOCULUM
CFU
GROWTH
19433*
29212*
25922*
100-1,000
100-1,000
1,000-2,000
good
good
inhibited
The Difco Manual
Uninoculated
tube
ATCC 29212
175
Egg Meat Medium
Section II
Ethyl Violet inhibit gram-positive bacilli and gram-positive cocci other

than enterococci. Monopotassium and Dipotassium Phosphates buffer
the medium. Sodium Chloride provides osmotic balance.
Formula
g
g
g
g
g
g
g
Precautions
TARGET ORGAN(S): Nerves, Lungs, Cardiovascular.
Storage
Bacto Egg Meat Medium
Intended Use
Bacto Egg Meat Medium is recommended for cultivating Clostridium
cultures used in detecting the sporicidal activity of disinfectants.

Dehydrated Appearance: Brown, free-flowing, homogeneous
pellets.
Solution:
15% solution, insoluble in distilled
or deionized water. Solution is a
light to medium amber, clear to very
slightly opalescent supernatant over
insoluble pellets.
Reaction of 15%
Solution at 25C:
pH 7.2 0.2
176
Procedure
EVA Broth
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Azide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4
Ethyl Violet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.00083
Expiration Date
Materials Provided
EVA Broth

Glassware
Autoclave
Incubator (35C)

Test Procedure
Results
Growth of enterococci.
References
1. Mallmann and Seligmann. 1950. Am. J. Pub. Health 40:286.
2. Litsky, Mallmann, and Fifield. 1953. Am. J. Pub. Health 43:873.
Packaging
EVA Broth
500 g
0606-17

Egg Meat Medium is a dehydrated medium containing particles of
meat, egg white and calcium carbonate.
The use of a combination meat and egg white culture medium was
reported by Rettger1 in his studies on Escherichia coli and Enterobacter
Cultural Response
Prepare Egg Meat Medium per label directions. Inoculate
tubes and incubate at 35 2C for 18-48 hours.
ORGANISM
Bacillus subtilis
ATCC
INOCULUM
CFU
GROWTH
19659*
3584*
100-1,000
100-1,000
good
good
The Difco Manual
Section II
Elliker Broth
aerogenes. Later, he described the use of this medium in studies of

intestinal putrefaction. 2 In 1923, Reddish and Rettger3 used the
medium in their detailed study of Clostridium putrificum and, the
following year, in a study of other spore-forming anaerobes.4
AOAC5 recommends Egg Meat Medium to propagate and maintain
cultures of Clostridium for use in testing sporicidal activity of liquid
and gaseous chemicals.

Beef muscle provides carbon, nitrogen, inorganic salts, vitamins, and
a variety of other nutrients to support bacterial growth. Egg whites are
a source of protein. Calcium carbonate helps neutralize acids and
displaces oxygen in the media.

Glassware
Garden soil extract (AOAC, 1995, 6.3.05, A.[a.][1.])5
Autoclave
1. Suspend 1.5 grams in 15 ml garden soil extract.5 Let stand at least
15 minutes to form a thoroughly wetted, even suspension.
2. Autoclave at 121C for 15 minutes. Allow to cool.
3. Avoid a rapid release of pressure after sterilization to prevent
expelling the medium from the test tubes.
Formula
Egg Meat Medium

Formula per liter
Test Procedure
Beef Muscle, from . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454 g

Egg White, from . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 eggs
Calcium Carbonate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Precautions
Storage
Expiration Date
Procedure
Materials Provided
Refer to AOAC5 for detailed procedures to determine presence or absence of

sporicidal activity of disinfectants against specified spore-forming bacteria.
Results
References
1. Rettger, L. F. 1903. An experimental study of the chemical
products of Bacillus coli communis and Bacillus lactis aerogenes.
Am. J. Physiol. 8:284.
2. Rettger, L. 1906. Studies on putrefaction. J. Biol. Chem. 2:71.
3. Reddish, G. F., and L. F. Rettger. 1923. Clostridium putrificum.
II. Morphological, cultural and biochemical study. J. Bact. 8:375.
4. Reddish, G. F., and L. F. Rettger. 1924. A morphological,
cultural and biochemical study of representative spore-forming
anaerobic bacteria. J. Bact. 9:13.
Packaging
Egg Meat Medium
500 g
0042-17
Egg Meat Medium
Bacto Elliker Broth
Intended Use
Bacto Elliker Broth is used for cultivating streptococci and lactobacilli,
particularly in dairy procedures.
Also Known As
Elliker Broth is also called Lactobacilli Broth.

Testing for lactic acid bacteria in dairy products may be useful for
various reasons.3 These include determining the cause of acid defects
in dairy products, evaluating lactic starter cultures, and controlling the
The Difco Manual
quality of cured cheese, cultured milks, and uncultured products.3

Lactic acid bacteria found in dairy products are primarily Streptococcus,
Lactococcus, Leuconostoc and Lactobacillus.3
Elliker Broth is prepared according to the formulation of Elliker,
Anderson and Hannesson,1 and modified by McLaughlin.2 This slightly
acidic medium contains nutrients to support the growth of streptococci
and lactobacilli.
A modification of Elliker Broth, Lactic (Elliker) Agar is recommended
for general purpose enumeration of lactic acid bacteria.3

Tryptone and Gelatin provide the nitrogen and amino acids in Elliker
Broth. Yeast Extract is the vitamin source in this formula. Dextrose,
Lactose and Saccharose are the fermentable carbohydrates. Sodium
177
Elliker Broth
Section II
Chloride maintains the osmotic balance of the medium, and ascorbic

acid is added to create a proper environment for organism growth.
Sodium Acetate is a selective agent against gram negative bacteria.
Formula
stored as directed. Do not use a product if it fails to meet the specifications

Procedure
Materials Provided
Elliker Broth
Formula Per Liter
Elliker Broth
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Saccharose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5
Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
g
g
g
g
g
g
g
g
g
Precautions
Storage
Expiration Date

Glassware
Autoclave
Incubator (35C)
Waterbath (45-50C)
Sterile tubes
1.
2.
3.
4.


Test Procedure
streptococci and lactobacilli, refer to standard methods in food testing.3,4,5
Results

this medium.

homogeneous.
Solution:
or deionized water on boiling.
Prepared Medium:
Light to medium amber, clear
Reaction of 4.85%
Solution at 25C:
pH 6.8 0.2
Cultural Response
Prepare Elliker Broth per label directions. Inoculate and
incubate at 35 2C for 18-48 hours except Streptococcus
cremoris which is incubated at 30 2C.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Lactobacillus casei
Lactobacillus delbrueckii subsp. lactis
Streptococcus cremoris
7469
8000
9596
100-1,000
100-1,000
100-1,000
good
good
good
178
References
1. Elliker, P. R., A. W. Anderson, and G. Hannesson. 1956. An agar
culture medium for lactic acid streptococci and lactobacilli. J. Dairy
Sci. 39:1611.
2. McLaughlin, C. B. 1946. Readily prepared medium for cultivation
of lactobacilli. J. Bacteriol. 51:560.
3. Frank, J. F., G. L. Christen, and L. B. Bullerman. 1993. Test for
groups of microorganisms, p. 271-286. In R. T. Marshall (ed.),
Standard methods for the examination of dairy products. 16th ed.
4. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compendium of methods for the microbiological association of food, 3rd.
ed. American Public Health Association, Washington, D.C.
Packaging
Elliker Broth
500 g
0974-17
The Difco Manual
Section II
Emerson YpSs Agar
Bacto Emerson YpSs Agar
Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Intended Use
Final pH 7.0 + 0.2 at 25C
Bacto Emerson YpSs Agar is used for cultivating Allomyces and other fungi.
Precautions

Emerson YpSs Agar is prepared according to the formula given by

Emerson.1 Emerson and Wilson2 used the medium at half strength for
streaking zygotes or zoospores to obtain single germlings.
Fungi are extremely successful organisms, as evidenced by their
ubiquity in nature.3 Of the estimated 250,000 species, fewer than 150
are known primary human pathogens.3 Opportunistic fungal pathogens
are increasing at an impressive rate relating directly to the expanding
size of the immunocompromised to patient population.3

Yeast Extract provides a source of trace elements, vitamins and amino
acids. Soluble Starch provides starch for hydrolysis, detoxification of
metabolic byproducts and as a carbon source. Dipotassium Phosphate
is a buffer. Magnesium Sulfate is a source of divalent cations and
sulfate. Bacto Agar is the solidifying agent.
Storage
Expiration Date
Procedure
Materials Provided
Emerson YpSs Agar
Formula
Emerson YpSs Agar

Formula Per Liter
Glassware
Autoclave
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 g

Bacto Soluble Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g

homogeneous.
Solution:
slight flocculent precipitate.
Prepared Medium:
flocculent precipitate.
Reaction of 4.05%
Solution at 25C:
pH 7.0 0.2
Prepare Emerson YpSs Agar per label directions. Inoculate and
Allomyces macrogynus
Allomyces reticulatus
Aspergillus niger
ATCC
INOCULUM
CFU
GROWTH
38327
42465
16404
9763
100-300
100-300
100-300
100-300
good
good
good
good
The Difco Manual

Test Procedure
Results
References
Cultural Response
ORGANISM

1. Lloydia. 1941. 4:77.

2. Mycologia. 1954. 46:393.
and classification of the fungi, p. 699-708. In Murray, P. R.,
Packaging
Emerson YpSs Agar
500 g
0739-17
179
Endo Agar
Section II
Formula
Bacto Endo Agar
Endo Agar
Formula Per Liter
Intended Use
Bacto Endo Agar is used for confirming the presence of coliform
organisms.

Endo1 developed a medium using a fuchsin sulfite indicator to differentiate lactose fermenting and lactose non-fermenting organisms.
Coliform organisms that ferment lactose produce red colonies and color
the surrounding medium. Typical reactions of this medium are not
caused by acid production but by the intermediate product
acetaldehyde, which reacts with sodium sulfite.2-3
Endo Agar was formerly a standard methods medium for the microbiological examination of water 4 and dairy products.5

Lactose-fermenting bacteria produce acetaldehyde. The aldehyde is
fixed by the sodium sulfite and in the presence of fuchsin forms red
colonies. A sheen is produced by rapid lactose fermenting organisms.
Lactose non-fermenting bacteria form clear, colorless colonies.
Endo Agar contains Bacto Peptone as a source of carbon, nitrogen,
vitamins and minerals. Lactose is the carbohydrate source. Basic
Fuchsin in the presence of Sodium Sulfite produces the red colonies.
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Basic Fuchsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sodium Sulfite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10
10
3.5
15
0.5
2.5
g
g
g
g
g
g
Precautions
AND SKIN. POSSIBLE RISK OF IRREVERSIBLE EFFECTS.
TARGET ORGAN(S): Liver, Thyroid.
Uninoculated
plate
Escherichia coli
ATCC 25922

Dehydrated Appearance: Medium purple, free-flowing, homogeneous.
Solution:
is pink, slightly opalescent, may
have a slight precipitate.
Prepared Medium:
Pink, slightly opalescent, may
Reaction of 4.15%
Solution at 25C:
pH 7.5 0.2
Cultural Response
Prepare Endo Agar per label directions. Inoculate plates and
incubate at 35 2C for 24 2 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
APPEARANCE
25922* 100-1,000
good
good
pink to red with

metallic sheen
colorless to
light pink
Escherichia coli
25923 1,000-2,000 marked to

complete inhibition
ATCC 14028
180
The Difco Manual
Section II
m Endo Agar LES

Test Procedure
Storage
Results

Rapid lactose fermenting organisms will produce red colonies that have
a metallic sheen. Slow lactose fermenting organisms will produce red
colonies. Lactose non-fermenting organisms will produce colorless
colonies.
Expiration Date
Procedure
Materials Provided
Endo Agar

Glassware
Petri dishes
Autoclave
Incubator (35C)
1.
2.
3.
4.
5.

Evenly disperse the precipitate when dispensing.
Use immediately.

If the medium is to be used the same day it is rehydrated, it does not
need to be autoclaved. Boil to dissolve completely before dispensing
into plates.
References
1. Endo, S. 1904. Uber ein Verfahren zum Nachweis der
Typhusbacillen. Centr. Bakt., Abt 1, Orig. 35:109-110.
2. Margolena, L. A., and P. A. Hansen. 1933. The nature of the
reaction of the colon organism on Endos medium. Stain Tech.
8:131-139.
3. Neuberg, C., and F. F. Nord. 1919. Anwendungen der
abfangmethode auf die bakteriengarungen. Biochem. Zeit.
96:133-174.
for the examination of dairy products, 13th ed. American Public
Packaging
Endo Agar
Bacto m Endo Agar LES
500 g
0006-17
Bacto m Endo Agar LES is used for enumerating coliforms in water by

membrane filtration.
with a metallic (golden) sheen within 24 hours incubation at 35C on

an Endo-type medium.
The U. S. Environmental Protection Agency specifies using m Endo Agar
LES in the total coliform methods for testing water using single-step,
two-step and delayed incubation membrane filtration methods.4,5
Also Known As
Intended Use
LES (Lawrence Experimental Station) Endo Agar

McCarthy, Delaney and Grasso 1 formulated Endo Agar LES
(Lawrence Experimental Station) for testing water for coliform
bacteria by a two-step membrane filter procedure using Lauryl Tryptose
Broth as a preliminary enrichment. They recovered higher numbers of
coliforms by this method compared with the one step technique using
m Endo Broth.
The American Public Health Association specifies using m Endo Agar
LES in the standard total coliform membrane filtration procedure for
testing drinking water2 and bottled water.3 It is also specified for use
in the completed phase of the standard total coliform fermentation
technique.2 The coliform bacteria are bacteria that produce a red colony
The Difco Manual
m Endo Agar LES contains tryptose, casitone and thiopeptone as

sources of carbon, nitrogen, vitamins and minerals. Yeast Extract
supplies B-complex vitamins, which stimulate bacterial growth.
Lactose is the carbohydrate. Phosphates are buffering agents. Sodium
Desoxycholate and Sodium Lauryl Sulfate are added as inhibitors.
Basic Fuchsin is a pH indicator. Sodium Sulfite is added to decolorize
the Basic Fuchsin solution. Bacto Agar is the solidifying agent.
Lactose-fermenting bacteria produce acetaldehyde that reacts with the
sodium sulfite and fuchsin to form red colonies. The development of
a metallic sheen occurs when the organism produces aldehydes with
the rapid fermentation of lactose. If the inoculum is too heavy, the
sheen will be suppressed. Lactose non-fermenting bacteria form clear,
colorless colonies.
181
m Endo Agar LES
Section II
Formula
m Endo Agar LES
Formula Per Liter
Bacto Casitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7
Thiopeptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4
Potassium Phosphate Dibasic . . . . . . . . . . . . . . . . . . . . . . . 3.3
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7
Sodium Lauryl Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05
Sodium Sulfite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.6
Bacto Basic Fuchsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g
g
g
g
g
g
g

Storage
Expiration Date
Precautions
Procedure

ORGAN(S): Liver, Thyroid.
Materials Provided

Dehydrated Appearance: Purple, free-flowing, homogeneous.
Solution:
or deionized water containing
2% ethanol on boiling. Solution
is pinkish-red, slightly opalescent
to opalescent with precipitate.
Prepared Medium:
Rose colored, slightly opalescent,
with precipitate.
Reaction of 5.1%
Solution at 25C:
pH 7.2 0.2
Cultural Response
Prepare m Endo Agar LES per label directions. Use the
membrane filter technique to inoculate filters and preincubate
on pads saturated with Lauryl Tryptose Broth (0241) at
35 2C for 1 1/2-2 hours. Transfer filters to plates of m Endo
Agar LES and incubate at 35 2C for 22 2 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
APPEARANCE
Escherichia
25922*
20-80
good
red
coli
with sheen
Salmonella
14028
20-80
good
pink
typhimurium
Staphylococcus 25923* 1,000-2,000 marked to
aureus
complete inhibition
182
m Endo Agar LES

Lauryl Tryptose Broth
95% Ethanol (Not denatured)
Glassware
Membrane filter apparatus
Membrane filter absorbent pads
Petri dishes, 60 mm
Incubator (35C)
m Endo Agar LES
20 ml ethanol (95% not denatured).
2. Boil to dissolve completely. Do Not Autoclave.
3. Dispense in 5-7 ml quantities into 60 mm sterile petri dishes.
1. Suspend 35.6 g in 1 liter of distilled or deionized water.

Collect samples and process according to recommended guidelines for
enumerating coliforms in water.2,4,5
Test Procedure
1.
2.
3.
4.
Place a membrane filter absorbent pad inside the cover.

Add 1.8-2.0 ml Lauryl Tryptose Broth to each pad.
Run the water sample through a membrane filter.
Place the filter, top side up, onto the pad containing Lauryl Tryptose
Broth. Use a rolling motion to avoid entrapping air bubbles.
5. Incubate at 35 2C for 11/2 -2 1/2 hours. Transfer the membrane
from the pad to the surface of the m Endo Agar LES medium in the
petri dish bottom, keeping the side on which the bacteria have been
collected facing upward.
The Difco Manual
m Endo Broth MF
Section II
6. Leave the filter pad in the lid and incubate the plates in the inverted
position at 35 2C for 22 2 hours.
7. Observe and count all colonies that are red and have a metallic sheen.
Results
All colonies that are red and have the characteristic metallic sheen are
considered coliforms. The sheen may cover the entire colony, may
only be in the center or may appear only around the edges.

1. Occasionally, noncoliform organisms may produce typical sheen
colonies. Coliform organisms may also occasionally produce
atypical colonies (dark red or nucleated colonies without sheen).
It is advisable to verify both colony types.2
Packaging
References
1. McCarthy, J. A., J. E. Delaney, and R. J. Grasso. 1961.
Measuring coliforms in water. Water Sewage Works. 108:238.
2. Eaton, A. D., L. S. Clesceri, and A. E. Greenberg (ed.), 1995.
Bacto m Endo Broth MF
3. Cowman, S., and R. Kelsey. 1992. Bottled water, p. 1031-1036.

In C. Vanderzant, and D. F. Splittstoesser (ed.), Compendium of
4. Bordner, R., and J. Winter (ed.), 1978. Microbiological
methods for monitoring the environment, water and wastes.
EPA-600/8-78-017. Environmental Monitoring and Support
Laboratory, Office of Research and Development, U. S.
Environmental Protection Agency, Cincinnati, OH.
5. Environmental Protection Agency. 1992. Manual for the
certification of laboratories analyzing drinking water.
EPA-814B-92-002. Office of Ground Water and Technical Support
Division, U. S. Environmental Protection Agency, Cincinnati, OH.
Intended Use
Bacto m Endo Broth MF is used for enumerating coliform organisms
in water by membrane filtration.
Also Known As
m Endo Medium. MF is a registered trademark of Millipore Filter.

Bacto m Endo Broth MF is prepared according to the formulation of
the Millipore Filter Corporation.1 for selectively isolating coliform
bacteria from water and other specimens using the membrane filtration
technique. The medium is a combination of the former m HD Endo
Medium and Lauryl Tryptose Broth.
The American Public Health Association specifies using m Endo Broth MF
in the standard total coliform membrane filtration procedure for testing
water2 and bottled water.3 APHA also specifies using m Endo Broth
MF in the delayed-incubation total coliform procedure by
adding sodium benzoate to make m Endo preservative medium.2
The coliform bacteria are defined as bacteria that produce a red
colony with a metallic sheen within 24 hours incubation at 35C on an
Endo-type medium.
The U. S. Environmental Protection Agency specifies using m Endo
Broth MF in the total coliform methods for testing water using singlestep, two-step and delayed incubation membrane filtration methods.4,5
m Endo Agar LES
100 g
500 g
0736-15
0736-17
100
500
2
10
0241-15
0241-17
0241-07
0241-08
g
g
kg
kg
Lactose is the carbohydrate. Phosphates are buffering agents. Sodium

Desoxycholate and Sodium Lauryl Sulfate are added as inhibitors.
Basic Fuchsin is a pH indicator. Sodium Sulfite is added to decolorize
the Basic Fuchsin solution. Bacto Agar is the solidifying agent.
Lactose-fermenting bacteria produce acetaldehyde that reacts with the
sodium sulfite and fuchsin to form red colonies. The development of a
metallic sheen occurs when the organism produces aldehydes with
the rapid fermentation of lactose. If the inoculum is too heavy, the sheen
will be suppressed. Lactose non-fermenting bacteria form clear, colorless colonies.
Formula
m Endo Broth MF
Formula Per Liter
Bacto Casitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Thiopeptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.5
Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . 4.375
Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . 1.375
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Sulfite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1
Bacto Basic Fuchsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.05
g
g
g
g
g
g
g
g
g
g
g
g

m Endo Broth MF contains Tryptose, Casitone and Thiopeptone as
supplies B-complex vitamins, which stimulate bacterial growth.
The Difco Manual
Precautions
183
m Endo Broth MF
Section II

ORGAN(S): Liver, Thyroid
Storage
Expiration Date
Procedure
Membrane filters
Membrane filter absorbent pads
Petri dishes, 60 mm
Incubator (35C)
20 ml ethanol (95%, not denatured).
2. Boil to dissolve completely. Do Not Autoclave.

Collect samples and process according to recommended guidelines for
enumerating coliforms in water.2,3,4,5
Test Procedure
1. Place a membrane filter absorbent pad inside a sterile 60 mm
petri dish.
2. Add 1.8-2.0 ml m Endo Broth MF to each pad.
3. Filter the water sample through a membrane filter.
4. Place filter top side up on the pad using a rolling motion to avoid
entrapping air bubbles.
6. Observe and count all colonies that are red and have a metallic sheen.
Materials Provided
m Endo Broth MF
Escherichia coli
ATCC 25922

95% Ethanol (Not denatured)
Glassware
Membrane filter apparatus

Dehydrated Appearance: Pinkish purple, free-flowing, homogeneous.
Solution:
water containing 2% ethanol on boiling.
Solution is pinkish-red, opalescent with precipitate.
Reaction of 4.8%
Solution at 25C:
pH 7.2 0.1
Cultural Response
ATCC 14028
Prepare m Endo Broth MF per label directions. Use the membrane filter
technique to inoculate filters. Incubate on pads saturated with m Endo Broth MF
at 35 2C for 24 2 hours.
ORGANISM
ATCC
INOCULUM
CFU
Escherichia coli
25922*
20-80
Salmonella typhimurium 14028
20-80
25923* 1,000-2,000
GROWTH
APPEARANCE
good
red with green metallic sheen
good
colorless to pink
marked to
complete inhibition
184
The Difco Manual
Section II
Enteric Fermentation Base
Results

In C. Vanderzant, and D. F. Splittstoesser (ed.). Compendium of
4. Bordner, R., and J. Winter (ed). 1978. Microbiological methods for
monitoring the environment, water and wastes. EPA-600/8-78-017.
Environmental Monitoring and Support Laboratory, Office of
Research and Development, U. S. Environmental Protection
Agency, Cincinnati, OH.
5. Environmental Protection Agency. 1992. Manual for the certification of laboratories analyzing drinking water. EPA-814B-92-002.
Office of Ground Water and Technical Support Division, U. S.
Environmental Protection Agency, Cincinnati, OH.
All colonies that are red and have the characteristic metallic sheen are
considered coliforms. The sheen may cover the entire colony, may only
be in the center or may appear only around the edges.

1. Occasionally, noncoliform organisms may produce typical sheen
colonies. Coliform organisms may also occasionally produce
atypical colonies (dark red or nucleated colonies without sheen). It
is advisable to verify both colony types.2
References
1. Fifield, C. W., and C. P. Schaufus. 1958. Improved membrane
filter medium for the detection of coliform organisms. J. Amer.
Water Works Assoc. 50:193.
Packaging
m Endo Broth MF
100 g
500 g
25 x 2 ml
0749-15
0749-17
0749-36
Bacto Enteric Fermentation Base
Intended Use
Bacto Enteric Fermentation Base is used with added carbohydrate
and indicator for differentiating microorganisms based on fermentation
reactions.
The fermentative properties of bacteria are valuable criteria in their

identification.1,2,3,4 A basal medium for determining the fermentation
reactions of microorganisms must be capable of supporting growth of

Solution:
Prepared Medium plain
+ Andredes Indicator: Light pinkish amber, clear without
precipitate.
Reaction of 1.8%
Solution at 25C:
pH 7.2 0.1
Cultural Response
Prepare Enteric Fermentation Base per label directions, with
addition of 1% Andrades Indicator, with and without 1%
dextrose. Inoculate each tube with one drop from an undiluted
suspension of the test organism. Incubate at 35 2C for
18-24 hours. Acid production is indicated by a change in color
from light amber to dark pink or red. Check for gas production
in at least 3% of the volume of the fermentation vial.
ORGANISM
Escherichia coli
Shigella flexneri
ATCC
GROWTH
25922*
14028*
12022*
good
good
good
PLAIN
w/ DEXTROSE
ACID/GAS
ACID/GAS
/
/
/
Uninoculated
tube
Escherichia coli
ATCC 25922
with Dextrose
Escherichia coli
ATCC 25922
plain
+/+
+/+
+/
The Difco Manual
185
test organisms and be free from fermentable carbohydrates. Enteric

Fermentation Base is prepared according to the formula described by
Edwards and Ewing.5,6

Beef Extract and Peptone provide the carbon and nitrogen sources
required for good growth of a wide variety of organisms. Sodium
Chloride maintains the osmotic balance of the medium. The
microorganisms tested are differentiated by their ability to ferment a
particular carbohydrate that has been added to the Enteric Fermentation
Base. The fermentation and resultant acid production are indicated by
a change in color of the pH indicator (Andrades indicator) which is
also added to the Enteric Fermentation Base.
Section II
CARBOHYDRATE
Adonitol
Arabinose
Cellobiose
Dextrose (Glucose)
Dulcitol
Glycerol*
Inositol
Lactose
Mannitol
Salicin
Sucrose
Xylose
FINAL
CONCENTRATION
ADD BEFORE
AUTOCLAVING
ADD AFTER
AUTOCLAVING
0.5%
0.5%
0.5%
1%
0.5%
0.5%
0.5%
1%
1%
0.5%
1%
0.5%
X
X
X
X
X
X
X
X
X
X
Formula
*Medium containing glycerol should be autoclaved for 10 minutes at 15 lbs pressure (121C).

Formula Per Liter
7. Dispense 3 ml amounts into test tubes containing inverted

fermentation vials (Durham tubes).
Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Precautions

recommended guidelines.7,8,9
specimen or sample.7,8,9
Test Procedure
Storage
For a complete discussion on identification of Enterobacteriaceae,

refer to the appropriate procedures outlined in the references.5,6,10

Results
Expiration Date
A positive result for gas includes production in at least 3% of the

volume of the fermentation tube. A positive reaction for gas production
is a change in color from light amber to dark pink or red.
Procedure
Materials Provided

Glassware
Autoclave
Incubator (35C)
Waterbath (45-50C)
Choice of filter-sterilized carbohydrate(s)
Andrades Indicator (0.5 g Acid Fuchsin in 100 ml water plus 16 ml
of 1.0 N Sodium Hydroxide)
1.
2.
3.
4.
5.
6.

Add 10 ml Andrades Indicator.
Cool to 45 to 50C in a waterbath.
Add appropriate amounts of sterile carbohydrates as indicated in
the table below.
186

1. Negative tubes remain colorless and should be observed regularly
for a total of 30 days.
References
Scotts diagnostic microbiology, 9th edition. Mosby-Year Book,
Inc., St. Louis, MO.
R. H. Yolken. (ed.). 1995. Manual of clinical microbiology,
6th edition. ASM Press, Washington, D.C.
3. Holt, J. G., N. R. Krieg, P. H. A. Sneath, J. T. Staley, and S. T.
Williams. 1994. Bergeys manual of determinative bacteriology,
9th edition. Williams & Wilkins, Baltimore, MD.
Enterobacteriaceae, 4th edition. Elsevier Science Publishing Co.,
Inc., New York, NY
5. Edwards, P. R., and W. H. Ewing. 1972. Identification of
Enterobacteriaceae, 3rd ed. Burgess Publishing Co., Minneapolis.
6. Balows, A., and W. J. Hausler. 1981. Diagnostic Procedures for
Bacteria, Mycotic and Parasitic Infections, 6th ed. American
The Difco Manual
Section II
m Enterococcus Agar
7. Baron, E. J., and S. M. Finegold. 1990. Bailey & Scotts Diagnostic Microbiology, 8th ed. C.V. Mosby Company, St. Louis, MO.
8. Gilligan, P. H. 1995. In P. R. Murray, E. J. Baron, M. A. Pfaller,
F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology, 6th ed. American Society of Microbiology, Washington, D.C.
H. D. Isenberg (ed.), Clinical microbiology procedures handbook,
Vol. 1. American Society for Microbiology, Washington, D.C.
Bacto m Enterococcus Agar
10. Isenberg, H. D. (ed.). 1992. Conventional tests (18 to 24 hours) Carbohydrate Fermentation Test, p. 1.19.28. Clinical microbiology
procedures handbook, vol.1. American Society for Microbiology.
Washington, D.C.
Packaging
500 g
1828-17
Intended Use
m Enterococcus Agar is used for isolating and enumerating enterococci in water and other materials by membrane filtration or pour
plate technique.
Also Known As
m Enterococcus Agar is also referred to as m Azide Agar

The enterococcus group is a subgroup of the fecal streptococci that
include E. faecalis, E. faecium, E. gallinarum, and E. avium. 1
Enterococci are differentiated from other streptococci by their ability
to grow in 6.5% sodium chloride, at pH 9.6, and at 10C and 45C.1
The enterococci portion of the fecal streptococcus group is a valuable
bacterial indicator for determining the extent of fecal contamination of
recreational surface waters.1 m Enterococcus Agar is used in standard
methods for the detection of fecal streptococcus and enterococcus
groups using the membrane filtration technique.1
m Enterococcus Agar was developed by Slanetz et al. 2 for the

enumeration of enterococci by the membrane filtration technique.
A modification of m Enterococcus Agar, adding triphenyltetrazolium
chloride (TTC), was described by Slanetz and Bartley3. This modified
medium proved to be a superior membrane filtration medium for the
enumeration of enterococci. Increased recovery and larger colonies
were obtained by incubating the inoculated membranes on the agar
surface instead of on pads saturated with liquid medium. The
membrane filtration method has the advantages of being simpler to
perform, not requiring confirmation and permitting a direct count of
enterococci in 48 hours. Burkwell and Hartman4 added 0.2% sodium
carbonate and 0.05% Tween 80 to m Enterococcus Agar to increase
the sensitivity for the direct plating method.

Tryptose provides the nitrogen, minerals and amino acids in
m Enterococcus Agar. Yeast Extract is the vitamin source and Dextrose
supplies carbon. Dipotassium Phosphate acts as a buffer for the

Solution:
upon boiling. Solution is light amber, very slightly to
slightly opalescent, without significant precipitate.
Prepared Medium:
Light amber, slightly opalescent, without precipitate.
Reaction of 4.2%
Solution at 25C:
pH 7.2 0.2
Cultural Response
Prepare m Enterococcus Agar per label directions. Inoculate medium using the
membrane filter technique. Incubate in humid atmosphere inoculated medium at
35 0.5C for 40-48 hours.
ORGANISM
ATCC
Enterococcus faecalis 19433

Enterococcus faecalis 29212*
Escherichia coli
25922*
INOCULUM
CFU
20-60
20-60
1,000
GROWTH
COLONY OF COLONY
ATCC 19433
good
pink to red
good
pink to red
marked to
complete inhibition
The Difco Manual
187
m Enterococcus Agar
Section II
medium. Sodium Azide is the selective agent to suppress the growth of

gram negative organisms. Bacto Agar is the solidifying agent.
Triphenyltetrazolium Chloride (TTC) is the dye used as an indicator of
bacterial growth. TTC is reduced to the insoluble formazan inside the
bacterial cell, resulting in the production of red colonies.
Formula
m Enterococcus Agar
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Sodium Azide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2,3,5-Triphenyl Tetrazolium Chloride . . . . . . . . . . . . . . . . 0.1
g
g
g
g
g
g
g
Precautions
2. HARMFUL. HARMFUL BY INHALATION AND IF
SYSTEM AND SKIN. Avoid contact with skin and eyes. Do not
tightly closed. TARGET ORGAN(S): Cardiovascular, Lungs,
Nerves.
Storage
Magnifying lens
1.
2.
3.
4.

DO NOT AUTOCLAVE.
Dispense into 9 x 50 mm Petri dishes to a depth of 4-5 mm
(approximately 4-6 ml).

Collect water samples as described in Standard Methods for the
Examination of Water and Wastewater, Section 9230 1 or by
laboratory policy.
Test Procedure
Membrane filtration procedure
1. Follow the membrane filtration procedure as described in Standard
Methods for the Examination of Water and Wastewater,
Section 9230C.1
2. Choose a sample size so that 20-60 colonies will result.
3. Transfer the filter to agar medium in a Petri dish, avoiding air
bubbles beneath the membrane.
4. Let plates stand for 30 minutes.
5. Invert plates and incubate at 35 0.5C for 48 hours.
Direct plating procedure
1. Inoculate medium with a specimen using the streak plate method.
2. Incubate plates at 35 2C for 24-48 hours.
Results1
Count all light and dark red colonies as enterococci. Count colonies
using a fluorescent lamp and a magnifying lens.


this medium.
Expiration Date
References

2. Slanetz, Bent, and Bartley. 1955. Public Health Rep. 70:67.
3. Slanetz, and Bartley. 1957. J. Bacteriol. 74:591.
4. Burkwell, and Hartman. 1964. Appl. Microbiol. 12:18.
Procedure
Materials Provided
m Enterococcus Agar

Glassware
Incubator (35C)
Fluorescent lamp
188
Packaging
m Enterococcus Agar
100 g
500 g
0746-15
0746-17
The Difco Manual
Section II
Eugon Agar & Eugon Broth
Bacto Eugon Agar

Bacto Eugon Broth
Intended Use
Bacto Eugon Agar and Eugon Broth are used for cultivating a wide
variety of microorganisms, particularly in mass cultivation procedures.
Also Known As
Eugon media are also referred to as Eugonic Agar and Eugonic Broth.

Eugon Agar
Solution:
Light amber, slightly opalescent,
precipitate may be visible.
Prepared Medium:
precipitate may be visible.
Reaction of 4.54%
Solution at 25C:
pH 7.0 0.2
Eugon Broth
Solution:
Light amber, clear, may have a
slight precipitate.
Prepared Medium:
Light amber, clear, may have a
slight precipitate.
Reaction of 3.04%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepared Eugon Agar and Eugon Broth per label directions.
Inoculate prepared medium and incubate for 18-48 hours (up
to 72 hours if necessary). Candida albicans and Aspergillus
niger should be incubated at 30 2C; all other cultures
should be incubated at 35 2C.
ORGANISM
Aspergillus niger
Brucella abortus
Candida albicans
Shigella flexneri
ATCC
INOCULUM
CFU
GROWTH
16404
4315
26790
9338
12022*
19615*
100-1,000
100-1,000
100-1,000
100-1,000
100-1,000
100-1,000
fair to good
good
good
good
good
good
The Difco Manual
Eugon Agar and Eugon Broth are prepared according to the formula
described by Vera.1 Eugon media were developed to obtain eugonic
(luxuriant) growth of fastidious microorganisms.2 These formulations
can be used with or without enrichment. Enriched with blood, Eugon
media support the growth of pathogenic fungi including Nocardia,
Histoplasma and Blastomyces. With the addition of Supplement B,
excellent growth of Neisseria, Francisella and Brucella is achieved.
The unenriched media support rapid growth of lactobacilli associated
with cured meat products, dairy products and other food.
Niven3 reported the use of Eugon Agar for the detection of lactic acid
in cured meats, and recommended it for investigating spoilage in meats.
Harrison and Hansen4 employed the medium for plate counts of the
intestinal flora of turkeys. Frank5 showed its usefulness in germinating
anaerobic spores pasteurized at 104C.
Eugon Agar is specified in the Compendium of Methods for the
Microbiological Examination of Food.6

Tryptose and Soytone provides the nitrogen, vitamins and amino acids
in Eugon Agar and Eugon Broth. The high concentration of Dextrose
is the energy source for rapid growth of bacteria. L-Cystine and
Sodium Sulfite are added to stimulate growth. Sodium Chloride maintains the osmotic balance of the media. The high carbohydrate content
along with high sulfur (cystine) content improves growth with
chromogenicity.2 Bacto Agar is the solidifying agent in Eugon Agar.
Formula
Eugon Agar
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.7
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Sodium Sulfite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g

Eugon Broth
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.7
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Sodium Sulfite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
g
g
g
g
g
g
Precautions
Storage
189
m FC Agar, m FC Broth Base & Rosolic Acid
Section II
Expiration Date
Results
Procedure
Materials Provided
Eugon Agar
Eugon Broth

Glassware
Autoclave
Incubator (35C)
Sterile Petri dishes or Sterile tubes
5% sterile defibrinated blood (optional)
Bacto Supplement B (optional)
1. Suspend the appropriate amount of medium 1 liter distilled or
deionized water:
Eugon Agar
45.4 g/l
Eugon Broth
30.4 g/l
4. OPTIONAL: When an enriched medium is being prepared, cool
to 50-55C prior to adding the desired enrichment. After the
enrichment is added, mix well.

Test Procedure
For a complete discussion on bacteria and fungi from clinical specimens,
refer to the appropriate procedures outlined in the references.7,8 For the
examination of bacteria and fungi in food refer to standard methods.6,9
Bacto m FC Agar
Bacto m FC Broth Base
Bacto Rosolic Acid
Intended Use
Bacto m FC Agar and Bacto m FC Broth Base are used with Bacto
Rosolic Acid in cultivating and enumerating fecal coliforms by the
membrane filter technique at elevated temperatures.
Also Known As
M-FC Medium
190

2. Eugon Agar is not recommended as a blood agar base for hemolytic
reactions because of its high sugar content.
3. It is suggested that Eugon Agar be prepared as required. Do not
melt and resolidify media containing enrichments.
References
1. Vera, H. D. 1947. The ability of peptones to support surface
growth of lactobacilli. J. Bacteriol. 54:14.
2. MacFaddin, J. D. 1985. Media for isolation-cultivationidentification-maintenance medical bacteria, p.301-303. vol. 1.
3. Niven. 1949. J. Bacteriol. 58:633.
4. Harrison, A. P., Jr., and P. A. Hansen. 1950. The bacterial flora
of the cecal feces of health turkeys. J. Bacteriol. 59:197.
5. Frank, H. A. 1955. The influence of various media on spore count
determinations of a putrefactive anaerobe. J. Bacteriol. 70:269.
6. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compendium of methods for the microbiological examination of food, 3rd
7. Isenberg, H. D. (ed.), 1992. Clinical microbiology procedures
R. H. Yolken (ed.), 1995. Manual of clinical microbiology, 6th ed.
Gaithersburg, MD.
Packaging
Eugon Agar
500 g
0589-17
Eugon Broth
500 g
0590-17

Geldreich et al.1 formulated a medium to enumerate fecal coliforms
(MFC) using the membrane filter (MF) technique without prior
enrichment. Fecal coliforms, i.e., those found in the feces of
warm-blooded animals, are differentiated from coliforms from
environmental sources by their ability to grow at 44.5 0.5C.2
Many Standard Methods membrane filtration procedures specify M-FC
medium for testing water. The American Public Health Association
(APHA) specifies M-FC medium and incubation at 44.5 0.5C in the
fecal coliform membrane filter procedure, the delayed-incubation
fecal coliform procedure, the two-layer agar method for recovering
injured fecal coliforms,2 and in the membrane filter method for fecal
coliforms in bottled water.3 The Association of Official Analytical
Chemists (AOAC) specifies m-FC Agar for detecting total coliforms
and fecal coliforms in foods.4
The Difco Manual
Section II
m FC Agar, m FC Broth Base & Rosolic Acid

Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Aniline Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
The U. S. Environmental Protection Agency specifies using M-FC

medium in fecal coliform methods for testing water by the direct MF
method or the delayed-incubation MF method.5,6

m FC Agar and m FC Broth Base contain Tryptose and Proteose
Peptone No. 3 as sources of carbon, nitrogen, vitamins and minerals.
Yeast Extract supplies B-complex vitamins that stimulate bacterial
growth. Lactose is a carbohydrate. Bile Salts No. 3 inhibits growth of
gram-positive bacteria. m FC Agar contains Bacto Agar as the
solidifying agent. The differential indicator system combines Aniline
Blue and Rosolic Acid.
g
g
g
g
g
g

m FC Broth Base
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.5
Aniline Blue (Water Blue) . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
Colonies of fecal coliforms are blue; non-fecal coliforms and other

organisms are gray to cream-colored.
Formula
g
g
g
g
g
g
g
m FC Agar
Formula Per Liter
Rosolic Acid
Rosolic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g/vial
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . . . 5 g

m FC Agar
Dehydrated Appearance: Beige with slight blue tint, free-flowing, homogeneous.
Solution:
on boiling. Without 1% rosolic acid: blue, very slightly
to slightly opalescent, may have a slight precipitate.
With 1% rosolic acid: cranberry red, slightly
Prepared Medium:
Without 1% rosolic acid: blue, slightly opalescent.
With 1% rosolic acid: cranberry red, slightly opalescent.
Reaction of 5.2%
Solution at 25C:
pH 7.4 0.2 (without 1% rosolic acid)
m FC Broth Base
Dehydrated Appearance: Beige with slight blue tint, free-flowing, homogeneous.
Solution:
3.7% solution, soluble in distilled or deionized water on boiling.
Without 1% rosolic acid: blue, slightly opalescent, may have a very fine precipitate.
With 1% rosolic acid: cranberry red, slightly opalescent, may have a very fine precipitate.
Reaction of 3.7%
Solution at 25C:
pH 7.4 0.2 (without 1% rosolic acid)
Rosolic Acid
Dehydrated Appearance: Dark reddish-brown with metallic green particles, free-flowing, fine crystalline powder.
Solution:
1.0% solution, soluble in 0.2 N NaOH. Solution is deep red, clear to very slightly opalescent.
Escherichia coli
ATCC 25922
Cultural Response
m FC Agar and m FC Broth Base
Prepare mFC Agar and mFC Broth Base per label directions with 1% Rosolic Acid. Using the membrane filter technique, inoculate
and incubate plates at 44.5 0.5C for 24 2 hours.
ORGANISM
Escherichia coli
ATCC
INOCULUM CFU
GROWTH
APPEARANCE
19433*
25922*
1,000-2,000
20-80
markedly to completely inhibited

good
blue
The Difco Manual
191
mFC Agar, mFC Broth Base & Rosolic Acid
Precautions
2. Rosolic Acid: IRRITANT. IRRITATING TO EYES, RESPIRATORY
SYSTEM AND SKIN. Avoid contact with skin and eyes. Do not breathe
Storage
Section II
4. Heat to boiling.
5. Cool before dispensing.

Collect samples and process according to recommended guidelines.2-6
Test Procedure
m FC Agar
1. Filter duplicate samples through separate membrane filters.
2. Transfer the filters to the surface of separate mFC Agar plates.
3. Place each plate in a separate waterproof plastic bags Submerge in
different waterbaths, one set at 35 2C and one set at 44.5 0.5C;
incubate for 24 2 hours.
4. Incubate one set of plates at 35C and one set at 44.5 0.5C for
24 2 hours.

Store Rosolic Acid at 15-30C. Rehydrated Rosolic Acid (1% solution)
is stable for 2 weeks if stored at 2-8C in the dark.
Results
Expiration Date
1. A few nonfecal coliform colonies may be observed on m FC media

due to the selective action of the elevated temperature and
the addition of the Rosolic Acid. It may be useful to elevate the
temperature to 45 0.2C to eliminate Klebsiella strains from the
fecal coliform group.2
Procedure
Materials Provided
m FC Agar
m FC Broth Base
Rosolic Acid

0.2 N sodium hydroxide
1 N hydrochloric acid
Glassware
Waterproof plastic bags
Waterbath (35C)
Waterbath (44.5 0.5C)
Rosolic Acid
1. Prepare a 1% solution, dissolving 1 gram in 100 ml 0.2 N NaOH.
m FC Agar
3. Add 10 ml of a 1% solution of Rosolic Acid in 0.2 N NaOH.
4. Continue heating for 1 minute. Do Not Autoclave.
5. If necessary, adjust to pH 7.4 with 1 N HCl.
m FC Broth Base
2. Add 1 ml of a 1% solution of Rosolic Acid in 0.2 N NaOH.
192
Colonies of fecal coliforms will be various shades of blue. Non-fecal

coliforms are gray to cream-colored.
References
1. Geldreich, E. E., H. F. Clark, C. B. Huff, and L. C. Best. 1965.
Fecal-coliform-organism medium for the membrane filter technique.
J. Am. Water Works Assoc. 57:208-214.
In C. Vanderzant, and D. F. Splittstoesser (ed.). Compendium of
4. Andrews, W. 1995. Microbial methods, p. 17.1-17-119. In Official
5. Bordner, R., and J. Winter (ed). 1978. Microbiological methods
for monitoring the environment. EPA-600/8-78-017. Environmental
Monitoring and Support Laboratory, Office of Research and
Development, U. S. Environmental Protection Agency, Cincinnati, OH.
6. Environmental Protection Agency. 1992. Manual for the certification
of laboratories analyzing drinking water. EPA-814B-92-002. Office
of Ground Water and Technical Support Division, U. S. Environmental
Protection Agency, Cincinnati, OH.
Packaging
m FC Agar
100 g
500 g
0677-15
0677-17
m FC Broth Base
100 g
500 g
0883-15
0833-17
6x1 g
3228-09
Rosolic Acid
The Difco Manual
Section II
m FC Basal Medium
Bacto m FC Basal Medium
Intended Use
Bacto m FC Basal Medium is used with MUG or BCIG for cultivating
and enumerating fecal coliforms by the membrane filter technique at
elevated temperatures.
m FC Basal Medium contains Tryptose and Proteose Peptone No. 3 as

sources of carbon, nitrogen, vitamins and minerals. Yeast Extract supplies
B-complex vitamins that stimulate bacterial growth. Bile Salts No. 3
inhibits the growth of gram-positive microorganisms. Bacto Agar is
the solidifying agent.
Formula

Ciebin et al.1 described a modification of m FC Medium called FC
Basal Medium, in which the chromogenic substrate 5-bromo-6-chloro-3indolyl--D-glucuronide (BCIG) is added for quantitative recovery of
Escherichia coli from untreated water samples to show fecal contamination
using membrane filter methods.
Standard method procedures use media with the fluorogenic substrate,
4-methylumbelliferyl -D-glucuronide (MUG) to enumerate E. coli by
membrane filter methods.2 Disadvantages of using MUG include the
requirement of ultra violet light, possible diffusion of fluorescence from
the colony to the surrounding medium and background fluorescence of
membrane filters. 3 Using BCIG in place of MUG to detect
-glucuronidase activity, gives visible blue colonies and an indigo-blue
complex that remains within the colony. Ciebin et al.1 found FC-BCIG
Medium comparable to standard MUG-based media for detection of
-glucuronidase activity of E. coli.
4
In another study, Ciebin et al. formulated DC Medium using FC Basal

Medium supplemented with lactose, BCIG and cefsulodin. It is a
differential coliform medium for the enumeration of coliforms and
E. coli in potable water using membrane filtration. Ciebin et al. compared
DC Medium to LES Endo Medium and FC-BCIG Medium. They found
DC Medium superior to LES Endo Medium in recovering coliforms
and equivalent to FC-BCIG Medium in recovering E. coli.
m FC Basal Medium
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
Precautions
Storage
Expiration Date

Solution:
3.95% solution, soluble in distilled or deionized water on
boiling. Solution is light amber, very slightly to slightly
opalescent, may have slight precipitate.
Prepared Medium:
Reaction of 3.95%
Solution at 25C:
pH 7.4 0.2
Cultural Response
Prepare m FC Basal Medium per label directions, with the addition of 0.01% MUG.
Using membrane filter technique, inoculate and incubate at 44.5 0.5C for 24 2 hours.
ORGANISM
ATCC
INOCULUM
CFU
Enterococcus faecalis 19433* 300-1,000

Escherichia coli
25922*
30-200
GROWTH
APPEARANCE
markedly to
good
Escherichia coli
ATCC 25922
blue-white fluorescence
The Difco Manual
193
Fildes Enrichment
Section II
Procedure
3. Count number of colonies and record observations. m FC-MUG

plates are read under long-wavelength (366 nm) ultraviolet light.
Materials Provided
m FC Basal Medium
Results
m FC-MUG
-glucuronidase-positive organisms produce a blue-white fluorescence.
-glucuronidase-positive organisms do not fluoresce.
MUG (4-methylumbelliferyl b-D-glucuronide)

BCIG (5-bromo-6-chloro-3-indolyl b-D-glucuronide)
1 N HCl
Glassware
Membrane filtration apparatus
Petri dishes, 60 mm
Waterproof plastic bags
Waterbath (35C)
Waterbath (44.5C)
m FC-BCIG
-glucuronidase-positive organisms produce a visible blue colony.
-glucuronidase-positive organisms produce a non-blue colony.
References
2. Add 100 mg MUG or BCIG, as desired, and boil to dissolve
completely. Do Not Autoclave.
4. Dispense into 60 mm sterile petri dishes.

Collect samples and process according to recommended guidelines
for water testing.2
Test Procedure
1. Filter duplicate water samples through membrane filtration apparatus.
2. Transfer each membrane to the surface of the m FC Basal Medium
plate. Place cultures in waterproof plastic bags. Submerge petri
dishes in waterbath. Incubate one set of plates at 35 2C and one
set at 44.5 0.5C for 24 2 hours.
Bacto Fildes Enrichment
1. Ciebin, B. W., M. H. Brodsky, R. Eddington, G. Horsnell, A.

Choney, G. Palmateer, A. Ley, R. Joshi, and G. Shears. 1995.
Comparative evaluation of modified m-FC and m-TEC medium
for membrane filter enumeration of Escherichia coli in water. Appl.
Environ. Microbiol. 61:3940-3942.
3. Farber, J. M. 1986. Potential use of membrane filters and a
fluorogenic reagent-based solid medium for the enumeration of
Escherichia coli in foods. Can. Inst of Food Sci. and Tech.
J. 19:34-37.
4. Ciebin, B. W., R. N. Schop, and M. H. Brodsky. 1997. DC
medium: a differential coliform medium for simultaneous enumeration of coliforms and Escherichia coli in water by membrane
filtration. Q-152. Abstr. 97th Annu. Meet. Am. Soc. for Microbiol.
1997. American Society for Microbiology, Washington, D.C.
Packaging
m FC Basal Medium
500 g
0698-17
Intended Use
Bacto Fildes Enrichment is used to enrich media for cultivating
Haemophilus influenzae.
Also Known As
Fildes Enrichment is also referred to as Fildes Broth.
Cultural Response
Prepare Heart Infusion Agar per label directions. Aseptically
add 5% Fildes Enrichment to the cooled medium at 50-55C
and pour plates. Inoculate and incubate at 35 2C under
increased CO2 for 18-24 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Haemophilus parainfluenzae
19418
7901
100-1,000
100-1,000
good
good
194
Fildes Enrichment, a sterile digest of sheep blood, is prepared according

to Fildes.1,2 This enrichment is recommended for use in liquid and solid
media for culturing Haemophilus influenzae and other fastidious
microorganisms requiring blood derivatives for optimal growth.
Body fluid specimens likely to harbor Haemophilus species should be
inoculated on 5% sheep blood agar, chocolate agar and a suitable
enrichment broth, such as Fildes Broth.3 Fildes-enriched gonococcal
medium, composed of gonococcal agar base containing 5% Fildes
Enrichment, 5% horse blood and 3 g of vancomycin per ml, is used
for lymph node aspirates and lesion scrapings from patients with
suspected chancroid.3
The Difco Manual
Section II
Fish Peptone No. 1
cedures established by laboratory policy or appropriate references.3,4
Fildes Enrichment is a rich source of factors that will promote growth

of fastidious microorganisms. The growth factors include hemin
(X factor) and nicotinamide adenine dinucleotide (NAD or V factor)
required by H. influenzae and other Haemophilus species.
Test Procedure
Formula
Body fluids and other clinical specimens inoculated in Fildes

Enrichment should be incubated for 7 days at 35-37C.3
Fildes Enrichment
A sterile digest of sheep blood.
Precautions
1. For In Vitro Diagnostic Use.
2. Follow proper, established laboratory procedure in handling and
Storage
Store Fildes Enrichment at 2-8C.
Expiration Date
Procedure
Materials Provided
Fildes Enrichment

Fildes Enrichment is a ready-to-use solution. Many factors in Fildes
Enrichment are heat labile. This enrichment cannot be heated and must
be added aseptically in the proper amounts to media that have been
sterilized in the autoclave and cooled to 50-55C.
Fildes Enrichment is usually employed in prepared media at a final

concentration of 5% for optimal results. Some formulas may require
higher or lower concentrations. Add Fildes Enrichment as required.
Results
Carefully examine clinical specimens incubated in Fildes Enrichment
for evidence of growth.3

may be encountered that fail to grow or grow poorly with this
enrichment.
2. Haemophilus species cannot be depended on to impart obvious
turbidity to broths even when they are present at densities exceeding
109 CFU/ml.3
References
1. Fildes. 1920. Br. J. Exp. Pathol. 1:129-130.
2. Fildes. 1921. Br. J. Exp. Pathol. 2:16-25.
3. Campos, J. M. 1995. Haemophilus, p. 556-565. In P. R. Murray,
1995. Manual of clinical microbiology, 6th ed. American Society
4. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures handbook, vol. 1. American Society for Microbiology,
Washington, D.C.
Packaging
Fildes Enrichment
6 x 5 ml
100 ml
6 x 100 ml
0349-57
0349-72
0349-73
Obtain and process specimens according to the techniques and pro-
Bacto Fish Peptone No. 1
Intended Use
Bacto Fish Peptone No. 1 is used in preparing microbiological
culture media.

Fish Peptone No. 1 is a non-bovine origin peptone. Fish Peptone No. 1
was developed by Difco Laboratories for pharmaceutical and vaccine
production to reduce Bovine Spongiform Encephalopathy (BSE) risk.
Fish Peptone No. 1 may substitute for other peptones, depending on
organism and production application.
The Difco Manual

Fish Peptone No. 1 is a non-mammalian/animal peptone used as a
nitrogen source in microbiological culture media.
Typical Analysis
Ash (%)
34.8
0.9
3.4
Loss on Drying (%)

pH, 1% Soln
3.4
6.9
Carbohydrate (%)
Total
<0.1

Total Nitrogen
Amino Nitrogen
10.6
3.2
AN/TN
30.2
195
Fish Peptone No. 1
Section II
Amino Acids (%)

Alanine
Arginine
Aspartic Acid
Cystine
Glutamic Acid
Glycine
Histidine
Isoleucine
Leucine
3.48
2.19
3.21
0.24
5.27
5.29
1.54
0.92
2.16
Lysine
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine
2.51
0.83
0.95
2.19
1.27
1.17
0.15
0.45
1.43
P. aeruginosa ATCC 27853

E. coli ATCC 25922
Y. ruckeri ATCC 29908
S. aureus ATCC 25923
S. cerevisiae ATCC 9763
Inorganics (%)
Calcium
Chloride
Cobalt
Copper
Iron
Lead
Magnesium
Manganese
0.020
9.326
<0.001
0.001
0.003
<0.001
0.017
<0.001
Phosphate
Potassium
Sodium
Sulfate
Sulfur
Tin
Zinc
3.848
4.183
9.351
1.004
1.629
<0.001
0.002
PABA
Pantothenic Acid
Pyridoxine
Riboflavin
Thiamine
Thymidine
95.0
63.2
7.2
26.8
NA
55.0
Vitamins (g/g)
Biotin
0.3
Cyanocobalamin
0.3
Folic Acid
1.5
Inositol
2820.0
Nicotinic Acid
603.0

Coliform
Salmonella
negative
negative
Standard Plate Count 18

Thermophile Count 379

Solution:
1% solution is light to medium amber,
clear to very slightly opalescent, may
Reaction of 1%
Solution at 25C:
pH 6.7 0.2
Storage
Expiration Date
Procedure

Refer to the final concentration of peptone in the formula of the
medium being prepared. Add Fish Peptone No. 1 as required.
Cultural Response
Prepare a 1% concentration of Fish Peptone No. 1 with the
addition of 0.5% sodium chloride. Inoculate test organisms and
incubate for 18-48 hours at 35 2C. Incubate Vibrio tubiashii
for 18-48 hours at 25 2C. Saccharomyces cerevisiae is
tested with the addition of 0.5% dextrose. Vibrio tubiashii is
tested with the addition of 1.5% sodium chloride.
ATCC
INOCULUM
CFU
GROWTH
6633
25922*
9763
25923*
19105
100-1,000
100-1,000
100-1,000
100-1,000
100-1,000
fair to good
good
good
good
good
*These cultures are available as BactrolTM Disks and should be

Bacillus subtilis is available as Bacto Subtilis Spore Suspension.
196

Fish Peptone No. 1
Bacillus subtilis
Escherichia coli
Vibrio tubiashii
Precautions
Materials Provided
ORGANISM
B. subtilis ATCC 6633

Test Procedure
See appropriate references for specific procedures on the medium
being prepared or the sample being analyzed.
Results

be encountered that fail to grow or grow poorly on prepared medium.
Packaging
Fish Peptone No. 1
500 g
10 kg
0551-17
0551-08
The Difco Manual
Section II
Fletcher Medium Base
Bacto Fletcher Medium Base
Intended Use
Bacto Peptone and Beef Extract provide the nitrogen, vitamins, carbon
and amino acids in Fletcher Medium Base. Sodium chloride maintains
the osmotic balance of the medium. Bacto Agar is the solidifying agent.
Bacto Fletcher Medium Base is used with sterile normal rabbit serum
for isolating and cultivating Leptospira.
Sterile normal rabbit serum is added to the formula to stimulate growth

of Leptospira.
Formula
In 1816, Adolf Weil described the first recognized infections of

Leptospirosis in humans.5 These cases were caused by Leptospira
interogans serovar icterohaemorrhagiae and the disease was subsequently named Weils Disease.5 Leptospirosis is a zoonotic disease,
having its reservoir in wild, domestic, and peridomestic animals.6
Infection usually results from direct or indirect exposure to the urine
of leptospiruric animals.6 Indirect exposure through contaminated
water and soil accounts for most sporadic cases.3 Direct exposure occurs
in pet owners, veterinarians and persons working with livestock.3
Leptospirosis is typically a biphasic illness.3,8 The infection is acute in
onset, with a flu- like syndrome persisting for 4 to 7 days.4 Onset of a
second immune phase, in which meningitis, skin rash, and hepatic
and renal involvement may be present, occurs within a few days.4
Fletcher Medium Base is prepared according to the formulation of
Fletcher.1 Myers et al.2 reported Fletcher Medium, with a basal agar
layer containing charcoal, to be superior to the standard medium for
the maintenance of leptospiral cultures. Fletcher Medium Base
prepared with sterile normal rabbit serum is specified for the isolation
of Leptospira.3,4,7

Formula Per Liter
0.3
0.2
0.5
1.5
g
g
g
g
Precautions
3. Leptospira species are BioSafety Level 2 pathogens. Handling clinical specimen material potentially infected with Leptospira species
should be performed in a Class II biological safety cabinet (BSC).7
Storage
Expiration Date

Solution:
2.5 g in 920 ml distilled or deionized
water, soluble upon boiling. Solution
is very light amber, clear to very
Prepared Medium:
Very light amber, very slightly to
Reaction of 2.5 g in
920 ml distilled water: pH 7.9 0.1 at 25C
Cultural Response
Prepare Fletcher Medium Base per label directions. Enrich
with sterile normal rabbit serum. Inoculate and incubate at
30 2C for up to 5 days.
ORGANISM
ATCC
INOCULUM
GROWTH
Leptospira interrogans
serovar australis
serovar canicola
Leptospira kirschneri
serovar grippotyphosa
23605
2-3 drops
good
23470
2-3 drops
good
23604
2-3 drops
good
The Difco Manual
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Procedure
Materials Provided

Glassware
Autoclave
Incubator (35C)
Waterbath (56C)
Sterile normal rabbit serum
1.
2.
3.
4.
5.

Aseptically add 80 ml sterile normal rabbit serum at 56C. Mix well.
Determine pH. If necessary, aseptically adjust to pH 7.9 0.1 with
1 N HCl or 1 N NaOH.

established by laboratory policy. Blood, cerebrospinal fluid (CSF) and
197
Folic AOAC Medium
urine are the specimens of choice for the recovery of leptospires from
patients with leptospirosis.3
Test Procedure7
1. Aseptically dispense into sterile screw-cap tubes in 5-7 ml amounts.
Store at room temperature overnight.
2. Inactivate the whole medium the day following its preparation by
placing the tubes in a water bath at 56C for 1 hour.
3. Allow the medium to cool before inoculation.
4. Growth is first seen in approximately 10 days at 35C (2 to 4 weeks
at 25C) as a cloud of minute granules that develop into
microcolonies just below the surface.
5. Gram stain is not satisfactory. The microcolonies can be fixed
with methanol and stained with Giemsa stain to show rod forms
at the edges.9
Results

this medium.
References
1. Fletcher, W. 1928. Recent work on leptospirosis, tsutsugamushi
disease and tropical typhus in the federated Malay States. Trans.
Roy. Soc. Trop. Med. Hyg. 21: 265-287.
Bacto Folic AOAC Medium
Section II
2. Myers, D. M., V. M. Varela-Diaz, and A. A. Siniuk. 1973.

Long-term survival of Leptospira in a biphasic culture medium
containing charcoal. Am. Soc. Microbiol. 25:514-516.
3. Kaufmann, A. F., and R. S. Weyant. 1995. Leptospiraceae,
p. 621-625. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover
and R. H. Yolken (ed.), Manual of clinical microbiology, 6th ed.
4. Koneman, E. W., S. D. Allen, V. R. Dowell, Jr., W. M. Janda,
H. M. Sommers, and W. C. Winn, Jr. 1988. Color atlas and
textbook of diagnostic microbiology, 3rd ed. J. B. Lippincott
Company, Philadelphia, PA.
5. Elliott, S. H. 1980. Discussion and clinical diagnosis of
Leptospirosis. J. Am. Med. Tech. 42:37-44.
6. Faine, S. (ed.). 1982. Guidelines for the control of Leptospirosis. W.
H. O. Offset publication no. 67. World Health Organization, Geneva.
8. Kelley, P. W. 1992. Leptospirosis, p. 1295-1301. In S. L. Gorbach,
J. G. Bartlett, and N. R. Blacklow (ed.), Infectious diseases. The
W. B. Saunders Co., Philadelphia, PA.
9. Weinman, D. 1981. Bartonellosis and anemias associated with
bartonella-like structures, p. 235-248. In A. Balows, and W. J.
Hausler, Jr. (ed.), Diagnostic procedures for bacterial, mycotic and
parasitic infections, 6th ed. American Public Health Association,
Washington, D.C.
Packaging
500 g
0987-17
Intended Use
Bacto Folic AOAC Medium is used for determining folic acid concentration by the microbiological assay technique.

Dehydrated Appearance: Off-white, free-flowing,
homogeneous.
Solution:
5.50% solution (single strength) and
11.0% solution (double strength),
soluble in distilled or deionized
water on boiling.
Solution is light amber, clear, may
Prepared Medium:
Very light amber, clear.
Reaction of 5.50%
Solution at 25C:
pH 6.7 0.1
Cultural Response
Prepare Folic AOAC Medium per label directions. This
medium should support the growth of E. hirae ATCC 8043
when prepared in single strength and supplemented with
folic acid.
198
Also Known As
AOAC is an abbreviation for Association of Official Analytical Chemists.

1. Maintenance Media: For carrying the stock culture to preserve
the viability and sensitivity of the test organism for its intended
purpose.
3. Assay Media: to permit quantitation of the vitamin under test.
Folic AOAC Medium is prepared for use in the microbiological assay
of folic acid according to the procedures of the Folic Acid Assay in
AOAC.1 Enterococcus hirae ATCC 8043 (Streptococcus faecium) is
the test organism in this assay.

Folic Acid AOAC Medium is a folic acid-free dehydrated medium
of E. hirae ATCC 8043. The addition of folic acid in specified
The Difco Manual
Section II
Folic AOAC Medium
increasing concentrations gives a growth response that can be

measured turbidimetrically or titrimetrically.
Formula
Folic AOAC Medium
Formula Per Liter
L-Asparagine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6
L-Tryptophane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
L-Cysteine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . 0.76
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Adenine Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Uracil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Xanthine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Thiamine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . 400
Calcium Pantothenate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800
Nicotinic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Glutathione . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
g
g
g
g
g
mg
mg
mg
mg
mg
mg
g
g
g
g
mg
mg
g
g
g
g
mg
mg
mg
Precautions
other chemicals must be used. Glassware is heated to 250C for at
Storage
Expiration Date
Procedure
Materials Provided
Folic AOAC Medium
The Difco Manual

Glassware
Autoclave
Stock culture of Enterococcus hirae ATCC 8043
Sterile tubes
Folic Acid
0.01 N NaOH
Dilute HCl
Spectrophotometer or Nephelometer
1.
2.
3.
4.
5.
6.
Suspend 11 grams in 100 ml distilled or deionized water.

Heat to boiling for 2-3 minutes.
Distribute 5 ml amounts into tubes, evenly dispersing the precipitate.

specific assay procedures. For assays, the samples should be diluted to
Test Procedure
Follow assay procedures as outlined in AOAC.1 It is essential that a
standard curve be set up for each separate assay. Autoclaving and
incubation conditions that can influence the standard curve readings
cannot always be duplicated. The standard curve is obtained by using
folic acid at levels of 0.0, 1, 2, 4, 6, 8 and 10 ng per assay tube (10 ml).
Folic AOAC Medium may be used for both turbidimetric and titrimetric
analysis. Turbidimetric readings should be taken after incubation at
35-37C for 16-18 hours. Titrimetric determinations are best made
following incubation at 35-37C for 72 hours.
The folic acid required for the preparation of the standard curve may
be prepared as follows:
A. Dissolve 50 mg dried folic acid in about 30 ml 0.01N NaOH
and 300 ml distilled water.
B. Adjust the pH reaction to 7.5 0.5 with diluted HCl solution.
Dilute to 500 ml with distilled water.
C. Add 2 ml of the solution to 50 ml distilled water. Adjust the
pH reaction to 7.5 0.5. Dilute to 100 ml with distilled water.
This yields a stock solution containing 2 mcg folic acid per ml.
D. Prepare the stock solution fresh daily.
The standard solution for the assay is made by diluting 1 ml of this
stock solution to 1 liter with distilled water. This solution contains 2 ng
folic acid per ml. Use 0.0, 0.5, 1, 2, 3, 4, and 5 ml per assay tube.
Some laboratories may wish to alter the concentration of folic acid
recommended above for the standard curve. This is permissible if the
concentration used is within the limits specified by AOAC.1
Results
disk or cup.
199
Folic Acid Assay Medium
Section II

3. The use of altered or deficient media may cause mutants

having different nutritional requirements that will not give a
satisfactory response.

methods of analysis of AOAC international, 16th ed. AOAC
1. The test organism used for inoculating an assay medium must

be cultured and maintained on media recommended for this
purpose.
References
Packaging
Folic AOAC Medium
100 g
0967-15*
*Store at 2-8C
Bacto Folic Acid Assay Medium
Intended Use
Bacto Folic Acid Assay Medium is used for determining folic acid
Folic Acid Assay Medium is a folic acid-free dehydrated medium

of E. hirae ATCC 8043. The addition of folic acid in specified
increasing concentrations gives a growth response that can be measured
turbidimetrically.

of vitamins. Three types of medium are used for this purpose:
1. Maintenance Medium: For carrying the stock culture to preserve the
2. Inoculum Medium: To condition the test culture for immediate use.
3. Assay Medium: To permit quantitation of the vitamin under test.
Folic Acid Assay Medium is used in the microbiological assay of folic
acid with Enterococcus hirae ATCC 8043 as the test organism. Folic
Acid Assay Medium is prepared according to the formula described by
Capps, Hobbs and Fox,1 modified with sodium citrate instead of
sodium acetate.

Dehydrated Appearance: Off white to very light beige,
Solution:
boiling for 2-3 minutes. Solution is
light amber, clear, may have a slight
precipitate.
Prepared Medium:
Very light amber, clear, may have a
very slight precipitate.
Reaction of 3.75%
Solution at 25C:
pH 6.8 0.2
Cultural Response
Prepare single-strength Folic Acid Assay Medium per label
directions. The medium should support the growth of E. hirae
ATCC 8043. The most effective range is 2-10 ng folic acid
per 10 ml tube.
200
Formula
Formula Per Liter
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
DL-Tryptophane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
Adenine Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Uracil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
g
g
g
g
g
mg
mg
mg
mg
mg
mg
mg
g
g
g
g
g
g
mg
mg
mg
Precautions
The Difco Manual
Section II

present.
Storage
Expiration Date
Procedure
Materials Provided

Glassware
Autoclave
Stock culture of Enterococcus hirae ATCC 8043
Sterile tubes
0.01 N NaOH
Dilute HCl
Folic Acid USP
Lactobacilli Broth AOAC
Centrifuge
Spectrophotometer
1.
2.
3.
4.
5.
6.


Prepare assay samples according to references given in the specific
assay procedures. Dilute the samples to approximately the same
concentration as the standard solution.
Test Procedure
Prepare stock cultures of E. hirae ATCC 8043 by stab inoculation of
Lactobacilli Agar AOAC. Incubate at 35-37C for 24-48 hours. Store
tubes in the refrigerator. Make transfers at monthly intervals. Prepare
the inoculum for assay by subculturing a stock culture of E. hirae
ATCC 8043 into a tube containing 10 ml of Lactobacilli Broth AOAC.
After incubation at 35-37C for 18-24 hours, centrifuge the cells under
The Difco Manual
aseptic conditions and decant the supernatant. Wash the cells three
times with 10 ml of sterile 0.85% saline. After the third wash, dilute
the cell suspension 1:100 with sterile 0.85% saline. Use one drop of
this latter suspension to inoculate each of the assay tubes.
It is essential that a standard curve be set up for each separate assay.
Autoclaving and incubation conditions that influence the standard
curve readings cannot always be duplicated. The standard curve is
obtained by using folic acid at levels of 0.0, 2, 4, 6, 8 and 10 ng per
10 ml assay tube. Turbidimetric readings should be made after
incubation at 35-37C for 18-24 hours. Refrigerate tubes for 15-30
minutes to stop growth before reading.
Prepare the folic acid stock solution required for the standard curve as
follows:
1. Dissolve 50 mg dried Folic Acid USP Reference Standard or
equivalent in about 30 ml of 0.01 N NaOH and 300 ml distilled
water.
2. Adjust to pH 7.5 0.5 with diluted HCl solution. Add distilled
water to give a volume of 500 ml.
3. Add 2 ml of the solution from step 2 to 50 ml distilled water.
Adjust the pH to 7.5 0.5 with HCl solution. Dilute to 100 ml with
distilled water to give a stock solution containing 2 mcg folic acid
per ml. Prepare the stock solution fresh daily.
Prepare the standard solution for the assay by diluting 1 ml of this
stock solution in 1 liter with distilled water. This solution contains 2 ng
folic acid per ml. Use 0.0, 0.5, 1, 2, 3, 4 and 5 ml per assay tube.
Following incubation, place the tubes in the refrigerator for 15-30
minutes to stop growth. The growth can be measured by a turbidimetric
method and the curve constructed from the values obtained. The most
effective assay range is between the levels of 2 and 10 ng folic acid per
10 ml tube.
Results
or cup.

response.
References
1. Capps, Hobbs, and Fox. 1948. J. Bacteriol. 55:869.
Packaging
100 g
0318-15
201
Folic Acid Casei Medium & Folic Buffer A, Dried
Bacto Folic Acid Casei Medium

Bacto Folic Buffer A, Dried
Section II
Intended Use
Bacto Folic Acid Casei Medium is used for determining folic acid
Bacto Folic Buffer A, Dried is prepared for use with Folic Acid Casei
Medium in the microbiological assay of serum folic acid.

Folic Acid Casei Medium is prepared for the microbiological assay of
folic acid, particularly folic acid in serum. Lactobacillus casei subsp.

Folic Acid Casei Medium
Dehydrated Appearance: Off-white, homogeneous, with a
tendency to clump.
Solution:
boiling 1-2 minutes. Single-strength
solution is light amber, clear, may
Prepared Medium:
Single-strength solution is very light
amber, clear, may have a very slight
precipitate.
Reaction of 4.7%
Solution at 25C:
6.7 0.1
Folic Buffer A, Dried
Dehydrated Appearance: White to off-white, free-flowing,
homogeneous.
Solution:
or deionized water.
Solution Appearance: Colorless to very light amber, clear.
Reaction of 1.54%
Solution at 25C:
pH 6.1 0.05
Cultural Response
Prepare Folic Acid Casei Medium per label directions. The
medium is tested by creating a standard curve using Folic Acid
at concentrations of 0 to 1.0 ng per 10 ml. This medium should
support the growth of L. casei subsp. rhamnosus ATCC 7469
when prepared in single strength and supplemented with
ascorbic acid and Folic Acid.
rhamnosus ATCC 7469 is used as the test organism in this assay.

Folic Acid Casei Medium is prepared according to the formulation
described by Flynn, Williams, ODell and Hogan1 and modified by
Baker et al.2 and Waters and Mollin.3
Total serum folic acid activity can vary depending on the disease state.
It has been reported that normal subjects have a mean serum folic acid
level of 9.9 ng per ml. Patients with uncomplicated pernicious anemia
have a mean serum folic acid level of 16.6 ng per ml while patients
with megaloblastic anemia have levels less than 4.0 ng per ml.
Folic Buffer A, Dried is used for preparing both the standard and the
serum specimen in the microbiological assay of folic acid.

Folic Acid Casei Medium is a folic acid-free dehydrated medium
of L. casei subsp. rhamnosus ATCC 7469. The addition of folic acid
in specified increasing concentrations gives a growth response that can
be measured turbidimetrically.
Formula
Formula Per Liter
Charcoal Treated Casitone . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
DL-Tryptophane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
L-Asparagine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6
L-Cysteine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Adenine Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Uracil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Xanthine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Glutathione (reduced) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Magnesium Sulfate, Anhydrous . . . . . . . . . . . . . . . . . . . . . 0.2
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Nicotinic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
g
g
g
g
g
g
g
g
mg
mg
mg
mg
g
mg
g
mg
mg
mg
mg
mg
mg
g
g
g
g
Final pH 6.7 0.1

Formula Per Liter
Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . 10.656 g
Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . 3.744 g
Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Final pH 6.1 0.05
202
The Difco Manual
Section II
Precautions
for at least 1 hour to burn off any organic residues that might be present.
Storage
Store Folic Acid Casei Medium and Folic Buffer A, Dried at 2-8C.
The dehydrated medium is very hygroscopic. Keep container tightly
closed.
Expiration Date
Procedure
Materials Provided

Glassware
Autoclave
Stock culture of Lactobacillus casei subsp. rhamnosus ATCC 7469
Ascorbic acid (25 mg)
Distilled and deionized water
0.01 N NaOH
0.05 N HCl
Folic Acid
Incubator (35-37C)
Micro Inoculum Broth
Centrifuge
Spectrophotometer
2. Add 50 mg ascorbic acid if standard and test samples are not prepared
in Folic Buffer A.
3. Boil for 1-2 minutes.
4. Dispense 5 ml amounts into tubes, evenly dispersing any precipitate.
5. Add standard or test samples.
6. Adjust tube volume to 10 ml with distilled or deionized water.
The Difco Manual
Folic Acid Casei Medium & Folic Buffer A, Dried
Folic Buffer A Dried

1. Dissolve the contents of one vial (15.4 grams) in 1 liter distilled or
deionized water.

Test Procedure
Preparation of Stock Cultures and Inoculum
Prepare stock cultures of the test organism, L. casei subsp. rhamnosus
ATCC 7469, by stab inoculation into prepared tubes of Lactobacilli Agar
AOAC. Incubate the cultures at 35-37C for 18-24 hours. Store cultures
in the refrigerator at 2-8C. Stock transfers are made at monthly intervals.
Prepare the inoculum for assay by subculturing from a stock culture of
L. casei subsp. rhamnosus into a tube containing 10 ml prepared
Micro Inoculum Broth. Incubate at 35-37C for 16-18 hours. Under
aseptic conditions, centrifuge the tubes to sediment the cells and decant
the supernatant. Wash the cells in 10 ml sterile single-strength Folic
Acid Casei Medium. Resediment the cells by centrifuging aseptically
and decant the supernatant. Repeat washing two more times. After the
third washing, resuspend the cells in 10 ml sterile single-strength
medium and dilute 1 ml with 99 ml of the same medium. One drop of
this suspension is used to inoculate each of the assay tubes. Read the
growth response of the assay tubes turbidimetrically after 18-24 hours
incubation at 35-37C. (Some laboratories use 0.85% saline instead of
the single-strength basal medium to wash and dilute the inoculum.)
Preparation of the Standard
It is essential that a standard curve be constructed for each separate
assay. Autoclave and incubation conditions can influence the standard
curve readings and cannot always be duplicated. The standard curve
may be obtained by using folic acid at levels of 0.0, 0.1, 0.2, 0.4, 0.6,
0.8 and 1 ng per assay tube (10 ml).
The folic acid required for preparation of the standard curve may be
prepared as follows:
Dissolve 50 mg dried folic acid in about 30 ml 0.01 N NaOH and 300
ml distilled water. Adjust to pH 7-8 with 0.05 N HCl and dilute to 500
ml with distilled water. Dilute 10 ml of this solution with 500 ml
distilled water. Further dilute 1 ml in 1 liter distilled water to make a
stock solution containing 2 ng per ml folic acid. Prepare the standard
solution containing 0.2 ng per ml folic acid by diluting 10 ml of stock
solution with 90 ml of Folic Buffer A, Dried solution. Use 0.0, 0.5, 1,
2, 3, 4 and 5 ml per assay tube.
Prepare the stock solution fresh daily.
Preservation of Serum Specimens
1. Allow the blood specimen to clot and the serum to separate from
the clot.
2. Aspirate the serum into a clean dry tube and centrifuge to remove
any cells that may be present. Avoid hemolysis. Dispense 5 ml
of each serum sample into clean dry test tubes and add 25 mg
ascorbic acid to each tube.
3. If the test is not begun immediately, place tubes in a freezer and
hold below -20C.
203
Fraser Broth
Section II
Preparation of Serum Specimen

1. Thaw the serum containing ascorbic acid.
2. Add 5 ml of the uniform sample to 45 ml rehydrated Folic Buffer
A, Dried.
3. Incubate the serum-buffer solution at 37C for 90 minutes. Autoclave
the incubated mixture at 121C for 2.5 minutes.
4. Remove the coagulated protein by centrifuging and transfer the
clear supernatant to a clean dry tube. The clear solution is the
sample to use in the folic acid assay.
Procedure for Total Folic Acid
1. Use 0.5, 1.0, 1.5 ml or other volumes of the prepared serum
extracts as described above.
2. Fill each assay tube with 5 ml of rehydrated Folic Acid Casei
Medium and sufficient distilled or deionized water to give a total
volume of 10 ml per tube.
3. Autoclave tubes at 121C for 5 minutes.
4. Add 1 drop of inoculum described under Preparation of Stock
Culture and Inoculum to each assay.
5. Incubate at 35-37C for 18-24 hours. Tubes are refrigerated for
15-30 minutes to stop growth before reading turbidimetrically.
interpolating the results with the values obtained on the standard curve,
taking into consideration the dilutions of the samples.

be cultured and maintained on media recommended for this
purpose.
response.
References
1. Flynn, Williams, ODell, and Hogan. 1951. Anal. Chem. 23:180.
2. Baker, Herbert, Frank, Pasher, Hunter, Wasserman, and
Sobotka. 1959. Clin. Chem. 5:275.
3. Waters and Molin. 1961. J. Clin. Pathol. 14:335.
Packaging
Results
The amount of folic acid in the test samples can be determined by
100 g
0822-15
6 x 15.4 g
3246-33
Fraser Broth
Bacto Fraser Broth Base . Fraser Broth Supplement
Intended Use
Bacto Fraser Broth Base is used with Bacto Fraser Broth Supplement
in selectively enriching and detecting Listeria.

First described in 1926 by Murray, Webb and Swann, 1 Listeria
monocytogenes is a widespread problem in public health and the food
industries. This organism has the ability to cause human illness
and death, particularly in immunocompromised individuals and
pregnant women.2 The first reported food-borne outbreak of listeriosis
was in 1985,3 and since then, microbiological and epidemiological
evidence from both sporadic and epidemic cases of listeriosis has
indicated that the principle route of transmission is via the consumption
of foodstuffs contaminated with Listeria monocytogenes.4
Implicated vehicles of transmission include turkey frankfurters,5
coleslaw, pasteurized milk, Mexican-style cheese, pat, and pickled pork
tongue. The organism has been isolated from commercial dairy and other
food processing plants, and is ubiquitous in nature, being present in a
wide range of unprocessed foods as well as in soil, sewage, silage
and river water.6
Bacto Fraser Broth Base and Bacto Fraser Broth Supplement are based
on the formulation of Fraser and Sperber.7 The medium is use in
the rapid detection of Listeria from food8 and environmental samples.
204
Many common food contaminants such as streptococci, enterococci,

Bacillus species, Escherichia coli, Pseudomonas aeruginosa and Proteus
vulgaris interfere with the isolation of Listeria monocytogenes.9
Listeria species grow over a pH range of 5.0-9.6, and survive in food
products with pH levels outside these parameters.10 Listeria spp. are
microaerophilic, gram-positive, asporogenous, non-encapsulated, nonbranching, regular, short, motile rods. Motility is most pronounced at 20C.
Identification of Listeria is based on successful isolation of the
organism, biochemical characterization and serological confirmation.

Bacto Tryptose, Bacto Beef Extract and Bacto Yeast Extract provide
nitrogen, vitamins and minerals. Sodium phosphate and potassium
phosphate are buffering agents. Differentiation is aided by including
ferric ammonium citrate in the final medium. Since all Listeria species
hydrolyze esculin, the addition of ferric ions to the medium will detect
the reaction. A blackening of the medium by cultures containing
esculin-hydrolyzing bacteria is the result of the formation of
6,7-dihydroxycoumarin that reacts with the ferric ions.7
Selectivity is provided by the presence of lithium chloride, nalidixic
acid and acriflavine in the formula. The high salt tolerance of Listeria
is used as a means to inhibit growth of enterococci.
The Difco Manual
Section II
Fraser Broth
Formula
Fraser Broth Base
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Esculin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Nalidixic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.02
Acriflavine HCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.024
g
g
g
g
g
g
g
g
g
g

Precautions
2. Fraser Broth Base:
TARGET ORGAN(S): Blood, Kidneys, Nerves

Fraser Broth Supplement:
AND SKIN. Avoid contact with skin and eyes. Do not breathe mist.
Storage
Store Bacto Fraser Broth Supplement at 2-8C.

Fraser Broth Base
Solution:
boiling. Solution is medium amber, clear to slightly
opalescent with a fine precipitate.
Prepared Tubes:
Medium amber, clear to slightly opalescent with
a fine precipitate.
Reaction of 5.5%
Solution at 25C:
pH 7.2 0.2
Bacto Fraser Broth Supplement
Cultural Response
Prepare Fraser Broth Base per label directions. Add Fraser Broth Supplement.
ORGANISM
Escherichia faecalis
Escherichia coli
ATCC
INOCULUM
CFU
29212* 1,000-2,000
25922* 1,000-2,000
19114
100-1,000
GROWTH
ESCULINE
REACTION

good
Uninoculated
tube
ATCC 19114
The Difco Manual
205
Fraser Broth
Expiration Date
Procedure
Materials Provided
Fraser Broth Base

Flasks with closure
Autoclave
Waterbath (45-50C)
Test tubes with closures
Incubator (30C)
Incubator (35C)
1. Suspend 55 grams of Fraser Broth Base in 1 liter of distilled or
deionized water.
4. Aseptically add 10 ml Fraser Broth Supplement. Mix well.

Test Procedure
To isolate Listeria monocytogenes from processed meats and poultry,
the following procedure is recommended by the U.S.D.A.8
1. Add 25 grams of test material to 225 ml of UVM Modified Listeria
Enrichment Broth and mix or blend thoroughly.
2. Incubate for 20-24 hours at 30C.
3. Transfer 0.1 ml of the incubated broth to Fraser Broth. Incubate at
35C for 26 2 hours.
4. At 24 and 48 hours, streak the Fraser Broth culture to Modified
Oxford Agar.
5. Incubate the Modified Oxford plates at 35C for 24-48 hours.
Results
1. Examine agar plates for suspect colonies. For further identification and confirmation of Listeria spp., consult appropriate
references.8,10,11,12
2. Rapid slide and macroscopic tube tests can be used for definitive
serological identification.

1. Since Listeria species other than L. monocytogenes can grow on
these media, an identification of Listeria monocytogenes must be
confirmed by biochemical and serological testing.11,12
206
Section II
2. Poor growth and a weak esculin reaction may be seen after 40 hours
incubation for some enterococci.
References
1. Murray, E. G. D., R. A. Webb, and M. B. R. Swann. 1926.
A disease of rabbits characterized by large mononuclear
leucocytosis caused by a hitherto undescribed bacillus Bacterium
monocytogenes (n. sp.). J. Path. Bact. 29:407- 439.
2. Monk, J. D., R. S. Clavero, L. R. Beuchat, M. P. Doyle, and
R. E. Brackett. 1994. Irradiation inactivation of Listeria
monocytogenes and Staphylococcus aureus in low- and high-fat,
frozen and refrigerated ground beef. J. Food Prot. 57:969-974.
3. Wehr, H. M. 1987. Listeria monocytogenes - a current dilemma
special report. J. Assoc. Off. Anal. Chem. 70:769-772.
4. Bremer, P. J., and C. M. Osborne. 1995. Thermal-death times of
Listeria monocytogenes in green shell mussels (Perna canaliculus)
prepared for hot smoking. J. Food Prot. 58:604-608.
5. Grau, F. H., and P. B. Vanderlinde. 1992. Occurrence, numbers,
and growth of Listeria monocytogenes on some vacuum-packaged
processed meats. J. Food Prot. 55:4-7.
6. Patel, J. R., C. A. Hwang, L. R. Beuchat, M. P. Doyle, and
R. E. Brackett. 1995. Comparison of oxygen scavengers for their
ability to enhance resuscitation of heat-injured Listeria
monocytogenes. J. Food Prot. 58:244-250.
food and environmental samples by esculin hydrolysis. J. Food
Prot. 51:762-765.
8. Lee, W. H., and D. McClain. 1994. Laboratory Communication
No. 57 (revised February 8, 1994), U.S.D.A., F.S.I.S. Microbiology
Division, Bethesda, MD.
9. Kramer, P. A., and D. Jones. 1969. Media selective for Listeria
monocytogenes. J. Appl. Bacteriol. 32:381-394.
10. Donnelly, C. W., R. E. Bracket, D. Doores, W. H. Lee, and
J. Lovett. 1992. Listeria, p. 637-663. In C. Vanderzant and D. F.
Washington, D.C.
11. Swaminathan, B., J. Rocourt, and J. Bille. 1995. Listeria,
p. 342-343. In P. R. Murray, E. J. Baron, M. A. Pfaller,
F. C. Tenover, and R. H. Yolken (ed.). Manual of clinical
Washington, D.C.
1993. Pathogens in milk and milk products. In R. T. Marshall (ed.),
Standard methods for the examination of dairy products, 16th ed.
Packaging
Fraser Broth Base
500 g
2 kg
6 x 10 ml
0219-17
0219-07
0211-60*
*Store at 2-8C
The Difco Manual
Section II
GC Medium Base, Supplement B, Supplement VX, Hemoglobin, Antimicrobic Vial CNV & Antimicrobic Vial CNVT
Bacto GC Medium Base . Bacto Supplement B

Bacto Supplement VX . Bacto Hemoglobin
Bacto Antimicrobic Vial CNV . Bacto Antimicrobic Vial CNVT
Intended Use
Bacto GC Medium Base is used with various additives in isolating and
cultivating Neisseria gonorrhoeae and other fastidious microorganisms.
Bacto Supplement B with Bacto Reconstituting Fluid B is used for
supplementing media to culture fastidious organisms, particularly
Neisseria gonorrhoeae and Haemophilus influenzae.
Bacto Supplement VX with Bacto Reconstituting Fluid VX is used to

culture fastidious microorganisms, particularly Neisseria gonorrhoeae.
Bacto Hemoglobin is used in preparing microbiological culture media.
Bacto Antimicrobic Vial CNV and Bacto Antimicrobic Vial CNVT
are sterile lyophilized preparations containing inhibitory agents to be
used in selective media for culturing Neisseria gonorrhoeae and
Neisseria meningitidis.
Also Known As
GC Medium is also referred to as Chocolate Agar Base, Chocolate

Agar, Enriched and GC Agar.
GC Medium Base
Solution:
medium amber, opalescent, may have
slight precipitate, ground glass
appearance.
Prepared Medium:
With Hemoglobin and Supplement:
chocolate brown, opaque.
Reaction of 3.6%
Solution at 25C:
pH 7.2 0.2
Hemoglobin
Dehydrated Appearance: Dark brown, fine, free-flowing.
Solution:
2% solution, insoluble in distilled or
deionized water; chocolate brown,
opaque with a dispersed precipitate.
Reaction of 2%
Solution at 25C:
pH 8.2 0.2
Supplement B
Lyophilized Appearance: Tan to reddish brown lyophilized
powder or cake.
Rehydrated Appearance: Medium to dark amber may have a
reddish tint, clear to slightly
opalescent solution.
Reconstituting Fluid:
Colorless, clear solution.
Sterility Test:
Satisfactory.
Reaction of
Solution at 25C:
pH 6.5-7.2
Supplement VX
Lyophilized Appearance: Pink, lyophilized powder.
Rehydrated Appearance: Pink, clear solution without precipitate.
Reconstituting Fluid:
Sterility Test:
Satisfactory.
Reaction of
Solution at 25C:
pH 0.75-2.5
The Difco Manual

In 1945, Johnston 1 described a medium that could successfully
produce colonies of N. gonorrhoeae in 24 rather than 48 hours. The
accelerated growth rates were primarily due to the decreased agar
content (solidity) of the media. GC Medium Base was introduced in
1947 with reduced agar content. While investigating the growth rate of
some gonococcal strains, a medium containing the growth factors
glutamine and cocarboxylase, was found to improve recovery.2,3 From
this discovery, Supplement B was developed. In a comparative study4
of 12 different media, an enriched Chocolate Agar prepared with
GC Medium Base, Hemoglobin and Supplement B proved superior for
isolating N. gonorrhoeae.
Supplement B w/ Reconstituting Fluid is a sterile yeast concentrate for
use in supplementing media for microorganisms with exacting growth
requirements. It is recommended for use in the preparation of chocolate
agar described by Christensen and Schoenlein.5
Supplement VX w/ Reconstituting Fluid is a sterile lyophilized
concentrate. Supplement B and Supplement VX are recommended for
enriching GC Medium Base, Proteose No. 3 Agar, Thayer-Martin
Medium and Modified Thayer-Martin Medium.
Hemoglobin, an autoclavable preparation of beef blood, is prepared
according to described by Spray.6 Hemoglobin provides hemin, which
is required by Haemophilus species and enhances growth of Neisseria
species.
In 1964, Thayer and Martin7 formulated a selective medium incorporating
the antibiotics polymyxin B and ristocetin into GC Agar with added
hemoglobin and yeast supplement B. Thayer and Martin8 improved
their medium by replacing the two original antibiotics with a new
microbial solution of colistin, vancomycin and nystatin (CVN). In
1970, Martin and Lester9 improved the new Thayer-Martin (TM) medium by increasing the agar and glucose content and by incorporating an
additional antibiotic, trimethoprim lactate (T) into the formulation. This
improved medium is called Modified Thayer-Martin (MTM) Medium.
Antimicrobic Vial CNV and Antimicrobic Vial CNVT are used in the
preparation of Thayer-Martin (TM) Medium and Modified ThayerMartin, respectively.
207
Martin and Lewis10 further improved selectivity of MTM by increasing

the concentration of vancomycin and replacing nystatin with
anisomycin for greater inhibition of yeasts; this is known as MartinLewis (ML) Agar Medium. Transgrow Medium is a transport medium
system incorporating either MTM or ML formulations.11

GC Medium Base is employed as the basal medium in the preparation
of Chocolate Agar Enriched, Thayer-Martin Medium and Modified
Thayer-Martin Medium.
Proteose Peptone No. 3 provides nitrogen, vitamins and amino acids in
GC Medium Base. Corn Starch absorbs any toxic metabolites that are
produced, Potassium Phosphate, Dibasic and Monobasic buffer the
medium. Sodium Chloride maintains osmotic balance. Bacto Agar is
Chocolate Agar is prepared from GC Medium Base with the addition
of 2% Hemoglobin. Hemoglobin provides hemin (X factor) required
for growth of Haemophilus and enhanced growth of Neisseria.
The growth rate of Neisseria and Haemophilus is improved with the
addition of 1% Supplement B or VX, providing the growth factors
glutamine and cocarboxylase. Supplement B contains yeast concentrate,
Section II
glutamine, coenzyme, cocarboxylase, hematin and growth factors.

Supplement VX is a sterile, defined lyophilized concentrate of essential
growth factors. Supplement VX supplies vitamins, amino acids,
coenzymes, dextrose and other factors to improve the growth of
Haemophilus and Neisseria species.
Antimicrobic Vial CNV and Antimicrobic Vial CNVT are antimicrobial
agents used as inhibitors in the selective media, Thayer-Martin
Medium and Modified Thayer-Martin Medium.
Formula
GC Medium Base
Formula Per Liter
Corn Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Potassium Phosphate, Dibasic . . . . . . . . . . . . . . . . . . . . . . . 4
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
g
g
g
g
g
g

ATCC 43069
Uninoculated
plate
Antimicrobic Vial CNV

Lyophilized Appearance: Pale yellow, dry cake or powder.
Rehydrated Appearance: Off-white to pale yellow, opalescent to
opaque even suspension.
Solubility:
Not completely soluble in distilled water,
but must be evenly suspendable.
Microbial Limits Test: Negative.
Antimicrobic Vial CNVT
Lyophilized Appearance: Pale yellow, dry cake or powder.
Rehydrated Appearance: Off-white to pale yellow, opalescent to
opaque even suspension.
Solubility:
Not completely soluble in distilled water,
but must be evenly suspendable.
Microbial Limits Test: Negative.
Cultural Response
GC Medium Base, Hemoglobin 2%, Supplement B or Supplement VX
Prepare Chocolate Agar with GC Medium Base, per label directions. Inoculate
and incubate at 35 2C under 5-10% CO2 for 18-48 hours.
ORGANISM
ATCC
INOCULUM CFU
GROWTH
10211
43069
30-300
30-300
good
good
Haemophilus parainfluenzae
ATCC 7901
GC Medium Base, Hemoglobin 2%, Supplement B or Supplement VX, Antimicrobic Vial CNV or CNVT
Prepare Thayer-Martin Medium or Modified Thayer-Martin Medium with GC Medium Base per label directions, enriched
with Antimicrobic Vial CNV or Antimicrobic Vial CNVT. Inoculate and incubate at 35 2C under CO2 for 18-48 hours.
ORGANISM
ATCC
Candida albicans
60193
Escherichia coli
25922*
43069
13090*
Neisseria sicca
9913*
Staphylococcus epidermidis 12228*
208
INOCULUM CFU
GROWTH
1,000
partial inhibition
1,000
100-1,000
good
100-1,000
good
100-1,000 marked to complete inhibition
1,000

be used as directed in Bactrol Disk Technical Information.
The Difco Manual
Section II
Hemoglobin
An autoclavable preparation of beef blood prepared according to the
procedure described by Spray.6
Expiration Date
Supplement B
Processed to preserve both the thermolabile and thermostable growth
accessory factors of fresh yeast, contains glutamine, coenzyme (V factor),
cocarboxylase and other growth factors, as well as hematin (X factor).
Supplement VX
Ingredients per 10 ml Vial
Adenine Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Cocarboxylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
L-Cysteine HCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Diphosphopyridine Nucleotide . . . . . . . . . . . . . . . . . . . . . 3.5
Ferric citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3
L-Glutamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Guanine HCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3
Thiamine HCI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.06
Vitamin B12 (Cyanocobalamin) . . . . . . . . . . . . . . . . . . . . . 0.2
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Procedure
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
Antimicrobial Vial CNV

A sterile, lyophilized preparation containing 7,500 g Colistin Sulfate,
12,500 units Nystatin and 3,000 g Vancomycin per 10 ml.
Antimicrobial Vial CNVT

A sterile lyophilized preparation containing 7,500 g Colistin Sulfate,
12,500 units Nystatin, 3,000 g Vancomycin and 5,000 g
Trimethoprim per 10 ml.
Precautions
2. Antimicrobic Vial CNV
SYSTEM AND SKIN REACTION. (US) MAY CAUSE HARM
tightly closed. TARGET ORGAN(S): Kidney, Ears, Lungs, Thorax.
HARMFUL. HARMFUL BY INHALATION AND IF SWALLOWED. (US) MAY CAUSE ALLERGIC EYE, RESPIRATORY
tightly closed. TARGET ORGAN(S): Lungs, Thorax.
Storage
Store GC Medium Base below 30C. The dehydrated medium is very
Store Hemoglobin below 30C. Store Hemoglobin 2% at 15-30C.
The Difco Manual
Store Supplements B and VX at 2-8C.

Store Antimicrobic Vials CNV and CNVT at 2-8C.
Materials Provided
GC Medium Base
Hemoglobin, Hemoglobin 2%
Supplement B or VX
Antimicrobial Vial CNV or CNVT

Glassware
Autoclave
Incubator (35C)
Waterbath (45-50C).
Supplement B
Supplement VX
1. Aseptically rehydrate Supplement B and Supplement VX with 10 ml
or 100 ml of the corresponding Reconstituting Fluid, as appropriate.
2. Rotate the vial to dissolve completely.
Hemoglobin
1. Place 10 grams of Hemoglobin in a dry beaker.
2. Measure 500 ml distilled or deionized water.
3. Add approximately 100 ml amounts to the Hemoglobin, stirring
well after each addition. Use a spatula to break up clumps.
4. Transfer to flasks as desired for autoclaving.
6. Cool to 45-50C.
7. Swirl flask to reestablish complete solution and add to an equal
amount of double-strength sterile agar base cooled to 45-50C.
Hemoglobin 2%
1. Shake the bottle to resuspend any sedimented hemoglobin before use.
Antimicrobial Vial CNV
Antimicrobial CNVT
1. Aseptically rehydrate Antimicrobial Vial CNV or Antimicrobial
CNVT with the appropriate amount of sterile distilled or deionized
water, as indicated on the product label.
2. Rotate the vial to dissolve completely.
Chocolate Agar, Enriched
1. Suspend 7.2 grams GC Medium Base in 100 ml distilled or
deionized water.
4. Aseptically add 100 ml Hemoglobin Solution 2%.
5. Aseptically add 2 ml Supplement B or Supplement VX. Mix well.
6. Dispense into sterile Petri dishes or tubes as desired.
209
Thayer-Martin Medium
deionized water.
4. Aseptically add 100 ml Hemoglobin Solution 2%.
5. Aseptically add 2 ml Supplement B or Supplement VX.
6. Aseptically add 2 ml rehydrated Antimicrobial Vial CNV to the medium.
Modified Thayer-Martin Medium
deionized water.
4. Aseptically add 100 ml Hemoglobin Solution 2% and 0.3 grams
dextrose to the medium.
5. Aseptically add 2 ml Supplement B or Supplement VX.
6. Aseptically add 2 ml of rehydrated Antimicrobial Vial CNVT to
the medium.

Test Procedure
For a complete discussion on the isolation and identification of Neisseria
and Haemophilus, consult the procedures outlined in the references.12,13,14
Results

2. GC Medium Base is intended for use with supplementation.
Although certain diagnostic tests may be performed directly on
this medium, biochemical and, if indicated, immunological testing
Consult appropriate references for further information.
3. Improper specimen collection, environment, temperature, CO2
level, moisture and pH can adversely affect the growth and viability
of the organism.
4. Inactivation or deterioration of antibiotics in Thayer-Martin or
Modified Thayer- Martin may allow growth of contaminants.
5. GC Medium Base has sufficient buffering capacity to offset the
very low pH of the small amount of Supplement VX added. The
pH of some media has to be adjusted with 1% NaOH after the
addition of Supplement VX.
References
1. Johnston, J. 1945. Comparison of gonococcus cultures read at 24
and 48 hours. J. Venera. Dis. Inform. 26:239.
2. Lankford, C. E., V. Scott, M. F. Cox, and W. R. Cooke. 1943.
Some aspects of nutritional variation of the gonococcus.
J. Bacteriol. 45:321.
210
Section II
3. Lankford, C. E., and E. E. Snell. 1943. Glutamine as a growth

factor for certain strains of Neisseria gonorrhoeae. J. Bacteriol.
45:421.
4. Carpenter, C. M., M. A. Bucca, T. C. Buck, E. P. Casman,
C. W. Christensen, E. Crowe, R. Drew, J. Hill, C. E. Lankford,
H. E. Morton, L. R. Peizer, C. I. Shaw, and J. D. Thayer. 1949.
Am. J. Syphil. Gonorrh. Vener. Dis. 33:164
5. Christensen and Schoenlein. 1947. Ann. Meeting CA Public
Health Assoc.
6. Spray. 1930. J. Lab. Clin. Med. 16:166.
7. Thayer, J. D., and J. E. Martin, Jr. 1966. Improved medium
selective for cultivation of N. gonorrhoeae and N. meningitidis.
Public Health Rep., 81:559.
8. Thayer, J. D., and A. Lester. 1971. Transgrow, a medium for
transport and growth of Neisseria gonorrhoeae and Neisseria
meningitidis. HSMHA Health Service Rep., 86:30.
9. Martin, J. E., and R. L. Jackson. 1975. A biological environmental chamber for the culture of Neisseria gonorrhoeae with a
new commercial medium. Public Health Rep., 82:361.
10. Martin, J. E., Jr., and J. S. Lewis. 1977. Anisomycin: improve
anti-mycotic activity in modified Thayer-Martin Medium. Public
Health Rep., 35:53.
& Wilkins, Baltimore, M.D.
12. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures handbook, vol 1. American Society for Microbiology, Washington, D.C.
American Society for Microbiology, Washington, D. C.
& Scotts diagnostic microbiology, 9th ed. Mosby-Year Book, Inc.,
St. Louis, MO.
Packaging
GC Medium Base
100
500
2
10
g
g
kg
kg
0289-15
0289-17
0289-07
0289-08
Hemoglobin
100
500
2
10
g
g
kg
kg
0136-15
0136-17
0136-07
0136-08
6 x 100 ml
3248-73
6 x 100 ml
100 ml
0276-60
0276-72
6 x 10 ml
100 ml
3354-60
3354-72
6 x 10 ml
3260-60
6 x 10 ml
100 ml
3198-60
3198-72
Hemoglobin 2% Solution
Supplement B
w/Reconstituting Fluid
Supplement VX
w/Reconstituting Fluid
The Difco Manual
Section II
GN Broth, Hajna
Bacto GN Broth, Hajna
Sodium Citrate and Sodium Desoxycholate inhibit growth of grampositive bacteria and of coliforms other than Salmonella and Shigella.
Dipotassium Phosphate and Monopotassium Phosphate buffer the
medium.
Intended Use
Bacto GN Broth, Hajna is used for isolating and cultivating gramnegative microorganisms.
Also Known As
Formula
Gram Negative (GN) Broth1

Hajna GN Broth1
Gram Negative Enrichment Broth1
GN Broth, Hajna
Formula Per Liter

Hajna2,3 formulated Gram Negative (GN) Broth as an enrichment
medium for enteric gram-negative bacilli, especially Salmonella and
Shigella, from clinical and non-clinical specimens. Croft and Miller4
demonstrated improved recovery of Shigella using GN Broth
enrichment compared to direct inoculation of agar media. Taylor and
Schelhart 5 reported improved recovery of both Salmonella and
Shigella when using GN Broth enrichment compared to direct
inoculation of agar media. Taylor and Schelhart6 showed GN Broth to
be superior to selenite enrichment medium for recovering Shigella.
GN Broth, Hajna is recommended as an enteric enrichment broth for
clinical specimens7,8 and as a nonselective enrichment broth for foods9
to recover Salmonella and Shigella.
GN Broth, Hajna contains Tryptose as a source of carbon, nitrogen,
vitamins and minerals. Dextrose and D-Mannitol are carbohydrates.
Dehydrated Appearance: Off-white to light tan, free-flowing,
homogeneous.
Solution:
or deionized water. Solution is light
amber, clear to very slightly
opalescent.
Prepared Medium:
opalescent.
Reaction of 3.9%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepare GN Broth, Hajna per label directions. Inoculate the
ATCC
25922*
14028*
12022*
19433*
INOCULUM
CFU
GROWTH
100-1,000
good
100-1,000
good
100-1,000
good
1,000-2,000 none to poor
The Difco Manual
g
g
g
g
g
g
g
g
Precautions
Expiration Date
Escherichia coli
Shigella flexneri
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Bacto D-Mannitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Storage
ORGANISM
The higher concentration of mannitol over dextrose favors growth

of mannitol-fermenting Salmonella and Shigella over mannitol
non-fermenting species, such as Proteus.
Procedure
Materials Provided
GN Broth, Hajna

Glassware
Autoclave
Incubator (35C)
2. Autoclave at 121C for 15 minutes. Avoid overheating.

Test Procedure
Results
Growth of gram-negative organisms, especially Salmonella and
Shigella species, is enhanced.
211
Gelatin & Gelatone
References
1. MacFaddin, J. F. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, vol 1, p. 357-359.
2. Hajna, A. A. 1955. A new specimen preservative for gram-negative
organisms of the intestinal group. Public Health Lab. 13:59-62.
3. Hajna, A. A. 1955. A new enrichment broth medium for gramnegative organisms of the intestinal group. Public Health Lab.
13:83-89.
4. Croft, C. C., and M. J. Miller. 1956. Isolation of Shigella
from rectal swabs with Hajna GN broth. Am. J. Clin. Path.
26:411-417.
5. Taylor, W. I., and D. Schelhart. 1967. Isolation of shigellae,
IV. Comparison of plating media with stools. Am. J. Clin. Path.
48:356-362.
Bacto Gelatin
Bacto Gelatone
Section II
6. Taylor, W. I., and D. Schelhart. 1968. Isolation of shigellae,

V. Comparison of enrichment broths with stools. Appl. Microbiol.
16:1383-1386.
7. Forbes, B. A., and P. A. Granato. 1995. Processing specimens for
bacteria., p. 265-267. In P. R. Murray, et al. (ed.), Manual of
Washington, D.C.
handbook, 1.10.8. American Society for Microbiology,
Washington, D.C.
Packaging
GN Broth, Hajna
500 g
0486-17
Intended Use
Bacto Gelatin is used in preparing microbiological culture media.
Bacto Gelatone is used in preparing microbiological culture media.
Also Known As
Gelatone is also referred to as Gelatin Peptone.

Gelatin is a protein of uniform molecular constitution derived chiefly
by the hydrolysis of collagen.1 Collagens are a class of albuminoids
found abundantly in bones, skin, tendon, cartilage and similar
animal tissues.1
Koch1 introduced gelatin into bacteriology when he invented the
gelatin tube method in 1875 and the plate method in 1881. This
innovation, a solid culture method, became the foundation for
investigation of the propagation of bacteria.1 However, gelatin-based
media were soon replaced by media containing agar as the
solidifying agent.
Gelatin is used in culture media for determining gelatinolysis
(elaboration of gelatinases) by bacteria. Levine and Carpenter2 and
Levine and Shaw3 employed gelatin media in their studies of gelatin
liquefaction. Garner and Tillett4 used culture media prepared with
gelatin to study the fibrinolytic activity of hemolytic streptococci.
Gelatin is a high grade gelatin in granular form which may be used as
a solidifying agent or may be incorporated into culture media for
various uses. Gelatin is used in Nutrient Gelatin, Motility
GI Medium, Motility Medium S, Stock Culture Agar and Dextrose
Starch Agar. Media containing gelatin are specified in Standard
Methods5,6 for multiple applications.
Gelatone, a granular pancreatic digest of gelatin, is deficient in
carbohydrates. It is distinguished by low cystine and tryptophan
212
content. Gelatone is used as an ingredient in media for fermentation

studies and, by itself, to support growth of non-fastidious microorganisms.

The melting point of a 12% concentration of Gelatin is between 28 and
30%C, which allows it to be used as a solidifying agent. Certain
microorganisms elaborate gelatinolytic enzymes (gelatinases)
which hydrolyze gelatin, causing liquefaction of a solidified
medium or preventing the gelation of a medium containing gelatin.
Gelatin is also used as a source of nitrogen and amino acids.
Gelatone is a peptone from gelatin obtained by digesting gelatin
with pancreatin.
Precautions
Storage
Expiration Date
Procedure
Materials Provided
Gelatin
Gelatone

Preparation varies depending on the medium being prepared.
The Difco Manual
Section II
Gelatin & Gelatone

Test Procedure
See appropriate references for specific procedures using Gelatin
or Gelatone.
Results
4. Garner and Tillett. 1934. J. Exp. Med. 60:255.

Gaithersburg, MD.
Packaging
Gelatin
100 g
500 g
10 kg
0143-15
0143-17
0143-08
Gelatone
500 g
0657-17
References
1. Gershenfeld, L., and L. F. Tice. 1941. Gelatin for bacteriological
use. J. Bacteriol. 41:645-652.
2. Levine and Carpenter. 1923. J. Bacteriol. 8:297.
3. Levine and Shaw. 1924. J. Bacteriol. 9:225.

Gelatin
Dehydrated Appearance: Light beige, free-flowing, homogeneous granules.
Solution:
12% solution, soluble in distilled or deionized water on
slight heating in a 50-55C waterbath. Solution is light amber,
clear to slightly opalescent, may have a slight precipitate.
Prepared Gel:
Very light amber, clear to slightly opalescent, may have
Reaction of 12%
Solution at 25C:
pH 6.8 0.2
Gelatone
Solution:
10% solution, soluble in distilled or deionized water:
1%-Very light to light amber, clear; 2%-Light to medium
amber, clear; 10%-Medium to dark amber, clear to
Reaction of 2%
Solution at 25C:
pH 6.3-7.6
Uninoculated
tube
Bacillus subtilis
ATCC 6633
Cultural Response
Gelatin
Prepare a 12% Gelatin solution in 0.8% Nutrient Broth and sterilize.
Inoculate and incubate at 35 2C under appropriate atmospheric
conditions for 18-48 hours or for up to two weeks for the gelatinase
test. To read gelatinase, refrigerate until well chilled and compare to
uninoculated tubes. Tubes positive for gelatinase will remain liquid.
ORGANISM
Escherichia coli
Bacillus subtilis
ATCC
INOCULUM
CFU
GROWTH
GELATINASE
25922*
11437
6633
100-1,000
100-1,000
100-1,000
good
good
good
+
+
Gelatone
Prepare a 2% Gelatone solution in 0.5% saline; adjust pH to
7.2-7.4; add 1.5% Bacto Agar, boil and sterilize. Inoculate
ORGANISM
Brucella suis
Escherichia coli
ATCC
INOCULUM
CFU
GROWTH
4314
25922*
25923*
100-1,000
100-1,000
100-1,000
good growth
good growth
good growth
Bacillus subtilis is available as Bacto Subtilis Spore Suspension.
The Difco Manual
213
Giolitti-Cantoni Broth Base & Potassium Tellurite Solution 3.5%
Section II
Bacto Giolitti-Cantoni Broth Base

Bacto Potassium Tellurite Solution 3.5%
Intended Use
Bacto Giolitti-Cantoni Broth Base is used with Bacto Potassium
Tellurite Solution 3.5% in enriching Staphylococcus aureus from
foods during isolation procedures.

1
Giolitti and Cantoni described a broth medium with added potassium

tellurite and a test procedure for enriching small numbers of staphylococci
in foods. Mossel et al2 recommended Giolitti-Cantoni Broth for
detecting Staphylococcus aureus in dried milk and other infant foods
where the organism should be absent from 1 g of test material.
bacilli. Potassium Tellurite Solution 3.5% supplies potassium tellurite,

which in combination with glycine, inhibits gram-positive bacteria
other than staphylococci.
Formula
Giolitti-Cantoni Broth Base
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto D-Mannitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Glycine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2
Sodium Pyruvate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
The International Dairy Federation (IDF) and American Public Health

Association recommend a procedure for detecting S. aureus in dairy
products using Giolitti-Cantoni Broth as an enrichment medium from
which selective media are inoculated.3, 4
g
g
g
g
g
g
g
g

Potassium Tellurite Solution 3.5%
Giolitti-Cantoni Broth Base contains Tryptone and Beef Extract as

supplies B-complex vitamins which stimulate bacterial growth.
D-Mannitol is the carbohydrate source. Sodium Pyruvate stimulates
growth of staphylococci. Lithium Chloride inhibits gram-negative
A filter-sterilized solution of potassium tellurite in distilled water.
Precautions

Solution:
Solution is medium amber, clear
Prepared Medium:
Medium amber, clear without
Reaction of 5.42%
Solution at 25C:
pH 6.9 0.2
Appearance:
Colorless, clear solution, may have
a fine precipitate.
Cultural Response
Prepare Giolitti-Cantoni Broth per label directions. Inoculate
per Test Procedure and incubate at 35 2C for 40-48 hours.
INOCULUM
CFU
ORGANISM
ATCC
Escherichia coli
Micrococcus luteus
25922* 1,000-2,000
10240 1,000-2,000
6538
100-1000
25923* 100-1000
214
Uninoculated
tube
GROWTH
APPEARANCE
inhibited
inhibited
good
good
no blackening
no blackening
blackening
blackening
Escherichia coli
ATCC 25922
ATCC 6538
The Difco Manual
Section II
HC Agar Base
2. Giolitti-Cantoni Broth Base

HARMFUL. MAY BE IRRITATING TO EYES, RESPIRATORY
SYSTEM AND SKIN. MAY CAUSE HARM TO THE UNBORN
CHILD. Avoid contact with skin and eyes. Do not breathe dust.
TARGET ORGANS: Blood, Kidneys, Nerves.
Potassium Tellurite Solution
WARNING! HARMFUL IF SWALLOWED. CAUSES IRRITATION.
Storage
Store dehydrated Giolitti-Cantoni Broth Base below 30C. The
Store Potassium Tellurite Solution 3.5% at 15-30C
Expiration Date
Procedure
Materials Provided

Glassware
Tubes 20 X 200 mm
Autoclave
Incubator (35C)
Sterile paraffin wax or sterile mineral oil
2.
3.
4.
5.
Warm gently to dissolve completely.

Dispense 19 ml amounts into 20 x 200 mm tubes.
Aseptically add 0.3 ml Potassium Tellurite Solution 3.5% per 19
ml tube or 0.03 ml when testing meat products or quality control
organisms.
6. Mix well.

Test Procedure
1. Inoculate 1 gram or 1 ml of test sample (0.1 gram or 0.1 ml when
testing meat or meat products) and 1 ml aliquots of each of a suitable decimal dilution series of the test sample into duplicate tubes.
2. Overlay each tube with 5 ml sterile molten paraffin wax to an
approximate height of 2 cm.
3. Incubate at 35 2C 40-48 hours.
4. Examine daily.
Results
Read tubes for blackening of the medium (a positive reaction) or no
blackening (a negative reaction). If blackening occurs, subculture to
Baird Parker Agar to confirm the isolation of S. aureus.
References
1. Giolitti, G., and C. Cantoni. 1966. A medium for the isolation of
staphylococci from foodstuffs. J. Appl. Bacteriol. 29:395-398.
2. Mossel, D. A. A., G. A. Harrewijn, and J. M. Elzebroek. 1973.
UNICEF.
3. International Dairy Federation. 1978. IDF Standard 60A:1978.
International Dairy Federation.
Packaging
1. Suspend 54.2 grams Giolitti-Cantoni Broth Base in 1 liter distilled

or deionized water.
Bacto HC Agar Base
500 g
25 ml
1809-17
1814-65
Intended Use
molds in cosmetic products that decreased incubation time to 3 days at

27.5 0.5C. HC Agar Base, based on the HC Agar formula of Mead
and ONeill, is supplemented with Polysorbate 80 to prepare HC Agar.
Bacto HC Agar Base, when supplemented with Polysorbate 80, is used

for enumerating molds in cosmetic products.

Methods for isolating molds from cosmetic products require
incubation for 5 to 7 days using traditional agar media.1 In 1986, Mead
and ONeill2 described a new medium, HC Agar, for enumerating
The Difco Manual
HC Agar Base contains Tryptone and Proteose Peptone as sources of

carbon, nitrogen, vitamins and minerals. Yeast Extract supplies B-complex
vitamins which stimulate bacterial growth. Dextrose provides a source
of fermentable carbohydrate. Ammonium Chloride and Magnesium
Sulfate provide essential ions. Disodium and Monopotassium Phosphates
215
HC Agar Base
Section II
buffer the pH to near neutrality. Sodium Carbonate inactivates low levels

of preservatives that are active at a more acidic pH (e.g., benzoic acid).
Chloramphenicol inhibits bacteria, including Pseudomonas aeruginosa
and Serratia marcescens, that are potential contaminants of cosmetic
products. Polysorbate 80 neutralizes preservatives and sequesters
surfactants that may be present in residual amounts from the product
sample.2 Bacto Agar is the solidifying agent.
Formula
HC Agar Base
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Ammonium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4
Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.06
Sodium Carbonate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g
g
g
g
g
HARM TO THE UNBORN CHILD. Avoid contact with skin and

container tightly closed. TARGET ORGAN(S): Blood, Eye/Ear,
Muscles, Nerves, Urogenital.
Storage
Expiration Date
Procedure
Precautions
Materials Provided

2. TOXIC. IRRITATING TO EYES, RESPIRATORY SYSTEM
AND SKIN. MAY CAUSE CANCER. POSSIBLE RISK OF
HC Agar Base

Dehydrated Appearance: Very light to light beige,
Solution:
Solution is medium to dark amber,
Prepared Medium:
Medium amber with yellow tint,
very slightly to slightly opalescent,
no significant precipitate.
Reaction of 5.45%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepare HC Agar Base per label directions. Supplement with
Polysorbate 80. Inoculate and incubate the plates at 27.5 0.5C
for 65-72 hours.
ORGANISM
ATCC
INOCULUM
CFU
Aspergillus niger
Serratia marcescens
16404
10145
13880
100-1000
1,000-2,000
1,000-2,000
GROWTH
good
none to poor
none to poor
216

Polysorbate 80
Glassware
Autoclave
Incubator (27.5 0.5C)
1.
2.
3.
4.
5.

Add 20 ml Polysorbate 80.
Dispense into petri dishes.

Collect specimens in sterile containers or with sterile swabs and
transport immediately to the laboratory in accordance with
Test Procedure
1. Process each specimen as appropriate for that specimen and
inoculate directly onto the surface of the medium.1 Inoculate
duplicate plates.
2. Incubate plates aerobically at 27.5 0.5C.
3. Examine plates for growth and recovery after 72 hours incubation.
4. Count mold colonies from duplicate plates and record average
count as mold count per gram or milliliter of sample.
The Difco Manual
Section II
m HCP Agar
Results
References
Mold cultures should yield good growth and recovery. Bacteria should
be inhibited.
1. Hitchins, A. D., T. T. Tran, and J. E. McCarron. 1995.

Microbiological methods in cosmetics, p. 23-01-23.12. In
Gaithersburg, MD.
2. Mead, C., and J. ONeill. 1986. A three-day mold assay for
cosmetics and toiletries. J. Soc. Cosmet. Chem. 37:49-57.

1. The 27.5 0.5C incubation temperature is critical for obtaining
statistically significant mold counts after three days using
this medium.
2. Nutritional requirements of organisms vary. Some strains may be
encountered that fail to grow or grow poorly on this medium.
Bacto m HPC Agar
Packaging
HC Agar Base
500 g
0685-17
Intended Use
Bacto m HPC Agar is used for enumerating heterotrophic organisms
in treated potable water and other water samples with low counts by
Also Known As
m HPC Agar is also known as m-Heterotrophic Plate Count Agar and
previously as membrane filter Standard Plate Count Agar, m-SPC Agar.

m HPC Agar was developed by Taylor and Geldreich in 1979 in their
pursuit of a suitable Standard Methods medium to use with the
membrane filter procedure.1 m HPC Agar was evaluated by many
investigators who reported it as a suitable alternate medium for
standard plate counts. 2,3,4 This medium is recommended for the
membrane filter method in the 19th edition of Standard Methods for
the Examination of Water and Wastewater.5
The volume of inoculum is limited with both pour and spread plate
techniques while the membrane filter method enables the use of large
samples, which is desirable for water with low counts.

In m HPC Agar, Bacto Peptone provides sufficient nitrogen and
carbon as well as other nutrients. Gelatin at 2.5% concentration
eliminates problems of liquefaction and spreading colonies. Bacto Agar
is a solidifying agent.
Formula
m HPC Agar
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Bacto Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
Precautions
The advantages of the membrane filter procedure over the standard

plate count method have been described by many investigators.6,7,8

Solution:
Soluble in distilled or deionized water on boiling. (Add
1% glycerol after boiling). Light amber, slightly opalescent
to opalescent, may have a precipitate.
Reaction of 6%
Solution at 25C:
pH 7.1 0.2 with 1% added glycerol and after autoclaving
5 minutes at 121-124C.
Prepared Plates:
Light amber, opalescent, may have a precipitate.
Cultural Response
Prepare m HPC Agar per label instructions. Dilute ten chlorinated water samples collected
as recommended by Standard Methods5 from different sources to yield 20-200 CFUs/10 ml.
Filter the dilutions through a membrane filter. Place the filters on m-HPC Agar plates and
incubate at 35 2C for 40-72 hours. Therecovery and morphology of bacteria on test medium
should be comparable to that of a reference lot.
water sample
The Difco Manual
217
m HPC Agar

Storage
Expiration Date
Procedure
Materials Provided
m HPC Agar

Dilution blanks
Pipettes or glass rods
Incubator (35C)
Stereoscopic microscope
Close fitting box or plastic bag containing moistened paper towels
Bacto Glycerol
1.
2.
3.
4.
5.
Suspend 6 grams in 100 ml distilled or deionized water.

Add 1 ml glycerol.
Autoclave at 121-124C for 5 minutes.
Dispense 5 ml aliquots into Petri dishes.

Water samples should be collected as described in Standard
Methods for the Examination of Water and Wastewater, Section 9060A.5
To minimize changes in bacterial population, water samples should be
tested as soon as possible after collection. The recommended
maximum elapsed time between collection and analysis of samples is
8 hours (maximum transit time of 6 hours, maximum processing time
of 2 hours). When analysis cannot begin within 8 hours, maintain
sample at a temperature below 4C but do not freeze. Maximum elapsed
time between collection and analysis must not exceed 24 hours.5
Test Procedure
1. The volume to be filtered will vary with the sample. Select a
maximum sample size to give 20 to 200 CFU per filter.
2. Filter appropriate volume through a sterile 47 mm, 0.45 m,
gridded membrane filter, under partial vacuum. Rinse funnel with
three 20 to 30 ml portions of sterile dilution water. Place filter on
agar in Petri dish.
3. Place dishes in close-fitting box or plastic bag containing
moistened paper towels.
4. Incubate at 35 0.5C for 48 hours. Duplicate plates may be
incubated at other conditions as desired.
218
Section II
Results
Count all colonies on the membrane when there are 2 or less colonies
per square. For 3 to 10 colonies per square, count 10 squares and
obtain average count per square. For 10 to 20 colonies per square,
count 5 squares and obtain average count per square. Multiply average
count per square by 100 and divide by the sample volume to give
colonies per milliliter. If there are more than 20 colonies per square,
record count as > 2,000 divided by the sample volume. Report
averaged counts as estimated colony-forming units. Make estimated
counts only when there are discrete, separated colonies.5

1. m HPC Agar is intended for use only with the membrane filter
method.
2. m HPC Agar is recommended for testing treated water.
3. Longer incubation times may be necessary to recover slowgrowing bacteria.
References
1. Taylor, R. H., and E. E. Geldreich. 1979. A new membrane filter
procedure for bacterial counts in potable water and swimming
pool samples. J. Amer. Water Works Assoc. 71:402-405.
2. Means, E. G., L. Hanami, H. F. Ridgway, and B. H. Olson.
1981. Evaluating mediums and plating techniques for enumerating
bacteria in water distribution systems. J. Amer. Water Works
Assoc. 73:585-590.
3. Nagy, L. A., and B. H. Olson. 1982. The occurrence of
filamentous fungi in drinking water distribution systems. Can. J.
4. Haas, C. N., M. A. Meyer, and M. S. Paller. 1982. Analytical
note: evaluation of the m-SPC method as a substitute for the
standard plate count in water microbiology. J. Amer. Water Works
Assoc. 74:322.
6. Lechevallier, M. W., R. J. Seidler, and T. M. Evans. 1980.
Enumeration and characterization of standard plate count bacteria
in chlorinated and raw water supplies. App. And Environ.
7. Stapert, E. M., W. T. Sokolski, and J. I. Northam. 1962. The
factor of temperature in the better recovery of bacteria from water
by filtration. Can. Journal Microbiol. 8:809-810.
8. Saleem, M., and R. L. Schlitzer. 1983. Comparative recovery
of bacteria from purified water by the membrane filter technique
and the standard plate count methods, p. 281. Abs. Ann.
Meeting, ASM.
Packaging
m HPC Agar
100 g
500 g
0752-15
0752-17
Glycerol
100 g
500 g
0282-15
0282-17
The Difco Manual
Section II
Heart Infusion Broth & Heart Infusion Agar
Bacto Heart Infusion Broth

Bacto Heart Infusion Agar
Intended Use
Bacto Heart Infusion Broth is used for cultivating fastidious
microorganisms.
Bacto Heart Infusion Agar is an infusion medium used for cultivating a
wide variety of fastidious microorganisms and as a base for preparing
blood agar.
Also Known As
Heart Infusion Broth may be used as the base in carbohydrate

fermentation tests.4
Several modifications of Heart Infusion media have been described.5
The addition of carbohydrates, blood or other ingredients result in
media used for a variety of purposes. The methodologies for the
multiple applications using Heart Infusion Agar and Heart Infusion
Broth are outlined in the references.

Infusion from Beef Heart and Tryptose supply the nutritional requirements for growth of microorganisms in Heart Infusion Media. Sodium
chloride maintains the osmotic balance of the medium, and Bacto Agar
is the solidifying agent. The addition of 5% sheep blood provides
additional growth factors and is used to determine hemolytic reactions.
Heart Infusion Broth is abbreviated as HIB, Heart Infusion Agar as HIA.
Formula
Heart Infusion Broth

Formula Per Liter
Heart Infusion Broth and Heart Infusion Agar are non-selective

general purpose media used for the isolation of nutritionally fastidious
microorganisms. One of the first media used for the cultivation of
bacteria was a liquid medium containing an infusion of meat. Huntoon1
using fresh beef heart and Bacto Peptone, prepared a hormone broth
to retain growth promoting substances. Highly pathogenic organisms,
such as meningococci and pneumococci, could be grown on infusion
medium without enrichments.1 The formulas for HIA and HIB contain
Tryptose, which is better suited to the nutritional requirements of
pathogenic bacteria than Bacto Peptone.
Heart Infusion Agar can be used as a base for the preparation of blood
agar in determining hemolytic reactions, and for mass cultivation of
microorganisms in the preparation of vaccines. Heart Infusion Media
are specified for the isolation of Vibrio cholerae and Vibrio species.2,3
Beef Heart, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . 500 g

Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g

Heart Infusion Agar
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
Precautions

Storage

Solution:
deionized water; light to medium
amber in color, clear.
Prepared Medium:
Reaction of 2.5%
Solution at 25C
pH 7.4 0.2
Heart Infusion Agar
Solution:
to medium amber, very slightly to
Prepared Medium:
Plain - Light to medium amber, slightly
opalescent with no precipitate. With
5% sheep blood - cherry red, opaque.
Reaction of 4%
Solution at 25C:
pH 7.4 0.2
The Difco Manual

Expiration Date
Procedure
Materials Provided
Heart Infusion Agar

Glassware
Autoclave
Incubator (35C)
Waterbath (45-50C)
219
Heart Infusion Broth & Heart Infusion Agar
Section II
Test Procedure

3. Cool to room temperature.
Heart Infusion Agar

OPTIONAL: To prepare blood agar, aseptically add 5% sterile
defibrinated blood to Heart Infusion Agar at 45-50C. Mix well.
4. Dispense into Petri dishes.
References
Results
1. Huntoon, F. M. 1918. Hormone Medium. A simple medium

employable as a substitute for serum medium. J. of Infect. Dis.
23:169-172.
2. Harmon, S. M., D. A. Kautter, D. A. Golden, and
E. J. Rhodehamel. 1995. p. 9.01-9.24. App. 3.24-3.25. FDA
Bacteriological Analytical Manual, 8th ed. AOAC International,
Arlington, VA.

Cultural Response
Prepare Heart Infusion Broth per label directions. Prepare Heart Infusion Agar with
and without 5% sheep blood. Inoculate and incubate at 35 2C for 18-48 hours.
ORGANISM
Escherichia coli
ATCC
INOCULUM CFU
GROWTH
25922*
25923
6305
19615*
100-1,000
100-1,000
100-1,000
100-1,000
good
good
good
good
Escherichia coli
ATCC 25922
ATCC 25923
Escherichia coli
ATCC 25922
Uninoculated
tube
Heart Infusion Agar

ORGANISM
ATCC
INOCULUM
CFU
Escherichia
25922* 100-1,000
coli
aureus
Streptococcus
6305 100-1,000
pneumoniae
Streptococcus
19615* 100-1,000
pyogenes
Streptococcus pyogenes ATCC 19615

All with blood on Heart Infusion Agar
220
GROWTH
HEMOLYSIS
GROWTH
w/5%
w/5%
PLAIN SHEEP BLOOD SHEEP BLOOD
good
good
beta
good
good
beta
fair
good
alpha
fair
good
beta
The Difco Manual
Section II
Hektoen Enteric Agar
3. Vanderzant, C. and D. F. Splittstoesser (ed.). 1992. p. 451-469.

1132. Compendium of Methods for the Microbiological
Examination of Food, 3rd ed. American Public Health Association,
Washington, D.C.
4. Ruoff, K. L. 1995. Streptococcus, p.305. In Murray, P.R., Baron
E.J., Pfaller, M.A. Tenover, F.C., and R.H. Yolken (ed.)., Manual
of Clinical Microbiology, 6th ed. American Society for Microbiology,
Washington, D.C.
5. Atlas, R. M. 1993. Handbook of Microbiological Media,
p. 426-431, CRC Press, Boca Raton, FL.
Bacto Hektoen Enteric Agar
Intended Use
Bacto Hektoen Enteric Agar is used for the isolating and differentiating
gram-negative enteric bacilli.
Also Known As
Hektoen Enteric Agar is also known as HE Agar or HEA.

Hektoen Enteric Agar was developed in 1967 by King and Metzger.1, 2
Compared to other enteric differentiating media commonly used in
clinical laboratories at that time, Hektoen Enteric Agar increased the
frequency of isolation of Salmonella and Shigella organisms. This was
accomplished by increasing the carbohydrate and peptone content of
Packaging
Heart Infusion Agar
100
500
2
10
g
g
kg
kg
0044-15
0044-17
0044-07
0044-08
100 g
500 g
2 kg
0038-15
0038-17
0038-07
the medium in order to counteract the inhibitory effects of the bile salts
and indicators. King and Metzger formulated a medium that
only slightly inhibited the growth of Salmonella and Shigella while
at the same time ensuring the adequate inhibition of gram-positive
microorganisms.
Hektoen Enteric Agar is used to isolate and differentiate Salmonella and
Shigella, which cause a variety of serious human gastrointestinal
illnesses.3 Salmonella is the most frequently reported cause of foodborne
outbreaks of gastroenteritis in the United States.4 Foods containing
poultry, eggs, or dairy products are the most frequent vehicles for
foodborne salmonellosis. For food samples, a variety of procedures
have been developed using Hektoen Enteric Agar as part of the multi-step
procedure to isolate Salmonella.5-8
ATCC 14028
Uninoculated
plate

Dehydrated Appearance: Light purplish beige, free-flowing,
homogeneous.
Solution:
7.6% solution soluble in distilled or
deionized water upon boiling.
Prepared Plates:
Green with yellowish cast,
Reaction of 7.6%
Solution at 25C:
pH 7.5 0.2
Cultural Response
Prepare Hektoen Enteric Agar per label directions. Inoculate
and incubate plates at 35 2C for 18-24 hours.
ORGANISM
Enterococcus
faecalis
Escherichia
coli
Salmonella
typhimurium
Shigella flexneri
ATCC
INOCULUM
CFU
29212* 1,000-2,000
25922*
100-1,000
14028*
100-1,000
12022*
100-1,000
GROWTH
COLONY
COLOR
markedly
inhibited
partial
salmon-orange,
inhibition may have bile ppt.
good
greenish blue,
w/black centers
good
greenish blue
Shigella flexneri
ATCC 12022
The cultures listed are the minimum that should be used for performance.
The Difco Manual
221
Section II
Novobiocin (15 mg/liter) can be added to Hektoen Enteric Agar to

inhibit growth of Citrobacter and Proteus colonies, which may
resemble those of Salmonella.9

Proteose Peptone is a source of nitrogen and other nutrients in Hektoen
Enteric Agar. Bile Salts and the dyes, brom thymol blue and acid
fuchsin, inhibit gram-positive organisms. Lactose, saccharose and
salicin are sources of fermentable carbohydrates. Ferric ammonium
citrate, a source of iron, allows production of hydrogen sulfide (H2S)
from sodium thiosulfate. H2S-positive colonies have black centers.
Yeast Extract provides vitamins and cofactors required for growth and
additional nitrogen and carbon. Bacto Agar is used as a solidifying agent.

Glassware
Autoclave
Incubator
Petri dishes
2 Heat to boiling with frequent agitation to dissolve completely.
Do not overheat. DO NOT AUTOCLAVE.

Formula
Test Procedure

Formula Per Liter

Bacto Bile Salts No. 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Bacto Salicin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Brom Thymol Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.065
Acid Fuchsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
g
g
g
g
g
g
g
g
g
g
g
g
Precautions
Storage
Expiration Date
Procedure
Materials Provided
222
Results

1. Do not autoclave this medium because excessive heat may alter
the ingredients.
2. Proteus species may resemble salmonellae or shigellae. Further
testing should be conducted to confirm the presumptive identification
of organisms isolated on this medium.
References
1. King, S., and W. I. Metzger. 1968. A new plating medium for the
isolation of enteric pathogens. Appl. Microbiol. 16:577-578.
2. King, S., and W. I. Metzger. 1968. A new plating medium for the
isolation of enteric pathogens. II. Comparison of Hektoen Enteric
Agar with SS and EMB Agar. Appl. Microbiol. 16:579-581.
3. Gray, L .D. 1995. Escherichia, Salmonella, Shigella, and Yersinia,
4. Centers for Disease Control. 1991. Summary of notifiable
diseases. Morbid. Mortal. Weekly Rep. 40 (53):3.
1992. Salmonella, p. 371-422. In Vanderzant, C., and D. F.
Washington, D.C.
1993. Pathogens in milk and milk products, p. 103-212.
In Marshall, R. T. (ed.), Standard methods for the examination
of dairy products. 16th ed. American Public Health Association,
Washington, D.C.
R. M. Amaguana. 1995. Salmonella, p. 5.01-5.20. In Bacteriological analytical manual, 8th ed. AOAC International,
Gaithersburg, MD.
8. Association of Official Analytical Chemists. 1996 official
methods of analysis of AOAC International, Supplement March
1996. AOAC International, Arlington, VA.
The Difco Manual
Section II
Hemoglobin
9. Hoben, D. A., D. H. Ashton, and A. C. Peterson. 1973. Some

observations on the incorporation of novobiocin into Hektoen
Enteric Agar for improved Salmonella isolation. Appl.
Packaging
100
500
2
10
g
g
kg
kg
0853-15
0853-17
0853-07
0853-08
Bacto Hemoglobin

Intended Use
Storage
Bacto Hemoglobin is used in preparing microbiological culture media.

Hemoglobin, an autoclavable preparation of beef blood, is prepared
according to the procedure described by Spray.1
Hemoglobin is used with GC Medium Base is in the preparation of
Chocolate Agar Enriched, Thayer-Martin Medium and Modified
Thayer-Martin Medium. Supplemented with Hemoglobin and Supplement
B or VX, the enriched media are used for the isolation and cultivation
of fastidious microorganisms, especially Neisseria and Haemophilus
species. With the exception of some laboratory-adapted strains of
Haemophilus aphrophilus, Haemophilus species require either
exogenous hemin (X factor), nicotinamide adenine dinucleotide (NAD)
(V factor), or both.2

Hemoglobin provides the hemin (X factor) required for growth of
Haemophilus and for enhanced growth of Neisseria species.
Formula
Store Hemoglobin below 30C. The dehydrated ingredient is very

Expiration Date
Procedure
Materials Provided
Hemoglobin

Glassware
Autoclave
GC Medium Base (for the cultivation of Neisseria and
Haemophilus species)
Supplement B or VX, depending on the medium being prepared
Antimicrobic Vial CNV or CNVT, depending on the medium
being prepared
Hemoglobin is obtained from beef blood, desiccated.
Precautions
1. Place 10 grams of Hemoglobin in a dry beaker.

2. Measure 500 ml distilled or deionized water.
3. Add the water in approximately 100 ml amounts, stirring well
after each addition. Use a spatula to break up clumps.
4. Transfer to flasks, as desired, for autoclaving.
6. Cool to 45-50C.
7. Swirl the flask to reestablish complete solution, then add to an equal
amount of double-strength sterile agar base cooled to 45-50C.

Dehydrated Appearance: Dark brown, fine, free-flowing.
Solution:
2% solution, insoluble in distilled or
deionized water. Solution is chocolate
brown, opaque with a dispersed
precipitate.
Reaction of 2%
Solution at 25C:
pH 8.2 0.2
Cultural Response
Prepare GC Medium enriched with 2% Hemoglobin and
Supplement B or VX per label directions. Inoculate and
incubate at 35 2C under 5-10% CO2 for 18-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
10211
43069
100-1,000
100-1,000
GROWTH
good
good
The Difco Manual
Test Procedure
Neisseria and Haemophilus species, refer to procedures outlined in
appropriate references.2,3,4
Results
References
1. Spray. 1930. J. Lab Clin. Med. 16:166.
223
Horse Serum, Desiccated
Section II
St. Louis, MO.
Bacto Horse Serum, Desiccated
Packaging
Hemoglobin
100
500
2
10
g
g
kg
kg
0136-15
0136-17
0136-07
0136-08
Intended Use
Bacto Horse Serum, Desiccated is used as an enrichment in bacteriological culture media.

Horse Serum, Desiccated is an enrichment prepared from filtersterilized, normal horse serum. When used in the preparation of
Mycoplasma Supplement, Horse Serum supplies cholesterol, a growth
stimulant for Mycoplasma.1 Loeffler2 used dextrose broth enriched with
horse serum for cultivating Corynebacterium diphtheriae.

Storage
Store Horse Serum, Desiccated and reconstituted Horse Serum at
2-8C.
Expiration Date
Procedure
A medium supplemented with horse serum or lysed horse blood is

usually sufficient to enhance the growth of fastidious anaerobes.3 Broth
media supplemented with horse serum are used in the microdilution
susceptibility testing of anaerobic bacteria.3
Materials Provided
Horse Serum, Desiccated provides essential nutritional factors that

stimulate organism growth.
Reagent
Horse Serum, Desiccated is sterile, lyophilized horse serum.
Precautions
Refer to the final concentration of Horse Serum, Desiccated specified

in the formula of the medium or enrichment being prepared. Add as
required.

Test Procedure
See appropriate references for specific procedures using Horse Serum,
Desiccated.1-3

Lyophilized Appearance: Brown, lyophilized cake or powder.
Solution:
Soluble in 10 ml distilled or
deionized water.
Rehydrated Appearance: Light to medium amber, clear to
Cultural Response
Prepare Tryptose Blood Agar Base with 10% Horse Serum
(rehydrated) per label directions. Inoculate and incubate at
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Streptococcus mitis
9895
6303*
100-1,000
100-1,000
good
good
224
Results
References
1. Taylor-Robinson, D. 1995. Mycoplasma and Ureaplasma,
2. Loeffler, F. 1887. Darauf theilte HeuLoeffer en einem Zweiten
Vortrag die ergebnisse seiner weiteren untersuchungen uber die
Diphtherie-Bacillen mit. Zentralb. Bacteriol. 2:105.
Packaging
12 x 10 ml
0261-61
The Difco Manual
Section II
ISP Medium 1, 2 & 4
Bacto ISP Medium 1 . Bacto ISP Medium 2

Bacto ISP Medium 4
Also Known As
Intended Use
ISP Medium 1 is also referred to as Tryptone Yeast Extract Broth.
Bacto ISP Medium 1, Bacto ISP Medium 2 and Bacto ISP Medium 4
are used for characterizing Streptomyces species according to the
International Streptomyces Project (ISP).1
ISP Medium 2 is also referred to as Yeast Malt Extract Agar.

ISP Medium 4 is also referred to as Inorganic Salts Starch Agar.

ISP Medium 1
Solution:
amber, clear to very slightly opalescent.
Prepared Medium:
opalescent, w/o significant precipitation.
Reaction of 0.8%
Solution at 25C:
pH 7.0 0.2
ISP Medium 2
Solution:
Prepared Medium:
opalescent, without precipitate.
Reaction of 3.8%
Solution at 25C:
pH 7.2 0.2
ISP Medium 4
Dehydrated Appearance: White to light beige, free-flowing,
homogeneous.
Solution:
deionized water on boiling; white to
off-white, opaque with precipitate.
Prepared Medium:
White to off-white, opaque, may have
a precipitate.
Reaction of 3.7%
Solution at 25C:
pH 7.2 0.2
Cultural Response
Prepare ISP Medium 1, ISP Medium 2 and ISP Medium 4 per
label directions. Inoculate tubes of prepared ISP Medium 1,
Inoculate prepared ISP Medium 2 and ISP Medium 4 with the
test organisms by placing a drop of inoculum near the edge of
the plate. Five parallel streaks across the plate are made from
this drop, followed by four perpendicular streaks. Incubate
inoculated plates at 30 2C for 48-96 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Streptomyces albus
Streptomyces lavendulae
3004
8664
100-1,000
100-1,000
good
good
ISP media were developed by Difco Laboratories for the International

Streptomyces Project (ISP) in order to select stable properties and
reproducible procedures for characterization of Streptomyces species.1

Tryptone and Yeast Extract are the nitrogen, vitamin, carbon and amino
acid source in ISP Medium 1.
Yeast Extract and Malt Extract provide nitrogen, amino acids and
vitamins in ISP Medium 2. Dextrose is the carbon source, and Bacto
Agar is the solidifying agent.
ISP Medium 4 is composed of many inorganic salts and Soluble Starch
to provide essential nutrients for organism growth. Bacto Agar is the
solidifying agent.
Formula
ISP Medium 1
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g

ISP Medium 2
Formula Per Liter
Bacto Malt Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
g
g
g
g

ISP Medium 4
Formula Per Liter
Magnesium Sulfate USP . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Ammonium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Calcium Carbonate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Ferrous Sulfate (FeSO47H2O) . . . . . . . . . . . . . . . . . . . . 0.001
Manganous Chloride (MnCl27H2O) . . . . . . . . . . . . . . . 0.001
Zinc Sulfate (ZnSO47H2O) . . . . . . . . . . . . . . . . . . . . . . 0.001
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
g
g
g
g
g
g
g
g
g
g
Precautions
The Difco Manual
225
Inositol Assay Medium
2. ISP Medium 4
AND SKIN. Avoid contact with skin and eyes. Do not breath dust.
TARGET ORGAN(S): Lungs, Intestines.
Section II
deionized water:
ISP Medium 1
8 g/l
ISP Medium 2
38 g/l
ISP Medium 4
37 g/l
4. Mix thoroughly while dispensing.

Test Procedure
Storage
For a complete discussion on the isolation and maintenance of

Streptomyces species refer to appropriate references.2,3
Results
Expiration Date
Procedure
References
Materials Provided
1. Shirling, E. B., and D. Gottlieb. 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16:313-340.
ISP Medium 1
ISP Medium 2
ISP Medium 4

Glassware
Autoclave
Incubator (30C)
Sterile tubes
Bacto Inositol Assay Medium

may be encountered that fail to grow or grow poorly on the medium.
Packaging
ISP Medium 1
500 g
0769-17
ISP Medium 2
500 g
0770-17
ISP Medium 4
500 g
0772-17
Saccharomyces cerevisiae ATCC 9080 (Saccharomyces uvarum) as

the test organism.
Intended Use
Bacto Inositol Assay Medium is used for determining inositol concentration by the microbiological assay technique.
Inositol Assay Medium is an inositol-free dehydrated medium

of S. cerevisiae ATCC 9080. The addition of inositol in specified
increasing concentrations gives a growth response that can be measured
turbidimetrically.

Inositol Assay Medium, a modification of the formula described by
Atkin et al.,1 is used in the microbiological assay of inositol using
226
Formula
Formula Per Liter
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 g
Potassium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Citric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g
The Difco Manual
Section II
Potassium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.85
Ferric Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
DL-Tryptophane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
L-Isoleucine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
L-Leucine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
L-Lysine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
L-Methionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
DL-Phenylalanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
L-Tyrosine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
L-Asparagine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8
DL-Aspartic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
DL-Serine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
Glycine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
DL-Threonine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4
L-Valine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
L-Histidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.124
L-Proline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
DL-Alanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4
L-Glutamic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6
L-Arginine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.48
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

g
g
g
g
mg
mg
mg
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
mg
mg
ware in microbiological assay procedures. Extremely small

present.
Storage
Expiration Date
Procedure
Materials Provided
Glassware
Autoclave
Stock culture of Saccharomyces cerevisiae ATCC 9080
Inositol
Sterile tubes
Centrifuge
Spectrophotometer
Precautions
2. Great care must be taken to avoid contamination of media or glass-

homogeneous.
Solution:
boiling. Light amber, clear, may have
Prepared Medium:
Light amber, clear, may have a slight
precipitate.
Reaction of 6.1%
Solution at 25C:
pH 5.2 0.2
Cultural Response
Prepare Inositol Assay Medium per label directions. Dispense
medium into 50 ml flasks with a titration from 0 to 10 g of
Inositol. Inoculate flasks with one drop of S. cerevisiae ATCC
9080 inoculum suspension (washed three times and diluted
1:1000). Incubate flasks at 25-30C for 20-24 hours. The curve
obtained from turbidimetric readings should be typical.
The Difco Manual
1.
2.
3.
4.
5.
6.

Boil to dissolve.
Dispense 5 ml amounts into flasks.

Assay samples are prepared according to references given in the specific
assay procedures. The samples should be diluted to approximately the
Test Procedure
Remove a loopful of culture from a stock culture slant of S. cerevisiae
ATCC 9080 and suspend it in 10 ml sterile 0.85% saline. Centrifuge
cells at moderate speed for 10 minutes. Decant the supernatant and
resuspend cells in 10 ml 0.85% sterile saline. Wash the cells three times
with 10 ml sterile 0.85% saline. After the third wash, resuspend the
227
KF Streptococcus Agar
Section II
cells in 10 ml 0.85% saline. Dilute 1 ml of the cell suspension in 1000 ml

of sterile 0.85% saline. This diluted suspension is the inoculum. Use
1 drop of inoculum suspension to inoculate each assay flask.
The concentrations of inositol required for the preparation of the standard
curve may be prepared by dissolving 200 mg inositol in 100 ml
distilled water. Mix thoroughly. Dilute 1 ml of this solution with 999 ml
distilled water to make a final solution containing 2 g inositol per ml.
Use 0.0, 0.5, 1, 2, 3, 4 and 5 ml per flask. Prepare this stock solution
fresh daily.
run. Autoclave and incubation conditions can impact the standard curve
readings and cannot always be duplicated. The standard curve is
obtained by using inositol at levels of 0.0, 1, 2, 4, 6, 8 and 10 g per
assay flask (10 ml).
Following inoculation, flasks are incubated at 25-30C for 20-24 hours.
Place flasks in the refrigerator for 15-30 minutes to stop growth. Growth
is measured turbidimetrically using any suitable spectrophotometer.

Results
1. Atkin, Schultz, Williams, and Frey. 1943. End. & Eng. Chem.,
Ann. Ed. 15:141.

or cup.

grown and maintained on media recommended for this purpose.
response.
References
Packaging
100 g
0995-15
Bacto KF Streptococcus Agar
Uninoculated
plate
ATCC 19433

Dehydrated Appearance: Light greenish-beige, free-flowing,
homogeneous.
Solution:
is light purple, very slightly to
Prepared Medium:
Light purple, very slightly to
Reaction of 7.64%
Solution at 25C:
7.2 0.2
Cultural Response
Prepare KF Streptococcus Agar per label directions. Inoculate
using the pour plate technique and incubate at 35 2C for 46-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
APPEARANCE
Enterobacter aerogenes 13048* 1,000-2,000

Escherichia coli
marked to
complete inhibition
19433*
30-300
good
red centers
29212*
30-300
good
red centers
25922* 1,000-2,000
marked to
complete inhibition
*These cultures are available as Bactrol Disks and are to be used as directed per Bactrol Disk Technical Information.
228
The Difco Manual
Section II
Intended Use
Storage
Bacto KF Streptococcus Agar is used with Bacto TTC Solution 1% in

isolating and enumerating fecal streptococci according to APHA.

Also Known As
Expiration Date
Kenner Fecal Streptococcus Agar

Kenner et al. developed KF Streptococcal Agar for use in detecting
streptococci in surface waters by direct plating or by the membrane
filtration method.1 These investigators compared the performance of
their formulation to other media used for enumerating fecal streptococci
and achieved greater recoveries with KF Streptococcal Agar.
Procedure
This medium is currently recommended for use in determining counts

of fecal streptococci in foods and water.2,3

Peptone provides a source of nitrogen, amino acids and carbon. Yeast
Extract is a source of trace elements, vitamins and amino acids. Maltose
and Lactose are fermentable carbohydrates and carbon sources. Sodium
Azide is a selective agent. Brom Cresol Purple is an indicator dye.
The addition of 1% triphenyltetrazolium chloride (TTC) causes
enterococci to develop a deep red color following reduction of
tetrazolium to an acid azo dye.
Formula
Formula Per Liter
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Glycerophosphate . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Maltose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Azide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
g
g
g
g
g
g
g
g
g
Materials Provided
TTC Solution 1%
Glassware
Incubator (35C)
Pipettes
Dilution blanks
2. Heat an additional 5 minutes. Avoid overheating which could
decrease the productivity of the medium. DO NOT AUTOCLAVE.
3. Add 10 ml TTC Solution 1% to the medium at 50C and mix well.
4. Pour medium into sterile Petri dishes if using the Membrane Filter
procedure. If using the Pour Plate technique, hold the liquid
medium at 45C.

Consult appropriate references for specific procedures using KF
Streptococcus Agar in the examination of waters and food.2,3 The pour
plate technique or the membrane filter procedure can be used for
detection and enumeration of enterococci.
Precautions
Test Procedure
1. For Laboratory Use

wash immediately with plenty of water. If inhaled, remove to
fresh air. If not breathing, give artificial respiration. If breathing is
Pour Plate Technique

1. Prepare appropriate dilutions of the test material.
2. Place the selected volume of sample in a Petri dish.
3. Pour 15 ml of the prepared medium at 45C into each plate.
4. Thoroughly mix the medium and sample to uniformly disperse the
organisms.
5. Allow the agar to solidify.
6. Incubate plates in the inverted position at 35 2C for 46-48 hours.
The Difco Manual
Membrane Filter Procedure

1. Filter a suitable volume of sample through a sterile membrane, as
directed.
2. Place the inoculated membrane filter on the solidified agar in the
Petri dish, inoculum side up.
3. Incubate the plates, inverted, at 35 2C for 46-48 hours.
229
KF Streptococcus Broth
Section II
Results

J. Lovett. 1992. Compendium of methods for the microbiological
Washington, D.C.
3. Bordner, R., and J. Winter. 1978. Microbiological methods for
monitoring the environment, water and wastes. EPA, Cincinnati, OH.
4. MacFadden, J. F. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, vol. I. Williams
& Wilkens, Baltimore, MD.
Enterococci will appear as red or pink colonies. The use of a stereoscopic

microscope with 15X magnification can aid in counting colonies.

1. Many strains of S. bovis and S. equinus are inhibited by azide.
2. Overheating may lower the pH, causing a decrease in the productivity of the medium.
References
Packaging
1. Kenner, B. A., H. F. Clark, and P. W. Kabler. 1961. Fecal

streptococci. I. Cultivation and enumeration of streptococci in
surface waters. Appl. Microbiol. 9:15.
Bacto KF Streptococcus Broth
500 g
0496-17
This medium currently is recommended for use in enumerating

enterococci in foods.5
Intended Use
Bacto KF Streptococcus Broth is used for isolating fecal streptococci.
Proteose Peptone No. 3 provides a source of nitrogen, amino acids and

carbon. Yeast Extract is a source of trace elements, vitamins and amino
acids. Maltose and Lactose are the fermentable carbohydrates and
carbon source. Sodium Azide is the selective agent. Brom Cresol Purple
is the indicator dye.
Also Known As
Kenner Fecal Streptococcus Broth

Kenner et al. developed KF Streptococcal Broth for the detection and
enumeration of enterococci in waters.1,2 They found that this formulation
was superior to other liquid media in the recovery of enterococci in
Most Probable Number (MPN) test systems. The medium is not
specific for presumptive identification of group D streptococci. Other
tests are required.2-4
The addition of 1% triphenyltetrazolium chloride, in the membrane

filter procedure, causes the enterococci to have a deep red color as a
result of tetrazolium reduction to an acid azo dye.

Dehydrated Appearance: Light greenish-beige, free-flowing, homogeneous.
Solution:
5.64% solution, soluble in distilled or deionized water with
frequent agitation on boiling. Solution is reddish to light
purple, clear to very slightly opalescent.
Prepared Tubes:
Purple, clear to very slightly opalescent.
Reaction of 5.64%
Solution at 25C:
7.2 0.2.
Cultural Response
Prepare KF Streptococcus Broth per label directions. Supplement with TTC
Solution 1%. Using the membrane filter technique, inoculate and incubate at 35 1C
in an atmosphere saturated with water vapor for 46-48 hours.
ORGANISM
Escherichia coli
ATCC
INOCULUM
CFU
GROWTH
APPEARANCE
13048*
19433*
29212*
25922*
300-1,000
30-200
30-200
300-1,000
inhibited
good
good
inhibited
red
red
ATCC 29212
230
The Difco Manual
Section II
Formula
Formula Per Liter
MPN Procedure
1. For an inoculum of 1 ml or less, suspend 56.4 g in 1 liter distilled
or deionized water.
For an inoculum of 10 ml, suspend 84.6 g in 1 liter distilled or
deionized water.
3. For an inoculum of 1 ml or less, dispense 10 ml amounts into
culture tubes.
For an inoculum of 10 ml, dispense 20 ml amounts into culture tubes.

Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Maltose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Azide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4
g
g
g
g
g
g
g
g
Precautions
Storage
Expiration Date
Procedure
Materials Provided

Glassware
Autoclave
Incubator (35C)
Pipettes
Culture tubes
TTC Solution 1%
Incubator (35C), saturated with water vapor
Sterile absorbent pads
The Difco Manual

1. Suspend 56.4 g in 1 liter distilled or deionized water.
3. Dispense 100 ml amounts into flasks and autoclave at 121C for
10 minutes.
4. Cool to 60C.
5. Add 1 ml TTC Solution 1% per 100 ml of medium.

Water or food samples should be collected and prepared according to
appropriate references.
Test Procedure
MPN Procedure
1. Inoculate tubes of the KF Streptococcus Broth with the appropriate
amount of inoculum.
2. Incubate tubes at 35 1C, with loosened caps, for 46-48 hours.
1. Place a sterile absorbent pad in each sterile Petri dish.
2. Saturate the pads with the sterile medium containing TTC.
3. Place an inoculated membrane filter, inoculated side up, on the
saturated pad.
4. Incubate at 35 1C in an atmosphere saturated with water vapor
for 46-48 hours.
Results
MPN Procedure
MPN tubes positive for enterococci are turbid with growth that appears yellow in color and does not produce foaming. When foaming
occurs, confirmation for enterococci should be made by Gram staining.
All red or pink colonies visible with 15x magnification are counted as
enterococci colonies.

1. Many strains of S. bovis and S. equinus are inhibited by azide.
2. The pH of KF Streptococcus Broth should be between 7.2 and 7.3.
If below 7.0, it should not be used.
3. Overheating may lower the pH, resulting in a decrease in productivity
of the medium.
231
KL Virulence Agar
References
streptococci. II. Quantification of streptococci in feces. Am. J. Public
Health 50:1553.
streptococci. I. Cultivation and enumeration of streptococci in surface
waters. Appl. Microbiol. 9:15.
3. MacFadden, J. F. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, vol. 1. Williams
Section II
4. Facklam, R. R., and M. D. Moody. 1970. Presumptive identification

of group D streptococci: the bile-esculin test. Appl. Microbiol.
20:245-250.
5. Donnelly, C. W., R. E. Bracket, D. Doores, W. H. Lee, and J.
Lovett. 1992. Compendium of Methods for the Microbiological
Examination of Foods, 3rd ed. American Public Health Association,
Washington, D.C.
Packaging
500 g
0997-17
KL Virulence Agar
Bacto KL Virulence Agar . KL Virulence Enrichment
KL Antitoxin Strips
Intended Use
Bacto KL Virulence Agar is used with Bacto KL Virulence
Enrichment, Bacto Chapman Tellurite Solution 1% and Bacto KL
Antitoxin Strips in differentiating virulent (toxigenic) from nonvirulent
strains of Corynebacterium diphtheriae.
Also Known As
KL Virulence Agar conforms with Klebs-Loeffler Virulence Agar.

Elek2 was the first to describe the agar plate diffusion technique for
demonstrating the in vitro toxigenicity (virulence) of Corynebacterium
diphtheriae. King, Frobisher, and Parsons 3 expanded on Eleks
technique and, by using a carefully standardized medium, obtained
results in agreement with animal inoculation tests. These authors
demonstrated that Difco Proteose Peptone possessed properties
essential for toxin production. Incorporating Difco Proteose Peptone
into the test medium assured consistent results. The authors used
rabbit, sheep and horse serum as enrichments, finding human serum to
be unsatisfactory. To overcome irregularities encountered in previous
formulations, Hermann, Moore, and Parsons1 refined the medium used
for the in vitro KL Virulence Test, simplifying the basal medium and
developing a nonserous enrichment. The medium and enrichment
described by these authors have been standardized for use in the
KL Virulence Test.
KL Virulence Agar and KL Virulence Enrichment are prepared
according to the formulation of Hermann, Moore and Parsons.1

Proteose Peptone provides the carbon and nitrogen sources required
for good growth of a wide variety of organisms and for toxin
production. Sodium Chloride maintains the osmotic balance of the
medium. Bacto Agar is incorporated as a solidifying agent.
KL Virulence Enrichment, composed of Casamino Acids, Glycerol and
Tween 80, provides a source of nonserous enrichment. Casamino
232
Acids is derived from acid-hydrolyzed casein that has low sodium

chloride and iron concentrations. The low iron concentration is
beneficial because iron is known to prevent the production of diph
theria toxin when present in more than minute amounts. Glycerol
(glycerine) contains no heavy metals and is used by bacteria as a source
of carbon. Tween 80 improves growth of certain strains of
Corynebacterium diphtheriae. Toxin produced by bacteria and diffused
into the medium is detected by precipitation with the antitoxin present
on the KL Antitoxin Strip. Chapman Tellurite Solution 1%
(1% potassium tellurite solution) inhibits gram-negative and most
gram-positive bacteria except Corynebacterium spp., Streptococcus
mitis, S. salivarius, enterococci, and possibly Staphylococcus
epidermidis. This permits direct testing of mixed primary cultures.
Formula
KL Virulence Agar
Formula Per Liter
Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g

KL Virulence Enrichment
Formula Per 100 ml
Bacto Casamino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Glycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 ml
Tween 80 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 ml
KL Antitoxin Strips
KL Antitoxin Strips are 1 x 7 cm filter paper strips containing antitoxin
to diphtheria toxin.
Precautions
The Difco Manual
Section II
KL Virulence Agar
Storage
Store KL Virulence Agar below 30C. The dehydrated medium is very

1. Suspend 37.5 grams of KL Virulence Agar in 1 liter distilled or

deionized water and heat to boiling to dissolve completely.
3. Cool in a water bath to 55-60C.
4. Aseptically dispense 10 ml of KL Virulence Agar into a Petri dish
containing 2 ml KL Virulence Enrichment and 0.5 ml Chapman
Tellurite Solution 1%; mix thoroughly.
5. Using aseptic technique, submerge a KL Antitoxin Strip or
equivalent beneath the agar prior to solidification.
Procedure
Materials Provided
For cases of suspected diphtheria, material for culture is obtained on

a swab from the inflamed membranes of the throat and nasopharynx,
or from wounds.4 Care must be taken not to contaminate the swab
with normal skin flora. The specimen should be immediately
transported to a laboratory and inoculated onto the proper media.
If the specimen is to be shipped to a laboratory, it should be placed
in a sterile tube or a special packet containing a desiccant such
as silica gel.5
Store KL Virulence Enrichment and KL Antitoxin Strips at 2-8C.

Store prepared plates at 2-8C.
Expiration Date
KL Virulence Agar
KL Antitoxin Strips

Glassware
Autoclave
Water bath (55-60C)
Incubator (35C)
Chapman Tellurite Solution 1%

KL Virulence Agar
Dehydrated Appearance: Light beige with some small dark specks,
free- flowing, homogeneous.
Solution:
is light to medium amber, slightly
opalescent, with a slight precipitate.
Reaction of 3.75%
Solution at 25C:
pH 7.8 0.2
Prepared Medium:
Light medium amber, slightly opalescent,
Appearance:
Colorless to very light amber, clear liquid.
KL Antitoxin Strips
Appearance:
White, filter paper strips, 1 x 7 cm.
Cultural Response
Prepare KL Virulence Agar per label directions, including KL
Virulence Enrichment, Chapman Tellurite Solution 1% and one
KL Antitoxin Strip per plate. Inoculate and incubate at 35 2C under
CO2 for up to 72 hours.
ORGANISM
ATCC
Corynebacterium diphtheriae Type gravis

8028
Corynebacterium diphtheriae Type intermedius
8032
25923*
+ = positive, line of precipitation at 45% angle to the strip
= negative, no line of precipitation
The Difco Manual
ATCC 8028
Precipitate lines are graphically enhanced for
demonstration purposes (see Results).
GROWTH
+
+
*This organism is available as a Bactrol culture and should
be used as directed.
233
Kligler Iron Agar
Test Procedure
Inoculate the medium by streaking a loopful of a 24-hour culture in
a single line across the plate perpendicular to (right angle to)
the antitoxin strip. (Do not touch the actual strip itself). As many as
eight cultures may be tested on a single plate.6 Place test isolates about
1 cm apart. Also inoculate a toxigenic (positive control) and a
nontoxigenic (negative control) C. diphtheriae strain approximately
1 cm on either side of the test isolates.6 Incubate the inverted plates
at 37C for 72 hours. Examine at 24-, 48- and 72-hour intervals.
Results
Toxigenic (virulent) cultures of C. diphtheriae will show fine lines
of precipitation at approximately 45 angles from the culture streak.
This line forms where toxin (from the bacteria) combines with
antitoxin from the strip. Primary precipitin lines form an arc of
identity with the precipitin line produced by an adjacent positive
control strain.7 Nontoxigenic strains of C. diphtheriae will show no
lines of precipitation.

1. Each test should include positive and negative controls.5
2. False-positive reactions may be seen after 24 hours as weak bands
near the antitoxin strip. These can be recognized when compared
with the positive control.8
3. Corynebacterium ulcerans and C. pseudotuberculosis may also
produce lines of toxin-antitoxin.9
Section II
3. King, E. O., M. Frobisher, Jr., and E. I. Parsons. 1949. The

in vitro test for virulence of Corynebacterium diphtheriae.
Am. J. Public Health 39:1314.
4. Clarridge, J. E., and C. A. Spiegel. 1995. Corynebacterium and
miscellaneous irregular gram-positive rods, Erysipelothrix and
Gardnerella, p. 357-378. In P. R. Murray, E. J. Baron, M. A. Pfaller,
F. C. Tenover, and R. H. Yolken (eds.), Manual of clinical
Washington, D.C.
5. Krech, T., and D. G. Hollis. 1991. Corynebacterium and related
organisms, p. 277-286. In A. Ballows, W. J. Hausler, Jr.,
K. Herrmann, H. D. Isenberg, and H. J. Shadomy (eds.), Manual of
Washington, D.C.
7. Washington, J. A., Jr. 1981. Laboratory procedures in clinical
microbiology. Springer-Verlag, New York, NY.
8. Lennette, E. H., A. Balows, W. J. Hausler, Jr., and J. P. Truant
(eds.). 1980. Manual of clinical microbiology, 3rd ed. American
9. Branson, D. 1972. Methods in clinical bacteriology. Charles
C. Thomas, Springfield, IL.
Packaging
References
KL Virulence Agar
1. Hermann, G. J., M. S. Moore, and E. I. Parsons. 1958. A

substitute for serum in the diphtheria in vitro test. Am. J. Clin.
Pathol. 29:181-183.
2. Elek, S. D. 1948. The recognition of toxicogenic bacterial strains
in vitro. Brit. Med. J. 1:493.
Bacto Kligler Iron Agar
KL Antitoxin Strips
500 g
0985-17
12 x 20 ml
0986-64
12 strips
6 x 1 ml
6 x 25 ml
3101-30
0299-51
0299-66
Bacto Kligler Iron Agar is used for differentiating pure cultures of

gram-negative bacilli based on the fermentation of dextrose and
lactose and production of hydrogen sulfide.
could be successfully combined with Russell double sugar medium for

the differentiation of the typhoid, paratyphoid and dysentery groups.
Bailey and Lacy4 simplified the formula by using phenol red as
the pH indicator instead of Andrade indicator. A similar medium
containing saccharose, Tryptone, ferrous sulfate and thiosulfate was
developed by Sulkin and Willett.5
Also Known As
Kligler Iron Agar is recommended for differentiation of enteric gramnegative bacilli from clinical specimens6-8 and food samples.9,10
Intended Use
Kligler Iron Agar is also known as KIA.

Kligler Iron Agar is a modification of Kliglers1 original formula. It
is recommended to identify pure cultures of colonies picked from
primary plating media, such as MacConkey Agar. Kliglers1 original
medium was a soft nutrient agar containing dextrose, Andrade
indicator and lead acetate. Russell2 devised a medium containing
glucose, lactose, and an indicator for the differentiation of
lactose-fermenting and nonlactose-fermenting gram negative bacilli.
Kligler3 found that lead acetate for the detection of hydrogen sulfide
234

Kligler Iron Agar combines the principles of Russell double sugar agar
and lead acetate agar into one medium. This combination permits
the differentiation of the gram-negative bacilli both by their ability to
ferment dextrose or lactose and to produce hydrogen sulfide. Beef
Extract, Yeast Extract, Bacto Peptone, and Proteose Peptone provide
nitrogen, vitamins and minerals. Ferrous sulfate and sodium thiosulfate
are the indicators of hydrogen sulfide production. Phenol red is the
pH indicator. Sodium chloride maintains the osmotic balance of the
medium. Bacto Agar is the solidifying agent.
The Difco Manual
Section II
Kligler Iron Agar
Formula
Procedure
Kligler Iron Agar

Formula Per Liter
Materials Provided
Kligler Iron Agar

Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Thiosulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.024
g
g
g
g
g
g
g
g
g
g
g

Tubes with closures
Autoclave
Incubator (35C)
Precautions
1.
2.
3.
4.

Dispense into tubes with closures.
Autoclave at 121C for 15 minutes. Cool in slanted position
with deep butts.
Storage
tubes at 2-8C.
Expiration Date

sterile swabs and transport immediately to the laboratory
following recommended guidelines.6-10
specimen or sample.6-10
The expiration date applies to the medium in its intact container when

Dehydrated Appearance: Pinkish beige, free flowing,
homogeneous.
Solution:
5.5% solution; soluble in distilled or
deionized water on boiling. Orange-red,
slightly opalescent with precipitate.
Prepared Tubes:
Slightly orange-red, slightly
opalescent, slight precipitate.
Reaction of 5.5%
Solution at 25C:
pH 7.4 0.2
Cultural Response
Prepare Kligler Iron Agar per label directions. Inoculate and
incubate tubes at 35C for 18-48 hours.
ORGANISM
ATCC
CFU
Citrobacter freundii 8090* undiluted

Escherichia coli
25922* undiluted
Proteus vulgaris
6380 undiluted
A = acid reaction (yellow)
+gas = cracks, splits or bubbles in medium
+H2S = black precipitate in butt
GROWTH
good
good
good
SLANT/
BUTT GAS
A/A
A/A
K/A
+
+
H2S
Uninoculated
tube
Citrobacter freundii
ATCC 8090
Escherichia coli
ATCC 25922
Proteus vulgatus
ATCC 6380
K = alkaline reaction (no color change)

gas = no cracks, splits, or bubbles in medium
H2S = no black precipitate in butt
The Difco Manual
235
Koser Citrate Medium
Test Procedure
1. Obtain a pure culture of the organism to be tested. Select
well-isolated colonies.
2. With an inoculating needle, pick the center of well-isolated
colonies obtained from solid culture media.
3. Stab the center of the medium into the deep of the tube to within
3-5 mm from the bottom.
4. Withdraw the inoculating needle, and streak the surface of the slant.
5. Loosen closure on the tube before incubating.
6. Incubate at 35C for 18-48 hours.
7. Read tubes for acid production of slant/butt, gas, and hydrogen
sulfide reactions.
Results
1. An alkaline slant-acid butt (red/yellow) indicates fermentation of
dextrose only.
2. An acid slant-acid butt (yellow/yellow) indicates fermentation of
dextrose and lactose.
3. An alkaline slant-alkaline butt (red/red) indicates that neither
dextrose nor lactose was fermented (non-fermenter).
4. Cracks, splits, or bubbles in the medium indicate gas production.
5. A black precipitate in the butt indicates hydrogen sulfide production.

1. H2S-producing organisms may produce a black precipitate to such
a degree that the reaction in the butt is completely masked. If H2S
is produced, dextrose is fermented even if it is not observed.11
2. Further biochemical tests and serological typing must be performed
for definite identification and confirmation of organisms.
3. Do not use an inoculating loop to inoculate a tube of Kligler Iron
Agar. While stabbing the butt, mechanical splitting of the medium
occurs, causing a false positive result for gas production.11
4. Best reactions are obtained on freshly prepared medium.
5. A pure culture is essential when inoculating Kligler Iron Agar. If
inoculated with a mixed culture, irregular observations may occur.
6. Hydrogen sulfide determinations using Kligler Iron Agar should
be limited to the members of the Enterobacteriaceae. Other
organisms may require more sensitive methods for detection of H2S
production.11
Bacto Koser Citrate Medium
Section II
7. Tubes should be incubated with caps loosened to allow a free

exchange of air, which is necessary to enhance the alkaline
condition on the slant.11
References
1. Kligler, I. J. 1917. A simple medium for the differentiation of
members of the typhoid-paratyphoid group. Am. J. Public Health.
7:1042-1044.
2. Russell, F. F. 1911. The isolation of typhoid bacilli from urine and
feces with the description of a new double sugar tube medium. J.
Med. Res. 25:217.
3. Kligler, I. J. 1918. Modifications of culture media used in the
isolation and differentiation of typhoid, dysentery, and allied
bacilli. J. Exp. Med. 28:319-322.
4. Bailey, S. F., and L. R. Lacy. 1927. A modification of the Kligler
lead acetate medium. J. Bacteriol. 13:183.
5. Sulkin, S. E., and J. C. Willett. 1940. A triple sugar-ferrous
sulfate medium for use in identification of enteric organisms.
J. Lab. Clin. Med. 25:649-653.
Isenberg, H.D. (ed.), Clinical microbiology procedures handbook,
St. Louis, MO.
10. Elliot, E. L., C. A. Kaysner, L. Jackson, and M. L. Tamplin.
1995. V. cholerae, V. parahaemolyticus, V. vulnificus, and other
Vibrio spp. In FDA bacteriological analytical manual, 8th ed.
Packaging
Kligler Iron Agar
500 g
0086-17
Intended Use
Bacto Koser Citrate Medium is used for differentiating Escherichia
coli from Enterobacter aerogenes based on citrate utilization.
Also Known As
Kosers Citrate Broth.

In 1923, the work of Koser demonstrated that coli-aerogenes bacteria
could be differentiated by their use of certain salts of organic acids.1
236
Koser found that the sodium salt of citric acid (sodium citrate) is used
as a source of carbon by E. aerogenes and not by E. coli. Biochemical
identification schemes for identifying E. coli frequently include
Koser citrate.
E. coli is an important member of the coliform group of bacteria. The
coliforms are described as aerobic and facultatively anaerobic gramnegative non-sporeforming bacilli that ferment lactose and form acid
and gas at 35C within 48 hours. Procedures to detect, enumerate and
presumptively identify coliforms are used in testing foods and dairy
products.2-5 Presumptive identification is confirmed by performing
biochemical tests that specifically identify E. coli.
The Difco Manual
Section II
Expiration Date
Bacto Koser Citrate Medium is prepared with chemically pure salts

and tested to determine that no sources of carbon (other than sodium
citrate) or nitrogen (other than ammonium salts) are present. Bacteria
that are able to use citrate as their carbon source will grow in the
medium and cause turbidity.
Formula
Materials Provided
Bacto Koser Citrate Medium

Formula Per Liter
Sodium Ammonium Phosphate . . . . . . . . . . . . . . . . . . . . .

Monobasic Potassium Phosphate . . . . . . . . . . . . . . . . . . . .
Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.5
1.0
0.2
3.0
g
g
g
g

Storage

Test Procedure
1. Transfer growth from a single colony or a loopful of liquid
suspension and inoculate the broth medium.
Results
Positive: Turbidity
Negative: Clear, no turbidity

References
Solution:
or deionized water. Solution is
colorless, clear.
Reaction of 0.57%
Solution at 25C:
pH 6.7 0.2
Cultural Response
Prepare medium per label directions. Inoculate and incubate
ATCC
INOCULUM
CFU
13048*
25922*
>1,000
>1,000
GROWTH
good
markedly to
The Difco Manual

2. Dispense required amount into test tubes.
1. The Koser citrate test is one of many biochemical tests required to

identify an isolate to genus and species.
Escherichia coli
Flask with closure

Autoclave
Precautions
ORGANISM
Procedure
1. Koser, S. A. 1923. Utilization of the salts of organic acids by the

colon- aerogenes group. J. Bacteriol. 8:493-520.
2. Vanderzant, C., and D. F. Splittoesser (ed.). 1992. Compendium
of methods for the microbiological examination of foods, 3rd ed.
5. Association of Official Analytical Chemists. 1995. Official methods
of analysis of AOAC International, 16th ed. AOAC International,
Arlington, VA.
Packaging
500 g
0015-17
237
LB Agar, Lennox
Section II
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
Bacto LB Agar, Lennox
Intended Use
Precautions
Bacto LB Agar, Lennox is used for maintaining and cultivating

recombinant strains of Escherichia coli.


LB Agar, Lennox, a nutritionally rich medium, was developed by
Lennox for the growth and maintenance of pure cultures of recombinant
strains of E. coli.1 These strains are generally derived from E. coli K12,
which are deficient in B vitamin production. This strain of E. coli
has been further modified through specific mutation to create an
auxotrophic strain that is not capable of growth on nutritionally deficient
media. LB Agar, Lennox provides all the nutritional requirements of
these organisms. LB Agar, Lennox contains half the sodium chloride
level of the Miller formulation of LB Agar.2 This allows the researcher
to select the optimal salt concentration for a specific strain.
Storage
Materials Provided
Peptides and peptones are provided by Tryptone. Vitamins (including

B vitamins) and certain trace elements are provided by Yeast Extract.
Sodium ions for transport and osmotic balance are provided by
Sodium Chloride. Bacto Agar is the solidifying agent.
Formula
LB Agar, Lennox
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g

Expiration Date
Procedure
LB Agar, Lennox

Autoclave
Waterbath (45-50C)
Petri dishes
Incubator (35C)

Solution:
is light beige, slightly opalescent.
Prepared Plates:
Medium amber, very slightly to
Reaction of 3.5%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepare LB Agar, Lennox per label directions. Inoculate and
incubate at 35 2C for 18- 24 hours.
ORGANISM
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
ATCC
INOCULUM
CFU
GROWTH
23724
33694
33849
39403
47014
53868
100-300
100-300
100-300
100-300
100-300
100-300
Good
Good
Good
Good
Good
Good
1.
2.
3.
4.
5.
Suspend 35 grams in 1 liter of distilled or deionized water.


Not applicable.
Test Procedure
Consult appropriate references for recommended test procedures.2
Results
After sufficient incubation, the medium should show growth as evidenced by formation of colonies and/or a confluent lawn of growth.
References
1. Lennox, E. S. 1955. Transduction of linked genetic characters of
the host by bacteriophage P1. Virology 1:190.
Seidman, J. A. Smith, and K. Struhl. 1994. Current protocols in
molecular biology, vol. 1. Current Protocols, New York, N.Y.
Packaging
LB Agar, Lennox
238
500 g
0401-17
The Difco Manual
Section II
LB Agar, Miller
Storage
Bacto LB Agar, Miller

very hygroscopic. Keep container tightly closed. Store prepared
medium at 2-8C.
Intended Use
Bacto LB Agar, Miller is used for maintaining and propagating
Escherichia coli in molecular microbiology procedures.

LB Agar, Miller is based on LB Medium as described by Miller for the
growth and maintenance of E. coli strains used in molecular microbiology
procedures.1-3 LB Agar, Miller is a nutritionally rich medium designed
for growth of pure cultures of recombinant strains. E. coli grows more
rapidly on this rich medium because it provides the cells with amino
acids, nucleotide precursors, vitamins and other metabolites that the
microorganism would otherwise have to synthesize.4
Expiration Date
Procedure
Materials Provided
LB Agar, Miller

Sodium ions for transport and osmotic balance are provided by sodium
chloride. Agar is added to the medium as a gelling agent.

Autoclave
Waterbath (45-50C)
Incubator (35C)
Formula
LB Agar, Miller
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g

Cool to 45-50C. Dispense into sterile Petri dishes.

Not applicable.
Test Procedure
Precautions
Consult appropriate references for recommended test procedures.3-6

Results
Growth should be evident by the appearance of colonies and/or a
confluent lawn on the surface of the medium.
References

Dehydrated Appearance: Very light to light tan, free-flowing,
homogeneous.
Solution:
is very light amber, slightly opalescent.
Prepared Plates:
Very light amber, slightly opalescent.
Reaction of 4.0%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepare Bacto LB Agar, Miller per label directions. Inoculate
plates and incubate at 35 2C for 18-24 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Escherichia coli
33526
100-1000
Good

The Difco Manual
1.
2.
3.
4.
1. Luria, S. E., and J. W. Burrous. 1955. Hybridization between

Escherichia coli and Shigella. J. Bacteriol. 74:461-476.
2. Luria, S. E., J. N. Adams, and R. C. Ting. 1960. Transduction of
lactose-utilizing ability among strains of E. coli and S. dysenteriae and
the properties of the transducing phage particles. Virology 12:348-390.
3. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY.
Seidman, J. A. Smith, and K. Struhl (ed.). 1994. Current
protocols in molecular biology. Greene Publishing Associates, Inc.,
Brooklyn, NY.
5. Sambrook, J., E. F. Fitsch, and T. Maniatis. 1989. Molecular
cloning: a laboratory manual, 2nd. ed. Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY.
6. Lennox, E. S. 1955. Transduction of linked genetic character of
the host by bacteriophage P1. Virology 1:190-206.
Packaging
LB Agar, Miller
500 g
2 kg
0445-17
0445-07
239
LB Broth, Lennox
Section II
Precautions
Bacto LB Broth, Lennox
Intended Use
Bacto LB Broth, Lennox is used for maintaining and propagating
Escherichia coli in molecular microbiology procedures.

LB Broth, Lennox is a nutritionally rich medium designed for growth
of pure cultures of recombinant strains. The formula is based on
L Broth described by Lennox for the growth and maintenance of
E. coli strains used in molecular microbiology procedures.1 E. coli
is grown to late log phase in LB Broth. Some plasmid vectors may
replicate to high copy numbers without selective amplification. Some
vectors may require selective amplification to reach high copy numbers.
Chloramphenicol can be added to inhibit host synthesis and, as a
result, prevent replication of the bacterial chromosome.2
LB Broth, Lennox contains ten times the sodium chloride level of Luria
Broth Base, Miller and one half of that found in LB Broth, Miller.3
This allows the researcher to select the optimal salt concentration for a
specific strain. If desired, the medium may be aseptically supplemented
with glucose to prepare the complete medium described by Lennox.

Peptides and peptones are provided by Tryptone. Vitamins (including B
vitamins) and certain trace elements are provided by Yeast Extract. Sodium
ions for transport and osmotic balance are provided by Sodium Chloride.
Formula
LB Broth, Lennox
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Expiration Date
Procedure
Materials Provided
LB Broth, Lennox

Tubes with closures
Autoclave
Incubator (35C)
1.
2.
3.
4.
5.

Allow to cool below 45C.
If desired, aseptically add 10 ml sterile 10% glucose solution and
mix thoroughly.
Test Procedure

Solution:
Prepared Medium:
Very light amber, clear to very
Reaction of 2.0%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepare LB Broth, Lennox per label directions. Inoculate and
ATCC
INOCULUM
CFU
GROWTH
53868 (DH5)
JM103
33694 (HB101)
100-300
100-300
100-300
Good
Good
Good
240

Not applicable
Escherichia coli
Escherichia coli
Escherichia coli
Storage
ORGANISM

Consult appropriate references for recommended test procedures.1,2,3
Results
References
the host by bacteriophage P1. Virology 1:190.
Laboratory Press, Cold Spring Harbor, New York.
Harbor Laboratory, Cold Spring Harbor, New York.
Packaging
LB Broth, Lennox
500 g
2 kg
10 kg
0402-17
0402-07
0402-08
The Difco Manual
Section II
LB Broth, Miller
Precautions
Bacto LB Broth, Miller
Intended Use
Bacto LB Broth, Miller (Luria-Bertani) is used for maintaining and
propagating Escherichia coli in molecular microbiology procedures.

LB Broth, Miller is based on LB Medium as described by Miller for the
growth and maintenance of Escherichia coli strains used in
molecular microbiology procedures.1-3 LB Broth, Miller is a nutritionally
rich medium designed for growth of pure cultures of recombinant
strains. Escherichia coli is grown to late log phase in LB Medium.
Some plasmid vectors replicate to a high copy number without
selective amplification. Some vectors do not replicate so freely, and
need to be selectively amplified. Chloramphenicol has been added to
inhibit host synthesis and as a result, prevents replication of the
bacterial chromosome.4
LB Broth, Miller contains twenty times the sodium chloride level of Luria
Broth Base, Miller and twice the level found in LB Broth,
Lennox.3-5 This allows the researcher to select the optimal salt concentration
for a specific strain.

sodium chloride.
Formula
LB Broth, Miller
Formula Per Liter

Storage
medium at 2-8C.
Expiration Date
Procedure
Materials Provided
LB Broth, Miller

Autoclave
Incubator (35C)

Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Test Procedure
Consult appropriate references for recommended test procedures.3,4
Results
Growth should be evident by the appearance of turbidity in the medium.
References
Dehydrated Appearance: Off-white to beige, free-flowing,

homogeneous.
Solution:
Prepared Tubes:
Reaction of 2.5%
Medium at 25C:
pH 7.0 0.2
Cultural Response
Prepare LB Broth, Miller per label directions. Inoculate the
tubes and incubate at 35 2C for 18-24 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Escherichia coli
33526
100-1,000
Good
The culture listed above is the minimum that should be used for
The Difco Manual
1. Luria, S. E., and J. W. Burrous. 1955. Hybridization between

Escherichia coli and Shigella. J. Bacteriol. 74:461-476.
2. Luria, S. E., J. N. Adams, and R. C. Ting. 1960. Transduction of
lactose- utilizing ability among stains of E. coli and S. dysenteriae
and the properties of the transducing phage particles. Virology.
12:348-390.
3. Miller, J. H. 1972. Experiments in molecular genetics. Cold
Spring Harbor Laboratory. Cold Spring Harbor, New York.
Laboratory, Cold Spring Harbor, New York.
the host by bacteriophage P1. Virology. 1:190-206.
Packaging
LB Broth, Miller
500 g
2 kg
0446-17
0446-07
241
LPM Agar Base & Moxalactam Antimicrobic Supplement
Section II
Bacto LPM Agar Base

Bacto Moxalactam Antimicrobic Supplement
Intended Use
LPM Agar Base is used with Bacto Moxalactam Antimicrobic
Supplement for isolating and cultivating Listeria monocytogenes.

industries. This organism can cause human illness and death,
particularly in immunocompromised individuals and pregnant women.2
The first reported food-borne outbreak of listeriosis was in 1985,3 and
since then, microbiological and epidemiological evidence from both
sporadic and epidemic cases of listeriosis has shown that the principal
route of transmission is via the consumption of foodstuffs
contaminated with Listeria monocytogenes.4
coleslaw, pasteurized milk, Mexican-style cheese, pat, and pickled
pork tongue. The organism has been isolated from commercial dairy
and other food processing plants, and is ubiquitous in nature, being
present in a wide range of unprocessed foods and in soil, sewage,
silage and river water.6

products with pH levels outside these parameters.7 Listeria spp.
are microaerophilic, gram-positive, asporogenous, non-encapsulated,
non-branching, regular, short, motile rods. Motility is most
pronounced at 20C.
The most common contaminating bacteria found in food sources
potentially containing Listeria are: streptococci, especially the
enterococci, micrococci and Bacillus species, Escherichia coli,
Pseudomonas aeruginosa and Proteus vulgaris.8
LPM Agar, a modification of McBride Listeria Agar, was developed
by Lee and McClain 9 to recover low numbers of Listeria
monocytogenes from samples with profusely mixed microflora. Its use
is recommended when testing food and dairy samples and clinical
specimens for Listeria.
Uninoculated
plate
ATCC 19114

LPM Agar Base
Dehydrated Appearance: Light tan, homogeneous, may have a
tendency to form soft lumps.
Solution:
Prepared Plates:
opalescent.
Reaction of 5.05%
Solution at 25C:
pH 7.3 0.2
Moxalactam Antimicrobic Supplement
Lyophilized Appearance: White to off-white cake (may be broken).
Solution:
Yellow tinted, clear solution when rehydrated
with 10 ml sterile distilled or deionized water.
Cultural Response
Prepare LPM Agar Base with Moxalactam Antimicrobic Supplement. Inoculate and
incubate plates at 35 2C for 18-48 hours.
ORGANISM
Escherichia coli
ATCC
INOCULUM
CFU
GROWTH
29212*
25922*
19114
1,000-2,000
1,000-2,000
100-1,000

good at 40-48 hours
242
The Difco Manual
Section II
LPM Agar Base & Moxalactam Antimicrobic Supplement
Storage
In LPM Agar, Tryptose and Beef Extract provide nitrogen, vitamins

and minerals. Sodium chloride maintains the osmotic balance of the
medium. Glycine anhydride is used for improved recovery of Listeria.
Lithium chloride, in an increased concentration, and phenylethanol are
incorporated to aid in suppression of both gram-positive and
gram-negative contaminants. Agar is a solidifying agent. Moxalactam
Antimicrobic Supplement is added to LPM Agar Base after autoclaving
to inhibit staphylococci, bacilli and Proteus species.
1. Store LPM Agar Base at 2-8C. The dehydrated medium is very

Store Moxalactam Antimicrobic Supplement at 2-8C.
Formula
Materials Provided
g
g
g
g
g
g
g

Formula per 10 ml
Moxalactam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg
Precautions
2. LPM Agar Base:
TARGET ORGAN(S): Blood, Face, Muscles, Nerves, Urogenital.
Moxalactam Antimicrobic Supplement:
MAY CAUSE ALLERGIC EYE, RESPIRATORY SYSTEM AND
SKIN REACTION. (US) Avoid contact with skin and eyes. Do not
tightly closed. TARGET ORGAN(S): Blood, Liver, Kidneys
The Difco Manual
Procedure
LPM Agar Base

Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Glycine Anhydride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Phenylethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Expiration Date
LPM Agar Base


Autoclave
Waterbath (45-50C)
Petri dishes
Incubator (35C)
1. Suspend 50.5 grams of LPM Agar Base in 1 liter of distilled or
deionized water.
4. Cool medium to 45-50C in a waterbath.
5. Aseptically add 10 ml Moxalactam Antimicrobic Supplement
rehydrated per label instructions with sterile distilled or
deionized water.
6. Mix well and dispense into Petri dishes.

recommended guidelines.7,10,11,12
2. Clinical specimens obtained from nonsterile sites, foods, and
specimens obtained from the environment should be selectively
enriched for Listeria spp. before being plated.10
specimen or sample.7,10,11,12
Test Procedure
Clinical specimens obtained from nonsterile sites should be selectively
enriched for Listeria spp. before being plated. Please refer to appropriate
references for the procedure to use with clinical specimens.10 For a
procedure for isolating Listeria from milk, milk products and food
samples, refer to an appropriate reference.7,11,12
Results
Observe colonies under oblique transmitted light. Listeria colonies
display a grey to blue color with a ground glass appearance.
243
Lactobacilli Agar AOAC & Lactobacilli Broth AOAC
References
3. Wehr, H. M. 1987. Listeria monocytogenes - a current dilemma.
Special Report. J. Assoc. Off. Anal. Chem. 70:769-772.
4. Bremer, P. J., and C. M. Osborne. 1995. Thermal-death times
of Listeria monocytogenes in green shell mussels (Perna
canaliculus) prepared for hot smoking. J. Food Prot. 58:604-608.
7. Donnelly, C. W., R. E. Bracket, D. Doores, W. H. Lee,
and J. Lovett. 1992. Listeria. In C. Vanderzant and D. F.
Splittstoesser (ed.). Compendium of methods for the microbio-
Bacto Lactobacilli Agar AOAC

Bacto Lactobacilli Broth AOAC
Section II
logical examination of foods, 3rd ed. American Public Health

9. Lee, W. H., and D. McClain. 1986. Improved Listeria
monocytogenes selective agar. Appl. Environ. Microbiol.
52:1215-1217.
Tenover, and R.H. Yolken (eds.). Manual of clinical microbiology,
1993. Pathogens in milk and milk products. In R. T. Marshall, ed.
12. Hitchins, A. D. 1992. Listeria monocytogenes, p. 141-151. FDA
Arlington, VA.
Packaging
LPM Agar Base
Moxalactam Antimicrobic
Supplement
500 g
2 kg
0221-17
0221-07
6 x 10 ml
0216-60
Intended Use
Bacto Lactobacilli Agar AOAC is used for maintaining stock cultures
used in the microbiological assays of vitamins and amino acids.
Bacto Lactobacilli Broth AOAC is used for preparing inocula used in
the microbiological assays of vitamins and amino acids.

1. Maintenance Media: For maintaining the stock culture to preserve
purpose.
Assay media contain all the factors necessary for optimal growth
of the test organism except the single essential vitamin to be
determined.
Lactobacilli Agar AOAC1 and Lactobacilli Broth AOAC1 are prepared
according to the formula recommended by Loy.2 Lactobacilli Agar
AOAC is used for maintaining stock cultures. Lactobacilli Broth AOAC
is used to prepare inocula of Lactobacillus leichmannii ATCC 7830,
Enterococcus faecium ATCC 8043, Lactobacillus plantarum
244
ATCC 8014, Lactobacillus casei ATCC 7469 and other organisms

used in the microbiological assay of B vitamins.
Lactobacillus species grow poorly on non-selective culture media and
require special nutrients. Mickle and Breed3 reported the use of tomato
juice in culture media for lactobacilli. Kulp,4 while investigating the
use of tomato juice on bacterial development, found that growth of
Lactobacillus acidophilus was enhanced.

Peptonized Milk and Yeast Extract provides the nitrogen, amino acids
and vitamins sources in Lactobacilli Agar AOAC and Lactobacilli Broth
AOAC. Dextrose is a carbon source to facilitate organism growth.
Tomato juice creates the proper acidic environment. Potassium
Phosphate Monobasic is a buffering agent. Tween 80 (Sorbitan
Monooleate Complex) acts as an emulsifier. Bacto Agar is a solidifying
agent in Lactobacilli Agar AOAC.
Formula
Formula Per Liter
Peptonized Milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Tomato Juice (100 ml) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
g
g
g
g
g
g
g

The Difco Manual
Section II
Lactobacilli Agar AOAC & Lactobacilli Broth AOAC

Formula Per Liter
Peptonized Milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Tomato Juice (100 ml) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Monobasic Potassium Phosphate . . . . . . . . . . . . . . . . . . . . . 2
Tween 80 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
g
g
g
g
g
g
Storage
Store the dehydrated medium below 2-8C. The dehydrated medium is
Expiration Date
Precautions
3. Take care to avoid contamination of media or glassware used for
Procedure
Materials Provided

Solution:
deionized water on boiling 2-3 minutes.
Prepared Medium:
Medium amber, opalescent when hot,
clearer when cooled to 45-50C.
Reaction of 4.8%
Solution at 25C:
pH 6.8 0.2
Solution:
or deionized water on boiling 2-3
minutes. Medium amber, clear, may
Prepared Medium:
Medium amber, opalescent when hot,
clear with a very slight precipitate
when cooled.
Reaction of 3.8%
Solution at 25C:
pH 6.8 0.2
Cultural Response
Prepare Lactobacilli Agar AOAC and Lactobacilli Broth AOAC
per label directions. Inoculate Lactobacilli Agar AOAC by
stabbing the medium with test organisms; incubate at 35 2C
for 18-48 hours. Inoculate Lactobacilli Broth AOAC with test
organisms and incubate at 35 2C for 18-24 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Enterococcus hirae
Lactobacillus casei
subsp. rhamnosus
subsp. delbrueckii
Lactobacillus plantarum
8043
100-1,000
good
7469
100-1,000
good
7830
8014
100-1,000
100-1,000
good
good
The Difco Manual

Scrupulously clean glassware free from detergents and other
chemical must be used.
Glassware
Autoclave
Incubator
Inoculating needle
0.9% NaCl
deionized water:
Lactobacilli Agar AOAC - 48 grams/liter
Lactobacilli Broth AOAC - 38 grams/liter
2. Heat to boiling 2-3 minutes to dissolve completely.

Samples are prepared for assay according to references given in the
specific assay procedures. The samples should be diluted to
Test Procedure
Stock Cultures
1. Prepare stock cultures in one or more tubes of sterile Lactobacilli
Agar AOAC.
2. Inoculate the medium using an inoculating needle.
4. Store at 2-8C.
5. Transfer at weekly or twice monthly intervals.
Inoculum
1. Subculture from a 16-24 hour stock culture into 10 ml Lactobacilli
Broth AOAC.
2. Incubate at 35-37C for 16-24 hours or as specified in specific
assay procedures.
3. Centrifuge the culture and decant the supernatant.
4. Resuspend cells in 10 ml of sterile 0.9% NaCl solution or sterile
single-strength basal assay medium.
245
Lactobacilli MRS Agar & Lactobacilli MRS Broth
5. Wash the cells by centrifuging and decanting the supernatant two

additional times unless otherwise indicated.
6. Dilute the washed suspension 1:100 with sterile 0.9% NaCl or
sterile single- strength basal assay medium or as indicated. Where
applicable, inoculum concentration should be adjusted according
to limits specified in AOAC1 or US Pharmacopeia.5
For a complete discussion on vitamin assay methodology refer to
appropriate procedures outlined in the references.1,5
Results
Refer to appropriate references for vitamin assay results.1,5

cultured and maintained on media recommended for that purpose.
2. Aseptic technique should be used throughout the vitamin assay
procedure.
3. The use of altered or deficient media may result in mutants with
response.
Bacto Lactobacilli MRS Agar

Bacto Lactobacilli MRS Broth
Section II
4. For a successful completion of these procedures, all conditions of

the assay must be adhered to meticulously.
References
2. Loy. 1958. J. AOAC. 4:61.
3. Mickle and Breed. 1925. Technical Bulletin 110, NY State
Agriculture Ex. Station.
Lactobacillus acidophilus. Science 76:17.
Packaging
100 g
100 g
0900-15*
0901-15*
*Store at 2-8C
Intended Use
Bacto Lactobacilli MRS Agar and Bacto Lactobacilli MRS Broth are
recommended for use in the isolation, enumeration and cultivation of
Lactobacillus species.
Also Known As
MRS is an abbreviation for the authors names, deMan, Rogosa and
Sharpe.

Lactobacilli MRS Agar and Lactobacilli MRS Broth are based on the
formulations of deMan, Rogosa and Sharpe.1 These media were shown
by the authors to support luxuriant growth of all lactobacilli from oral,
fecal, dairy and other sources.

Lactobacilli MRS Agar and Lactobacilli MRS Broth contain Peptone
and Dextrose. These ingredients supply nitrogen, carbon and other
elements necessary for growth. Polysorbate 80, Acetate, Magnesium
and Manganese provide growth factors for culturing a variety of
lactobacilli. The above ingredients may inhibit the growth of some
organisms other than lactobacilli.
Formula
Lactobacilli MRS Agar
Formula Per Liter
Bacto Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
246

Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Ammonium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Manganese Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g
g
g

Lactobacilli MRS Broth
Formula Per Liter
Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
g
g
g
g
g
g
g
g
g
g
Precautions
Storage
Store Lactobacilli MRS Agar and Lactobacilli MRS Broth at 2-8C.
The powders are very hygroscopic. Keep containers tightly closed.
The Difco Manual
Section II
Lactobacilli MRS Agar & Lactobacilli MRS Broth
Expiration Date
Procedure

the laboratory following recommended guidelines.2,3,4
2. Process each sample using procedures appropriate for that sample.2,3,4
Materials Provided
Test Procedure

Direct Counts
1. To obtain direct counts of lactobacilli, pour 15-20 ml sterile, molten
(45-50C) Lactobacilli MRS Agar into sterile Petri dishes containing
1 ml volumes of diluted test sample.
2. Distribute the inoculum throughout the medium by rotating the
plate in one direction and then in the reverse direction.
3. Allow the medium to solidify on a flat surface for 5-10 minutes.
4. Alternatively, plates of Lactobacilli MRS Agar can be used for direct recovery of organisms using the streak inoculation technique.
5. Incubate agar plates at 35C for 3 days, or at 30C for 5 days, in an
aerobic atmosphere supplemented with carbon dioxide.
Broth Enrichment
1. Samples can be inoculated directly into Lactobacilli MRS Broth.
2. Incubate broth tubes at 35C for 3 days, or at 30C for 5 days, in an
aerobic atmosphere.
3. Subculture growth in broth tubes to appropriate solid media.

Autoclave
Waterbath (45-50C)
Incubator (30 or 35C)

Solution:
deionized water upon boiling. Solution is
dark amber, clear to slightly opalescent.
Prepared Medium:
Reaction of 7.0%
Solution at 25C:
pH 6.5 0.2
Solution:
dark amber, clear to very slightly opalescent.
Reaction of 5.5%
Solution at 25C
pH 6.5 0.2
ATCC 7830
Cultural Response
Prepare Lactobacilli MRS Agar or Lactobacilli MRS Broth per label directions. Inoculate
Lactobacilli MRS Agar and incubate in a 5% CO2 atmosphere at 35C for 24- 72 hours. Inoculate Lactobacilli
MRS Broth and incubate at 35C for 24 hours.
ORGANISM
ATCC
Lactobacillus delbrueckii subsp. lactis 7830

9338
Lactobacillus species
11506
The Difco Manual
INOCULUM CFU
GROWTH
100-1,000
100-1,000
100-1,000
good
good
good
247
Lactose Broth
Section II
Results
Lactobacilli appear as large, white colonies embedded in or on
Lactobacilli MRS Agar or as turbidity in Lactobacilli MRS Broth.
Growth may be subcultured onto the appropriate media for use in
additional procedures. Refer to appropriate references for
recommendations on the culture of Lactobacillus spp.2,3,4

1. Organisms other than lactobacilli may grow in these media.
Isolates must be confirmed as lactobacilli by appropriate
biochemical testing.
References
1. deMan, J. C., M. Rogosa, and M. E. Sharpe. 1960. A medium
for the cultivation of lactobacilli. J. Appl. Bacteriol. 23:130.
Bacto Lactose Broth
2. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compendium of methods for the microbiological examination of foods.
3. MacFaddin, J. F. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, vol. 1. Williams & Wilkins,
Baltimore, MD.
Packaging
500 g
0882-17
500 g
2 kg
10 kg
0881-17
0881-07
0881-08
advantage to Salmonella over other bacteria.1 Lactose Broth is widely

used and is included in many Standard Methods procedures for testing
foods, dairy products and other materials.1,2,3,4
Intended Use
Bacto Lactose Broth is used for cultivating Salmonella and coliform
organisms in water, foods, dairy and pharmaceutical products.
In past years, Lactose Broth was recommended for detection of

coliforms in water5 and foods6 and for the confirmed phase in testing
dairy products.7
Lactose Broth is frequently used as a pre-enrichment medium when

testing foods and dairy products for Salmonella. In dried or processed
foods, salmonellae may be sublethally injured and in low numbers.
The presence of other bacteria as well as components of the food sample
may hinder growth and recovery of Salmonella. Pre-enrichment in a
nonselective medium such as Lactose Broth allows for repair of cell
damage, dilutes toxic or inhibitory substances, and provides a nutritional
Lactose Broth contains Beef Extract and Peptone as carbon and

nitrogen sources for general growth requirements. Lactose is a
carbohydrate source.
The purpose of a pre-enrichment medium is to provide a higher ratio
of Salmonella to non-Salmonella bacteria after incubation. Most

Dehydrated Appearance: Light beige to light tan, free-flowing, homogeneous.
Solution:
1.3% solution, soluble in distilled or deionized water with
slight warming. Light to medium amber, clear without
Prepared Medium:
Light to medium amber, clear without significant precipitate.
Reaction of 1.3%
Solution at 25C:
6.9 0.2
Cultural Response
Prepare Lactose Broth (dehydrated) per label directions. Inoculate and incubate
ORGANISM
Escherichia coli
Salmonella typhi
ATCC
INOCULUM
CFU
25922* 100-1,000
6539 100-1,000
GROWTH
GAS
ACID
good
good
*This culture is available as Bactrol Disks and should be used as directed in Bactrol Disks
248
Uninoculated
tube
Escherichia coli
ATCC 25922
The Difco Manual
Section II
non-Salmonella bacteria ferment lactose while Salmonella does not.

As lactose-fermenting bacteria metabolize lactose, the pH of the
medium decreases, creating a bacteriostatic effect on competing
microorganisms.
Formula
Bacto Lactose Broth
Formula Per Liter
Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Final pH 6.9 at 25C
Precautions
Storage
Expiration Date
Procedure
Materials Provided
Lactose Broth

Autoclave
Incubator (35C)
Tubes with closures
Fermentation vials
Disinfectant solution (for prepared Lactose Broth)
Lactose Broth (dehydrated)
3. Dispense into tubes containing inverted fermentation vials.
Lactose Broth
of food samples for isolation of Salmonella. Consult standard references

for specific instructions for each type of material being tested.1,2,3,4
1. Transfer a 25 gram or 25 ml sample of test material into a container.
Add 225 ml of sterile Lactose Broth. Mix as necessary to make a
homogeneous suspension. Incubate at 35C for 24 2 hours.
2. Transfer 1 ml of suspension to appropriate enrichment broths,
such as Tetrathionate Broth and Selenite Cystine Broth. Incubate
at 35C for 24 2 hours.
3. Transfer a loopful of suspension to appropriate selective agar
media, such as Hektoen Enteric Agar, XLD Agar and Bismuth
Sulfite Agar. Incubate at 35C for 24 2 hours.
Results
Pre-enrichment, selective enrichment and selective plating increase
the likelihood of isolating Salmonella from foods and other
materials.
References
1. Flowers, R. S., J. DAoust, W. H. Andrews, and J. S. Bailey.
1992. Salmonella, p. 371-442. In C. Vanderzant and D. F.
Splittstoesser (ed.), Compendium of methods for the microbiological examination of foods, 3rd ed. American Public Health
Marshall (ed.), Standard methods for the microbiological examination of dairy products, 16th ed. American Public Health Association,
Washington, D.C.
R. M. Amaguana. 1995. Salmonella, p. 5.01-5.20. In Bacteriological analytical manual, 8th ed. AOAC International,
Gaithersburg, MD.
4. Andrews, W. H. 1995. Microbial Methods, p. 1-119. In Official
6. American Public Health Association. 1976. Compendium of
methods for the microbiological examination of foods. American
Packaging
Lactose Broth
100
500
2
10
g
g
kg
kg
0004-15
0004-17
0004-07
0004-07
Lactose Broth
10 x 90 ml
9070-73

Test Procedure
Lactose Broth is used in the pre-enrichment phase of the preparation
The Difco Manual
249
Lactose Peptone Broth
Section II
Bacto Lactose Peptone Broth
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Brom Cresol Purple . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.02 g
Intended Use
Bacto Lactose Peptone Broth is used for the detection of coliform

organisms in water.

Lactose Peptone Broth is based on the Lactose Peptone Broth formula
described in German Standard Methods and German Drinking Water
Regulations.1 Lactose Peptone Broth is recommended as a non-selective
broth enrichment and detection medium for E. coli and other coliform
bacteria present in water. Lactose fermentation and gas production at
36 1C are used as the basis for this presumptive coliform test.

Lactose Peptone Broth contains Tryptone and Soytone which provide
the carbon and nitrogen sources required for good growth of a wide
variety of organisms. Lactose is provided as a source of fermentable
carbohydrate. Sodium Chloride is present in the medium to provide
a suitable osmotic environment. Brom Cresol Purple is used as
a colorimetric indicator to show the production of acid from the
fermentation of lactose.
Formula
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 g
Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
Precautions
Storage
medium at 2-8C.
Expiration Date
Procedure
Materials Provided

Durham tubes
Autoclave
Incubator (36 1C)

the laboratory following recommended guidelines.1
2. Process each sample using procedures appropriate for that sample.1

Dehydrated Medium: Light beige, free-flowing,
homogeneous.
Solution:
10.5% solution (triple-strength),
water. Solution is dark reddish-purple,
clear to slightly opalescent.
Prepared Tubes:
Dark reddish purple, clear to slightly
opalescent.
Reaction of 10.5%
Solution at 25C:
pH 7.4 0.2
Cultural Response
Prepare Lactose Peptone Broth per label directions. Inoculate
ORGANISM
Escherichia coli
Salmonella
typhimurium
ATCC
INOCULUM
CFU
25922* 10-100
14028* 100-1,000
LACTOSE
FERMENTATION
GAS
PRODUCTION
250
1. To prepare a triple strength solution, dissolve 105 grams in 1 liter
of distilled or deionized water. To prepare a single strength
solution, dissolve 35 grams in 1 liter of distilled or deionized water.
2. Dispense 50 ml into tubes or bottles containing a Durham tube.
Test Procedure1
Direct Broth Method
1. Add 100 ml of sample to 50 ml of triple strength Lactose
Peptone Broth.
3. Examine tubes or bottles for evidence of acid formation and gas
production.
Membrane Filtration Broth Method
1. Filter 100 ml of sample through a sterile 0.45 micron membrane filter.
2. Remove filter and place in 50 ml of single strength Lactose
Peptone Broth.
4. Examine tubes or bottles for evidence of acid formation and gas
production.
The Difco Manual
Section II
Results
References
Acid formation is demonstrated by a change in the color of the

medium from reddish-purple to yellow. Gas production is demonstrated
by the displacement of the medium from the Durham tube. Production
of both acid and gas is a presumptive indication of the presence of
coliform organisms.
Subculture presumptive positives onto Endo Agar and MacConkey
Agar. Incubate at 35 2C for 24 hours. Examine plates for the
presence of typical coliform colonies. Further biochemical testing is
necessary to confirm the presence and identify coliforms. Consult
appropriate references for further information on identification
of coliforms.2,3
1. DIN Deutsches Institut fr Normung. 1991. e.V.: Deutsche

Einheitsverfahren zur Wasser-, Abwasser-und Schlammunter
suchung: Mikrobiologische Verfahren (Gruppe k), Nachwels von
Escherichia coli und coliformen Keimen (K6). Reference Method
DIN 38411.
2. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and R.
H. Yolken (ed.). 1995. Manual of clinical microbiology, 6th ed.
St. Louis, MO.
Packaging
1. Detection of coliform bacteria in Lactose Peptone Broth using this

method is only a presumptive test.
Bacto Lauryl Tryptose Broth
Also Known As
Intended Use
Bacto Lauryl Tryptose Broth is used for detecting coliform organisms
in water and wastewater.

Solution:
Prepared Medium:
opalescent.
Reaction of 3.56%
Solution at 25C:
pH 6.8 0.2
Cultural Response
Prepare Lauryl Tryptose Broth per label directions. Inoculate
tubes containing inverted fermentation vials with the test
organisms and incubate at 35 2C for 24 2 hours.
ORGANISM
Enterobacter
aerogenes
Escherichia coli
Salmonella
typhimurium
Staphylococcus
aureus
ATCC
INOCULUM
CFU
GROWTH
13048*
30-100
good
25922*
14028*
30-100
30-100
good
good
GAS
PRODUCTION
25923* 300-1,000 markedly to

weak +
or
+
Gas production within 48 3 hours
The Difco Manual
500 g
0665-17
Lauryl Tryptose Broth is also referred to as Lauryl Sulfate Broth and is

abbreviated as LTB or LSB.

The coliform group comprises aerobic and facultatively anaerobic,
gram negative, nonspore-forming rods able to ferment lactose with
the production of acid and gas at 35C within 48 hours.1 Typically
these organisms are classified in the genera Escherichia, Enterobacter
and Klebsiella. Coliforms are often used as indicators of fecal
contamination in water, and bacterial contamination in food and
food products.
Lauryl Tryptose Broth is prepared according to the formula of
Mallmann and Darby.2 These researchers demonstrated the value of
Tryptose in the detection of coliform organisms.3 In a 2% concentration
of Tryptose, the growth rate during the early logarithmic growth
phase was increased when compared with Bacto Peptone.3 Mallmann
and Darby3 added phosphate buffers and sodium chloride. This
buffered tryptose lactose broth permitted slow lactose fermenters to
increase gas production in a shorter time frame. To improve the
methods used to isolate coliforms from water, Mallmann and Darby2
investigated many selective agents. Sodium lauryl sulfate gave the best
results for inhibition of organisms other than coliforms.2
In a comparative study of EC Medium and Lauryl Tryptose Broth,
Perry and Hajna4 reported both media to be highly sensitive and
specific for recovery of coliform bacteria from water, shellfish and
sewage. A positive presumptive test with either medium was more
dependable than the confirmed or completed test.
LTB is used in the presumptive phase of the Standard Total Coliform
Fermentation Technique in the examination of water. 5 Lauryl
Tryptose Broth is used in standard methods for coliform detection in
water and foods.1,5,6,7,8,9

Tryptose provides nitrogen, vitamins, minerals and amino acids in
Lauryl Tryptose Broth. Lactose is the fermentable carbohydrate for
251
Section II
coliforms. Potassium Phosphates are the buffering agents, and

Sodium Chloride is used to maintain the osmotic balance of the
medium. Sodium Lauryl Sulfate is the selective agent used to inhibit
organisms other than coliforms.
Formula
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Potassium Phosphate, Dibasic . . . . . . . . . . . . . . . . . . . . . 2.75
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Lauryl Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
g
g
g
g
g
g
Precautions
PREPARATION OF LAURYL TRYPTOSE BROTH5
Inoculum
mL
Dehydrated Lauryl
Amount of
Volume of
Tryptose Broth
Medium in Tube Medium+Inoculum Required
mL
mL
g/L
1
10
10
20
100
100
100
10 or more
10
20
10
50
35
20
Collect and process specimens according to laboratory policy or

standard methods.1,5,6,7,8,9
Test Procedure
Storage
Results
NOTE: Refrigerated Lauryl Tryptose Broth generally becomes cloudy

or forms precipitates. Incubate medium overnight at room temperature
(20C) before use to clear the medium.5
Expiration Date
35.6
71.2
53.4
106.8
106.8
137.1
213.6

11 or more
20
30
30
150
135
120
Follow the methods and procedures for the detection of coliform

organisms as described in standard methods.1,5,6,7,8,9
After incubation of the tubes at 35 2C for 24 hours, examine for
turbidity and gas production. If no gas has formed in the inverted
tube, reincubate and reexamine after 48 hours.5,6
Turbidity of the medium accompanied by formation of gas within
48 hours is a positive presumptive test for the presence of
coliforms.5,6 The result should be confirmed by additional standard
testing.1,5,6,7,8,9
Procedure

this medium.
Materials Provided
References
1. Christen, G. L., P. M. Davidson, J. S. McAllister, and L. A. Roth.

1992. Coliform and other indicator bacteria, p. 247-267. In R. T.
Marshal (ed.). Standard methods for the examination of dairy
products. 16th ed. American Public Health Association,
Washington, D.C.
2. Mallmann, W. L., and C. W. Darby. 1941. Uses of a lauryl
sulphate tryptose broth for the detection of coliform organisms.
Am. J. Public Health. 31:127.
3. Darby, C. W., and W. L. Mallmann. 1939. J. of Am. Water Works
Assoc. 31:689.
4. Perry and Hajna. 1944. Am. J. Public Health. 34:735.
5. Eaton, A. D., L. S. Clesceri, and A. E. Greenberg (ed.). Standard
methods for the examination of water and wastewater, 19th ed.

Glassware
Test tubes
Incubator (35C)
Fermentation vials
Autoclave
1.
2.
3.
4.

NOTE: Lauryl Tryptose Broth may be prepared in single strength
when examining 1 ml or less of water as an inoculum. For inocula
of 10 ml consult the table below.
252
The Difco Manual
Section II
Leptospira Medium EMJH

9. Bordner, R., and J. Winter (ed.). 1978. Microbiological methods
for monitoring the environment; water and wastes. Environmental
monitoring and support laboratory, U.S. Environmental Protection
Agency, Cincinnati, OH.
Packaging
100
500
2
10
g
g
kg
kg
0241-15
0241-17
0241-07
0241-08

Bacto Leptospira Medium Base EMJH . Bacto Leptospira
Enrichment EMJH
Intended Use
Bacto Leptospira Medium Base EMJH is used with Bacto Leptospira
Enrichment EMJH in cultivating Leptospira.

In 1816, Adolf Weil described the first recognized leptospiral
infections in humans. 1 These cases were caused by Leptospira
icterohaemorrhagiae and the disease was subsequently named Weils
Disease.1 Leptospirosis is a zoonotic disease, having its reservoir in
wild, domestic, and peridomestic animals. Infection usually results
from direct or indirect exposure to the urine of leptospiruric animals.2
Indirect exposure through contaminated water and soil accounts for

most sporadic cases. Direct exposure occurs in pet owners, veterinarians
and persons working with livestock.3
The basal medium and enrichment are prepared according to the
formulations described by Ellinghausen and McCullough4 as modified
by Johnson and Harris.5 They modified the formula by replacing rabbit
serum medium with Tween 80-albumin. Leptospira Medium EMJH
was used in cultivation studies of Leptospira.6
Leptospira Medium EMJH is recommended for the clinical isolation
of Leptospira.7,8

Leptospira Medium Base EMJH
Basal Solution:
2.3 grams of base in 900 ml distilled or deionized water;
soluble upon agitation; colorless, clear with no significant
precipitate.
Prepared Medium
w/Enrichment:
Very light to light amber, clear with no precipitate.
Reaction (Basal Medium)
at 25C:
pH 7.5 0.2
Leptospira Enrichment EMJH
Appearance:
Medium to dark amber, clear to very slightly opalescent
with no significant precipitate.
Cultural Response
Prepare the complete Leptospira Medium EMJH per label directions. Inoculate
tubes with undiluted Leptospira and incubate at 30 2C for up to 7 days.
ORGANISM
ATCC
INOCULUM CFU
GROWTH
Leptospira interrogans serovar australis

Leptospira interrogans serovar canicola
Leptospira interrogans serovar grippotyphosa
23605
23470
23604
one drop
one drop
one drop
good
good
good
Uninoculated
tube
ATCC 23605
The Difco Manual
253
Section II
Leptospira Medium Base EMJH contains ammonium chloride, a

nitrogen source, and thiamine, a growth factor. Sodium phosphate
dibasic and potassium phosphate monobasic are buffering agents.
Sodium chloride maintains the osmotic balance of this formula.
1. Dissolve 2.3 grams Leptospira Medium EMJH in 900 ml distilled

or deionized water.
3. Aseptically add 100 ml Leptospira Enrichment EMJH to the basal
medium at room temperature. Mix thoroughly.
Leptospira Enrichment EMJH contains albumin, polysorbate 80 and

additional growth factors for Leptospira.
Formula
Formula Per Liter
Sodium Phosphate Dibasic . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Potassium Phosphate Monobasic . . . . . . . . . . . . . . . . . . . . 0.3
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Ammonium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.25
Thiamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.005
g
g
g
g
g

A solution of albumin, polysorbate 80 and additional growth
factors for Leptospira.
Precautions
2. Leptospira Medium Base EMJH:
Storage
Store Leptospira Medium Base EMJH below 30C. The dehydrated
medium is very hygroscopic. Keep container tightly closed.
Store Leptospira Enrichment EMJH at 2-8%C.
Expiration Date
Procedure
Materials Provided
Leptospira Medium Base EMJH or

Glassware
Autoclave
Incubator (30C) (optional)
Sterile tubes
254

procedures established by laboratory policy. Blood, cerebrospinal fluid
(CSF) and urine are the specimens of choice for the recovery of
leptospires from patients with leptospirosis.5
Test Procedure
Culture Procedures7
Blood and Spinal Fluid
Freshly drawn blood is preferable; otherwise, use blood taken with
SPS, sodium oxalate or heparin.
1. Inoculate four 5 ml tubes of Leptospira Medium EMJH with
1-2 drops of fluid per tube.
2. Incubate in the dark at 28-30%C or at room temperature.
Urine
A total of 12 tubes will be inoculated for each urine specimen.
1. Prepare 1:10 and 1:100 dilutions of urine using Leptospira
Medium EMJH to dilute potential inhibitory substances.
2. Inoculate two 5 ml tubes each of Leptospira Medium EMJH with:
Urine undiluted, 1 drop per tube;
Urine diluted 1:10, 1 drop per tube;
Urine diluted 1:100, 1 drop per tube.
3. Duplicate the above inoculations using medium containing
200 g/ml 5- fluorouracil to inhibit contaminants.
4. Incubate the tubes in the dark at 28-30C or at room temperature.
Results7
1. Examine tubes weekly for signs of growth (turbidity, haze, or a
ring of growth).
2. Examine tubes microscopically each week. Take a small drop from
a few millimeters below the surface, and examine it with dark-field
illumination. Use 400X magnification.
3. Leptospires will be seen as tightly coiled spirochetes about 1 m
wide and 6-20 m long. Leptospires rotate rapidly on their long
axes and usually have hooked ends.
4. If the specimen is positive, subculture about 0.5 ml taken from the
area of growth to two tubes of fresh medium.

this medium.
References
1. Elliott, S. H. 1980. Discussion and clinical diagnosis of
Leptospirosis. J. Am. Med. Tech. 42:37-44.
The Difco Manual
Section II
Letheen Agar & Letheen Broth
2. Faine, S. (ed.). 1982. Guidelines for the control of leptospirosis. W.

H. O. Offset publication no. 67. World Health Organization, Geneva.
3. Kaufmann, A. F., and R. S. Weyant. 1995. Leptospiraceae,
p.621-625. In Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover
and R. H. Yolken (ed.). Manual of clinical microbiology, 6th ed.
4. Ellinghausen, Jr., H. C., and W. G. McCullough. 1965.
Nutrition of Leptospira pomona and growth of 13 other
serotypes: fractionation of oleic albumin complex (OAC) and a
medium of bovine albumin and polysorbate 80. Am. J. Vet.
Research 26:45-51.
5. Johnson, R., and V. G. Harris. 1967. Differentiation of pathogenic
of leptospires. J. Bacteriol. 94:27-31.
Bacto Letheen Agar

Bacto Letheen Broth
6. Rule, P. L., and A. D. Alexander. 1986. Gellan gum as a substitute

for agar in leptospiral media. J. Clin. Microbiol. 23:500-504.
Packaging
500 g
6 x 100 ml
0794-17
0795-73*
*Store at 2-8C
Intended Use
Bacto Letheen Agar is used for evaluating the bactericidal activity of
quaternary ammonium compounds.
Bacto Letheen Broth is used for determining the phenol coefficient of
cationic surface-active materials.
Also Known As
AOAC Letheen Agar/Broth and Trypticase Glucose Extract Agar
with Lecithin and Tween 80 are common terms for Letheen
Agar/Broth.

The value of a highly nutritional solid medium containing neutralizing
agents for quaternary ammonium compounds in sanitizers was
described by Weber and Black1 in 1948. The addition of lecithin
and Tween 80 to Tryptone Glucose Extract (TGE) agar resulted in
a medium that effectively neutralizes quaternary ammonium
compounds in the testing of germicidal activity. Letheen Agar is a
modification of TGE agar with the addition of lecithin and sorbitan
monooleate (Tween 80).
Letheen Broth was developed as a subculture medium for the
neutralization of quaternary ammonium compounds in disinfectant
testing. Quisno, Gibby and Foter,2 found that the addition of lecithin
and Tween 80 to F.D.A. Broth resulted in a medium that neutralized
high concentrations of quaternary ammonium salts. The resulting
medium, termed Letheen (a combination of Lecithin and Tween)
was easy to prepare and clear in appearance which aided in visual
inspection for growth. Letheen Broth is recommended by the
Official Methods of Analysis of the Association of Official Analytical
Chemists (AOAC)3 for use with disinfectants containing cationic
surface active materials.
Letheen Agar and Letheen Broth are specified for use by the American
Society for Testing Materials (ASTM) in the Standard Test Method for
Preservatives in Water Containing Cosmetics.4
The Difco Manual

Letheen Agar contains Beef Extract and Tryptone which provide the
carbon and nitrogen sources required for growth of a wide variety of
organisms. Dextrose is provided as a source of fermentable carbohydrate.
Bacto Agar is added as a solidifying agent. Lecithin and Sorbitan
Monooleate are added to neutralize surface disinfectants.2,5,6 Lecithin
is added to neutralize quaternary ammonium compounds and Sorbitan
Monooleate is incorporated to neutralize phenols, hexachlorophene,
formalin and, with lecithin, ethanol.7
Letheen Broth contains Peptamin and Beef Extract which provide the
carbon and nitrogen sources necessary for growth. Lecithin and Tween 80
are added as surface active disinfectant neutralizing agents. 2,5,6
Sodium Chloride is included to maintain osmotic balance.
Formula
Letheen Agar
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Sorbitan Monooleate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Lecithin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
g
g
g
g
g
g

Letheen Broth
Formula Per Liter
Bacto Peptamin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Lecithin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.7
Tween 80 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
g
g
g
g
g
Precautions
255
Letheen Agar & Letheen Broth
Section II
Storage
Procedure
Materials Provided
Expiration Date
Letheen Agar
Letheen Broth

Glassware
Distilled or deionized water|
Autoclave

Letheen Agar
Dehydrated Appearance: Tan, moist appearance, with a
tendency to clump.
Solution:
is light to medium amber, clear to
slight, fine precipitate.
Prepared Plates:
precipitate.
Reaction of 3.2%
Solution at 25C:
pH 7.0 0.2
Letheen Broth
Dehydrated Appearance: Tan, moist appearance, with a
tendency to clump.
Solution:
clear to slightly opalescent
(opalescent when hot), May have a
Prepared Tubes:
slight precipitate.
Reaction of 2.57%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepare Letheen Agar per label directions. Using the pour plate
technique, inoculate and incubate at 35 2C for 40-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Escherichia coli
11229
6538
100-1,000
100-1,000
good
good
Prepare Letheen Broth per label directions. Inoculate and

incubate at 35 2C for 40- 48 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Escherichia coli
Salmonella typhi
11229
6538
6539
100-1,000
100-1,000
100-1,000
good
good
good
256
1. Letheen Agar: Suspend 32 grams in 1 liter distilled or deionized

water. Boil to dissolve completely.
Letheen Broth: Suspend 25.7 grams in 1 liter distilled or deionized
water. Boil to dissolve completely.

Test Procedures
Letheen Agar and Letheen Broth are used in a variety of procedures.
Please consult appropriate references for further information.3,4
Results

1. The dehydrated Letheen Agar has a characteristic brown sugar
appearance. This does not indicate deterioration.
References
1. Weber, G. R., and L. A. Black. 1948. Relative efficiency of
quaternary inhibitors. Soap and Sanit. Chem. 24:134-139.
2. Quisno, R., I. W. Gibby, and M. J. Foter. 1946. A neutralizing
medium for evaluating the germicidal potency of the quaternary
ammonium salts. Am. J. Pharm. 118:320-323.
methods of analysis, 16th ed. Association of Official Analytical
Chemists, Washington, D.C.
4. American Society for Testing Materials. 1991. Standard test
method for preservatives in water-containing cosmetics, E 640-78.
Annual Book of ASTM Standards, Philadelphia, PA.
5. Erlandson, A. L., Jr., and C. A. Lawrence. 1953. Inactivating
medium for hexachlorophene (G-11) types of compounds and some
substituted phenolic disinfectants. Science 118:274-276.
6. Brummer, B. 1976. Influence of possible disinfectant transfer on
Staphylococcus aureus plate counts after contact sampling. App.
7. Favero (chm.). 1967. Microbiological sampling of surfaces-a state
of the art report. Biological Contamination Control Committee,
American Association for Contamination Control.
Packaging
Letheen Agar
500 g
0680-17
Letheen Broth
500 g
0681-17
The Difco Manual
Section II
Levine EMB Agar
Bacto Levine EMB Agar
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Eosin Y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4
Methylene Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.065
Intended Use
Bacto Levine EMB Agar is used for isolating and differentiating
lactose-fermenting from lactose-nonfermenting gram-negative
enteric bacilli.

Eosin methylene blue agar (EMB) was originally formulated by
Holt-Harris and Teague.1 The formulation contained eosin and methylene
blue as inhibitors and pH indicators, and the carbohydrates lactose and
sucrose. Levine2,3 modified the formulation by using lactose at an
increased concentration and omitting sucrose.
Levine EMB agar is used for detection and confirmation of coliforms,
specifically enteropathogenic E. coli in foods, dairy products and
other materials.4,5,6,7

Levine EMB Agar contains Bacto Peptone as a source of carbon
and nitrogen for general growth requirements and Lactose as the
carbohydrate. Eosin Y and Methylene Blue are pH indicators, as well
as inhibitors of microorganisms other than gram-negative bacilli.
However, some staphylococci, streptococci and yeast may grow as
small pinpoint colonies. Bacto Agar is the solidifying agent.
Formula
Levine EMB Agar
Formula Per Liter
g
g
g
g
Precautions
Storage
Expiration Date
Procedure
Materials Provided
Levine EMB Agar

Flask with closure
Autoclave
Incubator (35C)
Petri dishes
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Escherichia coli
ATCC 25922
Uninoculated
plate

Dehydrated Appearance: Reddish-pink free-flowing, homogeneous.
Solution:
deionized water on boiling; green to
wine red color with an orange cast,
slightly opalescent to opalescent.
May have a flocculent precipitate.
Prepared medium:
Wine red color with a green to orange
cast, slightly opalescent with a finely
dispersed flocculent precipitate.
Reaction of 3.75%
solution at 25C:
pH 7.1 0.2
Cultural Response
Prepare Levine EMB Agar per label directions. Inoculate medium
ORGANISM
Escherichia coli
ATCC
INOCULUM
CFU
25922* 100-1,000
GROWTH
COLONY COLOR
good
blue-black colonies, green

metallic sheen dark centers
good
colorless to amber
29212* 1,000-2,000 partially
colorless inhibited
ATCC 14028
The Difco Manual
257
Lima Bean Agar
1.
2.
3.
4.

Section II
2. The medium will support growth of most gram-negative bacilli but

some strains of Salmonella and Shigella may be inhibited.
References
E. coli colonies are typically dark-centered with or without a

metallic sheen.
Lactose fermenters:
Blue-black to brownish colored
colonies, may have dark centers
with or without metallic sheen.
Lactose non-fermenters:
Colorless or transparent, amber to
light-purple colonies.
Staphylococci and streptococci: Colorless pinpoint colonies.
Yeasts:
Colorless, dull pinpoint colonies;
may be spidery at edges.

for the isolation of Bacillus typhosa from stools. J. Infect. Dis. 18:596.
2. Levine, M. 1918. Differentiation of E. coli and A. aerogenes on a
simplified eosin-methylene blue agar. J. Infect. Dis. 23:43-47.
3. Levine, M. 1921. Bacteria fermenting lactose-the significance in
water analysis. Bull. 62. Iowa State College Eng. Exp. Sta., Ames, Iowa.
6. Hitchins, A. D., P. Feng, W. D. Watkins, S. R. Rippey, and
L. A. Chandler. 1995. Escherichia coli and the coliform bacteria.
7. Andrews, W. 1995. Microbial Methods, p. 1-119. In Official
Packaging
1. Levine EMB Agar is only moderately inhibitory. Some staphylococci,

streptococci, and yeasts may grow as small pinpoint colonies.
Perform microscopic examination and biochemical tests to
identify to genus and species.
Levine EMB Agar

material being tested.4,5,6,7
Test Procedure
Results
Bacto Lima Bean Agar
100
500
2
10
g
g
kg
kg
0005-15
0005-17
0005-07
0005-08
Intended Use
Bacto Lima Bean Agar is used for cultivating fungi.

Fungi are ubiquitous in nature.1 Of the estimated 250,000 species, fewer
than 150 are known to be primary human pathogens.1
Lima Bean Agar is prepared from an infusion of dry lima beans and is
solidified with 1.5% agar. The nutritive properties of lima beans and
the low pH of Lima Bean Agar create a suitable environment for the
growth of many fungi.

Infusion from Lima Beans is a source of nitrogen, carbon, amino acids
and vitamins. Bacto Agar is a solidifying agent.

Dehydrated Appearance: Light yellowish tan, free-flowing,
homogeneous.
Solution:
is light to medium amber, opalescent,
Prepared Media:
Light to medium amber, opalescent,
Reaction of 2.3%
Solution at 25C:
pH 5.6 0.2
258
Cultural Response
Prepare Lima Bean Agar per label directions. Inoculate and
incubate at 30 2 C for 40-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Aspergillus niger
Candida albicans
16404
9763
10231
100-1,000
100-1,000
100-1,000
good
good
good
The Difco Manual
Section II
Listeria Enrichment Broth
Formula
Lima Bean Agar
Formula Per Liter
Lima Bean, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . 62.5 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
Precautions
Storage
Expiration Date
Procedure
Materials Provided
Lima Bean Agar

Waterbath
1.
2.
3.
4.

Autoclave at 121 C for 15 minutes.

Test Procedure
Results
Refer to appropriate references and procedures.
References
and classification of the fungi, p. 699-708. In P. R. Murray, E. J.
Washington, D.C.
Packaging
Glassware
Autoclave
Lima Bean Agar
500 g
0117-17
Bacto Listeria Enrichment Broth
Intended Use
Bacto Listeria Enrichment Broth is used to selectively enrich Listeria
from food.

industries. This organism can cause human illness and death, particularly
in immunocompromised individuals and pregnant women.2 The first
reported food-borne outbreak of listeriosis was in 1985,3 and since then,
microbiological and epidemiological evidence from both sporadic and
epidemic cases of listeriosis has shown that the principal route of
transmission is via the consumption of foodstuffs contaminated with
Listeria monocytogenes.4
coleslaw, pasteurized milk, Mexican-style cheese, pat, and pickled
and other food processing plants, and is ubiquitous in nature, being
The Difco Manual

microaerophilic, gram-positive, asporogenous, non-encapsulated,
non-branching, regular, short, motile rods. Motility is most pronounced
at 20C.
potentially containing Listeria are: streptococci, especially the enterococci,
micrococci and Bacillus species, Escherichia coli, Pseudomonas
aeruginosa and Proteus vulgaris.8
Listeria Enrichment Broth is based on the formula developed by Lovett
et al.9 in which Tryptic Soy Broth is supplemented with Yeast Extract
for optimum growth of Listeria.

Tryptone, Soytone and Yeast Extract provide nitrogen, vitamins and
minerals. Dextrose is a carbohydrate source. Sodium Chloride
maintains the osmotic balance of the medium. Phosphate acts as a
buffer. Acriflavine HCl and Nalidixic Acid are added for selectivity
and Cycloheximide is used to inhibit growth of saprophytic fungi.
259
Section II
Formula
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Potassium Phosphate, Dibasic . . . . . . . . . . . . . . . . . . . . . . 2.5
Cycloheximide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05
Acriflavine HCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.015
Nalidixic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.04
g
g
g
g
g
g
g
g
g
Precautions
2. HARMFUL. POSSIBLE RISK OF IRREVERSIBLE EFFECTS.
POSSIBLE RISK OF HARM TO THE UNBORN CHILD. (US)
HARMFUL IF SWALLOWED. (EC) Avoid contact with skin and
container tightly closed. TARGET ORGAN(S): Cardiovascular,
Liver, Lungs, Nerves.

Dehydrated Appearance: Light beige, free flowing,
homogeneous.
Solution:
Solution is light to medium
yellowish amber with a faint green
ring at the surface, clear to very
Prepared Medium:
Light yellowish amber, clear to very
Reaction of 3.61%
Solution at 25C:
pH 7.3 0.2
Cultural Response
Prepare Listeria Enrichment Broth per label directions.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
29212* 2,000-10,000
suppressed at
18- 24 hours
Escherichia coli
25922* 2,000-10,000
marked to
complete inhibition
Listeria monocytogenes 19114
100-1000
good
Saccharomyces
25923 2,000-10,000
marked to
pastorianus
complete inhibition
260

Storage
medium at 2-8C.
Expiration Date
Procedure
Materials Provided

Autoclave
Incubator (30C)
1. Suspend 36.1 grams in l liter of distilled or deionized water.

2. Process each food sample, using procedures appropriate for that
sample.
Test Procedure
For food samples, use recommended laboratory procedures for isolating
Listeria.
Results
References
3. Wehr, H. M. 1987. Listeria monocytogenes - a current dilemma.
The Difco Manual
Section II
Litmus Milk

7. Conner, D. E., R. E. Brackett, and L. R. Beuchat. 1986.
Effect of temperature, sodium chloride, and pH on growth of Listeria
monocytogenes in cabbage juice. Appl. Environ. Microbiol. 52:59.
Bacto Litmus Milk

9. Lovett, J., D. W. Frances, and J. M. Hunt. 1987. Listeria
monocytogenes in raw milk: detection, incidence and pathogenicity.
J. Food Prot. 50:188-192.
10. McBride, M. E., and K. F. Girard. 1960. A selective method for
the isolation of Listeria monocytogenes from mixed bacterial
populations. J. Lab. Clin. Med. 55:153-157.
Packaging
500 g
10 kg
0222-17
0222-08
the genus Clostridium.1 This medium is also of value in the maintenance

and propagation of lactic acid bacteria. The reactions of litmus milk
are a valuable criterion in identification.1
Intended Use
Bacto Litmus Milk is used for differentiating microorganisms based
on acid production and coagulation or proteolysis of casein.

Litmus Milk has been used for many years to detect the metabolic
activities of microorganisms in milk and to aid in the identification of
bacterial species. It is especially useful in species differentiation within

Skim milk is a source of nutrients. The addition of a litmus indicator
to milk expands its usefulness as a differential medium. Litmus
incorporated in milk is both a pH indicator and an oxidationreduction indicator.

Dehydrated Appearance: Grayish-purple, free-flowing, homogenous.
Solution:
deionized. Solution is light purple
and opaque.
Prepared tubes:
Light purple and opaque
Reaction of 10%
Solution at 25C:
pH 6.8 0.2.
Cultural Response
Prepare Litmus Milk per label directions. Inoculate and incubate
at 35 2C for up to 7 days.
ORGANISM
ATCC
INOCULUM CFU
REACTION
IN LITMUS MILK
Bacillus subtilis
Lactobacillus casei
6633
12924
7469
1,000-2,000
1,000-2,000
1,000-2,000
K, D
R, C
A, R, C
A = Acid reactions:
K = Alkaline reactions:
R = Reduction:
C = Clot or Curd:
D = Digestion:
The Difco Manual
1.
2.
1.
2.
3.
1.
2.
3.
1.
2.
Pinkish-red medium.
Uninoculated
Bacillus subtilis Clostridium perfringens Lactobacillus casei
Due to fermentation of the carbohydrates lactose and glucose.
tube
ATCC 6633
ATCC 12924
ATCC 7469
Blue medium.
No fermentation of carbohydrates.
Organism attacks nitrogenous substances present in medium. The breakdown of lactalbumin by proteolytic enzymes form ammonia or basic amines.
White medium.
The enzyme reductase removes oxygen from the litmus, resulting in a decolorized, milky white appearance.
Reduction usually begins at the bottom of the tube.
Milk protein coagulation.
Coagulation due to either a precipitation of casein by acid formation or the conversion of casein to paracasein by the enzyme rennin resulting in a clear
watery fluid called whey.
1. Milk protein digested.
2. Clearing of medium and dissolution of clot by digestion of casein.
261
Litman Oxgall Agar
Section II
Formula
Litmus Milk
Formula Per Liter

Bacto Skim Milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 g

Bacto Litmus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.75 g
Precautions
Test Procedure
Results
Storage
Expiration Date
Procedure
Materials Provided
Litmus Milk
1. Avoid overheating during sterilization, as the milk sugar will

carmelize, resulting in discoloration giving an appearance atypical
of the sterile medium.
2. During the sterilization period, Litmus Milk is reduced to a white
colored base, however, upon cooling, the original color returns as
oxygen is absorbed.
3. Reactions observed in Litmus Milk are not sufficient to speciate.
Additional biochemical tests must be performed.1
References
1. MacFaddin, J. D. 1985. Media for isolation-cultivationidentification-maintenance medical bacteria, vol 1, p.275-284,
Packaging
Glassware
Autoclave
Litmus Milk
Bacto Littman Oxgall Agar
Intended Use
Bacto Littman Oxgall Agar is used for isolating and cultivating fungi,
especially dermatophytes.

In 1947, Littman1 described Littman Oxgall Agar which is neutral in
reaction and suitable for growth of pathogenic fungi. It is a selective
medium for the primary isolation of fungi.
Littman demonstrated that Littman Oxgall Agar is valuable for
culturing the dermatophytes. Molds and yeasts form nonspreading,
discrete colonies, that are easy to isolate in pure culture. He also
suggested that the medium be used for estimating the normal fungal
flora of feces, sputum and other human discharges. The medium
can also be used for single cell isolation of fungi and plate counts of
viable saprophytic fungi in foodstuffs and air.
In a comparative study, Littman 2 compared this medium with
Sabouraud Dextrose Agar using a large variety of pathogenic and
saprophytic fungi. He reported the isolation of three times as many
fungi from feces, sputum, skin scrapings and hair on Littman
Oxgall Agar and four times as many pathogenic dermatophytes on
the selective medium compared with Sabouraud Dextrose Agar.
262
500 g
0107-17

Bacto Peptone provides nitrogen, amino acids and carbon. Dextrose
is an additional carbon source. Oxgall restricts the spreading of fungus
colonies. Crystal Violet and streptomycin are selective bacteriostatic
agents. Bacto Agar is a solidifying agent.
Formula
Littman Oxgall Agar
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Oxgall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Crystal Violet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01
g
g
g
g
g
Precautions
AND SKIN. Avoid contact with skin and eyes. Do not breath dust.
The Difco Manual
Section II
Littman Oxgall Agar

Storage
Results
Test Procedure

Expiration Date
1. Although culture techniques are primary in the identification of

etiological agents of mycotic infections, they are not absolute.
Often identification must be accomplished by using one or more
of the following techniques to corroborate cultural findings: direct
microscopic examination of the specimen; animal inoculation;
biochemical determination; or serological procedures.
2. Do not use to culture Nocardia asteroides, Streptomyces, or any
other microorganism sensitive to streptomycin.3
Procedure
Materials Provided
Littman Oxgall Agar
References
1. Littman, M. L. 1947. Culture medium for primary isolation of

fungi. Science 106:109-111.
2. Littman, M. L. 1948. Growth of pathogenic fungi on a culture
medium. Am. J. Clin. Pathol. 18:409-420.
Glassware
Autoclave
Streptomycin
1.
2.
3.
4.
5.

Cool to 46C.
Add 30 mcg streptomycin per ml of medium.
Packaging
Littman Oxgall Agar
500 g
Uninoculated
plate
0294-17
ATCC 9763

Dehydrated Medium
Appearance:
Solution:
Reaction of 5.5%
Solution at 25C:
Grayish-blue, free-flowing, homogeneous.

blue, very slightly to slightly opalescent.
pH 7.0 0.2
Cultural Response
Prepare Littman Oxgall Agar per label directions with and
without the addition of streptomycin. Inoculate and incubate
plates at 25-30C for up to 72 hours.
ORGANISM
Candida albicans
Escherichia coli
Saccharomyces pastorianus
ATCC
10231
25922*
9763
9080
28185
INOCULUM
CFU
GROWTH
PLAIN
100-1,000
good
100-1,000
good
100-1,000
good
100-1,000
good
100-1,000 fair to good
GROWTH
w/STREPTOMYCIN
good
inhibited
good
good
fair to good
ATCC 28185
The Difco Manual
263
Liver Infusion Agar & Liver Infusion Broth
Section II
Bacto Liver Infusion Agar

Bacto Liver Infusion Broth
Intended Use
Bacto Liver Infusion Agar is used for cultivating Brucella and other
pathogenic organisms.
Bacto Liver Infusion Broth is used for cultivating a variety of
organisms, particularly Brucella and anaerobes.

Brucellosis is a zoonotic disease with a domestic animal reservoir.
Transmission by milk, milk products, meat and direct contact with
infected animals is the usual route of exposure.1
Most strains of Brucella will grow on chocolate or blood agar.
However, special media such as liver infusion, tryptose, tryptone or
brucella agar are preferred.2 The nutritive factors of Liver Infusion
media permit luxuriant growth of Brucella and other fastidious pathogens.

Infusion from Beef Liver and Proteose Peptone provide the nitrogen, amino
acids, vitamins and carbon sources in Liver Infusion media. Sodium
chloride maintains the osmotic balance. Bacto Agar is a solidifying agent.
Formula
Liver Infusion Agar
Formula Per Liter
Beef Liver, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . . 500
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
g
g
g
g
Liver Infusion Agar
Dehydrated Appearance: Dark beige to light tan, free-flowing,
homogeneous.
Solution:
5.5% solution, in distilled or
deionized water on boiling, medium
to dark amber, slightly opalescent to
opalescent.
Prepared Medium:
Medium to dark amber, slightly
opalescent.
Reaction of 5.5%
Solution at 25C
pH 6.9 0.2
Liver Infusion Broth
Solution:
or deionized water, medium to dark
opalescent with a few particles.
Prepared Medium:
Medium to dark amber, clear to very
slightly opalescent with a few particles.
Reaction of 3.5%
Solution at 25C
pH 6.9 0.2
Cultural Response
Prepare Liver Infusion Agar and Liver Infusion Broth per label
directions. Inoculate prepared medium and incubate under
5-10% CO2 at 35 2C for 18-48 hours, or up to 72 hours if
necessary. Incubate Clostridium under anaerobic conditions.
ORGANISM
ATCC
INOCULUM
CFU
Brucella abortus
Brucella melitensis
Brucella suis
4315
4309
4314
11437
100-1,000
100-1,000
100-1,000
100-1,000
GROWTH
good
good
good
good
264
For isolating Brucella strains from contaminated milk, crystal violet

(gentian violet) can be added to Liver Infusion Agar to suppress
gram-positive organisms.3 Five percent (5%) heated horse or rabbit
serum enhances growth of Brucella.4
Liver Infusion Agar at approximately half strength may be used to
prepare Endamoeba medium for cultivating Endamoeba histolytica.5
Liver Infusion Broth maintains a degree of anaerobiosis well
suited to support growth of anaerobic microorganisms, especially
Clostridium species.

Formula Per Liter
Beef Liver, infusion from . . . . . . . . . . . . . . . . . . . . . . . . . 500 g
Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Precautions
2. Brucella species are classified as Biosafety Level 3 pathogens. All
manipulations with live cultures and antigens must be confined to
a Class II biological safety cabinet (BSC).2
Storage
very hygroscopic. Keep containers tightly closed.
Expiration Date
stored as directed. Do not use if it fails to meet specifications for
Procedure
Materials Provided
Liver Infusion Agar

Glassware
Autoclave
Incubator (35C)
The Difco Manual
Section II
Liver Veal Agar
References
1. Liver Infusion Agar: Suspend 55 grams in 1 liter distilled or

deionized water and boil to dissolve completely.
Liver Infusion Broth: Dissolve 35 grams in 1 liter distilled or
deionized water.
3. Dispense Liver Infusion Agar into sterile Petri dishes or as desired.
Dispense Liver Infusion Broth into sterile tubes or as desired.

2. Carter, G. R. 1979. Diagnostic procedures in veterinary bacteriology
and mycology, 3rd ed. Charles C. Thomas, Springfield, IL.
3. MacFaddin, J. F. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, p. 802-806, vol. 1.
4. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey & Scotts
diagnostic microbiology, 9th ed. Mosby-Year Book, Inc., St. Louis, MO.
5. Cleveland, L. R., and E. P. Sanders. 1930. Encystation, multiple
fission without encystment, encystation, metacystic development,
and variation in a pure line and nine strains of Entamoeba
histolytica. Arch. Protietenkd. 70:223.

Test Procedure
Brucella, anaerobic microorganisms and other fastidious pathogens,
refer to the procedures described in Bailey & Scotts Diagnostic
Microbiology,4 Clinical Microbiology Procedures Handbook6 and
Manual of Clinical Microbiology.7
Results
Packaging

Liver Infusion Agar

Bacto Liver Veal Agar
Intended Use
500 g
500 g
10 kg
0052-17
0269-17
0269-08
Bacto Liver Veal Agar is used for cultivating anaerobic bacteria.
Spray1 described a procedure using the anaerobic culture dish for the
cultivation of these organisms. Liver Veal Agar is identical to the
medium described by Spray.2 Liver Veal Agar provides a rich supply of
nutrients for anaerobic and fastidious aerobic pathogens. The medium
supports excellent growth of sporulating anaerobes and can be used
for deep tube cultures.

Solution:
deionized water upon boiling;
medium to dark amber, opalescent,
Prepared Medium:
Medium to dark amber, opalescent;
Reaction of 9.7%
Solution at 25C:
pH 7.3 0.2
Cultural Response
Prepare Liver Veal Agar per label directions. Inoculate medium
and incubate at 35 2C under appropriate atmospheric
conditions. Incubate clostridia anaerobically, Neisseria under
increased CO2, and streptococci aerobically.
ORGANISM
Clostridium botulinum
Clostridium tetani
ATCC
INOCULUM
CFU
GROWTH
25763
10779
13090*
6305
100-1,000
100-1,000
100-1,000
100-1,000
good
good
good
good
The Difco Manual
Liver Veal Agar is specified in the FDA Bacteriological Analytical

Manual (BAM)3 and Compendium of Methods for the Microbiological
Examination of Food.4 Liver Veal Agar can be supplemented with 50%
egg yolk for the cultivation of anaerobic organisms.5

Infusion from Liver, Infusion from Veal, Proteose Peptone, Neopeptone,
Tryptone, Gelatin and Isoelectric Casein provide the rich nitrogen,
amino acids and vitamin content of the medium. Soluble Starch is
added to enhance the growth of anaerobes and Dextrose is a carbon
source. Sodium chloride maintains osmotic balance and Bacto Agar is
Formula
Liver Veal Agar
Formula Per Liter
Bacto Liver, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . . 50 g
Veal, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500 g
265
Loeffler Blood Serum

Bacto Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Isoelectric Casein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Neopeptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Nitrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Section II
g
g
g
g
g
g
g
g
g
g
Precautions
Storage
Expiration Date
Procedure
Materials Provided
Liver Veal Agar

Glassware
Autoclave
Incubator (35C)
Bacto Loeffler Blood Serum
Intended Use
Bacto Loeffler Blood Serum is used for cultivating Corynebacterium
diphtheriae from clinical specimens and in pure culture. The medium
is also used for demonstrating pigment production and proteolysis.
Also Known As
Loeffler Blood Serum is also referred to as Loefflers Serum Agar
Medium, Loefflers Coagulated Serum Slants and LAS.

Loeffler Blood Serum is employed in the cultural diagnosis of
diphtheria. Diphtheria is an acute infectious disease primarily of the
266


Test Procedure
For a complete discussion of the isolation and identification of anaerobic
bacteria and other fastidious aerobic pathogens, refer to the procedure
described in Clinical Microbiology Procedures Handbook6 and Manual
of Clinical Microbiology.7
Results

References
1. Spray, R. S. 1930. An improved anaerobic culture dish. J. Lab.
Clin. Med. 16:203.
2. Spray, R. S. 1936. Semisolid media for cultivation and
identification of the sporulating anaerobes. J. Bacteriol. 32:135.
5. Atlas, R. M. 1993. Handbook of microbiological media. CRC
Press, Boca Raton, FL.
Packaging
Liver Veal Agar
500 g
0059-17
upper respiratory tract but occasionally of the skin.1 It is caused by

toxigenic strains of Corynebacterium diphtheriae, of which there
are three biotypes: mitis, intermedius, and gravis.1 The signs and
symptoms of the disease are a pharyngeal membrane, sore throat,
malaise, headache and nausea.2 Death can result from respiratory
obstruction by the membrane or myocarditis caused by the toxin.2
Loeffler Blood Serum is a modification of the horse serum, dextrose
broth medium described by Loeffler3 for cultivating C. diphtheriae.
This lipid-rich medium supports rapid growth of C. diphtheriae and is
useful for demonstrating colonial pigmentation and the proteolytic
activities of anaerobes and other microorganisms. Loeffler Blood Serum
restores virulence and other identifying properties lost after prolonged
incubation or repeated subculturing.4 Colonies grown on Loeffler
medium exhibit excellent metachromatic granules under Gram stain.2
The Difco Manual
Section II
Cleveland and Sanders,5 and Spector6 used Loeffler Blood Serum in

media for the cultivation of Endamoeba histolytica. Thompson 7
hydrolyzed Loeffler Blood Serum with sodium hydroxide and added
it to a citrate agar for the isolation of C. diphtheriae. On Thompsons7
medium, growth of diphtheria bacilli was stimulated while other
respiratory flora were inhibited.
Expiration Date
Materials Provided
Beef Blood Serum provides the nitrogen, vitamins and amino acids
necessary to support the growth of corynebacteria in Loeffler Blood
Serum. Dextrose Broth is a source of fermentable carbohydrate and
maintains the osmotic equilibrium of the medium.
Formula
Procedure
Glassware
Autoclave
Incubator

Formula Per Liter
Beef Blood Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 parts
Dextrose Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 part
Precautions
1. Suspend 80 grams in 1 liter distilled or deionized water warmed to

42-45C. Check pH. Adjust to pH 7.1, if necessary.
2. Dispense into tubes having screw caps or other tightly sealing
closures. Slant the tubes in the autoclave. Close the door loosely.
3. Coagulate the medium in constantly flowing steam for 10 minutes.
Close the door tightly.
5. Allow autoclave pressure to fall to zero before removing tubes.
Storage
Store the dehydrated medium at 2-8%C. The dehydrated medium is
Both throat and nasopharyngeal specimens are necessary in cases

of respiratory illness. If cutaneous diphtheria is suspected, collect
skin, throat and nasopharynx specimens. Sterile silica gel is recommended for shipping clinical specimens when cultures are not
obtained on site.1
Test Procedure1
1. If the swab appears desiccated, was collected several days prior to

receipt, or is received in silica gel, place it into Todd-Hewitt Broth
supplemented with 3% sterile rabbit blood. Incubate the culture
overnight, then inoculate onto isolation media.
2. Inoculate the specimen onto cystine tellurite blood agar and blood
agar plates, and streak for isolation on a Loeffler Blood Serum
slant, leaving the swab on the slant during incubation.
3. Incubate aerobically at 35C.
4. After 2-4 hours, prepare and heat fix a smear from the Loeffler
Blood Serum slant. Flood the slide with Methylene Blue, Loeffler
for 1 minute, rinse with tap water, and blot dry. Examine the smear
for morphology typical of C. diphtheriae.
5. After 18-24 hours incubation, subculture onto a second plate of
cystine tellurite blood agar.

free-flowing.
8% Solution:
Soluble in distilled or deionized
water warmed to 42-45C.
Prepared Medium:
Coagulated in tubes - White to cream
colored, slightly transparent at apex
of slant.
Reaction of
8.0% Solution:
pH 7.1 0.2 at 25C
Cultural Response
Inoculate tubes with 30-300 CFU of test organism. Incubate
18-24 hours at 35 2C. Prepare slides from the growth, heat-fix,
and stain with Methylene Blue, Loeffler. Stained cells will
contain bipolar granules, club cells and some cells with
general granulation.
ORGANISM
ATCC
GROWTH
Corynebacterium diphtheriae type mitis

Corynebacterium diphtheriae type intermedius
Corynebacterium diphtheriae type gravis
8024
8032
8028
good
good
good
The Difco Manual
Results
Examine all plates at 24-48 hours for colonies typical of C. diphtheriae.
Subculture colonies that are catalase positive and exhibit typical
morphology onto blood agar to provide growth for identification
procedures.
Definitive identification of a C. diphtheriae isolate as a true pathogen
requires demonstration of toxin production.8
267
Lowenstein Medium Base; Lowenstein Medium, Gruft; Lowenstein Medium, Jensen; Lowenstein Medium, Jensen Deeps & Lowenstein Medium w/5% NaCl
For a complete discussion on the collection, isolation and identification

of Corynebacterium diptheriae and other Corynebacterium species,
refer to the appropriate procedures.
3.

2. Loeffler Blood Serum must be used in parallel with blood agar
and a tellurite- containing medium (cystine tellurite agar or
modified Tinsdale medium) for selection and differentiation of
Corynebacterium.2
3. Metachromatic granules that take up methylene blue are
characteristic of Corynebacterium; however, other microorganisms
may also display stained granules (e.g., Propionibacterium, some
Actinomyces, and pleomorphic streptococci strains) and resemble
corynebacteria. Additional culture, biochemical identification,
and toxigenicity tests must be performed for differentiation
and identification.4
References
1. Isenberg, H. D. (ed.) 1992. Clinical microbiology procedures
miscellaneous irregular gram-positive rods, Erysipelothrix, and
4.
5.
6.
7.
8.
Section II
Gardnerella, p. 357-377. In P.R. Murray, E.J. Baron, M.A. Pfaller,

F.C. Tenover and R.H. Yolken (ed.), Manual of clinical microbiology,
Loeffler, F. 1887. Darauf theilte HeuLoeffer en einem Zweiten
Vortrag die ergebnisse seiner weiteren untersuchungen uber die
Diphtherie-Bacillen mit. Zentralbl. Bacteriol. 2:105.
MacFaddin, J. D. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, p. 448-451,
Cleveland, L. R., and E. P. Sanders. 1930. Encystation, multiple
fission without encystment, metacystic development, and
variation in a pure line and nine strains of Entamoeba histolytica.
Arch. Protistenkd. 70:223.
Spector, B. K. 1932. A comparative study of cultural and
immunological methods of diagnosing infections with Entamoeba
histolytica. J. Prevent. Med. 6:117.
Thompson. 1929. J. Infect. Dis. 45:163.
Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Bailey &
St. Louis, MO.
Packaging
500 g
0070-17*
*Store at 2-8C
Bacto Lowenstein Medium Base . Bacto Lowenstein Medium,

Gruft . Bacto Lowenstein Medium, Jensen . Bacto Lowenstein
Medium, Jensen Deeps . Bacto Lowenstein Medium w/5% NaCl
Intended Use

Identity Specification
Lowenstein Medium Base
Dehydrated Appearance: Medium to dark green-blue, free
flowing, homogenous.
Solution:
6.2% solution containing 2% glycerol,
water on boiling. Dark blue-green,
opalescent, viscous.
Prepared Medium
(as Lowenstein Medium, Jensen): Pale green, smooth slant, opaque.
Lowenstein Medium, Gruft Tubes; Lowenstein Medium,
Jensen Tubes; Lowenstein Medium w/5% NaCl
Appearance:
Pale green, opaque, smooth slants.
Reaction of
Medium at 25C:
pH 6.8-7.4
Lowenstein Medium, Jensen Deeps
Appearance:
Pale green, opaque butts.
Reaction of
Medium at 25C:
pH 7.2 0.2
268
Bacto Lowenstein Media are prepared with fresh egg and glycerol to
isolate, cultivate and differentiate mycobacteria.
Lowenstein Medium, Jensen Deeps are used for determining the
catalase activity of mycobacteria.
Lowenstein Medium w/5% NaCl is used for differentiating mycobacteria
on the basis of NaCl tolerance.

tuberculosis each year.1 In 1985, the number of tuberculosis cases (TB) in
the United States began increasing. Prior to this time, the number of US
cases had been decreasing, reaching a low in 1984.2 Non-tuberculous
mycobacteria infections have also increased since 1985.3
The use of egg-based media for primary isolation of mycobacteria have
the following significant advantages:
1. Egg-based media support a wide variety of mycobacteria.
2. The growth of mycobacteria on egg media can be used for niacin testing.
A disadvantage of egg-based media is that contaminating proteolytic
organisms tend to liquefy the medium.3
The Difco Manual
Section II
Formula Per Liter
Lowenstein formulations are egg-based media that contain a moderate

amount of malachite green to suppress the growth of contaminating
organisms. These media are commonly used in the clinical laboratory
to isolate acid fast organisms from sterile and nonsterile sources.4
Bacto Lowenstein Medium, Jensen (LJ) is a modification of the Jensen
formulation for Lowenstein Medium.5 It contains salts and a moderate
concentration of malachite green to prevent the growth of most
contaminants and to allow early growth of mycobacteria.
Lowenstein Medium, Gruft is the Gruft modification of Lowenstein
Medium, Jensen.6 Ribonucleic acid is incorporated into the medium to
increase the isolation of mycobacteria. Penicillin and Nalidixic Acid
are added to decrease contamination.
The increased sodium chloride concentration in Lowenstein Medium,
Jensen w/5% NaCl helps to differentiate rapid-growing mycobacteria
from slow growers, which are inhibited in the presence of salt.
Glycerol is added as a carbon source.
Formula
g
g
g
g
g
g
ml
ml
ml
Lowenstein Medium, Jensen Deeps (per 1600 ml)

Formula Per Liter
Bacto Lowenstein Medium Base . . . . . . . . . . . . . . . . . . . 37.2
Bacto Glycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Distilled/Deionized Water . . . . . . . . . . . . . . . . . . . . . . . . 588
Homogenized Egg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1,000
g
ml
ml
ml
Lowenstein Medium Gruft (per 1600 ml)

Formula Per Liter
Bacto Glycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Homogenized Egg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1,000
Penicillin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80,000
Nalidixic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Ribonucleic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Lowenstein Medium w/5% NaCl (per 1600 ml)
The Difco Manual
Precautions
Storage
Store Lowenstein Medium Base below 30C.
Store prepared tubed media at 2-8C.
Expiration Date
stored as directed. Do not use product if it fails to meet specifications
Materials Provided
g
g
g
g
g
g
Lowenstein Medium, Jensen

Formula Per Liter
Magnesium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6
Potato Flour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Malachite Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4
Bacto Glycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Homogenized Egg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1,000
g
ml
ml
g
ml
Procedure

Formula Per Liter
Magnesium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6
Potato Flour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Malachite Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4

Bacto Glycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Homogenized Egg . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1,000
g
ml
ml
ml
units
mg
g

Lowenstein Medium, Gruft
Lowenstein Medium w/5% NaCl

Flasks
Screw capped tubes
Specimen digestant and decontaminant
Centrifuge
Inoculating Needles
Incubator
Fresh egg
Autoclave
Inspissator (optional)
Water bath (optional)
Glycerol
Tween-Hydrogen Peroxide 1:1
1. Suspend 37.2 grams of Lowenstein Medium Base in 600 ml
distilled or deionized water containing 12 ml of Glycerol and boil
with constant agitation.
3. Aseptically add the sterile base to 1 liter of a uniform suspension
of fresh eggs prepared under aseptic conditions. Swirl gently to
avoid introducing air into the suspension.
4. Dispense into sterile screw cap tubes or bottles. Arrange in a slanted
position.
5. Place in an inspissator, water bath or autoclave at 85C for 45
minutes to coagulate the medium.
269
Prepared Lowenstein Media

Prepared media are ready to use.

Test Procedure
Results
Observe for colonies that may or may not be pigmented. Colony
morphology depends on the species isolated.
Section II
molecular genetic insights. Clinical Microbiology Reviews 8:496-514.

2. Kleitmann, W. 1995. Resistance and susceptibility testing for
Mycobacterium tuberculosis. Clinical Microbiology Newsletter
17:65-69.
5. Enter. Bacteriol. Parasitnek. 1932. Abt. 1, 125:222.
6. J. Bact. 1965. 90:829.
Negative culture results do not rule out an active mycobacterial

infection. Some factors responsible for unsuccessful cultures are:
1. The specimen was not representative of the infectious material,
2. The mycobacteria were destroyed during digestion and decontamination of the specimen.
3. Gross contamination interfered with the growth of mycobacteria.
4. Proper aerobic and increased CO2 tension were not provided
during incubation.
Packaging
References
500 g
2 kg
0444-17
0444-07
Lowenstein Medium, Gruft
20 tubes
100 ubes
1417-39
1417-79
20 tubes
100 tubes
100 x 1 oz
1017-39
1017-79
1017-57
20 tubes
1423-39
20 tubes
1289-76
1. Musser, J. M. 1995. Antimicrobial resistance in Mycobacteria:
Cultural Response
Lowenstein Medium Base; Lowenstein Medium, Gruft Tubes;
Lowenstein Medium, Jensen Tubes
Prepare Lowenstein Medium Base (as Lowenstein Medium, Jensen) per label
directions or use prepared tubes. Inoculate and incubate at 35C under CO2 for
up to three weeks.
ATCC
ORGANISM
Escherichia coli
Mycobacterium tuberculosis H37Ra
25922*
6841
13950
12478
19981
25177
INOCULUM CFU
RECOVERY
1,000-2,000 partial inhibition

100-300
good
100-300
good
100-300
good
100-300
good
100-300
good
Tested on Lowenstein Medium, Gruft, only

Inoculate and incubate at 35C under CO2 for up to three weeks.
ORGANISM
ATCC
INOCULUM CFU
RECOVERY
Mycobacterium smegmatis
Mycobacterium tuberculosis
14468
25177
100-300
100-300
good
inhibited

Inoculate and incubate at 35C under CO2 for 2 weeks. Cap loosely for 7 days,
then tighten cap and incubate 1 week longer. Add 1 ml of Tween-Hydrogen
Peroxide reagent to the 2-week culture. Measure the height of the column of
bubbles after 5 minutes. (Tween-Hydrogen Peroxide reagent is a 1:1 final
solution of 30% hydrogen peroxide in distilled water and sterile, cooled 10%
Tween 80 in distilled water.)
ORGANISM
ATCC
INOCULUM CFU
RECOVERY
Mycobacterium gordonae
14470
25177
100-300
100-300
greater than 45 mm
less than 45 mm
270
Uninoculated
tube
Mycobacterium
fortuitum
ATCC 6841
The cultures listed are the minimum that should be

used for performance testing.
*These cultures are available as Bactrol Disks
The Difco Manual
Section II
Luria Agar Base, Miller
Bacto Luria Agar Base, Miller
Intended Use
Bacto Luria Agar Base, Miller is used for maintaining and propagating
Escherichia coli in molecular microbiology procedures with or
without added glucose.

Luria Agar Base, Miller, a nutritionally rich medium designed for
growth of pure cultures of recombinant strains, is based on the Luria
Broth Agar formula described by Miller.1 E. coli is grown to late log
phase in LB Medium. Some plasmid vectors replicate to a high copy
number and do not require selective amplification. Some vectors do
not replicate so freely and need to be selectively amplified. Chloramphenicol may be added to inhibit host synthesis and, as a result,
prevent replication of the bacterial chromosome.2
Luria Agar Base, Miller contains one tenth and one twentieth,
respectively, the sodium chloride level of the LB Agar, Lennox and
LB Agar, Miller formulations.1-3 This allows the researcher to select
the optimal salt concentration for a specific strain. The medium may
be aseptically supplemented with glucose, if desired.
Precautions
Storage
Expiration Date
Procedure
Materials Provided

Sodium Chloride. Bacto Agar is the solidifying agent.

Autoclave
Waterbath (45-50C)
Petri dishes
Incubator (35C)
20% glucose solution (optional)
Formula

Formula Per Liter
1.
2.
3.
4.
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g

Autoclave at 121C for 15 minutes. Cool to 45-50C in a waterbath.
If desired, aseptically add 10 ml sterile 20% glucose solution and
mix thoroughly.
Test Procedure
Dehydrated Appearance: Light tan, free flowing, homogeneous.

Solution:
Solution is light amber, very slightly
Prepared Medium:
Reaction of 3.05%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepare Luria Agar Base, Miller with 10 ml sterile 20% glucose
solution per label directions. Inoculate and incubate at 35 2C
for 18-24 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Escherichia coli
33526
100-300
Good

The Difco Manual
Results
Growth is evident in the form of isolated colonies and/or a confluent
lawn on the surface of the medium.
References
Harbor Laboratory. Cold Spring Harbor, New York.
Cold Spring Harbor, New York.
Packaging
500 g
2 kg
0413-17
0413-0
271
Luria Broth Base, Miller
Section II
Bacto Luria Broth Base, Miller

Intended Use
Bacto Luria Broth Base, Miller is used for maintaining and propagating
Escherichia coli in molecular microbiology procedures with or
without added glucose.
Precautions
Luria Broth Base, Miller is based on the Luria Broth formula described
by Miller for the growth and maintenance of Escherichia coli strains
used in molecular microbiology procedures.1
Luria Broth Base, Miller is a nutritionally rich medium designed for
growth of pure cultures of recombinant strains. Escherichia coli is
grown to late log phase in LB Medium. Some plasmid vectors may
replicate to high copy numbers without selective amplification. Some
vectors do not replicate so freely, and need to be selectively amplified.
Chloramphenicol can be added to inhibit host synthesis and as a result,
prevent replication of the bacterial chromosome.2
Luria Broth Base, Miller contains one tenth and one twentieth the
sodium chloride level of the Lennox and Miller formulations of LB
Agar, respectively.1,2,3 This allows the researcher to select the optimal
salt concentration for a specific strain. The medium may be aseptically
supplemented with glucose, if desired.
Storage

Peptides and peptones are provided by Bacto Tryptone. Vitamins
(including B vitamins) and certain trace elements are provided by
Bacto Yeast Extract. Sodium ions for transport and osmotic balance
are provided by sodium chloride.

Tubes with closures
Autoclave
Incubator (35C)
Formula

Formula Per Liter
1. Dissolve 15.5 grams in 1 liter of distilled or deionized water.

3. Cool to 45-50C. If desired, aseptically add 10 ml sterile 20%
glucose solution and mix thoroughly.
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g

medium at 2-8C.
Expiration Date
Procedure
Materials Provided
Not applicable.

Solution:
or deionized water. Solution is very
light to light amber, clear to very
Prepared Tubes:
Very light to light amber, clear to
Reaction of 1.55%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepare Luria Broth Base, Miller per label directions with
1 ml of 20% dextrose solution added to 100 ml of media.
Inoculate the tubes and incubate at 35 2C for 18- 24 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Escherichia coli
33526
100-1,000
Good

272
Test Procedure
Results
Growth is evident by the appearance of turbidity in the medium.
References
1. Miller, J. H. 1972. Experiments in molecular genetics. Cold
Spring Harbor Laboratory, Cold Spring Harbor, New York.
Laboratory, Cold Spring Harbor, New York.
Packaging
500 g
2 kg
0414-17
0414-07
The Difco Manual
Section II
Lysine Iron Agar
Bacto Lysine Iron Agar
production. Brom Cresol Purple, a pH indicator, is yellow at or below

pH 5.2 and purple at or above pH 6.8. Bacto Agar is a solidifying agent.
Intended Use
Formula
Bacto Lysine Iron Agar is used for differentiating microorganisms,

especially Salmonella, based on lysine decarboxylation/deamination
and H2S production.
Lysine Iron Agar

Formula Per Liter

Lysine Iron Agar is prepared according to the formulation of Edwards
and Fife1, who developed the medium to detect Salmonella arizonae
(formerly Arizona arizonae). Because S. arizonae ferments lactose so
rapidly, the authors found that the expected H2S production on triple
sugar iron agar was suppressed. Since S. arizonae strains are found
occasionally in outbreaks of food borne infection, it is important to be
able to detect them. By eliminating lactose and incorporating lysine,
Edwards and Fife devised a medium that differentiates enteric bacilli
based on their ability to decarboxylate or deaminate lysine and
produce abundant hydrogen sulfide. The medium is especially recommended for detecting rapid lactose-fermenting S. arizonae. It is specified
in Standard Methods for Salmonella testing.2,3,4,5,6

Lysine Iron Agar contains Bacto Peptone which provides carbon and
nitrogen sources required for good growth of a wide variety of organisms.
Yeast Extract provides vitamins and cofactors required for growth, as
well as additional sources of nitrogen and carbon. Dextrose is an energy
source. L-Lysine Hydrochloride is the substrate used to detect the lysine
decarboxylase and lysine deaminase enzymes. Ferric Ammonium
Citrate and Sodium Thiosulfate are indicators of hydrogen sulfide
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
L-Lysine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Thiosulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.04
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g
g
Precautions
Storage
Expiration Date

Solution:
reddish-purple, very slightly to slightly
opalescent without significant precipitate.
Prepared Medium:
Purple, slightly opalescent without
precipitate.
Reaction of 3.45%
Solution at 25C:
pH 6.7 0.2
Cultural Response
Prepare Lysine Iron Agar per label directions. Inoculate with
undiluted cultures and incubate at 35 2C for 18-48 hours.
ORGANISM
ATCC
Proteus mirabilis 25933

Salmonella
13314
arizonae
Salmonella
14028*
typhimurium
LYSINE
LYSINE
DECARBOXYLATION DEAMINATION
GROWTH
(BUTT)
(SLANT)
H 2S
(APEX OF
SLANT)
good
good
yellow
+ purple
+ red
purple
+ black
good
+ purple
purple
+ black
Uninoculated
tube
Proteus mirabilis
ATCC 25933
ATCC 14028
*Available as Bactrol Disks; use as directed in Bactrol Disks Technical Information.
The Difco Manual
273
Lysine Medium
Section II
Procedure
Materials Provided
1. Salmonella paratyphi A, unlike other Salmonella, does not

produce lysine decarboxylase and so produces an alkaline slant
and an acid butt.
2. H2S-producing Proteus species do not blacken the medium.2,7 It is,
therefore, suggested that Lysine Iron Agar be used in conjunction
with Triple Sugar Agar or other media to confirm differentiation.
3. The reaction of Morganella morganii may be variable after 24
hours incubation and may require longer incubation.7
Lysine Iron Agar

Glassware
Autoclave
Incubator (35C)
Inoculating needle
1.
2.
3.
4.

Dispense into tubes. Autoclave at 121C for 12 minutes.
Allow medium to cool in a position that will provide a short slant
and a deep butt.

Test Procedure
1. Using a straight needle, pick the center of a well-isolated colony
from a fresh, pure culture.
2. Inoculate by stabbing to the base of the butt and streaking the slant.
3. Cap the tube loosely to ensure aerobic conditions.
5. Examine at 18-24 and 40-48 hours for growth and color changes in
the butt and the slant of the medium and for blackening at the apex
of the slant.
Results
Lysine decarboxylase reaction:
Positive: Purple (alkaline) butt, purple slant.
Negative: Yellow (acid) butt, purple (alkaline) slant.
Lysine deaminase reaction:*
Positive: Red slant.
Negative: Purple slant.
Hydrogen sulfide reaction:
Positive: Blackened medium at the apex of the slant.
References
1. Edwards, P. R., and M. A. Fife. 1961. Lysine-iron agar in the
detection of Arizona cultures. Appl. Microbiol. 9:478.
3. Russell, S. F., J. -Y. DAoust, W. H. Andrews and J. S. Bailey.
Splittstoesser (eds.). Compendium of methods for the microbiological examination of food, 3rd ed. American Public Health Association,
Washington, D.C.
R. T. Marshall, (ed.), Standard methods for the examination of
Washington, D.C.
6. Andrews, W. H., G. A. June, P. S. Sherrod, T. S. Hammack,
and R. M. Amaguana. 1995. Salmonella, p. 5.01-5.20. In
Gaithersburg, MD.
7. Finegold, S. M., and W. J. Martin. 1982. Bailey and Scotts
diagnostic microbiology, 6th ed., p. 631. The CV Mosby Company,
St. Louis, MO.
Packaging
Lysine Iron Agar
* Proteus and Providencia cultures produce a red slant over a yellow (acid) butt.
Bacto Lysine Medium
Intended Use
Bacto Lysine Medium is used for isolating and enumerating wild
yeast contaminants in brewery pitching yeasts.

Walters and Thiselton1 formulated a liquid synthetic medium containing
lysine to study brewery yeasts. The medium separated yeasts into two
groups based on their ability to grow using L-lysine as a sole source of
nitrogen. Strains of brewery culture yeasts, Saccharomyces cerevisiae
and S. carlsbergensis, grew poorly or not at all in the medium (lysine
274
100 g
500 g
0849-15
0849-17
negative) while yeasts known as wild contaminants, including

S. pastorianus and S. cerevisiae var. turbidans, grew (lysine positive).
Morris and Eddy2 modified the formula by adding agar and confirmed
the work of Walters and Thiselton using quantitative results. They
showed a low level of infection of a pitching yeast with wild yeasts
could be determined. They also recommended the addition of antibiotics
to the medium for samples heavily contaminated with bacteria.

Lysine Medium contains Dextrose as the carbohydrate. L-Lysine is
the nitrogen source. Essential vitamins, salts, amino acids and other
nutrients are present in defined amounts. Bacto Agar is the solidifying
agent.
The Difco Manual
Section II
Lysine Medium
Formula
Storage
Lysine Medium
Formula Per Liter

Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44.5

Potassium Dihydrogen Phosphate . . . . . . . . . . . . . . . . . . 1.78
Calcium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.178
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.089
Adenine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.00178
DL-Methionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.000891
L-Histidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.000891
DL-Tryptophan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.000891
Boric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0000089
Zinc Sulphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0000356
Ammonium Molybdate . . . . . . . . . . . . . . . . . . . . . 0.0000178
Manganese Sulphate . . . . . . . . . . . . . . . . . . . . . . . . 0.0000356
Ferrous Sulphate . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0002225
Inositol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.02
Calcium Pantothenate . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.002
Aneurine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0004
Pyridoxine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0004
p-Aminobenzoic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0002
Nicotinic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0004
Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0002
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.000002
Folic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.000001
L-Lysine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.5
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
Precautions

Solution:
6.6% solution containing 1 ml 50%
potassium lactate solution per 100 ml
medium, soluble in distilled or deionized
water on gentle boiling; very light amber,
Prepared Medium:
Very light amber, slightly opalescent
Final reaction of pH-adjusted
6.6% solution at 25C: pH 4.8 0.2
Cultural Response
Prepare Lysine Medium per label directions. Inoculate and
incubate at 25 2C for 72 hours.
ATCC
INOCULUM
CFU
GROWTH
Pichia fermentans
10651
Saccharomyces pastorianus 2700
100-1,000
100-1,000
good
none to fair
ORGANISM
Expiration Date
Procedure
Materials Provided
Lysine Medium

Glassware
Petri dishes
Autoclave
Incubator (25C)
10% Lactic acid solution
50% Potassium lactate solution
1. Suspend 6.6 grams in 100 ml distilled or deionized water containing
1 ml 50% potassium lactate solution.
2. Boil gently to dissolve completely.
3. Cool to 50C.
4. Adjust to final pH using 10% lactic acid, if necessary.

Test Procedure
1. Wash and centrifuge the sample of pitching yeast three times with
distilled water.
2. Resuspend the pellet in distilled water to contain approximately
107 cells per ml.
3. Spread 0.2 ml of the cell suspension over the surface of the
prepared medium.
4. Incubate plates at 25C. Examine daily for growth.
Results
Count the number of colonies that develop and express the degree of
contamination as the number of wild cells per million cells of inoculum.

1. Use of a test inoculum having less than 104 cells may permit growth
of brewing yeast cells that can be confused with growth of wild
yeasts. Use of an inoculum that exceeds 104 cells will restrict
growth of the unwanted brewing yeasts to tiny microcolonies.2
References
1. Walters, L. S., and M. R. Thiselton. 1953. J. Inst. Brew.
59:401-404.
2. Morris, E. O., and A. A. Eddy. 1957. J. Inst. Brew. 63:34-35.
Packaging
Lysine Medium
The Difco Manual
500 g
1894-17
275
M9CA Medium
Section II
Bacto M9CA Medium
Intended Use
Bacto M9CA Medium is used for cultivating recombinant strains of
Escherichia coli.

M9CA is based on M9 Minimal Salts1 but with the addition of casamino
acids. The medium may be supplemented with an appropriate carbon
and energy source, such as dextrose. The Casamino Acids provide
nitrogen in a readily available form. The medium will support the
growth of wild-type and recombinant strains of E. coli. M9CA
contains salts that supply nitrogen, phosphorus, and trace minerals.

Casamino Acids make this a richer medium than M9 Minimal Salts,
providing all of the amino acids except tryptophan. Ammonium
Chloride provides a source of nitrogen. Sodium and Potassium Phosphates
buffer against pH changes due to carbohydrate metabolism. Dextrose,
aseptically added to the medium, is a carbon and energy source.
Magnesium Sulfate is a source of magnesium ions required in a variety
of enzymatic reactions, including DNA replication.

Storage
Expiration Date
Procedure
Formula
Materials Provided
M9CA Medium
Formula Per Liter
M9CA Medium
Bacto Casamino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Sodium Phosphate, Dibasic, Anhydrous . . . . . . . . . . . . . . 6.8
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
g
g
g
g
g
Precautions
homogeneous.
Solution:
to medium amber, clear.
Prepared Medium:
Light to medium amber, clear, no
Reaction of 1.53%
Solution at 25C:
pH 6.8 0.2
Cultural Response
Prepare M9CA Medium per label directions. Inoculate and
Escherichia coli (B)
ATCC
INOCULUM
CFU
GROWTH
23226
100-300
Good

276

Autoclave
Sterile 20% glucose solution
Sterile 1.0 M MgSO4 solution
Incubator (35C)
ORGANISM
1. Dissolve 15.3 grams in 1 liter of distilled or deionized water.

3. After cooling to below 50C, aseptically add 20 ml of filtersterilized 20% glucose solution and 2 ml of filter-sterilized 1M
Magnesium Sulfate solution. Mix well.

Test Procedure
Consult appropriate references for recommended test procedures.
Results
References
Packaging
M9CA Medium
Dextrose
500 g
500 g
0454-17
0155-17
The Difco Manual
Section II
M9 Minimal Salts, 5x
Bacto M9 Minimal Salts, 5x
Intended Use
Bacto M9 Minimal Salts, 5x is used in preparing M9 Minimal Medium
which is used for cultivating recombinant strains of Escherichia coli.

M9 Minimal Salts, 5x is a 5x concentrate that is diluted to a 1x
concentration and supplemented with an appropriate carbon and energy
source, such as dextrose, to provide a minimal, chemically defined
medium. The medium will support the growth of wild-type strains
of E. coli. M9 Minimal Salts is useful for maintaining positive selection
pressure on plasmids coding for the ability to produce essential
substances such as amino acids or vitamins. M9 Minimal Medium is
also used to maintain stocks of F containing bacteria for use with
M13. The medium can be supplemented with specific amino acids or
other metabolites, allowing for selection of specific auxotrophs.
Precautions
Storage
Store the dehydrated medium below 30C. The powder is very hygroscopic. Keep container tightly closed. Store prepared medium at 2-8C.
Expiration Date
Sodium Phosphate and Potassium Phosphate are present as buffering

agents. Ammonium Chloride is a source of nitrogen for cellular
systems. Sodium Chloride maintains isotonicity in the final medium.
Glucose may be added as a source of carbohydrate. Supplementing
the medium with magnesium and calcium increases the growth of
recombinants.
Formula
Formula Per Liter
Sodium Phosphate, Dibasic, Anhydrous . . . . . . . . . . . . . 33.9

Potassium Phosphate, Monobasic . . . . . . . . . . . . . . . . . . . 15
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
g
g
g
g
Procedure
Materials Provided

Autoclave
Sterile 20% glucose solution
Sterile 1.0 M MgSO4 solution
Sterile 1.0 M CaCl2 solution (optional)
Incubator (35C)
1. Dissolve 56.4 grams in 1 liter of distilled or deionized water. It is

recommended that this liter be separated into 200 ml aliquots.
3. To prepare M9 Minimal Medium, add 200 ml sterile M9 Minimal
Salts, 5x to 750 ml sterile distilled or deionized water, which has
been cooled to 45-50C. Adjust final volume to 1 liter.
4. Aseptically add 20 ml filter-sterilized 20% glucose solution, 2 ml
sterile 1.0 M magnesium sulfate (MgSO4) solution and, if desired,
0.1 ml sterile 1.0 M calcium chloride (CaCl2) solution. Mix well.
5. If desired, supplement with amino acids, as appropriate.

Solution:
colorless, clear.
Reaction of
5.64% Solution
(5x concentrate) at 25C: pH 6.8 0.2
Cultural Response
Prepare M9 Minimal Salts, 5x and dilute to 1x. Supplement
with glucose per label directions. Inoculate and incubate at
35C for 18-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
Escherichia coli
Escherichia coli
23226
39403
30-300
30-300
GROWTH
Good
Good

Test Procedure
Consult appropriate references for recommended test procedures.1-2
Results
Growth should be evident by the appearance of turbidity.
The Difco Manual
277
M17 Broth & M17 Agar
Section II
References
1. Davis, L. G., M. D. Dibner, and J. F. Battey. 1986. Basic
methods in molecular biology. Elsevier, New York, N.Y.
Bacto M17 Broth

Bacto M17 Agar

Laboratory, Cold Spring Harbor, N.Y.
Packaging
500 g
0485-17
Intended Use
Bacto M17 Broth is used for isolating and enumerating lactic
streptococci from yogurt, cheese starters and other dairy products.
Bacto M17 Agar is used for enumerating lactic streptococci in yogurt,
cheese starters and other dairy products.
M17 Broth
Dehydrated Appearance: Beige to medium tan, free-flowing,
homogeneous.
Solution:
or deionized water. Solution is lightmedium to medium amber, clear to
Prepared Medium:
Light medium to medium amber,
clear to very slightly opalescent, no
Reaction 3.725%
Solution at 25C:
pH 6.9 0.2
M17 Agar
Dehydrated Appearance: Beige to medium tan, free-flowing,
homogeneous.
Solution:
very slightly to slightly opalescent,
Prepared Medium:
opalescent, no significant
precipitate.
Reaction of 4.825%
Solution at 25C:
pH 6.9 0.2
Cultural Response
Prepare Nutrient Gelatin per label directions. Using a heavy
inoculum, inoculate by stabbing the tube and incubate at
35 2C for 18-48 hours or up to two weeks, if required. To
read gelatinase, refrigerate until well chilled and compare to
uninoculated tube. Tilt tubes carefully to test for liquefaction.
Tubes positive for gelatinase remain liquid.
ORGANISM
subsp. bulgaricus
Lactococcus lactis
subsp. cremoris
Streptococcus thermophilus
ATCC
INOCULUM
CFU
GROWTH
11842
100-1,000
none to poor
9625
19258
100-1,000
100-1,000
good
good
Lactic streptococci are acid-producing bacteria. They are nutritionally

fastidious and require complex culture media for optimum growth. One
study showed that in a synthetic medium, all strains had an obligate
requirement for at least six amino acids and three vitamins.1 These
homofermentative lactic streptococci produce large amounts of acid
and, in a culture medium without an adequate buffering system, the pH
decreases and adversely affects growth. Lowrie and Pearce 2
developed M16 Medium but it lacked a strong buffering system.
Terzaghi and Sandine3 worked with M16 Medium and demonstrated
that the rapid drop in pH that accompanies lactic streptococcal growth
can adversely affect colony size and phage plaque formation. They
modified M16 Medium using disodium--glycerophosphate as a
buffer and called it M17.
Shankar and Davies4 found that disodium--glycerophosphate in M17
Broth suppressed Lactobacillus bulgaricus and selectively isolated
Streptococcus thermophilus from yogurt. Similar results were
achieved using M17 Broth solidified with agar. The International
Dairy Federation recommends M17 Agar for isolating S. thermophilus
from yogurt.5 M17 Agar is a standard methods medium for isolating
lactic streptococci.6

M17 Broth and M17 Agar contain Tryptone, Soytone, and Meat Digest
as sources of carbon, nitrogen, vitamins and minerals. Yeast Digest
Disodium--Glycerophosphate buffers the medium as acid is produced
from fermentation of lactose. Ascorbic Acid stimulates growth of lactic
streptococci. Magnesium Sulfate provides essential ions for growth.
Bacto Agar is the solidifying agent in M17 Agar.
Formula
M17 Broth
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Meat Digest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Yeast Digest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Disodium--glycerophosphate . . . . . . . . . . . . . . . . . . . . . . 19
g
g
g
g
g
g
g
278
The Difco Manual
Section II
M Broth
M17 Agar
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Meat Digest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Ascorbic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Disodium--glycerophosphate . . . . . . . . . . . . . . . . . . . . . . 19
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
g
g
g
g
g
g
g
g
M17 Agar: Suspend 48.25 grams in 950 ml distilled or deionized

3. Cool to 50C.
4. Add 50 ml sterile 10% lactose solution. Mix well.

Test Procedure
Precautions
Results

References
Storage
Expiration Date
Procedure
Materials Provided
M17 Broth or
M17 Agar

Glassware
Petri Dishes (for M17 Agar)
Autoclave
Incubators (30C and 35C)
1. Reiter, B., and J. D. Oram. 1962. Nutritional studies on cheese

starters. I. vitamin and amino acid requirements of single strain
starters. J. Dairy Res. 29:63-77.
2. Lowrie and Pearce. 1971. J. Dairy Sci. Technol. 6:166.
3. Terzaghi, B. E., and W. E. Sandine. 1975. Improved medium for
lactic streptococci and their bacteriophages. Appl. Microbiol.
29:807-813.
4. Shankar, P. A., and F. L. Davies. 1977. A note on the suppression
of Lactobacillus bulgaricus in media containing -glycerophosphate
and application of such media to selective isolation of Streptococcus
thermophilus from yogurt. J. Soc. Dairy Tech. 30:28-30.
5. International Dairy Federation. 1981. Identification and enumeration of microorganisms in fermented milks. Joint IDF/ISO/
AOAC Group E44.
6. Vedamuthu, E. R., M. Raccach, B. A. Glatz, E. W. Seitz, and
M. S. Reddy. 1992. Acid-producing microorganisms, p. 225-238.
Packaging
M17 Broth
500 g
1856-17
1. M17 Broth: Dissolve 37.25 grams in 950 ml distilled or deionized

water.
M17 Agar
500 g
1857-17
Bacto M Broth
Intended Use
Bacto M Broth is used for cultivating Salmonella in foods and feeds
by the accelerated enrichment serology (ES) procedure.

M Broth, prepared according to the formula of Sperber and Diebel1,
contains all the nutrients necessary for good growth and flagella
development of Salmonella.
Fantasia, Sperber and Deibel2 compared the enrichment serology (ES)
procedure with the traditional procedure outlined in the
The Difco Manual
Bacteriological Analytical Manual3 (BAM) and reported excellent

agreement between the two. They found the ES procedure not only to
be faster and less complicated but also as accurate and sensitive
as the BAM procedure.
M Broth also conforms to the testing standards recommended
by Compendium of Methods for the Microbiological Examination
of Foods 4 (APHA) for the isolation and identification of
foodborne Salmonella.
Both monoclonal and polyclonal enzyme immunoassay (EIA)
methods have been described in AOAC Official Methods of Analysis5
using M Broth. These methods are screening procedures for the presence
of Salmonella and positive results must be confirmed by culture.
279
M Broth
Section II
Procedure
Yeast Extract is a source of B-complex vitamins. Tryptone provides

organic nitrogen. D- Mannose and Sodium Citrate are fermentation
energy sources. Mannose prevents fimbrial agglutination.1 Sodium
Chloride helps maintain osmotic equilibrium, while Dipotassium
Phosphate acts as a buffer. The inorganic salts stimulate bacterial
growth. Tween 80 is a surfactant and dispersing agent.
Materials Provided
Formula
M Broth
Formula per liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.5
Bacto D-Mannose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.04
Tween 80 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.75
g
g
g
g
g
g
g
g
g
g
Precautions
M Broth

Glassware
Autoclave
Lactose Broth
Selenite Cystine Broth
Tetrathionate Broth
Salmonella H Antisera Spicer-Edwards Set
Salmonella H Antiserum Poly D
Salmonella H Antiserum z6
Normal saline
NaCl
Formalin
50C waterbath
2. Heat to boiling for 1-2 minutes, stirring carefully.

Storage
Test Procedure

1. Prepare a 10% suspension of the test sample in Lactose Broth.

Incubate at 35 2C for 18-24 hours.
2. Transfer 1 ml of the above preenrichment culture to 9 ml of
Selenite Cystine Broth and 1 ml to 9 ml of Tetrathionate Broth.
Incubate both enrichment media at 35 2C for 24 hours.
Expiration Date

Dehydrated appearance: Beige, homogeneous with a tendency to lump.
Solution:
boiling for 1-2 minutes. Solution is light amber, clear to
very slightly opalescent, may have a slight precipitate.
Reaction of 3.62%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepare M Broth per label directions. Inoculate and incubate at 35 2C for
18-24 hours.
ORGANISM
Salmonella choleraesuis
ATCC
INOCULUM
CFU
12011 100-1,000
14028* 100-1,000
GROWTH
good
good
280
Uninoculated
tube
ATCC 12011
The Difco Manual
Section II
MIL Medium
3. Inoculate one 10 ml tube of M Broth, tempered to 35C, with

one drop from each of the above cultures. Incubate at 35 2C
for 6-8 hours.
4. Prepare a formalin-salt solution by adding 4.2 grams of NaCl and
3 ml of formalin to 100 ml of distilled water. Place one drop in
each of two Kahn tubes.
5. Carefully insert a pipette about 1 inch below the surface of the
M Broth culture and transfer 0.85 ml of culture to each of the above
Kahn tubes containing formalin-salt solution.
6. Prepare a pooled antiserum by combining together 0.5 ml each
of rehydrated Salmonella H Antiserum Poly D and Salmonella H
Antiserum z6 (Salmonella H Antisera Spicer-Edwards Set) in
11.5 ml of 0.85% NaCl.
7. Add 0.1 ml pooled Salmonella H Antiserum to one of the Kahn
tubes (above). Add 0.1 ml 0.85% NaCl solution to the other tube.
Shake the tubes gently. Incubate in a 50C water bath for 1 1/2 hours.
Results
Agglutination in the Kahn tube containing antiserum indicates the
presence of Salmonella. Agglutination in the Kahn tube containing
0.85% NaCl solution (control tube) indicates a rough culture which
should be streaked for isolation, passed through Motility GI Medium
to enhance flagella, and then retested with pooled antiserum.
Alternative Testing Procedures

Refer to AOAC International5 for screening procedures using enzyme
immunoassay or DNA hybridization to detect Salmonella antigen

in test samples.
References
1. Sperber, W. H. and R. H. Deibel. 1969. Accelerated procedure
for Salmonella detection in dried foods and feeds involving
only broth cultures and serological reactions. Appl. Microbiol.
17:533-539.
2. Fantasia, L. D., W. H. Sperber, and R. H. Deibel. 1969.
Comparison of two procedures for detection of Salmonella in food,
feed, and pharmaceutical products. Appl. Microbiol. 17:540-541.
3. Bacteriological Analytical Manual, 2nd ed. 1969. US HEW,
Washington, D.C.
4. Flowers, R. S., J.- Y. DAoust, W. H. Andrews, and J. S. Bailey.
Splittstoesser (ed.). Compendium of methods for the
microbiological examination of foods, 3rd ed. American Public
Packaging
M Broth
Bacto MIL Medium
Formula
Intended Use
MIL Medium
Formula Per Liter
Bacto MIL Medium is used for differentiating Enterobacteriaceae

based on motility, lysine decarboxylation, lysine deamination and
indole production.
Also Known As
MIL Medium conforms with Motility-Indole-Lysine Medium.

MIL Medium, prepared according to the formula of Reller and Mirrett,1
is a single culture medium that provides four differentiating biochemical
reactions. When used in conjunction with Triple Sugar Iron Agar (TSI)
and Urea Agar, as many as nine reactions are provided. This combination
enables reliable initial identification of Enterobacteriaceae. 2,3
Extensive testing of 890 enteric cultures by Reller and Mirrett1 gave
essentially the same results with MIL Medium as with the standard
motility, indole and lysine decarboxylase (Moeller) test media.

Bacto Peptone and Tryptone provide the carbon and nitrogen sources
required for good growth of a wide variety of organisms. Yeast Extract
provides vitamins and cofactors required for growth. Lysine Hydrochloride is present as a substrate to detect lysine decarboxylase or lysine
deaminase activity. Dextrose is an energy source. Ferric Ammonium
Citrate is an H2S indicator. Brom Cresol Purple is a pH indicator. Bacto
The Difco Manual
500 g
2 kg
0940-17
0940-07
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
L-Lysine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
g
g
g
g
g
g
g
g
Precautions
Storage
Expiration Date
281
MIL Medium
Section II
Procedure
Motility is indicated by a clouding of the medium or by growth extending

from the inoculation line.
Materials Provided
Lysine decarboxylase is indicated by a purple color throughout the

medium. This color may vary in intensity and may be bleached out to a
pale light color due to reduction of the indicator. Lysine-negative
cultures produce a yellow medium that may be purple or red on the
top. Tubes that show a purple reaction with a red color on top should
be incubated for a longer period of time.
MIL Medium

Glassware
Autoclave
Incubator (35C)
13 x 100 mm Screw-capped test tubes
Inoculating needle
SpotTest Indole Reagent Kovacs
After examining the medium for lysine deaminase, motility and lysine
decarboxylase reactions, add 3 or 4 drops of Indole Reagent Kovacs to
the top of each tube. The appearance of a pink to red color in the
reagent is interpreted as a positive indole test.
1.
2.
3.
4.

Dispense 5 ml amounts into 13 x 100 mm screw-capped test tubes.

Test Procedure
1. Using an inoculating needle, stab tubes with growth from an
18-24 hour pure culture.
2. Incubate the tubes at 35 2C for 18-24 hours.
3. After incubation, examine tubes for evidence of lysine deaminase,
motility and lysine decarboxylase reactions.
Results
Positive and negative reactions are based on 90% or more occurrences.

When an aberrant reaction occurs, subcultures should be plated on
differential media to ensure the purity of the culture.

1. Do not add Indole Reagent Kovacs until the final lysine deaminase,
lysine decarboxylase and motility results have been interpreted.
2. Occasionally, the indole test produces false-negative or falsely
weak reactions.3,4
References
1. Reller, L. B., and S. Mirrett. 1975. Motility-indole-lysine
medium for presumptive identification of enteric pathogens of
Enterobacteriaceae. J. Clin. Microbiol. 2:247-252.
Lysine deaminase is indicated by a red or red-brown color in the top

centimeter of the medium.

Solution:
is reddish purple, clear.
Prepared Medium:
Reddish purple, clear, semisolid.
Reaction of 3.65%
Solution at 25C:
pH 6.6 0.2
Cultural Response
Prepare MIL Medium per label directions. Inoculate and incubate
at 35 2C for 18-24 hours. After reading the lysine decarboxylase,
motility and lysine deaminase reactions, add Indole Reagent Kovacs
to determine the indole reaction.
ORGANISM
Escherichia coli
Providencia
alcalifaciens
Salmonella
enteritidis
Shigella flexneri
ATCC
LYSINE
DECARBOXYLASE
LYSINE
MOTILITY DEAMINASE
INDOLE
PRODUCTION
25922*
9886
+
+
13076
12022*
Uninoculated
tube
Escherichia coli
ATCC 25922
Shigella flexneri
ATCC 12022
All with Indole Reagent
282
The Difco Manual
Section II
MIO Medium

6th ed. American Society of Microbiology, Washington, D.C.
3. Baron, E. J., and S. M. Finegold. 1990. Bailey and Scotts
Diagnostic Microbiology, 8th ed. The C. V. Mosby Co.,
St. Louis, MO.
4. MacFaddin, J. F. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, vol.1. Williams

Bacto MIO Medium
Intended Use
Bacto MIO Medium is used for differentiating Enterobacteriaceae
based on motility, ornithine decarboxylase activity and indole production.
Also Known As
MIO Medium conforms with Motility Indole Ornithine Medium and
Ornithine Indole Motility (OIM) Medium.

Tests for indole production, motility and ornithine decarboxylase
activity play important roles in the identification of Enterobacteriaceae.
Ederer and Clark1 and Oberhofer and Hajkowski2 developed MIO
Medium which combines all three differentiating reactions in
onemedium. Ederer and Clark stressed the advantages of
MIO Medium in their extensive study comparing cultural reactions of
Enterobacteriaceae on MIO Medium with reactions on classic media.
Packaging
MIL Medium
500 g
1804-17
MIO Medium contains peptones which provide carbon and nitrogen.

Yeast Extract provides vitamins and cofactors required for growth
as well as additional sources of nitrogen and carbon. Dextrose
is an energy source. Bacto Agar is added to demonstrate motility.
The pH indicator, Brom Cresol Purple, facilitates detection of
decarboxylase activity.
Formula
MIO Medium
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto L-Ornithine HCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
g
g
g
g
g
g
g

Solution:
deionized water upon boiling; purple,
clear to slightly opalescent.
Prepared Medium:
Purple, slightly opalescent, semi-solid.
Reaction of 3.1%
Solution at 25C:
pH 6.5 0.2
Cultural Response
Prepare MIO Medium per label directions. Inoculate the
medium and incubate with caps loosened at 35 2C for
24-48 hours.
ORGANISM
Enterobacter
aerogenes
Escherichia coli
Klebsiella
pneumoniae
Proteus mirabilis
ORNITHINE
INDOLE DECARBOXYLASE
ATCC
GROWTH
MOTILITY
13048*
good
25922*
13883*
good
good
25933
good
Uninoculated
tube
ATCC 13048
Escherichia coli
ATCC 25922
Refer to appropriate references for typical motility, indole production and ornithine decarboxylase activity of various members of the Enterobacteriaceae.3,4,5,6
The Difco Manual
283
MYP Agar & Antimicrobic Vial P
Precautions
Storage
Section II
ornithine decarboxylase reaction; no color change indicates

a positive reaction. Motility is shown by clouding of the medium
or by growth extension from the inoculating line. Repeat the
reading at 40-48 hours.
4. Add 3-4 drops of SpotTest Indole Reagent Kovacs to each tube.
Record as indole positive if a pink or red color appears or as indole
negative if there is no color change.
Results
Expiration Date
Materials Provided
1. Do not add Kovacs reagent to the tubes until after final motility
and ornithine results have been interpreted.
2. To prepare the stored medium for use in motility studies, loosen
caps, heat to boiling and cool to 45-50C prior to inoculation.5
MIO Medium
References
1. Ederer, G. M., and M. Clark. 1970. Motility-Indole-Ornithine

medium. Appl. Microbiol. 2:849.
2. Oberhofer, T. R., and R. Hajkowski. 1970. Evaluation of
non-lactose-fermenting members of the Klebsiella-EnterobacterSerratia Division. I. Biochemical characteristics. Am. J. Clin.
Pathol. 54:720.
Enterobacteriaceae, 4th ed. Elsevier Science Publishing Co., Inc.,
New York, NY.
4. Krieg, N. R., and J. G. Holt (ed.). 1984. Bergeys manual of
systematic bacteriology, vol. 1. Williams & Wilkins, Baltimore, MD.
R. H. Yolken. (ed.). 1995. Manual of clinical microbiology.
Procedure
Glassware
Autoclave
Incubator (35C)
Inoculating wire
1.
2.
3.
4.

Dispense into test tubes.

Test Procedure
1. Pick isolated colonies with an inoculating wire and stab the
medium to the bottom of the tube.
2. Incubate with caps loosened at 35 2C for 24-48 hours.
3. Examine tubes at 24 hours for growth, color change and motility.
A color change from purple to yellow indicates a negative
Bacto MYP Agar

Bacto Antimicrobic Vial P
Packaging
MIO Medium
100 g
500 g
0735-15
0735-17
Also Known As
Mossel et al1 formulated Mannitol-Egg Yolk-Polymixin (MYP) Agar

to isolate and enumerate Bacillus cereus from foods. This medium
differentiates B. cereus from other bacteria based on its resistance to
polymixin, lack of mannitol fermentation, and presence of lecithinase.2,
3
B. cereus is commonly found in nature, on vegetables, and in some
processed foods.4 Under favorable circumstances the microorganism
grows to sufficient numbers and causes gastrointestinal illness.4
Outbreaks of food borne illness have been associated with boiled and
cooked rice, cooked meats and cooked vegetables. 5
MYP Agar is also known as Mannitol-Egg Yolk-Polymixin Agar.
MYP Agar is a recommended medium for testing foods.4, 5, 6
Intended Use
Bacto MYP Agar is used with Bacto Egg Yolk Enrichment 50%
and Bacto Antimicrobic Vial P for enumerating Bacillus cereus
from foods.
284
The Difco Manual
Section II
MYP Agar & Antimicrobic Vial P
2. Antimicrobic Vial P: MAY CAUSE ALLERGIC EYE,

RESPIRATORY SYSTEM AND SKIN REACTION. (US) MAY
BE HARMFUL IF ABSORBED OR INTRODUCED THROUGH
MYP Agar contains Beef Extract and Bacto Peptone as sources of

carbon, nitrogen, vitamins and minerals. D-Mannitol is the carbohydrate
source. Phenol Red is the pH indicator. Bacto Agar is the solidifying
agent. Egg Yolk Enrichment 50% provides lecithin. Antimicrobic Vial P
is Polymixin B which inhibits the growth of most other bacteria.
Bacteria that ferment mannitol produce acid products and form colonies
that are yellow. Bacteria that produce lecithinase hydrolyze the lecithin
and a zone of white precipitate forms around the colonies. B. cereus is
typically mannitol-negative (pink-red colonies) and lecithinase positive
(zone of precipitate around the colonies).
Formula
MYP Agar
Formula Per Liter
Storage

Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto D-Mannitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.025
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g

Store the desiccated and rehydrated Antimicrobic Vial P at 2-8C.
Expiration Date

Antimicrobic Vial P
30,000 units polymyxin B
Precautions
Uninoculated
plate
Bacillus cereus
ATCC 13061

MYP Agar
Solution:
is red, slightly opalescent.
Prepared Medium:
Red, slightly opalescent without
Reaction of 4.6 g/90 ml
distilled or deionized
water at 25C:
pH 7.2 0.1
Antimicrobic Vial P
Dehydrated Appearance: White cake or powder.
Cultural Response
Prepare MYP Agar per label directions. Supplement with Egg Yolk
Enrichment 50% and Antimicrobic Vial P (Polymyxin B). Inoculate
and incubate at 30 2C for 18-48 hours. Lecithinase reaction is read
as a zone of precipitate. Colonies that ferment mannitol are yellow.
ORGANISM
Bacillus cereus
Bacillus subtilis
ATCC
INOCULUM
CFU
13061 3-30 and 30-300

6633 3-30 and 30-300
27853*
1,000-2,000
GROWTH
good
good
inhibited
MANNITOL
LECITHINASE
FERMENTATION REACTION
Bacillus subtilis
ATCC 6633
The Difco Manual
285
MacConkey Broth
Section II
Procedure
Results
Materials Provided
Consult appropriate references.4,5,6
MYP Agar
Egg Yolk Enrichment 50%
Antimicrobic Vial P
References

Glassware
Petri dishes
Autoclave
Incubator (35C)
MYP Agar
1. Suspend 46 grams of MYP Agar in 900 ml distilled or deionized water.
3. Dispense 225 ml into 500 ml flasks.
5. Aseptically add 12.5 ml Egg Yolk Enrichment 50% and 4.1 ml
rehydrated Antimicrobic Vial P (25,000 units of polymyxin B).
Mix thoroughly.
Antimicrobic Vial P
1. Rehydrate with 5 ml sterile water.
1. Mossel, D. A. A., M. J. Koopman, and E. Jongerius. 1967.

Enumeration of Bacillus cereus in foods. Appl. Microbiol.
15:650-653.
2. Donovan, K. O. 1958. A selective medium for Bacillus cereus in
milk. J. Appl. Bacteriol. 21:100-103.
3. Coliner, A. R. 1948. The action of Bacillus cereus and related species on the lechithin complex of egg yolk. J. Bacteriol. 55:777-785.
4. Jeffery, E. J., and S. M. Harmon. 1995. Bacillus cereus, p. 14.
01-14.08. In Bacteriological analytical manual, 8th ed. AOAC
5. Harmon, S. M., J. M. Goepfert, and R. W. Bennett. 1992.
Bacillus cereus, p. 593-604. In C. Vanderzant, and D. F.
Washington, D.C.
6. Andrews, W. 1995. Microbial methods, p. 1-119. In Official methods
of analysis of AOAC International, 16th ed. AOAC International.
Arlington, VA.
Packaging
MYP Agar
Antimicrobial Vial P
Test Procedure
500 g
0810-17
6 x 10 ml
3268-60*
* Store at 2-8C
Bacto MacConkey Broth

Solution:
3.5% solution, soluble in distilled or deionized water;
purple, clear.
Prepared Tubes:
Purple, clear.
Reaction of 3.5%
Solution at 25C:
pH 7.3 0.1
Cultural Response
Prepare MacConkey Broth per label directions. Inoculate the medium and
ORGANISM
Escherichia coli
ATCC
INOCULUM
CFU
RECOVERY
MEDIUM
COLOR
29212* 1,000-2,000 markedly inhibited purple

25922* 100-1,000
good
yellow
GAS
The cultures listed are the minimum that should be used as for performance testing.
286
Uninoculated
tube
Escherichia coli
ATCC 25922
The Difco Manual
Section II
MacConkey Broth
Intended Use
Procedure
Bacto MacConkey Broth is used for cultivating gram-negative,

lactose-fermenting bacilli in water and foods as a presumptive test for
coliform organisms.
Materials Provided
Materials Provided But Not Required

MacConkey Broth is a modification of the original bile salt broth
recommended by MacConkey1 that contained 0.5% sodium taurocholate
and litmus as an indicator. In later publications, 2,3 MacConkey
suggested variations of this formulation using neutral red indicator
instead of litmus. Childs and Allen4 demonstrated the inhibitory effect
of neutral red and substituted the less inhibitory brom cresol purple.
Oxgall in the medium replaces the original sodium taurocholates
to inhibit growth of gram-positive organisms.

Peptone provides amino acids and other growth factors. Lactose is a
carbon energy source for gram-negative lactose-fermenting bacilli.
Oxgall inhibits the growth of gram-positive organisms. Brom Cresol
Purple is the indicator.
Formula
MacConkey Broth
Formula Per Liter
Bacto Oxgall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
MacConkey Broth
g
g
g
g
Glassware
Autoclave
Incubator (35C)
Tubes with closures
Rehydrate with proportionally less water when liquid inocula will
exceed 1 ml.

1. Collect specimens in sterile containers or with sterile swabs
and transport immediately to the laboratory in accordance with
3. Inoculate specimen as appropriate for that specimen.
4. Incubate tubes for 18-24 hours at 35 2C.
5. Examine tubes.
Test Procedure
Precautions
Results
1. For in Laboratory Use.

2. IRRITATING TO EYES, RESPIRATORY SYSTEM AND SKIN.
(US) Avoid contact with skin and eyes. Do not breathe dust. Wear
Lactose-fermenting organisms grow very well in MacConkey Broth

and produce acid, causing the medium to turn yellow. Gas is also
produced. Non-fermenting organisms produce good growth but will
not produce acid or gas.
References
Storage
1. MacConkey, A. 1901. Centr. Bakt. 29:740.

2. MacConkey, A. 1905. Lactose-fermenting bacteria in faeces.
J. Hyg. 5:333-379.
3. MacConkey, A. 1908. Bile salt media and their advantage in some
bateriological examinations. J. Hyg. 8:322-334.
4. Childs, E., and L. A. Allen. 1953. Improved methods for
determining the most probable number of Bacterium coli and of
Streptococcus faecalis. J. Hyg. Camb. 51:468-477.
Store MacConkey Broth below 30C. The powder is very hygoscopic.

Packaging
MacConkey Broth
500 g
0020-17
Expiration Date
The Difco Manual
287
MacConkey Media
Section II
MacConkey Media
Bacto MacConkey Agar . Bacto MacConkey Agar Base
Bacto MacConkey Agar CS . Bacto MacConkey Agar w/o CV
Bacto MacConkey Agar w/o Salt
Intended Use
MacConkey Media are selective and differential plating media mainly
used for the detection and isolation of gram-negative organisms from
clinical,1 dairy,2 food,3,4 water,5 pharmaceutical 6 and industrial7 sources.
Bacto MacConkey Agar is used for isolating and differentiating lactosefermenting from lactose nonfermenting gram-negative enteric bacilli.
Bacto MacConkey Agar Base is used with added carbohydrate in
differentiating coliforms based on fermentation reactions.
Bacto MacConkey Agar CS is used for isolating and differentiating

gram-negative enteric bacilli from specimens containing swarming
strains of Proteus.
Bacto MacConkey Agar w/o CV is used for isolating and differentiating
enteric microorganisms while permitting growth of staphylococci and
enterococci.
Bacto MacConkey Agar w/o Salt is used for isolating and differentiating
gram-negative bacilli while suppressing the swarming of most
Proteus species.
Also Known As
MacConkey Agar is also known as MAC.
MacConkey Agar
Dehydrated Appearance: Pink to pinkish beige, free-flowing,
homogenous.
Solution:
deionized water on boiling; reddish
purple, very slightly to slightly
opalescent.
Prepared Plates:
Pinkish red, slightly opalescent.
Reaction of 5.0%
Solution at 25C:
pH 7.1 0.2
MacConkey Agar Base
Dehydrated Appearance: Pink to pinkish beige, free-flowing,
homogenous.
Solution:
deionized water upon boiling; red,
Prepared Plates:
Red, slightly opalescent without
precipitate.
Reaction of 4.0%
Solution at 25C:
pH 7.1 0.2
MacConkey Agar CS
Dehydrated Appearance: Pinkish beige, homogenous, free-flowing.
Solution:
purple in color, slightly opalescent,
Prepared Plates:
Reddish purple, slightly opalescent,
Reaction of 5.0%
Solution at 25C:
pH 7.1 0.2
288
MacConkey Agar is based on the bile salt-neutral red-lactose agar of

MacConkey.8
The original MacConkey medium was used to differentiate strains of
Salmonella typhosa from members of the coliform group. Formula
modifications improved the growth of Shigella and Salmonella strains.
These modifications included the addition of 0.5% sodium chloride,
decreased agar content, and altered bile salts and neutral red concentrations. The formula improvements gave improved differential
reactions between these enteric pathogens and the coliform group.
MacConkey Agar contains crystal violet and bile salts that inhibit
gram-positive organisms and allow gram-negative organisms to grow.
Isolated colonies of coliform bacteria are brick red in color and may be
surrounded by a zone of precipitated bile. This bile precipitate is due
to a local pH drop around the colony due to lactose fermentation.
Colonies that do not ferment lactose (such as typhoid, paratyphoid and
dysentery bacilli) remain colorless. When lactose non-fermenters grow
in proximity to coliform colonies, the surrounding medium appears as
cleared areas.
MacConkey Agar Base is prepared without added carbohydrates,
which permits their addition either individually or in combination. It is
recommended that carbohydrates such as sucrose or lactose be added
in a concentration of 1% to the basal medium.
MacConkey CS (Controlled Swarming) contains carefully selected
raw materials to reduce the swarming of Proteus species which could
cause difficulty in isolating and enumerating other gram-negative
bacilli.
MacConkey Agar w/o CV (Crystal Violet) is a differential medium
that is less selective than MacConkey Agar. The lack of crystal violet
permits the growth of Staphylococcus and Enterococcus. Staphylococci
produce pale pink to red colonies and enterococci produce compact
tiny red colonies either on or beneath the surface of the medium.
The Difco Manual
Section II
MacConkey Media
MacConkey Agar w/o Salt is a differential medium that restricts

the swarming of Proteus species to aid in the detection and isolation
of enteric microorganisms. In addition, this medium does not contain
crystal violet, allowing Staphyloccocus and Enterococcus species
to grow.

Bacto Peptone and Proteose Peptone are sources of nitrogen and
other nutrients. Lactose is a fermentable carbohydrate. When lactose
is fermented, a local pH drop around the colony causes a color change
in the pH indicator (neutral red) and bile precipitation. Bile Salts, Bile
Salts No. 3 and Crystal Violet are selective agents that inhibit growth
of gram-positive organisms. Bacto Agar is a solidifying agent.
Formula
MacConkey Agar
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 g
MacConkey Agar w/o Salt

Dehydrated Appearance: Pinkish beige, free-flowing,
homogenous.
Solution:
reddish orange, slightly opalescent.
Prepared Plates:
Reddish orange, slightly opalescent.
Reaction of 4.7%
Solution at 25C:
pH 7.4 0.2
Cultural Response
Prepare MacConkey media per label directions. Inoculate and
MacConkey Agar
Enterococcus
faecalis
Escherichia
coli
Proteus
mirabilis
Salmonella
typhimurium
INOCULUM
CFU

MacConkey Agar Base
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Bacto Bile Salts, No. 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.03
Bacto Crystal Violet . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.001
g
g
g
g
g
g
g
MacConkey Agar CS
Formula Per Liter
MacConkey Agar w/o CV

Dehydrated Appearance: Pinkish beige, free-flowing,
homogenous.
Solution:
reddish orange, clear to very slightly
precipitate.
Prepared Plates:
Reddish orange, slightly opalescent
Reaction of 5.2%
Solution at 25C:
pH 7.4 0.2
ATCC
g
g
g
g
g
g
g
ORGANISM

Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Bile Salts, No. 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.03
GROWTH
APPEARANCE
BILE
PPT.
29212* 1,000-2,000 markedly to
25922* 100-1,000
good
pink
12453
100-1,000
good
colorless
14028* 100-1,000
good
colorless
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Bile Salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.03
g
g
g
g
g
g
g
g

Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Bile Salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
g
g
g
g
g
g

Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Bile Salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.075
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
g
g
g
g
g
Precautions
2. For MacConkey Agar w/o CV
The Difco Manual
289
MacConkey Media
Section II

Escherichia coli
ATCC 25922
Uninoculated
plate
MacConkey Agar Base

ORGANISM
ATCC
INOCULUM
CFU
GROWTH
APPEARANCE
Enterococcus 29212* 1,000-2,000 markedly to
faecalis
Escherichia
25922* 100-1,000
good
w/o lactose:
coli
colorless
w/lactose: pink
Proteus
12453 100-1,000
good
colorless
mirabilis
Salmonella
14028* 100-1,000
good
colorless
typhimurium
BILE
PPT.
+
+
MacConkey Agar CS
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
APPEARANCE
BILE
PPT.
Enterococcus 29212* 1,000-2,000 markedly to
faecalis
Escherichia
25922* 100-1,000
good
pink to red
/+
coli
Proteus
12453 100-1,000
good
colorless,
mirabilis
swarming markedly
to completely
inhibited
Salmonella
14028* 100-1,000
good
translucent,
typhimurium
colorless

ORGANISM
ATCC
INOCULUM
CFU

faecalis
Escherichia
25922* 100-1,000
coli
Proteus
12453 100-1,000
mirabilis
Salmonella
14028* 100-1,000
typhimurium
GROWTH
APPEARANCE
BILE
PPT.
good
red
good
pink or red
good
colorless
good
colorless
GROWTH
APPEARANCE
BILE
PPT.
good
red
good
pink to red
ATCC 14028
MacConkey Agar CS
Escherichia coli
ATCC 25922
Uninoculated
plate

ORGANISM
ATCC
INOCULUM
CFU

faecalis
Escherichia
25922* 100-1,000
coli
Proteus
12453 100-1,000
mirabilis
Salmonella
typhimurium
14028* 100-1,000
good
colorless;
swarming markedly
to completely
inhibited
good
colorless
290
Proteus mirabilis
ATCC 12453
ATCC 14028
The Difco Manual
Section II

Storage
Expiration Date
Procedure
Materials Provided
MacConkey Agar
MacConkey Agar Base
MacConkey Agar CS

Glassware
Autoclave
35C incubator
50C waterbath (optional)
Carbohydrate (lactose, sucrose, etc.) (optional)
For MacConkey Agar, MacConkey Agar CS, MacConkey Agar
w/o CV or MacConkey Agar w/o Salt:
MacConkey Agar
50 grams
MacConkey Agar CS
50 grams
52 grams
47 grams
2. Heat to boiling to dissolve completely. Avoid overheating.
3. Autoclave at 121C for 15 minutes. The media may be used without autoclave sterilization if the plates are to be inoculated on the
day of preparation.
4. Cool to 45-50C and dispense into sterile Petri dishes.
5. The surface of the medium should be dry when inoculated. Dry the
plates for 1-2 hours with the lids slightly ajar.
For MacConkey Agar Base:
1. Suspend 40 grams of medium in 1 liter distilled or deionized water.
3. Add 10 grams lactose or other desired carbohydrate before or after
sterilization, depending on heat lability.
4. Autoclave at 121C for 15 minutes. The media may be used without autoclave sterilization if the plates are to be inoculated on the
day of preparation. In this case, boiling the medium gently for 5
minutes is sufficient.
The Difco Manual
MacConkey Media
5. Cool to 45-50C and dispense into sterile Petri dishes.

6. The surface of the medium should be dry when inoculated. Dry the
plates for 1-2 hours with the lids slightly ajar.

For a complete discussion on the isolation and identification of enteric
organisms consult the appropriate references.
Test Procedure
For procedures on the isolation and identification of enteric organisms
consult the appropriate references.
Results
Lactose-fermenting organisms grow as pink to brick-red colonies
with or with out a zone of precipitated bile. Non-lactose fermenting
organisms grow as colorless or clear colonies.
Swarming by Proteus spp. is reduced on MacConkey Agar CS and
MacConkey Agar w/o Salt.
On MacConkey Agar w/o CV and MacConkey Agar w/o Salt, staphylococci produce pale pink to red colonies and enterococci produce
tiny red colonies; these organisms are inhibited on MacConkey Agar
and MacConkey Agar CS.

1. Although MacConkey media are selective primarily for gramnegative enteric bacilli, biochemical and, if indicated, serological
testing using pure cultures are recommended for complete
identification. Consult appropriate references for further
information. 1,3
2. Due to the selective properties of MacConkey Agar CS, some
strains of gram-negative enteric bacilli may be encountered that
fail to grow or grow poorly on this medium. Some strains of grampositive organisms may be encountered that are not inhibited
or only partially inhibited on this medium; some strains of
enterococci may grow on MacConkey Agar CS after prolonged
incubation.
3. Incubation of MacConkey Agar plates under increased CO2 has
been reported to reduce the growth and recovery of a number of
strains of gram-negative bacilli.9
4. For optimal performance, plates prepared from MacConkey Agar
CS should be incubated under aerobic conditions.
References
1. Gray, L. D. 1995. Escherichia, Salmonella, Shigella and
Yersinia, p. 450-456. In P. R. Murray, E. J. Baron, M. A, Pfaller,
F. C. Tenover, and R. H Yolken (ed.), Manual of clinical
Washington, D.C.
2. Flowers, R. S., W. Andrews, C. W. Donnelly, and E. Koenig
(H.M. Wehr, Tech. Comm.). 1992. Pathogens in milk and milk
products, p. 103-212. In R. T. Marshall, (ed.). Standard methods
for the examination of dairy products. 16th ed., American Public
291
MacConkey Sorbitol Agar
Section II

Coliforms-Escherichia coli and its Toxins, p. 325-369. In C.
Vanderzant, and D. F. Splittstoesser (ed.), Compendium of methods
for the microbiological examination of foods, 3rd ed. American
Public Health Association, Washington. D.C.
4. Food and Drug Administration. 1995. Bacteriological analytical
manual, 8th ed. AOAC International. Gaithersburg, MD.
5. Eaton, A. D., L. S. Clesceri, and A.E. Greenberg (ed.). 1995.
6. United States Pharmacopeial Convention, Inc. 1995. The United
8. MacConkey, A. 1905. Lactose-fermenting bacteria in feces.
J. Hyg. 5:333-379.
9. Mazura-Reetz, G. T. Neblett, and J. M. Galperin. 1979.

MacConkey Agar: CO2 vs. ambient incubation. Abst. Ann. Mtg.
American Society for Microbiology. C179.
Packaging
MacConkey Agar
100
500
2
10
g
g
kg
kg
0075-15-3
0075-17-1
0075-07-3
0075-08-2
MacConkey Agar Base
500 g
0818-17-3
MacConkey Agar CS
500 g
2 kg
10 kg
1818-17-1
1818-07-3
1818-08-2
500 g
0470-17-2
500 g
10 kg
0331-17-1
0331-08-2
Bacto MacConkey Sorbitol Agar
Intended Use
modifications used in MacConkey Agar improved the growth of

Shigella and Salmonella strains as well as the differential reactions
between these enteric pathogens and the coliform group. The
modifications included addition of 0.5% sodium chloride, decreased
agar content, and altered bile salts and neutral red concentrations.
Bacto MacConkey Sorbitol Agar is used for isolating and differentiating

enteropathogenic Escherichia coli serotypes.

The original MacConkey medium was used to differentiate strains of
Salmonella typhosa from members of the coliform group. Formula
Uninoculated
plate
Escherichia coli
O157:H7

Dehydrated Appearance: pinkish-beige, free flowing, homogeneous.
Solution:
purple, very slightly to slightly
opalescent.
Prepared Medium:
reddish-purple, slightly opalescent.
Reaction of 5.0%
Solution at 25C:
pH 7.1 0.2 at 25C
Cultural Response
Prepare MacConkey Sorbitol Agar per label directions.
Inoculate plates and incubate at 35 2C for 18-24 hours.
ORGANISM
ATCC
CFU
INOCULUM
RECOVERY
COLONY
COLOR
29212* 1,000-2,000 markedly
inhibited
Escherichia coli 0157:H7
100-1,000
good colorless
Escherichia coli
25922* 100-1,000
good pink-red
BILE
PPT
292
The Difco Manual
Section II
MacConkey Sorbitol Agar is a modification of the formula given by

Rappaport and Henig1 for isolating enteropathogenic Escherichia coli
serotypes 011 and 055. The usefulness of this medium in detecting
E. coli 0157:H7, a human pathogen associated with hemorrhagic colitis,
has been described.2,3,4
This medium employs d-sorbitol rather than lactose for isolating and
differentiating the enteropathogenic E. coli serotypes which tend to be
sorbitol negative. This medium can be used for clinical and food testing.1,5,6

Bacto Peptone and Proteose Peptone are nitrogen sources in the
medium. D-Sorbitol is a fermentable carbohydrate. Many hemorrhagic
E. coli strains will not ferment d-sorbitol and appear as colorless
colonies on MacConkey Sorbitol Agar. Bile salts and crystal violet are
selective agents that inhibit growth of gram-positive organisms.
Neutral red is a pH indicator. Bacto Agar is a gelling agent.

1. Collect specimens in sterile containers or with sterile swabs
and immediately transport to the laboratory in accordance with
3. Inoculate the specimen onto medium appropriate for that specimen.
4. Incubate plates for 18-24 hours at 35 2C.
5. Examine plates.
Test Procedure
Formula

Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.5
d-Sorbitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Bile Salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.03
5. Dry plates for 1-2 hours with the lids slightly ajar. The surface of
the medium should be dry when inoculated.
MacConkey Sorbitol Agar may be used without autoclave
sterilization if the plates are to be used on the day of preparation.
Boil the medium 2-3 minutes before pouring into Petri dishes and
dry before inoculation.
Results
g
g
g
g
g
g
g
g
Sorbitol-fermenting organisms produce pink colonies on MacConkey

Sorbitol Agar. Organisms that do not ferment sorbitol, such as
E. coli 0157:H7, are colorless.

1. The color of sorbitol-positive colonies can fade, making them hard

to distinguish from sorbitol-negative colonies.3
2. Upon prolonged incubation, strains of E. coli 0157:H7 can ferment
sorbitol.3
3. Strains of other organisms that do not ferment sorbitol may grow
on MacConkey Sorbitol Agar. It is necessary to select suspected
colonies for further identification.3
4. The sole use of this medium can cause the microbiologist to miss
other organisms that may be pathogenic.7
5. To isolate E. coli 0157:H7 from clinical specimens, inoculate fecal
specimens and rectal swabs on a small area of one quadrant and
streak for isolation. This will permit development of discrete colonies.
Expiration Date
References
1. Rappaport, F., and E. Henig. 1952. Media for the isolation and
differentiation of pathogenic Escherichia coli (serotypes 0111 and
055). J. Clin. Pathology. 5:361-362.
p. 450-456. In Murray, P.R., E. J. Baron, M.A. Pfaller, F. C.
3. Adams, S. 1991. Screening for verotoxin-producing Escherichia
coli. Clinical Lab Science 4(1):19-20.
4. March, S. B., and S. Ratnam. 1986. Sorbitol-MacConkey
medium for detection of Escherichia coli 0157:H7 associated with
hemorrhagic colitis. J. Clin. Microbiol. 23:869-872.
Coliforms-Escherichia coli and its toxins, p. 325-369. In
C. Vanderzant and D. F. Splittstoesser (ed.), Compendium of
American Public Health Association, Washington. D.C.
Precautions
Storage
Procedure
Materials Provided

Glassware
Autoclave
Incubator (35C)
1.
2.
3.
4.
Suspend 50 grams in 1 liter distilled or deionized water:

Heat to boiling to dissolve completely. Avoid overheating.
The Difco Manual
293
Malonate Broth
Section II
6. Hitchins, A. D., P. Feng, W. D. Watkins, S. R. Rippey, and L. A.

Chandler. 1995. Escherichia coli and the coliform bacteria.
7. Ewing, W. H., and P. R. Edwards. 1954. Isolation and preliminary
Bacto Malonate Broth
identification of Escherichia coli serotypes associated with cases

of diarrhea of the newborn. Public Health Lab. 12:75-81.
Packaging
500 g
0079-17
Formula
Malonate Broth
Formula Per Liter
Intended Use
Bacto Malonate Broth is used for differentiating Enterobacter from
Escherichia based on malonate utilization.

Malonate Broth, prepared according to the formula described by
Leifson1, is a liquid medium containing ammonium sulfate as the only
source of nitrogen and malonate as the only source of carbon. Leifson
was able to demonstrate that the Enterobacter group utilizes malonate
whereas the Escherichia group is unable to grow on the medium.
Malonate Broth is further described for differentiating Enterobacteriaceae in food and dairy products.2,3,4 In some cases, however, the
medium referenced is the modified Edwards and Ewing5 formulation
that contains yeast extract and dextrose. The modification permits
growth of organisms that would otherwise fail on the Leifson medium.

Malonate Broth contains Ammonium Sulfate, which is the sole source
of nitrogen in the medium; Sodium Malonate is the sole source of
carbon. Dipotassium Phosphate and Monopotassium Phosphate
provide buffering capability. Sodium Chloride maintains the osmotic
balance of the medium. Increased alkalinity resulting from malonate
utilization causes the indicator, Brom Thymol Blue, to change color
from green to blue.
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Sodium Malonate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Bacto Brom Thymol Blue . . . . . . . . . . . . . . . . . . . . . . . 0.025
g
g
g
g
g
g
Precautions

Dehydrated Appearance: Light green, free-flowing, homogeneous.
Solution:
deionized water. Solution is green, clear.
Reaction of 0.8%
Solution at 25C:
pH 6.7 0.2
Cultural Response
Prepare Malonate Broth per label directions. Inoculate
the medium with a loopful of test organism and incubate
ORGANISM
Escherichia coli
ATCC
GROWTH
APPEARANCE
13048*
25922*
good
poor to fair
blue
green
294
Uninoculated
tube
ATCC 13048
Escherichia coli
ATCC 25922
The Difco Manual
Section II
Malonate Broth Modified
Storage
Results

Malonate utilization is indicated by a change in the color of the medium

from green to blue:
Positive: Blue
Negative: Green
Expiration Date
The expiration date applies to the product in its intact container. Do not
use a product if it fails to meet specifications for identity and performance.

1. A slight bluing (blue-green) of the medium may occur after prolonged
incubation.6 In such cases, care should be taken in interpreting results.
Procedure
Materials Provided
References
Malonate Broth

Glassware
Autoclave
Incubator (35C)
3. Avoid introducing extraneous carbon and nitrogen.
Specimen Collection
Test Procedure
1. Inoculate tubes with a loopful of test organism.
3. Examine tubes for a change in the color of the medium from
green to blue.
1. Leifson, E. 1933. The fermentation of sodium malonate as a means

of differentiating Aerobacter and Escherichia. J. Bacteriol. 26: 329.
2. Bacteriological Analytical Manual. 1995. 8th ed. AOAC
International. Gaithersburg, MD.
Washington, D.C.
5. Edwards, P. R., and W. H. Ewing. 1962. Enterobacteriaceae. U.S.
Public Health Service Bulletin No. 734:19.
6. Oberhofer, T. R. 1985. Manual of nonfermenting gram-negative
bacteria. Churchill Livingstone, New York, NY.
Packaging
Malonate Broth
100 g
500 g
0395-15
0395-17
Bacto Malonate Broth Modified
Intended Use
Bacto Malonate Broth Modified is used for differentiating

Enterobacteriaceae based on malonate utilization.
Solution:
or deionized water with agitation.
Solution is green, clear.
Reaction of 0.93%
Solution at 25C:
pH 6.7 0.2
Cultural Response
Prepare Malonate Broth Modified per label directions.
Inoculate the medium with a loopful of undiluted organism
ORGANISM
Escherichia coli
Salmonella arizonae
ATCC
INOCULUM
COLOR OF
MEDIUM
13048*
25922*
13314
14028*
undiluted
undiluted
undiluted
undiluted
blue
green
blue
green
The Difco Manual
Also Known As
Malonate Broth Modified conforms with Malonate Broth, Ewing.

Malonate Broth Modified is essentially Malonate Broth to which Yeast
Extract and Dextrose have been added.1 These additional ingredients
initiate growth of some organisms that otherwise would fail to grow on
the unmodified medium and, thus, permit observation of those
organisms malonate activity.
Malonate utilization by microorganisms is indicated by an increase
in alkalinity and development of a deep blue color in the medium.
Malonate utilization forms a basis on which organisms can be
differentiated when testing food products for Enterobacteriaceae.2,3,4
It is useful in the differentiation of Escherichia coli from the
Klebsiella-Enterobacter groups and is considered especially valuable
in the differentiation of Salmonella. The majority of salmonellae do
not utilize malonate whereas Salmonella arizonae does.4,5,6
295
Section II
This medium may be used in conjunction with phenylalanine as

proposed by Shaw and Clarke7 to detect both malonate degradation
and phenylalanine deamination.
Procedure
Yeast Extract and Dextrose provide the vitamins and cofactors, as well
as minimal sources of carbon, required for good growth of a wide
variety of organisms. An organism that can utilize Sodium Malonate
as its carbon source while at the same time using Ammonium Sulfate
as its nitrogen source causes increased alkalinity due to the formation
of sodium hydroxide. This causes the indicator, Brom Thymol Blue, to
change color from green to blue. Some malonate-negative strains
produce a yellow color. This is due to fermentation of the dextrose
alone, producing increased acidity that causes the indicator to turn
yellow at pH 6.0. Dipotassium Phosphate and Monopotassium
Phosphate provide buffering capability. Sodium Chloride maintains the
osmotic balance of the medium.
Glassware
Autoclave
Incubator (35C)
Formula

Formula Per Liter
Test Procedure

Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Sodium Malonate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.25
Materials Provided
1. Suspend 9.3 grams in 1 liter distilled or deionized water and
agitate to dissolve completely.

g
g
g
g
g
g
g
g
Precautions
TARGET ORGAN(S): Lungs, Intestines
Storage
Expiration Date
Results
A color change of the medium to blue indicates that malonate has
been utilized.

1. Some malonate-positive organisms produce only slight alkalinity.
Compare any tube in question with an uninoculated malonate tube.
Any trace of blue color after a 48-hour incubation period denotes a
positive test. Before making a final negative interpretation, be sure
that test tubes have been incubated for 48 hours.7
References
1. Edwards, P. R. and W. H. Ewing. 1962. Enterobacteriaceae. U.S.
Public Health Service Bulletin No. 734:19.
4. Bacteriological Analytical Manual, 8th ed. 1995. AOAC
5. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures handbook, vol. 1. American Society for Microbiology. Washington, D.C.
R. H. Yolken (eds.). 1995. Manual of clinical microbiology,
Packaging
296
500 g
0569-17
The Difco Manual
Section II
Malt Agar
Bacto Malt Agar
Storage
Intended Use
Bacto Malt Agar is used for isolating and cultivating yeasts and molds
from food, and for cultivating yeast and mold stock cultures.

Malt media for yeasts and molds have been widely used for many
years. In 1919, Reddish1 prepared a satisfactory substitute for beer
wort from malt extract. Thom and Church2 used Reddishs medium
for their studies of the aspergilli. Malt Agar was also employed by
Fullmer and Grimes3 for their studies of the growth of yeasts on
synthetic media. Malt Agar is specified in Standard Methods4,5 for
the examination of yeasts and molds.
Principles Of The Procedure

Malt Agar contains Malt Extract which provides the carbon, protein and
nutrient sources required for the growth of microorganisms. Bacto Agar
is a solidifying agent. The acidic pH of Malt Agar allows for optimal
growth of molds and yeasts while restricting bacterial growth.
Formula
Malt Agar
Formula per liter
Bacto Malt Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
Expiration Date
Procedure
Materials Provided
Malt Agar

Glassware
Autoclave
Incubator
1.
2.
3.
4.

Autoclave at 121C for 15 minutes. Cool to room temperature.
To alter the medium to pH 4.5 or pH 3.5, add the quantity of sterile
85% lactic acid USP indicated on the label. (This quantity is specific
to the lot of product.) Do not reheat the medium after adding acid.
Precautions

Uninoculated
plate
Aspergillus niger
ATCC 16404

Solution:
is light to medium amber in color,
Prepared Medium:
Light to medium amber, very
slightly to slightly opalescent.
Reaction of a 4.5%
Solution at 25C:
5.5 0.2
Cultural Response
Prepare Malt Agar per label directions. Inoculate and incubate
plates at 30 2C for 40-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Aspergillus niger
Candida albicans
16404
10231
9763
100-1,000
100-1,000
100-1,000
good
good
good
The Difco Manual
Candida albicans
ATCC 10231
ATCC 9763
297
Malt Extract
Section II
Test Procedure
1. Do not heat the medium after addition of acid, as this will

hydrolyze the agar and reduce its solidifying properties.
3. Fulmer, E. I., and M. J. Grimes. 1923. The growth of yeasts on

synthetic agar media. Bacteriol., 8:585-588.
5. Association of Official Agricultural Chemists. 1995. Official
methods of analysis, 16th ed. Association of Official Agricultural
References
Packaging
1. Abs. Bact., 3:6, 1919.

2. Thom, C., and M. B. Church. 1926. The Aspergilli. Williams and
Wilkins Co., Baltimore, MD.
Malt Agar
Results
Bacto Malt Extract
500 g
10 kg
0024-17
0024-08
enumeration of yeasts, has Malt Extract as one of the main ingredients

in the formula. Several media containing Malt Extract are specified in
standard methods.1,2,3
Intended Use
Bacto Malt Extract is used for preparing microbiological culture
media for the propagation of yeasts and molds.

Malt Extract is obtained from barley. It is a useful ingredient of culture
media designed for the propagation of yeasts and molds. This product
is particularly suitable for yeasts and molds because it contains a high
concentration of carbohydrates, particularly maltose. The approximate
percentage of reducing sugars in Malt Extract is 60-63%. Malt Extract
is generally employed in culture media at concentrations between
10 to 100 grams per liter.
Malt Agar, a medium recommended for the detection and isolation of
yeasts and molds from dairy products, food and as a stock culture
contains Malt Extract. Wort Agar, used for the cultivation and

Malt Extract provides carbon, protein and nutrients for the isolation
and cultivation of yeasts and molds in bacterial culture media.
Precautions
Storage
Store the dehydrated ingredient below 30C. The product is very
Expiration Date
Procedure
Dehydrated Appearance: Medium tan, free-flowing,
homogeneous.
Solution:
deionized water; medium amber,
Reaction of 2%
Solution at 25C:
pH 4.5 -5.5
Cultural Response
Prepare 2% solution. Inoculate tubes with the test organisms.
Incubate tubes at 30 2C for up to three days.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Aspergillus niger
Candida albicans
16404
10231
100-1,000
100-1,000
good
good
The cultures listed are the minimum that should be used.
Materials Provided
Malt Extract

Refer to the final concentration of Malt Extract in the formula of the
medium being prepared. Add Malt Extract as required.

Test Procedure
See appropriate references for specific procedures using Malt Extract.
Results
298
The Difco Manual
Section II
Malt Extract Agar & Malt Extract Broth
References
Gaithersburg, MD.
Bacto Malt Extract Agar

Bacto Malt Extract Broth

Packaging
Malt Extract
100 g
500 g
10 kg
0186-15
0186-17
0186-08
Intended Use
Bacto Malt Extract Agar is used for isolating, cultivating and
enumerating yeasts and molds.
Bacto Malt Extract Broth is used for cultivating yeasts and molds.

Malt Extract Agar
Dehydrated Appearance: Off-white, free-flowing, homogeneous.
Solution:
Solution is very light amber, slightly
opalescent.
Prepared Medium:
Reaction of 3.36%
Solution at 25C:
pH 4.7 0.2
Malt Extract Broth
Dehydrated Appearance: Light beige to beige, free-flowing,
homogeneous.
Solution:
light amber to light amber, clear
Prepared Medium:
Light amber, clear without
Reaction of 1.5%
Solution at 25C:
pH 4.7 0.2
Cultural Response
Prepare Malt Extract Agar or Malt Extract Broth per
label directions. Inoculate and incubate at 30 2C for
18-72 hours.
ORGANISM
ATCC
INOCULUM
CFU
RECOVERY
Aspergillus niger
Candida albicans
16404
10231
9763
100-1,000
100-1,000
100-1,000
good
good
good
The Difco Manual

The use of malt and malt extracts for the propagation of yeasts and
molds is quite common. Reddish 1 described a culture medium
prepared from malt extract that was a satisfactory substitute for wort.
Thom and Church,2 following the formula of Reddish, used Malt
Extract as a base from which they prepared the complete media. Malt
Extract Broth is specified in standard methods for the examination of
yeasts and molds.3,4
Principles Of the Procedure

Malt Extract Agar contains Maltose as an energy source. Dextrin,
a polysaccharide derived from high quality starch, and Glycerol are
included as carbon sources. Peptone is provided as a nitrogen source.
Malt Extract Broth contains Malt Extract Base which provides
the carbon, protein, and nutrient sources required for growth of
microorganisms. Maltose is added as an energy source. Dextrose is
included as a source of fermentable carbohydrate. Yeast Extract
provides the vitamins and cofactors required for growth and additional
sources of nitrogen and carbon.
The acidic pH of Malt Extract Agar and Broth allow for the optimal
growth of molds and yeasts while restricting bacterial growth.
Formula
Malt Extract Agar
Formula per liter
Maltose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.75
Bacto Dextrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.75
Bacto Glycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.35
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.78
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g

Malt Extract Broth
Formula per liter
Malt Extract Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Maltose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.8
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
g
g
g
g
Precautions
299
Mannitol Salt Agar
Section II
Storage
Expiration Date
Results
Procedure
References
Materials Provided
1. Abs. Bact., 3:6, 1919.

2. Thom, C., and M. B. Church. 1926. The Aspergilli. Williams and
Wilkins Co., Baltimore.
3. MacFaddin, J. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, vol. 1. Williams
and Wilkins, Baltimore.
analytical manual, 8th ed. AOAC International. Gaithersburg, MD.
Malt Extract Agar

Malt Extract Broth

Glassware
Autoclave
Incubator
1. Malt Extract Agar: Suspend 33.6 grams in 1 liter of distilled or
deionized water. Heat to boiling to dissolve completely.
Malt Extract Broth: Dissolve 15 grams in 1 liter of distilled or
deionized water.
2. Autoclave at 121C for 15 minutes. Avoid overheating which could
cause a softer medium.
Bacto Mannitol Salt Agar
Test Procedure
Packaging
Malt Extract Agar
500 g
10 kg
0112-17
0112-08
Malt Extract Broth
500 g
10 kg
0113-17
0113-08
Intended Use
pH indicator from red to yellow. Typical pathogenic staphylococci

(coagulase-positive staphylococci) ferment mannitol and form yellow
colonies with yellow zones around the colonies. Typical non-pathogenic
staphylococci do not ferment mannitol and form red colonies.
Bacto Mannitol Salt Agar is used for isolating and differentiating

staphylococci.
Formula
Mannitol Salt Agar

Formula Per Liter
Chapman1 formulated Mannitol Salt Agar to isolate staphylococci by

inhibiting the growth of most other bacteria with a high salt concentration.
He added 7.5% sodium chloride to Phenol Red Mannitol Agar and noted
that pathogenic strains of staphylococci (coagulase-positive staphylococci)
grew luxuriantly and produced yellow colonies with yellow zones in
the surrounding medium. Nonpathogenic staphylococci produced small
red colonies with no color change to the surrounding medium.
Because Mannitol Salt Agar is selective, specimens from heavily
contaminated sources may be streaked onto this medium without
danger of overgrowth2. Mannitol Salt Agar is recommended for isolating
pathogenic staphylococci from clinical specimens,2 from cosmetics,3
and for microbial limit tests.4

Bacto D-Mannitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.025
g
g
g
g
g
g
Precautions
Storage
Mannitol Salt Agar contains Proteose Peptone No. 3 and Beef Extract
as sources of carbon, nitrogen, vitamins and minerals. D-Mannitol is
the carbohydrate source. Sodium Chloride, in high concentration,
inhibits most bacteria other than staphylococci. Phenol Red is the
pH indicator. Bacto Agar is the solidifying agent.
Bacteria that grow in the presence of a high salt concentration and
ferment mannitol produce acid products which turn the phenol red

300
Expiration Date
The Difco Manual
Section II
Mannitol Salt Agar
Procedure
Results
Materials Provided
Staphylococci will grow on this medium while the growth of most other
bacteria will be inhibited. Coagulase-positive staphylococci will
produce luxuriant growth of yellow colonies with yellow zones around
them. Coagulase negative staphylococci will produce small red
colonies with no color change to the medium surrounding them.
Mannitol Salt Agar

Glassware
Petri dishes
Autoclave
Incubator (35C)
References
1. Collect specimens as appropriate for the specimen and transport

guidelines.
1. Chapman, G. H. The significance of sodium chloride in studies of

staphylococci. J. Bacteriol. 50:201.
2. Kloos, W. E., and T. L. Bannerman. 1995. Staphylococcus and
Micrococcus. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C.
Microbiology methods for cosmetics, p. 23.01-23.12. In
Gaithersburg, MD.
Test Procedure
Packaging
Inoculate specimen onto medium as a primary isolation or inoculate

isolated colonies onto medium for differentiation.
Mannitol Salt Agar
100 g
500 g
Uninoculated
plate
0306-15
0306-17
ATCC 12228

Dehydrated Appearance: Light pink, free-flowing, homogeneous.
Solution:
is red, slightly opalescent.
Prepared Medium:
Pinkish red, slightly opalescent.
Reaction of 11.1%
Solution at 25C:
pH 7.4 0.2
Cultural Response
Prepare Mannitol Salt Agar per label directions. Inoculate and
incubate at 35 2C for 18-24 to 48 hours. A yellow zone
surrounding a colony indicates mannitol has been fermented.
ORGANISM
Enterobacter
aerogenes
Escherichia
coli
Proteus
mirabilis
Staphylococcus
aureus
Staphylococcus
epidermidis
The Difco Manual
ATCC
INOCULUM
CFU
GROWTH
13048* 1,000-2,000
APPEARANCE OF
MEDIUM AND COLONY
marked to
complete inhibition
25922* 1,000-2,000
marked to
complete inhibition
12453 1,000-2,000 partial inhibition
25923* 100-1,000
good
12228* 100-1,000
good
yellow colony
with yellow zone
red colony with no
zone of color change
ATCC 25923
301
Marine Agar 2216 & Marine Broth 2216
Section II
Bacto Marine Agar 2216

Bacto Marine Broth 2216
Intended Use
Bacto Marine Agar 2216 and Bacto Marine Broth 2216 are used for
cultivating heterotrophic marine bacteria.
Marine bacteria are present in nutrient sea water by the millions per ml
and are essential to the life cycle of all marine flora and fauna. The
enumeration and activity of marine bacteria are important to the food
industry for the conservation of marine life. Marine Agar 2216 and
Marine Broth 2216 are prepared according to the formula of ZoBell1
The media contain all of the nutrients necessary for the growth of
marine bacteria. The media contain minerals that nearly duplicate the
major mineral composition of sea water,2 in addition to Bacto Peptone
and Yeast Extract that provide a good source of nutrients.
In the use of Marine Agar 2216, the conventional pour plate and spread
plate techniques of enumeration are used. For the pour plate technique,
the agar must be cooled to 42C before inoculation because of the
thermo-sensitive nature of most marine bacteria. In the spread plate
technique, the agar is poured while hot and allowed to cool and
solidify before inoculation. This latter method was reported by Buck
and Cleverdon3 to give higher counts than the pour plate method
because of the increased growth of the thermo-sensitive bacteria.
Sizemore and Stevenson4 used Marine Agar 2216 routinely as the
upper nutrient layer of a marine agar-milk agar double-layer plate.
This two layer plate was developed for isolating proteolytic marine
bacteria. Marine Agar 2216 was also used in studies characterizing a
marine bacterium associated with Crassostrea virginica (the Eastern
Oyster).5

Bacto Peptone and Yeast Extract provide nitrogen, vitamins and minerals.
The high salt content helps to simulate sea water. Numerous minerals
are also included to duplicate the major mineral composition of sea
water. Bacto Agar is a solidifying agent.
Formula
Marine Agar 2216
Formula Per Liter
302
g
g
g
g
g
g

Marine Broth 2216
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Ferric Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.45
Sodium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.24
Sodium Bicarbonate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.16
Potassium Bromide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.08
Strontium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.034
Boric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.022

Sodium Silicate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.004
Sodium Fluoride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0024
Ammonium Nitrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0016
Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.008
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g
g
g
g
g
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Ferric Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19.45
Magnesium Chloride Dried . . . . . . . . . . . . . . . . . . . . . . . . 5.9
Sodium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.24
Sodium Bicarbonate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.16
Potassium Bromide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.08
Strontium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.034
Boric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.022
Sodium Silicate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.004
Sodium Fluoride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0024
Ammonium Nitrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0016
Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.008
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
Precautions
2. Marine Broth 2216
TARGET ORGAN(S): Kidneys, Nerves.
Storage
is very hygroscopic. Keep container tightly closed. Store prepared plates
at 2-8C.
Expiration Date
The Difco Manual
Section II
Marine Agar 2216 & Marine Broth 2216
Procedure
4. Marine Agar 2216: Cool agar to 45-50C in a waterbath.

5. Dispense.
Materials Provided
Marine Agar 2216
Marine Broth 2216

Test Procedure

Autoclave
Waterbath (45-50C)
Glassware
Incubator (20-25C)
Shaker
Results
References
Marine Agar 2216 - 55.1 grams;
Marine Broth 2216 - 37.4 grams.
1. ZoBell, C. E. 1941. Studies on marine bacteria. I. The cultural

requirements of heterotrophic aerobes. J. Mar. Res. 4:42-75.
2. Lyman, J., and R. H. Fleming. 1940. Composition of sea water. J.
Marine Res. 3:134.
3. Buck, J. D., and R. C. Cleverdon. 1960. The spread plate as a method
for the enumeration of marine bacteria. Limnol. Oceanogr. 5:78.
Uninoculated
plate
Vibrio harveyi
ATCC 14126

Marine Agar 2216
Dehydrated Appearance: Light beige with a few dark particles,
free flowing, homogeneous.
Solution:
opalescent with a precipitate.
Prepared Plates:
Light amber, slightly opalescent to
Reaction of 5.51%
Solution at 25C:
pH 7.6 0.2
Marine Broth 2216
Dehydrated Appearance: Light beige with a few dark particles,
free flowing.
Solution:
deionized water on boiling. Solution is light
amber, slightly opalescent with a precipitate.
Prepared Flasks:
Light amber, slightly opalescent with a precipitate.
Reaction of 3.74%
Solution at 25C:
pH 7.6 0.2
Cultural Response
Prepare Marine Agar 2216 per label directions. Inoculate and
incubate at 20-25C for 40-72 hours.
ORGANISM
ATCC
INOCULUM CFU
GROWTH
Vibrio fischeri
Vibrio harveyi
7744
14126
100-1,000
100-1,000
good
good
Prepare Marine Broth 2216 per label directions. Dispense 50 ml

amounts in 250 ml Erlenmeyer flasks. Inoculate and incubate at
20-25C on a shaker for 40-72 hours.
ORGANISM
ATCC
INOCULUM CFU
GROWTH
Vibrio fischeri
Vibrio harveyi
7744
14126
100-1,000
100-1,000
good
good
The Difco Manual
303
Maximum Recovery Diluent
Section II
4. Sizemore, R. K., and L. H. Stevenson. 1970. Method for the

isolation of proteolytic marine bacteria. Appl. Microbiol. 20:991-992.
5. Weiner, R. M., A. M. Segall, and R. R. Colwell. 1985.
Characterization of a marine bacterium associated with Crassostrea
virginica (the Eastern Oyster). Appl. Environ. Microbiol. 49:83-90.
Packaging
Marine Agar 2216
500 g
0979-17
Marine Broth 2216
500 g
0791-17
Bacto Maximum Recovery Diluent
Formula
Intended Use
Bacto Maximum Recovery Diluent is an isotonic diluent containing a
low level of peptone used for maintaining the viability of organisms
during dilution procedures.

Standard methods for the microbiological examination of foodstuffs
require sample dilution to be carried out accurately to estimate the
number of microorganisms. Diluents consisting of sterile saline,
phosphate buffer solutions and distilled water have all been shown to
have a lethal action on a wide range of organisms.1,2
The presence of low levels of peptone in the diluent at a pH of 7.0 0.2
affords protection for bacteria for at least one hour during the dilution
stage.3,4 The presence of peptone also allows accurate quantitative
procedures to be performed with minimal reductions in viable count
in the diluent.
Low levels of peptone help protect organisms in the diluent. Sodium
Chloride maintains proper osmotic pressure.
Formula per liter

Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Precautions
Storage
Expiration Date
Procedure
Materials Provided

Dehydrated Appearance: Cream, free-flowing, homogeneous.
Solution:
or deionized water, colorless, clear.
Prepared Medium:
Colorless, clear.
Reaction of 0.95%
Solution at 25C:
pH 7.0 0.2
Prepare the medium per label directions. Inoculate tubes with
the test organism. At time zero and after 30 minutes, subculture
1.0 ml aliquots into Tryptic Soy Agar, using the pour plate
technique. Incubate plates at 35C for 18-24 hours.
Escherichia coli
ATCC

2. Dispense into final containers and cap loosely.
25922*
25923*
Consult appropriate references for dilution procedures when testing

foods.1,2,3.4
Results
RECOVERY AFTER 30 MINUTES
no significant reduction
no significant reduction
References

304
Test Procedure
Cultural Response
ORGANISM
Glassware
Autoclave
1. DeMello, G. C., I. S. Danielson, and J. S. Kiser. 1951. The Toxic

effect of buffered saline on the viability of Brucella abortus.
J. Lab. Clin. Med. 37:579-583.
The Difco Manual
Section II
McBride Listeria Agar
2. Gunter, S. E. 1954. Factors determining the viability of selected

microorganisms in inorganic media. J. Bacteriol. 67:628-634.
3. Straka, R. P., and J. L. Stokes. 1957. Rapid destruction of bacteria
in commonly used diluents and its eliminations. Appl. Microbiol.
5:21-25.
4. Patterson, J. T., and J. A. Cassells. 1963. An examination of the
Bacto McBride Listeria Agar
value of adding peptone to diluents used in the bacteriological

testing of bacon curing brines. J. Appl. Bacteriol. 26:493-497.
Packaging
500 g
5 kg
1897-17
1897-03
Intended Use
Bacto McBride Listeria Agar is used for isolating Listeria
monocytogenes with or without the addition of blood.

reported food-borne outbreak of listeriosis was in 19853 and, since then,

Dehydrated Appearance: Light tan, homogeneous with
soft lumps.
Solution:
is light to medium amber, very
Prepared Medium
without Blood:
opalescent.
Prepared Medium
with Blood:
Cherry red, opaque.
Reaction of 4.6%
Solution at 25C:
pH 7.3 0.2
Cultural Response
Prepare McBride Listeria Agar per label directions. Inoculate
ORGANISM
ATCC
INOCULUM
CFU
RECOVERY
29212* 1,000-2,000
marked to
complete inhibition
Escherichia coli
25922* 1,000-2,000
marked to
complete inhibition
Listeria monocytogenes 19114 100-1,000
good
The Difco Manual

coleslaw, pasteurized milk, Mexican-style cheese, pat and pickled pork
tongue. The organism has been isolated from commercial dairy and
other food processing plants. Listeria species are ubiquitous in nature,
being present in a wide range of unprocessed foods, soil, sewage,
Listeria species grow over a pH range of 5.0-9.6 and survive in food
products with pH levels outside of these parameters.7 Listeria species
are microaerophilic, gram-positive, asporogenous, non-encapsulated,
at 20C.
potentially containing Listeria are streptococci, especially the
McBride Listeria Agar is prepared according to the formulation of
McBride and Girard,9 who originally described the medium and its use
in the selective isolation of Listeria from experimentally mixed
cultures. The medium has been recommended for isolating Listeria
from clinical specimens,10 raw milk11,12 and food samples.13
When the medium is used as a blood agar, a narrow zone of
beta-hemolysis may be evident around and under Listeria colonies.

Tryptose and Beef Extract provide nitrogen, vitamins and minerals.
Partial selectivity is provided by Lithium Chloride, Glycine and
Phenylethanol, which aid in suppressing both gram-positive and
gram-negative bacteria other than Listeria. If increased inhibition of
fungi is also needed, cycloheximide can be added after autoclaving.
Formula
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Glycine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Lithium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Phenylethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g
305
Precautions
ORGAN(S): Blood, Face, Muscles, Nerves, Urogenital.
Storage
Store the dehydrated medium at 2-8C. The powder is very hygroscopic.
Expiration Date
Procedure
Materials Provided

Autoclave
Waterbath (45-50C)
Defibrinated blood (optional)
Cycloheximide (optional)
Petri dishes
Incubator (35C)
1.
2.
3.
4.
5.

Cool medium to 45-50C in a waterbath.
To enhance selectivity and/or differentiation, aseptically add
cycloheximide (0.2 grams/liter) and/or sterile defibrinated blood
to the medium. Mix well.

306
Section II

Test Procedure
When testing clinical specimens for Listeria, inoculate directly onto
primary plating media and McBride Listeria Agar.10
When isolating Listeria from raw milk and food samples, refer to
appropriate references.12,13
Results
Observe colonies under oblique transmitted light. Listeria colonies
should display a grey to blue color with a ground glass appearance.
References
6. Patel, J. R., C. A. Hwang, L. R. Beuchat, M. P. Doyle, and R. E.
Brackett. 1995. Comparison of oxygen scavengers for their ability
to enhance resuscitation of heat-injured Listeria monocytogenes.
J. Food Prot. 58:244-250.
J. Lovett. 1992. Listeria, p. 637-663. In C. Vanderzant, and D. F.
Washington, D.C.
9. McBride, M. E., and K. F. Girard. 1960. A selective method for
the isolation of Listeria monocytogenes from mixed bacterial
populations. J. Lab. Clin. Med. 55:153-157.
10. Pezzlo, M. (ed.). 1992. Aerobic bacteria, p. 1.4.8. In H. D. Isenberg
(ed), Clinical microbiology procedures handbook, vol. 1. American
11. Hayes, P. S., J. C. Feeley, L. M. Graves, G. W. Ajello, and
D. W. Fleming. 1986. Isolation of Listeria monocytogenes from
raw milk. Appl. Environ. Microbiol. 51:438-440.
1993. Pathogens in milk and milk products. In R. T. Marshall (ed),
The Difco Manual
Section II
McClung Toabe Agar
Packaging
500 g
0922-17
McClung Toabe Agar

Bacto McClung Toabe Agar Base . Bacto Egg Yolk Enrichment 50%
Intended Use
Bacto McClung Toabe Agar Base is used with Bacto Egg Yolk
Enrichment 50% for isolating and detecting Clostridium perfringens
in foods based on the lecithinase reaction.

McClung and Toabe1 formulated a medium for isolating C. perfringens
from foods. With the addition of 50% egg yolk emulsion, C. perfringens
and a few other Clostridium species show the lecithinase reaction.
C. perfringens is found in raw meats, poultry, dehydrated soups and
sauces, raw vegetables and other foods and food ingredients, but
occurrences of food borne illness are usually associated with cooked
meat or poultry products.2 Spores of some strains that may resist heat
during cooking germinate and grow in foods that are not adequately
refrigerated.3 Enumerating the microorganism in food samples plays a
role in epidemiological investigation of outbreaks of food borne illness.2
McClung Toabe Agar Base contains Proteose Peptone as a source of

carbon, nitrogen, vitamins and minerals. Dextrose is the carbohydrate
source. Sodium Chloride maintains the osmotic balance of the medium.
Magnesium Sulfate provides divalent cations and sulfate. Sodium
Phosphate Dibasic and Potassium Phosphate Monobasic maintain
pH balance and provide a source of phosphates. Bacto Agar is the
solidifying agent. Egg Yolk Enrichment 50% provides egg yolk
lecithin. Lecithinase-producing clostridia, such as C. perfringens,
hydrolyze the lecithin and produce opaque halos.
McClung Toabe Agar Base

homogeneous.
Solution:
is light amber, opalescent, with a
precipitate.
Prepared Medium:
Light yellow, smooth, opaque.
Reaction of 7.5%
Solution at 25C:
pH 7.6 0.2
Appearance:
Canary yellow, opaque liquid with
a resuspendable precipitate.
Cultural Response
McClung Toabe Agar Base with Egg Yolk Enrichment 50%
Prepare McClung Toabe Agar Base with Egg Yolk Enrichment
50% per label directions. Inoculate and incubate the plates at
35 2C anaerobically for 18-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
LECITHINASE
REACTION
Clostridium
perfringens
Clostridium
perfringens
Staphylococcus
aureus
Staphylococcus
epidermidis
12919
100-1,000
good
opaque halo
12924
100-1,000
good
opaque halo
25923*
100-1,000
good
opaque halo
14990
100-1,000
good
none
The Difco Manual
Formula
Formula Per Liter
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
g
g
g
g
g
g
g

Sterile concentrated egg yolk emulsion
Precautions
Storage
Store the dehydrated McClung Toabe Agar Base medium below
30C. The dehydrated medium is very hygroscopic. Keep container
tightly closed.
307
Methyl Red and Voges-Proskauer Tests
Store the Egg Yolk Enrichment 50% at 2-8C.
Expiration Date
Section II

1. Ready for use.
2. Shake gently to resuspend precipitate.

Procedure
Test Procedure
Materials Provided

Results

Glassware
Petri dishes
Autoclave
Incubator, anaerobic (35C)
1. Suspend 75 grams of McClung Toabe Agar Base in 1 liter distilled
or deionized water.
3. Dispense 90 ml amounts into flasks.
5. Aseptically add 10 ml Egg Yolk Enrichment 50% to each flask of
prepared agar base.
6. Mix thoroughly.
7. Pour into sterile Petri dishes in approximately 15 ml amounts.
References
1. McClung, L. S., and R. Toabe. 1947. The egg yolk plate reaction
for the presumptive diagnosis of Clostridium sporogenes and certain
species of the gangrene and botulinum groups. J. Bact. 53:139.
2. Labbe, R. G., and S. M. Harmon. 1992. Clostridium perfringens,
p. 623-635. In C. Vanderzant, and D. F. Splittstoesser (ed.),
foods, 3rd ed. American Public Health Association, Washington, D.C.
3. Rhodehamel, E. J., and S. M. Harmon. 1995. Clostridium
perfringens, p. 16.01- 16.06. In Bacteriological analytical manual,
8th ed. AOAC International, Gaithersburg, MD.
Packaging
500 g
0941-17
12x10 ml
6x100 ml
3347-61*
3347-73*
*Store at 2-8C

Bacto MR-VP Medium . SpotTest Voges-Proskauer Reagent A
SpotTest Voges-Proskauer Reagent B
TM
Intended Use
Bacto MR-VP Medium is used for differentiating coliform organisms
based on the methyl red and Voges-Proskauer tests.
SpotTest Voges-Proskauer Reagents A and B are used for determining
the VP reaction of bacteria.
Also Known As
MR-VP Medium is also known as Methyl Red-Voges Proskauer
Medium.

1
In 1915, Clark and Lubs demonstrated that the colon-aerogenes family of bacteria could be divided into two groups based on their action in
308
a peptone and dextrose medium. When tested with the pH indicator

methyl red, the coli group produced high acidity while the
aerogenes group produced a less acid reaction. The test to detect
high-acid end products is known as the Methyl Red (MR) test. The test
to detect less-acid end products is based on the procedure described by
Voges and Proskauer in 1898.2 A color reaction occurs when certain
cultures, incubated in a medium containing peptone and dextrose, are
treated with potassium hydroxide and exposed to air. This reaction
detects the formation of acetylmethylcarbinol and is known as the
Voges-Proskauer (VP) Test.
The MR and VP tests appear in the identification scheme for the
Enterobacteriaceae, 3 which are important isolates in clinical microbiology,3 as well as in the microbiology of foods and dairy products.4,5
The Difco Manual
Section II
The MR and VP tests are used to complete and confirm the identification
of Escherichia coli. 4,5,6

MR-VP Medium contains Buffered Peptone as a carbon and nitrogen
source for general growth requirements. Dextrose is a fermentable
carbohydrate.
by the mixed acid pathway and produce acidic end products (pH < 4.4),
such as lactic, acetic and formic acids. Other bacteria metabolize
pyruvate by the butylene glycol pathway and produce neutral end
products (pH > 6.0), one of which is acetoin (acetylmethylcarbinol). In
the MR test, the pH indicator methyl red detects acidic end products.7
In the VP test, acetoin is oxidized in the presence of oxygen and
potassium hydroxide (KOH) to diacetyl, which produces a red color.8
Members of the Enterobacteriaceae convert glucose to pyruvate by

the Embden-Meyerhof pathway. Some bacteria metabolize pyruvate

MR-VP Medium
Dehydrated Appearance: Very light to light beige, free-flowing,
homogeneous.
Solution:
amber, clear.
Prepared Medium:
Light amber, clear.
Reaction of 1.7%
Solution at 25C:
pH 6.9 0.2
SpotTest Voges-Proskauer Reagent A
Appearance:
Yellow to dark amber, clear solution
inside a glass ampule contained in a
plastic dispenser.
Appearance:
Colorless, clear solution inside
a glass ampule contained in a
plastic dispenser.
Uninoculated
tube
ATCC 13048
Escherichia coli
ATCC 25922
MR-VP Medium
Cultural Response
MR-VP Medium, SpotTest Voges-Proskauer Reagent A
or SpotTest Voges-Proskauer Reagent B
Prepare MR-VP Medium per label directions. Inoculate
and incubate at 35 2C for 24-48 hours or up to 5 days.
Determine the methyl red and Voges-Proskauer test reactions.
ORGANISM
ATCC
INOCULUM
Enterobacter 13048* undiluted

aerogenes
Escherichia 25922* undiluted
coli
GROWTH
APPEARANCE
MR TEST
VP TEST
good
/yellow
+/red
good
+/red
/no change
Enterobacter
aerogenes
ATCC 13048
MR Test
The Difco Manual
Enterobacter
aerogenes
ATCC 13048
VP Test
Escherichia coli
ATCC 25922
MR Test
Escherichia coli
ATCC 25922
VP Test
309
The addition of -naphthol (SpotTest Voges-Proskauer Reagent A)

before KOH (SpotTest Voges-Proskauer Reagent B) enhances the
sensitivity of the test.8
Formula
MR-VP Medium
Formula Per Liter
Buffered Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 g
Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g

Formula Per Liter
-Naphthol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 g
Ethyl Alcohol (absolute) . . . . . . . . . . . . . . . . . . . . . . . . . 1,000 ml

Formula Per Liter
Potassium Hydroxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400 g
Distilled Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1,000 ml
Precautions
2. SpotTest Voges-Proskauer Reagent A
HIGHLY FLAMMABLE. IRRITANT. HIGHLY FLAMMABLE.
Avoid contact with skin and eyes. Do not breathe mist. Keep away
from sources of ignition. No smoking. Keep container tightly closed.
Target Organs: Liver, Blood.
CORROSIVE. CAUSES SEVERE BURNS. Avoid contact with
skin and eyes. Do not breathe mist. Wear suitable protective clothing.
Storage
Store the dehydrated MR-VP Medium below 30C. The powder is very
Store SpotTest Voges-Proskauer Reagents A and B at 15-30C.
Protect from light.
310
Section II
Expiration Date
Procedure
Materials Provided
MR-VP Medium

Glassware
Autoclave
Incubator (35 2C)
Test tubes, 13 x 100 mm
Methyl red indicator7
MR-VP Medium
2. Distribute into test tubes. Autoclave at 121C for 15 minutes.
Methyl Red Indicator
1. Dissolve 0.1 gram of methyl red in 300 ml of 95% ethyl alcohol.
2. Add sufficient distilled or deionized water to make 500 ml.

Test Procedure
1. Inoculate MR-VP Medium with growth from a single colony.
2. Incubate at 35 2C for 48 hours.
3. Test as follows.
Methyl Red Test
1. Transfer 2.5 ml of the MR-VP Medium culture to a tube (13 x 100 mm).
2. Add 5 drops of methyl red indicator and observe for a color change.
VP Test
1. Transfer 2.5 ml of the MR-VP Medium culture to a tube
(13 x 100 mm).
2. Add 0.3 ml (6 drops) of SpotTest Voges-Proskauer Reagent A
(5% -naphthol).
3. Add 0.1 ml (2 drops) of SpotTest Voges-Proskauer Reagent B
(40% KOH).
4. Gently agitate the tube and let stand for 10 to 15 minutes.
5. Observe for a color change.
Other Methods
SpotTest Voges-Proskauer Reagent A and Reagent B are suitable for
use in other modifications of the Voges-Proskauer test requiring the
use of these reagents.
The Difco Manual
Section II
Alternate VP Tube Method

1. Inoculate MR-VP Medium with the test organism and incubate at
2. Aseptically transfer 1 ml of the incubated MR-VP Medium culture
to a clean test tube.
3. Add 15 drops of Voges-Proskauer Reagent A followed by 5 drops
of Voges-Proskauer Reagent B.
4. Shake gently to aerate.
5. Examine for the appearance of a red color within 20 minutes.
6. If the 24-hour test is negative, repeat the test with a 48-hour culture
of the test organism. If equivocal results are obtained, repeat the
test with cultures incubated for 5 days at 25-30C.
7. VP reagents must be added in the order and the amounts specified

or a weak-positive or false-negative reaction may occur. A weakpositive reaction may be masked by a copper-like color which may
form due to the reaction of KOH and -naphthol.8
8. Read the VP test within 1 hour of adding the reagents. The KOH
and -naphthol may react to form a copper-like color, causing a
potential false-positive interpretation.8
9. Due to the possible presence of acetoin, diacetyl or related
substances in certain raw materials,9 the use of media low in
these substances (such as MR-VP Medium) is recommended for
this test.
Rapid Micro Method

1. Inoculate 0.2 ml of MR-VP Medium with the test organism.
2. Incubate for 4 hours at 35C.
3. Add 0.1 ml of 0.3% creatine solution.
4. Add 5 drops of Voges-Proskauer Reagent A followed by 2 drops of
Voges-Proskauer Reagent B.
5. Shake gently to aerate.
6. Examine for the appearance of a red color within 20 minutes.
1. Clark, W. M., and H. A. Lubs. 1915. The differentiation of bacteria

of the colon- aerogenes family by the use of indicators. J. Infect.
Dis. 17:160-173.
2. Voges, O., and B. Proskauer. 1898. Z. Hyg. 28:20-22.
3. Farmer, J. J., III. 1995. Enterobacteriaceae: Introduction and
identification, p. 438-449. In P. R. Murray, E. J. Baron, M. A.
Washington, D.C.
6 Association of Official Analytical Chemists. 1995. Bacteriological analytical manual, 8th ed. AOAC International,
Gaithersburg, MD.
handbook, sup. 1, 1.19.48. American Society for Microbiology,
Washington, D.C.
handbook, sup. 1, 1.19.58. American Society for Microbiology,
Washington, D.C.
9. Barritt, M. M. 1936. The intensification of the Voges-Proskauer
reaction by the addition of alpha-naphthol. J. Pathol. 42:441-454.
Results
Methyl Red (MR) Test
Positive:
Negative:
Bright red color.

Yellow-orange color.
Note: If the test is negative, continue to incubate the broth without

added reagent; repeat the test after an additional 18 to 24 hours incubation.
Voges-Proskauer (VP) Test
Positive:
Red color.
Negative:
No red color.

1. Results of the MR and VP tests need to be used in conjunction with
other biochemical tests to differentiate genus and species within
the Enterobacteriaceae.
2. A precipitate may form in the potassium hydroxide reagent solution.
This precipitate has not been shown to reduce the effectiveness of
the reagent.
3. Most members of the family Enterobacteriaceae give either a positive
MR test or a positive VP test. However, certain organisms such as
Hafnia alvei and Proteus mirabilis may give a positive result for
both tests.
4. Incubation time for the Methyl Red test cannot be shortened by
increasing the glucose concentration in the medium or by heavily
inoculating the broth.7
5. Incubate MR-negative tests for more than 48 hours and test again.
(See Results section.)
6. Read the VP test at 48 hours. Increased incubation may produce
acid conditions in the broth that will interfere with reading the
results.8
The Difco Manual
References
Packaging
MR-VP Medium
100 g
500 g
2 kg
0016-15
0016-17
0016-07
SpotTest Voges-Proskauer
Reagent A
50 x 0.75 ml
3558-26
SpotTest Voges-Proskauer
Reagent B
50 x 0.75 ml
3559-26
311
Micro Assay Culture Agar & Micro Inoculum Broth
Section II
Bacto Micro Assay Culture Agar

2.
Bacto Micro Inoculum Broth
Intended Use
Bacto Micro Assay Culture Agar is used for cultivating lactobacilli
and other organisms used in microbiological assays.
Bacto Micro Inoculum Broth is used for preparing the inoculum of
lactobacilli and other microorganisms used in microbiological assays
of vitamins and amino acids.

Three types of media are used in the microbiological assay of vitamins:
1. Maintenance Media, which preserve the viability and sensitivity of
the test culture for its intended purpose;

Micro Assay Culture Agar
Dehydrated Appearance: Light tan to tan, free-flowing,
homogeneous.
Solution:
Prepared Medium:
opalescent.
Reaction of 4.7%
Solution at 25C:
pH 6.7 0.2
Solution:
deionized water. Light to medium
amber in color, clear to very slightly
precipitate.
Prepared Medium:
Reaction of 3.7%
Solution at 25C:
pH 6.7 0.2
Cultural Response
Prepare Micro Assay Culture Agar and Micro Inoculum Broth
per label directions. Inoculate tubes with test organisms.
Incubate Micro Assay Culture Agar at 35 2C for 18-48 hours,
incubate Micro Inoculum Broth at 35 2C for 18-24 hours.
ORGANISM
ATCC
INOCULUM
CFU
Enterococcus hirae
804 100-1,000
Lactobacillus casei subsp. rhamnosus 7469 100-1,000
Lactobacillus delbrueckii subsp. lactis 7830 100-1,000
Lactobacillus plantarum
8014 100-1,000
312
GROWTH
good
good
good
good
Inoculum Media, which condition the test culture for immediate use; and,
3. Assay Media, which permit quantitation of the vitamin under test.
Assay media contain all factors necessary for optimal growth of
the test organism except the single essential vitamin to be determined.
Micro Assay Culture Agar is used for maintaining stock cultures of
lactobacilli and other test microorganisms. This medium is also used
for general cultivation of lactobacilli.
Micro Inoculum Broth is used for cultivating lactobacilli and
preparing the inoculum for microbiological assays.

Proteose Peptone No. 3 provides nitrogen and amino acids in both Micro
Assay Culture Agar and Micro Inoculum Broth. Yeast Extract is a vitamin
source. Dextrose is a carbon source. Monopotassium phosphate is a
buffering agent. Sorbitan monooleate complex (Micro Inoculum
Broth) and Polysorbate 80 (Micro Assay Culture Agar) act as emulsifiers.
Bacto Agar is a solidifying agent (Micro Assay Culture Agar).
Formula
Formula Per Liter
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Polysorbate 80 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
g
g
g
g
g
g

Formula Per Liter
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sorbitan Monooleate Complex . . . . . . . . . . . . . . . . . . . . . 0.1
g
g
g
g
g
Precautions
3. Take care to avoid contamination of media and glassware used
in microbiological assay procedures. Extremely small amounts
of foreign material may be sufficient to give erroneous results.
chemicals must be used.
Storage
Store the dehydrated media below 30C. The media are very
Expiration Date
The Difco Manual
Section II
Microbial Content Test Agar
Procedure
Materials Provided

Glassware
Autoclave
Incubator
Inoculating needle
0.9% NaCl
3. Dispense 10 ml amounts into 16-20 mm diameter tubes.
5. Agitate tubes prior to solidification to disperse the flocculent
precipitate.
2. Dispense 10 ml amounts into tubes of 16-20 mm diameter.
Stock Cultures
1. Prepare stock cultures in triplicate on Micro Assay Culture Agar,
inoculating tubes using a straight-wire inoculating needle.
2. Incubate tubes at 30-37C for 18-24 hours.
3. Store at 2-8C.
4. Transfer cultures at weekly or twice-monthly intervals.
Assay Inoculum
1. Subculture from a 16-24 hour stock culture of lactobacilli in Micro
Assay Culture Agar into a 10 ml tube of Micro Inoculum Broth.
2. Incubate at 35-37C for 16-24 hours or as specified in the assay
procedure.
3. Centrifuge the culture and decant the supernatant.
4. Resuspend cells in 10 ml of sterile 0.9% NaCl solution or sterile
single strength basal assay medium.
5. Wash the cells by centrifuging and decanting the supernatant two
additional times unless otherwise indicated.
6. Dilute the washed suspension 1:100 with sterile 0.9% single

strength basal assay medium or as indicated. Where applicable,
adjust inoculum concentration according to limits specified in
AOAC1 or US Pharmacopeia.2

Prepare samples for assay according to references given in the specific
assay procedure. Dilute assay samples to approximately the same
Test Procedure
For a complete discussion of vitamin assay methodology, refer to
appropriate procedures.1,2
Results
For test results on vitamin assay procedures, refer to appropriate
procedures.1,2

1. Test organisms used in assay procedures must be cultured and
maintained on media recommended for this purpose.
2. Follow assay directions exactly. The age, preparation and size of
inoculum are extremely important factors in obtaining a satisfactory
assay result.
3. Although other media and methods may be used successfully for
maintaining cultures and preparing inocula, uniformly good results
will be obtained if the methods described are followed exactly.
4. Aseptic technique should be used throughout the microbiological
assay procedure.
5. The use of altered or deficient media may create mutants
having different nutritional requirements. Such organisms will not
produce a satisfactory test response.
References
1. Association of Official Analytical Chemists. 1995. Official methods
Arlington, VA.
Packaging
100 g
500 g
0319-15
0319-17
500 g
0320-17
Bacto Microbial Content Test Agar
Intended Use
Bacto Microbial Content Test Agar is recommended for the detection
of microorganisms on surfaces sanitized with quaternary ammonium
compounds.
Also Known as
Tryptic Soy Agar with Lecithin and Polysorbate 80 (TSALT) and
Casein Soy Peptone Agar with Polysorbate 80 and Lecithin are
The Difco Manual
common terms for Microbial Content Test Agar. Tween 80 is also

known as Polysorbate 80.

Microbial Content Test Agar is a modification of Tryptic Soy Agar
with Lecithin and Tween 80. The formulation is recommended for
determining the sanitation efficiency of containers, equipment and work
areas (environmental monitoring). The Lecithin and Tween in the
formula inactivate some preservatives that may inhibit bacterial
313
Section II
growth, reducing preservative carryover. 1 The formulation is

recommended for the Aerobic Plate Count (Microbial Limit Test) for
water miscible cosmetic products containing preservatives.1
Precautions
Microbial Content Test Agar contains Tryptone and Soytone which

provide the carbon and nitrogen sources required for growth of a wide
variety of organisms. Lecithin and Polysorbate 80 are added to
neutralize surface disinfectants.2,3,4 Lecithin is added to neutralize
quaternary ammonium compounds. Polysorbate 80 is incorporated to
neutralize phenols, hexachlorophene, formalin and, with lecithin,
ethanol.5 Sodium Chloride provides osmotic equilibrium. Bacto Agar
has been incorporated into this medium as a solidifying agent.
Storage
Formula
Procedure

Formula Per Liter

Expiration Date
Materials Provided
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Lecithin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.7
Polysorbate 80 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g

Glassware
Autoclave

Dehydrated Appearance: Beige, free-flowing, homogeneous,
may appear moist.
Solution:
4.57% solution soluble in distilled
or deionized water on boiling with
frequent gentle swirling. At
approximately 50C, solution is
medium amber in color, slightly
opalescent, with a resuspendable
precipitate. At higher temperatures,
it is more opalescent.
Prepared Medium:
Medium amber, slightly opalescent,
Reaction of 4.57%
Solution at 25C
pH 7.3 0.2
Cultural Response
Prepare medium per label directions. Test Microbial Content
Test Agar in parallel with Plate Count Agar. Inoculate liquid
media with test organisms and pour plates. After the plates dry,
apply disks impregnated with varying dilutions of a quaternary
ammonium compound to the medium surface. Incubate plates
at 35 2C for 40-48 hours and inspect for zones of inhibition.
INOCULUM
CFU
ORGANISM
ATCC
Escherichia
coli
11229 100-1,000
Staphylococcus
aureus
6538P 100-1,000
GROWTH
smaller zone of inhibition

of growth compared to
Plate Count Agar
smaller zone of inhibition
of growth compared to
Plate Count Agar
Interpretation: The smaller zones of inhibition indicate neutralization

of quaternary ammonium compounds by Microbial Content Test Agar.
314
1. Suspend 45.7 g in 1 liter of distilled or deionized water.

2. Heat to boiling to dissolve completely with frequent careful
agitation to dissolve, 1-2 minutes.

Consult appropriate references.1,4
Test Procedures
Microbial Content Test Agar is used in a variety of procedures.
Consult appropriate references for further information.1,4
Results

may be encountered that fail to grow or grow poorly on the medium.
2. The effectiveness of preservative neutralization with this medium
depends on both the type and concentration of the preservative(s).
References
1. Orth, D. S. 1993. Handbook of Cosmetic Microbiology. Marcel
Dekker, Inc., New York, NY.
2. Quisno, R., I. W. Gibby, and M. J. Foter. 1946. A neutralizing
medium for evaluating the germicidal potency of the quaternary
ammonium salts. Am. J. Pharm. 118:320-323.
3 Erlandson, A. L., Jr., and C. A. Lawrence. 1953. Inactivating
medium for hexachlorophene (G-11) types of compounds and some
substituted phenolic disinfectants. Science 118:274-276.
4. Brummer, B. 1976. Influence of possible disinfectant transfer on
Staphylococcus aureus plate counts after contact sampling. App.
The Difco Manual
Section II
Middlebrook 7H9 Broth & 710 Agar, Mycobacteria 711 Agar, Middlebrook ADC Enrichment, Middlebrook OADC Enrichment & Enrichment w/WR1339 & Glycerol
5. Favero (chm.). 1967. Microbiological sampling of surfaces - a state

of the art report. Biological Contamination Control Committee,
American Association for Contamination Control.
Packaging
500 g
2 kg
0553-17
0553-07
Bacto Middlebrook 7H9 Broth . Bacto Middlebrook 7H10 Agar

Bacto Mycobacteria 7H11 Agar . Bacto Middlebrook ADC
Enrichment . Bacto Middlebrook OADC Enrichment . Bacto
Middlebrook OADC Enrichment w/WR 1339 . Bacto Glycerol
Intended Use
Bacto Middlebrook 7H9 Broth is used with Bacto Middlebrook ADC

Enrichment and Bacto Glycerol or Bacto Tween 80 for cultivating
pure cultures of mycobacteria and preparing the tubercle emulsion for
susceptibility testing.

tuberculosis each year.1-3
Because mycobacteria grow more rapidly in a broth medium, primary
culture of all specimens in a broth medium such as Middlebrook 7H9
Broth is recommended.6
There are two types of solid culture media for the primary isolation
of mycobacteria, those that have coagulated egg as a base
(Lowenstein formulations) and those that have an agar base
(Middlebrook formulations).
Bacto Middlebrook 7H10 Agar is used with Bacto Middlebrook OADC

Enrichment and Bacto Glycerol for isolating, cultivating and susceptibility testing of mycobacteria. The complete, prepared medium is also
available.
Bacto Middlebrook 7H10 Agar is also used with Bacto Middlebrook
OADC Enrichment w/WR 1339 to demonstrate cording in differentiating
Mycobacterium tuberculosis from atypical mycobacteria.
Bacto Mycobacteria 7H11 Agar is used with Bacto Middlebrook
OADC Enrichment and Bacto Glycerol for isolating, cultivating and
susceptibility testing of fastidious strains of mycobacteria. The complete,
prepared medium is also available.

Middlebrook 7H9 Broth
Dehydrated Appearance: Light beige, free-flowing, homogenous.
Solution:
amber, clear.
Prepared Medium:
Colorless to very light amber, clear.
Reaction of 0.47%
Solution at 25C:
6.6 0.2
Middlebrook 7H10 Agar
Dehydrated Appearance: Light beige with green tint,
free-flowing, homogenous.
Solution:
is light amber with a slight green tint,
Prepared Medium:
Light amber, slightly opalescent
Reaction of 1.9%
Solution at 25C:
pH 6.6 0.2
The Difco Manual
Egg-base media:
1. Support a wide variety of groups and species of mycobacteria;
2. Provide mycobacterial growth that can be used for niacin testing;
3. Have long shelf lives when refrigerated.3
Mycobacteria 7H11 Agar

Dehydrated Appearance: Light beige with a green tint,
free-flowing, homogenous.
Solution:
light yellowish green, slightly opalescent.
Prepared Medium:
Light amber with a greenish tint,
slightly opalescent, no precipitate.
Reaction of 0.21%
Solution:
pH 6.6 0.2
Middlebrook OADC Enrichment
Appearance:
Light amber, clear solution.
Middlebrook ADC Enrichment
Appearance:
Very light to light amber, clear solution.
Middlebrook OADC Enrichment w/WR 1339
Appearance:
Light amber, clear solution.
Glycerol
Identity Test:
ID positive; IR Spectrum comparable
to reference Glycerin.
Appearance:
Colorless, clear, syrupy liquid.
315
Section II
Cultural Response
Middlebrook 7H9 Broth with Middlebrook ADC Enrichment
35 2C under approximately 10% CO2 for up to 21 days.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
6841
13950
12478
25177
19981
100-300
100-300
100-300
100-300
100-300
good
good
good
good
good
Middlebrook 7H10 Agar with Middlebrook OADC Enrichment

Mycobacteria 7H11 Agar with Middlebrook OADC Enrichment
Prepare medium per label directions or use prepared tubes.
Inoculate and incubate at 35 2C under approximately 10%
CO2 for up to 21 days.
ATCC
ORGANISM
Escherichia coli
INOCULUM
CFU
GROWTH
25922* 1,000-2,000 markedly

inhibited
6841
100-300
good
13950
100-300
good
12478
100-300
good
25177
100-300
good
19981
100-300
good
Middlebrook 7H10 Agar with Middlebrook OADC Enrichment

w/WR 1339
35 2C under approximately 10% CO2 for up to three weeks.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
27294
100-1,000
good
*This culture is available as a Bactrol Disk and should be used
Agar-base media:
1. Tend not to liquefy in the presence of contaminating proteolytic
organisms;3
2. Are recommended for specimens from nonsterile sites7 because
colonies of mycobacteria can be viewed in a clear medium after
10-12 days incubation using a stereo microscope even if contaminating organisms are present;
3. Retain exact concentrations of added drugs because the medium is
solidified with agar rather than by inspissation of the egg. Also,
there is less drug inactivation when egg ingredients are absent.
Middlebrook 7H10 Agar is prepared according to Middlebrook, Cohn,
Dye, Russell and Levy.4 This medium contains a low concentration of
malachite green, which may be preferable for primary isolation.
Mycobacteria 7H11 Agar is a modification of Middlebrook 7H10 Agar
Special as recommended by Cohn, Waggoner and McClately.5 Cohn
et al. demonstrated that the addition of an enzymatic digest of casein
stimulates growth of the more fastidious strains of Mycobacterium
tuberculosis and provides improved susceptibility testing.

L-Glutamic Acid, Ammonium Sulfate, Biotin, Sodium Citrate and
Pyridoxine supply growth factors. Magnesium Sulfate, Ferric Ammonium Sulfate, Zinc Sulfate and Copper Sulfate are sources of trace ions.
Disodium Phosphate and Monopotassium Phosphate help to maintain
the pH of the medium. Malachite Green inhibits contaminating
organisms. Pancreatic Digest of Casein, a good source of nitrogen and
carbon, increases the recovery of isoniazid-resistant mycobacteria
on Mycobacteria 7H11 Agar. Glycerol enhances the growth of
Mycobacterium avium as well as other Mycobacterium species.7 Bacto
Middlebrook OADC and ADC Enrichments contain Dextrose and Oleic
Acid as carbon sources. Albumin Fraction V, Bovine and Catalase (Beef)
are growth factors. WR 1339, Triton encourages the demonstration
of cording in Mycobacterium tuberculosis.8
Formula
Formula Per Liter
Ammonium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
L-Glutamic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
Pyridoxine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.001
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0005
Ferric Ammonium Citrate . . . . . . . . . . . . . . . . . . . . . . . . 0.04
Calcium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0005
Zinc Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.001
Copper Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.001
g
g
g
g
g
g
g
g
g
g
g
g

Formula Per Liter
Uninoculated
tube
316
ATCC 6841
Ammonium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . .
Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0.5
1.5
1.5
0.4
g
g
g
g
The Difco Manual
Section II
Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.025

Zinc Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.001
Copper Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.001
L-Glutamic Acid (Sodium Salt) . . . . . . . . . . . . . . . . . . . . . 0.5
Pyridoxine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . 0.001
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0005
Malachite Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.00025
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g
g
g
g

g
g
g
g
g
g
g
g
g
g
g
g
Storage

Formula Per Liter
Pancreatic Digest of Casein . . . . . . . . . . . . . . . . . . . . . . . . . 1
L-Glutamic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4
Pyridoxine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.001
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0005
Ammonium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Malachite Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.001
g
g
g
ml

Formula Per Liter
Oleic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05
Albumin Fraction V, Bovine . . . . . . . . . . . . . . . . . . . . . . . . . 5
Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Catalase (Beef) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.004
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.85
Distilled water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
g
g
g
g
g
ml

Formula Per Liter
Oleic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05
Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Catalase (Beef) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.004
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.85
Distilled water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
WR 1339, Triton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.25
Glycerol
Formula Per Liter
Glycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 %
Precautions
2. Middlebrook 7H9 Broth
The Difco Manual
Expiration Date
Procedure
Materials Provided

Formula Per Liter
Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Catalase (Beef) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.003
Distilled water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

Store Middlebrook Enrichments at 2-8C.

Glycerol

Glassware
Autoclave
Incubator (35C)
Tween 80 (optional)
g
g
g
g
g
ml
g

1. Dissolve 4.7 grams in 900 ml of distilled or deionized water
containing 2 ml of Glycerol (or 0.5 g Tween 80, if desired).
3. Cool the medium to 45C.
4. Aseptically add 100 ml Middlebrook ADC Enrichment. Mix well.
1. Suspend 19 grams in 900 ml of distilled or deionized water
containing 5 ml Glycerol.
5. Aseptically add 100 ml Middlebrook OADC Enrichment or
Middlebrook OADC Enrichment w/WR 1339. Mix well.
317
Section II
Milk Agar

1. Suspend 21 grams in 900 ml distilled or deionized water containing
5 ml glycerol.
5. Aseptically add 100 ml Middlebrook OADC Enrichment. Mix well.

Test Procedure
1. Inoculate the specimen onto the medium.
2 Incubate tubes for up to eight weeks.
3 Examine tubes for growth.
Results

Mycobacterium tuberculosis. Clin. Microbiol. News. 17:65-69.
4. Middlebrook, G., M. L. Cohn, W. B. Dye, W. B. Russell, Jr.,
and D. Levy. 1960. Microbiologic procedures of value in
tuberculosis. Acta. Tubercul. Scand., 38:66.
5. Cohn, M. L., R. F. Waggoner, and J. K. McClatchy. 1968. The
7H11 Medium for the cultivation of mycobacteria. Am. Rev.
Resp. Dis., 98:295.
6. Tenover, F. C., J. T. Crawford, R. E. Huebner, L. J. Geiter,
C. R. Horsburgh, Jr., and R. C. Good. 1993. The resurgence
of tuberculosis: is your laboratory ready? J. Clin. Microbiol.
31:767-770.
7. Isenberg, H. D. (ed.). 1994. Clinical microbiology procedures handbook, sup. 1. American Society for Microbiology, Washington, D.C.
8. Am. Rev. Resp. Dis., 97:1133.

morphology is dependent on the species isolated.
Packaging
500 g
0713-17
500 g
20 tubes
100 tubes
0627-17
0627-39
0627-79
500 g
20 tubes
100 tubes
0838-17
0838-39
0838-79
Negative culture results do not rule out active infection by mycobacteria.

Some factors responsible for unsuccessful cultures are:
1. The specimen was not representative of the infectious material,
i.e., saliva instead of sputum;
2. The mycobacteria were destroyed during digestion and decontamination of the specimen;
3. Gross contamination interfered with the growth of the mycobacteria;
4. Proper aerobic and increased CO2 tension were not provided
during incubation.
References
molecular genetic insights. Clin. Microbiol. Rev. 8:496-514.
Bacto Milk Agar

w/ WR 1339
Glycerol
2 x 20 ml
0714-64
12 x 20 ml
6 x 100 ml
0722-64
0722-73
6 x 20 ml
0801-63
100 g
500 g
0282-15
0282-17
Leptospira species. Excretion of these organisms can increase the bulk

milk count by 105 organisms/ml.
Intended Use
1
Bacto Milk Agar is recommended by the British Standards Institute

and the International Dairy Federation for the enumeration of microorganisms in liquid milk, ice cream, dried milk and whey.

Liquid milk is a highly perishable foodstuff with a shelf life of only
5-10 days after pasteurization. Contamination of raw milk may arise
from either the soiled or diseased udder or inadequately cleaned milking
or storage equipment. Bovine mastitis or udder inflammation may
cause contamination with Staphylococcus aureus, Streptococcus
agalactiae, Escherichia coli or, more rarely, Yersinia enterocolitica and
318
Poor cleaning of the milking equipment may cause contamination with

micrococci, streptococci, coliforms or heat resistant Bacillus strains,
giving an increase of the bulk milk count of >5 x 104 organisms/ml.
Spoilage of pasteurized or raw milk by proteolytic psychrotrophic
bacteria can occur on prolonged storage below 7C.
Milk Agar conforms to the EEC Commission for the examination of
ice cream.2 Milk Agar is recommended for performing plate count tests
on milks, rinse waters, milk products and ice cream.3
Tryptone and Yeast Extract provide essential nutrients while Skim Milk
Powder is a source of casein. Dextrose is the carbon energy source.
The Difco Manual
Section II
Milk Agar
Proteolytic bacteria will be surrounded by a clear zone from the

conversion of casein into soluble nitrogenous compounds.1
Formula
Milk Agar
Formula Per Liter
Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Skim Milk Powder (antibiotic free) . . . . . . . . . . . . . . . . . . . 1
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.5
g
g
g
g
g
Precautions
1. For In Laboratory Use.

Hot plate
Autoclave
Dilution tubes containing 1/4-strength Ringers solution
Petri dishes
1% hydrochloric acid or 10% acetic acid
1. Suspend 22 grams in 1 liter distilled or deionized water and boil
gently to dissolve completely.
2. Dispense 10-12 ml per tube. Cap loosely.
4. Pour Milk Agar into Petri dishes for the spread plate technique or
allow tubes to cool to 45C for the pour plate technique.
Storage
Expiration Date
Procedure
Materials Provided
Milk Agar
Test Procedure
Total counts may be carried out using either pour plates or surface
counting techniques.
1. Prepare milk dilutions of 1/10, 1/100, 1/1,000 in 1/4-strength
Ringers solution. Use this inoculum within 15 minutes.
2. Pour Plates: Pipette 1 ml of each dilution into Petri dishes.
Add 10-12 ml of molten Milk Agar, cooled to 45C, and mix
thoroughly.
Spread Plates: Spread 1 ml of milk dilution over the surface of
the solidified medium in a Petri dish.
Results
Select plates containing 10-300 colonies. Results are expressed as

colonies per ml of product tested.

Solution:
Prepared Medium:
Reaction of 2.2%
Solution at 25C:
pH 6.9 0.1
Cultural Response
Prepare medium per label directions. Inoculate using the pour
plate technique and incubate at 30C for up to 72 hours.
ORGANISM
Lactobacillus casei
Lactococcus lactis
Streptococcus thermophilus
ATCC
INOCULUM
CFU
GROWTH
9595
19435
25923*
19258
30-300
30-300
30-300
30-300
good
good
good
good
Proteolytic psychrotrophic colonies may be enhanced by flooding the

plates with a solution of 1% hydrochloric acid or 10% acetic acid.
Pour off the excess acid solution and count the colonies surrounded
by clear zones.
References
1. Microbiological examination for dairy purposes. Diluents,
media and apparatus and their preparation and sterilisation.
BS4285, Sec. 1.2.
2. Klose, J. 1968. Susswaren. 14:778-782.
3. Dept. of Health. 1987. Memo.139/Foods.
Packaging
Milk Agar
500 g
5 kg
1859-17
1859-03

The Difco Manual
319
Minerals Modified Glutamate Broth
Section II
Bacto Minerals Modified Glutamate Broth
Intended Use
Bacto Minerals Modified Glutamate Broth is used for enumerating
coliform organisms in water.
Also Known As
Grays Minerals Modified Glutamate Broth

Gray1 described a simple formate-lactose-glutamate medium that could
be used as an alternative to MacConkey Broth for the presumptive
identification of coliform bacteria in water. Grays original medium
gave fewer false positive results than MacConkey Broth, was suitable
for use at 44C, and gave low volumes of gas.
The medium was improved2 by the addition of ammonium chloride
that, by replacing ammonium lactate, resulted in a doubling of the gas
volume. The addition of B complex vitamins, certain amino-acids and
magnesium ions resulted in an increased rate of fermentation. Comparative trials of the modified glutamate medium and MacConkey
Broth3 with chlorinated and unchlorinated waters showed that Grays
Minerals Modified Glutamate Broth gave significantly higher numbers
of positive results (acid and gas production) for coliform organisms
and Escherichia coli. This was especially apparent after 48 hours of
incubation but was also clearly seen with unchlorinated water samples
after only 24 hours incubation. For chlorinated water samples, results
with the two media were comparable. After 18-24 hours incubation,
Minerals Modified Glutamate Medium gave significantly fewer false

Dehydrated Appearance: Very light beige, free flowing,
homogeneous.
Solution:
deionized water on gentle warming.
Solution is purple, clear.
Reaction of 1.77%
Solution at 25C:
pH 6.7 0.1
(containing 0.25 grams of ammonium chloride per 100 ml)
Cultural Response
Prepare Minerals Modified Glutamate Broth per label
ORGANISM
Enterobacter
aerogenes
Enterococcus
faecalis
Escherichia
coli
Salmonella
typhimurium
ATCC
CFU
13048* 30-300
19433* 1,000
positive reactions. Clostridium perfringens, a common cause of false

positive reactions in MacConkey media, is unable to grow in a minimalglutamate based medium.
A major feature of Minerals Modified Glutamate Medium is its
superiority in initiating growth of Escherichia coli after exposure to
chlorine when incubated for 48 hours. In view of the known resistance
to chlorination of some viruses, the ability to isolate coliform bacteria
that survive marginal chlorination provides an additional safety factor
in water treatment.
In a comparison to Lauryl Tryptose Lactose Broth4, Minerals Modified
Glutamate Medium gave superior isolation of Escherichia coli after
48 hours incubation by the multiple tube method, especially in waters
containing small numbers of organisms. Minerals Modified Glutamate
Medium is the medium of choice for the detection of fecal contamination
in chlorinated drinking water supplies in Great Britain.4
Abbiss et al.5 compared Minerals Modified Glutamate Medium and
three other enrichment broths for the enumeration of coliform organisms
present in soft cheese, cooked meat and pat. Minerals Modified
Glutamate Medium was superior in sensitivity to Lauryl Sulfate
Tryptose Broth, MacConkey Broth and Brilliant Green Bile Broth.
Minerals Modified Glutamate Broth has been used in the modified
direct plate method for enumeration of Escherichia coli biotype 1 in
foods.6 According to this method, 15 grams of agar are added per liter
of single strength broth before autoclaving. The medium is poured in
12-15 ml amounts into sterile Petri dishes. This resuscitation agar is
used for the recovery of damaged cells from frozen or dried foodstuffs.
GROWTH/GAS PRODUCTION
Good growth and turbidity

Acid (yellow) production with gas
No visible growth
25922* 30-300
Good growth and turbidity

Acid (yellow) production with gas
14028* 30-300
Good growth and turbidity.
Negative for acid and gas.
Sodium Glutamate and Sodium Formate are the basis of a defined

minimal medium for the enumeration of coliform organisms in water.
Lactose is the carbohydrate source in Minerals Modified Glutamate
Broth. The addition of B complex vitamins, certain amino acids, and
magnesium ions allows an increased rate of fermentation. Phosphate
acts as a buffering agent. The addition of Ammonium Chloride allows
increased gas production by the test organism. Bromocresol Purple is
present as a pH indicator.
Formula
Formula Per Liter
Sodium Glutamate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4
Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Formate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.25
L-cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.02
L(-) Aspartic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.024
L(+) Arginine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.02
Thiamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.001
Nicotinic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.001
Pantothenic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.001
Magnesium Sulfate Heptahydrate . . . . . . . . . . . . . . . . . . . 0.1
Calcium Chloride Dihydrate . . . . . . . . . . . . . . . . . . . . . . 0.01
Bromocresol Purple . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01
g
g
g
g
g
g
g
g
g
g
g
g
g
g
Final Ph 6.7 0.1 at 25C
320
The Difco Manual
Section II
Precautions
Storage
Expiration Date
Procedure
1. 5 x 1 ml of sample into 5 x 5 ml of single-strength medium;

2. 5 x 1 ml of a 1:10 dilution of the sample into 5 x 5 ml of singlestrength medium.
Incubate the tubes at 35 2C. Examine after 18-24 hours incubation
and again at 48 hours,
Results
All tubes demonstrating acid production, indicated by the medium
turning yellow, and gas, either in the inverted fermentation vial or by
effervescence on shaking, may be regarded as presumptive positive
reactions. Each presumptive positive tube should be confirmed in
Brilliant Green Bile 2%, as well as with additional biochemical tests.
The most probable number of organisms in 100 ml of the original
water sample can be calculated using the following table.7
Materials Provided
Quantity of Water in Each Tube
Number of Tubes Used

Ammonium chloride
Fermentation vials
Autoclave
Incubator (35C)
1. Suspend 17.7 grams in distilled or deionized water, the amount of
water depending on the strength of medium desired:
Single-strength medium - 1 liter of water;
Double-strength medium - 500 ml of water.
2. Add 2.5 grams of ammonium chloride per liter and mix well.
3. Heat gently to dissolve completely.
4. Dispense into tubes as listed below and place an inverted fermentation vial into each tube:
Double strength medium - 1 x 5 ml;
Double strength medium - 5 x 10 ml;
Single strength medium - 10 x 5 ml.
5. Autoclave at 115-116C for 10 minutes. Before opening the
autoclave, allow the temperature to drop below 75C to avoid
entrapped air bubbles in the inverted fermentation vials.

Follow laboratory procedure for specimen collection.
Test Procedure
The multiple tube method is used for the enumeration of Escherichia
coli and coliform organisms using Minerals Modified Glutamate Broth.
For good quality water, inoculate the water sample into the medium in
the following volumes:
1. 50 ml of sample into 50 ml of double-strength medium;
2. 5 x 10 ml of sample into 5 x 10 ml of double-strength medium.
For more polluted waters, inoculate the water sample into the medium
in the following volumes:
The Difco Manual
Number of Tubes
Giving Positive
Reaction
50 ml
10 ml
Most Probable Number (MPN)

of coliforms in 100 ml in sample
0
0
0
0
0
0
1
1
1
1
1
1
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
4
5
7
2
3
6
9
16
+18

1. The performance of the medium is significantly affected by pH.
Avoid overheating the broth. Check the pH of each lot before
proceeding with testing.
2. Due to the nutritional requirements of the organisms, some organisms other than coliform bacteria may grow in the medium with
production of acid and gas. Test all presumptive-positive tubes to
confirm the presence of Escherichia coli.
References
1. Gray, R. D. 1959. Formate Lactose Glutamate: A chemically
defined medium as a possible substitute for MacConkey Broth in
the presumptive coliform examination of water. J. Hyg., Camb.
57:249-265.
2. Gray, R. D. 1964. An improved formate-lactose-glutamate medium
for the detection of Escherichia coli and other coliform organisms
in water. J. Hyg., Camb. 62:495-508.
3. P. H. L. S. Standing Committee on the Bacteriological
Examination of Water Supplies. 1968. Comparison of
MacConkey Broth, Teepol Broth and Glutamic Acid Media for
the enumeration of coliform organisms in water. J. Hyg., Camb.
65:67-82.
4. Joint Committee of the P. H. L. S. and the Standing Committee
of Analysts. 1980. A comparison between Minerals Modified
Glutamate Medium and Lauryl Tryptose Lactose Broth for the
enumeration of Escherichia coli and coliform organisms in water
by the multiple tube method. J. Hyg., Camb. 85:35-48.
321
Minimal Agar Davis & Minimal Broth Davis w/o Dextrose
Section II
5. Abbiss, J. S., J. M. Wilson, R. M. Blood, and B. Jarvis. 1981. A

comparison of Minerals Modified Glutamate Medium with other
media for the enumeration of coliforms in delicatessen foods.
J. Appl. Bact. 51:121-127.
6. Holbrook, R., J. M. Anderson, and A. C. Baird-Parker. 1980.
Modified Direct Plate Method for counting Escherichia coli in
foods. Food Technol. in Aust. 32:78- 83.
7. Departments of the Environment, Health & Social Security,

and P.H.L.S. 1982. The Bacteriological Examination of Drinking
Water Supplies, Report on Public Health and Medical Subjects
No. 71. HMSO, London.
Packaging
500 g
1850-17
Bacto Minimal Agar Davis

Bacto Minimal Broth Davis w/o Dextrose
Intended Use
Bacto Minimal Agar Davis is used for isolating and characterizing

nutritional mutants of Escherichia coli.
Minimal Agar Davis
Solution:
is medium amber, very slightly to
Prepared Medium:
Reaction of 2.66%
Solution at 25C:
pH 7.0 0.2
Minimal Broth Davis w/o Dextrose
Solution:
colorless, clear.
Prepared Medium:
Colorless, clear.
Reaction of 1.06%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Minimal Agar Davis
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Escherichia coli
Escherichia coli
6883
9637
100-1,000
100-1,000
good
good

ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Bacillus subtilis
Escherichia coli
Escherichia coli
6633
6883
9637
100-1,000
100-1,000
100-1,000
good
good
good
322
Bacto Minimal Broth Davis w/o Dextrose is used with added dextrose
in isolating and characterizing nutritional mutants of Escherichia coli
and Bacillus subtilis.

Lederberg1 described the Davis formulation for Minimal Agar Davis.
Minimal Broth Davis w/o Dextrose is the same formulation without
dextrose and agar. Both media support the growth of nutritional
mutants of E. coli while Minimal Broth Davis w/o Dextrose with added
dextrose also supports the growth of nutritional mutants of B. subtilis.
Lederberg1 described two techniques for isolating nutritional mutants
of E. coli, one by random isolation and the other by delayed enrichment.
Both Lederberg1 and Davis2 described a third technique using penicillin.
Nutritional mutants of B. subtilis can be isolated by these three
techniques and by a modification of the penicillin technique described
by Nester, Schafer and Lederberg.3
After the mutants are isolated, they are characterized biochemically by
growth in minimal broth supplemented with specific growth factors or
groups of growth factors. It is generally best to classify mutants
according to their requirements for amino acids, vitamins, nucleic acids
or other substances. This is done by supplementing the minimal
medium with Vitamin Assay Casamino Acids plus tryptophane, or a
mixture of water soluble vitamins, alkaline-hydrolyzed yeast, nucleic
acid or yeast extract, depending on the particular mutants desired. The
supplemented minimal broth is inoculated with a slightly turbid
suspension of the mutant colonies and incubated for 24 hours at 35C.
Growth with Vitamin Assay Casamino Acids indicates a vitamin
requirement. When a major growth factor group response in obtained,
the characterization is carried further by the same general procedure to
subgroups and finally to individual growth substances.

Minimal Agar Davis and Minimal Broth Davis w/o Dextrose contain
citrate and phosphates as buffers. Ammonium Sulfate is the carbon
source. Magnesium is a cofactor for many metabolic reactions.
Minimal Agar Davis contains Dextrose as the carbohydrate energy
source; Bacto Agar is the solidifying agent.
The Difco Manual
Section II
Minimal Agar Davis & Minimal Broth Davis w/o Dextrose
Formula
Minimal Agar Davis

Formula Per Liter
Minimal Agar Davis

Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g

Formula Per Liter
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
g
g
g
g
g
Precautions
2. Minimal Broth Davis w/o Dextrose
Storage
Expiration Date
Procedure
Materials Provided
Minimal Agar Davis

Glassware
Autoclave
Incubator (35C)
The Difco Manual

4. Aseptically add 10 ml 10% dextrose solution at room temperature.
5. Mix thoroughly.

Test Procedure
Random Technique
1. Irradiate a cell suspension of wild type E. coli.
2. Dilute the suspension 100-500 times.
3. Culture on a complete agar medium containing all the necessary
growth requirements.
4. Incubate the cultures at 35C for 24 hours.
5. Select isolated colonies and inoculate into Minimal Broth Davis
and a nutritionally complete broth.
7. Observe growth in both media.
Delayed Enrichment Method
1. Prepare plates of Minimal Agar Davis by pouring a 15-20 ml base
layer in a 95 mm sterile Petri dish followed by a 5 ml seed layer.
2. Inoculate with a diluted irradiated E. coli suspension.
3. Pour a 5-10 ml layer of uninoculated Minimal Agar Davis over the
seed layer.
4. Incubate for 24 hours or longer to allow for the growth of
prototroph cells (wild type cells).
5. Pour a layer of a complete agar medium over the minimal agar
medium to develop the mutant cells.
Penicillin Method
1. Wash an irradiated E. coli suspension with sterile saline and dilute
to 20 times the original volume in sterile minimal broth.
2. Dispense into tubes in desired amounts.
3. Add freshly prepared penicillin to each tube to give a final concentration of 200 units per ml.
4. Incubate at 35C for 4-24 hours on a shaker.
5. Spread 0.1 ml, 0.01 ml and 0.001 ml samples onto complete agar
plates.
7. Select isolated colonies and test for growth in minimal broth.
Bacillus subtilis Procedure
1. Grow cultures of Bacillus subtilis in Antibiotic Medium 3 for 18 hours.
2. Centrifuge to sediment the cells.
3. Aseptically decant the supernatant fluid.
323
Mitis Salivarius Agar & Chapman Tellurite Solution 1%
Section II
4. Resuspend the cells in minimal medium and centrifuge.

5. Decant the supernatant and resuspend the pellet in minimal
medium to give a cell concentration of about 2 x 108 cells per ml.
6. Irradiate the suspension with a low pressure mercury ultraviolet lamp
for a sufficient time to give a cell survival of 1 x 104 cells per ml.
7. Incubate the suspension at room temperature for 4-18 hours in the
minimal medium with appropriate substances added to allow for
the growth of desired mutants.
8. Wash the culture in sterile minimal medium.
9. Centrifuge and resuspend in the same medium.
10. Dilute 1 to 10 with sterile minimal medium.
11. Let stand for 60 minutes to starve the mutants.
12. Add penicillin to give a concentration of 2,000 units per ml.
13. Incubate 15 minutes.
14. Plate the culture on nutrient agar for colony isolation.
15. Identify the nutrition mutants by transferring colonies by replicate
plating onto plates of minimal agar which has been supplemented
with the appropriate nutritional substances.
Delayed Enrichment Method

Mutant colonies will grow as small colonies after the addition of the
complete medium which diffuses through the Minimal Agar.
Results
Packaging
Random Technique
Growth in the nutritionally complete medium and no growth in the
Minimal Broth indicates a mutant.
Minimal Agar Davis
500 g
0544-17
500 g
0756-17
Penicillin Method
Mutant colonies grow after the addition of penicillin.
B. subtilis Method
Mutant colonies grow on Nutrient Agar after the addition of penicillin.

1. Strains vary in their sensitivity to penicillin. Adjustments to
the time of treatment and concentration of penicillin may be
necessary.1
References
1. Lederberg, J. 1950. Isolation and characterization of biochemical
mutants of bacteria. Methods in Med. Res. 3:5-21.
2. Davis. 1949. Proc. Natl Acad. Sci. 35:1.
3. Nester, Schafer, and Lederberg. 1963. Genetics 48:529.
Bacto Mitis Salivarius Agar

Bacto Chapman Tellurite Solution 1%
Intended Use
Bacto Mitis Salivarius Agar is used with Bacto Chapman Tellurite
Solution 1% in isolating Streptococcus mitis, S. salivarius and
enterococci, particularly from grossly contaminated specimens.

Streptococcus mitis, Streptococcus salivarius and Enterococcus
species are part of the normal human flora. S. mitis and S. salivarius
are known as viridans streptococci. These organisms play a role in
cariogenesis and infective endocarditis and cause an increasing
number of bacteremias.1 Enterococci cause urinary tract infections,
wound infections and bacteremia.2 These organisms can colonize the
skin and mucous membranes.
streptococci. Trypan Blue gives the colonies a blue color. Bacto Agar
is the solidifying agent.
Formula
Mitis Salivarius Agar
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Trypan Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.075
Bacto Crystal Violet . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0008
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Chapman3,4,5 investigated methods for isolating streptococci and

formulated Mitis Salivarius Agar. The medium facilitates isolation
of S. mitis (Streptococcus viridans), S. salivarius (non-hemolytic
streptococci) and enterococci from mixed cultures.6
Precautions
Mitis Salivarius Agar contains Tryptose, Proteose Peptone No. 3 and

Proteose Peptone as sources of carbon, nitrogen, vitamins and minerals.
Dextrose and Saccharose are carbohydrate sources. Crystal Violet and
Potassium Tellurite (from Chapman Tellurite Solution 1%) inhibit most
gram-negative bacilli and most gram-positive bacteria except
324
g
g
g
g
g
g
g
g
g

Sterile 1% solution of Potassium Tellurite
1. Mitis Salivarius Agar: For Laboratory Use.

Chapman Tellurite Solution 1%: For Laboratory Use.
2. Chapman Tellurite Solution 1%
AND SKIN. Avoid contact with skin and eyes. Do not breathe mist.
The Difco Manual
Section II
Mitis Salivarius Agar & Chapman Tellurite Solution 1%

Storage
Store Chapman Tellurite Solution 1% at 15-30C.

Autoclave
Incubator (35C)
1. Suspend 90 grams of Mitis Salivarius Agar in 1 liter distilled or
deionized water.
4. Just prior to dispensing, add 1 ml Chapman Tellurite Solution 1%.
5. DO NOT HEAT THE COMPLETE MEDIUM.

Expiration Date
Test Procedure
Results
Procedure
Materials Provided

S. mitis produces small or minute blue colonies. These colonies may
become easier to distinguish with longer incubation. S. salivarius produces blue, smooth or rough gum drop colonies, 1-5 mm in diameter
depending on the number of colonies on the plate. Enterococcus species
form dark blue or black, shiny, slightly raised, 1-2 mm colonies.
1. If coliforms grown on the medium, they produce brown colonies.
Glassware
Petri dishes
ATCC 19433
Uninoculated
plate

Dehydrated Appearance: Bluish beige, free-flowing, homogeneous.
Solution:
deep royal blue, very slightly opalescent.
Prepared Medium:
Deep royal blue, slightly opalescent.
Reaction of 9.0%
Solution at 25C:
pH 7.0 0.2
Appearance:
Colorless, clear, may have a slight
precipitate.
Cultural Response
Prepare the complete medium per label directions. Inoculate and
INOCULUM
CFU
ORGANISM
ATCC
Escherichia coli
19433 100-1,000
25922* 1,000-2,000
GROWTH
APPEARANCE
good
blue black
partial to
brown, if any
complete inhibition
Staphylococcus aureus 25923* 1,000-2,000
partial to
complete inhibition
Streptococcus mitis
9895
100-1,000
good
blue
Streptococcus salivarius 9758
100-1,000
good
blue gum drop shape
The cultures listed are the minimum that should be used for performance testing
The Difco Manual
Streptococcus salvarius
ATCC 9758
*This culture is available as a Bactrol Disk and

should be used as directed in Bactrol Disks
325
Modified EC Medium & Novobiocin Antimicrobic Supplement
2. Molds will grow on the medium after two days incubation.

3. Erysipelothrix rhusiopathiae produces colorless, circular, convex
colonies.
4. Beta-hemolytic streptococci produce colonies that resemble S. mitis.
References
1. Facklam, R. R., and J. A. Washington II. 1991. Streptococcus
and related catalase-negative gram-positive cocci. p. 238-257. In
A. Balows, W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, and
H. J. Shadomy (ed.), Manual of clinical microbiology, 5th ed.
American Society for Microbiology. Washington, D.C.
2. Facklam, R.R., and D. F. Sahm. 1995. Enterococcus, p. 308-314.
R. H. Yolken (ed.), Manual of clinical microbiology, 6th ed.
Section II
3. Chapman, G. H. 1944. The isolation of streptococci from mixed

cultures. J. Bacteriol. 48:113.
4. Chapman, G. H. 1946. The isolation and testing of fecal
streptococci. Am. J. Dig. Dis. 13:105.
5. Chapman, G. H. 1947. Relationship of nonhemolytic and
viridans streptococci in man. Trans. N.Y. Acad. Sci. (Series 2) 10:45.
Packaging
500 g
0298-17
6 x 1 ml
6 x 25 ml
0299-51
0299-66
Bacto Modified EC Medium

Bacto Novobiocin Antimicrobic Supplement
Intended Use
Also Known As
Bacto Modified EC Medium is used with Bacto Novobiocin

Antimicrobic Supplement in the detection of Escherichia coli O157:H7
in meat and poultry products.
mEC+n or Modified E. coli Medium

Modified EC Medium
homogeneous.
Solution:
to medium amber, clear.
Prepared Medium:
Light to medium amber, clear, with
Reaction of 3.66%
Solution at 25C:
pH 6.9 0.2
Novobiocin Antimicrobic Supplement
Lyophilized Appearance: White cake.
Rehydrated Appearance: Colorless solution.
Cultural Response
Prepare Modified EC Medium per label directions. Add 10 ml
of Novobiocin Antimicrobic Supplement per liter. Inoculate
the tubes and incubate at 35 2C for 24 hours.
ORGANISM
ATCC
33186
Escherichia coli O157:H7 35150
INOCULUM CFU
GROWTH
1,000-2,000
10-100
none to poor
good
The cultures listed above are the minimum that should be used
326

Modified EC Medium and Novobiocin Antimicrobic Supplement are
based on the formula for modified EC broth with novobiocin (mEC+n)
as described by Okrend and Rose.1 In modifying the EC Medium
formula, Okrend and Rose reduced the Bile Salts No. 3 from 1.5 grams
per liter to 1.12 grams per liter and added 20 milligrams per liter of
sodium novobiocin. Okrend, Rose et al. reported that mEC+n was
useful in the enrichment and detection of E. coli O157:H7 from meats
and poultry products.2-4

Tryptone supports good growth of E. coli O157:H7 and is rich in
peptides and nitrogen. Lactose is an additional source of carbon for
organisms, such as E. coli, that can ferment this sugar. Potassium
Phosphate Dibasic and Monobasic are buffers that facilitate recovery
of injured cells. Sodium Chloride provides a suitable ionic environment
for growth of microorganisms.
Selectivity of the medium is achieved by the incorporation of Bile Salts
No. 3 into the base medium and by the addition of Sodium Novobiocin
to the complete medium. These agents suppress the growth of nuisance
organisms commonly found in foods. The Sodium Novobiocin is
provided in the freeze-dried state as Novobiocin Antimicrobic
Supplement. This supplement is rehydrated before use with sterile
Formula
Modified EC Medium
Formula per liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
The Difco Manual
Section II
Bacto Bile Salts No. 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.12
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Modified EC Medium & Novobiocin Antimicrobic Supplement

g
g
g
g
g

Formula per 10 ml vial
Sodium Novobiocin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg
Precautions
2. Modified EC Medium
difficult, give oxygen. Seek medical advice. If swallowed, seek
HARMFUL. HARMFUL BY INHALATION AND IF SWALLOWED. (EC) MAY CAUSE ALLERGIC EYE, RESPIRATORY
SYSTEM AND SKIN REACTION. Avoid contact with skin and
difficult, give oxygen. Seek medical advice. If swallowed, seek
medical advice immediately and show container or label.
Storage
1. Store Modified EC Medium below 30C. The dehydrated medium
2. Store Novobiocin Antimicrobic Supplement at 2-8C.
3. Store prepared medium at 2-8C.
Expiration Date
Procedure
Materials Provided
Modified EC Medium
The Difco Manual

EMB Agar
Sterile distilled or deionized water
Autoclave
Incubator (35C)
Incubator (42C)
Butterfields Phosphate Diluent
Phenol Red Sorbitol Agar with MUG
Rehydrate Novobiocin Antimicrobic Supplement with 10 ml sterile
1. Dissolve 36.6 grams Modified EC Medium in 1 liter of distilled or
deionized water.
4. Aseptically add 10 ml rehydrated supplement to 1 liter sterile basal
medium. Mix well.

sample.
Test Procedure
Many procedures and systems have been described for the use of
Modified EC Medium with Novobiocin in the selective and differential
enrichment of E. coli O157:H7 in meat and poultry samples. Please
consult appropriate references. 4-6 Listed below is the USDAs
recommended procedure for the enrichment and detection of E. coli
O157:H7 in meat and poultry samples using Modified EC Medium
with Novobiocin.2-4
1. Inoculate 25 grams of meat sample into 225 ml of Modified EC
Medium with Novobiocin in a stomacher bag. Blend or stomach as
required (i.e., 2 minutes) for thorough mixing.
3. Dilute cultures 10-fold in Butterfields Phosphate Diluent and
inoculate 0.1 ml of appropriate dilutions using a spread plate
technique onto MacConkey Sorbitol Agar (MSA) and MacConkey
Sorbitol Agar with BCIG (MSA-BCIG) agar plates.
4. Incubate plates at 42C for 24 hours.
5. Examine MSA plates for sorbitol-negative colonies (white)
and MSA-BCIG plates for sorbitol-negative, BCIG-negative
colonies (white).
6. Subculture sorbitol-negative colonies to respective plates of EMB
Agar and Phenol Red Sorbitol Agar containing MUG (PRS-MUG).
7. Incubate EMB and PRS-MUG Agar plates at 35C for 18-24 hours.
Examine plates for sorbitol fermentation, MUG reaction
(fluorescence), and typical E. coli growth on EMB Agar.
327
Modified Letheen Agar & Modified Letheen Broth
Results
Section II
1. Nutritional requirements may vary from strain to strain.
2. Okrend, A. J. G., B. E. Rose, and B. Bennett. 1990. A screening

method for the isolation of E. coli O157:H7 from ground beef.
J. Food Prot. 53:249-252.
3. Okrend, A. J. G., B. E. Rose, and C. P. Lattuada. 1990. Use of
5-bromo-4-chloro-3-indoxyl--D-glucuronide in MacConkey
Sorbitol Agar to aid in the isolation of E. coli O157:H7 from ground
beef. J. Food Prot. 53:941-943.
4. Okrend, A. J. G., B. E. Rose, and R. Matner. 1990. An improved
screening method for the detection and isolation of E. coli O157:H7
from meat, incorporating the 3M Petrifilm Test Kit-HEC-for
hemorrhagic Escherichia coli O157:H7. J. Food Prot. 53:936-940.
5. Hawkins, E. W., and L. E. Orme. 1995. Rapid testing methodology for Escherichia coli O157:H7 using commercially available
products. Proc. West. Sec., Amer. Soc. Animal Sci. vol. 46.
6. Johnson, R. P., R. J. Durham, S. T. Johnson, and L. A.
MacDonald. 1995. Detection of E. coli O157:H7 in meat by an
enzyme-linked immunosorbent assay, EHEC-Tek. Appl. Environ.
References
Packaging
1. Okrend, A. J. G., and B. E. Rose. 1989. Isolation and identification

of E. coli O157:H7 from meat. USDA Food Safety Inspection
Service. Rev. 3 of Laboratory Communication no. 38. E. coli O157:H7.
20 December 1989. U.S. Department of Agriculture, Washington, D.C.
Growth in Modified EC Medium with Novobiocin is demonstrated as

an increase in turbidity. Colonies of E. coli O157:H7 appear white on
MacConkey Sorbitol and MacConkey Sorbitol-BCIG Agars.
Fermentation of sorbitol in Phenol Red Sorbitol Broth is demonstrated
by the production of a yellow color in the medium. With sorbitol
non-fermenters, the color of the medium remains red to reddish
purple. Positive MUG reactions are demonstrated as a blue fluorescence in the medium under long-wave UV light. Colonies of E. coli on
EMB Agar appear blue-black to dark purple. A green metallic sheen
may also be present.
Cultures that are sorbitol-negative, MUG-negative, and produce
blue-black to dark purple colonies with a green metallic sheen on EMB
Agar are indicative of E. coli O157:H7. These cultures should be tested
serologically and with additional biochemical testing to confirm their
identity as E. coli O157:H7.
Limitations
Bacto Modified Letheen Agar

Bacto Modified Letheen Broth
Modified EC Medium
500 g
0340-17
6x10 ml
3197-60*
*Store at 2-8C.
Intended Use
Bacto Modified Letheen Agar and Bacto Modified Letheen Broth are
used for the microbiological testing of cosmetics.

Modified Letheen Agar and Modified Letheen Broth are based on
Letheen Agar, Modified and Letheen Broth, Modified as described in
the 7th edition of the U.S. FDA Bacteriological Analytical Manual.1
Letheen Agar, Modified and Letheen Broth, Modified are recommended
by the FDA for use in the microbiological testing of cosmetics.2

Beef Extract and Tryptone provide carbon and nitrogen sources required for good growth of a wide variety of bacteria and fungi. The
Tryptone level was increased in the Modified Letheen Agar and Broth
formulas to provide for better growth. Vitamins and cofactors, required
for growth as well as additional sources of nitrogen and carbon, are
provided by Yeast Extract. Sodium Chloride provides a suitable
osmotic environment. In Modified Letheen Broth, Sodium Chloride is
provided by the Letheen Broth component. Both media also contain
polysorbate 80, lecithin and sodium bisulfite to partially neutralize the
preservative systems commonly found in cosmetics. Bacto Agar is
included in Modified Letheen Agar as a solidifying agent.
328
Formula
Modified Letheen Agar
Formula Per Liter
Bacto Letheen Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
g
g
g
g
g
g
g

Modified Letheen Broth
Formula Per Liter
Bacto Letheen Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26.7
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
g
g
g
g
g
Precautions
The Difco Manual
Section II
Modified Letheen Agar & Modified Letheen Broth
Storage
Glassware
Autoclave
Incubators (35C, 30C)
Selective media
Expiration Date
stored as directed.
Do not use a product if it fails to meet specifications for identity and
performance.
Procedure
Materials Provided
1. Suspend the medium in 1 liter of distilled or deionized water:
Modified Letheen Agar - 59.1 grams;
Modified Letheen Broth - 43.8 grams.

Test Procedure 2
Dehydrated Appearance: Tan, homogeneous, appears moist
% Solution:
after cooling in approx. 45-50C
waterbath, clear to slightly
opalescent, may have slight fine
precipitate.
Prepared Medium:
Light-medium amber, slightly
opalescent, may have a slight fine
precipitate.
Reaction of 5.91%
Solution at 25C:
pH 7.2 0.2
Dehydrated Appearance: Tan, homogeneous, appears moist
% Solution:
after cooling, clear to slightly
opalescent, may have slight fine
precipitate.
Prepared Medium:
Medium-dark amber after cooling,
slightly opalescent, may have slight
fine precipitate.
Reaction of 4.38%
Solution at 25C:
pH 7.2 0.2
Cultural Response
Prepare Modified Letheen Agar or Broth per label directions.
Inoculate and incubate at 35C for 24-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH (AGAR/BROTH)
6538
25-100
Good
The culture listed above is the minimum that should be used for
The Difco Manual
1. Prepare and dilute samples in Modified Letheen Broth in accordance with established guidelines.
2. Using the spread plate technique, inoculate in duplicate 0.1 ml of
the diluted samples onto Modified Letheen Agar, Potato Dextrose
Agar (or Malt Extract Agar) containing chlortetracycline, Baird
Parker Agar (or Vogel-Johnson Agar, optional), Anaerobic Agar,
and a second set of Modified Letheen Agar plates.
3. Incubate one set of Modified Letheen Agar plates at 30 2C for
48 hours and the other set at 35 2C under anaerobic conditions
for 2-4 days. Incubate the Potato Dextrose Agar (or Malt Extract
Agar) plates at 30 2C for 7 days and the Baird Parker Agar
(or Vogel-Johnson Agar) plates, if inoculated, at 35 2C for
48 hours.
4. Incubate the diluted samples from step 1 at 35 2C for 7 days.
Subculture enriched samples onto Modified Letheen Agar only if
there is no growth on the primary Modified Letheen Agar plates.
Results
Examine plates for evidence of growth and characteristic colonial
morphology. Determine colony counts and subculture each colony type
onto Modified Letheen Agar and MacConkey Agar (also Baird Parker
or Vogel-Johnson Agar, if used in step 2).
Determine Gram reaction, cell morphology and catalase reactions.
Identify bacterial isolates in accordance with established procedures.2
References
1. Tomlinson, L. (ed.). 1992. FDA Bacteriological Analytical
Manual, 7th ed. AOAC International, Arlington, VA.
2. Hitchins, A. D, T. T. Tran, and J. E. McCarron. 1992. In L.A.
Tomlinson (ed.), FDA Bacteriological Analytical Manual, 7th Ed.
Packaging
500 g
0631-17-0
500 g
0630-17-0
329
Modified Listeria Enrichment Broth
Section II
Bacto Modified Listeria Enrichment Broth
Intended Use
Bacto Modified Listeria Enrichment Broth is used for selectively
enriching Listeria from raw and pasteurized milk according to the
International Dairy Federation.1

reported food-borne outbreak of listeriosis was in 1985.4 Since then,
coleslaw, pasteurized milk, Mexican-style cheese, pat and pickled
and other food processing plants. It is ubiquitous in nature, being

Dehydrated Appearance: Light beige, free flowing, homogeneous.
Solution:
is light to medium yellowish-amber
with a faint green ring at the surface,
Prepared Tubes:
Light yellowish-amber, clear to very
Reaction of 3.61%
Solution at 25C:
pH 7.3 0.2
Cultural Response
Prepare Modified Listeria Enrichment Broth per label directions.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
29212* 2,000-10,000 partially suppressed

at 18- 24 hours
Escherichia coli
25922* 2,000-10,000
marked to
complete inhibition
100-1,000
good
Saccharomyces
9080 2,000-10,000
marked to
pastorianus
complete inhibition
performance.
330

at 20C. Many common food contaminants such as streptococci,
enterococci, Bacillus species, Escherichia coli, Pseudomonas
aeruginosa and Proteus vulgaris interfere with the isolation of
Listeria Enrichment Broth is based on the formula developed by Lovett
et al.10 in which Tryptic Soy Broth was supplemented with yeast
extract for optimum growth of Listeria. Modified Listeria Enrichment
Broth is a modification of Listeria Enrichment Broth in which the
acriflavine content has been reduced from 15 mg to 10 mg per liter.
This modification reflects the lower concentration specified by the
International Dairy Federation1 for isolation of L. monocytogenes from
milk and milk products.

Modified Listeria Enrichment Broth contains Tryptone, Soytone and
Yeast Extract as nitrogen and vitamin sources. Dextrose provides an
energy source. Sodium Chloride maintains the osmotic balance of the
medium. Potassium Phosphate is a buffering agent. Cycloheximide is
incorporated to inhibit saprophytic fungi, while Nalidixic Acid inhibits
growth of gram-negative organisms. Acriflavine HCl is added at 10 mg
per liter to suppress growth of gram-positive bacteria.
Formula
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Cycloheximide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05
Acriflavine HCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01
Nalidixic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.04
g
g
g
g
g
g
g
g
g
Precautions
2. TOXIC. HARMFUL BY INHALATION AND IF SWALLOWED.
(EC) MAY CAUSE CANCER. POSSIBLE RISK OF HARM TO
THE UNBORN CHILD. Do not breathe dust. In case of accident
or if you feel unwell, seek medical advice immediately. (Show
label where possible.) Wear suitable protective clothing. Keep
container tightly closed. TARGET ORGAN(S): Blood, Cardiovascular, Face, Lungs, Nerves, Skin, Thorax.
The Difco Manual
Section II

air. If not breathing, give artificial respiration. If breathing is difficult, give oxygen. Seek medical advice. If swallowed, induce
or label.

of Listeria may be encountered that fail to grow or grow poorly on
this medium.
2. Modified Listeria Enrichment Broth is a partially selective medium.
Growth of some contaminating strains will be markedly but not
totally inhibited.
References
For dairy samples, the IDF1 selective enrichment method is as follows:

1. Add 25 ml liquid or 25 grams of solid test material to 225 ml
Modified Listeria Enrichment Broth and mix or blend thoroughly.
2. Incubate for 48 hours at 30C.1
3. At 48 hours, streak the Modified Listeria Enrichment Broth
culture onto plates of Oxford Medium or Palcam Medium.
4. Incubate the agar plates at 37C for 48 2 hrs.
1. International Dairy Federation. 1990. Milk and milk products detection of Listeria monocytogenes. IDF Provisional International
Standard No. 143. International Dairy Federation, Brussels.
monocytogenes (n. sp.). J. Path. Bact., 29:407- 439.
Washington, D.C.
10. Lovett, J., D. W. Frances, and J. M. Hunt. 1987. Listeria
monocytogenes in raw milk: detection, incidence and pathogenicity.
J. Food Prot. 50:188-192.
p. 342-343. In P.R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover,
and R. H. Yolken (ed.). Manual of clinical microbiology, 6th ed.
1993. Pathogens in milk and milk products. In R. T. Marshall (ed.).
Results
Packaging
1. Examine agar plates for typical Listeria colonies.

2. Consult appropriate references for selection of biochemical or
serological tests for confirmation of Listeria sp.1,8,11,12
Storage
medium at 2-8C.
Expiration Date
Procedure
Materials Provided

Autoclave
Incubator (30C)

1. Collect samples in sterile containers or with sterile swabs and
guidelines.1
2. For specific information about sample preparation and inoculation,
consult appropriate reference.1
Test Procedure
The Difco Manual
500 g
10 kg
0205-17
0205-08
331
Motility GI Medium
Section II
Bacto Motility GI Medium
Storage
Intended Use
Bacto Motility GI Medium is used for detecting motility of

microorganisms and for separating organisms in their motile phase.
Expiration Date

Motility GI Medium is prepared according to the formulation of
Jordan, Caldwell and Reiter.1 It is a semisolid gelatin-heart infusion
medium that is adaptable to use in both tubes and plates for motility studies.

Procedure
Materials Provided
Motility GI Medium
Heart Infusion Broth and Gelatin provide nitrogen, vitamins, and amino
acids. Bacto Agar is the solidifying agent. Motility is evidenced by the
presence of diffuse growth away from the line or spot of inoculation.
Nonmotile organisms grow only along the line of inoculation.
Formula
Motility GI Medium
Formula Per Liter

Glassware
Autoclave
Incubator (35C)
Waterbath (50-55C)
Bacto Heart Infusion Broth . . . . . . . . . . . . . . . . . . . . . . . . 25 g

Bacto Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53.4 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 g
Precautions
1.
2.
3.
4.
5.

If tubes are desired, dispense medium to a depth of 60-75 mm.
Cool tubes by placing in cold water up to the depth of the medium.
Cool flasks of medium to 50-55C; pour into sterile Petri dishes to
a depth of 1/8 inch or more and allow to solidify.

Solution:
medium amber, very slightly opalescent,
Prepared Medium:
Reaction of 8.14%
Solution at 25C:
pH 7.2 0.2
Cultural Response
Prepare Motility GI Medium per label instructions. Inoculate the
ORGANISM
Escherichia coli
ATCC
GROWTH
MOTILITY
25922*
13883*
good
good
332
Uninoculated
tube
Escherichia coli
ATCC 25922
ATCC 13883
The Difco Manual
Section II
Motility Medium S

Test Procedure
1. Inoculate with growth from an 18-24 hour pure culture.
2. If tubes are used, inoculate by stab inoculation. If plates are used, spot
the inoculum on the surface or stab just below the medium surface.
3. Incubate at a temperature and duration appropriate for the suspected
organism being tested.
4. Examine tubes or plates for growth and signs of motility.
Results
Motility is evidenced by the presence of diffuse growth away from the
line or spot of inoculation. Nonmotile organisms grow only along the
line of inoculation.

1. All weak or questionable motility test results should be confirmed
by flagella stain or by direct wet microscopy.2
2. Some flagellar proteins are not synthesized at higher temperatures.3
Bacto Motility Medium S
3. Some isolates of Yersinia enterocolitica demonstrate motility at 35C

while others may be nonmotile at 25C.2 The motility of Proteus is
also temperature dependent. This effect of temperature on motility
needs to be taken into account when deciding on a testing regimen.
4. Due to the temperature dependency of motility in some organisms,
a negative test tube or plate should be incubated an additional
5 days at a lower temperature of 22-25C.3
References
1. Jordan, E. O., M. E. Caldwell, and D. Reiter. 1934. Bacterial
motility. J. Bacteriol. 27:165.
2. DAmato, R. F., and K. M. Tomfohrde. 1981. Influence of
media on temperature-dependent motility test for Yersinia
enterocolitica. J. Clin. Microbiol. 14:347-348.
3. MacFaddin, J. F. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, vol. 1, Williams
Packaging
Motility GI Medium
500 g
0869-17
Intended Use
Bacto Motility Medium S is used with Bacto TTC Solution 1% in
detecting bacterial motility.
Also Known As
Motility Medium S is prepared according to the formula of Ball and

Sellers.1 The composition of the medium offers no more resistance to
motility during incubation than would a broth culture, yet it preserves
the stab line. The medium also permits further testing fornitrate
reduction, nitrogen gas production and gelatin liquefaction.
Motility Medium S conforms with Motility S Medium and MotilityNitrate Medium.

Solution:
boiling. Solution is medium amber, slightly opalescent,
Prepared Medium:
Reaction of 6.0%
Solution at 25C:
pH 7.3 0.2
Cultural Response
Prepare Motility Medium S with added TTC Solution 1% per label directions. Stab
inoculate the medium and incubate at 35 2C for 18-48 hours.
ORGANISM
Escherichia coli
Shigella flexneri
ATCC
GROWTH
MOTILITY
TTC
REDUCTION
25922*
13883*
12022
good
good
good
+
+
*These cultures are available as Bactrol disks and should be used according to the
technical information.
The Difco Manual
Uninoculated
tube
Escherichia coli
ATCC 25922
333
Motility Medium S
Section II

Infusion from Beef Heart, Gelatin and Tryptose provide nitrogen, vitamins
and amino acids to support growth of fastidious microorganisms.
Sodium Chloride maintains the osmotic balance of the medium and
also induces the swarming of Proteus species.2 Dipotassium Phosphate
provides buffering capability and has been shown to have a stimulatory
effect on the motility of Proteus species.1 Potassium Nitrate provides
trace elements necessary for bacterial growth. Organisms capable of
reducing nitrate, especially nitrate-reducing obligate aerobes1, exhibit
increased motility in the presence of 0.2% Potassium Nitrate. Bacto
Agar is a solidifying agent at a concentration that preserves the line of
inoculation. TTC (2,3,5-triphenyltetrazolium chloride) facilitates the
detection of motility. Growth along or out from the stab line is made
readily visible because formazan precipitates when TTC is reduced by
a microorganism.
Formula
Motility Medium S
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Potassium Nitrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
g
g
g
g
g
g
g
Precautions
Storage
Expiration Date
Procedure
Materials Provided
Motility Medium S

Glassware
Autoclave
Waterbath (60C)
Refrigerator (2-8C)
Incubator (35C)
TTC Solution 1%
1.
2.
3.
4.

Aseptically add 10 ml of TTC Solution 1% at 60C. Mix thoroughly.
334
5. Dispense into sterile test tubes.

6. Refrigerate at 2-8C until ready to use.

Test Procedure
1. Stab inoculate with growth from a pure 18-24 hour culture
immediately after removing the medium from the refrigerator.
3. Observe at 6, 24 and 48 hours.3 Examine tubes for signs of growth
and motility.
Results
Motility is evidenced by the presence of diffuse growth away from
the line or spot of inoculation. Nonmotile organisms grow only along
the line of inoculation.
Growth of microorganisms capable of reducing TTC will appear as
a red color along the stab line as well as in the areas into which the
cells have migrated.

1. All weak or questionable motility results should be confirmed by
flagella stain or by direct wet microscopy.4
2. Some flagellar proteins are not synthesized at higher temperatures.3
3. Some isolates of Yersinia enterocolitica demonstrate motility at 35C
while others may be nonmotile at 25C.4 The motility of Proteus
is also temperature dependent. This affect of temperature on motility
needs to be considered when deciding on a testing regimen.
4. Due to the fact that motility is temperature dependent in some
organisms, a test tube or plate yielding a negative result should be
incubated an additional 5 days at a lower temperature of 22-25C.3
5. The addition of tetrazolium salts to the medium may be inhibitory
to some bacteria.5
References
1. Ball, R. J., and W. Sellers. 1966. Improved motility medium.
Appl. Microbiol. 14:670-673.
2. Schneierson, S. S. 1961. Production of discrete nonswarming
colonies of Proteus on medium deficient in sodium chloride and
other salts. J. Bacteriol. 82:621-622.
4. DAmato, R. F., and K. M. Tomfohrde. 1981. Influence of media
on temperature-dependent motility test for Yersinia enterocolitica.
J. Clin. Microbiol. 14:347-348.
St. Louis, MO.
Packaging
Motility Medium S
TTC Solution 1%
TTC
500 g
30 ml
25 g
0761-17
3112-67*
0643-13
*Store at 2-8C
The Difco Manual
Section II
Motility Test Medium
Bacto Motility Test Medium
Intended Use
Motility Test Medium is used for detecting microbial motility.

In 1936, Tittsler and Sandholzer reported using a semisolid agar for
the detection of bacterial motility. 1 Motility Test Medium is a
modification of this formulation.
Bacterial motility is observed macroscopically by a diffuse zone of
growth spreading from the line of inoculation. Certain species
of motile bacteria will show diffuse growth throughout the entire
medium, while others may show diffusion from one or two points
only, appearing as nodular outgrowths along the stab. Tittsler
and Sandholzer reported that tubes incubated for one day gave
identical results with the hanging drop method and that incubation for
two days permitted them to demonstrate motility in an additional
4% of the cultures tested.1
Motility Test Medium is recommended for the detection of microbial
motility in food and dairy standard methods.2,3,4

Tryptose is a source of nitrogen, amino acids and carbon. Sodium
chloride provides essential ions while maintaining osmotic balance.
Agar is a solidifying agent used at a low concentration.
Formula
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Precautions
Storage
Expiration Date
Procedure
Materials Provided

Glassware
Autoclave
Incubator (35C)
Incubator (22-25C)
Inoculating needle
Beef Extract (optional)
(To obtain more luxuriant microbial growth, add 0.1-0.3% Beef
Extract prior to boiling the medium)

Dehydrated Media
Appearance:
Solution:
Reaction of 2%
Solution at 25C:
Light beige, free-flowing, homogeneous.

2% solution, soluble in distilled or deionized
water on boiling. Solution is light amber,
clear to slightly opalescent with no
pH 7.2 0.2
Cultural Response
Prepare Motility Test Medium per label directions. Inoculate by straight
stab of the test organisms and incubate at 35 2C for 18-48 hours.
ORGANISM
Escherichia coli
ATCC
GROWTH
MOTILITY
25922*
13883*
good
good
positive
negative
*These cultures are available as Bactrol Disks and should be used as directed
in Bactrol Disks Technical Information.
The Difco Manual
Uninoculated
tube
Escherichia coli
ATCC 25922
ATCC 13883
335
Mueller Hinton Medium & Mueller Hinton Broth
Section II
References
1. Tittsler, R. P., and L. A. Sandholzer. 1936. The use of semi-solid

agar for the detection of bacterial motility. J. Bacteriol. 31:575-580.
3. Marshall, R. T. (ed.). 1993. Standard methods for the examination of dairy products, 16th ed. American Public Health Association, Washington, D.C.
5. MacFaddin, J. D. 1985. Media for isolation-cultivationidentification-maintenance medical bacteria, p.538-543, vol 1.
Test Procedure
Inoculate tubes with a pure culture by stabbing through the center of
the medium with an inoculating needle to approximately one-half
the depth of the medium. Incubate at the proper temperature for
the organism under consideration and examine at 18-48 hours. If
negative, continue incubation at 22-25C for an additional 5 days.
Results
Motility is manifested macroscopically by a diffuse zone of growth
spreading from the line of inoculation. Certain species of motile
bacteria will show diffuse growth throughout the entire medium, while
others may show diffusion from one or two points only, appearing
as nodular growths along the stab line. Non-motile organisms grow
only along the line of inoculation.

1. Many organisms fail to grow deep in semisolid media, inoculating
pour plates may be advantageous.5
Bacto Mueller Hinton Medium

Bacto Mueller Hinton Broth
Packaging
100 g
500 g
0105-15
0105-17
Intended Use
Bacto Mueller Hinton Medium is used for antimicrobial susceptibility
testing of rapidly growing aerobic microorganisms by the disk
diffusion technique.
Bacto Mueller Hinton Broth is for antimicrobial susceptibility testing
of aerobic microorganisms by broth dilution methods.
Also Know As
Mueller Hinton media are abbreviated as M-H Agar and M-H Broth.

Mueller Hinton Medium duplicates the formula recommended by
Mueller and Hinton1 for the primary isolation of Neisseria species. In
the development of a simple transparent medium containing heat stable
ingredients, Mueller and Hinton selected pea meal extract agar.2 In their
modification, starch replaced the growth-promoting properties of
pea extract, acting as a protective colloid against toxic substances.
Tryptic digest of meat was substituted with casamino acids, technical.
Bauer, Kirby, Sherris and Tuck 3 recommended Mueller Hinton
Medium for performing antibiotic susceptibility tests using a single
disk of high concentration. Mueller Hinton Medium is used in the disk
diffusion method of susceptibility testing.7 Mueller Hinton Broth is
used for determining minimal inhibitory concentrations (MICs).4
Mueller Hinton Medium complies with requirements of the World
Health Organization.8 Mueller Hinton Medium is specified in the FDA
Bacteriological Analytical Manual 9 for food testing.
Mueller Hinton Medium is the recommended medium for testing most
commonly encountered aerobic and facultatively anaerobic bacteria.10
336
This unsupplemented medium has been selected by the National

Committee for Clinical Laboratory Standards (NCCLS) for several
reasons:11
It shows good batch-to-batch reproducibility.
It is low in sulfonamide, trimethoprim, and tetracycline inhibitors.
It gives satisfactory growth of most non-fastidious pathogens.
A large amount of data has been collected from antimicrobial
susceptibility tests with this medium.
A variety of supplements can be added to Mueller Hinton Medium.
For testing streptococci, supplementation with 5% defibrinated sheep
or horse blood is recommended.16 GC agar base with added 1% growth
supplement, is used for antimicrobial susceptibility testing of
Neisseria gonorrhoeae. Susceptibility testing of Haemophilus species
should be performed on Haemophilus Test Medium. Mueller Hinton
Medium should be supplemented with 2% NaCl for testing methicillin
or oxacillin against staphylococci.5 Mueller Hinton Medium with
Rabbit Serum is used for the cultivation and maintenance of
Corynebacterium species.6

Infusion from Beef and Casamino Acids, Technical provide nitrogen,
vitamins, carbon and amino acids in Mueller Hinton media. Starch is
added to absorb any toxic metabolites produced. Bacto Agar is the
solidifying agent.
The use of a suitable medium is essential for testing the susceptibility
of microorganisms to sulfonamides and trimethoprim. Antagonism to
sulfonamide activity is demonstrated by para-aminobenzoic acid
(PABA) and its analogs. Reduced activity of trimethoprim, resulting in
smaller growth inhibition zones and innerzonal growth, is demonstrated
on medium possessing high levels of thymidine. The PABA and
The Difco Manual
Section II
thymine/thymidine content of Mueller Hinton Medium and Mueller

Hinton Broth are reduced to a minimum, reducing the inactivation of
sulfonamides and trimethoprim.
Storage
Formula
Expiration Date
Mueller Hinton Medium

Formula Per Liter
Expiration date applies to the product in its intact container when stored
Beef, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300

Bacto Casamino Acids, Technical . . . . . . . . . . . . . . . . . . 17.5
Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
g
g
g
g
Store dehydrated media below 30C. The dehydrated media are very
Procedure
Materials Provided

Mueller Hinton Broth

Formula Per Liter
Beef, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 g

Bacto Casamino Acids, Technical . . . . . . . . . . . . . . . . . . 17.5 g
Bacto Soluble Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 g
Glassware
Autoclave
Incubator
Sterile 5% defibrinated blood (Optional)
Precautions

Dehydrated Appearance: Beige, homogeneous, free-flowing with
few dark specks.
Prepared Medium:
opalescent, no significant precipitation.
Reaction of 3.8%
Solution at 25C:
pH 7.3 0.1
Dehydrated Appearance: Light beige with a few dark specks,
Prepared Medium:
Very light amber, clear, may have
slight precipitation.
Reaction of 2.1%
Solution at 25C:
pH 7.3 0.1
Cultural Response
Mueller Hinton Medium: Prepare, inoculate and dispense
antibiotic disks following the procedure described by NCCLS.7,11
Typical test of Mueller Hinton Medium by agar diffusion method.
The cultures listed should have zone sizes near the middle of the
range of the concentration tested.7
Mueller Hinton Broth: Prepare and dispense into microdilution trays or microdilution tubes described by NCCLS.4 The cultures
listed should have MIC (endpoints) near the middle of the range of the concentration tested.4
ORGANISM
Escherichia coli
The Difco Manual
ATCC
29212*
25922*
27853*
25923*
*These organisms are available as Bactrol Disks and should be used as
337
1. Suspend 38 g of medium in 1 liter distilled or deionized water.
4. OPTIONAL: Supplement as appropriate.16 To supplement Mueller
Hinton Medium with sheep blood, aseptically add 5% sterile
defibrinated blood at 45-50C. Haemophilus Test Medium contains
15 mcg/ml NAD, 15 mcg/ml bovine hematin and 5 mg/ml yeast
extract. The medium recommended for testing Neisseria
gonorrhoeae consists of GC agar base with 10 ml/liter of the
following supplement: 1.1 g L-cystine, 0.03 g guanine HCl, 3 mg
thiamine HCl, 13 mg PABA, 0.01 g B12, 0.1 g cocarboxylase, 0.25 g
NAD, 1 g adenine, 10 g L- glutamine, 100 g glucose, 0.02 g ferric
nitrate, 1 l distilled or deionized water.
5. Pour cooled Mueller Hinton Medium into sterile Petri dishes on
a level, horizontal surface to give a uniform depth of about
4mm (60 to 70 ml of medium for 150 mm plates and 25 to 30 ml
for 100 mm plates) and allow to cool to room temperature.10
6. Check prepared Mueller Hinton Medium to ensure the final pH is
7.3 0.1 at 25C.
1. Suspend 21 g of medium in 1 liter distilled or deionized water.
2. Warm gently to dissolve.
4. Dispense Mueller Hinton Broth into sterile tubes.
5. Check prepared Mueller Hinton Broth to ensure the final
pH is 7.3 0.1 at 25C.

Test Procedure
For a complete discussion on antimicrobic susceptibility testing, refer
to the appropriate procedures outlined in the references.4,7,9,10,11,12
Results
Refer to appropriate references and procedures for results

may be encountered that fail to grow or grow poorly on these media.
2. Numerous factors can affect results: inoculum size, rate of growth,
medium formulation and pH, length of incubation and incubation
environment, disk content and drug diffusion rate, and measurement
of endpoints. Therefore, strict adherence to protocol is required to
ensure reliable results.12
3. Disk diffusion susceptibility testing is limited to rapidly growing
organisms. Drug inactivation may result from the prolonged
incubation times required by slow growers.12
4. Media containing excessive amounts of thymidine or thymine can
reverse the inhibitory effects of sulfonamides and trimethoprim,
causing zones of growth inhibition to be smaller or less distinct.10
5. Variation in the concentration of divalent cations, primarily
calcium and magnesium, affects results of aminoglycoside,
338
Section II
tetracycline, and colistin tests with P. aeruginosa isolates.13,14

A cation content that is too high reduces zones sizes, whereas a
cation content that is too low has the opposite effect.10
6. When Mueller Hinton Medium is supplemented with blood, the
zone of inhibition for oxacillin and methicillin may be 2 to 3 mm
smaller than those obtained with unsupplemented agar. 10
Conversely, sheep blood may markedly increase the zone diameters
of some cephalosporins when they are tested against enterococci.15
Sheep blood may cause indistinct zones or a film of growth within
the zones of inhibition around sulfonamide and trimethoprim disks.10
7. Mueller Hinton Medium deeper than 4 mm may cause false-resistant
results, and agar less than 4 mm deep may be associated with a
false-susceptibility report.10
8. A pH outside the range of 7.3 0.1 may adversely affect susceptibility
test results. If the pH is too low, aminoglycosides and macrolides
will appear to lose potency; others may appear to have excessive
activity.10 The opposite effects are possible if the pH is too high.10
9. When Mueller Hinton Medium is inoculated, no droplets of moisture
should be visible on the surface or on the petri dish cover.10
10. Mueller Hinton Medium should be inoculated within 15 minutes
after the inoculum suspension has been adjusted.10
11. The zone of inhibition diameters of some drugs, such as the
aminoglycosides, macrolides, and tetracyclines, are significantly
altered by CO2. Plates should not be incubated in increased CO2.10
References
1. Mueller, J. H., and J. Hinton. 1941. A protein-free medium for
primary isolation of gonococcus and meningococcus. Proc. Soc.
Exp. Biol. Med. 48:330-333.
2. Gordon and Hine. 1916. Br. Med. J. 678.
3. Bauer, A. L., W. M. M. Kirby, J. C. Sherris, and M. Turck. 1966.
Antibiotic susceptibility testing by a standardized single disc
method. Am. J. Clin. Pathol. 45:493-496.
Methods for dilution antimicrobial susceptibility tests for bacteria
that grow aerobically. Approved standard M7-A3. National
Committee for Clinical Laboratory Standards, Villanova, PA.
5. Huang, M., B., E. T. Gay, C. N. Baker, S. N. Banerjee, and
F. C. Tenover. 1993. Two percent sodium chloride is required for
susceptibility testing of staphylococci with oxacillin when using
agar-based dilution methods. J. Clin. Microbiol. 31:2683-2688.
Performance standards for antimicrobial disk susceptibility tests.
Approved standard M2-A5. National Committee for Clinical
8. World Health Organization. 1961. Standardization of methods
for conducting microbic sensitivity tests. Technical Report Series
No. 210, Geneva.
10. Wood, G. L., and J. A. Washington. 1995. Antibacterial
susceptibility tests: dilution and disk diffusion methods, p. 13271341. In Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover,
The Difco Manual
Section II
Muller Kauffmann Tetrathionate Broth Base

Evaluating production lots of dehydrated Mueller-Hinton agar.
Tentative standard M6-T. National Committee for Clinical
12. Isenberg, H. E. (ed.). 1992. Clinical microbiology procedures
13. Barry, A. L., G. H. Miller, C. Thornsberry, R. S. Hare,
R. N. Jones, R. R. Lorber, R. Ferraresi, and C. Cramer. 1987.
Influence of cation supplements on activity of netilmicin against
Pseudomonas aeruginosa in vitro and in vivo. Antimicrob. Agents
Chemother. 31:1514-1518.
14. Barry, A. L., L. B. Reller, G. H. Miller, J. A. Washington, F. D.
Schoenknecht, L. R. Peterson, R. S. Hare, and C. Knapp. 1992.
Revision of standards for adjusting the cation content of MuellerHinton broth for testing susceptibility of Pseudomonas aeruginosa
to aminoglycosides. J. Clin. Microbiol. 30:585-589.
15. Buschelman, B. J., R. N. Jones, and M. J. Bale. 1994. Effects of

blood medium supplements on activities of newer cephalosporins
tested against enterococci. J. Clin. Microbiol. 32:565-567.
Performance standards for antimicrobial disk susceptibility
tests-sixth edition. Approved Standard. M2-A6, Volume 7,
No.1 National Committee for Clinical Laboratory Standards,
Wayne, PA.
Packaging
100 g
500 g
2 kg
0757-15
0757-17
0757-07
100
500
2
10
0252-15
0252-17
0252-07
0252-08
g
g
kg
kg
Bacto Muller Kauffmann Tetrathionate Broth Base
Intended Use
Bacto Muller Kauffmann Tetrathionate Broth Base is used for enriching

Salmonella from water, foodstuffs and fecal samples prior to selective
isolation.
Muller1 recommended Tetrathionate Broth as a selective medium for

the isolation of Salmonella. Kauffmann2 modified the formula to
include oxbile and brilliant green as selective agents to suppress bacteria
such as Proteus spp.
The British Standard Specification specifies Brilliant Green
Tetrathionate Broth for isolating Salmonella from meat and meat products3
and from poultry and poultry products.4 It is also a recommended
selective broth for isolating Salmonella from animal feces and sewage
polluted water.5 Using more than one selective broth increases the
isolation of Salmonella from samples with multiple serotypes.6
Muller Kauffmann Tetrathionate Broth Base conforms with
ISO/DIS 3565.3

Dehydrated Appearance: Off-white to light beige, free-flowing,
homogeneous.
Solution:
10.58% solution, insoluble in distilled
or deionized water.
Prepared Medium:
Very pale green with white precipitate.
Cultural Response
Prepare Muller Kauffmann Tetrathionate Broth Base per
label directions, with the addition of 1.9 ml Iodine solution and
0.95 ml Brilliant Green solution per 100 ml of medium.
Inoculate and incubate at 42-43C for 18-24 hours. Subculture to
Brilliant Green Agar. Incubate at 35 2C for 18-24 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
APPEARANCE

good red colonies
Salmonella senftenburg 10384 100-1,000
good red colonies
(NCTC)
Escherichia coli
25922* 1,000-2,000 none to
poor
Proteus vulgaris
13315* 1,000-2,000 none to
poor

Muller Kauffmann Tetrathionate Broth Base contains Bacto Peptone
and Beef Extract as sources of carbon, nitrogen, vitamins and minerals.
Oxgall and added Brilliant Green are selective agents which inhibit
gram positive and other gram negative organisms. Calcium Carbonate
is the buffer. Sodium Thiosulfate is a source of sulfur.
Formula
Bacto Muller Kauffmann Tetrathionate Broth Base
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Calcium Carbonate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Sodium Thiosulphate (anhydrous) . . . . . . . . . . . . . . . . . . 38.1
Bacto Oxgall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7
g
g
g
g
g
g
Precautions
The Difco Manual
339

Storage
Expiration Date
Procedure
Materials Provided

Iodine solution (20 g iodine and 25 g potassium iodide in 100 ml water)
Brilliant Green solution (0.1 g Brilliant Green in 100 ml water)
Glassware
Autoclave
Incubator (43C)
Blender
Tetrathionate Broth
Selenite Brilliant Green Medium
Brilliant Green Agar Enrichment
Method of Preparation-Single Strength

1.
2.
3.
4.
5.

Boil gently.
Cool below 45C.
Add 19 ml iodine solution and 9.5 ml brilliant green solution.
Dispense into sterile tubes, mixing well to evenly dispense the
calcium carbonate.

Test Procedure
Meat and Meat Products
1. Weigh 25 grams of the sample into a sterile blender jar and add
225 ml of Buffered Peptone Water (1810) and macerate for sufficient
time to give 10,000-15,000 revolutions.
2. Transfer contents of the blender jar aseptically to a 500 ml flask.
Incubate at 37C 0.1C for 16-20 hours.
340
Section II
3. Transfer 10 ml samples to 100 ml Muller Kauffmann Tetrathionate

Broth and to 100 ml Selenite Brilliant Green Medium (0661).
4. Incubate Muller Kauffmann Tetrathionate Broth at 42-43C and
the Selenite Brilliant Green Enrichment at 37C.
5. Subculture broths after 18-24 hours and 48 hours onto Brilliant
Green Agar.
6. Incubate overnight.
7. Examine for the growth of typical colonies of Salmonella spp.
Sewage Polluted Natural Waters
This procedure is applicable to the isolation of Salmonella spp.
other than S. typhi.
1. Inoculate 25 ml aliquots of the sample into 25 ml of double strength
Buffered Peptone Water (1810). Incubate at 37C for 18 hours.
2. Transfer 1 ml samples into 10 ml of Muller Kauffmann
Tetrathionate Broth.
4. Subculture broths after 18-24 and 48 hours onto Brilliant Green
MacConkey Agar, prepared by adding 10 ml of a 0.33% (w/v)
aqueous solution of brilliant green to MacConkey Agar (0331) to
give a final concentration of 0.033 g/l.
5. Incubate at 37C overnight.
6. Examine for colonies typical of Salmonella spp.
Results
Salmonella spp. will produce red colonies with good growth.

1. The complete medium is unstable and should be used immediately.
It may be stored at 2-8C in the dark for no more than seven days.
2. Due to the nutritional requirements and inhibitory characteristics
of the organisms themselves, organisms other than salmonellae,
such as Morganella morganii and some Enterobacteriaceae may
grow in the medium.
3. Confirmatory tests, such as fermentation reactions and
seroagglutination should be carried out on all presumptive
Salmonella colonies that are recovered.
References
1. Muller, L. 1923. Un nouveau millieu denrichissement pour la
recherche du bacille typhique et des paratyphiques. C. R. Soc. Biol.
(Paris) 89:434-443.
2. Kauffmann, F. 1935. Weitere erfahrungen mit dem kombininierten
anreicherungsverfahren fur Salmonella bazillen. Ztschr. F. Hyg.
117:26-32.
3. International Organization for Standardization. Geneva. 1974.
(Draft International Standard ISO/DIS 3565).
4. A manual for recommended methods for the microbiological
examination of poultry and poultry products. 1982.
5. P.H.L.S. Monograph Series No. 8. 1974.
6. Harvey, R. W. S., and T. H. Price. 1976. Isolation of salmonellae
from sewage- polluted river water using selenite F and MullerKauffmann tetrathionate. J. Hyg. Camb. 77:333-339.
Packaging
Muller Kauffmann
Tetrathionate Broth Base
500 g
1853-17
The Difco Manual
Section II
Mycobiotic Agar
Formula
Bacto Mycobiotic Agar
Mycobiotic Agar
Formula Per Liter
Intended Use
Bacto Mycobiotic Agar is used for isolating pathogenic fungi.

Numerous media, such as Sabouraud Dextrose Agar, Sabouraud
Maltose Agar, Littman Oxgall Agar, Brain Heart Infusion Agar, and
Malt Agar have been used widely in culturing pathogenic fungi. The
Sabouraud media and Malt Agar are somewhat selective in nature
due to low pH, which may suppress bacterial growth. It is well known
that media for isolated pathogenic fungi can also be made selective by
the addition of antibiotics.1-8
Mycobiotic Agar contains cycloheximide and chloramphenicol
making it much more selective when compared to other fungal media.
This medium has proven useful in the isolation of the dermatophytes
and other pathogenic fungi from clinical specimens.9
Georg10 recommends the use of Mycobiotic Agar exclusively for
isolating dermatophytes (because none of the dermatophytes are
sensitive to cycloheximide or chloramphenicol) and in parallel to media
without antibiotics for isolating fungi which cause systemic disease.

Soytone provides carbon and nitrogen sources. Dextrose is a source
of carbon. Cycloheximide suppresses the growth of saprophytic
fungi. Chloramphenicol inhibits bacterial growth. Bacto Agar is the
solidifying agent.
Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Cycloheximide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Chloramphenicol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05
g
g
g
g
g
Precautions
2. TOXIC. TOXIC BY INHALATION AND IF SWALLOWED.
POSSIBLE RISK OF IRREVERSIBLE EFFECTS. POSSIBLE
RISK OF HARM TO THE UNBORN CHILD. Do not breathe dust.
In case of accident or if you feel unwell, seek medical advice
immediately (show the label where possible). Wear suitable
ORGAN(S): Eyes/Ears, Blood, Cardiovascular, Lymph Glands,
Muscles, Nerves, Urogenital
plenty of water and seek medical advice. After contact with skin, wash
immediately with plenty of water. If inhaled, remove to fresh air. If
not breathing, give artificial respiration. If breathing is difficult,
give oxygen. Seek medical advice. If swallowed, induce vomiting;
Storage

homogeneous.
Solution:
slightly opalescent, with no
Reaction of 3.56%
Solution at 25 C:
pH 6.5 0.2
Procedure
Materials Provided
Mycobiotic Agar
Cultural Response
Prepare Mycobiotic Agar per label directions. Inoculate and
incubate at 25-30C for 18-48 hours. For Trichophyton
mentagrophytes, inoculate a 1-2 week old undiluted Trichophyton culture directly onto a slant or plate. Trichophyton cultures
should be incubated up to 7 days.
ORGANISM
Expiration Date
ATCC
INOCULUM
CFU
Aspergillus niger
16404 100-1,000
Candida albicans
10231 100-1,000
Escherichia coli
25922* 1,000-2,000
Trichophyton mentagrophytes 9533
undiluted
GROWTH
inhibited
good
inhibited
good
Glassware
Autoclave
Petri dishes
Tubes with closures
3. Autoclave at 121C for 10 minutes. Avoid overheating, which will
decrease selectivity.

Test Procedure
The Difco Manual
341
Mycological Media
Results

1. Fungi that cause systemic disease may be sensitive to the antibiotics
cycloheximide and chloramphenicol. Primary isolation should
include the use of both non-selective and selective media. 11
Antibiotic-containing media should be incubated at room
temperature. Additional procedures may be required for complete
identification of pathogenic yeasts, particularly Candida albicans.
2. Although culture techniques are important in the identification of
etiological agents of mycotic infections, they are not absolute.
Identification must often be accomplished by using direct
microscopic examination of the specimen, animal inoculation,
biochemical determination, or serological procedures.
References
1. Leach, B. E., J. H. Ford, and A. J. Whiffen. 1947. Actidione, an
antibiotic from Streptomyces griseus. J. Am. Chem. Soc. 69:474.
2. Whiffen, A. J. 1948. The production, assay, and antibiotic activity
of actidione, an antibiotic from Streptomyces griseus. J. Bact. 56:283.
3. Phillips, G. B., and E. Hanel, Jr. 1950. Control of mold
contaminants on solid media by the use of actidione. J. Bacteriology
60:104-105.
4. Georg, L. K., L. Ajello, and M. A. Gordon. 1951. A selective medium for the isolation of Coccidioides immitis. Science 114:387-389.
Section II
5. Fuentes, C. A., F. Trespalacios, G. F. Baquero, and R.

Aboulafia. 1952. Effect of actidione on mold contaminants and
on human pathogens. Mycologia 44:170-175.
6. Georg. 1953. Arch. Dermatol. and Syphilol. 67:355.
7. Cooke, W. B. 1954. The use of antibiotics in media for the isolation of fungi from polluted water. Antibiotics and Chemotherapy
4:657-662.
8. Robinson, H. M., Jr., M. M. Cohen, R. C. V. Robinson, and
E. S. Bereston. 1956. Simplified office procedures for mycological
diagnosis. J. Am. Med. Assoc. 160:537-540.
9. Land, G. A. 1992. Culture media. In H. D. Isenberg, (ed.), Clinical microbiology procedures handbook, vol. 1, p. 6.7.1. American
10. Georg, L. K., E. S. McDonough, L. Ajello, and S. Brinkman.
dermatitidis and other fungi. J. Lab. & Clin. Med. 55:116-119.
Packaging
Mycobiotic Agar
100
500
2
10
g
g
kg
kg
0689-15
0689-17
0689-07
0689-08
Mycological Media
Bacto Mycological Agar . Bacto Mycological Agar w/Low pH
Intended Use
Bacto Mycological Agar is used for cultivating fungi at a neutral pH.
Bacto Mycological Agar w/Low pH is used for isolating and cultivating
fungi and aciduric bacteria.

The value of selective media for the initial cultivation of pathogenic
fungi has been demonstrated by numerous investigators.1,2,3 Earlier
media for fungi generally relied on an acid pH to make the media less
suitable for the growth of many bacteria.6 More recently developed
media use neutral or slightly alkaline reactions,4,5 antibiotics, bile salts
and dyes as selective agents against bacteria.
Mycological media are excellent basal media to which antifungal agents
may be added to study their affect on fungi. These media may be
supplemented with antibacterial substances to render them more
selective for the isolation and cultivation of fungi.
Mycological Agar and Mycological Agar w/Low pH are prepared
according to the formulation suggested by Huppert and Walker.7
Mycological Agar, which has a lower dextrose content than Sabouraud
Dextrose Agar, is recommended for the isolation and cultivation of
fungi from clinical specimens, foods,8 and cosmetics.9 This medium
may be adjusted to pH 4.0 after autoclaving by adding sterile lactic
acid or acetic acid.
342
Mycological Agar at a neutral pH is recommended for working with

pathogenic fungi. Mycological Agar w/Low pH is suitable for culturing
saprophytic yeasts and molds, and aciduric bacteria.

Soytone provides a source of carbon and nitrogen. Dextrose is an
additional source of carbon. Bacto Agar is the solidifying agent.
Formula
Mycological Agar
Formula Per Liter
Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g

Mycological Agar w/Low pH
Formula Per Liter
Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
The Difco Manual
Section II
Mycological Media
Precautions
Procedure
1. Mycological Agar: For Laboratory Use.

Mycological Agar w/Low pH: For Laboratory Use.
Materials Provided
Storage
Glassware
Autoclave
Antibacterial/antifungal agents
Expiration Date
Mycological Agar, Mycological Agar w/low pH

4. If desired, add antibacterial and antifungal agents after sterilizing
and cooling the medium to 45-50C.
Mycological Agar
homogeneous.
Solution:
Prepared Medium:
opalescent.
Reaction of 3.5%
Solution at 25C:
pH 7.0 0.2
homogeneous.
Solution:
Prepared Medium:
opalescent.
Reaction of 3.5%
Solution at 25C:
pH 4.8 0.2
Cultural Response
Mycological Agar, Mycological Agar w/Low pH
Prepare the medium per label directions. Inoculate and incubate
ORGANISM
ATCC
Aspergillus niger
Candida albicans
Penicillium abeanum
Saccharomyces
carlsbergensis
16404
10231
22346
9080
GROWTH
INOCULUM MYCOLOGICAL MYCOLOGICAL
CFU
AGAR
AGAR W/LOW PH
100-1,000
100-1,000
100-1,000
100-1,000
good
good
good
good
good
good
good
good
25923 100-1,000
good
inhibited
The Difco Manual
Mycological Agar
Test Procedure
Mycological Agar and Mycological Agar w/Low pH are used in a variety
of procedures. Consult appropriate references for further information.8,9
Results

1. Non-selective fungal media should be used concurrently with
selective media when isolating fungi due to the sensitivity of some
strains to cycloheximide and chloramphenicol.10,11,12
References
1.
2.
3.
4.
5.
6.
7.
Am. J. Publ. Health. 1951. 41:292.

Bull. D. Inst. Sieroteropl, Melan. 1926. 5:173.
Am. Rev. Resp. Dis. 1967. 95:1041.
A. J. Clin. Path. 1954. 24:621.
Rev. Latinoam Micobiol. 1958. 1:125.
A. J. Clin. Path. 1951. 21:684.
Huppert, M., and L. J. Walker. 1958. The selective and differential effects of cycloheximide on many strains of Coccidioides
immitis. Am. J. Clin. Pathol. 29:291.
9. Curry, A. S., J. G. Graf, and G. N. McEwen, Jr. 1993. CTFA
Microbiology Guidelines. The Cosmetic, Toiletry, and Fragrance
J. Lab. Clin. Med. 44:422.
11. McDonough, E. S., L. Ajello, L. K. Georg, and S. Brinkman.
dermatitidis and other fungi. J. Lab. Clin. Med. 55:116.
343
Neopeptone
Section II
12. McDonough, E. S., L. K. Georg, L. Ajello, and S. Brinkman.

1960. Growth of dimorphic human pathogenic fungi on media
containing cycloheximide and chloramphenicol. Mycopathol.
Mycol. Appl. 13:113.
Bacto Neopeptone
Intended Use
Bacto Neopeptone is used in preparing microbiological culture media.
Also Known As
Neopeptone is also referred to as Special Peptone.

Neopeptone is particularly well suited to the growth requirements of
fastidious miroorganisms. Certain delicate strains of microorganisms
are highly susceptible to the effects of bacteriostatic substances
frequently present in some peptones. The work of Dubos1 shows clearly

Tan, free-flowing granules.
1%, 2% and 10% solutions are soluble in
1%-Very light to light amber, clear to very
slightly opalescent, may have a precipitate.
2%-Light to medium amber, clear to very
slightly opalescent, may have a precipitate.
10%-Medium to dark amber, slightly
opalescent to opalescent, may have a
precipitate.
Reaction of 1%
Solution at 25C: pH 6.9 - 7.5
All solutions are prepared with the pH adjusted to 7.2 - 7.4.
SOLUTION
ORGANISM
Fermentable
2%
Escherichia
Carbohydrates
coli
Indole
0.1%
Escherichia
Production
coli
Acetylmethylcar- 0.1%
Enterobacter
binol Production
aerogenes
Hydrogen
1%
Salmonella
Sulfide
typhi
Toxicity
2% w/0.5% NaCl
Escherichia
& 1.5% Bacto Agar
coli
Toxicity
2% w/0.5% NaCl Staphylococcus
& 1.5% Bacto Agar
aureus
ATCC
RESULT
25922* negative
25922* positive
13048* positive
6539
25922*
positive
good
growth
25923* good
growth
344
500 g
2 kg
0405-17
0405-07
500 g
0305-17
that a peptone free from toxic factors can support the growth of
S. pneumococci from small inocula. Spray2 used Neopeptone in his
culture media for classification of sporulating anaerobes. Casman3
reported Neopeptone to be best suited for use in infusion base. Eldering
and Kendrick4 reported good results with Neopeptone in cultivating
Bordetella pertussis.
Neopeptone is valuable in culture media for the cultivation of
pathogenic fungi. Growth of these microorganisms is rapid and
colonial formation is uniform and typical for the various types. Bacto
Sabouraud Dextrose Agar and Bacto Sabouraud Maltose Agar are
prepared with Neopeptone.
Bacto Todd Hewitt Broth prepared with Neopeptone, is excellent for
growing Group A streptococci for serological typing. Several media
containing Neopeptone are specified in standard methods 5-7 for
multiple applications.
Neopeptone is an enzymatic digest of protein. Neopeptone contains a
wide variety of peptide sizes in combination with vitamins, nucleotides,
minerals and other carbon sources.
Typical Analysis
Ash (%)
7.0
1.2
0.3
Loss on Drying (%)

pH, 1% Soln
3.2
7.4
Carbohydrate (%)
Total
0.8

Total Nitrogen
Amino Nitrogen
Cultural Response
TEST
Mycological Agar
Dehydrated
Appearance:
Solution:
Packaging
13.7
3.3
AN/TN (%)
4.03
4.14
6.19
0.26
13.22
7.02
<0.01
0.36
3.65
Lysine
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine
0.012
0.344
<0.001
<0.001
<0.001
<0.001
0.006
<0.001
Phosphate
Potassium
Sodium
Sulfate
Sulfur
Tin
Zinc
23.8
Amino Acids (%)

Alanine
Arginine
Aspartic Acid
Cystine
Glutamic Acid
Glycine
Histidine
Isoleucine
Leucine
5.16
2.00
8.67
6.73
4.22
3.69
0.96
4.21
4.96
Inorganics (%)
Calcium
Chloride
Cobalt
Copper
Iron
Lead
Magnesium
Manganese
2.209
0.149
2.057
0.340
0.657
<0.001
<0.001
The Difco Manual
Section II
Neutralizing Buffer
Vitamins (g/g)
Results
Biotin
0.2
Cyanocobalamin
<0.1
Folic Acid
0.4
Inositol
3600.0
Nicotinic Acid
52.2
PABA
Pantothenic Acid
Pyridoxine
Riboflavin
Thiamine
Thymidine
2.9
16.0
2.3
1.3
<0.1
<14.0

References

1. Dubos, R. 1930. The bacteriostatic action of certain components

of commercial peptones as affected by conditions of oxidation and
reduction. J. Exp. Med. 52:331-345.
2. Spray, R. S. 1936. Semisolid media for cultivation and
identification of the sporulating anaerobes. J. Bacteriol. 32:135.
pneumococci and streptococci. Am. J. Clin. Pathol. 17:281-289.
4. Eldering, E., and P. L. Kendrick. 1936. Some practical considerations in B. pertussis vaccine preparation. Am. J. Public Health. 24:309.
Test Procedure
Packaging
See appropriate references for specific procedures using Neopeptone.
Neopeptone
Bacto Neutralizing Buffer
Intended Use
Coliform
Salmonella
Spore Count
negative
negative
175

Thermophile Count
75
Procedure
Materials Provided
Neopeptone

Refer to the final concentration of Neopeptone in the formula of the
medium being prepared. Add Neopeptone as required.

Dehydrated Appearance: Tan, free-flowing, homogeneous
Solution:
0.52% solution; soluble in distilled
Prepared tubes:
Very light to light amber, clear to
slightly opalescent without significant
precipitation
Reaction of 0.52%
Solution at 25C:
pH 7.2 0.2
Cultural Response
Prepare Bacto Neutralizing Buffer per label directions. Dilute
a disinfectant containing a quaternary ammonium compound
such as Roccal with Bacto Neutralizing Buffer from 1:2,500
to1:100,000. Inoculate the tubes with Staphylococcus aureus
ATCC 6538P. Prepare pour plates by transferring 1 ml from
each dilution to Bacto Tryptone Glucose Extract Agar (Product
Code 0002). Incubate the plates for 40-48 hours at 32C.
Record growth. Bacto Neutralizing Buffer inactivates the
bactericidal activity which the growth pattern should reflect.
500 g
10 kg
0119-17
0119-08
Bacto Neutralizing Buffer is recommended for detection of

microorganisms found on dairy and food equipment disinfected with
chlorine or quaternary ammonium compounds.

Bacto Neutralizing Buffer, a modification of the Standard Methods
buffered distilled water, has the ability to inactivate the bactericidal
and bacteriostatic effect of chlorine as well as quaternary ammonium
compounds. Neutralizing Buffer is recommended for use in the
microbiological examination of surfaces in the standard methods for
dairy and foods.1,2 Neutralizing Buffer is also recommended for the
digestion and decontamination of mycobacterial specimens.3

Monopotassium phosphate provides the buffering capability. Sodium
thiosulfate inactivates the effect of chlorine compounds. The aryl sulfonate
complex neutralizes the effects of quaternary ammonium compounds.
Formula
Neutralizing Buffer
Formula Per Liter
Monopotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . 0.0425 g
Sodium Thiosulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.16 g
Aryl Sulfonate Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g

The Difco Manual
345
Niacin Assay Medium
Section II
Precautions


Test Procedure
Storage
Expiration Date
Procedure
Materials Provided
Neutralizing Buffer

Glassware
Autoclave
Incubator
Tryptone Glucose Extract Agar
See appropriate standard methods1,2,3 for specific test methodologies.
Results
References
Washington, D.C.
2. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compendium of methods for the microbiological examination of foods, 3rd
3. Cernoch, P. L., R. K. Enns, M. A. Saubolle, and R. J.
Wallace, Jr. 1994. Cumitech 16A, Laboratory diagnosis of the
mycobacterioses. Coordinating ed., A. S. Weissfeld. American
Packaging
Neutralizing Buffer
100 g
0362-15
Bacto Niacin Assay Medium
Intended Use
Bacto Niacin Assay Medium is used for determining niacin concentration by the microbiological assay technique.
Dehydrated Appearance: Off-white, homogeneous, tendency

to clump.
Solution:
3.75% (single strength) or
7.5% (double strength) solution,
water on boiling 2-3 minutes.
Single strength solution is very
light amber, clear, may have a
slight precipitate.
Prepared Medium:
(Single strength) very light amber,
Reaction of 3.75%
Solution at 25C:
pH 6.7 0.2
Cultural Response
Prepare Niacin Assay Medium per label directions. Prepare
a standard curve using nicotinic acid reference standards at
0.0 to 0.25 g per 10 ml. The medium supports the growth
of L. plantarum ATCC 8014 when supplemented with
nicotinic acid.
346

Niacin Assay Medium is prepared according to the formula described
by Snell and Wright,1 modified by Krehl, Strong and Elvehjem2 and
Barton-Wright.3 Niacin Assay Medium is used in the microbiological
assay of nicotinic acid or nicotinamide (niacin) using Lactobacillus
plantarum ATCC 8014 as the test organism. The medium complies
with USP4 and AOAC.5

Niacin Assay Medium is a dehydrated medium free from nicotinic acid
and its analogs but containing all other nutrients and vitamins essential
for the cultivation of L. plantarum ATCC 8014. The addition of nicotinic
acid or its analogs in specified increasing concentrations gives a growth
response that can be measured turbidimetrically or titrimetrically.
The Difco Manual
Section II
Niacin Assay Medium
Formula
Procedure
Niacin Assay Medium

Formula Per Liter
Materials Provided

Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4
DL-Tryptophane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
Adenine Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Uracil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Pyridoxine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . 400
Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Niacin Assay Medium

g
g
g
g
g
mg
mg
mg
g
g
g
g
g
g
g
g
g
mg
mg
mg
Precautions
dust. Wear suitable protective clothing. Keep container tightly
closed. TARGET ORGAN(S): Kidney, Bladder.
3. Great care must be taken to avoid contamination of media or glassware in microbiological assay procedures. Extremely small amounts
Scrupulously clean glassware free from detergents and other chemicals must be used. Glassware must be heated to 250C for at least
1 hour to burn off any organic residues that might be present.
Storage
Expiration Date
The Difco Manual

Glassware
Autoclave
Stock culture of Lactobacillus plantarum ATCC 8014.
Sterile tubes
Centrifuge
Spectrophotometer
Lactobacilli Broth AOAC or Micro Inoculum Broth
Niacin
1.
2.
3.
4.
5.
6.

Boil for 2-3 minutes.
Adjust tube volumes to 10 ml with distilled or deionized water.

Test Procedure
Follow assay procedures as outlined in USP4 and AOAC.5
Stock cultures of the test organism L. plantarum ATCC 8014 are
prepared by stab inoculation of Lactobacilli Agar AOAC or Micro
Assay Culture Agar. After 24-48 hours incubation at 35-37C, the
cultures are kept refrigerated. Transfers are made in triplicate at
monthly intervals.
The inoculum for assay is prepared by subculturing a stock culture of
L. plantarum ATCC 8014 into 10 ml of Lactobacilli Broth AOAC or
Micro Inoculum Broth. After 18-24 hours incubation at 35-37C, the
cells are centrifuged under aseptic conditions and the supernatant
decanted. The cells are washed three times with 10 ml sterile 0.85%
saline. After the third wash, the cells are resuspended in 10 ml sterile
0.85% saline and finally diluted 1:100 with 0.85% sterile saline. One
drop of this latter suspension is used to inoculate each 10 ml
assay tube.
obtained by using niacin at levels of 0.0, 0.025, 0.05, 0.1, 0.15, 0.2
and 0.25 g niacin per assay tube (10 ml). Niacin Assay Medium may
be used for both turbidimetric and titrimetric analyses. Turbidimetric
readings should be made after 18-24 hours incubation at 35-37C.
Titrimetric determinations are best made following 72 hours incubation
at 35-37C.
The concentration of niacin required for the preparation of the standard
curve may be prepared by dissolving 0.05 grams of niacin in 1,000 ml
347
Nitrate Broth
Section II
distilled water, giving a stock solution of 50 g per ml. Dilute the stock
solution by adding 1 ml to 999 ml distilled water (50 ng/ml). Use 0.0,
0.5, 1, 2, 3, 4 and 5 ml of the 50 ng/ml solution per tube. Other standard
concentrations may be used provided the standard falls within the limits
specified by AOAC.5

response.
Results
References

or cup.
1. Snell and Wright. 1941. J. Biol. Chem. 13:675.

2. Krehl, Strong, and Elvehjem. 1943. Ind. & Eng. Chem., Ann.
Ed., 15:471.
3. Barton-Wright. 1944. J. Biochem. 38:314.
Convention, Inc., Rockville, MD.

Packaging
Bacto Nitrate Broth
identifying various types of bacteria. Certain bacteria reduce nitrates

to nitrites only, while others are capable of further reducing nitrite to
free nitrogen or ammonia.
Intended Use
Bacto Nitrate Broth is used for differentiating microorganisms based
on nitrate reduction.
Niacin Assay Medium
100 g
0322-15
Nitrites are colorless; however, in an acid environment, they will react

with alpha-naphthylamine to produce a pink or red color. When
nitrate-positive organisms reduce nitrates to nitrites, a pink color
develops in the broth medium when specific reagents are added.
Nitrate reduction is a valuable criterion for differentiating and

Dehydrated
Light to medium tan, free-flowing,
Medium Appearance: homogeneous.
Solution:
Reaction of 0.9%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepare Nitrate Broth per label directions. Inoculate and
ORGANISM
Acinetobacter calcoaceticus
Escherichia coli
ATCC
INOCULUM
CFU
GROWTH
NITRATE
REDUCTION
19606
13048*
25922*
14028*
100-1,000
100-1,000
100-1,000
100-1,000
good
good
good
good
+
+
+
*These cultures are available as Bactrol disks and should be used as directed.
348
Uninoculated
tube
ATCC 19606
Escherichia coli
ATCC 25922
The Difco Manual
Section II
Nutrient Agar
Nitrate-negative organisms, unable to reduce nitrates, yield no color

after the reagents are added. Nitrate-negative reactions are tested with
zinc dust to confirm the presence of unreduced nitrate.

Beef Extract and Peptone are sources of carbon, protein and nutrients.
Potassium Nitrate is a source of nitrate.
Formula
Nitrate Broth
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Potassium Nitrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Precautions
TARGET ORGAN(S): Blood, Nerves.
Incubator (35C)
0.8% Sulfanilic acid (SpotTest Nitrate Reagent A)
N,N-Dimethyl-alpha-naphthylamine (SpotTest Nitrate Reagent B)
Zinc dust (SpotTest Nitrate Reagent C)
1. Dissolve 9 grams of Nitrate Broth in 1 liter distilled or deionized water.

Not applicable
Test Procedure
1. Inoculate the medium with several colonies from a pure 18-24 hour
culture. Test an uninoculated control tube in parallel.
2. Incubate the tubes aerobically at 35 2C for 18-24 hours.
3. Test for nitrate by adding a few drops of 0.8% sulfanilic acid and
N,N-dimethyl- alpha-naphthylamine to each tube.
4. Observe for development of a distinct red or pink color within 1-2
minutes, indicating reduction of nitrate to nitrite.
5. If there is no color development, add a pinch of zinc dust (approximately 20 mg on an applicator stick) to the tube. If there is no color
development within 5-10 minutes, nitrate was reduced beyond
nitrite and the test result is positive.
Results
Development of a distinct red or pink color within 1-2 minutes indicates
reduction of nitrate to nitrite and is a positive test result.
1. The addition of too much zinc dust may cause a false-negative

reaction or a momentary color reaction.1
2. The nitrate test is very sensitive. An uninoculated nitrate control
should be tested with reagents to determine whether the medium is
nitrate free and that the glassware and reagents have not been
contaminated with nitrous oxide.1
3. The inoculum should not be taken from a liquid or broth suspension
of the organism.1
Procedure
References
Storage
Expiration Date
Materials Provided
Nitrate Broth

Glassware
Autoclave
1. MacFaddin, J. D. 1985. Media for isolation-cultivationidentification-maintenance medical bacteria, p.275-284, vol 1.

Packaging
Nitrate Broth
Bacto Nutrient Agar
500 g
0268-17
Intended Use
Bacto Nutrient Agar is used for cultivating a wide variety of microorganisms.

In the early 1900s the American Public Health Association (APHA)
The Difco Manual
suggested this formulation as a standard culture medium for use in

bacterial processing for water analysis.1 The name Nutrient Agar was
later adopted for the medium. In Standard Methods of Water Analysis2
and Standard Methods of Milk Analysis,3 the APHA advocated the
use of dehydrated media for bacterial examination of water and milk.
Nutrient Agar meets APHA and Association of Official Analytical
Chemists (A0AC) standard methods.4,5,6
349
Nutrient Agar
Section II
Nutrient Agar continues to be a widely used general purpose medium

for growing nonfastidious microorganisms. It is specified in many
standard methods procedures for examining foods, dairy products,
water and other materials.4,5,6,7
Procedure
Nutrient Agar contains Beef Extract and Bacto Peptone as carbon and
nitrogen sources for general growth requirements. Bacto Agar is added
as a solidifying agent.
Formula
Materials Provided
Nutrient Agar
Flask with closure
Autoclave
Petri dishes
Incubator
Nutrient Agar
Formula per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
1. Suspend 23 grams in 1 liter distilled or deionized water. Heat to

boiling to dissolve completely.
Process specimens according to established procedures for the type of

Precautions
1. For Laboratory Use
Test Procedure
Storage
1. Inoculate with growth from either a single colony on agar or a

loopful of broth and streak for isolation.
2. Incubate aerobically at 35C for 18 to 24 hours or longer if necessary.
Expiration Date
Results
The product is stable through the expiration date on the label when
stored as directed. Expiration date applies to the medium in its intact
container. Do not use if the medium is caked, discolored or shows other
signs of deterioration.
Good growth of nonfastidious organisms on Nutrient Agar will appear

as translucent colonies.

Solution:
medium amber, clear to slightly
opalescent, no significant precipitate.
Prepared medium:
Reaction of 2.3%
Solution at 25C:
pH 6.8 0.2
Cultural Response
Prepare Nutrient Agar per label directions. Inoculate medium
with the test organism and incubate at 35 2C for 18-48 hours.
ORGANISM
Escherichia coli
ATCC
INOCULUM
CFU
GROWTH
19433*
25922*
27853*
100-1,000
100-1,000
100-1,000
good
good
good
350
References
of water analysis, 3rd ed. American Public Health Association,
Washington, D.C.
of water analysis, 5th ed. American Public Health Association,
Washington, D.C.
of milk analysis, 4th ed. American Public Health Association,
Washington, D.C.
7. Vanderzant C., and D. F. Splittstoesser (ed.). 1992. Compendium
Packaging
Nutrient Agar
100 g
500 g
2 kg
0001-15
0001-17
0001-07
The Difco Manual
Section II
Nutrient Agar 1.5%
Bacto Nutrient Agar 1.5%
Precautions
Intended Use
Bacto Nutrient Agar 1.5% is used for cultivating a variety of microorganisms and with the addition of blood or other enrichment can be
used for cultivating fastidious microorganisms.
Storage
Expiration Date
Nutrient Agar 1.5% is a modification of Nutrient Agar, a formula

prepared according to APHA standards.1,2 This medium is a slightly
alkaline general purpose medium. Since this medium contains 0.8%
sodium chloride it can be used as a base for enrichment with blood, ascitic
fluid or other supplements for cultivating fastidious microorganisms.
Procedure
Materials Provided
Bacto Beef Extract and Bacto Peptone provide the nitrogen, vitamins,
amino acids and carbon sources in Nutrient Agar 1.5%. Sodium
chloride maintains the osmotic balance so that red blood cells will
not rupture when blood is added as supplement.1 Bacto Agar is the
solidifying agent.
Nutrient Agar 1.5%

Glassware
Autoclave
Incubator (35C)
Waterbath
Formula
Nutrient Agar 1.5%
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
Escherichia coli
ATCC 25922
ATCC 25923

Dehydrated Appearance: Beige to light tan, free-flowing, homogeneous.
Solution:
Prepared Medium:
Reaction of 3.1%
Solution at 25C:
pH 7.3 0.2
Cultural Response
Prepare Bacto Nutrient Agar 1.5% per label directions.
Inoculate and incubate the plates at 32 1C for 24 2 hours.
ORGANISM
ATCC

Streptococcus pyogenes 19615*
Escherichia coli
25922*
INOCULUM
CFU
100-1,000
100-1,000
100-1,000
100-1,000
GROWTH GROWTH W/15%

PLAIN
SHEEP BLOOD HEMOLYSIS
good
good
good
good
good
good
good
good
beta
alpha
beta
beta
ATCC 6305
ATCC 19615
The Difco Manual
351
Nutrient Agar with MUG
Section II
Results

4. To prepared an enriched medium, cool the sterile base to 45-50C
and add the desired enrichment. Mix thoroughly.
References
of methods for the microbiological examination of foods,

Test Procedure
Packaging
For a complete discussion of the isolation and identification of aerobic

and anaerobic microorganisms, refer to appropriate references.
Nutrient Agar 1.5%
500 g
0069-17
Bacto Nutrient Agar with MUG
Intended Use
the membrane from a total-coliform or fecal-coliform positive sample

to a Nutrient Agar substrate containing 4-methylumbelliferyl--Dglucuronide (MUG).1
Bacto Nutrient Agar with MUG is used for detecting and enumerating
Escherichia coli in water.
Mates and Shaffer3 used the membrane filter-Endo Agar method,

followed by incubation on Nutrient Agar with MUG, to detect and
enumerate E. coli within 4 hours of membrane transfer. E. coli was
recovered at a rate of 98% with no false-positive results.

Escherichia coli is a member of the fecal coliform group of bacteria.
The presence of E. coli is indicative of fecal contamination.1 Feng and
Hartman 2 developed a rapid assay for E. coli by incorporating
4-methylumbelliferyl--D-glucuronide (MUG) at a final concentration
of 100 g/ml into Lauryl Tryptose Broth. Nutrient Agar is similarly
modified with the addition of MUG. Rapid quantitation and verification
may be achieved with the membrane filtration procedure by transferring
Nutrient Agar with MUG is prepared according to the formula specified

by US EPA4 and Standard Methods.1
Uninoculated
plate
Escherichia coli
ATCC 25922

Solution:
Prepared Medium:
Light amber, clear to slightly
precipitate.
Reaction of 2.31%
Solution at 25C:
pH 6.8 0.2
Cultural Response
Prepare Nutrient Agar with MUG per label directions. After
incubation on mEndo Agar LES, aseptically transfer the membrane
to Nutrient Agar with MUG. Incubate 18-24 hours at 35 2C.
ORGANISM
Enterobacter
aerogenes
Escherichia coli
ATCC
INOCULUM GROWTH ON
CFU
mENDO LES
COLONY
COLOR
FLUORESCENCE
13048*
30-300
good
red
25922*
30-300
good
red w/sheen
352
The Difco Manual
Section II
Test Procedure
Beef Extract and Bacto Peptone are sources of nitrogen, vitamins,

carbon and amino acids. Bacto Agar is a solidifying agent. The
substrate, MUG (4-methylumbelliferyl--D-glucuronide), produces a
blue fluorescence when hydrolyzed by the enzyme -glucuronidase,
which is produced by most E. coli.
Follow the methods and procedures for water testing using mEndo
Agar LES in Standard Methods.1 After incubation on mEndo Agar LES,
aseptically transfer the membrane to Nutrient Agar with MUG. Incubate
18-24 hours at 35 2C. Expose the filter surface to longwave UV light.
Formula
Observe for fluorescence following incubation. Positive MUG reactions

exhibit a bluish fluorescence around the periphery of the colony under
longwave (approximately 366 nm) UV light.

Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
MUG (4-Methylumbelliferyl--D-glucuronide) . . . . . . . . 0.1
g
g
g
g
Precautions
Storage
Expiration Date
Procedure
Materials Provided

Glassware
Test tubes
Sterile pipettes
Incubator (35C)
Longwave UV lamp (approximately 366 nm)
mEndo Agar LES
Sterile membranes
Filter apparatus
Petri dishes (50 x 9 mm)
Sterile absorbent pad
1.
2.
3.
4.

Dispense into sterile 50 x 9 mm Petri dishes.
Results
Typical strains of E. coli (red with a green metallic sheen on mEndo

Agar LES) exhibit blue fluorescence on Nutrient Agar with MUG.
Non-E. coli coliforms may produce a metallic sheen but do not fluoresce.

this medium.
2. Glucuronidase-negative strains of E. coli have been encountered.5,6,7
Similarly, MUG-negative strains of E. coli have been reported in
this assay procedure but at a very low frequency.3
3. Strains of Salmonella and Shigella species that produce glucuronidase may infrequently be encountered.8 These strains must be
distinguished from E. coli on the basis of other parameters, i.e.,
gas production, lactose fermentation or growth at 44.5C.
References
2. Feng, P. C. S., and P. A. Hartman. 1982. Fluorogenic assays for
Microbiol. 43:1320-1329.
3. Mates, A., and M. Shaffer. 1989. Membrane filtration differentiation of E. coli from coliforms in the examination of water.
J. Appl. Bacteriol. 67:343-346.
4. Federal Register. 1991. National primary drinking water regulations; analytical techniques: coliform bacteria. Fed. Regist.
56:636-643.
5. Chang, G. W., J. Brill, and R. Lum. 1989. Proportion of -Dglucuronidase-negative Escherichia coli in human fecal samples.
Appl. Environ. Microbiol. 55:335-339.
6. Hansen, W., and E. Yourassowsky. 1984. Detection of glucuronidase in lactose fermenting members of the family
enterobacteriaceae and its presence in bacterial urine cultures.
7. Kilian, M., and P. Bulow. 1976. Rapid diagnosis of Enterobacteriaceae. Acta Pathol. Microbiol. Scand. Sect. B 84:245-251.
8. Damare, J. M., D. F. Campbell, and R. W. Johnston. 1985.
Simplified direct plating method for enhanced recovery of
Escherichia coli in food. J. Food Sc. 50:1736-1746.
Packaging
Collect water samples in accordance with recommended procedures.1
The Difco Manual
100 g
500 g
0023-15
0023-17
353
Nutrient Broth
Section II
Bacto Nutrient Broth

Nutrient Broth contains Beef Extract and Bacto Peptone as carbon
and nitrogen sources for general growth requirements.
Intended Use
Bacto Nutrient Broth is used for cultivating nonfastidious microorganisms.
Formula
Nutrient Broth
Formula per liter
In the early 1900s, the American Public Health Association (APHA)

suggested this formulation of a standard culture medium for use in
bacteriological procedures for water analysis.1 In Standard Methods
of Water Analysis 2 and Standard Methods of Milk Analysis, 3
the APHA advocated the use of dehydrated culture media for
bacteriological examination of water and milk.
Nutrient Broth, a widely used medium, is included in many standard
methods procedures. In Compendium of Methods for the Microbiological
Examination of Foods4 and Standard Methods for the Examination of
Dairy Products,5 Nutrient Broth is included as a satisfactory substitute
for the buffered rinse solution used in sampling equipment, containers
and air because it effectively neutralizes chlorine and quaternary
ammonium compounds. In Standard Methods for the Examination of
Water and Wastewater, Nutrient Broth is included in testing methods
for viruses using microporous filters.6
Nutrient Broth is used as a pre-enrichment medium when testing
certain foods and dairy products for Salmonella. In dried or processed
foods, salmonellae may be sublethally injured and in low numbers.
The presence of other bacteria and the components of the food
sample may hinder growth and recovery of Salmonella. Preenrichment
in a nonselective medium such as Nutrient Broth allows for repair
of cell damage, dilutes toxic or inhibitory substances, and provides
a nutritional advantage to Salmonella over other bacteria.4 Nutrient
Broth is included in many standard methods procedures for testing
foods, dairy products and other materials.4,5,7,8

Dehydrated Appearance: Medium tan, free-flowing,
homogeneous.
Solution:
amber, clear with no precipitate.
Prepared Medium:
Light to medium amber, clear with
no precipitate.
Reaction of 0.8%
Solution at 25C:
pH 6.8 0.2 at 25C
Cultural Response
Prepare Nutrient Broth per label directions. Inoculate medium
with the test organism and incubate at 35 2C for 18-24 hours.
Escherichia coli
ATCC
CFU
GROWTH
25922*
25923*
100-1,000
100-1,000
good
good
as directed in the Bactrol Disks Technical Information.
354
Precautions
Storage
Expiration Date
The product is stable through the expiration date on the label when
stored as directed. Expiry date applies to the medium in its intact
container. Do not use if the medium is caked, discolored or shows other
signs of deterioration.
Procedure
Material Provided
Nutrient Broth

Flask with closure
Autoclave
Containers, 225 ml
Incubator
ORGANISM

Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g

Test Procedure
Direct:
1. Inoculate the broth with specimen on a swab.
2. Incubate for 18-24 hours at 35 2C.
As a preenrichment medium when testing certain foods and dairy
products for Salmonella, consult appropriate references for specific
recommendations:4,5,7,8
1. Mix 25 grams of sample with 225 ml of Nutrient Broth.
3. Transfer a portion to one or more selective enrichment broths.
Results
Turbidity indicates growth.
References
of water analysis, 3rd ed. American Public Health Association,
Washington, D.C.
The Difco Manual
Section II
Nutrient Gelatin

of water analysis, 5th ed. American Public Health Association,
Washington, D.C.
of milk analysis, 4th ed. American Public Health Association,
Washington, D.C.
5. Marshall, R. T. (ed.) 1993. Standard methods for the microbiological examination of dairy products, 16th ed. American Public
Bacto Nutrient Gelatin
Intended Use
Bacto Nutrient Gelatin is used for detecting gelatin liquefaction by
proteolytic microorganisms.

Gelatin was the first gelling agent used to solidify culture media. The
advantages of a solid medium were the ability to perform plate counts
and to isolate microorganisms in pure culture. The disadvantages of
gelatin were the limitation of incubation at 20C, a temperature that is

Packaging
Nutrient Broth
100
500
2
10
g
g
kg
kg
0003-15-0
0003-17-8
0003-07-0
0003-08-9
lower than the optimum for growing many microorganisms, and the
fact that many organisms metabolize (liquefy) gelatin. Agar later
replaced gelatin as a solidifying agent.
Characterizing fermentative and non-fermentative gram-negative
bacilli includes the test for gelatin liquefaction. If the proteolytic
enzyme gelatinase is present, gelatin is hydrolyzed and loses its
gelling characteristic.1 Edwards and Ewing include this test in the
differentiation scheme for the Enterobacteriaceae.2 Procedures for
performing the standard tube method for gelatin liquefaction are available.2,3,4
Nutrient Gelatin contains Bacto Peptone and Beef Extract as carbon

and nitrogen sources for general growth requirements. Gelatin is the
substrate for determining if the microorganism has the proteolytic
enzyme to hydrolyze (liquefy) gelatin.
Formula
Dehydrated Appearance: Tan, free-flowing, fine granular.

Solution:
precipitate.
Prepared Medium:
precipitate.
Reaction of 12.8%
Solution at 25C:
pH 6.8 0.2
Cultural Response
Prepare Nutrient Gelatin per label directions. Using a heavy
inoculum, inoculate by stabbing the tube and incubate at
35 2C for 18-48 hours or up to two weeks, if required. To
read gelatinase, refrigerate until well chilled and compare to
uninoculated tube. Tilt tubes carefully to test for liquefaction.
Tubes positive for gelatinase remain liquid.
ORGANISM
ATCC
Escherichia coli
25922*
27853
RECOVERY
GELATINASE
good
good
Nutrient Gelatin
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Bacto Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 g
Precautions
Storage
Expiration Date
Procedure
Materials Provided
Nutrient Gelatin
The Difco Manual
355
NZCYM Broth, NZYM Broth & NZM Broth
Section II
Results
Flask with closure

Autoclave
Incubator (35C)
Inoculating needle
Refrigerator
Positive: Medium remains liquefied after refrigeration.

Negative: Medium becomes solid after refrigeration.
Uninoculated control tube: Medium becomes solid after refrigeration.
1.
2.
3.
4.

Warm to 50-55C to dissolve completely.
Dispense required amount in test tubes and place caps on tubes.

Test Procedure1
1. Using a sterile inoculating needle, touch several similar, well-isolated
colonies on agar and stab directly down the center of the tube to
approximately 10 mm from the bottom.
2. Incubate at 35 2C for 24-48 hours. Incubate an uninoculated
control tube with the test. Incubation may be extended to 14 days
for some organisms.
3. Examine at various intervals.
a. Transfer the tubes to a refrigerator or ice bath.
b. Do not shake the tubes when transferring from incubator
to refrigerator.
c. Gently invert the chilled tubes to test for solidity.

1. Use this method for detecting gelatinase only if the identification
procedure allows for incubation beyond 48 hours.
2. Gelatin is liquid at temperatures above 20C. If tubes are incubated
at 35C, they must be refrigerated in order to read for liquefaction.
Include an uninoculated tube in the test procedure for comparison.
3. Growth and liquefaction frequently occur only at the surface of the
tube. To prevent a false-negative interpretation, handle tubes
carefully when warm so that liquefied gelatin remains at the
surface of the tube.
References
New York, NY.
Packaging
Nutrient Gelatin
500 g
0011-17
Bacto NZCYM Broth

Bacto NZYM Broth, Bacto NZM Broth
Intended Use
Bacto NZCYM Broth, Bacto NZYM Broth, and Bacto NZM Broth are
used for cultivating recombinant strains of Escherichia coli.

NZCYM Broth was developed by Blattner et al. as an enriched medium
for cultivating recombinant strains of E. coli and propagating bacteriophage.1 E. coli grows rapidly in rich media, such as the NZ media,
which provide amino acids, vitamins and other metabolites the cell
would otherwise have to synthesize.2 Casein is supplied as Casein
Digest in NZ media, a different form than in the LB formulations,
providing a good nitrogen source.
The three variations of NZ Media allow the user to select a formulation
appropriate to the need.

Casein Digest, Yeast Extract and Casamino Acids provide the necessary
nutrients and cofactors required for excellent growth of recombinant
strains of E. coli. Due to its higher degree of digestion, Casamino
356
Acids is an excellent source of free amino acids. Sodium Chloride is

included in the medium to provide a suitable osmotic environment.
Magnesium Sulfate is a source of magnesium ions required in a variety
of enzymatic reactions, including DNA replication.
Formula
NZCYM Broth
Formula Per Liter
Bacto Casein Digest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Casamino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Magnesium Sulfate, Anhydrous . . . . . . . . . . . . . . . . . . . . 0.98
g
g
g
g
g

NZYM Broth
Formula Per Liter
Bacto Casein Digest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
The Difco Manual
Section II
NZCYM Broth, NZYM Broth & NZM Broth
Magnesium Sulfate, Anhydrous . . . . . . . . . . . . . . . . . . . . 0.98 g

NZM Broth
Formula Per Liter
Bacto Casein Digest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Magnesium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.94 g

NZCYM Broth
homogeneous.
Solution:
Prepared Tubes:
Reaction of 2.2%
Solution at 25C:
pH 7.0 0.2
NZYM Broth
homogeneous.
Solution:
Prepared Tubes:
Reaction of 2.1%
Solution at 25C:
pH 7.0 0.2
NZM Broth
homogeneous.
Solution:
Prepared Tubes:
Reaction of 1.6%
Solution at 25C:
pH 7.0 0.2
Cultural Response
NZCYM Broth, NZYM Broth or NZM Broth
ATCC
INOCULUM
CFU
GROWTH
23724
100-300
Good
The Difco Manual
Expiration Date
Materials Provided
NZCYM Broth
NZYM Broth
NZM Broth

Procedure
Precautions
ORGANISM
Storage

Tubes with closures
Autoclave
Incubator (35C)
1. Dissolve the medium in 1 liter of distilled or deionized water:
NZCYM Broth - 22 grams;
NZYM Broth - 21 grams;
NZM Broth - 16 grams.

Test Procedure
Consult an appropriate reference for recommended test procedures.3
Results
Growth should be evident in the form of turbidity.
References
1. Blattner, F. R., B. G. Williams, A. E. Blechl, K. DennistonThompson, H. E. Faber, L. A. Furlong, D. J. Grunwald,
D. O. Kiefer, D. D. Moore, J. W. Schumm, E. L. Sheldon,
and O. Smithies. 1977. Charon phages: Safer derivatives of
bacteriophage for DNA cloning. Science 196:161.
2. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore,
J. G. Seidman, J. A. Smith, and K. Struhl, (ed.). 1994. Current
protocols in molecular biology, vol. 1. Current Protocols,
New York, NY.
Cold Spring Harbor, NY.
Packaging
NZCYM Broth
NZYM Broth
NZM Broth
500 g
500 g
500 g
0404-17
0415-17
0435-17
357
OF Basal Medium
Section II
Bacto OF Basal Medium
Intended Use
Bacto OF Basal Medium is used with added carbohydrate
for differentiating gram-negative microorganisms based on oxidationfermentation patterns.
Also Known As
OF Basal Medium conforms with Oxidation Fermentation Basal Medium.

OF Basal Medium is based on the formula of Hugh and Leifson1, who
described the taxonomic significance of fermentative versus oxidative
metabolism of carbohydrates by gram-negative bacteria. When an
organism is inoculated into two tubes of OF Basal Medium containing
a carbohydrate and the medium in one tube is covered with petrolatum
to exclude oxygen, reactions of differential significance may be
observed. Fermentative organisms will produce an acid reaction in both
the covered and uncovered tubes. Oxidative organisms produce an acid
reaction in the uncovered tube while there is little or no growth and no
acid formation in the covered tube. Non-oxidative, non-fermentative
organisms yield no change in the covered tube and an alkaline reaction
in the uncovered tube. OF Basal Medium is listed in standard
procedures for the differentiation of gram-negative bacteria based on
the oxidation and fermentation of carbohydrates.2,3,4,5,6
A modification of OF Basal Medium was developed by Knapp and
Holmes7 to detect acid production from carbohydrates by Neisseria
spp. and Branhamella catarrhalis. The authors reduced the protein
concentration relative to the carbohydrate concentration, substituted
phenol red for brom thymol blue at a low concentration, and adjusted
the initial pH to 7.2. A selective and differential medium for Pseudomonas
cepacia, based on OF Basal Medium, was developed by Welch et al.8
by adding agar-agar, lactose, polymyxin B and bacitracin.

Tryptone provides nitrogen, vitamins and minerals. A high concentration
of carbohydrate is added to OF Basal Medium. This helps avoid
utilization of peptone by an aerobic organism, which would produce
an alkaline reaction and neutralize the slight acidity produced by
oxidation.9 Glucose is the carbohydrate commonly added to the
OF Basal Medium; lactose, maltose, mannitol, saccharose and xylose
may also be used. Sodium Chloride maintains the osmotic balance of
the medium and enhances growth of Brucella species.1 Dipotassium
Phosphate provides buffering capacity. Brom Thymol Blue acts as
a pH indicator, changing to yellow under acidic conditions. Bacto Agar
aids in the determination of motility and also helps evenly distribute
any acid produced at the surface of the medium.
Formula
OF Basal Medium
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
g
g
g
g
g

Dehydrated Appearance: Light beige with green tinge,
Solution:
0.94% solution, soluble on boiling in
distilled or deionized water. Solution
is green, clear, with no precipitate.
Prepared Medium :
Green, clear to very slightly
opalescent, with no precipitate.
Reaction of 0.94%
Solution at 25C:
pH 6.8 0.2
Cultural Response
Prepare OF Basal Medium per label directions. Inoculate
duplicate tubes, overlay one tube with 2 ml sterile mineral oil,
and incubate at 35C for 40-48 hours.
ORGANISM
Escherichia coli
Shigella flexneri
ATCC
19606
25922*
27853*
12022*
K = Alkaline reaction, green to blue medium

A = Acid reaction, yellow medium
G = Gas
PLAIN
OPEN CLOSED
K
K
K
K
K
K
K
K
w/1% DEXTROSE
OPEN CLOSED
A
AG
A
A
K
AG
K
A
Uninoculated
tube
Escherichia coli
ATCC 25922
Acinetobacter
calcoaceticus
ATCC 19606
Tubes above are closed, with Dextrose
Shigella flexneri
ATCC 12022
*These cultures are available as Bactrol Disks and are to be used as directed in Bactrol Disks Technical Information.
358
The Difco Manual
Section II
Precautions
Storage
Expiration Date
Procedure
Materials Provided
OF Basal Medium

Glassware
Autoclave
Sterile melted petrolatum (mineral oil)
Carbohydrates of choice: Glucose (dextrose), Lactose, Maltose,
Mannitol, Saccharose and/or Xylose
Incubator (35C)
1.
2.
3.
4.

Add 1% carbohydrate before or after sterilization, depending on
heat lability.
5. Dispense in test tubes.

Test Procedure
1. Inoculate duplicate test tubes by stabbing the medium to the
desired depth with an inoculating needle.
2. Overlay one tube of each set with 2 ml sterile mineral oil.
3. Incubate at a temperature appropriate for the test organism for a
minimum of 48 hours.
4. Examine the medium for gas production and a color change from
green to yellow.
Results
Alkaline reaction: green to blue medium
Acid reaction:
yellow medium
A color change of the medium in both the plain and overlay tubes,
with or without gas production, indicates fermentation of the
carbohydrate tested.
A color change in the plain tube, only, indicates oxidative metabolism
of the carbohydrate tested.
The Difco Manual
OF Basal Medium
No color change in both tubes of the set indicates the organism is

nonsaccharolytic for the carbohydrate tested.

1. The acid reaction produced by oxidative organisms is apparent at
the surface and gradually spreads throughout the medium. If the
oxidation is weak or slow, however, an initial alkaline reaction
at the surface of the open tube may persist for several days and
eventually convert to an acid reaction.
2. If an organism is unable to grow on OF Basal Medium, Cowan10
recommends adding either 2% serum or 0.1% yeast extract to each
carbohydrate tube.
3. Nonsaccharolytic organisms produce a slight alkalinity in the
open tube (blue-green color), while the covered tube will not
exhibit a color change (green).
References
1. Hugh, R. and E. Leifson. 1953. The taxonomic significance of
fermentative versus oxidative metabolism of carbohydrates by
various gram negative bacteria. J. Bacteriol. 66:24-26.
International. Gaithersburg, MD.
3. Vanderzant, C. and D. F. Splittstoesser (eds.). 1992. Compendium of methods for the microbiological examination of foods,
Washington, D.C.
Washington, D.C.
7. Knapp, J. S. and K. K. Holmes. 1983. Modified oxidationfermentation medium for detection of acid production from
carbohydrates by Neisseria spp. and Branhamella catarrhalis.
8. Welch, D. F., M. J. Muszynski, C. H. Pai, M. J. Marcon, M. M.
Hribar, P. H. Gilligan, J. M. Matsen, P. A. Ahlin, B. C. Hilman
and S. A. Chartrand. 1987. Selective and differential medium for
recovery of Pseudomonas cepacia from the respiratory tracts of
patients with cystic fibrosis. J. Clin. Microbiol. 25:1730-1734.
10. Cowan, S. T. 1974. Cowan and Steels manual for the identification
of medical bacteria, 2nd ed. Cambridge University Press,
Cambridge, MA.
Packaging
OF Basal Medium
Mineral Oil
100 g
500 g
30 ml
0688-15
0688-17
6663-30
359
OGYE Agar
Section II
OGYE Agar
Bacto OGYE Agar Base . Bacto Antimicrobic Vial
Oxytetracycline
Intended Use
Bacto OGYE Agar Base is for use with Bacto Antimicrobic Vial
Oxytetracycline in isolating and enumerating yeasts and molds in foods.
Also Known As
OGYE Agar is specified as a standard methods medium for use with

dairy products.2

OGYE Agar Base contains Yeast Extract to supply B-complex vitamins
which stimulate bacterial growth. Dextrose is the carbon energy source.
Bacto Agar is the solidifying agent. Antimicrobic Vial Oxytetracycline
inhibits the growth of bacteria.
OGY Agar Base

OGYA (Oxytetracycline-Glucose-Yeast (Extract) Agar)1

Acidified agar may be used for enumerating yeasts and molds in foods
and dairy products. However, in some cases, antimicrobics better
suppress bacterial growth and improve recovery of yeasts and molds.2, 3
Mossel et al.4, 5 described Oxytetracycline-Glucose Yeast Extract
(OGYE) Agar for selectively isolating and enumerating yeasts and
molds in foods. Mossel et al. demonstrated improved recovery compared
to acidified agar media.
Formula
OGYE Agar Base
Formula Per Liter
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 g

Antimicrobic Vial Oxytetracycline
Oxytetracycline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 mg
Precautions

OGYE Agar Base

Solution:
is medium amber, very slightly
Prepared Medium:
Reaction of 3.7%
Solution at 25C:
pH 7.0 0.2
Lyophilized Appearance: Yellow cake or powder
Cultural Response
Prepare OGYE Agar Base and Antimicrobic Vial
Oxytetracycline per label directions. Inoculate using pour
plate technique and incubate at 22 3C for up to 5 days.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Aspergillus niger
16404 100-1,000
good
Escherichia coli
25922* 1,000-2,000 inhibited
9763
100-1,000
good
Saccharomyces carlsbergensis 9080
100-1,000
good
(cerevisiae or uvarum)
S. cerevisiae ATCC 9080 is prepared to order.
360
Storage
very hygroscopic. Keep container tightly closed. Store the antimicrobic
supplement at 2-8C.
Expiration Date
Procedure
Materials Provided
OGYE Agar Base

Glassware
Petri dishes
Autoclave
The Difco Manual
Section II
Oatmeal Agar
4. Aseptically add 10 ml rehydrated Antimicrobic Vial Oxytetracycline to the medium. Mix well.
1. Aseptically add 10 ml sterile distilled or deionized to the Antimicrobic Vial Oxytetracycline.
2. Shake to dissolve contents.

Test Procedure
Results
References
1. MacFaddin, J. F. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, vol. 1. p. 579-582.
2. Frank, J. F., G. L. Christen, and L. B. Bullerman. 1993. Tests

for groups of microorganisms, p. 271-286. In R. T. Marshall (ed.),
Standard methods for the microbiological examination of
Washington, D.C.
and molds, p. 239-249. In C. Vanderzant, and D. F. Splittstoesser
(ed.), Compendium of methods for the microbiological
Washington, D.C.
4. Mossel, D. A. A., A. M. C. Kleynen-Semmeling, H. M. Vincentie,
H. Beerens, and M. Catsaras. 1970. Oxytetracycline-GlucoseYeast Extract Agar for selective enumeration of moulds and yeasts
in foods and clinical material. J. Appl. Bacteriol. 33:454-457.
5. Mossel, D. A. A., M. Visser, and W. H. J. Mengerink. 1962. A
comparison of media for the enumeration of moulds and yeasts in
food and beverages. Lab. Pract. 11:109-112.
Packaging
OGYE Agar Base
500 g
10 ml
1811-17
3267-59
Bacto Oatmeal Agar
Intended Use
Fungi are extremely successful organisms, as evidenced by their

ubiquity in nature.1 Of the estimated 250,000 species, fewer than 150
are known primary pathogens of humans.1
Bacto Oatmeal Agar is used for cultivating fungi, particularly for

macrospore formation.

Dehydrated Appearance: Beige, nonhomogeneous, may be
slightly lumpy.
Solution:
or deionized water on boiling with
frequent agitation. Solution is
off-white, opaque with
nonhomogeneous particles.
Prepared Medium:
Off-white, opaque appearance with
nonhomogeneous particles.
Reaction of 7.25%
Solution at 25C:
pH 6.0 0.2
Cultural Response
The detection of fungi is a great concern in the pharmaceutical, food

and cosmetic industry.

Oatmeal is a source of nitrogen, carbon, protein and nutrients. Bacto
Formula
Oatmeal Agar
Formula Per Liter
Oatmeal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.5 g
Prepare Oatmeal Agar per label directions. Inoculate and

ORGANISM
ATCC
INOCULUM
CFU
Aspergillus niger
Candida albicans
16404
10231
9763
100-1,000
100-1,000
100-1,000
GROWTH
good
good
good
The Difco Manual
Identification and classification of fungi is primarily based on the

morphologic differences in their reproductive structures. 2 Fungi
reproduce by producing spores.2 Large, multi-celled spores are called
macroconidia, macroaleuriospores or macrospores and are produced
by aerial sporulation.2
Precautions
Storage
361
Orange Serum Agar & Orange Serum Broth Concentrate 10X
Section II
Expiration Date

Procedure
Consult to appropriate references for specific procedures on the isolation and cultivation of fungi.
Materials Provided
Oatmeal Agar

Glassware
Autoclave
Incubator
1.
2.
3.
4.

Heat to boiling with constant agitation.
Test Procedure
Results
References
and classification of the fungi, p. 699-708. In P. R. Murray, E. J. Baron,
M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.). Manual of clinical
microbiology, 6th ed. American Society for Microbiology, Washington, D.C.
Packaging
Oatmeal Agar
500 g
0552-17
Bacto Orange Serum Agar

Bacto Orange Serum Broth Concentrate 10X
Intended Use
Formula
Bacto Orange Serum Agar is used for cultivating aciduric microorganisms, particularly those associated with spoilage of citrus products.
Bacto Orange Serum Broth Concentrate 10X is used for cultivating and
enumerating microorganisms associated with spoilage of citrus products.
Orange Serum Agar

Formula Per Liter

The low pH of fruit juices makes citrus fruit products susceptible
to spoilage by yeasts, molds and the bacteria Lactobacillus and
Leuconostoc.1 In the 1950s, Hays investigated spoilage in frozen
concentrated orange juice. He found that an agar medium containing
orange serum (juice) was superior to Lindegren Agar in isolating the
microorganisms responsible for spoilage causing a buttermilk off-odor.2
In a later comparative study, Murdock, Folinazzo and Troy found
Orange Serum Agar, pH 5.4 to be a suitable medium for growing
Leuconostoc, Lactobacillus and yeasts.3
Orange Serum Agar is included in recommended methods for examining
fruit beverages.1 Orange Serum Broth Concentrate 10X is used for small
samples to initiate growth of saprophytic and pathogenic fungi.4

Orange Serum Agar and Orange Serum Broth Concentrate 10X
contain Tryptone as carbon and nitrogen sources for general growth
requirements. Orange serum provides the acid environment favorable
to recovering acid-tolerant microorganisms. Yeast Extract supplies
B-complex vitamins which stimulate growth. Dextrose is the carbohydrate. Bacto Agar is the solidifying agent (Orange Serum Agar only).
362
Orange Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200

Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
ml
g
g
g
g
g

Orange Serum Broth Concentrate 10X
Formula Per Liter
Orange Serum Concentrate . . . . . . . . . . . . . . . . . . . . . . . 100
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
ml
g
g
g
g
Precautions
Storage
Store the dehydrated Orange Serum Agar and the Orange Serum Broth
Concentrate 10X at 2-8C.
The Difco Manual
Section II
Orange Serum Agar & Orange Serum Broth Concentrate 10X
The dehydrated Orange Serum Agar is very hygroscopic. Keep


Expiration Date

1. To prepare the single-strength medium, aseptically add 100 ml
Orange Serum Concentrate 10X to 900 ml sterile distilled or
deionized water and mix thoroughly.
2. Use aseptic technique to dispense 10 ml amounts into sterile test tubes.
Procedure
Materials Provided
Orange Serum Agar

Test Procedure
Orange Serum Agar
1. For plate count method, prepare serial 10-fold dilutions of the
test material.
2. Add 1 ml of test sample to sterile Petri dish.
3. Add 18-20 ml of sterile, molten agar (cooled to 45-50C) and swirl
plate gently to mix well.
4. Allow to solidify before incubating at 30C for 48 hours. Plates
can be held up to 5 days.
Orange Serum Broth Concentrate 10X is used for small samples to
initiate growth.

For Orange Serum Agar:
Flask with closure
Autoclave
For Orange Serum Broth Concentrate 10X:
Sterile test tubes
Sterile transfer pipettes, 10 ml
Orange Serum Agar
Results
Orange Serum Agar
Record colony morphology for each type of growth.
Turbidity indicates growth.
Orange Serum Agar
4.55% Solution:
slightly to slightly opalescent, may
Prepared Medium:
opalescent.
Reaction of 4.55%
Solution at 25C:
pH 5.5 0.2
Concentrate Appearance: Dark amber, clear solution.
Reaction of
Solution at 25C:
pH 5.6 0.2
Cultural Response
Prepare media per label directions. Inoculate medium and
incubate for 40-48 hours. Lactobacillus is incubated at 35 2C
and the remaining organisms are incubated at 30 2C.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Aspergillus niger
Leuconostoc mesenteroides
subsp. mesenteroides
Sacharomyes cerevisiae
16404
9338
100-1,000
100-1,000
good
good
23386
9763
100-1,000
100-1,000
good
good
1. Orange Serum Agar is not a differential medium. Perform

microscopic examination and biochemical tests to identify isolates
to genus and species if necessary.
2. If Orange Serum Agar is divided into aliquots and allowed to solidify,
remelt only once. Repeated heating may produce a softer medium.
References
1. Vanderzant, C., and D.F. Splittstoesser (ed.). 1992. Compendium
2. Hays, G. L. 1951. The isolation, cultivation and identification of
organisms which have caused spoilage in frozen concentrated
orange juice. Proc. Fla. State Hortic. Soc. 54:135-137.
3. Murdock, D. I., J. F. Folinazzo, and V. S. Troy. 1952. Evaluation
of plating media for citrus concentrates. Food Technol. 6:181-185.
Packaging
Orange Serum Agar
Orange Serum Broth
Concentrate 10X
500 g
6 x 100 ml
0521-17*
0518-73*
*Store at 2-8C
The Difco Manual
363
Oxford Medium Base, Oxford Antimicrobic Supplement & Modified Oxford Antimicrobic Supplement
Section II
Bacto Oxford Medium Base . Bacto Oxford Antimicrobic

Supplement . Bacto Modified Oxford Antimicrobic Supplement
Intended Use
Bacto Oxford Medium Base is used with either Bacto Oxford
Antimicrobic Supplement1 or Bacto Modified Oxford Antimicrobic
Supplement2 for isolating and differentiating Listeria monocytogenes.

monocytogenes is a widespread problem in public health and the
food industries. This organism can cause human illness and death,
The first reported food-borne outbreak of listeriosis was in 1985.5 Since
then, microbiological and epidemiological evidence from both sporadic
and epidemic cases of listeriosis has shown that the principal route of
and other food processing plants and is ubiquitous in nature, being
present in a wide range of unprocessed foods and in soil, sewage, silage
and river water.8
Listeria spp. grow over a pH range of 5.0-9.6 and survive in food products
with pH levels outside these parameters.9 Listeria spp. are microaerophilic, gram-positive, asporogenous, non-encapsulated, non-branching,
regular, short, motile rods. Motility is most pronounced at 20C.
potentially containing Listeria are streptococci, especially the
ATCC 19114
Uninoculated
plate

Oxford Medium Base
Solution:
medium amber, slightly opalescent with
a blue ring at the surface of the liquid.
Prepared Medium:
Light to medium amber, very slightly
Reaction of 5.75%
Solution at 25C:
pH 7.2 0.2
Oxford Antimicrobic Supplement
Appearance:
Yellow cake; yellow solution upon
rehydration.
Modified Oxford Antimicrobic Supplement
Appearance:
White to off-white cake; colorless solution
upon rehydration.
Cultural Response
Prepare Oxford Medium or Modified Oxford Medium per label
ORGANISM
Escherichia coli
ATCC
INOCULUM
CFU
RECOVERY ON
OXFORD MEDIUM
RECOVERY ON MODIFIED
OXFORD MEDIUM
29212*
25922*
19114
9080
1,000-2,000
1,000-2,000
100-1,000
1,000-2,000

good at 40-48 hours, black colonies

good at 40-48 hours, black colonies
(not tested)
*These cultures are available as Bactrol Disks and should be used as directed in Bactrol Disks Technical Information
364
The Difco Manual
Section II
Bacto Oxford Medium Base is prepared according to the formulation

of Curtis et al.11 who originally described the medium and its use in the
selective isolation of Listeria from mixed cultures.

Columbia Blood Agar Base combines Pantone, Bitone and Tryptic
Digest of Beef Heart which provide nitrogen, carbon, amino acids and
vitamins. Bacto Agar is the solidifying agent. Sodium Chloride
maintains the osmotic balance.
Ferric ammonium citrate aids in the differentiation of Listeria spp.
Since all Listeria spp. hydrolyze esculin, the addition of ferric ions to
the medium will detect the reaction. A blackening of the colony and
surrounding medium in cultures containing esculin-hydrolyzing
bacteria results from the formation of 6,7-dihydroxycoumarin which
reacts with the ferric ions.12
Selectivity is provided by the presence of Lithium Chloride in the
formula. The high salt tolerance of Listeria is used as a means to
markedly inhibit growth of enterococci.
Selectivity is increased by adding various antimicrobial agents to the
base. Incorporating these agents into Oxford Medium Base will
completely inhibit gram-negative organisms and most gram-positive
organisms after 24 hours of incubation. The most widely recognized
antimicrobial agent combinations are the Oxford Medium formulation11
and the Modified Oxford Medium formulation.2 The Oxford Medium
formulation contains cycloheximide, colistin sulfate, acriflavin,
cefotetan and fosfomycin (available as Oxford Antimicrobic Supplement). The Modified Oxford Medium formulation contains moxalactam
and colistin methane sulfonate or colistin sulfate (available as
Modified Oxford Antimicrobic Supplement).
Modified Oxford medium is recommended for isolating and identifying
Listeria monocytogenes from processed meat and poultry products.2
Oxford Medium is recommended for isolating Listeria from enrichment
broth cultures.13
Formula
g
g
g
g
g
mg
mg
mg
mg
mg

Colistin Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 mg
Moxalactam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg
The Difco Manual

Warning! HARMFUL IF SWALLOWED OR INHALED. MAY
CAUSE IRRITATION. MAY CAUSE BIRTH DEFECTS. Avoid
contact with eyes, skin and clothing. Do not breathe dust.
FIRST AID: If swallowed, induce vomiting. Call a physician.
Never give anything by mouth to an unconscious person. If inhaled,
remove to fresh air. If not breathing, give artificial respiration,
preferably mouth-to-mouth. If breathing is difficult, give oxygen.
Call a physician. In case of contact, immediately flush eyes with
plenty of water for at least 15 minutes. Call a physician. Flush skin
with water.
Storage
1. Store Oxford Medium Base below 30C dehydrated powder is very
Store Oxford Antimicrobic Supplement and Modified Oxford
Antimicrobic Supplement at 2-8C.
2. Store prepared media at 2-8C.
The expiration date applies to the product its intact container when
Procedure
Materials Provided
Oxford Medium Base

Acriflavine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Cefotetan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Colistin Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Cycloheximide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
Fosfomycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2. Oxford Medium Base
suitable protective clothing. Keep container tightly closed. Target
Organs: Blood, Kidneys, Nerves.
Expiration Date
Oxford Medium Base

Formula Per Liter
Bacto Columbia Blood Agar Base . . . . . . . . . . . . . . . . . . . 39
Esculin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Lithium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Precautions

Autoclave
Waterbath (45-50C)
Petri dishes
Incubator (35C)
365
1. Suspend 57.5 grams of Oxford Medium Base in 1 liter distilled or
deionized water.
4. Cool medium to 45-50C in a waterbath.
5. To prepare Oxford Medium: Aseptically rehydrate one vial of
Oxford Antimicrobic Supplement with 5 ml ethanol and 5 ml sterile
distilled water. Rotate in an end-over-end motion to dissolve
completely. Add 10 ml to 1 liter sterile Oxford Medium Base.
To prepare Modified Oxford Medium: Aseptically rehydrate one
vial of Modified Oxford Antimicrobic Supplement with 10 ml sterile distilled water. Rotate in an end-over-end motion to dissolve
completely. Add 10 ml to 1 liter sterile Oxford Medium Base.

recommended guidelines.1,2,9,13,14
2. Clinical specimens obtained from nonsterile sites, foods and
enriched for Listeria before being plated.14
3. Process each specimen using procedures appropriate for that
specimen or sample.1,2,9,13,14
Test Procedure
The USDA method2 involves enrichment of the food sample in UVM
Modified Listeria Enrichment Broth (one part sample to nine parts
broth) at 30C. After incubation, a portion of the enrichment mixture is
plated onto Oxford or Modified Oxford Medium.
The FDA method1 involves adding 25 ml of liquid or 25 grams of solid
material to 225 ml Listeria Enrichment Broth and incubating at 30C
for two days. After enrichment, the broth is plated onto Oxford Medium.
For further information when testing food samples or clinical
specimens for Listeria, consult appropriate references.1,2,9,13,14
Results
Select esculin-positive colonies and confirm their identity by further
biochemical testing. Use macroscopic tube and rapid slide tests for
definitive serological identification. For additional information, refer
to appropriate references.1,2,9,13,14

1. Since Listeria spp. other than L. monocytogenes can grow on these
media, an identification of L. monocytogenes must be confirmed
by biochemical and serological testing.14
2. Use freshly prepared antimicrobial agent solutions or aliquot
portions and store at -20C or below.
3. Poor growth and a weak esculin reaction may be seen after 40 hours
incubation for some enterococci.
References
Section II

No. 57 (revised May 24, 1989). U.S.D.A., F.S.I.S. Microbiology
Division, Beltsville, MD.
monocytogenes (n. sp.). J. Path. Bact. 29:407-439.
R. E. Brackett. 1995. Comparison of oxygen scavengers for
their ability to enhance resuscitation of heat-injured Listeria
Splittstoesser (ed.). Compendium of methods for the microbiological examination of foods, 3rd ed. American Public Health
11. Curtis, G. D. W., R. G. Mitchell, A. F. King, and J. Emma. 1989.
A selective differential medium for the isolation of Listeria
monocytogenes. Appl. Microbiol. 8:95-98.
food and environmental samples by esculin hydrolysis. J. Food
Prot. 51:762-765.
13. Chesemore, R. G. 1990. Bacteriological analytical manual,
Chapter 29 - Listeria isolation: culture medium substitution in
method of analysis. Federal Register 55(183):38753-4.
Tenover, R. H. Yolken (ed), Manual of clinical microbiology,
Packaging
Oxford Medium Base
500 g
2 kg
10 kg
0225-17
0225-07
0225-08
6 x 10 ml
0214-60
Modified Oxford Antimicrobic

Supplement
6 x 10 ml
0218-60
1. Hitchins, A. D. 1992. Listeria monocytogenes. Bacteriological

analytical manual, 8th ed. AOAC International, Arlington, VA.
366
The Difco Manual
Section II
PALCAM Medium
PALCAM Medium
Bacto PALCAM Medium Base . Bacto PALCAM
Antimicrobic Supplement
Intended Use
Bacto PALCAM Medium Base is used with Bacto PALCAM Antimicrobic Supplement in isolating and cultivating Listeria from foods.

PALCAM Medium Base and PALCAM Antimicrobic Supplement are
based on the PALCAM agar formulation of van Netten et al.,1 who
developed this selective and differential medium for use in the isolation
and enumeration of Listeria spp. from food samples. PALCAM
medium is recommended by AFNOR for use in the detection of
L. monocytogenes in foods,2 and by the IDF as an additional plating
medium for the detection of Listeria spp. in milk and milk products.3
PALCAM medium is recommended by Health Canada for the detection
of L. monocytogenes in food and environmental samples.4

Good growth of Listeria spp. is obtained by including Columbia
Blood Agar Base in PALCAM Medium Base. Columbia Blood Agar
Base provides the nutrients and cofactors required for good to excellent growth of Listeria. Selectivity of the complete medium is
achieved through the presence of Lithium Chloride, Polymyxin B

Sulfate and Acriflavine HCl, present in PALCAM Medium Base, and
Ceftazidime, provided by PALCAM Antimicrobic Supplement. These
agents effectively suppress growth of most commonly occurring
non-Listeria spp. of bacteria present in foods. The ceftazidime concentration is reduced from 20 mg/l to 8 mg/l for improved growth
and recovery of Listeria.
Differentiation on PALCAM Medium is based on esculin hydrolysis
and mannitol fermentation. All Listeria spp. hydrolyze esculin
as evidenced by a blackening of the medium. This blackening by
esculin-hydrolyzing bacteria results from the formation of 6,7
dihydroxycoumarin, which reacts with ferric ions that are present in
the medium as Ferric Ammonium Citrate. On occasion, organisms other
than Listeria, such as staphylococci or enterococci, may grow on this
medium. Mannitol and the pH indicator, Phenol Red, have been added
to differentiate mannitol-fermenting strains of these species from
Listeria based on mannitol fermentation. Mannitol fermentation is
demonstrated by a color change in the colony and/or the surrounding
medium from red or gray to yellow due to the production of acidic end
products.
Uninoculated
plate
ATCC 19114

PALCAM Medium Base
Solution:
deionized water on boiling; dark red,
with a slight precipitate.
Reaction of 6.8 %
Solution at 25C:
pH 7.2 0.2
PALCAM Antimicrobic Supplement
Lyophilized Appearance: White, free-flowing, homogeneous
powder.
Rehydrated Appearance: Colorless solution.
Cultural Response
Prepare PALCAM Medium per label directions. Inoculate and incubate
at 35 2C for 40-48 hours under microaerophilic conditions.
ATCC
INOCULUM
Escherichia coli
25922*
19114
1,000-2,000
100-1,000
25923*
29212*
ORGANISM
The Difco Manual
GROWTH
inhibited
good growth, gray-green
colonies with black precipitate
1,000-2,000
inhibited
1,000-2,000
inhibited

367
PALCAM Medium
Section II
Formula
Procedure
PALCAM Medium Base

Formula Per Liter
Materials Provided
Bacto Columbia Blood Agar Base . . . . . . . . . . . . . . . . . . . 39

Bacto Mannitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Esculin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Lithium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.08
Acriflavine HCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.005
Polymyxin B Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
g
g
g
g
g
g
g
g
g
g

Formula per 10 ml vial
Ceftazidime . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 mg
Precautions
3. PALCAM Medium Base:
ORGAN(S): Blood, Kidneys, Nerves.
4. PALCAM Antimicrobic Supplement:
MAY CAUSE ALLERGIC EYE, RESPIRATORY SYSTEM AND
tightly closed.
Storage
Store PALCAM Medium Base below 30C. The dehydrated medium
Store PALCAM Antimicrobic Supplement at 2-8C.
Store the rehydrated supplement and prepared medium at 2-8C.
Expiration Date
368
PALCAM Medium Base


Autoclave
Waterbath (45-50C)
Incubator (35C)
Depending on testing method:
Fraser Broth Base
Oxford Medium Base
Tryptic Soy Agar with 0.6% Yeast Extract
LPM Agar Base
1. Suspend 68 grams PALCAM Medium Base in 1 liter distilled or
deionized water and boil to dissolve completely.
3. Aseptically add 2 ml PALCAM Antimicrobic Supplement which
has been rehydrated with 10 ml sterile distilled or deionized water.
Mix well.

sample.
Test Procedure
A number of methods and incubation conditions may be used for
detecting and isolating Listeria on PALCAM Medium. In their original
work, van Netten et al. recommended incubation at 37C for 48 hours
under microaerophilic conditions.1 AFNOR, HPB and IDF methods
for detecting Listeria in foods and dairy products are listed below.
Consult guidelines appropriate to your country and sample type.
AFNOR Method for Foods2
1. Pre-enrich the sample in Demi-Fraser Broth. Incubate at 30C
for 18-24 hours. Subculture onto Oxford Medium or PALCAM
Medium.
2. Transfer 0.1 ml of the pre-enrichment culture into 10 ml of Fraser
Broth and incubate at 37C for 48 hours. Subculture onto Oxford
Medium or PALCAM Medium after 18-24 and 42-48 hours of
incubation.
3. After the required incubation, examine for presumptive Listeria
colonies.
The Difco Manual
Section II
PKU Test Agar, PKU Test Agar w/o Thienylalanine & Subtilis Spore Suspension No. 2
4. Confirm the identity of each presumptive Listeria isolate by

biochemical and/or serological testing.

IDF Method for Milk and Milk Products3

1. Enrich the sample in Modified Listeria Enrichment Broth.
Incubate at 30 1C for 48 2 hours.
2. Subculture onto Oxford Medium (and onto PALCAM Medium, if
desired). Incubate at 37 1C for 48 2 hours.
3. After the required incubation, examine for presumptive Listeria
colonies.
4. Subculture five presumptive colonies (or all of the colonies if there
are less than five) from each isolation medium onto Tryptic Soy
Agar with 0.6 % Yeast Extract.
Results
Health Canada Method for Foods and Environmental Samples4

1. Enrich the sample in Listeria Enrichment Broth (LEB). Incubate at
30C for 48 hours.
2. Transfer 0.1 ml of the primary enrichment broth culture into 9.9 ml
of modified Fraser Broth. Incubate at 35C for 24-48 hours. (If
desired, the LEB culture may also be streaked onto Oxford
Medium [OXA] and lithium chloride-phenylethanol-moxalactam
agar [LPM], modified Oxford medium [MOX] or PALCAM
medium [PAL]. Incubate LPM at 30C for 24-48 hours and OXA,
MOX and PAL at 35C for 24-48 hours.)
3. Examine modified Fraser broth for reactions. Subculture all positive
cultures (black, dark brown or dark green) after 24 and 48 hours
of incubation onto OXA and LPM, MOX or PAL, streaking for
isolation. Incubate LPM at 30C for 24-48 hours and OXA, MOX
and PAL at 35C for 24-48 hours. If desired, all negative modified
Fraser broth cultures (straw color) may be subcultured onto OXA
and LPM, MOX or PAL to facilitate recovery of esculin-negative
strains of L. monocytogenes.
4. Examine for presumptive Listeria colonies. Examine LPM under
oblique lighting positioned at a 45 angle relative to the surface of
the plate.
On PALCAM Medium, colonies of Listeria appear gray-green with

a black precipitate following inoculation and incubation at 35C
for 24-48 hours under aerobic or microaerophilic conditions.
Confirmation of the presence of Listeria is made following
subculture onto appropriate media and biochemical/serological
identification.2,3 Colonies of mannitol-fermenting organisms such
as staphylococci, which may grow on this medium, appear yellow
with a yellow halo.
References
1. Van Netten, P., I. Perales, A. Van de Moosalijk, G. D. W.
Curtis, and D. A. A. Mossel. 1989. Liquid and solid selective
differential media for the detection and enumeration of
L. monocytogenes and other Listeria spp. Int. J. of Food
2. Lassociation franaise de normalisation (AFNOR). 1993. Food
Microbiology- Detection of Listeria monocytogenes-Routine
Method, V 08-055. AFNOR, Paris.
3. International Dairy Federation. 1990. Milk and milk productsDetection of Listeria monocytogenes. IDF Provisional International
Standard no. 143. International Dairy Federation, Brussels.
4. Farber, J. M., D. W. Warburton, and T. Babiuk. 1994. Isolation
of Listeria monocytogenes from all food and environmental
samples. Health Protection Branch Ottawa, MFHPB-30.
Polyscience Publications, Quebec.
Packaging
PALCAM Medium Base
500 g
2 kg
3 x 10 ml
0636-17
0636-07
0637-57*
*Store at 2-8C
Bacto PKU Test Agar . Bacto PKU Test Agar w/o Thienylalanine
Bacto Subtilis Spore Suspension No. 2
Intended Use
Bacto PKU Test Agar is used with Bacto Subtilis Spore Suspension
No. 2 in estimating the phenylalanine level in blood.
Bacto PKU Test Agar w/o Thienylalanine is used with Bacto Subtilis
Spore Suspension No. 2 and -2-thienylalanine in estimating phenylalanine levels in blood.
Also Known As
The Guthrie Modified Bacterial Inhibition Assay (BIA) for PKU
Phenylketonuria (PKU) results from an inborn error of phenylalanine
metabolism. In this disease, phenylalanine hydroxylase deficiency
The Difco Manual
causes accumulation of the amino acid phenylalanine with subsequent

neurological damage.
In 1934, Folling1 reported the presence of a urine phenylalanine
metabolite in mentally retarded persons. Jervis2 established that
defective phenylalanine metabolism was the cause of the mental
retardation. Detection and management of PKU are possible by testing
infants for abnormal levels of phenylalanine or its metabolites. The
Guthrie bacterial inhibition assay (BIA), which estimates the level of
phenylalanine in the blood, is used for this purpose.3,4,5,6 Bacillus
subtilis ATCC 6633 growth is inhibited in minimal culture medium
containing -2-thienylalanine. Phenylalanine blocks the inhibition,
allowing the organism to grow.
369
In the PKU Test procedure, PKU Test Agar containing thienylalanine

or PKU Test Agar w/o Thienylalanine with added thienylalanine are
inoculated with a suspension of B. subtilis ATCC 6633. Filter paper
disks saturated with infant blood and control disks impregnated with
known concentrations of L-phenylalanine (2,4,6,8,12 and 20 mg%) are
applied to the surface of the medium. After incubation at 35C for
12-16 hours, the zones of growth around the test disks are compared
to the zones around the control disks. A growth zone around the test
disk comparable to the zone around the 4 mg% or higher disk is a
presumptive positive indication of phenylketonuria. A positive result
must be repeated using a duplicate test disk and a chemical or
spectrofluorometric procedure.7,8

PKU Test Agar and PKU Test Agar w/o Thienylalanine are defined
minimal media containing the factors necessary for B. subtilis growth
under appropriate conditions. -2-thienylalanine is an inhibitor
of B. subtilis growth. PKU Test Agar contains the inhibitor,
-2-thienylalanine; PKU Test Agar w/o Thienylalanine does not,
requiring the user to add -2-thienylalanine to the medium.
Phenylalanine supplied from a PKU-positive patient specimen will
overcome the inhibitory action of -2-thienylalanine.
Formula
PKU Test Agar
Formula Per Liter
L-Glutamic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
DL-Alanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Asparagine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0.5
0.5
0.5
10
15
g
g
g
g
g
Section II
Ammonium Nitrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Sodium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Manganese Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.005
Ferric Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.005
B2 Thienylalanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0033
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g
g
g
g

PKU Test Agar w/o Thienylalanine
Formula Per Liter
L-Glutamic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
DL-Alanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Ammonium Nitrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Sodium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Manganese Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.005
Ferric Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.005
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g
g
g
g
g
g
g
g

Bacillus Spore Suspension No. 2
Standardized, stable suspension of Bacillus subtilis ATCC 6633
containing 1.2 to 1.8 x 108 spores/ml.
Precautions
PKU Test Agar, PKU Test Agar w/o Thienylalanine
Dehydrated Appearance: Light beige to beige, free-flowing,
homogeneous.
Solution:
is light amber, very slightly to slightly
opalescent with a slight precipitate.
Prepared Medium:
Reaction of 5.0%
Solution at 25C:
pH 7.0 0.2
Subtilis Spore Suspension No. 2
Appearance:
White, opalescent, homogeneous
suspension.
Cultural Response
PKU Test Agar, PKU Test Agar w/o Thienylalanine
Prepare the final medium per label directions. Apply PKU
Standard Disks. Incubate at 35 + 2C for 12-16 hours. Measure
zones of growth around each PKU Standard Disk. Zones of
growth should increase in size comparable to the increasing
concentration of phenylalanine in the Standard Disks.
370

2. PKU Test Agar
HARMFUL. POSSIBLE RISK OF IRREVERSIBLE EFFECTS.
TARGET ORGAN(S): Skin, Lungs.
3. Bacillus Spore Suspension No. 2
CAUTION. While spore suspensions are not considered to be
pathogens, they are, nevertheless, live organisms. Never use mouth
pipetting. Always use some type of pipetting aid.
Storage
Store Subtilis Spore Suspension No. 2 at 2-8C.
The Difco Manual
Section II
Expiration Date
Procedure
Materials Provided
PKU Test Agar

-2-thienylalanine (use with PKU Test Agar w/o Thienylalanine)
PKU Standard Disks
Blood test forms with Lancet
Disk test pattern for 150 mm Petri dish
150 mm Petri dishes
Forceps
Alcohol sponges
Glassware
Autoclave
Incubator (35C)
5. Compare zones of growth around the patient disks to those around

the control disks to determine the approximate concentration of
phenylalanine in the blood.
Results
Growth zone diameters around the control disks are related to the
concentration of phenylalanine in the disks. A zone of growth may or
may not be present around the test disks depending on the presence or
absence of phenylalanine in the test specimen. The culture medium
outside the zones of growth will be comparable to an inoculated and
incubated plate to which no disks have been applied.
Compare the zone of growth around a test disk to the zone around the
standard disk containing 4 mg% phenylalanine. If the test zone is equal
to or larger than the 4 mg% control zone, the test result is a presumptive
positive and should be confirmed using a second sample. If the second
sample gives a similar result, determine the serum phenylalanine concentration by either a chemical10 or a spectrofluorometric procedure.
1. Obtain the sample at least 48 hours after the first milk feeding.
2. Collect a venous blood sample by heel puncture following established collection technique.9 Obtain sufficient blood to fill each
circle by a single application of the specimen card to the drop of
blood. Completely saturate the entire circle to ensure accuracy.
Allow the blood sample to air dry.
3. Punch a 1/4" disk from one of the blood spots and place it into a
labeled, clean, dry vial or place the entire specimen card on a wire
rack in the autoclave.
4 Autoclave the patient disks for exactly three minutes at 121C.
Remove the disks promptly after the temperature has dropped
below 100C. Do not use the disks until they are dry.
5. Follow manufacturers instructions for preparation of the control disks.
1. Collect the blood sample with care. The sample must saturate the
paper. Do not allow contact between the absorbent specimen card
and the collectors hands.
2. Autoclaved samples must be dry before use.
3. PKU Test Agar must not be overheated. Bring to a boil and mix
gently during heating. DO NOT AUTOCLAVE.
4. Do not add spores if the temperature of the medium is above 55C.
Distribute the spores uniformly in the medium without creating
bubbles.
5. Place the Petri dish on a horizontal surface while pouring the
medium to ensure an even depth of agar and a uniform distribution
of spores throughout the plate.
6. Test results at the 4 and 6 mg% levels are questionable and should
be repeated with a second test sample and the results confirmed by
a quantitative procedure.
7. Take care when opening ampules containing B. subtilis spores.
Autoclave the emptied ampules at 121-124C for 20 minutes.
8. Infants who are tested before 24 hours of age should have a repeat
test performed by 2 weeks of age.
9. A negative test of an infant on antibiotics should be reconfirmed
after antibiotic therapy is terminated. Antibiotics present in the
blood sample are usually inactivated by the autoclaving procedure,
but could be a source of error because some antibiotics will inhibit
the growth of B. subtilis.11
10. False-negative tests can result from the submission of an inadequate
sample, or if the patient has recently been exchange-transfused, or
if the patient has an insufficient dietary protein load.11
11. False-positive results can occur.12
Test Procedure
References
1. Prepare PKU Test Agar or PKU Test Agar w/o Thienylalanine per
label directions.
2. Dispense the final medium into 150 mm Petri dishes. Allow to solidify.
3. Using clean forceps, apply the autoclaved and dried test disks and
the prepared PKU Standard Disks, one of each concentration, to
the PKU Test Agar and press down gently.
1. Folling, A. 1934. Phenylpyruvic acid as a metabolic anomaly in

connection with imbecility. Z. Physiol. Chem. 227:169-176.
2. Jervis, G. 1953. Phenylpyruvic oligophrenia deficiency of phenylalanine oxidizing system. Proc. Soc. Exp. Biol. Med. 82:514-515.
3. Guthrie, R., and H. Tiechelmann. July 1960. London Conference on the Scientific Study of Mental Deficiency.
4. Guthrie, R. 1961. J. Am. Med. Assoc. 178:863.
2. Heat to boiling to dissolve completely. Simmer for 5 minutes.
3. PKU Test Agar w/o Thienylalanine, only: Add 1 ml of 0.33%
-2-thienylalanine solution per liter after simmering the medium;
mix thoroughly.
5. Aseptically add 1 ml Subtilis Spore Suspension No. 2 to each 150 ml
aliquot at 50-55C. Mix thoroughly to uniformly distribute the spores.
The Difco Manual
371
PPLO Media
Section II
5. Demain, A. L. 1958. J. Bact. 75:517.

6. Guthrie, R., and A. Susi. 1963. A simple phenylalanine method
for detecting phenylketonuria in large populations of newborn
infants. Pediatrics 32:338-343.
7. Ambrose, J. A., et al. 1967. Clin. Chem. Acta. 15:493.
8. Ambrose, J. A. 1969. Clin. Chem. 15:15.
Blood collection on filter paper for neonatal screening programs,
2nd ed.; Approved Standard. LA4-A2, vol. 12, no. 13. Wayne, PA.
10. LaDu, B. N., and P. J. Michael. 1960. J. Lab. Clin. Med. 55:491.
11. Nichols, Michael J. 1994. Tips on technology. MLO. 26:11-12.

12. Kirkman, H. N. , C. L. Carroll, E. G. Moore, et al. 1982.
Fifteen-year experience with screening for phenylketonuria with an
automated fluorometric method. Am. J. Hum. Genet. 34:743-752.
Packaging
PKU Test Agar
500 g
0980-17
500 g
0474-17
25 x 1
100 x 1
0981-36
0981-84
PPLO Media
Bacto PPLO Agar . Bacto PPLO Broth w/o CV . Bacto
Mycoplasma Supplement . Bacto Mycoplasma Supplement S
Intended Use
Bacto PPLO Agar when supplemented with Bacto Mycoplasma
Supplement or Bacto Mycoplasma Supplement S is used for isolating
and cultivating Mycoplasma.
Bacto PPLO Broth w/o CV when supplemented with Bacto Mycoplasma
Supplement or Bacto Mycoplasma Supplement S is used for isolating
and cultivating Mycoplasma.
Also Known As
PPLO is an abbreviation for pleuropneumonia-like organism.

Members of the class Mollicutes, Mycoplasma was first recognized
from a case of pleuropneumonia in a cow.11 The organism was designated
pleuropneumonia-like organism, or PPLO.11 Although some species
are normal human respiratory tract flora, M. pneumoniae is a major
cause of respiratory disease (primary atypical pneumonia, sometimes
called walking pneumonia).11 M. hominis, M. genitalium, and
Ureaplasma urealyticum are important colonizers (and possible
pathogens) of the human genital tract.11
PPLO Agar was described by Morton, Smith and Leberman.1 PPLO
Agar was used in a study of the growth requirements of Mycoplasma,2
along with the identification and cultivation of this organism.3,4,5
PPLO Broth w/o CV is prepared according to the formula described by
Morton and Lecci.2 Crystal Violet is omitted from this formula due to
its inhibitory action on some Mycoplasma. PPLO Broth w/o CV has
been used for the cultivation of Mycoplasma for research studies.6,7
Mycoplasma Supplement and Mycoplasma Supplement S are sterile
desiccated enrichments for use in PPLO media as described by
Hayflick.8 The supplements are prepared according to the formulations
of Chanock, Hayflick and Barile9 and Hayflick.10

Infusion from Beef Heart and Bacto Peptone provide the nitrogen,
vitamins, amino acids and carbon in PPLO Agar and PPLO Broth w/o
372
CV. Sodium Chloride maintains the osmotic balance of these formulations. Bacto Agar, a solidifying agent, is used in PPLO Agar at a
concentration slightly reduced from usual to ensure formation of the
largest possible colonies because the organisms grow into the agar with
only slight surface growth.12
PPLO media are supplemented with Mycoplasma Supplement or
Mycoplasma Supplement S because Mycoplasma spp. are fastidious in
their growth requirements.13
Mycoplasma Supplement contains fresh Yeast Extract and Horse
Serum. Yeast Extract provides the preformed nucleic acid precursors
that are required by Mycoplasma spp. 13 Horse Serum supplies
cholesterol, a growth stimulant.13
Mycoplasma Supplement S is a selective enrichment prepared by
adding Thallium Acetate and Penicillin to Mycoplasma Supplement.
Thallium Acetate and Penicillin are selective against gram-positive
and gram-negative bacteria.
Formula
PPLO Agar
Formula Per Liter
Bacto Beef Heart for Infusion, Infusion from . . . . . . . . . . 50
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
g
g
g
g

PPLO Broth w/o CV
Formula per Liter
Bacto Beef Heart for Infusion, Infusion from . . . . . . . . . . 50 g
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g

Mycoplasma Supplement
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01 g
Horse Serum, Desiccated . . . . . . . . . . . . . . . . . . . . . . . . . . 1.6 g
The Difco Manual
Section II
PPLO Media
Precautions
Mycoplasma Supplement S
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01
Horse Serum, Desiccated . . . . . . . . . . . . . . . . . . . . . . . . . . 1.6
Penicillin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55,000
Thallium acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
g
g
units
mg

PPLO Agar
Solution:
deionized water on boiling; solution
opalescent.
Prepared Medium:
Enriched w/30% Mycoplasma
Supplement: light to medium amber,
Reaction of 3.5%
Solution at 25C:
pH 7.8 0.2
PPLO Broth w/o CV
Solution:
deionized water; solution is light amber
and clear to very slightly opalescent.
Prepared Medium:
Light amber, clear.
Reaction of 2.1%
Solution at 25C:
pH 7.8 0.2

2. Mycoplasma Supplement S
SYSTEM AND SKIN REACTION. (US) IRRITATING TO EYES,
RESPIRATORY SYSTEM AND SKIN. (US) POSSIBLE RISK OF
IRREVERSIBLE EFFECTS. Avoid contact with skin and eyes. Do
tightly closed. Target Organs: Bladder, Nerves, Kidneys, Cardiovascular System.
Storage
PPLO Agar
PPLO Broth w/o CV
Store the lyophilized and rehydrated supplements at 2-8C.
Expiration Date
Lyophilized Appearance: Straw-colored, dried button, may be
dispersed.
Rehydrated Appearance: Light to dark straw-colored, clear to
slightly opalescent, readily soluble.
Lyophilized Appearance: Straw-colored, dried button, may be
dispersed.
Rehydrated Appearance: Light to dark straw-colored, clear to
slightly opalescent solution, readily
soluble.
Materials Provided
Cultural Response
PPLO Agar, PPLO Broth w/o CV
Prepare media enriched with 30% Mycoplasma Supplement or
Mycoplasma Supplement S per label directions. Inoculate
PPLO Broth w/o CV and incubate at 35 2C under 5-10%
CO2 for up to 7 days. Subculture to PPLO Agar and incubate
at 35 2C under 5-10% CO2 for up to 7 days. Examine
microscopically for growth on a daily basis.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Mycoplasma bovis
Mycoplasma gallinarum
25523
19708
100-1,000
100-1,000
good
good
The organisms listed are the minimum that should be used for
The Difco Manual
Procedure
PPLO Agar
PPLO Broth w/o CV

Glassware
Autoclave
Incubator (35C)
PPLO Agar
PPLO Broth w/o CV
1. PPLO Agar: Suspend 35 grams in 700 ml distilled or deionized
PPLO Broth w/o CV: Dissolve 21 grams in 700 mo distilled or
deionized water.
373
Pagano Levin Base
2. Autoclave at 121C for 15 minutes. Cool medium to 50-60C.

4. Aseptically add 300 ml Mycoplasma Supplement or 300 ml
Mycoplasma Supplement S to the sterile medium. Mix well.
1. Rehydrate with 30 ml sterile distilled or deionized water.
2. Rotate gently to dissolve.
3. Add 30 ml (the contents of one vial) to 70 ml sterile PPLO Agar or
PPLO Broth w/o CV.

Test Procedure
Mycoplasma spp. from clinical specimens, refer to appropriate
procedures outlined in the references.12,13,14
Results
PPLO Agar
PPLO colonies are round with a dense center and a less dense periphery,
giving a fried egg appearance on PPLO Agar. Vacuoles, large bodies
characteristic of Mycoplasma spp., are seen in the periphery.
Colonies vary in diameter from 10 to 500 microns (0.01-0.5 mm) and
penetrate into the medium.

2. Thallium acetate can partially inhibit some mycoplasmas.12
References
1. Morton, H. E., P. F. Smith, and P. R. Leberman. 1951. Venereal
diseases. Am. J. Syphilis Gonorrh. 35:361.
2. Morton, H. E., and J. G. Lecce. 1953. Selective action of thallium
acetate and crystal violet for pleuropneumonia like organisms of
human origin. J. Bacteriol. 66:646-649.
3. Chanock, R. M., W. D. James, H. H. Fox, H. C. Turner,
M. A. Mufson, and l. Hayflick. 1962. Growth of Eaton PPLO in
broth and preparation of complement fixing antigen. Soc. Exp.
Biol. Med. 110:884-889.
Bacto Pagano Levin Base
Intended Use
Bacto Pagano Levin Base is used with Bacto TTC Solution 1% and
neomycin in isolating and differentiating Candida spp.
Also Known As
Pagano Levin Base is also referred to as Pagano Levin Candida Test
Medium.
374
Section II
4. Craven, R. B., R. P. Wenzel, A. M. Calhoun, J. O. Hendley,

B. H. Hamory and J. M. Gwaltney, Jr. 1976. Comparison of the
sensitivity of two methods for isolation of Mycoplasma
pneumoniae. J. Clin. Microbiol. 4:225-226.
5. Gregory, J. E., and K. R. Cundy. 1970. Mycoplasma recovery
from the male genitourinary tract: voided urine versus the urethral
swab. Appl. Microbiol. 19:268- 270.
6. Adler, H. E., and A. J. Da Massa. 1967. Use of formalinized
Mycoplasma gallisepticum antigens and chicken erythrocytes in
hemagglutination and hemagglutination-inhibition studies. Appl.
7. Leland, D. S., M. A. Lapworth, R. B. Jones, and M. L. V. French.
1982. Comparative evaluation of media for isolation of Ureaplasma
urealyticum and genital Mycoplasma species. J. Clin. Microbiol.
16:709-714.
8. Hayflick, L. 1965. Tissue cultures and mycoplasmas. Tex. Rep.
Biol. Med. 23:285-303.
9. Chanock, R. M., L. Hayflick, and M. F. Barlie. 1962. Growth on
artificial medium of an agent associated with atypical pneumonia
and its identification as a pleuropneumonia-like organism. Proc.
Nat. Acad. Science 48:41.
10. Hayflick, L. 1968. Personal communication.
St. Louis, MO.
12. Kenny, G. E. 1985. Mycoplasmas, p. 407-411. In E. H. Lennette,
A. Balows, W. J. Hausler, Jr., and H. J. Shadomy (ed.). Manual of
Washington, D.C.
13. Taylor-Robinson, D. 1995. Mycoplasma and Ureaplasma,
Packaging
PPLO Agar
500 g
0412-17
PPLO Broth w/o CV
500 g
10 kg
0554-17
0554-08
6 x 30 ml
0836-68
6 x 30 ml
0837-68

Pagano Levin Base as described by Pagano, Levin, and Trejo1 is selective
for Candida. Candida spp. reduce TTC (2,3,5-triphenyltetrazolium
chloride) in the medium to produce colonies with various degrees of
color. Neomycin inhibits growth of most bacteria without appreciably
influencing the Candida. Gentamicin (50 g/ml) may also be added to
reduce bacterial populations according to Yamane and Saitoh. 2
Samaranayake, MacFarlane and Williamson3 found that modified
Pagano Levin Agar was far superior to the commonly used Sabouraud
Dextrose Agar in detecting multiple yeast species in a single sample.
The Difco Manual
Section II
Pagano Levin Base
Expiration Date
Bacto Peptone provides the carbon and nitrogen required for good
growth of a wide variety of organisms. Yeast Extract provides vitamins
and cofactors. Dextrose is an energy source. Bacto Agar is a solidifying
agent. TTC Solution 1%, added to the basal medium, facilitates the
differentiation of yeast colonies based on the color change that occurs
when a microorganism reduces TTC. Neomycin added to the base
inhibits the growth of most bacteria.
Formula
Pagano Levin Base

Formula Per Liter
Glassware
Autoclave
Incubator (25-30C)
TTC Solution 1%
Neomycin
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
Materials Provided
Pagano Levin Base
Precautions
Storage
medium at 2-8C.

Solution:
is light amber, very slightly to
Prepared Medium:
Plain - light amber, slightly
opalescent; with TTC and antibiotic light amber, milky.
Reaction of 6.6%
Solution at 25C:
pH 6.0 0.2
Cultural Response
Prepare Pagano Levin Agar per label directions. Inoculate and
incubate at 25-30C for up to 72 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Candida albicans
26790
100-1,000
good
Candida krusei
6121
100-1,000
good
Candida stellatoidea 36232 100-1,000

Escherichia coli
25922 1,000-2,000
good
inhibited
COLONY
COLOR
cream to
light pink
white,
spreading
light red
The Difco Manual
Procedure
1.
2.
3.
4.
5.

Cool to 50-55C.
Aseptically add 10 ml TTC Solution 1% (100 g TTC per ml of
medium and 500 g of neomycin per ml of medium). Mix thoroughly.

recommended guidelines.4-6
Test Procedure
1. Inoculate the surface of the medium with the specimen and
Results
C. albicans colonies appear cream-colored to light pink, smooth, round,
raised, opaque and glistening. Typical C. albicans colonies can be
confirmed on Chlamydospore Agar or Rice Extract Agar based on
chlamydospore production.
References
1. Pagano, J., J. D. Levin, and W. Trejo. 1958. Diagnostic medium
for differentiation of species of Candida. Antibiot. Annu. 19571958:137-143.
2. Yamane, N., and Y. Saitoh. 1985. Isolation and detection of
multiple yeasts from a single clinical sample by use of PaganoLevin agar medium. J. Clin. Microbiol. 21:276-277.
3. Samaranayake, L. P., T. W. MacFarlane, and M. I. Williamson.
1987. Comparison of Sabouraud Dextrose and Pagano-Levin Agar
Media for detection and isolation of yeasts from oral samples.
J. Clin. Microbiol. 25(1):162-164.
Vol. 1. American Society for Microbiology, Washington, D.C.
375
Panthenol Assay Medium & Panthenol Supplement
5. Baron, E. J., L. R. Peterson, S. M. Finegold. 1994. Bailey &

St. Louis, MO.
Section II

Packaging
Pagano Levin Base
500 g
0141-17
Bacto Panthenol Assay Medium

Bacto Panthenol Supplement
Intended Use
Bacto Panthenol Assay Medium is used with Bacto Panthenol Supplement in determining panthenol concentration by the microbiological
assay technique.

Panthenol Assay Medium and Panthenol Supplement, modifications
of the formulas of DeRitter and Ruben,1 are used in the microbiological

Panthenol Assay Medium
Solution:
distilled or deionized water on boiling.
Single-strength solution is light amber,
Prepared Medium
(Single-strength):
Reaction of 1.65%
Solution at 25C:
pH 6.0 0.2
Panthenol Supplement
Solution Appearance: Colorless to very, very light amber,
clear.
Reaction of
Solution at 25C:
pH 5.0-6.0
Cultural Response
Prepare Panthenol Assay Medium per label directions. Test the
medium by creating a standard curve using pantoic acid
reference standard at levels from 0.0 to 2.0 g per 10 ml. The
medium supports the growth of G. oxydans subsp. suboxydans
ATCC 621H when prepared in single strength and supplemented with Panthenol Supplement and pantoic acid.
376
assay of panthenol. Gluconobacter oxydans subsp. suboxydans ATCC

621H is the test organism used in this assay.

Panthenol Assay Medium with Panthenol Supplement added is a
panthenol-free medium containing all other nutrients and vitamins
essential for the cultivation of G. oxydans subsp. suboxydans ATCC
621H. The addition of pantoic acid in increasing specified concentrations
gives a growth response that can be measured turbidimetrically.
Formula
Formula Per Liter
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Bacto Vitamin Assay Casamino Acids . . . . . . . . . . . . . . . . . 2
Acid Digest of Casein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
L-Tryptophane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.15
Adenine Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Uracil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
-Alanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Liver Digest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.35
Nicotinic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Folic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Manganous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.16
g
g
g
g
g
g
mg
mg
mg
mg
mg
mg
mg
mg
mg
mg
g
g
g
mg
mg
g
g

Formula Per Liter
Bacto Glycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Lactic Acid USP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.68
Distilled or Deionized Water . . . . . . . . . . . . . . . . . . . . . . 71.5
g
g
g
ml
The Difco Manual
Section II
Panthenol Assay Medium & Panthenol Supplement
Precautions

2. Great care must be taken to avoid contamination of media and
results. Scrupulously clean glassware free from detergents and other
chemicals must be used. Glassware must be heated to 250C for at

specific assay procedure. For assays, the samples should be diluted to
Storage
Store Panthenol Assay Medium and Panthenol Supplement at 2-8C.
The dehydrated Panthenol Assay Medium is very hygroscopic. Keep
Expiration Date
Procedure
Materials Provided

Glassware
Autoclave
Stock culture of Gluconobacter oxydans subsp. suboxydans ATCC
621H
Sterile tubes
Panthenol
Pantoic Acid
Incubator (30 + 2C)
Shaker (160-300 rpm)
0.1 N NaOH
0.1 N HCl
Spectrophotometer
1. Suspend 33 grams in 900 ml distilled or deionized water.
2. Boil to dissolve.
3. Dispense 4.5 ml amounts into tubes, evenly dispersing any
precipitate.
5. Adjust tube volume to 9.5 ml with distilled or deionized water.
7. Aseptically add 0.5 ml Panthenol Supplement to each tube.
The Difco Manual
Test Procedure
Stock cultures of G. oxydans subsp. suboxydans ATCC 621H are
grown on Lactobacilli Agar AOAC and kept in the refrigerator.
Inoculum for assay is prepared by subculturing a stock culture of G.
oxydans ATCC 621H into 10 ml of single-strength Panthenol Assay
Medium supplemented with Panthenol Supplement and 4 g/ml pantoic
acid. Following incubation on a shaker (100 rpm) at 30 2C for
20-24 hours, centrifuge the culture under aseptic conditions. Decant
the supernatant and wash the cells three times with sterile 0.85%
saline. After the third wash, resuspend the cells in 10 ml sterile 0.85%
saline and adjust to a turbidity of 65-70% transmittance when read on
the spectrophotometer at 660 nm. Use one drop of this suspension to
inoculate each assay flask.
A standard curve must be constructed each time an assay is run.
Autoclave and incubation conditions can influence the standard curve
readings and cannot always be duplicated. The standard curve is
obtained by using pantoic acid at levels of 0.0, 0.2, 0.4, 0.6, 0.8, 1.0,
1.2, 1.6 and 2 g per assay flask.
The concentration of pantoic acid required for the preparation of the
standard curve may be prepared by the following procedure:
1. Dissolve 69.2 mg of pure panthenol in distilled water, adjust to pH
6.0 and dilute to 1 liter (1 ml contains the equivalent of 50 g of
pantoic acid).
2. Autoclave 8 ml of this solution with 8 ml 0.1 N NaOH at 121C for
30 minutes.
3. Cool, add distilled water, adjust to pH 6.0 with 0.1 N HCl and dilute
to 100 ml. This stock solution contains 4 g of pantoic acid per ml.
Prepare the standard solution by diluting 10 ml of the stock solution with
90 ml distilled water. This standard solution contains 0.4 g of pantoic
acid per ml. Use 0.0, 0.5, 1, 1.5, 2, 2.5, 3, 4 and 5 ml per flask (50 ml).
Following inoculation, the cultures are incubated on a suitable shaker
at approximately 100-300 rpm at 30 2C for 18-24 hours. Place
cultures in the refrigerator to stop growth. Measure the growth
turbidimetrically using a suitable spectrophotometer.
Results
or cup.

be cultured and maintained on a medium recommended for this
purpose.
377
Pantothenate Assay Medium

response.
Section II
References
1. DeRitter and Ruben. 1949. Anal. Chem. 21:823.
Packaging
100 g
12 x 20 ml
0994-15
0212-64
Bacto Pantothenate Assay Medium
Intended Use
Bacto Pantothenate Assay Medium is used for determining the
concentration of pantothenic acid and its salts by the microbiological
assay technique.

Pantothenate Assay Medium is a modification of the formula
described in the United States Pharmacopeia1 for the microbiological
assay of pantothenic acid and its salts using Lactobacillus plantarum
ATCC 8014 as the test organism. Pantothenate Assay Medium does
not contain Tween 80 (Sorbitan Monooleate Complex), which is
included in Pantothenate Medium AOAC USP.

Pantothenate Assay Medium is a dehydrated medium free from
pantothenic acid or pantothenate but containing all other nutrients and
vitamins essential for the cultivation of L. plantarum ATCC 8014.

Dehydrated Appearance: Very light beige, homogeneous with
a tendency to clump.
Solution:
3.65% (single-strength) or 7.3%
(double- strength) solution, soluble
Prepared Medium:
Reaction of 3.65%
Solution at 25C:
pH 6.7 0.1
Cultural Response
Prepare Pantothenate Assay Medium per label directions.
Prepare a standard curve using a pantothenic acid reference
standard at levels from 0.0 to 0.10 g per 10 ml. The medium
supports the growth of L. plantarum ATCC 8014 when
supplemented with calcium pantothenate.
378
The addition of calcium pantothenate in specified increasing concentrations gives a growth response that can be measured turbidimetrically
or titrimetrically.
Formula
Formula Per Liter
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4
DL-Tryptophane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
Adenine Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Uracil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Pyridoxine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
g
g
g
g
g
mg
mg
mg
g
g
mg
g
g
g
g
g
g
mg
mg
mg
Precautions
closed. TARGET ORGAN(S): Kidney, Bladder.
wash immediately with plenty of water. If inhaled, remove to
fresh air. If not breathing, give artificial respiration. If breathing is
3. Great care must be taken to avoid contamination of media or glassware in microbiological assay procedures. Extremely small amounts
The Difco Manual
Section II
4. Take precaution to keep sterilization and cooling conditions uniform

saline. After the third wash, resuspend the cells with sterile 0.85%
saline and adjust to a turbidity of 40-45% transmittance when read on
a spectrophotometer at 660 nm. Aseptically inoculate each assay tube
with one drop of the cell suspension.
Storage
Standard Curve


curve readings and cannot always be duplicated. The standard curve is
obtained by using calcium pantothenate solution at levels of 0.0, 0.01,
0.02, 0.03, 0.04, 0.05, 0.06, 0.08, and 0.1 g per assay tube (10 ml).
Turbidimetric determinations are made after 18-24 hours incubation at
35-37C. Construct a standard curve and determine the concentration
of the unknown by interpolation from the standard curve.
The concentration of pantothenic acid required for the preparation of
the standard curve may be prepared by dissolving 50 mg dried calcium
pantothenate in a solution containing approximately 500 ml distilled
water, 10 ml 0.2N acetic acid and 100 ml 0.2N sodium acetate. Dilute
to 1,150 ml with additional water to make the calcium pantothenate
concentration 43.47 g per ml; one ml equals 40 g pantothenic acid.
This solution is diluted by adding 25 ml to a solution containing 500
ml distilled water, 10 ml 0.2N acetic acid and 100 ml 0.2N sodium
acetate. Dilute to 1 liter with distilled water to make a stock solution
containing 1.0 g pantothenic acid per ml. The standard solution is
made by diluting 2 ml of the stock solution to 100 ml with distilled
water. This solution contains 0.02 g pantothenic acid per ml. Use 0.0,
0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0 and 5.0 ml per assay tube. Prepare the
stock solution fresh daily.
Expiration Date
Procedure
Materials Provided

Glassware
Autoclave
Sterile test tubes
Calcium Pantothenate
Incubator (35-37C)
Centrifuge
0.2 N Acetic acid
0.2 N Sodium acetate
Spectrophotometer
1.
2.
3.
4.
5.
6.


specific assay procedures. For assays, the samples should be diluted
to approximately the same concentration as the standard solution.
Test Procedure
Prepare stock cultures of L. plantarum ATCC 8014 in triplicate by stab
inoculation of Lactobacilli Agar AOAC. Incubate cultures for 18-24 hours
at 35-37C. Store the tubes at 2-8C. Prepare a fresh stock culture every
week. Do not use a culture older than 1 week for this assay.
Inoculum
Subculture from a stock culture of Lactobacillus plantarum ATCC
8014 to 10 ml of sterile single-strength Pantothenate Assay Medium
supplemented with 0.02 mcg pantothenate. Incubate for 18-24 hours at
35-37C. Centrifuge the cells under aseptic conditions and decant the
supernatant. Wash the cells three times with 10 ml sterile 0.85%
The Difco Manual
Results
disk or cup.

response.
References
Convention, Inc., Rockville, MD.
Packaging
100 g
0604-15
379
Pantothenate Assay Medium AOAC USP
Section II
Bacto Pantothenate Medium AOAC USP
Intended Use
Bacto Pantothenate Medium AOAC USP is used for determining the
concentration of pantothenic acid and pantothenate by the microbiological assay technique.
Also Known As
AOAC is an abbreviation for Association of Official Analytical
Chemists. USP is an abbreviation for United States Pharmacopeia.

Vitamin Assay Media are used in the microbiological assay of
vitamins. Three types of media are used for this purpose:
Pantothenate Medium AOAC USP is prepared for use in the microbiological assay of pantothenic acid and pantothenate according to the
procedures of Calcium Pantothenate Assay in USP1 and Pantothenate
Acid Assay in AOAC.2 Lactobacillus plantarum ATCC 8014 is the
test organism used in this assay.

Pantothenate Medium AOAC USP is a pantothenic acid/pantothenatefree dehydrated medium containing all other nutrients and vitamins
essential for the cultivation of Lactobacillus plantarum ATCC 8014. The
addition of calcium pantothenate in specified increasing concentrations
gives a growth response that can be measured turbidimetrically or

titrimetrically.
Formula
Pantothenate Medium AOAC USP
Formula Per Liter
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4
L-Tryptophane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Adenine Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Uracil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8
Nicotinic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
g
g
g
g
g
g
g
g
mg
mg
mg
mg
mg
mg
g
g
g
g
mg
g
g
Precautions
Dehydrated Appearance: Very light beige, homogeneous,
tendency to clump.
Solution:
Prepared Medium:
may have a very slight precipitate.
Reaction of 3.65%
Solution at 25C:
pH 6.7 0.1
Cultural Response
Prepare Pantothenate Medium AOAC USP per label directions.
Test the medium by creating a standard curve using a
pantothenic acid reference standard at 0.0 to 0.05 g per
10 ml. The medium supports the growth of Lactobacillus
plantarum ATCC 8014 when prepared in single strength and
supplemented with pantothenic acid.
380

results. Scrupulously clean glassware free from detergents and other
Storage
hygroscopic. Store in a container with calcium chloride or other desiccant. Keep container tightly closed.
Expiration Date
The Difco Manual
Section II
Procedure
Materials Provided

Glassware
Autoclave
Centrifuge
Sterile test tubes
Incubator (35-37C)
Spectrophotometer (660 nm)
Calcium Pantothenate USP
0.2 N Acetic Acid
0.2 N Sodium Acetate
Distilled water
1.
2.
3.
4.
5.
6.

Adjust tube volume to 10 ml.

specific assay procedure. The samples should be diluted to approximately
the same concentration as the standard solution.
Test Procedure
Follow the assay procedures as outlined in USP1 or AOAC.2
Prepare stock cultures of L. plantarum ATCC 8014 by stab inoculation of Lactobacilli Agar AOAC. Incubate stock cultures at 35-37C
( 0.5C) for 18-24 hours. Store the stock cultures at 2-8C. Prepare
fresh stab cultures every week. Do not use a culture more than one
week old for preparing the inoculum.
Subculture from a stock culture of Lactobacillus plantarum ATCC
8014 to a tube of sterile single-strength Pantothenate Medium AOAC
USP (10 ml) supplemented with 0.2 mcg pantothenate. Incubate for
18-24 hours at 35-37C. Centrifuge the cells under aseptic conditions
and decant the supernatant. Wash the cells three times with 10 ml sterile
0.85% NaCI. After the third wash, resuspend the cells with sterile
0.85% NaCI and adjust to a turbidity of 40-45% transmittance when
read on a spectrophotometer at 660 nm. Aseptically inoculate each
assay tube with one drop of the cell suspension.
Prepare solutions of Calcium Pantothenate USP Reference Standard
or pantothenic acid (or equivalent) according to USP1 or AOAC.2
Satisfactory results are obtained with the standard curve by using
pantothenic acid at levels of 0.0, 0.005, 0.01, 0.015, 0.02 and 0.025 g
per assay tube (10 ml) for the AOAC procedure. Calcium pantothenate
may be used at standard levels of 0.0, 0.01, 0.02, 0.03, 0.04 and 0.05
g per assay tube for the USP procedure. Pantothenate Medium AOAC
USP may be used for both turbidimetric and titrimetric analysis in the
AOAC procedure, and for turbidimetric analysis only for the USP
The Difco Manual
Pantothenate Assay Medium AOAC USP
procedure. Turbidimetric readings should be made after 18-24 hours

incubation at 35-37C ( 0.5C). Titrimetrically determinations are
made following 72 hours incubation at 35-37C ( 0.5C).
The concentration of pantothenic acid or calcium pantothenate required
for the preparation of the standard curve may be prepared as follows:
1. Dissolve 50 mg dried calcium pantothenate in 500 ml distilled
water, 10 ml 0.2 N acetic acid and 100 ml 0.2 N sodium acetate.
2. Dilute with additional water to make calcium pantothenate
concentration 43.47 g per ml for the AOAC procedure or dilute to
50 g per ml for the USP procedure. At 43.47 g per ml, one ml
should equal 40 g pantothenic acid.
Dilute further by adding 25 ml of this solution to 500 ml distilled
water, 10 ml 0.2 N acetic acid and 100 ml 0.2 N sodium acetate. Dilute
this solution to 1 liter with distilled water to make a stock solution
containing 1 g pantothenic acid per ml. The standard solution is made
by diluting 5 ml of the stock solution to 1000 ml distilled water to
obtain a solution containing 0.005 g pantothenic acid per ml. Use 0.0,
1, 2, 3, 4 and 5 ml per assay tube. For the USP procedure, dilute the 50 g
per ml solution with distilled water to make a standard concentration
of 0.01 g per ml. Other standard concentrations may be used provided
the standard falls within the limits specified by USP1 and AOAC.2
Results
or cup.

response.
References
2. Association of Official Analytical Chemists. 1995. Official methods of analysis of AOAC International, 16th ed. AOAC International,
Arlington, VA.
Packaging
100 g
0816-15
381
Peptamin
Section II
Bacto Peptamin
Intended Use
Bacto Peptamin is used in preparing microbiological culture media.
Also Known As
Peptamin is also referred to as Peptic Digest of Animal Tissue.

Dehydrated Appearance: Golden tan, free-flowing, granules.
Solution:
1%, 2% and 10% solutions, soluble
in distilled or deionized water.
1%-Very light amber, clear to very
slight precipitate.
2%-Light amber, clear to slightly
precipitate.
10%-Light to medium amber, clear
slight precipitate.
Reaction of 1%
Solution at 25C:
pH 7.0-7.6
Add inoculum density of organism. All solutions are prepared
with pH adjusted to 7.2-7.4.
ORGANISM
ATCC
RESULT
Fermentable
2%
Carbohydrates
Escherichia
coli
25922*
negative
Indole
Production
Escherichia
coli
25922*
positive
Acetyle0.1%
methylcarbinol w/0.5%
Production
Dextrose
Enterobacter
aerogenes
13048*
positive
Hydrogen
Sulfide
1%
Salmonella
typhi
6539
positive
Growth
Response
2% w/0.5% Brucella
NaCl, 0.1% suis
Agar, & 0.1%
Dextrose
4314
good
growth
Growth
Response
2% w/0.5% Escherichia
NaCl, 0.1% coli
Agar, & 0.1%
Dextrose
25922*
good
growth
Growth
Response
2% w/0.5% Staphylococcus 25923*

NaCl, 0.1% aureus
Agar, & 0.1%
good
growth
SOLUTION
0.1%

Peptamin provides nitrogen, amino acids, vitamins and carbon in
Precautions
Storage
Cultural Response
TEST
The development of Peptamin is the result of accumulated information

that no single peptone is the most suitable nitrogen source for
culturing fastidious bacteria. Extensive investigations were undertaken
at Difco Laboratories using peptic digests of animal tissue prepared
under varying digestion parameters.
Peptamin complies with the US Pharmacopeia XXIII (USP) 1
specification for peptic digest of animal tissue. Diluting and rinsing
solutions, Fluid A and Fluid D, contain 0.1% Peptamin. Fluid A and
Fluid D conform to the specifications of USP1 for diluting and rinsing
fluids in sterility tests.
Brucella media used for the cultivation of fastidious microorganisms
contain Peptamin as the nitrogen source. Peptamin is used in
Disinfectant Test Broth AOAC and Letheen Broth, media used for
testing disinfectants. Media containing Peptamin are specified in
standard methods for multiple applications.2,3,4

Expiration Date
Procedure
Materials Provided
Peptamin

Refer to the final concentration of Peptamin in the formula of the
medium being prepared. Add Peptamin as required.
Test Procedure
See appropriate references for specific procedures using Peptamin.
Results
382
The Difco Manual
Section II
Peptone & Peptone Bacteriological Technical
References
Gaithersburg, MD.

of food, 3rd ed. American Public Health Association,
Washington, D.C.
Packaging
Peptamin
500 g
0905-17
Bacto Peptone
Bacto Peptone Bacteriological Technical
Intended Use
Carbohydrate (%)
Bacto Peptone and Bacto Peptone Bacteriological Technical are used

in preparing microbiological culture media.
Bacto Peptone, an enzymatic digest of protein, was first introduced

commercially in 1914 and became the standard Peptone for the
preparation of bacteriological culture media. The importance of
Peptone as a nutritive source in culture media was demonstrated by
studies of Klinger.5 The nutritive value of Peptone is largely dependent
upon the amino acid content that supplies essential nitrogen.
Many studies have used Bacto Peptone in culture media preparation.6,7,8,9
In a study by Morton, Smith and Leberman,10 Bacto Peptone was
reported to be superior to other peptones in a medium recommended for
the isolation and cultivation of pleuropneumonia-like organisms. Bacto
Peptone has been shown to be a satisfactory enrichment, replacing
serum, for cell proliferation.11 Peptone is routinely recommended for
culture media preparation. Several media containing Peptone are
specified in standard methods1,2,3,4 for multiple applications.
Peptone Bacteriological Technical can be used as the nitrogen source
in microbiological culture media when a standardized peptone is not
essential. Although it has not been as carefully standardized as other
peptones, certain parameters such as solubility, clarity, pH and other
growth supporting properties are monitored to permit its use as a
nitrogen source.

Bacto Peptone and Peptone Bacteriological Technical are enzymatic
digests of protein. Bacto Peptone contains nitrogen in a form that is
readily available for bacterial growth. Both products have a high
peptone and amino acids content and only a negligible quantity of
proteoses and more complex nitrogenous constituents.
Typical Analysis
Bacto Peptone

15.5
3.1
AN/TN
8.67
6.76
5.60
0.20
10.21
15.59
0.58
1.45
3.01
Lysine
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine
0.008
1.086
<0.001
<0.001
0.004
<0.001
0.007
<0.001
Phosphate
Potassium
Sodium
Sulfate
Sulfur
Tin
Zinc
20.0
Amino Acids (%)

Alanine
Arginine
Aspartic Acid
Cystine
Glutamic Acid
Glycine
Histidine
Isoleucine
Leucine
3.42
1.19
1.81
8.80
2.87
1.81
0.36
0.64
2.35
Inorganics (%)
Calcium
Chloride
Cobalt
Copper
Iron
Lead
Magnesium
Manganese
0.445
0.203
1.759
0.244
0.410
<0.001
0.001
Vitamins (g/g)
Biotin
0.2
Cyanocobalamin
<0.1
Folic Acid
0.3
Inositol
2400.0
Nicotinic Acid
21.9
PABA
Pantothenic Acid
Pyridoxine
Riboflavin
Thiamine
Thymidine
<0.5
5.9
1.7
3.9
<0.1
413.0

Coliform
Salmonella
Spore Count
negative
negative
90

Thermophile Count
13
Precautions
The Difco Manual
6.9
Total Nitrogen
Amino Nitrogen
Ash (%)
4.4
Clarity, 1% Solution (NTU) 0.5
0.5
Total
Loss on Drying (%)

pH, 1% Solution
3.0
7.0

383
Peptone & Peptone Bacteriological Technical
Section II
Storage
Store the products below 30C. The dehydrated product is very hygroscopic. Keep container tightly closed.
Bacto Peptone
Solution:
1%, 2% and 10% solutions are soluble
1%- Light amber, clear, no precipitate;
2%- Light to medium amber, clear,
no precipitate;
10%- Medium to dark amber, clear
to very slightly opalescent, may
have a very slight precipitate.
Reaction of 1%
Solution at 25C:
pH 6.8-7.2
Expiration Date
Peptone Bacteriological Technical

Dehydrated Appearance: Tan, free-flowing, granules.
Solution:
1%- Very light to light amber, clear;
2%- Light-medium amber, clear;
10%- Medium-dark amber, clear to
Reaction of 1%
Solution at 25C:
pH 6.3-7.6
SOLUTION
ATCC
ORGANISM
Escherichia
coli
Escherichia
coli
Enterobacter
aerogenes
Salmonella
typhi
Escherichia
coli
INOCULUM RESULT
25922*
negative
25922*
positive
13048*
positive
6539
positive
25922* 100-1,000 good

growth

aureus
growth

Prepare 2% Peptone Bacteriological Technical in 0.5% saline
and adjust to pH 7.2-7.4; add 1.5% Bacto Agar, boil and
sterilize. Inoculate and incubate at 35 2C for 18-48 hours.
ORGANISM
Escherichia coli
ATCC
INOCULUM CFU
RESULT
25922*
25923*
100-1,000
100-1,000
good growth
good growth
384
Materials Provided
Bacto Peptone

Refer to the final concentration of Bacto Peptone or Peptone Bacteriological Technical in the formula of the medium being prepared. Add
Bacto Peptone or Peptone Bacteriological Technical as required.

See appropriate references for specific procedures using Bacto Peptone
or Peptone Bacteriological Technical.
Bacto Peptone
All solutions are adjusted to pH 7.2-7.4.
Fermentable 2%
Carbohydrates
Indole
0.1%
Production
Acetylmethyl- 0.1%
carbinol
with 0.5%
Production Dextrose
Hydrogen
1%
Sulfide
Production
Growth
2% with
Response
1.5% Agar
and 0.5%
NaCl
Growth
2% with
Response
1.5% Agar
and 0.5%
NaCl
Procedure
Test Procedure
Cultural Response
TEST
Results
References
3rd ed. American Public Health Association, Washington D.C.
Gaithersburg, MD.
Washington, D.C.
5. Klinger, I. J. 1917. The effect of hydrogen ion concentration on
the production of precipitates in a solution of peptone and its
relation to the nutritive value of media. J. Bacteriol. 2:351-353.
6. Spray, R. S. 1929-1930. J. Lab. Clin. Med. 15:179.
7. Stainsby and Nicholls. 1932. J. Lab. Clin. Med. 17:530.
8. Huntoon, F. M. 1918. Hormone medium. A simple medium
employable as a substitute for serum medium. J. Infect. Dis.
23:169-172.
The Difco Manual
Section II
Peptone Iron Agar
9. Jones and Wise. 1926. J. Bacteriol. 11:359.

10. Morton, H. E., P. F. Smith, and P. R. Leberman. 1951. Venereal
diseases. Am. J. Syphilis Gonorr. 35:361.
11. Rutzky, L. P. 1981. Peptone growth factors for serial cell
proliferation in the absence of serum. Cambridge University Press.
Bacto Peptone Iron Agar
Packaging
Bacto Peptone
100
500
2
10
g
g
kg
kg
500 g
0118-15
0118-17
0118-07
0118-08
0885-17
Intended Use
Bacto Peptone Iron Agar is used for detecting hydrogen sulfide
production by microorganisms.

Levine and co-workers1,2 described a medium containing Proteose
Peptone and ferric citrate for detection of hydrogen sulfide production
by coliform bacteria. They demonstrated that such a medium served to
differentiate strains that were Voges-Proskauer negative, methyl-red positive and citrate positive from other members of the Enterobacteriaceae.
Levine reported that ferric citrate was a much more sensitive indicator
of hydrogen sulfide production than lead acetate, producing a medium
that gave definite reactions within 12 hours. Peptone Iron Agar is a
modification of Levines original formula in which Bacto Peptone has
been included with Proteose Peptone and the more soluble ferric
ammonium citrate is used in place of ferric citrate.
Tittsler and Sandholzer3 compared Peptone Iron Agar with lead
acetate agar for the detection of hydrogen sulfide and found that
Peptone Iron Agar had the advantage of giving earlier reactions and
clearer results.
Bacto Peptone and Proteose Peptone are nitrogen sources in Peptone

Iron Agar. Ferric Ammonium Citrate and Sodium Thiosulfate are used
to detect H2S production. Sodium Glycerophosphate is a buffering
compound. Bacto Agar is a solidifying agent.
Formula
Peptone Iron Agar
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Sodium Glycerophosphate . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
Precautions

Solution:
is light amber, very slightly to slightly
opalescent.
Prepared Medium:
Reaction of 3.6%
Solution at 25C:
pH 6.7 0.2
Cultural Response
Prepare Peptone Iron Agar per label directions. Inoculate and
ORGANISM
Escherichia coli
ser. enteritidis
ATCC
INOCULUM
CFU
GROWTH
H 2S
PRODUCTION
25922*
undiluted
good
13076
undiluted
good
+ (black)
Uninoculated
Escherichia coli
tube
ATCC 25922
The Difco Manual
ATCC 13076
385
Peptone Water
Storage
medium at 2-8C.
Section II
3. Once isolated colonies of coliform bacteria are obtained on solid

plated media, follow the test procedure below.
Test Procedure
Procedure
1. Obtain a pure culture of a test organism. Pick the center of a single

colony with an inoculating needle.
2. Inoculate a tube of Peptone Iron Agar by the stab method. Stab
the needle to within 1/4 to 1/2 inch of the bottom. Withdraw the
needle following the initial line of inoculation.
3. Incubate tubes at 35C for 18-48 hours.
4. Read tubes for growth and hydrogen sulfide production.
Materials Provided
Results
Peptone Iron Agar
Any blackening of the medium along the line of inoculation or

throughout the butt indicates hydrogen sulfide production.
For a complete discussion of the identification of coliform bacteria,
refer to the appropriate references.4,5,6
Expiration Date

Test tubes with closures or Petri dishes
Autoclave
Waterbath (45-50C)
Incubator (35C)
3. If preparing tubes, dispense the medium in 10 ml amounts. If
preparing plates, leave the medium in the flask.
5. If preparing tubes, cool in an upright position. If pouring plates,
cool the medium to 45-50C. Dispense into sterile Petri dishes.

guidelines.4,5,6
2. For specific information about specimen preparation and inoculation for isolation of coliform bacteria, consult appropriate
references.4,5,6
Bacto Peptone Water
Intended Use
Bacto Peptone Water is used for cultivating non-fastidious organisms,
for studying carbohydrate fermentation patterns, and for performing
the indole test.

The formulation of Peptone Water makes it useful for cultivating nonfastidious organisms.1 This non-selective medium has been used as a
basal medium for biochemical tests such as carbohydrate fermentation
patterns and production of indole.1,2
386
References
1. Levine, M., R. Vaughn, S. S. Epstein, and D. Q. Anderson.
1932. Some differential reactions in the colon-aerogenes group of
bacteria. Proc. Soc. Exp. Biol. Med. 29:1022-1024.
2. Levine, M., S. S. Epstein, and R. H. Vaughn. 1934. Differential
reactions in the colon group of bacteria. Am. J. Publ. Health
24:505-510.
3. Tittsler, R. P., and L. A. Sandholzer. 1937. Advantages of
peptone iron agar for the routine detection of hydrogen sulphide
production. Am. J. Publ. Health 27:1240-1242.
4. Pezzlo, M. (ed.). 1994. Aerobic bacteriology, p. 1.0.1.-1.20.47. In
Packaging
Peptone Iron Agar
500 g
0089-17

Peptone Water contains Peptone as a source of carbon, nitrogen,
vitamins and minerals. Sodium Chloride maintains the osmotic
balance of the medium.
Formula
Peptone Water
Formula Per Liter
Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Precautions
The Difco Manual
Section II
Peptone Water

1% Phenol Red solution

Indole Test strips
Storage

1. Dissolve 15 grams in 1 liter distilled or deionized water with

warming and frequent agitation.
Expiration Date
Procedure
Materials Provided
Peptone Water
Glassware
Autoclave
Incubator (35C)
Tubes with closures
Fermentation tubes
Carbohydrate solutions

Test Procedure
For Performing Carbohydrate Fermentation
1. Inoculate tubes with test organism.
2. Incubate tubes at 35 2C for 18-48 hours.
3. Observe for color change.

Dehydrated Appearance: Cream-white to light tan,
Solution:
deionized water on warming with
frequent agitation. Solution is light
Reaction of 1.5%
Solution at 25C:
pH 7.2 0.2
Cultural Response
Growth/Indole Reaction
Prepare Peptone Water per label directions. Inoculate and
incubate at 35 2C for 18-48 hours. Indole reaction is read
using Indole Test Strips (1627).
ORGANISM
Escherichia coli
ATCC
INOCULUM
CFU
GROWTH
INDOLE
REACTION
25922*
undiluted
good
positive
Carbohydrate Fermentation
Prepare Peptone Water per label directions with the addition
of phenol red and dextrose. Inoculate and incubate at 35 2C
for 18-48 hours.
ORGANISM
For Determining Carbohydrate Fermentation Patterns

1. Add 1.8 ml 1% phenol red solution to 1 liter rehydrated Peptone
Water. Mix thoroughly.
2. Dispense into test tubes containing inverted Durham vials.
4. Aseptically add sufficient sterile carbohydrate solution to yield a
1% final concentration. Rotate each tube to thoroughly distribute
the carbohydrate.
ATCC
INOCULUM
CFU
Escherichia coli
25922* 100-1,000
ACID
GROWTH PRODUCTION
good
good
positive
positive
For Performing the Indole Test

1. Using aseptic technique, suspend an Indole Test Strip 10 mm above
the surface of a 24 or 48 hour culture.
2. Incubate at 37C for 5-30 minutes.
Results
For Determining Carbohydrate Fermentation Patterns
Acid is produced when carbohydrates are fermented. This is indicated
by a yellow color in the medium. Gas production is indicated by the
presence of gas bubbles in the fermentation tube.
For performing the Indole Test
Observe for the formation of a violet color on the strip which indicates
a positive test for indole production.

1. Medium is pink in color when hot but becomes colorless upon cooling.
2. Vibrio spp. should not be incubated longer than 18-20 hours. Longer
incubation may cause the development of suppressed forms.3
References
2. Balows, A., W. J. Hausler, K. L. Herrmann, H. D. Isenberg,
3. Finegold, S. M., and W. Martin. 1982. Bailey and Scotts
diagnostic microbiology, 6th ed. St. Louis
Packaging
Peptone Water
The Difco Manual
500 g
1807-17
387
Phenol Red Agar Media
Section II

Bacto Phenol Red Agar Base . Bacto Phenol Red Lactose Agar
Bacto Phenol Red Mannitol Agar
Intended Use
vantage of a solid fermentation medium is that it permits observation

of fermentation reactions under both aerobic and anaerobic conditions.5,6
Deep tubes can provide sufficiently anaerobic conditions for the growth
of obligate anaerobic bacilli. Any gas formation that occurs during a
reaction is indicated by splitting of the agar or accumulation of gas
bubbles in the base.
Bacto Phenol Red Agar Base is used with added carbohydrate in differentiating pure cultures of bacteria based on fermentation reactions.
Bacto Phenol Red Lactose Agar is used for differentiating pure
cultures of bacteria based on lactose fermentation reactions.
Bacto Phenol Red Mannitol Agar is used for differentiating pure
cultures of bacteria based on mannitol fermentation reactions.
Phenol Red Agar Base supports excellent growth of many fastidious

bacteria. It is a basal medium free of any fermentable carbohydrates
that could give erroneous interpretations. With the exception of the
omitted carbohydrate, it is a complete medium prepared with Phenol
Red as an indicator of reaction changes. Phenol Red Agar Base
permits the user to prepare any quantity of medium needed, adding to

Phenol Red Agar Base with added carbohydrate is well suited for the
study of fermentation reactions of microorganisms.1,2,3,4 However, while
liquid media are generally employed in studying fermentation reactions,
many bacteriologists prefer a solid medium for this purpose. One ad-

Dehydrated Appearance: Pink, homogeneous, free-flowing.
Solution:
Phenol Red Agar Base: 3.1% solution,
upon boiling. Solution is orange-red
to red, clear to slightly opalescent.
Phenol Red Lactose Agar: 4.1% solution,
soluble in distilled or deionized water upon
boiling. Solution is orange-red to red, slightly
opalescent without significant precipitate.
Phenol Red Mannitol Agar: 4.1%
solution, soluble in distilled or deionized
water upon boiling. Solution is orange-red
to red, very slightly opalescent.
Prepared Medium:
Red to orange-red, slightly opalescent.
Reaction of the
Solutions at 25C:
pH 7.4 0.2
Cultural Response
Uninoculated
tube
Prepare media per label directions. Inoculate and incubate at

ORGANISMS
Alcaligenes faecalis
Escherichia coli
Shigella flexneri
ORGANISMS
ATCC
GROWTH
8750
25922*
13883*
12022*
good
good
good
good
ATCC
Escherichia coli
25922*
388
GROWTH
good
good
good
PHENOL RED AGAR BASE

(w/o CARBOHYDRATES)
ACID
GAS
PHENOL RED
LACTOSE AGAR
ACID
GAS

w/1% MALTOSE
ACID
GAS
+
+
PHENOL RED
MANNITOL AGAR
ACID
GAS
+
+
+
+
+
+
+
Typical positive
reaction with
acid and gas

w/1% SUCROSE
ACID
GAS
Typical negative
reaction with
positive growth

w/1% DEXTROSE
ACID
GAS
+
+
+
+
+

The Difco Manual
Section II
different portions any fermentable substance to be tested. Usually a

1% final concentration of a test carbohydrate is added. An entire series
of carbohydrate agars can be made up readily, conveniently, and
economically. Phenol Red Lactose Agar and Phenol Red Mannitol
Agar already contain the specified carbohydrate.

Proteose Peptone No. 3 and Beef Extract provide the carbon and
nitrogen required for good growth in a wide variety of organisms.
Sodium Chloride maintains the osmotic balance of the medium. Bacto
Agar is the solidifying agent. Phenol Red serves as a pH indicator,
turning from red-orange to yellow when acid is produced during
fermentation of the carbohydrate.
Glassware
Autoclave
Incubator (35C)
Choice of carbohydrates to be added to the basal medium
Tubes with closures
g
g
g
g
g
g
1. Suspend 31 grams in 900 ml distilled or deionized water and boil

3. Cool the medium to 45-50C.
4. Aseptically add 100 ml of a sterile 10% carbohydrate solution (w/v).
5. Dispense into sterile tubes with closures.
Phenol Red Lactose Agar

Formula Per Liter

Phenol Red Mannitol Agar
Formula Per Liter
g
g
g
g
g
g
Precautions
Storage
medium at 15-30C.
Expiration Date
The Difco Manual
g
g
g
g
g

D-Mannitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.025
Phenol Red Agar Base


1. Suspend 31 grams in 1 liter distilled or deionized water and boil to
2. When preparing 1% carbohydrate fermentation agars, dissolve 10
grams of the desired carbohydrate in the basal medium prior to
sterilization
4. Cool the medium to 45-50C.
OR

Formula Per Liter

Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.025
Materials Provided
Formula
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.025
Procedure

1. Suspend 41 grams of the selected medium in 1 liter distilled or
deionized water.
3. Dispense into tubes. Autoclave at 121C for 15 minutes.

Test Procedure
1. Inoculate the sterile medium by stabbing into the butt and streaking
the surface of the slant.
If desired, inoculate obligate anaerobic bacteria into melted
medium that has been cooled to 45C. Allow the agar to solidify
prior to incubation.
2. Incubate at 35 2C for 4-48 hours (or anaerobically for
24-72 hours).
3. Examine periodically for growth, acid production and gas formation.
Results
Fermentation of the carbohydrate is indicated by a change in the color
of the medium from red to canary yellow. Gas formation is indicated
by the collection of gas bubbles in the base or by splitting of the agar.
389
Phenol Red Carbohydrate Media

1. The addition of some carbohydrates to the basal medium may cause
an acid reaction. To restore the original pH (and color of the
medium), add 0.1 N sodium hydroxide on a drop-by-drop basis. Take
care not to make the medium too alkaline, which would prevent
fermentation from occurring within the usual incubation period.
2. When inoculating tubes, stab gently and do not use a loop. Rough
stabbing or using a loop to stab may give the false appearance of
gas production when mechanical splitting of the medium is what
actually occurred.
References
6th edition. American Society for Microbiology, Washington, D.C.
Section II

Enterobacteriaceae, 4th edition. Elsevier Science Publishing Co.,
Inc., New York, NY.
5. Bacteriological Analytical Manual, 8th edition. 1995. AOAC
Packaging
500 g
0098-17
500 g
0100-17
500 g
0103-17

Bacto Phenol Red Broth Base . Bacto Phenol Red Dextrose
Broth . Bacto Phenol Red Lactose Broth . Bacto Phenol Red
Mannitol Broth . Bacto Phenol Red Saccharose Broth
Intended Use
Phenol Red Carbohydrate Media are basal media used with added
carbohydrates in differentiating pure cultures of bacteria based on
fermentation reactions.

The fermentative properties of bacteria are valuable criteria in their
identification.1,2,3,4 A basal medium for determining the fermentation
reactions of microorganisms must be capable of supporting growth of
test organisms and be free from fermentable carbohydrates. Vera5 used
a fermentation test medium employing the pH indicator phenol red
and obtained highly accurate results.
Phenol Red Broth Base is recommended for use to determine the ability
of organisms to ferment various carbohydrates.6,7,8,9 Different fermentable
substances may be added in any desired concentration. The concentration
of carbohydrate generally employed for testing fermentation reactions
of bacteria is 0.5 to 1%. Some investigators prefer to use 1% rather
than 0.5% to ensure against reversion of the reaction due to depletion
of the carbohydrate.
Phenol Red Broth Base is an excellent substrate for streptococci, as
well as for other less fastidious bacteria, the growth promotion of the
medium can be greatly improved for fastidious, microaerophilic, and
obligately anaerobic strains by the addition of a small amount of Bacto
Agar (0.1-0.2%). A medium containing this small quantity of agar may
be heated it to the boiling point to drive out the dissolved air. The tubes
390
are then cooled to below 40C, without excessive agitation, just prior
to inoculation. The fermentation reaction of gonococci may be determined
by using 0.8% Bacto Agar and adding 5% sterile fresh rabbit serum to
the sterile Phenol Red Broth Base containing the selected carbohydrate.
Coagulase Plasma EDTA can be added to Phenol Red Mannitol Broth
to prepare Coagulase Mannitol Broth. This medium is useful in determining
the ability of Staphylococcus aureus to ferment mannitol and to
coagulate plasma.10

Proteose Peptone No. 3 and Beef Extract provide the carbon and nitrogen
sources required for good growth of a wide variety of organisms. Sodium
Chloride maintains the osmotic balance of the medium. Phenol Red
serves as an indicator, turning from red-orange to yellow when acid is
produced during fermentation of the added carbohydrates.
Formula
Phenol Red Broth Base
Formula Per Liter
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.018
g
g
g
g
The Difco Manual
Section II
Phenol Red Saccharose Broth

Formula Per Liter
Phenol Red Dextrose Broth

Formula Per Liter
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.018
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
g
g
g
g
g

Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.018
g
g
g
g
g

Phenol Red Carbohydrate Broths contain the above ingredients with
5 g/liter of the specified carbohydrate.
Phenol Red Lactose Broth

Formula Per Liter
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.018
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
g
g
g
g
g
Precautions
Storage
Phenol Red Mannitol Broth

Formula Per Liter
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.018
Bacto Mannitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
g
g
g
g
g
very hygroscopic. Keep containers tightly closed. Store the prepared
media at 2-8C.
Expiration Date
when stored as directed. Do not use a product if it fails to meet specifications
Procedure
Materials Provided


Solution:
orange-red to red, clear.
Prepared Media:
Orange-red to red, clear.
Reactions of 1.6%
Solution at 25C:
pH 7.4 0.2
Solution:
Prepared Media:
Reactions of 2.1%
Solution at 25C:
pH 7.4 0.2
Solution:
Prepared Media:
Reactions of 2.1%
Solution at 25C:
pH 7.4 0.2
The Difco Manual

Glassware
Autoclave
Incubator (35C)
Carbohydrates (as needed)
Tubes with closures
Fermentation tubes
2. Distribute into tubes. To detect gas production, place inverted
fermentation tubes (Durham tubes) in the tubes of medium.
When preparing 0.5-1% carbohydrate fermentation broths, dissolve
5-10 grams of the desired carbohydrate in the basal medium prior to
sterilization, or dissolve 16 grams of Phenol Red Broth Base in 900 ml
distilled or deionized water and aseptically add 100 ml of a sterile
5-10% carbohydrate solution (w/v) after sterilizing and cooling the
basal medium.
391
Section II
Phenol Red Dextrose Broth, Phenol Red Lactose Broth,

Phenol Red Mannitol Broth, Phenol Red Saccharose Broth
1. Suspend 21 grams of the appropriate Phenol Red Carbohydrate
Broth in 1 liter distilled or deionized water and stir to
2. For better growth of fastidious organisms (such as streptococci,
pneumococci, and gonococci) add 1 gram of Bacto Agar per liter
of medium and dissolve by boiling prior to sterilizing.
3. Dispense into tubes. To detect gas production, place inverted
fermentation tubes in the tubes of medium.
If the media are not used the same day they are sterilized, prior to use,
place the medium in flowing steam or a boiling water bath for a few
minutes to drive off dissolved gases. Allow to cool without agitation.
2. Incubate at 35 2C for 4-18 hours with caps loosened.

3. Examine tubes for growth, acid production, and gas production
(if fermentation vials are used).
Results
A yellow color of the medium indicates a positive reaction for
carbohydrate fermentation. If fermentation vials are used, bubbles in
the inverted vials are an indication of gas production. The presence of
a single bubble is recorded as positive for the production of gas.10

1. The addition of some carbohydrates to the basal medium may
result in an acid reaction. In this case, it is suggested that
0.1N sodium hydroxide be added drop by drop to restore the original
color. Take care not to make the medium too alkaline for true
fermentation to occur within the usual incubation period.
2. To ensure accuracy of interpretation, uninoculated control tubes
and/or inoculated Phenol Red Broth Base control tubes should be
run in parallel with the fermentation tests.

Test Procedure
1. Inoculate tubes with one drop of a diluted pure culture.

Solution:
deionized water. Solution is orange-red
to red, clear.
Prepared Media:
Reactions of 2.1%
Solution at 25C:
pH 7.4 0.2
Solution:
deionized water. Solution is orange-red
to red, clear.
Prepared Media:
Reactions of 2.1%
Solution at 25C:
pH 7.4 0.2
Uninoculated
tube
Cultural Response
Prepare media per label directions. Inoculate and incubate at 35 2C
for 18-48 hours.
ORGANISM
ATCC
GROWTH
BASE
A G
DEXTROSE
A G
LACTOSE
A G
MANNITOL
A G
SACCHAROSE
A G
8750
good
Escherichia coli
25922*
good
+ +
+ +
+ +
13883*
good
+ +
+ +
+ +
+ +
Shigella flexneri
12022*
good
Typical positive
reaction with
acid and gas
Typical negative
reaction with
positive growth
A = Acid
G = Gas
*These cultures are available as Bactrol Disks and should be used as directed in the Bactrol Disks Technical Information.
392
The Difco Manual
Section II
Phenylalanine Agar
References
2. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover,
and R. H. Yolken. (ed.). 1995. Manual of clinical microbiology, 6th edition. American Society for Microbiology,
Washington, D.C.
Enterobacteriaceae, 4th edition. Elsevier Science Publishing
Co., Inc., New York, NY.
5. Vera, H. D. 1950. Relation of peptones and other culture
media ingredients to accuracy of fermentation tests. Am. J.
Public Health 40:1267.
Bacto Phenylalanine Agar
7. Vanderzant, C., and D. F. Splittstoesser. 1992. Compendium of

Public Health Assoc., Washington, D.C.
8. Association of Official Analytical Chemists. 1995 official
methods of analysis of AOAC International. AOAC International,
Arlington, VA.
9. Franson, M. A. H., A. D. Eaton, L. S. Clesceri, and A. E.
Greenberg. 1995. Standard methods for the examination of water and
wastewater. American Public Health Association, Washington, D.C.
Packaging
500 g
0092-17
500 g
0093-17
500 g
0094-17
500 g
0097-17
500 g
0095-17
Also Known As
Phenylalanine Agar is also known as Phenylalanine Deaminase Medium.
Intended Use
Bacto Phenylalanine Agar is used for differentiating Proteus

and Providencia species from other Enterobacteriaceae based on
deamination of phenylalanine.
Buttiaux, Osteux, Fresnoy and Moriamez1 developed a method to

differentiate members of the Proteus and Providencia groups from

Solution:
on boiling. Solution is light amber, very slightly to
slightly opalescent without significant precipitate.
Prepared Medium:
Light amber, slightly opalescent without precipitate.
Reaction of 2.3%
Solution at 25C:
7.3 0.2
Cultural Response
Prepare Phenylalanine Agar per label directions. Inoculate the medium and
ORGANISM
Proteus vulgaris
Providencia alcalifaciens
ATCC
INOCULUM
CFU
13048* 100-1,000
13315* 100-1,000
9886 100-1,000
GROWTH
REACTION
good
good
good
+
+
The Difco Manual
Uninoculated
tube with
reagent
Proteus vulgaris
ATCC 13315
393
Phenylalanine Agar
Section II
other Enterobacteriaceae based on the ability of Proteus and

Providencia to deaminate phenylalanine to phenylpyruvic acid by
enzymatic activity.2 Bynae modified this method by incorporating
phenylalanine in the medium used to grow the organisms. Ewing,
Davis and Reavis4 simplified the Bynae formulation by omitting
proteose peptone. Phenylalanine Agar is prepared according their
formula.

Glassware
Autoclave
Incubator (35C)
SpotTest Ferric Chloride Reagent (3557) or 8-12% ferric chloride
0.1 N HCl
Phenylalanine Agar is used to differentiate Proteus, Providencia and

Morganella (originally classified in the genus Proteus) from other
members of the family Enterobacteriaceae. In addition, some strains
of Enterobacter agglomerans, Enterobacter sakazakii, Rahnella
aquatilis, Tatumella ptyseos and a few nonfermenting gram-negative
bacilli are also capable of deaminating phenylalanine.4,5
Test Procedure
Phenylalanine Agar contains DL-Phenylalanine which serves as a

substrate for deamination to phenylpyruvic acid. After incubation,
phenylpyruvic acid is detected by the addition of ferric chloride
reagent. The ferric ions chelate the phenylpyruvic acid and form a green
color. 5 Yeast Extract provides vitamins and cofactors required
for growth as well as additional sources of nitrogen and carbon.
Dipotassium Phosphate provides buffering capability. Sodium
Chloride maintains the osmotic balance of the medium. Bacto Agar is
1. Inoculate the medium and incubate at 35C for 18-24 hours.

2. After recording the growth response, add 3-5 drops of SpotTest
Ferric Chloride Reagent to each tube.
3. Examine for color development within 1-5 minutes. A dark green
color indicates a positive reaction.
Formula
Phenylalanine Agar
Formula Per Liter
1. A positive phenylalanine reaction should be interpreted quickly

because the green color disappears within 10 minutes after
addition of ferric chloride solution. Adding additional reagent
usually regenerates the color.

Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
DL-Phenylalanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
g
g
g
g
g
Precautions
Storage
1.
2.
3.
4.

Dispense into tubes. Autoclave at 121C for 15 minutes.
Allow medium to solidify in a slanted position.
Results
Positive: Dark green
Negative: No color change
2. Certain species rapidly deaminate phenylalanine, allowing for a

positive test result within 4 hours of incubation.4
References
1. Buttiaux, R., R. Osteux, R. Fresnoy and J. Moriamez. 1954.
Les proprits biochimiques charactristiques du gengre Proteus:
Inclusion souhaitable des Providencia dans celui-ca. Ann. Inst.
Pasteur 87:357-386.
Expiration Date
3. Ewing, W. H., B. R. Davis, and R. W. Reavis. 1957. Phenylalanine and malonate media and their use in enteric bacteriology.
Public Health Lab. 15:153.

handbook, vol 1. American Society for Microbiology,
Washington, D.C.
Procedure

Materials Provided
Phenylalanine Agar
Packaging
Phenylalanine Agar
394
100 g
500 g
0745-15
0745-17
The Difco Manual
Section II
Phenylethanol Agar
Bacto Phenylethanol Agar
Formula
Phenylethanol Agar
Formula Per Liter
Intended Use
Bacto Phenylethanol Agar is used for isolating staphylococci and
streptococci from specimens containing gram-negative organisms.
Also Known As
Phenylethanol Agar is also referred to as Phenylethyl Alcohol
(PEA) Agar.
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Phenylethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
g
g
g
g
g
Precautions

Brewer and Lilley1,2 reported that the addition of phenylethanol to a
nutritive medium will permit growth of gram-positive organisms but
markedly to completely inhibit growth of gram-negative organisms
found in the same specimen. Phenylethanol Agar inhibits the swarming
of Proteus spp. and can be used to selectively isolate anaerobic bacteria
from clinical specimens with mixed flora. Phenylethanol Agar is
specified for use in several reference methods.3,4,5

Tryptose and Beef Extract provide the nitrogen and carbon required
for good growth of a wide variety of organisms. Sodium Chloride
maintains the osmotic balance. Bacto Agar is a solidifying agent.
Phenylethanol is bacteriostatic for gram-negative bacteria and
inhibits DNA synthesis. Optional addition of 5% defibrinated sheep
blood to the basal medium can enhance microorganism recovery on
the medium.

ORGAN(S): Eyes, Face, Urogenital.
ATCC 25923
Uninoculated
plate

Dehydrated Appearance: Beige, homogeneous with soft clumps.
Solution:
amber, very slightly to slightly opalescent.
Prepared Medium:
Without blood - light amber, slightly
opalescent;
With blood - cherry red, opaque.
Reaction of 3.55%
Solution at 25C:
pH 7.3 0.2
Cultural Response
Prepare Phenylethanol Agar with 5% sterile defibrinated sheep
blood per label directions. Inoculate and incubate at 35 2C under
5-10% CO2 for 18-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
Proteus mirabilis
12453
1,000-2,000
25923* 100-1,000
100-1,000
Streptococcus pyogenes 19615* 100-1,000
RECOVERY
HEMOLYSIS
partial
inhibition
growth
growth
growth
N/A
beta
alpha
beta
ATCC 19615
The Difco Manual
395
Phytohemagglutins
Section II
Storage
Results
Store the dehydrated medium at 2-8C. The dehydrated medium is

medium at 2-8C.
Examine plates for growth and hemolysis. Perform additional biochemical testing to identify the organism.
Expiration Date
Procedure
Materials Provided
Phenylethanol Agar

Glassware
Autoclave
Incubator (35C)
1.
2.
3.
4.

defibrinated blood to the cooled medium at 45-50C. Mix well.

Test Procedure
1. Inoculate plates with test specimens. Streak to obtain isolated
colonies.
2. Incubate plates at 35 2C under 5-10% CO2 for 18-24 hours and,
if necessary, 40-48 hours.

1. Some gram-positive cocci may be slightly inhibited and may
require further incubation (to 48 hours) for sufficient growth to be
evident.6
2. Subculture gram-positive colonies onto Tryptic Soy Agar (TSA),
Selenite Broth and other biochemical media for definitive
identification.6
3. Pseudomonas aeruginosa is not inhibited on this medium.7
References
1. Brewer, J. H., and B. D. Lilley. 1949. Paper presented at the
December meeting of the Maryland Association of Medical and
Public Health Laboratories.
2. Lilley, B. D., and J. H. Brewer. 1953. The selective antibacterial
action of phenylethylalcohol. J. Pharm. Assoc. 42:6.
3. Baron, E. J., and S. M. Finegold. 1990. Bailey & Scotts
diagnostic microbiology, 8th ed. The C.V. Mosby Company,
St. Louis, MO.
4. Isenberg, H. D. 1992. Clinical microbiology procedures handbook.
6th ed. ASM Press, Washington, D.C.
7. Washington, J. A., Jr. 1981. Laboratory procedures in clinical
microbiology. Springer-Verlag, New York.
Packaging
Phenylethanol Agar
100 g
500 g
2 kg
0504-15
0504-17
0504-07
Phytohemagglutinins
Bacto Phytohemagglutinin M . Bacto Phytohemagglutinin P
Intended Use
Bacto Phytohemagglutinin M and Bacto Phytohemagglutinin P are

used for the isolation of lymphocytes and nucleated erythrocytes from
blood and marrow. They are also used for initiating mitosis in lymphocytes
for chromosomal analysis.
Hemagglutination
Phytohemagglutinins M or P were originally used for hemagglutination
techniques.1,2 Phytohemagglutinins were then used with dextran3 and
fibrinogen 4 to produce excellent yields of morphologically and
physiologically intact lymphocytes in a suspension with no hemolysis.
Also Known As
Phytohemagglutinin M is also known as PHA-M. Phytohemagglutinin P
is also known as PHA-P.
396
Phytohemagglutinin M or P have been used to agglutinate the erythrocytes

of all human blood groups, and those of many animals such as rabbit,
The Difco Manual
Section II
Phytohemagglutinins
dog, cat, chicken, duck, mouse, rat, sheep, horse, pig, frog and guinea
pig. Phytohemagglutinin has been used to obtain the plasma suspension
of trypanosomes from the blood of infected rats.5
Phytohemagglutinin P is a sterile, desiccated, purified, highly potent

protein phytohemagglutinin from which the polysaccharide moiety has
been removed.
Mitogenic Activity
Nowell6 discovered that phytohemagglutinin M initiates mitosis in
cultures of lymphocytes isolated from peripheral blood. Later,
phytohemagglutinin P was also shown to possess this property. The
application of this technique is important in the characterization of
chromosomes. A procedure using phytohemagglutinin-stimulated
lymphoblasts has been used to cultivate human immunodeficiency
virus type 1 (HIV-1) from infected individuals by cocultivation
cultures.7 Human T-lymphocytes have been activated by phytohemagglutinin to the blastic killer-cell state in preparation for in-vivo
immunotherapy trials in donor cancer patients.8
Precautions
A simplified procedure for lymphocyte mitogenesis was developed by

Moorhead, Nowell, Mellman, Batipps and Hungerford,9 in which the
cultures were routinely allowed to incubate for 3 days (65-70 hours).
Their method incorporated the hypotonic treatment developed by
Hughes10 and Hsu and Pomerat.11 The flame drying of slides by Scherz12
and the staining procedure by Rothfels and Siminovitch13 were helpful
contributions in this procedure. Staining of chromosomes by one of
many methods produces characteristic bands. For more information
on chromosome staining, please refer to appropriate references.14-17
Store desiccated Phytohemagglutinin M and Phytohemagglutinin P at

2-8C. The rehydrated solutions are stable for at least 2 weeks at -20C.
Phytohemagglutinin M
Phytohemagglutinin P
Both Phytohemagglutinin M and P will agglutinate the erythrocytes of

all human blood types, and those of animals. The rehydrated P-form
has approximately 40 times more hemagglutinating potency than the
M-form. Both forms will also stimulate the lymphocytes of peripheral
blood to undergo mitosis in vitro.
Reagents
Phytohemagglutinin M is a stable, nontoxic, desiccated
mucophytohemagglutinin.

Phytohemagglutinin M or P
Lyophilized Appearance: White, porous lyophilized cake.
Solution Appearance: Contents of 1 vial, soluble in 5 ml
sterile distilled or deionized water
within 2 minutes. Solution is
colorless, clear to slightly opalescent.
Performance Response
When reconstituted with 5 ml sterile distilled or deionized
water, 0.1 ml Phytohemagglutinin M or 0.01 ml Phytohemagglutinin P is added to 7 ml RPMI #1640 Medium containing
the lymphocytes from 5 ml heparinized human blood. The
mitogenicity test is performed using the above components
and procedures with 4 samples of human blood. A mitotic
index of at least 75 should be obtained from the lymphocytes
of each of the four samples of blood. A total of at least 400
should be obtained from the sum of all four cultures.
The Difco Manual

2. Observe universal blood and body fluid precautions in the handling
and disposing of specimens.18.19
3. Practice the following routine laboratory safety procedures:
Do not pipette by mouth.
Use aseptic technique and established laboratory procedures in
handling and disposing of infectious materials.
Storage
Expiration Date
Procedure
Materials Provided

Sterile syringe
Sterile test tube
30 units sterile heparin dissolved in 0.85% sterile saline
RPMI Medium #1640
700 units of Penicillin
700 g Streptomycin
Colchicine (10-5 Molar)
Hanks Balanced Salt Solution
Methanol, reagent grade
Glacial Acetic Acid, reagent grade
Deionized water
Giemsa Stain
Pipettes, 0.1 ml, 1 ml, 5 ml
Water aspirator
Pasteur pipettes
Centrifuge
Incubator, 35C
Microscope slides
Microscope (12.5X eyepiece with 10X low power, 40X high dry, and
100X oil immersion objectives)
Reagent Preparation
Phytohemagglutinin M or the sterile Phytohemagglutinin P is rehydrated
by adding 5 ml of sterile distilled or deionized water, or equivalent,
and rotating gently to mix contents thoroughly. The solutions are
approximately 1% in 0.85% saline. Both solutions contain approximately
50 mg protein per 5 ml.
397
Phytohemagglutinins

For each culture, a 5 ml sample of blood is adequate. Draw the blood
with a sterile syringe and immediately place in a sterile screw-capped
test tube containing 30 units of sterile heparin and mix thoroughly.
Dissolve the heparin in 1 ml of a sterile 0.85% saline solution before
collecting the specimen. Start the agglutination and mitotic procedures
immediately, or they may be postponed for at least 24 hours, if the
specimen is stored at 2-8C.
Observe aseptic technique from the collection of the blood sample
until the addition of the colchicine.
Test Procedure
Lymphocyte Separation and Inoculation
1. Transfer 5 ml of blood containing 30 units of heparin to a sterile
screw-capped test tube under aseptic conditions.
2. Add either 0.1 ml of rehydrated Phytohemagglutinin M or 0.0025 ml
of Phytohemagglutinin P to the 5 ml of heparinized blood, and mix
the contents by inverting several times.
3. Let the erythrocytes agglutinate at 25C for 15-30 minutes.
4. Centrifuge the tube at 500 rpm for 2 minutes. Excessive centrifuging must be avoided to prevent sedimentation of the lymphocytes.
5. Transfer the hazy plasma-lymphocyte suspension (about 2 ml) by
means of a sterile Pasteur pipette to 7 ml of a culture medium
consisting of RPMI #1640 Medium, 700 units of Penicillin, 700
g Streptomycin, and either 0.1 ml of Phytohemagglutinin M,
if the erythrocytes have been agglutinated with the M-form, or 0.01
ml of Phytohemagglutinin P, if the erythrocytes have been agglutinated with the P-form. The optimal concentration of lymphocytes
in the culture is 1.0-1.2 X 106 per ml. If Phytohemagglutinin P and
aseptic conditions are used, the antibiotics may be omitted.
Incubation of Culture
6. Incubate the culture in a vertical position at 35 2C with
occasional swirling for 3-4 days. Care should be taken to maintain
proper incubation temperature. A significant increase in mitotic
index is often obtained by incubating 4 days instead of 3. It is very
important to always maintain the proper pH range in the culture.
The phenol red indicator should not become more acidic than a
light amber nor more alkaline than a light pink. If the indicator
becomes amber, loosen the cap for an hour or so to allow the
escape of CO2. This precaution is often most necessary at the
beginning and end of the incubation.
7. End the mitosis by the addition of 1 ml of 105 molar colchicine,
and continue the incubation at 35 2C for another 4-6 hours. The
exposure of cells to the colchicine should not be less than 4 hours
or more than 6 hours.
Harvesting and Fixation of Cells
8. Transfer the entire culture to a graduated conical centrifuge tube
(15 ml) and centrifuge for 6-8 minutes at 600-800 rpm.
9. Carefully aspirate off the supernatant fluid.
10. Add 5 ml of warm (35 2C) Hanks Balanced Salt Solution and
resuspend the cells in the centrifuge tube with a Pasteur pipette.
11. Centrifuge at 600-800 rpm for 6-8 minutes.
12. Carefully aspirate off the supernatant with the pipette and add 1 ml
of Hanks Balanced Salt Solution.
13. Resuspend the packed cells with the Pasteur pipette.
398
Section II
14. Add 3 ml of warm (35 2C) distilled water, in 1 ml portions,

with momentary agitation after each addition to produce a
hypotonic solution.
15. Incubate the suspension at 35 2C for 10 minutes only. The
exposure of the cells to this hypotonic, diluted Hanks Balanced
Salt Solution should not exceed 10 minutes.
16. Centrifuge the lymphocyte solution at 600-800 rpm for 6-8 minutes.
17. Carefully aspirate off the supernatant.
18. Add slowly, without disturbing the button of cells, 4 ml of freshly
prepared fixative consisting of 1 part glacial acetic acid and 3 parts
methanol (reagent grade only).
19. Let the cells soak in the fixative for 15-30 minutes. Cells should be
treated gently during this stage of fixation. At this point, cells may
be stored overnight at 2-8C.
20. Resuspend with the Pasteur pipette.
21. Centrifuge at 600-800 rpm for 6-8 minutes, and carefully remove
the supernatant by aspiration.
22. Resuspend the cells in 4 ml fresh fixative with the Pasteur pipette,
and centrifuge at 600-800 rpm for 6-8 minutes. Repeat this step
again if necessary to disperse clumps of cells.
23. Carefully aspirate the supernatant.
24. Add 0.5-1.0 ml of fresh fixative to the button of cells and resuspend with the Pasteur pipette to get a hazy suspension.
Preparation of Slides
25. Label clean microscope slides and place them in clean, chilled
distilled water.
26. In rapid succession, shake the excess water off a chilled slide, wipe
the water off its underside, add 3-4 drops of the cell suspension by
means of the Pasteur pipette, tip the slide several times to spread
the suspension, and ignite the fixative by bringing it momentarily
in contact with a flame. When the fixative is burned off, wave the
slide vigorously to hasten drying. The slide should not get hot, but
drying should be accomplished as rapidly as possible.
Staining of Slides
Slides may be stained with Giemsa, orcein or other stains according to
the method of Rothfels and Siminovitch.14 The procedure using
Giemsa is given below.
27. Dilute the 1 ml of stock Giemsa Stain (20X stock) with 19 ml of
distilled water. The 1 ml of stock Giemsa Stain should be used the
same day it is diluted 20-fold with water.
28. Place the slides in a small staining dish or Petri dish and cover
them with 20 ml of the staining solution for 10-20 minutes.
29. Rinse the slides gently in distilled water and air dry.
30. Examine the slides under the microscope. The mitotic spreads may
be scanned at a total magnification of 125X, examined more closely
at 500X, or photographed under oil immersion at 1,000X. Slides
may be protected by cover slips and made permanent by conventional procedures.
Alternatively, the chromosomes may be treated by staining procedures
to show G-banding. Refer to appropriate references for alternative
staining procedures.18
Results
A mitotic index of at least 30 may be expected from the lymphocytes
from the heparinized peripheral blood of a healthy individual.
The Difco Manual
Section Ii
Plate Count Agar & Standard Methods Agar

1. For mitotic investigations, avoid the following:
Anticoagulants containing oxalates or phenols
Cytotoxic antibiotics, drugs or heavy metals (Penicillin and
Streptomycin are acceptable.)
Hypertonic and hypotonic media except for the intentional
swelling of the chromosomes
Irradiation of the patient or culture, which can produce breaks
in the chromosomes.
Some plastic materials cause cytotoxic effects.
References
1. Li, J. G., and E. E. Osgood. 1949. A method for the rapid
separation of leukocytes and nucleated erythrocytes from blood
or marrow with a phytohemagglutinin from red beans (Phaseolus
vulgaris). Blood 4:670-675.
2. Takikawa, K., T. Ito, J. Kato, T. Yoshida, H. Kondo, and
I. Miyata. 1957. Studies on the isolation of granules and
mitrochondria of leukocytes. Acta Haemat. 18:179-184.
3. Chen, H. P., and G. K. Palmer. 1958. A method for isolating
leukocytes. Am. J. Clin. Pathol. 30:567-569.
4. Skoog, W. A., and W. S. Beck. 1956. Studies on the fibrinogen,
dextran, and phytohemagglutinin methods of isolating leukocytes.
Blood 11:436-54.
5. Yaeger, R. G. 1960. A method of isolating trypanosomas from
blood. J. Parasitol. 46:288.
6.. Nowell, P. C. 1960. Phytohemagglutinin: an initiator of mitosis in
cultures of normal human leukocytes. Cancer Research 20:462-468.
7. Clarke, L. M. (ed.). 1992. Viruses, Rickettsiae, Chlamydiae, and
Mycoplasmas, p. 8.1.1-8.26.21. In H. D. Isenberg, (ed.), Clinical
microbiology procedures manual, vol. 2. American Society for
8. Frenster, J. H. 1976. Phytohemagglutinin-activated autochthonous
lymphocytes for systemic immunotherapy of human neoplasms.
Ann. NY. Acad. Sci. 277:45- 51.
9. Moorhead, P. S., P. C. Nowell, W. J. Mellman, D. M. Batipps,
and D. A. Hungerford. 1960. Chromosome preparations of
leukocytes cultured from human peripheral blood. Exp. Cell.
Res. 20:613.
10. Hughes, A. 1952. Some effects of abnormal tonicity on dividing

cells in chick tissue cultures. Quart. J. Microscopic Sci. 93:207.
11. Hsu, T. C., and C. M. Pomerat. 1953. Mammalian chromosomes
in vitro II. A method for spreading the chromosomes of cells in
tissue culture. J. Hered. 44:23-29.
12. Scherz, R. G. 1962. Blaze drying, by igniting the fixative, for
improved spreads of chromosomes in leukocytes. Stain. Tech.
37:386.
13. Rothfels, K. H., and L. Siminovitch. 1958. An air-drying technique
for flattening chromosomes in mammalian cells grown in vitro.
Stain Tech. 33:73-77.
14. Bird, B. R., and F. T. Forrester. 1981. Basic Laboratory Techniques in Cell Culture. U. S. Department of Health and Human
Services, CDC, Atlanta, GA.
15. Freshney, R. I. 1983. Culture of animal cells: A manual of basic
technique. Alan R. Liss, Inc., New York, NY.
16. Jones Brando, L. V. 1995. Cell culture systems, p. 158-165. In
Yolken (eds.), Manual of clinical microbiology, 6th ed. American
17. Gustashaw, K. M. 1991. Chromosome stains. In M. J. Barch (ed.),
The ACT Cytogenetics Laboratory Manual, 2nd ed. The Association of Cytogenetic Technologists, Raven Press, Ltd., New York,
NY.
18. Centers for Disease Control. 1988. Update: universal precautions
for prevention of transmission of human immunodeficiency virus,
hepatitis B virus, and other blood borne pathogens in health-care
settings. Morbidity and Mortality Weekly Reports 37:377-382,
387-388.
19. Occupational Safety and Health Administration, U.S.
Department of Labor. 1991. 29 CFR part 1910. Occupational
exposure to blood borne pathogens; final rule. Federal Register
56:64175-64182.
Packaging
Phytohemagglutinin M
5 ml
6 x 5 ml
0528-56*
0528-57*
Phytohemagglutinin P
5 ml
6 x 5 ml
3110-56*
3110-57*
*Store at 2-8C
Bacto Plate Count Agar

Bacto Standard Methods Agar
Intended Use
Bacto Plate Count Agar is a Standard Methods medium used for
enumerating aerobic bacteria in water, wastewater, foods and dairy
products.1,2,3,4,5 This medium is also recommended as a general plating
medium for determining bacterial populations.
Also Known as
Standard Methods Agar and Tryptone Glucose Yeast Agar are alternate
names for Plate Count Agar.
The Difco Manual

Plate Count Agar was developed by Buchbinder, Baris and Goldstein6
in 1953 at the request of the American Public Health Association.
Results showed that a dehydrated milk-free medium containing 0.25%
Yeast Extract, 0.5% Tryptone, 0.1% Dextrose and 1.5% Agar per liter
approximated the productivity of Tryptone Glucose Extract Agar with
added milk. Buchbinder et al. recommended that a dehydrated culture
medium be used in preparing the standard plate count medium rather
than preparing the medium from ingredients. Bacto Plate Count Agar
is prepared with the same ingredients originally suggested by
Buchbinder et al.7 Combinations of Yeast Extract and Tryptone have
been used in media for the examination of dairy products for the presence
of thermophilic organisms since 1928.8,9 This formula is specified in
Standard Methods for the Examination of Water and Wastewater,1
399
Plate Count Agar & Standard Methods Agar
Section II
Standard Methods for the Examination of Dairy Products,2 Compendium

of Methods for the Microbiological Examination of Foods3 and the
Association of Official Analytical Chemists (AOAC)4 and the FDA
Bacteriological Analytical Manual.5
Expiration Date
Procedure
Plate Count Agar contains Tryptone and Yeast Extract which provide
the carbon and nitrogen sources required for growth of a wide variety
of organisms. Dextrose is a source of fermentable carbohydrate
(energy source). Bacto Agar is a solidifying agent.
The expiration date applies to the product in its intact container product
Materials Provided
Plate Count Agar
Standard Methods Agar
Formula
Plate Count Agar
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose (Glucose) . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
Precautions
Storage
Store Plate Count Agar below 30C. The powder is very hygroscopic.
Store Standard Methods Agar at 15-30C.
Glassware
Autoclave
Plate Count Agar
1. Suspend 23.5 grams in 1 liter of distilled or deionized water.
Standard Methods Agar (prepared)
1. Loosen the caps on the bottles prior to heating.
2. Heat the medium in the autoclave for 7 minutes to melt the agar. A
small solidified mass remains that can be melted by swirling the hot
agar. Cycle time depends on the number of bottles in the chamber.
Uninoculated
plate
Pasteurized milk

Plate Count Agar
Dehydrated Medium: Light beige, homogeneous, free-flowing.
Solution:
slightly opalescent, no precipitate.
Prepared Medium:
no precipitate.
Reaction of 2.35%
Solution at 25C:
7.0 0.2
Cultural Response
Plate Count Agar (dehydrated)
Prepare Plate Count Agar per label directions. Inoculate with serial
dilutions (30-300 CFU/ml) of pasteurized and raw milk samples using
the pour plate method (standard plate count) and incubate at 32 1C
for 48 hours. Statistical analysis of data should yield counts comparable
to an approved lot of medium.
Standard Methods Agar (prepared)
Melt Standard Methods Agar and aseptically dispense into Petri dishes.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Lactobacillus acidophilus
11506
25923
30-300
30-300
good
good
400
The Difco Manual
Section II
m Plate Count Broth

4.
Test Procedure
1. Perform serial dilutions on samples (food, water) to be tested
using the heterotrophic (standard) plate count method. Select
dilutions that will yield plates with counts of 30-300 colonies.
2. Dispense a portion of each test dilution (e.g., 0.1 ml, 1.0 ml) into
separate sterile Petri dishes.
3. Add 10-12 ml of tempered (45C) Plate Count Agar to Petri dishes
containing test dilutions.
4. Swirl the dishes to thoroughly mix the agar and test dilution.
5. Allow plates to cool and solidify.
6. Incubate at 32 1C for 48 hours.
Results
Count colonies on all plates containing 30-300 colonies. Calculate
bacterial count per milliliter of sample by multiplying the average
number of colonies per plate by the reciprocal of the dilution used.
Report the count as CFU/ml.
References
2. Marshall, R. T. (ed.).1993. Standard methods for the examination
Washington, D.C.
3 Vanderzant, C., and D. F. Splittstoesser (ed.).1992. Compendium
5.
6.
7.
8.
9.

Association of Official Agricultural Chemists. 1995. Official
Bandler, R., M. E. Stack, H. A. Koch, V. H. Tournas, and P. B.
Mislivec. 1995. Yeasts, molds, and mycotoxins, p. 18.01-18.03.
Arlington VA.
Buchbinder, L., Y. Baris, and L. Goldstein. 1953. Further
studies on new milk-free media for the standard plate count of dairy
products. Am. J. Public Health 43:869- 872.
Buchbinder, L., Y. Baris, E. Alff, E. Reynolds, E. Dillon,
V. Pessin, L. Pincus, and A. Strauss.1951. Studies to formulate
new media for the standard plate count of dairy products. Pub
Health Rep. 66:327-340.
Prickett, P. S. 1928. Thermophillic and thermoduric microorganisms with special reference to species isolated from milk: V.
Description of spore-forming types. Technical Bulletin. NY
State Agri. Exp. Station 147:5-58.
Breed, R. S., P. A. Down, G. C. Supplee, P. S. Prickett, and G. J.
Hucker. 1932. Methods for use in the bacteriological examination
of dry milk and related powders. J. Dairy Sci. 15:383-389.
Packaging
Plate Count Agar
100
500
2
10
10 x 500
g
g
kg
kg
ml
0479-15
0479-17
0479-07
0479-08
9081-80
Bacto m Plate Count Broth
Intended Use
Bacto m Plate Count Broth is used for enumerating microorganisms

by membrane filtration.
Dehydrated Appearance: Light beige to beige, free flowing
homogeneous.
Solution:
amber, clear to slightly opalescent,
Reaction of 1.7%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepare m Plate Count Broth per label directions. Inoculate
and incubate the plates at 35 2C for 18-24 hours.
ORGANISM
Escherichia coli
ATCC
INOCULUM
CFU
RECOVERY
25922*
25923*
20-80
20-80
good to excellent
good to excellent
The above cultures are the minimum used for performance testing.
*These organisms are available as Bactrol Disks and are to be
The Difco Manual
Also Known As
m Plate Count Broth is also referred to as m TGY Broth, m Tryptone
Glucose Yeast Broth, or m Standard Methods Broth.

m Plate Count Broth is a nonselective general-purpose medium
for determining bacterial counts from food and water samples using
the membrane filtration procedure. This medium has the same
formulation as Plate Count Agar except that agar has been omitted
and the ingredients are employed in twice the concentration as in the
solid medium.1

Yeast Extract is a source of trace elements, vitamins and amino acids.
Tryptone provides carbon and nitrogen for bacterial metabolism.
Dextrose is a fermentable carbohydrate and carbon source.
401
Potato Dextrose Agar & Potato Dextrose Broth
Section II
Formula
m Plate Count Broth

Formula Per Liter


Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g
Precautions
Storage
Expiration Date
Procedure
Materials Provided
m Plate Count Broth

Glassware
Autoclave
Incubator (35 2C)
Pipettes
Bacto Potato Dextrose Agar

Bacto Potato Dextrose Broth
Intended Use
Bacto Potato Dextrose Agar is used for culturing yeasts and molds
from food and dairy products. Bacto Potato Dextrose Broth is used for
cultivating yeasts and molds.

Potato Dextrose Agar is a general purpose medium for yeasts and molds
that can be supplemented with acid or antibiotics to inhibit bacterial
growth. It is recommended for plate count methods for foods, dairy
products1,2,3,4 and for testing cosmetics.3 It can be used for growing
clinically significant yeasts and molds.5 The nutritionally rich base
(potato infusion) encourages mold sporulation and pigment production
in some dermatophytes.6
402

Water samples should be collected and prepared according to recommended guidelines.2,3,4
Test Procedure
1. Place a sterile absorbent pad in each 50 x 9 mm Petri dish.
2. Saturate the pad with approximately 2.0-2.4 ml of prepared medium.
3. Place an inoculated membrane filter, inoculated side up, on the
saturated pad.
4. Incubate in a 35 2C incubator for 18-24 hours.
Results
After incubation, count the colonies on the surface of the filter.
The colonies can be subcultured to appropriate media for identification,
if desired.
References
1. MacFadden, J. F. 1985. Media for isolation-cultivationidentification- maintenance of medical bacteria. vol. 1. Williams
2. Eaton, A. D., L. S. Cleseri, and A. E. Greenberg (ed.). 1995
3. Hitchins, A. D. 1992. FDA Bacteriological Analytical Manual,
7th ed. AOAC International, Arlington, VA.
Packaging
m Plate Count Broth
100 g
500 g
0751-15
0751-17
Potato Dextrose Broth is a general purpose broth medium for yeasts

and molds formulated as is Potato Dextrose Agar, but without agar.

Potato Dextrose Agar and Potato Dextrose Broth contain an infusion
from potatoes and Dextrose which encourage luxuriant fungal growth.
Bacto Agar is added to Potato Dextrose Agar as the solidifying agent.
Many standard procedures call for lowering the pH of Potato Dextrose
Agar to 3.5 0.1 to inhibit bacterial growth. The label on each
container of the medium specifies the amount of sterile tartaric acid
(10%) to add to the sterile medium. Do not reheat the acidified
medium because heating in the acid state will hydrolyze the agar.
Formula
Potato Dextrose Agar
Formula per liter
Potatoes, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . . . 200 g
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g

The Difco Manual
Section II
Potato Dextrose Agar & Potato Dextrose Broth
Potato Dextrose Broth

Formula per liter
Potatoes, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . . . 200 g

Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Flask with closure

Autoclave
Sterile tartaric acid, 10% solution (optional)
Precautions


4. To alter the pH of the medium to 3.5 0.1, add the amount of
sterile 10% tartaric acid specified on the label. Do not reheat the
medium after adding the acid.
Storage
Store the dehydrated medium below 30C. The powder is very hygroscopic.
Expiration Date
Procedure

1. Suspend 24 grams in 1 liter distilled or deionized water and warm
slightly to dissolve completely.
Materials Provided
Potato Dextrose Agar or Potato Dextrose Broth
Candida albicans
ATCC 10231
Aspergillus niger
ATCC 16404

Solution:
light amber, very slightly opalescent.
Prepared Medium:
Reaction of 3.9%
Solution at 25C:
pH 5.6 0.2
Solution:
deionized water upon slight warming;
very light amber, clear.
Prepared Medium:
Very light amber, clear.
Reaction of 2.4%
Solution at 25C:
pH 5.1 0.2
ATCC 9763
Cultural Response
Prepare Potato Dextrose Agar per label directions. Inoculate with
test organisms. Incubate plates at 30 2C for up to 7 days.
ORGANISM
ATCC
INOCULUM
CFU
RECOVERY
Aspergillus niger
Candida albicans
16404
10231
9763
9533
100-1000
100-1000
100-1000
undiluted
good
good
good
good
ATCC 9533

Prepare Potato Dextrose Broth per label directions. Inoculate
medium and incubate at 30 2C for 48 hours.
ORGANISM
ATCC
INOCULUM
CFU
RECOVERY
Aspergillus niger
Candida albicans
16404
10231
9763
100-1000
100-1000
100-1000
good
good
good
The Difco Manual
403
Potato Infusion Agar
Section II
References
2. Frank, J. F., G. L. Christen, and L. B. Bullerman (G. H.
Richardson, Tech. Comm.) 1993. Tests for groups of microorganisms. p. 271-286. In Marshall, R.T. (ed.). Standard methods
for the microbiological examination of dairy products, 16th ed.
3 Association of Official Analytical Chemists. 1995. Bacteriological
5. Dixon, D. M., and R. A. Fromtling. 1995. Morphology,
taxonomy, and classification of the fungi, p. 699-708. In Murray,
P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken
6. MacFaddin, J. F. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, vol 1. Williams
Test Procedure
Pour plate method1,3
1. Add 1 ml of test sample to a sterile Petri dish.
2. Add the specified amount (10 or 20 ml) of sterile, molten agar
(cooled to 45- 50C) and swirl gently to mix well. Allow to solidify.
3. Incubate at 22-25C or 30-32C (depending on the method being
followed) for 5 days or longer.
For complete information, refer to Standard Methods procedures in
the References section.
Results
Yeasts will grow as creamy to white colonies. Molds will grow as fuzzy
colonies of various colors. Count the number of colonies and consider
the dilution factor (if the test sample was diluted) in determining the
yeast and/or mold counts per gram or milliliter of material.
Growth is indicated as turbidity.
Packaging

1. Heating Potato Dextrose Agar after acidifying hydrolyzes the agar
and may destroy the solidifying properties.
2. Potato Dextrose Agar is not a differential medium. Perform microscopic examination and biochemical tests to identify isolates to
genus and species if necessary.
Bacto Potato Infusion Agar
100 g
500 g
2 kg
0013-15
0013-17
0013-07
500 g
10 kg
0549-17
0549-08
Intended Use
Bacto Potato Infusion Agar is used for cultivating Brucella, especially
in mass cultivation procedures.
Dehydrated Appearance: Medium tan, free-flowing, homogeneous.
Solution:
4.9% solution, soluble in 2% glycerol
solution upon boiling. Medium amber,
slightly opalescent, with a slight
precipitate.
Prepared Medium:
Medium amber, slightly opalescent to
Reaction of 4.9%
Solution at 25C:
pH 6.8 0.2C
Cultural Response
Prepare Potato Infusion Agar per label directions. Inoculate
prepared medium and incubate at 35 2C under approximately
5-10% CO2 for up to 72 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Brucella abortus
Brucella melitensis
Brucella suis
4315
4309
4314
100-1,000
100-1,000
100-1,000
good
good
good
404
Potato Infusion Agar is prepared according to the formula used by

Stockman and MacFadyean for the isolation of Brucella abortus.
Brucellosis is a zoonotic disease with a domestic-animal reservoir.1
Transmission by milk, milk products, meat and direct contact with
infected animals is the usual route of exposure.1
Tryptose agar w/ 5% bovine serum, with or without antibiotics,
remains a standard plating medium for the isolation of brucellae.1 Most
strains of Brucella spp. will grow on chocolate and blood agar, and the
addition of 5% heated horse or rabbit serum enhances growth
on all media.2 Potato Infusion Agar permits luxuriant growth of
characteristic colonies of B. abortus from infected materials, and may
be used with excellent results in mass cultivation of Brucella in the
preparations of vaccines and antigens.

Infusion from potatoes, Beef Extract and Proteose Peptone provide
the nitrogen, vitamins and amino acids in Potato Infusion Agar.
Dextrose and Glycerol are used as a carbon source in this formula.
The Difco Manual
Section II
Presence-Absence Broth
Sodium chloride maintains the osmotic balance of the medium.

Waterbath (45-50C)
Formula

Formula Per Liter

2% Glycerol.
Potatoes, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . . . 200

Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
Precautions
3. Brucella spp. are classified as Biosafety Level 3 pathogens. All
manipulations with live cultures and antigens must be confined
to a Class II biological safety cabinet (BSC).1

Specimens should be collected in sterile containers or with sterile swabs
and transported immediately to the laboratory in accordance with
Test Procedure
1. Incubate plates at 35 2C in 5-10% CO2 for 10 days.1 For a
complete discussion on the inoculation and identification of
Brucella spp., consult appropriate references.
Results
Storage
Limitations


2. Best results are obtained on freshly prepared medium with a
moist surface.
Expiration Date
Procedure
Material Provided
Material Required But Not Provided

Glassware
Autoclave
Incubator (35C)
References
Murray, P.R., E.J. Baron, M.A. Pfaller, F.C. Tenover, and R.H.
2. Baron, E. J., L. R. Peterson and S. M. Finegold. 1994. Bailey &
Scotts Diagnostic microbiology, 9th ed. Mosby-Year Book, Inc.,
St. Louis, MO.
Packaging
500 g
0051-17
Bacto Presence-Absence Broth
Intended Use
Bacto Presence-Absence Broth is used for detecting coliforms in
treated water.
Also Known As
Presence-Absence Broth is abbreviated as P-A Broth.

The Presence-Absence (P-A) test is a presumptive detection test for
coliforms in water. The test is a simple modification of the multiple-tube
procedure.1 One test sample, 100 ml, is inoculated into a single culture
bottle to obtain qualitative information on the presence or absence of
The Difco Manual
coliforms based on the presence or absence of lactose fermentation.1

This test is based on the principle that coliforms and other pollution
indicator organisms should not be present in a 100 ml water sample.2-8
Comparative studies with the membrane filter procedure indicate that
the P-A test may maximize coliform detection in samples containing
many organisms that could overgrow coliform colonies and cause
problems in detection.1 The P-A test is described in standard methods
for water testing1 and by US EPA.9

Beef Extract, Peptone and Tryptose provides the nitrogen, vitamins
and amino acids in Presence-Absence Broth. Lactose is the carbon
405
Section II
source in the formula. The Potassium Phosphates provide buffering

capacity; Sodium Chloride maintains the osmotic balance of the medium.
Sodium Lauryl Sulfate is the selective agent, inhibiting many organisms
except coliforms. Brom Cresol Purple is used as an indicator dye;
lactose-fermenting organisms turn the medium from purple to yellow
with or without gas production.
Expiration Date
Formula
Materials Provided
Presence-Absence Broth (single-strength)

Formula Per Liter
stored as directed. Do not use a product if it fails to meet specifications for identity and performance.
Procedure

Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.46
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.83
Potassium Phosphate, Dibasic . . . . . . . . . . . . . . . . . . . . . 1.35
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.46
Brom Cresol Purple . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0085
Glassware
Screw-cap dilution bottle with capacity > 150 ml
Incubator (35C)
g
g
g
g
g
g
g
g
g
Precautions
1. To prepare triple-strength medium, suspend 91.5 grams in 1 liter

2. Warm gently to dissolve completely.
3. Dispense 50 ml amount into screw-cap 250 ml milk dilution
bottles.
4. Autoclave at 121C for 12 minutes, with the total autoclave time
not to exceed 30 minutes.

Collect water samples as described in recommended procedures.1,9
Storage

Solution:
water; purple, clear to very slightly opalescent without
Prepared Medium:
Purple, clear to very slightly opalescent without
Reaction of 3.05%
Solution at 25C:
pH 6.8 0.2
Cultural Response
Prepare Presence-Absence Broth in triple strength solution (9.15%). Sterilize
in 50 ml quantities in milk dilution bottles with capacity greater than 150 ml.
Add 100 ml of drinking water after medium is sterilized and cooled to room
temperature. Inoculate bottles with the test organisms. Incubate bottles for
18-48 hours at 35C.
ORGANISM
Escherichia coli
Escherichia coli
ATCC
INOCULUM
CFU
GROWTH
RESULTS
29212*
25922*
13762
27853*
100-1,000
100-1,000
100-1,000
100-1,000
moderate
good
good
poor to moderate
slight yellow to purple

yellow color w/ or w/o gas production
yellow color w/ or w/o gas production
no color change
406
The Difco Manual
Section II
Proteose No. 3 Agar
Test Procedure

2. The P-A test is only a presumptive test for coliforms.
3. Confirmation and differentiation of coliforms detected by the
P-A test may be achieved by use of appropriate confirmatory
media, incubation times and temperatures as outlined in appropriate
references.1,9
4. Extending the P-A test incubation period to 72 or 96 hours will
allow isolation of other indicator organisms. However, indicator
bacteria isolated after 48 hours incubation may not be considered
for regulatory purposes.
2. Weiss, J. E., and C. A. Hunter. 1939. Simplified bacteriological

examination of water. J. Am. Water Works Assoc. 31:707-713.
3. Clark, J. A. 1968. A presence absence (P-A) test providing
sensitive and inexpensive detection of coliforms, fecal coliforms,
and fecal streptococci in municipal drinking water supplies. Can.
J. Microbiol. 14:13-18.
4. Clark, J. A. 1969. The detection of various bacteria indicative of
water pollution by a presence-absence (P-A) procedure. Can. J.
5. Clark, J. A., and L. T. Vlassoff. 1973. Relationships among
pollution indicator bacteria isolated from raw water and distribution
systems by the presence-absence (P-A) test. Health Lab. Sci.
10:163-172.
6. Clark, J. A., and J. E. Pagel. 1977. Pollution indicator bacteria
associated with municipal raw and drinking water supplies. Can. J.
7. Clark, J. A. 1980. The influence of increasing numbers of
nonindicator organisms upon the detection of indicator organisms
by the membrane filter and presence-absence tests. Can. J.
8. Clark, J. A., C. A. Burger, and L. E. Sabatinos. 1982.
Characterization of indicator bacteria in municipal raw water,
drinking water and new main water samples. Can J. Microbiol.
28:1002-1013.
9. Federal Register. 1989. National primary drinking water
regulations; total coliforms (including fecal coliforms and E. coli).
Fed regist. 54:27544-27568.
References
Packaging
1. Inoculate 50 ml of the sterile triple strength P-A Broth with 100 ml

of the water sample.
2. Invert the bottle a few times to achieve an even distribution of the
medium throughout the test sample.
3. Incubate at 35 0.5C.
4. Inspect for acid and gas production after 24 and 48 hours of
incubation.
Results
A distinct yellow color indicates lactose fermentation, an acid reaction.
Gas production can be observed by a foaming reaction when the bottle
is gently shaken. Any amount of gas and/or acid is a positive presumptive
test requiring confirmation.1 Report results as positive or negative for
coliforms per 100 ml of sample.

Bacto Proteose No. 3 Agar
Intended Use
Bacto Proteose No. 3 Agar is used with added enrichment in isolating
and cultivating Neisseria and Haemophilus.

Proteose No. 3 Agar, introduced in 1938, is used for isolating Neisseria
gonorrhoeae. When enriched with Hemoglobin and Supplement B,2,3
Proteose No. 3 Agar recovers gonococci in a manner comparable to
more complex media, ranking only slightly lower than GC Medium at
24 hours.
Chocolate agar may be prepared from Proteose No. 3 Agar with the
addition of 2% Hemoglobin. Hemoglobin provides X factor
(hemin), required for growth of Haemophilus and enhanced growth
of Neisseria.
The growth rate of Neisseria and Haemophilus spp. may be improved
with the addition of 1% Supplement B or VX, which provide the growth
factors glutamine and cocarboxylase.
The Difco Manual
500 g
2 kg
0019-17
0019-07

Proteose Peptone No. 3 provides nitrogen, vitamins and amino acids.
Dextrose is a carbon source. Sodium Chloride maintains the osmotic
balance in the medium, which is buffered by Disodium Phosphate.
Proteose Peptone No. 3 Agar is intended for use with supplementation
by 2% hemoglobin and Supplement B or Supplement VX.
Formula
Proteose No. 3 Agar
Formula Per Liter
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
Precautions
407
Proteose No. 3 Agar
Section II

Storage
2.
3.
4.
5.
6.

Aseptically add 500 ml sterile 2% Hemoglobin solution. Mix well.
Add 10 ml of Supplement B or Supplement VX. Mix thoroughly.
Dispense into sterile Petri dishes or as desired.
Expiration Date

Test Procedure
Haemophilus or Neisseria spp., refer to the procedures outlined in the
references.4,5,6
Procedure
Materials Provide
Proteose No. 3 Agar
Results
Glassware
Autoclave
Incubator (35C)
Waterbath (45-50C)
Hemoglobin (2%)
Supplement B or Supplement VX
1. Suspend 45 grams in 500 ml liter distilled or deionized water.

this medium
2. Proteose No. 3 Agar is intended for use with supplementation.
Although certain diagnostic tests may be performed directly on
this medium, biochemical and, if indicated, immunological testing
Consult appropriate references for further information.
References
Solution:
9% (double strength) solution, soluble
boiling with frequent agitation. Light
to medium amber in color, opalescent
with a slight flocculent precipitate.
Prepared Medium
(Single-strength):
Light amber, opalescent with a slight
flocculent precipitate, firmly solid.
Reaction of 9%
Solution at 25C:
pH 7.3 + 0.2
Cultural Response
Prepare Proteose Agar No. 3 per label directions. Inoculate
and incubate at 35 2C under approximately 5-10% CO2
for 18-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Neisseria sicca
10211
43070
13102
9913*
100-1,000
100-1,000
100-1,000
100-1,000
good
good
good
good
408
1. Carpenter, C. M., M. A. Bucca, T. C. Buck, E. P. Casman, C.

W. Christensen, E. Crowe, R. Drew, J. Hill, C. E. Lankford, H.
E. Morton, L. R. Peizer, C. S. Shaw, and J. D. Thayer. 1949.
Evaluation of twelve media for the isolation of the gonococcus.
Am. J. Syphil. Gonorrh. Vener. Dis. 33:164
2. Lankford, C. E., V. Scott, M. F. Cox, and W. R. Cooke. 1943.
Some aspects of nutritional variation of the gonococcus.
3. Lankford, C. E., and E. E. Snell. 1943. Glutamine as growth factor
for certain strains of Neisseria gonorrhoeae. J. Bacteriol. 45:410.
6. Baron, E. J., L. R. Petersons, and S. M. Finegold. 1994. Bailey
St. Louis, MO.
Packaging
Proteose No. 3 Agar
Hemoglobin 2% Solution
500 g
0065-17
6 x 100 ml
3248-73
6 x 10 ml
100 ml
0276-60
0276-72
Supplement VX w/Reconstituting Fluid 6 x 10 ml

100 ml
3354-60
3354-72
Supplement B w/Reconstituting Fluid
The Difco Manual
Section II
Proteose Peptones
Proteose Peptones
Bacto Proteose Peptone . Bacto Proteose Peptone No. 2
Bacto Proteose Peptone No. 3
Intended Use
Bacto Proteose Peptone is used in preparing microbiological culture

media and in producing bacterial toxins.
Bacto Proteose Peptone No. 2 is used in preparing microbiological
culture media.
Bacto Proteose Peptone No. 3 is used in preparing microbiological
culture media.
Difco Laboratories conducted extensive investigations to optimize

peptone production. Studies of peptic digests of animal tissue
prepared under varying digestion parameters led to the development
of Proteose Peptone, Proteose Peptone No. 2 and Proteose Peptone
No. 3. Data accumulated during these studies demonstrated that
no one peptone is the most suitable nitrogen source for every
microbiological application.

Proteose Peptone
Solution:
soluble in distilled or deionized water:
1%-Light amber, clear to very
slight precipitate;
slight precipitate;
10%-Medium to dark amber, clear
slight precipitate.
Nitrogen (Kjeldahl Method): 12.4-14.5%
Amino Nitrogen
(Modified Sorensen Method):
2.0-3.75%
Reaction of 1%
Solution at 25C:
pH 6.6-7.6
Solution:
1%-Light to medium amber, clear,
no precipitate;
2%-Medium amber, clear, no
precipitate;
10%-Medium to dark amber, slightly
opalescent to opalescent with
precipitate.
Amino Nitrogen
4.1-5.3%
Reaction of 1%
Solution at 25C:
pH 7.2-7.6
The Difco Manual
Proteose Peptone was originally developed to produce a diphtheria

toxin of high and uniform potency. Its suitability for this purpose was
quickly established. Proteose Peptone is used in preparing toxin for
diphtheria antitoxin, toxin-antitoxin mixtures, and for toxoid. Many
studies support use of Proteose Peptone in culture media for diphtheria
toxin production.1,2,3,4
Proteose Peptone is exceptionally valuable in the production of
bacterial toxins, including toxins of Corynebacterium diphtheriae,
Clostridium botulinum, Pneumococcus, Salmonella pullorum and
scarlet fever toxin.5,6,7,8 Proteose Peptone has many properties that
account for its suitability in culturing fastidious pathogens, including
its nitrogenous components, buffering range and high proteose
content. These elements create an environment suitable for the
maintenance of virulence and the elaboration of bacterial by-products.
For this reason, stock cultures are well preserved on media containing
Proteose Peptone.
Proteose Peptone No. 2 was originally developed for use in media
intended for producing diphtheria toxin. Interest was renewed by
Bunney and Thomas 9 through their study of diphtheria toxin
production in a semisynthetic medium. Proteose Peptone No. 2 is used
in media for producing bacterial toxins and for cultivating a wide
range of bacterial species.
Proteose Peptone No. 3, a modification of Proteose Peptone, is used in
preparing chocolate agar for propagating Neisseria species and chocolate
tellurite agar for propagating Corynebacterium diphtheriae. While
investigating the nutritional values of the Proteose Peptones, Proteose
Peptone No. 3 was found to provide superior nutrition for fastidious
microorganisms. It can replace the meat infusion-peptone combination
in infusion media. Proteose Peptone No. 3 supports growth of streptococci, staphylococci, meningococci, pneumococci, gonococci and
other microorganisms requiring a highly nutritious substrate. Proteose
No. 3 Agar, prepared with Proteose Peptone No. 3 as its major source
of nitrogen, vitamins and amino acids, is used with added enrichments
for isolating and cultivating Neisseria and Haemophilus.

Proteose Peptone is an enzymatic digest of protein high in proteoses.
Proteose Peptone No. 2 and Proteose Peptone No. 3 are enzymatic
digests of protein.
409
Proteose Peptones
Section II
Typical Analysis
PROTEOSE
PEPTONE
PROTEOSE
PEPTONE
PROTEOSE
PEPTONE NO. 2
PROTEOSE
PEPTONE NO. 3
11.1
1.4
0.9
3.1
7.2
12.7
1.5
0.6
3.5
7.2
11.4
2.2
0.5
4.0
7.2
<0.1
1.3
1.4
14.0
2.9
20.7
12.6
5.0
39.7
13.2
3.5
26.5
6.50
5.12
7.28
0.87
11.95
9.68
2.01
3.04
5.66
5.33
1.97
2.86
5.93
3.49
3.14
0.60
2.35
3.76
6.08
5.47
7.45
0.40
10.57
10.84
<0.01
1.00
3.57
5.22
1.51
7.94
5.31
4.64
3.90
0.94
1.92
4.73
5.99
5.49
6.92
1.12
12.38
9.26
1.74
2.65
5.70
5.02
1.86
2.72
4.94
3.65
3.32
0.59
1.96
3.62
Ash (%)
Clarity, 1% Solution (NTU)
Loss on Drying (%)
pH, 1% Solution
Carbohydrate (%)
Total

Total Nitrogen
Amino Nitrogen
AN/TN
Amino Acids (%)

Alanine
Arginine
Aspartic Acid
Cystine
Glutamic Acid
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine
PROTEOSE
PEPTONE NO. 2
PROTEOSE
PEPTONE NO. 3
Inorganics (%)
Calcium
Chloride
Cobalt
Copper
Iron
Lead
Magnesium
Manganese
Phosphate
Potassium
Sodium
Sulfate
Sulfur
Tin
Zinc
0.021
4.510
<0.001
<0.001
0.002
<0.001
0.027
<0.001
0.872
0.685
3.677
0.162
0.812
<0.001
0.002
0.024
3.644
<0.001
<0.001
<0.001
<0.001
0.024
<0.001
1.674
0.815
3.956
0.232
0.698
<0.001
0.003
0.023
3.581
<0.001
<0.001
0.002
<0.001
0.027
<0.001
1.447
0.982
3.815
0.232
0.975
<0.001
0.007
Vitamins (g/g)
Biotin
Choline (as Choline Chloride)
Cyanocobalamin
Folic Acid
Inositol
Nicotinic Acid
PABA
Pantothenic Acid
Pyridoxine
Riboflavin
Thiamine
Thymidine
0.1
2300.0
<0.1
0.4
5000.0
79.9
4.2
20.0
1.1
<0.1
1.2
99.7
0.3
4500.0
<0.1
0.5
4700.0
157.1
1.2
47.0
4.0
6.4
1.6
1319.0
0.4
3700.0
<0.1
0.3
8900.0
124.2
<0.5
20.0
1.3
6.8
0.1
659.6

Coliform
Salmonella
Spore Count
Thermophile Count
negative
negative
393
443
73
negative
negative
75
1450
<50
negative
negative
890
915
25

Dehydrated Appearance: Golden tan, free-flowing granules.
Solution:
1%-Very light amber, clear to very
slightly opalescent, may have a slight
precipitate;
2%-Light amber, clear to slightly
precipitate;
slight precipitate.
Amino Nitrogen
2.25-4.85%
Reaction of 1%
Solution at 25C:
pH 7.0-7.6
410
The values presented above are typical. This information is for

broad comparison use only and is not indicative of the makeup of
any particular lot of material. No guarantee is made, either
expressed or implied, that any specific lot of product will match the
values presented.
Precautions
Storage
Store below 30C. The dehydrated ingredient is very hygroscopic. Keep
Expiration Date
The Difco Manual
Section II
Proteose Peptones
Procedure
Results
Materials Provided
Proteose Peptone
H2S Test Strips
Indole Test Strips
KL Antitoxin Strips
References

Refer to the final concentration of Proteose Peptone, Proteose Peptone
No. 2 or Proteose Peptone No. 3 in the formula of the medium being
prepared. Add as required.
1.
2.
3.
4.
5.
6.
7.
8.
9.
Hewitt. 1930. Biochem. J. 24:984.

Bunney. 1930. J. Immunol. 20:71.
Kirkbride, Berthelsen and Clark. 1931. J. Immunol. 21:1.
Hazen and Heller. 1932. J. Bacteriol. 23:195.
Kirkbride and Wheeler. 1926. J. Immunol. 11:477.
Nelson. 1927. J. Infect. Dis. 41:9.
Kneeland and Dawes. 1932. J. Exp. Med. 55:735.
Hanks and Rettger. 1932. J. Immunol. 22:283.
Bunney and Thomas. 1936. J. Immunol. 31:95.
Packaging
Proteose Peptone
500 g
10 kg
0120-17
0120-08

500 g
10 kg
0121-17
0121-08
Test Procedure
500 g
2 kg
10 kg
0122-17
0122-07
0122-08
See appropriate references for specific procedures using Proteose

Peptone, Proteose Peptone No. 2 or Proteose Peptone No. 3.
Cultural Response
Proteose Peptone, Proteose Peptone No. 2 and Proteose Peptone No. 3
For each Test specified, prepare a Test Solution of the desired Proteose Peptone and, if necessary, adjust to pH 7.2-7.4; sterilize, inoculate
and incubate according to standard test procedure.
TEST
INOCULUM
RESULT
Escherichia coli
Escherichia coli
25922*
25922*
1 drop, undiluted
1 drop, undiluted
Acetylmethylcarbinol
Production (AMC)
Hydrogen Sulfide
Production
Growth Response
0.1% w/ 0.5%
dextrose
1%
13048*
1 drop, undiluted
Salmonella typhi
6539
1 drop, undiluted
2% w/ 0.1% agar, 0.5%

NaCl and 0.1% dextrose
2% w/ 0.1% agar, 0.5%
2% w/ 0.1% agar, 0.5%
Brucella suis
4314
undiluted
negative; red color

positive; pink color on
Indole Test Strip
positive; pink color upon
adding reagents
positive; brownish blackening
of H2S Test Strip
good growth
Growth Response
ORGANISM
ATCC
Fermentable Carbohydrate 2%
Indole Production
0.1%
Growth Response
TEST SOLUTION
25923*
100-1,000 CFU
good growth
Escherichia coli
25922*
100-1,000 CFU
good growth
Proteose Peptone
Prepare KL Virulence Agar from individual ingredients using 2 grams of the test Proteose Peptone; sterilize, add KL Virulence
Enrichment and dispense into Petri dishes containing KL Antitoxin Strips. Inoculate with a loopful of surface growth and incubate
at 35 2C for 72 hours. Examine at 24, 48 and 72 hours.
TEST
ORGANISM
ATCC
RESULT
Toxin Production
Toxin Production
Toxin Production
Corynebacterium diphtheriae Type intermedius

Corynebacterium diphtheriae Type gravis
Corynebacterium diphtheriae Type mitis
8032
8028
8024
precipitin line
precipitin line
precipitin line
The Difco Manual
411
Pseudomonas Agar Media
Section II

Bacto Pseudomonas Agar F . Bacto Pseudomonas Agar P
Intended Use
Pseudomonas Agar F is used with Bacto Glycerol for detecting and

differentiating Pseudomonas aeruginosa from other pseudomonads
based on fluorescein production.
Pseudomonas Agar F and Pseudomonas Agar P, patterned after the

formulations described by King, Ward and Raney,1 are modified to
USP specifications.2
Pseudomonas Agar F enhances the production of fluorescein by
Pseudomonas and inhibits the formation of pyocyanin. Pseudomonas
Agar P, in contrast, enhances the production of pyocyanin and inhibits
the formation of fluorescein. Both pigments diffuse from Pseudomonas
colonies into the medium in which they grow. Fluorescein elaborated
on Pseudomonas Agar F is a fluorescent yellow color, while pyocyanin elaborated on Pseudomonas Agar P is a blue color.
Some Pseudomonas strains elaborate both pigments, while others
Pseudomonas Agar P is used with Bacto Glycerol for detecting and

differentiating Pseudomonas aeruginosa from other pseudomonads
based on pyocyanin production.
Also Known As
Pseudomonas Agar F is known as Pseudomonas Agar Medium for
Detection of Fluorescein.
Pseudomonas Agar P is also known as Pseudomonas Agar Medium for
Detection of Pyocyanin.
ATCC 9027
ATCC 9027

Pseudomonas Agar F
Solution:
3.8% solution with 1% Glycerol, soluble
boiling. Solution is light to medium
opalescent.
Prepared Medium:
opalescent, without precipitate.
Reaction of 3.8%
Solution at 25C:
pH 7.0 0.2
Pseudomonas Agar P
Solution:
4.64% solution with 1% Glycerol,
upon boiling. Solution is light to medium
Prepared Medium:
Light to medium amber, slightly opalescent,
Reaction of 4.64%
Solution at 25C:
pH 7.0 0.2
Pseudomonas
Agar F
Pseudomonas
Agar P
Cultural Response
Prepare medium per label directions. Inoculate and incubate at 35 2C for 18-24 hours.
ORGANISM
Pseudomonas cepacia
ATCC
GROWTH
9027
27853*
25609
good
good
good
PIGMENT PRODUCTION
PSEUDOMONAS AGAR F PSEUDOMONAS AGAR P
greenish yellow
greenish yellow
no pigment
blue
blue
no pigment
*This culture is available as a Bactrol Disk and should be used as directed in Bactrol Disks Technical Information.
412
The Difco Manual
Section II
elaborate only one of the two. When Pseudomonas Agar F and

Pseudomonas Agar P are used together, they provide for easy and rapid
identification of most Pseudomonas strains as specified in the FDA
Bacteriological Analytical Manual.3

Pseudomonas Agar F
Tryptone and Proteose Peptone No. 3 provide carbon and nitrogen
sources required for good growth and also aid in fluorescein production.
Phosphate stimulates fluorescein production and has an inhibitory effect
on pyocyanin. Dipotassium Phosphate increases the phosphorus
content over that supplied by the peptones. Magnesium Sulfate provides
necessary cations for the activation of fluorescein production. Bacto
Agar is a solidifying agent. Glycerol, added during preparation of the
medium, is a carbon source.
Pseudomonas Agar P
Bacto Peptone provides the carbon and nitrogen sources required for
good growth. Glycerol is a carbon source. Magnesium Chloride and
Potassium Sulfate stimulate pyocyanin production. Bacto Agar is a
solidifying agent.
Pseudomonas Agar F
Formula Per Liter
10
10
1.5
1.5
15
g
g
g
g
g
20
1.4
10
15
g
g
g
g

Pseudomonas Agar P
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Magnesium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Potassium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Precautions
Storage
media at 2-8C.
Expiration Date
Procedure
Materials Provided
Pseudomonas Agar F
Pseudomonas Agar P
The Difco Manual
Glassware
Autoclave
Incubator (35C)
Tubes with closures
Bacto Glycerol
1. Suspend the medium in 1 liter distilled or deionized water containing
10 grams of Glycerol:
Pseudomonas Agar F - 38 grams;
Pseudomonas Agar P - 46.4 grams.

Test Procedure
1. Obtain the inoculum from a pure 18-24 hour culture of Pseudomonas.
2. Inoculate plates or agar slants by streaking the surface.
Formula
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . .
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Results
Examine colonies under ultraviolet light (Woods lamp).4 Take care
when using UV illumination because it may have a bactericidal effect.
Be sure there is good growth before placing the culture under UV light.
Pseudomonas Agar F: Positive result is indicated by a light, bright
greenish-yellow color diffusing into the agar with a fluorescent zone
surrounding the growth.
Pseudomonas Agar P: Positive result is indicated by a blue pigment
that diffuses into the agar.

1. Occasionally, a Pseudomonas culture is encountered that will
produce small amounts of pigment in the medium. When this
happens, a yellow-green color will appear on Pseudomonas Agar F
or a blue-green color on Pseudomonas Agar P. If a blue-green color
occurs on Pseudomonas Agar P, confirmation of the presence of
pyocyanin can be made by extraction with chloroform (CHCl3).4
2. The formation of nonpigmented colonies does not completely rule
out a Pseudomonas aeruginosa isolate.
3. A pyocyanin-producing Pseudomonas strain will usually also
produce fluorescein. It must, therefore, be differentiated from other
simple fluorescent pseudomonads by other means. Temperature can
be a determining factor as most other fluorescent strains will not
grow at 35C. Rather, they grow at 25-30C.4
References
1. King, E. O., M. K. Ward, and D. E. Raney. 1954. Two simple
Clin. Med. 44:301.
2. The United States Pharmacopeia. 1995. The United States pharmacopeia, 23rd ed. United States Pharmacopeial Convention,
Rockville, MD.
413
Pseudomonas Isolation Agar
Section II

4. MacFaddin, J. F. 1985. Media for isolation-cultivationidentification-maintenance medical bacteria, vol. 1. Williams
Packaging
Pseudomonas Agar F
100 g
500 g
0448-15
0448-17
Pseudomonas Agar P
500 g
0449-17
Bacto Pseudomonas Isolation Agar
Intended Use
Bacto Pseudomonas Isolation Agar is used with added glycerol in

isolating Pseudomonas and differentiating Pseudomonas aeruginosa
from other pseudomonads based on pigment formation.
Bacto Peptone provides the carbon and nitrogen necessary for bacterial
growth. Magnesium Chloride and Potassium Sulfate promote
production of pyocyanin. Irgasan, an antimicrobial agent, selectively
inhibits gram-positive and gram-negative bacteria other than
Pseudomonas spp. Bacto Agar is a solidifying agent. Glycerol serves
as an energy source and also helps to promote pyocyanin production.

Pseudomonas aeruginosa is an opportunistic pathogen that can infect
eyes, ears, burns and wounds.2,4 It is also a leading cause of hospital
acquired infections. Patients undergoing antibiotic therapy are especially
susceptible to infection by Pseudomonas aeruginosa.
Pseudomonas Isolation Agar is prepared according to a slight modification of the Medium A formulation of King, Ward and Raney.1 It is
especially useful for isolating Pseudomonas from clinical specimens
such as stools, wounds and urine. 2 Pseudomonas Isolation Agar
includes Irgasan, a potent broad spectrum antimicrobial that is not
active against Pseudomonas.3 As well as being selective, Pseudomonas
Isolation Agar is formulated to enhance the formation of the blue or
blue-green pyocyanin pigment by Pseudomonas aeruginosa. The
pigment diffuses into the medium surrounding growth.
Formula
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Potassium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Irgasan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.025
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.6
g
g
g
g
g

Uninoculated
plate
ATCC 27853

Dehydrated Appearance: Very light beige, homogeneous,
free-flowing.
Solution:
distilled or deionized water containing
2% glycerol. Solution is light to
Prepared Medium:
Light amber, slightly opalescent, firm.
Reaction of 4.5%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepare Pseudomonas Isolation Agar per label directions.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
APPEARANCE
Escherichia coli
25922* 1,000-2,000 marked to

complete inhibition
Pseudomonas aeruginosa 10145 100-1,000
good
green to blue-green
Pseudomonas aeruginosa 27853* 100-1,000
good
green to blue-green
414
The Difco Manual
Section II
Purple Broth Base & Purple Agar Base
Precautions
Test Procedure

1. Inoculate the medium using the streak plate method to obtain

isolated colonies.
Storage
Results

Examine for the presence of good growth. Pseudomonas aeruginosa

colonies will be green to blue-green with pigment that diffuses into
the medium.
Expiration Date
Procedure
Materials Provided

Glassware
Autoclave
Incubator (35C)
Glycerol
2. Add 20 ml of Glycerol.

Bacto Purple Broth Base

Bacto Purple Agar Base

1. Some strains of Pseudomonas aeruginosa may fail to produce
pyocyanin.6
2. Non-Pseudomonas aeruginosa strains that are not completely
inhibited on this medium may be encountered and must be
differentiated from Pseudomonas aeruginosa. Consult appropriate
references.2,5
References
1. King, E. O., M. K. Ward, and D. E. Raney. 1954. Two simple
& Clin. Med. 44(2):301-307.
2. Baron, E. J., and S. M. Finegold. 1990. Bailey & Scotts Diagnostic Microbiology, 8th ed. C.V. Mosby Company, St. Louis, MO.
3. Furia and Schenkel. 1968. Soap and Chemical specialties. January.
4. Gilligan, P. H. 1995. In P. R. Murray, E. J. Baron, M. A. Pfaller,
F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical
microbiology, 6th ed. American Society of Microbiology,
Washington, D.C.
6. Gaby, W. L., and E. Free. 1931. J. Bacteriol. 22:349.
Packaging
500 g
0927-17
Glycerol
100 g
500 g
0282-15
0282-17
Intended Use
Bacto Purple Broth Base and Purple Agar Base are used with added
carbohydrate in differentiating pure cultures of bacteria, particularly
of enteric organisms, based on fermentation reactions.

Purple Broth Base and Purple Agar Base are carbohydrate-free
fermentation media that are preferred by some bacteriologists because
of their slightly acid reaction (pH 6.8). When supplemented with carThe Difco Manual
bohydrates, these media are useful in obtaining accurate fermentation

reactions in the identification of Enterobacteriaceae and other
microorganisms. The concentration of carbohydrate generally employed
for testing the fermentation reactions of bacteria is 0.5 or 1%. Some
investigators prefer to use 1% rather than 0.5% to insure against reversion
of the reaction due to depletion of the carbohydrate by some microorganisms. Purple Broth Base with added carbohydrates is specified in
several standard methods.1,2,3,4

Proteose Peptone No. 3 and Beef Extract provide the carbon and nitrogen
sources required for good growth of a wide variety of organisms.
Sodium Chloride maintains the osmotic balance of the medium. Brom
Cresol Purple serves as an indicator, assuming a yellow color when
415
Section II
acid is produced during the fermentation of the added carbohydrate. In

Purple Agar Base, the Bacto Agar serves as a solidifying agent.
Formula

Bacto Brom Cresol Purple . . . . . . . . . . . . . . . . . . . . . . . . 0.02 g
Final pH at 25C 6.8 0.2
Purple Broth Base

Formulas Per Liter

Purple Broth Base
Dehydrated Appearance: Light tan with grayish-green cast,
Solution:
deionized water. Solution is purple,
Prepared Tubes:
Purple, clear to very slightly opalescent.
Reaction of 1.6%
Solution at 25C:
pH 6.8 0.2
Purple Agar Base
Dehydrated Appearance: Light tan with grayish-green cast,
Solution:
is purple, very slightly to slightly
opalescent.
Prepared Medium:
Purple, slightly opalescent.
Reaction of 3.1%
Solution at 25C:
pH 6.8 0.2
Uninoculated
tube
Typical positive
growth with
acid and gas reaction
Uninoculated
tube
Escherichia coli
ATCC 25922
Typical negative
growth with
acid and gas reaction
Cultural Response
Purple Broth Base
Prepare Purple Broth Base per label directions with 1.0% Dextrose.
Inoculate and incubate the tubes at 35 2C for 18-48 hours. A color
change to yellow indicates acid production, and the appearance of
bubbles in the inverted fermentation vial indicates gas production.
ORGANISM
Escherichia coli
Salmonella
typhimurium
ATCC
INOCULUM
CFU
RECOVERY
REACTION w/1% DEXTROSE

ACID
GAS
8750 100-1,000
25922* 100-1,000
good
good
14028* 100-1,000
good
Purple Agar Base

Prepare Purple Agar Base per label directions with 1.0% Dextrose.
Inoculate tubes with test organisms by stabbing the butt of the tube
and streaking the slant. Incubate at 35 2C for 18-48 hours. A color
change to yellow indicates acid production, and the appearance of
bubbles indicates gas production.
ORGANISM
Escherichia coli
Salmonella
typhimurium
ATCC
INOCULUM
CFU
RECOVERY
REACTION w/1% DEXTROSE

ACID
GAS
8750 1,000-2,000
25922* 1,000-2,000
good
good
14028* 1,000-2,000
good
Purple Agar Base
Escherichia coli
ATCC 25922
+ Dextrose
416
The Difco Manual
Section II
Purple Agar Base

Formulas Per Liter
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
Final pH at 25C 6.8 0.2
Precautions
Storage
Expiration Date
when stored as directed. Do not use a product if it fails to meet specifications for identity and performance.
Procedure
Materials Provided
Bacto Purple Broth Base
Bacto Purple Agar Base

Glassware
Autoclave
Incubator (35C)
Choice of carbohydrates
Fermentation vials (Purple Broth Base)
Purple Broth Base
1. Suspend 16 grams in 1 liter distilled or deionized water and heat to
boiling to dissolve completely.
To prepare fermentation broths, add 0.5-1% carbohydrate before
or after sterilization, depending on heat lability. Dispense into tubes
containing inverted fermentation vials.
Purple Agar Base
1. Suspend 31 grams in 1 liter distilled or deionized water and boil to
2. To prepare 0.5-1% carbohydrate fermentation agars, dissolve 5-10
grams of the desired carbohydrate in the basal medium prior to
sterilization.
OR
1. Dissolve 31 grams in 900 ml distilled or deionized water and
boil to dissolve completely.
The Difco Manual

3. Cool the basal medium to 45-50C
4. Aseptically add 100 ml sterile 5-10% carbohydrate solution (w/v).

Test Procedure
1. Inoculate tubes using a light inoculum from an 18-24 hour pure
culture. To inoculate Purple Broth Base tubes, use a loopful of
inoculum. For Purple Agar Base tubes, stab with an inoculating
needle to within 1/4 inch from the bottom of the tube.
2. Incubate tubes for 24-72 hours at 35 2C in an aerobic or anaerobic
atmosphere, depending on the organisms being tested.
3. Examine tubes daily for acid production and gas formation. Hold
negative tubes for a total of 30 days.
Results
A yellow color is a positive reaction for fermentation of the carbohydrate. Bubbles in the inverted fermentation vials are an indication of
gas production. Even the presence of a single bubble is significant to
record as positive.5

1. The addition of some carbohydrates to the media may result in an
acid reaction. In this case, it is suggested that the proper pH be
restored by adding sterile 0.1N sodium hydroxide dropwise.
2. Avoid excessive heating or prolonged heat exposure of media to
avoid hydrolysis of the carbohydrates.
3. Tubes should be tightly stoppered during the incubation period for
fermentation studies of the enteric group to avoid reversion caused
by rapid depletion of the carbohydrate(s).5
References
examination of dairy products. American Public Health Assoc.,
Washington, D.C.
Public Health Assoc., Washington, D.C.
4. Association of Official Analytical Chemists. 1995 Official
methods of analysis of AOAC International. AOAC International,
Arlington, VA.
Packaging
Purple Broth Base
500 g
0227-17
Purple Agar Base
500 g
0228-17
417
Purple Lactose Agar
Section II
Bacto Purple Lactose Agar
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Intended Use
Bacto Purple Lactose Agar is used for cultivating coliform organisms;
for differentiating lactose-fermenting from lactose-nonfermenting
organisms.

Purple Lactose Agar is a modification of Litmus Lactose Agar,
described by Wurtz.1 In Purple Lactose Agar, brom cresol purple
replaces litmus, which is less selective and less stable.
Purple Lactose Agar is used for detecting coliforms and in differential
studies based on the fermentation of lactose. Tests used to differentiate
Enterobacteriaceae determine the organisms ability to use a
carbohydrate with the production of acid metabolic end products.2
Colonies of lactose-fermenting organisms are differentiated from
lactose non-fermenters by a color change of the indicator from
blue-purple (alkaline) to yellow (acid). If gas is produced during
fermentation of the carbohydrate, bubbles will appear in the medium.2
g
g
g
g
Precautions
Storage
Expiration Date
Procedure

Beef Extract and Bacto Peptone provide the nitrogen, vitamins and
amino acids in Purple Lactose Agar. Bacto Lactose is the carbohydrate
used in the fermentation reaction. Bacto Agar is the solidifying agent.
Bacto Brom Cresol Purple is the pH indicator.
Formula
Purple Lactose Agar
Formula Per Liter
Materials Provided
Purple Lactose Agar

Glassware
Autoclave
Incubator

Dehydrated Appearance: Light beige with greenish cast,
free-flowing and homogeneous.
2.8% Solution:
Soluble in distilled or deionized water on
boiling. Solution is purple, clear to very
Reaction of
2.8% Solution:
pH 6.8 0.1 at 25C
Cultural Response
Inoculate the agar slant by stabbing the butt and streaking with an
inoculating needle. Incubate tubes at 35 2C for 18-48 hours.
Acid production is indicated by a yellow color.
ORGANISM
Escherichia coli
Salmonella typhi
ATCC
GROWTH
ACID
(YELLOW)
GAS
13048*
25922*
19430
25923*
good
good
good
good
+
+
+
+
418
Uninoculated
tube
Escherichia coli
ATCC 25922
Salmonella typhi
ATCC 19430
The Difco Manual
Section II
Pyridoxine Y Medium
1.
2.
3.
4.

Dispense into sterile tubes.
2. Medium is slightly acid (pH 6.8) and positive reactions may be

slower than with phenol red carbohydrate medium.5
References
1. Wurtz. 1897. Technique Bacteriologique, Masson, Paris.

St. Louis, MO.
handbook, vol 1. American Society for Microbiology, Washington, D.C.
5. MacFaddin, J. D. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, vol 1. Williams
Packaging

Purple Lactose Agar

Test Procedure
For a complete discussion on the expected reactions of specific
Enterobacteriaceae species, refer to Manual of Clinical Microbiology,3
Clinical Microbiology Procedures Handbook 4 and Bailey & Scotts
Diagnostic Microbiology.2
Results
Bacto Pyridoxine Y Medium
Intended Use
Bacto Pyridoxine Y Medium is used for determining pyridoxine

500 g
0082-17

Pyridoxine Y Medium is patterned after the formulation of Campling
and Nixon,1 and modified by Hurley2 and Parrish, Loy and Kline.3 This
medium is used in the microbiological assay of pyridoxine using
Saccharomyces cerevisiae ATCC 9080 (Saccharomyces uvarum) as
the test organism.
Pyridoxine Y Medium is free from pyridoxine, but contains all other

nutrients and vitamins essential for the growth of S. cerevisiae
ATCC 9080. The addition of pyridoxine in specified increasing
concentrations gives a growth response that can be measured
turbidimetrically or titrimetrically.
Dehydrated Appearance: White to off-white, fine, free-flowing,

homogeneous.
Solution:
2.65% (single strength) 5.3% (double
strength) solution, soluble in distilled
or deionized water upon boiling for
2-3 minutes. Solution is almost
colorless to very light amber, clear,
Prepared Medium:
Single strength solution is colorless
to very light amber, clear, may have
Reaction of 2.65%
Solution at 25C:
pH 4.4 0.2
Cultural Response
Prepare Pyridoxine Y Medium per label directions. This medium
should support the growth of Saccharomyces cerevisiae ATCC
9080 when prepared in single strength and supplemented with a
mixture containing 1 ng per ml each of pyridoxal hydrochloride,
pyridoxamine hydrochloride and pyridoxine hydrochloride.
The Difco Manual
Formula
Pyridoxine Y Medium
Formula Per Liter
L-Asparagine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
L-Histidine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . 20
DL-Methionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
DL-Tryptophane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
DL-Isoleucine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
DL-Valine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Nicotinic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
Biotin Salt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
g
mg
mg
mg
mg
mg
g
g
g
g
mg
419
Pyridoxine Y Medium
Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Inositol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Boric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Magnesium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Potassium Iodide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Ammonium Molybdate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Copper Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Zinc Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500
Section II
g
mg
g
g
g
g
g
g
g
g
g
g
g
Precautions
other chemicals must be used. Glassware is heated to 250C for
at least 1 hour to burn off any organic residues that might be
present.
throughout assay.
closed.
Storage
Expiration Date
Procedure
Materials Provided
Pyridoxine Y Medium

Glassware
Autoclave
Stock culture of Saccharomyces cerevisiae ATCC 9080
Sterile tubes
420

Pyridoxal HCl
Pyridoxamine 2 HCl
Pyridoxine HCl
Shaker (100 rpm)
Incubator (25-30C)
Centrifuge
Ethyl alcohol
Spectrophotometer
1.
2.
3.
4.
5.
6.
Dissolve 5.3 grams in 100 ml distilled or deionized water.

Boil 2- 3 minutes to dissolve completely.
Dispense 5 ml amounts into flasks, evenly dispersing the precipitate.
Steam at 100C for 10 minutes.

Assay samples are prepared according to specific assay procedures.
For assays, the samples should be diluted to approximately the same
Test Procedure
Stock cultures of S. cerevisiae ATCC 9080 are carried on Lactobacilli
Agar AOAC. Following incubation at 25-30C (held constant
within 0.5C) for 18-24 hours, store the cultures in the dark at 2-8C.
Prepare fresh slant cultures every week. Do not use stock cultures for
preparing the inoculum if more than one week old. Inoculum for assay
is prepared by subculturing a stock culture of S. cerevisiae ATCC 9080
into a tube (10 ml) of single strength Pyridoxine Y Medium containing
1 ng per ml each of pyridoxal hydrochloride, pyridoxamine
dihydrochloride and pyridoxine hydrochloride. After 18-24 hours
incubation at 25-30C (held constant within 0.5C), centrifuge the
cells under aseptic conditions and decant the liquid supernatant. Wash
the cells 3x with 10 ml sterile 0.85% saline. After the third wash,
resuspend in 10 ml sterile single strength medium and adjust to a
turbidity of 45-50% transmittance when read on the spectrophotometer
at 660 nm.
It is essential that a standard curve be set up for each separate assay.
Conditions of steaming and temperature of incubation which influence the standard curve readings cannot always be duplicated. Obtain
the standard curve by using pyridoxine hydrochloride at levels of 0, 1,
2, 4, 6, 8 and 10 ng per flask (10 ml).
The concentrations of pyridoxine hydrochloride required for the
preparation of the standard curve may be prepared as follows:
A. Dissolve 50 mg dried pyridoxine hydrochloride in about 100 ml in
HCL solution.
B. Dilute 500 ml with in HCL.
C. Further dilute by adding 2 ml to 998 ml distilled water to make a
stock solution containing 200 ng pyridoxine hydrochloride per ml.
Prepare the stock solution fresh daily.
To make the standard solution, dilute 1 ml of stock solution with 99 ml
distilled water, to make a solution containing 2 ng pyridoxine
hydrochloride per ml. Use 0.0, 0.05, 1, 2, 3, 4 and 5 ml per assay tube.
The Difco Manual
Section II
R2A Agar
Following inoculation, incubate the tubes on a shaker (about 100 rpm)

at 25-30C for 22 hours. Steam in the autoclave for 5 minutes to stop
growth. Measure the growth turbidimetrically using a spectrophotometer
at any specific wavelength between 540 and 660 nm.
Results
1. Prepare a standard concentration response curve by plotting the response
readings against the amount of standard in each tube, disk or cup.

response.
References
1. Campling, and Nixon. 1954. J. Physiol. 126:71.
2. Hurley. 1960. J. AOAC. 43:43.
3. Parrish, Loy, and Kline. 1956. J. AOAC. 39:157.
Packaging
Pyridoxine Y Medium

grown and maintained on a medium recommended for this purpose.
*Store at 2-8C
Bacto R2A Agar
Intended Use
Bacto R2A Agar is used for enumerating heterotrophic organisms in
treated potable water.

R2A Agar was developed by Reasoner and Geldreich1 for bacteriological
plate counts of treated potable water. A low nutrient medium, such as
R2A Agar, in combination with a lower incubation temperature and
longer incubation time stimulates the growth of stressed and
chlorine-tolerant bacteria.1 Nutritionally rich media, such as Tryptone
Glucose Yeast Extract Agar (TGEA) or Plate Count Agar (PCA),

homogeneous.
Solution:
Solution is light amber in color,
slightly opalescent, with a slight
precipitate.
Prepared Plates:
Light amber in color, slightly
opalescent, with a slight precipitate.
Reaction of 1.82%
Solution at 25C:
7.2 0.2.
Cultural Response
Prepare R2A Agar per label directions. Inoculate with tap water
samples using the streak plate method and/or the membrane
filter method. Incubate at 35 2C for 40-72 hours. Recovery
is typical compared to an approved control lot and greater than
parallel plates of Plate Count Agar.
The Difco Manual
100 g
0951-15*
support the growth of fast-growing bacteria but may suppress slow

growing or stressed bacteria found in treated water. When compared with
TGEA and PCA, R2A Agar has been reported to improve the recovery
of stressed and chlorine-tolerant bacteria from drinking water systems.2,3,4
R2A Agar is recommended in Standard Methods for the Examination
of Water and Wastewater5 for pour plate, spread plate and membrane
filter methods for heterotrophic plate counts.

Yeast Extract provides a source of trace elements and vitamins. Proteose
Peptone No. 3 and Casamino Acids provide nitrogen, vitamins, amino
acids, carbon and minerals. Dextrose serves as a carbon source. Soluble
Starch aids in the recovery of injured organisms by absorbing toxic
metabolic by-products. Sodium Pyruvate increases the recovery of
stressed cells. Potassium Phosphate is used to balance the pH and provide
phosphate. Magnesium Sulfate is a source of divalent cations and sulfate.
Formula
R2A Agar
Formula Per Liter
Bacto Casamino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Soluble Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Sodium Pyruvate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g
g
g
Precautions
421
R2A Agar
Section II
Storage

1. R2A Agar is intended for use only with treated potable water since
it is recommended for compromised bacteria.
2. Use of the pour plate method is discouraged because recovery of
stressed bacteria may be compromised by the heat shock (44-46C)
and low oxygen tension that are part of the procedure.6,7
3. Incubation time longer than indicated above may be necessary to
recover additional slow-growing bacteria.
4. R2A Agar performs best with the spread plate technique; however,
that procedure is limited to a small sample volume.
5. Fast-growing bacteria may produce smaller size colonies on
R2A Agar than on nutritionally rich media.
6. R2A Agar is a low nutrient medium intended for culturing
compromised microorganisms. Good growth of standard, healthy
control organisms does not necessarily reflect the ability of the
medium to recover stressed organisms. Each new lot of medium
should be performance tested against a previous lot of R2A Agar
using tap water.
Expiration Date
Procedure
Materials Provided
R2A Agar

Autoclave
Petri dishes
Membrane filter equipment and filters
Dilution blanks
Pipettes or glass rods
Incubator (20, 28 or 35C)
Colony counter
References

Water samples should be collected as described in Standard Methods
for the Examination of Water and Wastewater, Section 9060A.5
To minimize changes in bacterial population, water samples should be
tested as soon as possible, but at least within six hours of collection if
the sample has not been refrigerated or within 30 hours if refrigerated.
Test Procedure
1. Prepare test dilutions for heterotrophic plate count.
2. Plate the test sample and dilutions by the spread plate, pour plate or
membrane filter method. Do not exceed 1 ml of sample or dilution
per spread or pour plate. The volume of test sample to be filtered for
the membrane filter technique will vary.
3. Maintain proper humidity during prolonged incubation:
INCUBATION
TEMPERATURE
MINIMUM INCUBATION
TIME 3
OPTIMAL INCUBATION
TIME 3
35C
20 or 28C
72 hours
5 days
5-7 days
7 days
Results
Count colonies on spread or pour plates demonstrating 30-300
colonies per plate or 20-200 colonies when using the membrane
filter method. Compute bacterial count per ml of sample by multiplying
the average number of colonies per plate by the reciprocal of the
appropriate dilution.
1. Reasoner, D. J., and E. E. Geldreich. 1979. A new medium for

the enumeration and subculture of bacteria from potable water.
Abstracts of the Annual Meeting of the American Society for
Microbiology 79th Meeting, Paper No. N7.
2. Fiksdal, L., E. A. Vik, A. Mills, and T. Staley. 1982. Non-standard
methods for enumerating bacteria in drinking water. Journal
AWWA. 74:313-318.
3. Kelly, A. J., C. A. Justice, and L. A. Nagy. 1983. Predominance
of chlorine tolerant bacteria in drinking water systems. Abstracts
of the Annual Meeting of the American Society for Microbiology
79th Meeting, Paper No. Q122.
4. Means, E. G., L. Hanami, H. F. Ridgway, and B. H. Olson.
1981. Evaluating mediums and plating techniques for enumerating bacteria in water distribution systems. Journal AWWA.
53:585-590.
6. Van Soestberger, A. A., and C. H. Lee. 1969. Pour plates or streak
plates? Appl. Microbiol. 18:1092.
7. Klein, D. A., and S. Wu. 1974. Stress: a factor to be considered
in heterotrophic microorganism enumeration from aquatic
environments. Appl. Microbiol. 27:429.
Packaging
R2A Agar
100 g
500 g
2 kg
1826-15
1826-17
1826-07
Report counts as colony forming units (CFU) per ml and report variables
of incubation such as temperature and length of time.
422
The Difco Manual
Section II
Raka-Ray No. 3 Broth & Raka-Ray No. 3 Medium
Bacto Raka-Ray No. 3 Broth

Bacto Raka-Ray No. 3 Medium
Intended Use
Bacto Raka-Ray No. 3 Broth and Medium are recommended for the
isolation of lactic acid bacteria encountered in beer and the brewing
process.

Spoilage organisms are often seriously detrimental to beer flavor.
Lactic acid bacteria including lactobacilli and pediococci which can
cause spoilage are physiologically very diverse..
Raka-Ray No. 3 Broth and Medium were developed from a formulation
suggested by Saha, Sondag, and Middlekauff1 who tested a range of
ingredients for their ability to stimulate growth of lactic acid bacteria.
Tween 80, liver extract, maltose, N-acetyl glucosamine and yeast
extract were found to stimulate growth. Tomato juice, free fatty acids
and lyophilized beer solids (all of which are found in several media
formulations for lactic acid bacteria) were inhibitory.
In comparative studies using in-process beer samples, Raka-Ray
media gave higher colony counts for lactobacilli than Tomato Juice

Raka-Ray No. 3 Broth
Solution:
deionized water with 1% Tween 80.
Solution is medium to dark amber, clear.
Reaction of 5.89%
Solution at 25C:
pH 5.4 0.2
Raka-Ray No. 3 Medium
Solution:
deionized water with 1% Tween 80
upon boiling. Solution is medium to dark
Reaction of 7.49%
Solution at 25C:
pH 5.4 0.2
Cultural Response
Prepare Raka-Ray No. 3 Broth or Medium with selective agents
per label directions. Inoculate and incubate anaerobically at
27-30C for 18-48 hours.
ORGANISM
Escherichia coli
Lactobacillus brevis
Lactobacillus buchneri
ATCC
INOCULUM
CFU
GROWTH
25922* 1,000-2,000 none to poor

367
30-300
good
11307
30-300
good
8042
30-300
good
Agar, W-L Differential Agar and Universal Beer Agar, with larger
colonies developing after 2-4 days of anaerobic incubation1.2
Raka-Ray No. 3 Medium yields larger lactic acid bacterial colonies
than Universal Beer Agar.3 Raka-Ray No. 3 Medium also suppressed
the growth of non-lactic acid, facultative bacteria such as Aerobacter
aerogenes and Flavobacterium proteus that are often associated with
lactic beer spoilage organisms.3
Raka-Ray No. 3 Medium is also recommended by the European
Brewing Congress Analytical Microbiologica for enumeration of
lactobacilli and pediococci4. The broth and agar may be made more
selective by the addition of 3 grams of 2-phenylethanol and 3 mg of
cycloheximide (Actidione) dissolved in a small quantity of acetone per
liter of medium before autoclaving. Yeasts and gram-negative bacteria
are suppressed, facilitating enumeration of the lactic bacterial flora.
Polysorbate 80, Liver Digest, Maltose and other sugars, N-Acetyl
Glucosamine and Yeast Extract stimulate the growth of lactobacilli.
The optional addition of cycloheximide provides increased selectivity
against yeasts and gam-negative bacteria.
Formula
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Liver Digest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Maltose Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Fructose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Betaine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Di-ammonium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
L-Aspartic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
Magnesium Sulphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.98
Manganese Sulphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.42
N-Acetyl Glucosamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Potassium Glutamate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
g
g
g
g
g
g
g
g
g
g
g
g
g
g

Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Liver Digest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Maltose Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Fructose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Betaine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Di-ammonium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Potassium Aspartate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
Magnesium Sulphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.98
Manganese Sulphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.42
N-Acetyl Glucosamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Potassium Glutamate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g

The Difco Manual
423
Rappaport-Vassiliadis Medium Semisolid
Precautions
Storage
Section II
2-phenylethanol and 3 mg cycloheximide (Actidione) per liter

before autoclaving. Do not overheat.
5. Pour 15-20 ml of Raka-Ray Medium into each Petri dish and allow
to solidify.

Expiration Date
Test Procedure

Overlay Technique for Enumeration of Lactic Acid Bacteria

1. Inoculate 0.1 ml of the beer sample onto well-dried plates
containing 15-20 ml Raka-Ray No. 3 Medium. Five replicates of
each sample are recommended.
2. Spread over the surface of the medium using a sterile glass rod.
3. Overlay the surface with 4 ml of the molten sterilized medium
cooled to 50C.
4. Incubate plates at 27-30C in an anaerobic (H2/CO2) atmosphere.
Results

Tween 80
2-phenylethanol
Actidione
Acetone
Autoclave
Waterbath (50C)
Petri dishes
Sterile tubes
Anaerobic chamber
Lactobacilli are visible after 48 hours incubation as smooth, moist

colonies that are 1 mm in diameter. Incubate the medium for a total
of 7 days to allow development of slow-growing Pediococcus strains.
If the number of colonies on each plate exceeds 300, the sample should
be diluted 1:10 in sterile physiological saline and retested.
Procedure
Materials Provided
1. Suspend 58.9 grams in 1 liter of distilled or deionized water containing 10 ml Tween 80. Dispense into tubes with closures.
1. Suspend 74.9 grams in 1 liter of distilled or deionized water
containing 10 ml Tween 80.
3. To increase the selectivity of the medium, add 3 grams of
References
1. Saha, R. B., R. J. Sondag, and J. E. Middlekauff. 1974. An
improved medium for the selective culturing of lactic acid bacteria.
Proceedings of the American Society of Brewing Chemists.
9th Congress, 9-10.
2. VanKeer, C., L. Van Melkebeke, W. Vertriest, G. Hoozee, and
E. Van Schoonenberghe. 1983. Growth of Lactobacillus species
on different media. J. Inst. of Brewing 89:360-363.
3. Report of the Technical Subcommittee. 1976. Microbiological
Controls. J. Am. Soc. of Brewing Chemists 34:93-94.
4. European Brewing Congress Analytica Microbiologica. 1981.
J. Inst. of Brewing 87:314.
Packaging
500 g
500 g
1865-17
1867-17

Bacto Rappaport-Vassiliadis (MSRV) Medium Semisolid
Modification . Novobiocin Antimicrobic Supplement
Intended Use
Bacto Rappaport-Vassiliadis (MSRV) Medium Semisolid Modification

is is used with Bacto Novobiocin Antimicrobic Supplement in rapidly
detecting motile Salmonella in feces and food products.
Rappaport-Vassiliadis (MSRV) Medium Semisolid Modification is a

modification of Rappaport-Vassiliadis enrichment broth for detecting
motile Salmonella in feces and food products. The original work on
424
The Difco Manual
Section II

Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.34
Magnesium Chloride Anhydrous . . . . . . . . . . . . . . . . . . 10.93
Malachite Green Oxalate . . . . . . . . . . . . . . . . . . . . . . . . 0.037
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7
MSRV medium showed that a semi-solid medium in Petri dishes could

be used as a rapid and sensitive means of isolating motile Salmonella
from food products following pre-enrichment or selective enrichment.1,2
The semisolid medium allows motility to be detected as halos of growth
around the original point of inoculation.
g
g
g
g
g
The medium is recommended by the European Chocolate Manufacturers

Association. A collaborative study performed with support of the
American Cocoa Research Institute (ACRI) and the Canadian Chocolate
Manufacturers Association (CCMA) resulted in first action adoption of
the MSRV method by AOAC International.3

Formula per 10 ml
Sodium Novobiocin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 mg
Precautions
MSRV Medium may be used as a plating medium for isolating

Salmonella spp. (other than S. typhi and S. paratyphi type A) from
stool specimens with high sensitivity and specificity.4,5

2. Rappaport-Vassiliadis (MSRV) Medium Semisolid Modification
TARGET ORGAN(S): Nerves, Kidneys.

Rappaport-Vassiliadis (MSRV) Medium Semisolid Modification
contains Tryptose and Casein Hydrolysate as carbon and nitrogen
sources for general growth requirements. Magnesium Chloride raises
the osmotic pressure in the medium. Novobiocin (Novobiocin
Antimicrobic Supplement) and Malachite Green inhibit organisms
other than Salmonella. The low pH of the medium combined with the
Novobiocin, Malachite Green and Magnesium Chloride select for highly
resistant Salmonella spp. Bacto Agar is the solidifying agent.

HARMFUL. HARMFUL BY INHALATION AND IF SWALLOWED. (EC) MAY CAUSE ALLERGIC EYE, RESPIRATORY
SYSTEM AND SKIN REACTION. Avoid contact with skin and
Formula
Formula per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.59 g
Casein Hydrolysate (Acid) . . . . . . . . . . . . . . . . . . . . . . . . 4.59 g
Uninoculated
plate
ATCC 14028

Dehydrated Appearance: Pale green, homogeneous, free-flowing.
Solution:
deionized water upon boiling. Blue, clear
Prepared Medium:
Blue, slightly opalescent, no
significant precipitate, semisolid.
Reaction of 3.16%
Solution at 25C:
pH 5.2 0.2
Cultural Response
Prepare Rappaport-Vassiliadis (MSRV) Medium Semisolid
Modification per label directions. Inoculate using three drops
(approximately 0.1 ml) at discreet locations on the plate and incubate
at 42 0.5C for 18-24 hours.
ORGANISM
ATCC
INOCULUM
CFU
Citrobacter freundii
8090
1,000-2,000
27853* 1,000-2,000
14028* 100-1,000
Salmonella senftenberg (NCTC) 10384
100-1,000
GROWTH
HALO/
MOTILITY
marked to
complete inhibition
none
good
good
+
+
The Difco Manual
425

Storage
Store Rappaport-Vassiliadis (MSRV) Medium Semisolid Modification
below 30C. The powder is very hygroscopic. Keep container
tightly closed.
Store Novobiocin Antimicrobic Supplement at 2-8C.
Expiration Date
Section II
Results
Positive: Growth of migrated cells is visible as a gray-white, turbid
zone extending out from the inoculated drop. Test sample
is considered presumptively positive for motile Salmonella.
Negative: Medium remains blue-green around the drops, with no
gray-white, turbid zone extending out from the drop. Test
sample is considered negative for motile Salmonella.
To confirm a presumptive identification of Salmonella:3
Rapid serologic confirmation
1. Inoculate M Broth with growth from migration edge on MSRV plate.
2. Incubate at 35C for 4 to 6 hours (until turbid). M-broth culture
can be held for up to 24 hours at 35C.
3. Test with Salmonella O and H antisera.

Culture confirmation
1. Transfer a loopful of growth from the migration edge on MSRV
plate onto Hektoen Enteric Agar and streak for isolation.
3. From colonies of Hektoen agar that show colony appearance
typical of Salmonella (green colonies with black centers), perform
biochemical tests to confirm the identification.
Procedure
Materials Provided
Flask with closure

Autoclave
Incubator (35C)
Waterbath
The combination of malachite green, magnesium chloride and a low

pH may inhibit certain Salmonella, such as S. typhi and S. choleraesuis.
Isolation techniques should include a variety of enrichment broths
and isolation media.
1. DeSmedt, J. M., R. Bolderdijk, H. Rappold, and

D. Lautenschlaeger. 1986. Rapid Salmonella detection in foods
by motility enrichment on a modified semi-solid RappaportVassiliadis medium. J. Food Prot. 49:510-514.
2. DeSmedt, J. M., and R. Bolderdijk. 1987. Dynamics of
Salmonella isolation with modified semi-solid Rappaport-Vassiliadis
medium. J. Food Prot. 50:658- 661.
3. DeSmedt, J. M., R. Bolderdijk, and J. Milas. 1994. Salmonella
detection in cocoa and chocolate by motility enrichment on
modified semi-solid Rappaport-Vassiliadis medium: a collaborative
study. J. AOAC Int. 77:365-373.
4. Dusch, H., and M. Altwegg. 1995. Evaluation of five new plating
media for isolation of Salmonella sp. J. Clin. Micro. 33:802-804.
5. Aspinall, S. T., M. A. Hindle, and D. N. Hutchinson. 1992.
Improved isolation of Salmonellae from faeces using a semi-solid
Rappaport-Vassiliadis Medium. Eur. J. Clin. Microbiol. Infect.
Dis. 11:936-939.
and R. M. Amaguana. 1995. Salmonella. p. 5.01-5.20. In FDA
Gaithersburg, MD.
References
1. Suspend 31.6 grams of Rappaport-Vassiliadis (MSRV) Medium

Semisolid Modification in 1 liter distilled or deionized water.
2. Heat to boiling to dissolve completely. Do not autoclave.
3. Cool to 50C.
4. Aseptically add 10 ml Novobiocin Antimicrobic Supplement,
rehydrated per label instructions with sterile distilled or deionized
water. Mix well.

Test Procedure3,6
Pre-enrichment
1. Add 25 grams of cocoa or chocolate to 225 ml of sterile reconstituted nonfat dry milk with 0.45 ml of a 1% aqueous brilliant green
dye solution; mix well.6
2. Incubate at 35C for 20 2 hours.3
Selective Enrichment3
3. Inoculate 10 ml Tetrathionate Broth (prewarmed to 35C) with
1 ml of the pre-enrichment culture.
4. Incubate at 35C for 8 0.5 hours.
3
Motility Enrichment on MSRV

5. After selective enrichment incubation, mix the broth culture.
Inoculate 3 drops at separate spots on an MSRV plate.
6. Incubate at 42 0.5C for 16 0.5 hours.
426
Packaging
Rappaport-Vassiliadis (MSRV)
Medium Semisolid Modification
500 g
1868-17
6 x 10 ml
3197-60*
*Store at 2-8%C
The Difco Manual
Section II
Rappaport-Vassiliadis R10 Broth
Bacto Rappaport-Vassiliadis R10 Broth
Intended Use
Bacto Rappaport-Vassiliadis R10 Broth is used for selectively enriching

Salmonella from meat and dairy products, feces and sewage polluted
water.
Rappaport-Vassiliadis R10 Broth is also known as RV Enrichment

Broth or R10 Broth.
Rappaport-Vassiliadis R10 Broth contains Tryptone as carbon and

nitrogen sources for general growth requirements. Magnesium
Chloride raises the osmotic pressure in the medium. Malachite Green
is inhibitory to organisms other than salmonellae. The low pH of the
medium (5.1 0.2 at 25C), combined with the presence of malachite
green and magnesium chloride, select for the highly resistant
Salmonella spp.
Formula
Rappaport et al1 formulated an enrichment medium for Salmonella that

was modified by Vassiliadis et al.2 The Rappaport formulation,
designated R25/37C, recommended incubation at 37C; the
Vassiliadis modification, designated R10/43C, had a reduced level of
malachite green and recommended incubation at 43C. Later work by
Peterz showed that incubation at 41.5 0.5C for 24 hours improved
recovery of Salmonella spp.3

Formula Per Liter
Rappaport-Vassiliadis R10 Broth is a selective enrichment medium that

is used following pre-enrichment of the specimen in a suitable
pre-enrichment medium. It has gained approval for use in analyzing
milk and milk products,4 raw flesh foods, highly contaminated foods
and animal feeds.5,6
Also Known As
This medium selectively enriches for salmonellae because bacteria,

including other intestinal bacteria, are typically resistant to or inhibited
by malachite green, high osmotic pressure and/or low pH. S. typhi and
S. choleraesuis are sensitive to malachite green and may be inhibited.

Dehydrated Appearance: Pale green to green, free-flowing,
homogeneous.
Solution:
or deionized water upon gentle
heating; blue, clear.
Reaction of 2.66%
Solution at 25C:
pH 5.1 0.2
Cultural Response
Prepare Rappaport-Vassiliadis R10 Broth per label directions.
Inoculate and incubate at 41.5 0.5C for 18-48 hours.
Subculture to Brilliant Green Agar and incubate at 35 2C
for 18-24 hours.
ORGANISM
ATCC
INOCULUM
CFU
Escherichia coli
25922* 1,000-2,000
13076
14028*
100-1,000
100-1,000
RECOVERY
markedly
inhibited
good
good
The Difco Manual
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.54

Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2
Magnesium Chloride Anhydrous . . . . . . . . . . . . . . . . . . . 13.4
Malachite Green Oxalate . . . . . . . . . . . . . . . . . . . . . . . . 0.036
g
g
g
g
g
Precautions
TARGET ORGAN(S): Nerves, Kidneys.
air. If not breathing, give artificial respiration. If breathing is difficult, give oxygen. Seek medical advice. If swallowed seek medical advice immediately and show this container or label.
Storage
Expiration Date
stored as directed. Do not use if it fails to meet specifications for identity and performance.
Procedure
Materials Provided

Flask with closure
Containers suitable for 10 ml aliquots
Autoclave
427
Reinforced Clostridial Medium
1. Suspend 26.6 grams in 1 liter distilled or deionized water. Heat
gently to dissolve.
2. Dispense 10 ml amounts into suitable containers. Sterilize at
115-116C for 15 minutes.
Specimen Preparation
Consult an appropriate reference for specific instructions related to the
type of product being tested.4,5,6
Test Procedure
Water and Sewage Samples
For isolating Salmonella (other than S. typhi) from water and associated
materials such as sewage liquor, sewage sludge, digested sludge and
pressed sludge cake.
1. Concentrate the sample by filtering it through a plug of sterile
absorbent cottonwool inserted in the neck of a large sterile funnel
or through a Whatman No. 17 absorbent pad.
Pre-enrichment
2. Using aseptic technique, transfer the cottonwool plug or the pad to
100 ml of a suitable pre-enrichment medium such as Buffered
Peptone Water.
3. Incubate at 37 0.5C for 18-24 hours.
Selective Enrichment
4. Inoculate 10 ml of Rappaport-Vassiliadis R10 Broth with 0.1 ml of
the pre-enrichment culture. Inoculate 10 ml of Muller-Kauffman
Tetrathionate Broth with 1 ml of the pre-enrichment culture.
5. Incubate Rappaport-Vassiliadis R10 Broth at 41.5 0.5C. Incubate
Muller- Kauffman Tetrathionate Broth at 42 1C for 48 hours.
Results
6. After incubation, subculture both selective enrichment broths to
Brilliant Green Agar and XLD Agar. Incubate at 35 2C for
18-24 hours.
7. Examine for typical Salmonella colonies. Confirm identification
of isolates by biochemical and serologic tests.
Milk and Foods
For isolating Salmonella (other than S. typhi) from milk and milk products,4 raw flesh foods, highly contaminated foods and animal feeds.5,6
Pre-enrichment
1. Add 25 grams or a 25 ml sample of the specimen to 225 ml of
pre-enrichment medium. Consult appropriate references for the
type of product being tested.4,5,6
2. Incubate at 35C for 24 2 hours5,6 or at 37C for 16-20 hours,4
depending on the referenced procedure being followed.
Section II
Selective Enrichment
1. Inoculate 10 ml of Rappaport-Vassiliadis R10 Broth with 0.1 ml of
pre-enrichment culture. Inoculate 10 ml of another selective
enrichment medium such as Tetrathionate Broth or Selenite
Cystine Broth with 1 ml of the pre-enrichment culture.4,5,6
2. Incubate Rappaport-Vassiliadis R10 Broth at 41.5 0.5C4 for
24 2 hours. Incubate the other selective enrichment broths
appropriately.
Results
1. After incubation, subculture Rappaport-Vassiliadis R10 Broth and
the other selective enrichment broths to selective agar media and
incubate at 35 2C for 24 2 hours.4,5
2. Examine for typical Salmonella colonies. Confirm identification
of isolates by biochemical and serologic tests.

The combined inhibitory factors of this medium (malachite green,
magnesium chloride, low pH) may inhibit certain Salmonella, such as
S. typhi and S. choleraesuis. Isolation techniques should include a
variety of enrichment broths and isolation media.
References
1. Rappaport, F., N. Konforti, and B. Navon. 1956. A new enrichment medium for certain salmonellae. J. Clin. Pathol. 9:261-266.
2. Vassiliadis, P., D. Trichopoulos, A. Kalandidi, and
E. Xirouchaki. 1978. Isolation of salmonellae from sewage with
a new procedure of enrichment. J. Appl. Bacteriol. 44:233-239.
3. Peterz, M., C. Wiberg, and P. Norberg. 1989. The effect of
incubation temperature and magnesium chloride concentration on
growth of salmonella in home-made and commercially available
dehydrated Rappaport-Vassiliadis broths. J. Appl. Bacteriol.
66:523-528.
4. International Dairy Federation. 1995. Milk and milk products:
detection of Salmonella. IDF Standard 93B:1005. Brussels,
Belgium.
and R. M. Amaguana. 1995. Salmonella. p. 5.01-5.20. In FDA
Gaithersburg, MD.
6. Andrews, W. H. (ed.). 1995. Microbial methods, p.1-119. In
Official methods of analysis of AOAC International, 16th ed.
Packaging
500 g
1858-17
Bacto Reinforced Clostridial Medium
Intended Use
Bacto Reinforced Clostridial Medium is used for cultivating and

enumerating clostridia, other anaerobes, and other species of bacteria
from foods and clinical specimens.
Reinforced Clostridial Medium is a semisolid medium formulated by

Hirsch and Grinstead.1 Their work demonstrated that the medium
outperformed other media in supporting growth of clostridia from small
inocula and produced higher viable cell counts.1 Barnes and Ingram2
428
The Difco Manual
Section II
used the medium to dilute vegetative cells of Clostridium perfringens.

Barnes et al3 used a solid (agar) version of the medium to enumerate
clostridia in food. The medium is a non-selective enrichment
medium and grows various anaerobic and facultative bacteria when
incubated anaerobically.4
Precautions
Storage

Reinforced Clostridial Agar contains Tryptose and Beef Extract as
Dextrose is the carbohydrate source. Sodium Chloride maintains the
osmotic balance. In low concentrations, Soluble Starch detoxifies
metabolic by-products. Cysteine Hydrochloride is the reducing agent.
Sodium Acetate acts as a buffer. The small amount of Bacto Agar makes
the medium semisolid.

Expiration Date
Procedure
Materials Provided
Formula

Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Soluble Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Cysteine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
g
g
g
g
g
g
g
g
g
Glassware
Autoclave
Incubator (35C, anaerobic conditions)

Test Procedure
Dehydrated Appearance: Light tan, free-flowing,
homogeneous.
Solution:
is medium amber, slightly opalescent.
Upon cooling medium becomes
more opalescent.
Reaction of 3.8%
Solutionat 25C:
pH 6.8 0.2
Cultural Response
Prepare Reinforced Clostridial Medium per label directions.
ORGANISM
Clostridium botulinum
ATCC
INOCULUM
CFU
GROWTH
23745
25763
13124*
100-1,000
100-1,000
100-1,000
good
good
good
The Difco Manual
Results
References
1. Hirsch, A., and E. Grinstead. 1954. Methods for the growth and
enumeration of anaerobic spore formers from cheese, with
observations on the effect of nisin. J. Dairy Res. 21:101-110.
2. Barnes, E. M., and M. Ingram. 1956. The effect of redox potential
on the grown Clostridium welchii strain isolated from horse muscle.
J. Appl. Bacteriol. 19:117- 128.
3. Barnes, E. M., J. E. Despaul, and M. Ingram. 1963. The behavior
of a food poisoning strain of Clostridium welchii in beef. J. Appl.
Bacteriol. 26:415.
Packaging
500 g
1808-17
429
Riboflavin Assay Medium
Section II
Bacto Riboflavin Assay Medium
Intended Use
Riboflavin Assay Medium is used for determining riboflavin concentration by the microbiological assay technique.

1. Maintenance Media: For maintaining the stock culture to preserve the
Riboflavin Assay Medium is a modification of the medium described
by Snell and Strong.1 It is recommended for use in the microbiological
assay of riboflavin following the methodology outlined by the U.S.
Food and Drug Administration2 using Lactobacillus casei subsp.
rhamnosus ATCC 7469 as the test organism.
DL-Tryptophane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
Adenine Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Uracil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Xanthine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Magnesium Sulfate USP . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Manganese Sulfate Monohydrate . . . . . . . . . . . . . . . . . . . . 20
Sodium Chloride USP . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Pyridoxal Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Folic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800
Nicotinic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
g
g
mg
mg
mg
mg
g
mg
mg
mg
mg
mg
mg
g
g
g
g
g
Precautions
Riboflavin Assay Medium is free from riboflavin but contains all other
nutrients and vitamins essential for the growth of Lactobacillus casei
subsp. rhamnosus ATCC 7469. The addition of riboflavin in specified
measured turbidimetrically or titrimetrically.

3. Take great care to avoid contamination of media or glassware for
Scrupulously clean glassware, free from detergents and other
chemicals, must be used.
4. Take precautions to keep sterilizing and cooling conditions
uniform throughout assay. Glassware is heated to 250C for at least
1 hour to burn off any organic residues that might be present.
Formula
Formula Per Liter
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
L-Asparagine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6
g
g
g
g
g
g

Solution:
2.4% solution (single strength) and
4.8% (double strength), soluble in
distilled or deionized water on
boiling. Light to medium amber,
Prepared Medium:
Light amber, clear, may have a very
slight precipitate.
Reaction of 2.4%
Solution at 25C:
pH 6.8 0.2
Cultural Response
Prepare Riboflavin Assay Medium per label directions. The
medium supports the growth of L. casei subsp. rhamnosus
ATCC 7469 when prepared in single strength and supplemented
with riboflavin. Measure growth response turbidimetrically
with increasing concentrations of riboflavin to produce a
standard curve.
430
Storage
Expiration Date
Procedure
Materials Provided

Glassware
Autoclave
Stock culture of Lactobacillus casei subsp. rhamnosus ATCC 7469
Sterile tubes
Lactobacilli Agar AOAC or Micro Assay Culture Agar
Lactobacilli Broth AOAC or Micro Inoculum Broth
Riboflavin USP
Spectrophotometer
The Difco Manual
Section II
Rice Extract Agar
1.
2.
3.
4.
5.
6.


Test Procedure
1
Follow assay procedures as outlined in AOAC. Levels of riboflavin

used in the determination of the standard curve should be prepared
according to this reference or according to the following procedure.
Stock Cultures
Stock cultures of L. casei subsp. rhamnosus ATCC 7469 are prepared
by stab inoculation into 10 ml of Lactobacilli Agar AOAC. After
24-48 hours incubation at 35-37C, the stock cultures are kept in the
refrigerator. Transfers are made at monthly intervals in triplicate.
influence the standard curve readings, cannot be duplicated exactly

from assay to assay. The standard curve is obtained by using Riboflavin
USP Reference Standard or equivalent at levels of 0.0, 0.025, 0.05,
0.075, 0.1, 0.15, 0.2 and 0.3 g riboflavin per assay tube (10 ml).
The concentration of riboflavin required for the preparation of the
standard curve may be prepared by dissolving 0.1 g of Riboflavin USP
Reference Standard or equivalent in 1,000 ml of distilled water by
heating, giving a stock solution of 100 g per ml. Dilute the stock
solution by adding 1 ml to 999 ml distilled water. Use 0.0, 0.25, 0.5,
0.75, 1, 1.5, 2 and 3 ml of the diluted stock solution per tube. Prepare
the stock solution fresh daily.
Results
disk or cup.
thirds of the values do not vary by more than 10%.
Inoculum
Inoculum for assay is prepared by subculturing a stock culture of
L. casei subsp. rhamnosus ATCC 7469 into 10 ml of Lactobacilli Broth
AOAC or Micro Inoculum Broth. Following incubation for 16-24 hours
at 35-37C, the culture is centrifuged under aseptic conditions and the
supernatant liquid decanted. After washing 3 times with 10 ml sterile
0.85% saline, the cells are resuspended in 10 ml sterile 0.85% saline.
The cell suspension is then diluted with sterile 0.85% saline, to a
turbidity of 35-40% transmittance when read on the spectrophotometer
at 660 nm. One drop of this latter suspension is then used to inoculate
each of the assay tubes.
Riboflavin Assay Medium may be used for both turbidimetric and
titrimetric determinations. Turbidimetric readings should be made
after 18-24 hours incubation at 35-37C, where as titrimetric
determinations are best made after 72 hours incubation at 35-37C.
Using Riboflavin Assay Medium, the most effective assay range is
between 0.025 and 0.15 g riboflavin.
Standard Curve
run. Conditions of autoclaving and temperature of incubation, which
Bacto Rice Extract Agar

3. The use of altered or deficient media may cause mutants having different nutritional requirements that will not give a satisfactory response.
5. Maintain pH below 7.0 to prevent loss of riboflavin.
References
1. Snell and Strong. 1939. Ind. and Eng. Chem. 11:346.
2. Association of Analytical Chemists. 1996. U.S. Food and Drug
Administration methods or the microbiological analysis of selected
nutrients. AOAC Internationl, Gaithersburg, MD.
Packaging
100 g
0325-15*
*Store at 2-8C
Intended Use
Bacto Rice Extract Agar is used for differentiating Candida albicans
and other Candida spp. based on chlamydospore formation.

Rice Extract Agar is prepared according to the formulation of
Taschdjian.1 Chlamydospores were observed consistently and in
abundance upon this medium 17-24 hours after inoculation with
Candida albicans. The morphology of both the pathogenic and
The Difco Manual
nonpathogenic Candida agreed in every respect with that of the

cultures grown on corn meal agar; corn meal agar was the medium
most routinely used in laboratories at that time, but was time-consuming
and laborious to prepare. In later studies Taschdjian2 and Kelly and
Funigiello3 showed that the addition of Tween 80 (polysorbate 80) to
Rice Extract Agar enhanced chlamydospore formation by C. albicans.
The addition of 2% dextrose enhanced pigment production by
Trichophyton rubrum permitting the differentiation between this
dermatophyte and Trichophyton mentagrophytes.
Taubert and Smith4 recommended Rice Extract Agar for use in the
diagnosis of vulvovaginal candidiasis. A cotton-tipped applicator was
431
Rice Extract Agar
Section II
used for obtaining a specimen and then rolled on the surface of a rice
extract agar plate; a cover glass was then applied to the agar, covering
most of the inoculum.
Procedure
The Rice Extract provides the sole source of nutrients in the medium.
This lack of nutrients together with the oxygen-deficient culture
conditions (covering the inoculum with a cover glass) creates a deficient
environment that induces the formation of specific morphological
forms (chlamydospores and pseudomycelia in particular) in some
yeasts. The addition of Tween 80 further stimulates chlamydospore
formation due to its content of oleic acids. Bacto Agar is incorporated
into the medium as a solidifying agent.
Glassware
Autoclave
Formula
Rice Extract Agar
Formula Per Liter
Materials Provided
Rice Extract Agar
1.
2.
3.
4.

Aseptically dispense medium into sterile Petri dishes.

White Rice, Extract from . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g

Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Test Procedure

1. Inoculate the plates by cutting through the surface of the agar with
an inoculating wire.
2. Cover the inoculated area with a sterile cover slip.
3. Invert plates and incubate at 23-25C for 18-72 hours.
4. Examine for chlamydospores microscopically using approximately
100X magnification and by focusing upon the line of inoculation.
Storage
Results
Precautions

After 24 to 48 hours most strains of C. albicans and C. stellatoidea

will have formed typical chlamydospores.5
Expiration Date
1. Further studies should be performed to confirm the results obtained.

2. Tween 80 enhances chlamydospore production in many species
of Candida. It is therefore necessary to use additional media for
species identification.6
3. High temperatures for incubation should be avoided as chlamydospores are not formed at 37C.
References
Solution:
Solution is light amber, opalescent
with precipitation.
Prepared Medium:
Colorless to light amber, opaque,
precipitate.
Reaction of 2.5%
Solution at 25C:
pH 7.1 0.2
Cultural Response
Prepare Rice Extract Agar per label directions. Inoculate and
incubate at 23-25C for 18-72 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
CHLAMYDOSPORES
Candida albicans
Candida albicans
10231
26790
30-300
30-300
good
good
+
+
1. Taschdjian, C. L. 1953. A simple prepared identification medium

for Candida albicans. Mycologia 45:474.
2. Taschdjian, C. L. 1957. Routine identification of Candida
albicans: Current methods and a new medium. Mycologia 49:332.
3. Kelly, J. P., and F. Funigiello. 1959. Candida albicans: A study of
media designed to promote chlamydospore production. J. Lab. Clin.
Med. 53:807-809.
4. Taubert, H. D., and A. G. Smith. 1960. The clinical use of
Taschdjians medium in the diagnosis of vulvovaginal candidiasis.
J. Lab. Clin. Med. 55:820- 828.
5. Cooper, and Silva-Hutner. 1985. In Lennette, Balows, Hausler,
and Shadomy (ed.), Manual of clinical microbiology, 4th ed. ASM,
Washington, D.C.
Packaging
Rice Extract Agar
432
500 g
0899-17
The Difco Manual
Section II
Rogosa SL Agar & Rogosa SL Broth
Bacto Rogosa SL Agar

Bacto Rogosa SL Broth
feces, vaginal specimens and foodstuffs.3,4 The low pH and high

acetate concentrations effectively suppress other bacterial flora allowing lactobacilli to flourish.
Intended Use
Bacto Rogosa SL Agar and Bacto Rogosa SL Broth are used for
cultivating oral, vaginal and fecal lactobacilli.
Also Known As
Rogosa SL Agar is also known as RMW Agar.

Rogosa SL Agar and Broth are a modification of media described by
Rogosa, Mitchell and Wiseman.1,2 These media are used for isolation,
enumeration and identification of lactobacilli in oral bacteriology,

Rogosa SL Agar
Dehydrated Appearance: Beige, homogeneous with soft clumps.
Solution:
Solution is light amber, slightly
opalescent and may have a slight
precipitate.
Reaction of 7.5%
Solution at 25C:
pH 5.4 0.2
Rogosa SL Broth
Dehydrated Appearance: Beige, appears moist, slightly lumpy.
Solution:
Reaction of 6.0%
Solution at 25C:
pH 5.4 0.2
Cultural Response
Rogosa SL Agar
Prepare Rogosa SL Agar per label directions. Inoculate and
Rogosa SL Broth
Prepare Rogosa SL Broth per label directions. Inoculate and
ORGANISM
Lactobacillus casei
ATCC
9595
4797
25923*
INOCULUM
CFU
GROWTH
100-1,000
good
100-1,000
good
1,000-2,000
marked to
complete inhibition
The Difco Manual
Tryptone provides carbon and nitrogen. Yeast Extract is a source of

trace elements, vitamins and amino acids. Dextrose, Arabinose and
Saccharose are carbohydrate sources that provide carbon. Sodium
Acetate and Ammonium Citrate inhibit streptococci, molds and other
oral microbial flora and restrict swarming. Monopotassium Phosphate
provides buffering capability. Magnesium Sulfate, Manganese Sulfate
and Ferrous Sulfate are sources of inorganic ions. Sorbitan Monooleate
(Polysorbate 80) acts as a surfactant. Bacto Agar is a solidifying agent.
Formula
Rogosa SL Agar
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Arabinose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.03
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g
g
g
g
g
g
g

Rogosa SL Broth
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Arabinose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.03
g
g
g
g
g
g
g
g
g
g
g
g
Precautions
Storage
Store dehydrated media at 2-8C. The dehydrated media are very
433
Rose Bengal Agar
Section II
Expiration Date
Test Procedure
Procedure
Materials Provided
Rogosa SL Agar
Rogosa SL Broth

Glassware
Glacial acetic acid
Incubator (35C)
Rogosa SL Agar
3. Add 1.32 ml glacial acetic acid and mix well.
4. Boil 2-3 minutes. DO NOT AUTOCLAVE.
Rogosa SL Broth
2. Add 1.32 ml glacial acetic acid and mix well.
3. Boil 2-3 minutes. DO NOT AUTOCLAVE.
Results

The salt in the formulation makes the media not suitable for isolation
of dairy lactobacilli; e.g., L. lactis, L. bulgaricus and L. helveticus.4
References
1. Rogosa, M., J. A. Mitchell, and R. F. Wiseman. 1951. A
selective medium for the isolation and enumeration of oral and
fecal lactobacilli. J. Bacteriol. 62:132.
2. Rogosa, M., J. A. Mitchell, and R. F. Wiseman. 1951. A
selective medium for the isolation and enumeration of oral and
fecal lactobacilli. J. Dental Res. 30:682.
3. Vedamuthu, E. R., M. Raccach, B. A. Glatz, E. W. Seitz, and M.
S. Reddy. 1992. Acid-producing Microorganisms. In C. Vanderzant
and D. F. Splittstoesser (ed.), Compendium of methods for the microbiological examination of foods, 3rd ed. American Public Health
Assoc., Washington, D.C.
Packaging
Rogosa SL Agar
500 g
10 kg
0480-17
0480-08
Rogosa SL Broth
500 g
0478-17

Rose Bengal Agar

Bacto Rose Bengal Agar Base . Bacto Rose Bengal
Antimicrobic Supplement C
Intended Use
Bacto Rose Bengal Agar Base is used with Bacto Rose Bengal Antimicrobic Supplement C in isolating and enumerating yeasts and molds.
Also Known As
Rose Bengal Agar is also known as Rose Bengal Chloramphenicol Agar
and Rose Bengal-Malt Extract Agar.

A number of methods have been described for the selective isolation
of fungi from environmental materials and foodstuffs containing mixed
populations of fungi and bacteria. The use of media with an acid pH
that selectively inhibits the growth of bacteria and thereby promotes
the growth of fungi has been widely employed.1,2,3 A number of investigators have reported, however, that acidified media may actually
inhibit fungal growth,4,5 fail to completely inhibit bacterial growth,5
and have little effect in restricting the size of mold colonies.6 Smith
434
and Dawson7 used Rose Bengal in a neutral pH medium for the selective
isolation of fungi from soil samples. Chloramphenicol, streptomycin,
oxytetracycline and chlortetracycline have been used for the improved,
selective isolation and enumeration of yeasts and molds from soil,
sewage and foodstuffs.4,8,9,10,11
Rose Bengal Agar Base supplemented with Rose Bengal Antimicrobic
Supplement C is a modification of the Rose Bengal Chlortetracycline
Agar formula of Jarvis.11 Instead of chlortetracycline, chloramphenicol
is employed in this medium as a selective supplement. Of the antibiotics
most frequently employed in media of neutral pH, chloramphenicol
is recommended because of its heat stability and broad antibacterial
spectrum.12 Rose Bengal Agar is recommended in standard methods for
the enumeration of yeasts and molds from foodstuffs and water.12,13,14,15

Soytone provides the carbon and nitrogen sources required for good
growth of a wide variety of organisms. Dextrose is an energy source.
The Difco Manual
Section II
Rose Bengal Agar
Monopotassium Phosphate provides buffering capability. Magnesium

Sulfate provides necessary trace elements. Rose Bengal is included as
a selective agent that inhibits bacterial growth and restricts the size
and height of colonies of the more rapidly growing molds. The restriction in growth of molds aids in the isolation of slow-growing fungi by
preventing overgrowth by more rapidly growing species. Rose Bengal
is taken up by yeast and mold colonies, thereby facilitating their
recognition and enumeration. Rose Bengal Antimicrobic Supplement C
is a lyophilized antimicrobic supplement containing chloramphenicol
which inhibits bacteria. Bacto Agar is the solidifying agent.
Formula
Rose Bengal Agar Base
Formula Per Liter
Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Rose Bengal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
2. Rose Bengal Antimicrobic Supplement C

TOXIC. MAY CAUSE CANCER. MAY CAUSE HERITABLE
GENETIC DAMAGE. POSSIBLE RISK OF HARM TO THE
UNBORN CHILD. MAY CAUSE SENSITIZATION BY INHALATION AND SKIN CONTACT. Wear suitable protective clothing, gloves and eye/face protection. In case of accident or if you
feel unwell, seek medical advice immediately. (Show label where
possible.) Do not breathe dust. Keep container tightly closed.
Target Organs: Blood, Bone Marrow.
wash immediately with plenty of water. If swallowed seek medical
advice immediately and show this container or label. If inhaled,
remove to fresh air. If not breathing, give artificial respiration. If
breathing is difficult, give oxygen. Seek medical advice.
Storage
Store Rose Bengal Agar Base dehydrated below 30C. The dehydrated
medium is very hygroscopic. Keep container tightly closed. Store the
prepared medium at 2-8C.
Store Rose Bengal Antimicrobic Supplement C at 2-8C. Do not open
or rehydrate vials until ready to use. Store rehydrated vials at 2-8C
and use within 24 hours.

Rose Bengal Antimicrobic Supplement C
Formula Per 2 ml Vial
Chloramphenicol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05 g
Precautions
Aspergillus niger
ATCC 1015
Uninoculated
plate

Dehydrated Appearance: Beige to faint pink, free-flowing, homogeneous.
Solution:
water on boiling. Solution is reddish pink,
Complete
Prepared Medium:
Bright pink, very slightly to
Reaction of 3.2%
Solution at 25C:
pH 7.2 0.2
Lyophilized Appearance: Lyophilized white cake, may be dispersed.
Rehydrated Appearance: Colorless, clear.
Solubility:
Soluble in 2 ml ethanol.
Cultural Response
Prepare Rose Bengal Agar per label directions. Inoculate using the pour plate
technique (for Aspergillus niger, inoculate the surface of an agar slant) and
incubate aerobically at 25-30C for up to 7 days.
ORGANISM
ATCC
INOCULUM CFU
RECOVERY
COLONY COLOR
Aspergillus niger
Candida albicans
Escherichia coli
Micrococcus luteus
1015
10231
25922*
10240
100-300
100-300
1,000-2,000
1,000-2,000
good
good
inhibited
inhibited
white
pink
The Difco Manual
Candida albicans
ATCC 10231

*This culture is available as a Bactrol Disk and should be
435
Rose Bengal Agar
Expiration Date
Procedure
Materials Provided

Glassware
Autoclave
Incubator (25C)
Ethanol (reagent grade)
Bent glass rods
1. Rose Bengal Antimicrobic Supplement C: To rehydrate, aseptically add 2 ml of ethanol per vial of dehydrated supplement and
invert several times to dissolve the powder.
2. Rose Bengal Agar Base: To rehydrate, suspend 16 grams in 500 ml
4. Sterilize the basal medium at 121C for 15 minutes and then cool
to 45-50C.
5. Aseptically add 2 ml of the rehydrated Rose Bengal Antimicrobic
Supplement C to 500 ml of cooled agar base. Mix thoroughly.
6. Dispense into sterile Petri dishes and allow to dry overnight at room
temperature (21-25C).

Collect specimens in sterile containers and transport immediately to
the laboratory in accordance with recommended guidelines. 12,13
Prepare samples for dilution plating inoculation. It is recommended that
yeast and molds be enumerated by a surface spread-plate technique
rather than with pour plates.12 The spread-plate technique provides
maximal exposure of cells to atmospheric oxygen and eliminates heat
stress from molten agar.12
Test Procedure
1. Inoculate 0.1 ml of appropriate dilutions in duplicate on the solidified
agar. Spread over the entire surface using a sterile bent glass rod.
2. Incubate plates at 25-30C for up to 7 days.
Results
Colonies of yeast appear pink due to the uptake of rose bengal. Count
plates containing 15 to 150 colonies and report the counts as colony
forming units (CFU) per gram or ml of sample.

1. Although this medium is selective primarily for fungi, microscopic
examination is recommended for presumptive identification.
Biochemical testing using pure cultures is required for complete
identification.
2. Due to the selective properties of this medium and the type of
specimen being cultured, some strains of fungi may be encountered
436
Section II
that fail to grow or grow poorly on the complete medium; similarly,

some strains of bacteria may be encountered that are not inhibited
or only partially inhibited.
3. Care should be taken not to expose this medium to light since photodegradation of rose bengal yields compounds that are toxic to fungi.
References
1. Waksman, S. A. 1922. A method for counting the number of fungi
in the soil. J. Bacteriol. 7:339-341.
2. Koburger, J. A. 1976. Yeasts and molds, p. 225-229. In M. L. Speck
(ed.), Compendium of methods for the microbiological examination
of foods. American Public Health Association, Washington, D.C.
3. Mossel, D. A. A., M. Visser, and W. H. J. Mengerink. 1962. A
comparison of media for the enumeration of moulds and yeasts in
foods and beverages. Lab Practice 11:109-112.
4. Martin, J. P. 1950. Use of acid, rose bengal and streptomycin in
the plate method for estimating soil fungi. Soil Sci. 69:215-232.
5. Koburger, J. A. 1972. Fungi in foods. IV. Effect of plating
medium pH on counts. J. Milk Food Technol. 35:659-660.
6. Tyner, L. E. 1944. Effect of media compositions on the numbers
of bacterial and fungal colonies developing in Petri plates. Soil
Sci. 57:271-274.
7. Smith, N. R., and V. T. Dawson. 1944. The bacteriostatic action
of rose bengal in media used for the plate counts of soil fungi. Soil
Sci. 58:467-471.
8. Cooke, W. B. 1954. The use of antibiotics in media for the isolation of
fungi from polluted water. Antibiotics and Chemotherapy 4:657-662.
9. Papavizas, G. C., and C. B. Davey. 1959. Evaluation of various
media and antimicrobial agents for isolation of soil fungi. Soil Sci.
88:112-117.
10. Overcast, W. W., and D. J. Weakley. 1969. An aureomycin-rose
bengal agar for enumeration of yeast and mold in cottage cheese.
J. Milk Technol. 32:442-445.
11. Jarvis, B. 1973. Comparison of an improved rose bengalchlortetracycline agar with other media for the selective isolation
and enumeration of molds and yeasts in foods. J. Appl. Bact.
36:723-727.
and Molds. In C. Vanderzant and D. F. Splittstoesser (eds.),
foods, 3rd ed. American Public Health Assoc., Washington, D.C.
of dairy products, 16th ed. American Public Health Assoc.,
Washington, D.C.
14. Eaton, A.D., L.S. Clesceri, and A.E. Greenberg (ed.). 1995.
15. MacFaddin, J. F. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria. Williams
Packaging
Rose Bengal Antimicrobic
Supplement C
500 g
10 kg
1831-17
1831-08
6 x 2 ml
3352-54
The Difco Manual
Section II
SABHI Agar Base
Bacto SABHI Agar Base
Intended Use
maintaining osmotic balance. Disodium Phosphate provides buffering

capacity. Bacto Agar is a solidifying agent. Chloromycetin, when
added, is a broad spectrum antibiotic that inhibits a wide variety of
gram-negative bacteria.
Bacto SABHI Agar Base is for use with chloromycetin and blood
(optional) in isolating and cultivating pathogenic fungi.
Formula
SABHI Agar Base

Formula Per Liter
Sabouraud 1 formulated Sabouraud Dextrose Agar as a general

purpose medium for the recovery of dermatophytes. Brain Heart
Infusion is a highly nutritive medium used for cultivating a variety of
fastidious organisms and medically important fungi.2 SABHI Agar
Base, prepared according to the formulation of Gorman3, combines
the ingredients from both Sabouraud Dextose Agar and Brain Heart
Infusion. It is particularly useful for maximum recovery of Blastomyces
dermatitidis and Histoplasma capsulatum from body tissues and
fluids and as a primary recovery medium for saprophytic and
pathogenic fungi.4
Gorman reported that the addition of blood to this medium increased
recovery and conversion to the yeast phase of H. capsulatum and
B. dermatitidis.3,5 Selectivity can be obtained by adding chloromycetin
or other antimicrobics to the medium.5

Bacto Neopeptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.25
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g
g
Precautions
Storage
Infusions from Calf Brains and Beef Heart are sources of carbon,
protein and nutrients. Proteose Peptone and Neopeptone are sources
of nitrogen, amino acids and carbon. Dextrose is an additional
carbon source. Sodium Chloride provides essential ions while

Aspergillus niger
ATCC 16404
Uninoculated
plate

Solution:
is medium amber, slightly opalescent
Reaction of 5.9%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepare SABHI Agar Base according to the label directions and
100 mg/ml of chloromycetin, with and without 10% sheep blood.
Inoculate and incubate tubes at 30 2C for up to 7 days.
ORGANISM
Aspergillus niger
Candida albicans
Escherichia coli
ATCC
INOCULUM
CFU
16404 100-1,000
10231 100-1,000
25922* 1,000-2,000
9763
100-1,000
Staphylococcus
25923* 1,000-2,000
aureus
Trichophyton mentagrophytes 9533
100-1,000
GROWTH WITH
AND WITHOUT BLOOD
good
good
marked to
complete inhibition
good
marked to
complete inhibition
good
ATCC 9763
The Difco Manual
437
SF Medium
Section II
Expiration Date
2. Incubate SABHI tubes/plates at 30 2C for up to 7 days.
Results
Procedure
Observe SABHI tubes/plates for growth and record colony morphology.
SABHI Agar Base
1. Non-selective fungal media should be used concurrently with

selective media when isolating fungi due to the sensitivity of some
strains to antibiotics.5
References
Glassware
Autoclave
OPTIONAL: Chloromycetin or other sterile antimicrobics
OPTIONAL: Defibrinated sheep blood
1. Sabouraud, R. 1892. Ann. Dermatol. Syphilol. 3:1061.

2. Dixon, D. M., and R. A Fromtling. 1995. In P. R. Murray, E. J.
Baron, M. A. Pfaller, F .C. Tenover, and R. H. Yolken (ed.),
3. Gorman, J. W. 1967. Sabhi, a new culture medium for pathogenic
fungi. Am. J. Med. Technol. 33:151.
St. Louis, MO.
handling, p. 19-32. In P. R. Murray, E. J. Baron, M. A. Pfaller,
F. C. Tenover, and R. H. Yolken, (ed.). Manual of clinical
Washington, D.C.
Materials Provided
1.
2.
3.
4.
5.

Cool medium to 50-55C.
OPTIONAL: To prepare selective medium, aseptically add 1 mL
chloromycetin solution (100 mg/ml) to 1 liter of sterile medium.
6. OPTIONAL: To prepare blood agar, aseptically add sterile sheep
blood at a concentration of 10% (e.g. 100 ml blood to 900 ml of
sterile medium).

Test Procedure
Packaging
SABHI Agar Base
1. Inoculate SABHI tubes/plates with specimen.
Bacto SF Medium
500 g
2 kg
0797-17
0797-07
inhibitor, sodium azide. Second, it detects whether an organism can

ferment the carbohydrate, dextrose, producing a pH color change.
Intended Use
Bacto SF Medium is used for isolating and cultivating fecal

streptococci from milk, water, sewage and feces.
Tryptone is a source of carbon, nitrogen, vitamins and minerals.

Dextrose is a fermentable carbohydrate. Sodium Chloride maintains
the osmotic balance of the medium. Sodium Azide inhibits cytochrome
oxidase of gram-negative bacteria. Brom Cresol Purple is a pH
indicator. Phosphates buffer the medium.
Group D enterococci will grow in the presence of azide and ferment
glucose. This produces an acid pH that changes the color of the
medium from purple to yellow.
Also Known As
Streptococcus Faecalis Medium

Hajna and Perry1 specified the formulation of SF Broth, a medium
that is selective for fecal streptococci when incubated at 45.5C.
SF Broth has been used for testing water and other materials for fecal
contamination.2,3,4 Detection of fecal streptococci is used as an indicator
of pollution.
SF medium is used to differentiate Group D enterococci from Group D
non-enterococci and other Streptococcus spp. that are not Group D.
SF Medium is differential in two ways. First, it differentiates based on
whether an organism has the ability to grow in the presence of the
438
Formula
SF Medium
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
g
g
g
g
The Difco Manual
Section II
SF Medium
Sodium Azide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5 g
Bacto Brom Cresol Purple . . . . . . . . . . . . . . . . . . . . . . . 0.032 g

Storage
Expiration Date

Dehydrated Appearance: Light beige to gray, may have a
light greenish tint, free-flowing,
homogeneous.
Solution:
deionized water. Solution is purple,
clear with no precipitate.
Prepared Tubes:
Purple, clear with no precipitate.
Reaction of 3.6%
Solution at 25C:
pH 6.9 0.2
Cultural Response
Prepare SF Medium per label directions. Inoculate and
incubate at 45 0.5C 18-24 and 40-48 hours.
Escherichia coli
Streptococcus bovis
19433*
27270
25922*
33317
INOCULUM
CFU
GROWTH
ACID
REACTION
1,000-2,000 good yellow (acid)

1,000-2,000 good yellow (acid)
1,000-2,000 inhibited no change
1,000-2,000 none to no change
poor
The Difco Manual
SF Medium
Glassware
Autoclave
Incubator (45 0.5)
1. Suspend 36 grams in 1 liter distilled or deionized water. Rehydrate
with proportionally less water when liquid inocula will exceed 1 ml.

Test Procedure1
1. Inoculate SF Medium with a heavy inoculum from a pure 18-24
hour culture of the test organism.
2. Incubate at 45 0.5C for 18-48 hours.
3. Read tubes for growth and acid production at 18-24 hours and
40-48 hours.
Results
A positive result is indicated by growth (turbidity) in the medium with
the production of a yellowish-brown color (acid production). A
negative reaction is indicated by poor or no growth and no color change
in the medium.
ATCC
Materials Provided
Precautions
ORGANISM
Procedure
1. Pure cultures of Streptococcus spp. should be inoculated into SF Broth.

2. Group D streptococci include both enterococcal and nonenterococcal strains. Consult appropriate references for further
identification of Group D streptococci.5,6,7,8
References
1. Hajna, A. A., and C. A. Perry. 1943. Comparative study of
presumptive and confirmative media for bacteria of the coliform
group and fecal streptococci. Am. J. Public Health 33:550-556.
2. Facklam, R. R. 1972. Recognition of group D streptococcal
species of human origin by biochemical and physiological tests.
streptococci. I. Cultivation and enumeration of streptococci in
surface water. Appl. Microbiol. 9:15.
4. Shattock, P. M. F. Enterococci, p. 303-319. In J. C. Ayers,
A. A. Kraft, H. E. Snyder, and H. W. Walker (eds.), Clinical and
biological hazards in food. University Press, Ames, Iowa.
439
SFP Agar
Section II
7. Flowers, R. S., W. Andrews, C. W. Donnelly, and E. Koenig. 1993.
Pathogens in milk and milk products, p. 103-212. In R. T. Marshall,
(ed.), Standard methods for the examination of dairy products, 16th
8. Eaton, A. D., L. S. Clesceri, and A. E. Greenberg (eds.). 1995.

Packaging
SF Medium
500 g
0315-17
SFP Agar
Bacto SFP Agar Base . Bacto Egg Yolk Enrichment 50%
Bacto Antimicrobic Vial K . Bacto Antimicrobic Vial P
Intended Use
Bacto SFP Agar Base is used with Bacto Egg Yolk Enrichment 50%,
Bacto Antimicrobic Vial P and Bacto Antimicrobic Vial K in detecting
and enumerating Clostridium perfringens in foods.
Shahidi Ferguson Perfringens (SFP) Agar Base is prepared according

to the formulation of Shahidi and Ferguson.1 With the addition of 50%
egg yolk emulsion, both the lecithinase reaction and the sulfite
reaction can identify Clostridium perfringens. The selectivity of the
medium is due to the added kanamycin and polymixin B.
Also Known As
Tryptose Sulfite Cycloserine (TSC) Agar
Clostridium perfringes
ATCC 12919
Uninoculated
plate

SFP Agar Base
Solution:
medium to dark amber, slightly opalescent.
Prepared Medium (Final): Canary yellow, opaque.
Reaction of 4.7%
Solution at 25C:
pH 7.6 0.2
Appearance:
Canary yellow, opaque solution with
a resuspendable precipitate.
Antimicrobic Vial K
Rehydrated Appearance: Colorless, clear solution.
Antimicrobic Vial P
Rehydrated Appearance: Colorless, clear solution.
ATCC 12924
Cultural Response
SFP Agar
Prepare the SFP Agar base layer and cover layer per label directions, inoculating
the base layer. Incubate at 35 2C under anaerobic conditions for 18-48 hours.
ORGANISM
ATCC
INOCULUM CFU
GROWTH
COLOR OF COLONIES
12919
12924
30-300
30-300
good
good
black with halo

black with halo
440

The Difco Manual
Section II
SFP Agar

sauces, raw vegetables and other foods and food ingredients, but
occurrences of food borne illness are usually associated with cooked
meat or poultry products.2 Spores of some strains that may resist heat
during cooking germinate and grow in foods that are not adequately
refrigerated.3 Enumerating the microorganism in food samples plays a
role in the epidemiological investigation of outbreaks of food borne
illness.2
SFP Agar (with added kanamycin and polymixin B) is comparable to
Tryptose Sulfite Cycloserine (TSC) Agar, which uses cycloserine as
the inhibitory component.2, 4

SFP Agar Base contains Tryptose and Soytone as sources of carbon,
vitamins which stimulate bacterial growth. Ferric Ammonium
Citrate and Sodium Sulfite are H2S indicators. Clostridia reduce sulfite
to sulfide, which reacts with iron to form a black iron sulfide precipitate. Antimicrobic Vial P contains Polymyxin B and Antimicrobic
Vial K contains Kanamycin; both are inhibitors to organisms other
than Clostridium spp. Egg Yolk Enrichment 50% provides egg yolk
lecithin which some clostridia hydrolyze. Bacto Agar is the
solidifying agent.
Formula
SFP Agar Base
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Ferric Ammonium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Bisulfite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Storage
Store SFP Agar Base below 30C. The dehydrated medium is very
Store Egg Yolk Enrichment 50%, Antimicrobic Vial K and Antimicrobic
Vial P at 2-8C.
Expiration Date
Procedure
g
g
g
g
g
g

Sterile concentrated egg yolk emulsion
Antimicrobic Vial K
25,000 mcg Kanamycin per 10 ml vial
Antimicrobic Vial P
30,000 units Polymyxin B per 10 ml vial
Precautions
2. Antimicrobic Vial K
tightly closed.
The Difco Manual
Antimicrobic Vial P
MAY BE HARMFUL IF ABSORBED OR INTRODUCED
THROUGH SKIN. (US) MAY CAUSE ALLERGIC EYE, RESPIRATORY SYSTEM AND SKIN REACTION. (US) Avoid contact
clothing. Keep container tightly closed.
Materials Provided (one of the following)

SFP Agar Base
Antimicrobic Vial K
Antimicrobic Vial P

Glassware
Petri dishes
Autoclave
SFP Agar Base
Base Layer:
4. Add 100 ml Egg Yolk Enrichment 50%, 10 ml of rehydrated
Antimicrobic Vial P (30,000 units polymyxin B sulfate) and 4.8 ml
rehydrated Antimicrobic Vial K (12 mg kanamycin).
5. Mix thoroughly.
Cover Layer:
2. Prepare as above, except omit Egg Yolk Enrichment 50%.
441
SIM Medium
Section II

1. Ready for use.
2. Shake gently to resuspend precipitate.
Antimicrobic Vial K
1. Aseptically add 10 ml sterile distilled or deionized water to the
Antimicrobic Vial K.
2. Shake to dissolve contents.
Antimicrobic Vial P
1. Aseptically add 10 ml sterile distilled or deionized water to the
Antimicrobic Vial P.
2. Rotate in an end-over-end motion to dissolve contents.

Test Procedure
References
1. Shahidi, S. A., and A. R. Ferguson. 1971. New quantitative,
qualitative, and confirmatory media for rapid analysis of food for
Clostridium perfringens. Appl. Microbiol. 21:500-506.
p. 623-635. In C. Vanderzant, and D. F. Splittstoesser (ed.). Compendium of methods for the microbiological examination of foods,
4. Andrews, W. 1995. Microbial methods, p. 1-119. In Official methods
Arlington, VA.
Packaging
500 g
0811-17
Antimicrobic Vial K
6 x 10 ml
3339-60
Results
Antimicrobic Vial P
6 x 10 ml
3268-60
12 x 10 ml
6 x 100 ml
3347-61
3347-73
Bacto SIM Medium
Formula
SFP Agar Base
Intended Use
SIM Medium is used for differentiating Salmonella and Shigella
species based on hydrogen sulfide production, indole formation
and motility.
Also Known As
Sulfide Indole Motility Medium

Semisolid media have been used extensively in the determination
of bacterial motility throughout the history of bacteriology.1 The
production of hydrogen sulfide, indole formation and motility are
useful diagnostic tests in the identification of Enterobacteriaceae,
especially Salmonella and Shigella. In 1940, Sulkin and Willett2
showed motility, hydrogen sulfide production and carbohydrate
fermentation by members of the Salmonella and Shigella groups.
They called attention to the brush- like growth or motility of the
typhoid organisms. Green and co-workers3 used SIM medium to detect motility in a large series of cultures of typhoid organisms.

Bacto Peptone provides nitrogen, amino acids and additional carbon.
Beef Extract is a source of carbon, protein and nutrients. Peptonized
Iron and Sodium Thiosulfate are indicators of hydrogen sulfide
production. Bacto Agar is a solidifying agent.
Nitrate Broth
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Peptonized Iron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
g
g
g
g
g
Precautions
Storage
Store dehydrated medium below 30C. The dehydrated medium is
Expiration Date
Procedure
Materials Provided
SIM Medium

Glassware
442
The Difco Manual
Section II
SIM Medium
Autoclave
Inoculating needle
1.
2.
3.
4.

Dispense the medium into tubes to an approximate depth of 3 inches.

Test Procedure
1. Using an inoculum from the growth of a pure culture at 18-24 hours,
stab with an inoculating needle two-thirds into the medium.
Carefully ensure the needle is withdrawn through the same stab line.
2. Incubate aerobically at 35 2C for 18-24 hours.
3. Observe for motility, H2S and Indole production.
4. Add 3-4 drops of SpotTest Indole Reagent Kovacs.
Results
Motility and H 2S production should be determined before the
addition of reagents for determination of indole production. Motility
is observed as a diffuse growth outward from the stab line or turbidity
of the medium. H2S production is shown by a blackening along the
stab line. Indole production is seen as the production of a red color
after the addition of 3-4 drops of SpotTest Indole Reagent Kovacs.
1. Do not take inoculum from liquid or broth suspensions because

growth initiation will be delayed.4
2. Reactions are not sufficient to speciate organisms. Additional
biochemical and serological tests are required for confirmation.5
3. When using Ehrlichs reagent for indole test, 1 ml. of chloroform
must be added prior to adding the reagent.6
References
1. Tittsler, R. P., and L. A. Sandholzer. 1936. The use of semi-solid
agar for the detection of bacterial motility. Journal of
Bacteriology. 31:575.
2. Sulkin and Willett. 1940. J. Lab. Clin. Med. 25:649.
3. Greene, R. A., E. F. Blum, C. T. DeCoro, R. B. Fairchild,
M. T. Kaplan, J. L. Landau, and T. R. Sharp. 1951. Rapid
methods for the detection of motility. J. Bact. 62:347.
4. Sosa. 1943. Dr. Carlos G. Malbran. Rev. Inst. Bact. 11:286.
6. Koneman, E. W., S. D. Allen, V. R. Dowell, Jr., W. M. Janda, H.
M. Sommers, and W. C. Winn, Jr. 1988. Color Atlas and
textbook of diagnostic microbiology, p. 147. 3rd ed. J. B. Lippincott
Company, Philadelphia.
Packaging
SIM Medium
500 g
0271-17

Dehydrated Media
Appearance:
Solution:
Reaction of 3.6%
Solution at 25 C:
Beige, homogeneous, free-flowing.

medium amber, clear to slightly opalescent.
pH 7.3 0.2
Cultural Response
Prepare SIM Medium per label instructions. Dispense 15 ml of
medium into standard size tubes. Inoculate using a straight needle
with a single stab to the center through two-thirds of the medium.
Incubate tubes at 35 2C for 18-24 hours and read for growth,
H2S production and motility. Add 3-4 drops of SpotTest Indole
Reagent Kovacs. Indole production is indicated by a red color
after reagent addition.
ORGANISM
ATCC
Escherichia coli
25922*
Salmonella typhi
6539
Shigella flexneri
12022*
GROWTH
H 2S
MOTILITY
INDOLE
good
good
good
good
+
+
+
+
+
Uninoculated
tube
Escherichia coli
ATCC 25922
with indole reagent
ATCC 14028
with indole reagent
The Difco Manual
443
SOB Medium
Section II
Precautions
Bacto SOB Medium
Intended Use
Bacto SOB Medium is used for cultivating recombinant strains of
Escherichia coli.

SOB Medium was developed by Hanahan1 as a nutritionally rich growth
medium for preparation and transformation of competent cells. Transformation requires making perforations in the bacterium (i.e., making the
cells competent) to allow the introduction of foreign DNA into the cell.
To survive this process, competent cells need a rich, isotonic environment.
SOC Medium, used in the final stage of transformation, may be
prepared by aseptically adding 20 ml of a filter-sterilized 20% solution
of glucose (dextrose) to the sterile SOB Medium. This addition provides
a readily available source of carbon and energy in a form E. coli can
use in mending the perforations and for replication.2

Tryptone and Yeast Extract provide sources of nitrogen and growth
factors which allow the bacteria to recover from the stress of transformation and grow well. Sodium Chloride and Potassium Chloride
provide a suitable osmotic environment. Magnesium Sulfate is a source
of magnesium ions required in a variety of enzymatic reactions,
including DNA replication.
SOB Medium
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Magnesium Sulfate, Anhydrous . . . . . . . . . . . . . . . . . . . . . 2.4
Potassium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.186
g
g
g
g
g

Expiration Date
Procedure
Materials Provided
SOB Medium
Autoclave
Incubator 35C
Waterbath 45-50C (optional)
Filter-sterilized 20% solution of glucose (dextrose) (optional)
3. If desired, SOC Medium can be prepared by adding 20 ml of a
filter-sterilized 20% glucose solution cooled to 45-50C.

homogeneous.
Solution:
Prepared Medium:
Reaction of 2.8%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepare SOB Medium per label directions. Inoculate and
Storage
Formula
ORGANISM

ATCC
INOCULUM
CFU
GROWTH
53868
100-300
Good


Test Procedure
Results
References
1. Hanahan, D. 1983. Studies on transformation of Escherichia coli
with plasmids. J. Mol. Biol. 166:557.
Packaging
SOB Medium
444
500 g
0443-17
The Difco Manual
Section II
SPS Agar
Bacto SPS Agar
Bacto SPS Agar is used for detecting and enumerating Clostridium

perfringens in food.
stimulate bacterial growth. Ferric Citrate and Sodium Sulfite are

H2S indicators. Clostridia reduce the sulfite to sulfide which reacts with
the iron from ferric citrate to form a black iron sulfide precipitate.
Tween 80 is a dispersing agent. Polymyxin B Sulfate and Sulfadiazine
are inhibitors to organisms other than Clostridium spp. Sodium
Thioglycollate is a reducing agent. Bacto Agar is the solidifying agent.
Also Known As
Formula
SPS Agar is also known as Sulfite Polymixin Sulfadiazine Agar or

Perfringens Selective Agar.
SPS Agar
Formula Per Liter
Intended Use

In the 1950s, Mossel 1 and Mossel et al. 2 proposed media for
enumerating anaerobic sulfite-reducing clostridia in foods. Angelotti
et al.3 modified the formula as Sulfite Polymixin Sulfadiazine (SPS)
Agar and used it to quantitate C. perfringens in foods.
sauces, raw vegetables and other foods and food ingredients.
Occurrences of food borne illness from C. perfringens are usually
associated with cooked meat or poultry products.4 Spores of some
strains that may resist heat during cooking germinate and grow in foods
that are not adequately refrigerated.5 Enumerating the microorganism
in food samples plays a role in epidemiological investigation of
outbreaks of food borne illness.4

SPS Agar contains Tryptone as a source of carbon, nitrogen, vitamins
and minerals. Yeast Extract supplies B-complex vitamins which
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Ferric Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Sodium Sulfite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Sodium Thioglycollate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
Tween 80 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05
Sulfadiazine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.12
Polymyxin B Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g
g
g
Precautions
Uninoculated
plate
ATCC 12919

Solution:
light to medium amber, slightly
opalescent.
Reaction of 4.1%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepare SPS Agar per label directions. Inoculate and incubate
the plates at 35 2C anaerobically for 18-24 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
APPEARANCE
Clostridium
perfringens
Clostridium
sporogenes
Escherichia
coli
Salmonella
typhimurium
Staphylococcus
aureus
12919
100-1,000
good
11437
100-1,000
25922*
100-1,000
black
colonies
black
colonies
14028*
100-1,000
25923*
100-1,000
none
to fair
marked to
complete inhibition
marked to
complete inhibition
fair
white
to good
colonies
ATCC 25923
The Difco Manual
445
SS Agar
Section II
Storage
4. Incubate anaerobically at 35 2C for 24-48 hours.

Results
Expiration Date
Procedure
Materials Provided
SPS Agar

Glassware
Petri dishes
Autoclave

Consult appropriate standard methods.4, 5.
Test Procedure
1. Dispense inoculum into sterile Petri dish.
2. Pour medium cooled to 50-55C over the inoculum.
3. Gently but thoroughly mix the inoculum and medium. Allow to
solidify on a flat surface.
Bacto SS Agar
Intended Use
Bacto SS Agar is used for isolating Salmonella and some Shigella.
Also Known As
SS Agar is also known as Salmonella-Shigella Agar.

domesticated animals. Infection with non-typhi Salmonella often
causes mild, self-limiting illness. Typhoid fever, caused by S. typhi, is
characterized by fever, headache, diarrhea, and abdominal pain, and
can produce fatal respiratory, hepatic, splenic, and/or neurological damage.1 These illnesses result from the consumption of raw, undercooked
or improperly processed foods contaminated with Salmonella.
Shigella spp. cause classic bacillary dysentery (shigellosis), which is a
descending intestinal illness characterized by abdominal pain, fever,
and watery diarrhea. Shigella dysenteriae can cause a severe form of
446
Clostridium perfringens will grow as black colonies with good growth.
The high degree of selectivity of SPS Agar may inhibit some strains of
C. perfringens while other strains that grow may fail to produce
distinguishing black colonies.4
References
1. Mossel, R. S. 1959. Enumeration of sulfite-reducing clostridia
occurring in foods. J. Sci. Food Agric. 19:662.
2. Mossel, D. A. A., A. S. DeBruin, H. M. J. van Diepen,
C. M. A. Vendrig, and G. Zoutewelle. 1956. The enumeration of
anaerobic bacteria, and of Clostridium species in particular, in
foods. J. Appl. Microbiol. 19:142.
3. Angelotti, R., H. E. Hall, M. J. Foster, and K. M. Lewis.
1962. Quantitation of Clostridium perfringens in foods. Appl.
Microbiol. 10:193.
p. 623-635. In C. Vanderzant, and D. F. Splittstoesser (ed.),. Compendium of methods for the microbiological examination of foods,
Packaging
SPS Agar
100 g
500 g
0845-15*
0845-17*
*Store at 2-8C
dysentery that has been reported to have fatality rates of up to 20%.

Most cases of shigellosis are individual cases due to person-to-person
transmission. When associated with outbreaks, the disease usually is
transmitted by contaminated food and/or water.1
SS Agar is a modification of the Desoxycholate Citrate Agar described
by Leifson.2 SS Agar was found to be superior to other media for the
isolation of Salmonella and Shigella spp.3 Ewing and Bruner found
SS Agar to have the advantage that large amounts of inoculum could
be used when isolating Salmonella or Shigella from clinical samples.4
Caudill5 reported on the satisfactory use of SS Agar in isolation of
Shigella organisms. Hormaeche and his co-workers6 used SS Agar with
other media for isolation of Shigella as the causative agent of infantile
summer diarrhea.
The use of SS Agar is recommended for testing clinical specimens for
the presence of Salmonella and some Shigella spp.1,7 For food testing,
consult appropriate references on the use of SS Agar.8

In SS Agar, Bacto Bile Salts No. 3 and Brilliant Green are
complementary in inhibiting gram-positive bacteria, most coliform
bacteria, and the swarming phenomenon of Proteus spp., while allowing
The Difco Manual
Section II
SS Agar
Salmonella spp. to grow. Sodium thiosulfate and ferric citrate allow

the detection of hydrogen sulfide by the production of colonies with
black centers. Lactose is the carbohydrate present in SS Agar. Neutral
red and brilliant green are present as pH indicators.

Formula
Storage
SS Agar
Formula Per Liter

very hygroscopic. Keep container tightly closed. Store prepared plates
at 2-8C.

Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5
Ferric Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5
Brilliant Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.33
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.025
g
g
g
g
g
g
g
g
mg
g
Expiration Date
Procedure
Materials Provided
SS Agar
Precautions

Waterbath (45-50C)
Petri dishes
Incubator (35C)
ATCC 14028
Uninoculated
plate

Dehydrated Appearance: Very light buff to pink, free flowing,
homogeneous.
Solution:
is red-orange, very slightly to slightly
opalescent.
Prepared Plates:
Red-orange, slightly opalescent.
Reaction of 6.0%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepare SS Agar per label directions. Inoculate and incubate
plates at 35 2C for 18-24 hours and 48 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
29212* 1,000-2,000 partial inhibition
Escherichia coli
25922* 1,000-2,000 partial inhibition
good
Shigella flexneri
12022* 100-1,000
fair to good
APPEARANCE
colorless
pink to red
colorless
w/black centers
colorless
Shigella flexneri
ATCC 12022
The Difco Manual
447
Sabouraud Media
2. Heat to boiling for no more than 2-3 minutes to dissolve completely.

Avoid overheating. DO NOT AUTOCLAVE.
4. Dispense into sterile Petri dishes. Allow the surface of the medium
to air dry for two hours by leaving the lids ajar.

Test Procedure
For isolation of Salmonella and Shigella spp. from clinical specimens,
inoculate fecal samples and rectal swabs onto one quadrant of a SS Agar
plate and streak for isolation. This will permit the development of
discreet colonies. Incubate plates at 35C. Examine at 24 hours and
again at 48 hours for colonies resembling Salmonella or Shigella spp.
Note: SS Agar is inhibitory to some strains of Shigella spp. For
additional information about specimen preparation and inoculation of
clinical specimens, consult appropriate references.1,7
For testing food samples, consult appropriate references.8
Results
Enteric organisms are differentiated by their ability to ferment lactose.
Salmonella and Shigella spp. are lactose non-fermenters and form
colorless colonies on SS Agar. Salmonella spp. that are H2S positive
produce colonies with black centers. Some Shigella spp. are inhibited
on SS Agar.
Coliforms are partially inhibited on SS Agar. E. coli produces pink to
red colonies and may have some bile precipitation. Colonies of
Enterobacter aerogenes appear cream to pink in color. Citrobacter and
Proteus spp. may grow on SS Agar and produce colonies with gray to
black centers due to H2S production. Enterococcus faecalis is partially
inhibited on SS Agar; colonies of E. faecalis are colorless.

1. SS Agar is a highly selective medium. For this reason, it is not
recommended as the sole medium for primary isolation of
Shigella.1,2,9 Some strains of Shigella may not grow.
Section II
2. A few nonpathogenic organisms may grow on SS Agar. These

organisms can be differentiated by their ability to ferment lactose.10
References
2. Leifson, E. 1935. New culture media based on sodium desoxycholate
for the isolation of intestinal pathogens and for the enumeration of
colon bacilli in milk and water. J. Pathol. Bacteriol. 40:581.
3. Rose, H. M., and M. H. Kolodny. 1942. The use of SS
(Shigella-Salmonella) Agar for the isolation of Flexner Dysentery
bacilli from the feces. J. Lab. Clin. Med. 27:1081-1083.
4. Ewing, W. H., and D. W. Bruner. 1947. Selection of Salmonella
and Shigella cultures for serologic classification. Am. J. Clin.
Pathol. 17:1-12.
5. Caudill, F. W., R. E. Teague, and J. T. Duncan. 1942. A rural
shiga dysentery epidemic. JAMA 119:1402-1406.
6. Hormaeche, E., N. L. Surraco, C. A. Peluffo, and P. L. Aleppo.
1943. Causes of infantile summer diarrhea. Am. J. Dis. Child.
66:539-551.
Isenberg, H. D. (ed.), Clinical microbiology procedures handbook,
8. Vanderzant, C., and D. F. Splittstoesser (ed.) 1992. Compendium of methods for the microbiological examination of foods,
9. Taylor, W. I., and B. Harris. 1965. Isolation of shigellae. II.
Comparison of plating media and enrichment broths. Am. J. Clin.
Pathol. 44:476.
Packaging
SS Agar
100
500
2
10
g
g
kg
kg
0074-15
0074-17
0074-07
0074-08
Sabouraud Media
Bacto Sabouraud Agar Modified . Bacto Sabouraud Dextrose Agar
Sabouraud Dextrose Broth . Bacto Sabouraud Maltose Agar
Bacto Sabouraud Maltose Broth . Bacto Fluid Sabouraud Medium
Intended Use
Bacto Sabouraud Agar Modified is used for cultivating fungi at a neutral pH.
Bacto Sabouraud Dextrose Agar and Broth and Bacto Sabouraud
Maltose Agar and Broth are used for culturing yeasts, molds and
448
aciduric microorganisms.
Bacto Fluid Sabouraud Medium is used for cultivating yeasts, molds
and aciduric microorganisms and for detecting yeasts and molds in
normally sterile materials.
The Difco Manual
Section II
Sabouraud Media

Sabouraud Agar Modified
homogeneous.
Solution:
precipitate.
Prepared Medium:
precipitate.
Reaction of 5.0%
Solution at 25C:
pH 7.0 0.2
Sabouraud Dextrose Agar
Solution:
slightly to slightly opalescent
Prepared Medium:
Reaction of 6.5%
Solution 25C:
pH 5.6 0.2
Sabouraud Dextrose Broth
homogeneous.
Solution:
amber, clear.
Prepared Medium:
Light amber, clear.
Reaction of 3.0%
Solution at 25C:
pH 5.6 0.2
Sabouraud Maltose Agar
homogeneous.
Solution:
is light amber, slightly opalescent,
Prepared Medium:
Reaction of 6.5%
Solution at 25C:
pH 5.6 0.2
Sabouraud Maltose Broth
Solution:
amber, clear to slightly opalescent.
Prepared Medium:
opalescent.
Reaction of 5.0%
Solution at 25C:
pH 5.6 0.2
Sabouraud Agar Modified is a modification of the Sabouraud Dextrose

Agar formulation devised by Raymond Sabouraud for his dermatophyte
studies.1 Sabouraud Agar Modified, used for the recovery of dermatophytes2, contains reduced dextrose (2%) and has a neutral pH (7.0).
The selectivity of the medium can be improved with the addition of
antibiotics, such as chloramphenicol to inhibit bacterial growth and
cycloheximide to inhibit saprophytic fungi.3,4
Sabouraud Dextrose Agar and Sabouraud Dextrose Broth are modifications of the Dextrose Agar described by Sabouraud.1 They are used
for cultivating pathogenic fungi, particularly those associated with skin
infections. The high dextrose concentration and acidic pH make these
media selective for fungi.5 Georg6 demonstrated that the addition of
cycloheximide, streptomycin and penicillin to Sabouraud Dextrose
Agar produces an excellent medium for the primary isolation of
dermatophytes. Sabouraud Dextrose Agar is also used for determining
the microbial content of cosmetics7 and for the mycological evaluation
of food.8 Sabouraud Dextrose Agar is available in the dehydrated form
and prepared in 200 ml amounts. In the prepared form, Sabouraud
Dextrose Agar is used for pouring plates.
Sabouraud Maltose Agar is a modification of Sabouraud Dextrose Agar
with maltose substituted for dextrose. It is a selective medium due to
the acid pH. Davidson, Dawding and Buller9 reported that Sabouraud
Maltose Agar was a satisfactory medium in their studies of the infections
caused by Microsporon audouini, M. lanosum and Trichophyton
gypseum. Davidson and Dawding10 also used this medium in isolating
T. gypseum from a case of tinea barbae.
Sabouraud Maltose Broth is a modification of Sabouraud Dextrose
Broth in which maltose is substituted for dextrose. It is selective due to
its acid pH and is used for the detection of fungi.
Fluid Sabouraud Medium is employed in sterility test procedures for
determining the presence of molds, yeasts and aciduric microorganisms.
The acid reaction of the final medium is inhibitive to a large number of
bacteria and makes the medium particularly well suited for cultivating
fungi and acidophilic microorganisms.

Sabouraud Agar Modified, Sabouraud Dextrose Agar, and Sabouraud
Dextrose Broth contain Neopeptone which provides the carbon and
nitrogen required for growth of a wide variety of organisms. Dextrose
is included as an energy source. Bacto Agar is incorporated into the
agar media as a solidifying agent.
Sabouraud Maltose Agar and Sabouraud Maltose Broth contain
Neopeptone which provides the carbon and nitrogen sources required
for growth of a wide variety of organisms. Maltose is included in the
medium as an energy source. Sabouraud Maltose Agar contains Bacto
Agar as the solidifying agent.
Fluid Sabouraud Medium contains Casitone and Peptamin which provide
nitrogen, vitamins, minerals and amino acids. Dextrose is an energy source.
Formula
Formula Per Liter
Bacto Neopeptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g

The Difco Manual
449
Sabouraud Media
Section II

Formula Per Liter
Fluid Sabouraud Medium

Formula Per Liter
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
Bacto Casitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Bacto Peptamin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g

Formula Per Liter
Precautions
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g

Storage

Formula Per Liter
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
Store dehydrated Sabouraud media below 30C. The dehydrated

Store prepared Sabouraud Dextrose Agar at 15-30C.
Expiration Date

Formula Per Liter
Bacto Maltose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 g
Candida albicans
ATCC 60193
Uninoculated
plate

Solution:
deionized water. Solution is light amber,
Prepared Medium:
Reaction of 3.0%
Solution at 25C:
pH 5.7 0.2
Cultural Response
Sabouraud Agar Modified, Sabouraud Maltose Agar,
Sabouraud Maltose Broth, Fluid Sabouraud Mediumm
Sabouraud Dextrose Agar, Sabouraud Dextrose Broth
Prepare dehydrated medium per label directions. Inoculate and
incubate at 30 2C for 18-48 hours or up to 7 days if necessary.
ORGANISM
ATCC
INOCULUM
CFU
RECOVERY
Aspergillus niger
Candida albicans
16404
10231
9763
100-1,000
100-1,000
100-1,000
good
good
good
Sabouraud Dextrose Agar (prepared)

Melt medium and aseptically dispense into plates. Inoculate and incubate
at 30 2C for 18-48 hours, except Aspergillus niger which is incubated
at room temperature for 3-5 days.
ORGANISM
ATCC
INOCULUM
CFU
Aspergillus niger
ATCC 16404
RECOVERY
Aspergillus niger
16404 100-1,000
good
Candida albicans
10231 100-1,000
good
9763
100-1,000
good
The cultures listed are the minimum that should be used for perfoemance testing.
450
The Difco Manual
Section II
Procedure
Materials Provided
Sabouraud Dextrose Agar (dehydrated or prepared)
Fluid Sabouraud Medium. (For Laboratory Use)

Glassware
Autoclave
Incubator
Sterile Petri dishes or tubes with closures
Dehydrated Media
1. Suspend the indicated amount of dehydrated medium in 1 liter of
distilled or deionized water and boil to dissolve completely. Avoid
overheating which could cause a softer medium.
Sabouraud Agar Modified - 50 grams
Sabouraud Dextrose Agar - 65 grams
Sabouraud Maltose Agar - 65 grams
Dissolve the indicated amount of dehydrated medium in 1 liter of
Sabouraud Dextrose Broth - 30 grams
Sabouraud Maltose Broth - 50 grams
Fluid Sabouraud Medium - 30 grams
Prepared Sabouraud Dextrose Agar
Melt the agar to pour into plates by one of the following methods.
Loosen the bottle caps, then autoclave bottles at 121C for 3 minutes
to melt the agar. A small solidified mass may remain that can be
melted by swirling the hot agar. Autoclave time depends on the
number of bottles in the chamber.
NOTES: Autoclave small batches to limit darkening of the medium.
Long cycles have a tendency to shrink the clear label material.
Heat bottles in boiling water. Time will vary; it may take up to
40 minutes to melt the agar.
Microwave the bottles to melt the agar. Time will vary with the microwave and the number of bottles to be melted. When microwaving,
boiling over is a significant problem with smaller bottles.
Sabouraud Media
2. Avoid overheating a medium with an acidic pH because this often

causes a soft medium.
References
1. Sabouraud, R. 1892. Ann. Dermatol. Syphilol. 3:1061.
3. Larone, D. H. 1995. Medically important fungi, a guide to identification, 3rd ed. American Society for Microbiology, Washington, D.C.
4. Wentworth, B. B. (ed.) 1988. Diagnostic procedures for mycotic
and parasitic infections, 7th ed. American Public Health Association,
Washington, D.C.
5. Jarett, L., and A. C. Sonnenwirth (ed.) 1980. Gradwohls
clinical laboratory methods and diagnosis, 8th ed. CV Mosby.
J. Lab. Clin. Med., 44:422-428.
CTFA Microbiology Guidelines. The Cosmetic, Toiletry, and
8. Beuchat, L. R., J. E. Corry, A. D. King, Jr., and J. I. Pitt (ed.).
1986. Methods for the mycological examination of food. Plenum
Press, New York.
9. Davidson, A. M., E. S. Dowding, and A. H. R. Buller. 1932.
Hyphal fusions in dermatophytes. Can. J. Res. 6:1.
10. Davidson, A. M., and E. S. Dowding. 1932. Tinea barbae of the
upper lip. Arch. Dermatol. Syphilol. 26:660.
13. MacFaddin J. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, vol. 1. Williams
and Wilkins, Baltimore.
Packaging
0109-17
0109-15
0109-08
0109-07
9074-76
500 g
100 g
2 kg
0382-17
0382-15
0382-07
500 g
2 kg
0110-17
0110-07
500 g
0429-17
500 g
0642-17
Test Procedure
Results

1. Antimicrobial agents incorporated into a medium to inhibit bacteria
may also inhibit certain pathogenic fungi.
The Difco Manual
50
100
10
2
10 x 200
0747-17
0747-07
g
g
kg
kg
ml

500 g
2 kg
451
Schaedler Agar & Schaedler Broth
Section II
Bacto Schaedler Agar

Bacto Schaedler Broth
Intended Use
Bacto Schaedler Agar is used with or without blood in cultivating and
enumerating anaerobic and aerobic microorganisms.
Bacto Schaedler Broth is used for cultivating anaerobic and aerobic
microorganisms with or without added blood or enrichment.

Schaedler Agar and Schaedler Broth are prepared according to the
formulation described by Schaedler, Dubos and Costello1 and modified

Schaedler Agar
Solution:
deionized water on boiling. Light to
Prepared Medium:
Reaction of 4.19%
Solution at 25C
pH 7.6 0.2
Schaedler Broth
Solution:
deionized water on boiling 1-2
minutes. Light to medium amber,
clear to slightly opalescent, may have
a very slight black precipitate.
Prepared Medium:
slightly opalescent, may have a very
slight black precipitate.
Reaction of 2.84%
Solution at 25C:
pH 7.6 0.2
Cultural Response
Prepare Schaedler Agar or Schaedler Broth per label directions.
Prereduce Schaedler Broth prior to inoculation with anaerobic
organisms. Inoculate medium; incubate at 35 2C for 18-48
hours under aerobic or anaerobic conditions, depending on the
requirements of the inoculum.
ORGANISM
Clostridium novyi B
ATCC
INOCULUM
CFU
GROWTH
25285*
8482
27606
19615*
100-1,000
100-1,000
100-1,000
100-1,000
good
good
good
good
Incubate anaerobically.
452
by Mata, Carrillo and Villatoro.2 Modifications include reduced dextrose

to avoid interference with hemolytic reactions and reduced yeast
extract to avoid darkening of the medium3 as well as adjusted sodium
chloride and peptone concentrations. Schaedler Broth is the same
formulation as Schaedler Agar but with the agar omitted.
While studying the gastrointestinal flora of mice, Schaedler et al.1
formulated a medium to recover both aerobic and anaerobic microorganisms. Mata et al.2 used a modification of the Schaedler formula to
study human fecal microflora.
Stalons, Thornsberry and Dowell4 evaluated nine broth media in varied
carbon dioxide atmospheres for their ability to support growth of
anaerobic bacteria. Schaedler Broth in an atmosphere of 5% CO2,
10% hydrogen and 85% nitrogen exhibited the fastest and highest
growth response.
Anaerobic bacteria cause a variety of human infections including
endocarditis, meningitis, wound infections following bowel surgery
or trauma, and bacteremia.5,6 Since anaerobes vary in their sensitivity
to oxygen and nutritional requirements7, appropriate collection, culture
medium and incubation are vital to recovery.7 Schaedler media are
suitable for standard procedures used in cultivating anaerobic bacteria.7,8,9

Tryptic Soy Broth, Proteose Peptone No.3 and Yeast Extract provide
the vitamins, nitrogen and amino acids in Schaedler media. Dextrose
is a carbon source, and Tris (Hydroxymethyl) Amino Methane is used
to buffer the medium. Hemin (X factor) stimulates growth. Bacto Agar
is the solidifying agent in Schaedler Agar.
The following supplements can be added to Schaedler media.
Sheep, horse or rabbit blood (5%) - for enrichment and for detecting
hemolysis and pigment production.9
Vitamin K1 (1%) - to promote growth of some pigmented Prevotella
and Porphyromonas spp. (formerly known as Bacteroides).9
Colistin and nalidixic acid (0.01 grams/liter, each) (Schaedler
CNA agar) - for selectively isolating anaerobic gram-positive cocci.10
Kanamycin (0.01 grams/liter) and vancomycin (7.5 mg/liter)
(Schaedler KV Agar) - for selectively isolating anaerobic
gram-negative bacteria.10
Formula
Schaedler Agar
Formula Per Liter
Bacto Tryptic Soy Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Proteose Peptone No.3 . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Tris (Hydroxymethyl) Amino Methane . . . . . . . . . . . . . . . . 3
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4
Hemin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5
g
g
g
g
g
g
g
g

Schaedler Broth
Formula Per Liter
Bacto Tryptic Soy Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Proteose Peptone No.3 . . . . . . . . . . . . . . . . . . . . . . . . 5 g
The Difco Manual
Section II
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Tris (Hydroxymethyl) Amino Methane . . . . . . . . . . . . . . . . 3
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4
Hemin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01
Schaedler Agar & Schaedler Broth

g
g
g
g
Precautions
Storage
Expiration Date
Procedure
Materials Provided
Schaedler Agar
Schaedler Broth

Glassware
Autoclave
Incubator (35C)
Sterile defibrinated sheep, horse or rabbit blood (optional)
deionized water:
Schaedler Agar - 41.9 grams/liter;
Schaedler Broth - 28.4 grams/liter.
2. OPTIONAL: Add 1 ml of 1% vitamin K1 in absolute ethanol.
3. Heat to boiling for 1-2 minutes to dissolve completely.

Test Procedure
For a complete discussion of aerobic and anaerobic bacteria from
clinical specimens, refer to the appropriate procedures outlined in the
references. 7,8,9 For the examination of bacteria in food, refer to
standard methods.11,12,13
Results
The Difco Manual

isolate recovered to ensure that the organism is an anaerobe.7
5. Because of the high dextrose concentration in Schaedler Agar when
it is supplemented with 5% blood, beta-hemolytic streptococci may
produce a hemolytic reaction that is similar to alpha hemolysis.
References
1. Schaedler, R. W., R. Dubos, and R. Costello. 1965. The
development of the bacterial flora in the gastrointestinal tract of
mice. J. Exp. Med. 122:59.
2. Mata, L. J. , C. Carrillo, and E. Villatoro. 1969. Fecal
microflora in healthy persons in the preindustrial region. Appl.
Microbiol. 17:596.
3. MacFaddin, J. D. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, p. 695-699, vol. 1.
4. Stalons, D. R., C. Thornsberry, and V. R. Dowell, Jr. 1974.
Effect of culture medium and carbon dioxide concentration on
growth of anaerobic bacteria commonly encountered in clinical
specimens. Appl. Microbiol. 27:1098-1104.
and H. J. Shadmony (ed.). 1991. Manual of clinical microbiology,
Charles C. Thomas, Springfield, Il.
Book, Inc. St. Louis, MO.
Packaging
Schaedler Agar
500 g
0403-17
Schaedler Broth
500 g
0534-17
453
Selenite Broth
Section II
Bacto Selenite Broth
Intended Use
Bacto Selenite Broth is used for enriching Salmonella spp. during
isolation procedures and for isolating Salmonella in foods.
Also Known As
Selenite Broth is also referred to as Selenite F (Fecal) Broth.

Selenite Broth is used as a selective enrichment for the cultivation
of Salmonella spp. that may be present in small numbers and
competing with intestinal flora. Salmonella organisms are also injured
in food-processing procedures, including exposure to low temperatures,
sub-marginal heat, drying, radiation, preservatives or sanitizers.1
Although injured cells may not form colonies on selective media, they
can cause infection if ingested.2 Salmonella spp. cause many types of
infections, from mild self-limiting gastroenteritis to life-threatening
typhoid fever.3 The most common form of Salmonella disease is
self-limiting gastroenteritis with fever lasting less than 2 days and
diarrhea lasting less than 7 days.3
The formula of Selenite Broth is described by Leifson 4 as Selenite F
Broth. Guth,5 according to Handel and Theodorascu, observed that
Escherichia coli was more susceptible to the toxicity of sodium selenite

Dehydrated Appearance: Off-white, free-flowing,
homogeneous.
Solution:
or deionized water on boiling; very
light amber, clear to very slightly
precipitate.
Prepared Medium:
slight precipitate.
Reaction of 2.3%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepare Selenite Broth per label directions. Incubate inoculated
medium at 35 2C for 18-24 hours. After incubation,
subculture onto MacConkey Agar plates and incubate plated
media at 35 2C for 18-24 hours.
ORGANISM
ATCC
CFU
Escherichia
coli
25922* 100-1,000
Salmonella
typhimurium
14028* 100-1,000
GROWTH
partial to
marked
inhibition
good
MACCONKEY
AGAR
pink
w/bile ppt
colorless
454
than S. typhi. Guth employed sodium selenite as a selective agent in

agar medium and enrichment broth for the isolation of S. typhi from
feces. Leifson4 extended Guths observations and developed a selenite
agar and selenite broth for the isolation of typhoid and paratyphoid
bacilli from clinical specimens.
Leifson 4 found the selenite broth was not sufficiently toxic to
completely inhibit fecal coliforms and enterococci. These organisms
were inhibited during the first 8 -12 hours but increased rapidly after
this time period. Salmonella spp. multiply fairly rapidly after
inoculation. It is suggested that selenium toxicity may be a reaction
with sulphur and sulphydryl groups in certain strains of bacteria.6,7
There have been many modifications of Selenite Broth from the original
formula described by Leifson. Selenite Cystine Broth is used as a
selective enrichment broth recommended by AOAC 8 and USP 9 for
detecting Salmonella in food, dairy products and other materials of
sanitary importance. Selenite Brilliant Green Sulfa (SBS) Enrichment
Broth and Selenite Brilliant Green Mannitol (SBM) Enrichment Broth
have also been used for the cultivation of Salmonella.10
Selenite Broth conforms with APHA11 and is specified in Clinical
Microbiology Procedures Handbook 12 and Manual of Clinical
Microbiology.13

Bacto Tryptone provides the nitrogen, vitamins and amino acids in
Selenite Broth. Bacto Lactose is a fermentable carbohydrate. Selenite
is reduced by organism growth. A rise in pH decreases the selective
activity of the selenite. The acid produced by lactose fermentation helps
to maintain a neutral pH. Sodium selenite inhibits the growth of
gram-positive bacteria and many gram-negative bacteria. Sodium
phosphate is a buffering agent.
Formula
Selenite Broth
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Sodium Selenite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Sodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
g
g
g
g
Precautions
2. Very TOXIC. FATAL IF INHALED OR SWALLOWED.(US)
VERY TOXIC BY INHALATION AND IF SWALLOWED.(EC)
DANGER OF CUMULATIVE EFFECTS.(EC) IRRITATING TO
EYES, RESPIRATORY SYSTEM AND SKIN. Avoid contact with
skin and eyes. Do not breathe dust. Wear suitable protective
clothing. Keep container tightly closed. TARGET ORGAN(S):
Lungs, Kidneys, Spleen, Liver.
wash immediately with plenty of water. If skin irritation persists,
seek medical advice. If inhaled, remove to fresh air. If not breathing,
give artificial respiration. If breathing is difficult, give oxygen.
Seek medical advice. If swallowed, induce vomiting; seek medical
The Difco Manual
Section II

2. Sorrells, K. M., M. L. Speck, and J. A. Warren. 1970.

Pathogenicity of Salmonella gallinarum after metabolic injury by
freezing. Appl. Microbiol. 19:39- 43.
Tenover and R. H. Yolken (ed.), Manual of clinical microbiology,
4. Leifson, E. 1936. New selenite selective enrichment medium for
the isolation of typhoid and paratyphoid (Salmonella) bacilli. Am.
J. Hyg. 24:423.
5. Guth, F. 1916. Centr. Bakt. I Abt. Orig. 77:487.
6. Weiss, K. F., J. C. Ayres, and A. A. Kraft. 1965. Inhibitory action
of selenite on Escherichia coli, Proteus vulgaris, and Salmonella
thompson. J. Bacteriol. 90:857-862.
7. Rose, M. J., N. K. Enriki, and J. A. Alford. 1971. Growth and
survival of enterobacteria in selenite-cystine broth containing
thiosulfate. J. Food Sci. 36:590-593.
10. MacFaddin, J. F. 1985. Media for isolation-cultivationidentification- maintenance of medical bacteria, p. 701-705, vol 1,
11. Russell, S. F., J.-Y. DAoust, W. H. Andrews, and J. S. Bailey.
1992. Salmonella, p. 371-422. In Vanderzant, C., and D. F.
Splittstoesser (ed.) Compendium of methods for the microbiological
Washington, D.C.
handbook, vol. 1, American Society for Microbiology,
Washington. D.C.
R. H. Yolken (ed.) 1995. Manual of clinical microbiology, 6th ed.

Packaging
References
Selenite Broth
Storage
Expiration Date
Procedure
Material Provided
Selenite Broth

Glassware
Incubator
Sterile tubes
2. Heat to boiling to pasteurize.
3. Avoid overheating. DO NOT AUTOCLAVE.

Test Procedure
of Salmonella species refer to the appropriate procedures outlined
in the references.
Results

identification of salmonellae in foods. J. Food Prot. 44:385-386.
Bacto Selenite Cystine Broth
100 g
500 g
10 kg
0275-15
0275-17
0275-08
Intended Use
Bacto Selenite Cystine Broth is used for selectively enriching
Salmonella in food and water.

Selenite Cystine Broth is the formulation by Leifson1 with cystine
added. Leifson determined that Selenite Broth favored the growth of
The Difco Manual
Salmonella while reducing growth of fecal coliforms and enterococci.1

The growth and recovery of Salmonella in food samples can be
hindered by non-Salmonella bacteria, substances indigenous to the food
sample, and in dried, processed food, the Salmonella may be present in
low numbers and in a injured condition.2 Using protocols that involve
preenrichment, selective enrichment and selective plating increase
the likelihood of recovering Salmonella. In most standard method
procedures Selenite Cystine Broth is recommended in the selective
enrichment step.2,3,4,5,6 As a selective enrichment medium, Selenite
455
Section II
Cystine Broth is formulated to allow the proliferation of Salmonella and

while inhibiting the growth of competing non-Salmonella bacteria.2

Selenite Cystine Broth contains Tryptone as a source of carbon,
nitrogen, vitamins and minerals. Lactose is the carbohydrate. Sodium
Acid Selenite inhibits gram-positive bacteria and most enteric
gram-negative bacteria except Salmonella. L-cystine is a reducing agent.
Formula
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Acid Selenite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01
g
g
g
g
g
Precautions
2. VERY TOXIC. FATAL IF INHALED OR SWALLOWED. (US)
VERY TOXIC BY INHALATION AND IF SWALLOWED. (EC)
DANGER OF CUMULATIVE EFFECTS. (EC) IRRITATING TO
EYES, RESPIRATORY SYSTEM AND SKIN. Avoid contact with
skin and eyes. Do not breathe dust. Wear suitable protective

Solution:
deionized water on boiling.
Prepared Medium:
slightly opalescent, may have a slight
precipitate.
Reaction of 2.3%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Escherichia
coli
Salmonella
typhimurium
Shigella sonnei
ATCC
INOCULUM
CFU
25922* 100-1,000
GROWTH
APPEARANCE
partial to
complete
inhibition
pink with bile

precipitate
14028* 100-1,000
good
9290* 100-1,000 fair to good
colorless
colorless
456
Storage
Expiration Date
Procedure
Materials Provided

Glassware
Autoclave
Incubator (35C)
Tetrathionate Broth
XLD Agar
MacConkey Agar
1.
2.
3.
4.

Dispense into tubes to a depth of 60 mm.
DO NOT AUTOCLAVE. Use immediately.
Prepare Selenite Cystine Broth per label directions. Inoculate

and incubate the tubes at 35 2C for 24 2 hours and
subculture on MacConkey Agar plates.
ORGANISM
TARGET ORGAN(S): Lungs, Kidneys, Spleen, Liver.

wash immediately with plenty of water. If skin irritation persists,
seek medical advice. If inhaled, remove to fresh air. If not
breathing, give artificial respiration. If breathing is difficult, give
oxygen. Seek medical advice. If swallowed, induce vomiting; seek
Test Procedure4,5
1. Prepare sample according to food type.
2. Inoculate into recommended pre-enrichment broth.
3. Transfer 1 ml of mixture to 10 ml Selenite Cystine Broth and to
10 ml Tetrathionate Broth.
5. Mix and streak 3 mm loopful (10 l) of sample from both broths
onto Bismuth Sulfite Agar, Xylose Lysine Desoxycholate Agar,
Hektoen Enteric Agar or MacConkey Agar.
6. Incubate plates at 35C for 24 2 hours.
7. Examine plates for the presence of colonies that are typical for
Salmonella spp.
The Difco Manual
Section II
Simmons Citrate Agar
Results
Marshall (ed.), Standard methods for the microbiological

R. M. Amaguana. 1995. Salmonella, p. 5.01-5.20. In Bacteriological
5. Andrews, W. 1995. Microbial methods, p. 1-119. In Official

1. A brick red precipitate may appear if Selenite Cystine Broth is
overheated during preparation or exposed to excessive moisture
during storage.
References
1. Leifson, E. 1936. New selenite selective enrichment medium for
the isolation of typhoid and paratyphoid (Salmonella) bacilli.
Am J. Hyg. 24:423-432.
Washington, D.C.
3. Flowers, R. S., W. H. Andrews, E. W. Donnely, and E. Koenig.
Bacto Simmons Citrate Agar
Intended Use
Bacto Simmons Citrate Agar is used for differentiating Enterobacteriaceae
based on citrate utilization.

Koser1 first developed a liquid medium for differentiating coliforms
from fecal coliforms. Fecal coliforms were unable to use citrate as the
sole source of carbon and inorganic ammonium salt as a sole source of
nitrogen. Non-fecal coliforms, such as Enterobacter aerogenes or
Salmonella enteritidis could use citrate in such a medium with resultant
alkalinity. The liquid medium had the disadvantage of appearing turbid
when large inocula were used although no growth had taken place.
This observation led Simmons2 to devise a solid medium that eliminated
the problem with turbidity.
Packaging
100
500
2
10
g
g
kg
kg
0687-15
0687-17
0687-07
0687-08
dihydrogen phosphate and sodium citrate as their sole sources of nitrogen

and carbon will grow on this medium and produce a color change from
green (neutral) to blue (alkaline).
Formula
Formula Per Liter
Ammonium Dihydrogen Phosphate . . . . . . . . . . . . . . . . . . . 1
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g
Precautions
Simmons Citrate Agar is a modification of Kosers medium to which

brom thymol blue and 1.5% agar have been added. Organisms able to
metabolize the citrate grow luxuriantly. The medium is alkalinized and
changes from its initial green to deep blue in 24-48 hours. E. coli either
do not grow at all on this medium, or grow so sparsely that no change
in reaction is apparent.
Simmons Citrate Agar is recommended for differentiation of enteric
gram-negative bacilli from clinical specimens,3,4 water samples,5 and
food samples.6-9

The ammonium dihydrogen phosphate is the sole source of nitrogen

in Simmons Citrate Agar. Magnesium is a cofactor for a variety of
metabolic reactions. Phosphate acts as a buffer. Sodium citrate is the
sole source of carbon in this medium. Sodium chloride maintains the
osmotic balance of the medium. Agar is the solidifying agent. Brom
thymol blue is the pH indicator. Organisms that can utilize ammonium
The Difco Manual
Storage
very hygroscopic. Keep container tightly closed. Store prepared tubes
at 2-8C.
Expiration Date
Procedure
Materials Provided
457
Section II
Results

Tubes with closures
Autoclave
Incubator (35C)
A positive reaction is indicated by growth on the slant with an

intense blue color (alkaline reaction). A negative reaction is indicated
by no growth to poor growth without change in color (medium
remains green).
1. When inoculating a variety of biochemicals, flame the inoculating

loop or needle before streaking Simmons Citrate Agar or inoculate
Simmons Citrate Agar first to avoid a false positive result.10
2. Some citrate positive organisms require 48 hours or longer
incubation for a pH change to occur.10
1.
2.
3.
4.

Autoclave at 121C for 15 minutes. Cool in a slanted position with
long slant and short butt.

Test Procedure
1. Obtain a pure culture of the organism to be tested.
2. With an inoculating needle or loop, pick the center of a
well-isolated colonies obtained from solid culture media.
3. Streak only the surface of the slant with a light inoculum.
4. Loosen the closure on the tube.
References
1. Koser, S. A. 1923. Utilization of the salts of organic acids by the
colon-aerogenes group. J. Bacteriol. 8:493.
2. Simmons, J. S. 1926. A culture medium for differentiating
organisms of typhoid- colon aerogenes groups and for isolation of
certain fungi. J. Infect. Dis. 39:209.
Isenberg, H.D. (ed.), Clinical microbiology procedures handbook,
4. Baron, E. J., L. R. Peterson, S. M. Finegold. 1994. Bailey &
St. Louis, MO.

Dehydrated Appearance: Mustard yellow to yellow-green,
free flowing, homogeneous.
Solution:
is forest green, slightly opalescent,
Prepared Tubes:
Forest green, slightly opalescent, may
Reaction of 2.42%
Solution at 25C:
pH 6.8 0.2
Cultural Response
Prepare Simmons Citrate Agar per label directions. Inoculate
with 1 l of a dilution equivalent to a 0.5 McFarland Standard
and incubate the tubes at 35 2C for 18-48 hours.
ORGANISM
ATCC
Enterobacter aerogenes 13048*

Escherichia coli
25922*
GROWTH
COLONY COLOR
good
none to poor
good
blue
green
blue
Uninoculated
tube
ATCC 13048
Escherichia coli
ATCC 25922
458
The Difco Manual
Section II
Skim Milk
7. FDA Bacteriological Analytical Manual, 8th ed. AOAC

61:38917-38925.
Packaging
100 g
500 g
0091-15
0091-17
Bacto Skim Milk
fermentation and for detecting proteolytic enzymes that hydrolyze casein

(milk protein) and cause coagulation (clot formation).3
Intended Use
Bacto Skim Milk is used for preparing microbiological culture media

and for differentiating organisms based on coagulation and proteolysis
of casein.
Skim Milk is a source of lactose and casein. In the differential test medium, Litmus Milk, lactose fermentation is detected by the pH indicator,
litmus. Hydrolysis of casein is detected by visible formation of a clot.
Formula
Skim Milk is soluble, spray-dried skim milk. When prepared in a 10%

solution, it is equivalent to fresh skim milk.
Skim Milk can be used to prepare skim milk agar for detecting proteolytic
microorganisms in foods1, including dairy products.2 It can also be used to
prepare litmus milk, a differential test medium for determining lactose
Skim Milk
Formula Per Liter
Skim Milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 g
Precautions

homogeneous.
Solution:
10% solution, soluble in distilled
Solution is white, opalescent. After
autoclaving, solution is off-white to
beige, opaque.
Reaction of 10%
Solution at 25C:
pH 6.3 0.2
Chemical Test:
Positive reaction with 3,5-dinitro
salicylic acid.*
Storage
* Place 2 drops of a 2% solution of Skim Milk on filter paper and air dry.
Dispense 3 drops of 0.5% 3,5-dinitro salicylic acid in 4% sodium
hydroxide over the spot. Heat to 105C for 5 minutes and note color
development. A positive test is indicated by development of a brown color.
Materials Provided
Cultural Response
Prepare Skim Milk per label directions. Inoculate with a
drop or loopful of undiluted culture and incubate the tubes
at 35 2C for 1-7 days.
ORGANISM
Lactobacillus casei
Escherichia coli
ATCC
GROWTH
APPEARANCE
9595
25922*
12919
good
good
good
acid, reduction, curd

acid, reduction, curd
stormy fermentation
The Difco Manual

Expiration Date
Procedure
Skim Milk

Glassware
Autoclave
Incubator (35C)
1. Dissolve 100 grams in 1 liter distilled or deionized water (with
warming, if necessary).

459
Snyder Test Agar
Section II
Test Procedure
Results

Skim Milk supports growth of many microorganisms. Perform
microscopic examination and other biochemical tests to identify
isolates to the genus and species level, if necessary.
References
1. Lee, J. S., and A. A. Kraft. 1992. Proteolytic microorganisms,
p. 193-198. In C. Vanderzant and D. F. Splittstoesser (ed.).
Bacto Snyder Test Agar

for groups of microorganisms, p. 271-286. In Marshall, R. T. (ed.)
Standard methods for the microbiological examination of dairy
products, 16th ed. American Public Health Association,
Washington, D.C.
3. MacFaddin, J. F. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, p. 440-445. Williams
Packaging
Skim Milk
500 g
0032-17
subsurface carious lesion becomes a clinical cavity with extension of

the decay into the dentine.2
Intended Use
Bacto Snyder Test Agar is used for estimating the relative number of
lactobacilli in saliva based on acid production.
Also Known As
Snyder 3,4 described a test procedure for determining, by colorimetric

analysis, the rate and amount of acid produced by microorganisms in
saliva. The procedure uses an agar medium that is known as Snyder
Test Agar. Alban5 simplified the procedure, used it extensively and
reported it to be more accurate than Snyders original procedure.
BCG Dextrose Agar1
Snyder Test Agar contains Tryptose as a source of carbon, nitrogen,

vitamins and minerals. Dextrose is the carbohydrate. Brom Cresol
Green is the pH indicator. Bacto Agar is the solidifying agent.
Tooth decay (dental caries) is a localized, progressive demineralization

of the hard tissues of the crown and root surfaces of teeth. Streptococcus
mutans and possibly lactobacilli ferment dietary carbohydrates that
produce acids that cause the demineralization. The organisms reside
in dental plaque, which is a gelatinous material that adheres to the
surfaces of teeth. Demineralization of the tooth alternates with periods
of remineralization. If demineralization exceeds remineralization, a
Microorganisms that use the dextrose in the medium acidify the

medium and the pH indicator, brom cresol green, changes color from
blue-green to yellow.

Dehydrated Appearance: Light green, free-flowing, homogeneous.
Solution:
on boiling. Solution is dark emerald green, slightly
opalescent.
Prepared Medium:
Dark emerald green, slightly opalescent.
Reaction of 6.5%
Solution at 25C:
pH 4.8 0.2
Cultural Response
Prepare Snyder Test Agar per label directions. Inoculate and incubate the
tubes at 35 2C for 18-72 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
ACID
PRODUCTION
Lactobacillus casei
9595
9338
100-1,000
100-1,000
good
good
+
+
460
Uninoculated
tube
ATCC 9338
The Difco Manual
Section II
Snyder Test Agar
Formula
3. Rotate the inoculated tubes to mix the inoculum uniformly with

the medium and allow to solidify in an upright position.
4. Incubate at 35C. Observe color at 24, 48 and 72 hours.
Snyder Test Agar

Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Brom Cresol Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.02
g
g
g
g
g
Final pH 4.8 0. 2 at 25C
Precautions
Storage
Expiration Date
Procedure
Materials Provided
Alban Modication
1. Collect enough unstimulated saliva to just cover the medium in the
tube. When specimen collection is difficult, dip a sterile cotton
swab into the saliva under the tongue or rub on tooth surfaces and
place the swab just below the surface of the medium.
2. Incubate the inoculated tubes and an uninoculated control at 35C.
3. Examine tubes daily for four days.
4. Observe daily color change compared to control tube.
Results
Snyder Procedure
Observe tubes for a change in color of the medium from bluish-green
(control) to yellow. A positive reaction is a change in color so that
green is no longer dominant. Record as ++ to ++++. A negative
reaction is no change in color or only a slight change. Green is still
dominant. Record as 0 to +.
Interpretation:
CARIES ACTIVITY
Marked
Moderate
Slight
Negative
24
HOURS INCUBATION
48
72
Positive
Negative
Negative
Negative
Positive
Negative
Negative
Positive
Negative
Snyder Test Agar

Glassware
Petri dishes
Autoclave
Incubator (35C)
Waterbath (45C)
Cotton Swab
Paraffin

Specimens should be collected preferably before breakfast, lunch, or
dinner, and before the teeth are brushed. This procedure can be done
just before lunch or dinner.
Test Procedure
Snyder Procedure 3,4
1. Collect specimens of saliva in a sterile container while patient is
chewing paraffin for 3 minutes.
2. Shake specimens thoroughly and transfer 0.2 ml to a tube of sterile
Snyder Test Agar melted and cooled to 45C. (Prepared medium
in tubes is heated in a boiling water bath for 10 minutes and
cooled to 45C.
The Difco Manual
Data summarizing the correlation between the Snyder colorimetric test

and Lactobacillus counts on specimens of saliva collected routinely
are tabulated.
Alban Modification
a. No color change
b. Color beginning to change to yellow from top of medium down (+)
c. One half of medium yellow (++)
d. Three fourths of medium yellow (+++)
e. The entire medium is yellow (++++)
The final report is a composite of the daily readings, for example;
+ ++ +++. The readings indicate the rapidity and amount of acid
production.

1. The data indicate only what is happening at the time the specimen
was collected.
2. At least two specimens collected with 2-4 days must be obtained
to establish a base-line or reference point.
3. Only when two or more specimens have been cultured can any
reliability or prediction be obtained.
4. The clinician must study enough cases by use of periodic laboratory
data to establish the value of significance for the purpose intended.
References
1. MacFaddin, J. F. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, p. 713-715. vol. 1.
461
Soytone & Soytone No. 2
Section II
2. Lewis, D. W., and A. I. Ismail. 1995. Periodic health examination,

1995 update: 2. Prevention of dental caries. Canadian Medical
Association Journal 152:836- 846.
3. Snyder. 1941. J. Dent. Res. 20:189.
4. Snyder. 1941. J. Am. Dent. Assoc. 28:44.

5. Alban. 1970. J. Dent. Res. 49:641.
Packaging
Snyder Test Agar
Bacto Soytone
Bacto Soytone No. 2
500 g
0247-17
Intended Use
Bacto Soytone and Bacto Soytone No. 2 are enzymatic digests of
soybean meal.
Also Known As
Soytone is also known as Peptone S and Peptone Soya.
Soytone and Soytone No. 2 are enzymatic hydrolysates of soybean

meal prepared under controlled conditions for use in microbiological
procedures. They are recommended for use in media for the cultivation of a large variety of organisms, including fungi and microbiological assay media. The nitrogen source in Soytone and Soytone No. 2
contains the naturally occurring high concentrations of vitamins and
carbohydrates of soybean. Media supplemented with blood produce
typical bacterial hemolytic patterns with Soytone and Soytone No. 2
as the main source of nitrogen.
Soytone, Soytone No. 2

Dehydrated Appearance: Light to medium tan, free-flowing,
homogenous.
Solution:
deionized water. Light to medium
opalescent.
Reaction of 1%
Solution at 25C:
pH 7.0 0.5
Cultural Response
TEST
SOLUTION OF
SOYTONE OR
SOYTONE NO.2 ORGANISM
ATCC
Fermentable 2%
Escherichia 25922*
Carbohydrate
coli
Indole
0.1%
Escherichia 25922*
Production
coli
Acetylmethyl- 1%
Enterobacter 13048*
carbinol
w/0.5%
aerogenes
Production NaCl and
0.5% dextrose
Hydrogen
1%
Salmonella 14028*
Sulfide
typhimurium
Production
TEST
Growth
Response
Growth
Response
Growth
Response
1.5% agar
SOLUTION OF
SOYTONE OR
SOYTONE NO.2
2% w/0.5%
NaCl and
1.5% agar
2% w/0.5%
NaCl and
1.5% agar
2% w/0.5%
NaCl and
SOYTONE SOYTONE NO.2

RESULT
RESULT
positive negative
Soytone is an enzymatic digest of soybean meal.
positive
positive
Soytone No. 2 is a papaic digestion of soybean meal.
positive
positive
Typical Analysis
Soytone
positive
positive
Ash (%)
12.0
1.2
Loss on Drying (%)

pH, 1% Solution
4.6
7.2
Carbohydrate (%)
ORGANISM
ATCC
Brucella suis
4314
Escherichia
coli
RESULT
good growth
25922* good growth
Staphylococccus 25923* good growth

aureus
*These culture are available as Bactrol Disks and should be used as
462
Soytone No. 2 minimizes Bovine Spongiform Encephalopathy (BE)

risk in vaccine production because the enzyme used is also of plant
origin.
Total
24.0

Total Nitrogen
Amino Nitrogen
9.4
3.1
AN/TN
33.0
Amino Acids (%)

Alanine
Arginine
Aspartic Acid
Cystine
Glutamic Acid
Glycine
Histidine
Isoleucine
Leucine
2.46
3.82
7.27
1.45
12.76
2.51
1.24
2.37
4.03
Lysine
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine
3.45
0.86
2.46
2.92
2.87
2.17
0.47
1.93
2.65
The Difco Manual
Section II
Spirit Blue Agar & Lipase Reagent
Inorganics (%)
Calcium
Chloride
Cobalt
Copper
Iron
Lead
Magnesium
Manganese
0.055
0.165
<0.001
<0.001
0.008
<0.001
0.161
<0.001
Phosphate
Potassium
Sodium
Sulfate
Sulfur
Tin
Zinc
0.820
2.220
3.404
2.334
1.660
<0.001
0.001
Procedure
Materials Provided
Soytone
Soytone No. 2

Vitamins (g/g)
Biotin
0.2
Cyanocobalamin
<0.1
Folic Acid
3.0
Inositol
2100.0
Nicotinic Acid
19.1
PABA
Pantothenic Acid
Pyridoxine
Riboflavin
Thiamine
Thymidine
9.0
13.0
11.0
<0.1
1.2
113.2

Coliform
Salmonella
Spore Count

negative
negative
10

Thermophile Count
38
<3
Precautions
Refer to the final concentration of Soytone or Soytone No. 2 in the
formula of the medium being prepared. Add Soytone or Soytone No. 2
as required.

Test Procedure
See appropriate references for specific procedures using Soytone or
Soytone No. 2.
Results
Storage
Packaging
Store Soytone and Soytone No. 2 below 30C. The products are very
Soytone
500 g
10 kg
0436-17
0436-08
Expiration Date
Soytone No. 2
500 g
10 kg
0508-17
0508-08
Bacto Spirit Blue Agar

Lipase Reagent
Intended Use
Bacto Spirit Blue Agar is for use with Bacto Lipase Reagent or other
lipid source for detecting and enumerating lipolytic microorganisms.

In 1941, Starr1 described a lipid emulsion medium for detecting
lipolytic (lipase-producing) microorganisms to which he added the dye,
spirit blue. Other dyes as indicators of lipolysis were toxic to many
microorganisms. Spirit blue did not have toxic effects. When testing
samples of dairy products, air and sewage on Spirit Blue Agar, Starr
obtained accurate counts of lipolytic microorganisms and total
microbial counts on the same medium.
Lipolytic microorganisms, such as psychrotrophic bacteria, molds or
yeasts, can adversely affect the flavor of milk and high fat dairy
products. Spirit Blue Agar is a recommended medium for testing milk
and dairy products.2
The Difco Manual
Lipase Reagent, a mixture of tributyrin and Polysorbate 80, is

recommended as the lipid source. Other lipoidal emulsions may be
prepared from cottonseed meal, cream, Wesson oil and olive oil. A
satisfactory emulsion can be prepared by dissolving 10 grams gum
acacia or 1 ml Tween 80 in 400 ml warm distilled water, adding 100
ml cottonseed or olive oil and agitating vigorously to emulsify.

Spirit Blue Agar contains Tryptone as a source of carbon, nitrogen,
vitamins and minerals. Yeast Extract supplies B-complex vitamins
which stimulate bacterial growth. Spirit Blue is the indicator of lipolysis.
Lipase Reagent contains tributyrin, a true fat and the simplest triglyceride
occurring in natural fats and oils. It is a good substrate when testing for
lipolytic microorganisms because some microorganisms that hydrolyze
tributyrin will not hydrolyze other triglycerides or fats containing
longer chain fatty acids.2
463
Spirit Blue Agar & Lipase Reagent
Section II
Formula
Procedure
Spirit Blue Agar

Formula Per Liter
Materials Provided
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Spirit Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.15
Spirit Blue Agar

Lipase Reagent
g
g
g
g

Glassware
Petri dishes
Autoclave
Incubator (35C)

Lipase Reagent
A ready-to-use lipid suspension, containing a mixture of
tributyrin and Polysorbate 80.
Precautions
Storage
Store the Lipase Reagent at 15-30C.
1. Suspend 35 grams of Spirit Blue Agar in 1 liter distilled or

deionized water.
4. Aseptically add 30 ml Lipase Reagent or other lipid source and
mix thoroughly.

Collect specimens in sterile containers and transport immediately to
the laboratory following recommended guidelines.
Expiration Date
Uninoculated
plate
ATCC 25923

Spirit Blue Agar
Dehydrated Appearance: Grayish-beige, free-flowing, homogeneous.
Solution:
is royal blue, slightly opalescent.
Prepared Medium:
plain - royal blue, opalescent
plain + 3% Lipase reagent - pale blue,
opalescent
Reaction of 3.5%
Solution at 25C:
pH 6.8 0.2
Lipase Reagent
Appearance:
White, opaque emulsion
Cultural Response
Prepare Spirit Blue Agar per label directions, with the addition of 3% Lipase
Reagent after sterilization. Inoculate and incubate at 35 2C for up to 72 hours.
ORGANISM
Proteus mirabilis
ATCC
INOCULUM
CFU
25933
25923*
6538
12228*
100-1,000
100-1,000
100-1,000
100-1,000
GROWTH HALO/LIPOLYSIS
good
good
good
good
no halo
halo
halo
halo
ATCC 12228
464
The Difco Manual
Section II
m Staphylococcus Broth
Test Procedure
References
1. Inoculate organism onto medium.

2. Incubate plates at 35 2C for up to 72 hours.
Lipolytic microorganisms metabolize the lipid in the medium and
form colonies with halos indicating lipolysis.
1. Starr, M. P. 1941. Spirit blue agar: a medium for the detection of

lipolytic microorganisms. Science 93:333-334.
2. Frank. J. F., G. L. Christen, and L. B. Bullerman. 1993. Tests for
groups of microorganisms, p. 276-277. In R. T. Marshall (ed.), Standard methods for the microbiological examination of dairy products,
Packaging
1. Because the nutritional requirements of organisms vary, some

this medium.
Spirit Blue Agar
100 g
500 g
0950-15
0950-17
Lipase Reagent
6 x 20 ml
0431-63
Results
Bacto m Staphylococcus Broth
Intended Use
Bacto m Staphylococcus Broth is used for isolating staphylococci by
the membrane filtration technique.

Solution:
deionized water on warming. Solution
is light amber, clear to slightly
precipitate.
Prepared Medium:
precipitate.
Reaction of 10.4%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepare m Staphylococcus Broth per label directions. Use the
membrane filtration technique with the test organisms. Inoculate
and incubate at 35 2C under humid conditions for 40-48
hours. Plates are read for recovery and pigment production.
Mannitol fermentation is detected by adding a drop of Brom
Thymol Blue to the site where a colony was removed. Yellow
color indicates a positive result for Mannitol fermentation.
ORGANISM
ATCC
INOCULUM
MANNITOL
PIGMENT
CFU
GROWTH FERMENTATION PRODUCTION
Escherichia
25922* 20-200 inhibited
coli
Staphylococcus 25923* 20-200 good
aureus
epidermidis
Staphylococci, along with other bacteria, are indicators of recreational

water quality.4 Indicators of health risk include normal skin flora that
are likely to be shed, such as Pseudomonas, Streptococcus, and
Staphylococcus.5 These organisms account for a large percentage of
swimming pool-associated illness.4
The coagulase-positive species, Staphylococcus aureus, is well documented
as a human opportunistic pathogen.3 Coagulase-negative Staphylococcus
spp. are a major component of the normal microflora of humans.3 Staphylococci are widespread in nature, though they are mainly found
living on the skin, skin glands, and mucous membranes of mammals
and birds.3
Chapman1 added 7.5% NaCl to Phenol Red Mannitol Agar to achieve
a selective medium for staphylococci. While studying this medium
formulation, Chapman 2 developed Staphylococcus Medium 110.
m Staphylococcus Broth is patterned after the formula of Staphylococcus
Medium 110.
m Staphylococcus Broth, with the addition of sodium azide, is specified
for Recreational Waters in Standard Methods for the Examination of
Water and Wastewater.4

Tryptone provides the nitrogen, amino acids and minerals in
m Staphylococcus Broth. Yeast Extract is the vitamin source in this
formula. Lactose and Mannitol are the carbohydrates for bacterial
growth. Dipotassium Phosphate is the buffering agent. The high
concentration of Sodium Chloride permits this medium to be selective
for staphylococci.
N/A
Formula
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Bacto Mannitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
g
g
g
g
g
g

The Difco Manual
465
Staphylococcus Medium 110
Section II
Precautions
Test Procedure

1. Follow the membrane filtration procedure described in Standard

Methods, Section 9213,4 or as described by laboratory procedures.
2. Use 2.0-2.5 ml of medium to saturate the paper pads on which the
inoculated membrane is placed.
Storage
Expiration Date
Procedure
Materials Provided

Membrane filter
Autoclave
Glassware
Incubator (35C)
Sterile tubes
Paper pads
Results
Observe tubes for growth, indicating a positive reaction. Inoculate tubes
showing turbidity to the appropriate medium for confirmation of
Staphylococcus.

2. m Staphylococcus Broth is used in sequence with an additional
medium for confirmation. If necessary, confirm positive isolates
using biochemical reactions.
References

2. Warm to dissolve completely.
NOTE: When autoclave sterilization is not practical, boil medium for
5 minutes.
1. Chapman. 1945. J. Bacteriol. 50:201.

2. Chapman. 1946. J. Bacteriol. 51:409.
Micrococcus, p. 282-298. In P. R. Murray, E. J. Baron, M. A.
5. Seyfried, P. L., R. S. Tobin, N. E. Brown, and P. F. Ness. 1985.
A prospective study of swimming-related illness. II. Morbidity and
the microbiological quality of water. Amer. J. Public Health
75:1071.
Packaging
Collect water samples as described in Standard Methods, Section 92134

or as specified by laboratory procedures.
100 g
500 g
0649-15
0649-17
Bacto Staphylococcus Medium 110
Intended Use
Also Known As
adding 7.5% NaCl to Phenol Red Mannitol Agar to make a selective

isolation medium for staphylococci using a high salt content. Further
studies by Chapman5 led to the development of Staphylococcus
Medium 110. This medium is included in standard methods procedures
for selectively isolating pathogenic staphylococci from foods.6
Staphylococcus Medium 110 is also known as Staphylococcus Agar

No. 110 (Staphy-110, S-110) and Stone Gelatin Agar.1
Bacto Staphylococcus Medium 110 is used for isolating and differentiating

staphylococci based on mannitol fermentation, pigment formation and
gelatinase activity.

Stone2 described a culture medium on which food-poisoning staphylococci
gave a positive gelatinase test. Chapman, Lieb and Curcio3 later
reported that pathogenic staphylococci strains typically ferment
mannitol, form pigment and produce gelatinase. Chapman4 suggested
466
Staphylococcus Medium 110 contains Tryptone as a source of carbon,

vitamins which stimulate bacterial growth. Sodium Chloride, in high
concentration, inhibits most bacteria other than staphylococci. Lactose
and D-Mannitol are the carbohydrates. Gelatin is included for testing
liquefaction. Bacto Agar is the solidifying agent.
The Difco Manual
Section II
Pathogenic staphylococci (coagulase-positive staphylococci) typically

resist the high salt concentration and form colonies with a yellow-orange
pigment. These organisms typically ferment mannitol and produce acid,
and liquefy gelatin, producing zones of clearing around the colonies.
Precautions
Formula
Storage

Formula Per Liter

Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Bacto D-Mannitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g
g
Expiration Date
Procedure
Materials Provided

Dehydrated Appearance: Very light beige to beige,
Solution:
Solution is light amber, slightly
opalescent to opalescent, with
heavy precipitate.
Prepared Medium:
to opalescent.
Reaction of 14.9%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepare Staphylococcus Medium 110 per label directions.
Inoculate the plates and incubate the plates at 35 2C for
18-48 hours.
To test for mannitol fermentation, remove a colony from the
medium, add a drop of 0.04% brom thymol blue to the plate,
and observe for the formation of a yellow color (positive
reaction).
To test for gelatinase reaction, flood the plate with 5 ml of
saturated ammonium sulfate solution and incubate at 35 2C
for 10 minutes. Observe for a zone of clearing around the
colonies (positive reaction).
ORGANISM
ATCC
INOCULUM
CFU
GROWTH PIGMENT** Gelatinase Mannitol
Escherichia
coli
25922* 100-300 marked

to complete
inhibition
aureus
epidermidis
N/A
Glassware
Petri dishes
Autoclave
Incubator (35C)
0.04% Bromthymol blue
Saturated ammonium sulfate solution
1.
2.
3.
4.


Collect specimens or food samples in sterile containers or with sterile
swabs and transport immediately to the laboratory following
Test Procedure
Consult appropriate references for procedures concerning selection and
enumeration of staphylococci.6
Results
Growth of pathogenic staphylococci produces colonies with yelloworange pigment.
N/A

+
**Pigment is seen as a yellow to orange color.

The Difco Manual

1. Enterococcus faecalis may grow on Staphylococcus Medium 110

as tiny colonies with mannitol fermentation. Differentiate these
organisms from staphylococci with the Gram stain and catalase test.
2. Suspected staphylococci must be subcultured to Nutrient Broth,
Blood Agar, BHI Broth, or Tryptose Phosphate Broth for coagulase
testing as the high salt content of Staphylococcus Medium 110 may
interfere with results.
3. Pigment production is not a reliable criterion for differentiation of
staphylococcal spp.
467
Starch Agar
Section II
References
1. MacFaddin, J. F. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, vol. 1. p. 722-726.
2. Stone, R. V. 1935. A cultural method for classifying staphylococci
as of the food poisoning type. Proc. Soc. Exptl. Biol. Med.
33:185-187.
3. Chapman, G. H., C. W. Lieb, and L. G. Curcio. 1937. Isolation
and cultural differentiation of food-poisoning staphylococci. Food
Research. 2:349.
4. Chapman, G. H. 1945. The significance of sodium chloride in
studies of staphylococci. J. Bacteriol. 50:201.
Bacto Starch Agar
Bacto Starch Agar is used for cultivating microorganisms being tested

for starch hydrolysis.

In 1915,1 Vedder formulated Starch Agar for cultivating Neisseria.
Since then, other media have been developed that are superior to Starch
Agar for the isolation of Neisseria spp, including enriched GC
Medium Base. Starch Agar is used in differentiating microorganisms
based on the starch hydrolysis test.
500 g
2 kg
10 kg
0297-17
0297-07
0297-08

Beef Extract provides the nitrogen, vitamins, carbon and amino acids
in Starch Agar. Starch reacts with Grams Iodine to give a blue color.
Organisms hydrolyzing starch through amylase production will
produce a clearing around the isolate while the remaining medium is
blue. Bacto Agar is a solidifying agent.
Formula
Starch Agar
Formula Per Liter
homogeneous.
Solution:
precipitate.
Prepared Medium:
Reaction of 2.5%
Solution at 25C:
pH 7.5 0.2
Cultural Response
Inoculate with a single streak of undiluted test organism and
ATCC
RECOVERY
STARCH HYDROLYSIS
6633
25922*
25923*
19615*
good
good
good
good
positive
negative
negative
negative
468

Bacto Soluble Starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 g
Bacillus subtilis
Escherichia coli
Packaging
Starch Agar Medium for Pseudomonas 2 and Starch Agar with

Bromcresol Purple3 are modifications of Starch Agar used for the
differentiation of Gardnerella vaginalis.
Intended Use
ORGANISM
5. Chapman, G. H. 1946. A single culture medium for selective

isolation of plasma-coagulating staphylococci and for improved
testing of chromogenesis, plasma coagulation, mannitol fermentation
and the Stone reaction. J. Bacteriol. 51:409.
Precautions
Storage
Store the dehydrated medium below 30C. The powders are very hygroscopic. Keep container tightly closed.
Expiration Date
Procedure
Materials Provided
Starch Agar

Glassware
Autoclave
Incubator (35C)
Gram Iodine
The Difco Manual
Section II
Stock Culture Agar
1.
2.
3.
4.
5.

medium.
Dissolve 25 grams in 1 liter distilled or deionized water.

Cool to 45-50C.

Test Procedure
Starch Hydrolysis Test
Flood the surface of a 48-hour culture on Starch Agar with Gram Iodine.
For a complete discussion of the collection, isolation and identification
of microorganisms, refer to appropriate references.4,5
Results
References
1. Vedder. 1915. J. Infect. Dis. 16:385.
3. MacFaddin, J. D. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, p. 727-729,
Starch hydrolysis (+) is indicated by a colorless zone surrounding

colonies. A blue or purple zone indicates that starch has not been
hydrolyzed (-).
Packaging
Bacto Stock Culture Agar
Intended Use
Bacto Stock Culture Agar is used for maintaining stock cultures of
bacteria, particularly streptococci.

Dehydrated Appearance: Light tan, free-flowing,
homogeneous.
Solution:
Solution is medium amber,
opalescent.
Prepared Medium:
Medium amber, opalescent.
Reaction of 5%
Solution at 25C:
pH 7.5 0.2
Cultural Response
Prepare Stock Culture Agar per label directions. Inoculate
undiluted broth cultures of the test organisms by stabbing
the medium with an inoculating needle. Incubate at 35C
for 18-48 hours.
ORGANISM
ATCC
25923*
6305
19615*
GROWTH
good
good
good
Starch Agar
500 g
0072-17
Ayers and Johnson1 reported a medium that gave luxuriant growth and
extended viability of streptococci and other organisms. The success of
their medium can be attributed to its semisolid consistency, added
casein, buffered environment and dextrose, which serves as a readily
available source of energy. This study reported that pathogenic
streptococci remained viable for at least four months at room temperature
(24C) in the medium. Organisms such as Streptococcus pneumoniae,
Mycobacterium spp. and others, grew well on their medium. Stock
Culture Agar is prepared to duplicate the medium described by
Ayers and Johnson.1
Stock Culture Agar may also be prepared with L-asparagine
(1 gram/liter) for the maintenance of pathogenic and non-pathogenic
bacteria, especially streptococci.2

Infusion from Beef Heart, Proteose Peptone, Gelatin and Isoelectric
Casein provide the nitrogen, vitamins and amino acids in Stock
Culture Agar. Dextrose is a carbon source. Disodium phosphate is a
buffering agent. Sodium citrate acts as a preservative. Bacto Agar is a
solidifying agent.
Formula
Stock Culture Agar
Formula Per Liter
Bacto Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Isoelectric Casein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5
g
g
g
g
g
g
g
g

The Difco Manual
469
Sulfite Agar
Section II
Precautions

1.
2.
3.
4.
Storage


Expiration Date
Test Procedure
Results
Procedure
Materials Provided
Stock Culture Agar

References
Glassware
Autoclave
Incubator (35C)
Waterbath (45-50C)
L-aspargine (optional)
1. Ayers, S. H., and W. T. Johnson. 1924. Studies of the streptococci.

Bacto Sulfite Agar
Packaging
Stock Culture Agar
500 g
0054-17
Bacto Sulfite Agar is used for detecting thermophilic, H2S-producing

anaerobes, particularly in foods.
Sulfite Agar contains Tryptone as a source of carbon, nitrogen, vitamins

and minerals. Sodium Sulfite, upon reduction, produces hydrogen
sulfide. Bacto Agar is the solidifying agent.
Iron nails or iron strips will combine with any dissolved oxygen in the
medium and provide an anaerobic environment.
Formula
Intended Use
Sulfide spoilage of foods is due to three factors: high spore counts, the
heat resistance of the spores, and subjecting the finished product to
elevated temperatures. The last factor may occur if the processed food
is not cooled adequately.3
Clark and Tanner1 described the thermophilic organisms that cause
spoilage in canned foods as flat-sour spoilage organisms, thermophilic
anaerobes and sulfide-spoilage organisms. They used Sulfite Agar to
study sulfide-spoilage organisms in sugar and starch.
Both beet and cane sugar can carry spores of the thermophilic bacteria
that are spoilage agents.2 Desulfotomaculum nigrificans, first classified
as Clostridium nigrificans, causes spoilage in non-acid canned foods
such as vegetables and infant formula.3 The growth of D. nigrificans
occurs in the range of pH 6.2-7.8, with the best growth occurring at
pH 6.8-7.3. Scanty growth can be observed at pH 5.6. The reaction of
most vegetables, except corn and peas, falls below pH 5.8, so sulfide
spoilage is rare.3
Sulfite Agar is a recommended Standard Methods medium for isolating
D. nigrificans.2, 3
470
Sulfite Agar
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Sodium Sulfite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Final pH 7.6 0. 2 at 25C
Precautions
Storage
Expiration Date
The Difco Manual
Section II
Sulfite Agar
Procedure
Starch and Flour

1. Place 20 grams of starch or flour in a dry, sterile, graduated 250 ml
Erlenmeyer flask.
2. Add sterile water to the 100 ml mark, swirling occasionally.
3. Close the flask with a sterile rubber stopper.
4. Shake well to obtain a uniform, lump-free suspension. Add sterile
glass beads to the sample mixture to aid in thoroughly mixing
during shaking.
Materials Provided
Sulfite Agar

Glassware
Autoclave
Incubator (35C)
Iron nails or strips
Nonfat Dry Milk

1. Place 10 grams of nonfat dry milk in a sterile, graduated 250 ml
Erlenmeyer flask.
2. Add .02N sodium hydroxide to the 100 ml mark.
3. Shake to completely dissolve.
4. Autoclave at 5 pounds pressure for 10 minutes.
5. Cool immediately.

Dry Sugar
1. Place 20 grams of dry sugar in a dry, sterile, graduated 250 ml
Erlenmeyer flask closed with a rubber stopper.
2. Add sterile water to the 100 ml mark and shake to dissolve.
3. Replace the stopper with a sterile cotton plug, bring the solution
rapidly to a boil, and continue boiling for 5 minutes.
4. Replace evaporated liquid with sterile water.
5. Cool immediately in cold water.
Liquid Sugar
Prepare as for dry sugar except determine the amount of liquid sugar
needed on the basis of %Brix in order to be equivalent to 20 grams of
dry sugar.2
Cream
1. Mix 2 grams of gum tragacanth and 1 gram of gum arabic in
100 ml of water in an Erlenmeyer flask.
2. Sterilize at 121C for 20 minutes.
3. Transfer 20 ml of cream sample to a sterile, graduated 250 ml
Erlenmeyer flask.
4. Add sterilized gum mixture to the 100 ml mark.
5. Shake carefully using a sterile rubber stopper.
6. Loosen the stopper. Autoclave at 5 pounds pressure for 5 minutes.
Soy Protein Isolates
1. Prepare a 10% suspension of soy protein isolate in sterile 0.1%
peptone water in milk dilution or similar bottles.

Dehydrated Appearance: Very light beige, free-flowing, homogeneous.
Solution:
deionized water upon boiling. Light amber,
Prepared Medium:
Light amber, very slightly to
Reaction of 3.1%
Solution at 25C:
pH 7.6 0.2
Cultural Response
Prepare Sulfite Agar per label directions. Inoculate molten medium,
solidify, and incubate aerobically at 55 2C for 18-48 hours.
ORGANISM
ATCC
INOCULUM
SULFITE
CFU
GROWTH REDUCTION
10149 30-100
Clostridium thermosaccharolyticum 7956 30-100
Desulfotomaculum nigrificans
19858 30-100
good
good
good
+
+
*This culture is available as Bactrol Disks and should be used as directed in
The Difco Manual
Uninoculated
tube
Bacillus
stearothermophilus
ATCC 10149
Desulfotomaculum
nigrificans
ATCC 19858
471
Synthetic Broth AOAC
2. Adjust to pH 7.0 0.1.

3. Autoclave at 5 pounds pressure for 20 minutes.
Test Procedure
Sugar
1. Divide 20 ml of heated sugar solution among 6 screw-cap tubes
(20 x 150 mm) containing approximately 10 ml of freshly autoclaved,
still molten Sulfite Agar and a nail.
2. Cool and solidify immediately in cold water.
3. Preheat the tubes to 50-55C.
Starch and Flour
1. Divide 20 ml of the starch or flour suspension among 6 screw-cap
tubes (20 x 150 mm) containing approximately 10 ml of freshly
autoclaved, still molten Sulfite Agar and a nail.
2. Swirl the tubes several times to ensure even dispersion of the
starch or flour in the medium. Heat in a boiling water bath for
15 minutes, continuing to swirl the tubes.
Nonfat Dry Milk
1. Transfer 2 ml of nonfat dry milk solution to each of 2 screw cap
tubes (20 X 150 mm) containing freshly autoclaved, still molten
Sulfite Agar and a nail.
2. Gently swirl several times.
5. Incubate at 50-55C for 24-48 3 hours.
6. Count colonies of D. nigrificans and report on the basis of a
10 gram sample.
Soy Protein Isolates
1. Add 1 ml of soy protein isolate suspension to each of 10 tubes
containing freshly autoclaved, still molten Sulfite Agar and a nail.
If using already prepared medium, heat the tubes immediately
before inoculation to eliminate oxygen.
2. Mix tubes.
3. Solidify in an ice water bath.
4. Overlay with Vaspar.
Section II
5. Preheat the tubes to 55C.

6. Incubate at 55C for 14 days. Take preliminary counts at 48 hours,
7 days and 14 days in case tubes become completely blackened.
7. Count the blackened areas for each tube and report as the number
of spores per gram of soy isolate.
Results
Hydrogen sulfide production from the reduction of sulfite causes a
blackening of the medium.
Sulfide spoilage spores should be present in not more than 2 of 5
samples tested (40%) with not more than 5 spores per 10 gram in any
one sample.4

1. Nails or iron strips should be cleaned in hydrochloric acid and
rinsed well to remove any rust before being placed into tubes of
medium.
2. If iron nails or iron strips are not available, substitute 10 ml of 5%
ferric citrate solution.
3. Spoiled peas may not show discoloration but will show blackening
with a dark- colored brine.
4. Spangling of the enamel may occur as a result of the interaction of
dissolved hydrogen sulfide with the iron of the container.
References
1. Clark, F. M., and F. W. Tanner. 1937. Thermophilic canned-food
spoilage organisms in sugar and starch. Food Res. 2:27-39.
2. Andrews, W. 1995. Microbial methods, p. 1-119. In Official
3. Donnelly, L. S., and R. R. Graves. 1992. Sulfide spoilage
sporeformers, p. 317- 323. In C. Vanderzant and D. F.
Splittstoesser (ed.). Compendium of methods for the microbiological examination of foods, 3rd ed. American Public Health
4. NCA Research Laboratories. 1968. Laboratory Manual for Food
Canners and Processors, vol. 1, p. 104. Natl. Canners Assn. (Now,
Natl. Food Processors Assn.) AVI Inc., Westport, CT.
Packaging
Sulfite Agar
500 g
0972-17
Bacto Synthetic Broth AOAC
Principles Of The Procedure
Intended Use
The chemically-defined ingredients in Synthetic Broth AOAC provide

nitrogen, carbon, vitamins and minerals required for bacterial growth.
Bacto Synthetic Broth AOAC is used for maintaining disinfectant test

cultures.
Formula

Synthetic Broth AOAC is a chemically defined broth recommended
by the Association of Official Analytical Chemists (AOAC).1 It
contains all the nutrients essential for growth of the test cultures used
in determining the phenol coefficients of disinfectants.
472

Formula Per Liter
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05 g
DL-Methionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.37 g
L-Arginine HCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4 g
The Difco Manual
Section II
DL-Histidine HCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.3

L-Lysine HCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.85
L-Tyrosine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.21
DL-Threonine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
DL-Valine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
L-Leucine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8
DL-Isoleucine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.44
Glycine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.06
DL-Serine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.6
DL-Alanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.43
L-Glutamic Acid HCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3
L-Aspartic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.45
DL-Phenylalanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.26
DL-Tryptophan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05
L-Proline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.05
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Magnesium Sulfate Anhydrous Reagent . . . . . . . . . . . . . 0.05
Potassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5
Thiamine HCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01
Nicotinamide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
Storage
Expiration Date
Procedure
Materials Provided
Precautions
closed.

Dehydrated Appearance: White, homogeneous, free-flowing.
Solution:
is colorless and clear with no
precipitate.
Prepared Medium:
Colorless and clear with no
precipitate.
Reaction of 1.7%
Solution at 25C:
pH 7.1 0.1
Cultural Response
Prepare Synthetic Broth AOAC per label directions. Inoculate
and incubate the tubes at 35 2C for 18-24 hours.
ORGANISM
ATCC
APPROXIMATE
INOCULUM CFU
Salmonella typhi
15442
10708
6539
6538
100
100
100
100
GROWTH
good
good
good
good
The Difco Manual
Glassware
Autoclave
Incubator (35)
20 x 150 mm tubes with closures
Sterile 10% dextrose solution
1.
2.
3.
4.
5.

Boil for 1-2 minutes.
Dispense 10 ml amounts into 20 x 150 mm culture tubes.
Before inoculating, aseptically add 0.1 ml sterile 10% dextrose
solution to each tube.

Test Procedure
Results

Not applicable
References
Packaging
500 g
10 kg
0352-17
0352-08
473
m T7 Agar
Section II
Bacto m T7 Agar
Intended Use
Standard Methods procedures to recover injured total coliform bacteria

from treated water specify m T7 Agar.9 Stressed organisms can be
present in treated drinking water and wastewater, saline waters and
relatively clean surface waters.9
Bacto mT7 Agar is used for recovering injured coliforms from treated
water by membrane filtration.

Selective media used with the membrane filter method do not
adequately recover injured coliforms.1,2,3,4 McFeters et al. studied the
influences of diluents, media and procedures in recovering injured
coliform bacteria and found improved recovery using Tergitol 7 Agar.5
LeChevallier et al. modified Tergitol 7 Agar and developed a new
medium, m T7 Agar, for improved recovery of injured coliforms from
drinking water.6 In a later study, LeChevallier et al.7 evaluated mT7
Agar as a fecal coliform medium and found optimum recovery using
preincubation at 37C for 8 hours followed by incubation at 44.5C for
12 hours.7 The authors found that incorporation of 0.1 g of penicillin G
per ml, aseptically added to the medium after autoclaving, prevented
growth of gram-positive cocci that may break through. Later, they
found that 1.0 g/ml of penicillin G provided far better inhibition of
gram-positive organisms without interfering with the recovery of
coliforms. LeChevallier and McFeters reported the work of five collaborating laboratories testing coliform recovery from contaminated
surface water and sewage samples.8 They found m T7 Agar to be more
effective than m Endo Agar in recovering coliforms.

Dehydrated Appearance: Yellow-green to blue-green,
free-flowing, homogeneous, may
have a slightly moist appearance
and/or tendency to form soft lumps.
Solution:
Reddish purple, slightly opalescent
Prepared Medium:
Reddish purple, slightly opalescent
Reaction of 4.86%
Solution at 25C:
pH 7.4 0.2
Cultural Response
Prepare mT7 Agar per label directions. Inoculate with test
organisms diluted in 10 ml of water. Incubate at 35 2C for
8 hours and then at 44.5C for an additional 12 hours.
ORGANISM
Escherichia coli
Escherichia coli
ATCC
INOCULUM
CFU (approx.)
25922*
13762
19433
27853*
100
100
100
100
GROWTH
COLONY
COLOR
good
yellow
good
yellow
poor to fair
poor to fair
474
The ingredients of m T7 Agar support growth of injured coliforms.

Proteose Peptone No. 3 provides nitrogen and amino acids. Yeast
Extract is a vitamin source and Lactose provides carbon. Tergitol 7
and Polyoxyethylene Ether W-1 are selective agents at optimal
concentrations that will not affect recovery of injured coliforms. Brom
Cresol Purple and Brom Thymol Blue are indicators of lactose
fermentation. The combination of dyes provides a good differential
reaction as well as additional inhibition to noncoliform bacteria. Bacto
Penicillin G (1.0 g/ml), aseptically added to the medium after autoclaving, prevents growth of gram-positive cocci without interfering
with recovery of coliforms.8
Formula
m T7 Agar
Formula Per Liter
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Tergitol 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.4
Polyoxyethylene Ether W-1 . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Brom Thymol Blue . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
Bacto Brom Cresol Purple . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
ml
g
g
g
g
Precautions
Storage
Store prepared plates containing penicillin G at 2-8C and use within
1 week after preparation.6
Expiration Date
Procedure
Materials Provided
m T7 Agar

Glassware
Autoclave
The Difco Manual
Section II
TAT Broth Base & TAT Broth

Sterile 47 mm 0.45 m gridded membrane filters
Sterile Petri dishes 50 x 9 mm
Pipettes
Dilution bottles
Incubator or waterbath (37C and 45C )
Penicillin G (1.0 g/ml)
2. The procedure for enumerating fecal coliforms with m T7 Agar

requires two incubation temperatures.
3. The addition of penicillin G is required for better inhibition of
gram-positive bacteria.
4. m T7 Agar may recover other coliforms in addition to E. coli. Some
drinking water samples contain so many non-coliform bacteria that
confluent growth may occur. Care must be taken to distinguish
yellow colonies from background growth.9
References
1.
2.
3.
4.
1. Maxcy, R. B. 1970. Non-lethal injury and limitations of recovery

of coliform organisms on selective media. J. Milk Food Technol.
33:445-448.
2. Scheusner, D. L., F. F. Busta, and M. L. Speck. 1971. Inhibition
of injured Escherichia coli by several selective agents. Appl.
3. Grabow, W. O. K., and M. du Preez. 1979. Comparison of
mEndo LES, MacConkey and Teepol media for membrane filtration
counting of total coliform bacteria in water. Appl. Environ.
4. Hoadley, A. W., and C. M. Cheng. 1974. Recovery of indicator
bacteria on selective media. J. Appl. Bacteriol. 37:45-57.
5. McFeters, G. A. , S. C. Cameron, and M. W. LeChevallier. 1982.
Influence of diluents, media and membrane filters on detection of
injured waterborne coliform bacteria. Appl. Environ. Microbiol.
43:97-103.
6. LeChevallier, M. W., S. C. Cameron, and G. A. McFeters. 1983.
New medium for improved recovery of coliform bacteria from
drinking water. Appl. Environ. Microbiol. 45:484-492.
7. LeChevallier, M. W., P. E. Jajanoski, A. K. Camper, and G. A.
McFeters. 1984. Evaluation of m-T7 agar as a fecal coliform
bacteria from drinking water. Appl. Environ. Microbiol. 48:371-375.
8. LeChevallier, M. W., and G. A. McFeters. 1985. Enumerating
injured coliforms in drinking water. Research and Technology.
J. AWWA. 77:81-87.

To prepare a more selective medium, aseptically add 1.0 g
penicillin G per ml to the sterile medium cooled to 45C.
5. Dispense 4-5 ml amounts into 50 x 9 mm Petri dishes.
Note: Stock solutions of 0.1 mg/ml of penicillin G (sodium salt) can
be filter sterilized, frozen in aliquots, and stored for up to 6 months.
(One international or USP penicillin unit is equivalent to 0.6 g of
benzylpenicillin sodium).

Water samples should be collected as described in Standard Methods
for the Examination of Water and Wastewater.9
Test Procedure
For a complete discussion of stressed organisms in water testing, refer
to the membrane filter procedure for the coliform group as described
in Standard Methods for the Examination of Water and Wastewater.9
Incubate inoculated plates at 37C for 8 hours and then at 44.5C for
an additional 12 hours. This procedure has been found to produce
consistently higher fecal coliform counts with mT 7 Agar.7
Results
After incubation, count all yellow, smooth, convex colonies as
coliforms with the aid of a stereoscopic microscope.

medium.
Bacto TAT Broth Base

Bacto TAT Broth
Packaging
mT7 Agar
100 g
0018-15
Intended Use
Bacto TAT Broth Base with added Tween 20 and Bacto TAT Broth are
used for cultivating microorganisms from highly viscous or gelatinous
materials.
Also Known As
TAT (Tryptone-Azolectin-Tween) Broth Base is also referred to as
Fluid Casein Digest-Soy Lecithin Polysorbate 20 Medium.
The Difco Manual

TAT Broth Base with the addition of Tween 20 is recommended for
sterility testing of viscous materials, such as salves or ointments. It is
especially adapted to the sterility testing of cosmetics. Cosmetics
and pharmaceutical products are subject to contamination during
manufacturing and use by consumers.1 Preservatives are used in
aqueous products to make them self-sterilizing for vegetative bacteria,
yeasts and molds.1
TAT Broth Base is an enrichment medium developed to isolate and
cultivate microorganisms. TAT Broth Base conforms to the formula
specified by US Pharmacopeia for use in Microbial Limit Tests.2
475
TAT Broth Base & TAT Broth
Section II
Expiration Date
Tryptone provides the nitrogen, vitamins, amino acids and carbon in

TAT Broth Base. Azolectin and Tween 20 neutralize preservatives in
the cosmetics or pharmaceutical products, allowing bacteria to grow.
Formula
Procedure
TAT Broth Base

Formula Per Liter
Materials Provided
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Azolectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g

TAT Broth
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Azolectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Tween 20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 ml
Precautions
Storage
TAT Broth Base
Solution:
2.5% solution with 4% Tween 20;
solution is light amber, clear to very
slightly opalescent with a very slight
precipitate.
Prepared Medium:
opalescent.
Reaction of 2.5%
Solution w/ 4% Tween
20 at 25C:
pH 7.2 0.2
Cultural Response
TAT Broth
Prepare TAT Broth Base per label directions or use prepared
TAT Broth. Inoculate and incubate at 35 2C for 18-48 hours.
Salmonella typhi

Tween 20 (for dehydrated TAT Broth Base)
Glassware
Autoclave
Waterbath (50-60C)
Sterile test tubes
TAT Broth Base (dehydrated)
2. Add 40 ml Tween 20.
3. Heat to 50-60C.
4. Let stand 15-30 minutes with occasional agitation to dissolve completely.
TAT Broth (prepared)
1. In an area adjacent to the clean room, remove bottles from their boxes.
2. Follow careful aseptic technique when uncapping bottles for testing.
ORGANISM
TAT Broth Base (dehydrated)

TAT Broth (prepared)
ATCC
INOCULUM
CFU
GROWTH
27853*
6539
25923*
100-1,000
100-1,000
100-1,000
good
good
good
as directed in Bactrol Disk Technical Information.

Test Procedure
1. Add one gram or one ml of an undiluted sample to 40 ml of
complete medium and agitate to obtain an even suspension.
2. Incubate tubes at 35 2C for 18-48 hours.
For a complete discussion on sterility testing refer to appropriate
procedures in USP.2
Results
Tubes or bottles exhibiting growth should be subcultured for identification.

References
1. Orth, D. S. 1993. Handbook of cosmetic microbiology. Marcel
Dekker, Inc., New York, N.Y.
States pharmacopeia, 23rd ed. Microbial limits tests, p. 1681-1686.
The United States Pharmacopeial Convention Inc., Rockville, MD.
Packaging
TAT Broth Base
TAT Broth
476
500 g
10 x 90 ml
0984-17
9072-73
The Difco Manual
Section II
TB Hydrolysis Reagent
Bacto TB Hydrolysis Reagent
Intended Use
Bacto TB Hydrolysis Reagent is used for differentiating mycobacteria
on their ability to hydrolyze Polysorbate 80. TB Hydrolysis Reagent is
also used for differentiating Moraxella catarrhalis from Neisseria spp.
Also Known As
Tween hydrolysis is a common term for the TB Hydrolysis Reagent test.

Wayne, Doubek and Russell 1 differentiated various species and
subgroups of acid-fast bacilli using the Tween 80 hydrolysis test.
Kubica and Dye2 used the test to differentiate clinically significant from
clinically insignificant mycobacteria. In 1970, Runyan, Kubica, Morse,
Smith and Wayne3 defined a positive reaction as one that occurs in
less than five days. A doubtful reaction was defined as one that occurs in
five to ten days. A negative reaction was defined as one occurring after
ten days.
In 1973, Kubica4 recognized the greater reliability of a 10-day Tween
hydrolysis test for separation of clinically insignificant from clinically
significant members of both the scotochromagenic and nonphotochromogenic mycobacteria. These observations were later confirmed
by Wayne et al.5 citing that a 10-day reading was regarded as a better
end point for the Tween hydrolysis test.
In 1990, Weiner and Penha5 described the differentiation of Moraxella
catarrhalis from other Moraxella and Neisseria spp. using TB
Hydrolysis Reagent.

Polysorbate 80 binds to the neutral red indicator, causing the solution
to be amber colored. If the mycobacterial lipase splits the Polysorbate 80,
it can no longer complex with the neutral red indicator which then
exhibits its normal red color at pH 7. The intensity of the red depends
upon how much Polysorbate 80 is split.
Some mycobacteria possess a lipase capable of splitting Polysorbate 80
into oleic acid and polyoxyethylated sorbitol, modifying the solution
from yellow to pink. The differential criterion of this test is based on
the relative time necessary for a particular species or subgroup to
hydrolyze the compound. Most M. kansasii2 strains and clinically
insignificant species are positive in five days4,5 or less, while clinically
significant species may be negative even after three weeks. Mycobacterium
tuberculosis generally yields a positive reaction in 10-20 days.
Moraxella catarrhalis hydrolyzes Tween 80 after 24 hours of incubation,
producing a clear change in color from amber to pink-red. Other
Moraxella and Neisseria spp. remain negative after an additional
24 hours of incubation.6
Formula
TB Hydrolysis Reagent is a sterile, phosphate-buffered solution of
Tween 80 and neutral red.
Precautions
1. CAUTION: Laboratory acquired infection is always a distinct
possibility when handling and processing specimens containing
Mycobacterium tuberculosis. Laboratory procedures with specimens
containing M. tuberculosis should be performed in a properly
equipped laboratory (i.e., under a Class 1 negative pressure or
Class 2 laminar flow biological safety cabinet) and by personnel
thoroughly familiar with proper techniques. For detailed information,
consult the appropriate references.7,8,9
Storage
Store TB Hydrolysis Reagent at 2-8C.
Expiration Date
Reagent Appearance: Reddish-amber solution
Cultural Response
The TB Hydrolysis Test is performed on cultures which are
inoculated and incubated at 35 2C for 5-10 days for
Mycobacterium sp. and 18-48 hours for Moraxella and
Neisseria spp.
Procedure
Materials Provided
ORGANISM
ATCC
TIME TO
COLOR CHANGE
REACTION
Mycobacterium gordonae
Moraxella (Branhamella)
catarrhalis
Neisseria sicca
14470
12478
19981
25238
5 days
5 days
10 days
24 hours
positive
positive
negative
positive
13 x 75 mm screw cap test tubes

Bacteriological loop
Incubator (35C)
White paper or porcelain
9913
48 hours
negative
Positive - any change in reagent color from the original color

Negative - no change in color

transport immediately to the laboratory according to recommended
guidelines.7
The Difco Manual
477
TCBS Agar
Section II
2. Process each specimen, using procedures appropriate for that sample.7

3. Test actively metabolizing 3-4 week old pure cultures of
Mycobacterium, or an 18-24 hour isolate of Moraxella spp.
Carefully exclude underlying culture medium.
Test Procedure
1. Prepare and sterilize 13 x 75 mm screw cap test tubes containing
1 ml distilled or deionized water. Cool to room temperature.
2. Add two drops TB Hydrolysis Reagent, taking care not to touch
the glass dropper, which could contaminate the reagent and cause
aberrant test results.
3. Transfer one loopful of test culture to the tube. Thoroughly emulsify
the culture in the reagent.
4. When testing Mycobacterium spp.:
a. use a known positive (M. kansasii ATCC 12478) and negative
(uninoculated tube and M. scrofulaceum) control in parallel with
the test culture to ascertain the validity of test results.
b. incubate at 35 2C in the dark with caps tight for 5-10 days.
c. read tubes at 5 and 10 days for any change in color in a strong
light against a white background.
5. When testing Moraxella and Neisseria spp.:
a. use Moraxella catarrhalis ATCC 25238 for a positive control,
and Neisseria sicca ATCC 9913 as a negative control.
b. incubate at 35 2C in the dark with caps tight for 18-48 hours.
c. read tubes at 24 and 48 hours.
6. Do not shake the tubes. Examine the liquid, not the sedimented
cells. Compare the color of the liquid with the control tube color.
7. Record results.
8. Upon completion of the test, follow proper established laboratory
procedures in disposing of infectious materials.
Results
For Mycobacterium spp. - A positive reaction is indicated by a color
change of the solution from amber to pink or red in 5 days or less. A
doubtful reaction is a color change in 5 to 10 days. A negative reaction
is no color change after 10 days.
For Moraxella and Neisseria spp. - A positive reaction is a color
change of the solution from amber to red or pink after 24 hours of
Bacto TCBS Agar
Intended Use
Bacto TCBS Agar is used for isolating and cultivating Vibrio cholerae
and other enteropathogenic vibrios.
Also Known As
TCBS Agar is an abbreviation for Thiosulfate-Citrate-Bile-Sucrose
Agar. TCBS is also called Vibrio Selective Agar.

TCBS Agar, prepared according to the formula of Kobayashi et al.1, is
a modification of the selective medium from Nakanishi.2 All Vibrio
spp. that are pathogenic to humans, except V. hollisae, will grow on
TCBS Agar. This medium is recommended for isolating Vibrio spp.
478
incubation. A negative reaction is no color change after 48 hours

of incubation.
References
1. Wayne, L. G., J. R. Doubek, and R. L. Russell. 1964. Classification and identification of mycobacteria. 1. Tests employing
Tween 80 as substrate, Am. Rev. Respir. Dis. 90:588-597.
2. Kubica, G. P., and W. E. Dye. 1967. Laboratory methods for
clinical and public health, mycobacteriology, p. 44. National
Communicable Disease Center, Atlanta, Georgia.
3. Runyon, E. H., A. G. Karlson, G. P. Kubica, and L. G. Wayne.
1974. Mycobacterium, p. 165. In E. H. Lennette, E. H. Spaulding,
and J. P Truant (ed.), Manual of clinical microbiology, 2nd ed.
4. Kubica, G. P. 1973. Differential identification of mycobacteria.
Am. Rev. Respir. Dis. 107:9-21.
5. Wayne, L. G., et al. 1974. Highly reproducible techniques for use in
systematic bacteriology in the genus Mycobacterium: tests for pigment,
urease, resistance to sodium chloride, hydrolysis of Tween 80, and
beta-galactosidase. Int. J. Syst. Bacteriol. 24:412-419.
6. Weiner, M., and P. D. Penha. 1990. Evaluation of Bacto TB
Hydrolysis Reagent (Tween 80) for the identification of
Branhamella catarrhalis. J. Clin. Microbiol. 28:126-127.
8. Strain, B. A., and D. M. Grochel. 1995 Laboratory safety and
infectious waste management, p. 75-85. In P. R. Murray, E. J.
Washington, D.C.
9. Kent, P. T., and G. P. Kubica. 1985. Public Health
Mycobacteriology, p. 5-20. Centers for Disease Control,
Atlanta, Georgia.
Packaging
5 ml
3192-56*
*Store at 2-8C
from stool specimens3 and is specified in Standard Methods as

Thiosulfate-Citrate-Bile-Sucrose Agar for food testing.4,5
TCBS Agar is highly selective, meets the nutritional requirements of
Vibrio spp., and allows vibrios to compete with intestinal flora.
All members of the genus are able to grow in media containing
increased salt concentrations and some species are halophilic.6 Vibrios
are natural inhabitants of sea water. 6 Human disease has been
associated with ingestion of contaminated water and consumption of
contaminated shellfish or seafood.
V. cholerae is the etiologic agent of a secretory diarrhea spread by the
fecal-oral route.3 Infections may be asymptomatic, mild, or severe.3
If not treated, patients with severe cholera may die within 5 hours as a
result of massive fluid and electrolyte loss.3
Seven cholera pandemics have been reported since 1817.7 In 1993,
the first reports of epidemic cholera due to a new serogroup,
The Difco Manual
Section II
TCBS Agar
non-01 cholerae, appeared.8,9 This strain was designated V. cholerae

0139 and given the synonym Bengal.3

Yeast Extract and Proteose Peptone No. 3 provide the nitrogen,
vitamins, and amino acids in TCBS Agar. Sodium Citrate, Sodium
Thiosulfate and Oxgall are selective agents which provide an alkaline
pH to inhibit gram-positive organisms and suppress coliforms.
The pH of the medium is increased to enhance growth of Vibrio
cholerae because this organism is sensitive to acid environments.
Saccharose is a fermentable carbohydrate, and Sodium Chloride
stimulates growth. Sodium Thiosulfate is a sulfur source and acts with
Ferric Citrate as an indicator to detect hydrogen sulfide production.
Brom Thymol Blue and Thymol Blue are pH indicators. Bacto Agar is
Formula
Precautions
Storage
TCBS Agar
Formula Per Liter
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Thiosulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Oxgall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Ferric Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Thymol Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.04
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Expiration Date
g
g
g
g
g
g
g
g
g
g
g
Procedure
Materials Provided
TCBS Agar
Vibrio cholerae
ATCC 15748

Dehydrated Appearance: Light tan with greenish cast, free-flowing,
homogeneous.
Solution:
8.9% solution, soluble on boiling
in distilled or deionized water.
Solution is forest green and
Prepared Medium:
Green, slightly opalescent.
Reaction of 8.9%
Solution at 25C:
pH 8.6 0.2
Cultural Response
Prepare TCBS Agar per label directions. Inoculate the medium
with 10 microliters (l) of a heavy suspension and incubate at
ORGANISM
INOCULUM
ATCC (HEAVY SUSPENSION) GROWTH
Escherichia coli
25922*
Vibrio cholerae El Tor
15748
Vibrio parahaemolyticus
10 l
10 l
10 l
COLONY
COLOR
inhibited
good
yellow
good
blue green
*This culture is available as BactrolTM Disks and should be used as directed in Bactrol Disks Technical Information.
The Difco Manual
479
m TEC Agar
Section II
References
Glassware
Incubator (35C)
Waterbath (45-50)
1. Kobayashi, T., S. Enomoto, R. Sakazaki, and S. Kuwahara.

1963. A new selective medium for pathogenic vibrios, TCBS
(modified Nakanishis agar). Jpn. J. Bacteriol. 18:387.
2. Nakanishi, Y. 1963. An isolation agar medium for cholerae and
enteropathogenic halophilic vibrios. Modern Media 9:246.
3. McLaughlin, J. C. 1995. Vibrio, p. 465-476. In P, R. Murray, E. J.
Washington, D.C.
6. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Vibrio
and related species, Aeromonas, Plesiomonas, Campylobacter,
Helicobacter, and others, p. 429-444. Bailey & Scotts diagnostic
microbiology, 9th ed. Mosby-Year Book, Inc., St. Louis, MO.
7. Colwell, R. R. 1996. Global climate and infectious disease: the
cholera paradigm. Science 274:2025-2031.
8. Bhattacharya, M. K., S. K. Bhattacharya, S. Garg, P. K. Saha,
D. Dutta, G. B. Nair, B. C. Deb, and K. P. Das. 1993. Outbreak
of Vibrio cholerae non-01 in India and Bangladesh. Lancet
341:1346-1347.
9. Ramamurthy, T., S. Garg, R. Sharma, S. K. Bhattacharya,
G. B. Nair, T. Shimada, T. Takeda, T. Karasawa, H. Kurazano,
A. Pal, and Y. Takeda. 1993. Emergence of novel strain of Vibrio
cholerae with epidemic potential in southern and eastern India.
Lancet 341:703-704.
10. Kelly, M. T., F. W. Hickman-Brenner, and J. J. Farmer III.
1992. Vibrio, p. 384- 395. In A. Balows, W. J.Hausler, Jr., K. L.
Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.)., Manual of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C.
11. MacFaddin, J. D. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, vol 1, p. 763-767.
12. Bottone, E. J., and T. Robin. 1978. Vibrio parahaemolyticus;
Suspicion of presence based on aberrant biochemical and
morphological features. J. Clin. Microbiol. 8:760.
13. Morris, G. K., M. H. Merson, I. Huq, A. Kibrya, and R. Black.
1979. Comparison of four plating media for isolating Vibrio
cholerae. J. Clin. Microbiol. 9:79.
1.
2.
3.
4.

Heat to boiling to dissolve completely. DO NOT AUTOCLAVE.
Cool to 45-50C.

procedures established by laboratory policy. If any delay in culturing
is anticipated, addition of the specimen to Cary-Blair transport
medium is essential because Vibrio spp. are particularly susceptible to
drying.3 Inoculation into alkaline peptone water is acceptable if
subculture will be within 6-8 hours.10
Test Procedure
For a complete discussion of the isolation and identification of Vibrio
cholerae and other enteropathogenic vibrios, refer to the procedures
Results
After 18-24 hours of incubation at 35C, sucrose-fermenting vibrios
(V. cholerae, V. alginolyticus, V. harveyi, V. cincinnatiensis, V. fluvialis,
V. furnissii, V. metschnikovii, some V. vulnificus) appear as
medium-sized, smooth, opaque, thin-edged yellow colonies on TCBS
Agar.6 The other clinically important vibrios and most V. vulnificus do
not ferment sucrose and appear green.6

2. Further tests are necessary for identification and confirmation of
Vibrio spp.11
3. On initial isolation, V. parahaemolyticus may be confused
with Aeromonas hydrophila, Plesiomonas shigelloides and
Pseudomonas species.12
4. Sucrose-fermenting Proteus species produce yellow colonies which
may resemble those of Vibrio.11
5. TCBS is an unsatisfactory medium for oxidase testing of
Vibrio spp.13
6. A few strains of V. cholerae may appear green or colorless on TCBS
due to delayed sucrose fermentation.11
Bacto m TEC Agar
Packaging
TCBS Agar
100 g
500 g
0650-15
0650-17
Also Known As
m TEC is an abbreviation for membrane (medium) Thermotolerant E. coli.
Intended Use
Bacto m TEC Agar is used for isolating, differentiating and rapidly
enumerating thermotolerant Escherichia coli from water by membrane
filtration and an in situ urease test.
480

Escherichia coli is widely used as an indicator of fecal pollution in
water. There are many procedures for enumerating E. coli based on its
The Difco Manual
Section II
m TEC Agar
ability to grow at elevated temperatures and produce indole from

tryptophane.1,2 The determination of indole production in conjunction
with the most-probable-number procedure often requires the use of
another medium and additional incubation time.
The membrane filter procedure has been recognized by Standard
Methods for the Examination of Water and Wastewater as an alternate
test procedure.3 In 1981, Dufour et al. developed a simple, accurate,
nonlethal membrane filter technique for the rapid enumeration of
E. coli.4 This medium, m TEC Agar, quantifies E. coli within 24 hours
without requiring subculture and identification of isolates. The authors
reported that they were able to recover E. coli from marine, estuarine
and fresh water samples.
Brom Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.08 g

Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
Final pH 7.3 + 0.2 at 25C
Precautions
Storage
Expiration Date

m TEC Agar contains sufficient nutrients to support the growth of
E. coli. Proteose peptone is a source of nitrogen, amino acids, carbon
and amino acids. Yeast Extract provides trace elements, vitamins and
amino acids. Potassium Phosphate Monobasic and Potassium
Phosphate Dibasic offer buffering capabilities. Lactose is a fermentable
carbohydrate and carbon source. Sodium Lauryl Sulfate and Sodium
Desoxycholate are selective against gram-positive bacteria. Brom
cresol purple and brom phenol red are indicator components and Bacto
Agar solidifies the medium.
Formula
m TEC Agar
Formula Per Liter
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5
Potassium Phosphate Dibasic . . . . . . . . . . . . . . . . . . . . . . . 3.3
Sodium Lauryl Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
Brom Cresol Purple . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.08
g
g
g
g
g
g
g
g
g
The expiration date applies to the product is its intact container when
Procedure
Materials Provided
m TEC Agar

Autoclave
Pipettes
Dilution blanks
35C incubator
44.5C waterbath or incubator
Waterproof plastic bags if water bath is used
Urea
Phenol Red

Dehydrated Appearance: Green to grayish tan, free-flowing and homogeneous.
Solution:
on boiling. Solution is deep purple with red cast,
Reaction of 4.53%
Solution at 25C:
pH 7.3 + 0.2
Cultural Response
Prepare m TEC Agar per label directions. Inoculate and incubate the plates at
35C for two hours. Transfer plates and incubate at 44.5 + 0.5C for approximately
22 + 2 hours. After incubation, filters are removed and placed over pads, saturated with
approximately 2 ml of urease substrate. Yellow to yellow-brown colonies (urease negative)
are counted after 15-20 minutes.
ORGANISM
ATCC
INOCULUM
CFU
RECOVERY
APPEARANCE
Escherichia coli
8739
20-80
good
yellow to yellow-brown colonies
Escherichia coli
ATCC 8739
The culture listed is the minimum that should be used for performance testing.
The Difco Manual
481
TPEY Agar Base
Section II
m TEC Agar
1. Suspend 45.3 g in 1 liter distilled or deionized water.
4. Dispense 4-5 mls amounts into 50 x 10 mm Petri dishes and allow
to solidify.
Urea Substrate
1. Combine 2 g urea and 10 mg phenol red in 100 ml distilled water.
2. Adjust pH to 5.0 + 0.2.
3. Store at 2-8C. Use within one week.
Note: Other methods may recommend an alternative pH.3,6 Prepare
substrate according to recommended guidelines.
1. The 35C incubation step is required to resuscitate stressed

organisms. The 44.5C incubation temperature is required to
inhibit non-thermotolerant organisms.
2. The urease test is required to presumptively identify E. coli.
3. Choose a water sample size that will result in 20-80 colonies per
filter. Plates containing more than 80 colonies are not recommended because high counts may not provide accurate urease test
results.
4. Do not trap air bubbles underneath the filter.

Water samples should be collected and prepared in accordance to
recommended guidelines.3,6
Test Procedure
1. Follow the membrane filter procedure described in Standard
Methods for the Examination of Water and Wastewater.3
2. Incubate inoculated plates for 2 hours at 35C to resuscitate
injured cells.
3. Transfer the plates to a 44.5 + 0.5C waterbath or incubator and
incubate for 22 + 2 hours.
4. Transfer countable filters to pads saturated with urea substrate.
5. After 15-20 minutes, count all yellow to yellow-brown colonies
with the aid of a stereoscopic microscope.
Results
References
1. Mara, D. D. 1973. A single medium for the rapid detection of
Escherichia coli at 44C. J. Hyg. 71:783-785.
2. Pugsley, A. P., L. J. Evision, and A. James. 1973. A simple
technique for the differentiation of Escherichia coli in water
examination. Water RES. 7:1431-1437.
3. Eaton, A. D., L. S. Cleseri, and A. E. Greenberg (ed.). 1995.
Standard methods for the examination of water and wastewater.
4. Dufour, A. P., E. R. Strickland, and V. J. Cabelli. 1981.
Membrane filter method for enumerating Escherichia coli. Appl.
Environ. Microbiol. 41:1152- 1158.
5. Dufour, A. P., and V. J. Cabelli. 1975. Membrane filter procedure
for enumerating the component genera of the coliform group in
seawater. Appl. Microbiol. 29:826-833.
6. 1996 Annual Book of ASTM Standards, Water and
Environmental Technology (PCN: 01-110296-16). ASTM,
WestConshohocken, PA.
Yellow to yellow-brown colonies (urease negative) may be presumptively

identified as E. coli.
Packaging
Bacto TPEY Agar Base
Intended Use
Bacto TPEY Agar Base is used with Bacto EY Tellurite Enrichment
and Bacto Antimicrobic Vial P in detecting and enumerating
coagulase-positive staphylococci.
Also Known As
TPEY Agar Base conforms with Tellurite-Polymyxin-Egg Yolk Agar
Base.

1,2
TPEY Agar Base is prepared according to the formulation of Crisley.

The complete medium is prepared by aseptically adding EY Tellurite
Enrichment and Antimicrobic Vial P (polymyxin B) to the sterile TPEY
Agar Base. This medium permits the isolation and enumeration of
coagulase-positive staphylococci from a variety of specimens such as
food products, air, dust and soil. Coagulase-negative staphylococci and
other organisms are markedly to completely inhibited.
m TEC Agar
100 g
0334-15
Lithium Chloride, Potassium Tellurite and Polymyxin B inhibit a wide

variety of microorganisms, including coagulase-negative staphylococci.
Yeast Extract provides vitamins and cofactors required for growth, as
well as additional sources of nitrogen and carbon. Tryptone provides
nitrogen, vitamins and amino acids. Mannitol is an energy source.
Sodium Chloride maintains the osmotic balance. Bacto Agar is
incorporated into the medium as a solidifying agent.
Formula
TPEY Agar Base
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Mannitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
g
g
g
g
g
g
Precautions
482
The Difco Manual
Section II
TPEY Agar Base

AND SKIN. (US) MAY CAUSE HARM TO THE UNBORN
CHILD. (US) Avoid contact with skin and eyes. Do not breathe
closed. TARGET ORGAN(S): Blood, Kidneys, Nerves.
Storage
Expiration Date
1. Suspend 60 grams of TPEY Agar Base in 900 ml distilled or
deionized water.
4. Aseptically add 100 ml of EY Tellurite Enrichment warmed to
room temperature and 10 ml of rehydrated Antimicrobic Vial P.
Mix thoroughly.
Alternatively, use 100 ml of a 30% egg yolk emulsion, 10 ml of
Chapman Tellurite Solution 1% and 0.4 ml of filter sterilized 1%
polymyxin B solution.
5. Pour 15-17 ml amounts into sterile Petri dishes.

Consult appropriate references.3
Materials Provided
Results
TPEY Agar Base

Antimicrobic Vial P (Polymyxin B)

Solution:
60 grams per 900 ml solution, soluble
boiling. Prior to adding enrichment,
solution is light to medium amber,
opalescent.
Prepared Medium:
Yellowish-beige, opaque.
Reaction of 6.0%
Solution at 25C:
pH 7.2 0.2
Cultural Response
Prepare TPEY Agar Base per label directions. Inoculate and
ATCC
INOCULUM
CFU
GROWTH
COLONY
APPEARANCE HALO
Escherichia
25922* 1,000-2,000 marked to
coli
complete inhibition
good
black
aureus
Staphylococcus 14990 100-1,000
poor to fair
black
epidermidis
Zone of precipitation/clearing around the colony.
The Difco Manual
Glassware
Autoclave
Waterbath (50-55C)
Incubator (35C)
Test Procedure
Procedure
ORGANISM
Coagulase-positive staphylococci form black or dark-gray colonies due

to the reduction of colorless tellurite to free tellurium. Three types of
egg yolk precipitation reactions are produced by coagulase-positive
staphylococci:
1. a discrete zone of precipitated egg yolk around and beneath the
colonies;
2. a clear zone or halo surrounding the colonies with a possible zone
of precipitate beneath the colonies; and,
3. no zone or halo around the colonies, but a precipitate beneath the
colonies.

1. Mannitol-positive and/or tellurite-positive staphylococcal strains
that are coagulase-negative are occasionally found. Definitive
identification of S. aureus, therefore, should be based primarily on
the coagulase reaction, with mannitol fermentation and tellurite
reduction being used only for confirmation.3,5
2. The prepared medium becomes less inhibitory to coagulase-negative
strains of staphylococci if it is stored for longer than one week.2
3. Graves and Frazier4 showed that Bacillus spp. able to grow
on TPEY Agar produce an antibiotic that inhibits growth of
staphylococci.
References
1. Crisley, F. D., R. Angelotti, and M. J. Foter. 1964. Multiplication of Staphylococcus aureus in synthetic cream fillings and pies.
Public Health Rep. 79:369.
2. Crisley, F. D., J. T. Peeler, and R. Angelotti. 1965. Comparative
evaluation of five selective and differential media for the detection
and enumeration of coagulase-positive staphylococci in foods.
483
TSA Blood Agar Base, Tryptic Soy Blood Agar Base No. 2 & Tryptic Soy Blood Agar Base EH
3. Koneman, E. W., S. D. Allen, V. R. Dowell, Jr., and

H. M. Sommers. 1979. Color atlas and textbook of diagnostic
microbiology, 2nd ed. J. B. Lippincott Company, Philadelphia, PA.
4. Graves, R. R., and W. C. Frazier. 1963. Food microorganisms
influencing the growth of Staphylococcus aureus. Appl. Microbiol.
11:513.
Section II
5. MacFaddin, J. F. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, Vol. 1. Williams &
Wilkins, Baltimore, M.D.
Packaging
TPEY Agar Base
500 g
0556-17
Bacto TSA Blood Agar Base . Bacto Tryptic Soy Blood Agar
Base No. 2 . Bacto Tryptic Soy Blood Agar Base EH
Intended Use
where clear and distinct hemolytic reactions are of prime importance.
Bacto TSA Blood Agar Base is used with blood in isolating and
cultivating fastidious microorganisms where clear and distinct
hemolytic reactions are of prime importance.
Bacto Tryptic Soy Blood Agar Base EH is used with blood in isolating
and cultivating fastidious microorganisms from specimens where clear
and distinct hemolytic reactions are of prime importance.
Bacto Tryptic Soy Blood Agar Base No. 2 is used with blood in
isolating and cultivating fastidious microorganisms from specimens
Escherichia coli
ATCC 25922
ATCC 25923

TSA Blood Agar Base
Solution:
deionized water upon boiling, medium
Prepared Medium:
With 5% sheep blood - bright
medium red, opaque, firm.
Reaction of 4.0%
Solution at 25C:
pH 7.3 0.2
Tryptic Soy Blood Agar Base No. 2
Solution:
medium amber, slightly opalescent
Prepared Medium:
With 5% sheep blood - bright cherry
red, opaque.
Reaction of 4.0%
Solution at 25C:
pH 7.3 0.2
Tryptic Soy Blood Agar Base EH
Solution:
precipitate.
Prepared Medium:
With 5% sheep blood - bright cherry
red, opaque.
Reaction of 4.0%
Solution at 25C:
pH 7.3 0.2
484
ATCC 6305
ATCC 19615
Cultural Response
Prepare TSA Blood Agar per label directions. Inoculate and
incubate at 35C under 5-10% CO2 for 18-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
Escherichia coli
25922* 100-1,000
25923* 100-1,000
Streptococcus pneumoniae 6305 100-1,000
19615* 100-1,000
GROWTH HEMOLYSIS
good
good
good
good
beta
alpha
beta
The Difco Manual
Section II
TSA Blood Agar Base, Tryptic Soy Blood Agar Base No. 2 & Tryptic Soy Blood Agar Base EH
Also Known As
Blood Agar Base is abbreviated as BAB. Tryptic Soy Agar is abbreviated
as TSA, and is referred to as Soybean-Casein Digest Agar Medium, USP.

Blood Agar Bases are typically supplemented with 5-10% sheep, rabbit
or horse blood for use in isolating, cultivating and determining
hemolytic reactions of fastidious pathogenic microorganisms.
In 1919, Brown 1 experimented with blood agar formulations to
determine their affects on colony formation and hemolysis. Growth of
pneumococci was noticeably influenced when a medium contained
peptone manufactured by Difco.
Tryptic Soy Agar is based on the Soybean-Casein Digest formula
specified by US Pharmacopeia.2 Tryptic Soy Agar is a general purpose
medium used for multiple applications, e.g., maintaining culture collections, performing colony counts3 and testing bacterial contaminants in
cosmetics.4 TSA Blood Agar Base was developed to achieve good growth
and to improve hemolytic reactions of pathogenic microorganisms.
Tryptic Soy Blood Agar Base No. 2 and Tryptic Soy Blood Agar Base
EH represent further improvements to TSA Blood Agar Base. TSA
Blood Agar Base No. 2 provides clearer hemolytic reactions with
group A streptococci while TSA Blood Agar Base EH provides
dramatic, enhanced hemolysis.
Blood Agar Base media are specified in Standard Methods5,6 for
food testing.

Blood Agar Base media formulations have been prepared using specially
selected raw materials to support good growth of a wide variety of
fastidious microorganisms. TSA Blood Agar Base contains two peptones,
Pancreatic Digest of Casein and Papaic Digest of Soybean Meal, which
provide nitrogen, carbon, amino acids and vitamins. Agar is a
solidifying agent; Sodium Chloride maintains osmotic balance.
Tryptic Soy Blood Agar Base No. 2 and Tryptic Soy Blood Agar
Base EH are similar in composition to TSA Blood Agar Base.
The formulations have been modified through the use of peptones
(Tryptone H and Tryptone H Plus) developed at Difco Laboratories to
improve and enhance hemolysin production while minimizing
antagonism or loss in activity of streptococcal hemolysins. Both basal
media contain Soytone for additional nitrogen, Agar as a solidifying
agent, and Sodium Chloride to maintain osmotic balance.
Supplementation with blood (5-10%) provides additional growth
factors for fastidious microorganisms and is the basis for determining
hemolytic reactions. Hemolytic patterns may vary with the source of
animal blood or type of basal medium used.7
Blood agar bases are relatively free of reducing sugars, which have
been reported to adversely influence the hemolytic reactions of
beta-hemolytic streptococci.8
Formula
TSA Blood Agar Base
Formula Per Liter
Pancreatic Digest of Casein . . . . . . . . . . . . . . . . . . . . . . . . 15
Papaic Digest of Soybean Meal . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

The Difco Manual
g
g
g
g

Formula Per Liter
Bacto Tryptone H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g

Formula Per Liter
Bacto Tryptone H Plus . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
Precautions
Storage
Expiration Date
Procedure
Materials Provided
TSA Blood Agar Base

Glassware
Autoclave
Incubator (35C)
TSA Blood Agar Base - 40 grams;
Tryptic Soy Blood Agar Base No. 2 - 40 grams;
Tryptic Soy Blood Agar Base EH - 40 grams.

Collect specimens in sterile containers or with sterile swabs and transport
485
TT Broth Base Hajna
Section II
Test Procedure
References

growth will display the most reliable hemolytic reactions owing to
the activity of both oxygen-stable and oxygen-labile streptolysins.7
3. Examine the medium for growth and hemolytic reactions after
18-24 and 48 hours incubation.
4. Four types of hemolysis on blood agar media have been described:9
a. Alpha hemolysis () is the reduction of hemoglobin to meth
emoglobin in the medium surrounding the colony, causing a
greenish discoloration of the medium.
b. Beta hemolysis () is the lysis of red blood cells, producing a
c. Gamma hemolysis () indicates no hemolysis. No destruction
of red blood cells occurs and there is no change in the medium.
d. Alpha-prime hemolysis (` ) is a small zone of complete hemoly
sis that is surrounded by an area of partial lysis.
1. Brown, J. H. 1919. The use of blood agar for the study of

streptococci, NY Monograph No. 9. The Rockefeller Institute for
Medical Research.
2. The United States Pharmacopeia (USP XXIII) and The
National Formulary (NF 18). 1995. Sterility tests, p. 1686-1690.
3. Swanson, K. J., F. F. Busta, E. H. Peterson, and M. G.
Johnson. 1992. Colony count methods, p. 75-95. In Vanderzant,
C., and D. F. Splittstoesser (ed.). Compendium of methods for
the microbiological examination of food, 3rd ed. American
4. Curry, A. S., G. G. Joyce, and G. N. McEwen Jr. 1993. CTFA
Microbiology guidelines. The Cosmetic, Toiletry and Fragrance
Association, Inc. Washington, D.C.
App. 3.08-3.09, Bacteriological analytical manual, 8th ed. AOAC
foods, 3rd ed., p.1175. American Public Health Association,
Washington, D.C.
pneumococci and streptococci. Am. J. Clin. 17:281-289.
9. Isenberg, H. D. (ed.). 1992. Interpretation of aerobic bacterial
growth on primary culture media, p. 1.6.1-1.6.7, Clinical
microbiology procedures handbook, vol. 1. American Society for
Microbiology, Washington D.C.
& Scotts diagnostic microbiology, 9th ed. p. 415. Mosby-Year

1. TSA Blood Agar Base media are intended for use with blood
supplementation. Although certain diagnostic tests may be performed
directly on this medium, biochemical and, if indicated, immunological testing using pure cultures are recommended for complete
identification. Consult appropriate references for further information.
4. Colonies of Haemophilus haemolyticus are beta-hemolytic on
horse and rabbit blood agar and must be distinguished from colonies
of beta-hemolytic streptococci using other criteria. The use of sheep
blood has been suggested to obviate this problem since sheep blood
is deficient in pyridine nucleotides and does not support growth of
H. haemolyticus.10
reactions of beta-hemolytic streptococci.7 For optimal performance,
incubate blood agar base media under increased CO2 or anaerobic
conditions.
Bacto TT Broth Base Hajna
Intended Use
Bacto TT Broth Base Hajna is used for enriching Salmonella from food
and dairy products prior to isolation procedures.
Also Known As
TT Broth Base Hajna is also referred to as Tetrathionate Broth Base Hajna.
486
Packaging
TSA Blood Agar Base
500
2
10
500
2
10
500
2
10
g
kg
kg
g
kg
kg
g
kg
kg
0026-17
0026-07
0026-08
0027-17
0027-07
0027-08
0028-17
0028-07
0028-08

TT Broth Base Hajna is used as a selective enrichment for the cultivation of Salmonella spp. Salmonella organisms can be injured in foodprocessing procedures. These procedures include exposure to low
temperatures, sub-marginal heat, drying, radiation, preservatives and
sanitizers.1 Although injured cells may not form colonies on selective
media, they can cause disease if ingested.2 Salmonella spp., in particular,
cause many types of infections from mild self-limiting gastroenteritis
The Difco Manual
Section II
TT Broth Base Hajna
to life-threatening typhoid fever.3 The most common form of Salmonella disease is self-limiting gastroenteritis with fever lasting less than
2 days and diarrhea lasting less than 7 days.3
Formula
TT Broth Base Hajna
Formula Per Liter
TT Broth Base Hajna conforms to the formulation of Hajna and

Damon.4 The medium is a modification of the enrichment described
by Kauffmann5 and Knox.6 Hajna and Damon4 developed a new
broth containing yeast extract, peptone, carbon sources and the
selective agents, sodium desoxycholate and brilliant green (replacing
Bile Salts).

Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Bacto D-Mannitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Brilliant Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01
TT Broth Base Hajna is used in testing Salmonella in egg processing

plants.7 It is specified in the Compendium of Methods for the Microbiological Examination of Foods.8
g
g
g
g
g
g
g
g
g
Precautions
Tryptose provides nitrogen and amino acids. Yeast Extract supplies

growth factors and vitamins. Dextrose and Mannitol are fermentable
carbohydrates. Selectivity is accomplished by the combination of
Sodium Thiosulfate and tetrathionate, suppressing coliform organisms.6
Tetrathionate is formed in the medium by the addition of a solution
containing iodine and potassium iodide. Organisms containing the
enzyme tetrathionate reductase will proliferate in this medium.

FIRST AID:
In case of contact with eyes, rinse immediately with plenty of
water and seek medical advice. After contact with skin, wash
immediately with plenty of water. If inhaled, remove to fresh air. If
not breathing, give artificial respiration. If breathing is difficult,
Sodium Desoxycholate and Brilliant Green are selective agents that

suppress coliform bacteria and inhibit gram-positive organisms.
Calcium Carbonate is a neutralizer that absorbs toxic metabolites.
Storage
Dehydrated Appearance: Beige to very light green,
Solution:
9.15% solution, insoluble in distilled
green, slightly opalescent with a
heavy white precipitate.
Prepared Medium:
Light green, slightly opalescent with
a heavy white precipitate.
Reaction of 9.15%
Solution at 25C:
pH 7.6 0.2 (after addition of the
iodine solution)
Prepare TT Broth Base Hajna with 4% iodine solution per
label directions. Inoculate and incubate at 35 2C for 18-24
hours. After incubation, plate the inoculated broth onto
MacConkey Agar and incubate at 35 2C for 18-24 hours.
Escherichia
coli
Salmonella
typhimurium
ATCC
INOCULUM
CFU
25922* 100-1,000
14028* 100-1,000
GROWTH
COLONY COLOR ON
MACCONKEY AGAR
none
to poor
good
pink with
bile ppt.
colorless
used a directed in Bactrol Disks Technical Information.
The Difco Manual
Expiration Date
Procedure
Materials Provided
TT Broth Base Hajna
Cultural Response
ORGANISM

Glassware
Autoclave
Incubator (35C)
Waterbath (45-50C)
Iodine crystals
Potassium iodide
MacConkey Agar
1.
2.
3.
4.

Cool below 50C.
Add 40 ml iodine solution (5 grams iodine crystals and 8 grams
potassium iodide dissolved in 40 ml distilled or deionized water)
and mix well.
487
Tellurite Blood Solution
Section II
5. Dispense into sterile tubes while keeping suspension well mixed.

6. Do not heat the medium after adding iodine.

Obtain and process specimens according to the techniques and procedures established by laboratory policy. For a complete discussion on
the collection, isolation and identification of Salmonella, refer to the
appropriate procedures outlined in the references.
Results
References
2. Sorrells, K. M., M. L. Speck, and J. A. Warren. 1970.
Pathogenicity of Salmonella gallinarum after metabolic injury by
freezing. Appl. Microbiol. 19:39- 43.
3. Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover,
and R. H. Yolken (ed.). 1995. Manual of clinical microbiology,
Bacto Tellurite Blood Solution
Intended Use
Bacto Tellurite Blood Solution is used with Bacto Proteose No. 3 Agar
and Bacto Dextrose in isolating Corynebacterium diphtheriae.

Tellurite Blood Solution is used in the cultural diagnosis of diphtheria,
an acute infectious disease primarily of the upper respiratory tract but
4. Hajna, A. A., and S. R. Damon. 1956. New enrichment and

plating medium for the isolation of Salmonella and Shigella
organisms. Appl. Microbiol. 4:341.
5. Kauffman, F. 1930. Ein kombiniertes Anreicherungsverfahren fur
Typhus-und-Paratyphusbazillen. Zentralb. Bakteriol. Parasitenkd.
Infektionskr. Hyg. Abr. I Orig. 113:148.
6. Knox, R., P. H. Gell, and M. R. Pollack. 1942. Selective media
for organisms of the Salmonella group. J. Pathol. Bacteriol.
54:469-483.
7. Catalano, C. R., and S. J. Knable. 1994. Incidence of
Salmonella in Pennsylvania egg processing plants and destruction
by high pH. J. Food Prot. 57:587-591.
Washington, D.C.
Packaging
TT Broth Base Hajna
500 g
2 kg
0491-17
0491-07
occasionally of the skin.1 Diphtheria is caused by toxigenic strains of

C. diphtheriae, of which there are three biotypes; mitis, intermedius
and gravis.1 The symptoms of the disease are development of a
pharyngeal membrane, sore throat, malaise, headache and nausea.2
Death can result from respiratory obstruction by the membrane or from
myocarditis caused by the toxin.2
Tellurite Blood Solution is a mixture of defibrinated bloods with added
potassium tellurite. This solution is used to markedly reduce growth of
commensal organisms encountered when isolating Corynebacterium
diphtheriae.
Tellurite Blood Solution contains defibrinated blood for essential

nutrients to enhance the growth of C. diphtheriae. Potassium Tellurite
is a selective agent that inhibits the growth of commensal organisms.
Solution Appearance: Dark, red-brown liquid.
Cultural Response
Prepare Proteose No. 3 Agar with Dextrose (1.5 grams per
liter) and 5% Tellurite Blood Solution. Heat to 70-80C to
chocolatize the medium. Inoculate and incubate at 35 2C
for 18-48 hours.
ORGANISM
ATCC
Corynebacterium
8028
diptheriae type gravis
Corynebacterium
8032
diptheriae type intermedius
Corynebacterium
8024
diptheriae type mitis
Escherichia coli
25922*
Streptococcus pyogenes 19616*
Tellurite Blood Solution is a combination of approximately 95%

defibrinated, lysed horse and bovine blood from which most of the
cellular debris has been removed. One-percent (1%) Potassium Tellurite
is added.
INOCULUM
CFU
GROWTH
COLOR
100-1,000
good
black
Precautions
100-1,000
good
black
100-1,000
good
black

1,000-2,000 inhibited
488
Reagent
Storage
Store Tellurite Blood Solution at 2-8C.
Expiration Date
The Difco Manual
Section II
Tellurite Glycine Agar
Procedure
Results
Materials Provided

1. Definitive identification of a strain of C. diphtheriae as a true

pathogen requires demonstration of toxin production.3
References
1. Shake Tellurite Blood Solution before use.

2. Refer to the final concentration of Tellurite Blood Solution in the
formula of the medium being prepared. Add Tellurite Blood
Solution as required.

miscellaneous irregular gram-positive rods, Erysipelothrix, and
Gardnerella, p. 357-377. In P. R. Murray, E. J. Baron, M. A. Pfaller,
F. C. Tenover, and R. H. Yolken (ed.). Manual of clinical microbiology, 6th ed. American Society for Microbiology, Washington, D.C.
St. Louis, MO.

Both throat and nasopharyngeal specimens are necessary in cases of
respiratory illness. If cutaneous diphtheria is suspected, collect skin,
throat and nasopharynx specimens. Use sterile silica gel for shipping
clinical specimens when cultures are not taken locally.
Test Procedure
For a complete discussion of the collection, isolation and identification
of C. diphtheriae and other Corynebacterium spp., refer to
appropriate procedures in the references.1,2
Packaging
6 x 25 ml
0139-66

Bacto Tellurite Glycine Agar . Bacto Chapman Tellurite
Solution 1%
Uninoculated
plate
ATCC 25923

Solution:
deionized water upon boiling. Medium
amber, opalescent with precipitation.
Prepared Medium:
Medium amber, opalescent with
precipitation.
Reaction of 6.25%
Solution at 25C:
pH 7.2 0.2
Cultural Response
Prepare Tellurite Glycine Agar per label directions and enrich
with Chapman Tellurite Solution 1%. Incubate inoculated medium
ORGANISM
Escherichia coli
ATCC
INOCULUM
CFU
GROWTH
25922* 1,000-2,000 inhibited

25923* 100-1,000
good
12228* 100-1,000
good
COLONY
COLOR
black
The Difco Manual
489
Section II
Intended Use
Bacto Tellurite Glycine Agar is used with Bacto Chapman Tellurite
Solution 1% for isolating coagulase-positive staphylococci.

The coagulase-positive species Staphylococcus aureus is well
documented as a human opportunistic pathogen.3 Foods are examined
for the presence of S. aureus and/or its enterotoxins to confirm that
S. aureus is the causative agent of foodborne illness, to determine
whether a food is the source of staph food poisoning, and to
determine post-processing contamination.4
Ludlam2 described a selective medium for the isolation of staphylococci.
This medium was alkaline in reaction, contained mannitol, and lithium
chloride with potassium tellurite as the selective agents. Zebovitz,
Evans and Niven1 modified Ludlams medium by adding glycine as a
selective agent and adjusting the reaction of the basal medium to
pH 7.2 instead of pH 9.6.
Tellurite Glycine Agar is prepared according to the formula of Zebovitz,
Evans and Niven.1 The medium permits the isolation of coagulase-positive
staphylococci from food, air, dust, soil and clinical specimens.
Coagulase-negative staphylococci and other bacteria are markedly to
completely inhibited.
Chapman Tellurite Solution 1%: MAY BE IRRITATING TO

EYES, RESPIRATORY SYSTEM AND SKIN.(US) Avoid contact
with skin and eyes. Do not breathe mist. Wear suitable protective
Storage
Store Tellurite Glycine Agar dehydrated medium below 30C. The
Store Chapman Tellurite Solution 1% at 15-30C.
Expiration Date
Procedure

Tryptone and Soytone are sources of nitrogen and amino acids in
Tellurite Glycine Agar. Yeast Extract is a vitamin source in this
formulation. D-Mannitol is a source of fermentable carbohydrate
for coagulase-positive staphylococci. Lithium chloride, glycine and
potassium tellurite are the selective agents. Dipotassium phosphate is
used to buffer the medium. Bacto Agar is the solidifying agent.
Materials Provided
Bacto Tellurite Glycine Agar
Bacto Chapman Tellurite Solution 1%
Chapman Tellurite Solution is a sterile 1% solution of potassium tellurite,

a differential agent. Coagulase-positive staphylococci reduce tellurite
and produce black colonies.5
Glassware
Autoclave
Incubator (35C)
Formula

Formula Per Liter
1.
2.
3.
4.

Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Glycine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto D-Manniol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.5
g
g
g
g
g
g
g
g
Precautions
2. Tellurite Glycine Agar: HARMFUL. IRRITATING TO EYES,
RESPIRATORY SYSTEM AND SKIN. MAY CAUSE HARM TO
THE UNBORN CHILD. Avoid contact with skin and eyes. Do not
tightly closed. TARGET ORGAN(S): Blood, Kidneys, Nerves.
490

Aseptically add 10 ml Chapman Tellurite Solution 1% to the
medium at 50-55C. Mix well.

Test Procedure
coagulase-positive staphylococci from clinical specimens refer to
appropriate procedures.3,6 For the examination of staphylococci in
foods refer to standard methods.4,7
Results
Coagulase-positive staphylococci produce black colonies within 24 hours
of incubation at 35C.
The Difco Manual
Section II
Tergitol 7 Agar & Tergitol 7 Broth

2. An occasional coagulase-negative staphylococci may produce
small gray colonies, not readily confused with black coagulasepositive colonies.
References
1. Zebovitz, E., J. B. Evans, and C. F. Niven Jr. 1955. Tellurite glycine
agar: A selective plating medium for the quantitative detection
of coagulase-positive staphylococci. J. Bacteriol. 70:686-690.
2. Ludlam. 1949. Monthly Bull. Ministry of Health. 8:15.
3. Kloos, W. E., and T. L. Bannerman. 1995. Staphylococcus
and Micrococcus, p. 282 - 298. In Murray, P. R., E. J. Baron, M. A.
Pfaller, F. C. Tenover, and R. H. Yolken (ed.). Manual of clinical
Washington, D.C.
Bacto Tergitol 7 Agar

Bacto Tergitol 7 Broth

Gaithersburg, M.D.
5. MacFaddin, J. D. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, vol. 1, Williams &
Wilkins, Baltimore, M.D.
Packaging
500 g
0617-17
6 x 1 ml
6 x 25 ml
0299-51
0299-66
Intended Use
Bacto Tergitol 7 Agar and Bacto Tergitol 7 Broth are selective media
used for enumerating and differentiating coliform bacteria.
Also Known As
Bacto Tergitol 7 Agar and Bacto Tergitol 7 Broth are also know as
T7 Agar and T7 Broth, respectively.

Tergitol 7 Agar and Broth, prepared according to the formula published
by Chapman, are selective for Escherichia coli and members of the
coliform group.1 Chapman reported that the addition of Tergitol 7 to
an agar medium consisting of Proteose Peptone No. 3, Yeast Extract,
lactose, and brom thymol blue permitted unrestricted development of
all coliform bacteria and inhibited development of gram-negative spore
formers as well as gram-positive microorganisms. Counts of coliform
organisms on Tergitol 7 Agar plates were found to be 30% higher than
on some other selective media.
Chapman2 modified his original Tergitol 7 Agar formula by adding
40 mg of triphenyltetrazolium chloride (TTC) per liter. This medium
was found to be helpful in the early recognition and identification of
Escherichia coli. Confirmation of the presence of E. coli was possible
after only 10 hours incubation at 35C. Chapman also reported that
Tergitol 7 Agar with added TTC gave a selective medium suitable for
the isolation of Candida and other fungi. Candida growing on this
medium produce white, circular, convex, entire colonies about 1 mm
in diameter in 24 hours. Candida colonies may appear pale blue
because of the color of the medium, while yeasts produce red colonies.
Tergitol 7 Agar with TTC was shown to be useful in routine water
analysis and the examination of foods.3,4 The medium conforms with
the recommendations of the APHA.5
The Difco Manual

Tergitol 7 (sodium heptadecyl sulfate) inhibits growth of gram-positive
microorganisms and spore-forming gram-negative microorganisms, as
well as the swarming of Proteus, while allowing for superior recovery
of coliforms. Lactose fermentation is indicated by a color change of
the pH indicator, brom thymol blue. Lactose-fermenting microorganisms
produce yellow colonies. Escherichia coli produces yellow colonies
with yellow zones, while Enterobacter and Klebsiella colonies are
greenish-yellow. Nonfermenting organisms, such as Salmonella and
Shigella, produce colonies surrounded by blue zones.
When TTC is added to the medium, it serves as an indicator of bacterial
growth. TTC is rapidly reduced to insoluble red formazan by most
lactose-fermenting organisms except Escherichia coli, Enterobacter
and Klebsiella species. In the presence of TTC, lactose fermenters,
which includes the coliforms, produce greenish-yellow colonies with
yellow zones, while lactose nonfermenters produce red colonies
surrounded by blue zones.
Proteose Peptone No. 3 provides the carbon and nitrogen sources
required for good growth of a wide variety of organisms. Vitamins and
cofactors required for growth, as well as additional sources of nitrogen
and carbon, are provided by yeast extract. The Agar incorporated into
Tergitol 7 Agar serves as a solidifying agent.
Formula
Tergitol 7 Agar
Formula Per Liter
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Tergitol 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
g
g
g
g
g
g
491
Tergitol 7 Agar & Tergitol 7 Broth
Section II
Tergitol 7 Broth
Formula Per Liter


Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Tergitol 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
g
g
g
g
g
Storage
Expiration Date
Precautions
Uninoculated
plate
Escherichia coli
ATCC 25922

Tergitol 7 Agar
Solution:
is green, slightly opalescent.
Prepared Plates:
Green, slightly opalescent without
precipitate.
Reaction of 3.3%
Solution at 25C:
pH 6.9 0.2
Tergitol 7 Broth
Dehydrated Appearance: Beige, may have slight greenish tint,
Solution:
Prepared Tubes:
Green, slightly opalescent.
Reaction of 1.8%
Solution at 25C:
pH 6.9 0.2
ATCC 14028
Cultural Response
Prepare medium per label directions. Inoculate Tergitol 7 Agar plates
with test organisms. Inoculate Tergitol 7 Broth tubes and leave caps
loosened. Incubate at 35 2C for 18-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
19433 100-1,000 none to poor
Escherichia coli
25922* 100-1,000
good
good
ACID
PRODUCTION
N/A
+
+ = positive, yellow colony or medium

= negative, blue colony or medium as directed.
The cultures listed are the minimum that should be used for performance
testing.
Uninoculated
tube
492
Escherichia coli
ATCC 25922
ATCC 14028
The Difco Manual
Section II
Terrific Broth
Procedure
Materials Provided
Tergitol 7 Agar
Tergitol 7 Broth

Glassware
Autoclave
Incubator (35C)
OPTIONAL: Bacto TTC Solution 1% or Bacto TTC
1. Suspend medium in 1 liter distilled or deionized water:
Tergitol 7 Agar-33 grams;
Tergitol 7 Broth-18 grams.
4. OPTION: Cool Tergitol 7 Agar to 50C. Add 4 ml of either TTC
Solution 1% or a filter-sterilized 1% solution of TTC.

Test Procedure
Results

1. Since the medium with TTC permits growth of coliform organisms,
this fact must be taken into consideration in the isolation of
Candida from specimens.
Bacto Terrific Broth
2. Pour plates do not give satisfactory results.

3. Allow plates to dry with lids slightly ajar for 1-2 hours after
dispensing.6
4. Reduction of TTC is an irreversible reaction that produces an
insoluble formazan compound.
References
1. Chapman, G. H. 1947. A superior culture medium for the
enumeration and differentiation of coliforms. J. Bacteriol. 53:504.
2. Chapman, G. H. 1951. A culture medium for detecting and
confirming Escherichia coli in ten hours. Am. J. Public Health
41:1381.
3. Kulp, W., C. Mascoli, and O. Tavshanjian. 1953. Use of
tergitol-7 triphenyl tetrazolium chloride agar as the coliform
confirmatory medium in routine sanitary water analysis. Am.
J. Public Health 43:1111.
4. Mossel, D. A. A. 1962. An ecological investigation on the usefulness
of two specific modifications of Eijkmans test as an element of
the methods for the detecting of faecal contamination of foods.
J. Appl. Bacteriol. 25:20.
5. Speck, Marvin L. (ed.). 1992. Compendium of methods for the
Packaging
Tergitol 7 Agar
500 g
0455-17
Tergitol 7 Broth
500 g
0912-17
TTC Solution 1%
30 ml
3112-67*
*Store at 2-8C
Intended Use
Bacto Terrific Broth is used with Bacto Glycerol in cultivating recombinant strains of Escherichia coli.

homogeneous.
Solution:
Prepared Medium:
Reaction of 4.76%
Solution at 25C:
pH 7.2 0.2
The Difco Manual

Terrific Broth is a highly enriched medium developed by Tartoff and
Hobbs to improve yield in plasmid bearing E. coli.1 Recombinant
strains have an extended growth phase in the medium. The addition of
Cultural Response
Prepare Terrific Broth per label directions. Inoculate and
incubate the tubes at 35 2C for 18-24 hours.
ORGANISM
ATCC
INOCULUM CFU
GROWTH

23724
33694
33849
39403
47014
53868
100-1,000
100-1,000
100-1,000
100-1,000
100-1,000
100-1,000
good
good
good
good
good
good
493
Section II
extra Tryptone and Yeast Extract in the medium allows higher plasmid
yield per volume. Glycerol is used as the carbohydrate source in this
formulation. Unlike glucose, glycerol is not fermented to acetic acid.
Procedure
Tryptone and Yeast Extract provide necessary nutrients and cofactors

for excellent growth of recombinant strains of E. coli. The Yeast Extract
concentration is increased to allow for elevated cell yields.
Potassium Phosphates are added to provide potassium for cellular
systems and prevent cell death due to a drop in pH. Glycerol is added
as a carbon and energy source.

Autoclave
Incubator (35C)
Glycerol
Formula
Terrific Broth
1. Dissolve 47.6 grams in 1 liter of distilled or deionized water. Add
4 ml of Glycerol to the medium.
Terrific Broth
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Potassium Phosphate, Dibasic . . . . . . . . . . . . . . . . . . . . . .
Potassium Phosphate, Monobasic . . . . . . . . . . . . . . . . . . .
Materials Provided
12
24
9.4
2.2
g
g
g
g

Test Procedure
Precautions
Results

Storage
Expiration Date
References
1. Tartoff, K. D., and C. A. Hobbs. 1987. Improved media for
growing plasmid and cosmid clones. Bethesda Research
Laboratories Focus 9:12.
Laboratory, Cold Spring Harbor, N.Y.
Packaging
Terrific Broth
500 g
0438-17
Glycerol
100 g
500 g
0282-15
0282-17
Bacto Tetrathionate Broth Base
Intended Use
Bacto Tetrathionate Broth Base is used for enriching Salmonella species
during isolation procedures.
Also Known As
Tetrathionate Broth (Base) can be abbreviated as TT Broth (Base).

Tetrathionate Broth Base is used as a selective enrichment for the
cultivation of Salmonella species that may be present in small numbers
and compete with intestinal flora. Salmonella organisms may also be
injured in food-processing procedures, which include exposure to low
temperatures, sub-marginal heat, drying, radiation, preservatives and
sanitizers.1 Although injured cells may not form colonies on selective
media, they can, if ingested, cause disease.2 Salmonella species cause
494
many types of infections, from mild self-limiting gastroenteritis to

life-threatening typhoid fever.3 The most common form of Salmonella
disease is self-limiting gastroenteritis with fever lasting less than two
days and diarrhea lasting less than 7 days.3
Mueller4 demonstrated the effectiveness of Tetrathionate Broth for
enriching typhoid and paratyphoid bacilli while inhibiting coliform
organisms. Using modified Muellers broth, Kauffmann5,6 increased
the number of positive isolates. Tetrathionate Broth was used in studies for the poultry industry7,8 and in a collaborative study for rapid screening of Salmonella in food.9
Modifications of Tetrathionate Broth Base include TT Broth w/Brilliant
Green, TT Broth Base, Hajna, Mueller Kauffmann Tetrathionate Broth
Base and Tetrathionate with Novobiocin.10
Tetrathionate Broth Base is specified in standard methods12,13,14,15 for
Salmonella testing. Tetrathionate Broth is used in processing fecal
cultures for bacteria.16
The Difco Manual
Section II

Proteose Peptone provides the nitrogen, carbon, vitamins and amino
acids in Tetrathionate Broth Base. Selectivity is accomplished by the
combination of Sodium Thiosulfate and tetrathionate, which suppresses
commensal intestinal organisms.17 (Tetrathionate is formed in the
medium upon addition of the iodine and potassium iodide solution.)
Organisms containing the enzyme tetrathionate reductase will proliferate
in the medium. Bile Salts, a selective agent, suppresses coliform
bacteria and inhibits gram-positive organisms. Calcium Carbonate
neutralizes and absorbs toxic metabolites.
Formula

Storage

Formula Per Liter
Bacto Bile Salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
g
g
g
g
Store Tetrathionate Broth Base dehydrated below 30C. The dehydrated

medium is very hygroscopic. Keep container tightly closed. Store the
prepared medium at 2-8C.
Expiration Date
Precautions
2. Tetrathionate Broth Base:
Procedure
Materials Provided
Glassware
Iodine solution (see Method of Preparation)
Incubator (35C)
Sterile tubes
Dehydrated Appearance: White to off-white, may have a slight

greenish tint, free-flowing,
homogeneous.
Solution:
4.6% solution, insoluble in distilled or
deionized water. Suspension is milky
white, opaque. On standing, supernatant
is nearly colorless to light yellow
over a heavy white precipitate.
Prepared Medium:
Nearly colorless to light yellow
supernatant over a heavy white
precipitate.
Reaction of 4.6%
Solution at 25C:
pH 8.4 0.2 (measured before
iodine solution is added).
Cultural Response
Prepare Tetrathionate Broth Base per label directions and enrich
with 2% iodine solution. Inoculate with 100-1,000 CFUs of test
organism and incubate at 35 2C for 18-24 hours. Subculture
onto MacConkey Agar and incubate at 35 2C for 18-24 hours.
ORGANISM
ATCC
GROWTH
COLOR OF COLONY ON
MACCONKEY AGAR
Escherichia 25922* little or no increase

coli
in the number of colonies
Salmonella 14028*
good
typhimurium
pink w/bile
precipitate
colorless
The Difco Manual
1.
2.
3.
4.

Heat to boiling. DO NOT AUTOCLAVE.
Cool to below 60C.
potassium iodide in 20 ml water).
5. DO NOT REHEAT MEDIUM. DO NOT AUTOCLAVE.
6. Dispense into sterile tubes. Use immediately.

Test Procedure
Salmonella, refer to appropriate procedures outlined in the references.
Results

495
m Tetrathionate Broth Base
References
2. Sorrells, K. M., M. L. Speck, and J. A. Warren. 1970. Pathogenicity of Salmonella gallinarum after metabolic injury by freezing.
Appl. Microbiol. 19:39- 43.
4. Muller, L. 1923. Un nouveau milieu denrichissement pour la
recherche du bacille typhique et des paratyphiques. C. R. Soc. Biol.
89:434. Paris.
5. Kauffmann, F. 1930. Ein kombiniertes anreicherungsverfahren
fur typhus-und-paratyphusbacillen. Zentralb. Bakteriol.
Parasitenke. Infektionskr. Hyg. Abt.I orig. 113:148.
Anreicherungsverfahren fur Salmonellabacillen. Z. Hyg.
7. Jones, F. T., R. C. Axtell, D. V. Rives, S. E. Scheideler, F. R.
Tarver, Jr., R. L. Walker, and M. J. Wineland. 1991. A survey of
Salmonella contamination in modern broiler production. J. Food
Prot. 54:502-507.
8. Barnhart, H. M., D. W. Dressen, R. Bastien, and O. C.
Pancorbo. 1991. Prevalence of Salmonella enteritidis and other
serovars in ovaries of layer hens at time of slaughter. J. Food Prot.
54:488-492.
9. Eckner, K. F., W. A. Dustman, M. S. Curiale, R. S. Flowers,
and B. J. Robison. 1994. Elevated-temperature, colorimetric,
monoclonal, enzyme-linked immunosorbent assay for rapid
screening of Salmonella in foods: collaborative study. J. Assoc.
Off. Anal. Chem. 77:374-383.
Section II
10. MacFaddin, J. D. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, p. 751-754,

R. M. Amaguana. 1995. Salmonella. p 5.01-5.20. In Bacteriological
analytical manual, 8th ed. AOAC International. Gaithersburg, MD.
1992. Salmonella, p.371-422. In Vanderzant, C. and D. F.
examination of food, 3rd ed. American Public Health Association,
Washington, D.C.
Marshall (ed.) Standard methods for the examination of dairy products. 16th ed., American Public Health Association, Washington, D.C.
15. Federal Register. 1991. Animal and plant health inspection
service: chicken affected by Salmonella enteritidis, final rule. Fed.
Regist. 56:3730-3743.
16. Isenberg, H. D. (ed.). 1992. Clinical microbiology procedures handbook, vol. 1, American Society for Microbiology, Washington, D. C.
17. Knox, R., P. H. Gell, and M. R. Pollack. 1942. Selective
media for organisms of the Salmonella group. J. Pathol. Bacteriol.
54:469-483.
Packaging
500 g
2 kg
0104-17
0104-07
Bacto m Tetrathionate Broth Base
Intended Use
Bacto m Tetrathionate Broth Base is used for selectively enriching
Salmonella by membrane filtration prior to isolation procedures.

Salmonella spp. cause many types of infections, from mild self-limiting
gastroenteritis to life-threatening typhoid fever.2 The most common
form of Salmonella disease is self- limiting gastroenteritis with fever
lasting less than two days and diarrhea lasting less than 7 days.2
Tetrathionate Broth, in single strength and without calcium carbonate,
was used by Kabler and Clark1 for the preliminary enrichment of
Salmonella other than S. typhi. Their investigation found that
approximately 80% of Salmonella species recovered were from mixed
cultures and that most coliforms were suppressed. The presence of
calcium carbonate in the medium gave poor, erratic results. The
authors1 reported favorable results for enrichment of S. typhimurium
in the membrane filtration technique. This study used a 3-hour
preliminary incubation on pads saturated with Tetrathionate Broth
followed by 15 hours incubation on m Brilliant Green Broth.
496
m Tetrathionate Broth Base has the same formulation as Tetrathionate

Broth Base, except that calcium carbonate has been omitted.1

Proteose Peptone provides nitrogen, vitamins, amino acids and carbon
in m Tetrathionate Broth Base. Selectivity is achieved by the combination
of sodium thiosulfate and tetrathionate, which suppresses commensal
intestinal organisms.3 Tetrathionate is formed in the medium by the
addition of iodine and potassium iodide solution. Organisms containing
the enzyme tetrathionate reductase will proliferate in the medium. Bile
Salts, a selective agent, is added to suppress coliform bacteria and
inhibit gram-positive organisms.
Formula
Formula Per Liter
Bacto Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Bacto Bile Salts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Sodium Thiosulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 g

The Difco Manual
Section II
Precautions
TARGET ORGAN(S): Lungs.
Storage

Iodine solution (see Method of Preparation, #4)
Incubator (35C)
m Brilliant Green Broth
1.
2.
3.
4.

Cool medium to below 60C.
potassium iodide dissolved in 20 ml distilled or deionized water).
Use the complete medium containing iodine within 24 hours.
5. Do not heat the medium after adding the iodine solution.
6. Dispense 2 ml amounts of medium onto sterile absorbent pads in
50-60 mm Petri dishes.

Obtain and process samples according to the techniques and procedures established by laboratory policy.
Use rehydrated medium within 24 hours.
Expiration Date
Test Procedure
1. Perform membrane filtration with the inoculum to be tested.
Procedure
Escherichia coli
ATCC 25922
Materials Provided

Glassware

Dehydrated Appearance: Light beige with greenish cast,
Solution:
amber, clear, may have some precipitate.
Prepared Medium:
Light amber, clear, may have some precipitate.
Reaction of 3.6%
Solution at 25C:
pH 8.0 0.2 (before adding iodine solution)
ATCC 14028
Cultural Response
Prepare m Tetrathionate Broth per label directions. Inoculate and incubate at 35 2C
in a humid atmosphere for approximately 3 hours. Transfer filters to pads containing
m Brilliant Green Broth and continue incubation to a total of 18-24 hours.
ORGANISM
Escherichia coli
ATCC
INOCULUM
CFU
GROWTH
COLONY COLOR ON
m BRILLIANT GREEN BROTH
25922*
14028*
30-300
30-300
good
good
yellow to green
pink to red
*These cultures are available as Bactrol Disks and should be used as directed in Bactrol Disks
The Difco Manual
497
Thermoacidurans Agar
Section II
2. Place the membrane filter on a pad soaked with m Tetrathionate

Broth (with iodine) and incubate at 35 2C for 3 hours.
3. Aseptically transfer the filter to a pad soaked with 2 ml m Brilliant
Green Broth in a 50-60 mm Petri dish.
4. Incubate at 35 2C for an additional 15-21 hours (total incubation
to 18-24 hours) in a humid atmosphere.
Results
Examine for growth. Salmonella species produce pink to red colonies.

Bacto Thermoacidurans Agar
Intended Use
Bacto Thermoacidurans Agar is used for isolating and cultivating
Bacillus coagulans (Bacillus thermoacidurans) from foods.

Stern et al. 1 described a medium for isolating B. coagulans
(B. thermoacidurans) which causes flat sour spoilage in tomato
juice. Flat sour spoilage of canned foods can caused by Bacillus
coagulans (Bacillus thermoacidurans). Bacterial growth results in a
0.3-0.5 drop in pH, while the ends of the can remain flat. B. coagulans
is a soil microorganism that can be found in canned tomato
products and dairy products. Conditions favorable to multiplication
of the organism can result in spoilage of the food product.2
Thermoacidurans agar can also be used to isolate mesophilic
spore forming anaerobes (Clostridium spp.) from foods. 2 These
microorganisms tolerate high heat, grow in the absence of oxygen
References
1. Kabler and Clark. 1952. Am. J. Public Health 42:390.
3. Knox, R., P. H. Gell, and M. R. Pollack. 1942. Selective media
for organisms of the Salmonella group. J. Pathol. Bacteriol.
54:469-483.
Packaging
500 g
0580-17
and grow over the range of temperatures used in canned and

processed foods. They are of primary importance in spoilage of
low-acid foods packed in hermetically sealed containers.2

Thermoacidurans Agar contains Proteose Peptone to provide the
carbon and nitrogen for general growth requirements. Yeast
Extract supplies B-complex vitamins which stimulate bacterial
growth. Dextrose is the carbohydrate source. Bacto Agar is a
solidifying agent.
Formula
Formula Per Liter
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
g
g
g
g
g
Precautions


Solution:
amber, opalescent without
Prepared Medium:
Light amber, opalescent without
precipitate.
Reaction of 3.9%
Solution at 25C:
pH 5.0 0.2
Cultural Response
Prepare Thermoacidurans Agar per label instructions. Inoculate
and incubate the plates at 55 1C for 18-48 hours.
TEST ORGANISM
ATCC
INOCULUM
CFU
Bacillus coagulans
7050
100-1,000
GROWTH
good
The culture listed is the minimum that should be used for performance
testing.
498
Storage
Expiration Date
Procedure
Materials Provided

Glassware
The Difco Manual
Section II
Thiamine Assay Medium & Thiamine Assay Medium LV
Autoclave
Incubator (55C)
Petri dishes
Results

Microorganisms other than B. coagulans may grow on this medium.
Perform microscopic examination and biochemical tests to identify to
genus and species if necessary.
References
1. Stern, Hegarty, and Williams. 1942. Food Research 7:186.

Test Procedure
Packaging
Thermoacidurans Agar
500 g
0303-17
Bacto Thiamine Assay Medium

Bacto Thiamine Assay Medium LV
Intended Use
Bacto Thiamine Assay Medium is used for determining thiamine
Bacto Thiamine Assay Medium LV is used for determining
thiamine concentration by the microbiological assay technique using
Lactobacillus viridescens ATCC 12706.
Also Known As
Thiamine is also know as Vitamin B1.

of vitamins. Three types of medium are used for this purpose:
1. Maintenance Medium: For carrying the stock culture to preserve the
2. Inoculum Medium: To condition the test culture for immediate use.
3. Assay Medium: To permit quantitation of the vitamin under test.
Assay media contain all factors necessary for optimal growth of the
test organism except the single essential vitamin to be determined.
Thiamine Assay Medium is prepared according to the formula by Sarett
and Cheldelin.1 Lactobacillus fermentum ATCC 9338 is used as the
test organism in the microbiological assay of thiamine.
Thiamine Assay Medium LV, patterned after APT medium, was
described by Deibel, Evans and Niven2 for the microbiological assay
of thiamine using Lactobacillus viridescens ATCC 12706.
Nutritional studies by Evans and Niven3 on the heterofermentative
lactobacilli that cause greening in cured meat products indicated that
thiamine was an essential vitamin for growth of these organisms.
Deibel, Evans and Niven4 described APT medium for lactobacilli
cultivation. They reported that lactobacilli required at least 10 ng
thiamine per ml for growth in contrast to 0.2 to 3 ng per ml for
thiamine-requiring streptococci, leuconostocs and staphylococci. Further,
they suggested that lactobacilli requiring large amounts of thiamine
The Difco Manual
might be employed in microbiological assay procedures. In 1957,2 these

authors described a medium for the microbiological assay of thiamine
using Lactobacillus viridescens ATCC 12706 as the test organism.
This medium is known as Thiamine Assay Medium LV.

Thiamine Assay Medium and Thiamine Assay Medium LV are free
from thiamine, but contain all other nutrients and vitamins essential
for the growth of the test organisms. The addition of thiamine in specified
measured turbidimetrically.
Formula
Thiamine Assay Medium
Formula Per Liter
Thiamine-Free Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Bacto Vitamin Assay Casamino Acids . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
Adenine Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Uracil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Folic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.8
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
g
g
g
g
g
mg
mg
mg
g
g
g
g
g
g
g
g
g
g
mg
mg
mg
499
Section II
Precautions
Thiamine Assay Medium LV

Formula Per Liter
Thiamine-Free Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . 10
Thiamine-Free Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Sodium Citrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Dipotassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.08
Tween 80 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
g
g
g
g
g
g
g
g
g
g


3. Great care to avoid contamination of media or glassware must be
taken in microbiological assay procedures. Extremely small amounts
of foreign material may be sufficient to give erroneous results. Scrupulously clean glassware free from detergents and other chemicals
must be used. Glassware must be heated to 250C for at least 1 hour
to burn off any organic residues that might be present.
Storage
Store the dehydrated media at 2-8C. The dehydrated medium is very

Dehydrated Medium: Beige, homogeneous, tendency to
clump.
Solution:
distilled or deionized water on boiling
2-3 minutes. 4.25% solution is light
amber, clear, may have a slight precipitate.
Prepared Medium:
4.25% solution is light amber, clear,
Reaction of 4.25%
Solution at 25C:
pH 6.5 0.2
Expiration Date

Dehydrated Appearance: Beige, homogeneous, tendency to
clump.
Solution:
distilled or deionized water on boiling
2-3 minutes. 4.2% solution is light
amber, clear, may have a slight
precipitate.
Prepared Medium:
4.2% solution is light amber, clear,
Reaction of 4.2%
Solution at 25C:
pH 6.0 0.2
Glassware
Autoclave
Spectrophotometer or nephelometer
Centrifuge
Incubator, 30C and 35C
Sterile tubes and caps
Sterile 0.85% NaCl
Stock culture of Lactobacillus viridescens ATCC 12706 or
Stock culture of Lactobacillus fermentum ATCC 9338
APT Agar
APT Broth
Thiamine hydrochloride
Cultural Response
Prepare single-strength Thiamine Assay Medium per label
directions. Prepare a standard curve using a thiamine hydrochloride reference standard at 0.0 to 0.05 g per 10 ml.
Inoculate with Lactobacillus fermentum ATCC 9338 and
incubate with caps loosened at 35-37C for 16-18 hours. Read
percent transmittance using a spectrophotometer at 660 nm.
Prepare single-strength Thiamine Assay Medium LV per label
directions. Prepare a standard curve using a thiamine hydrochloride reference standard at 0.0 to 25.0 g per 10 ml.
Inoculate with Lactobacillus viridescens ATCC 12706 and
incubate with caps loosened at 30 2C for 16-20 hours. Read
percent transmittance using a spectrophotometer at 660 nm.
500
Procedure
Materials Provided
1. Suspend the medium in 100 ml distilled or deionized water:
Thiamine Assay Medium - 8.5 grams;
Thiamine Assay Medium LV - 8.4 grams.
2. Boil 2-3 minutes to dissolve completely.
3. Dispense 5 ml amounts into tubes, evenly dispersing the precipitate.
5. Adjust tube volume to 10 ml with distilled or deionized water.
The Difco Manual
Section II

Prepare assay samples according to references given in the specific
assay procedures. The samples should be diluted to approximately the
Test Procedure
Prepare stock cultures of the test organism, Lactobacillus fermentum
ATCC 9338, by stab inoculation on Lactobacilli Agar AOAC or
Micro Assay Culture Agar. After 24-48 hours incubation at 35-37C,
keep the tubes in the refrigerator. Make transfers in triplicate at monthly
intervals.
Prepare the inoculum by subculturing a stock culture of the test
organism in 10 ml of Lactobacilli Broth AOAC or Micro Inoculum
Broth. After 16-18 hours incubation at 35-37C, centrifuge the cells
under aseptic conditions and decant the supernatant liquid. Wash the
cells three times with 10 ml sterile 0.85% NaCl. After the third wash,
resuspend the cells in 10 ml sterile 0.85% NaCI. Add 0.5 ml of this
suspension to 100 ml sterile 0.85% NaCl. Use one drop of the resulting
suspension to inoculate the assay tubes.
A standard curve should be run with each assay because conditions of
heating and incubation temperature that influence the standard curve
readings cannot always be duplicated.
The tubes for the Thiamine Assay Medium standard curve contain
0.0, 0.005, 0.01, 0.015, 0.02, 0.03, 0.04 and 0.05 g of thiamine
hydrochloride per 10 ml tube. The most effective assay range for
Thiamine Assay Medium is between 0.005 and 0.03 g thiamine.
Prepare the stock solution of thiamine required for the preparation of
the standard curve in Thiamine Assay Medium as follows:
1. Dissolve 0.1 gram of thiamine hydrochloride in 1,000 ml of
distilled water (100 g/ml).
2. Add 1 ml of the solution in Step 1 to 99 ml distilled water (1 g/ml).
3. Add 1 ml of the solution in Step 2 to 99 ml distilled water to give a
final concentration of 10 ng (0.010 g/ml). Use 0.0, 0.5, 1, 1.5, 2,
3, 4 and 5 ml of this final solution per tube. Prepare fresh stock
solution daily.
After 20-24 hours incubation at 35-37C, L. fermentum ATCC 9338 is
capable of using the pyrimidine and thiazole moieties of the thiamine
molecule. It is essential that the growth response be measured turbidimetrically prior to this time. Incubate the tubes at 35-37C for 16-18
hours, then place in the refrigerator for 15-30 minutes to stop growth.
The growth can then be measured by any suitable nephelometric method.
Prepare stock cultures of the test organism, L. viridescens ATCC 12706,
by stab inoculation on APT Agar or Lactobacilli Agar AOAC. After
24-48 hours incubation at 30 2C, keep the tubes in the refrigerator.
Make transfers in triplicate at monthly intervals.
Prepare the inoculum by subculturing a stock culture of the test organism
to 10 ml APT Broth or Lactobacilli Broth AOAC. After 16-20 hours
incubation at 30 2C, centrifuge the cells under aseptic conditions
and decant the supernatant liquid. Wash the cells three times with 10 ml
sterile 0.85% NaCl. After the third wash, resuspend the cells in 10 ml
sterile 0.85% NaCl. Add 1 ml of this cell suspension to 100 ml sterile
0.85% NaCl. Use one drop of this suspension to inoculate the assay tubes.
The Difco Manual
A standard curve should be run with each assay because conditions of

heating and incubation temperature that influence the standard curve
readings cannot always be duplicated.
The standard curve for Thiamine Assay Medium LV is obtained by
using thiamine at levels of 0.0, 1, 2.5, 5, 7.5, 10, 15, 20 and 25 ng of
thiamine hydrochloride per 10 ml tube. This is obtained by using 0.0,
0.2, 0.5, 1, 1.5, 2, 3, 4 and 5 ml of the standard solution, which contains
5 ng (0.005 g) thiamine hydrochloride per ml. The most effective
assay range is between 2.5 and 20 ng per tube.
The solution for preparing the standard curve for Thiamine Assay
Medium LV may be prepared as follows:
1. Dissolve 50 mg of thiamine hydrochloride in 500 ml distilled
water (100 g/ml).
2. Add 1 ml of the solution in Step 1 to 99 ml distilled water (1 g/ml).
3. Add 1 ml of the solution in Step 2 to 199 ml distilled water to give
a final concentration of 5 ng (0.005 g) per ml.
Following incubation of L. viridescens ATCC 12706 at 30 2C for
16-20 hours, the growth response is measured turbidimetrically.
Results
Thiamine Assay Medium and Thiamine Assay Medium LV
or cup.

2. Aseptic technique should be used throughout the microbiological
assay procedure.
different nutritional requirements which will not give a satisfactory
response.
4. For successful results, all conditions of the assay must be followed
exactly.
References
1. Sarett and Cheldelin. 1944. J. Biol. Chem. 155:153.
2. Deibel, Evans, and Niven. 1957. Paper presented 57th general
meet. Soc. Am. Bacteriol. Detroit, MI.
3. Evans and Niven. 1951. J. Bacteriol. 62:599.
4. Diebel, Evans, and Niven. 1955. Bacteriol. Proc.
Packaging
100 g
0326-15
100 g
0808-15
501
Thioglycollate Media
Section II
Bacto Fluid Thioglycollate Medium . Bacto NIH Thioglycollate
Broth . Bacto Brewer Thioglycollate Medium . Bacto Fluid
Thioglycollate Medium w/Beef Extract . Bacto Fluid
Thioglycollate Medium w/K Agar . Bacto Thioglycollate Medium
w/o Dextrose . Bacto Thioglycollate Medium w/o Dextrose or
Indicator . Bacto Thioglycollate Medium w/o Indicator
Intended Use
Bacto Fluid Thioglycollate Medium is used for detecting microorganisms
in normally sterile materials. Fluid Thioglycollate Medium conforms
to the formula specified by the US Pharmacopeia XXIII (USP)1, the
Code of Federal Regulations (21 CFR)2 and European Pharmacopeia
for sterility testing of pharmaceutical products, biologics and devices.
Bacto NIH Thioglycollate Broth is used in detecting microorganisms
in normally sterile, turbid or viscous materials. This formula conforms
with USP Alternate Thioglycollate Medium.1
Bacto Brewer Thioglycollate Medium is for detecting microorganisms
in normally sterile materials.
Bacto Fluid Thioglycollate Medium w/Beef Extract is used in
cultivating microorganisms from normally sterile biological products.
Bacto Fluid Thioglycollate Medium w/K Agar is for detecting
microorganisms in normally sterile materials containing mercurial
preservatives when clarity and early visualization of growth are desired.
Bacto Thioglycollate Medium w/o Dextrose and Thioglycollate
Medium w/o Dextrose or Indicator are used for detecting microorganisms in normally sterile materials, especially those containing
mercurial preservatives. These media formulations may be used with
added carbohydrates for fermentation studies.
Bacto Thioglycollate Medium w/o Indicator is for detecting microorganisms in normally sterile materials, especially those containing
mercurial preservatives, when no oxidation-reduction indicator is
required.
Also Known As
Fluid Thioglycollate Medium is often referred to as Thioglycollate
Medium and abbreviated as FTM.
NIH Thioglycollate Medium is also known as USP Thioglycollate
Medium Alternative and Alternate Fluid Thioglycollate.
Thioglycollate Medium w/o Indicator and Thioglycollate Medium w/o
Dextrose or Indicator have been called Thioglycollate Fermentation
Media.

Quastel and Stephenson4 found that the presence of a small amount of
a compound containing an -SH group (cysteine, thioglycollic acid,
502
glutathione) permitted aerobic growth of Clostridium sporogenes in

tryptic digest broth.
Falk, Bucca and Simmons5 pointed out the advantages of using small
quantities of agar (0.06-0.25%) in detecting contaminants during
sterility testing of biologicals. The value of combining a small amount
of agar and a reducing substance was demonstrated by Brewer.6
Brewers experiments revealed that in a liquid medium containing
0.05% agar, anaerobes grew equally well in the presence or absence
of sodium thioglycollate. Marshall, Gunnish and Luxen7 reported
satisfactory cultivation of anaerobes in Brewers Thioglycollate
Medium in the presence of a mercurial preservative. Nungester, Hood
and Warren 8 and Portwood 9 confirmed the neutralization of the
bacteriostatic effect of mercurial compounds by sodium thioglycollate.
Malin and Finn10 reported the commonly used medium containing
thioglycollate is inhibitory to some organisms in the presence of a
carbohydrate. In 1941, the National Institutes of Health specified the
use of two thioglycollate media in sterility testing, the Brewer Formula
and the Linden Formula.11 The Linden Formula was later referred to as
Modified Brewer Thioglycollate Medium in which meat infusion was
replaced by plant (soy) peptones.12
Fluid Thioglycollate Medium is prepared according to the formula
in the FDA Bacteriological Analytical Manual (BAM)13 and AOAC
Official Methods of Analysis14 for the examination of food, and for
determining the phenol coefficient and sporicidal effects of disinfectants.
Fluid Thioglycollate Medium is also specified for sterility checks on
banked blood.15
Fluid Thioglycollate Medium w/ Beef Extract is recommended by
Animal and Plant Health Inspection Service, USDA,3 in the detection
of viable bacteria in live vaccines. Thioglycollate Medium w/o Dextrose
and Thioglycollate Medium w/o Dextrose or Indicator may be used
with added carbohydrates for fermentation studies.
Thioglycollate Medium w/o Indicator is the medium of choice for
diagnostic work because the lack of indicator avoids possible toxicity
to organisms.13 This medium supports a minimal inoculum with early
visibility of growth.
When used as an enrichment broth to support plated media,
thioglycollate media are often supplemented with hemin and vitamin
K1.17 Several modifications of this medium, usually with the addition
The Difco Manual
Section II
Solution:
Fluid Thioglycollate Medium
Solution:
deionized water on boiling. Appearance
of solution immediately after
sterilization - light amber, clear.
Prepared Medium:
Appearance of solution immediately
after sterilization - light amber, clear.
After cooling to room temperature,
light amber, slightly opalescent with
pink upper layer. If pink layer is greater
than 10% of the tube, the medium may be
restored once by heating on a steambath
until the pink color disappears.
Reaction of 2.98%
Solution at 25C:
pH 7.1 0.2
Brewer Thioglycollate Medium
Solution:
4.05 % solution, soluble in distilled or
deionized water on boiling; medium amber,
clear to very slightly opalescent with upper
10% or less medium green on standing.
Prepared Medium:
Medium amber, clear to very slightly
opalescent with upper 10% or less
medium green.
Reaction of 4.05%
Solution at 25C:
pH 7.2 0.2
NIH Thioglycollate Broth
Solution:
clear to very slightly opalescent, may have
Prepared Medium:
Reaction of 2.9%
Solution at 25C:
pH 7.1 0.2
Fluid Thioglycollate Medium w/Beef Extract
Solution:
3.47% solution, soluble upon boiling
for 1-2 minutes. Immediately after
sterilization, appearance is medium to
dark amber, clear, becoming medium to
dark amber with upper 10% or less of
medium pink and slightly opalescent.
Prepared Medium:
Medium to dark amber with upper 10%
or less medium pink, slightly opalescent.
Reaction of 3.47%
Solution at 25C:
pH 7.2 0.2
Fluid Thioglycollate Medium w/K Agar
The Difco Manual
Prepared Medium:
Reaction of 2.9%
Solution at 25C:

deionized water upon boiling. Appearance of
solution immediately after sterilization-light
amber, clear. After cooling to room
temperature and shaking, solution becomes
pink with slight opalescence.
Appearance of solution immediately
after sterilization - light amber, clear.
After cooling to room temperature, light
amber, with some opalescence with
upper 10% or less of medium pink.
pH 7.2 0.2
Thioglycollate Medium w/o Dextrose

Solution:
clear to very slightly opalescent. Upper
10% or less medium green on standing.
Prepared Medium:
Light amber, very slightly opalescent with
upper 10% or less medium turning green
on cooling.
Reaction of 2.4%
Solution at 25C:
pH 7.2 0.2
Thioglycollate Medium w/o Indicator
Solution:
amber, clear without significant precipitate.
After cooling to room temperature, solution
may exhibit slight opalescence.
Prepared Medium:
Appearance of solution immediately after
sterilization- light amber, clear without
significant precipitate. After cooling to
room temperature, solution may exhibit
slight opalescence.
Reaction of 2.9%
Solution at 25C:
pH 7.2 0.2
Thioglycollate Medium w/o Dextrose or Indicator
Solution:
amber, clear with no significant precipitate.
After cooling to room temperature-light,
very slightly to slightly opalescent with no
Prepared Medium:
Immediately after sterilization-light, clear
with no significant precipitate. After
cooling to room temperature - light, very
slightly to slightly opalescent with no
Reaction of 2.4%
Solution at 25C:
pH 7.2 0.2
503
Section II
of sodium polyanetholesulfonate (SPS), are used in the inoculum of

blood cultures specifically, for the isolation of anaerobes.18
preservatives, making thioglycollate media useful in testing material

which contains heavy metals.
The methodologies for the multiple applications using thioglycollate

medium are outlined in the references.
Resazurin or Methylene Blue are oxidation indicators. In the oxidized

state, methylene blue appears green, resazurin turns pink. In the
reduced state both compounds are colorless. Bacto Agar eliminates the
need for seals because it retards dispersion of CO2, diffusion of oxygen
and reducing substances. 12 Substituting K Agar and Potassium
Chloride for Bacto Agar and Sodium Chloride in Fluid Thioglycollate
Medium w/K Agar produces a medium with greater clarity to facilitate
earlier visual recognition of growth.

Thioglycollate media support the growth of a large variety of fastidious
microorganisms having a wide range of growth requirements. The
nitrogen source, provided by Casitone, Infusion from Beef, Proteose
Peptone, Beef Extract, Pancreatic Digest of Casein varies with the
formula. Yeast Extract is added as a source of vitamins.
Dextrose is included in the formulations because many organisms show

earlier and more vigorous growth. Sodium chloride is used to maintain
the osmotic balance of the media. Potassium Chloride and Dipotassium
Phosphate are used as buffering agents.
Sodium Thioglycollate, Thioglycollic Acid and L-Cystine lower the

oxidation-reduction potential of the medium by removing oxygen to
maintain a low Eh. By creating an environment with a low Eh, the
reducing agents prevent the accumulation of peroxides which can be
toxic to some organisms. The sulfhydryl groups (-SH) of these
compounds also neutralize the antibacterial effect of mercurial
Cultural Response
Brewer Thioglycollate Medium (0236), Thioglycollate Medium w/o Indicator (0430),
Thioglycollate Medium w/o Dextrose (0363), Thioglycollate Medium w/o Dextrose
or Indicator (0432).
Medium was prepared per label directions. Tubes were inoculated with the
test organisms and incubated at 35 2C for 18-48 hours.
ORGANISM
ATCC
INOCULUM CFU
GROWTH
Clostridium novyi
25923
7659
11437
25285
10-100
10-100
10-100
10-100
good
good
good
good
Fluid Thioglycollate Medium w/K Agar (0607), Fluid Thioglycollate Medium

w/Beef Extract (0697), NIH Thioglycollate Broth (0257).
Medium was prepared per label directions. Tubes were inoculated with the
test organisms and incubated at 30-35C for up to 7 days.
ORGANISM
ATCC
INOCULUM CFU
GROWTH
Bacillus subtilis
Candida albicans
6633
8482
10231
19404
10-100
10-100
10-100
10-100
good
good
good
good
Uninoculated
tube
USP and EP Growth Promotion Procedure1

Fluid Thioglycollate Medium. Prepare FTM per label directions. Inoculum
of 10-100 CFU were used and incubated for up to 7 days at temperature
specified.
ORGANISM
Bacillus subtilis
Candida albicans
Candida albicans
Clostridium novyi
ATCC
INOCULUM
CFU
GROWTH
INC. TEMP
6633*
8482*
10231*
2091*
19404*
6538P*
25923
7659
13124
10-100
10-100
10-100
10-100
10-100
10-100
10-100
10-100
10-100
growth must be evident

good
good
good
good
30-35C
30-35C
20-25C
20-25C
30-35C
30-35C
30-35C
30-35C
30-35C
ATCC 8482
Mercurial Neutralization
This test is performed by recovering the test
organisms in Fluid Thioglycollate Medium
after exposure to 1% Merthiolate
ORGANISM
ATCC
6538P
19615**
INOCULUM
CFU
RECOVERY
1,000
1,000
good
good

**These cultures are available as Bactrol Disks
*Pharmacopeia growth promotion
504
The Difco Manual
Section II
Formula

Formula per liter

Formula Per Liter
Bacto Casitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.75
Resazurin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.001
g
g
g
g
g
g
g
g

Formula Per Liter
Bacto Casitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.25
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.75
Methylene Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.002
g
g
g
g
ml
g
g
g
g
g
g
g
g
g
g

Formula Per Liter
Bacto Casitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
g
g
g
g
g
g

Formula per liter
Bacto Casitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
K Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.45
Resazurin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.001
g
g
g
g
g
g
g

Formula Per Liter
Pancreatic Digest of Casein . . . . . . . . . . . . . . . . . . . . . . . . 15
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.75
Resazurin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.001
g
g
g
g
g
g
g
g
g

Formula Per Liter

Formula Per Liter
Beef, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Methylene Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.002

Bacto Casitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.25
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.75
g
g
g
g
g
ml
g
g

Bacto Casitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.25
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.75
g
g
g
g
ml
g
Precautions
1. For Laboratory Use:
2. Do not reheat the media more than once, because continued reheating
gives rise to toxicity. Do not to reheat NIH Thioglycollate Broth.
3. When testing human serum, treat all specimens as infectious agents.
Storage
1. Store the dehydrated medium below 30C. The dehydrated medium
2. Store prepared media at 15-30C.
The Difco Manual
505
3. For Fluid Thioglycollate Medium, Fluid Thioglycollate Medium

w/Beef Extract and Fluid Thioglycollate Medium w/K Agar,
if more than 30% of the medium is pink prior to use, reheat once
(100C) to drive off absorbed oxygen.
For Brewer Thioglycollate Medium, if more than 20% of the
medium is green prior to use, reheat once (100C).
After prolonged storage, reheat Thioglycollate Medium w/o
Indicator, Thioglycollate Medium w/o Dextrose or Indicator
and Thioglycollate Medium w/o Dextrose only once in flowing
steam or a boiling water bath to drive off dissolved gases.
Expiration Date
Procedure
Materials Provided

Glassware
Autoclave
Incubator (35C)
Waterbath
Sterile tubes
deionized water:
29.8 g
40.5 g
29 g
34.7 g
29 g
24 g
29 g
Thioglycollate Medium w/o Dextrose or Indicator 24 g
3. Dispense as desired, using only clean, rust-free equipment.

Test Procedure
For a complete discussion on the isolation and identification of bacteria
and yeasts, refer to appropriate procedures outlined in the references.
506
Section II
Results
Typically growth is visually observed in the media. Gram negative
bacilli tend to grow diffusely, gram positive cocci exhibit puff-ball
type growth and strict aerobes, such as pseudomonads and yeast, tend
to grow in a thin layer on the surface of the broth.
References
States pharmacopeia, 23rd ed., p.1686-1690. The United States
Pharmacopeial Convention Inc. Rockville, MD.
2. Federal Register. 1992. General biological products standards.
Fed. Regist. 21:610.12.
3. Federal Register. 1992. Detection of viable bacteria and fungi
except in live vaccines. Fed. Regist. 21:113.26.
4. Quastel and Stephenson. 1926. J. Biochem. 20:1125.
5. Falk, C. R., H. Bucca, and M. P. Simmons. 1939. A comparative
study of the use of varying concentrations of agar in the test
medium used to detect contaminants in biologic products.
6. Brewer, J. H. 1940. Clear liquid mediums for the aerobic cultivation of anaerobes. J. Amer. Med. Assoc. 115:598-600.
7. Marshall, M. S., J. B. Ginnish, and M. P. Luxen. 1940. Test for
the sterility of biologic products. Proc. Soc. Exp. Biol. Med.
43:672.
8. Nungester, W. J., M. N. Hood, and M. K. Warren. 1943. Use
of thioglycollate media for testing disinfectants. Proc. Soc. Exp.
Biol. Med. 52:287.
9. Portwood, L. 1944. Observations of the failure of sterility test
media to support the growth of laboratory contaminants.
10. Malin, B., and R. K. Finn. 1957. The use of a synthetic resin in
anaerobic media. J. Bacteriol. 62:349-350.
11. Linden. 1941. NIH fluid thioglycollate medium for the sterility test.
12. MacFaddin, J. F. 1985. Media for isolation-cultivationidentification maintenance of medical bacteria, vol.1, p. 755-762.
Rhodehamel. 1995. Clostridium perfringens, p.16.01-16.06. In
Gaithersburg, MD.
Methods of Analysis of AOAC International, 16th ed. AOAC
15. Federal Register. 1992. Additional standard for human blood and
blood products. Fed Regist. 21:640.2.17.
16. Forbes, B. A., and P. A. Granato. 1995. Processing specimens
for bacteria, p. 267. In Murray, P. R., Baron E. J., Pfaller,
M. A., Tenover, F .C., and R. H. Yolken (ed.), Manual of clinical
Washington D.C.
17. Isenberg, H. D. (ed.) 1992. Processing and interpretation of blood
cultures, p.1.7.1 - 1.7.2. Clinical microbiology procedures handbook,
vol.1, American Society for Microbiology, Washington, D.C.
The Difco Manual
Section II
Thiol Medium & Thiol Broth
18. Baron, E. J., L. R. Peterson, and S. M. Finegold. 1994. Cultivation

and isolation of viable pathogens, p. 91,. Bailey & Scotts diagnostic
microbiology, 9th ed. Mosby-Year Book, Inc. St. Louis, MO.

w/ Beef Extract
Packaging

w/K Agar
100
500
2
10
g
g
kg
kg
0256-15
0256-17
0256-07
0256-08
500 g
10 kg
0697-17
0697-08
500 g
2 kg
0607-17
0607-07
500 g
0363-17
500 g
0257-17

or Indicator
500 g
0432-17
500 g
10 kg
0236-17
0236-08
500 g
0430-17
Bacto Thiol Medium

Bacto Thiol Broth
Intended Use
Bacto Thiol Medium is used for cultivating organisms from body
fluids and other materials containing penicillin, streptomycin or
sulfonamides.
Bacto Thiol Broth is used for cultivating organisms from body
fluids and other materials containing penicillin, streptomycin or
sulfonamides.

While studying Vibrio fetus cultivation, Huddleson1 found that vibrios
remained viable in Thiol Medium at room temperature for at least 150
days without transfer. Christensen2 tested Thiol Medium for the ability
to neutralize penicillin and streptomycin. Ten milliliters (10 ml) of
Thiol Medium can inactivate up to 100 units of penicillin and up to
1,000 micrograms of streptomycin, producing luxuriant growth of
staphylococci and other organisms from dilute inocula in 24 hours.
3
Szawatkowski and Shanson and Barnicoat reported Thiol Broth to

be superior in supporting the growth of Bacteroides species in blood
cultures. Thiol Broth was used to study the optimum incubation
period of blood culture broths. 5 Media containing thiol and
thioglycollate are recommended for recovery of nutritionally variant
streptococci (NVS).6
Thiol Broth has the same formulation as Thiol Medium, omitting the
agar. Thiol is cited in Clinical Microbiology Procedures Handbook7 as
a medium specific for anaerobic bacteria in blood cultures.

Proteose Peptone No. 3 and Yeast Extract provide nitrogen, vitamins
and amino acids in Thiol media. Dextrose is a carbon source. Sodium
Chloride maintains osmotic balance. Para-aminobenzoic Acid is a
preservative. Thiol Complex is rich in sulfhydryl (-SH) groups, which
neutralize the bacteriostatic and bactericidal effects of penicillin,
streptomycin and sulfonamides. Thiol Medium contains 0.1% Bacto
Agar to maintain an Eh potential that facilities anaerobic growth
and aids in dispersion of reducing substances and CO2 formed in the
environment.8
The Difco Manual
Formula
Thiol Medium
Formula Per Liter
Bacto Proteose Peptone No.3 . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Thiol Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
g
g
g
g
g
g
g

Thiol Broth
Formula Per Liter
Bacto Proteose Peptone No.3 . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Thiol Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
g
g
g
g
g
g
Precautions
Storage
Store the dehydrated medium below 30%C. The dehydrated medium
Expiration Date
Use Thiol media within four days of preparation.
Procedure
Materials Provided
Thiol Medium
Thiol Broth
507
Thiol Medium & Thiol Broth
Section II

3. Dispense as desired, using clean, rust-free equipment to prevent
precipitate formation.
Glassware
Autoclave
Incubator (35C)
Thiol Medium - 30 grams;
Thiol Broth - 29 grams.

Thiol Medium
homogeneous.
Solution:
deionized water on boiling. Very
slightly opalescent when hot,
opalescent after cooling.
Prepared Medium:
Very light amber; very slightly to
slightly opalescent when hot,
opalescent after cooling.
Reaction of 3%
Solution at 25C:
pH 7.1 0.2
Thiol Broth
homogeneous.
Solution:
deionized water on boiling; very
light to light amber, clear to slightly
opalescent.
Prepared Medium:
Very light amber, clear to slightly
opalescent.
Reaction of 2.9%
Solution at 25C:
pH 7.1 0.2
Cultural Response
Prepare Thiol Medium and Thiol Broth per label directions.
Test 5 unit, 100 unit and 1,000 unit concentrations of
penicillin and 100 g, 1,000 g and 10,000 g concentrations
of streptomycin. Inoculate and incubate at 35 2C for
18-48 hours.
Staphylococcus
aureus
Streptococcus
pyogenes
ATCC
INOCULUM
CFU
GROWTH
GROWTH
w/o ANTIBIOTICS w/ANTIBIOTICS
25923* 100-1,000
good
good
19615* 100-1,000
good
good
Antibiotic concentrations up to 100 units of penicillin or 1000 g
of streptomycin.
508
Test Procedure
For a complete discussion on processing and interpretation of blood
cultures and body fluids from clinical specimens, refer to appropriate
references.7,9
Results
ORGANISM

2. Strict reliance on blood culture bottles containing Thiol Broth is not
recommended for aerobic microorganisms. Always use an aerobic
medium, for example, a vented Tryptic Soy Broth, for optimum
isolation of the broad spectrum of microorganisms that can cause
bacteremia or septicemia.
References
1. Huddleson, I. F. 1948. A satisfactory medium for the isolation,
cultivation, and maintenance of viability of Vibrio fetus (bovine).
2. Christensen, C. W. 1947. Presented at the Michigan Branch, Society
of American Bacteriologists, Detroit, MI., December 12, 1947.
3. Szawatkowski, M. V. 1976. A comparison of three readily available
types of anaerobic blood culture media. Med. Lab. Sci. 33:5-12.
4. Shanson, D. C., and M. Barnicoat. 1975. An experimental
comparison of Thiol broth with Brewers thioglycollate for anaerobic
blood cultures. J. Clin. Pathol. 28:407-409.
5. Murray, P. R. 1985. Determination of the optimum incubation
period of blood culture broths for the detection of clinically
significant septicemia. J. Clin. Microbiol. 21:481-485.
6. Donnelly, J. P. 1994. Nutritionally variant streptococci and B6.
Infect. Dis. Alert 6:109-112.
Packaging
Thiol Medium
500 g
0307-17
Thiol Broth
500 g
10 kg
0434-17
0434-08
The Difco Manual
Section II
Tinsdale Agar
Tinsdale Agar
Bacto Tinsdale Base . Bacto Tinsdale Enrichment Desiccated
Intended Use
Bacto Tinsdale Base is used with Bacto Tinsdale Enrichment Desiccated
in isolating and differentiating Corynebacterium diphtheriae.
Also Known As
Tinsdale Base (TIN) is also called Tinsdale Selective Medium, Tinsdale
Tellurite Medium and Tellurite Agar.

Tinsdale Base, supplemented with Tinsdale Enrichment, is employed
in the cultural diagnosis of diphtheria. Diphtheria, an acute infectious
disease primarily of the upper respiratory tract but occasionally of the
skin,1 is caused by toxigenic strains of Corynebacterium diphtheriae.
The three biotypes are mitis, intermedius and gravis.1 The signs and
symptoms of the disease are a pharyngeal membrane, sore throat,
malaise, headache and nausea.2 Death can result from respiratory
obstruction by the membrane or myocarditis caused by the toxin.2
Tinsdale3 developed a serum-cystine-thiosulfate-tellurite agar medium

for the primary isolation and differentiation of C. diphtheriae. This
formulation distinguished between C. diphtheriae and diphtheroids
which exhibited similar characteristics. The differential principle is
based on the capacity of C. diphtheriae to produce a brown or black
halo around the colonies.
Billings 4 simplified Tinsdale Basal Medium by using Proteose
Peptone No. 3 as a nutrient source. This modification improved the
differential qualities and recovery of Corynebacterium diphtheriae.
Tinsdale Base and Tinsdale Enrichment are prepared according to
the Billings4 modification. Moore and Parsons5 confirmed the halo
formation of C. diphtheriae with one exception; C. ulcerans occasionally
produced colonies similar to C. diphtheriae and required biochemical
identification.
ATCC 8028
Uninoculated
plate

Tinsdale Base
Solution:
opalescent to opalescent, without
Prepared Medium:
opalescent to opalescent without
precipitate.
Reaction of 4.5%
Solution at 25C:
pH 7.4 0.2
Tinsdale Enrichment Desiccated
Lyophilized Appearance: Light to dark tan cake; variations may occur.
Solution:
Soluble in distilled or deionized water. Solution
is light to dark amber, clear to opalescent, may have
Cultural Response
Prepare Tinsdale Agar per label directions. Inoculate and incubate at
ORGANISM
ATCC
Corynebacterium diphtheriae type gravis 8028

Corynebacterium diphtheriae type mitis
8024
13883*
19615*
INOCULUM
CFU
GROWTH
APPEARANCE
100-1,000
good
brown with halos
100-1,000
good
brown with halos
100-1,000 marked to complete inhibition
100-1,000
fair
brown to black without halos
The Difco Manual
509
Tinsdale Agar
Tinsdale Enrichment Desiccated contains Bovine Serum, Sodium

Hydroxide, L-Cystine, Sodium Thiosulfate and Potassium Tellurite in
the quantity and proportion described by Billings.4

Proteose Peptone No. 3 provides the nitrogen, vitamins, carbon and
amino acids in Tinsdale Base. Sodium Chloride maintains the osmotic
balance of the medium. Bacto Agar is the solidifying agent.
Tinsdale Enrichment contains Bovine Serum, which provides essential
growth factors. Sodium Hydroxide maintains the pH. L-Cystine and
Sodium Thiosulfate are H2S indicators. Potassium Tellurite is a selective
agent. The formation of black to brown halos surrounding the colony
result from the reduction of potassium tellurite to metallic tellurite.
Stabbing the medium with an inoculating needle accentuates darkening
of the medium by C. diphtheriae.
Formula
Tinsdale Base
Formula Per Liter
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Section II
1. Suspend 45 grams of Tinsdale Base in 1 liter distilled or deionized
water.
5. Rehydrate Tinsdale Enrichment with 15 ml sterile distilled or
deionized water and rotate in an end-over-end motion to dissolve
completely.
6. Aseptically add 15 ml rehydrated Tinsdale Enrichment to each 100
ml of Tinsdale Base at 50-55C. Mix well.

1. Both throat and nasopharyngeal specimens are necessary in cases
of respiratory illness. If cutaneous diphtheria is suspected, collect
skin, throat and nasopharyngeal specimens. Sterile silica gel is recommended for shipping when clinical specimens are not cultured
locally.
Test Procedure

1. For a complete discussion on the collection, isolation and identification of Corynebacterium diphtheriae and other Corynebacterium
species, refer to the appropriate procedures outlined in the references.
2. Inoculate plates with the test organisms in a manner to obtain discrete
colonies and stab the medium several times with an inoculating needle.
3. Definitive identification of a strain of C. diphtheriae as a true
pathogen requires demonstration of toxin production.6 Characteristic
colonies of C. diphtheriae may be inoculated directly onto KL
Virulence Agar enriched with KL Virulence Enrichment and
containing KL Antitoxin Strips for toxigenicity tests.
Storage


2. Tinsdale Agar is not suitable as a primary plating medium, since it
may not support the growth of some strains of C. diphtheriae.1
3. Corynebacterium ulcerans, Corynebacterium pseudotuberculosis
and (rarely) Staphylococcus species may produce a characteristic
halo on Tinsdale Agar.1
4. Do not read Tinsdale Agar early because several organisms may
exhibit slight browning on this medium in 18 hours.1
5. Incubation at 5-10% CO2 retards the development of halos on
Tinsdale Agar.1
6. On media containing tellurite, diphtheria bacilli are shorter and
stain more uniformly; however, granules are less readily observed
than when grown on Loefflers medium.7
7. Further biochemical tests may be necessary to distinguish
between C. diphtheriae and C. ulcerans due to similar reactions on
this medium.
pH 7.4 0.2 at 25C

Contains Bovine Serum, L-Cystine, Sodium Hydroxide, Sodium
Thiosulfate and Potassium Tellurite at pH 8.0-10.0 at 25C.
Precautions
Store Tinsdale Enrichment Desiccated at 2-8C.
Expiration Date
Procedure
Materials Provided
Tinsdale Base

Glassware
Autoclave
Incubator (35C)
Waterbath (45-50C)
510
References
The Difco Manual
Section II
Todd Hewitt Broth

miscellaneous irregular gram-positive rod, Erysipelothrix, and
Gardnerella, p. 357-377. In P. R. Murray, E. J. Baron, M. A.
Washington, D.C.
3. Tinsdale, G. F. W. 1947. A new medium for the isolation and
identification of C. diphtheriae based on the production of hydrogen
sulphide. J. Pathol. Bacteriol. 59:461-466.
4. Billings, E. 1956. An investigation of Tinsdale Tellurite medium:
its usefulness and mechanisms of halo-formation. M.S. thesis.
University of Michigan, Ann Arbor, MI.
Bacto Todd Hewitt Broth
Intended Use
Bacto Todd Hewitt Broth is used for cultivating streptococci,
pneumococci and other fastidious organisms; for cultivating group A
streptococci prior to serological typing.
Also Known As
Todd Hewitt Broth can be abbreviated as THB.
5. Moore, M. S., and E. I. Parsons. 1958. A study of a modified

Tinsdales medium for the primary isolation of Corynebacterium
diphtheriae. J. Infect. Dis. 102:88.
St. Louis, MO.
7. Bailey, R. W., and E. G. Scott. 1966. Diagnostic microbiology,
2nd ed., p. 213. The C. V. Mosby Company, St. Louis, MO.
Packaging
Tinsdale Base
500 g
0786-17
6 x 15 ml
0342-33
according to the formula described by Updyke and Nickle2 who

compared media for type specific extract production of group A
streptococci. This study was performed using Todd Hewitt Broth
prepared with infusion of fresh beef heart as a control. Results showed
Todd Hewitt Broth was particularly satisfactory for growth of group
A streptococci for serological typing.
Elliott3 reported that Todd Hewitt Broth prepared with neopeptone was
excellent for growing group A streptococci for the production of
type specific M substance. This is possible because proteinase is not
produced in this medium.
Moody, et al.4 used Todd Hewitt Broth in the fluorescent-antibody

identification of group A streptococci from throat cultures. Todd Hewitt
Broth is recommended as an enrichment medium for the growth of
streptococcal cells in the identification of groups A and B by IF staining.5
Todd Hewitt Broth was used as an enrichment broth for group A
streptococci in a comparison study of a rapid antigen test.6
Principles of the Procedures
Todd Hewitt Broth was originally developed for the production of

antigenic streptococcal hemolysin.1 Todd Hewitt Broth is prepared

homogeneous.
Solution:
or deionized water; light to
medium amber, clear with no
Prepared Medium:
Light to medium amber, clear with
Reaction of 3.0%
Solution at 25C:
pH 7.8 0.2
Cultural Response
Prepare Todd Hewitt Broth per label directions. Incubate
inoculated tubes at 35 2C for 18-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
13090*
25923*
6303*
19615*
100-1,000
100-1,000
100-1,000
100-1,000
good
good
good
good
The Difco Manual
Infusion from Beef Heart and Neopeptone provides the nitrogen,

vitamins and amino acids in Todd Hewitt Broth. Dextrose is the carbon
source, and a stimulant for hemolysin production. 7 Disodium
Phosphate and Sodium Carbonate act as buffers to aid in neutralizing
acid production from dextrose fermentation, and protect hemolysin
from inactivation.7 Sodium Chloride maintains the osmotic balance of
the medium.
Formula
Todd Hewitt Broth
Formula Per Liter
Bacto Neopeptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Sodium Carbonate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
g
g
g
g
g
g
Precautions
511
Tomato Juice Media
Storage
Expiration Date
Procedure
Materials Provided
Todd Hewitt Broth

Glassware
Autoclave
Incubator (35C)
Sterile tubes

Test Procedure
For a complete discussion on the isolation, identification and serological
procedures of fastidious microorganisms, refer to the procedures
described in appropriate references.4,5,8,9
Results
Section II
2. Todd Hewitt Broth cannot be used unbuffered for bile solubility

testing10
References
1. Todd, E. W., and L. F. Hewitt. 1932. A new culture medium for
the production of antigenic streptococcal haemolysin. J. Pathol.
Bacteriol. 35:973.
2. Updyke, E. L., and M. I. Nickle. 1954. A dehydrated medium for
the preparation of type specific extracts of group A
streptococci. Appl. Microbiol. 2:117.
3. Elliott. 1945. J. Exp. Med. 81:573.
4. Moody, M. D., A. C. Siegel, B. Pittman, and C. C. Winter. 1963.
Fluorescent- antibody identification of group A streptococci from
throat swabs. Am. J. Public Health, 53:1083.
5. Facklam, R. R., and R. B. Carey. 1985. Streptococci and
Aerococci, p. 154-175. In, E. H.Lennette, A. Balows, W. J.
Hausler, Jr., and H. J. Shadomy (ed.), Manual of clinical
Washington, D.C.
6. Bourbeau, P. P., B. J. Heiter, J. P. Anhalt, and D. W. Naumovitz.
1993. Comparison of direct specimen testing utilizing testpack
strep A with testing of specimens following a two-hour broth
enrichment. Diagn. Microbiol. Infect. Dis. 17:93-96.
R. H. Yolken (ed.). 1995. Manual of clinical microbiology, 6th
ed. American Society for Microbiology, Washington, D.C.
Packaging
Todd Hewitt Broth

100
500
2
10
g
g
kg
kg
0492-15
0492-17
0492-07
0492-08
Tomato Juice Media

Bacto Tomato Juice Agar . Bacto Tomato Juice Agar Special
Bacto Tomato Juice Broth
Intended Use
Bacto Tomato Juice Agar is used for cultivating and enumerating

Lactobacillus species.
In 1925, Mickle and Breed1 reported the use of tomato juice in culture
media used for cultivating lactobacilli. Kulp2 investigated the use
of tomato juice on bacterial development and found that the growth
of L. acidophilus was enhanced. Tomato Juice Agar, prepared according
to Kulp and Whites3 modification, is especially useful in cultivating
L. acidophilus from clinical specimens and foodstuffs.4
Bacto Tomato Juice Agar Special is used for cultivating and enumerating lactobacilli and other acidophilic microorganisms from
saliva and other specimens.
Bacto Tomato Juice Broth is used for cultivating yeasts and other
aciduric microorganisms.
512
The Difco Manual
Section II
Tomato Juice Media
Tomato Juice Agar Special is recommended for the direct plate count
of lactobacilli from saliva and for cultivation of other acidophilic
microorganisms. The number of lactobacilli in saliva is an index of
a predisposition to dental caries as described by Jay.5, 6 Many dentists
use the direct count of lactobacilli for the diagnosis of caries.
The acidic pH of Tomato Juice Agar Special encourages growth of
lactobacilli while inhibiting growth of accompanying bacteria. This
medium is more selective for lactobacilli than Tomato Juice Agar.
Tomato Juice Broth is recommended for use in cultivating and
isolating yeasts, lactobacilli and other aciduric microorganisms from
clinical specimens and foods.

Tomato Juice Agar and Tomato Juice Agar Special
Tomato Juice is a source of carbon, protein and nutrients. Peptone
provides a source of nitrogen, amino acids and carbon. Peptonized Milk
contains lactose as an energy source. Bacto Agar is a solidifying agent.
Tomato Juice Broth
Tomato Juice is a source of carbon, protein and nutrients. Yeast Extract
is a source of trace elements, vitamins and amino acids. Dipotassium
Phosphate and Monopotassium Phosphate provide buffering
capability. Magnesium Sulfate, Ferrous Sulfate and Manganese
Uninoculated
plate
Lactobacillus casei
ATCC 9595

Tomato Juice Agar
Solution:
medium to dark amber, very slightly
Reaction of 5.1%
Solution at 25C:
pH 6.1 0.2
Tomato Juice Agar Special
Solution:
medium to dark amber, slightly opalescent.
Reaction of 6.0%
Solution at 25C:
pH 5.0 0.2
Tomato Juice Broth
Solution:
4.1% solution, soluble in distilled or deionized water.
Solution is dark amber, clear without significant precipitate.
Reaction of 4.1%
Solution at 25C:
pH 6.7 0.2
Cultural Response
Tomato Juice Agar
Prepare Tomato Juice Agar per label directions. Inoculate using
the pour plate technique and incubate at 35 2C for 40-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Lactobacillus casei
4356
9595
4797
100-1,000
100-1,000
100-1,000
good
good
good

Prepare Tomato Juice Agar Special per label directions. Inoculate
and incubate at 35 2C for 18-48 hours (72 hours if necessary).
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Lactobacillus casei
4356
9595
4797
100-1,000
100-1,000
100-1,000
good
good
good
The Difco Manual
Tomato Juice Broth

Prepare Tomato Juice Broth per label directions. Inoculate and
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Lactobacillus casei
Saccharomyces carlsbergensis
9595
4797
9080
9763
100-1,000
100-1,000
100-1,000
100-1,000
good
good
good
good
513
Tomato Juice Media
Section II
Sulfate provide inorganic ions. Sodium Chloride is a source of

essential ions that maintain the osmotic balance of the medium.
Autoclave
Formula
Tomato Juice Agar

Formula Per Liter
Tomato Juice Agar

1. Equilibrate the medium to room temperature before opening.
Tomato Juice (400 ml) . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Peptonized Milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20
10
10
11
g
g
g
g

Formula Per Liter
Tomato Juice (400 ml) . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Peptonized Milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20
10
10
20
g
g
g
g
Tomato Juice (400 ml) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01
g
g
g
g
g
g
g
g
g

Tomato Juice Broth
Formula Per Liter

1. Equilibrate the medium to room temperature before opening.
Tomato Juice Broth

Test Procedure
Results
References
1. Mickle and Breed. 1925. Technical Bulletin 110. NY State

Agriculture Exp. Station.
2. Kulp, W. L. 1927. Scientific apparatus and laboratory methods.
An agar medium for plating L. acidophilus and L. bulgaricus.
Science 66:512-513.
L. acidophilus. Science 76:17-18.
4. MacFaddin, J. D. 1985. Media for isolation-cultivationidentification- maintenance of medical bacteria, vol 1, p. 776-778.
5. Jay, P., and S. Gordon (ed). 1938. Bacteriology and immunology
of dental caries and dental science and dental art. Lea
and Febiger, Philadelphia, PA.
6. Jay, P., W. J. Pelton, and J. M. Wisan. 1949. Dentistry in public
health. W. B. Saunders Company, Philadelphia, PA.
Procedure
Packaging
Materials Provided
Tomato Juice Agar
500 g
0031-17
Tomato Juice Agar

Tomato Juice Broth
500 g
0389-17
Tomato Juice Broth
500 g
10 kg
0517-17
0517-08
Precautions
1. Tomato Juice Agar: For Laboratory Use.
Tomato Juice Agar Special: For Laboratory Use.
Tomato Juice Broth: For Laboratory Use.
Storage
Expiration Date

Glassware
514
The Difco Manual
Section II
Transport Media
Transport Media
Bacto Transport Medium Amies . Bacto Transport Medium
Amies w/o Charcoal . Bacto Transport Medium Stuart . Bacto
Cary-Blair Transport Medium
Intended Use
Bacto Transport Medium Amies, Transport Medium Amies w/o Charcoal
and Transport Medium Stuart are used for collecting, transporting and
preserving microbiological specimens.
Bacto Cary-Blair Transport Medium is used for collecting, transporting
and preserving microbiological specimens, particularly those containing
Vibrio cholerae.

Transport media are chemically defined, semisolid, non-nutritive,
phosphate buffered media that provide a reduced environment.

Transport Medium Amies
Dehydrated Appearance: Black, free-flowing, homogeneous.
Solution:
is black, opaque.
Prepared Vials:
Black, opaque, semi-solid.
Reaction of 2.0%
Solution at 25C:
pH 7.3 0.2
Transport Medium Amies w/o Charcoal
Solution:
is colorless to very light amber,
opalescent with precipitate.
Prepared Vials:
Colorless to very light amber,
opalescent with precipitate, semi-solid.
Reaction of 1.0%
Solution at 25C:
pH 7.3 0.2
Transport Medium Stuart
Dehydrated Appearance: Bluish white, free-flowing,
homogeneous.
Solution:
is light amber, opalescent with a
bluish upper layer.
Prepared Vials:
Light amber, opalescent with blue
upper layer, without precipitate,
semi-solid.
Reaction of 1.41%
Solution at 25C:
pH 7.4 0.1
Transport media are formulated to maintain the viability of microorganisms without significant increase in growth.
In 1948, Moffett, Young and Stuart described a medium for transporting
gonococcal specimens to the laboratory.1 Stuart, Toshach and Patsula
improved this formulation, introducing what is now known as Stuarts
Transport Medium.2 The ability of Stuarts medium to maintain the
viability of gonococci during transport3,4 led other researchers to explore
its use with a variety of specimens. This medium is currently recommended for throat, vaginal, and wound samples.
In 1964, Cary and Blair modified Stuarts medium by substituting
inorganic phosphates for glycerophosphate and raising the pH to 8.4.5
The modified medium was effective in maintaining the viability of
Salmonella and Shigella6,7 in fecal samples. Due to its high pH,
Cary-Blair Transport Medium is also effective in maintaining the
viability of Vibrio cultures for up to four weeks.8 Cary-Blair Transport
Medium is currently recommended for fecal and rectal samples.
Amies9 confirmed Cary and Blairs observations that an inorganic salt
buffer was superior to the glycerophosphate. He further modified the
formulation by using a balanced salt solution containing inorganic
phosphate buffer, omitting the methylene blue, and adding charcoal.
This modified medium yielded a higher percentage of positive
cultures than the transport medium of Stuart. Transport Medium Amies,
available with and without charcoal, is recommended for throat,
vaginal, and wound samples.

In the formulations, potassium chloride, calcium chloride, magnesium
chloride and sodium chloride provide essential ions that help maintain
osmotic balance while controlling permeability of bacterial cells.
Monopotassium phosphate and Disodium phosphate provide buffering
capabilities. Sodium thioglycollate suppresses oxidative changes and
provides a reduced environment. Sodium glycerophosphate is a buffer
for use with calcium chloride. Methylene blue is a colorimetric
pH indicator of the oxidation-reduction state. Charcoal neutralizes fatty
acids that are toxic to microorganisms. Bacto Agar is a solidifying agent.
Formula
Formula Per Liter
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Charcoal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
g
g
g
g
g
g
g
g
g

The Difco Manual
515
Transport Media
Section II
Sodium Thioglycollate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 g

Formula Per Liter
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
g
g
g
g
g
g

homogeneous.
Solution:
Solution is colorless with a very
light amber tint, very slightly to
slight, fine precipitate.
Prepared Vials:
Colorless to whitish gray, opalescent
without significant precipitate,
semi-solid.
Reaction of 1.27%
Solution at 25C:
pH 8.4 0.2
Cultural Response
Transport Medium Amies, Transport Medium
Amies w/o Charcoal and Transport Medium Stuart
Prepare media per label directions. Inoculate sterile swabs with
suspensions of test organisms containing 1,000-10,000 CFU/
0.1 ml. Place swabs in the medium and incubate at room
temperature for 18-24 hours. Remove swabs, streak on
prepared chocolate agar plates and incubate appropriately.
All cultures should be viable.
ORGANISM
Haemophilus influenzae Type b
Neisseria meningitidis Group B
Streptococcus pyogenes Group A
ATCC
25285*
10211
13090*
43069
6305
19615*

Prepare Cary-Blair Transport Medium per label directions.
Inoculate sterile swabs with suspensions of test organisms
containing 1,000-10,000 CFU/0.1 ml. Place in the medium, and
incubate at room temperature for up to 48 hours. Remove
swabs, streak on prepared TSA with 5% Sheep Blood plates
and incubate appropriately. All cultures should be viable.
ORGANISM
ATCC
Shigella dysenteriae
Vibrio cholerae biotype eltor
Vibrio parahaemolyticus EB 101
13076
13313
15748
516

Formula Per Liter
Bacto Methylene Blue . . . . . . . . . . . . . . . . . . . . . . . . . . 0.002
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
g
g
g
g
g

Formula Per Liter
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
g
g
g
g
g
Precautions
2. Transport Medium Amies: IRRITANT. IRRITATING TO EYES,
and eyes. Do not breathe dust. Wear suitable protective clothing.
Transport Medium Amies w/o Charcoal: IRRITANT. IRRITATING
TO EYES, RESPIRATORY SYSTEM AND SKIN. Avoid contact
Transport Medium Stuart: IRRITANT. IRRITATING TO EYES,
and eyes. Do not breathe dust. Wear suitable protective clothing.
Cary-Blair Transport Medium: IRRITANT. IRRITATING TO
EYES, RESPIRATORY SYSTEM AND SKIN. Avoid contact
The Difco Manual
Section II
Storage
Store dehydrated media below 30C. The dehydrated media are very
Expiration Date
Procedure
Materials Provided
Transport Medium Amies, Transport Medium Amies w/o Charcoal,
Transport Medium Stuart, or Cary-Blair Transport Medium.

Glassware
Autoclave
Incubator
1. Transport Medium Amies: Suspend 20 grams in 1 liter distilled
or deionized water. Invert vials just before solidification to
uniformly distribute the charcoal.
Transport Medium Amies w/o Charcoal: Suspend 10 grams in
1 liter distilled or deionized water.
Transport Medium Stuart: Suspend 14.1 grams in 1 liter
Cary-Blair Transport Medium: Suspend 12.7 grams in 1 liter
3. Dispense into 6-8 ml capacity screw-cap vials to within 5mm of
the top. Cap tightly.

Refer to appropriate references for specimen collection and primary
isolation technique recommendations.10,11,12
Test Procedure
1. Insert specimen swab(s) into the upper third of the medium in the
transport container.
2. Cut or break off the protruding portion of the swab stick. Tightly
screw the lid on the bottle or vial.
The Difco Manual
Transport Media
3. Label the bottle or vial and send to the laboratory with minimum
delay. Specimens may be refrigerated until ready for shipment.
4. Submit to laboratory within 24 hours for culture and analysis.
Results
Survival of bacteria in a transport medium depends on many factors
including the type and concentration of bacteria in the specimen, the
formulation of the transport medium, the temperature and duration of
transport, and inoculation to appropriate culture media within 24 hours.
Optimal growth and typical morphology can only be expected following
direct inoculation and appropriate cultivation.

1. Specimens taken from transport media will not exhibit the optimal
or comparative growth as expected from direct inoculation and
cultivation. These media do, however, provide an adequate degree
of preservation for those specimens which cannot be forwarded
immediately to the laboratory for prompt evaluation.
2. Viability of cells will diminish over time and some degree of
multiplication or growth of contaminants can occur during
prolonged periods of transit. This is particularly true of fecal
specimens that contain substantial numbers of coliform organisms.
3. The condition of the specimen received by the laboratory for
culture is a significant variable in recovery and final identification
of the suspect pathogen. An unsatisfactory specimen (overgrown
by contaminants, containing non-viable organisms, or having the
number of pathogens greatly diminished) can lead to erroneous or
inconclusive results.
References
1. Moffett, M., J. L. Young, and R. D. Stuart. 1948. Centralized
gonococcus culture for dispersed clinics; the value of a new
transport medium for gonococci and trichomonas. Brit. Med. J.
2:421-424.
2. Stuart, R. D., S. R. Toshach, and T. M. Patsula. 1954. The
problem of transport of specimens for culture of gonococci. Can.
J. Public Health 45:73-83.
3. Stuart, R. D. 1946. The diagnosis and control of gonorrhea by
bacteriological cultures. Glasgow M. J. 27:131-143.
4. Stuart, R. D. 1959. Transport medium for specimens in public
health bacteriology. Public Health Reports 74:431-438.
5. Cary, S. G., and E. B. Blair. 1964. New transport medium for
shipment of clinical specimens. J. Bacteriol. 88:96-98.
6. Cary, S. G., M. S. Matthew, M. H. Fusillo, and C. Harkins.
1965. Survival of Shigella and Salmonella in a new transport
medium. Am. J. Clin. Path. 43:294- 296.
7. Neuman, D. A., M. W. Benenson, E. Hubster, and Thi Nhu Tuan.
1971. N. Am. J. Clin. Path. 57:33-34.
8. Kelly, M. T., F. W. Hickman-Brenner, and J. J. Farmer III. 1991.
Vibrio, p. 384- 395. In A. Balows, W. J. Hausler, Jr., K. L.
Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.), Manual of
clinical microbiology, 5th ed.. American Society for Microbiology,
Washington D.C.
9. Amies, C. R. 1967. A modified formula for the preparation of
Stuarts transport medium. Can. J. Public Health 58:296-300.
517
Trichophyton Agars
Section II

10. Miller, J. M., and H. T. Holmes. 1995. Specimen collection,

transport, and storage, p. 19-31. In P. R. Murray, E. J. Baron,
M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of
clinical microbiology, 6th ed. American Society of Microbiology,
Washington, D.C.
11. Isenberg, H. D., F. D. Schoenknecht, and A. von Graevenitz.
1979. Cumitech 9, Collection and processing of bacteriological
specimens. Coord. Ed., S. J. Rubin. American Society for
Packaging
500 g
0996-17
Transport Medium Amies w/o Charcoal 500 g
0832-17
500 g
0621-17
500 g
0505-17
Trichophyton Agars
Bacto Trichophyton Agar 1 . Trichophyton Agar 2 . Trichophyton
Agar 3 . Trichophyton Agar 4 . Trichophyton Agar 6
Trichophyton Agar 7
their colonies resemble each other so closely that they cannot be
identified by morphological criteria. These species are differentiated

as indicated in Table 1 by their growth in the absence or presence of
inositol, thiamine, or a combination. T. verrucosum grows faster at 37C
than at room temperature while T. schoenleinii and T. concentricum
are not stimulated by incubation at 37C.
Group 2 includes T. tonsurans, T. mentagrophytes and T. rubrum. These
species usually produce microconidia, but only occasionally produce
macroconidia. Their colonial forms and pigments are so variable that
differentiation by these means is inadequate. Ajello and Georg2 based
differentiation on their nutritional behavior on Trichophyton Agars 1
and 4 and by in vitro hair cultures. T. mentagrophytes forms perforating
organs within 2-3 weeks in human hair samples suspended in water;
T. rubrum does not.
Intended Use
Bacto Trichophyton Agars are used for differentiating Trichophyton
species based on differing nutritional requirements.

Trichophyton Agar media are prepared according to the formulations
of Georg and Camp1 and are based on the nutritional requirements of
various species. The growth patterns of the different species are given
in Table 1.1 (see Table 1 below)
The authors divided the dermatophytes into four groups. Group 1
includes T. verucosum, T. schoenleinii and T. concentricum. Organisms
within this group seldom produce spores or distinctive pigments and
Table 1. Growth patterns of Trichophyton species on Trichophyton Agars.1
DERMATOPHYTE
TESTED
T. verrucosum
T. schoenleinii
T. concentricum
T. tonsurans
T. mentagraphytes
T. rubrum
T. ferrugineum
T. violaceum
T. megninii
T. gallinae
T. equinum*
STRAINS STUDIED
(1% REPORTING)
100 (84%)
100 (16%)
50
19 (50%)
19 (50%)
70
50
50
14
13
13
7
13
0
0
4+
4+
2+
to 1+
4+
4+
4+
to 1+
0
4+
4+
2+
TRICHOPHYTON AGARS
3
4
4+
4+
4+
4+
4+
0
4+
4+
4+
0
4+
4+
4+
4+
4+
4+
4+
4+
4+
* T. equinum will grow to a 4+ reaction on Trichophyton Agar 5, which is not commercially available from Difco Laboratories. Trichophyton Agar 5 is equivalent
to Trichophyton Agar 1 (product code 0877) with added nicotinic acid (200 g per liter).
518
The Difco Manual
Section II
Trichophyton Agars
Group 3 includes T. violaceum. It seldom produces microconidia but

does develop characteristically pigmented colonies. T. violaceum has a
similar nutritional pattern as T. tonsurans; however, it grows very
slowly even in the presence of thiamine and produces a glabrous colony
without spores. T. tonsurans grows rapidly in the presence of thiamine
and shows numerous microconidia.
Group 4 includes T. megninii and T. equinum. Both can be identified
solely from nutritional requirements. T. megninii requires histidine,
as indicated on Trichophyton Agar 6 in Table 1. T. equinum requires
nicotinic acid, as indicated in the table.
T. gallinae is differentiated morphologically as well as culturally from
T. megninii and T. equinum. T. gallinae grows on Trichophyton Agar 1
or 6 without added vitamins.

Trichophyton Agars 1, 2, 3 and 4
Vitamin Assay Casamino Acids is the source of nutrients. Dextrose
provides carbon. Monopotassium Phosphate provides buffering
capability. Magnesium Sulfate is a source of divalent cations and
sulfate. Bacto Agar is a solidifying agent. Where included in the
formulations for Trichophyton Agars 2,3 and 4, Inositol and Dextrose

provide carbon; Thiamin is present for organisms requiring the
vitamin for growth.
Trichophyton Agars 6 and 7
Ammonium Nitrate is a source of nitrogen. Dextrose provides carbon.
Monopotassium Phosphate provides buffering capability. Magnesium
Sulfate is a source of divalent cations and sulfate. Bacto Agar is a
solidifying agent. Histidine Hydrochloride is present in Trichophyton
Agar 7 for organisms requiring the amino acid histidine.
Formula
Trichophyton 1
Formula Per Liter
Bacto Vitamin Assay Casamino Acids . . . . . . . . . . . . . . . .
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5
40
1.8
0.1
15
g
g
g
g
g

Trichophyton Agars 1, 2, 3, 4, 6 and 7
Dehydrated Appearance: White to off-white, free-flowing, homogeneous.
Solution:
5.9% solution, soluble in distilled or deionized water upon
boiling. Solution is light to medium amber, slightly
opalescent.
Reaction of 5.9%
Solution at 25C:
pH 6.8 0.2
Cultural Response
Trimchophyton Agars 1, 2 and 3
at 30 2C for up to 2 weeks.
ORGANISM
ATCC
GROWTH
AGARS 1 & 2
GROWTH
AGAR 3
Trichophyton concentricum
Trichophyton schoenleinii
Trichophyton verrucosum
9358
4822
34470
good
good
none to poor
good
good
good
Uninoculated
tube
Trichophyton Agar 4
ORGANISM
ATCC
GROWTH
Trichophyton rubrum
Trichophyton verrucosum
Trichophyton violaceum
28188
34470
8376
good
none to poor
good
Trichophyton Agar 6
ORGANISM
ATCC
GROWTH
Microsporum gallinae
Trichophyton megninii
22243
12106
good
none to poor
The Difco Manual
Trichophyton
schoenleinii
ATCC 4822
Trichophyton Agar 7
ORGANISM
ATCC
GROWTH
Microsporum gallinae
Trichophyton megninii
12108
12106
good
good
519
Trichophyton Agars
Section II
Expiration Date
Trichophyton 2
Formula Per Liter
Bacto Vitamin Assay Casamino Acids . . . . . . . . . . . . . . . .
Bacto Inositol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.5
50
40
1.8
0.1
15
g
mg
g
g
g
g
Bacto Vitamin Assay Casamino Acids . . . . . . . . . . . . . . . . 2.5

Bacto Inositol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Thiamin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 g
g
mg
g
g
g
g
Trichophyton Agars 1, 2, 3, 4, 6 and 7

Glassware
Autoclave
Trichophyton 4
Formula Per Liter
Bacto Vitamin Assay Casamino Acids . . . . . . . . . . . . . . . . 2.5
Thiamin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
Test Procedure1,2,4
1. Inoculate the media by placing a small particle (approximately
1 mm square) of a colony on each medium.
2. Incubate at 30C for up to 2 weeks.
Results
Examine the media, comparing the amount of growth on each. A small
amount of growth is documented as a + and heavy growth as 4+.
Trichophyton 6
Formula Per Liter
1.5
40
1.8
0.1
15
g
g
g
g
g
1.5
30
40
1.8
0.1
15
g
mg
g
g
g
g

Trichophyton 7
Formula Per Liter
Precautions
Storage
Store dehydrated Trichophyton Agars below 30C. The dehydrated
520
Materials Provided
Ammonium Nitrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Histidine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Procedure
Trichophyton 3
Formula Per Liter
Ammonium Nitrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. It is important that pure cultures from a medium that is not vitamin
enriched, such as Sabouraud Dextrose Agar of Mycobiotic Agar,
be used for the inoculum.
2. If cultures are contaminated with bacteria, the cultures should be
grown on a medium containing antibiotics, such as Mycobiotic
Agar or Brain Heart CC Agar, for several generations to eliminate
the bacteria. Many bacteria synthesize vitamins and may invalidate
the test results.
3. When inoculating Trichophyton Agars, take care not to carry over
growth substances from primary cultures to the tube media used in
the differential tests. Inocula transferred to the nutrition tubes
should be very small.
References
1. Georg, L. K., and L. B. Camp. 1957. Routine nutritional tests
for the identification of dermatophytes. J. Bact. 74:113-121.
2. Ajello, L., and L. K. Georg. 1957. In vitro hair cultures for differentiating between atypical isolates of Trichophyton mentagrophytes
and Trichophyton rubrum. Mycopath. ef. Mycol. Appl. 7:3-17.
handling, p. 19- 32. In P. R. Murray, E. J. Baron, M. A. Pfaller, F.
C. Tenover, and R. H. Yolken, (ed.). Manual of clinical microbiology,
The Difco Manual
Section II
Triple Sugar Iron Agar
4. McGinnis, M. R., and L. Pasarell. 1992. Mycology, p. 6.11.6-6.11.7.

In H. D. Isenberg (ed.), Clinical Microbiology Procedures Handbook, vol. 1. American Society for Microbiology, Washington, D.C.
Trichophyton Agar 2
500 g
0874-17
Trichophyton Agar 3
500 g
0965-17
Trichophyton Agar 4
500 g
0197-17
Packaging
Trichophyton Agar 6
500 g
0524-17
Trichophyton Agar 7
500 g
0955-17
Trichophyton Agar 1
500 g
0877-17
Bacto Triple Sugar Iron Agar
Intended Use
Bacto Triple Sugar Iron Agar is used for differentiating gram-negative
enteric bacilli based on fermentation of dextrose, lactose and sucrose
and on hydrogen sulfide production.
Also Known As
Triple Sugar Iron Agar is also known as TSI.

In 1911, Russell1 described the use of two sugars in an agar medium
to differentiate gram-negative organisms of intestinal origin. Lead or
iron salts were added to the Russell medium to detect the presence
of hydrogen sulfide. Kligler2,3 added lead acetate to Russell Double
Sugar Agar, resulting in a medium that was capable of differentiating
typhoid, paratyphoid and dysentery. A modification of this medium,
Kligler Iron Agar, using phenol red as an indicator and iron salts to
detect hydrogen sulfide production, was developed.
Krumweide and Kohn4 modified Russell Double Sugar Agar by the

addition of sucrose to the medium. This modification allowed for an
earlier detection of coliform organisms that ferment lactose slowly,
since many of these organisms attack sucrose more readily than
lactose. The added sucrose also permitted the exclusion of certain
coliform and Proteus organisms that can attack sucrose, but not
lactose, in a 24-48 hour incubation period.
In 1940, Sulkin and Willet5 described a triple sugar ferrous sulfate
medium for use in the identification of enteric organisms. Difco
Laboratories concurrently developed a similar medium by adding 1%
sucrose to Kligler Iron Agar with phenol red as the indicator. Hajna6
described a similar medium for the identification of bacteria of the
intestinal group.
Triple Sugar Iron Agar is essentially the formula originally described
by Sulkin and Willet.5 Tryptone has been replaced by a combination
of Bacto Peptone and Proteose Peptone. Yeast Extract has been
added, and Phenol Red is used as an indicator instead of Brom
Thymol Blue.

Solution:
red, very slightly opalescent, may have
a slight dark precipitate.
Prepared Tubes:
Red, slightly opalescent, may have a
slight precipitate.
Reaction 6.5%
Solution at 25C:
pH 7.4 0.2
Cultural Response
Prepare Triple Sugar Iron Agar per label directions. Inoculate
and incubate the tubes at 35C for 18-24 hours.
ORGANISM
Escherichia coli
Shigella flexneri
ATCC
CFU
GROWTH
SLANT
/BUTT
25922*
9027
13076
12022*
undiluted
undiluted
undiluted
undiluted
good
good
good
good
A/A
K/K
K/A
K/A
A = acid reaction (yellow)

+gas = cracks, splits or bubbles
in medium
+H2S = black precipitate in butt
The Difco Manual
GAS H2S
K = alkaline reaction (no color change)

gas = no cracks, splits or bubbles
in medium
H2S = no black precipitate in butt
Uninoculated
tube
Escherichia coli
ATCC 25922
Salmonella
enteritidis
ATCC 13076
Shigella flexneri
ATCC 12022
521
Section II
Triple Sugar Iron Agar is recommended for differentiation of enteric

gram negative bacilli from clinical specimens,7,8,9 dairy samples10, and
food samples.11,12,13,14 Its use is also recommended for microbial limits
testing of Escherichia coli and Salmonella spp.15

Beef Extract, Yeast Extract, Bacto Peptone, and Proteose Peptone
provide nitrogen, vitamins, and minerals. Triple Sugar Iron Agar
contains three carbohydrates (dextrose, lactose and sucrose). When
these carbohydrates are fermented, the resulting production of acid
is detected by the phenol red indicator. The color changes that result
are yellow for acid production and red for alkalinization. Sodium
thiosulfate is reduced to hydrogen sulfide. Hydrogen sulfide then
reacts with an iron salt yielding the typical black iron sulfide. Sodium
chloride maintains the osmotic balance of the medium. Bacto Agar is
Formula
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sucrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.024
g
g
g
g
g
g
g
g
g
g
g
g

Tubes with closures
Inoculating needle
Autoclave
Incubator (35C)
1.
2.
3.
4.

Autoclave at 121C for 15 minutes. Cool in slanted position
with deep butts.

sterile swabs and transport immediately to the laboratory
following recommended guidelines.7-14
Test Procedure
well-isolated colonies.
2. With an inoculating needle, pick the center of well-isolated
colonies obtained from solid culture media.
3. Stab the center of the medium into the deep of the tube to within
3 - 5 mm from the bottom.
4. Withdraw the inoculating needle, and streak the surface of the slant.
5. Loosen closure on the tube before incubating.
7. Read tubes for acid production of slant/butt, gas, and hydrogen
sulfide reactions.
Precautions
Results

1. An alkaline slant-acid butt (red/yellow) indicates fermentation of

dextrose only.
2. An acid slant-acid butt (yellow/yellow) indicates fermentation of
dextrose, lactose and/or sucrose.
3. An alkaline slant-alkaline butt (red/red) indicates that neither
dextrose nor lactose was fermented (non-fermenter).
4. Cracks, splits, or bubbles in the medium indicate gas production.
5. A black precipitate in the butt indicates hydrogen sulfide production.
Storage
tubes at 2-8C.
Expiration Date
Procedure
Materials Provided

522

1. Hydrogen sulfide production may be evident on Kligler Iron Agar
but negative on Triple Sugar Iron Agar. Studies by Bulmash and
Fulton15 showed that the utilization of sucrose could suppress the
enzymatic mechanisms responsible for H2S production. Padron
and Dockstader16 found that not all H2S-positive Salmonella are
positive on TSI.
2. Sucrose is added to TSI to eliminate some sucrose-fermenting
non-lactose fermenters such as Proteus and Citrobacter spp.17
3. Further biochemical tests and serological typing must be performed
for definite identification and confirmation of organisms.
The Difco Manual
Section II
Tryptic Nitrate Medium
4. Do not use an inoculating loop to inoculate a tube of Triple Sugar

Iron Agar. While stabbing the butt, mechanical splitting of the
medium occurs, causing a false positive result for gas production.17
5. A pure culture is essential when inoculating Triple Sugar Iron Agar.
If inoculated with a mixed culture, irregular observations may occur.
6. Tubes should be incubated with caps loosened. This allows a free
exchange of air, which is necessary to enhance the alkaline
condition on the slant.17
References
1. Russell, F. F. 1911. The isolation of typhoid bacilli from urine and
feces with the description of a new double sugar tube medium.
J. Med. Res. 25:217.
2. Kligler, I. J. 1917. A simple medium for the differentiation of
members of the typhoid-paratyphoid group. Am. J. Public Health
7:1042-1044.
3. Kligler, I. J. 1918. Modifications of culture media used in the
isolation and differentiation of typhoid, dysentery, and allied
bacilli. J. Exp. Med. 28:319-322.
4. Krumwiede, C. and L. Kohn. 1917. A triple sugar modification
of the Russell Double Sugar medium. J. Med. Res. 37:225.
5. Sulkin, S. E., and J. C. Willett. 1940. A triple sugar-ferrous
sulfate medium for use in identification of enteric organisms.
J. Lab. Clin. Med. 25:649-653.
6. Hajna, A. A. 1945. Triple-sugar iron agar medium for the
identification of the intestinal group of bacteria. J. Bacteriol.
49:516-517.
7. Pezzlo, M. (ed.). 1994. Aerobic bacteriology, p. 1.0.0-1.20.47.
In H. D. Isenberg, (ed.), Clinical microbiology procedures handbook, vol. 1. American Society for Microbiology, Washington, D.C.
St. Louis, MO.

Marshall (ed.), Standard methods for the examination of dairy
Washington, D.C.
of foods, 3rd ed. American Public Health Association,
Washington, D.C.
International, Gaithersburg, M.D.
61:38917-38925.
15. Bulmash, J. M. and M. D. Fulton. 1964. Discrepant tests for
hydrogen sulfide. J. Bacteriol. 88:1813.
16. Padron, A. P. and W. B. Dockstader. 1972. Selective medium
for hydrogen sulfide production. Appl. Microbiol. 23:1107.
Packaging
Bacto Tryptic Nitrate Medium
Formula
Intended Use

Formula Per Liter
Bacto Tryptic Nitrate Medium is used for differentiating microorganisms based on nitrate reduction.

Tryptic Nitrate Medium is a differential, semi-solid, general purpose
medium that supports growth of aerobes as well as facultative and
obligate anaerobes.1 The formulation includes potassium nitrate which
can be reduced by certain organisms to either nitrite or nitrogen gas.
Nitrate reduction can be detected by various test methods and is used
in differentiating organisms from clinical samples, foods and dairy
products.1,2,3,4,5

Tryptose is a source of nitrogen, amino acids, and vitamins. Dextrose
provides carbohydrates. Potassium Nitrate provides the basis for nitrate
reduction. Disodium Phosphate is a buffering agent. The low agar
content, which allows varying degrees of anaerobiosis in the medium,
supports growth of organisms with various oxygen requirements.
The Difco Manual
100 g
500 g
2 kg
0265-15
0265-17
0265-07
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Disodium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g
Potassium Nitrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Precautions
Storage
523
Section II
Expiration Date
Test Procedure
1. Obtain a pure culture of the organism to be tested from a solid

culture medium. Select well-isolated colonies.
2. Inoculate a tube of Tryptic Nitrate Medium and incubate at 35 2C
for 18-24 hours.
3. Read tubes for growth.
4. Test for nitrate reduction using SpotTest Nitrate Reagents A, B
and C or equivalents per reagent instructions.
Results

Autoclave
Incubator (35C)
SpotTest Nitrate Reagents A, B and C or equivalents
1. After adding Nitrate Reagents A and B:

Positive nitrate reduction reaction: Development of a red-violet
color within 1-2 minutes indicates that nitrate has been reduced
to nitrite.
Presumptive negative nitrate reduction reaction: Lack of color
development denotes an absence of nitrite in the medium; this
should be confirmed by addition of Nitrate Reagent C (zinc dust).
2. After adding Nitrate Reagent C:

Positive nitrate reduction reaction: Lack of color development
indicates that nitrate has been reduced to nitrogen gas.
Negative nitrate reduction reaction: Development of a red-vio
let color within 5- 10 minutes indicates that unreduced nitrate is
still present.
Procedure
Materials Provided
1.
2.
3.
4.


1. Collect specimens or samples in sterile containers or with sterile
swabs and transport immediately to the laboratory in accordance
with recommended guidelines.1-7
sample.1-7

1. This medium is not recommended for indole testing of coliforms
and other enterics.1

Dehydrated Appearance: Beige, free flowing, homogeneous.
Solution:
opalescent.
Prepared Medium:
Light amber, slightly opalescent;
semisolid.
Reaction of 2.5%
Solution at 25C:
pH 7.2 0.2
Cultural Response
Prepare Tryptic Nitrate Medium per label directions. Inoculate
and incubate at 35 2C for 18-24 hours; incubate Clostridium
sporogenes anaerobically.
ORGANISM
Escherichia coli
INOCULUM
ATCC
NITRATE
CFU
11437 100-1,000
25922* 100-1,000
25923* 100-1,000
GROWTH REDUCTION
good
good
good
+
+
Uninoculated
tube
Escherichia coli
ATCC 25922
ATCC 11437
524
The Difco Manual
Section II
Tryptic Soy Agar
References
Pathogens in milk and milk products, p. 103-212. In R. T. Marshall
Bacto Tryptic Soy Agar
(ed.), Standard methods for the examination of dairy products,

6. Murray, P. R., E. J. Baron, M. A., Pfaller, F. C. Tenover, and
and Scotts diagnostic microbiology, 9th ed. Mosby-Year Book,
Packaging
500 g
0367-17
SpotTest Nitrate Reagent A
50 x 0.75 ml
3554-26
SpotTest Nitrate Reagent B
50 x 0.75 ml
3555-26
SpotTest Nitrate Reagent C
50 x 1 g
3556-26
Intended Use
Bacto Tryptic Soy Agar is used for isolating and cultivating fastidious
microorganisms and, with blood, in determining hemolytic reactions.
Also Known As
Tryptic Soy Agar (TSA) conforms with Soybean-Casein Digest Agar
Medium, USP.
In 1955, Leavitt et al.1 demonstrated that Tryptic Soy Agar supports

excellent growth of a both aerobic and anaerobic microorganisms.
Tryptic Soy Agar is a general purpose medium used for multiple
applications, e.g., as a blood culture medium, as maintenance medium
for culture collections, in colony count methods2, and for testing
bacterial contaminants in cosmetics.3
Escherichia coli
ATCC 25922
ATCC 25922

Solution:
is light amber, slightly opalescent.
Plates Prepared
Without Blood:
Light amber, slightly opalescent,with
Plates Prepared
With Blood:
Cherry red, opaque with no hemolysis.
Reaction of 4.0%
Solution at 25C :
pH 7.3 0.2
Cultural Response
Prepare Tryptic Soy Agar per label instructions. Inoculate and
incubate the plates at 35 2 under approximately 5-10% CO2
for 18-24 hours.
ORGANISM
Escherichia coli
ATCC
INOCULUM
CFU
GROWTH
HEMOLYSIS
25922*
25923*
6305
19615*
100-1,000
100-1,000
100-1,000
100-1,000
good
good
good
good
beta
alpha
beta
ATCC 6305
ATCC 19615
CAMP test: Perform Camp test using Staphylococcus aureus ATCC 33862 or ATCC 25923. Streptococcus agalactiae ATCC 12386 positive reaction
(arrow head area of clearing) Streptococcus pyogenes ATCC 19615 negative reaction (no arrow head formation)
The Difco Manual
525
Tryptic Soy Agar
Section II
TSA is recommended in multiple water and wastewater applications.4

Tryptic Soy Agar conforms to the formula specified by US
Pharmacopeia for use in Microbiological Tests 5, Antimicrobial
Preservatives-Effectiveness and Microbial Limits Test.
Clinically, Tryptic Soy Agar is used in the differentiation of
Haemophilus species, because it does not contain the X and V
factors required for Haemophilus growth. With the addition of
Differentiation Disks V, X and VX, Haemophilus growth can be
observed around the appropriate disk.
Tryptic Soy Agar is a nutritious base to which a variety of supplements
can be added. Addition of 5% sterile, defibrinated sheep, horse or
rabbit blood provides an excellent general purpose medium that
allows the growth of yeast species, staphylococci, enterococci and
gram-negative bacilli.6 TSA blood agars may be used to determine
hemolytic reactions of bacteria. TSA supplemented with lecithin and
polysorbate 80 is the formula for Microbial Content Test Agar
(also known as TSALT) used in environmental monitoring.7 For
the examination of foods, Tryptic Soy Agar is supplemented
with 3% NaCl for the isolation of Vibrio species and halophilic
microorganisms.8 Tryptic Soy Agar supplemented with 0.6% yeast
extract is used for the isolation of Listeria monocytogenes and
cultivation of a wide variety of heterotrophic microorganisms. 8
Addition of colistin and nalidixic acid to TSA is used for the
selective isolation of gram-positive cocci. 9 Gunn et al. 10 used
trimethoprim and sulfamethoxazole (SxT) supplementation to inhibit
normal flora on throat specimens, allowing Groups A and B
streptococci to grow well. Addition of iron salt and sodium thiosulfate
to TSA aids in the identification of non-fermentating gram-negative
bacilli, and with nitro blue tetrazolium (0.5% aqueous solution)
allows for the selective isolation of Corynebacterium diphtheriae.11
Chocolate Agar for culturing Haemophilus influenzae and related
organisms may be prepared by adding 1% Hemoglobin and
Supplement B to Tryptic Soy Agar.
The methodologies for multiple applications using Tryptic Soy Agar
are outlined in the references.

Storage
Expiration Date
Procedure
Materials Provided
Tryptic Soy Agar

Glassware
Autoclave
Incubator (35C)
Defibrinated blood (optional)
1.
2.
3.
4.


Test Procedure
Results
Tryptone and Soytone provide nitrogen, vitamins and minerals. The

natural sugars from the soybean promote bacterial growth. Sodium
References
Formula
Tryptic Soy Agar
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Pancreatic Digest of Casein
Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Papaic Digest of Soybean Meal
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Precautions
526
g
g
g
g
1. Leavitt, J. M., I. J. Naidorf and P. Shugaevsky. 1955. The

undetected anaerobe in endodontics; a sensitive medium for
detection of both aerobes and anaerobes. The NY J. Dentist.
25:377-382.
2. Swanson, K. J., F. F. Busta, E. H. Peterson and M. G. Johnson.
1992. Colony Count Methods, p.75-95. In C. Vanderzant,
and D. F. Spittstoesser (ed.), Compendium of methods for the
3. Curry, A. S., G. G. Joyce, and G. N. McEwen, Jr. 1993. CTFA
Microbiology guidelines. The Cosmetic, Toiletry, and Fragrance
Association, Inc., Washington, D.C.
4. Greenberg, A. E., L. S. Clesceri, and A. D. Eaton (ed). 1995.
The Difco Manual
Section II
Tryptic Soy Broth & Tryptic Soy Broth w/o Dextrose
5. The United States Pharmacopeia. 1995. Microbiological tests,

p. 1681-1686. The United States pharmacopeia, 23rd ed. United
States Pharmacopeial Convention, Rockville, MD.
Etiological agents recovered from clinical material, p. 244. Bailey
& Scotts Diagnostic Microbiology, 9th ed. Mosby-Year Book, Inc.,
St. Louis, MO.
7. Orth, D. S. 1993. Handbook of cosmetic microbiology. Marcel
Dekker, Inc., New York, NY.
9. Ellner, P. 1966. J. of Clin. Pathol. 45:502-504.
10. Gunn, B. A., D. K. Ohashi, C. A. Gaydos, and E. S. Holt. 1977.

Selective and enhanced recovery of group A and B streptococci
from throat cultures with sheep blood containing sulfamethoxazole
and trimethoprim. J. Clin. Microbiol. 5:650-655.
Packaging
Tryptic Soy Agar
100
500
2
10
g
g
kg
kg
0369-15
0369-17
0369-07
0369-08
Bacto Tryptic Soy Broth

Bacto Tryptic Soy Broth w/o Dextrose
Intended Use
Bacto Tryptic Soy Broth is used for cultivating a wide variety of
microorganisms. Tryptic Soy Broth conforms with the formula
specified in the US Pharmacopeia XXIII (USP)1 and the Code of
Federal Regulations (21 CFR)2 for sterility testing of pharmaceutical
products, biologics and devices.
Bacto Tryptic Soy Broth w/o Dextrose, a low carbohydrate formulation of Tryptic Soy Broth, is used for cultivating fastidious and
non-fastidious microorganisms.
Also Known As
Tryptic Soy Broth is commonly referred to as Soybean-Casein Digest

Tryptic Soy Broth
Solution:
amber, clear.
Prepared Medium:
Light amber, clear.
Reaction of 3.0%
Solution at 25C:
pH 7.3 0.2
Tryptic Soy Broth w/o Dextrose
Solution:
deionized water; light amber, clear to
Prepared Medium:
opalescent.
Reaction of 2.75%
Solution at 25C:
pH 7.3 0.2
The Difco Manual
Medium, USP, and Fluid Soybean-Casein Digest Medium; it is

abbreviated as TSB.

Tryptic Soy Broth is a general purpose medium used for isolating
fastidious and non-fastidious microorganisms. Tryptic Soy Broth was
originally developed for use without blood in determining the
effectiveness of sulfonamides against pneumococci and other
organisms.3 Tryptic Soy Broth is often used to support growth of
non-typical isolates such as Brucella.4 Clostridia and non-sporulating
anaerobes grow luxuriantly in this broth when incubated under
anaerobic conditions. Garrison5 and Hedgecock6 used TSB to support
growth of Histoplasma capsulatum. Mashimo and Ellison 7
supplemented this medium with agar to enhance growth of anaerobic
organisms. With the addition of 6.5% NaCl, TSB can be used for the
selective growth of group D streptococci.
Tryptic Soy Broth was chosen by the USDA Animal and Plant Health
Inspection Service for detecting viable bacteria in live vaccines.9 It is
used in the coliphage detection procedure, a proposed methodology in
Standard Methods for the Examination of Water and Wastewater.10 TSB
is recommended for testing bacterial contaminants in cosmetics11 and
complies with established standards12,13 in the food industry.
TSB is recommended by the National Committee for Clinical Laboratory
Standards (NCCLS)14 for inoculum preparation when performing the
disk diffusion sensitivity test, also known as the Kirby-Bauer method.
The rich nutritional base of Tryptic Soy Broth is often modified to
provide varying growth environments. With the addition of
1% Supplement B, TSB will support growth of Neisseria, Haemophilus
influenzae and other related organisms. The medium is used as an
enrichment broth in clinical applications and is an excellent blood
culture medium when supplemented with SPS and CO2.15
Tryptic Soy Broth w/o Dextrose, a modification of TSB, is a basal
medium to which carbohydrates may be added for use in fermentation
studies. Agar may be added (0.5-1.0 grams/liter) to enhance anaerobic
growth.16 Phenol red and other indicators may also be added.
527
Tryptic Soy Broth & Tryptic Soy Broth w/o Dextrose
Section II

Tryptone and Soytone are nitrogen sources in Tryptic Soy Broth and
Tryptic Soy Broth w/o Dextrose. Dextrose is a carbon energy source
that facilitates organism growth. Sodium chloride maintains osmotic
balance, while dipotassium phosphate is a buffering agent.
Storage
Dextrose is omitted from the formula for Tryptic Soy Broth w/o
Dextrose to permit use of the medium in fermentation studies. The
carbohydrate concentration used most frequently in fermentation
reactions is 0.5% or 1%.
Expiration Date
Formula
Procedure
Tryptic Soy Broth

Formula Per Liter
Materials Provided
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Bacto Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
g
g
g
g
g
Tryptic Soy Broth

g
g
g
g

Glassware
Autoclave
Incubator (35C)
Sterile tubes

Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Bacto Soyton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Dipostassium Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5

Tryptic Soy Broth - 30 grams;
Tryptic Soy Broth w/o Dextrose - 27.5 grams.
Precautions
User Quality Control Cont.
Cultural Response
Prepare Tryptic Soy Broth or Tryptic Soy Broth w/o Dextrose per label directions.
Inoculate medium and incubate at 35 2C for 18-48 hours or up to 76 hours if necessary.
ATCC
INOCULUM
CFU
GROWTH
13090*
12228*
6305
19615*
10-100
10-100
10-100
10-100
fair to good
good
good
good
ORGANISM
USP and EP Growth Promotion1

Prepare Tryptic Soy Broth per label directions. Inoculate medium and
incubate at the temperature specified for up to 7 days.
ORGANISM
Bacillus subtilis
Candida albicans
Candida albicans
ATCC
INOCULUM
CFU
INCUBATION
TEMPERATURE
RECOVERY
6633
2091
10231
19404
6538P
10-100
10-100
10-100
10-100
10-100
20-25C
20-25C
20-25C
30-35C
30-35C

Uninoculated
tube
Staphylococcus
epidermidis
ATCC 12228
528
The Difco Manual
Section II
Tryptone

Test Procedure
Methodologies for the multiple applications using Tryptic Soy Broth
and Tryptic Soy Broth w/o Dextrose are outlined in the references.
Results
References
2. Federal Register. 1992. General biological products standards.
Fed. Regist. 21:610.12.
3. McCullough, N. B. 1949. Laboratory tests in the diagnosis of
brucellosis. Amer. J. of Public Health 39:866-869.
Microorganisms encountered in the blood, p. 205. Bailey & Scotts
diagnostic microbiology, 9th ed. Mosby-Year Book, Inc.,
St. Louis, MO.
5. Garrison, R. G. 1961. Studies of the respiratory activity of
Histoplasma capsulatum. J. of Infect. Dis. 108:120-124.
6. Hedgecock, L. W. 1971. Effect of vaccines prepared from
Histoplasma capsulatum and other yeast on experimental
tuberculosis. J. Bacteria. 82:115- 123.
7. Mashimo, P. A., and S. A. Ellison. 1959. Simple method for the
isolation of anaerobic oral vibrios. J. Bacteria. 78:636-639.
8. Sherman, J. M., and P. Stark. 1961. Streptococci which grow at
high temperatures. J. Bacteria. 22:275-285.
9. Federal Register. 1992. Detection of viable bacteria and fungi

except in live vaccine. Fed. Regist. 21:113.26.
10. Greenberg, A. E., L. S. Clesceri, and A. D. Eaton (eds). 1992.
Coliphage detection, 9,22-23. Standard methods for the
11. Curry, A. S., G. G. Joyce, and G. N. McEwen, Jr. 1993. CTFA
Microbiology guidelines. The Cosmetic, Toiletry, and Fragrance
Association, Inc., Washington, D.C.
13. Cunnif, P. 1995. Official methods of analysis AOAC International,
Performance standards for antimicrobial disk susceptibility tests,
M2-A5, vol. 13, no. 24. National Committee for Clinical
15. Isenberg, H. D. (ed.). 1992. Processing and interpretation of blood
cultures, p. 1.7.1-1.7.2. Clinical microbiology procedures handbook, vol. 1. American Society for Microbiology, Washington, D.C.
16. MacFaddin, J. D. 1985. Media for isolation-cultivationidentification-maintenance of medical bacteria, p. 797, vol. 1.
Packaging
Tryptic Soy Broth
100
500
2
10
g
g
kg
kg
0370-15
0370-17
0370-07
0370-08
500 g
10 kg
0862-17
0862-08
Bacto Tryptone
Intended Use
Also Known As
Bacto Tryptone is used in preparing microbiological culture media.

Tryptone is also referred to as Peptone C, Peptone 50 and Tryptone T.
homogeneous powder.
Solution:
1%-Very light to light amber, clear
without precipitate;
without precipitate;
10%-Medium to dark amber, clear to
slight precipitate.
Amino Nitrogen
(Modified Sorensen Method): 4.0-6.6%
Reaction of 2%
Solution at 25C:
pH 6.9-7.4
The Difco Manual

Tryptone is a pancreatic digest of casein used as a nitrogen source in
culture media formulated for isolating and cultivating fastidious and
nonfastidious bacteria and fungi.
Tryptone was developed by Difco Laboratories while investigating a
peptone particularly suitable for the elaboration of indole by bacteria.
The high tryptophane content of Tryptone makes it valuable for use in
detecting indole production.1,2,3 The absence of detectable levels of carbohydrates in Tryptone makes it a suitable peptone in differentiating
bacteria on the basis of their ability to ferment various carbohydrates.
Several media containing Tryptone are specified in standard
methods4,5,6,7 for multiple applications.

Tryptone is a pancreatic digest of casein especially rich in tryptophane.
Casein, a milk protein, is a rich source of amino acid nitrogen.
529
Tryptone
Section II
Typical Analysis

Coliform
Salmonella
Spore Count
Thermophile Count
Ash (%)
6.8
1.3
Loss on Drying (%)

pH, 1% Solution
3.7
7.2
Carbohydrate (%)
Total
7.7

Total Nitrogen
Amino Nitrogen
13.0
5.2
AN/TN 20.7
2.86
3.03
6.11
0.42
17.05
1.75
2.02
4.40
7.11
Lysine
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine
0.013
0.186
<0.001
<0.001
<0.001
<0.001
0.017
<0.001
Phosphate
Potassium
Sodium
Sulfate
Sulfur
Tin
Zinc
40.0
6.70
2.57
3.71
7.45
4.29
3.58
0.71
1.42
5.00
Inorganics (%)
Calcium
Chloride
Cobalt
Copper
Iron
Lead
Magnesium
Manganese
2.669
0.229
2.631
0.241
0.740
<0.001
0.003
Materials Provided
Tryptone

Refer to the final concentration of Tryptone in the formula of the
medium being prepared. Add Tryptone as required.

Test Procedure
See appropriate references for specific procedures using Tryptone.
Results
See appropriate references and procedures for results.
Vitamins (g/g)
Biotin
0.1
Cyanocobalamin
<0.1
Folic Acid
0.3
Inositol
1400.0
Nicotinic Acid
97.8
The values presented are typical. This information is for broad

comparison use only and is not indicative of the makeup of any
particular lot of material. No guarantee is made, either expressed or
implied, that any specific lot of product will match the values presented.
Procedure
Amino Acids (%)

Alanine
Arginine
Aspartic Acid
Cystine
Glutamic Acid
Glycine
Histidine
Isoleucine
Leucine
negative
negative
73
870
8
PABA
Pantothenic Acid
Pyridoxine
Riboflavin
Thiamine
Thymidine
3.7
5.3
0.6
<0.1
0.4
93.4
References
1. J. Bacteriol. 1933. 25:623.
2. Pure Culture Study of Bacteria. 1947. No. 3
3. Centr. Bakt. , I Abt. 1915. 76:1.
Cultural Response
For each Test specified, prepare a Test Solution of Tryptone and, if necessary, adjust to pH 7.2-7.4; sterilize, inoculate and incubate
according to standard test procedure.
TEST
TEST SOLUTION
INOCULUM
RESULT
Escherichia coli
Escherichia coli
25922*
25922*
1 drop, undiluted
1 drop, undiluted
Production
Hydrogen Sulfide
Production
Growth Production
13048*
1 drop, undiluted
6539
1 drop, undiluted
negative; red color

positive; pink color on
Indole Test Strip
positive; pink color upon
adding reagents
positive; brownish blackening
of H2S Test Strip
good growth
0.1% w/ 0.5%
dextrose
1%
ORGANISM
ATCC
Fermentable Carbohydrates 2%
Indole Production
0.1%
Salmonella typhi
2% w/1.5% agar
Escherichia coli
25922*
100-1,000 CFU
and 0.5% NaCl
Growth Production
2% w/1.5% agar
25923*
100-1,000 CFU
good growth
and 0.5% NaCl
530
The Difco Manual
Section II
Tryptone Glucose Extract Agar & m TGE Broth

analytical manual , 8th ed. AOAC International, Gaithersburg, MD.

Washington, D.C.
Packaging
Tryptone
100
500
2
10
g
g
kg
kg
0123-15
0123-17
0123-07
0123-08
Bacto Tryptone Glucose Extract Agar

Bacto m TGE Broth
Also Known As
Tryptone Glucose Extract Agar is also known as Yeast Dextrose Agar

and may be abbreviated as TGEA.
Intended Use
Bacto Tryptone Glucose Extract Agar is used for cultivating and
enumerating microorganisms in water and dairy products.
Bacto m TGE Broth is used for enumerating microorganisms by
m TGE is an abbreviation for membrane Tryptone Glucose Extract.

Uninoculated
plate
Pasturized
milk

Dehydrated Appearance: Light to medium tan, free-flowing,
homogeneous.
Solution:
Prepared Medium:
opalescent, no precipitate.
Reaction of 2.4%
Solution at 25C:
pH 7.0 0.2
m TGE Broth
Solution:
deionized water. Solution is medium amber,
Prepared Medium:
Medium amber, clear to very slightly opalescent.
Reaction of 1.8%
Solution at 25C:
pH 7.0 0.2
Cultural Response
Prepare Tryptone Glucose Extract Agar per label directions in
parallel with a reference control. Inoculate with pasteurized
and raw milk samples using the pour plate technique and
incubate at 32 1C for 47-49 hours. Recovery of bacteria
from the milk samples should be comparable for both the test
and reference lots.
m TGE Broth
Prepare mTGE Broth per label directions. Inoculate using the
membrane filter technique and incubate in a humid atmosphere
ORGANISM
Escherichia coli
ATCC
INOCULUM CFU
GROWTH
25922*
25923*
100-1,000
100-1,000
good
good
The Difco Manual
531
Tryptone Glucose Extract Agar & mTGE Broth
Section II
Procedure
In the 1930s, Bower and Hucker1 developed a medium for detecting

bacteria in milk and other dairy products. Many investigators compared
the performance of Tryptone Glucose Skim Milk Agar to Nutrient Agar
for estimating bacteria in milk and other dairy products.2,3,4 Prickett5
used a glucose agar containing Tryptone to study thermophilic bacteria
in milk. This medium, described in Standard Methods of Milk Analysis6,
was prepared in the dehydrated form as Yeast Dextrose Agar. The
American Public Health Association (APHA) adopted Tryptone
Glucose Extract Agar for use in testing milk and dairy products in
1948.7 For many years, Tryptone Glucose Extract Agar with added milk
remained the Standard Methods medium for dairy products8 and was
also adopted for testing water.9 Currently, the APHA specifies using
Tryptone Glucose Extract Agar for the heterotrophic plate count
procedure in testing bottled water.10
Materials Provided
m TGE Broth is a nonselective nutrient medium for the determination

of bacterial counts by the membrane filter method. The broth has the
same formulation as Tryptone Glucose Extract Agar, except that the broth
contains no agar and the ingredients are at twice the concentration.
Tryptone Glucose Extract Agar (TGEA)

m TGE Broth
Tryptone Glucose Extract Agar and m TGE Broth contain Beef Extract
and Tryptone as sources of carbon, nitrogen, vitamins and minerals.
Dextrose (Glucose) is a carbohydrate. Tryptone Glucose Extract Agar
contains Bacto Agar as a solidifying agent.
Glassware
Autoclave
Petri dishes (Tryptone Glucose Extract Agar)
Sterile membranes (m TGE Broth)
Filter apparatus (m TGE Broth)
Sterile absorbent pads (m TGE Broth)
Incubator (TGEA - 32 1C; mTGE Broth - 35C)

Test Procedure

Formula Per Liter
g
g
g
g

m TGE Broth
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g
Precautions
Storage
Expiration Date
532
Collect samples in accordance with laboratory procedures.10
Formula
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose (Glucose) . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

m TGE Broth
1. Use the appropriate culture method, as follows:

TGEA - pour plate method;
m TGE - membrane filtration.
2. Incubate the inoculated medium in a humid atmosphere, as follows:
TGEA - 32 1C for 47-49 hours;
m TGE - 35 2C for 18-24 hours.
Results
Count total colonies and record results.

References
1. Bowers and Hucker. 1935. Tech. Bull. 228. NY State Agr. Exp. Sta.
2. Yale. 1938. Am. J. Pub. Health 28:148.
3. Proc. 36th Cong. Intern. Assoc. Ice Cream Manufacturers.
1936. 2:132.
4. Dennis and Weiser. 1937. J. Dairy Science 20:445.
5. Prickett. 1928. Tech. Bull. 147. NY State Agr. Exp. Sta.
6. Standard Methods of Milk Analysis, 6th ed. 1934.
The Difco Manual
Section II
Tryptone Water

Packaging
Bacto Tryptone Water
differentiation of bacteria and the identification of E. coli isolated from

food and water samples.2,3
Intended Use
Bacto Tryptone Water is recommended for use in the detection of

Escherichia coli in food and water samples based on indole production.
Tryptone Water is similar to Tryptone Broth, containing both Tryptone

(1%) and Sodium Chloride. Due to its high tryptophan content,
Tryptone is suitable for use in detecting indole production by bacteria.
Tryptophan is hydrolyzed and deaminated to produce indole, pyruvic
acid and ammonia.4 Indole can then be detected by the addition of either Kovacs or Ehrlichs Reagent, which contain an aldehyde group.
The aldehyde group combines with indole to produce a red color in the
alcohol layer. Sodium Chloride is added to the medium to provide a
suitable osmotic environment.

Tryptone Water is based on the Tryptone Water formula described in
AFNOR and ISO Standards.1 In these procedures, Tryptone Water is
used with Brilliant Green Bile 2% to determine the most probable
number (MPN) of E. coli present in meat and meat products. Growth
and gas production in Brilliant Green Bile 2% and indole production
in Tryptone Water following incubation of both media at 44 1C is
used as the basis for this presumptive E. coli test.
Tryptone Water may also be used for differentiation of bacteria based
on indole production. Production of indole using pure cultures in
tryptophan containing media is recommended as an aid in the
100 g
500 g
2 kg
0002-15
0002-17
0002-07
100 g
500 g
0750-15
0750-17
m TGE Broth
Formula
Tryptone Water
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Precautions
Solution:
1.5 % solution, soluble in distilled or
opalescent with no significant
precipitate.
Prepared Medium:
slightly opalescent with no significant
precipitate.
Reaction of 1.5%
Solution at 25C:
pH 7.3 0.2
Cultural Response
Prepare Tryptone Water per label directions. Inoculate and
incubate the tubes at 35 2C for 18-24 hours. Add 0.5 ml
SpotTest Indole Reagent Kovacs to the tubes to test for indole
production. Formation of a red color denotes a positive indole test.
ORGANISM
Escherichia coli
Enterobacter cloacae
Storage
Store the dehydrated medium below 30C. The powder is very hygroscopic.
Keep container tightly closed. Store prepared medium at 2-8C.
Expiration Date
Procedure
Materials Provided
Tryptone Water
ATCC
INOCULUM
CFU
INDOLE
PRODUCTION
GROWTH
25922*
13047
100-300
100-300
positive
negative
good
good
The Difco Manual


Autoclave
Incubator (30 1C)
Incubator (44 1C)
Diluent
533
Tryptose
Section II

1. Collect food and water samples in sterile containers and transport
immediately to the laboratory following recommended guidelines.1-4
2. Process each food and water sample using procedures appropriate
for that sample.1-4
Test Procedure
Presumptive Test For E. coli in Meats and Meat Products1
1. Suspend one part sample in 9 parts diluent. Homogenize sample.
2. Dilute homogenate in duplicate using serial 10-fold dilutions to
10-6 using 1 ml of material to be diluted to 9 ml of diluent. Mix
each dilution thoroughly.
3. Transfer 1 ml of homogenate (step 1) to 6 tubes containing 10 ml
Brilliant Green Bile 2% and group in 2 sets of 3 tubes each. If the
number of coliforms is expected to be high, transfer 10 ml of
homogenate into 6 tubes containing double strength Brilliant Green
Bile 2%.
4. Transfer 1 ml from each of the dilutions prepared in step 2 into
each of 3 tubes containing 10 ml Brilliant Green Bile 2%.
5. Incubate Brilliant Green Bile 2% tubes (prepared in steps 3 and 4)
at 30 1C for 48 2 hours. Subculture all tubes containing gas,
using 1 drop of inoculum, into Tryptone Water and Brilliant Green
Bile 2%.
6. Incubate tubes prepared in step 5 at 44 1C for 48 2 hours.
Examine and record the tubes of Brilliant Green Bile 2% tubes

containing gas. Add 0.5 ml SpotTest Indole Reagent Kovacs to the
Tryptone Water tubes. Observe tubes for the formation of a red ring at
the top of medium indicating indole production. Record the tubes for
positive indole production. Determine the MPN (Most Probable
Number) of E. coli present in the sample based on the number of tubes
that are positive for both gas and indole. Consult the appropriate
3-tube MPN table.2
Indole Determination Using Pure Cultures
Examine tubes for the formation of a red ring at the top of the tube
indicating indole production.

1. Detection of E. coli in meats using Tryptone Water is a presumptive test. If confirmatory testing is required, please consult
appropriate references.
2. Indole testing is recommended as an aid in the differentiation of
microorganisms based on indole production. For complete
identification of the organism, further biochemical evaluation
is necessary.
References
Indole Determination Using Pure Cultures

1. Inoculate Tryptone Water using a light inoculum of an 18-24 hour
pure culture.
2. Incubate the tubes at 35 2C with loosened caps for 18-24 hours.
3. Add 0.5 ml of SpotTest Indole Reagent Kovacs directly to the
tube and agitate. Allow tubes to stand for 5-10 minutes.
1. International Organization for Standardization: Meat and

meat products. Detection and enumeration of presumptive coliform
bacterial and presumptive E. coli (Reference method ISO/DIS
3811-1979).
Standard Methods for the Examination of Water and Wastewater,
4. MacFaddin, J. F. Biochemical Test for Identification of Medical
Bacteria, 3rd ed. 1985. Williams and Wilkens, Baltimore, MD.
Results
Packaging
Presumptive Test For E. coli in Meats and Meat Products
Tryptone Water
Bacto Tryptose
Intended Use
Bacto Tryptose is an enzymatic digest of protein for use in preparing
Also Known As
Tryptose is also referred to as Polypeptone Peptone.

properties. Tryptose was originally developed as a peptone particularly
adapted to the growth requirements of Brucella. An agar medium
containing Tryptose, sodium chloride and dextrose, without liver or
other infusions, was shown to be an excellent medium for propagation
534
500 g
0644-17
of these organisms.1 Culture media prepared with Tryptose were

superior to the meat infusion peptone media previously used for the
cultivation of Brucella, streptococci, pneumococci, meningococci and
other fastidious bacteria.2,3
An agar medium prepared with 2% Tryptose and 0.5-0.8% sodium
chloride, without tissue infusion, is an excellent blood agar base. The
growth of organisms on Tryptose Blood Agar Base is luxuriant and the
zones of hemolysis produced are distinct and clear. Huddleson1 used a
broth containing 2% Tryptose as an enrichment medium in the
isolation of Brucella from clinical specimens.

properties. The digestive process in Tryptose results in assorted
peptides, including those of higher molecular weight.
The Difco Manual
Section II
Tryptose
Typical Analysis
Ash (%)
9.7
0.8
2.3
Loss on Drying (%)

pH, 1% Soln
3.2
7.4
7.1

Total Nitrogen
Amino Nitrogen
13.4
4.4
AN/TN
32.5
Amino Acids (%)

Alanine
Arginine
Aspartic Acid
Cystine
4.45
4.65
6.34
0.44
Lysine
Methionine
Phenylalanine
Proline
4.64
1.92
7.52
6.33

Solution:
1, 2 and 10% solutions are soluble in
1% - Light amber, clear.
2% - Medium amber, clear to slightly
opalescent.
10% - Medium to dark amber, very
slightly opalescent to opalescent, may
have precipitation.
Reaction of 1%
Solution at 25C:
pH 7.1 to 7.5
Cultural Response
TEST
SOLUTION
ORGANISM
Fermentable
Carbohydrates
Indole
Production
Hydrogen
Sulfide
Production
Growth
Response
2%
Escherichia
coli
Escherichia
coli
Enterobacter
aerogenes
Salmonella
typhi
Growth
Response
Growth
Response
Growth
Response
0.1%
0.1% w /0.5%
dextrose
1%
13.92
2.84
<0.01
0.34
3.67
Serine
Threonine
Tryptophan
Tyrosine
Valine
4.09
3.55
0.62
2.21
1.93
0.001
0.886
<0.001
<0.001
0.002
<0.001
0.022
<0.001
Phosphate
Potassium
Sodium
Sulfate
Sulfur
Tin
Zinc
2.144
0.679
3.410
0.308
0.737
<0.001
0.005
Inorganics (%)
Carbohydrate (%)
Total
Glutamic Acid
Glycine
Histidine
Isoleucine
Leucine
ATCC
RESULT
Calcium
Chloride
Cobalt
Copper
Iron
Lead
Magnesium
Manganese
Vitamins (g/g)
Biotin
0.2
Choline (as Choline Chloride)2700.0
Cyanocobalamin
<0.1
Folic Acid
0.4
Inositol
5400.0
Nicotinic Acid
47.4
PABA
Pantothenic Acid
Pyridoxine
Riboflavin
Thiamine
Thymidine
11.4
16.0
1.4
4.3
0.1
769.0

Coliform
Salmonella
Spore Count
negative
negative
875

Thermophile Count 100
Precautions
Storage
Store the dehydrated ingredient below 30C. The dehydrated ingredient is very hygroscopic. Keep container tightly closed.
25922* negative
Expiration Date
25922* positive
13048* positive
6539
positive
4314
good
growth
Procedure
Materials Provided
Tryptose
2% w/ 0.5%
NaCl, 0.1% agar,
and 0.1% dextrose
2% w/ 0.5%
NaCl, 0.1% agar,
and 0.1% dextrose
2% w/ 0.5%
NaCl, 0.1% agar,
and 0.1% dextrose
2% w/ 0.5%
NaCl, 0.1% agar,
and 0.1% dextrose
Brucella
suis

Staphylococcus 25923*
aureus
good
growth
Streptococcus
pneumoniae
6303*
good
growth
Refer to the final concentration of Tryptose in the formula of the

medium being prepared. Add Tryptose as required.
Streptococcus
pyogenes
19615*
good
growth
The Difco Manual
Test Procedure
Results
References
1. Huddleson, I. F. 1939. Brucellosis in man and animals. 14.
Oxford University Press, Oxford.
535
Tryptose Agar & Tryptose Broth
Section II
2. Casman, E. P. 1942. A dehydrated medium to supplement meat

infusion as a base for blood agar. J. Bacteriol. 43:33.
Packaging
Bacto Tryptose Agar

Bacto Tryptose Broth
Intended Use
Bacto Tryptose Agar is used for cultivating a wide variety of fastidious
microorganisms, particularly for isolating Brucella according to
Huddleson and Castaeda.
Bacto Tryptose Broth is used for cultivating Brucella and other

Tryptose Agar
Dehydrated Appearance: Light beige, homogeneous,
free-flowing.
Solution:
distilled or deionized water; light
opalescent, without significant
precipitate.
Prepared Medium:
Reaction of 4.1%
Solution at 25C:
pH 7.2 0.2
Tryptose Broth
Solution:
deionized water; light amber, clear,
Prepared Medium:
Light amber, clear without
Reaction of 2.6%
Solution at 25C:
pH 7.2 0.2
Cultural Response
Prepare Tryptose Agar and Tryptose Broth per label
directions. Inoculate and incubate at 35 2C under 5-10%
CO2 for 40-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Brucella abortus
Brucella melitensis
Brucella suis
4315
4309
6597
100-1,000
100-1,000
100-1,000
good
good
good
Tryptose
100
500
2
10
g
g
kg
kg
0124-15
0124-17
0124-07
0124-08
Tryptose media, prepared without extract or infusion of meat, are

recommended for the cultivation and isolation of pathogenic and
saprophytic bacteria. Historically, it was considered necessary to
include meat extract or infusion as a nutritional supplement in
culture media. Tryptose was developed while studying the growth
requirements of Brucella. Huddleson1 found tryptose media to be
equal or superior to meat infusion media, providing uniformity for the
cultivation and differentiation of fastidious organisms.
Tryptose media are particularly well suited for the isolation of Brucella
from blood. Castaeda2 studied the isolation of Brucella species using
a broth containing 2% Tryptose and 2% sodium citrate. Sodium citrate
serves as an anticoagulant and assists in inactivating complement in
the blood specimen.
Tryptose Broth can be used as a complete basal medium or
supplemented with enrichments. Huddleson3 used a broth containing
2% Tryptose as an enrichment medium in the isolation of Brucella
from clinical specimens. McCullough et al. reported that addition of
thiamine, dextrose and iron salts increased growth of Brucella suis.4
Addition of 0.1% agar to Tryptose Broth can increase growth of aerobes
and anaerobes in liquid media. Blood agar may be prepared by adding
5% sterile, defibrinated sheep, horse or rabbit blood to the sterile medium.
The high productivity of tryptose media in the isolation and cultivation
of Brucella supports use of these formulas as general purpose media,
especially when avoidance of animal tissue products is desired.
Tryptose Agar with 5% bovine serum, with or without antibiotics,
remains a standard plating medium for the isolation of brucellae.5
For isolation of Brucella stains from contaminated milk, crystal violet
(gentian violet) can be added to Tryptose Agar to suppress
gram-positive organisms.6 Tryptose media can be supplemented with
thiamine or citrate for the cultivation and maintenance of fastidious
aerobic and facultative microorganisms.7
Tryptose Agar is specified in the Compendium of Methods for the
Microbiological Examination of Food. 8 Tryptose media are
recommended in FDA Bacteriological Analytical Manual for
serological testing.9

In Tryptose media, Tryptose is a source of nitrogen and carbon.
Dextrose is a source of carbohydrate. Sodium Chloride maintains
osmotic balance. Bacto Agar is the solidifying agent in Tryptose Agar.
Formula
Tryptose Agar
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
536
The Difco Manual
Section II
Tryptose Broth
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 g
Precautions
Storage
Expiration Date
Procedure
Materials Provided
Tryptose Agar
Tryptose Broth

Glassware
Autoclave
Incubator (35C)
Sterile Petri dishes and/or tubes
Bacto Agar (optional)
Tryptose Agar
3. Distribute into tubes, bottles or flasks.
5. To prepare blood agar, aseptically add 5% sterile defibrinated
sheep, horse or rabbit blood. Dispense into sterile Petri dishes.
Tryptose Broth
2. Distribute into tubes, bottles or flasks.

Specimens should be collected in sterile containers or with sterile
swabs and transported immediately to the laboratory in accordance with
recommended guidelines outlined in the references.
Test Procedure
Methodologies for the multiple applications using tryptose media are
The Difco Manual
Tryptose Agar & Tryptose Broth
Results

1. Tryptose media are general purpose, non-selective media. Although
certain diagnostic tests may be performed directly on the medium,
biochemical and, if indicated, immunological testing using pure
cultures are recommended for complete identification.
2. When preparing blood agar, hemolytic reactions of some strains
of group D streptococci have been shown to be affected by
differences in animal blood.
reactions of beta-hemolytic streptococci. 10 For optimal
performance, incubate tryptose media supplemented with blood
under increased CO2 or anaerobic conditions.
4. Dextrose has been shown to inhibit hemolysin production by
some organisms.
References
1. Huddleson, I. F. 1943. Brucellosis in man and animals. Rev. Ed.
The Commonwealth Fund, New York.
2. Castaeda, M. R. 1947. A practical method for routine blood
cultures in brucellosis. Proc. Soc. Exp. Biol. Med. 64:114-115.
3. Huddleson, I. F. 1939. Brucellosis in man and animals.
14. Oxford University Press, Oxford.
4. McCullough, W. G., R. C. Mills, E. J. Herbst, W. G. Roessler,
and C. R. Brewer. 1947. Studies on the nutritional requirements
of Brucella suis. J. Bacteriol. 53:5-15.
5. Moyer, N. P., and L. A. Holcomb. 1995. Brucella. In P. R. Murray,
7. Atlas, R. M. 1995. Handbook of microbiology media for the
examination of food, p. 266-268. CRC Press, Boca Raton, FL.
Rhodehamel. 1995. FDA Bacteriological analytical manual, 8th ed.
Packaging
Tryptose Agar
500 g
2 kg
10 kg
0064-17
0064-07
0064-08
Tryptose Broth
500 g
10 kg
0062-17
0062-08
537
Tryptose Blood Agar Base & Tryptose Blood Agar Base w/Yeast Extract
Section II
Bacto Tryptose Blood Agar Base

Bacto Tryptose Blood Agar Base w/Yeast Extract
Intended Use
Bacto Tryptose Blood Agar Base is used with blood in isolating, cultivating
and determining the hemolytic reactions of fastidious microorganisms.
Bacto Tryptose Blood Agar Base w/Yeast Extract is used with or without
blood in cultivating a wide variety of microorganisms.
Also Known As

Investigations of the nutritive properties of Tryptose demonstrated that
culture media prepared with this peptone were superior to the meat
infusion peptone media previously used for the cultivation of Brucella,
streptococci, pneumococci, meningococci and other fastidious bacteria.
Casman1,2 reported that a medium consisting of 2% Tryptose, 0.3%
Beef Extract, 0.5% NaCl, 1.5% Bacto Agar and 0.03% dextrose equaled
fresh beef infusion base with respect to growth of organisms. The small
amount of carbohydrate was noted to interfere with hemolytic reactions,
unless the medium was incubated in an atmosphere of carbon dioxide.
Tryptose Blood Agar Base and Tryptose Blood Agar Base w/ Yeast
Extract are nutritious infusion-free basal media typically supplemented
with 5-10% sheep, rabbit or horse blood for use in isolating, cultivating
and determining hemolytic reactions of fastidious pathogenic
microorganisms. Without enrichment, these bases can be used as general
purpose media. Tryptose Blood Agar Base is specified in FDA
Bacteriological Analytical Manual.4 Tryptose Blood Agar Base w/ Yeast
Extract was formulated to provide a base with additional nutrients to
improve the growth of fastidious organisms.
Escherichia coli
ATCC 25922
ATCC 25923

Tryptose Blood Agar Base
Solution:
amber, slightly opalescent.
Prepared Medium:
Light amber, slightly opalescent. With
5% sheep blood-cherry red, opaque.
Reaction of 3.3%
Solution at 25C:
pH 7.2 0.2
Tryptose Blood Agar Base w/ Yeast Extract
Solution:
deionized water on boiling; light to medium
Prepared Medium:
Light to medium amber, slightly opalescent.
Reaction of 3.4%
Solution at 25C:
pH 7.3 0.2
ATCC 6305
ATCC 19615
Cultural Response
Prepare Tryptose Blood Agar Base and Tryptose Blood Agar Base w/ Yeast Extract with and without 5% sterile defibrinated
sheep blood per label directions. Inoculate and incubate at 35 2C under 5-10% CO2 for 18-48 hours.
ORGANISM
Escherichia coli
ATCC
INOCULUM
CFU
GROWTH
w/o BLOOD
GROWTH
W/BLOOD
HEMOLYSIS
18-48 H
25922*
13090*
25923*
6305
19615*
100-1,000
100-1,000
100-1,000
100-1,000
100-1,000
good to excellent
fair to good
good
fair to good
fair to good
good to excellent
good
good
good
good
beta
N/A
beta
alpha
beta
538
The Difco Manual
Section II
Tryptose Blood Agar Base & Tryptose Blood Agar Base w/Yeast Extract
Tryptose is the source of nitrogen, carbon and amino acids in Tryptose

Blood Agar Base and Tryptose BAB w/ Yeast Extract. Beef Extract
provides additional nitrogen. Bacto Agar is the solidifying agent.
Sodium Chloride maintains osmotic balance. Yeast Extract supplies
additional vitamins and cofactors for growth.

deionized water.
33 g
Tryptose Blood Agar Base w/ Yeast Extract 34 g
Supplementation with 5-10% blood provides additional growth factors

for fastidious microorganisms, and is used to determine hemolytic
patterns of bacteria.
Formula

Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g

Tryptose Blood Agar Base w/Yeast Extract
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Test Procedure
g
g
g
g
g
Precautions
Storage
Expiration Date
Procedure
Materials Provided
Tryptose Blood Agar Base w/ Yeast Extract

Glassware
Autoclave
Incubator (35C)
5% sterile defibrinated blood (optional)
The Difco Manual

properly collected and transported to the laboratory.7 Obtain and process
specimens according to the procedures established by institutional
policy. Specimens should be collected in sterile containers or with sterile
swabs, and transported immediately to the laboratory in accordance
with recommended guidelines outlined in the references.
1. Process each specimen as appropriate, and inoculate directly onto
the surface of the medium. Streak for isolation with an inoculating
loop, then stab the agar several times to deposit beta-hemolytic
streptococci beneath the agar surface. Subsurface growth will
display the most reliable hemolytic reactions of both oxygen-stable
and oxygen-labile streptolysins.3
2. Incubate plates aerobically, anaerobically or under conditions of
increased CO2 (5-10%) in accordance with established laboratory
procedures.
3. Examine plates for growth and hemolytic reactions after 18-24 and
48-hour incubation. Four different types of hemolysis on blood agar
methemoglobin in the medium surrounding the colony. This
causes a greenish discolorization of the medium.
b. Beta ()-hemolysis is the lysis of red blood cells, resulting in a
c. Gamma ()-hemolysis indicates no hemolysis. No destruction
of red blood cells occurs, and there is no change in the medium.
d. Alpha-prime (` )-hemolysis is a small zone of complete
hemolysis that is surrounded by area of partial lysis.

1. Blood Agar Base Media are intended for use with blood
supplementation. Although certain diagnostic tests may be
performed directly on this medium, biochemical and, if indicated,
immunological testing using pure cultures are recommended for
complete identification. Consult appropriate references for further
information.
539
Tryptose Phosphate Broth
Section II
4. Colonies of Haemophilus haemolyticus are beta-hemolytic on horse

and rabbit blood agar, and must be distinguished from colonies on
beta-hemolytic streptococci using other criteria. The use of sheep
blood has been suggested to obviate this problem since sheep blood
is deficient in pyridine nucleotides and does not support growth of
H. haemolyticus.6
5. The atmosphere of incubation has been shown to influence
hemolytic reactions of beta-hemolytic streptococci.3 For optimal
performance, incubate blood agar base media under increased CO2
or anaerobic conditions.
6. Hemolytic patterns may vary with the source of animal blood or
type of base medium used.3

Rhodehamel. 1995. FDA Bacteriological analytical manual, 8th
ed. AOAC International, Arlington, VA.
St. Louis, MO.
References
Packaging
1. Casman, E. P. 1942. A dehydrated medium to supplement meat

infusion as a base for blood agar. J. Bacteriol. 43:33.
500 g
2 kg
0232-17
0232-07

w/ Yeast Extract
500 g
0662-17
Bacto Tryptose Phosphate Broth
Intended Use
Bacto Tryptose Phosphate Broth is used for cultivating fastidious

microorganisms.
Tryptose Phosphate Broth is an infusion-free buffered medium

recommended for the cultivation of fastidious, pathogenic microorganisms. In a study by Waisbren, Carr, and Dunnett,1 Tryptose Phosphate
Broth was used in the tube method for antibiotic sensitivity testing.
Also Known As
Tryptose Phosphate Broth is abbreviated as TPB.

Solution:
2.95% solution, soluble in distilled or deionized water; light
amber, may be very slightly opalescent with a very
slight precipitate.
Prepared Medium:
Light amber, clear to very slightly opalescent, may have a
Reaction of 2.95%
Solution at 25C:
pH 7.3 0.2
Cultural Response
Prepare Tryptose Phosphate Broth per label directions. Inoculate and incubate at
35 2oC for 18-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
13090*
12228*
6305
19615*
100-1,000
100-1,000
100-1,000
100-1,000
good
good
good
good
Uninoculated
tube
540
Streptococcus
pneumoniae
ATCC 6305
The Difco Manual
Section II
UBA Medium
Tryptose Phosphate Broth is valuable in tissue culture procedures,

as shown by Ginsberg, Gold and Jordan. 2 The proteose content
of Tryptose Phosphate Broth is considered to be a stimulating factor
for cells. Tryptose Phosphate Broth is specified in the FDA
Bacteriological Analytical Manual for cell culture procedures.3

Tryptose provides carbon and nitrogen. Dextrose is a carbon source.
Sodium Chloride maintains osmotic balance. Buffering capacity is
provided by Disodium Phosphate.
The addition of 0.1-0.2% agar to Tryptose Phosphate Broth facilitates
anaerobic growth and aids in dispersion of reducing substances and
CO2 formed in the environment.4 The low agar concentration provides
suitable conditions for both aerobic growth in the upper zone and for
microaerophilic and anaerobic growth in the lower zone.

Glassware
Autoclave
Incubator (35C)
Tubes with closures
Bacto Agar (optional)
2. If a medium containing 0.1% agar is desired, add 1 gram of Bacto
Agar. Heat to boiling to dissolve completely.
Formula


Formula Per Liter
Test Procedure
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
g
g
g
g
Results
Precautions


References
1. Waisbren, B. A., M. S. Carr, and J. Dunnett. 1951. The tube

dilution method of determining bacterial sensitivity to antibiotics.
Am. J. Clin. Pathol. 21:884.
2. Ginsberg, Gold, and Jordan. 1955. Proc. Soc. Exp. Biol. Med. 89:66.
Rhodehamel. 1995. FDA Bacteriological analytical manual,
Procedure
Packaging
Materials Provided
Storage
Expiration Date
Bacto UBA Medium
Intended Use
Bacto UBA Medium is used for cultivating microorganisms of
significance in the brewing industry.
Also Known As
UBA is also referred to as Universal Beer Medium or Universal
Beer Agar.
The Difco Manual
500 g
2 kg
10 kg
0060-17
0060-07
0060-08

UBA Medium is a basal medium to which beer is added. It is based
on the formula developed by Kozulis and Page1 who compared it with
other media commonly used in breweries for detecting microbial
contamination. 2 The characteristics of UBA Medium are closer
to the natural environmental conditions found in the typical brewery
than other media studied. UBA Medium supports growth of more
varieties of lactic acid bacteria and yields larger colonies in a shorter
time than traditional brewers media. Due to the presence of beer
541
UBA Medium
Section II
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 g
in the medium, it is selective for growth of microorganisms that have

adapted themselves to existent conditions in the brewery. The presence of hop constituents and alcohol inhibits growth of many airborne
microorganisms not adapted to this environment.3
Precautions
UBA Medium supports growth of Lactobacillus, Pediococcus,

Acetobacter and yeast strains which may be found contaminating the
wort and beer.

Storage

Peptonized Milk contains lactose as an energy source. Tomato Juice
is a source of carbon, protein and nutrients. Dextrose provides
additional carbon. Dipotassium and Monopotassium Phosphate
provide buffering capability. Magnesium Sulfate, Ferrous Sulfate and
Manganese Sulfate are sources of ions that stimulate metabolism.
Sodium Chloride maintains the osmotic balance of the medium. Bacto

Formula
Materials Provided
UBA Medium
Formula Per Liter
UBA Medium

Bacto Peptonized Milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Tomato Juice (244 ml) . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.1
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.006
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.006
Manganese Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.006
g
g
g
g
g
g
g
g
g
g
Expiration Date
Procedure

Glassware
Autoclave
1. Suspend 62 grams in 750 ml distilled or deionized water or
halogen-free tap water.
3. Add 250 ml of commercial beer (not degassed) and mix well.
Test Procedure

free-flowing.
Solution:
Reaction of 6.2%
Solution at 25C:
pH 6.3 0.2
Cultural Response
Prepare UBA Medium per label directions. Inoculate the
medium and incubate at 30 2C for up to 3 days.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Acetobacter pasteurianus
12879
9338
8081
100-1,000
100-1,000
100-1,000
good
good
good
Results
References
1. Kozulis, J. A., and H. E. Page. 1968. A new universal beer agar
medium for the enumeration of wort and beer microorganisms.
Proc. Am. Soc. Brew. Chem. 1968:52-58.
2. Murphy, D. T., and L. T. Saletan. 1970. Use of microbiological
media in the brewery. Tech. Q. Master Brew. Assoc. Am. 7:182-187.
Packaging
UBA Medium
500 g
0856-17
542
The Difco Manual
Section II
UVM Modified Listeria Enrichment Broth
Bacto UVM Modified Listeria Enrichment Broth
Intended Use
Bacto UVM Modified Listeria Enrichment Broth is used for rapidly
isolating Listeria monocytogenes.

industries. This organism can cause human illness and death,
The first reported food-borne outbreak of listeriosis was in 1985,3 and
since then, microbiological and epidemiological evidence from both
sporadic and epidemic cases of listeriosis has shown that the principal
route of transmission is via the consumption of foodstuffs
contaminated with Listeria monocytogenes.4
and other food processing plants and is ubiquitous in nature, being
at 20C.

Solution:
is light to medium amber with a faint
bluish-green ring at the surface, very
slightly opalescent with a fine
precipitate.
Reaction of 5.2%
Solution at 25C:
pH 7.2 0.2
Cultural Response
Prepare UVM Modified Listeria Enrichment Broth per label
directions. Inoculate tubes and incubate at 35 2C for
18-48 hours.
ORGANISM
Escherichia faecalis
ATCC
INOCULUM
CFU
GROWTH
29212* 1,000-2,000
suppressed at
18-24 hours
Escherichia coli
25922* 1,000-2,000
marked to
complete inhibition
100-1,000
good
The Difco Manual

potentially containing Listeria are: streptococci, especially the
UVM Modified Listeria Enrichment Broth is a modification of the
formula described by Donnelly and Baigent.9 It is used for selective
enrichment of Listeria spp. from food7,11 and clinical specimens.10

Tryptose, Beef Extract and Yeast Extract in UVM Modified Listeria
Enrichment Broth provide nitrogen, vitamins and minerals. Sodium
chloride maintains the osmotic balance of the medium. Phosphate acts
as a buffering agent. Nalidixic acid inhibits growth of gram-negative
organisms. Acriflavine hydrochloride inhibits many gram-positive
bacteria. Esculin is hydrolyzed by Listeria species.
Formula
Formula Per Liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Esculin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Nalidixic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.02
Acriflavine HCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.012
g
g
g
g
g
g
g
g
g
Precautions
closed.
Storage
medium at 2-8C.
543
Universal Preenrichment Broth
Expiration Date
Section II

monocytogenes and Staphylococcus aureus in low-and high-fat,
6. Patel, J. R., C. A. Hwang, L. R. Beuchat, M. P. Doyle, and R. E.
Brackett. 1995. Comparison of oxygen scavengers for their
J. Lovett. 1992. Listeria, p. 637-663. In C. Vanderzant, and
D. F. Splittstoesser (ed.), Compendium of methods for the
9. Donnelly, C. W., and G. J. Baigent. 1986. Method for flow
cytometric detection of Listeria monocytogenes in milk. Appl.
10. Swaminathan, B., J. Rocourt, and J. Bille. 1995. Listeria.
In P. R. Murray, et al. (ed), Manual of clinical microbiology,
No. 57 (revised May 24, 1989). U.S.D.A., F.S.I.S. Microbiology
Division, Bethesda, MD.
12. Hayes, P. S., L. M. Graves, B. Swaminathan, G. W. Ajello, G.
B. Marcolm, R. E. Weaver, R. Ransom, K. Deaver, B. D.
Plikaytis, A. Schuchat, J. D. Wenger, R. W. Pinner, C. V.
Broome, and The Listeria Study Group. 1992. Comparison of
three selective enrichment methods for the isolation of Listeria
monocytogenes from naturally contaminated foods. J. Food. Prot.
55:952-959.
References
Packaging

monocytogenes (n. sp.). J. Path. Bact. 29:407-439.
UVM Modified Listeria

Enrichment Broth
Procedure
Materials Provided

Autoclave
Incubator (30C)
Incubator (35C)

recommended guidelines.7,10,11,12
2. Clinical specimens obtained from nonsterile sites, foods and
enriched for Listeria species before being plated.10
specimen or sample.7,10,11,12
Test Procedure
The USDA method11 involves enrichment of the specimen in UVM
Modified Listeria Enrichment Broth (one part sample in nine parts
broth) at 30C. After incubation, a portion of the enrichment mixture is
added to an enrichment broth or plated onto the final isolation agar.7
For further information when testing food samples or clinical
specimens, refer to appropriate references.7,10,11,12
Results
500 g
2 kg
10 kg
0223-17
0223-07
0223-08
Bacto Universal Preenrichment Broth
Intended Use
Bacto Universal Preenrichment Broth is used for recovering sub-lethally

injured Salmonella and Listeria from food products.
Traditional methods for recovering Salmonella and Listeria from food

products require separate preenrichment media for each microorganism.1, 2
544
The Difco Manual
Section II
Some broth media recommended for preenrichment contain antibiotic

inhibitors3 or have insufficient buffering capacity which hinder recovery
of sublethally injured cells.3, 4 ,5
Bailey and Cox3 formulated Universal Preenrichment Broth to permit
simultaneous resuscitation of sublethally injured Salmonella and Listeria.
The broth medium provides sufficient buffering capacity to prevent
rapid decreases in pH and allows for repair of injured cells that might
be sensitive to low pH values or inhibitory substances.

Universal Preenrichment Broth contains Tryptone and Proteose Peptone
as sources of carbon, nitrogen, vitamins and minerals. Sodium and
Potassium Phosphates buffer the medium. Sodium Chloride, Magnesium
Sulfate and Ferric Ammonium Citrate provide essential ions. Dextrose
is an energy source. Sodium Pyruvate helps stimulate the metabolism
of stressed organisms.
Formula
Formula Per Liter
Precautions
2. MAY BE IRRITATING TO EYES, RESPIRATORY SYSTEM AND
Storage
Expiration Date
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Potassium Phosphate Monobasic . . . . . . . . . . . . . . . . . . . . 15
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Sodium Pyruvate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
g
g
g
g
g
g
g
g
g
Procedure
Materials Provided

Solution:
warming. Solution is light to medium amber, slightly
opalescent to opalescent, may have precipitate.
Prepared Medium:
Light to medium amber, slightly opalescent to opalescent,
may have precipitate.
Reaction of 3.8%
Solution at 25C:
pH 6.3 0.2
Cultural Response
Prepare Universal Preenrichment Broth per label directions. Inoculate and
incubate the tubes at 35 2C for 18-24 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
19115
13076
14028*
10-100
10-100
10-100
good to excellent
good to excellent
good to excellent
Uninoculated
tube
The Difco Manual
Salmonella
typhimurium
ATCC 14028
545
Urea Agar Base, Urea Agar Base Concentrate, Urea Broth & Urea Broth Concentrate
Section II
References
Glassware
Autoclave
Incubator (35C)
1. Vanderzant, C., and D.F. Splittstoesser (ed.). 1992. Compendium

1.
2.
3.
4.

Heat gently to dissolve completely.

Test Procedure
Substitute Universal Preenrichment Broth for preenrichment
media as specified for Salmonella and Listeria 1,2 and follow
recommended procedures.
Results
Gaithersburg, MD.
3. Bailey, J. S., and N. A. Cox. 1992. Universal preenrichment broth
for the simultaneous detection of Salmonella and Listeria in foods.
J. Food Protect. 55:256-259.
4. Bailey, J. S., D. L. Fletcher, and N. A. Cox. 1990. Efficacy of
enrichment media for recovery of heat-injured Listeria
5. Juven, B. J., N. A. Cox, J. S. Bailey, J. E. Thomson, O. W. Charles,
and J. V. Shutze. 1984. Recovery of Salmonella from artificially
contaminated poultry feed in non-selective and selective broth
media. J. Food Prot. 47:299-302.
Packaging
500 g
0235-17
Salmonella and Listeria demonstrate good growth and recovery

following preenrichment in this broth.
Bacto Urea Agar Base . Bacto Urea Agar Base Concentrate

Bacto Urea Broth . Bacto Urea Broth Concentrate

Urea Agar Base
Dehydrated Appearance: Light orange-red to orange-red, homogeneous,
inherently lumpy.
Solution:
deionized water. Solution is orange, clear.
Prepared Agar Medium: Reddish orange, very slightly opalescent.
Reaction of 2.9%
Solution at 25C:
pH 6.8 0.1
Urea Agar Base Concentrate
Solution:
29% solution is reddish orange, clear liquid.
Reaction of 29%
Solution at 25C:
pH 6.75 0.15
Urea Broth
Dehydrated Appearance: Light orange to light pink, homogeneous,
inherently lumpy.
Solution:
deionized water. Solution is
orange-yellow, clear.
Reaction of 3.87%
Solution at 25C:
pH 6.8 0.1
546
Uninoculated
tube
Proteus vulgaris
ATCC 13315
Escherichia coli
ATCC 25922
The Difco Manual
Section II
Intended Use
Bacto Urea Agar Base, when combined with Bacto Agar, is used for
differentiating microorganisms based on urease activity.
Bacto Urea Agar Base Concentrate is a sterile 10X solution of Urea
Agar Base which, when combined with Bacto Agar, is used for preparing
Urea Agar.
Bacto Urea Broth is used for differentiating microorganisms, particularly
Proteus species, based on urease production.
Bacto Urea Broth Concentrate is a sterile 10X solution of Urea Broth
ready to use as recommended. It is suggested for laboratories that
require only small amounts of medium.
Also Known As
Urea Agar Base is also known as Urea Agar Base, Christensen or
Christensens Urea Agar.
Urea Broth is also referred to as Stuarts Urea Broth.

Christensen1 devised a urea agar medium containing peptone and
dextrose that had a reduced buffer content. The medium supported a
more vigorous growth of many of the gram-negative enteric bacilli and
readily permitted observation of urease production.
Ewing2 used Urea Agar as a differential medium in the examination of
many cultures from stool specimens. Urea Agar may be used as a
screening medium (along with Triple Sugar Iron Agar) for the selection
of Salmonella and Shigella cultures for serologic classification.3 Qadri
et al.4 developed a spot test for the rapid detection of urease activity
by applying diluted Urea Agar Base Concentrate to filter paper and
inoculating the paper with a loopful of 24-48 hour culture. Ureasepositive results were obtained within 2 minutes. When combined with
results of other rapid screening methods, Urea Agar is the most
common way to detect the production of urease by yeasts.5 Urea Agar

Base Concentrate has also been used in differentiating mycobacteria
species.6
Urea Broth, prepared according to the formula of Stuart, Van Stratum
and Rustigian7 is a highly buffered urea medium that provides all the
essential growth requirements for Proteus. Stuart et al.7 noted that by
decreasing the amount of buffer in their standard medium to one-tenth
or one-hundredth of the original concentration, the incubation time for
Proteus could be decreased from 12-48 hours to 2-4 hours. When the
amount of buffer is decreased, however, other organisms capable of
urease production give a positive test. Rustigian and Stuart8 used urea
decomposition as a limiting characteristic for the identification of
Proteus strains from other members of the family Enterobacteriaceae.
Ferguson and Hook9 reported that urease production could be used to
differentiate between members of the Proteus and Salmonella groups.
The medium is positive for Proteus, Morganella morganii, Providencia
rettgeri and a few Providencia stuartii strains.
The detection of urease production is an important differential test in
microbiology and is outlined in standard references.10-16

Bacto Peptone provides carbon and nitrogen required for good growth
of a wide variety of organisms. Yeast Extract provides vitamins and
cofactors required for growth and as an additional source of nitrogen
and carbon. Dextrose is included as an energy source. Sodium Chloride
maintains the osmotic balance of the medium. Potassium Phosphate,
Monobasic and Potassium Phosphate, Dibasic provide buffering
capability. Urea provides a source of nitrogen for those organisms
producing urease. This is indicated by a color change of the pH indicator,
Phenol Red, from yellow (pH 6.8) to red to pink-red (pH 8.1).
Urea Broth Concentrate

Appearance:
38.7% solution is reddish-orange,
clear liquid.
Reaction of 38.7%
Solution at 25C:
pH 6.8 0.2
Cultural Response
Urea Agar Base and Urea Agar Base Concentrate
Prepare Urea Agar per label directions. Inoculate and incubate at
Urea Broth and Urea Broth Concentrate
Prepare Urea Broth per label directions. Inoculate and incubate
ATCC
UREASE PRODUCTION
Escherichia coli
25922*
Proteus vulgaris
13315*
negative, no color change

in the medium
positive, red or cerise medium
ORGANISM
Uninoculated
tube
Proteus vulgaris
ATCC 13315
Escherichia coli
ATCC 25922
Urea Broth
The Difco Manual
547
Formula
Urea Agar Base

Formula Per Liter
Glassware
Autoclave
Refrigerator (2-8C)
Incubator (35C)
Bacto Agar
Filter sterilization apparatus
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Urea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.012
g
g
g
g
g
g
Urea Agar Base
Equilibrate this medium to room temperature before opening.

A liquid, 10X concentrate of Urea Agar Base.
The presence of urea in this medium renders it inherently lumpy. This

condition will not adversely affect a properly stored medium.
Urea Broth
Formula Per Liter
Bacto Urea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01
Section II
g
g
g
g
g

A liquid, 10X concentrate of Urea Broth.
Precautions
2. Urea Broth: IRRITANT. IRRITATING TO EYES, RESPIRATORY
SYSTEM AND SKIN. Avoid contact with skin and eyes. Do not
tightly closed.
Storage
1. Dissolve 29 grams in 100 ml distilled or deionized water.

2. Filter sterilize. DO NOT BOIL OR AUTOCLAVE.
3. Suspend 15 grams of Bacto Agar in 900 ml distilled or deionized
water.
6. Cool to 50-55C.
7. Aseptically add 100 ml of the filter sterilized Urea Agar Base to
the cooled Bacto Agar. Mix thoroughly. DO NOT HEAT THE
COMPLETE MEDIUM.
8. Distribute in sterile test tubes. Slant the tubes to have a butt about
2 cm in depth and a slant about 3 cm in length.
If crystals have formed in the concentrate prior to preparing the final
medium, place the tube(s) in a water bath at 40-50C for a few
moments. Agitate to dissolve the crystals.
1. Suspend 1.5 grams of Bacto Agar in 90 ml distilled or deionized
water.
4. Cool to 50-55C. Aseptically add 10 ml of Urea Agar Base
Concentrate.
5. Mix thoroughly; dispense into tubes and slant.
hygroscopic. Keep containers tightly closed. Store the prepared media
also at 2-8C.
Urea Broth
Equilibrate this medium to room temperature before opening.
Store Urea Agar Base Concentrate and Urea Broth Concentrate at 2-8C.
The presence of urea in this medium renders it inherently lumpy. This

condition will not adversely affect a properly stored medium.
Expiration Date
The expiration date applies to the products in their intact container
Procedure
Materials Provided
Urea Agar Base
Urea Broth
548
1. Dissolve 38.7 grams in 1 liter distilled or deionized water. Mix

thoroughly to dissolve completely.
2. Filter sterilize. DO NOT BOIL OR AUTOCLAVE THE MEDIUM.
3. Aseptically distribute 3 ml amounts into sterile test tubes (14 x
125 mm or equivalent).
If crystals have formed in the concentrate prior to preparing the final
medium, place the tube(s) in a water bath at 40-50C for a few
moments. Agitate to dissolve the crystals.
Do no heat Urea Broth above 50C during preparation or sterilization.
The Difco Manual
Section II
1. To prepare 100 ml of final medium, sterilize 90 ml of distilled or

deionized water at 121-124C for 15 minutes.
2. Cool to 50-55C. Aseptically add 10 ml of Urea Broth Concentrate.
Mix thoroughly.
3. Distribute 3 ml amounts into sterile test tubes (14 x 125 mm).

Test Procedure
Urea Agar
1. Use a heavy inoculum of growth from a pure 18-24 hour culture.
Inoculate by streaking back and forth over the entire slant surface.
Do not stab the butt because it serves as a color control.
2. Incubate tubes with loosened caps at 35 2C.
3. Observe reactions after 6 and 24 hours and every day thereafter for
a total of 6 days.1 Longer periods of incubation may be necessary.
Urea Broth
1. Inoculate with a heavy inoculum, using a straight needle or a drop
from an 18-24 hour culture. Shake tube gently to resuspend the
bacteria.
2. Incubate aerobically at 35 2C.
3. Record reactions after 8, 12, 24 and 48 hours of incubation.
Results
Urea Agar
Positive: The production of urease is indicated by an intense red or
cerise color on the slant which may penetrate into the butt.
Negative: No color change of the medium.
Urea Broth
Positive: The production of urease is indicated by an intense red or
cerise color throughout the broth.
Negative: No color change of the broth.

Urea Agar Base
1. The alkaline reaction produced in this medium after prolonged
incubation may not be caused by urease activity. False positive
reactions may occur due to the utilization of peptones (especially
in slant agar by Pseudomonas aeruginosa, for example) or other
proteins which raise the pH due to protein hydrolysis and the
release of excessive amino acid residues. To eliminate possible
protein hydrolysis, perform a control test with the same test
medium without urea.17
2. Do not heat or reheat the medium because urea decomposes
very easily.
3. Urea Agar detects rapid urease activity of only the urease-positive
Proteus species. For results to be valid for the detection of Proteus,
the results must be read within the first 2 to 6 hours after incubation.
Urease-positive Enterobacter, Citrobacter or Klebsiella, in contrast,
hydrolyze urea much more slowly, showing only slight penetration
of the alkaline reaction into the butt of the medium in 6 hours and
requiring 3 to 5 days to change the reaction of the entire butt.
The Difco Manual
Urea Broth
1. To rule out false positives due to protein hydrolysis (as opposed to
urea hydrolysis) that may occur in the medium after prolonged
incubation, perform a control test with the same test medium
without urea.17
2. Do not heat or reheat the medium because urea decomposes
very easily.
3. The high buffering system in this medium masks urease activity in
organisms that are delayed positive. This medium is therefore
recommended for the detection of urease activity in all Proteus
spp., Providencia rettgeri and urease- positive Providencia
stuartii.1 M. morganii slowly hydrolyzes urea and may require
approximately a 36 hour incubation for a strong urease-positive
reaction to occur.1 If in doubt as to a result, compare with an
uninoculated tube or incubate for an additional 24 hours.
4. Variations in the size of the inoculum can affect the time required
to reach positive (alkaline, pH 8.1) results. The accepted standard
inoculum is 0.1 ml.1
References
1. Christensen, W. B. 1946. Urea decomposition as a means of
differentiating Proteus and paracolon cultures from each other and
from Salmonella and Shigella types. J. Bacteriol. 52:461.
2. Ewing, W. H. 1946. An additional Shigella paradysenteriae
serotype. J. Bacteriol. 51:433-445.
3. Ewing, W. H., and D. W. Bruner. 1947. Selection of Salmonella
and Shigella cultures for serologic classification. Am. J. Clin.
Path. 17:1-12.
4. Qadri, S. M. Hussain, S. Zubairi, H. P. Hawley, and E. G.
Ramirez. 1984. Simple spot test for rapid detection of urease
activity. J. Clin. Microbiol. 20(6):1198-1199.
Scotts Diagnostic Microbiology, 9th edition. Mosby-Year Book,
6. Kent, P. T., and G. P. Kubica. 1985. Public health
mycobacteriology - A guide for the level III laboratory. U.S.
Public Health Service, Atlanta, GA.
7. Stuart, C. A., E. Van Stratum, and R. Rustigian. 1945. Further
studies on urease production by Proteus and related organisms.
8. Rustigian, R., and C. A. Stuart. 1941. Decomposition of urea by
Proteus. Proc. Soc. Exptl. Biol. Med. 47:108-112.
9. Ferguson, W. W., and A. E. Hook. 1943. Urease activity
of Proteus and Salmonella organisms. J. Lab. Clin. Med.
28:1715-1719.
American Public Health Assoc., Washington, D.C.
11. Marshall, R. T. (ed.) 1993. Standard methods for the examination
of dairy products, 16th ed. American Public Health Assoc.,
Washington, D.C.
549
VJ Agar
Section II

R. H. Yolken. 1995. Manual of clinical microbiology, 6th ed. ASM
Press, Washington, D.C.
16. Ewing, W. H. 1986. Edwards and Ewings Identification of
New York, NY.
Packaging
Urea Agar Base
100 g
500 g
Urea Agar Base Concentrate 10X

Urea Broth
500 g
Bacto VJ Agar
12 x 10 ml
12 x 10 ml
0283-15
0283-17
0284-61
0272-17
0280-61
To isolate coagulase-positive, mannitol fermenting staphylococci,

Vogel and Johnson3 modified Tellurite-Glycine Agar by Zebovitz
et al.1 by increasing the mannitol content and adding a pH indicator.
Vogel-Johnson (VJ) Agar selects and differentiates the coagulasepositive staphylococci which ferment mannitol and reduce tellurite.2
Intended Use
Bacto VJ Agar is used with Bacto Chapman Tellurite Solution 1% for
isolating coagulase-positive, mannitol-fermenting staphylococci.
VJ Agar is specified as a standard methods medium for cosmetics,4,5

pharmaceutical articles6 and nutritional supplements.6
Also Known As
VJ Agar is also known as Vogel and Johnson Agar, Modification of
Tellurite-Glycine Agar1, and Tellurite-Glycine-Phenol Red Agar Base2

VJ Agar contains Tryptone as a source of carbon, nitrogen, vitamins
and minerals. Yeast Extract supplies B-complex vitamins which
stimulate bacterial growth. Mannitol is the carbohydrate. Chapman

Coagulase-positive staphylococci, primarily Staphylococcus aureus,
are among the microorganisms that can cause spoilage or chemical
changes in cosmetic products.4
Uninoculated
palate
ATCC 25923

Dehydrated Appearance: Pink, homogenous, free-flowing.
Solution:
is red, slightly opalescent with a
white precipitate.
Prepared Medium:
Red, slightly opalescent, may have
slight white precipitate.
Reaction of 6.0%
Solution at 25C:
pH 7.2 0.1
Cultural Response
Prepare VJ Agar per label directions with the addition of
Chapman Tellurite Solution, 1%. Inoculate and incubate the plates
ORGANISM
Escherichia coli
ATCC
TELLURITE
GROWTH
MANNITOL
REDUCTION
FERMENTATION
marked to
(no change) (no change)
complete inhibition
Proteus mirabilis
25933
partial to
+ (black)
(red)
complete inhibition
good
+ (black)
+ (yellow)
Staphylococcus
epidermidis
25922*
12228*
none to fair
(translucent
to black)
ATCC 25923
with Potassium Tellurite
(red)
550
The Difco Manual
Section II
Veal Infusion Agar & Veal Infusion Broth
Tellurite Solution 1% contains Potassium Tellurite which, along with

Lithium Chloride and Glycine, inhibits most microorganisms except
the staphylococci. Phenol Red is the pH indicator. Bacto Agar is the
solidifying agent.
Petri dishes
Autoclave
Incubator (35C)
Formula
Bacto VJ Agar
Formula Per Liter
VJ Agar
4. Add 20 ml Chapman Tellurite Solution 1%. Mix well.
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Mannitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Glycine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.025
g
g
g
g
g
g
g
g
Precautions
TARGET ORGAN(S): Blood, Kidneys, Nerves.
Storage
Store the dehydrated VJ Agar below 30C. The dehydrated medium is
Expiration Date
Procedure
Materials Provided

Test Procedure
Results
Coagulase-positive strains of S. aureus reduce tellurite and form black
colonies on the medium. These strains typically ferment mannitol and
exhibit yellow halos around the black colonies.
References
1. Zebovitz, E., J. B. Evans, and C. F. Niven, Jr. 1955. TelluriteGlycine Agar: a selective plating medium for the quantitative
detection of coagulase-positive staphylococci. J. Bacteriol. 70:686.
3. Vogel, R. A., and M. Johnson. 1960. A modification of the
Tellurite-Glycine medium for use in the identification of
Staphylococcus aureus. Public Health Lab. 18:131.
Microbiological methods for cosmetics, p. 23.01-23.11. In
Gaithersburg, MD.
CTFA microbiology guidelines. The Cosmetic, Toiletry, and
VJ Agar
Packaging
VJ Agar

Glassware
Bacto Veal Infusion Agar

Bacto Veal Infusion Broth
The Difco Manual
100 g
500 g
0562-15
0562-17
Intended Use
Bacto Veal Infusion Agar is used for cultivating fastidious microorganisms
with or without added enrichment.
Bacto Veal Infusion Broth is used for cultivating fastidious microorganisms.
551
Veal Infusion Agar & Veal Infusion Broth
Section II
The nutritive factors of Veal Infusion media permit luxuriant growth of

fastidious microorganisms. Veal Infusion Agar may be used as a base
with blood, ascitic fluid, serum or other enrichments. Veal Infusion
media are specified for use in the examination of food.1,2 Veal Infusion
Agar is specified in AOAC Official Methods of Analysis for culturing
eggs and egg products, and as a maintenance medium for E. coli.3
Veal Infusion Broth is recommended for culturing E. coli in the AOAC
procedure for invasiveness of mammalian cells.3
Infusion from Lean Veal and Proteose Peptone No. 3 provides the
nitrogen, vitamins, carbon and amino acids in Veal Infusion media.
Sodium Chloride maintains the osmotic balance of the formulations.
Bacto Agar is the solidifying agent in Veal Infusion Agar.
Veal Infusion Agar

homogeneous.
Solution:
deionized water on boiling. Light to
Prepared Medium:
Reaction of 4.0%
Solution at 25C:
pH 7.4 0.2
Veal Infusion Broth
homogeneous.
Solution:
deionized water, very light amber,
Prepared Medium:
slightly opalescent with no more
than very slight precipitation.
Reaction of 2.5%
Solution at 25C:
pH 7.4 0.2
Cultural Response
Prepare Veal Infusion Agar per label directions with and
without 5% sterile defibrinated sheep blood. Inoculate medium
with the test organisms. Incubate inoculated plates at 35 2C
for 18-48 hours under approximately 10% CO2.
Prepare Veal Infusion Broth per label directions. Inoculate
tubes with the test organisms. Incubate inoculated tubes at
ATCC
INOCULUM
CFU
GROWTH
13090*
12228*
9895
6305
100-1,000
100-1,000
100-1,000
100-1,000
good
good
good
good
552
g
g
g
g
Streptococcus mitis
Veal Infusion Agar

Formula Per Liter
Lean Veal, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . . 500
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
ORGANISM
Formula
Veal Infusion Broth

Formula Per Liter
Lean Veal, Infusion from . . . . . . . . . . . . . . . . . . . . . . . . . 500 g
Precautions
Storage
Expiration Date
Procedure
Materials Provided
Veal Infusion Agar
Veal Infusion Broth

Glassware
Autoclave
Incubator (35C)
deionized water:
Veal Infusion Agar
40 g/l
Veal Infusion Broth
25 g/l
2. Heat to boiling to dissolve completely (Veal Infusion Agar).
The Difco Manual
Section II
Veillonella Agar


by laboratory policy.
Test Procedure
For a complete discussion on the examination of fastidious microorganisms in food refer to the procedures outlined in the references.1,2,3

Bacto Veillonella Agar
Intended Use
Bacto Veillonella Agar is used with added vancomycin in isolating
Veillonella.

Veillonella Agar is prepared according to the formula described by
Rogosa 1,2 as modified by Rogosa, Fitzgerald, MacKintosh and
Beaman.3 Rogosas2 experiments with oral specimens from humans
Dehydrated Appearance: Beige, free-flowing, homogeneous
with small dark particles.
Solution:
Prepared Medium:
Pink, slightly opalescent without
precipitate.
Reaction of 3.6%
Solution at 25C
pH 7.5 0.2
Cultural Response
Prepare Veillonella Agar per label directions. Using the pour
plate technique, inoculate plates with 1 ml of the diluted test
organisms and 1 ml of the specimen. Pour 20 ml medium per
plate, mix well. Incubate plates at 35 2C anaerobically for
18-48 hours.
Veillonella criceti
Veillonella dispar
Veillonella ratti
ATCC
INOCULUM
CFU
GROWTH
17747
17748
17746
19615*
100-1,000
100-1,000
100-1,000
100-1,000
good
good
good
inhibited
The Difco Manual

2. Vanderzant, C., and D. F Splittstoesser. (ed.). 1992. Compendium
Packaging
Veal Infusion Agar
500 g
0343-17
Veal Infusion Broth
500 g
10 kg
0344-17
0344-08
and rats demonstrated the medium to be highly selective for Veillonella

species. Streptomycin was originally employed as the selective agent.
Later, Rogosa et al.3 demonstrated vancomycin to be superior to
streptomycin in reducing growth of extraneous organisms without
restricting growth of Veillonella.
Veillonella parvula is part of the normal human fecal flora.4 V. parvula,
V. atypica and V. dispar are flora colonizing the oral cavity.4 Veillonella
species have been encountered in patients with bite wound, head,
neck, oral and miscellaneous soft tissue infections.5 Veillonella species
are anaerobic gram negative diplococci and appear as clumps of
diplococci when stained.
ORGANISM
References
Tryptone and Yeast Extract provide the nitrogen, vitamins, amino

acids and carbon in Veillonella Agar. Sodium Thioglycollate is
a reducing agent, and lowers the oxidation-reduction potential of the
medium by removing oxygen to maintain a low pH. Basic Fuchsin
and Vancomycin are the selective agents. Sodium Lactate, 60%
provides nutrients and selective properties. Bacto Agar is the
solidifying agent.
Formula
Veillonella Agar
Formula Per Liter
Bacto Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Sodium Thioglycollate . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.75
Bacto Basic Fuchsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.002
Sodium Lactate, 60% . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
ml
g
Precautions
Storage
553
Violet Red Bile Agar
Section II
Expiration Date

isolate recovered to ensure the organism is an anaerobe.6
Procedure
Materials Provided
Veillonella Agar

Glassware
Autoclave
Incubator (35C)
Tween 80 (optional)
Vancomycin
1.
2.
3.
4.
5.

OPTIONAL: Add 1 gram Tween 80, if desired.
Add 7.5 mcg vancomycin per ml of sterile medium at 50-55C.
Mix thoroughly.

properly collected and transported to the laboratory.6 Obtain and process
specimens according to the techniques and procedures established by
institutional policy.
Test Procedure
1. Rogosa1,2,3 recommends that one ml of the diluted specimen be
added to a sterile Petri dish.
2. Pour approximately 20 ml of medium to the Petri dish, and rotate
to mix well with the inoculum.
3. Incubate plates anaerobically at 35 2C for 40-48 hours; 72 hours
if necessary.
For a complete discussion on Veillonella species from clinical specimens,
refer to the appropriate procedures outlined in the references.4,6,7 For
the examination of anaerobic bacteria in food refer to standard methods.8, 9, 10
Bacto Violet Red Bile Agar
Intended Use
Bacto Violet Red Bile Agar is used for enumerating coliform organisms
in dairy products.
Also Known As
Violet Red Bile Lactose Agar
554
References
1. Rogosa, M. 1955. Nutrition of the Vellonella. J. Dent. Res.
34:721-722.
2. Rogosa, M. 1956. A selective medium for the isolation and
enumeration of the Veillonella from the oral cavity. J. Bacteriol.
72:533-536.
3. Rogosa, M., R. J. Fitzgerald, M. E. MacKintosh, and
A. J. Beaman. 1958. Improved medium for selective isolation of
Veillonella. J. Bacteriol. 76:455-456.
5. Summanen, P., E. J. Baron, D. M. Citron, C. Strong,
H. M. Wexler, and S. M. Finegold. 1993. Wadsworth anaerobic
bacteriology manual, 5th ed. Star Publishing Co., Belmont, CA.
10. Marshall, R. T. (ed.). 1992. Standard methods for the microbiological examination of dairy products, 16th ed. American Public Health
Association, Washington. D.C.
Packaging
Veillonella Agar
500 g
0917-17

The coliform group of bacteria includes aerobic and facultatively
anaerobic gram-negative non-sporeforming bacilli that ferment lactose
and form acid and gas at 35C within 48 hours. Members of the
Enterobacteriaceae comprise the majority of the group but other
lactose fermenting organisms may also be included.
Procedures to detect, enumerate and presumptively identify coliforms
are used in testing foods and dairy products.1,2,3 One method for
performing the presumptive test for coliforms uses Violet Red Bile
Agar (VRBA). If typical coliform colonies appear, they are tested
further to confirm their identification as coliforms.
The Difco Manual
Section II

Violet Red Bile Agar (VRBA) contains Bacto Peptone to provide
carbon and nitrogen sources for general growth requirements.
Yeast Extract supplies B-complex vitamins which stimulate
bacterial growth. Bile Salts No. 3 and Crystal Violet inhibit most
gram-positive microorganisms. Lactose is the carbohydrate source
and Neutral Red is the pH indicator. Bacto Agar is the solidifying agent.
Formula
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.03
g
g
g
g
g
g
g
g

Procedure
Materials Provided

Flask with closure
Autoclave
Incubator (35C or 32C for dairy products)
2. Heat to boiling to dissolve completely. Do not boil for more than
2 minutes. DO NOT AUTOCLAVE.

Test Procedure
Precautions
Storage
Expiration Date
Presumptive test for coliforms using solid medium:

1. Transfer a 1 ml aliquot of test sample to a Petri dish.
2. Add 10 ml of Violet Red Bile Agar (at 48C) and swirl to mix.
3. Allow medium to solidify before incubating at 35C for 18 to
24 hours; use 32C for dairy products.
4. Examine for purple-red colonies, 0.5 mm in diameter (or larger),
surrounded by a zone of precipitated bile acids.
5. Continue with confirmatory testing of typical coliform colonies.1.2,3
Uninoculated
plate
ATCC 13048

Dehydrated Appearance: Reddish-beige, homogeneous, free-flowing.
Solution:
4.15% solution, reddish-purple, very
Prepared plates:
Reddish-purple, slightly opalescent.
Reaction of 4.15%
solution at 25C:
7.4 0.2
Cultural Response
Prepare Violet Red Bile Agar per label directions. Inoculate
and incubate the plates at 32 1C for 24 2 hours.
ATCC
INOCULUM
CFU
GROWTH
COLONY COLOR
Enterobacter
aerogenes
13048*
30-300
good
Escherichia
coli
25922*
30-300
good
red, may have

slight red precipitate
around colonies
deep red
with red precipitate
around colonies
ORGANISM
Staphylococcus 25923* 1,000-2,000

markedly to
aureus
Escherichia coli
ATCC 25922
The Difco Manual
555
Violet Red Bile Agar with MUG
Section II
Results
References
Lactose fermenters: Purple-red colonies, with or without a zone of

precipitate around the colonies
Lactose non-fermenters: Colorless to transparent colonies
Gram-positive cocci: Colorless, pinpoint colonies
1. Christen, G. L., P. M. Davidson, J. S. McAllister, and

L. A. Roth. 1993. Coliform and other indicator bacteria, p. 247-252.
In Marshall, R. T. (ed.). Standard methods for the microbiological
Vanderzant, C., and D. F. Splittstoesser (ed.). Compendium of
L. A. Chandler. 1995. Escherichia coli and the coliform bacteria,

1. Violet Red Bile Agar may not be completely inhibitory to grampositive organisms. Perform Gram stain and biochemical tests as
necessary to identify isolates.
2. The medium will grow gram-negative bacilli other than members
of the Enterobacteriaceae. Perform biochemical tests to identify
isolates to genus and species.
3. Boiling the medium for longer than 2 minutes can decrease the
ability to support growth.
4. Plates of Violet Red Bile Agar should not be incubated longer than
24 hours because microorganisms that are only partially
inhibited may grow after extended incubation.
5. For optimum performance, prepare and use the medium within
24 hours.
Packaging
100 g
500 g
2 kg
0012-15
0012-17
0012-07
Bacto Violet Red Bile Agar with MUG
Intended Use
Formula
Bacto Violet Red Bile Agar with MUG is used for enumerating
Escherichia coli and total coliform bacteria in food and dairy products.

Formula Per Liter
Also Known As
VRBA with MUG

Violet Red Bile Agar is specified in Standard Methods procedures
to enumerate coliforms in food and dairy products.1,2,3 In 1982, Feng
and Hartman developed a rapid fluorogenic assay for Escherichia coli
by incorporating 4-methylumbelliferyl--D-glucuronide (MUG) into
Lauryl Tryptose Broth.4 Incorporating MUG into Violet Red Bile Agar
permits the detection of E. coli among the coliform colonies.2, 3
Standard Methods procedures specify Violet Red Bile Agar with MUG
for detecting E. coli in food and dairy products by fluorescence.1,2,3

Violet Red Bile Agar contains Bacto Peptone as a source of carbon,
vitamins which stimulate bacterial growth. Bile Salts No. 3 and
Crystal Violet inhibit gram-positive bacteria. Lactose is a carbohydrate
source. Neutral Red is a pH indicator. MUG (4-methylumbelliferyl-D-glucuronide) is a substrate used for detecting glucuronidase
activity. Bacto Agar is a solidifying agent.
E. coli produces the enzyme glucuronidase which hydrolyzes MUG
to yield a fluorogenic compound detectable with long-wave UV
light (366 nm). Typical strains of E. coli (red colonies surrounded by
a bile precipitate) exhibit blue fluorescence. Non-E. coli coliforms
may produce red colonies with zones of precipitated bile but they
are MUG negative.
556

Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.03
MUG (4-methylumbelliferyl--D-glucuronide) . . . . . . . . 0.1
g
g
g
g
g
g
g
g
g
Precautions
Storage
Expiration Date
Procedure
Materials Provided
The Difco Manual
Section II
under long-wave fluorescent light, MUG-positive colonies are

surrounded by a bluish fluorescent halo. MUG-negative colonies lack
the fluorescent halo.
Glassware
Petri dishes
Autoclave
Incubator (32C)
Waterbath (45C)
E. coli colonies are red surrounded by a zone of precipitated bile and

fluoresce blue under long-wave UV light.
Salmonella and Shigella strains that produce glucuronidase may be
encountered infrequently but these are generally lactose negative and
appear as colorless colonies which may fluoresce.
2. Heat to boiling and boil no more than 2 minutes to dissolve
completely. DO NOT AUTOCLAVE.
3. Cool to 45C.

1. Glucuronidase-negative strains of E. coli have been encountered.5,6,7
Similarly, glucuronidase-positive strains of E. coli that do not
fluoresce have been reported.8
2. Strains of Salmonella and Shigella that produce glucuronidase may
infrequently be encountered.9 These strains must be distinguished
from E. coli on the basis of other parameters, e. g., gas production,
lactose fermentation or growth at 44.5C.

Collect specimens in sterile containers or with sterile swabs and transport immediately to the laboratory in accordance with recommended
guidelines.1,2,3
Test Procedure
References
1. Process each specimen as appropriate for that specimen.1,2,3

2. Incubate plates at 35C for 22-26 hours.
3. Examine plates for growth and fluorescence.
1. Christen, G. L., P. M. Davidson, J. S. McAllister, and L. A.

Roth. 1993. Coliform and other indicator bacteria, p. 247-269. In
examination of dairy products, 16th ed. American Public
Results
Coliform organisms form purplish-red colonies that are generally
surrounded by a reddish zone of precipitated bile. When examined
Uninoculated
plate
Escherichia coli
ATCC 25922

Dehydrated Appearance: Reddish beige, free-flowing, homogeneous.
Solution:
is reddish purple, slightly opalescent,
without significant precipitate. Very
slight surface material may be present.
Prepared Medium:
Reddish purple, very slightly to slightly
Reaction of 4.16%
Solution at 25C:
pH 7.4 0.2
Cultural Response
Prepare Violet Red Bile Agar with MUG per label directions.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
COLONY COLOR
APPEARANCE
Escherichia
25922* 30-300
good
deep red,
fluorescence
coli
with a bile ppt.
Enterobacter
13048* 30-300
good
pink, may
no fluorescence
aerogenes
have a bile ppt.
Staphylococcus 25923* 1,000
marked to
aureus
complete inhibition
The Difco Manual
557
Violet Red Bile Glucose Agar
Section II

Coliforms-Escherichia coli and its toxins, p. 325-369. In
4 Feng, P. C. S., and P. A. Hartman. 1982. Fluorogenic assays for
Microbiol. 43:1320-1329.
5. Chang, G. W., J. Brill, and R. Lum. 1989. Proportion of
-D-glucuronidase- negative Escherichia coli in human fecal
samples. Appl. Environ. Microbiol. 55:335-339.
6. Hansen, W., and E. Yourassowsky. 1984. Detection of

-glucuronidase in lactose fermenting members of the family
Enterobacteriaceae and its presence in bacterial urine cultures.
J. Clinical Microbiol. 20:1177-1179.
7. Kilian, M., and P. Bulow. 1976. Rapid diagnosis of
Enterobacteriaceae. Acta Pathol. Microbiol. Scand. Sect. B
84:245-251.
8. Mates, A., and M. Shaffer. 1989. Membrane filtration
differentiation of E. coli from coliforms in the examination of
water. J. Appl. Bacteriology 67:343-346.
9. Damare, J. M., D. F. Campbell, and R. W. Johnston. 1985.
Simplified direct plating method for enhanced recovery of
Escherichia coli in food. Journal of Food Science 50:1736-1746.
Packaging
500 g
0029-17
Bacto Violet Red Bile Glucose Agar
Intended Use
Bacto Violet Red Bile Glucose Agar is used for detecting and enumerating
Enterobacteriaceae in foods and dairy products.
The Enterobacteriaceae group includes lactose-fermenting coliform

bacteria, nonlactose-fermenting strains of E. coli, and nonlactosefermenting species such as Salmonella and Shigella. When examining
Also Known As
Violet Red Bile Glucose Agar is also known as VRBGA.
Uninoculated
plate
Escherichia coli
ATCC 25922

Dehydrated Appearance: Pink-beige, free-flowing, homogeneous.
Solution:
reddish-purple, very slightly to slightly
opalescent, without significant precipitate.
Prepared Medium:
Reddish-purple, very slightly to
Reaction of 4.15%
Solution at 25C:
pH 7.4 0.2
Cultural Response
Prepare Violet Red Bile Glucose Agar per label directions.
Using the pour plate method, inoculate and incubate at 35 2C
for 18-24 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
APPEARANCE
Acinetobacter baumanii
Escherichia coli
19606 1,000-2,000 none to poor
25922* 30-300
good
red colonies with
red-purple halos
Salmonella typhimurium 14028* 30-300
good
red colonies with
red-purple halos
Staphylococcus aureus 25923* 1,000-2,000 none to poor
Salmonella typhimurium ATCC 14028
558
The Difco Manual
Section II
some foods, it is desirable to detect Enterobacteriaceae rather than the

coliform bacteria.1,2
Enterobacteriaceae are glucose-fermenting bacteria. Mossel et al 3
modified Violet Red Bile Agar, suggested by MacConkey4, that contains
lactose by adding glucose to improve the recovery of Enterobacteriaceae.
Later work by Mossel et al.5,6 demonstrated that lactose could be omitted,
resulting in the formulation known as Violet Red Bile Glucose Agar.

Violet Red Bile Glucose Agar contains Bacto Peptone as a source of
vitamins which stimulate bacterial growth. Glucose is a carbohydrate.
Bile Salts No. 3 and Crystal Violet inhibit gram positive bacteria. Glucose
fermenters produce red colonies with red-purple halos in the presence
of Neutral Red, a pH indicator. Bacto Agar is a solidifying agent.

Glassware
Petri dishes
Autoclave
Incubator (35C)
2. Heat to boiling and boil for no more than 2 minutes to dissolve
completely.

Formula
Test Procedure

Formula Per Liter
This medium can be used in spread or pour plate procedures, with or

without an overlay. In addition, this medium can be used as an overlayer
for spread plates to both prevent swarming colonies and to provide
semi-anaerobic conditions that suppress the growth of nonfermentative
gram negative organisms. Stab inoculation procedures can also be used
with this medium.

Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.03
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g
g
Precautions
2. IRRITATING TO EYES, RESPIRATORY SYSTEM AND SKIN.
protective clothing. Keep container tightly closed.
After contact with skin, wash immediately with plenty of water. If
inhaled, remove to fresh air. If not breathing, give artificial respiration. If breathing is difficult, give oxygen. Seek medical advice. If
swallowed seek medical advice immediately and show this
container or label.
Results
Enterobacteriaceae ferment glucose, produce acid products and form
red to dark purple colonies surrounded by red-purple halos.

1. When used in the pour plate procedure, the medium should be
freshly prepared, tempered to 47C, and used within 3 hours.
References
1. Draft Standard Methods for Microbiological Examination of Meat

Products. Part 3: Detection and enumeration of Enterobacteriaceae.
BS5393: Part 3 1977 ISO/DIS 5552.
2. Mossel, D. A. A. 1985. Media for Enterobacteriaceae. Int. J. Food
Microbiol. 2:27.
3. Mossel, D. A. A., W. H. J. Mengerink, and H. H. Scholts. 1962.
Use of a modified MacConkey agar medium for the selective
growth and enumeration of Enterobacteriaceae. J. Bacteriol.
84:381.
4. MacConkey, A. 1905. Lactose-fermenting bacteria in faeces.
J. Hyg. 5:333-378.
5. Mossel, D. A. A., I. Eelderink, M. Koopmans, and F. van
Rossem. 1978. Lab Practice 27:1049-1050.
6. Mossel, D. A. A., I. Eelderink, M. Koopmans, and F. van
Rossem. 1979. Influence of carbon source, bile salts and incubation temperature on recovery of Enterobacteriaceae from foods
using macconkey-type agars. J. Food Protect. 42:470-475.
Procedure
Packaging
Materials Provided
Storage
Expiration Date
500 g
1866-17
The Difco Manual
559
Vitamin B12 Assay Medium
Section II
Bacto Vitamin B12 Assay Medium
Intended Use
Formula
Bacto Vitamin B12 Assay Medium is used for determining vitamin B12

Formula Per Liter

1. Maintenance Media: For carrying the stock culture to preserve
purpose.
Vitamin B12 Assay Medium is prepared according to the formula
described by Capp, Hobbs and Fox.1 This medium is used in the
microbiological assay of vitamin B12 using Lactobacillus delbrueckii
subsp. lactis ATCC 4797 or 7830 (Lactobacillus leichmannii).

Vitamin B12 Assay Medium is a vitamin B12-free medium containing
all other nutrients and vitamins essential for the cultivation of
L. delbrueckii subsp. lactis ATCC 4797 or 7830. To obtain a standard
curve, USP Cyanocobalamin Reference is added in specified increasing
concentrations providing a growth response that can be measured
titrimetrically or turbidimetrically.

Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Sodium Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
L-Cystine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
DL-Tryptophane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2
Adenine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Guanine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Uracil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Xanthine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Folic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Sorbitan Monooleate Complex . . . . . . . . . . . . . . . . . . . . . . 2
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Ferrous Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
g
g
g
g
g
mg
mg
mg
mg
mg
mg
mg
g
mg
g
g
g
g
g
g
g
mg
mg
mg
Precautions

other chemicals must be used. Glassware must be heated to
250C for at least 1 hour to burn off any organic residues that
might be present.
Dehydrated Appearance: Very light to light beige,

homogeneous, with a tendency
to clump.
Solution:
3.8% solution (single-strength),
7.6% (double strength), soluble in
distilled or deionized water on
boiling 2-3 minutes. Solution
(single strength) is light amber,
Prepared Medium:
Reaction of 3.8%
Solution at 25C:
pH 6.3 0.2
Cultural Response
Prepare Vitamin B12 Assay Medium per label directions.
Dispense into tubes with a titration from 0 to 0.25 ng of
USP Cyanocobalamin Reference Standard. Inoculate with
L. delbrueckii subsp. lactis ATCC 4797 and incubate at
35-37C for 18-24 hours. Turbidimetric measurements are
taken using a spectrophotometer. The curve is then constructed
from the values obtained.
560
Storage
Expiration Date
The Difco Manual
Section II
Procedure
Materials Provided

Glassware
Autoclave
Stock culture of Lactobacillus delbrueckii subsp. lactis
ATCC 4797 or 7830
Centrifuge
0.1 N NaOH
Cyanocobalamin USP
Spectrophotometer or nephalometer
Lactobacilli Agar AOAC or B12 Culture Agar USP
Lactobacilli Broth AOAC or B12 Inoculum Broth USP
1.
2.
3.
4.
5.
6.

Boil 2-3 minutes.

Test Procedure
Stock cultures of the test organism, L. delbrueckii subsp. lactis
ATCC 4797 or 7830, are prepared by stab inoculation of Lactobacilli
Agar AOAC or B12 Culture Agar USP. Following incubation at 37C
for 24-48 hours, the tubes are stored in the refrigerator. Transfers are
made at 2 week intervals.
Inoculum for the assay is prepared by subculturing a stock of
L. delbrueckii subsp. lactis ATCC 4797 or 7830 into a tube containing
10 ml of Lactobacilli Broth AOAC or B12 Inoculum Broth USP. After
incubation at 35-37C for 18-24 hours, the cells are centrifuged under
aseptic conditions and the supernatant liquid decanted. The cells are
washed by resuspending in 10 ml of sterile 0.85% saline solution and
centrifuging. The washing is repeated for a total of 3 times. Finally the
cells are resuspended in 10 ml of sterile 0.85% saline. The cell suspension
is then diluted 1:100 with sterile 0.85% saline. One drop is used to
inoculate each assay tube.
run. Conditions of autoclaving and temperature of incubation that
influence the standard curve readings cannot always be duplicated.
The concentrations required for the preparation of the standard curve
are obtained by adding sufficient 25% ethanol to an accurately weighed
amount of USP Cyanocobalamin Reference Standard (resulting in a
solution containing 1.0 g of cyanocobalamin per ml). This stock
solution is stored in the refrigerator and should be used within 60 days.
The Difco Manual
In the preparation of the standard curve, further dilutions of this stock

solution (1 /ml) are made as follows:
A. Add 1 ml stock solution to 99 ml distilled water (1 ml = 10 ng).
B. Add 1 ml of the solution from step A to 199 ml distilled water
(1 ml = 0.05 ng).
An acceptable standard curve can be obtained by using the USP
Cyanocobalamin Reference Standard at levels of 0.0, 0.025, 0.05, 0.1,
0.15, 0.2 and 0.25 ng per assay tube. This is accomplished by adding 0,
0.5, 1, 2, 3, 4 and 5 ml of the 0.05 ng/ml solution per assay tube and
sufficient distilled or deionized water to make 10 ml volume per tube.
A standard concentration is used which, after incubation, gives a
transmittance value at the 5 ml level of not less than that which
corresponds to a dry cell weight of 1.25 mg (see USP2 for method of
calibration of a spectrophotometer and determination of dry cell
weight). For the titrimetric method, a standard concentration should be
used which, after incubation, will give a titration at the 5 ml level of
8-12 ml 0.1N sodium hydroxide.
Inoculate and incubate at 35-37C for 18-24 hours. For turbidimetric
determinations, place tubes in a refrigerator at 2-8C for 15-20 minutes
to stop growth. The growth can be measured by a nephelometric
method. Titrimetric determinations of growth are made after incubation
at 37C for 72 hours. The curve is then constructed from the
values obtained.
Results
or cup.

response.
References
1. Capps, Hobbs, and Fox. 1949. J. Biol. Chem. 178:517.
Packaging
100 g
0360-15*
*Store at 2-8C
561
WL Nutrient Medium, WL Nutrient Broth & WL Differential Medium
Section II
Bacto WL Nutrient Medium . Bacto WL Nutrient Broth

Bacto WL Differential Medium
Intended Use
Bacto WL Nutrient Medium and Bacto WL Nutrient Broth are used for
cultivating yeasts, molds and bacteria encountered in brewing and
industrial fermentation processes.
Bacto WL Differential Medium is used for isolating bacteria
encountered in brewing and industrial fermentation processes.
Also Known As
WL Nutrient Medium is also referred to as Wallerstein Laboratory
Medium.
WL Nutrient Broth is also referred to as Wallerstein Laboratory
Nutrient Broth.
WL Differential Medium is also referred to as Wallerstein Laboratory
Differential Medium.

WL Nutrient Media were developed by Green and Gray1,2 in their study
of various fermentation processes. An exhaustive study examining the
methods of fermentation control procedures in worts, beers, liquid
yeasts and similar fermentation products led to the development of
WL Nutrient Media.

WL Nutrient Medium and WL Differential Medium
Dehydrated
Media Appearance: Light beige with a greenish tint,
Solution:
blue to greenish blue, slightly opalescent
Prepared Media:
Blue to greenish blue, slightly
precipitate.
Reaction of 8.0%
Solution at 25 C: pH 5.5 0.2
WL Nutrient Broth
Dehydrated
Media Appearance: Light beige with a greenish tint,
Solution:
deionized water. Solution is blue, clear
Prepared Medium: Blue, clear without precipitation.
Reaction of 6.0%
Solution at 25C:
pH 5.5 0.2
562
At a pH of 5.5, counts of viable bakers yeast may be made on the WL

Nutrient Medium. By adjusting the pH to 6.5, the medium is suitable
for obtaining counts of bakers and distillers yeast. The medium can
support the growth of bacteria, but unless the number of yeast cells is
small the bacteria may not be detected. Due to this limitation, Green
and Gray developed WL Differential Medium that inhibits the growth
of yeasts without inhibiting the growth of bacteria present in beers.
WL Nutrient Agar and WL Differential Medium are used simultaneously as a set or three plates. One plate is prepared from WL
Nutrient Agar and two plates from WL Differential Medium.3 The WL
Nutrient Agar plate is incubated aerobically to obtain a total count
of mainly yeast colonies. A differential agar plate is incubated
aerobically for growth of acetic acid bacteria, Flavobacterium, Proteus
and thermophilic bacteria. Another differential agar plate is incubated
anaerobically for growth of lactic acid bacteria and Pediococcus.

Casitone provides nitrogen, amino acids, and carbon. Dextrose is the
source of carbohydrate. Monopotassium Phosphate buffers the media.
Potassium Chloride, Calcium Chloride and Ferric Chloride are
essential ions and help to maintain osmotic balance. Magnesium
Sulfate and Manganese Sulfate are sources of divalent cations. Brom
Cresol Green is a pH indicator.
Agar is the solidifying agent in WL Nutrient Medium and WL
Differential Medium.
Actidione (cycloheximide) inhibits yeasts and molds in WL Differential
Medium.
Formula
WL Nutrient Medium
Formula Per Liter
Bacto Casitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Ferric Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0025
Manganese Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0025
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Brom Cresol Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.022
g
g
g
g
g
g
g
g
g
g
g

WL Nutrient Broth
Formula Per Liter
Bacto Casitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
g
g
g
g
g
g
g
The Difco Manual
Section II
WL Nutrient Medium, WL Nutrient Broth & WL Differential Medium
Ferric Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0025 g

Manganese Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0025 g
Brom Cresol Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.022 g
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Brom Cresol Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.022 g
Actidione . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.004 g
WL Differential Medium
Formula Per Liter
Precautions

Bacto Casitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Ferric Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0025
Manganese Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.0025
g
g
g
g
g
g
g
g
g

Storage
Uninoculated
plate
Escherichia coli
ATCC 25922
Cultural Response
WL Nutrient Medium
Prepare WL Nutrient Medium per label directions. Inoculate and
incubate for 40-48 hours at 35 2C for bacteria and at 30 2C
for yeasts.
ORGANISM
Escherichia coli
ATCC
INOCULUM
CFU
GROWTH
25922*
9338
9763
100-1,000
100-1,000
100-1,000
fair to good
fair to good
good
ATCC 9338
WL Nutrient Broth
Prepare WL Nutrient Broth per label directions. Inoculate and
incubate for 40-48 hours at 35 2C for bacteria and at 30 2C
for yeasts.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
ACID
Escherichia coli
25922* 100-1,000 fair to good +
Lactobacillus fermentum 9338 100-1,000 fair to good +
Saccharomyces cerevisiae 9763 100-1,000
good
+
Acid + = positive, yellow
Escherichia coli
ATCC 25922
Lactobacillus
fermentum
ATCC 9338
Saccharomyces
cerevisiae
ATCC 9763
The Difco Manual
+
sl. +
+
Acid = negative, no color change
Prepare WL Differential Medium per label directions. Inoculate
and incubate for 40-48 hours at 35 2C for bacteria and at
30 2C for yeasts.
ORGANISM
GAS
Escherichia coli
ATCC
25922*
9338
9763
INOCULUM
CFU
GROWTH
100-1,000
500-1,000
1,000-2,000
good
good
inhibited
563
XL Agar Base & XLD Agar
Section II
Expiration Date
OPTIONAL: Add fermentation tubes before sterilizing to assess

gas production.
Procedure
Materials Provided
WL Nutrient Medium
WL Nutrient Broth
Test Procedure
Results
Glassware
Autoclave
Petri dishes
Tubes with closures
Fermentation tubes
References
WL Nutrient Medium and WL Differential Medium
OPTIONAL: To adjust the pH to 6.5, add the amount of 1% sodium
carbonate solution specified on the product label to the rehydration
water before dissolving the medium.
WL Nutrient Broth
1. Green, S. R., and P. P. Gray. 1950. Paper read at American Society

of Brewing Chemists Meeting. Wallerstein Lab. Commun. 12:43.
2. Green, S. R., and P. P. Gray. 1950. A differential procedure
applicable to bacteriological investigation in brewing. Wallerstein
Lab. Commun. 13:357.
Packaging
WL Nutrient Medium
WL Nutrient Broth
500
10
500
500
g
kg
g
g
0424-17
0424-08
0471-17
0425-17
Bacto XL Agar Base . Bacto XLD Agar
Uninoculated
plate
ATCC 14028

XL Agar Base
Solution:
is red, very slightly opalescent.
Prepared Plates:
Red, slightly opalescent.
Reaction of 4.7%
Solution at 25C:
pH 7.4 0.2
XLD Agar
Solution:
Solution is red, very slightly opalescent.
Prepared Plates:
Red, slightly opalescent.
Reaction of 5.7%
Solution at 25C:
pH 7.4 0.2
564
Shigella flexneri ATCC 12022
The Difco Manual
Section II
Intended Use
Bacto XL Agar Base is used with or without selective agents for
isolating, differentiating and enumerating enteric bacteria.
Bacto XLD Agar is used for isolating and differentiating gram-negative
enteric bacilli, especially Shigella and Providencia.
Also Known As
XL Agar with added brilliant green is referred to as XLBG Agar.

XL (Xylose Lysine) Agar Base was developed by Taylor1 for isolating
and differentiating gram-negative enteric bacilli. The medium is
nonselective, allowing growth of most enteric bacteria. XL Agar is
recommended for performing counts of enteric organisms.
XL Agar Base can be supplemented with sodium thiosulfate and ferric
ammonium citrate and, further, with sodium desoxycholate (2.5 grams
per liter) to make XLD Agar. XL Agar Base can be made selective for
Salmonella by adding brilliant green (1.25 ml of a 1% aqueous solution
per liter) prior to autoclaving. The resulting XLBG Agar inhibits
coliforms and Shigella and is recommended for isolating Salmonella
following selenite or tetrathionate enrichment in food analysis.1
XLD Agar was developed principally for isolating Shigella and
Providencia. It has been shown to be more effective than other enteric
differential media.2,3,4

Yeast Extract provides sources of nitrogen and carbon, as well as
vitamins and cofactors required for growth. Xylose, Lactose, and
Sucrose (Saccharose) provide sources of fermentable carbohydrate.

Xylose is fermented by most enteric organisms except Shigella and
Providencia. Lysine is added to differentiate Salmonella. As xylose is
exhausted, Salmonella organisms decarboxylate lysine causing a
reversion to alkaline conditions. Alkaline reversion by other
lysine-positive organisms is prevented by excess acid production from
fermentation of lactose and sucrose.
Sodium Thiosulfate and Ferric Ammonium Citrate allow visualization
of hydrogen sulfide production under alkaline conditions. Acidic
conditions inhibit the reaction. Phenol Red is an indicator. Sodium
Chloride maintains osmotic balance in the medium. Bacto Agar is a
solidifying agent.
Sodium Desoxycholate in XLD Agar inhibits growth of gram-positive
organisms.
Formula
XL Agar Base
Formula Per Liter
L-Lysine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Xylose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.75
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5
Bacto Saccharose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.08
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
g
g
g
g
g
g
g
g

Uninoculated
plate
Providencia alcalifaciens
ATCC 9886
Cultural Response
XL Agar Base
Prepare XL Agar Base per label directions. Inoculate the medium
ATCC
ORGANISM
Escherichia coli
Salmonella
typhimurium
Shigella flexneri
INOCULUM
CFU
GROWTH
APPEARANCE
29212* 100-1,000 poor to fair yellow

25922* 100-1,000
good
yellow
14028* 100-1,000
good red w/black
centers
12022* 100-1,000
good
red
XLD Agar
Prepare XLD Agar per label directions. Inoculate the medium
ATCC
INOCULUM
CFU
Enterococcus
faecalis
Escherichia coli
29212*
1,000-2,000
Salmonella
typhimurium
Shigella flexneri
14028*
ORGANISM
25922*
12022*
GROWTH
APPEARANCE
partial
inhibition
1,000-2,000
partial yellow, may have
inhibition a bile precipitate
100-1,000
good
red w/black
centers
100-1,000
good
red
ATCC 14028
Shigella flexneri
ATCC 12022
The organisms listed are the minimum used for performance testing.
*These cultures are available as Bactrol Disks and are to be used as directed in Bactrol Disks Technical Information.
The Difco Manual
565
Section II
XLD Agar
Formula Per Liter
L-Lysine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Xylose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.75
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.08
g
g
g
g
g
g
g
g
g
g
g
Precautions
2. XLD Agar:
Storage
Store the dehydrated media below 30C. The dehydrated medium is
Expiration Date
Procedure
Materials Provided
XL Agar Base
XLD Agar

Glassware
Autoclave
Incubator
Petri dishes
34% sodium thiosulfate, sterile (XL Agar)
4% ferric ammonium citrate, sterile (XL Agar)
1% brilliant green (XLBG)
XL Agar Base
(OPTIONAL: To prepare XLBG Agar, add 1.25 ml aqueous
1% solution of brilliant green.)
566
2.
3.
3.
4.

Cool to 55-60C.
Aseptically add 20 ml sterile aqueous 34% solution of sodium
thiosulfate and 4% solution of ferric ammonium citrate.
Mix thoroughly.
XLD Agar
DO NOT AUTOCLAVE.
3. Cool to 55-60C. Dispense as desired.

XLD Agar is listed in several procedures as a plating medium for the
testing of food samples.8,9,10,11 Refer to the appropriate references
regarding specimen preparation.
Test Procedure
Feces or rectal swabs may be plated directly; selective enrichment
broths, such as Selenite Broth or Tetrathionate Broth, may be used prior
to streaking.5,6,7
Results
Degradation of xylose, lactose and sucrose generates acid products,
causing a color change in the medium from red to yellow.
Hydrogen sulfide production under alkaline conditions causes
colonies to develop black centers. This reaction is inhibited by the acid
conditions that accompany carbohydrate fermentation.
Lysine decarboxylation in the absence of lactose and sucrose
fermentation causes reversion to an alkaline condition and the color of
the medium changes back to red.

1. Red, false-positive colonies may occur with some Proteus and
Pseudomonas species.
2. Incubation in excess of 48 hours may lead to false-positive results.
3. S. paratyphi A, S. choleraesuis, S. pullorum and S. gallinarum
may form red colonies without black centers, thus resembling
Shigella species.
4. Some Proteus strains will give black-centered colonies on XLD
Agar.
References
1. Taylor, W. I. 1965. Isolation of shigellae. I. Xylose lysine agars;
new media for isolation of enteric pathogens. Am. J. Clin. Pathol.
44(4):471-475.
2. Rollender, W., O. Beckford, R. D. Belsky, and B. Kostroff. 1969.
Comparison of xylose lysine deoxycholate agar and MacConkey
agar for the isolation of Salmonella and Shigella from clinical
specimens. Tech. Bull. Reg. Med. Tech. 39(1):8-10.
3. Pollock, H. M., and B. J. Dahlgren. 1974. Clinical evaluation of
enteric media in the primary isolation of Salmonella and Shigella.
Appl. Microbiol. 27(1):197-201.
The Difco Manual
Section II
XLT4 Agar Base & XLT4 Agar Supplement
4. Bhat, P., and D. Rajan. 1975. Comparative evaluation of

desoxycholate citrate medium and xylose lysine desoxycholate
medium in the isolation of shigellae. Am. J. Clin. Pathol. 64:399-404.
8. Smith, J. L., and R. L. Buchanan. 1992. Shigella, p. 423-431. In
C. Vanderzant and D. F. Splittstoesser (ed.), Compendium of
methods for the microbiological examination of food, 3rd ed.
Bacto XLT4 Agar Base

Bacto XLT4 Agar Supplement
Marshall, (ed.), Standard methods for the examination of dairy

Washington, D.C.
and R. M. Amaguana. 1995. Salmonella, p. 5.01-5.20. In
Gaithersburg, MD.
Packaging
XL Agar Base
500 g
0555-17
XLD Agar
100
500
2
10
0788-15
0788-17
0788-07
0788-08
g
g
kg
kg
Intended Use
Bacto XLT4 Agar Base is used with Bacto XLT4 Agar Supplement in
isolating non-typhi Salmonella.

Base Solution:
red, very slightly to slightly opalescent.
XLT4 Agar Supplement: Colorless to slightly yellow, clear,
slightly viscous solution.
Prepared Medium:
Reddish-orange, very slightly to
Reaction of Final
Medium at 25C:
pH 7.4 0.2
Cultural Response
Prepare XLT4 Agar per label directions. Inoculate and incubate
ORGANISM
Escherichia coli
Proteus mirabilis
ATCC
CFU
INOCULUM
GROWTH
COLONIAL
MORPHOLOGY
29212* 1,000 markedly inhibited
25922* 1,000 partially inhibited yellow colonies

25933
1,000
inhibited
14028* 100-1,000
good
yellow to red colonies
with black centers
25923* 1,000
inhibited
ATCC 14028
on XLT4 Agar Base with XLT4 Supplement
The Difco Manual
567
XLT4 Agar Base & XLT4 Agar Supplement
Section II

Numerous media have been developed for isolating and differentiating
enteric pathogens. The majority were designed to recover a broad
spectrum of enteric pathogens.1 Consequently, overgrowth of nuisance
or contaminating organisms can be a major problem when recovery of
a specific organism or species is desired. This is particularly true for
Salmonella isolation media where overgrowth of Proteus, Providencia
and Pseudomonas can dramatically interfere with the detection and
isolation of Salmonella.
In 1990, Miller and Tate described a new medium, XLT4 Agar, for
isolating Salmonella.1 The authors established the selectivity of XLT4
Agar using pure cultures of a variety of enteric organisms. They also
evaluated its sensitivity in detecting and isolating Salmonella using
fecal-contaminated farm samples containing high numbers of competing
bacteria. In follow-up studies, Miller2,3 and Tate4 reported that XLT4
Agar significantly improved the recovery of non-typhi Salmonella from
chicken and farm environmental drag-swab samples.
XLT4 Agar can be used clinically to screen stool samples for nontyphoid Salmonella.5,6

XLT4 Agar Base contains Proteose Peptone No. 3 as a source of complex
nitrogen compounds. Yeast Extract is added as a source of vitamins
and other cofactors. Differentiation of Salmonella from other organisms
that also grow on this medium is based on fermentation of Xylose,
Lactose and Sucrose, decarboxylation of Lysine, and the production of
hydrogen sulfide. Hydrogen sulfide production is detected by the addition
of ferric ions. Sodium Thiosulfate is added as a source of inorganic
sulfur. Sodium Chloride maintains the osmotic balance of the medium.
Bacto Agar is the solidifying agent. Phenol Red is added as an indicator
of pH changes resulting from fermentation and decarboxylation
reactions. XLT4 Agar Supplement is added to inhibit growth of
non-Salmonella organisms.
Store XLT4 Agar Base below 30C. The dehydrated medium is very
hygroscopic. Keep container tightly closed. Store prepared medium
at 2-8C.
Store XLT4 Agar Supplement at 15-30C.
Expiration Date
Materials Provided
XLT4 Agar Base

Formula Per Liter
g
g
g
g
g
g
g
g
g
g
g

XLT4 Agar Supplement
A 27% solution (approximate) of the surfactant 7-ethyl-2-methyl-4-undecanol
hydrogen sulfate, sodium salt, formerly produced by Union Carbide under
the tradename of Tergitol 4.
Precautions
2. XLT4 Agar Base
568
Storage
Procedure
Formula
L-Lysine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Xylose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.75
Bacto Lactose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Bacto Phenol Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.08
CORROSIVE. CAUSES BURNS. HARMFUL BY INHALATION, IN CONTACT WITH SKIN AND IF SWALLOWED. Avoid
contact with skin and eyes. Do not breathe mist. Wear suitable
protective clothing, gloves and eye/face protection. Keep container
tightly closed.
XLT4 Agar Base


Waterbath (45-50C)
Petri dishes
Incubator (35C)
1. Suspend 59 grams of XLT4 Agar Base in 1 liter distilled or
deionized water.
2. Add 4.6 ml XLT4 Agar Supplement.
3. Heat to boiling to dissolve completely. Avoid overheating. DO
NOT AUTOCLAVE. Cool to 45-50C in a waterbath.

guidelines.7-10
The Difco Manual
Section II
YM Agar & YM Broth

sample.7,8,10,11
1. XLT4 Agar is intended for detecting and isolating Salmonella based

on selectivity and colonial characteristics. Presumed Salmonella
colonies must be confirmed by biochemical and/or immunological
methods. Consult appropriate references for further information.5,7,8,12
of Salmonella may be encountered that fail to grow or grow poorly
on this medium.
3. Non-Salmonella strains that are not completely inhibited on this
medium may be encountered and must be differentiated from
Salmonella. Consult appropriate references.7,8,10,12
4. Freshly inoculated plates and plates held over several days may
develop multicolored, metallic looking crystals/flecks on the
surface. These crystals/flecks do not interfere with the performance
of the medium.
2. Miller, R. G., C. R. Tate, E. T. Mallinson, and J. A. Schemer.

1991. Xylose-Lysine-Tergitol 4: An improved selective agar medium
for the isolation of Salmonella. Poultry Science 70:2429-2432.
3. Miller, R. G., C. R. Tate, E. T. Mallinson, and J. A. Schemer.
1992. Erratum. Xylose-Lysine-Tergitol 4: An improved selective
agar medium for the isolation of Salmonella. Poultry Science
71:398.
4. Tate, C. R., R. G. Miller, and E. T. Mallinson. 1992. Evaluation
of two isolation and two non-isolation methods for detecting
naturally occurring salmonellae from broiler flock environmental
drag-swab samples. J. Food Prot. 55:964-967.
5. Dusch, H., and M. Altwegg. 1994. Evaluation of Xylose-LysineTergitol 4 (XLT4) Agar and Modified Semisolid Rappaport
Vassiliadis (MSRV) Medium for the isolation of non-typhoid
salmonellae from stool samples. Abstr. Annu. Meet. Am. Soc.
Microbiol. C5:557.
6. Dusch, H., and M. Altwegg. 1995. Evaluation of five new plating
media for the isolation of Salmonella species. J. Clin. Microbiol.
33:802-804.
and R. M. Amaguana. 1995. Salmonella. In FDA Bacteriological
analytical manual, 8th ed. AOAC International, Arlington, VA.
8. Vanderzant, C., and D. F. Splittstoesser (ed.) 1992. Compendium of methods for the microbiological examination of foods,
transport, and storage, p. 19-31. In P. R. Murray, et al. (ed). Manual of
Washington, D.C.
10. Pezzlo, Marie (ed). 1992. Aerobic bacteria. In H. D. Isenberg (ed),
Clinical microbiology procedures handbook, vol. 1. American
11. Forbes, B. A., and P. A. Granato. 1995. Processing specimens for
bacteria, p. 265-281. In P. R. Murray, et al. (ed), Manual of clinical
12. Gray, L. D. 1995. Escherichia, Salmonella, Shigella and Yersinia.
In P. R. Murray, et al. (ed), Manual of clinical microbiology, 6th
ed. American Society for Microbiology, Washington, D.C.
References
Packaging
1. Miller, R. G., and C. R. Tate. 1990. XLT4: A highly selective

plating medium for the isolation of Salmonella. The Maryland
Poultryman April:2-7.
XLT4 Agar Base
500 g
0234-17
l00 ml
0353-72
Bacto YM Agar
Bacto YM Broth
Also Known As
Test Procedure
1. Inoculate a suitable Salmonella enrichment broth (such as
Tetrathionate Broth) and incubate at 35C for 18-24 hours.
2. Following enrichment, subculture onto XLT4 Agar. Streak for
isolation.
3. Incubate plates aerobically at 35 2C. Examine for growth after
18-24 and 48 hours incubation.
Results
Typical Salmonella colonies (H2S-positive) appear black or blackcentered with a yellow periphery after 18-24 hours of incubation. Upon
continued incubation, the colonies become entirely black or pink to
red with black centers.
Colonies of H2S-negative Salmonella strains appear pinkish-yellow.
Most Citrobacter colonies that grow on this medium are yellow without
evidence of blackening. Growth of Enterobacter aerogenes and
Escherichia coli is markedly inhibited; colonies that do grow appear
yellow without evidence of blackening. Growth of Proteus,
Pseudomonas, Providencia, Alteromonas putrefaciens, Yersinia
enterocolitica and Acinetobacter calcoaceticus is markedly to
completely inhibited on XLT4 Agar. Shigella species are partially
inhibited and colonies appear red.
Intended Use
Bacto YM Agar and YM Broth are used for cultivating yeasts, molds
and other aciduric microorganisms.
The Difco Manual
YM is an abbreviation for Yeast Extract and Malt Extract.

YM Agar and YM Broth are prepared according to the formulation
published by Wickerham.1,2,3 Wickerham suggested that YM Broth
acidified to pH 3.0-4.0 be used as an enrichment medium for
yeasts from populations also containing bacteria and molds. To favor
isolation of fermentative species, add a layer of sterile paraffin oil
569
YM Agar & YM Broth
Section II
1 cm deep on the surface of the inoculated broth. Incubate the culture

until growth appears and then streak onto YM Agar to obtain isolated
yeast colonies. To isolate fermentative and oxidative strains, place
acidified inoculated YM Broth on a rotary shaker for 1 or 2 days. This
favors yeast recovery while preventing the sporulation of molds.
Media selectivity may be enhanced through acidification or through
addition of selective agents. YM Broth may be acidified prior to
sterilization. YM Agar should be sterilized without pH adjustment
and sterile acid added to the sterile molten medium cooled to
45-50C. Acidified YM Agar should not be heated. Antibiotics may
be aseptically added to the sterile media. Other fungistatic materials,

YM Agar
Solution:
slightly opalescent, without
Prepared Plates:
Reaction of 4.1%
Solution at 25C:
pH 6.2 0.2
YM Broth
Solution:
precipitate.
NOTE:
At pH adjusted to 3.0-4.0, medium
becomes slightly opalescent.
Prepared Tubes:
Light - medium amber, clear to very
slightly opalescent without significant
precipitate.
Reaction of 2.1%
Solution at 25C:
pH 6.2 0.2
Cultural Response
Prepare 2 sets of YM Agar plates or YM Broth tubes (one set
pH 6.2, one set adjusted to pH 3.0-4.0) per label directions.
ORGANISM
Aspergillus niger
Candida albicans
Escherichia coli
Lactobacillus casei
Saccharomyces
cerevisiae
ATCC
INOCULUM
CFU
GROWTH
PH 3.0-4.0
16404 100-1,000
10231 100-1,000
25922* 100-1,000
GROWTH
PH 6.2

Malt Extract is a source of carbon, protein and nutrients. Bacto Peptone
is an additional source of carbon and provides nitrogen and amino
acids. Dextrose provides carbon. Bacto Agar is a solidifying agent.
Formula
YM Agar
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
YM Broth
Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
g
g
g
g
Precautions
Storage
Expiration Date
Procedure
Materials Provided
YM Agar
YM Broth
good
good
good
7469
good
Glassware
Autoclave
Antibiotics
Sterile 10% HCl, Tartaric Acid or 10% Citric Acid
9763
100-1,000
good
good
g
g
g
g
g
good
good
markedly to
100-1,000
poor to fair
570
such as sodium propionate and diphenyl may be added to YM Agar

to eliminate molds and permit the enumeration of yeasts to
mixed populations.
YM Agar
The Difco Manual
Section II
YPD Agar & YPD Broth
YM Broth
Optional (for Agar or Broth): If desired, acidify the medium
to pH 3.0-4.0 by adding sterile 10% HCl, Tartaric Acid or
10% Citric Acid. Selective agents, e.g., penicillin (20 units per ml
final concentration) or streptomycin (40 micrograms per ml final
concentration) may be added to the medium after sterilization
using aseptic technique.
Test Procedure
1. Inoculate YM Agar plates or YM Broth tubes with sample to evaluated
for the presence of yeasts, molds, or aciduric microorganisms.
Results
Examine the plates or tubes for growth. Record YM Agar results as
Bacto YPD Agar

Bacto YPD Broth
colony forming units (CFU) per volume of sample. Record YM Broth

results as growth or no growth.

1. Acidified YM Agar should not be overheated.
References
1. 1951. U. S. Dept. Agricult. Tech. Bull. No. 1029.
2. 1939. J. Tropical Med. Hyg. 42:176.
3. Jong, S. C., and M. J. Edwards. 1991. American Type Culture
Collection Catalog of filamentous fungi, 18th ed. American Type
Collection, Rockville, MD.
Packaging
YM Agar
500 g
0712-17
YM Broth
500 g
10 kg
0711-17
0711-08
Intended Use
Bacto YPD Agar and Bacto YPD Broth are used for maintaining and
propagating yeasts in molecular microbiology procedures.
Also Known As
YPD media are also known as Yeast Extract-Peptone-Glucose media

and may be abbreviated as YEPD.
YPD Agar
Solution:
Prepared Medium:
opalescent.
Reaction of 6.5%
Solution at 25C:
pH 6.5 0.2
YPD Broth
Solution:
opalescent.
Prepared Medium:
Reaction of 5.0%
Solution at 25C:
pH 6.5 0.2
Cultural Response
Prepare YPD Agar or YPD Broth per label directions. Inoculate
and incubate the plates or tubes at 25 2C for 42-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Kluyveromyces lactis
8563
18790
9080
100-1,000
100-1,000
100-1,000
good
good
good
The Difco Manual

General methods in yeast genetics specify using yeast extractpeptone-glucose (YPD) medium for cultivating Saccharomyces
cerevisiae and other yeasts.1 Yeasts grow well on a minimal medium
containing only glucose and salts. The addition of protein and yeast
cell extract hydrolysates allow faster growth so that during exponential
or log-phase growth, the cells divide every 90 minutes.1

YPD Agar and YPD Broth contain Bacto Peptone as a source of
vitamins which stimulate bacterial growth. Dextrose is the carbohydrate
source. YPD Agar contains Bacto Agar as the solidifying agent.
Formula
YPD Agar
Formula Per Liter
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10
20
20
15
g
g
g
g

YPD Broth
Formula Per Liter
Bacto Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
Bacto Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
571
Yeast Extract
Section II
Precautions
Storage
Expiration Date
Procedure
Materials Provided
1. Suspend 65 grams of YPD Agar in 1 liter of distilled or deionized

water.
YPD Broth

Test Procedure
Results
Growth of colonies on the agar or in the broth (turbidity).
References
YPD Agar
YPD Broth

Glassware
Autoclave
Incubator (25C)
1. Ausubel, F. M. , R. Brent, R. E. Kingston, D. D. Moore, J. G.

Seidman, J. A. Smith, and K. Struhl. 1994. Current Protocols in
Molecular Biology., Current Protocols, Brooklyn, NY.
Packaging
YPD Agar
500 g
2 kg
0427-17
0427-07
YPD Broth
500 g
2 kg
0428-17
0428-07
YPD Agar
Yeast Extract
Bacto Yeast Extract . Bacto Yeast Extract, Technical
Bacto Autolyzed Yeast
Intended Use
Bacto Yeast Extract, Bacto Yeast Extract, Technical and Bacto Autolyzed Yeast are used in preparing microbiological culture media.

Yeast Extract is the water soluble portion of autolyzed yeast. The
autolysis is carefully controlled to preserve the naturally occurring
B-complex vitamins. Yeast Extract is prepared and standardized for
bacteriological use. It is an excellent stimulator of bacterial growth
and used in culture media in place of, or in addition to, beef extract.
Yeast Extract is generally employed in the concentration of 0.3-0.5%.
Yeast Extract has been used successfully in culture media for studies
of bacteria in milk and other dairy products. The advantage of Yeast
Extract for this purpose is documented by Prickett1 on the thermophilic
and thermoduric bacteria of milk. Since publication of Pricketts1 study,
Yeast Extract has been used more frequently in the study of bacterial
flora in milk. Hutner2 used this product in a stock broth for streptococci. Partansky and McPherson3 used Yeast Extract in combination
with Bacto Malt Extract and Bacto Agar for testing mold resistant
properties of oil paints.
572
Yeast Extract is an excellent source of B-complex vitamins and is

often used to supply these factors in bacteriological culture media. Snell
and Strong4 used Yeast Extract for the preparation of yeast supplement
in their medium for riboflavin assay. It has been a valuable ingredient
for carrying stock cultures, and for preparation of inocula of lactobacilli
for microbiological assay of vitamins. This product is also of value in
the assay of antibiotics. A growth substance, B factor for Norcardia,
can be isolated from Yeast Extract.5 Yeast Extract supplies this factor
necessary for a rifampin mutant to product rifampin.5
Several media containing Yeast Extract are specified in standard methods
for multiple applications.6,7,8,9
Yeast Extract, Technical is a water soluble portion of autolyzed yeast
containing vitamin B complex. Yeast Extract, Technical is used in bacterial culture media when a standardized yeast extract is not essential. It
demonstrates acceptable clarity and growth promoting characteristics.
Autolyzed Yeast is a desiccated product containing both the soluble
and insoluble portions of autolyzed bakers yeast. It is recommended
for preparation of yeast supplements used in microbiological assay for
riboflavin and pantothenic acid.10,11
The Difco Manual
Section II
Yeast Extract

Yeast Extract is typically prepared by growing bakers yeast,
Saccharomyces sp., in a carbohydrate-rich plant medium. The yeast

Yeast Extract
Solution:
1% solution - soluble in distilled or
medium amber, clear, may have a
2% solution-medium amber, clear,
Reaction of 1%
Solution at 25C:
pH 6.6 0.2
Dehydrated Appearance: Light to medium beige, free-flowing,
homogeneous.
Solution:
1% solution - soluble in distilled or
medium amber in color, clear to
very slightly opalescent, may have
a precipitate.
Autolyzed Yeast
Dehydrated Appearance: Medium to dark brown, homogenous,
free-flowing.
Solution:
1% solution - not completely soluble
boiling. Solution is amber, opaque,
Reaction of 1%
Solution at 25C:
pH 4.9-6.3
Cultural Response
Yeast Extract
Prepare a solution containing 1% Yeast Extract and 0.5%
sodium chloride. Adjust the pH to 7.2 0.2 using dilute NaOH.
Inoculate tubes with the test organisms and incubate at 35 2C
for 18-48 hours.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
13090*
25923*
6305
100-1,000
100-1,000
100-1,000
fair to good
good
good

Prepare a solution containing 2% Yeast Extract with 0.5%
sodium chloride. Adjust the pH to 7.2 0.2 using dilute NaOH.
Inoculate tubes with the test organisms and incubate at 35 2C
for 18-24 hours.
ORGANISM
Escherichia coli
ATCC
INOCULUM
CFU
GROWTH
25922*
19615*
100-1,000
100-1,000
good
good
The Difco Manual
is harvested, washed and resuspended in water, where is undergoes

autolysis, i.e., self digestion using yeasts enzymes. Yeast Extract is
the total soluble portion of this autolytic action. The autolytic activity
is stopped by a heating step. The resulting Yeast Extract is then filtered
clear and subsequently made a powder by the spray drying process.
Yeast Extract, Yeast Extract, Technical and Autolyzed Yeast provide
vitamins, nitrogen, amino acids and carbon in microbiological culture
media.
Typical Analysis
Yeast Extract
Ash (%)
11.2
1.5
2.7
Loss on Drying (%)

pH, 1% Soln
3.1
6.7
Carbohydrate (%)
Total
17.5

Total Nitrogen
Amino Nitrogen
10.9
6.0
AN/TN
5.36
3.02
6.69
0.74
14.20
3.25
1.20
3.23
4.69
Lysine
Methionine
Phenylalanine
Proline
Serine
Threonine
Tryptophan
Tyrosine
Valine
0.013
0.380
<0.001
<0.001
<0.001
<0.001
0.075
<0.001
Phosphate
Potassium
Sodium
Sulfate
Sulfur
Tin
Zinc
55.0
Amino Acids (%)

Alanine
Arginine
Aspartic Acid
Cystine
Glutamic Acid
Glycine
Histidine
Isoleucine
Leucine
5.15
1.05
2.53
2.60
2.84
2.95
1.36
1.20
3.79
Inorganics (%)
Calcium
Chloride
Cobalt
Copper
Iron
Lead
Magnesium
Manganese
3.270
3.195
1.490
0.091
0.634
<0.001
0.011
Vitamins (g/g)
Biotin
3.3
Cyanocobalamin
<0.1
Folic Acid
1.5
Inositol
1400.0
Nicotinic Acid
597.9
PABA
Pantothenic Acid
Pyridoxine
Riboflavin
Thiamine
Thymidine
763.0
273.7
43.2
116.5
529.9
17.5

Coliform
Salmonella
Spore Count
negative
negative
9

Thermophile Count
60
<5
Precautions
573
Yeast Extract Glucose Chloramphenicol Agar
Section II
Storage
References
Store the dehydrated ingredient below 30C. The dehydrated ingredient

1.
2.
3.
4.
5.
Expiration Date
Procedure
Materials Provided
Yeast Extract
Autolyzed Yeast

Refer to the final concentration of Yeast Extract, Yeast Extract, Technical
or Autolyzed Yeast in the formula of the medium being prepared. Add
Yeast Extract, Yeast Extract, Technical or Autolyzed Yeast as required.

Test Procedure
Prickett. 1928. Tech. Bull. 147. NY Agr. Exp. Sta.

Hutner. 1938. J. Bacteriol. 35:429.
Partansky and McPherson. 1940. Ind. Eng. Chem., Anal. Ed. 12:443.
Snell and Strong. 1939. Ind. Eng. Chem., Anal. Ed. 11:346.
Kawaguchi, T., T. Asahi, T. Satoh, T. Uozumi, and T. Beppu.
1984. B-factor, an essential regulatory substance inducing the production of rifamycin in a Nocardia sp. J. Antibiot. 37:1587-1594.
6. Vanderzant, C., and D. F. Splittstoesser (ed.). 1992. Compendium of methods for the microbiological examination of food, 3rd
Washington, D.C.
Packaging
Yeast Extract
100
500
2
10
g
g
kg
kg
0127-15
0127-17
0127-07
0127-08
500 g
10 kg
0886-17
0886-08
Autolyzed Yeast
500 g
10 kg
0229-17
0229-08
See appropriate references for specific procedures using Yeast Extract,

Yeast Extract, Technical or Autolyzed Yeast.
Results

encountered that fail to grow or grow poorly on prepared medium.
Bacto Yeast Extract Glucose Chloramphenicol Agar
Intended Use
Bacto Yeast Extract Glucose Chloramphenicol Agar is a selective agar
recommended by the International Dairy Federation1,2 for enumerating
yeasts and molds in milk and milk products.
Also Known As
Yeast Extract Glucose Chloramphenicol Agar is also known as YGC Agar.

The antibiotic method for enumerating yeasts and molds in dairy
products has become the method of choice, replacing the traditional
acidified method.2 The use of antibiotics for suppressing bacteria results
in better recovery of injured fungal cells, which are sensitive to an acid
environment, and in less interference from precipitated food particles
during the counting.3-7
Yeast Extract Glucose Chloramphenicol Agar is a nutrient medium that
inhibits the growth of organisms other than yeasts and molds due to
574
the presence of Chloramphenicol. When a sample contains predominantly

yeasts and/or injured yeasts, the use of Yeast Extract Glucose
Chloramphenicol Agar may offer some advantage.2 After incubation at
25C, colonies are counted and yeast colonies are distinguished from
molds by colony morphology.

Yeast Extract provides basic nutrients. Glucose is a carbon energy
source. Chloramphenicol inhibits bacterial growth.
Formula
Formula per liter
Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
g
g
g
g

The Difco Manual
Section II
Precautions
2. TOXIC. MAY CAUSE CANCER. POSSIBLE RISK OF HARM
tightly closed. TARGET ORGAN(S): Blood, Nerves, Lymph
Glands, Eyes.
Storage
Expiration Date
Procedure
Materials Provided
Glassware
Autoclave

Solution:
Reaction of 3.81%
Solution at 25C:
pH 6.6 0.2
Prepare the medium per label directions. Inoculate by the pour
plate technique and incubate at 25 2C for up to 4 days.
Aspergillus niger
Candida albicans
Escherichia coli
ATCC
16404
10231
25922*
9763
INOCULUM
CFU
GROWTH
30-300
good
30-300
good
30-300
good

The Difco Manual
2. Boil gently to dissolve completely.
3. Dispense 10-12 ml aliquots into tubes or other final containers and
cap loosely.
Test Procedure
1. Prepare initial sample dilutions using 10 grams or 10 ml of sample in
90 ml of diluent, as listed below:
SAMPLE
10 grams or 10 ml
Milk
Liquid milk product
Dried Milk
Whey powder
Buttermilk powder
Lactose
Casein
Cheese
Butter
Edible ice
Custard dessert
Fermented milk
Yogurt
DILUENT 90 ml
PREPARATION
1/4-strength Ringers solution
Mix.
Shake at 47C.
2% dipotassium phosphate
solution
2% sodium citrate solution
Shake at 47C.
Shake.
Shake at 47C.
Shake at 47C.
2. Add 10 ml from the initial dilution prepared above (#1) to 90 ml of

1/4-strength Ringers solution. One milliliter (1 ml) of this dilution
corresponds to 0.01 gram/ml of sample.
3. Prepare further dilutions by adding 10 ml of the 0.01 gram/ml dilution
above (#2) to 90 ml of diluent.
4. Pipette 1 ml of each dilution into two Petri dishes.
5. Pour 10 ml of sterile molten agar (cooled to 45C) into each dish.
Mix thoroughly.
6. Incubate at 25C for 4 days.
Results
1. Select plates containing 10-300 colonies and count the colonies.
Distinguish yeasts from molds by colony morphology.
2. Express results as yeasts and molds per gram or per milliliter.
References
Cultural Response
ORGANISM
Petri dishes
Ringers solution
2% Dipotassium phosphate
2% Sodium citrate
1. International Dairy Federation. Standard Method ISO/DIS 6611.

for groups of microorganisms, p. 281-283. In R. T. Marshall, (ed.),
3. Beuchat, L. R. 1979. Comparison of acidified and antibioticsupplemented potato dextrose agar from three manufacturers for
its capacity to recover fungi from foods. J. Food Prot. 42:427-428.
4. Cooke, W. B., and A. R. Brazis. 1968. Occurrence of molds and
yeasts in dairy products. Mycopathol. Mycol. Appl. 35:281-289.
575
Yeast Media
Section II
5. Koburger, J. A. 1970. Fungi in foods: 1. Effect of inhibitor and

incubation temperature on enumeration. J. Milk Food Technol.
33:433-434.
6. Koburger, J. A. 1973. Fungi in foods: 5. Response of natural
populations to incubation temperatures between 12 and 32C.
J. Milk Food Technol. 36:434- 435.
7. Overcase, W. W., and D. J. Weakley. 1969. An aureomycin-rose

bengal agar for enumeration of yeast and mold in cottage cheese.
J. Milk Food Technol. 32:442- 445.
Packaging
Yeast Extract Glucose
Chloramphenicol Agar
500 g
5 kg
1900-17
1900-03
Yeast Media
Bacto Yeast Morphology Agar . Bacto Yeast Carbon Base
Bacto Yeast Nitrogen Base . Bacto Yeast Nitrogen Base w/o
Amino Acids . Bacto Yeast Nitrogen Base w/o Amino Acids and
Ammonium Sulfate
Intended Use

Yeast Morphology Agar
Solution:
is very light amber, very slightly to
Prepared Medium:
Reaction of 3.5%
Solution at 25C:
pH 5.6 0.2
Yeast Carbon Base
Solution:
1.17% (single-strength) and 11.7%
(10X) solution, soluble in distilled or
deionized water with slight warming.
Single-strength solution is colorless
to very light amber, clear after
filter-sterilization.
Prepared Medium:
Colorless to very light amber, clear,
no precipitate.
Reaction of 1.17%
Solution at 25C:
pH 5.5 0.2
Yeast Nitrogen Base
Solution:
(10X) solution, soluble in distilled or
deionized water with agitation.
Single-strength solution is almost
colorless and clear; 10X solution is
yellow and clear.
Prepared Medium:
Colorless, clear without precipitate.
Reaction of 0.67%
Solution at 25C:
pH 5.4 0.2
576
Bacto Yeast Morphology Agar is used for classifying yeasts based on

colonial characteristics and cell morphology.
Bacto Yeast Carbon Base is used for classifying yeasts based on nitrogen
assimilation.
Bacto Yeast Nitrogen Base is used for classifying yeasts based on carbon
assimilation.
Bacto Yeast Nitrogen Base w/o Amino Acids is used for classifying
yeasts based on amino acid and carbohydrate requirements.
Bacto Yeast Nitrogen Base w/o Amino Acids and Ammonium Sulfate
is used for classifying yeasts based on carbon and nitrogen requirements.

Yeasts are unicellular, eukaryotic, budding cells that are generally
round-to-oval or elongate in shape.1 They multiply principally by the
production of blastoconidia (buds).1 Yeast colonies are moist and
creamy or glabrous to membranous in texture.1 Yeasts are considered
opportunistic pathogens.1
The yeast media cited are prepared according to the formulas of
Wickerham.2,3,4,5,6
Yeast Carbon Base tests the ability of yeasts to assimilate nitrogen by
the addition of various nitrogen sources. The inclusion of vitamins aids
in the utilization of nitrogen-containing compounds by certain yeasts
which cannot assimilate these compounds in the absence of vitamins.
Yeast Nitrogen Base is a suitable medium for studying strains of yeast
that require certain vitamins.
Yeast Nitrogen Base w/o Amino Acids, which lacks the amino acids
histidine, methionine and tryptophane, and Yeast Nitrogen Base w/o
Amino Acids and Ammonium Sulfate, which lacks amino acids and
ammonium sulfate, are prepared according to Guenters7 modification
of Wickerhams Yeast Nitrogen Base formulation.
These media are included in many applications for the study of yeasts
in molecular genetics.8,9

Yeast Morphology Agar contains all essential nutrients and vitamins
necessary for the cultivation of yeasts, including a source of carbohydrate.
The Difco Manual
Section II
Yeast Media
Yeast Carbon Base contains all essential nutrients and vitamins necessary
for the cultivation of yeasts except a source of nitrogen.
Yeast Nitrogen Base contains all essential nutrients and vitamins
necessary for the cultivation of yeasts except a source of carbohydrate.
Yeast Nitrogen Base w/o Amino Acids contains all essential vitamins
and inorganic salts necessary for the cultivation of yeasts except histidine,
methionine, tryptophane and a source of carbohydrate.
Yeast Nitrogen Base w/o Amino Acids and Ammonium Sulfate contains
all essential nutrients and vitamins necessary for the cultivation of yeasts
except amino acids and a source of nitrogen and carbohydrate.
Formula
Formula per Liter
Nitrogen Sources
Ammonium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 g
Asparagine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5 g
Carbon Source
Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Amino Acids
L-Histidine Monohydrochloride . . . . . . . . . . . . . . . . . . . . 10
LD-Methionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
LD-Tryptophan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Vitamins
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Folic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Inositol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2,000
Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Uninoculated
plate
g
mg
mg
mg
g
g
g
g
g
g
g
g
g
Candida albicans
ATCC 10231
Yeast Nitrogen Base w/o Amino Acids

Solution:
0.67% (single strength) or 6.7% (10X)
water with agitation. Single-strength
solution is colorless to very pale
yellow and clear; 10X solution
is yellow and clear.
Prepared Medium:
Reaction of 0.67%
Solution at 25C:
pH 5.4 0.2
Yeast Nitrogen Base w/o Amino Acids and Ammonium Sulfate
Dehydrated Appearance: Light yellowish-beige, free-flowing,
homogeneous.
Solution:
0.17% (single-strength) and 1.7% (10X)
water. Single-strength solution is
colorless to very pale yellow and clear;
10X solution is yellow and clear.
Prepared Medium:
Reaction of 0.17%
Solution at 25C:
pH 4.5 0.2
Cultural Response
Prepare Yeast Morphology Agar per label directions. Inoculate
using the pour plate technique and incubate at 25-30C for
18-48 hours. Also, inoculate by the Dolman technique (streak
and point) and incubate at 25-30C for 6-7 days.
ORGANISM
ATCC
GROWTH
DOLMAN
PLATE TEST
Kloeckera apiculata
Candida albicans
9774
9080
10231
good
good
good
hyphae
ATCC 9080
Yeast Carbon Base (with and without 5% ammonium sulfate)

Yeast Nitrogen Base (with and without 5% dextrose)
Yeast Nitrogen Base w/o Amino Acids (with and without 5% dextrose,
0.02% DL-methionine, 0.02% DL-tryptophane and 0.015% L-histidine)

(with and without 5% dextrose, 5% ammonium sulfate, 0.02%
DL-methionine, 0.02% DL-tryptophane and 0.01% L-histidine)
Prepare the medium per label directions with and without the
supplements indicated above. Inoculate and incubate at 25-30C
for 2-5 days.
ORGANISM
ATCC
Kloeckera apiculata
9774
9080
GROWTH WITHOUT GROWTH WITH

SUPPLEMENT(S)
SUPPLEMENT(S)
none to poor
none to poor
good
good
The Difco Manual
577
Yeast Media
Compounds Supplying Trace Elements
Boric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500
Copper Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Ferric Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Sodium Molybdate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Zinc Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
Salts
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Section II
g
g
g
g
g
g
g
g
g
g
g
g

Yeast Carbon Base
Formula per Liter
Carbon Source
Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Amino Acids
L-Histidine Monohydrochloride . . . . . . . . . . . . . . . . . . . . . 1
LD-Methionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
LD-Tryptophan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Vitamins
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Folic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Inositol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2,000
Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Boric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500
Copper Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Ferric Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Zinc Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
Salts
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
g
mg
mg
mg
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g

Yeast Nitrogen Base
Formula per Liter
Nitrogen Sources
Amino Acids
L-Histidine Monohydrochloride . . . . . . . . . . . . . . . . . . . . 10
LD-Methionine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
LD-Tryptophan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Vitamins
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
578
Folic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Inositol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2,000
Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Boric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500
Copper Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Ferric Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Zinc Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
Salts
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g

Formula per Liter
Nitrogen Sources
Vitamins
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Folic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Inositol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2,000
Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Boric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500
Copper Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Ferric Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Zinc Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
Salts
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g
g

g
mg
mg
mg
g
g

Formula per Liter
Vitamins
Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Folic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Inositol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2,000
Niacin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
g
g
g
g
g
g
The Difco Manual
Section II
Yeast Media
Pyridoxine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . 400 g

Riboflavin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 g
Thiamine Hydrochloride . . . . . . . . . . . . . . . . . . . . . . . . . 400 g
Boric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500
Copper Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Ferric Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Zinc Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
g
g
g
g
g
g
g
Salts
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1
g
g
g
g
Precautions
2. Yeast Morphology Agar
Yeast Nitrogen Base
AND SKIN. HARMFUL IF SWALLOWED. (EC) Avoid contact
AND SKIN. HARMFUL IF SWALLOWED. (EC) Avoid contact
Storage
Expiration Date
Procedure
Materials Provided
Yeast Carbon Base
The Difco Manual
Yeast Nitrogen Base


Glassware
Filter sterilization equipment
Sterile test tubes
Spectrophotometer
Petri dishes
Pipettes
Dextrose or an equivalent carbohydrate (Yeast Nitrogen Base)
Nitrogen source (Yeast Carbon Base)
1. Suspend 35 grams of Yeast Morphology Agar in 1 liter distilled or
deionized water.
4. Pour the sterile medium into plates to a depth of approximately 1.5 mm.
5. Allow the plates to stand at room temperature.
Yeast Carbon Base
1. Prepare a 10X solution by dissolving 11.7 grams of Yeast Carbon
Base and a nitrogen source in 100 ml distilled or deionized water.
NOTE: When using potassium nitrate, an important nitrogencontaining compound in nitrogen assimilation testing, add 0.78 grams.
2. Warm to dissolve, if necessary. Mix well.
3. Filter-sterilize the solution.
4. Store at 2-8C.
5. Prepare the final medium by aseptically pipetting 0.5 ml of the
10X solution into 4.5 ml of sterile distilled water.
6. Mix the solution thoroughly by shaking before inoculation.
Yeast Nitrogen Base
1. Prepare a 10X solution by dissolving 6.7 grams of Yeast Nitrogen
Base and 5 grams of Dextrose or an equivalent amount of other
carbohydrate in 100 ml distilled or deionized water.
2. Warm slightly to dissolve. Mix well.
3. Filter-sterilize the solution.
4. Store at 2-8C.
1. Prepare a 10X solution by dissolving 6.7 grams of Yeast Nitrogen
Base w/o Amino Acids and 5 grams of Dextrose or an equivalent
amount of other carbohydrate and 5-10 mg% of the desired amino
acid in 100 ml of distilled or deionized water.
2. Filter sterilize the solution.
3. Store at 2-8C.
579
Yeast Media

1. Prepare a 10X solution by suspending 1.7 grams of Yeast Nitrogen
Base w/o Amino Acids and Ammonium Sulfate and nitrogen and
carbon sources, as required, in 100 ml distilled or deionized water.
2. Filter sterilize the solution.
3. Store at 2-8C.
10X solution into 4.5 ml sterile distilled water.

Procedure
Inoculate plates using the Dolman technique (as follows) described by
Wickerham and Rettger.1 This is an excellent method for studying the
hyphae of filamentous yeasts.
1. Near one side of the plate (from the relative positions of 10 oclock
to 2 oclock), lightly inoculate a single streak taken from a slant
culture.
2. In addition to the single streak, inoculate two points near the other
side of the plate (at the 4 oclock and 8 oclock positions).
3. Cover a central section of the streak inoculation and one point
inoculation with cover glasses, as follows:
a. With forceps, remove a cover glass from absolute alcohol, drain
momentarily, and burn off excess alcohol by passing over a low
flame.
b When the cover glass has cooled, place one edge on the agar
and allow it to fall across the central portion of the inoculated
streak. Place a second cover glass over one point inoculation.
4. Incubate at 25-30C for 6-7 days.
5. After incubation, observe with a high dry objective.
Yeast Carbon Base, Yeast Nitrogen Base, Yeast Nitrogen Base
w/o Amino Acids, Yeast Nitrogen Base w/o Amino Acids and
Ammonium Sulfate
1. Inoculate the prepared tubed medium very lightly with the test
organism.
2. Incubate at 25C for 6-7 days.
3. After incubation (6-7 days and, if necessary, 20-24 days), shake
the tubes to suspend growth.
4. Read for growth.
Section II
Yeast Carbon Base, Yeast Nitrogen Base, Yeast Nitrogen Base

w/o Amino Acids, Yeast Nitrogen Base w/o Amino Acids and
Ammonium Sulfate
Measure growth turbidimetrically at 660 nm wavelength using a spectrophotometer. Turbidimetric readings on assay tubes should be comparable to the control.

may be encountered that fail to grow or grow poorly on a medium.
2. Yeasts grown on a rich medium may carry a reserve of nitrogen in
the form of protein. Possible errors due to this reserve are eliminated
by making two serial transfers in the complete medium. When the
first transfer is seven days old, the culture is shaken and one loopful
is transferred to a second tube of the complete medium containing
the same source of nitrogen. If a positive test is obtained when the
second culture is seven days old, the organism being tested assimilates
this particular nitrogen source.
References
(ed.). Manual of clinical microbiology, 6th ed. American Society
2. Wickerham, L. J. 1951. Taxonomy of yeasts. Technical bulletin
No. 1029, U. S. Dept Agriculture.
3. Wickerham, L. J. 1939. J. Tropical Med. Hyg. 42:176.
4. Wickerham, L. J. 1946. A critical evaluation of the nitrogen
assimilation tests commonly used in the classification of yeasts.
5. Wickerham, L. J. 1948. J. Bacteriol. 56:363.
6. Wickerham, L. J. 1943. J. Bacteriol. 46:501.
7. Guenter. Personal Communication.
8. Sherman F., G. R. Fink, and J. B. Hicks. 1986. Methods in yeast
genetics. Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.
9. Brownstein, B. H., G. A. Silverman, R. D. Little, D. T. Burke,
S. J. Korsmeyer, D. Schlessinger, and M. V. Olson. 1989.
Isolation of single-copy human genes from a library of yeast
artificial chromosomes clones. Science. 244:1348-1351.
10. Warren, N. G., and H. J. Shadomy. 1991. Yeasts of medical
importance, p. 617- 629. In A. Balows, W. J. Hausler, Jr., K. L.
Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.). Manual of
Washington, D.C.
Carbon Assimilation Test

Refer to the procedure described in the Manual of Clinical Microbiology.10
Packaging
100 g
0393-15
Nitrogen Assimilation Test

Refer to the procedure described in the Manual of Clinical Microbiology.10
Yeast Carbon Base
100 g
0391-15
Yeast Nitrogen Base
100 g
0392-15
Results
100 g
2 kg
10 kg
0919-15
0919-07
0919-08

and Ammonium Sulfate
100 g
10 kg
0335-15
0335-08

Using the high-dry objective, observe for hyphae of filamentous
yeasts.
580
The Difco Manual
Section II
Yersinia Selective Agar

Bacto Yersinia Selective Agar Base . Bacto Yersinia
Salmonella-Shigella Agar, Schiemann found that CIN Agar provided
Antimicrobic Supplement CN and
better inhibition of normal enteric organisms and provided improved
direct recovery of Y. enterocolitica from feces.3 Schiemann later modified

his original formula by substituting 0.5 grams of deoxycholate for
the bile salts mixture and by reducing the content of novobiocin to
2.5 mg/liter for improved growth of strains of Y. enterocolitica
serogroup 0:8.5 The concentration of cefsulodin in the antimicrobic
supplement was reduced from that described by Schiemann to further
improve growth and recovery of Y. enterocolitica.
Intended Use
Bacto Yersinia Selective Agar Base is used with Bacto Yersinia
Antimicrobic Supplement CN in isolating and cultivating
Yersinia enterocolitica.
Also Known As
Yersinia Selective Agar is also known as CIN Agar, Modified or
Cefsulodin-Irgasan-Novobiocin Agar, Modified.

Selectivity of Yersinia Selective Agar Base is due to the presence of
bile salts, crystal violet and Irgasan, which markedly inhibit growth
of gram-positive and many gram-negative organisms. Supplementation
with Yersinia Antimicrobic Supplement CN (Cefsulodin and Novobiocin)
improves inhibition of normal enteric organisms. Differentiation is
based on mannitol fermentation. Organisms capable of fermenting

Yersinia enterocolitica is a significant enteric pathogen6 and can be
food- or waterborne.7
Yersinia Selective Agar is a selective and differential medium that
supports good growth of Y. enterocolitica and some other Yersinia species.
The formulation is based on the Cefsulodin-Irgasan-Novobiocin (CIN)
Agar formulation of Schiemann.2-5 In comparison with MacConkey Agar
Yersinia enterocoliticia
ATCC 9610
Uninoculated
plate

Yersinia Selective Agar Base
Dehydrated Appearance: Light pinkish beige, free-flowing, homogeneous.
Solution:
deionized water upon boiling. Reddish
purple, very slightly to slightly opalescent.
Prepared Medium:
Reddish orange, very slightly to
Reaction of a 5.95%
Solution at 25C:
pH 7.4 0.2
Yersinia Antimicrobic Supplement CN
Dehydrated Appearance: Lyophilized, white, homogeneous cake.
Solution:
Soluble on rehydration with distilled or
deionized water, colorless, clear.
Reaction of
Solution at 25C:
pH 5.2-6.3
Cultural Response
Prepare Yersinia Selective Agar according to label directions. Inoculate and
incubate at 30 2C for 18-24 hours or at 22-25C for 48 hours.
ORGANISM
Escherichia coli
Yersinia enterocolitica
ATCC
INOCULUM
CFU
GROWTH
29212*
25922*
27853*
9610
2,000-10,000
2,000-10,000
2,000-10,000
100-1,000

good
APPEARANCE
colorless with dark pink centers;

bile precipitate may be present
The Difco Manual
581
Section II
mannitol produce a pH decrease around the colony which allows

absorption of neutral red, giving the colony a red color. Due to the
localized pH decrease, a zone of precipitated bile may also be present.
Organisms that do not metabolize mannitol to acid end products will
form colorless, translucent colonies.
Formula
Glassware
Incubator (22-25C or 30 2C)
Autoclave

Formula Per Liter
Bacto Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Mannitol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Sodium Deoxycholate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Sodium Cholate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.5
Sodium Chloride . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Sodium Pyruvate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Magnesium Sulfate Heptahydrate . . . . . . . . . . . . . . . . . . . 10
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5
Neutral Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Crystal Violet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Irgasan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
g
g
g
g
g
g
g
g
mg
g
mg
mg
mg

Formula Per 10 ml Vial
Cefsulodin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 mg
Novobiocin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 mg
Precautions
2. MAY CAUSE ALLERGIC EYE, RESPIRATORY SYSTEM AND
tightly closed. Target Organs: Liver, Blood.

1. To rehydrate the supplement, aseptically add 10 ml sterile distilled
or deionized water to the vial.
2. Invert the vial gently several times to dissolve the powder.
1. Suspend 59.5 grams in 1 liter distilled or deionized water and boil
2. Autoclave at 121C for 15 minutes. Avoid overheating.
3. Cool the medium to 45-50C. Aseptically add 10 ml rehydrated
Yersinia Antimicrobic Supplement CN to the medium. Mix well.
4. Dispense into Petri dishes.

All specimens should be collected in sterile containers in accordance
with recommended guidelines and should be transported immediately
to the laboratory. For specific information on collection and storage of
specimens consult appropriate references.
Test Procedure
For a complete discussion on the isolation and identification of Yersinia,
consult appropriate references.
Results
Y. enterocolitica colonies appear translucent or translucent with dark
pink centers. Colony edges are entire or irregular. After 48 hours
incubation, colonies appear dark pink with a translucent border and
may be surrounded by a zone of precipitated bile.
Growth of non-Yersinia organisms is markedly to completely inhibited.
1. Yersinia Selective Agar Base and Yersinia Antimicrobic Supplement

CN are intended for use in the preparation of Yersinia Selective Agar.
Although this medium is selective for Yersinia, biochemical testing
using pure cultures is necessary for complete identification.
2. Due to the selective properties of the medium, some Yersinia strains
may be encountered that fail to grow or grow poorly on the complete
medium. Some strains of normal enteric organisms may be encountered that are not inhibited or are only partially inhibited on the
complete medium, such as Citrobacter freundii, Serratia
liquefaciens and Enterobacter agglomerans.
3. Growth of Yersinia frederiksenii, Y. kristensenii, Y. pseudotuberculosis
and Y. intermedia is not inhibited on the complete medium. Colonies
of these organisms must be differentiated from Y. enterocolitica on
the basis of additional characteristics.
Procedure
References
Materials Provided

Storage
Store Yersinia Selective Agar Base dehydrated below 30C. The
dehydrated medium is very hygroscopic. Keep container tightly closed
Store Yersinia Antimicrobic Supplement CN lyophilized and rehydrated
at 2-8C. Do not open or rehydrate vials until ready to use. Use the
rehydrated product within 24 hours.
Expiration Date

582
The Difco Manual
Section II
2. Schiemann, D. A. 1979. Synthesis of a selective agar medium for

Yersinia enterocolitica. Can. J. Microbiol. 35:1298-1304.
3. Schiemann, D. A. 1980. Yersinia enterocolitica: Observations on
some growth characteristics and response to selective agents. Can.
J. Microbiol. 26:1232-1240.
4. Devenish, J. A., and D. A. Schieman. 1981. An abbreviated
scheme for identification of Yersinia enterocolitica isolated from
food enrichments on CIN (cefsulodin-irgasan-novobiocin) agar.
Can. J. Microbiol. 27:937-941.
5. Schiemann, D. A. 1982. Development of a two step enrichment
procedure for recovery of Yersinia enterocolitica from food. Appl.
8. Food and Drug Administration. 1995. Bacteriological analytical
manual, 8th ed. AOAC International, Gaithersburg, MD.
Packaging
500 g
10 kg
1817-17
1817-08
Yersinia Antimicrobic Supplement CN 6 x 10 ml
3196-60
The Difco Manual
583
Section III
ATS Medium
Bacto ATS Medium
Formula
ATS Medium
Formula Per Liter
Intended Use
Bacto ATS Medium is used for the isolation and cultivation of
mycobacteria.
Egg Yolk Suspension . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500 ml

Glycerol Extract of Potatoes . . . . . . . . . . . . . . . . . . . . . . 500 ml
Malachite Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.2 ml
Final pH 6.4-7.5 at 25C
Also Known As
ATS Medium is also known as American Trudeau Society Medium.
Precautions


tuberculosis each year. 1 During the mid 1980s the number of
tuberculosis (TB) cases in the U.S. began increasing. Before this time,
the number of cases in the U.S. had been decreasing, reaching a low in
1984. Non-tuberculous mycobacteria infections have also increased
since the mid 1980s.2
Two types of semi-solid culture media are available for the isolation of
mycobacteria: egg-based media and agar-based media. Most formulations
for the isolation of mycobacteria include malachite green, which is
used to inhibit contaminating organisms.
Storage
Expiration Date
Procedure
Materials Provided

ATS Medium is prepared according to the formula described by the
committee on evaluation of Laboratory Procedures of the American
Trudeau Society.3 ATS Medium is an egg-based medium containing a
small amount of Malachite Green. Due to the low concentration of
Malachite Green, this formulation permits the relatively early detection
of mycobacteria colonies. This medium is well suited to specimens
that are not heavily contaminated such as cerebral spinal fluid (CSF)
and pleural fluid.4,5 When present, contaminating microorganisms may
liquefy the medium.
ATS Medium

Incubator

3. Inoculate the specimen into medium.
Test Procedure
1. Incubate tubes for up to eight weeks.

Results
Dehydrated Appearance: Not applicable

Prepared Medium:
Light green, opaque
Reaction of
Medium at 25C:
pH 6.4 -7.5


Some factors that are responsible for unsuccessful cultures include
the following:
Cultural Response
Inoculate and incubate at 35 2C under CO2 for up to
three weeks.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Mycobacterium tuberculosis H37Ra
6841
13950
12478
25177
19981
100-1,000
100-1,000
100-1,000
100-1,000
100-1,000
good
good
good
good
good
These cultures are the minimum that should be used for

The Difco Manual
1. The specimen was not representative of the infectious material, i.e.

saliva instead of sputum.
2. The mycobacteria were destroyed during digestion and
3. Gross contamination interfered with the growth of the mycobacteria.
4. Proper aerobic CO2 tension was not provided during incubation.
References
Molecular genetic insights. Clin. Microbiol. Rev. 8:496-514.
587
HYcheck
Section III

Mycobacterium tuberculosis. Clin. Microbiol. News 17:65-69.
3. Woodruff, C. E., D. Crombie, J. S. Woolley, E. Medlar, and
W. Steeken. 1946. Report of the committee on evaluation of
laboratory procedures. Am. Rev. Tuberc. 54:428-432.
R. H. Yolken (ed.), Manual of clinical microbiology, 6th ed.

Packaging
ATS Medium
100 tubes
1019-79*
Bacto Dubos Albumin Broth
See page 163 of Section II for complete listing of this product.
HYcheck
HYcheck D/E Neutralizing Agar . HYcheck for Disinfection
Control . HYcheck for Enterobacteriaceae . HYcheck Plate
Count Agar With TTC . HYcheck for Total Count . HYcheck for
Yeasts and Molds . HYcheck for Yeasts and Molds with TTC
Intended Use
HYcheck is a hygiene contact slide which is used for assessing the
microbiological contamination of surfaces or fluids.

Monitoring the microbial flora of environmental surfaces, walls, ceilings,
and equipment is an important stage in achieving good manufacturing
practices in factories handling foods, cosmetics or pharmaceuticals.1,2,3
To maintain good hygiene standards in hotels and restaurant kitchens,
microbiological contamination must also be monitored.4 Methods to

HYcheck D/E Neutralizing Agar
Medium:
Appearance:
Lavender
Microbial Limits Test: Satisfactory
pH at 25C:
7.6 0.2
HYcheck for Disinfection Control
Media:
Appearance: Lavender
Microbial
Limits Test:
Satisfactory
pH at 25C:
7.6 0.2
Tryptic Soy Agar

Light amber
Satisfactory
7.3 0.2
588
monitor the environmental flora have been described using either swabbing
techniques5 or contact plates.6 Contact slides were created to monitor
the microbial flora of liquids (e.g. urine, milk) and equipment surfaces
in the clinical and food industries.1 Contact slides are statistically
comparable to swab and contact plates for surface sampling.1 The
HYcheck contact slides were developed for the testing of fluids and
surfaces for microbial cleanliness.
HYcheck is a double sided, hinged plastic paddle containing two agar
surfaces. The agar surface extends above the paddle allowing for contact
with test surfaces. The hinged paddle allows the agar surface to be
easily held against each test area during sampling. The surface area of
the paddle is clearly divided into seven units of one centimeter each to
allow direct counting of microbial density per unit area.
The HYcheck range of hygiene control slides consists of seven media
combinations designed to meet the various needs for monitoring different
types of microbial contamination.
HYcheck D/E Neutralizing Agar has both sides coated with
D/E Neutralizing Agar, a medium developed by Dey and Engley7 to
neutralize a broad spectrum of disinfectants and preservative antimicrobial
chemicals. D/E Neutralizing Agar neutralizes higher concentrations of
residual antimicrobials, when compared with other standard neutralizing
formulas, such as Letheen media, Thioglycollate media, and Neutralizing
Buffer.8,9 Complete neutralization of disinfectants is important because
disinfectant carryover can result in a false no growth result. D/E
Neutralizing media effectively neutralize the inhibitory effects of
disinfectant carryover,10,11 allowing differentiation between bacteriostasis
and true bactericidal actions of disinfectant chemicals.
The Difco Manual
Section III
HYcheck
HYcheck for Enterobacteriaceae

Media:
Violet Red Bile
Tryptic Soy Agar
Glucose Agar
Appearance:
Reddish purple
Light amber
Microbial
Limits Test:
Satisfactory
Satisfactory
pH at 25C:
7.4 0.2
7.3 0.2
HYcheck Plate Count Agar with TTC
Medium:
Plate Count Agar with 0.01% TTC
Appearance:
Light amber
Microbial Limits Test: Satisfactory
pH at 25C:
7.0 0.2
HYcheck for Total Count
Media:
Plate Count Agar
Appearance:
Light amber
Microbial
Limits Test:
Satisfactory
pH at 25C:
7.0 0.2
HYcheck for Yeasts and Molds
Media:
Rose Bengal
Appearance:
Rose pink
Microbial
Limits Test:
Satisfactory
pH at 25C:
7.2 0.2
HYcheck for Yeast and Molds with TTC
Media:
Rose Bengal
Appearance:
Rose pink
Microbial
Limits Test:
Satisfactory
pH at 25C:
7.2 0.2
Plate Count Agar

with 0.01% TTC
Light amber
Aspergillus niger NCPF 2275

Bacillus subtilis
Candida albicans
Escherichia coli
Pseudomonas /nosa
The Difco Manual
GROWTH
ON VRBGA
ORGANISM
ATCC
GROWTH
ON PCA W/TTC
19433*
25922*
13315
14028*
25923*
good
good
good
good
poor
Light amber
ORGANISM
Tryptic Soy Agar

with 0.01% TTC
Light amber
Satisfactory
7.3 0.2
ATCC
GROWTH ON D/E AGAR
6633
2091
25922*
27853*
25923*
12228*
good
good
good
good
good
good
good
good
good
good
good
good
good
good


Satisfactory
7.3 0.2
GROWTH
ON TSA
13048*
good
19433* none to poor
25922*
good
good
14028*
good
25931*
good
25923* none to poor
Tryptic Soy Agar
Escherichia coli
Proteus vulgaris
ATCC
19433*
25922*
13315
14028*
25923*
GROWTH
GROWTH
ON PCA ON PCA W/TTC
good
good
good
good
good
good
good
good
good
poor

Inoculate and incubate at 30 2oC for 18-48 hours.
ORGANISM
ATCC
Candida albicans
2091
Escherichia coli
25922*
Saccharomyces cerevisiae NCYC 1211
Serratia marcescens
8100
25923*
19615*
GROWTH
ON RBCA
GROWTH
ON TSA
good
good
none to poor
good
none to poor
none to poor
none to poor
good
good
good
good
good
good
good
HYcheck for Yeasts and Molds with TTC

ORGANISM

ORGANISM
Escherichia coli
Proteus mirabilis NCTC 11938
Shigella sonnei
ATCC
Satisfactory
7.0 0.2

Bacillus subtilis
Candida albicans
Escherichia coli
ORGANISM
Escherichia coli
Proteus vulgaris
Cultural Response (approx inoculum 30-300 CFU)
ORGANISM

ATCC
GROWTH
ON D/E
GROWTH
ON TSA
6633
2091
25922*
27853*
25923*
12228*
good
good
good
good
good
good
good
good
good
good
good
good
good
good
ATCC
Candida albicans
2091
Escherichia coli
25922*
Saccharomyces cerevisiae NCYC 1211
Serratia marcescens
8100
25923*
19615*
GROWTH
GROWTH
ON RBCA ON TSA W/TTC
good
good
none to poor
good
none to poor
none to poor
none to poor
good
good
good
poor
good
poor
good
589
HYcheck
Section III
HYcheck for Disinfection Control has side one coated with

D/E Neutralizing Agar (D/E) (see above), and side two coated with
Tryptic Soy Agar (TSA). In 1955, Leavitt et al.12 demonstrated that
Tryptic Soy Agar supports excellent growth of a both aerobic and
anaerobic microorganisms. Tryptic Soy Agar is a general purpose
medium that is recommended in multiple water and wastewater
applications.13

mercurials. Sodium Thiosulfate neutralizes iodine and chlorine.
Sodium Bisulfite neutralizes formaldehyde and glutaraldehyde. Lecithin
neutralizes quaternary ammonium compounds and Polysorbate 80
neutralizes phenols, hexachlorophene, formalin and, with lecithin,
ethanol. Brom Cresol Purple is a colorimetric indicator. Bacto Agar is
HYcheck for Enterobacteriaceae has side one coated with Violet Red
Bile Glucose Agar and side two coated with Tryptic Soy Agar, a general
purpose growth medium. Violet Red Bile Glucose Agar is a selective
medium used for the enumeration of Enterobacteriaceae in foods.
Coliform bacteria have long been used as an index of fecal contamination
in waters, and their presence in milk is used as an index of sanitation in
milk processing.14 The presence of Enterobacteriaceae, coliforms,
Salmonellae, Klebsiella or Citrobacter, in raw foodstuffs is an indicator
of fecal contamination. Their presence after processing may indicate a
failure in the manufacturing process.
Tryptic Soy Agar (TSA) - side two

HYcheck Plate Count Agar with TTC has both sides coated with
Plate Count Agar with TTC (0.01% 2,3,5-Triphenyl Tetrazolium
Chloride).
HYcheck for Total Count has side one coated with Plate Count Agar
and side two coated with Plate Count Agar with 0.01% TTC. Plate
Count Agar is used for enumerating bacteria in water, wastewater, food
and dairy products.13,15-18 TTC is a redox indicator that is colorless in
the oxidized form. TTC is reduced to insoluble triphenylformazan by
certain actively metabolizing bacteria, resulting in a red color in the
presence of bacterial growth.
There are two HYcheck products for yeasts and molds: 1) HYcheck
for Yeasts and Molds has side one coated with Rose Bengal
Chloramphenical Agar and side two coated with Tryptic Soy Agar;
2) HYcheck for Yeasts and Molds with TTC has side one coated
with Rose Bengal Chloramphenical Agar and side two coated with
Tryptic Soy Agar with 0.01% TTC. Rose Bengal Chloramphenical Agar
is recommended in the selective isolation and enumeration of yeasts
and molds from environmental materials and foodstuffs. The pH of the
medium is near neutrality for improved growth and recovery of acid
sensitive strains. 19-21

Violet Red Bile Glucose Agar (VRBGA) - side one
Yeast Extract provides vitamins, cofactors, nitrogen and carbon. Glucose
provides a source of fermentable carbohydrate. Bacto Agar is a
solidifying agent.
Tryptic Soy Agar - side two
HYcheck Plate Count Agar (PCA) with TTC

Tryptone and Yeast Extract provide carbon and nitrogen. Dextrose
provides a source of fermentable carbohydrate. TTC is a redox indicator.

Plate Count Agar - side one
Tryptone and Yeast Extract provide carbon and nitrogen. Dextrose provides
a source of fermentable carbohydrate. Bacto Agar is a solidifying agent.
Plate Count Agar with TTC - side two
a source of fermentable carbohydrate. TTC is a redox indicator. Bacto
Rose Bengal Chloramphenicol Agar (RBCA) - side one

Soytone provides carbon and nitrogen. Dextrose provides a source of
fermentable carbohydrate. Rose Bengal and Chloramphenicol inhibit
bacterial growth and restrict size and height of rapidly growing mold
colonies. Bacto Agar is a solidifying agent.
Tryptone provides carbon and nitrogen. Yeast Extract provides vitamins,

cofactors and additional nitrogen and carbon. Dextrose provides
mercurials. Sodium Thiosulfate neutralizes iodine and chlorine. Sodium
Bisulfite neutralizes formaldehyde and glutaraldehyde. Lecithin
neutralizes quaternary ammonium compounds and Polysorbate 80
neutralizes phenols, hexachlorophene, formalin and, with lecithin,
ethanol. Brom Cresol Purple is a colorimetric indicator. Bacto Agar is

D/E Neutralizing Agar (D/E) - side one
Tryptone provides carbon and nitrogen. Yeast Extract provides vitamins,
cofactors and additional nitrogen and carbon. Dextrose provides
590

a source of fermentable carbohydrate. Bacto Agar is a solidifying agent.

Rose Bengal Chloramphenicol Agar - side one
Soytone provides carbon and nitrogen. Dextrose provides a source of
fermentable carbohydrate. Rose Bengal suppresses bacterial growth
and restricts size and height of rapidly growing mold colonies.
Chloramphenicol inhibits bacteria. Bacto Agar is a solidifying agent.
The Difco Manual
Section III
HYcheck

a source of fermentable carbohydrate. TTC is a redox indicator. Bacto
103
104
105
Precautions
1. Do not touch agar surface.
2. Do not use if there are signs of dehydration or contamination.
Storage
Store HYcheck slides at 2-15C.
Expiration Date
Candida albicans ATCC 60193

on Rose Bengal Chloramphenicol Agar
Procedure
103
Materials Provided
104
105
(One type is provided per package.)

HYcheck for Yeasts and Molds with TTC.
Test Procedure
Surfaces
1. Loosen cap and remove HYcheck slide from the container.
2. Examine for dehydration or contamination.
3. Hold terminal spike against surface to be tested.
4. Press down on the spike to bend the paddle around the hinge line.
5. Gently lower the slide and press agar into contact with the test surface.
6. Apply firm and even pressure on the test surface for a few seconds.
7. Repeat procedure using the second agar surface on an area adjacent
to the initial test site.
8. Replace slide in the container and close tightly.
9. Incubate in an upright position at indicated temperature.
Liquids
1. Loosen cap and remove HYcheck Slide from the container.
2. Examine for dehydration or contamination.
3. Immerse slide into test fluid so that agar surface becomes totally
covered (if insufficient liquid is available, pour over surface of
the slide).
4. Allow to drain.
5. Replace slide in the container and close tightly.
6. Incubate in an upright position at indicated temperature.
Aspergillus niger ATCC 1015

on Rose Bengal Chloramphenicol Agar
103
104
105
106
Escherichia coli ATCC 11229

on Tryptic Soy Agar with 0.01% TTC
Results
The following photos are reactions to Candida albicans, Aspergillus

niger and Escherichia coli.
1. Do not use the HYcheck Slide if it is contaminated or the agar

medium is significantly dehydrated.
The Difco Manual
591
Petragnani Medium
References
1. Restaino, L. 1994. HYcheck Slides versus contact plates compared
to the swab technique. Dairy, Food and Environ. Sanit. 14:528-530.
2. Scott, E., S.F. Bloomfield, and C.G. Barlow. 1984. A comparison
of contact plate and calcium alginate swab techniques for quantitative assessment of bacteriological contamination of environmental
surfaces. J. Appl. Bact. 56:317- 320.
3. Thomas, M. E. M., E. Piper, and I. M. Mauer. 1972. Contamination of an operating theatre by Gram negative bacteria.
Examination of water supplies, cleaning methods and wound
infections. J. Hygiene 70:63-73.
4. Baird, R. M. 1981. Cleaning and disinfection of the hospital
pharmacy. S.A.B. Technical Series Number 16. Disinfectants: their
use and evaluation of effectiveness.
5. Griffiths, W. E. 1978. Contact slides for use in environmental
hygiene studies. Environ. Health 86:36-37.
6. Cain, R. M., and H. Steele. 1953. The use of calcium alginate
soluble wool for the examination of cleansed eating utensils. Can.
J. Pub. Health 44:464-467.
7. Dey, B. P., and F. B. Engley, Jr. 1970. A universal neutralizing
medium for antimicrobial chemicals. Presented at the Chemical
Specialties Manufacturing Association (CSMA) Proceedings
56th mid year.
8. Dey, B. P., and F. B. Engley, Jr. 1983. Methodology for recovery
of chemically treated Staphylococcus aureus with neutralizing
medium. Appl. Environ. Microbiol. 45:1533-1537.
9. Dey, B. P., and F. B. Engley, Jr. 1978. Environmental sampling
devices for neutralization of disinfectants. Presented at the 4th
International Symposium on Contamination Control.
10. Dey, B. P., and F. B. Engley, Jr. 1994. Neutralization of antimicrobial chemicals by recovery media. J. Microbiol. Methods
19:51-58.
11. Dey, B. P., and F. B. Engley, Jr. 1995. Comparison of Dey and
Engley (D/E) neutralizing medium to letheen medium and standard
methods medium for recovery of Staphylococcus aureus from
sanitized surfaces. J. Ind. Microbiol. 14:21-25.
12. Leavitt, J. M., I. J. Naidorf, and P. Shugaevsky. 1955. The undetected anaerobe in endodontics; a sensitive medium for detection
of both aerobes and anaerobes. The N.Y. J. Dentist. 25:377-382.
Section III
13. Greenberg, A. E., L. S. Clesceri and A. D. Eaton (ed.). 1995.

14. International Dairy Federation. Milk and milk products-count
of coliform bacteria. International Dairy Federation Standard
FIL-IDF 73:1974.
15. Swanson, K. J., F. F. Busta, E. H. Peterson, and M. G. Johnson.
1992. Colony Count Methods, p.75-95. In C. Vanderzant, and D. F.
Washington, D.C.
Washington, D.C.
17. Association of Official Agricultural Chemists. 1995. Official
18. Bandler, R., M. E. Stack, H. A. Koch, V. H. Tournas, and P. B.
Mislivec. 1995. Yeasts, molds and mycotoxins, p. 18.01-18.03.
In FDA Bacteriological Manual, 8th ed. AOAC International,
Arlington, VA.
19. Martin, J. P. 1950. Use of acid, rose bengal and streptomycin in
the plate method for estimating soil fungi. Soil Sci. 69:215-232.
20. Koburger, J. A. 1972. Fungi in foods. IV. Effect of plating
medium pH on counts. J. Milk Food Technol. 35:659-660.
21. Jarvis, B. 1973. Comparison of an improved rose bengalchlortetracycline agar with other media for the selective isolation
and enumeration of molds and yeasts in foods. J. Appl. Bact.
36:723-727.
Packaging
20 units
9041-36
20 units
9039-36
20 units
9037-36
20 units
9045-36
20 units
9053-36
20 units
9038-36
HYcheck for Yeasts and Molds with TTC 20 units
9046-36
Bacto Petragnani Medium
Intended Use
Bacto Petragnani Medium is used for isolating and cultivating
mycobacteria.

health problem. Almost three million people worldwide die of tuberculosis each year.1 During the mid 1980s. the number of tuberculosis
(TB) cases in the U.S. began increasing. Before this time, the number
592
of cases in the U.S. had been decreasing, reaching a low in 1984.2

Non-tuberculous mycobacterial infections have also increased since
the mid 1980s.3
Two types of semi-solid culture media are available for the isolation
of mycobacteria, egg-based media and agar-based media. Most
formulations for the isolation of mycobacteria include malachite
green, which is used to inhibit contaminating organisms.
Petragnani Medium is an egg-based medium that is a modification of
Petragnani4 medium described by Norton, Thomor and Broom.5 The
formulation contains a large amount of malachite green which
The Difco Manual
Section III
Petragnani Medium
inhibits the growth of contaminating organisms. This medium is well

suited to specimens that are from nonsterile areas that may be heavily
contaminated.3, 6

Whole Milk, Whole Eggs and Egg Yolks are protein sources. Potatoes
and Potato Flour are starches that provide a carbohydrate source. Glycerol
is a carbon source. Malachite Green inhibits contaminating organisms.
Formula
Petragnani Medium
Formula Per Liter
Whole Milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 900
Potato Flour . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Potato . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500
Whole Eggs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1200
Egg Yolks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
Bacto Glycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Bacto Malachite Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2
ml
g
g
ml
ml
ml
g
pH 7.2 0.2 at 25C
Precautions
Procedure
Materials Provided
Petragnani Medium

Specimen decontaminant and digestant
Buffer
Bovine albumin
Centrifuge
Inoculating Needles
CO2 Incubator (35C)
Supplied ready to use

3. Inoculate the specimen onto the medium.

Test Procedure
Storage
Results
Store prepared medium at 2-8C

Expiration Date

Prepared Appearance: Light to medium green, opaque, smooth
slants with no visible contamination.
Reaction of
Medium at 25C:
pH 7.2 0.2
Cultural Response
Inoculate and incubate at 35 2C under CO2 for up to 21 days.
ORGANISM
ATCC
INOCULUM
CFU
GROWTH
Escherichia coli
25922* 1,000-2,000
Mycobacterium
tuberculosis H37Ra
6841
13950
12478
partial to
complete inhibition
100-1,000
good
100-1,000
good
100-1,000
good
25177
100-1,000
good
These cultures are the minimum that should be used for

The Difco Manual
1. Incubate tubes for up to eight weeks.


Some factors that are responsible for unsuccessful cultures are:
The specimen was not representative of the infectious material,
The mycobacteria were destroyed during digestion and
Gross contamination interfered with the growth of the
mycobacteria.
Proper aerobic CO2 tension was not provided during incubation.
References
Molecular genetic insights. Clin. Microbiol. Rev. 8:496-514.
Mycobacterium tuberculosis. Clin. Microbiol. News. 17:65-69.
4. Rend. d. adunanze dell, accad. med. fis. florentina sperimentale,
77:101, 1923.
593
Petragnani Medium
Section III
5. Norton, J. F., G. J. Thomas, and N. H. Broom. 1932. Laboratory

tests for tubercle bacilli by culture methods. Am. Rev. Tuberc.,
25:378.
6. Isenberg, H. D. (Ed.). 1994. Clinical microbiology procedures handbook, sup. 1. American Society for Microbiology, Washington, D.C.
Packaging
Petragnani Medium
594
100 tubes
1010-79
The Difco Manual
Section IV
Acridine Orange Stain & SpotTest Acridine Orange Stain
Bacto Acridine Orange Stain

SpotTest Acridine Orange Stain
Intended Use
Bacto Acridine Orange Stain and SpotTest Acridine Orange Stain

are used for detecting microorganisms in direct smears by the fluorescent staining technique.
Acridine orange is a fluorochromatic dye that binds to the nucleic acids of

bacteria and other cells.11 Under UV light, Acridine Orange stains RNA
and single-stranded DNA orange; double-stranded DNA appears green.
Formula
Fluorochromatic staining of microorganisms using acridine orange was

first described by Strugger and Hilbrich in 19421 and has been used in
the microscopic examination of soil and water.2,3 Acridine orange
possesses differential staining properties with regard to clinical
materials when prepared at a low pH.4 Bacteria stain bright orange and
are differentiated from human cells and tissue debris which stain pale
green to yellow.
Acridine Orange Stain

Formula Per Liter
Acridine orange staining is a simple, rapid, inexpensive alternative to

blind subcultures.5 The stain is more sensitive than the Gram stain for
detecting microorganisms in clinical materials at concentrations of
approximately 1 x 104 colony-forming units per ml.6
Acridine orange at a low pH has been used for the detection of
Trichomonas vaginalis7 and Neisseria gonorrhoeae8 in clinical materials
and for the enumeration of mycoplasmas.9 The stain may be useful in
the rapid screening of normally sterile specimens, such as cerebrospinal fluid where few organisms may be present, and in the rapid
examination of blood smears or smears containing proteinaceous
material, where differentiation of organisms from background material
may be difficult.10
Acridine Orange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.1 g

Acetate Buffer, 0.5M . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 liter
Precautions
Storage
Store at 15-30C. Acridine Orange Stain is light sensitive. Protect
from light.
Expiration Date
Procedure
Materials Provided
Ampule Crusher

Solution:
The solution should be clear, orange, and
without evidence of a precipitate.
Reaction at 25C: pH 3.5-4.0
Prepare slides of the test organisms and sheep blood stained
using Acridine Orange Stain or SpotTest Acridine Orange
Stain. Examine slides using a fluorescent microscope at
1000X magnification.
Escherichia coli
ATCC
STAINED BACTERIA
25922*
33186
orange to red-orange rods

orange to red-orange cocci
Background for both organisms: staining is hazy black or green;

red blood cell ghosts stain pale green or have a green periphery.
The Difco Manual
Glass microscope slides

Methanol
Fluorescent microscope suitable for use with Acridine Orange
Cultural Response
ORGANISM
Not applicable

Not applicable
Preparation, Staining, and Examination of Smears

1.
2.
3.
4.
5.
Prepare a smear of the specimen to be stained on a clean glass slide.

Allow to air dry.
Fix smear with 50% or 100% methanol for 1 to 2 minutes.
Drain excess methanol and allow smear to dry.
If using SpotTest Acridine Orange Stain, hold the dispenser
upright with the tip pointing in an outward direction. Using the
provided ampule crusher, squeeze gently to crush the glass ampule
inside the dispenser. Invert and squeeze slightly to dispense the
stain on a per drop basis.
597
Gram Stain Sets and Reagents
6. Flood the slide with Acridine Orange Stain for 2 minutes.

7. Rinse thoroughly with tap water and allow to dry.
8. Smears may be initially examined at 100X to 400X magnification
using a fluorescent microscope. Findings should be confirmed by
examination at 1000X with an oil immersion objective.
Results
Bacteria and fungi stain bright orange. The background appears
black to yellow green. Human epithelial and inflammatory cells and
tissue debris stain pale green to yellow. Activated leukocytes will
stain yellow, orange or red depending on the level of activation and
the amount of RNA produced. Erythrocytes either do not stain or
stain pale green.

1. Acridine Orange staining provides presumptive information on the
presence and identification of microorganisms in the specimen.
Because microorganisms seen in smears, including nonviable
organisms, may arise from external sources (i.e., specimen collection
devices, slides or water used for rinsing), all positive smears should
be confirmed by culture.
2. Approximately 104 colony-forming units per ml are required for
detection by the Acridine Orange staining method.
3. Acridine orange staining does not distinguish between gram-positive
and gram-negative organisms. The gram reaction may be determined
by performing the Gram stain procedure directly over the acridine
orange stain after removing the immersion oil with xylene.12
4. Nuclei or granules from disintegrated, activated leukocytes may
resemble cocci at lower magnifications (e.g., 100X-400X). They
may be distinguished on the basis of morphology at higher magnifications (e.g., 1000X).
5. Certain types of debris may fluoresce in Acridine Orange stained
smears. This debris may be distinguished from microorganisms on
the basis of morphology when viewed at higher magnification.
References
1. Strugger, S., and P. Hilbrich. 1942. Die fluoreszenzmikroskopische
unterscheidung lebender und toten bakterienzeillen mit hilfe des
akridinorangefrbung. Deut. Teirarztl. Wochscher. 50:121-130.
Section IV
2. Strugger, S. 1948. Fluorescence microscope examination of

bacteria in soil. Can. J. Research 26:188-193.
3. Jones, J. F., and B. M. Simon. 1975. An investigation of errors in
direct counts of aquatic bacteria by epifluorescence microscopy,
with reference to a new method for dyeing membrane filters.
J. Appl. Bacteriol. 39:317-329.
4. Kronvall, G., and E. Myhre. 1977. Differential staining of bacteria
in clinical specimens using acridine orange buffered at low pH.
Acta. Path. Microbiol. Scand. Sect. B 85:249-254.
5. McCarthy, L. R., and J. E. Senne. 1980. Evaluation of acridine
orange stain for detection of microorganisms in blood cultures.
6. Lauer, B. A., L. B. Reller, and S. Mirrett. 1981. Comparison of
acridine orange and Gram stains for detection of microorganisms
in cerebrospinal fluid and other clinical specimens. J. Clin.
7. Greenwood, J. R., and K. Kirk-Hillaire. 1981. Evaluation of
acridine orange stain for detection of Trichomonas vaginalis in
vaginal specimens. J. Clin. Microbiol. 14:699.
8. Forsum, U., and A. Halln. 1979. Acridine orange staining of
urethral and cervical smears for the diagnosis of gonorrhea. Acta.
Dermatovener 59:281-282.
9. Rosendal, S., and A. Valdivieso-Garcia. 1981. Enumeration of
mycoplasmas after acridine orange staining. Appl. Environ.
Microbiol. 41:1000-1002.
R. H. Yolken (eds.). 1995. Manual of clinical microbiology,
11. Kasten, F. H. 1967. Cytochemical studies with acridine orange
and the influence of dye contaminants in the staining of nucleic
acids. Internat. Rev. Cytol. 21:141- 202.
12. Baron, E. J., and S. M. Finegold. 1990. Bailey & Scotts diagnostic
microbiology, 8th ed. The C. V. Mosby Company, St. Louis, MO.
Packaging
SpotTest
1 x 250 ml
6 x 250 ml
3336-75
3336-76
50 x 0.75 ml
3561-26
Bacto Gram Stain Sets and Reagents

Gram Stain Set . Gram Stain Set (with Stabilized Iodine)
3-Step Gram Stain Set-S . 3-Step Gram Stain Set-T
Intended Use
Bacto Gram Stain Sets and reagents are used to stain microorganisms
from cultures or specimens by the differential Gram method.

The Gram stain was devised in 1884 by Christian Gram1 in an attempt
to differentiate bacterial cells from infected tissue. Although Gram
598
observed what is now called the Gram reaction, he did not recognize
the taxonomic value of his technique.2
The Hucker3 modification of the Gram stain is now used to differentiate
intact, morphologically similar bacteria into two groups based on cell
color after staining. In addition, cell form, size and structural details
are evident. Such preliminary information provides important clues
to the type of organism(s) present, the further techniques required
The Difco Manual
Section IV
to characterize them, and the therapy to initiate while awaiting test

results.

The Gram stain procedure consists of 4,5,6 :
1. Staining a fixed smear with crystal violet;
2. Applying iodine as a mordant;
3. Decolorizing the primary stain with alcohol/acetone; and,
4. Counterstaining with safranin or basic fuchsin.
A crystal violet-iodine complex forms in the protoplast (not the cell
wall) of all organisms stained by this procedure. Organisms able to
retain this dye complex after decolorization are classified as
gram-positive while those that can be decolorized and
counterstained are classified as gram-negative.2,4,5,6
Positive Blood Culture Bottle
Specimen containing numerous
gram-negative rods with shape and
size of enteric rods. The culture
grew Klebsiella pneumoniae.
Generally, the cell wall is nonselectively permeable. It is theorized that

during the Gram stain procedure, the cell wall of gram-positive cells
is dehydrated by the alcohol in the decolorizer and loses permeability,
thus retaining the primary stain. However, the cell wall of
gram-negative cells has a higher lipid content and becomes more
permeable when treated with alcohol, resulting in loss of the primary stain.
The principles of the 3-Step Gram Stain procedure are identical to
the 4-step procedure described above. However, the decolorizing and
counterstaining steps have been combined into one reagent.
The molecular basis for the Gram stain has not yet been determined.
Formula
Reagents are provided in two sizes, a 250 ml plastic dispensing bottle
with a dropper cap and a one-gallon container with a dispensing tap.
Standardization may include adjustment to meet performance
specifications.
3329-Gram Crystal Violet
PRIMARY STAIN
Aqueous solution of Crystal Violet.
3331-Gram Iodine
MORDANT
(Working solution prepared from Gram Diluent and Gram Iodine 100X)
Ground Beef
Sample containing E. coli: H7
and Staphylococcus aureus.
Iodine Crystals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 g

Potassium Iodide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.6 g
Distilled Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 liter
3342-Stabilized Gram Iodine

MORDANT
Polyvinylpyrrolidone-Iodine Complex . . . . . . . . . . . . . . 100 g
Potassium Iodide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 g
Distilled Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 liter
3330-Gram Decolorizer
DECOLORIZER
Upon disruption or removal of the cell wall, the protoplast of
gram-positive (as well as gram-negative) cells can be decolorized and
the gram-positive attribute lost. Thus, the mechanism of the Gram
stain appears to be related to the presence of an intact cell wall able to
act as a barrier to decolorization of the primary stain.

Run controls daily using 18-24 hour cultures of known grampositive and gram-negative microorganisms. It is very important
that controls be included in each staining run, preferably on the
same slide. When performing the Gram stain on a clinical
specimen, particularly when the results will be used as a guide
to the selection of a therapeutic agent, such a control system
furnishes assurance that the iodine solution is providing proper
mordant activity and that decolorization was performed properly.
ORGANISM*
Escherichia coli
* Available as Bactrol Disks.
ATCC
EXPECTED RESULTS
25923
25922
gram-positive cocci
gram-negative rods
Acetone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 ml
Isopropanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750 ml
3332-Gram Safranin
COUNTERSTAIN
Safranin O Powder (pure dye) . . . . . . . . . . . . . . . . . . . . . . . 4 g
Denatured Alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 ml
Distilled Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800 ml
3343-Gram Basic Fuchsin

COUNTERSTAIN
Basic Fuchsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.08
Phenol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6
Isopropyl Alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5
Distilled Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 993
g
g
ml
ml
3335-3-Step Gram Safranin-S

DECOLORIZER/COUNTERSTAIN
Alcohol-based solution of safranin.*
3341-3-Step Gram Safranin-T

Alcohol-based solution of safranin.*
* Patent Pending
The Difco Manual
599
Precautions
2. 3329-Gram Crystal Violet
POSSIBLE RISK OF IRREVERSIBLE EFFECTS. US Avoid
contact with skin and eyes. Do not breathe spray. Wear suitable
3331-Gram Iodine 100X
HARMFUL BY INHALATION, IN CONTACT WITH SKIN AND
IF SWALLOWED. MAY CAUSE HARM TO THE UNBORN
CHILD. Avoid contact with skin and eyes. Do not breathe fumes.
3342-Stabilized Gram Iodine
HARMFUL IN CONTACT WITH SKIN. IRRITATING TO EYES,
RESPIRATORY SYSTEM AND SKIN. POSSIBLE RISK OF
HARM TO THE UNBORN CHILD.US Avoid contact with skin and
eyes. Do not breathe fumes. Wear suitable protective clothing. Keep
3330 - Bacto Gram Decolorizer
HIGHLY FLAMMABLE. IRRITATING TO EYES, RESPIRATORY SYSTEM AND SKIN. Avoid contact with skin and eyes.
Do not breathe mist or vapor. Wear suitable protective clothing.
Keep container tightly closed. Keep away from sources of ignition.
No smoking.
3332 - Bacto Gram Safranin
FLAMMABLE. EC HARMFUL BY INHALATION AND IF
SYSTEM AND SKIN. POSSIBLE RISK OF IRREVERSIBLE
EFFECTS.US Avoid contact with skin and eyes. Do not breathe
vapor. Wear suitable protective clothing. Keep container tightly
closed.
3335 - Bacto 3-Step Gram Safranin-S
HIGHLY FLAMMABLE. HARMFUL BY INHALATION AND
IF SWALLOWED. IRRITATING TO EYES, RESPIRATORY
SYSTEM AND SKIN. POSSIBLE RISK OF IRREVERSIBLE
EFFECTS.US POSSIBLE RISK OF HARM TO THE UNBORN
CHILD.US Avoid contact with skin and eyes. Do not breathe mist.
Keep away from sources of ignition. No smoking.
3341 - Bacto 3-Step Gram Safranin-T
HIGHLY FLAMMABLE. HARMFUL BY INHALATION,
IN CONTACT WITH SKIN AND IF SWALLOWED. IRRITATING
TO EYES, RESPIRATORY SYSTEM AND SKIN. POSSIBLE
RISK OF IRREVERSIBLE EFFECTS. US POSSIBLE RISK
OF HARM TO THE UNBORN CHILD.US Avoid contact with
skin and eyes. Do not breathe mist. Wear suitable protective
clothing. Keep container tightly closed. Keep away from sources
of ignition. No smoking.
FIRST AID:
water and seek medical advice.
Gram Iodine 100X: Take off immediately all contaminated clothing.
After contact with skin, wash immediately with plenty of water.
600
Section IV
If inhaled, remove to fresh air. If not breathing, give artificial

respiration. If breathing is difficult, give oxygen. Seek medical
advice.
If swallowed seek medical advice immediately and show this
container or label.
3. Studies demonstrate that the traditional Gram Iodine working
solution (Gram Iodine 100X dissolved in Gram Diluent) is
relatively unstable and may cause variability in the Gram stain
when sufficient iodine is no longer available to the solution.
Protect the iodine solution from undue exposure to air and heat.
Include controls in all staining runs or at least once daily (see
USER QUALITY CONTROL) to ensure that the solution is
providing proper mordant activity.
Storage
Store Gram Stain reagents at 15-30C.
Expiration Date
stored as directed.
Use the traditional Gram Iodine working solution within three months
of preparation, not exceeding the Expiry of either component.

1. Apply the test specimen to a clean glass slide in a manner that will
yield a thin, uniform smear. Emulsify colonies from an 18-24 hour
culture in saline to obtain the proper density.
2. Allow the smear to air dry.
3. Fix the smear to the slide using one of the following techniques:
A. Heat fix by passing the slide through a low flame 2-3 times.
Cool the slide to room temperature before staining. NOTE: Do not
overheat the slide; excessive heating will cause atypical staining.
B. Methanol fix6,7 the slide by flooding with absolute methanol
for 1-2 minutes and rinse with tap water before staining. NOTE:
For proper fixation, store absolute methanol in a brown screwcapped bottle and replenish the working supply every two weeks.
Reagent Preparation
Prepare the traditional Gram Iodine working solution by adding an
entire 2.5 ml ampule of Gram Iodine 100X to 250 ml Gram Diluent or
an entire 40 ml vial of Gram Iodine 100X to 1 gallon of Gram Diluent;
mix thoroughly.
4-Step Staining Procedure4

Materials Provided
4-Step Technique
Gram Crystal Violet
Gram Iodine or Bacto Stabilized Gram Iodine
Gram Decolorizer
Gram Safranin or Bacto Gram Basic Fuchsin

Microscope slides
Bunsen burner or methanol
The Difco Manual
Section IV
Swabs
Blotting paper
Microscope with oil immersion lens
Bactrol Gram Slide
Bactrol Disks
1. Flood the fixed smear with primary stain (Gram Crystal Violet) and
stain for 1 minute.
2. Remove the primary stain by gently washing with cold tap water.
3. Flood the slide with mordant (either Gram Iodine or Stabilized Gram
Iodine) and retain on the slide for 1 minute.
4. Remove the mordant by gently washing with tap water.
5. Decolorize (Gram Decolorizer) until solvent running from the slide
is colorless (30-60 seconds).
6. Wash the slide gently in cold tap water.
7. Flood the slide with counterstain (either Gram Safranin or Gram
Basic Fuchsin) and stain for 30-60 seconds.
8. Wash the slide with cold tap water.
9. Blot with blotting paper or paper towel or allow to air dry.
10. Examine the smear under an oil immersion lens.
3-Step Staining Procedure

Materials Provided
3-Step Stabilized Iodine Technique
Gram Crystal Violet
Stabilized Gram Iodine
3-Step Gram Safranin-S
3-Step Traditional Iodine Technique

Gram Crystal Violet
Gram Iodine
3-Step Gram Safranin-T

Microscope slides
Bunsen burner or methanol
Swabs
Blotting paper
Microscope with oil immersion lens
Bactrol Gram Slide
Bactrol Disks
1. Flood the fixed smear with primary stain (Gram Crystal Violet)
and stain for 1 minute.
2. Remove the primary stain by gently washing with cold tap water.
3. Flood the slide with mordant (Stabilized Gram Iodine or Gram
Iodine [traditional formulation]) and retain on the slide for 1 minute.
(Refer to LIMITATIONS OF THE PROCEDURE, #5.)
4. Wash off the mordant with decolorizer/counterstain (3-Step Gram
Safranin-S or 3-Step Gram Safranin-T). (NOTE: Do not wash off
iodine with water.) Add more decolorizer/counterstain solution to
the slide and stain 20-50 seconds.
5. Remove the decolorizer/counterstain solution by gently washing
the slide with cold tap water.
The Difco Manual
6. Blot with blotting paper or paper towel or allow to air dry.

7. Examine the smear under an oil immersion lens.
Results
REACTION
Gram-positive
Gram-negative
4-STEP
TECHNIQUE
USING
GRAM SAFRANIN
4-STEP
TECHNIQUE
USING
BASIC FUCHSIN
3-STEP TECHNIQUE
USING EITHER
GRAM SAFRANIN-S
OR GRAM SAFRANIN-T
Purple-black
cells
Pink to red
cells
Bright purple to
purple-black cells
Bright pink to
fuchsia cells
Purple-black
to purple cells
Red-pink to
fuchsia cells

1. The Gram stain provides preliminary identification information
only and is not a substitute for cultural studies of the specimen.
2. Prior treatment with antibacterial drugs may cause gram-positive
organisms from a specimen to appear gram-negative.
3. Use of an 18-24 hour culture is advisable for best results since
fresh cells have a greater affinity than old cells for most dyes. This
is particularly true of many spore formers, which are strongly
gram-positive when examined in fresh cultures but which later
become gram-variable or gram-negative.
4. The Gram stain reaction, like the acid-fast reaction, is altered by
physical disruption of the bacterial cell wall or protoplast. The cell
walls of gram-positive bacteria interpose a barrier which prevents
leaching of the dye complex from the cytoplasm. Cell walls of
gram-negative bacteria contain lipids soluble in organic solvents,
which are then free to decolorize the cytoplasm. Therefore, a
microorganism that is physically disrupted by excess heating will
not react to Gram staining as expected.
5. 3-Step Gram Safranin-S is intended for use with stabilized iodine.
3-Step Gram Safranin-T is intended for use with traditional iodine.
Unsatisfactory results may occur if other combinations of iodine
and 3-Step Gram Safranin are used.
6. Over time, a fine precipitate may develop in Gram Basic Fuchsin,
3-Step Gram Safranin-S and 3-Step Gram Safranin-T. Product
performance will not be affected.
References
1. Fortschr. Med., 1884, 2:185
2. Donnelly, J. P. 1962. The secrets of Grams stain. Infec. Dis. Alert.
15:109-112.
3. N.Y. Agr. Exp. Sta. Tech. Bull., 1923. 93.
4. Bartholomew, J. W. 1962. Variables influencing results, and the
precise definition of steps in gram staining as a means of
standardizing the results obtained. Stain Technol. 37:139-155.
5. Kruczak-Filipov, P., and R. G. Shively. 1992. Gram Stain
procedure, p. 1.5.1-1.5.18. In H.D. Isenberg (ed.), Clinical
Microbiology Procedures Handbook, vol. 1. American Society for
6. Murray, P. R. (ed.). 1995. Manual of Clinical Microbiology, 6th
ed. American Society of Microbiology, Washington, D.C.
7. Mangels, J. I., M. E. Cox, and L. H. Lindley. 1984. Methanol
fixation. An alternative to heat-fixation of smear. Diag. Microbiol.
Infect. Dis. 2:129-137.
601
TB Stain Sets and Reagents
Section IV
Packaging
Gram Crystal Violet
PRIMARY STAIN
6 x 250 ml
1 gallon
3329-76
3329-83
Gram Iodine
MORDANT
6 x 250 ml
1 gallon
3331-76
3331-83

MORDANT
6 x 250 ml
1 gallon
3342-76
3342-83
Gram Decolorizer
DECOLORIZER
6 x 250 ml
1 gallon
3330-76
3330-83
Gram Safranin
COUNTERSTAIN
6 x 250 ml
1 gallon
3332-76
3332-83
Gram Basic Fuchsin

COUNTERSTAIN
6 x 250 ml
1 gallon
3343-76
3343-83

6 x 250 ml
1 gallon
3335-76
3335-83

6 x 250 ml
1 gallon
3341-76
3341-83
Gram Stain Set

Contents: Gram Crystal Violet
Gram Iodine
Gram Decolorizer
Gram Safranin
4 x 250
250
250
250
250
ml
ml
ml
ml
ml
3328-32
Gram Stain Set

(with Stabilized Iodine)
Gram Decolorizer
Gram Safranin
4 x 250 ml
3338-32
250
250
250
250
ml
ml
ml
ml
3-Step Gram Stain Set-S

3 x 250
250
250
250
ml
ml
ml
ml
3334-3
3-Step Gram Stain Set-T

Gram Iodine
3 x 250
250
250
250
ml
ml
ml
ml
3337-32
50 slides
3140-26
Bactrol Gram Slide
Bacto TB Stain Sets and Reagents

TB Stain Set K . TB Stain Set ZN . TB Fluorescent Stain Set M
TB Fluorescent Stain Set T
Intended Use
TB Fluorescent Stain Set T uses the acid-fast fluorescent procedure

described by Truant, Brett and Thomas.4,12
Bacto TB Stain Sets are used to stain smears prepared from specimens
suspected of containing mycobacteria for early presumptive diagnosis
of mycobacterial infection.
Also Known As
TB Stain Set K is also known as the Kinyoun Stain.
TB Stain Set ZN is also known as the Ziehl-Neelsen Stain.
TB Fluorescent Stain Set M is also known as the Morse Stain.
TB Fluorescent Stain Set T is also known as the Truant Stain.

The microscopic staining technique is one of the earliest methods
devised for detecting the tubercle bacillus and it remains a standard
procedure.1-7 The unique acid-fast characteristic of mycobacteria makes
the staining technique valuable in early presumptive diagnosis, and
provides information about the number of acid-fast bacilli present.
Fluorescent microscopy offers many advantages over classic methods
for detecting mycobacteria because of its speed and simplicity, the ease
of examining the slide, and the reliability and superiority of the method.8
TB Stain Set K uses the Kinyoun (cold) acid-fast procedure described
by Kinyoun.4,9
TB Stain Set ZN uses the Ziehl-Neelsen (hot) acid-fast procedure
described by Kubica and Dye.4,10
TB Fluorescent Stain Set M uses the auramine O acid-fast fluorescent
procedure described by Morse, Blair, Weiser and Sproat.4,11
602
The lipid content of the cell wall of acid fast bacilli makes staining
of these organisms difficult. In acid fast stains, the phenol allows
penetration of the primary stain, even after exposure to acid-alcohol
decolorizers. For an organism to be termed acid fast, it must resist
decolorizing by acid-alcohol. A counterstain is then used to emphasize
the stained organisms, so they may be easily seen microscopically.
When using Stain Set K, acid fast bacilli (AFB) appear red against a
green background if Brilliant Green K is used as the counterstain or
red against a blue background if Methylene Blue is the counterstain.
When using Stain Set ZN, AFB appear red against a blue background
because Methylene Blue is used as the counterstain.
When using Stain Set M, AFB have a bright yellow-green fluorescence.
When using Stain Set T, AFB have a reddish-orange fluorescence.
Formula
3326-TB Stain Set K
Formulas per Liter
3321-TB Carbolfuchsin KF
Basic Fuchsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Phenol USP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Isopropanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Ethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Distilled Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750
g
g
ml
ml
ml
The Difco Manual
Section IV
3318-TB Decolorizer
Hydrochloric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 ml
Denatured Ethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 970 ml
3327-TB Brilliant Green K

Brilliant Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 g
Sodium Hydroxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.02 g
Distilled Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1000 ml
3324-TB Stain Set ZN

Formulas per Liter
3313-TB Carbolfuchsin ZN
Basic Fuchsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.7
Phenol USP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Isopropanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Distilled Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 905
g
g
ml
ml
3318-TB Decolorizer

It is recommended that a positive and negative control slide, such as
Bactrol TB Slide, be included with each batch of slides stained with
acid fast stains.
3313-TB Carbolfuchsin ZN
Appearance:
Reddish-purple suspension with no visible
precipitate.
3314-TB Decolorizer TM
Appearance:
Colorless, clear suspension.
3323-TB Fluorescent Stain Set M

Formulas per Liter
g
g
ml
ml
ml
3317-TB Auramine-Rhodamine T
Appearance:
Red, viscous solution.
Hydrochloric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 ml
Isopropanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 700 ml
3318-TB Decolorizer
Appearance:
3315-TB Potassium Permanganate
3319-TB Methylene Blue

Appearance:
Blue solution with no visible precipitation.
Appearance:
Reddish purple suspension.
Potassium Permanganate . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
3325-TB Fluorescent Stain Set T

Formulas per Liter
3327-TB Brilliant Green

Appearance:
Green solution.
Stain Value
Stain Bactrol TB Slides (3139) using the appropriate TB
stain procedure. Examine slides using a light or fluorescent
microscope at a total magnification of 1000X (oil immersion).
TB STAIN SET K
TB STAIN SET K
USING TB
USING TB
ATCC BRILLIANT GREEN METHYLENE BLUE
TB
STAIN
SET ZN
Dark pink Dark pink

to red
to red
Blue
Blue
Blue
Blue
TB FLUORESCENT
STAIN SET M
TB FLUORESCENT
STAIN SET T
ORGANISM
ATCC
Positive Control
M. tuberculosis H37 Ra
25177
Bright
yellow-green
fluorescence
Reddish, orange
fluorescence
Negative Control
S. aureus
K. pneumoniae
25923
13883
No fluorescence
No fluorescence
No florescence
No florescence
The Difco Manual
Methylene Blue USP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 g

Ethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 ml
Auramine O . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Phenol USP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Glycerine USP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Isopropanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
Distilled Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 650
3316-TB Auramine M
Appearance:
Yellow suspension.
Positive Control
M. tuberculosis H37 Ra 25177 Dark pink
to red
Negative Control
S. aureus
25923
Green
K. pneumoniae
13883
Green
3316-TB Auramine M

Appearance:
Purple solution.
ORGANISM
Hydrochloric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 ml
Denatured Ethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 970 ml
Auramine O . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Rhodamine B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Phenol USP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Glycerine USP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 600
Isopropanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
Distilled Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260
g
g
g
ml
ml
ml
Hydrochloric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 ml
Isopropanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 700 ml

Potassium Permanganate . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 g
Precautions
2. 3313-TB Carbolfuchsin ZN
TOXIC IN CONTACT WITH SKIN AND IF SWALLOWED.EC
CAUSES BURNS. EC POSSIBLE RISK OF IRREVERSIBLE
603
EFFECTS.EC Avoid contact with skin and eyes. Do not breathe mist.
HIGHLY FLAMMABLE. CAUSES BURNS. Avoid contact with
skin and eyes. Do not breathe mist. Wear suitable protective
clothing. Keep container tightly closed. Keep away from sources
of ignition. No smoking.
Avoid contact with skin and eyes. Do not breathe mist. Wear suitable
3316-TB Auramine M
FLAMMABLE. IRRITATING TO EYES, RESPIRATORY SYSTEM AND SKIN. POSSIBLE RISK OF IRREVERSIBLE
EFFECTS.US Avoid contact with skin and eyes. Do not breathe mist.
FLAMMABLE. IRRITATING TO EYES, RESPIRATORY SYSTEM AND SKIN. TOXIC IN CONTACT WITH SKIN AND IF
SWALLOWED.EC CAUSES BURNS.EC POSSIBLE RISK OF
IRREVERSIBLE EFFECTS. Avoid contact with skin and eyes. Do
not breathe mist. Wear suitable protective clothing. Keep container
tightly closed. Keep away from sources of ignition. No smoking.
3318-TB Decolorizer
HIGHLY FLAMMABLE. HARMFUL BY INHALATION AND
IF SWALLOWED. IRRITATING TO EYES, RESPIRATORY
SYSTEM AND SKIN.US POSSIBLE RISK OF IRREVERSIBLE
FLAMMABLE. IRRITATING TO EYES, RESPIRATORY SYSTEM AND SKIN. HARMFUL BY INHALATION AND IF
SWALLOWED. POSSIBLE RISK OF IRREVERSIBLE
EFFECTS.US Avoid contact with skin and eyes. Do not breathe
vapors. Wear suitable protective clothing. Keep container tightly
closed.
FLAMMABLE. IRRITATING TO EYES, RESPIRATORY SYSTEM AND SKIN. HARMFUL BY INHALATION AND IF
SWALLOWED. EC POSSIBLE RISK OF IRREVERSIBLE
FIRST AID:
water and seek medical advice.
After contact with skin, wash immediately with plenty of water.
604
Section IV
If inhaled, remove to fresh air. If not breathing, give artificial

respiration. If breathing is difficult, give oxygen. Seek medical
advice.
If swallowed seek medical advice immediately and show this
container or label.
Storage
Store TB Stain Sets and reagents at 15-30C. Reagents that have been
removed from the packing carton should be stored in the dark.
Expiration Date
Procedure
Materials Provided
TB Stain Set K*
TB Stain Set ZN*
TB Fluorescent Stain Set M*
TB Fluorescent Stain Set T*
Bactrol TB Slides
*Individual reagents available separately. See Packaging.

Microscope slidesnew or cleaned in acid dichromate solution
Staining rack
Microscope with oil immersion lens, OR
Fluorescent microscope
(See #8 of each fluorescent staining procedure for a complete
description of the appropriate assembly required.)

1. Acid fast stains may be performed on any type of clinical specimen
suspected of containing mycobacteria.4-5 Smears from sputum and
other respiratory tract secretions are usually made from concentrated
specimens. For procedures used in concentrating specimens for
acid fast bacilli, please consult appropriate references.4-7
2. Apply a thin smear of the specimen directly on a clear microscope
slide.
3. Allow smear to air dry.
4. Fix the smear to the slide by passing the slide through a low flame
2-3 times, avoiding excessive heat.
Test Procedure
Kinyoun Stain
TB Stain Set K
1. Place slides on a staining rack and flood with TB Carbolfuchsin
KF for 4 minutes. Do not heat.
2 Wash gently in running water.
3. Decolorize with TB Decolorizer for 3-5 seconds, or until no more
red color appears in washing.
4. Wash gently in running water.
5. Counterstain with either TB Brilliant Green K or TB Methylene
Blue (available separately) for 30 seconds.
The Difco Manual
Section IV

7. Air dry. If using TB Methylene Blue, dry over gentle heat.
Ziehl-Neelsen Stain
TB Stain Set ZN
1. Place slides on a staining rack and flood with TB Carbolfuchsin
ZN. Heat gently to steaming and allow to steam for 5 minutes.
3. Decolorize with TB Decolorizer for 3-5 seconds or until no more
red color appears in washing.
5. Counterstain with either TB Methylene Blue or TB Brilliant
Green K for 30 seconds.
7. Dry over gentle heat.
Morse Stain
Fluorescent Stain Set M
1. Place slides on a staining rack and flood with TB Auramine M for
15 minutes.
3. Decolorize with TB Decolorizer TM for 30-60 seconds.
4. Wash slides gently in running water.
5. Counterstain with TB Potassium Permanganate for 2 minutes.
7. Air dry.
8. Examine under a microscope fitted, as described by Morse et al.,11
with an incandescent bulb, a KG 1 heat filter, a 3-4 mm thick BG
excitation filter, an ordinary substage condenser and a No. 51 bright
field or GG barrier filter.
Truant Stain
1. Place slides on a staining rack and flood with TB AuramineRhodamine T that has been thoroughly shaken prior to use. Leave
undisturbed for 20-25 minutes at room temperature.
3. Decolorize with TB Decolorizer TM for 2-3 minutes.
4. Wash gently in running tap water.
5. Counterstain with TB Potassium Permanganate for 4-5 minutes.
7. Blot lightly. Dry in air or very gently over a flame.
8. Examine under a microscope fitted, as described by Truant et al.,13
with 25X objective, an HBO L2 bulb heat filter, a BG 12 primary
filter and OG 1 barrier filter.
Results

1. A positive staining reaction provides presumptive evidence of the
presence of M. tuberculosis in the specimen. A negative staining
reaction does not necessarily indicate that the specimen will be
culturally negative for M. tuberculosis. For positive identification
of M. tuberculosis, cultural methods must be employed.
The Difco Manual
2. Rapidly growing mycobacteria may retain acid-fast stains to a

varying degree. Most rapidly growing mycobacteria will not
fluoresce in fluorochrome-stained smears.4
3. Organisms other than mycobacteria, such as Rhodococcus spp.,
Nocardia spp., Legionella micdadei, and the cysts of
Cryptosporidium spp. and Isospora spp., may display various
degrees of acid-fastness.4
4. When decolorizing with acid-alcohol, avoid under-decolorization.
It is difficult to over-decolorize acid-fast organisms.
5. During the counterstaining step with potassium permanganate,
timing is critical. Quenching the fluorescing bacilli occurs when
counterstaining for a longer period of time.4
6. If fluorochrome stained slides cannot be observed immediately,
they may be stored at 2-8C in the dark for up to 24 hours. This is
required to prevent fading of the fluorescence.4
7. Prolonged counterstaining in non-fluorochrome stains may mask
the presence of acid-fast bacilli. Use of brilliant green may help to
minimize this problem.4
References
1. Ziehl, F. 1882. Zur Frbung des Tuberkelbacillus. Dtsch. Med.
Wochenschr. 8:451.
2. Neelsen, F. 1883. Ein Casuistischer Beitrag zur Lehre von der
Tuberkulose. Centralbl. Med. Wiss. 21:497-501.
3. National Tuberculosis Association. 1961. Diagnostic Standards
and Classification of Tuberculosis. National Tuberculosis Association, New York, NY.
4. Master, R. N. 1992. Mycobacteriology, p. 3.0.1-3.16.4. In
Isenberg, H. D. (ed.), Clinical 2microbiology procedures handbook,
5. Nolte, F. S., and B. Metchock. 1995. Mycobacterium, p. 400-437.
Yolken (eds.), Manual of clinical microbiology, 6th ed. American
St. Louis, MO.
7. Kent, P. T., and G. P. Kubica. 1985. Public Health
Mycobacteriology: a guide for the Level III laboratory, p. 57-68.
U.S. Department of Health and Human Services, Centers for
Disease Control, Atlanta, GA.
8. Taylor, R. D. 1966. Modification of the Brown and Brenn Gram
Stain for the differential staining of gram-positive and gramnegative bacteria in tissue sections. Am. J. Clin. Pathol. 46:472-4.
9. Kinyoun, J. J. 1915. A note on Uhlenhuths method for sputum
examination for tubercle bacilli. Am. J. Pub. Health. 5:867-70.
10. Kubica, G. P., and W. E. Dye. 1967. Laboratory methods for
clinical and public health Mycobacteriology. U.S.P.H. Serv.
Publication No., 1547; Superintendent of Documents, U.S.
Government Printing Office, Washington, D.C.
11. Fitzsimmons General Hospital. 1968. Mycobact. Lab. Methods.
Rept. No. 17., May, 1968.
12. Truant, J. P., W. A. Brett, and W. Thomas. 1962. Fluorescence
microscopy of tubercle bacilli stained with auramine and
rhodamine. Bull. Henry Ford Hosp. 10:287-296.
605
Section IV
13. Willis, H. S., and M. M. Cummings. 1952. Diagnostic and

Experimental Methods in Tuberculosis, 2nd ed. Charles C. Thomas,
Springfield, IL.
Packaging
TB Stain Set K
Contains:
TB Carbolfuchsin KF
TB Decolorizer
TB Brilliant Green K
TB Stain Set ZN
Contains:
TB Carbolfuchsin ZN
TB Decolorizer
TB Methylene Blue
TB Fluorescent Stain Set M
Contains:
TB Auramine M
TB Decolorizer TM
TB Potassium Permanganate
Contains:
TB Auramine-Rhodamine T
TB Decolorizer TM
3 x 250 ml
3326-32
250
250
250
3 x 250
ml
ml
ml
ml
3324-32
250
250
250
3 x 250
ml
ml
ml
ml
3323-32
250
250
250
3 x 250
ml
ml
ml
ml
3325-32
250 ml
250 ml
250 ml
TB Auramine M
6 x 250 ml
3316-76
TB Auramine-Rhodamine T
6 x 250 ml
3317-76
TB Brilliant Green K
6 x 250 ml
3327-76
TB Carbolfuchsin KF
6 x 250 ml
3321-76
TB Carbolfuchsin ZN
6 x 250 ml
3313-76
TB Decolorizer
6 x 250 ml
3318-76
TB Decolorizer TM
6 x 250 ml
3314-76
TB Methylene Blue
6 x 250 ml
3319-76
6 x 250 ml
3315-76
Bactrol TB Slides
606
50 slides
3139-26
The Difco Manual
Section V
Bordetella Antigens and Antiserum
Bacto Bordetella Antigens and Antiserum

Bordetella Pertussis Antiserum . Bordetella Parapertussis
Antiserum . Bordetella Pertussis Antigen
Intended Use
Bacto Bordetella Pertussis Antiserum and Bacto Bordetella
Parapertussis Antiserum are used in the slide agglutination test for
identifying Bordetella pertussis and Bordetella parapertussis.
Bacto Bordetella Pertussis Antigen is used to demonstrate a positive
quality control test in the slide agglutination test.

All members of the genus Bordetella are respiratory pathogens of
warm-blooded animals. Two species, B. pertussis and B. parapertussis,
are uniquely human pathogens. These organisms adhere to, multiply
among and remain localized in the ciliated epithelial cells of the
respiratory tract. B. pertussis is the major cause of whooping cough or
pertussis. B. parapertussis is associated with a milder, less frequently
occurring form of the disease.1 Person-to-person transmission occurs
by the aerosol route. Pertussis is a highly contagious disease that, more
than 90% of the time, attacks unimmunized populations.2 Toxin
production remains the major distinction between B. pertussis and
B. parapertussis.
Classic pertussis caused by B. pertussis occurs in three stages. The
first (catarrhal) stage is characterized by nonspecific symptoms similar
to a cold or viral infection. The disease is highly communicable during
this stage, which lasts 1-2 weeks. During the second (paroxysmal)
stage, the cough increases in intensity and frequency. This stage
is marked by sudden attacks of severe, repetitive coughing, often
culminating with the characteristic whoop. The whooping sound is
caused by the rapid inspiration of air after the clearance of mucusblocked airways.3 This stage may last 1-4 weeks. The beginning of the
convalescent stage is marked by a reduction in frequency and severity
of coughing spells. Complete recovery may require weeks or months.

Bordetella Pertussis Antiserum
Bordetella Parapertussis Antiserum
Lyophilized Appearance: Light gold to amber, button to
powdered cake.
Rehydrated Appearance: Light gold to amber, clear liquid.
Bordetella Pertussis Antigen
Appearance:
Light gray to white suspension, may
settle upon standing.
Rehydrate Bordetella Pertussis and Parapertussis Antiserum
per label directions. Test as described (see Test Procedure).
Bordetella Pertussis Antigen or known positive and negative
control cultures must give appropriate reactions.
The Difco Manual
Despite the availability of an effective whole-cell vaccine, pertussis

remains a disease of worldwide distribution because many developing
nations do not have the resources for vaccinating their populations.4
Major outbreaks have occurred even in developed nations such as Great
Britain and Sweden. Pertussis is endemic in the United States, with
most disease occurring as isolated cases. There has been a shift in the
age group affected by the disease. In the past, children in the 1-5 year
age group were more prone to pertussis. Since adults do not receive
booster vaccinations, children less than one year of age2 have become
more susceptible because of a decrease in passively transferred maternal
antibodies.
Bordetella are tiny, gram-negative, strictly aerobic coccobacilli that
occur singly or in pairs and may exhibit a bipolar appearance. While
some species are motile, B. pertussis and B. parapertussis are nonmotile.
They do not produce acid from carbohydrates. B. pertussis will not
grow on common blood agar bases or chocolate agar, while
B. parapertussis will grow on blood agar and sometimes on chocolate
agar. Media for primary isolation must include starch, charcoal, ionexchange resins or a high percentage of blood to inactivate inhibitory
substances.3 B. pertussis may be recovered from secretions collected
from the posterior nasopharynx, bronchoalveolar lavage and
transbronchial specimens.

Identification of Bordetella species includes isolation of the microorganisms, biochemical identification and serological confirmation.
Serological confirmation involves the reaction in which the microorganism (antigen) reacts with its corresponding antibody. The in vitro
reaction produces macroscopic clumping called agglutination. The
desired homologous reaction is rapid, does not dissociate (has high
avidity), and binds strongly (has high affinity).
Because a microorganism (antigen) may agglutinate with an antibody
produced in response to some other species, heterologous reactions
are possible. These are weak in strength or slow in formation. Such
unexpected and, perhaps, unpredictable reactions may lead to some
confusion in serological identification. Therefore, a positive homologous
agglutination reaction should support the morphological and biochemical
identification of the microorganism.
Homologous reactions are rapid and strong. Heterologous reactions
are slow and weak.
Reagents
Bordetella Pertussis and Parapertussis Antisera are lyophilized,
polyclonal rabbit antiglobulins containing approximately 0.04%
Thimerosal as a preservative. When rehydrated and used as described,
each 1 ml vial of Bordetella Pertussis or Parapertussis Antiserum
diluted 1:10 contains sufficient reagent for 200 slide tests.
609
Bordetella Antigens and Antiserum
Section V
Bordetella Pertussis Antigen is a ready-to-use suspension of killed,

whole organisms adjusted to a density approximating two times a
McFarland Barium Sulfate Standard No. 3 (9 x 108 organisms per ml).
Bordetella Pertussis Antigen contains 0.04% Thimerosal. When used
as described, each 5 ml vial contains sufficient reagent to perform
approximately 140 slide tests.
Because antigen density may vary, it is adjusted to ensure optimum
performance when the antigen is standardized with hyperimmune sera
obtained from laboratory animals.
are very small, white, glistening, convex, entire and usually exhibit
tiny zones of hazy hemolysis. Colonies of B. parapertussis are usually
larger than those of B. pertussis, may have a slightly brown color, and
do not have a glistening surface. For specific recommendations for
culture and identification, consult appropriate references.3,5 Determine
that a pure culture of the microorganism has been obtained and that
biochemical test reactions are consistent with the identification of
the organism as Bordetella. After these criteria are met, serological
identification can proceed.
Precautions
Test Procedure

2. Bordetella Pertussis Antiserum
Bordetella Parapertussis Antisera
The Packaging of This Product Contains Dry Natural Rubber.
3. Bordetella Pertussis Antigen is not intended for use in the immunization of humans or animals.
1. Bordetella Antiserum: On an agglutination slide, dispense

2 separate drops (approximately 35 l, each) of the antiserum to be
tested, the first to be used for the test isolate and the second for the
positive control.
2. Negative control: Dispense 1 drop of sterile 0.85% NaCl solution.
3. Test isolate: Transfer a loopful of isolated colony from a solid agar
medium to the first drop of antiserum and a second loopful to the
negative control (0.85% NaCl solution).
4. Positive control: As appropriate, add 1 drop of Bordetella Pertussis
Antigen or a small amount of a known B. pertussis or B. parapertussis
culture to the second drop of antiserum.
5. Mix each test and control serum reaction area using separate
applicator sticks.
6. Rotate the slide for 1 minute and read for agglutination.
Storage
Store lyophilized and rehydrated Bordetella Pertussis Antiserum and
Bordetella Parapertussis Antiserum at 2-8C.
Store Bordetella Pertussis Antigen at 2-8C.
Expiration Date
Procedure
Materials Provided

Agglutination slides
Applicator sticks
Sterile 0.85% NaCl solution
Inoculating loop
Reagent Preparation
Equilibrate all materials to room temperature before performing the
test. Ensure that all glassware and pipettes are clean and free of
detergent residues.
Bordetella Pertussis and Parapertussis Antiserum: To rehydrate, add
1 ml sterile distilled or deionized water and rotate gently to completely
dissolve the contents. Dilute the rehydrated antiserum 1:10 with
sterile 0.85% NaCl solution. Dilute only enough for 1-2 days testing
requirements.
Bordetella Pertussis Antigen is ready to use.

Isolation of Bordetella from clinical specimens requires the use of
certain media such as Bordet-Gengou Agar. Colonies of B. pertussis
610
Results
1. Read and record results as follows.
4+ 100% agglutination; background is clear to slightly hazy.
3+ 75% agglutination; background is slightly cloudy.
2+ 50% agglutination; background is moderately cloudy.
1+ 25% agglutination; background is cloudy.
No agglutination.
2. Positive control: Should produce 3+ or greater agglutination.
Negative control: Should produce no agglutination.
Positive test result: Agglutination of 3+ or greater within 1 minute.

1. Correct interpretation of serological reactions depends on culture
purity as well as morphological characteristics and biochemical
reactions that are consistent with identification of the microorganism
as a Bordetella species.
2. Serological methods alone cannot identify the isolate as Bordetella.
3. Excessive heat from external sources (hot bacteriological loop,
burner flame, light source, etc.) may prevent making a smooth
suspension of the microorganism or may cause evaporation or
precipitation of the test mixture. False-positive reactions may occur.
4. Rough culture isolates do occur and will agglutinate spontaneously
causing agglutination of the negative control (autoagglutination).
Smooth colonies must be selected and tested in serological procedures.
5. Prolonged exposure of reagents to temperatures other than those
specified is detrimental to the products.
6. A rehydrated Bordetella Antiserum that is cloudy or develops a
precipitate during use should be discarded.
The Difco Manual
Section V
Brucella Antigens and Antisera
7. Some Hemophilus species will grow on Bordetella isolation media

and may cross-react with B. pertussis antisera. Rule out X- and
V-factor dependence using Differentiation Disks V, X and VX.5
8. Shake the antigen vial well before use to obtain a smooth, uniform
suspension. Occasionally, a Bordetella suspension may settle out
during storage.
9. Bordetella antigen will display irreversible autoagglutination if, at
anytime during shipment or storage, it is subjected to freezing
temperatures. Do not allow to freeze.
References
recent experience and a review of the literature. Am. J. Dis. Child
131:560-563.
2. Bass, J. W., and S. R. Stephenson. 1987. The return of pertussis.
Pediatr. Infect. Dis. J. 6:141-144.
3. Marcon, M. J. 1995. Bordetella, p. 566-573. In P. R. Murray,

4. Wright, P. F. 1991. Pertussis in developing countries: definition
of the problem and prospects for control. Rev. Infect. Dis.
13:S228-S234.
5. Pezzlo, M. 1992. Aerobic bacteriology, p. 1.0.1-1.20.47. In H. D.
Isenberg (ed.) Clinical microbiology procedures handbook, vol. 1.
6. Rose, N. R., H. Friedman, and J. L. Fahey (ed.). 1986. Manual
of clinical laboratory immunology, 3rd ed. American Society for
Packaging
1 ml
2309-50
1 ml
2310-50
5 ml
2585-56
Bacto Brucella Antigens and Antisera

Brucella Abortus Antigen (Slide) . Brucella Abortus Antigen (Tube)
Brucella Melitensis Antigen (Slide) . Brucella Suis Antigen (Slide)
Brucella Abortus Antiserum . Febrile Negative Control
Intended Use
Bacto Brucella Abortus Antigen (Slide) and (Tube), Brucella Melitensis
Antigen (Slide) and Brucella Suis Antigen (Slide) are used in the detection
of antibodies by the slide and tube agglutination tests (as indicated).
Bacto Brucella Abortus Antiserum is used to demonstrate a positive
quality control test reaction in the slide and tube agglutination tests.
Bacto Febrile Negative Control is used to demonstrate a negative
quality control test reaction in the slide agglutination test.

Brucellae are intracellular parasites that, upon invasion, produce fever
in their host. Consequently, they are often called Febrile Antigens.
Brucellae also cause localized infection of bone, tissue and organ
systems in humans.1,2 These organisms are intracellular pathogens of
the reticuloendothelial system. They form granulomatous masses in
various organs.
Brucellosis is the disease state caused by these organisms. The disease
has an abrupt onset, usually three to four weeks after exposure.
Symptoms of the disease include fever, arthralgia, malaise, chills and
sweating. Approximately 70% of patients with acute brucellosis have
a tube agglutination titer of 1:160 or greater.3 Osteomyelitis is the most
frequent complication in humans.4,5
Most cases of brucellosis are due to exposure to animals, including
cattle, sheep, goats and swine, or to laboratory cultures of Brucella
species.3 Patient history usually includes exposure to livestock or to meat
processing. Routes of infection are nasopharyngeal, gastrointestinal,
The Difco Manual
conjunctival, respiratory and through abraded skin.5 Sporadic episodes

of food-associated brucellosis have been caused by B. melitensis.6,7
The human immune response to a particular microorganism results in
measurable antibody production which, in some cases, can assist in
completing the patients clinical diagnosis. In blood samples, the
antibody titer during the initial phase of the infection (acute) is
compared to the antibody titer 7-14 days later (convalescent). A high
acute phase antibody titer (1:320) and paired acute and convalescent
samples that show an increase in antibody titer are helpful in assisting
the diagnosis of brucellosis.
A preliminary test using either the rapid slide test and/or the macroscopic
tube test may be performed on the initial serum specimen and reported
to the physician at that time. An aliquot of the serum should be transferred
to a sterile test tube, sealed tightly, and kept in the freezer. When the
second serum is obtained, it should be run in parallel with the original
specimen. In this manner, the original serum will serve as a control.
Any difference in titer will be more credible because the bias associated
with the performance of the test and determining the endpoint will be
reduced.
Brucellae are small, nonmotile, nonencapsulated gram-negative
coccobacilli. The organisms grow aerobically and their growth is
enhanced by incubation in CO2.
Six species of Brucella have been recognized. Among these, B. abortus
(cattle), B. suis (swine), B. melitensis (goats and sheep) and B. canis
(dogs) are infective for humans, although B. canis infections in humans
are rare.8
611
The rapid slide procedure is a screening test designed to detect

agglutinins. The tube test is a confirmatory procedure designed to
quantitate febrile agglutinins. It is, therefore, necessary that any
positive results obtained in the screening (slide test) of specimens be
confirmed by a tube test. The tube agglutination test is used clinically
in the United States.9,10,11
Certain organisms may share cross-reacting antigens leading to the
production of heterologous antibodies. These heterologous antibodies
may react with one or more antigens in a febrile antibody test procedure,
causing low-level antibody titers that may not singly be indicative of
disease. Cross reactions between Brucella and Francisella tularensis,
Yersinia enterocolitica and Vibrio cholerae can occur.

Agglutination tests involving the use of Brucella antigens detect the
presence of antibodies that react with the test antigen. The serological
procedure involves serially diluting the patient serum and then adding
a standard volume of antigen. The endpoint of the test is the last dilution of the serum that shows a specific amount of agglutination. The
end point, reported as a dilution of the serum, is called the patients
antibody titer.
Reagents
Brucella Abortus Antigen (Slide), Brucella Melitensis Antigen
(Slide) and Brucella Suis Antigen (Slide) are ready-to-use, chemically
inactivated and stabilized suspensions of Brucella abortus 1119-3,12
Brucella melitensis and Brucella suis, respectively. The slide antigens
Section V
contain approximately 2% packed cells and 20% glycerin, as well as

0.5% phenol and approximately 0.002% crystal violet and 0.005%
brilliant green as preservatives. When used as described, each 5 ml vial
contains sufficient reagent for 20 slide tests.
Brucella Abortus Antigen (Tube) is a ready-to-use suspension of
Brucella abortus 1119-312 adjusted to a density approximating a
Brucella Abortus Antigen (Tube) contains 0.5% phenol as a preservative but does not contain dye. When used as directed, each 25 ml vial
contains sufficient reagent for 6 tests.
Because antigen density may vary, density is adjusted to ensure optimum
obtained from laboratory animals. Variation in antigen color intensity
is normal and will not affect the outcome of the test.
Brucella Abortus Antiserum is a lyophilized, polyclonal rabbit antiserum containing approximately 0.04% Thimerosal as a preservative.
Brucella Abortus Antiserum is unabsorbed. Serological cross-reactions
occur in unabsorbed sera from Brucella species because B. abortus, B.
suis and B. melitensis are antigenically related, containing common A
(abortus) and M (melitensis) substances. (Monospecific sera prepared
by absorption produce weak, unstable reagents that make interpretation
of agglutination results difficult.)
When rehydrated and used as described, each 3 ml vial of Brucella
Abortus Antiserum contains sufficient reagent for 19 slide tests or
30 tube tests.
Febrile Negative Control is a standard protein solution containing

approximately 0.04% Thimerosal as a preservative. When used as
described, each 3 ml vial contains sufficient reagent for 32 slide tests.
Precautions
Brucella Abortus Antigen (Slide), Brucella Suis Antigen

(Slide), Brucella Melitensis Antigen (Slide)
Appearance:
Turquoise, blue-violet suspension.
Brucella Abortus Antigen (Tube)
Appearance:
Light gray to white suspension.
Brucella Abortus Antiserum
powdered cake.
Febrile Negative Control
Lyophilized Appearance: Colorless to light gold, button to
powdered cake.
Rehydrated Appearance: Colorless to light gold, clear liquid.
Rehydrate Brucella Abortus Antiserum and Febrile Negative
Control per label directions. Perform the slide or tube
agglutination test using Brucella Abortus Antigen (Tube),
Brucella Abortus (Slide), Brucella Suis (Slide), or Brucella
Melitensis (Slide). Dilute both positive and negative controls
in the same proportion as the patients serum and process in
the same manner, following appropriate procedure.
An antigen is considered satisfactory if it does not agglutinate
with the negative control and yields a 2+ reaction at a titer of
1:80 or more with the positive control.
612

2. Brucella Abortus Antiserum
3. Observe universal blood and body fluid precautions in the
handling and disposing of specimens.13,14
4. Brucella Antigens are not intended for use in the immunization of
humans or animals.
Storage
Store Brucella Antigens (Slide) and (Tube) at 2-8C.
Store lyophilized and rehydrated Brucella Abortus Antiserum at 2-8C.
Store lyophilized and rehydrated Febrile Negative Control at 2-8C.
Expiration Date
Procedure
Materials Provided
Brucella Abortus Antigen (Slide)
Brucella Suis Antigen (Slide)
The Difco Manual
Section V
Brucella Melitensis Antigen (Slide)


Slide Test
Agglutination slides with 5 squares
Applicator sticks
Serological pipettes, 0.2 ml
Tube Test
Culture tubes, 12 x 75 mm, and rack
Waterbath, 35-37C
Serological pipettes, 1 ml and 5 ml
Reagent Preparation
Brucella Abortus Antigen (Slide) and (Tube), Brucella Suis Antigen
(Slide) and Brucella Melitensis Antigen (Slide) are ready to use.
Equilibrate all materials to room temperature prior to performing
the tests. Ensure that all glassware and pipettes are clean and free of
detergent residues.
Brucella Abortus Antiserum: To rehydrate, add 3 ml sterile 0.85%
NaCl solution and rotate gently to completely dissolve the contents.
The rehydrated antiserum is considered a 1:2 working dilution.
Febrile Negative Control: To rehydrate, add 5 ml sterile distilled or
deionized water and rotate gently to completely dissolve the contents.

Collect a blood specimen by aseptic venipuncture. After the specimen
has clotted, centrifuge to obtain the serum required for the test. Serum
specimens must be clear, free of hemolysis and show no visible
evidence of bacterial contamination (turbidity, hemolysis or particulate
matter). Consult appropriate references for more information on the
collection of specimens.15,16
Store serum specimens at room temperature for no longer than 4 hours;
for prolonged storage, keep at 2-8C for up to 5 days or maintain below
-20C. Serum specimens must not be heated; heat may inactivate or
destroy certain antibodies.
Slide Test
Use the slide test only as a screening test. Confirm positive results with
the tube test.
In some cases of brucellosis, sera may display a prozone reaction, the
inability of an antigen to react at higher serum antibody concentrations.
It is advisable to run all 5 serum dilutions of the rapid slide test, rather
than just one dilution, to eliminate the possibility of missing positive
reactions due to the prozone phenomenon.
1. Test serum: Using a 0.2 ml serological pipette, dispense 0.08, 0.04,
0.02, 0.01 and 0.005 ml of each test serum into a row of squares on
the agglutination slide.
The Difco Manual
2. Positive control: Using a 0.2 ml serological pipette, dispense 0.08,

0.04, 0.02, 0.01 and 0.005 ml of Brucella Abortus Antiserum into a
row of squares on the agglutination slide.
3. Negative control: Using a 0.2 ml serological pipette, dispense 0.08,
0.04, 0.02, 0.01 and 0.005 ml of Febrile Negative Control into a
4. Antigen: Shake the vial of Brucella Antigen (Slide) well to ensure
a smooth, uniform suspension. Dispense 1 drop (35 l) of antigen
in each drop of test serum, positive control and negative control.
5. Mix the rows of test and control serum, using a separate applicator
stick for each row. Start with the most dilute mixture (0.005 ml)
and work to the most concentrated (0.08 ml).
7. The final dilutions in squares 1-5 correspond to tube dilutions of
1:20, 1:40, 1:80, 1:160 and 1:320, respectively.
Tube Test
1. In a rack, prepare a row of 8 culture tubes (12 x 75 ml) for each test
serum, including a positive control row for the Brucella Abortus
Antiserum and an antigen control row for the Febrile Negative
Control Serum.
2. Dispense 0.9 ml of sterile 0.85% NaCl solution in the first tube of
each row and 0.5 ml in the remaining tubes.
3. Test serum: Using a 1 ml serological pipette, dispense 0.1 ml of
serum in the first tube in the row and mix thoroughly. Transfer
0.5 ml from tube 1 to tube 2 and mix thoroughly. Similarly, continue
transferring 0.5 ml through tube 7, discarding 0.5 ml from tube 7
after mixing. Proceed in like manner for each serum to be tested.
Tube 8 is the antigen control tube and contains only sterile 0.85%
NaCl solution.
4. Positive control: Using a 1 ml serological pipette, dispense 0.1 ml
of Brucella Abortus Antiserum in the first tube in the row and mix
thoroughly. Transfer 0.5 ml from tube 1 to tube 2 and mix
thoroughly. Similarly, continue transferring 0.5 ml through tube 7,
discarding 0.5 ml from tube 7 after mixing. Tube 8 is the antigen
control tube and contains only sterile 0.85% NaCl solution.
5. Antigen Control: Shake the vial of Brucella Abortus Antigen
(Tube) to ensure a smooth, uniform suspension. Add 0.5 ml of
antigen to all 8 tubes in each row and shake the rack to mix the
suspensions.
6. Final dilutions in tubes 1-7 are 1:20, 1:40, 1:80, 1:160, 1:320, 1:640
and 1:1280, respectively.
7. Incubate in a waterbath at 35-37C for 48 3 hours.
8. Remove from the waterbath. Avoid excessive shaking before
reading the reactions, either when the tubes are in the waterbath
or when removing them from the waterbath.
9. Read and record results.
Results
No agglutination.
613
Section V
2. Positive control: Should produce 2+ or greater agglutination at a

1:80 dilution.
Negative controlRapid Slide Test, only: Should produce no
agglutination.
Antigen controlMacroscopic Tube Test, only: Should show no
agglutination in tube #8 of each row.
If results for either the positive or the negative control are not as
specified, the test is invalid and results cannot be reported.
Test serum: The titer is the highest dilution that shows 2+
agglutination.
Refer to Table 1 and Table 2 for examples of test reactions.
3. The Rapid Slide Test is a screening test, only; results must be confirmed using the Macroscopic Tube Test.
Table 1. Sample Rapid Slide Test reactions.
SERUM (ml)
CORRELATED
TUBE DILUTION
0.08
1:20
0.04
1:40
0.02
1:80
0.01
1:160
0.005
1:320
Serum titer
REACTIONS
SPECIMEN 1
SPECIMEN 2
SPECIMEN 3
3+
2+
1+
1:40
4+
4+
3+
3+
1+
1:160
4+
3+
2+
+
1:80
Table 2. Sample Macroscopic Tube Test reactions.

REACTIONS
SERUM DILUTION
SPECIMEN 1
SPECIMEN 2
SPECIMEN 3
1:20
1:40
1:80
1:160
1:320
1:640
1:1280
Serum titer
4+
4+
3+
2+
1+
1:160
3+
2+
1+
1:40
4+
4+
4+
4+
3+
2+
1+
1:640
Interpretation
For a single serum specimen, a titer of 1:80 is a weak positive that
suggests infection, but not necessarily a recent infection.3,17
A two-dilution increase in the titer of paired serum specimens (from the
acute to the convalescent serum) is significant and suggests infection.
A one-dilution difference is within the limits of laboratory error.
Past history in the use of Brucella suspensions has produced a pattern
of titers that are considered significant. A titer of 1:80 is considered
a weakly positive result while most patients with acute undulant fever
demonstrate a titer of 1:160 or greater.
Limitations of the Procedure18,19

1. The slide test is intended for screening only and results should be
confirmed by the tube test. Slide test dilutions are made to detect a
prozone reaction and do not represent true quantitation of the
614
antibody. A serum specimen with a prozone reaction shows no

agglutination because of excessively high antibody concentrations.
To avoid this occurrence, all 5 slide test serum dilutions should be run.
The detection of antibodies in serum specimens may complete the
clinical picture of brucellosis. However, isolation of the causative
agent from patient specimens may be required. A definitive
diagnosis must be made by a physician based on patient history,
physical examination and data from all laboratory tests.
3. The accuracy and precision of the tests can be affected not only by
test conditions but also by the subjectivity of the person reading
the endpoint.
4. Cross-reactions may occur due to antigenic similarities to other
organisms. A definite serological relationship exists between
Brucella and Francisella tularensis. Cross-reactions may also
occur between Brucella-positive sera and Proteus OX19 antigen,
Vibrio cholerae or Yersinia enterocolitica serotype 9.20
5. While a single serum specimen showing a positive reaction at a
1:80 dilution suggests infection, it is not diagnostic. An antibody
titer greater than 1:160 may occur in healthy individuals with a
past history of the disease.
6. To test for a significant rise in antibody titer, at least two specimens
are necessary, an acute specimen obtained at the time of initial
symptoms and a convalescent specimen obtained 7 to 14 days later.
A two-dilution increase in titer is significant and suggests infection.
8. Exposure to temperatures below 2C can cause autoagglutination.
Antigens must be smooth, uniform suspensions. Examine antigen
vials for agglutination before use. Agglutinated suspensions are
not usable and should be discarded.
9. Adhering to the recommended time and temperature of incubation
is important when performing the tube test. For best results, locate
the waterbath in an area free of mechanical vibration.
10. Serum specimens from patients suffering from acute brucellosis
demonstrate little or no antibody titer during the first 10 days of
the disease.
11. Serological interpretation of an agglutinin titer in vaccinated individuals should be avoided since antibody levels may persist for
years.
12. Individuals who have recovered from brucellosis may demonstrate
a nonspecific agglutinin response upon infection with an etiological agent of a heterologous febrile species.
References
1. Moyer, N. P., and L. A. Holcomb. 1988. Brucellosis, p. 143-154.
In A. Balows, W. J. Hausler, Jr., M. Ohashi, and A. Tubano (ed.),
Laboratory diagnosis and infectious diseases: principles and
practice, vol. 1. Springer and Verlag, New York, NY.
2. Smith, L. D., and T. A. Ficht. 1990. Pathogenesis of Brucella.
Crit. Rev. Microbiol. 17:209-230.
P, R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H.
Society for Microbiology, Washington, D. C.
The Difco Manual
Section V
Candida Albicans Antiserum
4. Potasman, I., L. Even, M. Banai, E. Cohen, D. Angel, and

M. Jaffe. 1991. Brucellosis: an unusual diagnosis for a seronegative patient with abscesses, osteomyelitis, and ulcerative
colitis. Rev. Infect. Dis. 13:1039-1042.
5. Young, E. J. 1983. Human brucellosis. Rev. Infect. Dis. 5:821-842.
6. Arnow, P. M., M. Smaron, and V. Ormiste. 1984. Brucellosis in
a group of travelers to Spain. J. Am. Med. Assoc. 251:505-507.
7. Centers for Disease Control. 1983. Brucellosis - Texas. Morbid.
Mortal. Weekly Rep. 32:548-553.
8. Rose, N. R., H. Friedman, and J. L. Fahey, (ed.). 1986. Manual
of clinical immunology, 3rd ed. American Society for Microbiology,
Washington, D. C.
9. Moyer, N. P., G. M. Evans, N. E. Pigott, J. D. Hudson, C. E.
Farshy, J. C. Feeley, and W. J. Hausler, Jr. 1987. Comparison of
serologic screening tests for brucellosis. J. Clin. Microbiol.
25:1969-1972.
10. Peiris, V., S., Fraser, M. Fairhurst, D. Weston, and
E. Kaczmarski. 1992. Laboratory diagnosis of Brucella infection:
some pitfalls. Lancet 339:1415.
11. Young, E. J. 1991. Serologic diagnosis of human brucellosis:
analysis of 214 cases by agglutination tests and review of the
literature. Rev. Infect. Dis. 13:359-372.
12. Spink, W. W., N. D. McCullough, L. M. Hutchings, and C. K.
Mingle. 1954. A standardized antigen for agglutination technique
for human brucellosis. Report No. 3 of the National Research
Council, Committee on Public Health Aspects of Brucellosis. Am.
J. Patho. 24:496-498.
hepatitis B virus, and other bloodborne pathogens in health-care
387-388.

Department of Labor. 1991. 29 CFR part 1910. Occupational
exposure to bloodborne pathogens; final rule. Federal Register
56:64175-64182.
Isenberg (ed.), Clinical microbiology procedures handbook, vol.
16. Miller, J. M., and H. T. Holmes. 1995. Specimen collection, transport
and storage. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C.
17. Hausler, W. J. Jr., and F. P. Knontz. 1970. Brucellosis. In H. L.
Bodily, E. L. Updyke, and J. O. Mason (ed.), Diagnostic
procedures for bacterial, mycotic and parasitic infections, 5th ed.
American Public Health Association, New York, NY.
18. Alton, G. G., L. M. Jones, and D. E. Peitz. 1975. Laboratory
techniques in brucellosis. World Health Organization Monogr.
Ser No. 55.
19. McCullough, N. D. 1976. Immune response to Brucella, p. 304311. In N. R. Rose and H. Friedman (ed.), Manual of clinical
immunology. American Society for Microbiology, Washington, D.C.
20. Ahovnen, P., E. Jansson, and K. Aho. 1969. Marked crossagglutination between brucellae and a subtype of Yersinia
enterocolitica. Acta Pathol. Microbiol. Scand. 75:291-295.
Packaging
5 ml
2909-56
25 ml
2466-65
5 ml
2916-56
Brucella Melitensis Antigen (Slide)

Brucella Suis Antigen (Slide)
5 ml
2915-56
3 ml
2871-47
3 ml
3239-56
Bacto Candida Albicans Antiserum
Intended Use
Bacto Candida Albicans Antiserum is used in the slide agglutination
test for identifying Candida albicans.

Candida albicans is an opportunistic pathogen. Infection with this
organism will usually arise from an endogenous source in a compromised
host. Candidiasis caused by C. albicans presents as superficial infections
of the skin, oral thrush, systemic and disseminated infections involving
most internal organs, and mucocutaneous candidiasis.1 Vaginitis caused
by C. albicans is the most common type of yeast infection.
C. albicans, a saprophyte, appears in large numbers throughout the
oral-gastrointestinal tract of many warm-blooded vertebrates.1 It is
rarely isolated from normal skin. Person-to-person transmission of
candidiasis can occur.
Candida albicans appears to possess many virulence attributes that
may promote successful parasitism. These attributes include rapid
The Difco Manual
germination upon seeding tissue from the bloodstream,3 protease

production,4 complement protein-binding receptor,5,6 surface variation
and hydrophobicity.7
Candida albicans will grow on Sabauroud Dextrose Agar as white to
cream-colored, butyrous colonies. C. albicans can be isolated from
blood agar as a colony with short marginal extensions. Microscopically,
C. albicans produces budding yeast cells, pseudohyphae or true
hyphae. The organism may be identified by the production of germ
tubes or chlamydospores. Identification of Candida albicans includes
both biochemical and serological confirmation.8

Serological confirmation requires that the microorganism (antigen)
react with its corresponding antibody. This in vitro reaction produces
macroscopic clumping called agglutination. The desired homologous
reaction is rapid, does not dissociate (has high avidity), and bonds
strongly (has high affinity).
615

produced in response to another species, heterologous reactions are
possible. These are weak in strength or slow in formation. Such
unexpected and, perhaps, unpredictable reactions may lead to some
Agglutination of the somatic antigen in the slide test appears as a firm
granular clumping. Homologous reactions occur rapidly and are strong
(3+). Heterologous reactions are slow and weak.
Reagents
Candida Albicans Antiserum is a lyophilized, polyclonal rabbit antiserum
containing approximately 0.04% Thimerosal as a preservative.
When rehydrated and used as described, each 3 ml vial contains
sufficient Candida Albicans Antiserum for 60 tests.
Precautions
2. The Packaging of This Product Contains Dry Natural Rubber.
Storage
Store lyophilized and rehydrated Candida Albicans Antiserum at 2-8C.
Expiration Date
Procedure
Materials Provided

Applicator sticks
0.85% Sodium Chloride
Inoculating loop

powdered cake.
Rehydrate Candida Albicans Antiserum per label directions.
Perform the slide agglutination test using appropriate positive
and negative control cultures. The negative control should
show no agglutination. Homologous cultures should give a
3+ or greater agglutination.
616
Section V
Reagent Preparation
Equilibrate all materials to room temperature before performing the tests.
Ensure that all glassware and pipettes are clean and free of residues
such as detergents.
Candida Albicans Antiserum: To rehydrate, add 3 ml sterile 0.85%
NaCl solution and rotate gently to dissolve the contents completely.

Candida albicans can be recovered on Tryptic Soy Agar with 5% Sheep
Blood, Sabouraud Dextrose Agar or Brain Heart Infusion Agar. For
specific recommendations on isolation of Candida albicans from
clinical specimens, consult appropriate references.9,10,11
Test Procedure
1. Culture the test organism on Sabouraud Dextrose Agar at room
temperature.
2. Candida Albicans Antiserum: Dispense one drop at one end of a
microscope slide.
3. 0.85% NaCl solution: Dispense one drop at the other end of the
same slide.
4. Test organism: Place a partial loopful of a smooth homologous
culture between the drops of antiserum and NaCl solution but not
in direct contact with either.
5. Using an applicator stick, suspend the culture in the drop of NaCl
solution and check for autoagglutination. If there is no autoagglutination, mix the culture suspension with the drop of antiserum.
6. Rotate the slide by hand for one minute and read immediately for
agglutination. Record results.
Results
No agglutination.
3. Negative control: Should produce no agglutination. If agglutination occurs, the culture is rough and cannot be tested. Subculture
to a non-inhibitory medium, incubate and test the organism again.
4. Test isolate: 3+ or greater agglutination is a positive result.
5. Partial (less than 3+) or delayed agglutination should be considered negative.

1. Serological techniques employing Candida Albicans Antiserum for
the identification of Candida albicans serve as corroborative
evidence in the determination of the organism as the etiological
agent of the disease. Final identification cannot be made without
consideration of morphological, serological, and biochemical
characterization.
2. Excessive heat from external sources (hot bacteriological loop, burner
flame, light source, etc.) may prevent making a smooth suspension
of the microorganism or cause evaporation or precipitation of the
test mixture. False-positive reactions may occur.
The Difco Manual
Section V
Coagulase Plasma & Coagulase Plasma EDTA
3. Rough culture isolates do occur and will agglutinate spontaneously,

4. Agglutination reactions of 3+ or greater in the slide test are
interpreted as positive reactions. Cross-reactions resulting in a 1+
or 2+ agglutination are likely since somatic antigens are shared
among such organisms as Candida tropicalis, Candida kefyr and
Candida stellatoidea.
6. Discard any Candida Albicans Antiserum that is cloudy or has a
precipitate after rehydration or storage.
References
1. Ahearn, D. G., and R. L. Schlitzer. 1981. Yeast Infections,
p. 991-1012. In A. Balows, and W. J. Hausler (ed.), Diagnostic
2. Odds, F. C. 1988. Candida and candidosis, 2nd ed. Bailliere
Tindall, London, England.
3. Hazen, K. C., D. O. Brawner, M. H. Riesselman, J. E. Cutler,
and M. A. Jutila. 1991. Differential adherence of hydrophobic and
hydrophilic Candida albicans yeast cells to mouse tissues. Infect.
Immun. 59:907-912.
4. Kwon-Chung, K. J., D. Lehman, C. Good, and P. T. Magee.
1985. Genetic evidence for the role of extracellular proteinase in
virulence of Candida albicans. Infect. Immun. 49:571-575.
Bacto Coagulase Plasma

Bacto Coagulase Plasma EDTA
Intended Use
Bacto Coagulase Plasma1 and Bacto Coagulase Plasma EDTA1-8 are
used for detecting coagulase activity by staphylococci.
Bacto Coagulase Plasma is used for detecting the production of germ
tubes by Candida albicans.2

Coagulase Detection
Identification of staphylococci is based on microscopic examination,
colonial morphology, and cultural and biochemical characteristics.
Staphylococci associated with acute infection (S. aureus in humans
and S. intermedius and S. hyicus in animals) can clot plasma. The most
widely used and generally accepted criterion for identification of these
pathogenic organisms is based on the presence of the enzyme coagulase.1 The ability of Staphylococcus to produce coagulase was first reported by Loeb9 in 1903. Coagulase binds plasma fibrinogen, causing
the organisms to agglutinate or plasma to clot. Two different forms of
coagulase can be produced, free and bound. Free coagulase is an extracellular enzyme produced when the organism is cultured in broth.
Bound coagulase, also known as clumping factor, remains attached to
the cell wall of the organism. The tube test can detect the presence of
The Difco Manual
5. Calderone, R. A., L. Linehan, E. Wadsworth, and A. L.

Sandberg. 1988. Identification of C3d receptors on Candida
albicans. Infect. Immun. 252-258.
6. Gilmore, B. J., E. M. Retsinas, J. S. Lorenz, and M. K.
Hostetter. 1988. An iC3b receptor on Candida albicans:
structure, function, and correlates for pathogenicity. J. Infect.
Dis. 257:38-46.
7. Hazen, K. C., and P. M. Glee. Cell surface hydrophobicity and
medically important fungi. Curr. Top. Med. Mycol., in press.
8. Rosenthal, S. A., and D. Furnari. 1958. Slide agglutination as a
presumptive test in the laboratory diagnosis of Candida albicans.
J. Invest. Derm. 31:251-253.
and other yeasts of medical importance. In P. R. Murray,
10. Land, G. A. 1992. Mycology, p. 6.0.1.-6.12.4. In H. D. Isenberg
(ed.), Clinical microbiology procedures handbook, vol. 1. American
St. Louis, MO.
Packaging
3 ml
2281-47
both bound and free coagulase. The slide test can detect only bound
coagulase.10 Isolates that do not produce clumping factor must be tested
for the ability to produce extracellular coagulase (free coagulase).
The tube test has traditionally been the standard in determining coagulase activity. The slide test is unreliable in the identification of some
strains of oxacillin-resistant S. aureus.11,12 False-positive results are
sometimes obtained with the slide test when testing S. saprophyticus,13
S. schleiferi, S. lugdunensis, S. intermedius,4 S. hyicus3 and micrococci.11,14 In addition, colonies used for testing must not be picked from
media containing high concentrations of salt (for example, mannitolsalt agar), because autoagglutination and false-positive results may
occur.1 Slide tests must be read quickly, because false-positive results
may appear with reaction times longer than 10 seconds. Isolates that
autoagglutinate cannot be reliably tested with the slide coagulase
method. Finally, 10-15% of S. aureus strains may yield a negative
result, which requires that the isolates be reexamined by the tube test.
Coagulase Plasma and Coagulase Plasma EDTA are recommended for
performing the tube coagulase test. The inoculum used for testing must
be pure because a contaminant may produce false results after
prolonged incubation. For the coagulase test, Coagulase Plasma EDTA
is superior to citrated plasma because citrate-utilizing organisms such
as Pseudomonas species, Serratia marcescens, Enterococcus faecalis
and strains of Streptococcus will clot citrated plasma in 18 hours.15
Germ Tube Development
C. albicans is usually associated with an animal host. It appears in
large numbers as a saprophyte throughout the oral-gastrointestinal tract
617
of many warm-blooded vertebrates.16 It is rarely isolated from normal

skin. Person-to-person transmission of candidiasis can occur. Usually,
candidiasis caused by C. albicans is endogenous in origin and develops
with stress or debilitation of the host.16
C. albicans is the species most commonly isolated from patients with
nearly all forms of candidiasis.17 This organism is an opportunistic pathogen and appears to possess many virulence attributes that may promote
successful parasitism. These attributes include rapid germination
upon seeding tissue from the bloodstream,18 protease production,19
complement protein-binding receptor,20,21 and surface variation and
hydrophobicity.22
C. albicans will grow on Sabouraud Dextrose Agar as white to cream
colored, creamy colonies. It can be isolated from blood agar as a colony
with short marginal extensions. Microscopically, C. albicans produces
budding yeast cells, pseudohyphae or true hyphae.
One of the simplest and most valuable tests for the rapid presumptive
identification of C. albicans is the germ tube test.23 Smith and Elliott
recommended the use of rabbit coagulase plasma.2 The test is considered
presumptive because not all isolates of C. albicans will be germ
tube-positive and false positives may be obtained despite well-trained
staff.24 Ferrigno, Ramirez and Robison recommended testing for germ
tube production with citrated plasma.25

Coagulase Plasma
Lyophilized Appearance: Off-white to cream colored, dried
button or fluffy powder.
Rehydrated Appearance: Off-white to cream to light rose
colored, opaque liquid.
Coagulase Plasma EDTA
Lyophilized Appearance: Off-white to cream colored, dried
button or fluffy powder.
Rehydrated Appearance: Off-white to cream to light rose
colored, opaque liquid.
Rehydrate Coagulase Plasma or Coagulase Plasma EDTA per
label directions. Perform the Coagulase Test or the Germ Tube
Test procedure as described (see Test Procedure).
Staphylococcus saprophyticus
Candida albicans
Candida tropicalis
ATCC
GERM TUBE
COAGULASE TEST DEVELOPMENT
25923*
Clot in tube
3647
Clot in tube
12228* No clot in tube
15305 No clot in tube
18804
Germ tube
development
750
No germ tube
development
618

Coagulase Detection
S. aureus produces two types of coagulase, free and bound. Free coagulase is an extracellular enzyme produced when the organism is cultured in broth. Bound coagulase, also known as the clumping factor,
remains attached to the cell wall of the organism.
In the tube test, free coagulase liberated from the cell acts on prothrombin
in the coagulase plasma to give a thrombin-like product. This product
then acts on fibrinogen to form a fibrin clot.3
The tube test is performed by mixing an overnight broth culture or
colonies from a noninhibitory agar plate into a tube of rehydrated
coagulase plasma. The tube is incubated at 37C. The formation of a
clot in the plasma indicates coagulase production.
The germ tube test involves suspending suspected colonies of yeast in
a tube of Coagulase Plasma. The tube is incubated at 37C for 2-4
hours. The cells are then observed microscopically for short, hyphal
extensions from the yeast cells called germ tubes. Germ tubes are easily differentiated from blastoconidial germination; germ tubes have no
constriction at their juncture with the yeast cell while blastoconidial
germination does produce a constriction. C. albicans usually produces
germ tubes under specified test conditions within 2 hours. Other species
of Candida do not produce germ tubes, except for an occasional
isolate of Candida tropicalis.4
Reagents
ORGANISM
Section V
Coagulase Plasma is lyophilized rabbit plasma to which sodium

citrate has been added as the anticoagulant.
Coagulase Plasma EDTA is lyophilized rabbit plasma to which EDTA
(ethylenediaminetetraacetic acid) has been added as the anticoagulant.
EDTA is not utilized by bacteria. Coagulase Plasma EDTA does not
give false-positive reactions with bacteria that utilize citrate.
Precautions
Storage
Store unopened Coagulase Plasma and Coagulase Plasma EDTA at
2-8C.
Store reconstituted plasma at 2-8C for up to 5 days, or aliquot in 0.5 ml
amounts, freeze promptly and store at -20C for up to 30 days. Do not
thaw and refreeze.
Expiration Date
Procedure
Materials Provided
Coagulase Plasma
The Difco Manual
Section V

Bacteriological inoculating loop
Sterile 1 ml pipettes
Sterile Pasteur pipettes
Sterile serological pipettes, 1, 5, and 10 ml
Incubator (37C)
Culture tubes, 12 x 75 mm
Timer
Waterbath (35-37C)
BHI broth or noninhibitory agar (Coagulase Detection)
Sabouraud Dextrose Agar (Germ Tube Development)
Reagent Preparation
Rehydrate Coagulase Plasma and Coagulase Plasma EDTA by adding
sterile distilled or deionized water to the vial as indicated below. Mix
by gentle end-over-end rotation of the vial.
PRODUCT SIZE
STERILE
DISTILLED WATER
3 ml
15 ml
25 ml
3 ml
15 ml
25 ml
APPROXIMATE
NUMBER OF TESTS
6
30
50

1. Collect specimens or samples in sterile containers or with sterile
swabs and transport immediately to the laboratory according to
recommended guidelines.1,3-8
sample.1,3-8
Coagulase Detection
1. Obtain a pure culture of the organism to be tested. Select wellisolated colonies.
2. Determine that the test culture has characteristics of S. aureus as
listed below. Consult appropriate references for further identification
of S. aureus.1,3-8
Morphology (media dependent):
Blood Agar Base
Opaque, yellow to orange,
w/5% Sheep Blood
with hemolysis.
DNase Test Agar
Clearing of green dye.
w/Methyl Green
Mannitol Salt Agar
Yellow to orange, surrounded
by yellow zones.
Staphylococcus Medium 110 Yellow to orange.
Black.
VJ Agar
Black, surrounded by
yellow zones.
Baird Parker Agar
Grey to black shiny colonies
surrounded by zones of clearing.
Gram Stain:
Gram-positive cocci occurring
in grape-like clusters or,
occasionally, in chains.
Catalase Test:
Positive.
Mannitol Fermentation:
Positive.
3. Using a bacteriological loop, transfer a well-isolated colony from
a pure culture into a tube of sterile Brain Heart Infusion broth.
Incubate for 18-24 hours or until a dense growth is observed.
The Difco Manual
Alternatively, 2-4 colonies (1 loopful) taken directly from a

noninhibitory agar plate may be used as an inoculum instead of a
broth culture.
well-isolated colonies grown on Sabouraud Dextrose Agar for
48-72 hours.
Test Procedure
Coagulase Test
1. Using a sterile 1 ml pipette, add 0.5 ml of rehydrated Coagulase
Plasma or Coagulase Plasma EDTA to a 12 x 75 mm test tube
supported in a rack.
2. Using a sterile 1 ml serological pipette, add 2 drops of the overnight
broth culture of the test organism to the tube of plasma or, using a
sterile bacteriological loop, thoroughly emulsify 2-4 colonies
(1 loopful) from a noninhibitory agar plate in the tube of plasma.
3. Mix gently.
4. Incubate in a waterbath at 35-37C for up to 4 hours.
5. Examine the tube for coagulation hourly until a clot is evident or
until 4 hours have elapsed. If no clot has formed within 4 hours,
reincubate and examine after 24 hours.
Examine by gently tipping the tube. Avoid shaking or agitating the
tube, which could cause breakdown of the clot and, consequently,
doubtful or false-negative test results.
6. Record results.
Germ Tube Test
1. Using a sterile 1 ml pipette, add 0.5 ml of the rehydrated Coagulase
Plasma (citrated) to a 12 x 75 mm test tube in a rack.
2. Touch the tip of a sterile Pasteur pipette to a yeast colony growing
on a Sabouraud Dextrose Agar plate.
3. Gently emulsify the cells in the tube of rehydrated plasma.
4. Incubate the mixture in a waterbath at 37C for 2-4 hours.
5. Examine 1 drop of the incubated mixture microscopically for
germ tubes.
6. Record results.
Results
Coagulase Test
Any degree of clotting in Coagulase Plasma or Coagulase Plasma
EDTA is considered a positive test.
Germ Tube Test
The development of short, lateral hyphal filaments (germ tubes) on the
individual yeast cells with no constriction at the point of attachment is
considered a positive test.

1. The slide agglutination technique for determining the coagulase
activity of staphylococci is not recommended because false-positive reactions may occur with some strains when animal plasmas
are used. In addition, spontaneous agglutination may occur when
rough cultures are used. Because 10-15% of S. aureus isolates may
yield a negative result when this test is employed, all negative slide
reactions must be confirmed by the tube test.
619
2. Some species of organisms utilize citrate in their metabolism and

will yield false-positive reactions for coagulase activity. Normally,
this would not cause problems since the coagulase test is performed
almost exclusively on staphylococci. However, it is possible that
bacteria which utilize citrate may contaminate Staphylococcus
cultures on which the coagulase test is being performed. These
contaminated cultures may, upon prolonged incubation, give
false-positive results due to citrate utilization.3
3. When checking results of the Coagulase Test, observe tubes hourly
during the first 4 hours of incubation. Some strains of S. aureus
produce fibrinolysin, which may lyse clots. If the tubes are not
read until 24 hours of incubation, reversion to a false-negative may
occur.26
4. Do not use plasmas if a heavy precipitate or clot has formed before
inoculation.
References
Micrococcus, p. 282-298. In P. R. Murray, P. R., E. J. Baron, M. A.
Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology, 6th ed. American Society for Microbiology, Washington, D.C.
2. Smith, R., and L. P. Elliott. 1983. Are there better ways to
diagnose candidiasis? Diag. Med. May-June:91-93.
St. Louis, MO.
6. FDA Bacteriological Analytical Manual. 1995. 8th ed. AOAC
Pathogens in milk and milk products, p. 103-212. In R. T. Marshall
9. Loeb, L. 1903. The influence of certain bacteria on the coagulation
of the blood. J. Med. Res. 10:407-419.
10. Kloos, W. E., and J. H. Jorgensen. 1985. Staphylococci, p. 143153. In E. H. Lennette, A Balows, W. J. Hausler, Jr., and H. J.
Shadomy (ed.), Manual of clinical microbiology, 4th ed. American
11. Hall, G. S., K. Pratt, G. Woods, and C. C. Knapp. 1988. Differentiation of Staphylococcus aureus from other Micrococcaceae:
comparison of Staphaurex and the slide coagulase test with the
tube coagulase test. Lab. Med. 19:817-820.
12. Smith, S. M., and C. Berezny. 1986. Comparative evaluation of
identification systems for testing methicillin-resistant strains of
Staphylococcus aureus. J. Clin Microbiol. 24:173-176.
620
Section V
13. Gregson, D. B., D. E. Low, M. Skulnick, and A. E. Simor. 1988.

Problems with rapid agglutination methods for identification of
Staphylococcus aureus when Staphylococcus saprophyticus is
being tested. J. Clin Microbiol. 26:1398-1399.
14. Hinnebusch, C., D. Glenn, and D. A. Bruckner. 1992. Potential
misidentification of Staphylococcus species when using rapid identification tests that detect clumping factor. Abstr. General Meeting,
Amer. Soc. Microbiol., C-485, p. 498. American Society for Microbiology, Washington, D.C.
15. Bayliss, B. G., and E. R. Hall. 1965. Plasma coagulation by
organisms other than Staphylococcus aureus. J. Bacteriol.
89:101-104.
16. Ahearn, D. G., and R. L. Schlitzer. 1981. Yeast Infections,
p. 991-1012. In A. Balows and W. J. Hausler (ed.), Diagnostic
American Public Health Association, Washington, D.C .
17. Odds, F. C. 1988. Candida and candidiasis, 2nd ed. Bailliere
Tindall, London, England.
18. Hazen, K. C., D. O. Brawner, M. H. Riesselman, J. E. Cutler,
and M. A. Jutila. 1991. Differential adherence of hydrophobic and
hydrophilic Candida albicans yeast cells to mouse tissues. Infect.
Immun. 59:907-912.
19. Kwon-Chung, K. J., D. Lehman, C. Good, and P. T. Magee.
1985. Genetic evidence for the role of extracellular proteinase in
virulence of Candida albicans. Infect. Immun. 49:571-575.
20. Calderone, R. A., L. Linehan, E. Wadsworth, and A. L.
Sandberg. 1988. Identification of C3d receptors on Candida
albicans. Infect. Immun. 56:252-258.
21. Gilmore, B. J., E. M. Retsinas, J. S. Lorenz, and M. K.
Hostetter. 1988. An iC3b receptor on Candida albicans: structure,
function, and correlates for pathogenicity. J. Infect. Dis. 257:38-46.
22. Hazen, K. C., and P. M. Glee. Cell surface hydrophobicity and
medically important fungi. Curr. Top. Med. Mycol., in press.
24. Dealler, S. F. 1991. Candida albicans colony identification in
5 minutes in a general microbiology laboratory. J. Clin Microbiol.
29:1081-1082.
25. Ferrigno, R. G., J. M. Ramirez, and D. Robison. 1983. EDTA
interference in germ-tube production. Diag. Med. 6:10.
26. Kloos, W. E., and D. W. Lambe, Jr. 1991. Staphylococcus, p. 222237. In A. Balows, W. J. Hausler, Jr., K. L. Herrmann, H. D.
Isenberg, and H. J. Shadomy (ed.), Manual of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C.
Packaging
Coagulase Plasma
6 x 3 ml
6 x 15 ml
6 x 25 ml
0286-46
0286-86
0286-66
6 x 3 ml
6 x 15 ml
6 x 25 ml
0803-46
0803-86
0803-66
The Difco Manual
Section V
E. Coli Antisera
Bacto E. Coli Antisera

E. Coli O Antiserum O157 . E. Coli H Antiserum H7
Intended Use
Bacto E. Coli O Antiserum O157 and E. Coli H Antiserum H7 are
used for identifying Escherichia coli O157:H7.

E. coli O157:H19 was described in 1972 as a causative agent of diarrhea
in swine.1 The H19 flagellar antigen was the common antigen for
serogroup O157. An H7 variant of somatic group O157 has been
incriminated in severe hemorrhagic colitis.
E. coli O157:H7 is a foodborne pathogen that can cause potentially
fatal enteric-related disease in humans.2,3,4,5,6,7,8 This disease is characterized by sudden onset of severe cramps and abdominal pain, followed
by a watery stool that may become markedly bloody. A series of 107
outbreaks involving 387 persons was traced to imported Camembert
cheese in the United States in 1971.9 E. coli O157:H7 was recognized
as a cause of hemorrhagic colitis in 19825, and hemolytic uremic
syndrome in 1983.10 The 1982 outbreak was derived from ingested
hamburgers.5,11
The incidence of disease caused by this organism has increased
significantly over the past decade.4,12 The largest outbreak of E. coli
O157:H7 disease occurred during January 1993, in Washington State,
where more than 600 patients with hemorrhagic colitis were confirmed.2
The source of the outbreak was identified as undercooked hamburger
at multiple outlets of the same fast food restaurant chain.
E. coli O157:H7 is an enteric pathogen that requires only a low inoculum
to cause disease. Transmission is usually via high volume food items
whose preparation is not always under stringent control and is served

to a target audience (children and the elderly) most at risk for complications of illness. The organism has been isolated from several foods,
including undercooked hamburger, drinking water, new potatoes, turkey
roll, raw milk and apple cider. Serotyping of the entero-hemorrhagic
E. coli is useful in the epidemiological documentation of the spread of
a particular strain in a foodborne outbreak.12

E. Coli O Antiserum O157 is used in the tube technique for O antigen
titration. E. Coli H Antiserum H7 is used in the tube agglutination technique for detecting H antigens. These antisera are used to confirm the
presence of E. coli O157:H7 after selective isolation. For isolation of
this organism from food, use MacConkey Sorbitol Agar.13 MacConkey
Sorbitol Agar has also been used successfully to isolate E. coli O157:H7
from stool specimens. However, high levels of contaminating coliform
organisms will mask the O157 strains.14
Procedures for serological confirmation by E. Coli O Antiserum O157
and E. Coli H Antiserum H7 require a pure culture of the test organism
isolated on Veal Infusion Agar or other enriched solid medium. The
serological technique is based on the reaction of a specific antiserum
with its homologous antigen. While the specificity of serological methods
is not absolute, serotyping of E. coli, taken with biochemical characteristics, can provide an accurate identification of the etiological agent.
Reagents
E. Coli O Antiserum O157 and E. Coli H Antiserum H7 are lyophilized,
polyclonal rabbit antisera containing approximately 0.04% Thimerosal
as a preservative.
Precautions
E. Coli O Antiserum O157
powdered cake.
E. Coli H Antiserum H7
powdered cake.
Rehydrated Appearance: Straw colored, clear solution.
Cultural Response
Rehydrate E. Coli O Antiserum O157 and E. Coli H Antiserum H7 per
label directions. Test as described (see Test Procedure). Known positive and negative control cultures must give appropriate reactions.

Storage
Store lyophilized and rehydrated E. Coli Antisera at 2-8C.
Expiration Date
Procedure
ORGANISM
ATCC
REACTION
Materials Provided
E. coli O157:H7
E. coli O111:K58:H21
35150
29552
Positive
Negative

These strains may be used for Quality Control. All cultures should be
serologically validated before use.
The Difco Manual

Veal Infusion Agar
Motility GI Medium
621
E. Coli Antisera
Test tubes (12 x 75 mm) or other suitable test tubes and rack
Formalin
1 ml serological pipettes
McFarland Standard No. 3
Waterbath (50 2C)
E. Coli Antiserum: To rehydrate, add 3 ml sterile 0.85% NaCl solution
and rotate gently to dissolve contents completely. The rehydrated
antiserum is considered a 1:2 working dilution.
Tube Technique for O Antigen Titration
1. To prepare pure cultures of the test organism, plate the organism
on Veal Infusion Agar and incubate at 35 2C for 16-18 hours.
2. Suspend some growth from the solid medium in 0.85% NaCl
solution to give a homogeneous suspension.
3. Heat the bacterial suspension in a boiling water bath for 30-60
minutes. The culture should be homogeneous. Precipitation
indicates a rough culture and the suspension should be discarded.
4. Allow the suspension to cool; dilute with 0.85% NaCl solution to
a density approximating that of a McFarland Barium Sulfate
Standard No. 3.
5. Add formalin to a final concentration of 0.5% by volume.
6. In a rack, prepare a row of 8 culture tubes (12 x 75 mm) for each
test suspension
7. Dispense 0.9 ml of 0.85% NaCl solution in the first tube of each
row and 0.5 ml in the remaining tubes.
8. E. Coli O Antiserum O157: Prepare serial dilutions using the
rehydrated antiserum, which is already at a 1:2 working dilution.
Dispense 0.1 ml of antiserum in the first tube in the row and mix
thoroughly. Transfer 0.5 ml from tube 1 to tube 2 and mix
discarding 0.5 ml from tube 7 after mixing. Proceed in like manner
for each suspension to be tested. Tube 8 is the antigen control tube
and contains only sterile 0.85% NaCl solution.
This procedure yields antiserum dilutions of 1:20-1:1280.
9. Heated bacterial suspension: Add 0.5 ml to each of the 8 tubes.
Final antiserum dilutions are 1:40-1:2560.
8. Incubate in a waterbath at 50 2C for 18-20 hours. Read for
agglutination.
Tube Agglutination Technique for H Antigen Detection
1. Prepare an actively motile culture of the suspect E. coli culture by
several successive transfers in Motility GI Medium. At least 2-3
passages through Motility GI Medium are necessary before
attempting to establish the presence and identity of H antigens.
Fresh isolates of E. coli generally have poorly developed flagella.
2. Inoculate a loopful of the Motility GI Medium culture into a
tube of Veal Infusion Broth. Incubate 6-8 hours at 35 2C or
overnight, if necessary.
3. Inactivate the culture by adding formalin to a final concentration
of 0.3% (0.3 parts formaldehyde per 100 parts of the Veal Infusion
Broth culture). If necessary, adjust the density of the suspension
with formalinized saline to approximate a McFarland Barium
Sulfate Standard No. 3. This broth culture will be used as the
test antigen in step 6.
622
Section V
4. E. Coli H Antiserum H7: Prepare a 1:500 dilution by adding

0.2 ml of rehydrated antiserum, which is already a 1:2 working
dilution, to 49.8 ml of 0.85% NaCl solution.
5. Pipette 0.5 ml of the antiserum dilution into a test tube.
6. Test antigen: Add 0.5 ml to the above dilution and shake well.
The resulting antiserum dilution will be 1:1,000.
7. Incubate the tube in a 50 2C waterbath for 1 hour and read for
agglutination.
Results
Observe test results with indirect lighting against a dark background.
Record as follows.
4+ 100% agglutination of cells; supernatant fluid is clear to
very slightly hazy.
3+ 75% agglutination of cells; supernatant fluid is slightly
cloudy.
2+ 50% agglutination of cells; supernatant fluid is moderately
cloudy.
1+ 25% agglutination of cells; supernatant fluid is cloudy.
Less than 25% agglutination of cells.
No agglutination.
E. Coli O157: Cultures showing 2+ or greater agglutination at a
dilution of 1:320 or greater are considered positive.
E. Coli H7: Tubes showing 2+ or greater agglutination are considered
positive.

1. Final identification of E. coli O157 is based on biochemical
reactions and the presence of the O antigen.
2. The test organism must be identified to at least the genus level and,
in some cases, to the species level biochemically before serotyping
E. coli.
3. If the antiserum is cloudy after rehydration, check its bacterial
purity and the pH of the saline. Discard any serum that is cloudy
and/or has a precipitate unless it has been clarified and shown to
react properly with known control cultures.
4. Adhere strictly to the time limitations in both tests.
5. Exposure of the organism or plate to heat from external sources (a
hot bacteriological loop, burner flame, light source, etc.) may
result in either a culture that cannot be suspended readily or evaporation and/or precipitation of the test mixture. These conditions
may cause false-positive reactions.
6. The test culture must be checked in a saline control for smoothness.
Stock cultures and, sometimes, isolated cultures may be rough and
will agglutinate spontaneously in a normal serum. Therefore, it is
necessary to select smooth colonies for serological testing.
7 In E. coli serology, as in any serological test, known positive and
negative control cultures should be employed.
8. Antisera should not be subjected to repeated freezing and thawing.
Such treatment is detrimental to antibody content.
References
1. Furowicz, A. J., and F. Orskov. 1972. Two new Escherichia coli
antigens, O150 and O157, and one new K antigen, K93, in strains
isolated from veterinary diseases. Acta. Pathol. Microbiol. Scand.
Sect. B. 80:441-444.
The Difco Manual
Section V
FA Bordetella Pertussis & Parapertussis
2. Centers for Disease Control. 1993. Emerging infectious diseases.

Morb. Mort. Wkly. 42:257-260.
3. Glass, K. A., J. M. Leffelholtz, J. P. Ford, and M. P. Doyle. 1992.
Fate of Escherichia coli O157:H7 as affected by pH or sodium
chloride and in fermented, dry sausage. Appl. Environ. Microbiol.
58:2513-2516.
4. Padhye, N. V., and M. P. Doyle. 1992. Escherichia coli O157:H7
epidemiology, pathogenesis, and methods for detection in food.
J. Food Prot. 55:555-565.
5. Riley, L. W., R. S. Remis, and S. D. Helgerson, et al. 1983.
Hemorrhagic colitis associated with a rare Escherichia coli
serotype. N. Engl. J. Med. 308:681-685.
6. Samadpour, M., L. Grimm, B. Desai, D. Alfi, J. E. Ongerth,
and P. I. Tarr. 1993. Molecular epidemiology of Escherichia coli
O157:H7 strains using bacteriophage A -restriction fragment length
polymorphism analysis: application to a multistate foodborne outbreak and a day care center cluster. J. Clin. Microbiol. 31:3179-3183.
7. Tarr, P. I. 1994. Review of 1993 Escherichia coli O157:H7
outbreak: western United States. Dairy, Food, and Environmental
Sanitation 14:372-373.
9. Marrier, R., J. G. Wells, R. C. Swanson, W. Callahan, and

I. J. Mehlman. 1973. An outbreak of enteropathogenic E. coli
foodborne disease traced to imported French cheese. Lancet
2:1376-1378.
Marshall (ed.), Standard methods for the examination of dairy products, 16th ed. American Public Health Association. Washington, D.C.
11. Morbidity and Mortality Weekly Reports. November, 1982.
12. Lior, H. 1994. Escherichia coli O157:H7 and verotoxigenic
Escherichia coli (VTEC). Dairy, Food, and Environ. Sanit. 14:378-382.
p. 4.01-4.29. FDA bacteriological analytical manual, 8th ed. AOAC
Coliforms - E. coli and its toxins, p. 325-369. ln C. Vanderzant and
D. F. Splittstoesser (ed.), Compendium of methods for the microbiological examination of foods, 3rd ed. American Public Health
Packaging
3 ml
2970-47
3 ml
2159-47
Bacto FA Bordetella Pertussis

Bacto FA Bordetella Parapertussis
Intended Use
Bacto FA Bordetella Pertussis and Bacto FA Bordetella Parapertussis

are used for identifying Bordetella pertussis and Bordetella
parapertussis by the direct fluorescent antibody technique.
All members of the genus Bordetella are respiratory pathogens of

warm-blooded animals. B. pertussis and B. parapertussis are two
uniquely human species. These organisms adhere to, multiply among
and remain localized in the ciliated epithelial cells of the respiratory
tract. B. pertussis is the major cause of whooping cough or pertussis.
Bordetella parapertussis is associated with a milder, less frequently
occurring form of the disease.1 Person-to-person transmission occurs
by the aerosol route.
Pertussis is a highly contagious disease that attacks unimmunized
populations in more than 90% of cases.2 Toxin production remains the
major distinction of B. pertussis.
Classic pertussis caused by B. pertussis occurs in three stages. The
first or catarrhal stage is characterized by nonspecific symptoms similar
to a cold or viral infection. The disease is highly communicable during
this stage, which lasts 1-2 weeks. In the second or paroxysmal stage,
the cough increases in intensity and frequency. This stage is marked by
sudden attacks of severe, repetitive coughing, often cumulating with
the characteristic whoop that is caused by a rapid inspiration of air
after the clearance of mucus-blocked airways.3 This stage may last 1-4
weeks. The beginning of the convalescent stage is marked by a reduction
in the frequency and severity of coughing spells. Complete recovery
may require weeks or months.

FA Bordetella Pertussis, FA Bordetella Parapertussis
Lyophilized appearance: Yellow button to powdered cake.
Rehydrated appearance: Yellow, clear solution.
Rehydrate FA Bordetella and FA Bordetella Parapertussis per
label directions. Perform the fluorescent antibody staining
procedure using appropriate known Bordetella pertussis and
Bordetella parapertussis cultures as homologous and
heterologous controls. The positive control should produce a
4+ reaction using the working dilution of the conjugate. The
negative control should not exceed a 1+ reaction using the
working dilution of the conjugate.
The Difco Manual
623
Despite the availability of an effective whole-cell vaccine, pertussis

remains a disease of worldwide distribution because many developing
nations do not have the resources for vaccinating their populations.4
Major outbreaks have occurred even in developed nations such as Great
Britain and Sweden. Pertussis is endemic in the United States, with
most disease occurring as isolated cases. A shift in the age group
affected by the disease has occurred. In the past, children in the 1-5
year age group were more prone to pertussis. Children less than one
year of age2 have become more susceptible to the disease because of a
decrease in passively transferred maternal antibodies, since adults do
not receive booster vaccinations.
Bordetella are tiny gram-negative coccobacilli occurring singly or in
pairs and they may exhibit a bipolar appearance. They are strict aerobes
and some members are motile. B. pertussis and B. parapertussis are
nonmotile and produce no acid from carbohydrates. B. pertussis will
not grow on common blood agar bases or chocolate agar, whereas
B. parapertussis will grow on blood agar and sometimes chocolate agar.
Media for primary isolation must include starch, charcoal, ion-exchange resins or a high percentage of blood to inactivate inhibitory
substances.3 B. pertussis may be recovered from secretions collected
from the posterior nasopharynx, bronchoalveolar lavage and
transbronchial specimens.
The direct fluorescent antibody test (DFA) has long been used for the
rapid, direct detection of B. pertussis and B. parapertussis in nasopharyngeal specimens with varying degrees of success.5,6,7 Eldering,
Eveland and Kendrick8,9 and Holwerda and Eldering10 showed the
usefulness of the FA procedure, although a complete correlation
between the agglutination method and FA technique was not obtained.
Nonetheless, the FA procedure could detect both smooth and rough
cultures of B. pertussis and B. parapertussis and could also be applied
to direct specimens. Further data showed little or no cross reactions
between conjugates prepared from B. pertussis and B. parapertussis
cultures. The disadvantage of this procedure is that technical skill and
experience are required for the technician to perform and read the test.
The DFA should always be used with, not as a replacement for,
culture.11,12 It has been suggested that laboratories proficient in DFA and
culture for pertussis should obtain a DFA sensitivity of 60% or greater
and a specificity of at least 90% over time compared with culture.12
B. pertussis and B. parapertussis are slow growing organisms,
developing in 3-4 days.
By employing the fluorescent antibody technique, the time required to
detect these organisms can be significantly reduced. The FA procedure
may be applied to direct nasopharyngeal smears or may be used to
identify young cultures of B. pertussis or B. parapertussis.
Principles of The Procedure

The direct FA technique involves preparation of a smear from the clinical
specimen on a glass slide. Nasopharyngeal swabs are obtained from
the patient and inoculated into 0.5 ml of Casamino Acids solution.12
Smears are made from this solution and stained with a specific antibody labeled with a fluorescent marker (fluorescein isothiocyanate or
FITC) directed against B. pertussis or B. parapertussis. After incubation
with the antibody preparation, smears are washed in phosphate-buffered
saline, air dried, cover slipped with fluorescent-antibody mounting
fluid, and examined under a fluorescent microscope.
624
Section V
Reagents
FA Bordetella Pertussis and FA Bordetella Parapertussis are
lyophilized, polyclonal, fluorescein-conjugated chicken antisera. They
have been prepared according to modifications of the methods of
Eldering, Eveland and Kendrick8,9 and Holwerda and Eldering. 1
Approximately 0.02% Thimerosal is added as a preservative.
Precautions
2. FA Bordetella Pertussis
FA Bordetella Parapertussis
and disposing of specimens.13,14
Storage
Store lyophilized FA Bordetella Pertussis and FA Bordetella
Parapertussis at 2-8C.
Aliquots of the titered conjugate may be prepared in small vials, frozen
in the undiluted state and stored below -20C for optimal stability. The
conjugate should not be exposed to repeated freezing and thawing.
Expiration Date
Procedure
Materials Provided
FA Bordetella Pertussis

FA Buffer, Dried
FA Mounting Fluid pH 7.2
1% Casamino Acids
Staining Tray
Fluorescent microscope assembly:
Lamps:
HBO-50, HBO-100, HBO-200 or Xenon
XBO-150; 6X 5A Tungsten
Excitation wavelength: 365 nm
Ocular:
10X
Objective:
10X, 40X (Fluorite)
Filters:
BG-12 or KP490, K515 or K530
Condenser:
Dark-field D1.20-1.40
Microscope slides
95% ethanol
Staining jar
Cover slips
McFarland Barium Sulfate Standard #3
The Difco Manual
Section V
Reagent Preparation
Test Procedure

test. Ensure that all glassware and pipettes are clean and free of
detergent residues.
1. Add several drops (one drop equals ~35 l) of the appropriate

FA Bordetella conjugate to the fixed smear.
2. Spread the conjugate over the surface of the smear.
3. Place the slide in a Staining Tray or moisture chamber.
4. Incubate at room temperature for 30 minutes.
5. Remove excess conjugate and place the slide in a staining jar
containing FA Buffer for 10 minutes with 2 changes of the buffer
followed by 1 rinse in distilled water for 2 minutes.
6. Remove the slide; allow to drain and air dry or blot with bibulous
paper.
7. Add a small drop (~35 l) of FA Mounting Fluid pH 7.2 to the
center of the stained area and mount with a cover slip.
8. Examine each smear using a fluorescent microscope with an
excitation wavelength of 365 nm under a 40X or 100X objective.
Record the absence or presence and degree of fluorescence.
FA Bordetella Pertussis and FA Bordetella Parapertussis: To

rehydrate, add 5 ml sterile distilled or deionized water and rotate
gently to completely dissolve the contents.
The working dilution of the conjugate should be determined shortly
after rehydration. The titer of a conjugate varies with the technique used,
the fluorescent microscope and filter used, and the age of the bulb.
The conjugate should be titrated using a known culture of B. pertussis
or B. parapertussis homologous to the conjugate. (Dilutions of the
conjugate are made in FA Buffer.) The titer is determined as follows:
DILUTION OF CONJUGATE
FLUORESCENCE
1:5
4+
1:10
4+
1:20
4+
1:40
4+
1:80
2+
In this example, the last 4+ fluorescence is found in a 1:40 dilution of

the conjugate. One less dilution is chosen for a margin of safety. The
working dilution in this case is, therefore, 1:20.

Direct Nasopharyngeal Smears
1. Obtain a nasopharyngeal swab and emulsify it in 0.5 ml of sterile
1% Casamino Acids.12
2. Hold the specimen in Casamino Acids solution for no more than
2 hours.
3. Smear the emulsified specimen on a clean microscope slide.
4. Allow the smear to air dry and fix it by gentle heating or by a
1 minute immersion in 95% ethanol.
Culture Isolates
1. Isolation of Bordetella from clinical specimens requires the use of
certain media such as Bordet-Gengou Agar. Colonies of B. pertussis on Bordet-Gengou Agar or Charcoal Agar are very small, white,
opaque, convex and entire. For specific recommendations, consult
appropriate references.3,12 Determine that a pure culture of the
microorganism has been obtained and that biochemical test reactions are consistent with the identification of the organism as a
Bordetella species. After these criteria are met, serological
identification can be performed.
2. Pick appropriate colonies and emulsify in approximately 2 ml sterile
distilled or deionized water. Adjust cell density to approximate a
McFarland Barium Sulfate Standard #3.
3. Smear the emulsified specimen on a clean microscope slide.
4. Allow the smear to air dry and fix it by a 1 minute immersion in
95% ethanol. Remove slides and allow them to air dry.
Control Slides
Prepare positive and negative control slides using appropriate homologous antigens, following the procedure listed under Culture Isolates.
The Difco Manual
Results
1. Read and record results based on the intensity of fluorescence, as
follows:
4+ Maximum fluorescence; brilliant yellow-green peripheral staining.
3+ Bright yellow-green peripheral staining.
2+ Definite, but dull, yellow-green peripheral staining.
1+ Barely visible peripheral staining.
Complete absence of yellow-green peripheral fluorescence.
2. Positive control: Should show a 4+ reaction using the working
dilution of the conjugate.
Negative control: Should not exceed a 1+ reaction using the
working dilution of the conjugate.
Test smears: A 2+ fluorescence should be considered a positive
result.
3. If the positive control is less than 3+, or if the negative control
exceeds 1+, the conjugate may have deteriorated or the pH of the
FA Buffer or FA Mounting Fluid may have changed. Repeat the
test with new reagents.

1. At the Routine Test Dilution (RTD) of both FA Bordetella Pertussis
and FA Bordetella Parapertussis, the reaction should be brilliant
and specific with smooth strains of homologous cultures. Usually,
there are no cross reactions with the heterologous strains at the
RTD. With some heterologous strains, a 1+ reaction may occur. Such
a minimal reaction should not interfere with the interpretation of
the test results.
Some strains of Bordetella bronchiseptica, on the other hand, may
cross react with both conjugates in varying degrees, giving 1+ to
4+ reactions with a small population of the cells in the smear. The
majority of the cell population, however, should not stain at more
than a 2+ intensity.
2. Some experience is required to grade the intensity of the fluorescence
and to ignore the occasional nonspecific staining of gram-negative
diplococci, gram-positive cocci and diphtheroid-like rods.12
3. When testing cultural isolates, the density of the positive control
should be adjusted to give 4+ fluorescence with the homologous
conjugate. The density of culture isolates should be comparable to
the positive control in order to standardize fluorescence.
625
FA Product Accessories and Reagents
4. All glassware employed in the preparation, testing and storage

of these reagents must be free of detergents or other harmful
residues.
5. The fluorescent antibody technique can provide only presumptive
identification of B. pertussis or B. parapertussis. A negative result
should not be considered conclusive as this type of reaction may
occur when only a few organisms are present in the specimen.
Final identification can be made only after consideration of cultural,
morphological and serological characteristics.
References
recent experience and a review of the literature. Am. J. Dis. Child
131:560-563.
2. Bass, J. W., and S. R. Stephenson. 1987. The return of pertussis.
Pediatr. Infect. Dis. J. 6:141-144.
3. Marcon, M. J. 1995. Bordetella, p. 566-573. In P. R. Murray, E. J.
Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.),
Microbiology, Washington, D. C.
4. Wright, P. F. 1991. Pertussis in developing countries: definition
of the problem and prospects for control. Rev. Infect. Dis.
13:S228-S234.
5. Strebel, P. M., S. L. Cochi, K. M. Farizo, B. J. Payne, S. D.
Hanauer, and A. L. Baughman. 1993. Pertussis in Missouri:
evaluation of nasopharyngeal culture, direct fluorescent antibody
testing, and clinical case definitions in the diagnosis of pertussis.
Clin. Infect. Dis. 16:276-285.
6. Halperin, S. A., R. Bortolussi, and A. J. Wort. 1989. Evaluation
of culture, immunofluorescence and serology for the diagnosis of
pertussis. J. Clin Microbiol. 27:752-757.
Section V
7. Onorato, I. M., and S. G. F. Wassilak. 1987. Laboratory diagnosis

of pertussis: the state of the art. Pediatr. Infect. Dis. J. 6:145-151.
8. Kendrick, P. L., Eldering, G., and W. C. Eveland. 1961. Fluorescent antibody techniques. Am. J. Diseases Children 101:149-154.
9. Eldering, G., W. C. Eveland, and P. L. Kendrick. 1962.
Fluorescent antibody staining and agglutination reactions in
Bordetella pertussis cultures. J. Bacteriol. 83:745-749.
10. Holwerda, J., and G. Eldering. 1963. Culture and fluorescent
antibody methods in diagnosis of whooping cough. J. Bacteriol.
86:449-451.
11. Gilchrist, M. J. R. 1991. Bordetella, p. 471-477. In A. Balows, W.
J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy
Isenberg (ed.) Clinical microbiology procedures handbook, vol. 1.
387-388.
14. Occupational Safety and Health Administration, U.S. Department of Labor. 1991. 29 CFR, part 1910. Occupational exposure
to bloodborne pathogens; final rule. Federal Register 56:6417564182.
Packaging
FA Bordetella Pertussis
5 ml
2359-56
5 ml
2378-56
Bacto FA Product Accessories and Reagents

FA Buffer, Dried . FA Mounting Fluid pH 7.2 . FA Mounting
Fluid pH 9 . Staining Tray
Intended Use
Bacto FA Buffer, Dried is used in fluorescent antibody (FA) staining
procedures.
Bacto FA Mounting Fluid pH 7.2 is used in FA procedures to mount
specimens on slides at pH 7.2.
Bacto FA Mounting Fluid pH 9 is used in FA procedures to mount
specimens on slides at pH 9.
The Staining Tray is used in FA staining procedures.

FA Buffer, Dried is a phosphate buffer-NaCl mixture which, upon
rehydration, yields a 0.85% NaCl solution buffered to pH 7.2. It is
used for making dilutions of rehydrated FA Globulins for test
626
purposes, as well as for washing slides in FA staining procedures. It is

also recommended as a general purpose phosphate buffered saline.
FA Mounting Fluids are buffered glycerine preparations used in
fluorescent antibody procedures as a semipermanent mounting medium
for specimens on slides. With mounting media, the cover slip should
be pressed down firmly to reduce hazy images. Add a very small drop
of mounting fluid and avoid forming bubbles.
Mounting media may be adjusted to any pH compatible with the
fluorescence of the fluorochrome to be used. For FITC procedures,
the pH should not be lower than 7.0 because fluorescence decreases
rapidly below this pH. FITC preparations may be mounted at pH of
9.0 to increase the intensity of fluorescence;1 however, nonspecific
staining is also increased.2 The pH of a mounting fluid decreases with
The Difco Manual
Section V
time because of oxidation of the glycerol and absorption of CO2 by

the mounting fluid.3
The Staining Tray provides a moist, dark incubation chamber for
FA conjugated slides.

Slides are stained using fluorescent antibody procedures. After
removal of excess conjugate or serum, a drop of the appropriate
FA Mounting Fluid pH 7.2 or FA Mounting Fluid pH 9 is added. A
cover slip is applied, taking care not to form bubbles when the cover
slip is added.
FA Product Accessories and Reagents
Storage
Store dehydrated FA Buffer, Dried below 30C. Upon rehydration, store
FA Buffer, Dried at 2-8C.
Store FA Mounting Fluid pH 7.2 and FA Mounting Fluid pH 9 at
15-30C.
Reagents
Expiration Date
FA Buffer, Dried is a phosphate buffer-NaCl mixture which, upon

rehydration, yields a 0.85% NaCl solution buffered to pH 7.2.
FA Mounting Fluid pH 7.2 and FA Mounting Fluid pH 9 are standardized,

reagent grade glycerin adjusted to pH 7.2 and 9.0, respectively.
Precautions
2. FA Buffer, Dried
closed.
Procedure
FA Buffer, Dried
Refer to appropriate procedures for FA Bordetella, Fluorescent
Treponemal Antibody Testing (FTA-ABS), FA Streptococcus Group A
or FA Rhodamine Counterstain.
Refer to appropriate procedures for FA Bordetella or FTA-ABS.
FA Mounting Fluid pH 9
Refer to appropriate procedures for FA Streptococcus Group A or
FA Rhodamine Counterstain.

FA Buffer, Dried
Dehydrated Appearance: White, free flowing, homogeneous.
Solution:
solution, soluble in distilled or
deionized
water. Solution is colorless, clear.
Reaction of 1%
Solution at 25C:
pH 7.2 0.05
Appearance:
Colorless, clear, free from lint.
pH at 25C:
7.2 0.1
Appearance:
Colorless, clear, free from lint.
pH at 25C:
9.0 0.1
Perform the fluorescent antibody procedure using an appropriately titrated conjugate. Rinse off excess conjugate. Add a
cover slip with an appropriate FA Mounting Fluid. Read
smears with a fluorescent microscope. The FA Mounting Fluid
pH 7.2 or FA Mounting Fluid pH 9 must not show quenching
of fluorescence and must show 4+ fluorescence for all slides
tested with the homologous antigen.
The Difco Manual
1. An acid pH will cause a marked decrease in fluorescence.

2. Fresh mounting fluid should be used. The fluid should be discarded
if any color or turbidity appears.2
References
1. Pital, A., and S. L. Janowitz. 1963. Enhancement of staining
intensity in the fluorescent-antibody reaction. J. Bacteriol.
86:888-889.
2. Cherry, W. B. 1974. Immunofluorescence techniques, p. 29-44.
In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual
of clinical microbiology, 2nd ed. American Society for Microbiology,
Washington, D.C.
3. Anhalt, J. P. 1981. Fluorescent antibody procedures and
counterimmuno-electrophoresis, p. 249-277. In J. Washington
(ed.), Laboratory procedures in clinical microbiology. SpringerVerlag Inc, New York, NY.
Packaging
FA Buffer, Dried
6x10 ml
100 g
10 kg
2314-33
2314-15
2314-08
6x5 ml
2329-57
6x5 ml
3340-57
Staining Tray
1 tray
5251-31
627
FA Streptococcus Group A
Section V
Bacto FA Streptococcus Group A
Intended Use
Reagents
Bacto FA Streptococcus Group A is used for identifying Group A

Streptococcus by the direct fluorescent antibody technique.
FA Streptococcus Group A conjugate has been cross-absorbed to

remove cross reactivity known to exist with serogroups C and G.
Normally occurring Staphylococcus agglutinins have been blocked by
unconjugated normal rabbit serum or by an unconjugated Staphylococcus immune serum. Approximately 0.02% Thimerosal is used as a
preservative.

Streptococcus pyogenes (Group A streptococcus) is the most common
cause of bacterial pharyngitis in children. Symptoms include fever,
pharyngeal erythema and edema, tonsillar exudate and enlarged cervical
lymph nodes. Physical findings alone cannot distinguish between
Group A streptococcal pharyngitis and pharyngitis caused by other
agents such as viruses or mycoplasma. Other infections caused by
Group A streptococci include scarlet fever, impetigo and skin infections
that range from mild to severe with toxic shock symptoms and tissue
necrosis.
The working dilution of the conjugate should be determined upon

rehydration. The titer of a conjugate varies with the technique, the fluorescent microscope, the filter and the age of the bulb. The working
dilution may vary from laboratory to laboratory. Dilutions of the conjugate are made in rehydrated FA Buffer. The titer is determined as
follows:
DILUTION OF CONJUGATE
Streptococci are facultatively anaerobic gram-positive cocci. They are

catalase negative and may be alpha, beta or non-hemolytic. Lancefield
divided the streptococci into serological groups according to the groupspecific somatic carbohydrate they possessed.1,2,3 The Lancefield groups
have quite different clinical significance. There may be biochemical
and hemolytic differences within the same serological group.
Moody, Ellis and Updyke4 showed that group-specific conjugates could
be prepared from the antiserum used in the Lancefield precipitin test.
This led to the development of the direct fluorescent antibody technique
for the identification of Streptococcus groups. The Group A conjugate,
prepared according to the method of Moody, Ellis and Updyke4 and
Moody, Siegel, Pittman and Winter5 , is used for the detection and
identification of group A Streptococcus.6-8

The direct FA technique involves the preparation of a smear from the
clinical specimen on a glass slide. Smears are ethanol-fixed and stained
with a specific antibody labeled with a fluorescent marker (fluorescein
isothiocyanate or FITC) directed against Group A Streptococcus. The
antigen-antibody reaction is then observed microscopically, using a
suitable wave length of light compatible with the fluorescent marker
employed.

Lyophilized Appearance: Yellow button to powdered cake.
Rehydrated Appearance: Yellow, clear solution.
Rehydrate FA Streptococcus Group A per label directions.
Perform the fluorescent antibody staining procedure using a
known culture of Group A Streptococcus as the homologous
control. The positive control should produce a 4+ reaction
in the 1:20 or greater dilution of the conjugate with the
homologous antigen.
628
FLUORESCENCE
1:5
4+*
1:10
4+
1:20
4+
1:40
4+
1:80
2+
*4+ fluorescence is defined as brilliant yellow-green cocci with sharp

cell outlines and nonstaining centers.
In this example, the last 4+ fluorescence is found in a 1:40 dilution of
the conjugate. Use one dilution lower for a margin of safety. The
working dilution, therefore, in this case is 1:20.
Precautions
Storage
Store lyophilized FA Streptococcus Group A at 2-8C.
Aliquots of the titered conjugate should be prepared in small vials,
frozen in the undiluted state and stored below -20C for optimal stability.
Prepare only a sufficient amount of diluted conjugate for each days use.
Expiration Date
Procedure
Materials Provided

FA Buffer, Dried
The Difco Manual
Section V
Staining Tray
Lamps:
HBO-50, HBO-100, HBO-200 or
Xenon XBO-150; 6X 5A Tungsten
Excitation Wavelength:
365 nm
Ocular:
10X
Objective:
10X, 40X (Fluorite)
Filters:
BG-12 or KP490, K515 or K530
Condenser:
95% Ethanol
McFarland Barium Sulfate Standard #3
Glass slides
Cover slips
Sterile swabs
Culture tubes, 12 x 75 mm
Coplin jars
Todd Hewitt Broth
Incubator, 35 2C
Reagent Preparation
tests. Ensure that all glassware and pipettes are clean and free of
residues such as detergent.
FA Streptococcus Group A: To rehydrate, add 5 ml sterile distilled or

Specimens submitted to the laboratory for the detection of streptococci
must be obtained under proper medical guidance. Suitable specimens
are those collected from the nose, nasopharyngeal area and throat, skin,
wounds, pus, blood, cerebrospinal fluid and urine. When collecting a
throat specimen, it is imperative that the sample is collected properly
to obtain an adequate amount of material. Improperly obtained specimens will yield cultures that contain minimal numbers of streptococci.
For specific information on specimen collection and preparation,
consult appropriate references.9,10
Note: Specimens containing streptococci survive well at 4EC on tightly
capped blood agar slants but survive poorly in a broth medium.
1. Place a swab having a throat or other specimen, excluding urine,
into a tube containing 1 ml Todd Hewitt Broth and incubate at
35 2C for 2-5 hours. For urine samples, proceed to step #3.
2. Drain the swab against the side of the tube and place it into another
sterile tube for storage in a refrigerator, if desired.
3. Centrifuge tubes containing known and unknown cultures for 10
minutes at 1,500-2,000 rpm to sediment cells.
4. Pour off supernatant liquid (autoclave before discarding) and
resuspend the cells in 2 ml distilled or deionized water. Adjust
density to approximately a McFarland Barium Sulfate Standard #3.
5. Prepare duplicate smears of the same culture on prepared microscope slides.
6. Allow the smear to air dry.
7. Fix the smear by placing the slide in 95% ethanol for 1 minute.
The Difco Manual
8. Remove the slide and allow to air dry.

Note: Smears may also be prepared from specimens grown on blood
agar and suspected of being Streptococcus because of their growth
characteristics. The isolation medium recommended for this species is
any one of several blood agar bases containing 5% sterile, defibrinated
sheep blood. Hemolytic reactions should be determined from a pure
culture before serological examination. Sheep blood plates are recommended because they exhibit clear-cut reactions for streptococci. For
antigen preparation, Todd Hewitt Broth is recommended.
Test Procedure
1. Add several drops of a predetermined working dilution of FA
Streptococcus Group A conjugate to the smear on one end of a
microscope slide. Distribute it evenly over the entire smear with
an applicator stick so as not to disturb the smear.
2. Place the slide in a Staining Tray or moist chamber. Incubate at
room temperature for 30 minutes.
3. Drain off excess conjugate and place in a Coplin jar containing FA
Buffer solution. Let stand for 10 minutes with 2 changes of buffer
and a final rinse of distilled water.
4. Remove the slide; allow it to drain and air dry.
5. Add one small drop of FA Mounting Fluid pH 9 to the slide and
mount with a glass cover slip.
6. Examine using a fluorescent microscope with an excitation wavelength
of 365 nm under a 40X or 100X objective.
7. Read and record the amount of fluorescence.
Results
1. Read and record results as follows:
4+ Maximum fluorescence; brilliant yellow-green; clear-cut
cell outline; sharply defined cell center.
3+ 75% fluorescence; less brilliant yellow-green; clear-cut
cell outline; sharply defined cell center.
2+ 50% fluorescence; definite but dim; cell outline less well
defined.
Negligible or complete lack of fluorescence (negative).
2. A 4+ fluorescence in the unknown smear with the homologous
conjugate is evidence that the unknown organism is homologous
to Streptococcus Group A conjugate.

1. Some experience is required to grade the intensity of the fluorescence.
2. Discard the conjugate if contaminated.
3. The conjugate should not be subjected to repeated freezing and
thawing. Such treatment is detrimental to the antibody content.
4. All glassware employed in the preparation, testing and storage of
these reagents must be free of detergents or other harmful residues.
5. FA Buffer showing turbidity or mold growth should be discarded.
References
1. Lancefield, R. C. 1933. A serological differentiation of human and
other groups of haemolytic streptococci. J. Exp. Med. 57:571-595.
2. Lancefield, R. C. 1928. The antigenic complex of Streptococcus
haemolyticus. I. Demonstration of a type of specific substance in
extracts of Streptococcus haemolyticus. J. Exp. Med. 47:91-103.
629
FTA-ABS Test Reagents
Section V
3. Lancefield, R. C. 1938. A micro precipitin-technique for

classifying hemolytic streptococci, and improved methods for
producing antisera. Proc. Soc. Exp. Biol. Med. 38:473-478.
4. Moody, M. D., E. C. Ellis, and E. L. Updyke. 1958. Staining
bacterial smears with fluorescent antibody. IV. Grouping
streptococci with fluorescent antibody. J. Bacteriol. 75:553-560.
5. Moody, M. D., A. C. Siegel, B. Pitmann, and C. C. Winter. 1963.
Fluorescent-antibody identification of group A streptococci from
throat swabs. Am. J. Publ. Hlth. 53:1083-1092.
6. Warfield, M. A., R. H. Page, W. W. Zuelzer, and C. S. Stulberg.
1961. Immunofluorescence in diagnostic bacteriology. II. Identification of Group A Streptococci in throat smears. Am. J. Dis. Child.
101:160-163.
7. Peeples, W. J., D. W. Spielman, and M. D. Moody. 1961. Field
application of fluorescent antibody technique for identification of
group A streptococcus. Pub. Health Rep. 76:651-654.
8. Estela, L. A., and H. E. Shuey. 1963. Comparison of fluorescent

antibody, precipitin, and bacitracin disk methods in the identification
of group A streptococci. Amer. J. Clin. Pathol. 40:591-597.
transport, and storage, p. 19-32. In P. R. Murray, E. J. Baron, M. A.
Pfaller, F. C. Tenover, R. H. Yolken (ed.), Manual of clinical microbiology, 6th ed. American Society for Microbiology, Washington, D. C.
10. Isenberg, H. D. (ed.) 1992. Clinical microbiology procedures handbook, vol. 2. American Society for Microbiology, Washington, D. C.
Packaging
FA Buffer, Dried
Staining Tray
5
6 x 10
100
10
6x5
1
ml
ml
g
kg
ml
tray
2318-56
2314-33
2314-15
2314-08
3340-57
5251-31
Bacto FTA-ABS Test Reagents

FTA Serum Reactive . FTA Antigen . FTA Serum Non-Reactive
FTA Sorbent . FTA Sorbent Control . FA Human Globulin
Antiglobulin (Rabbit) . Tween 80 . FA Buffer, Dried
Intended Use
The FTA-ABS Test (Fluorescent Treponemal Antibody Absorption) is
an indirect immunofluorescent procedure for detecting human antibody
against Treponema pallidum, the causative agent of syphilis. The test
uses the following reagents: FTA Antigen, FTA Serum Reactive, FTA
Serum Non-Reactive, FTA Sorbent, FTA Sorbent Control, FA Human
Globulin Antiglobulin (Rabbit), Tween 80, FA Buffer, Dried and FA
Mounting Fluid pH 7.2.
The persistent reactivity of the FTA-ABS Test to a treated case of syphilis,
sometimes for life, minimizes its use for following the response to
therapy as well as making it unreliable for detecting new untreated
cases in epidemiological investigations.
Bacto FTA Reagents are not FDA cleared (approved) for use in
testing (i.e., screening) blood or plasma donors.12
During the secondary stage, most serology tests for syphilis are reactive
and treponemes may be found in the lesions by using dark-field
microscopy. The latent period, which is asymptomatic, may last for
years. Serological tests are usually reactive in the early latent period
but reactivity in non-treponemal tests decreases during the late latent
period. Symptoms of the tertiary or late stage of syphilis may occur
10-20 years after initial infection. Approximately 71% of patients in
the tertiary stage of syphilis have reactive non-treponemal tests.2.3 In
the tertiary stage, treponemal tests will usually be reactive and are the
only basis for diagnosis. The lesions in tertiary syphilis will have few
treponemes. Neurosyphilis is a complication of tertiary syphilis.
Since the clinical manifestations of syphilis can be confused with other
infectious diseases or with noninfectious conditions that cause skin
lesions, proper diagnosis must be based on microscopic examination
of lesion material and serological test results.2
Treponema pallidum is the causative agent of syphilis, a chronic

infection with many clinical manifestations. These manifestations
occur in distinct stages and detection of each stage requires different
laboratory tests.
The FTA test was introduced in 1957 by Deacon, Falcone and Harris.4
Certain difficulties were encountered with respect to sensitivity versus
specificity. In its original form using a 1:5 dilution of patient serum,
the test yielded many false-positive reactions. There seemed to be a cross
reaction of the treponemal antigen with antibodies to group antigens
that are common to all treponemes. The titer of sera containing the
nonspecific group antibodies ranged from 1:5 to 1:100.
During the primary stage, treponemes present in the characteristic

lesion, a chancre, are detectable by dark-field microscopy1 or by the
Direct Fluorescent Antibody Test for Treponema pallidum (DFA-TP).
In 1960, Deacon, Freeman and Harris5 introduced a modified procedure, the FTA-200 test, which used a 1:200 dilution of patient serum.
By increasing the dilution of the serum, nonspecific antibodies were
630
The Difco Manual
Section V
diluted beyond their titer and could no longer interfere with the test.
However, testing a highly diluted serum decreased the sensitivity of
the test. Low antibody titer, which occurs during primary syphilis, was
not detected.
by the Centers for Disease Control and Prevention (CDC). Other standard
treponemal tests include Fluorescent Treponemal Antibody-Absorption
Double Staining Test (FTA-ABS DS) and the Micro Hemagglutination
Assay for Antibodies to Treponema pallidum (MHA-TP).
Deacon and Hunter6 showed that appropriate absorption could eliminate

or block the reactivity of nonspecific antibodies. This absorption produced the FTA-ABS test, an improved test using a 1:5 serum dilution.7
Treponemal antigen tests, such as the FTA-ABS test, are used as confirmatory tests in diagnostic problem cases, such as with patients for
whom the clinical, historical or epidemiological evidence of syphilis
disagrees with nontreponemal tests. The FTA-ABS test is more
sensitive than the VDRL test in primary, late latent and tertiary syphilis.
However, the persistent reactivity of the FTA-ABS test to a treated
case of syphilis, sometimes for life, minimizes its use for following
response to therapy. Therefore, the FTA-ABS test is also unreliable in
detecting new untreated cases in epidemiological investigations. The
test should not be used as a routine screening procedure.3,8
The likelihood of obtaining a reactive FTA-ABS test result in various
stages of untreated syphilis has been reported as follows:2
The FTA-ABS test is a standard diagnostic test for syphilis as defined

FTA Antigen
Lyophilized Appearance: White button to powdered cake.
Rehydrated Appearance: White to off-white, slightly
opalescent liquid.
FTA Serum Reactive
Lyophilized Appearance: Off-white to light amber, button to
powdered cake.
Rehydrated Appearance: Light gold to slightly amber liquid.
FTA Serum Non-Reactive
Lyophilized Appearance: Off-white to light amber, button to
powdered cake.
FTA Sorbent
Lyophilized Appearance: Light amber to dark brown, button
to powdered cake.
Rehydrated Appearance: Gold to brown liquid.
FTA Sorbent Control:
Lyophilized Appearance: Off-white to light amber, button
to powdered cake.
FTA Human Globulin Antiglobulin (Rabbit)
Lyophilized Appearance: Light yellow to yellow-orange,
button to powdered cake.
Rehydrated Appearance: Yellow-green to yellow-orange
liquid.
Control Pattern
Rehydrate and dilute reagents per directions (see Reagent
Preparation). Test as described. Tests failing to exhibit the
following control results are unsatisfactory and should not
be reported.8,13
SERUM TESTED
EXPECTED
FLUORESCENCE INTERPRETATION
Reactive Control Serum - Unabsorbed

Reactive Control Serum - Absorbed
Minimally Reactive Control Serum
Nonreactive Control Serum
Nonspecific Serum Control - Unabsorbed
Nonspecific Serum Control - Absorbed
Nonspecific Staining Control - Unabsorbed
Nonspecific Staining Control - Absorbed
The Difco Manual
4+
3+ to 4+
Reactive
Reactive
1+
N
Reactive
Nonreactive
2+ to 4+
N to
Reactive
Nonreactive
N
N
Nonreactive
Nonreactive
STAGE OF UNTREATED SYPHILIS
% REACTIVE
Primary
Secondary
Latent
Tertiary (Late)
84
100
100
96

Patient serum is diluted 1:5 in sorbent and layered on a microscope
slide fixed with T. pallidum. If the patients serum contains antibodies,
these antibodies will coat the treponemes on the slide. Fluoresceinlabeled anti-human immunoglobulin is added. It combines with the
patient antibodies already adhering to the T. pallidum and produces
fluorescein-stained spirochetes that can be observed with a fluorescent
microscope.7,9
Reagents
FTA Antigen (also known as T. pallidum antigen) is a lyophilized,
standardized, killed suspension of Treponema pallidum (Nichols
strain).
FTA Serum Reactive is lyophilized, standardized syphilitic human
sera containing 0.02% Thimerosal as a preservative. It is used to make
Reactive Control Serum (4+) - Unabsorbed, Reactive Control Serum
(4+) - Absorbed, and Minimally Reactive Control Serum (1+). It is
used as a positive control in the FTA-ABS test.
FTA Serum Non-Reactive is lyophilized, standardized, non-syphilitic
human sera containing 0.02% Thimerosal as a preservative. It is used
to make Nonreactive Control Serum (N). It is used as a negative
control in the FTA-ABS test.
FTA Sorbent is a lyophilized, standardized extract of the nonpathogenic
Reiters treponeme (T. phagedenis) prepared from broth culture. It is used
to remove antibodies against nonpathogenic treponemes during preparation of the test specimen, Reactive Control Serum (4+) - Absorbed
and Nonspecific Staining Control - Absorbed.
FTA Sorbent Control is lyophilized, standardized, non-syphilitic
human sera containing 0.02% Thimerosal as a preservative. It is used
to make Nonspecific Control Serum - Unabsorbed, which demonstrates
at least 2+ nonspecific reactivity at a 1:5 dilution in FA Buffer, and
Nonspecific Control Serum - Absorbed, which demonstrates essentially
no reactivity at a 1:5 dilution in FTA Sorbent.
631
FA Human Globulin Antiglobulin (Rabbit) is lyophilized, fluorescein-conjugated (FITC) antihuman globulin containing 0.02%
Thimerosal as a preservative. It is used to show the presence of
human syphilitic antibodies on the treponemal antigen.
Tween 80 is Polysorbate 80, U.S.P. It is used to prepare 2% Tween 80,
which acts as a dispersing agent.
FA Buffer, Dried is phosphate buffered saline (PBS) which, upon
rehydration, yields a 0.85% NaCl solution buffered to pH 7.2. FA
Buffer is used in preparing Reactive Control Serum (4+) - Unabsorbed,
Minimally Reactive Control Serum (1+), Nonreactive Control Serum
(N) and Nonspecific Staining Control - Unabsorbed.
FA Mounting Fluid pH 7.2 is standardized, reagent grade glycerin
adjusted to pH 7.2 for use in mounting specimens on slides to be viewed
under the fluorescent microscope.
Precautions
2. FTA Serum Reactive
FTA Sorbent Control
WARNING! POTENTIAL BIOHAZARDOUS REAGENTS.
Each donor unit used in the preparation of these reagents was tested
by an FDA-approved method for the presence of the antibody to
human immunodeficiency virus (HIV) as well as for hepatitis B surface antigen and found to be negative (were not repeatedly reactive).
Because no test method can offer complete assurance that HIV,
hepatitis B virus or other infectious agents are absent, these reagents
should be handled at the Biosafety Level 2 as recommended for
any potentially infectious human serum or blood specimen.10,11
3. FTA Antigen
FTA Serum Reactive
FTA Sorbent
FTA Sorbent Control
FA Human Globulin Antiglobulin (Rabbit)
4. FA Buffer, Dried
5. Observe universal blood and body fluid precautions in handling
and disposing of specimens.
Storage
Store unopened products as specified below:
FTA Antigen
2-8C
FTA Serum Reactive
2-8C
2-8C
FTA Sorbent
2-8C
632
Section V
FTA Sorbent Control

FA Human Globulin
Antiglobulin (Rabbit)
Tween 80
FA Buffer, Dried
2-8C
2-8C in the dark
15-30C
Below 30C
15-30C
Expiration Date
Rehydrated FTA Antigen stored at 2-8C is stable for 1 week.
Rehydrated FA Buffer showing turbidity or mold growth should be
discarded.
Discard 2% Tween 80 that exhibits a precipitate or pH change.
Procedure
Materials Provided
FTA Antigen
FTA Serum Reactive
FTA Sorbent
FTA Sorbent Control
Tween 80
FA Buffer, Dried
FA Mounting Fluid, pH 7.2

Timer
Serological pipettes, 0.2 ml, 5 ml, 1 ml
Micropipettors delivering 10-200 l
Test tubes, 12 x 75 mm
Water bath (56C)
Vortex mixer
Platinum loop, 2 mm, 26 gauge
Slides, plain or frosted, 1 x 3 inch, 1 mm thick, inscribed with 2 x 1
cm circles
Staining dish with removable slide carriers
Slide board or holder
Moisture chamber
Acetone
Bibulous paper
Distilled water
Incubator, 35-37C
Oil, Immersion
Cover slips, No. 1, 22 mm square
Lamps:
HBO-50, HBO-100, HBO-200 or Xenon
XBO-150; 6X 5A Tungsten
Ocular:
10X
Objective:
10X, 40X (Fluorite)
Filters:
BG-12 or KP490, K515 or K530
Condenser:
Reagent Preparation
FTA Antigen: Rehydrate with 1 ml distilled or deionized water and
rotate to completely dissolve the contents. This solution will yield
The Difco Manual
Section V
approximately 3.5 x 107 treponemes per ml. Mix thoroughly with a

disposable pipette and rubber bulb, drawing the suspension into and
expelling it from the pipette 8-10 times to break treponemal clumps
and ensure an even distribution of treponemes. Confirm the even
distribution by dark-field examination. Use FTA Antigen in its entirety
to prepare antigen smears on the day it is rehydrated. Approximately
200-300 slides may be prepared with 1 ml of antigen.
To prepare FTA Antigen smears:
1. Wipe inscribed slides with clean gauze and, if necessary, alcohol
to remove dust particles.
2. Using a platinum wire loop (2 mm, 26 gauge), smear 1 loopful of
reconstituted FTA Antigen within the 2 circles. Air dry at room
temperature for at least 15 minutes.
3. Immerse the dry slide into acetone for 10 minutes to fix the
treponemal antigen smear to the slide; air dry. Fix no more than
50 slides per 200 ml of acetone.
4. Use slides immediately or store at or below -20C after acetone
fixation. Thaw before use; do not refreeze. Use within 1 year, but
only if satisfactory results are obtained with test controls.
FTA Serum Reactive: Rehydrate with 5 ml distilled or deionized
water and rotate gently to completely dissolve the contents. Aliquot in
0.4 ml amounts and store at or below -20C. Do not refreeze thawed
aliquot. Approximately 12 tests may be obtained per 5 ml vial. This
serum should be heated at 56C for 30 minutes before use.
FTA Serum Non-Reactive: Rehydrate with 5 ml distilled or deionized
0.4 ml amounts and store at or below -20C. Approximately 90-100
tests may be obtained per 5 ml vial. This serum should be heated at
56C for 30 minutes before use.
FTA Sorbent: Rehydrate with 5 ml distilled or deionized water and
rotate gently to completely dissolve the contents. Store at 2-8C or
aliquot and store at -20C. The quantity of FTA Sorbent used for each
test sample or serum is 0.2 ml. The quantity of FTA Sorbent needed for
3 controls is 0.6 ml. Approximately 20-25 tests may be performed with
5 ml of FTA Sorbent.
FTA Sorbent Control: Rehydrate with 0.5 ml distilled or deionized
0.25 ml amounts and store at or below -20C. For each test, 0.1 ml of
FTA Sorbent Control is needed. Approximately 2 tests may be
performed per 0.5 ml vial because of evaporation from heating. This
serum should be heated at 56C for 30 minutes before using.
FA Human Globulin Antiglobulin (Rabbit): Rehydrate with 1 ml or
5 ml distilled or deionized water, depending on label directions. Aliquot
in 0.5 ml amounts and store at or below -20C. Each lot is supplied
with a dilution titer. Since conditions and equipment differ from one
laboratory to another, it is necessary to titer and test a new lot of conjugate with the fluorescent microscope assembly currently in use.3,8,13
1. Prepare serial dilutions in 2% Tween 80, including the titer specified
on the vial.
2. Test each dilution per the Test Procedure with Reactive Control
Serum (4+) and Nonspecific Staining Control.
3. Test a known lot of reagent using the Reactive Control Serum (4+),
Minimally Reactive Control Serum (1+) and Nonspecific Staining
Control as controls of the reagents and test conditions.
The Difco Manual
4. During further testing, use the dilution that produces 1 doubling

dilution lower than the 4+ endpoint. The 4+ endpoint is the highest
dilution of conjugate yielding 4+ fluorescence with the Reactive
Control Serum (4+).
FA Buffer, Dried: Dissolve 10 grams in 1 liter of distilled or deionized
water and rotate gently to completely dissolve the contents. Store at
2-8C. Use the solution if it is free of mold growth and turbidity.
Tween 80: Heat the bottle of Tween 80 and a flask containing 98 ml
FA Buffer to 56C in a water bath. Add 2 ml of Tween 80 to the buffer
and rinse the pipette thoroughly in the buffer. Check the pH and adjust
to pH 7.2 with 1N NaOH. Discard if a precipitate develops or the pH
changes.

Test serum: Collect patient (test) serum according to recommended
procedures.2,3,8,9,13 Store specimens at room temperature for up to
4 hours or at 2-8C for up to 5 days; serum specimens may be frozen at
or below -20C.
Test and control sera: Equilibrate the sera to room temperature, then
heat at 56C for 30 minutes. Reheat previously heated sera for 10
minutes on the day of testing. Cool to room temperature before testing.
Bacterial contamination or excessive hemolysis may render a specimen
unsuitable for testing. Such specimens should not be tested.
Test Procedure
This procedure conforms with those published by the U. S. Department
of Health, Education and Welfare14 and with subsequent procedures
published by the American Public Health Association.9,13
1. FTA Antigen smears: Obtain previously prepared smears, thaw
and dry if appropriate, and identify the frosted end of the slides to
correspond with each test and control serum to be tested.
2. Prepare the following test and control sera in appropriately identified
tubes no more than 30 minutes before testing and mix thoroughly
(at least 8 times):
Test Serum (1:5): Dilute 0.05 ml (50 l) of heated (or reheated)
test serum in 0.2 ml (200 l) FTA Sorbent.
Reactive Control Serum (4+) - Unabsorbed: Dilute 0.05 ml (50 l)
FTA Serum Reactive in 0.2 ml (200 l) FA Buffer (PBS).
Reactive Control Serum (4+) - Absorbed: Dilute 0.05 ml (50 l)
FTA Serum Reactive in 0.2 ml (200 l) FTA Sorbent.
Minimally Reactive Control Serum (1+): Dilute FTA Serum
Reactive, as indicated on the label, in FA Buffer (PBS) to yield a
1+ fluorescence. The minimal degree of fluorescence that can be
reported as reactive is 1+ fluorescence.
Nonreactive Control Serum (N) (1:40): Prepare a 1:40 dilution
of FTA Serum Non-Reactive by adding 0.05 ml (50 l) of serum
to 1.95 ml FA Buffer (PBS).
Nonspecific Serum Control - Unabsorbed (2+ nonspecif ic
reactivity): Dilute 0.05 ml (50 l) FTA Sorbent Control in 0.2 ml
(200 l) FA Buffer (PBS).
Nonspecific Serum Control - Absorbed (nonreactive, - to ):
Dilute 0.05 ml (50 l) FTA Sorbent Control in 0.2 ml (200 l)
FTA Sorbent.
Nonspecific Staining Control - Unabsorbed: Use 0.03 l (30 ml)
FA Buffer (PBS) undiluted.
Nonspecific Staining Control - Absorbed: Use 0.03 ml (30 l)
FTA Sorbent undiluted.
633
Section V
3. FTA Antigen smears: Cover the previously identified FTA Antigen

smears with 0.03 ml (30 l) of the corresponding test or control serum
prepared above, making certain that the entire smear is covered.
4. Place the slides in a moist chamber to prevent evaporation and
incubate at 35-37C for 30 minutes.
5. Place the slides in a slide carrier and rinse as follows:
Rinse in running FA Buffer for 5 seconds.
Soak in FA Buffer for 5 minutes.
Agitate by dipping in and out of the buffer 30 times.
Repeat the soaking and agitation in fresh buffer.
Rinse in running distilled water for 5 seconds.
Gently blot dry with bibulous paper.
6. FA Human Globulin Antiglobulin (Rabbit): Dilute the antiglobulin
to its working titer (determined above) using 2% Tween 80 in
FA Buffer.
7. FTA Antigen smears: Cover each test and control smear with
approximately 0.03 ml (30 l) of diluted FA Human Globulin
Antiglobulin (Rabbit). Spread uniformly to cover the entire smear.
8. Repeat steps 4 and 5.
9. Mount the slides immediately using a small drop of FA Mounting
Fluid pH 7.2 and apply a cover slip, being careful not to trap air
bubbles in the mounting fluid.
10. Immediately examine the slides microscopically for intensity of
fluorescence using the microscope assembly described above. If it
is necessary to delay reading, store the slides in the dark and read
within 4 hours. Results are valid only if the quality control pattern
is satisfactory.
11. Verify the presence of treponemes on the nonreactive control slides
by dark-field microscopy.
Results
Using the 1+ serum control as a reading standard, record the intensity
of fluorescence of the treponemes and report as follows. Retest all
specimens with an initial test fluorescence of 1+. When a specimen
initially read as 1+ yields a retest reading of 1+ or greater, it is reported
as reactive. All other results are reported as nonreactive. Retesting
nonreactive specimens is not necessary.
Without historical or clinical evidence of treponemal infection, equivocal
test results (see below) suggest the need for testing a second specimen
obtained 1-2 weeks after the initial specimen.
INTENSITY OF
FLUORESCENCE
INITIAL TEST
RESULT
RETEST
RESULT
REPORT
Moderate to strong
Equivalent to 1+ control
2+ to 4+
1+
1+
1+
to <1+
NA
>1+
1+
<1+
NA
NA
Reactive
Reactive
Reactive minimal*
Nonreactive
Nonreactive
Nonreactive
Visible staining but <1+

None or vaguely visible,
not distinct
Moth eaten or beaded
Atypical
*Equivocal result.

1. When the treponemal test results and the clinical opinion disagree,
repeat the treponemal test and obtain additional clinical and
historical information. If the disagreement persists, send the specimen
to a reference laboratory such as the local state health department
634
2.
3.
4.
5.
for additional confirmatory tests. The final diagnosis depends on

the clinical judgment of a specialist very experienced in sexually
transmitted diseases.2,3
The test should not be used to follow the response to therapy nor
can it be relied on to detect new, untreated cases in epidemiological
investigations.
Atypical fluorescence and false-positive results have been
associated with patients having active systemic, discoid and
drug-induced varieties of lupus erythematosus 13-17 and other
autoimmune diseases.
Elderly patients may exhibit unexplained FTA-ABS reactions.
At times, deciding whether a reading is weak or vaguely visible
may be difficult. The ability to make this distinction is critical,
since a nonreactive (vaguely visible to none) serum is not retested.
References
1. Creighton, E. T. 1990. Dark field microscopy for the detection
and identification of Treponema pallidum, p. 49-61. In S. A. Larsen,
E. F. Hunter, and S. J. Kraus (ed.), Manual of tests for syphilis,
8th ed. American Public Health Association, Washington, D. C.
2. Janda, W. M. (ed.). 1992. Immunology, p. 9.7.1-9.7.20. In H. D.
Isenberg (ed.), Clinical microbiology procedures handbook,
vol. 2. American Society for Microbiology, Washington, D. C.
3. Norris, S. J., and S. A. Larsen. 1995. Treponema and other
host-associated spirochetes, p. 636-651. In P. R. Murray, E. J.
Washington, D. C.
4. Deacon, W. E., V. H. Falcone, and A. Harris. 1957. A fluorescent
test for treponemal antibodies. Proc. Soc. Exp. Biol. and Med.
96:477-480.
5. Deacon, W. E., V. H. Falcone, and A. Harris. 1960. Fluorescent
treponemal antibody test. A modification based on quantitation
(FTA-200). Proc. Soc. Exp. Biol. and Med. 103:827-829.
6. Deacon, W. E., and E. M. Hunter. 1962. Treponemal antigens as
related to identification and syphilis serology. Proc. Soc. Exp. Biol.
and Med. 110:352-356.
7. Hunter, E. F., W. E. Deacon, and P. E. Meyer. 1964. An improved
FTA test for syphilis; the absorption procedure (FTA-ABS). Publ.
Hlth. Report 79:410-412.
8. Turgeon, M. L. 1990. Immunology and serology in laboratory
medicine. The C. V. Mosby Company, St. Louis, MO.
9. Wentworth, B. B., and F. N. Judson. 1984. Laboratory methods
for the diagnosis of sexually transmitted diseases. American
settings. Morbid. Mortal. Weekly Rep. 37:377-382, 387-388.
11. Occupational Safety and Health Administration, U. S. Department of Labor. 1991. 29CFR, part 1910. Occupational exposure to
bloodborne pathogens; final rule. Federal Register 56:64175-64182.
12. Johnson, R. M. Letter. July 1, 1994. Department of Health
& Human Services, Public Health Service, Food and Drug
Administration, Rockville, MD.
The Difco Manual
Section V
13. Larsen, S. A., E. F. Hunter, and S. J. Kraus. 1990. A manual of tests for
syphilis. American Public Health Association, Washington, D.C.
14. U.S. Department of Health, Education and Welfare. 1969.
Manual of tests for syphilis; PHS Publication No. 411. US
Government Printing Office, Washington, D.C.
15. Kraus, S. J., J. R. Haserick, and M. A. Lantz. 1970. Fluorescent
treponemal antibody absorption test reactions in lupus erythematosus. N. Engl. J. Med. 282:1287-1290.
16. Goldman, J. N., and M. A. Lantz. 1971. FTA-ABS and VDRL
slide test reactivity in a population of nuns. J.A.M.A. 217:53-55.
17. Shore, R. N., and J. A. Faricelli. 1977. Borderline and reactive
FTA-ABS results in lupus erythematosus. Arch. Dermatol.
113:37-41.
18. Monson, R. A. 1973. Biological false-positive FTA-ABS test in
drug-induced lupus erythematosus. J.A.M.A. 224:1028-1030.
19. Anderson, B., and M. T. Stillman. 1978. False-positive FTA-ABS

in hydralazine-induced lupus. J.A.M.A. 239:1392-1493.
Packaging
FA Buffer, Dried
FA Human Globulin Antiglobulin
(Rabbit)
FTA Antigen
FTA Serum Reactive
FTA Sorbent
FTA Sorbent Control
Tween 80
6 x 10
100
1
5
6x5
1
5
5
5
6 x 0.5
6x5
g
g
ml
ml
ml
ml
ml
ml
ml
ml
ml
2314-33
2314-15
2449-50
2449-56
2329-57
2344-50
2440-56
2439-56
3259-56
3266-49
3118-57
FTA-ABS Test Procedure

Abbreviated Schematic
STEP 1
Prepare sera and
reagents.
STEP 2
Dilute sera.
STEP 3
Add test and control sera
to appropriate FTA
Antigen smears.
STEP 4
Add conjugate to the
FTA Antigen smears.
STEP 5
Record reactions of test
and control sera. Verify
that control sera provided
the expected results.
Apply 0.03 ml
conjugate to the smear.
Incubate, rinse and
mount slide. Examine
microscopically.
Dependent on
antibody status
of test serum.
FTA Antigen
Rehydrate with 1 ml
distilled or deionized
water. Prepare smears.
Fix with acetone. Use as
FTA Antigen smear.
FTA Antigen Smear

Thaw, dry and identify
sufficient FTA Antigen
smears to correspond with
each of the test and
control sera to be tested.
Heat at 56C for

30 minutes or reheat
previously heated
serum for 10 minutes.
Dilute 1:5 by adding

0.05 ml serum
to 0.2 ml in
FTA Sorbent.
Test (Patient) Serum

Apply 0.03 ml test
serum to an FTA
Antigen smear.
Incubate smear. Rinse.
FTA Serum Reactive

Rehydrate with
5 ml distilled
or deionized water.
Heat at 56C for
30 minutes.

0.05 ml FTA Serum
Reactive to 0.2 ml
FA Buffer.
Reactive Control Serum (4+) Unabsorbed

Apply 0.03 ml
Apply 0.03 ml
Reactive Control Serum
Unabsorbed to an FTA
Incubate, rinse and
Antigen smear.
microscopically.

0.05 ml FTA Serum
Reactive to 0.2 ml
FTA Sorbent.
Reactive Control Serum (4+) Absorbed

Apply 0.03 ml
Apply 0.03 ml
Reactive Control Serum
Absorbed to an FTA
Incubate, rinse and
Antigen smear.
microscopically.
Dilute FTA Serum

Reactive to 1+
fluorescence (labeled
titer) in FA Buffer.
Minimally Reactive Control Serum (1+)

Apply 0.03 ml
Apply 0.03 ml
Minimally Reactive
Control Serum to an
Incubate, rinse and
FTA Antigen smear.
microscopically.
The Difco Manual
4+ Reactive
3+ to 4+ Reactive
1+ Reactive
635
FTA Serum Nonreactive

Rehydrate with
5 ml distilled
or deionized water.
Heat at 56C for
30 minutes.
FTA Sorbent Control
Rehydrate with
0.5 ml distilled
or deionized water.
Heat at 56C for
30 minutes.
FA Buffer, Dried
Dissolve 10 grams
in 1 liter distilled
or deionized water.
FTA Sorbent
Rehydrate with
5 ml distilled
or deionized water.
Section V

0.05 ml FTA Serum
Nonreactive to
1.95 ml FA Buffer.
Nonreactive Control Serum

Apply 0.03 ml
Apply 0.03 ml
Nonreactive Control
Serum to an FTA
Incubate, rinse and
Antigen smear.
microscopically.

0.05 ml FTA
Sorbent Control to
0.2 ml FA Buffer.
Nonspecific Serum Control Unabsorbed

Apply 0.03 ml
Apply 0.03 ml
Nonspecific Serum
Control - Unabsorbed to
Incubate, rinse and
an FTA Antigen smear.
microscopically.

0.05 ml FTA
Sorbent Control to
0.2 ml FA Buffer.
Nonspecific Serum Control Absorbed

Apply 0.03 ml
Apply 0.03 ml
Nonspecific Serum
Incubate, rinse and
microscopically.
Use 0.03 ml FA Buffer

0.05 ml FTA Serum
Nonreactive to
1.95 ml FA Buffer.
Nonspecific Staining Control Unabsorbed

Apply 0.03 ml
Apply 0.03 ml
Nonspecific Staining
Incubate, rinse and
microscopically.
Use 0.03 ml FTA Sorbent

undiluted as the
diluent (above) and as
the Nonspecific Staining
Control - Absorbed
Nonspecific Staining Control Absorbed

Apply 0.03 ml
Apply 0.03 ml
Nonspecific Staining
Control -Absorbed to
Incubate, rinse and
microscopically.
N Nonreactive
2+ to 4+ Reactive
N to Nonreactive
N Nonreactive
N Nonreactive

Rehydrate with 1 ml
Dilute to labeled
or 5 ml distilled
titer with
or deionized water.
2% Tween. Use
Determine titer if
as Conjugate.
a new lot.
Tween 80
Heat Tween 80 and
FA Buffer to 56C.
Add 2 ml Tween 80
to 98 ml FA Buffer.
Adjust to pH 7.2.
636
The Difco Manual
Section V
Febrile Antigen Set
Bacto Febrile Antigen Set
Contains: Brucella Abortus Antigen (Slide) . Proteus OX19 Antigen

(Slide) . Salmonella O Antigen Group D . Salmonella H Antigen a
Salmonella H Antigen b . Salmonella H Antigen d . Febrile Positive
Control Polyvalent . Febrile Negative Control
Intended Use
Bacto Febrile Antigen Set is used in the detection of febrile antibodies
by the slide and tube agglutination tests.

Agglutination tests have been used in diagnosing certain febrile illnesses
since the early 1900s. Patients experiencing febrile symptoms,
including fever, chills, malaise and fatigue, were considered likely to
have typhoid fever, brucellosis, rickettsial infection (either typhus or
spotted fever) or tularemia. The agents of these infections are difficult
or unlikely to be isolated by routine laboratory methods but do cause
detectable increases in antibody levels in the patients serum.

Brucella Abortus Antigen (Slide), Proteus OX19 Antigen (Slide),
Salmonella O Antigen Group D, Salmonella H Antigen a,
Salmonella H Antigen b, Salmonella H Antigen d
Appearance:
Turquoise-blue-violet suspension.
Febrile Positive Control Polyvalent
Lyophilized appearance: Light gold to amber, button to
powdered cake.
Rehydrated appearance: Light gold to amber, clear liquid.
Lyophilized appearance: Colorless to light gold, button to
powdered cake.
Rehydrated appearance: Colorless to light gold, clear liquid.
Rehydrate Febrile Positive Control Polyvalent and Febrile
Negative Control per label directions. Perform the slide or tube
agglutination test using an appropriate Febrile Antigen and
positive and negative controls diluted in the same proportion
as a patient serum.
A Febrile Antigen is considered satisfactory if it does not
agglutinate with the negative control and shows 2+ or greater
agglutination with the positive control at the following dilution:
Brucella Abortus Antigen
Proteus OX19 Antigen
Salmonella O Antigen Group D
Salmonella H Antigen a
Salmonella H Antigen b
Salmonella H Antigen d
The Difco Manual
1:80
1:160
1:80
1:80
1:80
1:80
Febrile Antigen is a term generally referring to bacterial suspensions

representative of many species of microorganisms pathogenic to man
which are accompanied by a fever in the host. A battery of febrile antigens
evolved as the febrile antigen or febrile agglutinin test. The febrile
antigen test is based on the Widal test (Salmonella somatic O and
flagellar H antigens), the Weil-Felix test (antigens of selected Proteus
strains), and the Brucella abortus antigen test.1,2,3 In some situations,
the Francisella tularensis antigen test is included in the battery.
DISEASE
ASSOCIATED FEBRILE ANTIGEN
Brucellosis
Brucella abortus
Rocky Mountain spotted fever Proteus OX19
Typhus
Proteus OX19
Typhoid fever
Typhoid fever
Paratyphoid fever
Paratyphoid fever
In 1896, Widal introduced techniques for testing patients serum for
antibodies in cases of typhoid fever.1 The Widal test was used diagnostically in two ways. First, it was considered diagnostic when a single
high titer of antibodies occurred during the first week of illness. In
addition, it was diagnostic if a greater than fourfold titer rise existed
in serum samples taken 1 to 2 weeks apart.2,4,5,6 The Widal test was
developed to include Salmonella typhi and other species of Salmonella
detected by a variety of O and H antigens. S. typhi and S. paratyphi
A and B are the major pathogens in this group that can produce
clinically distinct systemic illness. The Widal test for antibodies to the
O antigens of Salmonella serotypes most likely to cause typhoid fever
(usually S. typhi and S. paratyphi A and B) can be useful in diagnosing
typhoid fever when other methods have failed.7
The Weil-Felix test became popular in the 1920s after it was observed
that certain strains of Proteus would agglutinate early convalescentphase sera from patients with suspected rickettsial disease.3 Proteus
antigens (OX2, OX19 and OXK) will cross-react in predictable
patterns, although the reactions are not highly sensitive or specific.
Diagnosis of the cause of febrile disease cannot be based solely on the
analysis of serum samples for antibody response. Many factors may
affect measurable antibody levels. For example, the patients immune
response can be affected by age, immune status, general state of health
and previous immunizations.
may react with one or more antigens in an antibody test procedure
resulting in low-level antibody titers that may not, when used alone,
suggest disease. Cross reactions can occur among species of
Francisella and Brucella, among various species of Salmonella, and
637
Febrile Antigen Set
between Brucella species and Yersinia enterocolitica or Vibrio cholerae.

Antibodies produced in response to a Proteus infection can react with
Proteus OX19 and be misinterpreted as rickettsial antibodies.
The rapid slide test is the most widely used procedure employing
febrile antigens because of the simplicity with which the results may
be reported. Negative slide test reactions can usually be reported as
such if all five serum dilutions have been used. Although the slide test
is not quantitative, running the series of dilutions is necessary to detect
agglutinin content of a serum that might be overlooked for a prozone
phenomenon where higher concentrations of the serum may yield
negative results, but a dilution of the serum is positive. This often
occurs in sera containing Brucella agglutinins and, to a lesser extent,
typhoid agglutinins.
The macroscopic tube test2 should be used to confirm the presence of
antibodies demonstrated by the slide technique and to quantitate their
titer in suspect sera. When quantitative determinations of Rickettsia or
Brucella agglutinins are necessary, tube antigens are used.

Agglutination tests involving the use of febrile antigens determine the
procedure involves serially diluting the patient serum, then adding a
standard volume of antigen. The end point of the test is the last dilution of
the serum that shows a specific amount of agglutination. The end point
converted to a dilution of the serum is called the patients antibody titer.
Reagents
Antigens
1. Febrile Antigens are ready-to-use, whole cell suspensions of the
organisms listed below. Proteus OX19 Antigen (Slide) contains
20% glycerin.
Brucella Abortus Antigen (Slide) - Brucella abortus
Proteus OX19 Antigen (Slide) - Proteus vulgaris OX19
Salmonella O Antigen Group D - Salmonella typhi O901
Salmonella H Antigen a - Salmonella paratyphi A
Salmonella H Antigen b - Salmonella paratyphi B
Salmonella H Antigen d - Salmonella typhi H901
2. Slide test: The Febrile Antigens (Brucella Abortus Antigen (Slide),
Proteus OX19 Antigen (Slide), Salmonella O Antigen Group D,
Salmonella H Antigen a, Salmonella H Antigen b and Salmonella
typhi H901) are used in the slide test and contain sufficient
reagent for 20 slide tests.
Tube test: Salmonella O and H Antigens may also be used in the
tube test and contain sufficient reagent for 25 tube tests.
Brucella Abortus Antigen (Slide) and Proteus OX19 Antigen (Slide)
are used only in the slide test. When confirmation of the slide test
and quantitation are required, Brucella Abortus Antigen (Tube) and
Proteus OX19 (Tube) may be purchased as separate products.
3. Antigen Density: Salmonella O and H Antigens are adjusted to a
density approximating 20 times a McFarland Barium Sulfate
Standard No. 3 (1.8 x 1010 organisms per ml). These antigens are
used undiluted for the slide test and diluted 1:20 for the tube test.
Because antigen density may vary, it is adjusted for optimum
performance when standardized with hyperimmune sera obtained
from laboratory animals.
Variation in antigen color intensity is normal and will not affect
test performance.
638
Section V
4. Febrile Antigens contain the following preservatives:

Brucella Abortus Antigen (Slide): 0.5% phenol, and approximately
0.002% crystal violet and 0.005% brilliant green.
Proteus OX19 Antigen (Slide): 0.25% formaldehyde, and approximately 0.002% crystal and 0.005% brilliant green.
Salmonella O Antigen Group D: 0.5% phenol, and approximately
Salmonella H Antigens a: 0.5% formaldehyde, and approximately
Salmonella H Antigens b: 0.5% formaldehyde, and approximately
Salmonella H Antigens d: 0.5% formaldehyde, and approximately
Antisera
1. Febrile Positive Control Polyvalent is lyophilized, polyclonal,
polyvalent goat antisera containing approximately 0.04% Thimerosal
as a preservative. It contains antibodies for all of the components
of the Febrile Antigen Set. Each vial contains sufficient reagent for
32 slide tests or 50 tube tests using single antigens or for approximately 5 slide tests when using all of the antigens in the set.
2. Febrile Negative Control is a lyophilized, standard protein solution
Each vial contains sufficient reagent for 32 slide tests using single
antigens or for approximately 5 slide tests using all of the antigens
in the set.
Precautions
3. Proteus OX19 Antigen (Slide)
POSSIBLE RISK OF IRREVERSIBLE EFFECTS. Avoid contact
with skin and eyes. Do not breathe mist. Wear suitable
ORGAN(S): Eyes, Kidneys, Lungs, Skin.
5. Febrile Antigens are not intended for use in the immunization of
humans or animals.
Storage
Store Febrile Antigens at 2-8C.
Store lyophilized and rehydrated Febrile Positive Control Polyvalent
at 2-8C.
The Difco Manual
Section V
Expiration Date
The expiration date applies to a product in its intact container when
Procedure
Materials Provided
Febrile Antigen Set:
Proteus OX19 Antigen (Slide)

Slide Test
Agglutination slides, 5 squares, 1 each
Applicator sticks
Sterile deionized water or equivalent
Tube Test
Culture tubes 12 x 75 mm and rack
Waterbath, 35-37C and 50 2C
Refrigerator, 2-8C
Reagent Preparation
Febrile Antigens are ready to use.
Febrile Positive Control Polyvalent: To rehydrate, add 5 ml sterile
distilled or deionized water and rotate gently to completely dissolve
the contents.
Febrile Negative Control: To rehydrate, add 5 ml sterile deionized water,
or equivalent, and rotate gently to completely dissolve the contents.

Collect a blood specimen by aseptic venipuncture. Serum is required
for the test. Store serum specimens at room temperature for no longer
than 4 hours; for prolonged storage, keep at 2-8C for up to 5 days or
maintain at or below -20C. Serum specimens must be clear, free of
hemolysis and show no visible evidence of bacterial contamination (turbidity, hemolysis or particulate matter). Refer to appropriate references
for more information on collection of specimens.10,11 Serum specimens
must not be heated. Heat may inactivate or destroy certain antibodies.
An increase in titer over a period of time is the best indicator of active
infection. The accuracy and precision of the tests can be affected not
only by test conditions, but also by the subjectivity of the person reading
the endpoint.
The Difco Manual
Febrile Antigen Set
A preliminary test using either the rapid slide test and/or the macroscopic tube test may be performed on the initial serum specimen and
reported to the physician at that time. An aliquot of the serum should
be transferred to a sterile test tube, sealed tightly, and kept in the freezer.
When the second serum is obtained, it should be run in parallel with
the original specimen. In this manner, the original serum will serve as
a control and any difference in titer will be more credible, since the
bias associated with the performance of the test and determining the
endpoint will be reduced.
Test Procedure
Slide Test
Use the slide test only as a screening test; confirm positive results
with the tube test. Test each Febrile Antigen separately, repeating steps
1-6 for each Antigen.
1. Test Serum: Using a 0.2 ml serological pipette, dispense 0.08, 0.04,
the agglutination slide.
0.04, 0.02, 0.01 and 0.005 ml of Febrile Positive Control Polyvalent into a row of squares on the agglutination slide.
4. Febrile Antigen: Gently shake the vial of antigen to ensure a
smooth, uniform suspension. Place one drop (35 l) of antigen
suspension in each drop of test serum, positive control and
negative control.
5. Mix each row of test and control serum, using a separate applicator
stick for each row. Start with the most dilute mixture (0.005 ml)
and work to the most concentrated (0.08 ml).
7. The final dilutions in squares 1-5 correspond with tube dilutions of
1:20, 1:40, 1:80, 1:160, 1:320, respectively.
Results
No agglutination.
2. Positive control: Should show 2+ or greater agglutination at the
following dilutions:
1:80
1:160
1:80
1:80
1:80
1:80
3. Negative control: Should show no agglutination.
4. Test specimens: The serum titer is that dilution which shows 2+
or greater agglutination. See Table 1.
639
Febrile Antigen Set
Section V

SERUM (ml)
CORRELATED
TUBE DILUTION
0.08
1:20
0.04
1:40
0.02
1:80
0.01
1:160
0.005
1:320
Serum titer
9. Read and record results.

REACTIONS
SPECIMEN 1
SPECIMEN 2
SPECIMEN 3
3+
2+
1+
1:40
4+
4+
3+
3+
1+
1:160
4+
3+
2+
+
1:80
Tube Test
Salmonella O Antigen Group D and Salmonella H Antigens a, b and d
in the Febrile Antigen Set are used for both slide and tube agglutination tests. Brucella Abortus Antigen (Slide) and Proteus OX19 Antigen
(Slide) are intended only for slide tests. When confirmation of the slide
test and quantitation is required, separate tube test antigens, Brucella
Abortus Antigen (Tube) and Proteus OX19 (Tube), may be purchased
separately.
Results
No agglutination.
2. Salmonella Antigens used in tube agglutination procedures detect
antibodies to either O (somatic) antigens or H (flagellar) antigens
and these antibodies give different reactions. An O antigen and the
corresponding antibody give a coarse, compact agglutination that
may be difficult to disperse. An H antigen and its corresponding
antibody give a loose flocculent agglutination. Do not vigorously
shake tubes containing H antigens. Characteristic O and H agglutination is illustrated below.
Each Febrile Antigen must be tested separately. Repeat steps 1-10 for
each antigen.
Prepare a 1:20 dilution of each antigen to be tested by adding 1 part of
antigen to 19 parts of sterile NaCl solution.
1. Prepare a row of 8 culture tubes (12 x 75 ml) for each test serum,
including a row for the Febrile Positive Control Polyvalent.
2. 0.85% NaCl solution: Dispense 0.9 ml in the first tube of each
row and 0.5 ml in the remaining tubes.
test serum in the first tube in the row and mix thoroughly. Transfer
0.5 ml from tube 1 to tube 2 and mix thoroughly. Similarly, continue
after mixing. Tube 8 is the antigen control tube and contains only
sterile 0.85% NaCl solution.
of Febrile Positive Control Polyvalent in the first tube in the row
and mix thoroughly. Transfer 0.5 ml from tube 1 to tube 2 and mix
5. Febrile Antigen: Add 0.5 ml of the diluted antigen suspension to
all 8 tubes in each row and shake the rack to mix.
6. The final dilutions in tubes 1-7 are 1:20, 1:40, 1:80, 1:160, 1:320,
1:640 and 1:1280, respectively.
7. Incubate as specified (eg., in a waterbath or refrigerator):
Brucella Abortus Antigen:
35-37C for 48 3 hours.
Proteus OX19 Antigen:
35-37C for 2 hours, then at
Salmonella O Antigen Group D: 50 2C for 17 1 hours.
Salmonella H Antigens a:
50 2C for 1 hour.
Salmonella H Antigens b:
50 2C for 1 hour.
Salmonella H Antigens d:
50 2C for 1 hour.
8. Remove from incubation. Avoid excessive shaking before reading
the reactions, either when the tubes are incubating or when
removing them from incubation.
640
Somatic O Agglutination
Flagellar H Agglutination
3. Positive control: Should show a 2+ or greater agglutination at the
following dilutions:
1:80
1:160
1:80
Salmonella H Antigens a, b and d
1:80
4. Antigen control: Tube 8 of each row should show no agglutination.
5. Test serum: The serum titer is that dilution which shows a 2+ or
greater agglutination. See Table 2.
REACTIONS
SERUM DILUTION
SPECIMEN 1
SPECIMEN 2
SPECIMEN 3
1:20
1:40
1:80
1:160
1:320
1:640
1:1280
Serum titer
4+
4+
3+
2+
1+
1:160
3+
2+
1+
1:40
4+
4+
4+
4+
3+
2+
1+
1:640
The Difco Manual
Section V
Febrile Antigen Set
Interpretation
1. Compare results:
DISEASE
ASSOCIATED FEBRILE ANTIGEN
SIGNIFICANT TITER
Brucellosis
Brucella Abortus
Rocky Mountain
spotted fever*
Proteus OX19
Typhus*
Proteus OX19
Typhoid fever
Salmonella O Antigen Group D**
Typhoid fever
Salmonella H Antigen d**
Paratyphoid fever
Salmonella H Antigen a**
Paratyphoid fever
Salmonella H Antigen b**
1:160
1:160
1:160
1:80
1:80
1:80
1:80
* Rocky Mountain spotted fever cannot be differentiated from typhus by this test.
** Antibodies produced in response to other Salmonella species can cross-react.
2. Single serum specimen: A significant titer suggests infection.

3. Pair of serum specimens (acute and convalescent): A two-dilution
increase in titer is significant and suggests infection. A one-dilution
difference is within the limits of laboratory error.
4. Positive control and antigen control: If results are not as described,
the test is invalid and results cannot be reported.

1. The slide test is intended for screening only and should be confirmed
by the tube test. Slide test dilutions are made to detect a prozone
reaction and do not represent true quantitation of the antibody. A
serum specimen with a prozone reaction shows no agglutination
because of excessively high antibody concentrations. To avoid this
occurrence, all 5 serum dilutions in the slide test should be run.
2. Detection of antibodies in serum specimens may complete the
clinical picture of a patient having an infection. However, isolation
of the causative agent from patient specimens may be required. A
definitive diagnosis must be made by a physician based on
patient history, physical examination and data from all laboratory tests.
3. Cross-reacting heterologous antibodies are responsible for many
low-titer reactions. Infections with other organisms, vaccinations
and history of disease may result in a low level of antibody titer.
Antimicrobial therapy may suppress antibody production.
Cross reactions between antigens and antibodies of B. abortus and
F. tularensis, Y. enterocolitica or V. cholerae can occur.
Rocky Mountain spotted fever and typhus cannot be differentiated by
this test because species of Rickettsia cause cross-reacting antibodies.
Infections with Proteus species can cause cross-reacting antibodies.
Cross-reactions between antigens and antibodies of various
Salmonella species can occur.
Previous immunizations with typhoid vaccine or previous infection
with Salmonella species sharing common antigens with S. typhi
can cause elevated antibody titers for prolonged periods. Other
non-typhoid febrile illnesses may cause elevation of cross-reacting
antibodies.
4. While a single serum specimen showing a significant titer suggests
infection, it is not diagnostic.
are necessary: an acute specimen, obtained at the time of initial
The Difco Manual
symptoms, and a convalescent specimen, obtained 7 to 14 days

later. A two-dilution difference in the titers is a significant increase
in antibody level and suggests infection.
Antigens must be smooth, uniform suspensions; before use, examine
antigen vials for agglutination. Suspensions with agglutination are
8. Discard rehydrated Febrile Positive Control Polyvalent or Febrile
Negative Control that is cloudy or has a precipitate anytime during
its period of use.
References
1. Widal, F. 1896. Serodiagnostic de la fivre typhoide. Sem. Med.
16:259.
2. Spink, W. W., N. D. McCullough, L. M. Hutchings, and
C. K. Mingle. 1954. A standardized antigen for agglutination
technique for human brucellosis. Report no. 3 of the National
Research Council, Committee on Public Health Aspects of
Brucellosis. Am. J. Pathol. 24:496-498.
3. Weil, E., and A. Felix. 1916. Zur serologischen Diagnosis des
Fleckfiebers. Wien. Klin. Wochenschr. 29:33-35.
4. Miller, L. E., H. R. Ludke, J. E. Peacock, and R. H. Tomar.
1991. Manual of laboratory immunology, 2nd ed. Lea & Febiger.
5. Rose, N. R., H. Friedman, and J. L. Fahey (eds.). 1986. Manual
7. Sack, R. B., and D. A. Sack. 1992. Immunologic methods for the
diagnosis of infections by Enterobacteriaceae and Vibrionaceae,
p. 482-488. In N. R. Rose, E. C. De Macario, J. L. Fahey, H.
Friedman, and G. M. Penn (eds.), Manual of clinical laboratory
immunology, 4th ed. American Society for Microbiology,
Washington, D. C.
387-388.
Department of Labor. 1991. 29 CFR, part 1910. Occupational
56:64175-64182.
10. Pezzlo, M. 1992. Aerobic bacteriology, p. 1.0.1-1.20.47. In
transport and storage. In P. R. Murray, E. J. Baron, M. A. Pfaller,
Washington, D.C.
641
Francisella Tularensis Antigens and Antisera
Section V
Packaging
Febrile Antigen Set
Contains:
8 x 5 ml
2407-32
Available separately:
Proteus OX19 Antigen (Tube)
5
25
5
25
5
5
5
5
5
5
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
2909-56
2466-65
2234-56
2247-65
2842-56
2844-56
2845-56
2847-56
3238-56
3239-56
Bacto Francisella Tularensis Antigens and Antisera

Francisella Tularensis Antigen (Slide) . Francisella Tularensis
Antigen (Tube) . Francisella Tularensis Antiserum
the organism. The organisms are gram negative, stain faintly, and have
Intended Use
Bacto Francisella Tularensis Antigens (Slide) and (Tube) are used in
the detection of antibodies by the slide and tube agglutination tests.1,2
Bacto Francisella Tularensis Antiserum is used to demonstrate a positive
quality control test reaction in the slide and tube agglutination tests.
Bacto Febrile Negative Control is used to demonstrate a negative quality
control test reaction in the slide agglutination test.

Two species of the genus Francisella exist, Francisella tularensis and
Francisella novicidia.3 The latter species occurs rarely and is not known
to infect humans.
F. tularensis is the causative agent of tularemia in humans. The disease
was first described in humans in 1907.1 It is a zoonotic disease transmitted to humans by direct contact with wild animals or bites of insect
vectors such as ticks and biting flies. Wild animals such as rabbits,
beavers, muskrats, domestic mammals and birds are involved in disease
transmission.
The organism directly invades the skin, conjunctiva or mucosa of the
oropharynx from blood or tissue of the infected animal. Indirect transmission includes bites of insect vectors, inhalation of contaminated
feces or soil, or ingestion of contaminated water or poorly cooked meat.
Patients experience a rapid onset of febrile symptoms including
malaise, chills, fever and fatigue. Several forms of the infection occur,
each with additional characteristic symptoms. F. tularensis is a pathogenic microorganism that, upon invasion, produces a fever in its host.
Consequently, it is often called a Febrile Antigen.
For growth on culture media, F. tularensis requires both blood and cystine
or cysteine. Gram stains of cultural isolates aid in the identification of
642
extremely small coccoid cells that are often hard to visualize even at
1,000X magnification.2
The human immune response to a particular microorganism results in
measurable antibody production that can sometimes help in completing
the patients clinical diagnosis. In blood samples, the antibody titer
during the initial (acute) phase of the infection is compared to the
antibody titer 7-14 days later (convalescent). Antibody titers that are
high initially in the acute phase (1:160) or an acute or con valescent
pair of samples that shows an increase in antibody titer are helpful in
the diagnosis of tularemia.4,5,6
Certain organisms may share cross-reacting antigens, leading to the
may react with one or more antigens in a febrile antibody test procedure,
producing low-level antibody titers. A titer of less than 1:20 is not
considered diagnostic because nonspecific cross-reactions are common
at this level.1 Cross-reactions between Francisella and Brucella can
occur.

Agglutination tests involving the use of Francisella antigens detect the
a standard volume of antigen. The endpoint of the test is the last dilution
of the serum that shows a specific amount of agglutination. The end
point, reported as a dilution of the serum, is called the patients
antibody titer.
The Difco Manual
Section V
Reagents
Precautions
Francisella Tularensis Antigen (Slide) is a ready-to-use suspension

of Francisella tularensis containing 20% glycerin, as well as 0.5%
phenol, approximately 0.2% crystal violet and approximately 0.5%
brilliant green as preservatives. When used as described, each 5 ml vial
contains sufficient reagent for 20 slide tests.

2. Francisella Tularensis Antigen (Tube)
POSSIBLE RISK OF IRREVERSIBLE EFFECTS. Avoid contact
with skin and eyes. Do not breathe mist. Wear suitable
protective clothing. Keep container tightly closed. Target Organs:
Eyes, Kidneys, Lungs, Skin.
4. Biosafety level 2 precautions are recommended when handling
specimens suspected of containing F. tularensis.9
5. Francisella Tularensis Antigens are not intended for use in the
immunization of humans or animals.
Francisella Tularensis Antigen (Tube) is a ready-to-use suspension

of Francisella tularensis adjusted to a density approximating a
Francisella Tularensis Antigen (Tube) contains 0.5% formalin but does
not contain dye. When used as described, each 25 ml vial contains
sufficient reagent for 6 tests.
Because antigen density may vary, density is adjusted to ensure optimum
obtained from laboratory animals. Variation in antigen color intensity
is normal and will not affect the outcome of the test.
Francisella Tularensis Antiserum is a lyophilized, polyclonal rabbit
antiserum containing approximately 0.04% Thimerosal as a
preservative. When rehydrated and used as described, each 3 ml vial
contains sufficient reagent for 19 slide tests or 30 tube tests.
Febrile Negative Control is a standard protein solution containing
approximately 0.02% Thimerosal as a preservative. When used as
described, each 3 ml vial contains sufficient reagent for 32 slide tests.
Storage
Store Francisella Tularensis Antigens (Slide) and (Tube) at 2-8C.
Store lyophilized and rehydrated Francisella Tularensis Antiserum at
2-8C.
Expiration Date
Francisella Tularensis Antigen (Slide)

Appearance:
Blue-violet suspension.
Francisella Tularensis Antigen (Tube)
Appearance:
Francisella Tularensis Antiserum
powdered cake.
powdered cake.
Rehydrate Francisella Tularensis Antiserum and Febrile
Negative Control per label directions. Perform the Rapid
Slide Test using Francisella Tularensis Antigen (Slide) or the
Macroscopic Tube Test using Francisella Tularensis Antigen
(Tube). Dilute both positive and negative controls in the same
proportion as a patient serum and process in the same manner,
following appropriate procedure.
An antigen is considered satisfactory if it fails to agglutinate
with the negative control and reacts to a titer of 1:160 or more
with the positive control.
The Difco Manual
Procedure
Materials Provided

Slide Test
Agglutination slides with five 1-inch squares
Applicator sticks
Tube Test
Waterbath, 35-37C
643
Section V
Reagent Preparation
Francisella Tularensis Antigen (Slide) and Francisella Tularensis
Antigen (Tube) are ready to use.
detergent residues.
Francisella Tularensis Antiserum: To rehydrate, add 3 ml sterile 0.85%
NaCl solution and rotate gently to dissolve the contents completely.
deionized water and rotate gently to dissolve the contents completely.
4.
5.
6.

has clotted, centrifuge to obtain the serum required for the test. Serum
specimens must be clear, free of hemolysis and show no visible
matter). Consult appropriate references for more information on
collection of specimens.2,10
for prolonged storage, keep at 2-8C for up to 5 days or maintain below -20C. Serum specimens must not be heated; heat may inactivate
or destroy certain antibodies.
Slide Test
the tube test.
an agglutination slide.
0.04, 0.02, 0.01 and 0.005 ml of Francisella Tularensis Antiserum
into a row of squares on the agglutination slide.
4. Antigen: Shake the vial of Francisella Tularensis Antigen (Slide)
thoroughly to ensure a smooth, uniform suspension. Dispense 1
drop (35 l) of antigen in each drop of test serum, positive control
and negative control.
5. Mix each row of test serum and control serum, using a separate
applicator stick for each row. Start with the most dilute mixture
(0.005 ml) and work to the most concentrated (0.08 ml).
7. The final dilutions in squares 1-5 correspond approximately to tube
dilutions of 1:20, 1:40, 1:80, 1:160 and 1:320, respectively.
Tube Test
1. In a rack, prepare a row of 8 culture tubes (12 x 75 mm) for each
test serum and a positive control row for the Francisella Tularensis
Antiserum.
2. Dispense 0.9 ml of sterile 0.85% NaCl solution in the first tube of
serum in the first tube in the row and mix thoroughly. Transfer 0.5
644
7.
8.
9.
ml from tube 1 to tube 2 and mix thoroughly. Similarly, continue

after mixing. Proceed in like manner for each serum to be tested.
Positive control: Using a 1 ml serological pipette, dispense 0.1 ml
of Francisella Tularensis Antiserum in the first tube in the row and
mix thoroughly. Transfer 0.5 ml from tube 1 to tube 2 and mix
discarding 0.5 ml from tube 7 after mixing.
Antigen control: Tube 8 is the antigen control tube and contains
only sterile 0.85% NaCl solution.
Antigen: Shake the vial of Francisella Tularensis Antigen (Tube)
to ensure a smooth, uniform suspension. Add 0.5 ml of antigen to
all 8 tubes in each row and shake the rack to mix the suspensions.
Final dilutions in tubes 1-7 are 1:20, 1:40, 1:80, 1:160, 1:320, 1:640
Incubate in a waterbath at 35-37C for 22 2 hours.
Remove from the waterbath. Avoid excessive shaking before reading the reactions, when the tubes are in the waterbath, or when
removing them from the waterbath.
Results
No agglutination.
2. Positive control: Should produce 2+ or greater agglutination at a
1:160 dilution.
Negative control - Rapid Slide Test, only: Should produce no
agglutination.
Antigen control - Macroscopic Tube Test, only: Should produce
no agglutination in tube #8 of each row.
If results for either the positive or negative control are not as
specified, the test is invalid and results cannot be reported.
Test serum: The titer is the highest dilution that shows 2+
agglutination.
Refer to Table 1 and Table 21 for examples of test reactions.
3. The Rapid Slide Test is a screening test, only; results must be
confirmed using the Macroscopic Tube Test.
SERUM (ml)
CORRELATED
TUBE DILUTION
0.08
1:20
0.04
1:40
0.02
1:80
0.01
1:160
0.005
1:320
Serum titer
REACTIONS
SPECIMEN 1
SPECIMEN 2
SPECIMEN 3
3+
2+
1+
1:40
4+
4+
3+
3+
1+
1:160
4+
3+
2+
+
1:80
The Difco Manual
Section V

REACTIONS
SERUM DILUTION
SPECIMEN 1
SPECIMEN 2
SPECIMEN 3
1:20
1:40
1:80
1:160
1:320
1:640
1:1280
Serum titer
4+
4+
3+
2+
1+
1:160
3+
2+
1+
1:40
4+
4+
4+
4+
3+
2+
1+
1:640
Interpretation
For a single serum specimen, a titer of 1:160 at 2+ or greater suggests
infection.1
A 2-dilution increase in the titer of paired serum specimens (from the
acute to the convalescent serum) is significant and suggests infection.
A 1-dilution difference is within the limits of laboratory error.

1. The slide test is intended for screening only and results should be
confirmed by the tube test. Slide test dilutions are made to detect a
prozone reaction and do not represent true quantitation of the antibody. A serum specimen with a prozone reaction shows no
agglutination because of excessively high antibody concentrations.
To avoid this occurrence, all five serum dilutions (slide test) should
be run.
2. The detection of antibodies in serum specimens may complete the
clinical picture of tularemia. However, isolation of the causative
agent from patient specimens may be required. A definitive diagnosis must be made by a physician based on patient history, physical
examination and data from all laboratory tests.
low titer reactions. Cross-reactions between antigens and antibodies
of Brucella species and Francisella tularensis can occur. Infections
with other organisms, vaccinations and a history of disease may
cause low antibody titers. Antimicrobial therapy may suppress
antibody production.
4. While a single serum specimen showing a titer of 1:160 suggests
5. To test for a significant rise in antibody titer, at least two
specimens are necessary, an acute specimen obtained at the time
of initial symptoms and a convalescent specimen obtained 7 to
14 days later. A two-dilution increase in titer is significant and
suggests infection.
7. Exposure to temperatures below 2C can cause antigen autoagglutination. Antigens must be smooth, uniform suspensions.
Examine antigen vials for agglutination before use. Agglutinated
suspensions are not usable and should be discarded.
The Difco Manual
8. Adhering to the recommended time and temperature of incubation

is important when performing this test. For best results, locate the
waterbath in an area free of mechanical vibration.
References
1. Stewart, S. J. 1995. Francisella, p. 545-548. In P. R. Murray, E. J.
3. Holt, J. G., N. R. Krieg, P. H. Sneath, J. T. Staley, and S. T.
9th ed. Williams & Wilkins, Baltimore, MD.
5. Rose, N. R., H. Friedman, and J. L. Fahey (ed.). 1986. Manual
of clinical immunology, 3rd ed. American Society for Microbiology,
Washington, D. C.
387-388.
56:64175-64182.
9. U. S. Department of Health and Human Services. 1988.
Biosafety in microbiological and biomedical laboratories,
2nd ed. U. S. Department of Health and Human Services
publication no. 88-8395. U. S. Government Printing Office,
Washington, D. C.
transport and storage. In P. R. Murray, E. J. Baron, M. A.
Washington, D. C.
Packaging
5 ml
2240-56
5 ml
25 ml
2251-56
2251-65
3 ml
2241-47
3 ml
3239-56
645
Haemophilus Influenzae Antisera
Section V
Bacto Haemophilus Influenzae Antisera

Haemophilus Influenzae Antiserum Poly . Haemophilus
Influenzae Antiserum Type a . Haemophilus Influenzae
Antiserum Type b . Haemophilus Influenzae Antiserum Type c
Haemophilus Influenzae Antiserum Type d . Haemophilus
Influenzae Antiserum Type e . Haemophilus Influenzae
Antiserum Type f
Intended Use
Bacto Haemophilus Influenzae Antisera are used in slide agglutination
tests for serotyping Haemophilus influenzae.

H. influenzae was first described by Pfeiffer1 in 1892 from patients
during an influenza pandemic. Pittman2 described the six capsular
serotypes of H. influenzae in 1931. He recognized that members of
serotype b were most likely to cause invasive infections.
H. influenzae is part of the normal respiratory flora of humans and
many animal species. Often, the organism becomes an opportunistic
secondary invader, usually following viral influenza. This organism
can cause a variety of diseases from chronic respiratory infections to
meningitis. Most of the H. influenzae isolates associated with meningitis
possess the serotype b capsule.3 Serotype b is believed to cause more
than 90% of all Haemophilus infections in children less than six years
of age. Although the incidence of H. influenzae type b infections has
been drastically reduced by the introduction of effective vaccines,
Haemophilus species remain important causes of a wide range of
human infections.

Haemophilus Influenzae Antiserum Poly
Haemophilus Influenzae Antiserum Type a
Haemophilus Influenzae Antiserum Type b
Haemophilus Influenzae Antiserum Type c
Haemophilus Influenzae Antiserum Type d
Haemophilus Influenzae Antiserum Type e
Haemophilus Influenzae Antiserum Type f
powdered cake.
Rehydrated Appearance: Light gold to amber liquid.
Rehydrate Haemophilus Influenzae Antisera per label
directions. Test as described (see Test Procedure). Known
positive and negative control cultures must give appropriate
reactions.
646
H. influenzae is a nonmotile, facultative anaerobe requiring both factor

X (hemin) and factor V (NAD) for in vitro growth. In microscopic
morphology, the organism is a pleomorphic gram-negative coccobacillus
and sometimes forms threads or filaments.
The presence of a polysaccharide capsule is a major virulence factor
for strains of H. influenzae that cause systemic infection. H. influenzae
is divided into serological groups a, b, c, d, e and f based on capsular
polysaccharides. Most encapsulated strains that cause infection belong
to serotype b.1 The encapsulated strains are referred to as typeable
strains. Nonencapsulated or non-typeable strains may also cause
infection. Infections caused by nonencapsulated strains are usually
related to the upper respiratory tract.
Antigenic similarities exist between H. influenzae and many unrelated
bacteria. H. influenzae serotype b shares cross-reacting antigens with
Streptococcus pneumoniae serotypes 6, 15a, 29 and 35a, Escherichia coli,
and several species of Staphylococcus, Streptococcus and Bacillus.
The Quellung (swelling) reaction has also been used for recognition of
encapsulated (typeable) strains of H. influenzae.1,4 The principle of this
antigen-antibody reaction is not agglutination as in the slide technique,
but an apparent increase in capsular size due to deposition of antibody
on the cell surface. If the Quellung reaction is performed, one must be
aware that these organisms are often found in the nonencapsulated state,
which are untypable. In addition, capsulated strains of type e generally
possess small capsules. Such strains should be defined serologically
employing the slide agglutination test, only. Consult an appropriate
reference for details of the Quellung reaction.4

Identification of H. influenzae includes isolation of the microorganism,
biochemical identification and serological confirmation.
Serological confirmation involves the reaction in which the microorganism (antigen) reacts with its corresponding antibody. This in vitro
desired homologous reaction is rapid, does not dissociate (high
avidity), and binds (high affinity).
produced in response to other species, heterologous reactions are
possible. These are weak in strength or slow in formation. Such unexpected and, perhaps, unpredictable reactions may lead to some
The Difco Manual
Section V

are slow and weak.
Reagents
Haemophilus Influenzae Antisera are lyophilized, polyclonal rabbit
antisera containing approximately 0.02% Thimerosal as a preservative.
When rehydrated and used as described, each 1 ml vial of Haemophilus
Influenzae Antiserum contains sufficient reagent for 20 slide tests.
Precautions
3. Follow established laboratory procedure in handling and disposing
of infectious materials.
Storage
Store lyophilized and rehydrated Haemophilus Influenzae Antisera
at 2-8C.
Expiration Date
Procedure
Materials Provided

Applicator sticks
Reagent Preparation
Haemophilus Influenzae Antiserum: To rehydrate, add 1 ml sterile
distilled or deionized water and rotate to completely dissolve the contents.
Equilibrate all materials to room temperature prior to performing
detergent residues.

H. influenzae can be recovered from clinical specimens on chocolate
agar. For specific recommendations, consult appropriate references.1,5
Determine that a pure culture of the microorganism has been obtained
and that biochemical test reactions are consistent with the identification
of the organism as H. influenzae. After these criteria are met, serological
The Difco Manual
Haemophilus Influenzae Antisera
Testing the Isolate for Autoagglutination

1. From the test culture on chocolate agar, transfer a loopful of growth
to a drop of sterile 0.85% NaCl solution on a clean slide and
emulsify the organism.
2. Rotate the slide for one minute and then observe for agglutination.
3. If agglutination (autoagglutination) occurs, the culture is rough and
cannot be tested. Subculture to chocolate agar, incubate, and test
the organism again as described in steps 1 and 2.
If no agglutination occurs, proceed with testing the organism.
Test Procedure
Test culture isolates with Haemophilus Influenzae Poly for presumptive
identification, then test with monospecific antisera.
1. Dispense 1 drop of the Haemophilus Influenzae Antiserum to be
tested on an agglutination slide.
2. Transfer a loopful of growth of the test organism to the drop of
antiserum and mix thoroughly.
3. Rotate the slide for one minute and read for agglutination.
4. Repeat this procedure for known positive and negative control
cultures.
Results
Observe test results and record agglutination as follows:
4+
3+
2+
1+
100% agglutination; background is clear to slightly hazy.

75% agglutination; background is slightly cloudy.
50% agglutination; background is moderately cloudy.
25% agglutination; background is cloudy.
No agglutination.
Positive control: Should produce 3+ or greater agglutination.

Positive test result: Agglutination of 3+ or greater within one minute.

as H. influenzae.
2. Serological methods alone cannot identify the isolate as
H. influenzae.
burner flame, light source, etc.) may prevent a smooth suspension
of the microorganism or may cause evaporation or precipitation of
the test mixture. False-positive reactions may occur.
4. Rough culture isolates occur and will agglutinate spontaneously
5. H. influenzae has antigenic similarities to several unrelated bacteria.
Cross-reactions can occur between H. influenzae and strains
of S. pneumoniae, Escherichia coli and several species of
Staphylococcus, Streptococcus and Bacillus.
6. Haemophilus Influenzae Antisera have been tested using undiluted
cultures taken from agar media. These antisera have not been tested
using antigen suspensions in NaCl solution or other diluents. If the
647
Listeria Antigens & Antisera
Section V
user employs a variation of the recommended procedure, each lot

of antiserum must be tested with known control cultures to verify
that expected reactions are obtained under the modified procedure.
8. A rehydrated Haemophilus Influenzae Antiserum that is cloudy or
develops a precipitate during use should be discarded.

4. Cruse, J. M., and R. E. Lewis. 1995. Illustrated Dictionary of
Immunology, p. 253. CRC Press, Inc., Boca Raton, FL.
5. Pezzlo, M. 1992. Aerobic bacteriology, p. 1.0.1.-1.20.47. In
References
Packaging

2. Pittman, M. 1931. Variation and type specificity in the bacterial
species Hemophilus influenzae. J. Exp. Med. 53:471-495.
3. Insel, R., and P. Anderson. 1986. Haemophilus influenzae Type b:
assays for the capsular polysaccharide and for antipolysaccharide
antibody. In N. R. Rose, H. Friedman, and J. L. Fahey (ed.), Manual
1 ml
2237-50
1 ml
2250-50
1 ml
2236-50
1 ml
2789-50
1 ml
2790-50
1 ml
2791-50
1 ml
2792-50
Bacto Listeria Antigens and Antisera

Listeria O Antiserum Type 1 . Listeria O Antiserum Type 4
Listeria O Antiserum Poly . Listeria O Antigen Type 1 (Slide)
Listeria O Antigen Type 1 (Tube) . Listeria O Antigen Type 4 (Slide)
Listeria O Antigen Type 4 (Tube)
Intended Use
Bacto Listeria O Antisera Types 1, 4, and Poly are used for identifying
Listeria monocytogenes in the macroscopic tube and rapid slide tests.
Bacto Listeria O Antigens Types 1 and 4 (Tube) and (Slide) are used
as positive controls in the macroscopic tube and rapid slide tests,
respectively.

industries. This organism can cause human illness and death, particularly in immunocompromised individuals and pregnant women.2 The
first reported food-borne outbreak of listeriosis was in 19853 and, since
then, microbiological and epidemiological evidence from both sporadic
and epidemic cases of listeriosis has shown that the principal route of
Implicated vehicles of transmission include turkey frankfurters,5 coleslaw,
pasteurized milk, Mexican-style cheese, pat and pickled pork tongue.
The organism has been isolated from commercial dairy and other food
processing plants and is ubiquitous in nature, being present in a wide
range of unprocessed foods and in soil, sewage, silage and river water.6
648

products with pH levels outside these parameters.7 Listeria species are
at 20C.
potentially containing Listeria are streptococci, enterococci, micrococci,
Bacillus species, Escherichia coli, Pseudomonas aeruginosa and
Proteus vulgaris.8
Identification of Listeria is based on successful isolation of the organism,
biochemical characterization and serological confirmation.
Strains of Listeria species are divided into serotypes based on cellular (O)
and flagellar (H) antigens.9 Thirteen serotypes of L. monocytogenes are
known. Most human disease is caused by serotypes 1/2a, 1/2b and 4b.10

Identification of Listeria monocytogenes includes both biochemical and
serological confirmation. Serological confirmation requires that the
microorganism (antigen) react with its corresponding antibody. This in
vitro reaction produces macroscopic clumping called agglutination.
The desired homologous reaction is rapid, does not dissociate (high
avidity) and bonds strongly (high affinity).
The Difco Manual
Section V

possible. These are characterized as weak in strength or slow in formation.
Such unexpected and perhaps unpredictable reactions may lead to some
confusion in serological identification. A positive homologous agglutination reaction should support the morphological and biochemical
granular clumping. Homologous reactions occur rapidly and are strong
(3+). Heterologous reactions form slowly and are weak.
The agglutination of the somatic antigen in the tube tests appears as a
loose flocculation that can easily be resuspended. Homologous reactions
using Listeria O Antisera should exceed a titer of 2+ at 1:320.
Reagents
Listeria O Antisera Types 1, 4, and Poly are lyophilized, polyclonal
rabbit antisera containing approximately 0.04% Thimerosal as a
preservative. The antisera are prepared according to procedures
recommended by Gray.11 Listeria O Antisera Types 1 and 4 are specific
for the respective serotypes of L monocytogenes while Listeria O
Antiserum Poly contains agglutinins for L. monocytogenes serotypes
1 and 4.
Listeria O Antigens Types 1 and 4 (Tube) and (Slide) are suspensions
of appropriate L. monocytogenes serotypes containing 0.3% formaldehyde
as a preservative. When used according to the suggested procedure,
the reagents will yield the following:

REAGENT
Listeria O Antiserum
Listeria O Antigen (Slide)
Listeria O Antigen (Tube)
VIAL
1 ml
5 ml
25 ml
NUMBER OF TESTS
10 tube tests, 400 slide tests

100 slide tests
5 tube tests
Precautions
2. Listeria O Antiserum Type 1
Listeria O Antiserum Type 4
Listeria O Antiserum Poly
3. Listeria O Antigen Type 1 (Slide)
Listeria O Antigen Type 4 (Slide)
POSSIBLE RISK OF IRREVERSIBLE EFFECTS. (US) Avoid
Storage
Store lyophilized and rehydrated Listeria O Antisera at 2-8C.
Store Listeria O Antigen (Slide) and (Tube) at 2-8C.
Expiration Date

powdered cake.
Appearance:
White, liquid suspension.
Rehydrate Listeria O Antiserum per label directions. Perform
the slide or tube agglutination test using appropriate Listeria
O Antigens (Slide) or (Tube).
Slide test: An antiserum is considered satisfactory if it
demonstrates a 3+ or greater reaction at 1:80 with a 1:5 dilution
of the homologous antigen.
Macroscopic tube test: An antiserum is considered satisfactory
if it demonstrates a 3+ or greater reaction with the 1:320
dilution of the homologous antigen.
The Difco Manual
Procedure
Materials Provided

Rapid Slide Test
FA Buffer, Dried
Applicator sticks
Waterbath, 80-100C
Formaldehyde
Droppers
649
Macroscopic Tube Test

FA Buffer, Dried
McFarland Barium Sulfate Standard No. 3
Serological pipettes, 1 ml
Waterbath, 50C
Refrigerator, 2-8C
Formaldehyde
Reagent Preparation
residues such as detergents.
Listeria O Antisera: To rehydrate, add 1 ml sterile distilled or deionized
water to each vial. Rotate gently to dissolve contents completely.
Listeria O Antigens (Slide) and (Tube) are ready to use.

From clinical specimens, Listeria can be recovered on selective
differential media such as McBride Listeria Agar, Oxford Agar,
Modified Oxford Agar, LPM Agar or Palcam Medium. For specific
recommendations on isolation of Listeria from clinical specimens,
consult appropriate references.10,12,13 Determine that a pure culture of
the microorganism has been obtained and that biochemical test reactions
are consistent with the identification of the organism as Listeria
monocytogenes. After these criteria are met, serological identification
can be performed.
From food or dairy samples, Listeria can be recovered when samples
are processed to recover injured microorganisms and prevent overgrowth of competing microorganisms. Consult appropriate references
for recommended procedures for the isolation of Listeria from
foods.7,14,16 Having followed an established protocol, isolate a pure culture of the microorganism and confirm that biochemical test
reactions are consistent with the identification of the organism as
Listeria monocytogenes. After these criteria are met the serological
Test Procedure
Rapid Slide Test
1. FA Buffer, Dried: Rehydrate per label directions.
2. Test isolate: Suspend growth from a solid agar medium in FA Buffer.
3. Heat the organism suspension at 80-100C (in a waterbath) for
1 hour.
4. Centrifuge the suspension and remove the bulk of the supernatant
fluid.
5. Resuspend the organism in the remaining portion of liquid.
6. Listeria Antiserum: On an agglutination slide, dispense 2 separate
drops of the desired antiserum diluted 1:20 in NaCl solution. The
first drop will be used for the test isolate and the second for the
negative control.
7. Organism suspension: Add 1 drop of heated organism to the first
drop of antiserum.
8. Negative control: Dispense 1 drop of FA Buffer on the agglutination
slide. Add one drop of organism suspension from step 5.
650
Section V
9. Positive control: Add one drop of homologous Listeria O Antigen

(Slide) to the second drop of antiserum.
10. Rotate the slide for 1-2 minutes and read for agglutination.
Slide Test Results

No agglutination.
2. Positive control: Should show a 3+ or greater agglutination.
3. Negative control: Should show no agglutination. If agglutination
occurs, the culture is rough and cannot be tested. Subculture to a
non-inhibitory medium, incubate and test the organism again.
4. Test isolates: 3+ or greater agglutination is a positive result.
5. A partial (less than 3+) or a delayed agglutination reaction should
be considered negative.
Macroscopic Tube Test

1. Test isolate: Suspend growth of the test organism from a solid agar
medium in FA Buffer. Adjust to a density approximating that of a
McFarland Barium Sulfate Standard No. 3.
2. Prepare a row of 9 culture tubes (12 x 75 mm) for each serum
suspension to be tested, including the positive control.
3. Formalized FA Buffer: Dispense 0.9 ml formalized FA Buffer
(0.3 ml formaldehyde per 300 ml FA Buffer) to the first tube in
each row and 0.5 ml to the remaining tubes.
4. Listeria O Antiserum: Using a 1 ml serological pipette, add 0.1 ml
of the desired antiserum to tube 1 in each row and mix thoroughly.
Transfer 0.5 ml from tube 1 to tube 2 and mix thoroughly. In like
manner, continue transferring 0.5 ml through tube 8, discarding
0.5 ml from tube 8 after mixing. Tube 9 is an antigen control tube.
Upon addition of the test suspension, final dilutions will be 1:20
through 1:2560 for tubes 1 through 8, respectively.
5. Test Suspension: Add 0.5 ml of the test suspension to each of
9 tubes.
6. Positive control: Add 0.5 ml of an appropriate Listeria O Antigen
to each of 9 tubes containing antiserum.
7. Negative control: Add 0.5 ml of the test suspension to a tube
containing FA Buffer.
8. Shake the rack to mix. Incubate in a 50C waterbath for 2 hours.
Refrigerate overnight. Read for agglutination the following
morning.
Tube Test Results

No agglutination.
2. Positive control: Should show 2+ or greater agglutination at 1:320.
3. Antigen control: Tube 9 of each row should show no agglutination.
The Difco Manual
Section V
4. If results of the positive control or antigen control are not as described, the test is invalid and results cannot be read.
5. Test serum: The titer is that dilution which shows a 2+ or greater
agglutination at 1:320.

1. Serological techniques employing Listeria O Antisera serve as
corroborative evidence for the identification of Listeria
monocytogenes. Final identification cannot be made without
consideration of morphological, serological and biochemical
characterization.
suspension of the microorganism or cause evaporation or precipitation of the test mixture. False-positive reactions may occur.
3. Rough culture isolates occur and will agglutinate spontaneously,
Smooth colonies must be selected and tested in serological
procedures.
4. Agglutination reactions of 3+ or greater in the slide test are interpreted as positive reactions. Cross-reactions resulting in a 1+ or 2+
agglutination are likely since there are somatic antigens shared
among different organisms such as staphylococci, enterococci and
Bacillus species.10
6. Exposure of Listeria O Antigens to temperatures below 2C can
result in autoagglutination. Antigens must be smooth uniform
suspensions; examine antigen vials for agglutination before use.
Suspensions with agglutination are not usable and should be
discarded.
7. It is important in this test to use the recommended time and
temperature of incubation. Also, care should be taken to make certain
that the waterbath is in a location free of mechanical vibration.
8. Discard any Listeria O Antiserum that is cloudy or has a precipitate
after rehydration or storage.

J. Lovett. 1992. Listeria, p. 637-663. In C. Vanderzant and
D. F. Splittstoesser (ed.), Compendium of methods for the
9. Seeliger, H. P. R., and K. Hohne. 1979. Serotyping of Listeria
monocytogenes and related species, p. 31-49. In T. Bergen and
J. R. Norris (ed.), Methods in microbiology, vol. 13. Academic
Press, London, England.
p. 342-343. In P. R. Murray, Baron, Ffaller, Tenover and Yolken
11. Gray, M. L., and A. H. Killinger. 1966. Listeria monocytogenes
infections. Bacteriol. Rev. 30:309-382.
12. Pezzlo, M. 1994. Aerobic bacteriology, p. 1.0.1.-1.20.47. In H. D.
St. Louis, MO.
14. Hitchins, A. D. 1995. Listeria monocytogenes, p. 10.01-10.13.
In FDA Bacteriological analytical manual, 8th ed. AOAC
1992. Pathogens in milk and milk products. In Marshall, R. T.,
Packaging
References
1 ml
2300-50
1. Murray, E. G. D., R. A. Webb, and M. B. R. Swann. 1926. A

disease of rabbits characterized by large mononuclear leucocytosis
caused by a hitherto undescribed bacillus Bacterium monocytogenes
(n. sp.). J. Path. Bact. 29:407-439.
1 ml
2301-50
1 ml
2302-50
5 ml
2303-56
25 ml
2305-65
5 ml
2304-56
25 ml
2306-65
6 x 10 g
100 g
10 kg
2314-33
2314-15
2314-08
The Difco Manual
FA Buffer, Dried
651
Neisseria Meningitidis Antisera
Section V
Bacto Neisseria Meningitidis Antisera

Neisseria Meningitidis Antiserum Group A . Neisseria Meningitidis
Antiserum Group B . Neisseria Meningitidis Antiserum Group C
Neisseria Meningitidis Antiserum Group D . Neisseria Meningitidis
Antiserum Group W135 . Neisseria Meningitidis Antiserum
Group X . Neisseria Meningitidis Antiserum Group Y . Neisseria
Meningitidis Antiserum Group Z . Neisseria Meningitidis Antiserum
Group Z . Neisseria Meningitidis Antiserum Poly (Groups A, B, C, D)
Neisseria Meningitidis Antiserum Poly 2 (Groups X, Y, Z)
Intended Use
Bacto Neisseria Meningitidis Antisera are used in the slide agglutination
test for serotyping Neisseria meningitidis.

Neisseria meningitidis is found in the oropharynx and nasopharynx of
humans. Because the organism survives poorly in the environment,
humans are the primary reservoir. In asymptomatic persons, the carrier
state lasts for various periods, usually several weeks. The microorganism
is transmitted from person-to-person by direct contact with respiratory
secretions or airborne droplets.1,2

Neisseria Meningitidis Antiserum Poly
Neisseria Meningitidis Antiserum Poly 2
Neisseria Meningitidis Antiserum A
Neisseria Meningitidis Antiserum B
Neisseria Meningitidis Antiserum C
Neisseria Meningitidis Antiserum D
Neisseria Meningitidis Antiserum X
Neisseria Meningitidis Antiserum Y
Neisseria Meningitidis Antiserum Z
Neisseria Meningitidis Antiserum W135
Lyophilized Appearance: Light gold to amber button to
powdered cake.
Rehydrated Appearance: Light gold to amber liquid.
Rehydrate Neisseria Meningitidis Antisera per label directions.
Test as described (see Test Procedure). Known positive and
negative control cultures must give appropriate reactions.
652
In some colonized persons, the organism spreads from the nasopharynx

through the bloodstream to produce meningococcemia, meningitis or
both. Meningococcemia is characterized by a petechial or purpuric skin
rash. In fulminating infections (Waterhouse-Friderichsen syndrome),
widespread coagulation and fulminant sepsis occur, resulting in shock
and, usually, death.3 Persons with inherited complement deficiencies
are at greater risk for acquiring systemic meningococcal infections and
may experience repeated episodes.2
Typical human specimens for isolating the organism are cerebrospinal
fluid (CSF), blood, skin lesions (in cases where petechiae occur) and
nasopharyngeal swabs. N. meningitidis occurs in the cervix and vagina
of females and can cause serious pelvic disease. Other sources for the
organism are the anal canal and, in males, the urethra.
N. meningitidis is divided into serological groups based on the presence
of either capsular or outer membrane protein antigens. Among the
currently recognized groups are A, B, C, D, 29E, H, I, K, L, X, Y, Z,
Z and W135. Groups A, B, C, Y, and W135 are most frequently implicated in systemic disease.4 Classically, group A and C strains cause
epidemic meningococcal disease.3 Group B strains have been associated
with sporadic infections. Other serogroups are sporadically isolated
from carriers and patients with disease.
N. meningitidis are gram-negative cocci, usually occurring in pairs
called diplococci. They are strict aerobes and produce the enzyme,
cytochrome oxidase. The growth of N. meningitidis is enhanced by a
CO2-enriched atmosphere.
The Quellung reaction (capsular swelling) has been performed for
serotyping N. meningitidis. However, capsules have not been demonstrated
in strains of serogroup B organisms. In addition, the Quellung reaction
is very nonspecific. N. meningitidis should be defined serologically by
the slide agglutination test rather than by the Quellung reaction.

Identification of N. meningitidis includes isolation of the microorganism,
biochemical identification and serological confirmation.
The Difco Manual
Section V
desired homologous reaction is rapid, has at least a 3+ reaction, does
not dissociate (high avidity), and binds (high affinity).
possible. These are characterized as weak in strength or slow in formation. Such unexpected and, perhaps, unpredictable reactions may
lead to some confusion in serological identification. Therefore, a
positive homologous agglutination reaction should support the
morphological and biochemical identification of the microorganism.
are slow and weak.
Reagents
Neisseria Meningitidis Antisera are lyophilized, polyclonal rabbit
Neisseria Meningitidis Antisera Poly and Group D are absorbed for
detection of Group D; Neisseria Meningitidis Antisera Poly 2, Z,
W135, A, B, C, X, Y and Z are not absorbed for detection of Group D,
which is rarely isolated.
Neisseria Meningitidis Antisera detect the following antigenic groups:
ANTISERUM
ANTIGENIC GROUP(S) DETECTED
Poly
A, B, C, D
Poly 2
X, Y, Z
W135
W135
A
A
B
B
C
C
D
D
X
X
Y
Y
Z
Z, Z
Z
Z
When rehydrated and used as described, each 1 ml vial of Neisseria
Meningitidis Antiserum contains sufficient reagent for 20 slide tests.
Precautions
Storage
Store lyophilized and rehydrated Neisseria Meningitidis Antisera at 2-8C.
Expiration Date
Procedure
Materials Provided
The Difco Manual
Neisseria Meningitidis Antiserum A

Neisseria Meningitidis Antiserum B
Neisseria Meningitidis Antiserum C
Neisseria Meningitidis Antiserum D
Neisseria Meningitidis Antiserum X
Neisseria Meningitidis Antiserum Y
Neisseria Meningitidis Antiserum W135

Applicator sticks
Reagent Preparation
Neisseria Meningitidis Antisera: To rehydrate, add 1 ml sterile distilled
or deionized water and rotate gently to completely dissolve the contents.
Equilibrate all materials to room temperature prior to performing the
detergent residues.

N. meningitidis can be recovered on blood agar or chocolate agar.
Determine that the test organism has the following characteristics of
N. meningitidis:
Morphology:
grayish-white, opaque, smooth,
butyrous, non-pigmented
Gram stain:
gram-negative diplococcus
Oxidase:
positive
Catalase:
positive
ONPG reaction:
negative
Nitrate reduction:
negative
Glucose:
positive
Maltose:
positive
Sucrose:
negative
Fructose:
negative
Lactose:
negative
of the organism as N. meningitidis. For more detailed information on
the biochemical identification of N. meningitidis, consult appropriate
references.3,5 After these criteria are met, serological identification can
proceed.
Testing the Isolate for Autoagglutination

1. From the test culture on chocolate agar, transfer a loopful of growth to
a drop of sterile 0.85% NaCl solution on a clean slide and emulsify
the organism.
2. Rotate the slide for one minute and then observe for agglutination.
If agglutination (autoagglutination) occurs, the culture is rough and
cannot be tested. Subculture to chocolate agar, incubate, and test
the organism again as described in steps 1 and 2.
If no agglutination occurs, proceed with testing the organism.
653
Section V
Choosing Antisera to Test

1. Test the organism first with Neisseria Meningitidis Antisera Poly,
Poly 2 and Group W135.
2. Depending on the reaction, continue testing as follows.
If agglutination occurs with
Test with
Neisseria Meningitidis Antiserum: Neisseria Meningitidis Antiserum:
Poly
Groups A, B, C, D
Poly 2
Groups X, Y, Z, Z (See NOTE.)
Group W135
No further testing is required.
NOTE: N. meningitidis Group Z organisms may agglutinate
monospecific Neisseria Meningitidis Antiserum Group Z. However, N.
meningitidis Group Z organisms will not agglutinate Neisseria
Meningitidis Antiserum Group Z. The expected agglutination
reactions of Neisseria Meningitidis Antiserum Groups Z and Z with
test organisms are:
Test Organism
N. meningitidis Group Z
N. meningitidis Group Z
Neisseria Meningitidis Antiserum

Group Z
Group Z
3+
+
3+
Test Procedure
1. Neisseria Meningitidis Antiserum: Dispense 1 drop of the antiserum to be tested on an agglutination slide.
2. Test isolate: Transfer a loopful of growth to the drop of antiserum
and mix thoroughly.
4. Repeat this procedure for known positive and negative cultures.
Results
No agglutination.
Test isolate: A positive test result is defined as agglutination of 3+
or greater within one minute.

purity, as well as morphological characteristics and biochemical
as N. meningitidis.
2. Serological methods alone cannot identify the isolate as
N. meningitidis. Organisms unrelated to Neisseria, yet capable of
causing meningitis, and other species of Neisseria can cross-react
with meningococcal antisera. Cultural isolation must precede
serological examination.
the test mixture. False-positive reactions may occur.
654
4. Rough culture isolates occur and will agglutinate spontaneously,

5. N. meningitidis Group A and N. meningitidis Group C may crossreact due to the presence of common capsular polysaccharides.
6. Group Z meningococci may agglutinate group Z antiserum. Group
Z meningococci will not agglutinate group Z antiserum.
7. Neisseria Meningitidis Antisera have been tested using undiluted
cultures taken from agar media. These antisera have not been tested
using antigen suspensions in NaCl solution or other diluents. If the
user employs a variation of the recommended procedure, each lot
of antiserum must be tested with known control cultures to verify
that expected reactions are obtained under the modified procedure.
9. A rehydrated Neisseria Meningitidis Antiserum that is cloudy or
develops a precipitate during use should be discarded.
References
1. Given, K. F., B. W. Thomas, and A. G. Johnston. 1977. Isolation
of Neisseria meningitidis from the urethra, cervix, and anal canal:
further observations. Br. J. Vener. Dis. 53:109-112.
2. Janda, W. M., M. Bohnhoff, J. A. Morello, and S. A. Lerner.
1980. Prevalence and site-pathogen studies of Neisseria
meningitidis and N. gonorrhoeae in homosexual men. JAMA
244:2060-2064.
3. Knapp, J. S., and R. J. Rice. 1995. Neisseria and Branhamella,
4. Zollinger, W. D., B. L. Brandt, and E. C. Tramont. 1986.
Immune response to Neisseria meningitidis, p. 346-352. In N. R.
Rose, H. Friedman, and J. L. Fahey (ed.), Manual of clinical laboratory immunology, 3rd ed. American Society for Microbiology,
Washington, D.C.
Packaging
1 ml
2232-50
1 ml
2910-50
Neisseria Meningitidis Antiserum Group A
1 ml
2228-50
Neisseria Meningitidis Antiserum Group B
1 ml
2229-50
Neisseria Meningitidis Antiserum Group C
1 ml
2230-50
Neisseria Meningitidis Antiserum Group D
1 ml
2231-50
Neisseria Meningitidis Antiserum Group X
1 ml
2880-50
Neisseria Meningitidis Antiserum Group Y
1 ml
2881-50
Neisseria Meningitidis Antiserum Group Z
1 ml
2891-50
Neisseria Meningitidis Antiserum Group Z
1 ml
2252-50
Neisseria Meningitidis Antiserum Group W135 1 ml
2253-50
The Difco Manual
Section V
Proteus Antigens and Antisera
Bacto Proteus Antigens and Antisera . The Weil-Felix Test

Proteus OX2 Antigen (Slide) . Proteus OX2 Antigen (Tube)
Proteus OX19 Antigen (Slide) . Proteus OX19 Antigen (Tube)
Proteus OXK Antigen (Slide) . Proteus OXK Antigen (Tube)
Proteus OX2 Antiserum . Proteus OX19 Antiserum
Proteus OXK Antiserum . Febrile Negative Control
Intended Use
Bacto Proteus OX2, OX19 and OXK Antigens (Slide) and (Tube) are
used for detecting antibodies by the slide and tube agglutination tests.
Rickettsiae cause a variety of human diseases that share symptoms such

as chills, fever, malaise and myalgia. These symptoms occur suddenly,
usually within 3 to 14 days after exposure. Patients frequently have a
rash and may have mild pulmonary symptoms. The rickettsiae are
obligate intracellular bacteria and multiply within arthropods (lice,
ticks, fleas, etc.) which may serve as the vectors of infection.
The spotted fevers are caused by species of Rickettsia, with Rocky
Mountain spotted fever caused by Rickettsia rickettsii being well
known. Epidemic and murine typhus are caused by R. prowazekii
and R. typhi, respectively. Scrub typhus is caused by Orientalis
tsutsugamushi. For a complete discussion of the rickettsiae, consult
an appropriate reference.1-5
Because rickettsial diseases develop as a febrile illness, patient diagnosis
has frequently involved measurements of antibody response. The
Weil-Felix test became popular in the 1920s after it was observed that
certain strains of Proteus would agglutinate early-convalescent-phase
sera from patients with suspected rickettsial disease.6 Proteus antigens
(OX2, OX19 and OXK) will cross-react in predictable patterns,
although the reactions are not highly sensitive or specific. Rickettsiae
are pathogenic microorganisms that, upon invasion, produce a fever in
their host. Proteus antigens are often called Febrile Antigens because
they are used to detect the response to a rickettsial infection.
The human immune response to a particular microorganism results
in measurable antibody production that in some cases can help in
completing the patients clinical diagnosis. In blood samples, the
antibody titer during the initial phase of the infection (acute) is compared
to the antibody titer 7 to 14 days later (convalescent). Antibody titers
that are high initially in the acute phase or an acute or convalescent
pair of samples that shows an increase in antibody titer are helpful in
the diagnosis of disease.
Diagnosis of the cause of febrile disease cannot be based solely on
may react with one or more antigens in an antibody test procedure
resulting in low-level antibody titers that may not, as a single result,
suggest disease. The Weil-Felix test is not specific for rickettsial diseases.
Bacto Proteus OX2, OX19 and OXK Antisera are used in the quality
control testing of Proteus OX2, OX19 and OXK Antigens in slide and
tube agglutination tests.

Proteus OXK Antigen (Slide)
Appearance:
Turquoise-blue-violet suspension.
Proteus OXK Antigen (Tube)
Appearance:
Proteus OX2 Antiserum
Proteus OXK Antiserum
powdered cake.
powdered cake.
Rehydrate Proteus OX Antisera and Febrile Negative Control
per label directions. Perform the slide or tube agglutination
test using Proteus OX Antigen (Slide) or (Tube). Both positive
and negative controls are diluted in the same proportion as a
patient serum and processed in the same manner following
procedures for the rapid slide test or the macroscopic tube test
(see Test Procedure).
An antigen is considered satisfactory if it does not agglutinate
with the negative control, and if it reacts 2+ or greater at a titer
of 1:160 or more with the positive control.
The Difco Manual
655

Agglutination tests involving the use of Proteus antigens determine
the presence of antibodies that react with the test antigen. The serological procedure involves serially diluting the patient serum and then
adding a standard volume of an antigen. The end point of the test is the
last dilution of the serum that shows a specific amount of agglutination.
The end point converted to a dilution of the serum is called the patients
antibody titer.
Reagents
Antigens
1. Proteus Antigens are ready to use, nonmotile strains of the
organisms listed below. Proteus Antigen (Slide) contains 20%
glycerin. Each vial of Proteus Antigen (Slide) contains sufficient
reagent for 33 slide tests. Each vial of Proteus Antigen (Tube)
contains sufficient reagent for 6 tube tests.
Proteus OX2 Antigen (Slide) and (Tube) - Proteus vulgaris OX2
Proteus OX19 Antigens (Slide) and (Tube) - Proteus vulgaris OX19
Proteus OXK Antigen (Slide) and (Tube) - Proteus mirabilis OXK
2. Concentration of Antigen: Antigen density may vary because it is
adjusted for optimum performance when standardized with
hyperimmune sera obtained from laboratory animals.
Variation in color intensity is normal and will not affect test
performance.
3. Proteus antigens contain the following preservative(s):
Proteus OX2, OX19 and OXK Antigens (Slide): 0.5%
formaldehyde, and approximately 0.002% crystal violet and
0.005% brilliant green.
Proteus OX2, OX19 and OXK Antigens (Tube): 0.25%
formaldehyde.
Antisera
1. Proteus Antisera are lyophilized, polyclonal rabbit antisera
containing approximately 0.04% Thimerosal as a preservative. Each
vial contains sufficient reagent for 19 slide tests or 30 tube tests.
2. Febrile Negative Control is a standard protein solution containing
0.02% Thimerosal as a preservative. Each vial of Febrile Negative
Control contains sufficient reagent for 32 slide tests.
Precautions
3. Proteus OX2 Antigen (Slide)
656
Section V

4. Proteus OX2 Antiserum
6. Proteus Antigens are not intended for use in the immunization of
humans or animals.
Storage
Store Proteus OX2, OX19 and OXK Antigens (Slide) and (Tube) at
2-8C.
Store lyophilized and rehydrated Proteus OX2, OX19 and OXK
Antisera at 2-8C.
Expiration Date
Procedure
Materials Provided

Slide test
Applicator sticks
Tube Test
Waterbath, 35-37C
The Difco Manual
Section V
Reagent Preparation
Proteus OX2, OX19 and OXK Antigens (Slide) and (Tube) are ready
to use.
Proteus OX2, OX19 and OXK Antisera: To rehydrate, add 3 ml sterile
0.85% NaCl solution and rotate gently to completely dissolve the
contents. The rehydrated antiserum is considered a 1:2 working dilution.

maintain below -20C.
Serum specimens must be clear, free of hemolysis and show no visible
matter). Consult appropriate references for more information on
collection of specimens.9,10 Serum specimens must not be heated. Heat
may inactivate or destroy certain antibodies.
Test Procedure
Slide Test
the tube test.
1. Test serum: Using a 0.2 ml serological pipette, dispense 0.08,
0.04, 0.02, 0.01 and 0.005 ml of serum into a row of squares on the
agglutination slide.
0.04, 0.02, 0.01 and 0.005 ml of Proteus Antiserum into a row of
squares on the agglutination slide.
4. Proteus Antigen: Shake the vial of antigen well to ensure a
smooth, uniform suspension. Add one drop (approximately 35 l)
of antigen to each drop of diluted test serum, positive control and
negative control.
5. Mix each row of test sera and control sera, using a separate
7. The final dilutions in squares 1-5 correspond with tube dilutions of
1:20, 1:40, 1:80, 1:160, 1:320, respectively.
Results
No agglutination.
The Difco Manual
2. Positive control: Should show 2+ or greater agglutination at 1:160.

4. If results for either the positive or negative controls are not as
described, the test is invalid and results cannot be read.
5. Test specimens: The serum titer is that dilution which shows 2+ or
greater agglutination.
6. The slide test is a screening test, only; results must be confirmed
with the tube test.
Tube Test
1. Prepare a row of 8 culture tubes (12 x 75 ml) for each test serum,
including a row for the appropriate Proteus Antiserum.
2. Sterile 0.85% NaCl solution: Dispense 0.9 ml in the first tube of
3. Test serum: Using a 1 ml serological pipette, add 0.1 ml of the
serum in the first tube in the row and mix thoroughly. Transfer
0.5 ml from tube 1 to tube 2 and mix thoroughly. In like manner,
continue transferring 0.5 ml through tube 7, discarding 0.5 ml from
tube 7 after mixing. Tube 8 is the antigen control tube and contains
4. Positive control: Using a 1 ml serological pipette, add 0.1 ml of
the appropriate Proteus Antiserum to the first tube in the row and
mix thoroughly. Transfer 0.5 ml from tube 1 to tube 2 and mix
thoroughly. Continue transferring 0.5 ml through tube 7, discarding
0.5 ml from tube 7 after mixing. Tube 8 is the antigen control tube
and contains only sterile 0.85% NaCl solution.
5. Proteus Antigen: Shake the vial of antigen to ensure a smooth,
uniform suspension. Add 0.5 ml of the antigen to each of the
8 tubes in each row and shake the rack to mix the suspensions.
Final dilutions in tubes 1-7 are 1:20, 1:40, 1:80, 1:160, 1:320, 1:640
6. Incubate in a waterbath at 35-37C for 2 hours; then refrigerate at
7. Remove from incubation. Avoid excessive shaking before reading
the reactions either when the tubes are incubated or when removing
them from the incubation.
8. Read and record the results.
Results
No agglutination.
2. Positive control: Should show a 2+ or greater agglutination at
1:160.
3. Antigen control (tube 8 of each row): Should show no agglutination.
4. If results of the positive control or antigen control are not as
described, the test is invalid and results cannot be read.
5. For each test serum, the serum titer is that dilution which shows
2+ or greater agglutination.
657
Section V
Interpretation1
9. Rehydrated Proteus OX2, OX19 and OXK Antisera that is cloudy

or has a precipitate during use should be discarded.
Compare results:
PROTEUS PROTEUS PROTEUS
OX2
OX19
OXK
DISEASE
AGENT
Epidemic Typhus*
Murine Typhus*
Scrub Typhus
Rocky Mountain
Spotted Fever**
Other Spotted Fevers**
R. prowazekii
R. typhi
O. tsutsugamushi
+
+
+
+
R. rickettsii
Rickettsia sp.
+
+
+
+
*In cases of epidemic and murine typhus, the strength of the antibody agglutination
with Proteus OX19 is usually stronger (4+) than the agglutination with Proteus
OX2 (2+).
**In cases of spotted fevers, antibodies may agglutinate either or both strains of
Proteus OX19 or OX2, and the strength of agglutination may vary from 1+ to 4+.
For a single serum specimen, a titer of 1:160 is suggestive of infection.

A pair of serum specimens (acute and convalescent) showing a twodilution difference in the titers is a significant increase in antibody
level and is suggestive of infection. A one dilution difference is within
the limits of laboratory error.

1. The slide test is for screening only and results should be confirmed by performing the tube test. The slide test dilutions are
made to detect a prozone reaction and do not represent true
quantitation of the antibody. A serum specimen with a prozone
reaction shows no agglutination because of excessively high
antibody concentrations. To avoid this occurrence, all 5 serum
dilutions (slide test) should be run.
2. The detection of antibodies in serum specimens may complete the
clinical picture of a patient having infection. However, the isolation
of the causative agent from patient specimens may be required. A
definitive diagnosis must be made by a physician based on patient
history, physical examination and data from all laboratory tests.
low titer reactions. Infections with other organisms, vaccinations
and past history of disease may result in low level of antibody titers.
The Weil-Felix test is not specific for rickettsial diseases. Rickettsia
species cause cross-reacting antibodies, and infections with
Proteus species can also cause cross-reacting antibodies.
are necessary, an acute specimen obtained at time of initial symptoms and a convalescent specimen obtained 7 to 14 days later. A
two-dilution difference in titer is a significant increase in antibody
level and is suggestive of infection.
6. The Weil-Felix test does not differentiate between epidemic and
murine typhus.
8. Exposure of the antigen reagents to temperatures below 2C can
result in autoagglutination. Antigens must be smooth, uniform
suspensions. Examine antigen vials for agglutination before use.
Suspensions with agglutination are not usable and should be discarded.
658
References
1. Eisemann, C. S., and J. V. Osterman. 1986. Rickettsiae,
p. 593-599. In N. R. Rose, H. Friedman, and J. L. Fahey, (ed.),
Manual of clinical laboratory immunology, 3rd ed. American
2. McDade, J. E. 1991. Rickettsiae, p. 1036-1044. In A. Balows (ed.),
3. Olson, J. G, and J. E. McDade. 1995. Rickettsia and Coxiella,
p. 678-685. In P. R. Murray, E. J. Baron, M A. Pfaller, F. C. Tenover,
6. Weil, E., and A. Felix. 1916. Zur serologischen Diagnosis des
Fleckfiebers. Wien. Klin. Wochenschr. 29:33-35.
387-388.
56:64175-64182.
Washington, D.C.
Packaging
5 ml
2243-56
25 ml
2248-65
5 ml
2234-56
25 ml
2247-65
5 ml
2244-56
25 ml
2249-65
3 ml
2245-47
3 ml
2235-47
3 ml
2246-47
5 ml
3239-56
The Difco Manual
Section V
QC Antigens Salmonella
Bacto QC Antigens Salmonella

QC Antigen Salmonella O Group A . QC Antigen Salmonella O
Group B . QC Antigen Salmonella O Group C1 . QC Antigen
Salmonella O Group C2 . QC Antigen Salmonella O Group D
QC Antigen Salmonella O Group E1 . QC Antigen Salmonella O
Group E2 . QC Antigen Salmonella O Group E4 . QC Antigen
Salmonella O Group F . QC Antigen Salmonella O Group G1
QC Antigen Salmonella O Group H . QC Antigen Salmonella O
Group I . QC Antigen Salmonella Vi

Intended Use
Bacto QC Antigens Salmonella are used in the quality control testing
of Salmonella Antisera by the slide agglutination test.

QC Antigens Salmonella O Groups A, B, C1, C2, D, E1, E2,
E4, F, G1, H, I and Salmonella Vi
Appearance:
Liquid, light gray to white, may
settle on standing.
powdered cake.
Cultural Response
Rehydrate the Salmonella antiserum per label directions.
Perform the slide agglutination test using an appropriate
QC Antigen Salmonella as the homologous (positive) or
heterologous (negative) control. The homologous control
should produce 3+ or greater agglutination. The heterologous
control should not produce agglutination. Infrequently,
a +/- reaction will occur.
The following chart lists the identifying antigen(s) of various
Salmonella Antisera and the recommended homologous QC
Antigen(s) Salmonella (positive control). To demonstrate a
heterologous (negative control) reaction, use a QC Antigen
Salmonella that contains antigens unrelated to those in the
homologous control.
The Difco Manual
Salmonella species cause a variety of human diseases called salmonelloses. The range of disease is from mild self-limiting gastroenteritis
to a more severe form, possibly with bacteremia to typhoid fever, which
can be severe and life-threatening. Severe disease and bacteremia are
associated primarily with S. choleraesuis, S. paratyphi A and S. typhi,
while most of the other 2300 or more strains are associated with
gastroenteritis. The severity of the diarrheal disease depends on the
virulence of the strain and the condition of the human host.
Salmonellae are found in nature and occur in the intestinal tract of
many animals, both wild and domestic. The microorganism can spread
to man through environmental contact or from eating contaminated
meat or vegetable food products.
The genus Salmonella is in the family Enterobacteriaceae. Salmonellae
are facultatively anaerobic, gram-negative bacilli that typically are
oxidase negative, lactose negative, H2S positive and produce gas.
of both the somatic or cell wall (O) antigens and the flagellar (H)
antigens. The antigenic formula provides the O antigen(s) first, followed
by the H antigen(s). In characterizing serotypes of Salmonella, the
somatic O heat-stable antigens are identified first and are numbered 1-67
using Arabic numerals. The numbers are not completely continuous
because certain strains were reclassified to other genera and the
antigenic Arabic numbers were deleted from the scheme.
Serogroups, which represent the organization of the Salmonella
strains based on the antigen(s) shared in common, are designated by
the letters A-Z. After exhausting the alphabet, the serogroups were
numbered beginning with the numeral 51 (the serogroup Z organism
having antigen number 50). While one somatic antigen identifies
each serogroup, certain other antigens may be shared among several
serogroups.
659
Section V
The use of Salmonella antisera in the serological identification of

Salmonella requires the use of quality control test suspensions to
verify that the antisera are performing as expected. Most laboratories
are required to test antisera with positive and negative controls prior
to use. 1,2 QC Antigens Salmonella are designed as homologous
controls for testing the efficacy of the Salmonella grouping antisera
employed in routine laboratory procedures.
Serological procedures that confirm the identification of an organism

are usually agglutination reactions. Agglutination reactions may be
either homologous or heterologous. Homologous reactions occur
between a microorganism (antigen) and the corresponding antibody.
These reactions occur rapidly and are strong. Heterologous reactions
occur when a microorganism (antigen) reacts with an antibody

SALMONELLA
ANTISERUM
QC ANTIGEN SALMONELLA
HOMOLOGOUS CONTROL(S)
Poly A-I & Vi
A, B, D, E1, E2, E4, F,

G1, H, I, Vi
A, B, D, E1, E2, E4
C1, C2, F, G1, H
I
A
B
B
C1
C2
D
E 1, E 2 , E4
E1
E2
E4
F
G1
G1
H
I
Vi
A
B
B
B
C1
C2
D
E1
E2
E4
G1
H
Note: Parentheses ( ) indicate that the antigen is poorly developed or

agglutinates weakly. For a complete and current explanation of the
classification of Salmonella, consult appropriate references.3,4,5
660
Heterologous reactions may be unexpected and unpredictable and may

lead to confusion in serological identification. Therefore, only strongly
positive homologous agglutination reactions should be regarded as
significant.
Reagents
Poly A
Poly B
Poly C
Group A Factors 1, 2, 12
Group B Factors 1, 4, 5, 12
Group B Factors 1, 4, 12, 27
Group C1 Factors 6, 7
Group C2 Factors 6, 8
Group D1 Factors 1, 9, 12
Group E Factors 1, 3, 10, 15, 19, 34
Group E1 Factors 3, 10
Group E2 Factors 3, 15
Group E4 Factors 1, 3, 19
Group F Factor 11
Group G Factors 13, 22, 23, (36), (37)
Group G1 Factors 13, 22, (36), (37)
Group H Factors 1, 6, 14, 24, 25
Group I Factor 16
Vi
Factor 2
Factor 4
Factors 4, 5
Factor 5
Factor 7
Factor 8
Factor 9
Factor 10
Factor 15
Factor 19
Factor 22
Factor 14
produced in response to some other species or serotype. These

reactions occur slowly and are weak.
HOMOLOGOUS
IDENTIFYING
ANTIGEN(S)
QC ANTIGEN
SALMONELLA
ORGANISM USED FOR

ANTIGEN PREPARATION
O Group A
O Group B
O Group C1
O Group C2
O Group D
O Group E1
O Group E2
O Group E4
O Group F
O Group G1
O Group H
O Group I
Vi
S. paratyphi A var. Durazzo, Factors 2, 12

S. typhimurium Factors 1, 4, [5], 12
S. choleraesuis factors 6, 7
S. newport factors 6, 8
S. gallinarum factors 1, 9, 12
S. anatum factors 3, 10
S. newington factors 3, 15
S. senftenberg factors 1, 3, 19
S. rubislaw factor 11
S. poona factors [1], 13, 22, [36], [37]
S. carrau factors 6, 14, 24
S. hvittingfoss factor 16
Citrobacter ballerup O29
2
4, 5
7
8
9
10
15
19
11
22
14
16
Vi
Note: Brackets [ ] indicate that the antigen may be absent.
Underlining indicates that the O antigen has been lysogenized in that

strain.
These antigen suspensions are ready to use. QC Antigens Salmonella
O are preserved with 0.5% phenol USP; QC Antigen Salmonella Vi
contains 0.01% Thimerosal. When used as described, each vial of QC
Antigen Salmonella has sufficient reagent for 20 slide tests.
Febrile Negative Control is a lyophilized standard protein solution,
containing approximately 0.04% Thimerosal as a preservative. When
used as described, each vial of Febrile Negative Control has sufficient
Precautions
1. For In Vitro Diagnostic use.
2. QC Antigens Salmonella
4. QC Antigens Salmonella are not to be used for immunization of
humans or animals.
Storage
Store QC Antigens Salmonella at 2-8C.
Expiration Date
The Difco Manual
Section V
Procedure
Materials Provided

Applicator sticks
Reagent Preparation
QC Antigens Salmonella are ready to use.
Before using QC Antigens Salmonella, examine the Salmonella
antisera (Poly, Group or Factor) chosen for use. The antisera must meet
all product specifications.
Test Procedure
1. Positive control: Dispense 1 drop (35 Fl) of the Salmonella
Antiserum to be tested on an agglutination slide. Add 1 drop of
the QC Antigen Salmonella chosen as the positive control and
mix thoroughly.
2. Negative control: Dispense 1 drop of Febrile Negative Control on
the agglutination slide. Add 1 drop of the QC Antigen Salmonella
chosen as the positive control and mix thoroughly.
3. Rotate the slide for 1 minute and read for agglutination. Results
must be read within 1 minute.
Results
No agglutination.
2. Positive control: Should show 3+ or greater agglutination.
3. Negative control: Should show no agglutination. Rarely, a +/reaction is possible.
3. The density of QC Antigens Salmonella is adjusted so that they

give negative reactions with heterologous Salmonella Group
Antisera. However, an antiserum may have a particular avidity for
a given factor and agglutinate with another QC Antigen Salmonella
having the common factor. For example, Salmonella O Group E1
Antiserum Factors 3, 10 will agglutinate QC Antigen Salmonella
O Group E 1 (3, 10) but may also agglutinate QC Antigen
Salmonella O Group E2 (3, 15) if the antiserum is of high titer for
factor 3.
4. Exposure to temperatures below 2C can result in autoagglutination.
Antigens must be smooth, uniform suspensions. Examine the antigen
vial for agglutination before use. Suspensions with agglutination
are not usable and should be discarded.
References
Washington, D. C.
3. Ewing, W. H. (ed.). 1986. Edwards and Ewings identification of
New York, NY.
Kauffmann-White Scheme. U. S. Dept. Health and Human
Services, Public Health Service, Centers for Disease Control and
5. Popoff, M. Y., and L. LeMinor. 1997. Antigenic formulas of the
Salmonella serovars. WHO Collaborating Centre for Reference and
Research on Salmonella. Institut Pasteur, Paris, France.
Packaging
QC Antigen Salmonella O Group A
1 ml
2130-50
QC Antigen Salmonella O Group B
1 ml
2131-50
QC Antigen Salmonella O Group C1
1 ml
2132-50
QC Antigen Salmonella O Group C2
1 ml
2133-50
QC Antigen Salmonella O Group D
1 ml
2134-50
QC Antigen Salmonella O Group E1
1 ml
2135-50
1 ml
2136-50
1 ml
2137-50

2. QC Antigens Salmonella will react with their corresponding
homologous Salmonella polyvalent, group or factor antiserum.
Some single factor antisera may give weaker reactions than polyvalent or grouping antisera due to specificity of the single factor
antiserum for the identifying antigen(s).
QC Antigen Salmonella O Group F
1 ml
2138-50
QC Antigen Salmonella O Group G1
1 ml
2139-50
QC Antigen Salmonella O Group H
1 ml
2140-50
QC Antigen Salmonella O Group I
1 ml
2141-50
QC Antigen Salmonella Vi
1 ml
2142-50
5 ml
3239-56
The Difco Manual
661
QC Antigens Shigella
Section V
Bacto QC Antigens Shigella

QC Antigen Shigella Group A . QC Antigen Shigella Group A1
QC Antigen Shigella Group B . QC Antigen Shigella Group C
QC Antigen Shigella Group C1 . QC Antigen Shigella Group C2
QC Antigen Shigella Group D . QC Antigen Alkalescens-Dispar
Group 1
Intended Use
Bacto QC Antigens Shigella and Bacto QC Antigen AlkalescensDispar Group 1 are used in the quality control testing of Shigella
Antisera Poly and Alkalescens-Dispar Antiserum Poly by the slide
agglutination test.

Appearance:
QC Antigen Alkalescens-Dispar Group 1

Appearance:
powdered cake.
Rehydrate Shigella Antiserum Poly and Alkalescens-Dispar
Antiserum Poly per label directions. Perform the slide
agglutination test using an appropriate QC Antigens Shigella
or Alkalescens-Dispar Group 1.
The following chart lists the QC Antigens Shigella or QC
Antigen Alkalescens-Dispar Group 1 recommended as the
homologous (positive) control antigen. The homologous
control antigen has certain identifying antigen(s) in common
with the antiserum.
ANTISERUM
QC ANTIGEN
HOMOLOGOUS CONTROL
Shigella Antiserum Poly Group A

Shigella Antiserum Poly Group A1
Shigella Antiserum Poly Group B
Shigella Antiserum Poly Group C
Shigella Antiserum Poly Group C1
Shigella Antiserum Poly Group D
Alkalescens-Dispar Antiserum Poly
Shigella Group A
Shigella Group A1
Shigella Group B
Shigella Group C
Shigella Group C1
Shigella Group C2
Shigella Group D
Alkalescens-Dispar Group 1
662
Shigella species cause the diarrheal disease known as shigellosis (classic

bacillary dysentery) in humans. The range of illness is from mild
diarrhea to severe dysentery characterized by abdominal cramps and
frequent passage of bloody, mucoid stools. While the disease is usually
self-limiting, it can be life threatening to the young, the elderly and
malnourished persons. Shigella species are carried primarily in humans
and are not generally distributed in nature. While transmission is usually
direct person-to-person or through contaminated water supplies, food
borne outbreaks do occur.
The genus Shigella belongs to the family Enterobacteriaceae. Shigella
oxidase negative, lactose negative, H2S negative and do not produce
gas. Shigella and Escherichia are genetically related. Certain strains of
E. coli may resemble Shigella biochemically because both can be lactose
negative, nonmotile and non-gas-producing. These anaerogenic,
nonmotile types have historically been called the Alkalescens-Dispar
group and are presently classified as E. coli.1-6
Serological testing with polyvalent and group specific antisera should
be used to confirm the identification of isolates that are morphologically
and biochemically identified as Shigella species. Shigella are nonmotile,
so serological identification is based on somatic (O) antigens.
However, some strains have envelope antigens that prevent agglutination
in somatic antisera. Heating the suspension at 100C for 15-60
minutes destroys these interfering antigens. The four named species or
serotypes of Shigella are S. dysenteriae (10 serovars), S. flexneri (six
serovars), S. boydii (15 serovars) and S. sonnei. For a complete and
current explanation of the classification of Shigella, consult appropriate
references.1
Shigella Antisera Poly and Alkalescens-Dispar Antiserum Poly are used
in the serological identification of Shigella species and the AlkalescensDispar (A-D) Group. QC Antigens Shigella and Alkalescens-Dispar
Group 1 are designed as positive controls for testing the efficacy of the
Shigella grouping antisera used in laboratory procedures.
QC Antigens Shigella and QC Antigen Alkalescens-Dispar Group 1
may also be used as negative controls by using a heterologous antigen
(possessing no common antigen) with a given test serum. However,
cross reactivity may occur. Consult appropriate references for further
details on cross reactivity.1
The Difco Manual
Section V

Serological procedures that confirm the identification of an organism
are usually agglutination reactions. Agglutination reactions may be
either homologous or heterologous. Homologous reactions occur
between a microorganism (antigen) and the corresponding antibody.
These reactions occur rapidly and are strong. Heterologous reactions
occur when a microorganism (antigen) reacts with an antibody
produced in response to another species or serotype. These reactions
occur slowly and are weak.
Heterologous reactions may be unexpected and unpredictable and may
lead to confusion in serological identification. Therefore, only strongly
positive homologous agglutination reactions should be regarded as
significant.
Storage
Store QC Antigens Shigella and QC Antigen Alkalescens-Dispar
Group 1 at 2-8C. Prolonged exposure of reagents to temperatures other
than those specified is detrimental to the products.
Expiration Date
Procedure
Reagents
QC ANTIGEN
ORGANISM IDENTITY
Shigella Group A
Shigella Group A1
Shigella Group B
Shigella Group C
Shigella Group C1
Shigella Group C2
Shigella Group D
Shigella dysenteriae type 1

Shigella dysenteriae type 8
Shigella flexneri type 6
Shigella boydii type 3
Shigella sonnei
E. coli A-D Group 1

contain killed whole organisms preserved in 0.5% formaldehyde. They
are ready to use.
When used as described, each vial of antigen contains sufficient
Precautions
2. QC Antigen Shigella Group A
QC Antigen Shigella Group A1
QC Antigen Shigella Group B
QC Antigen Shigella Group C
QC Antigen Shigella Group C1
QC Antigen Shigella Group D
The Difco Manual
4. QC Antigens Shigella and QC Antigen Alkalescens-Dispar

Group 1 are not to be used for immunization of humans or
animals.
Materials Provided

Applicator sticks
Reagent Preparation
are ready to use.
Test Procedure
1. Positive control: Dispense 1 drop (35 l) of the Shigella Antiserum
or Alkalescens-Dispar Antiserum Poly to be tested on an agglutination slide. Add 1 drop of the appropriate QC Antigen Shigella or
QC Antigen Alkalescens-Dispar Group 1 chosen as the positive
control and mix thoroughly.
2. Negative control: Dispense 1 drop of sterile 0.85% NaCl solution
or Febrile Negative Control on the agglutination slide. Add 1 drop
of the appropriate QC Antigen Shigella or QC Antigen AlkalescensDispar Group 1 and mix thoroughly.
Results
No agglutination.
3. Negative control: Should show no agglutination. Rough reactions
can occur. If so, repeat the test using Febrile Negative Control.
663
Salmonella Antisera
Section V

the test mixture. False-positive reactions can occur.
Antigens must be smooth uniform suspensions. Examine antigen
vials for agglutination before use. Suspensions with agglutination
are not usable and should be discarded.
3. Allow the QC Antigens, the antisera and all equipment used to be
at room temperature at the time of testing. The test reagents, if
cold, may cause false-negative reactions.
4. Shake the antigen well before use to suspend the organisms.
4. Pezzlo, M. (ed.). 1994. Aerobic Bacteriology, p. 1.0.1-1.20.47. In

p. 6.01-6.06. In FDA Bacteriological Analytical Manual, 8th ed.
6. Smith, J. L. 1992. Shigella, p. 423-431. In C. Vanderzant and
D. F. Splittstoesser (eds.), Compendium of methods for the
Packaging
QC Antigen Shigella Group A
1 ml
2100-50
References
QC Antigen Shigella Group A1
1 ml
2101-50
1. Ewing, WH. (ed.). 1986. Edwards and Ewings identification of

New York, NY.
St. Louis, MO.
QC Antigen Shigella Group B
1 ml
2102-50
QC Antigen Shigella Group C
1 ml
2103-50
1 ml
2104-50
1 ml
2105-50
QC Antigen Shigella Group D
1 ml
2106-50
1 ml
2116-50
5 ml
3239-56
Bacto Salmonella Antisera

Salmonella O Antisera . Salmonella H Antisera . Salmonella H
Antisera Spicer-Edwards
Intended Use
Bacto Salmonella O Antisera are used in agglutination tests for the
identification of Salmonella by somatic (O) antigens.
Bacto Salmonella H Antisera are used in tube agglutination tests for
the identification of Salmonella by flagellar (H) antigens.

Salmonella O Antisera
powdered cake.
Salmonella H Antisera
powdered cake.
664
Bacto Salmonella H Antisera Spicer-Edwards are used in tube

agglutination tests for screening and identifying the most commonly
encountered salmonellae by flagellar (H) antigens.

Salmonella species cause a variety of human diseases called salmonelloses. The range of disease is from mild self-limiting gastroenteritis to
more severe forms, possibly with bacteremia or typhoid fever, which
can be life-threatening. Severe disease and bacteremia are associated
primarily with three serovars of S. enterica subsp. enterica
(Cholerasuis, Paratyphi A and Typhi) while most of the other 2,300 or
more strains are associated with gastroenteritis. The severity of the
diarrheal disease depends upon the virulence of the strain and the
condition of the human host.
Salmonella is found in nature and occurs in the intestinal tract of
many animals, both wild and domestic. The microorganism can spread
to man from contact with the environment or from eating meat or
vegetable food products.
The Difco Manual
Section V
Salmonella Antisera
Table 1. Differentiation of the genus Salmonella from other genera.1
Rehydrate Salmonella O, Salmonella Vi, and Salmonella H
Antisera per label directions. Perform the slide agglutination
test using appropriate Salmonella O and Vi Antisera and QC
Antigens Salmonella O Groups A -I and Vi.
The chart below includes the QC Antigens Salmonella O
recommended as homologous (positive) control antigens. (The
homologous control antigen has certain identifying antigen(s)
in common with the antiserum.) For a negative (heterologous)
antigen control, use a QC Antigen Salmonella containing
antigens unrelated to those in the homologous control.
Homologous control: Should show 3+ or greater agglutination.
Negative control: Should show no agglutination. Rarely, a +/
reaction is possible.
For Salmonella H Antisera, maintain stock cultures of known
serological identification, and prepare antigen positive and
negative controls by using known serotypes and following the
procedure described above in Tube Test Preparation.
SALMONELLA O ANTISERUM
Poly A-I & Vi
QC ANTIGEN SALMONELLA O
HOMOLOGOUS CONTROL
Poly A
Poly B
Poly C
Groups A,B,C1,C2,D,E1,E2,
E4,F,G1,H,I,Vi
Groups A,B,D,E1,E2,E4
Groups C1,C2,F,G1,H
Group I
Group A Factors 1,2,12

Group B Factors 1,4,5,12
Group B Factors 1,4,12,27
Group C1 Factors 6,7
Group C2 Factors 6,8
Group D1 Factors 1,9,12
Group E Factors 1,3,10,15,19,34
Group E1 Factors 3,10
Group E2 Factors 3,15
Group E4 Factors 1,3,19
Group F Factor 11
Group G Factors 13,22,23,(36),(37)
Group G1 Factors 13,22,(36),(37)
Group H Factors 1,6,14,24,25
Group I Factor 16
Vi
Group A
Group B
Group B
Group C1
Group C2
Group D
Groups E1,E2,E4
Group E1
Group E2
Group E4
Group F
Group G1
Group G1
Group H
Group I
Group Vi
Factor 2
Factor 4
Factors 4,5
Factor 5
Factor 7
Factor 8
Factor 9
Factor 10
Factor 15
Factor 19
Factor 22
Factor 14
Group A
Group B
Group B
Group B
Group C1
Group C2
Group D
Group E1
Group E2
Group E4
Group G1
Group H
Note: Parentheses ( ) enclosing the designation for an antigen indicate that

the antigen is poorly developed or agglutinates weakly agglutinates. For a
complete and current explanation of the classification of Salmonella, consult
appropriate references.1,2,3,8,11
The Difco Manual
Salmonella
Test
Indole production
Citrate, Simmons
H2S production
Urea hydrolysis
Lysine decarboxylase
Ornithine
decarboxylase
D-Adonitol,
acid production
L-Arobinose,
acid production
L-Rhamnose,
acid production
D-Sorbitol,
acid production
D-Xylose,
acid production
Acetate utilization
+
[+]
d
[]
Citrobacter
amolonaticus diversus freundii
Edwardsiella
+
+
+
+
+
[+]
[+]
+
+
+
[+]
d
[]
+
[+]
[+]
[+]
[+]
90-100% positive
76-89% positive
26-75% positive
11-25% positive
0-10% positive
All Salmonella serovars belong to two species: S. bongori, which

contains 18 serovars, and S. enterica, which contains the remaining 2,300
or more serovars divided among six subspecies.2,3
The six subspecies of S. enterica are:
(The legitimate species name for S. enterica is S. choleraesuis.
the species name, S. enterica.4 LeMinor and Popoff5 published a
request to the Judicial Commission to use S. enterica as a species name.
The Commission ruled that S. choleraesuis is the legitimate name.6,7
S. enterica is used in many countries and is favorably accepted as the
species name.8,9 The Centers for Disease Control has adopted this
designation until the problem of naming this species is resolved.2 )
Nomenclature and classification of these bacteria are constantly
changing.1 Salmonella and the former Arizona should be considered a
single genus, Salmonella.9 It is recommended that laboratories report
the names of Salmonella serovars for the subspecies enterica. The
serovar names are no longer italicized and the first letter is capitalized.
For example, the strain that used to be identified as Salmonella
typhimurium is now known as Salmonella Typhimurium.
665
Salmonella Antisera
Section V
Serovars of other subspecies of S. enterica (except some in the

subspecies salamae and houtenae) and those of S. bongori are not named
and are designated by their antigenic formula. For the most recent
information on nomenclature, consult appropriate references.1,7,10-13
Results are for 48-hours incubation. Tests were performed at 35-37C.

biochemical characterization and serotyping. However well-defined
the serology of Salmonella, the use of serological procedures does not
supersede cultural isolation and biochemical characterization. Any
serological results obtained before biochemical identification must be
considered as presumptive identification only. Consult to appropriate
references for complete identification of Salmonella.1,2,3,8,11-14

The antigenic formula lists the O antigen(s) first, followed by the
H antigen(s). The major antigens are separated by colons and the
components of the antigens separated by commas. For example, the
antigenic formula for Salmonella Typhimurium is Salmonella
1,4,5,12:i:1,2. This means that the strain has O antigen factors 1,4,5 and
12, the flagella phase 1 antigen i, and flagella phase 2 antigens 1 and 2.
Characterizing the Serotypes of Salmonella

Salmonella O Antigens: The somatic (O) heat-stable antigens are
identified first. The O antigens are numbered 1-67 using Arabic
numerals. The numbers are not completely continuous because certain
strains were reclassified to other genera and the antigenic Arabic
numbers were deleted from the schema.
Table 2a. Differentiation of Salmonella species, subspecies, and

some serovars.1,2
Table 2b. Differentiation of Salmonella species, subspecies and

some serovars.1,2
Salmonella enterica
Test
Citrate,
Simmons
H 2S
production
Lysine
decarboxylase
Ornithine
decarboxylase
Motility
KCN, growth
Malonate
utilization
D-Glucose,
gas
L-arobinose,
acid
Dulcitol,
acid
Lactose, acid
Maltose, acid
Melibiose,
acid
L-Rhamnose,
acid
D-Sorbitol
Trehalose,
acid
D-Xylose,
acid
Mucate, acid
Tartrate,
Jordans
ONPG
Salmonella
bongori
subsp.
arizonae
subsp.
subsp.
enterica diaizonae
subsp.
houtenae
subsp.
indica
[+]
+
+
+
+
[+]
+
[+]
[]
+
+
+
+
[+]
+
+
+
+
[]
+
[+]
+
+
+
+
+
+
+
+
+
+
+
+
d
[]
+
+
+
[+]
d
666
90-100% positive
76-89% positive
26-75% positive
subsp.
salamae
Test
Citrate,
Simmons
H 2S
production
Lysine
decarboxylase
Ornithine
decarboxylase
Motility
KCN, growth
Malonate
utilization
D-Glucose,
gas
L-arobinose,
acid
Dulcitol,
acid
Lactose, acid
Maltose, acid
Melibiose,
acid
L-Rhamnose,
acid
D-Sorbitol
Trehalose,
acid
D-Xylose,
acid
Mucate, acid
Tartrate,
Jordans
ONPG
[]
0
Salmonella enterica
subsp. enterica
serovar
serovar
Serovar
serovar Serovar
Choleraesuis Gallinarum Paratyphi A Pullorum Typhi
[]
[+]
+
d
+
+
+
+
+
+
[+]
+
+
[]
[+]
+
+
[+]
[+]
+
d
[+]
d
+
[]
11-25% positive
10% positive
The Difco Manual
Section V
Serogroups represent the organization of Salmonella strains based on

the antigen(s) shared in common and are designated by the letters A-Z.
After exhausting the alphabet, the serogroups were numbered beginning
with No. 51 (the serogroup Z organism having antigen No. 50). While
one somatic antigen identifies each serogroup, certain other antigens
may be shared among several serogroups.
Most organisms contain antigens in common that will cause crossreactions in an unabsorbed or partially absorbed antiserum. One
somatic antigen identifies a serogroup and is shared in common by
all members of a given serogroup. For example, serogroup A is
represented by three members, Salmonella Paratyphi A (somatic antigens
1,2,12), Salmonella Kiel (somatic antigens 1,2,12), and Salmonella
Nitra (somatic antigens 2,12). All three members of this serogroup
contain antigens 2 and 12 in common. Serogroup B is represented by
many organisms consisting having different combinations of somatic
antigens 1,4,5,12 and 27. Serogroup D organisms contain somatic
antigens 1,9,12, etc.
In the above example, all three serogroups A, B, and D contain
antigens 1 and 12. An antiserum prepared from a 1,2,12 culture, if not
absorbed, will react with cultures of serogroups B and D in varying
degrees depending on the concentration of the commonly shared 1 and
12 factors. This must be taken into consideration when choosing an
antiserum to be used in the examination of the salmonellae.
Several different antisera are available. Some represent group antigens.
Others are single factor sera, which should be used when testing for an
identifiable antigen in a given serogroup. Such a single factor serum is
not called a group serum, though it contains the group identifiable
agglutinin. (It has been recommended by the CDC that the term group
be applied only to those sera possessing all the major agglutinins found
in that group.)
In unabsorbed antisera, cross-reactions occur if strains sharing some
like antigens are tested, even when they are in separate serogroups
based on the major group antigen(s) they possess. Unabsorbed antisera
are available as group antisera containing all factors in that group.
In absorbed antisera, cross reactions are less likely and are weaker.
Absorbed antisera are available as factor specific antisera.
Flagellar Salmonella H Antigens: The flagellar (H) antigens are heat
labile and are usually associated with motility. Cultures are ordinarily
flagellated and actively motile, although flagellated cultures can be
nonmotile. H antigen characterization is done after the serogroup of
the strain is determined. The H antigens of Salmonella are designated
by letters of the alphabet, a-z, followed by z, z1, z2, etc., and by Arabic
numerals. H antigens exist in 2 phases, phase 1 and phase 2. Phase 1
antigens are expressed in letters a-z, etc., and the phase 2 antigens are
most often expressed in Arabic numerals. Older cultures may express
both phases of a diphasic serotype, but recent clinical isolates more
often express only one phase. Phase reversal may be necessary to
isolate both phases of a diphasic culture. Consult an appropriate
reference for more detailed information.8
A pure H antiserum cannot be prepared without some somatic content.
However, since H antigens are highly antigenic, the serum derived
from motile cultures may be used at a dilution that reduces somatic
agglutination below the detection level.
Salmonella Vi Antigen: The Vi Antigen is a heat-labile envelope
antigen that may surround a cell wall and mask somatic antigen activity.
Microorganisms having the Vi Antigen will not agglutinate in O antisera.
The Difco Manual
Salmonella Antisera
Using Salmonella Antisera

Salmonella O Antisera: The recommended serological Identification
scheme begins with Salmonella O Antisera Poly A through Poly G,
which contain the following:
SALMONELLA POLY GROUP ANTISERA
SOMATIC GROUPS PRESENT
Bacto Salmonella O Antiserum Poly A

Bacto Salmonella O Antiserum Poly B
Bacto Salmonella O Antiserum Poly C
Bacto Salmonella O Antiserum Poly D
Bacto Salmonella O Antiserum Poly E
Bacto Salmonella O Antiserum Poly F
Bacto Salmonella O Antiserum Poly G
A,B,D,E1,(E2,E3),*E4,L
C1,C2,F,G,H
I,J,K,M,N,O
P,Q,R,S,T,U
V,W,X,Y,Z
5155
5661
*Strains of groups E2 and E3 are lysogenized by phage 15, then by phage 34.
These strains are now classified into group E1.3
If agglutination occurs, use individual Salmonella O Group Factor

Antisera to determine the specific serogroup to which the isolate
belongs. For efficiency, test first with individual Salmonella O Group
Factor Antisera.
If agglutination does not occur with Poly A or B, test the isolate with
Salmonella O Antiserum Vi. If positive, heat and retest. If agglutination
does not occur with Salmonella O Antiserum Vi, the isolate is not
likely to be Salmonella. Results should be examined. If questions
exist, the isolate should be sent to a reference laboratory.
If agglutination does not occur with Poly C, D, E, F, and G, the isolate
is not likely to be Salmonella.
Table 3. Schema for using Salmonella O Antisera Poly Groups A, B,
C, D, E, F and G.
Test with
Test Result
Salmonella O antisera Poly Groups A, B, C, D, E, F and G

+
with
with
Poly A or B
Poly C, D, E,
F and G
Test with
Individual
Salmonella O
Antisera
Test Result
+ with one
Salmonella O
Antiserum
(required)
Test
Conclusion
or Next
Action
Determine the
Salmonella H
Antigen
Vi Antiserum
Heat and retest Test isolate

with individual
is not a
Salmonella O Salmonella
Antisera
Test isolate
is not a
Salmonella
Salmonella O Antiserum Poly A-I and Vi: This antiserum detects

factors 1-16, 19, 22-25, 34 and Vi. This combination of factors
represents the most frequently isolated Groups A-I and the Vi antigens
and is used to screen possible Salmonella isolates.
A positive reaction indicates that further serological testing is needed
to identify the isolate using Salmonella O Group Factor Antisera. The
most common serogroups are B, D and C1. For efficiency, first use
the Salmonella O Group Factor Antisera for these serogroups.
If the isolate is positive with Salmonella O Antiserum Poly A-I and Vi
but negative with Poly A-Poly G, test the isolate with Salmonella Vi
Antiserum. If positive with Salmonella Vi Antiserum, heat and retest
using individual Salmonella O Antisera. If negative with Salmonella
667
Salmonella Antisera
Section V
Vi Antiserum, the isolate is not likely to be Salmonella. Results

should be examined. If questions exist, the isolate should be sent to a
reference laboratory.
A negative reaction indicates the isolate is not in serogroups A-I. If
the biochemical reactions are consistent with Salmonella, a serogroup
other than A-I is possible. Further testing with antisera for other
serogroup antigens is necessary.
Table 4. Schema for using Salmonella O Antiserum Poly A-I & Vi.
Salmonella O Antisera Poly A-I and Vi
+
Test with
Test Result
Test with
Individual Salmonella O Antisera
Test Result
Salmonella H Antisera Spicer-Edwards: Salmonella H Antisera

Spicer-Edwards is used for screening and identifying the most commonly
encountered Salmonella using a combination of polyvalent and single
complex antisera.
Table 5. Identification of Salmonella H using Salmonella H Antisera
Spicer-Edwards.
Test Result
Spicer-Edwards
Determine the
Salmonella H
Antigen
Heat and retest Test isolate

with individual
is not a
Salmonella O Salmonella
Antisera
May be a
Salmonella
detectable
by use of
Salmonella
O Antisera
Poly C, D,
E, F or G
Salmonella O Group Factor Antisera and Single Factor Antisera:

Use selected Salmonella O Group Factor Antisera. Cross reactions may
occur between serogroups that share O antigens. Consider this partial
list of Salmonella O Group Factor Antisera as an example:
Salmonella O Antiserum Group A Factors 1, 2, 12
Salmonella O Antiserum Group B Factors 1, 4, 5, 12
Salmonella O Antiserum Group B Factors 1, 4, 12, 27
Factors 1 and 12 occur in combination with other antigens and may
cause cross-reactions. The strength of the reactions will help in interpretation. Rapidly forming 3+ or greater agglutination indicates a
homologous reactions.
Use selected Salmonella O Factor Antisera. Absorbed antisera specific
for an identifiable antigen in a given serogroup is used to identify the
isolate further. In the example above, Salmonella O Factor Antisera
could be used:
Salmonella O Antiserum Factor 2
Salmonella O Antiserum Factors 4, 5
Salmonella O antiserum Factor 5
Polyvalent Salmonella H Antisera: Further identification of a
Salmonella isolate includes the characterization of the flagellar antigens.
Agglutination with the following Polyvalent H Antisera can be done:
SALMONELLA POLY GROUP ANTISERA
H Antigen(s)
e,h
G Complex*
Z4 Complex**
z10
z29
Table 6. Identification of Salmonella H using Salmonella H Antisera.

H Antigen(s)
e,n,x,
e,n,z15
I,v
I,w
I,z13
I,z28
1,2
1,5
1,6
1,7
EN Complex
L Complex
1 Complex
FLAGELLAR ANTIGENS PRESENT
Salmonella H Antiserum Poly a-z Groups EN,G,L,Z4, 1 complexes and

a-k,r-z,z6,z10,z29
Salmonella H Antiserum Poly A Groups a,b,c,d,i,z10,z29
Salmonella H Antiserum Poly B Groups eh,en,enx,enz15, G complex
Salmonella H Antiserum Poly C Groups k,l,r,y,z,z4
Salmonella H Antiserum Poly D Groups z35,z36,z37,z38,z39,z41,z42
Salmonella H Antiserum Poly E 1 complex z6
668
Unabsorbed and Absorbed Salmonella H Antisera: Complete

identification of a Salmonella isolate involves analysis of phase 1 and
phase 2 antigens using H antisera. For the complex pattern of analysis
and procedures, consult appropriate references.8
Salmonella Vi Antiserum
Test with
Test
Conclusion
or Next
Action
Absorbed H antisera specific for single antigens or a complex of

antigens can be used to identify the isolate further.
* The G complex component of Salmonella H Antisera Spicer-Edwards

1 and 4 reacts with antigens f,g; f,g,s; f,g,t; g,m; g,m,q; g,m,s; g,m,s,t;
g,m,t; g,p; g,p,s; g,p,u; g,q; g,s,t; g,t; m,p,t,u; and m,t.
** The Z4 Complex component reacts with z4,z23; z4,z24; and z4, z32.
Note that no antigen is positive with all four Salmonella H Antisera

Spicer-Edwards Any antigen that reacts with all four sera should be
checked for smoothness.
The Difco Manual
Section V
Extent of Serological Identification Necessary

Complete serological characterization of Salmonella is not required
for successful detection of the microorganism when it occurs as a
pathogen. The use of adequate isolation procedures and differential
biochemical tests is of primary importance. Possible Salmonella isolates can be presumptively identified with a minimum of serological
identification. Isolates can be sent to laboratories that perform the level
of testing necessary to completely identify the microorganism.
For a further discussion of the serological identification of Salmonella,
consult appropriate references.1,2,3,8,11

Identification of Salmonella species includes both biochemical and
serological identification. Serological confirmation involves the
procedure in which the microorganism (antigen), reacts with its
corresponding antibody. This in vitro reaction produces macroscopic
clumping called agglutination. The desired homologous reaction is
rapid, does not dissociate (high avidity) and bonds strongly (high affinity).
possible. These are characterized as weak in strength or slow in
formation. Such unexpected and perhaps unpredictable reactions may
lead to some confusion in serological identification. Therefore, a
positive homologous agglutination reaction should support the
morphological and biochemical identification of the microorganism.
granular clumping. Homologous reactions are rapid and strong (3+).
Heterologous reactions are slow and weak.
Agglutination of the flagellar antigens in the tube test appears as a
loose flocculation that can easily be resuspended.
Reagents
Salmonella O, H, and Vi Antisera are lyophilized, polyclonal rabbit
Salmonella O Poly Antisera are polyvalent antisera. Each antiserum
is specific for certain serogroup antigens. When properly rehydrated
and used as recommended, each vial of Salmonella O or Vi Antisera
contains sufficient reagent for 60 tests. Salmonella O Antisera Poly
A-I and Vi is prepared with representative strains of these serogroups
and is not absorbed. It may cross-react with other antisera because of
shared common O antigens.
Salmonella O Group Antisera are specific for the major factors present
in the serogroup. Salmonella O Factor Antisera are specific for the
factors of the individual serogroups. When using Salmonella O Group
Antisera, cross-reactions are possible because serogroups may share
non-major group antigens. Salmonella O Factor Antisera are absorbed
as necessary to render each antiserum as specific as practical without
reducing the homologous reactions to an unsatisfactory level.
Salmonella H Poly Antisera are polyvalent antisera. Each antiserum
is specific for certain flagellar antigens. Each vial of Salmonella H
Antiserum contains sufficient reagent to perform between 150-1500
tests, depending on the antiserum used. Salmonella H Antisera are
either absorbed or unabsorbed specifically for either phase 1 or phase 2
antigens. Salmonella H Antisera Spicer-Edwards are pooled, polyvalent antisera and additional adjunctive antisera to identify the more
commonly occurring H antigens.
The Difco Manual
Salmonella Antisera
Precautions
Storage
Store lyophilized and rehydrated Salmonella O, H and Vi antisera
at 2-8C.
Prolonged exposure of reagents to temperatures other than those
specified is detrimental to the products. Discard any antiserum that
becomes cloudy during storage.
Expiration Date
Procedure
Materials Provided
Salmonella Vi Antiserum
(See Packaging.)

Slide Test
0.85% NaCl solution, sterile
Agglutination slides with 1 inch squares
Applicator sticks
Boiling waterbath
Centrifuge
Tube Test
0.85% NaCl solution, sterile
Waterbath, 50 2C
Serological pipettes, 1 ml
Formaldehyde
Reagent Preparation
Salmonella O, H and Vi Antisera: To rehydrate, add 3 ml of sterile
0.85% NaCl solution and rotate gently to completely dissolve the
contents. Rehydrated antisera are considered a 1:2 dilution. Subsequent
Salmonella O Antisera dilutions are based on this as a starting dilution.
The H antisera are further diluted for use.

Clinical specimens: Salmonella can be recovered from selective
differential media such as Hektoen Enteric Agar or XLD agar. For
specific recommendations, consult appropriate references.11,12 Determine that a pure culture of the microorganism has been obtained and
669
Salmonella Antisera
that biochemical test reactions are consistent with the identification of

the organism as a Salmonella species. After these criteria are met,
serological identification can be performed.
Food samples: Salmonella can be recovered when samples are
processed to recover injured microorganisms and prevent overgrowth
of competing microorganisms. Consult appropriate references for
recommended procedures for isolation of Salmonella from foods.13,14
and that biochemical test reactions are consistent with the identification of the organism as a Salmonella species. After these criteria have
been met, serological identification can be performed.
Slide Test Procedure

Salmonella O and Vi Antisera
Use this procedure to test the isolate with each selected antiserum.
1. Salmonella Antiserum: Add Dispense 1 drop (3 l) of each
antiserum to be tested on an agglutination slide.
2. Negative control: Dispense 1 drop of 0.85% sterile NaCl solution
on an agglutination slide.
3. Test isolate: From a solid agar medium, transfer a portion of a
loopful of an isolated colony to each of the two reaction areas
above and mix thoroughly.
4. Positive control: Dispense 1 drop of each Salmonella O Antiserum
to be tested on an agglutination slide. Add 1 drop of an appropriate
QC Antigen Salmonella.
5. Rotate the slides for 1 minute and read for agglutination. Results
must be read within 1 minute.
Slide Test Results

2.
3.
4.
5.
6.

No agglutination.
Positive control: Should show a 3+ or greater agglutination.
Negative control: Should show no agglutination. If agglutination
occurs, the culture is rough and cannot be tested. Subculture to a
non-inhibitory medium, incubate and test the organism again.
Test isolates: 3+ or greater agglutination is a positive result.
A partial (less than 3+) or delayed agglutination reaction should
be considered negative.
If a positive reaction occurs with Salmonella Vi Antiserum, follow
this procedure:
Prepare a dense suspension of the isolate from an agar medium
in 3-5 ml of sterile 0.85% NaCl solution.
Heat in a boiling waterbath for 30-60 minutes and cool. The
suspension should not precipitate after heating. If this occurs,
select another colony for testing.
Centrifuge at 1,000 rpm for 10-15 minutes.
Aspirate and discard the supernatant.
Resuspend the sediment in 0.5 ml sterile 0.85% NaCl solution.
Retest a drop of the sediment with Salmonella O Group
Antisera, as outlined.
670
Section V
7. If the heated culture continues to react with Salmonella Vi Antiserum and not with the Salmonella O Antisera, the isolate may
not be Salmonella. Test the isolate further to determine if it is
correctly identified.
8. If an H antigen identification is required, proceed to the next
section.
9. When a negative reaction is obtained with Salmonella O Antiserum Poly A-I and Vi in the above procedure, the organism is presumptively negative for Salmonella that belong to serogroups A-I.
Biochemical tests should be performed to confirm this negative
result. If biochemical tests prove the organism to be a Salmonella,
a serogroup beyond serogroup A-I is probably involved.
If the organism reacts with Poly A-I and Vi but does not react
with the specific somatic antiseraa, it should be checked with
Salmonella Vi Antiserum by the above procedure.
Tube Test Preparation

1. 0.6% formalized saline: Prepare by adding 6 ml formaldehyde
per 1,000 ml of sterile 0.85% NaCl solution.
2. Test organism: It is often necessary to increase the motility of the
test organism. To accomplish this, make several consecutive
transfers in Motility GI Medium.
Inoculate the tube slightly below the surface of the medium
using the stab method.
Incubate at 35-37C for 18-20 hours.
Transfer only those organisms that have migrated to the bottom
of the tube.
When the organism successfully travels 50-60 mm through the
medium in 18-20 hours, it is ready for use.
An infusion broth such as Veal Infusion Broth is recommended
for cultivating the motile Salmonella prior to testing. It should
be inoculated and incubated at 35C for 24 hours. Brain Heart
Infusion Broth may be used with incubation at 35C for 4-6 hours.
If Tryptic Soy Broth is used, incubate at 35C for 24 hours.
Prepare the test organism suspension by using equal volumes
of broth culture and 0.6% formalized saline. The final density
of this test suspension should be that of a McFarland Barium
Sulfate Standard No. 3.
3. Positive control: Commercially prepared QC Salmonella H antigens are not available. The user must maintain stock cultures of
known serological identification for use in quality control. Prepare
the antigen by using known serotypes and following the procedure
described above. (See Test organism, above.)
4. Salmonella H Antisera: Rehydrated antisera is considered a 1:2
working dilution. Prepare dilutions as follows and use on the day
prepared. Discard any unused portion.
Most Salmonella H Antisera: A 1:1,000 final dilution is used.
Prepare by adding 0.1 ml rehydrated antiserum to 25 ml of
0.85% NaCl solution.
Salmonella H Antisera x, z 13, z15 and z 28: A 1:500 final
dilution is used. Prepare by adding 0.1 ml rehydrated antiserum
to 12.5 ml of 0.85% NaCl solution.
Salmonella H Antiserum Poly a-z: A 1:100 final dilution is
used. Prepare by adding 0.1 ml rehydrated antiserum to 2.5 ml
of 0.85% NaCl solution.
The Difco Manual
Section V
Salmonella Antisera
Tube Test Procedure
Salmonella H Antiserum:
1. Prepare a 12 x 75 ml culture tube for each organism to be tested.
2. Diluted antiserum: Dispense Add 0.5 ml of diluted antiserum in
each tube.
3. Test isolate: Add 0.5 ml to the appropriate tube.
4. Positive control: Add 0.5 ml of antigen positive control to a tube
containing 0.5 ml of antiserum.
5. Negative control: Add 0.5 ml of 0.85% NaCl solution to a tube
containing 0.5 ml of test isolate.
6. Incubate all tubes in a waterbath at 50 2C for 1 hour.
7. Read for flocculation (agglutination).
8. Repeat the Tube Test using a phase-reversed test organism. (See
the procedure for phase reversal below.)
1. Complete O and H antigen characterization of a Salmonella isolate

is required for final identification. Due to the complexity of the
laboratory procedures, identification with polyvalent antisera may
be sufficient for most laboratories.
2. Possible Salmonella isolates having inconsistencies in biochemical
reactions and O and H antigen tests should be referred to a reference laboratory for further testing.
4. Rough culture isolates do occur and will agglutinate spontaneously, causing agglutination of the negative control reaction
(autoagglutination). Smooth colonies must be selected and tested
in serological procedures.
5. In the slide agglutination procedure for O antigen testing, it is
recommended that several colonies be tested and that unabsorbed
polyvalent antisera be used followed by absorbed single factor
antisera. For example, colonies of a 1,2,12 culture on an agar plate
will have varying degrees of each antigen. A 1,2,12 antiserum
absorbed of 1 and 12 antibodies will be highly specific but will
show weak or no agglutination with colonies that have less of
antigen 2 and more of antigens 1 and 12. Using unabsorbed
Salmonella O Antiserum Group Factors 1,2,12 to test several
suspicious colonies on a plate followed by testing with absorbed
Salmonella O Antiserum Factor 2 gives the needed balance of
sensitivity and specificity.
6. The slide agglutination antisera (Salmonella O Antisera) have been
prepared for use in identifying cultures already defined biochemically. Such cultures are taken from an agar medium using a bacterial
loop. A portion of the isolated colony is emulsified in a drop of
antiserum. It is recommended that more than one colony be tested.
The sera have been tested and absorbed using this method. They
have not been tested employing an antigen suspension in NaCl
solution or alcohol-treated cultures. If variations in the recommended procedures are to be used, the investigator is advised to
test each lot of antiserum with known positive control cultures to
ensure its proper homologous and heterologous reactions under
their test conditions.
7. Agglutination reactions of 3+ or greater are interpreted as positive
reactions. Cross-reactions resulting in a 1+ or 2+ agglutination are
likely since there are somatic antigens shared among different
groups as non-major group antigens.
8. The tube agglutination technique is recommended for H antigen
testing because cross-reactions with somatic antigens may occur
at the dilutions used in the slide technique.
9. No attempt has been made to absorb or test for O antibodies in
H antisera.
10. In the tube test, make certain that the proper dilution is prepared
for a given antiserum. Various dilutions are used for various sera.
The information is given under Tube Test Preparation. Also, it is
important in this test to use the recommended time and temperature
of incubation. Make certain that the waterbath is in a location free
of mechanical vibration.
Phase Reversal:
1. Prepare Motility GI Medium phase reversal medium according to
directions.
2. Prepare the antiserum opposite to the phase desired. For example,
incubating Salmonella Typhimurium phase 1[i] in GI Motility
Medium containing i antiserum allows the growth and spread of
S. Typhimurium phase 2 [1,2].
3. Add 1 ml of a 1:10 dilution of antiserum to 25 ml of sterile GI
Motility Medium and mix well. Pour into a sterile Petri dish and
allow to solidify.
4. Inoculate by punching the edge of the solidified medium.
5. Incubate at 35-37C for 24 hours.
6. Transfer growth from the spreading edge opposite the inoculation
site to a liquid medium for testing according to steps under Tube
Test Procedure Salmonella H Antisera.
7. If motility is not acceptable, pass through GI Motility Medium again.
Tube Test Procedure

Salmonella H Antiserum Spicer-Edwards
1. Prepare the test organism and the 1:2 antiserum dilution as
described above in Tube Test Preparation.
2. Final 1:1,000 dilution of antiserum: Prepare by adding 0.1 ml
of rehydrated antiserum (1:2 working dilution) to 25 ml of 0.85%
NaCl solution.
3. Prepare 4 culture tubes (12 x 75 ml) for each test organism.
4. Salmonella H Antisera Spicer-Edwards 1-4: Add 0.5 ml of the
diluted antiserum to the culture tubes.
5. Test organism: Add 0.5 ml to each tube.
6. Incubate tubes in waterbath at 50 2C for 1 hour.
7. Remove from the waterbath. Avoid excessive shaking when the tubes
are in the waterbath or when removing them from the waterbath
prior to reading the reactions.
8. Read for flocculation (agglutination).
Tube Test Results

Compare results with the flocculation (agglutination) patterns for the
Spicer-Edwards schemae. (Table 5).
The Difco Manual
671
Salmonella Antisera
11. There may exist common antigens between various O serogroups

of Salmonella. As an example, Salmonella O Antiserum Poly A
contains, among others, agglutinins for factor 1, since cultures
possessing factor 1 were used in immunization. It may be expected
that this polyvalent antiserum will react with cultures other than
those contained in O serogroups A, B, D, E, and L due to the
common 1 antigen (those organisms in Group G1, G2, H, R, T, etc.,
which contain factor 1).
12. Salmonella O Antiserum Poly A-I & Vi has been prepared with
representative members of those somatic groups and has not been
absorbed. It is obvious that this serum may and will react with
higher O groups of Salmonella.
References
1. Holt, J. G., N. R. Krieg, P. H. Sneath, J. T. Staley, and S. T.
3. Popoff, M. Y., and L. LeMinor. 1997. Antigenic Formulas of the
Salmonella Serovars. WHO Collaborating Centre for Reference
and Research on Salmonella. Institut Pasteur, Paris, France.
5. LeMinor, L., and M. Y. Popoff. 1987. Request for an opinion.
37:465-468.
6. Wayne, L. G. 1991. Judicial Commission of the International Committee on Systematic Bacteriology. Int. J. Syst. Bacteriol. 41:185-187.
8. Ewing, W.H. 1986. Edwards and Ewings Identification of
Enterobacteriaceae, 4th ed. Elisevier Science Publishing Co., Inc.,
New York, NY.
37:361-363.
10. Farmer III, J. J., III, A. C. McWhorter, D. J. Brenner, and G.
D. Morris. 1984. The Salmonella-Arizona group of Enterobacteriaceae: nomenclature, classification and reporting. Clin.
Microbiol. Newsl. 6:63-66.
12. Isenberg, H. D. (ed.) 1992. Clinical microbiology procedures
Washington, D.C.
Gaithersburg, MD.
672
Section V

Salmonella. In C. Vanderzant, C., and Splittstoesser, D.F. (eds.),
Packaging
Salmonella H Antiserum a
Salmonella H Antiserum b
Salmonella H Antiserum c
Salmonella H Antiserum d
Salmonella H Antiserum eh
Salmonella H Antiserum f
Salmonella H Antiserum h
Salmonella H Antiserum I
Salmonella H Antiserum k
Salmonella H Antiserum m
Salmonella H Antiserum p
Salmonella H Antiserum r
Salmonella H Antiserum s
Salmonella H Antiserum t
Salmonella H Antiserum w
Salmonella H Antiserum x
Salmonella H Antiserum y
Salmonella H Antiserum z
Salmonella H Antiserum EN Complex
Salmonella H Antiserum G Complex
Salmonella H Antiserum L Complex
Salmonella H Antiserum Z4 Complex
Salmonella H Antiserum Poly a-z
Salmonella H Antiserum
Poly A (a,b,c,d,i,z10,z29)
Salmonella H Antiserum Poly B
(eh,en,enx,enz15, and G Complex)
Poly C (k,l,r,y,z1,z4)
Poly D (z35,z36,z37,z38,z39,z41,z42)
Poly E (1 Complex, z6)
Single Factor 2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
2820-47
2821-47
2822-47
2823-47
2273-47
2544-47
2545-47
2824-47
2274-47
2546-47
2548-47
2275-47
2550-47
2551-47
2554-47
2555-47
2276-47
2277-47
2473-47
2279-47
2556-47
2557-47
2558-47
2561-47
2280-47
2562-47
2270-47
2269-47
2271-47
2278-47
2406-47
2539-47
3 ml
2540-47
3 ml
2541-47
3 ml
2542-47
3 ml
2543-47
3 ml
2474-47
The Difco Manual
Section V
Single Factor 5
Single Factor 6
Single Factor 7
Spicer-Edwards 1
Spicer-Edwards 2
Spicer-Edwards 3
Spicer-Edwards 4
Salmonella H Antiserum 1 Complex
Salmonella O Antiserum Factors 4,5
Salmonella O Antiserum Group A
Factors 1,2,12
Salmonella O Antiserum Group B
Factors 1,4,5,12
Factors 1,4,12,27
Salmonella O Antiserum Group C1
Factors 6,7
Factors 6,8
Factors (8), 20
Salmonella O Antiserum Group D1
Factors 1,9,12
Factors (9),46
The Difco Manual
Salmonella Antisera
3 ml
2475-47
3 ml
2476-47
3 ml
2477-47
3 ml
2265-47
3 ml
2266-47
3 ml
2267-47
3 ml
2268-47
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
ml
2272-47
2814-47
2659-47
2815-47
2660-47
2816-47
2817-47
2818-47
2257-47
2779-47
2661-47
2258-47
2259-47
2662-47
2663-47
2664-47
2666-47
2667-47
2512-47
2947-47
3 ml
2948-47
3 ml
2973-47
3 ml
2949-47
3 ml
2950-47
3 ml
3016-47
3 ml
2951-47
3 ml
3017-47
Salmonella O Antiserum Group E

Factors 1,3,10,15,19,34
Salmonella O Antiserum Group E1
Factors 3,10
Factors 3,15
Factors (3),(15),34
3 ml
2819-47
3 ml
2952-47
3 ml
2954-47
3 ml
3018-47

Factors 1,3,19
Salmonella O Antiserum Group F
Factor 11
Salmonella O Antiserum Group G
Factors 13,22,23,(36),(37)
3 ml
3019-47
3 ml
2260-47
3 ml
3029-47
Salmonella O Antiserum Group G1

Factors 13,22,(36)
Factors 1,13,23,(37)
Salmonella O Antiserum Group H
Factors 1,6,14,24,25,47
Salmonella O Antiserum Group I
Factor 16
Salmonella O Antiserum Group J
Factor 17
3 ml
2261-47
3 ml
3020-47
3 ml
2262-47
3 ml
2263-47
3 ml
2517-47
Salmonella O Antiserum Group K

Factor 18
Salmonella O Antiserum Group L
Factor 21
Salmonella O Antiserum Group M
Factor 28
3 ml
2518-47
3 ml
2519-47
3 ml
2520-47
3 ml
2521-47
3 ml
2522-47
3 ml
3 ml
2264-47
2534-47
3 ml
2535-47
3 ml
2536-47
3 ml
2537-47
3 ml
2538-47
3 ml
2645-47
3 ml
2646-47
3 ml
2827-47
Salmonella O Antiserum Group N

Factor 30
Salmonella O Antiserum Group O
Factor 35
Salmonella O Antiserum Poly A-I & Vi
Salmonella O Antiserum Poly A
(A,B,D,E1,E2,E3,E4,& L)
Salmonella O Antiserum Poly B
(C1,C2,F,G, & H)
Salmonella O Antiserum Poly C
(I,J,K,M,N,& O)
Salmonella O Antiserum Poly D
(P,Q,R,S,T,& U)
Salmonella O Antiserum Poly E
(V,W,X,Y, & Z)
Salmonella O Antiserum Poly F
(Groups 51-55)
Salmonella O Antiserum Poly G
(Groups 56-61)
Salmonella O Antiserum Vi
673
Salmonella, Antigenic Scheme
Section V

Update of the Kauffmann-White Scheme
The Centers for Disease Control1 has modified the Kauffmann-White2
Antigenic Scheme originally proposed by Ewing.3 The updated scheme
is used with Difco Salmonella Antisera as an aid in the serological
identification of Salmonella.
All of the Salmonella serovars belong to two species: S. bongori
containing 18 serovars and S. enterica containing the remaining 2300+
serovars divided among 6 subspecies.1,4
The six subspecies of S. enterica are:
The legitimate species name for the above subspecies is S. choleraesuis.
the species name S. enterica.5 LeMinor and Popoff6 published a
request for the use of S. enterica as a species name to the Judicial
Commission. The Judicial Commission ruled that S. choleraesuis is
the legitimate name.7,8 S. enterica is used in many countries and is
favorably accepted as the species name.3,9 The Centers for Disease
Control has adopted this designation until the problem of naming this
species is resolved.1
Nomenclature and classification of these bacteria are ever changing.13
Salmonella and the former Arizona should be considered a single
genus - Salmonella.9 It is recommended that laboratories report names
of Salmonella serovars for the subspecies enterica. These serovar
names are no longer italicized.1,4 The first letter of the serovar name
begins with a capital letter. For example, the strain that used to be
identified as Salmonella typhimurium is now known as Salmonella
Typhimurium.
Serovars of other subspecies of S. enterica (except some in subspecies
salamae and houtenae) and those of S. bongori are not named, and are
designated by their antigenic formula. For the most recent information
on nomenclature, consult appropriate references.1,4,8,10-14
The O antigen groups were first designated by letters of the alphabet.
Additional antigens were later delineated. Since each letter of the
alphabet was already used to describe an O antigen group, numbers
51 to 67 were used to describe the next O antigen groups.
674
O ANTIGEN GROUP
O ANTIGEN FACTORS PRESENT
A
B
C1
C2
C3
D1
D2
D3
E1
E2
E3
E4
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
V
W
X
Y
Z
51
52
53
54
55
56
57
58
59
60
61
62
63
65
66
67
1,2,12
4,12; 1,4,5,12; or 1,4,12,27
6,7,[Vi] or 6,7,14
6,8
8; or 8,20
1,9,12
9,46
1,9,12,46,27
3,10
3,15
3,15,34
1,3,19
11
13,22 or 13,23
6,14; 6,14,24; or 1,6,14,25
16
17
18
21
28
30
35
38
30
40
41
42
43
44
45
47
48
50
51
52
53
54
55
56
57
58
59
60
61
62
63
65
66
67
The Difco Manual
Section V
When writing an antigenic formula, list the O antigen(s) first followed

by the H antigen(s). Separate the major antigens by colons and the
components of the antigens by commas. For example, the antigenic
formula for Salmonella Typhimurium is Salmonella 1,4,5,12:i:1,2. This
means that the strain has O antigen factors 1,4,5 and 12, the flagella
phase 1 antigen i, and flagellar phase 2 antigens 1 and 2.
biochemical characterization and serotyping. Any serological
results obtained before biochemical identification must be considered
as presumptive identification only. Refer to appropriate references for
complete identification of Salmonella.1,3,4,10,12-15
The Kauffmann-White Scheme is presented in two forms. Appendix A
contains a list of Salmonella Serotypes by O Group. Appendix B
contains an alphabetical list of Salmonella Serotypes.
References
1. McWhorter-Murlin, A. C. and F. W. Hickman-Brenner. 1994.
Identification and Serotyping of Salmonella and an update of the
2. Kauffmann, F. 1969. Enterobacteriaceae, 2nd ed. Munksgaard,
Copenhagen.
New York, NY.
4. Popoff, M. Y. and L. LeMinor. 1997. Antigenic Formulas of the
Salmonella Serovars. WHO Collaborating Centre for Reference
and Research on Salmonella. Institut Pasteur, Paris, France.
6. LeMinor, L. and M. Y. Popoff. 1987. Request for an opinion.
37:465-468.
7. Wayne, L. G. 1991. Judicial Commission of the International
Committee on Systematic Bacteriology. Int. J. Syst. Bacteriol.
41:185-187.
37:361-363.
11. Farmer, J. J., III, A. C. McWhorter, D. J. Brenner, and
G. D. Morris. 1984. The Salmonella-Arizona group of
Enterobacteriaceae: nomenclature, classification and reporting.
Clin. Microbiol. Newsl. 6:63-66.
12. Isenberg, H. D. (ed.) 1992. Clinical microbiology procedures handbook, vol. 2. American Society for Microbiology, Washington, D.C.
13. Holt, J. G., N. R. Krieg, P. H. Sneath, J. T. Staley, S. T.
The Difco Manual

bacteriological analytical manual, 8th edition. AOAC International,
Gaithersburg, MD.
Salmonella. In Vanderzant, C. and D. F. Splittstoesser, (eds.),
16. Popoff, M, Y., and L. LeMinor. 1992. Antigenic formulas of
the Salmonella serovars, 6 th revision. WHO Collaborating
Centre for Reference and Research on Salmonella. Pasteur
Institute, Paris, France.
17. Rohde, R. 1979. Serological integration of all known Arizona
species into the Kauffmann-White scheme. Zentralbl. Bakteriol.
Hyg, I. Abt. Orig. A. 243:148-176.
18. LeMinor, L. 1984. Genus III. Salmonella. In N. R. Krieg, (ed.),
Bergeys manual of systematic bacteriology, vol. 1. The Williams
and Wilkins Co., Baltimore, MD.
Appendix A
Kauffmann-White Scheme
List of Salmonella Serotypes by O Group
(Updated 1994)
Appendix A contains a list of Salmonella Serotypes by O Group. The
serotypes are sorted by O group first, then by Phase I and Phase 2 of
the H antigens. The z antigens do not appear in the correct numerical
order - z4 will be listed after z10 because the 1 of 10 is read first, and
will appear after z29.
Key:
IP Institut Pasteur. See reference #16 in
Kauffmann-White Scheme monograph published
by WHO Collaborating Centre for Reference and
Research on Salmonella.
Ar. Arizona antigenic formula
Rohde Refer to reference #17 in Kauffmann-White
Scheme monograph by R. Rohde. He
incorporated all known Arizona serotypes
into the Kauffmann-White Scheme.
Bergey Refer to reference #18 in Kauffmann-White
Scheme monograph by L. LeMinor for
Bergeys Manual of Systematic Bacteriology.
Underlined
Numbers Numbers that are underlined in a serotype
represent somatic factors determined by phage
conversion. They are present if the culture is
lysogenized by the corresponding converting
phage.
[ ] O or H factors may be present or absent
without relation to phage conversion.
( ) O or H factor is weakly agglutinable.
R phases Abnormal specificities of H antigens that were
described by Kauffmann. They are uncommon.
675

SUBSPECIES O ANTIGEN GROUP
676
Section V
SEROTYPE
Paratyphi A
Nitra
Kiel
Koessen
O ANTIGENS
PHASE 1
PHASE 2
1,2,12
2,12
1,2,12
2,12
4,12
4,12
1,4,[5],12
4,[5],12
4,12,27
4,[5],12
1,4,[5],12
1,4,[5],12,27
1,4,[5],12,27
1,4,5,27
1,4,12
1,4,12,27
1,4,12,27
4,12,27
1,4,5,12
a
g,m
g,p
l,v
a
a
a
a
a
a
a
a
a
a
a
b
b
b
b
b
b
b
[1,5]
1,5
1,6
e,n,x
1,2
1,5
1,5
[1,7]
e,n,x
e,n,x
[e,n,x]
e,n,z15
l,w
z39
z6
[1,2],
(tartrate +)
1,5
1,5
1,6
1,7
e,n,x
I
I
I
I
II
I
I
I
I
I
I
II
II
I
I
II
I
I
I
A
A
A
A
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
I
II
I
I
I
B
B
B
B
B
Limete
Canada
Uppsala
Abony
1,4,12,27
4,12
4,12,27
4,12,27
1,4,5,12
Abortusbovis
1,4,12,27
e,n,x
Sladun
1,4,12,27
e,n,x
II
I
I
I
B
B
B
B
Sofia
Wagenia
Wien
Abortuscanis
1,4,12,27
1,4,12,27
1,4,12,27
4,5,12
b
b
b
b
[e,n,x]
e,n,z15
l,w
Rz5
I
I
B
B
Tripoli
Paratyphi B
1,4,12,27
1,4,[5],12
b
[b]
z6
[1,2]
I
I
I
B
B
B
Legon
Abortusovis
Altendorf
1,4,12,27
4,12
4,12
c
c
c
1,5
1,6
1,7
Womba
4,12,27
1,7
Abortusequi
Kisangani
Fulica
Hessarek
Arechavaleta
Bispejerg
Makoma
Tinda
Huettwilen
Nakuru
Schleissheim
Java
NOTE
IP calls Java, Paratyphi B var. Java. Java is often

monophasic in the U.S. May possess H phase Rz33.
IP combined Sladun (1,4,12,27:b:e,n,x) with

Abony to form Abony 1,4,[5],12,27:b:e,n,x
(gelatin neg.).
Gelatin pos., mucate pos.1-4 days. Abortusbovis was
combined with Abony (1,4,5,12:b:e,n,x), gelatin
neg. The name Abortusbovis was dropped.
IP combined Sladun with Abony (1,3,4,12:
b:e,n,x) to form Abony 1,4,[5],12,27:b:e,n,x.
Sladun is now called Abony var. O 27+.
The name Sladun has been dropped.
Abortuscanis was combined with Paratyphi B

(1,4,[5],12:b:1,2) in 1938 and the name
Abortuscanis was dropped.
Paratyphi B is tartrate neg.; Paratyphi B var.
Java (CDC calls this S. ser. Java) is often
monophasic (1,4,5,12:b:-) and is tartrate pos.
Paratyphi B and Java may possess H phase Rz33.
IP combined Womba (4,12,27:c:1,7) with

Altendorf to form Altendorf 4,12,27:c:1,7.
IP combined Womba with Altendorf (4,12:c:1,7)
to form Altendorf 4,12,27:c:1,7. Womba is
now called Altendorf var. 027+. The name
Womba has been dropped.
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
I
I
I
I
I
B
B
B
B
B
Bissau
Jericho
Hallfold
Bury
Cairo
4,12
1,4,12,27
1,4,12,27
4,12,27
1,4,12,27
c
c
c
c
d
e,n,x
e,n,z15
l,w
z6
1,2
Stanley
4,5,12
1,2
I
I
I
I
II
I
I
I
I
B
B
B
B
B
B
B
B
B
Eppendorf
1,4,12,27
Brezany
1,4,12,27
Schwarzengrund 1,4,12,27
Sarajane
1,4,[5],12,27
Kluetjenfelde
4,12
Duisburg
1,4,12,27
Mons
1,4,12,27
Ayinde
1,4,12,27
Salinatis
4,12
d
d
d
d
d
d
d
d
d,e,h
1,5
1,6
1,7
e,n,x
e,n,x
e,n,z15
l,w
z6
d,e,n,z15
I
I
I
I
I
I
II
II
II
I
I
II
I
I
I
II
II
II
II
I
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
Saintpaul
Reading
Eko
Kaapstad
Chester
Sandiego
Caledon
Bechuana
Joenkoeping
1,4,[5],12
1,4,[5],12
4,12
4,12
1,4,[5],12
4,[5],12
4,12
1,4,12,27
4,12
1,4,[5],12
1,4,[5],12
4,[5],12
4,12
4,[5],12
4,12
4,12
4,12
1,4,12,27
1,4,12,27
4,5,12
e,h
e,h
e,h
e,h
e,h
[e,h]
e,n,x
e,n,x
(f),g
f,g
f,g,s
f,g,t
g,m
g,m,s
g,m,t
g,m,t
g,m,t
g,[m],[s],t
g,[m],t
g,s,t
1,2
[1,5]
1,6
1,7
e,n,x
e,n,z15
1,2,7
1,[5],7
[1,2]
[1,2]
z6,z42
z39
e,n,x
[1,5]
Kingston
1,4,12,27
g,s,t
[1,2]
I
I
I
II
B
B
B
B
Budapest
Travis
Tennyson
1,4,12,27
4,[5],12
4,5,12
4,12
g,t
g,z51
g,z51
g,z62
1,7
e,n,z15
The Difco Manual
Makumira
Derby
Agona
Essen
Hato
California
NOTE
IP combined Cairo with Stanley (4,5,12:d:1,2)

to form Stanley 1,4,[5],12,27:d:1,2. The name
Cairo has been dropped.
IP combined Cairo (1,4,12,27:d:1,2) with
Stanley to form Stanley 1,4,[5],12,27:d:1,2
IP states that Salinatis was combined with

Duisburg (1,4,12,27:d:e,n,z15). This is incorrect;
IP should have stated that it was combined
with Sandiego (4,[5],12:e,h:e,n,z15), because
Salinatis loses the d and becomes Sandiego.
Not in IP book
IP calls this monophasic var. of Bechuana.
IP combined Joenkoeping with Kingston

(1,4,12,27:g,s,t:-) to form Kingston
1,4,[5],12,27:g,s,t:-. The name Joenkoeping
has been dropped.
IP combined Kingston with Joenkoeping (4,5,12:
g,s,t:-) to form Kingston 1,4,[5],12,27:g,s,t:[1,2].
Kingston may possess H phase Rz27 or Rz43.
677

678
I
I
I
I
I
I
II
I
I
I
II
I
I
I
I
I
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
I
II
I
II
I
I
I
II
I
I
I
I
I
I
I
II
I
I
I
I
I
I
I
I
I
I
I
I
I
I
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
Section V
SEROTYPE
Typhimurium
var. Copenhagen
Typhimurium
Lagos
Agama
Farsta
Tsevie
Gloucester
Tumodi
Massenya
Neumuenster
Ljubljana
Texas
Fyris
Azteca
Bredeney
Kimuenza
Brandenburg
Clackamas
Mono
Togo
Kilwa
Ayton
Haduna
Kubacha
Kano
Vom
Tyresoe
Kunduchi
Reinickendorf
Banana
Madras
Heidelberg
Bradford
Winneba
Remo
Bochum
Southampton
Africana
Drogana
Coeln
Trachau
Finaghy
O ANTIGENS
PHASE 1
PHASE 2
1,4,12
1,2
1,4,5,12
1,4,[5],12
4,12
4,12
4,12
1,4,12,27
4,12,27
1,4,12
1,4,12,27
1,4,12,27
1,4,12,27
4,12,27
4,[5],12
4,[5],12
4,[5],12,27
1,4,12,27
i
i
i
i
i
i
i
i
k
k
k
k
k
l,v
l,v
l,v
1,2,[7]
1,5
1,6
e,n,x
e,n,z15
l,w
z35
z6
1,5
1,6
1,6
e,n,x
e,n,z15
1,2
1,5
1,7
1,4,12,27
l,v
1,4,12,27
l,v
1,4,[5],12,27
l,v
1,4,12,27
l,v
4,12
l,v,[z13]
4,12
l,w
4,12
l,w
4,12
l,w
1,4,12,27
l,w
4,12
l,z13,[z28]
1,4,12,27
l,z13,z28
1,4,12,27
l,z13,z28
1,4,12,27
l,[z13],[z28]
4,12
l,[z13],z28
1,4,[5],12,27 l,[z13],[z28]
4,12
l,z28
4,12
l,z28
1,4,[5],12
m,t
4,[5],12
m,t
1,4,[5],12
r
4,12,27
r
4,12
r
1,4,12,27
r
4,[5],12
r
1,4,12,27
r
4,12
r,i
1,4,12,27
r,(i)
4,[5],12
y
4,12,27
y
4,12
y
e,n,x
e,n,x
e,n,z15
z39
1,6
1,5
1,6
e,n,x
z6
1,6
1,7
e,n,x
e,n,z15
1,5
[1,2]
e,n,x
[1,5]
e,n,z15
1,2
1,5
1,6
1,7
l,w
z6
l,w
e,n,z15
1,2
1,5
1,6
NOTE
Bredeney may possess H phase Rl,z40

instead of l,v.
IP does not get z13
Banana may possess H phase Rz45.
IP calls Drogana r,i.
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
1,4,12,27
1,4,12,27
y
y
1,7
e,n,x
I
I
B
B
Teddington
Ball
Ruki
4,5,12
e,n,x
Dalat
4,5,27
e,n,x
I
I
I
I
II
I
I
II
I
II
I
I
II
I
I
I
I
I
I
I
I
I
I
II
I
I
I
I
I
I
II
I
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
B
Jos
Kamoru
Shubra
Kiambu
Entebbe
Haifa
Ituri
Tudu
Albert
Tokoin
Mura
Vellore
Fortune
Brancaster
Helsinki
Pasing
Tafo
Yaounde
Sloterdijk
Tejas
Wilhelmsburg
Durbanville
Jaja
1,4,12,27
4,12,27
4,[5],12
4,12
1,4,12,27
4,12
1,4,12
4,12
4,12
1,4,12,27
1,4,[5],12
1,4,12
4,12
1,4,12,27
1,4,[5],12
1,4,12
4,12
4,12
4,12
1,4,12
1,4,12,27
1,4,12,27
1,4,12,27
1,4,12
4,12
1,4,12,27
1,4,12,27
1,4,12,27
4,12
1,4,[5],12,27
1,4,12,27
4,12,27
y
y
z
z
z
z
z
z
z
z
z
z
z
z
z10
z10
z10
z10
z10
z10
z10
z10
z29
z29
z35
z35
z35
z35
z36
z38
[z39]
z4,z23
e,n,z15
z6
1,2
1,5
1,5
1,6
1,7
1,7
e,n,x
e,n,x
e,n,z15
l,w
z39
z6
1,2
1,5
1,6
e,n,x
e,n,z15
l,w
z35
z6
[e,n,x]
1,5
1,7
e,n,z15
z6
[e,n,z15]
1,[5],7
Stanleyville
1,4,[5],12
z4,z23
[1,2]
I
I
I
B
B
B
Vuadens
Kalamu
Thayngen
4,12,27
4,[5],12
1,4,12,27
z4,z23
z4,z24
z41
z6
[1,5]
1,(2),5
The Difco Manual
Loubomo
Indiana
Neftenbach
Nordenham
Koenigstuhl
Preston
NOTE
IP combined Ball with Ruki (4,5,12:y:e,n,x)

and Dalat (4,5,27:y:e,n,x) to form Ball
1,4,[5],12,27:y:e,n,x.
IP combined Ruki with Ball (1,4,12,27:y:e,n,x)
and Dalat (4,5,27:y:e,n,x) to form Ball 1,4,[5],
12,27:y:e,n,x. The name Ruki has been dropped.
Dalat was combined with Ball. The name
Dalat has been dropped.
IP combined Jaja with Stanleyville

(1,4,[5],12:z4,z23:[1,5]) to form Stanleyville
1,4,[5],12,27:z4,z23:[1,5]. Jaja is now called
Stanleyville var. O 27+. The name Jaja has
been dropped.
IP combined Jaja (4,12,27:z4,z23:-) with
Stanleyville to form Stanleyville
1,4,[5],12,27:z4,z23:[1,2].
679

680
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
1,4,12,27
6,7
6,7
6,7,14
6,7
6,7
6,7,14
6,7
6,7,14
6,7
6,7
6,7
6,7,14
z41
a
a
a
a
a
a
a
a
a
a
b
e,n,z15
1,6
1,5
1,5
1,6
1,7
e,n,x
e,n,z15
l,w
z42
z6
z6
I
II
I
II
I
I
I
I
I
II
I
II
I
B
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
Maska
I
I
I
I
I
II
I
I
C1
C1
C1
C1
C1
C1
C1
C1
Brazzaville
Edinburg
Adime
Koumra
Lockleaze
Bloemfontein
Georgia
Ohio
6,7
6,7
6,7
6,7
6,7,14
6,7
6,7
6,7
b
b
b
b
b
b
b
b
1,2
1,5
1,6
1,7
e,n,x
[e,n,x]:z42
e,n,z15
l,w
C1
Nienstedten
6,7,14
[l,w]
I
II
I
I
I
I
C1
C1
C1
C1
C1
C1
Kotte
Leopoldville
Hissar
Choleraesuis
Decatur
6,7
6,7
6,7,14
6,7,14
6,7
6,7
b
b
b
c
c
c
z35
z39
z6
1,2
1,5
1,5
I
I
C1
C1
Paratyphi C
Typhisuis
6,7,[Vi]
6,7
c
c
1,5
1,5
I
I
I
I
I
I
I
I
I
C1
C1
C1
C1
C1
C1
C1
C1
C1
Choleraesuis var. Kunzendorf

Birkenhead
6,7
Schwabach
6,7
Namibia
6,7
Kaduna
6,7,14
Kisii
6,7
Kambole
6,7
Isangi
6,7,14
Mission
6,7
6,7
c
c
c
c
d
d
d
d
c
1,6
1,7
e,n,x
e,n,z15
1,2
1,[2],7
1,5
1,5
C1
Kivu
1,6
Sanjuan
Umhlali
Austin
Oslo
Denver
Coleypark
Calvinia
Damman
Nissii
6,7
NOTE
Nissii was combined with Nienstedten

(6,7,14:b:l,w) as a monophasic variant of
Nienstedten. Nienstedten is now called a variant
of Ohio by IP. The name Nissii has been dropped.
IP combined Nienstedten (6,7,14:b:[l,w]) with

Ohio to form Ohio 6,7,14:b:[l,w}. Ohio may
possess H phase Rz59.
Nienstedten was combined with Nissii (6,7,14:b:-)
and called Nienstedten; then IP combined
Nienstedten with Ohio (6,7:b:l,w) to form
Ohio (6,7,14:b:[l,w]). Nienstedten is now
called Ohio var. O 14+ by IP.
H2S negative
IP has dropped Decatur and calls it dulcitol
positive, mucate positive variant of Choleraesuis.
Typhisuis is a bioserotype found in pigs. It is
like Choleraesuis except tartrate negative.
[1,5] H2S positive
Mission was combined with Isangi 6,7,14:d:1,5.

The name Mission has been dropped.
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
C1
Amersfoort
6,7
e,n,x
C1
Omderman
6,7,14
e,n,x
I
I
C1
C1
Gombe
Eimsbuettel
6,7,14
6,7,14
d
d
e,n,z15
l,w
C1
Livingstone
6,7
l,w
I
II
I
I
I
I
I
I
I
C1
C1
C1
C1
C1
C1
C1
C1
C1
Wil
6,7
6,7
6,7,14
6,7
6,7
6,7
6,7
6,7,14
6,7,14
d
d
d
e,h
e,h
e,h
e,h
e,h
f,g
l,z13,z28
z42
z6
1,2
1,5
1,6
1,7
e,n,z15
C1
Rissen
6,7
f,g
I
I
II
I
II
I
II
I
I
II
IV
I
I
I
I
I
I
I
I
I
I
I
I
I
I
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
Eingedi
Afula
6,7
6,7
6,7
6,7,14
6,7
6,7,14
6,7
6,7
6,7
6,7
6,7
6,7
6,7,14
6,7
6,7
6,7
6,7
6,7
6,7,14
6,7,14
6,7
6,7
6,7
6,7
6,7
f,g,t
f,g,t
(g),m,[s],t
g,m,[p],s
g,m,[s],t
g,m,[t]
g,[m],s,t
g,s,[t]
g,t
g,t
g,z51
g,z51
i
i
i
i
i
i
i
k
k
k
k
k
k
1,2,7
e,n,x
[1,5]
[1,2,7]
e,n,x
[z42]
[1,6]
e,n,x:z42
1,5
1,2
1,5
1,6
1,7
e,n,z15
l,w
z6
1,2
1,6
1,7
e,n,x
e,n,z15
R1,10
The Difco Manual
Nieukerk
Larochelle
Lomita
Norwich
Nola
Braenderup
Ardwick
Montevideo
Othmarschen
Menston
Riggil
Alamo
Augustenborg
Oritamerin
Garoli
Lika
Athinai
Norton
Stuttgart
Galiema
Daytona
Baiboukoum
Singapore
Escanaba
Cardiff
NOTE
IP combined Omderman (6,7,14:d:e,n,x) with

Amersfoort to form Amersfoort 6,7,14:d:e,n,x.
IP combined Omderman with Amersfoort
(6,7:d:e,n,x) to form Amersfoort 6,7,14:d:e,n,x.
Omderman is now called Amersfoort
var. O 14+ by IP.
IP combined Eimsbuettel with Livingstone
(6,7:d:l,w) to form Livingstone 6,7,14:d:l,w.
Eimbuettel is now called Livingstone
var. O 14+ by IP.
IP combined Eimsbuettel (6,7,14:d:l,w) with
Livingstone to form Livingstone 6,7,14:d:l,w.
IP combined Ardwick with Rissen (6,7:f,g:-)

to form Rissen 6,7,14:f,g:-. Ardwick is now
called Rissen var. O 14+ by IP.
IP combined Ardwick (6,7,14:f,g:-) with
Rissen for form Rissen 6,7,14:f,g:-.
IP combined Cardiff that contains H phase

R1,10 with Thompson (6,7,14:k:1,5).
681

Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
6,7
6,7
6,7,14
k
(k)
[k]
[z6]
z:[z55]
[1,5]
l,v
l,v
l,v
l,v
l,v
l,v
l,v
l,v
l,v
1,2
1,5
1,6
1,7
e,n,x
e,n,z15
z35
z53
z6
II
IIIa
I
C1
C1
C1
I
I
I
I
I
I
I
IIIb
I
C1
C1
C1
C1
C1
C1
C1
C1
C1
Concord
Irumu
Mkamba
Kortrijk
Bonn
Potsdam
Coromandel
Gdansk
6,7
6,7
6,7
6,7
6,7
6,7,14
6,7
6,7
6,7
C1
Gelsenkirchen
6,7,14
l,v
z6
I
I
II
II
I
I
I
I
I
II
II
II
I
I
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
Gabon
Colorado
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
l,w
l,w
l,w
l,w
l,z13
l,z13
l,z13,z28
l,z13,z28
l,z13,[z28]
l,z28
l,z28
l,z28
m,p,t,[u]
m,t
1,2
1,5
1,5,7
z42
1,5
e,n,x
1,7
z6
e,n,z15
1,5:[z42]
e,n,x
z6
C1
Thielallee
6,7,14
m,t
II
I
I
I
I
I
I
I
I
I
I
I
I
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
Winston
Oakey
Virchow
Infantis
Nigeria
Colindale
Papuana
Grampian
Richmond
Bareilly
Oyonnax
Gatow
6,7
6,7
6,7
6,7
6,7,14
6,7
6,7
6,7
6,7
6,7
6,7,14
6,7
6,7
m,t
m,t
m,t
r
r
r
r
r
r
y
y
y
y
1,6
z64
1,2
1,5
1,6
1,7
e,n,z15
l,w
1,2
1,5
1,6
1,7
682
Thompson
Nessziona
Kenya
Strathcona
Makiso
Neukoelin
Heilbron
Haelsingborg
Oranienburg
NOTE
(Ar. 27:22:31:[37])
R1,10 (6,7:k:R1,10) with Thompson.
(Ar. 27:23:25)
IP combined Gelsenkirchen (6,7,14:l,v:z6)
with Gdansk to form Gdansk 6,7,14:l,v:z6.
IP combined Gelsenkirchen with Gdansk
(6,7:l,v:z6) to form Gdansk 6,7,14:l,v:z6.
Gelsenkirchen is now called Gdansk
var. O 14+ by IP.
IP combined Theilallee (6,7,14:m,t:-) with

Oranienburg to form Oranienburg 6,7,14:m,t:-.
Oranienburg may possess H phase Rz57.
IP combined Thielallee with Oranienburg
(6,7:m,t:-) to form Oranienburg 6,7,14:m,t:-.
Theilallee is now called Oranienburg
var. O 14+ by IP.
Infantis may possess H phase Rz49.
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
NOTE
6,7
6,7,14
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7,14
6,7,14
6,7
6,7
6,7
6,7
6,7,14
6,7
6,7
6,7
6,7
6,7
6,7,14
y
y
z
z
z
z
z
z
z
z
z
z
z10
z10
z10
z10
z10
z10
z10
z10
z10
z10
z29
z29
z35
z35
z36
z36
z36
z38
e,n,x
e,n,z15
1,2
1,5
1,5
1,6,[7]
e,n,x
e,n,z15
l,w
z39
z42
z6
1,2
1,5
1,6
1,7
e,n,x
e,n,z15
l,w
z35
z35
z6
[1,2,7]
1,6
e,n,z15
e,n,z15
z42
Hartford may possess H phase Rz50 or Rz67.

Mikawasima may possess H phase Rz47 or Rz50.
I
I
I
I
II
I
I
I
I
II
II
II
I
I
I
I
I
I
I
I
II
I
II
I
I
I
IV
I
II
I
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
Hartford
Mikawasima
Chile
Poitiers
Tosamanga
Oakland
Cayar
Businga
Bruck
C1
Lille
6,7
z38
I
II
IV
I
I
I
I
I
IV
II
VI
I
I
II
II
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
Rumford
Gilbert
Roterberg
Obogu
Planckendael
Goma
Aequatoria
Somone
Kralendyk
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
z38
z39
z4,z23
z4,z23
z4,z23
z4,z23
z4,z23
z4,z24
z4,z24
z4,z24
z41
z41
z41
z42
z42
1,2
1,5,7
1,5
1,6
z6
[e,n,z15]
z42
1,7
l,w
z35
1,7
e,n,x:1,6
The Difco Manual
Oysterbeds
Menden
Inganda
Eschweiler
Ngili
Djugu
Mbandaka
Jerusalem
Omuna
Redba
Tennessee
Tienba
Palime
Argentina
Tampico
Bacongo
Bornum
Hillsborough
Tamilnadu
Sullivan
IP combined Bornum with Lille (6,7:z38:-) to

form Lille 6,7,14:z38:-. Bornum is now called
Lille var. O 14+ by IP.
IP combined Lille with Bornum (6,7,14:z38:-)
to form Lille 6,7,14:z38:-.
683

684
I
II
I
C1
C1
C2
II
I
I
I
I
I
II
I
II
II
I
I
I
I
I
II
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
I
I
I
I
I
I
I
I
C2
C2
C2
C2
C2
C2
C2
C2
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
6,7
6,7
6,8
z44
z6
1,7
1,2
Stourbridge
Eboko
Sanga
Gatuni
Konstanz
Presov
Shipley
Bukuru
Banalia
Tounouma
Wingrove
Utah
Bronx
Belfast
Belem
Santiago
Quiniela
Alexanderpolder
Tado
Mexicana
6,8
6,8
6,8
6,8
6,8
6,8
6,8
6,8
6,8
6,8
8,20
8
6,8
8,20
6,8
6,8
6,8
6,8
8
6,8
8
6,8
8,20
6,8
6,8
8,20
6,8
6,8
6,8
6,8
6,8
8,20
6,8
8
8,20
6,8
a
a
a
a
a
a
a
a
a
a
b
b
b
b
b
b
b
b
b
b
b
b
b
b
b
c
c
c
c
c
c
c
c
c
d
1,5,7
1,2
1,5
1,6
1,7
e,n,x
e,n,x
e,n,z15
z39
z52
[z6]
1,2
1,2
1,5
1,5
1,5
1,6
1,7
1,7
e,n,x
e,n,x
e,n,z15
e,n,z15
l,w
z6
z6
1,2
1,5
1,6
1,7
e,n,x
e,n,x
e,n,z15
l,w
z6
1,2
Muenchen
Virginia
Manhattan
Yovokome
Dunkwa
Portanigra
Sterrenbos
Herston
6,8
8
6,8
8,20
6,8
8,20
6,8
6,8
d
d
d
d
d
d
d
d
1,2
[1,2]
1,5
1,5
1,7
1,7
e,n,x
e,n,z15
Bulovka
Cape
Newport var.
Puerto Rico
Valdosta
Doncaster
Curacao
Nordufer
Narashino
Leith
Tulear
Be
Djelfa
Skansen
Korbol
Nagoya
NOTE
Mexicana was combined with Muenchen.

The name Mexicana has been dropped.
The Difco Manual
Section V
SEROTYPE
I
II
I
I
C2
C2
C2
C2
Labadi
I
I
I
I
I
I
I
II
I
I
I
II
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
II
I
I
I
II
I
II
I
I
I
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
Ferruch
Kottbus
Cremieu
Atakpame
Tshiongwe
Rechovot
Sandow
The Difco Manual
Bardo
Newport
Emek
Chincol
Reubeuss
Alminko
Nanergou
Lindenburg
Bargny
Takoradi
Warnow
Malmoe
Bonariensis
Aba
Cyprus
Magherafelt
Kentucky
Kallo
Blockley
Haardt
Schwerin
Charlottenburg
Litchfield
Pakistan
Loanda
Manchester
Holcomb
Edmonton
Amherstiana
Fayed
Hiduddify
Breukelen
Bassa
Yokoe
O ANTIGENS
PHASE 1
PHASE 2
8,20
6,8
8
6,8,20
d
d
e,h
e,h
z6
z6:z42
1,2
1,2
8
6,8
6,8
8,20
6,8
8,20
6,8
6,8
8,20
6,8
8,20
6,8
8,20
6,8
6,8
8,20
6,8
6,8
6,8
6,8
6,8
6,8
8,20
8,20
6,8
6,8
8
6,8
6,8
6,8
8
6,8
6,8
6,8
6,8
6,8
8
6,8
6,8
6,8
6,8
6,8
6,8
8,20
e,h
e,h
e,h
e,h
e,h
e,h
f,g
f,g,m,t
g,m,s
g,m,[s]
g,m,t
g,m,t
g,s,t
g,s,t
i
i
i
i
i
i
i
i
i
i
k
k
k
k
k
l,v
l,v
l,v
l,v
l,v
l,v
l,v
l,(v)
l,w
l,w
l,z13,z28
l,z28
l,z13,[z28]
m,t
m,t
1,5
1,5
1,[6]
1,7
e,n,z15
z6
e,n,z15
[e,n,x]
[e,n,x]
1,7
1,2
1,5
1,5
1,6
1,7
e,n,x
e,n,z15
l,w
l,w
z6
1,2
1,5
1,5
e,n,x
e,n,z15
1,2
1,2
1,5
1,7
e,n,x
e,n,x
e,n,z15
1,6
1,2
z6:z42
1,5
e,n,x
e,n,z15
-
NOTE
Newport may possess H phase Rz50 or Rz58

or Rz78 or R1,12
Blockley may possess H phase Rz58
685

686
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
II
II
I
I
I
I
I
I
C2
C2
C2
C2
C2
C2
C2
C2
Baragwanath
Germiston
Bsilla
Hindmarsh
Akanji
Noya
Goldcoast
Pikine
6,8
6,8
6,8
8,20
6,8
8
6,8
8,20
m,t
m,t
r
r
r
r
r
r
1,5
e,n,x
1,2
1,5
1,7
1,7
l,w
z6
I
I
I
I
I
C2
C2
C2
C2
C2
Cocody
Hidalgo
Bovismorbificans
Brikama
Altona
8,20
6,8
6,8
8,20
8,20
r,i
r,i
r,[i]
r,[i]
r,[i]
e,n,z15
e,n,z15
1,5
l,w
z6
I
I
I
I
II
I
I
I
I
I
I
I
I
I
II
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
II
II
II
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
Giza
Brunei
Tananarive
Bulgaria
8,20
8,20
6,8
6,8
6,8
8
6,8
6,8
8
6,8
6,8
8,20
8,20
6,8
6,8
6,8
8
6,8
8,20
8,20
6,8
6,8
8,20
6,8
6,8
8
8,20
6,8
8,20
8,20
6,8
6,8
6,8
8
y
y
y
y
y
y
y
y
y
y
y
y
y
z
z
z
z
z
z
z10
z10
z10
z10
z10
z10
z10
z10
z10
z10
z10
z10
z29
z29
z29
1,2
1,5
1,5
1,6
1,6:z42
1,7
1,7
e,n,x
e,n,x
e,n,z15
l,w
l,w
z6
1,5
1,5
e,n,z15
e,n,z15
l,w
z6
1,2
1,2
1,5
1,5
1,7
e,n,x
e,n,x
e,n,z15
e,n,z15
l,w
z6
z6
1,5
e,n,x
e,n,x:z42
Alagbon
Inchpark
Daarle
Sunnycove
Praha
Benue
Sindelfingen
Kralingen
Mowanjum
Kalumburu
Phaliron
Kuru
Daula
Bazenheid
Zerifin
Mapo
Paris
Cleveland
Hadar
Istanbul
Chomedey
Glostrup
Remiremont
Molade
Wippra
NOTE
Pikine was combined with Altona (8,20:r,[i]:z6).

The name Pikine has been dropped.
Pikine (8,20:r:z6) was combined with Altona

and called Altona.
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
C2
D1
D1
Tamale
Uno
Kolda
Yarm
Angers
Apeyeme
Bellevue
Lezennes
Breda
Chailey
Dabou
Corvallis
Albany
Duesseldorf
Tallahassee
Diogoye
Aesch
Gallinarum
Pullorum
8,20
6,8
8,20
6,8
8,20
8,20
8
6,8
6,8
6,8
8,20
8,20
8,20
6,8
6,8
8,20
6,8
1,9,12
1,9,12
z29
z29
z35
z35
z35
z38
z4,z23
z4,z23
z4,z23
z4,z23
z4,z23
z4,z23
z4,z24
z4,z24
z4,z32
z41
z60
[e,n,z15]
[e,n,z15]
1,2
1,2
z6
1,7
1,7
e,n,x
[e,n,z15]
l,w
[z6]
z6
1,2
D1
Miami
1,9,12
1,5
D1
Sendai
1,9,12
1,5
II
I
I
I
II
I
II
II
I
I
II
II
II
I
I
I
I
I
I
I
I
I
II
I
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
9,12
9,12
1,9,12
1,9,12
1,9,12
9,12
9,12
1,9,12
1,9,12
1,9,12
1,9,12
1,9,12
1,9,12
9,12
9,12
9,12
9,12
9,12
9,12,[Vi]
1,9,12
9,12
9,12
9,12
9,12
a
a
a
a
a
a
a
a
b
b
b
b
b
c
c
c
c
c
d
d
d
d
d
d
1,5
1,6
1,7
e,n,x
e,n,x
e,n,z15
z39
z42
1,2
1,5
e,n,x
z39
z6
1,5
1,6
1,7
e,n,z15
z6
1,5
1,6
1,7
e,n,x
e,n,z15
The Difco Manual
Os
Saarbruecken
Lomalinda
Durban
Onarimon
Frintrop
Mjimwema
Suederelbe
Blankenese
Goeteborg
Ipeko
Elokate
Alabama
Ridge
Typhi
Ndolo
Tarshyne
Eschberg
Rhodesiense
Bangui
NOTE
Albany may possess H phase Rz45.
Gallinarum must be identified biochemically.

IP combined Pullorum with Gallinarum
(1,9,12:-:-). They must be identified biochemically.
Miami must be differentiated from Sendai
with biochemical tests. Miami is pos. for H2S,
citrate, and tartrate; Sendai is neg.
Sendai must be differentiated from Miami
with biochemical tests. Sendai is neg. for H2S,
citrate, and tartrate; Miami is pos.
Typhi may possess H phase Rj or Rz66.
687

688
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
d
d
d
e,h
e,h
e,h
e,h
e,n,x
e,n,x
[f],g,m,[p],[t]
[f],g,t
g,m,q
g,m,s,t
z35
z39
z6
1,2
1,5
1,7
e,n,z15
1,[5],7
1,6
[1,7]
1,5
I
II
I
I
I
I
I
II
II
I
I
I
II
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
Jaffna
Enteritidis
Berta
Blegdam
Muizenberg
1,9,12
9,12
9,12
9,12
1,9,12
9,12
9,12
9,12
9,12
1,9,12
1,9,12
9,12
9,12
II
II
D1
D1
Kuilsrivier
Manica
1,9,12
1,9,12
g,m,s,t
g,m,s,t
e,n,x
z42
II
I
I
I
I
II
II
D1
D1
D1
D1
D1
D1
D1
Dublin
Naestved
Rostock
Moscow
Neasden
Hamburg
1,9,12
1,9,12,[Vi]
1,9,12
1,9,12
9,12
9,12
1,9,12
g,m,[s],t
g,p
g,p,s
g,p,u
g,q
g,s,t
g,t
[1,5,7]:[z42]
e,n,x
I
II
I
I
I
I
I
I
I
I
II
I
I
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
Goettingen
Italiana
9,12
1,9,12
9,12
9,12
1,9,12
9,12
9,12
1,9,12
9,12
9,12
9,12
9,12
9,12
g,z51
g,z62
g,z63
i
k
k
l,v
l,v
l,v
l,v
l,v
l,v
l,v
1,5
1,5
1,5
1,6
1,2
1,5
1,7
e,n,x
e,n,x
e,n,z15
R1,11
II
I
II
I
I
I
I
D1
D1
D1
D1
D1
D1
D1
Victoria
Daressalaam
Itami
Miyazaki
Napoli
Javiana
9,12
1,9,12
1,9,12
9,12
9,12
1,9,12
1,9,12
l,v
l,w
l,w
l,z13
l,z13
l,z13
l,z28
z39
1,5
e,n,x
1,5
1,7
e,n,x
1,5
Zega
Bournemouth
Eastbourne
Westafrica
Israel
Lindrick
Newmexico
Antarctica
Seremban
Claibornei
Goverdhan
Mendoza
Panama
Kapemba
Zaiman
NOTE
IP combined Muizenberg with Hamburg

(1,9,12:g,t:-) and Manica (1,9,12:g,m,s,t:z42)
to form S. II 1,9,12:g,[m],[s],t:[1,5,7]:[z42].
IP combined Manica with Hamburg
(1,9,12:g,t:-) and Muizenberg (9,12:g,m,s,t:1,5)
to form S. II 1,9,12:g,[m],[s],t:[1,5,7]:[z42].
IP combined Hamburg with Manica

(1,9,12:g,m,s,t:z42) and Muizenberg
(9,12:g,m,s,t:1,5) to form S. II
1,9,12:g,m,[s],t:[1,5,7]:[z42].
Panama may possess H phase R1,11
IP combined Italiana that contains H phase

R1,11 with Panama (1,9,12:l,v:1,5). The
name Italiana has been dropped.
The Difco Manual
Section V
II
I
II
II
I
II
II
I
I
I
I
I
I
I
II
II
II
I
I
I
II
II
I
I
II
I
I
I
II
I
I
I
I
I
I
II
I
I
I
I
I
I
I
I
I
I
I
I
II
The Difco Manual
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
SEROTYPE
Kotu
Pensacola
Jamaica
Camberwell
Campinense
Lome
Powell
Lawndale
Kimpese
Stellenbosch
Hueningen
Angola
Portland
Ruanda
Treguier
Canastel
Penarth
Elomrane
Wynberg
Wangata
Natal
Ottawa
Franken
Baildon
Doba
Cheltenham
Zadar
Worb
Lundby
Bamboye
Kolar
Linguere
Itutaba
Ontario
Quentin
Strasbourg
Olten
Plymouth
Bergedorf
Waedenswil
Guerin
O ANTIGENS
PHASE 1
PHASE 2
9,12
9,12
9,12
9,12
1,9,12
1,9,12
1,9,12
9,12
9,12
9,12
9,12
9,12
1,9,12
9,12
1,9,12
9,12
1,9,12
9,12
9,12
1,9,12
9,12
1,9,12
9,12
1,9,12
1,9,12
1,9,12
9,12
1,9,12
1,9,12
1,9,12
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
l,z28
l,z28
l,z28
m,t
m,t
m,t
m,t
r
r
r
r
y
z
z
z
z
z
z10
z10
z10
z29
z29
z35
z38
z39
z4,z23
z4,z24
z41
z42
z60
a
a
b
b
b
b
b
b
b
c
d
d
d
d
d
e,h
e,h
e,h
e,n,x
1,5:[z42]
1,6
e,n,x
[1,2]
1,5
z39
1,5
1,7
e,n,z15
z6
1,7
1,5
1,6
1,7
z39
z6
1,5
e,n,z15
z6
[1,5]
e,n,x
z6
1,7
[1,7]
1,5
1,[5],7
z67
e,n,x
e,n,z15
1,5
1,6
e,n,x
e,n,x
l,w
z35
z6
z6
1,5
1,6
1,7
e,n,z15
z6
1,2
1,5
z6
1,5,7
NOTE
689

I
I
I
II
I
II
I
I
I
I
I
I
I
I
I
I
II
I
I
I
I
I
I
I
II
II
I
I
I
I
II
I
II
I
I
I
I
I
II
I
I
II
I
II
II
II
II
II
II
690
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D2
D3
D3
D3
D3
D3
D3
Section V
SEROTYPE
Wernigerode
Hillingdon
Macclesfield
Duivenhoks
Gateshead
Mathura
Potto
Marylebone
Cochin
Ceyco
India
Geraldton
Toronto
Ackwepe
Sangalkam
Deckstein
Shoreditch
Sokode
Benin
Irchel
Nantes
Mayday
Haarlem
Bambylor
Lishabi
Inglis
Mahina
Louisiana
Ouakam
Hillegersberg
Basingstoke
Trimdon
Fresno
Ekotedo
Ngaparou
Maarssen
Wuppertal
Zuerich
O ANTIGENS
PHASE 1
PHASE 2
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,46
1,9,12,46,27
9,12,46,27
1,9,12,46,27
1,9,12,46,27
1,9,12,46,27
1,9,12,46,27
f,g
g,m
g,m,s,t
g,[m],[s],t
g,s,t
g,z62
i
i
k
k
k
l,v
l,v
l,v
l,w
m,t
m,t
r
r
r
y
y
y
y
z
z
z
z10
z10
z10
z10
z10
z10
z29
z35
z35
z35
z38
z39
z4,z23
z4,z24
z4,z24
z41
c
g,t
l,z13,z28
y
z10
z10
1,(2),7
[e,n,x]
e,n,z15
z6
1,2
1,5
z35
1,5
1,6
e,n,x;[z44]
e,n,x
1,7
e,n,z15
z6
1,7
e,n,x
l,w
z6
1,5
e,n,x
e,n,z15
1,7
e,n,x
e,n,z15
z39
z6
z6
1,5
e,n,z15
z6
1,7
z39:z42
z39
e,n,x
z39
z39
1,5
e,n,x
NOTE
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
z10
z4,z24
a
a
z39
[1,5]
1,2
1,5
II
II
I
I
D3
D3
E1
E1
Aminatu
Goelzau
1,9,12,46,27
1,9,12,46,27
3,10
3,10
E1
Oxford
3,10
1,7
I
II
I
II
I
I
E1
E1
E1
E1
E1
E1
Masembe
Matroosfontein
Galil
3,10
3,10
3,10
3,10
3,10
3,10
a
a
a
a
b
b
e,n,x
e,n,x
e,n,z15
z39
1,2
1,5
I
I
I
II
I
I
I
II
I
I
I
I
E1
E1
E1
E1
E1
E1
E1
E1
E1
E1
E1
E1
Allerton
Huvudsta
Benfica
Wilmington
Asylanta
Gbadago
Ikayi
3,10
3,10
3,10
3,10
3,10,[15]
3,10
3,10,[15]
3,10
3,10
3,10
3,10,[15]
3,10,[15]
b
b
b
b
b
b
b
b
b
c
c
c
1,6
1,7
e,n,x
e,n,x
e,n,z15
l,w
z35
z39
z6
1,2
1,5
1,6
I
I
I
I
I
I
E1
E1
E1
E1
E1
E1
Pramiso
Agege
Anderlecht
Okefoko
Stormont
Shangani
3,10
3,10
3,10
3,10
3,10
3,10
c
c
c
c
d
d
1,7
e,n,z15
l,w
z6
1,2
1,5
I
I
I
E1
E1
E1
Lekke
Onireke
Souza
3,10
3,10
3,10
d
d
d
1,6
1,7
e,n,x
II
I
I
I
I
I
E1
E1
E1
E1
E1
E1
Madjorio
Birmingham
Maron
Weybridge
Vejle
3,10
3,10
3,10
3,10
3,10
3,10
d
d
d
d
d
e,h
e,n,x
e,n,z15
l,w
z35
z6
1,2
E1
Muenster
3,10
e,h
1,5
E1
Anatum
3,10
e,h
1,6
The Difco Manual
Kalina
Butantan
Yaba
Epicrates
Westminster
NOTE
IP combined Clichy (3,15:a:1,5) with Goelzau

to form Goelzau 3,10,[15]:a:1,5.
IP combined Khartoum (3,15,34:a:1,7) with
Oxford to form Oxford 3,10,[15],[15,34]:a:1,7.
Masembe may possess H phase Rz5.
IP combined Rosenthal (3,15:b:1,5) and

unnamed 3,15,34:b:1,5 with Butantan to form
Butantan 3,10,[15],[15,34]:b:1,5.
CDC has no 3,10:b:z35.
Ikayi Var. O 15+ was described after E1 and

E2 were combined.
IP combined Pankow (3,15:d:1,5) with

Shangani to form Shangani 3,10,[15]:d:1,5.
IP combined Eschersheim (3,15:d:e,n,x) with

Souza to form Souza 3,10,[15]:d:e,n,x.
IP combined Goerlitz (3,15:e,h:1,2) with Vejle

to form Vejle 3,10,[15]:e,h:1,2.
IP combined Muenster with Newhaw (3,15:e,h:1,5)
and Arkansas (3,15,34:e,h:1,5) to form
Muenster 3,10,[15],[15,34]:e,h:1,5.
IP combined Newington (3,15:e,h:1,6) and
Minneapolis (3,15,34:e,h:1,6) with Anatum to
form Anatum 3,10,[15],[15,34]:e,h:1,6.
691

692
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
3,10
e,h
1,7
E1
Nyborg
I
I
I
E1
E1
E1
Newlands
Lamberhurst
Meleagridis
3,10,[15,34]
3,10
3,10
e,h
e,h
e,h
e,n,x
e,n,z15
l,w
I
II
I
I
I
I
E1
E1
E1
E1
E1
E1
Sekondi
Chudleigh
Alfort
Regent
Suberu
Amsterdam
3,10
3,10
3,10
3,10
3,10
3,10
e,h
e,n,x
f,g
f,g,[s]
g,m
g,m,s
z6
1,7
e,n,x
[1,6]
II
I
E1
E1
Parow
Westhampton
3,10,[15]
3,10
g,m,s,t
g,s,t
II
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
E1
E1
E1
E1
E1
E1
E1
E1
E1
E1
E1
E1
E1
E1
E1
E1
Islington
Bloomsbury
Cuckmere
Amounderness
Truro
Bessi
Falkensee
Hoboken
Yeerongpilly
Wimborne
Zanzibar
Serrekunda
Yundum
Marienthal
Newrochelle
Nchanga
3,10
3,10
3,10
3,10
3,10
3,10
3,10
3,10
3,10
3,10
3,10,[15]
3,10
3,10
3,10
3,10
3,10
g,t
g,t
i
i
i
i
i
i
i
k
k
k
k
k
k
l,v
1,5
1,2
1,5
1,7
e,n,x
e,n,z15
l,w
z6
1,2
1,5
1,7
e,n,x
e,n,z15
l,w
1,2
I
I
E1
E1
Sinstorf
London
3,10
3,10
l,v
l,v
1,5
1,6
E1
Give
3,10
l,v
1,7
II
I
I
II
I
I
I
I
E1
E1
E1
E1
E1
E1
E1
E1
Ruzizi
Sinchew
Fuhlsbuettel
Assinie
Freiburg
Uganda
Fallowfield
3,10
3,10
3,10
3,10
3,10
3,10
3,10,15
3,10
l,v
l,v
l,v
l,v
l,w
l,z13
l,z13
l,z13,z28
e,n,x
e,n,z15
z35
z6
z6
1,2
1,5
e,n,z15
NOTE
IP combined Selandia (3,15:e,h:1,5) with

Nyborg to form Nyborg 3,10,[15]:e,h:1,7.
IP combined Meleagridis with Cambridge

(3,15:e,h:l,w) and Wildwood (3,15,34:e,h:l,w)
to form Meleagridis 3,10,[15],[15,34]:e,h:l,w.
IP combined Drypool (3,15:g,m,s) and

Drypool var. O 34+ with Amsterdam to form
Amsterdam 3,10,[15],[15,34]:g,m,s:-.
IP combined Halmstad (3,15:g,s,t:-) and Canoga
(3,15,34:g,s,t:-) with Westhampton to form
Westhampton 3,10,[15],[15,34]:g,s,t:-. Westhampton
may possess H phase Rz37 or Rz43 or Rz45.
IP combined Nchanga with Nancy (3,15:l,v:1,2)

to form Nchanga 3,10,[15]:l,v:1,2.
IP combined London with Portsmouth
(3,15:l,v:1,6) to form London 3,10,[15]:l,v:1,6.
IP combined Newbrunswick (3,15:l,v:1,7) and
Menhaden (3,15,34:l,v:1,7) with Give to form
Give 3,10,[15],[15,34]:[d],l,v:[d],1,7. Give
may possess H phase d; Rl,z40; or Rz77.
Assinie may possess H phase Rz45.
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
3,10
3,10
3,10
3,10
3,10
3,10
3,10
3,10,15,34
3,10
3,10
3,10
3,10
3,10
l,z13,z28
l,z28
l,z28
l,z28
l,z28
l,z28
m,t
m,t
m,t
r
r
r
r
z6
1,5
1,7
e,n,x
e,n,x
z39
1,5
[1,6]
e,n,x
1,5
1,7
e,n,z15
z6
I
II
I
I
II
II
II
I
II
I
I
I
I
E1
E1
E1
E1
E1
E1
E1
E1
E1
E1
E1
E1
E1
Hoghton
I
I
I
E1
E1
E1
Seegefeld
Dumfries
Rutgers
3,10
3,10
3,10
r,[i]
r,[i]
Rl,z40
1,2
1,6
1,7
E1
Amager
3,10
1,2
E1
Orion
3,10
1,5
I
I
I
I
I
E1
E1
E1
E1
E1
Mokola
Ohlstedt
Bolton
Langensalza
Stockholm
3,10
3,10
3,10
3,10
3,10
y
y
y
y
y
1,7
e,n,x
e,n,z15
l,w
z6
I
II
I
I
II
I
II
I
I
I
E1
E1
E1
E1
E1
E1
E1
E1
E1
E1
Fufu
Alexander
Harleystreet
Huddinge
Finchley
Clerkenwell
Tafelbaai
Landwasser
Okerara
Lexington
3,10
3,10
3,10
3,10
3,10
3,10
3,10
3,10
3,10
3,10
z
z
z
z
z
z
z
z
z10
z10
1,5
1,5
1,6
1,7
e,n,x
l,w
z39
z6
1,2
1,5
I
I
I
I
I
II
I
II
E1
E1
E1
E1
E1
E1
E1
E1
Harrisonburg 3,10,[15],[15,34]
Coquilhatville
3,10
Kristianstad
3,10
Biafra
3,10
Jedburgh
3,10,[15]
3,10
Everleigh
3,10
3,10
z10
z10
z10
z10
z29
z29
z29
z29
1,6
1,7
e,n,z15
z6
e,n,x
e,n,x
The Difco Manual
Joal
Lamin
Westpark
Southbank
Stikland
Ughelli
Elisabethville
Simi
Weltevreden
NOTE
IP combined Lanka 3,15:r:z6 with Weltevreden

to form Weltevreden 3,10,[15]:r:z6.
Rutgers has been dropped from the scheme

and the H phase Rl,z40 is now considered an
R phase of Give.
IP combined Tuebingen (3,15:y:1,2) with
Amager to form Amager 3,10,[15]:y:1,2.
Amager may possess H phase Rz45.
IP combined Binza (3,15:y:1,5) and
Thomasville (3,15,34:y:1,5) with Orion to
form Orion 3,10,[15],[15,34]:y:1,5.
IP combined Tournai (3,15:y:z6) with

Stockholm to form Stockholm 3,10,[15]:y:z6.
IP combined Lexington with Manila (3,15:z10:1,5)

and Illinois (3,15,34:z10:1,5) to form Lexington
3,10,[15],[15,34]:z10,1,5. Lexington may
693

694
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
z35
z35
z35
z36
z38
z38
z39
z4,z23
z4,z23
z4,z24
z4,z24
z69
a
1,7
l,w
z6
z42
[z6]
1,[5],7
[1,7]
z6
1,7
1,5
I
I
I
I
II
I
II
I
I
I
II
I
I
E1
E1
E1
E1
E1
E1
E1
E1
E1
E1
E1
E1
E2
Zongo
Shannon
Cairina
Macallen
Mpila
Bolombo
Winchester
Adabraka
Wagadugu
Florian
Pietersburg
Clichy
3,10
3,10
3,10
3,10
3,10
3,10
3,10
3,10
3,10
3,10,[15]
3,10
3,10,[15,34]
3,15
E2
Rosenthal
3,15
1,5
E2
Pankow
3,15
1,5
E2
Eschersheim
3,15
e,n,x
E2
Goerlitz
3,15
e,h
1,2
E2
Newhaw
3,15
e,h
1,5
E2
Newington
3,15
e,h
1,6
E2
Selandia
3,15
e,h
1,7
E2
Cambridge
3,15
e,h
l,w
E2
Drypool
3,[15],[15,34]
g,m,s
E2
Halmstad
3,15
g,s,t
E2
Nancy
3,15
l,v
1,2
NOTE
IP combined Clichy with Goelzau (3,10:a:1,5)

to form Goelzau 3,10,[15]:a:1,5. Clichy is now
called Goelzau var. O 15+ by IP.
IP combined Rosenthal and unnamed 3,15,34:b:1,5
with Butantan (3,10:b:1,5) to form Butantan
3,10,[15],[15,34]:b:1,5. Rosenthal is now
called Butantan var. O 15+ by IP.
IP combined Pankow with Shangani (3,10:d:1,5)
to form Shangani 3,10,15:d:1,5. Pankow is
now called Shangani var. O 15+ by IP.
IP combined Eschersheim with Souza (3,10:d:e,n,x)
to form Souza 3,10,[15]:d:e,n,x. Eschersheim
is now called Souza var. O 15+ by IP.
IP combined Goerlitz with Vejle (3,10:e,h:1,2)
to form Vejle 3,10,15:e,h:1,2. Goerlitz is now
called Vejle var. O 15+ by IP.
IP combined Newhaw and Arkansas
(3,15,34:e,h:1,5) with Muenster (3,10:e,h:1,5)
to form Muenster 3,10,[15],[15,34]:e,h:1,5.
Newhaw is now called Muenster var. O 15+ by IP.
IP combined Newington and Minneapolis
(3,15,34:e,h:1,6) with Anatum (3,10:e,h:1,6) to
form Anatum 3,10,[15],[15,34]:e,h:1,6.
Newington is now called Anatum var. O 15+ by IP.
IP combined Selandia with Nyborg (3,10:e,h:1,7)
to form Nyborg 3,10,[15]:e,h:1,7. Selandia is
now called Nyborg var. O 15+ by IP.
IP combined Cambridge and Wildwood
(3,15,34:e,h:l,w) with Meleagridis (3,10:e,h:l,w)
Cambridge is now called Meleagridis
var. O 15+ by IP.
IP combined Drypool 3,15:g,m,s:- and Drypool
var. O34+ with Amsterdam (3,10:g,m,s:-) to
form Amsterdam 3,10,[15],[15,34]:g,m,s:-.
Drypool is now called Amsterdam var. O 15+
or O 15+, 34+ by IP.
IP combined Halmstad and Canoga (3,15,34:g,s,t:-)
with Westhampton (3,10:g,s,t:-) to form
Westhampton 3,10,[15],[15,34]:g,s,t:-. Halmstad
is now called Westhampton var. O 15+ by IP.
IP combined Nancy with Nchanga (3,10:l,v:1,2)
to form Nchanga 3,10,[15]:l,v:1,2. Nancy is
now called Nchanga var. O 15+ by IP.
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
E2
Portsmouth
3,15
l,v
1,6
E2
Newbrunswick
3,15
l,v
1,7
E2
Kinshasa
3,15
l,z13
1,5
E2
Lanka
3,15
z6
E2
Hamilton
3,15
Rz27
E2
Tuebingen
3,15
1,2
E2
Binza
3,15
1,5
E2
Tournai
3,15
z6
E2
Manila
3,15
z10
1,5
E3
Khartoum
3,15,34
1,7
E3
Arkansas
3,15,34
e,h
1,5
E3
Minneapolis
3,15,34
e,h
1,6
E3
Wildwood
3,15,34
e,h
l,w
E3
Drypool
3,15,34
g,m,s
E3
Canoga
3,15,34
g,s,t
The Difco Manual
NOTE
IP combined Portsmouth with London (3,10:l,v:1,6)

to form London 3,10,[15]:l,v:1,6. Portsmouth
is now called London var. O 15+ by IP.
IP combined Newbrunswick and Menhaden
(3,15,34:l,v:1,7) with Give (3,10:l,v:1,7) to
form Give 3,10,[15],[15,34]:[d],l,v:[d],1,7.
Newbrunswick is now called Give var. O 15+ by IP.
IP combined Kinshasa with Uganda (3,10:l,z13:1,5)
to form Uganda 3,10,[15]:l,z13:1,5. Kinshasa
is now called Uganda var. O 15+ by IP.
IP combined Lanka with Weltevreden (3,10:r:z6)
to form Weltevreden 3,10,[15]:r:z6. Lanka is
now called Weltevreden var. O 15+ by IP.
IP combined Hamilton with Goerlitz
(3,15:e,h:1,2) and Vejle (3,10:e,h:1,2) to form
Vejle 3,10,15:e,h:1,2:Rz27. Hamilton is now
called Vejle var. Rz27+. The name Hamilton
has been dropped.
IP combined Tuebingen with Amager (e,10:y:1,2)
to form Amager 3,10,[15]:y:1,2. Tuebingen is
now called Amager var. O 15+ by IP.
IP combined Binza and Thomasville
(3,15,34:y:1,5) with Orion (3,10:y:1,5) to
form Orion 3,10,[15],[15,34]:y:1,5. Binza is
now called Orion var. O 15+ by IP.
IP combined Tournai with Stockholm
(3,10:y:z6) to form Stockholm 3,10,[15]:y:z6.
Tournai is now called Stockholm var. O 15+ by IP.
IP combined Manila and Illinois (3,15,34:z10:1,5)
with Lexington (3,10:z10:1,5) to form
Lexington 3,10,[15],[15,34]:z10:1,5. Manila is
now called Lexington var. O 15+ by IP.
IP combined Khartoum with Oxford (3,10:a:1,7)
to form Oxford 3,10,[15],[15,34]:a:1,7.
Khartoum is now called Oxford var. O 15+ by
IP. CDC has no 3,15:a:1,7. Khartoum was
found by IP with colonies containing O 3,15.
IP combined Arkansas and Newhaw (3,15:e,h:1,5)
with Muenster (3,10:e,h:1,5) to form Muenster
3,10,[15],[15,34]:e,h:1,5. Arkansas is now
callled Muenster var. O 15+, 34+ by IP.
IP combined Minneapolis and Newington
(3,15:e,h:1,6) with Anatum (3,10:e,h:1,6) to form
Anatum 3,10,[15],[15,34]:e,h:1,6. Minneapolis
is now called Anatum var. O 15+ by IP.
IP combined Wildwood and Cambridge
(3,15:e,h:l,w) with Meleagridis (3,10:e,h:l,w)
Wildwood is now called Meleagridis
var. O 15+, 34+ by IP.
IP combined Drypool (3,15:g,m,s:-) and Drypool
var. O 34+ with Amsterdam (3,10:g,m,s:-) to
or var. O 15+,34+ by IP.
IP combined Canoga and Halmstad (3,15:g,s,t:-)
Westhampton 3,10,[15],[15,34]:g,s,t:-. Canoga
is now called Westhampton var. O 15+, 34+ by IP.
695

696
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
E3
Menhaden
3,15,34
l,v
1,7
E3
Thomasville
3,15,34
1,5
E3
Illinois
3,15,34
z10
1,5
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
Niumi
Juba
Gwoza
Alkmaar
Gnesta
Visby
Tambacounda
Kande
Broughton
Chittagong
Accra
Eastglam
Bida
Madiago
Ahmadi
Liverpool
Tilburg
Niloese
Vilvoorde
Hayindogo
Sanktmarx
Sao
Calabar
Rideau
Bilu
Petahtikva
Maiduguri
Kouka
Dessau
Senftenberg
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,10,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
(1),3,10,(19)
1,3,19
1,3,19
1,3,19
1,3,15,19
1,3,19
a
a
a
a
b
b
b
b
b
b
b
c
c
c
d
d
d
d
e,h
e,h
e,h
e,h
e,h
f,g
f,g,t
f,g,t
f,g,t
g,m,[t]
g,s,t
g,[s],t
1,5
1,7
e,n,z15
l,w
1,5
1,6
e,n,x
e,n,z15
l,w
z35
z6
1,5
1,6
1,7
1,5
e,n,z15
l,w
z6
1,5
1,6
1,7
e,n,z15
l,w
1,(2),7
1,7
e,n,z15
I
I
I
I
I
I
I
E4
E4
E4
E4
E4
E4
E4
Stratford
Chichester
Machaga
Avonmouth
Zuilen
Taksony
Bethune
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
i
i
i
i
i
[i]
k
1,2
1,6
e,n,x
e,n,z15
l,w
z6
1,7
NOTE
IP combined Menhaden with Give (3,10:l,v:1,7)

and Newbrunswick (3,15:l,v:1,7) to form Give
3,10,[15],[15,34]:[d],l,v:1,7. Menhaden is now
called Give var. O 15+, 34+ by IP.
IP combined Thomasville and Binza (3,15:y:1,5)
with Orion (3,10:y:1,5) to form Orion
3,10,[15],[15,34]:y:1,5. Thomasville is now
called Orion var. O 15+ by IP.
IP combined Illinois and Manila (3,15:z10:1,5)
with Lexington (3,10:z10:1,5) to form Lexington
3,10,[15],[15,34]:z10:1,5. Illinois is now called
Lexington var. O 15+, 34+ by IP.
Tilburg may possess H phase Rz49.
Senftenberg may possess H phase Rz37 or Rz43

or Rz45 or Rz46. Simsbury (1,3,19:Rz27:-) is now
considered an H phase Rz27 of Senftenberg.
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
Ngor
Parkroyal
Svedvi
Fulda
Westerstede
Winterthur
Lokstedt
Stuivenberg
Bedford
Tomelilla
Kindia
Cannstatt
Yalding
Fareham
Simsbury
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
l,v
l,v
l,v
l,w
l,z13
l,z13
l,z13,z28
l,z13,z28
l,z13,z28
l,z28
l,z28
m,t
r
r,i
Rz27
1,5
1,7
e,n,z15
1,5
[1,2]
1,6
1,2
1,5
e,n,z15
1,7
e,n,x
e,n,z15
l,w
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
II
I
I
VI
I
II
I
I
II
I
I
I
VI
VI
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
E4
F
F
F
F
F
F
F
F
F
F
F
F
F
F
Gatineau
Thies
Cannonhill
Kinson
Slade
Krefeld
Korlebu
Kainji
Lerum
Schoeneberg
Carno
Hongkong
Dallgow
Llandoff
Ochiogu
Ilugun
Sambre
1,3,19
1,3,19
1,3,15,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,19
1,3,10,19
1,3,19
11
11
11
11
11
11
11
11
11
11
11
11
11
11
y
y
y
y
y
y
z
z
z
z
z
z
z10
z29
z38
z4,z23
z4,z24
a
a
a
a
a,[d]
a
a
a
b
b
b
b
b
1,5
1,7
e,n,x
e,n,x
e,n,z15
l,w
1,5
1,6
1,7
e,n,z15
l,w
z6
e,n,z15
[z6]
[e,n,z15]
z6
1,5
1,2
1,5
1,5
1,7
[d]:e,n,z15
e,n,z15
l,z13,z28
z6:z42
1,2
1,5
1,6
1,7
e,n,x
The Difco Manual
Gallen
Marseille
Toowong
Montgomery
Luciana
Epinay
Glencairn
Atento
Leeuwarden
Wohlen
Srinagar
NOTE
IP combined Simsbury with Senftenberg

1,3,19:g,[s],t:-. Simsbury is now considered an
R phase of Senftenberg. The name Simsbury
has been dropped.
697

I
I
I
II
I
I
I
I
I
I
I
I
II
I
IV
I
I
I
I
I
I
I
I
I
I
I
IIIb
I
I
I
I
IIIb
IIIb
I
I
I
II
I
II
I
I
I
I
I
I
I
I
I
II
698
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
Section V
SEROTYPE
Pharr
Chiredzi
Woodinville
Ati
Gustavia
Chandans
Pennsylvania
Findorff
Chingola
Adamstua
Redhill
Grabouw
Missouri
Mundsburg
Aberdeen
Brijbhumi
Heerlen
Veneziana
Pretoria
Abaetetuba
Sharon
Colobane
Kisarawe
Mannheim
Amba
Stendal
Maracaibo
Fann
Bullbay
Glidji
Connecticut
Osnabrueck
Huila
Moers
Lincoln
Senegal
Rubislaw
Volta
Euston
Solt
Jalisco
Herzliya
Crewe
Maroua
O ANTIGENS
PHASE 1
PHASE 2
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
b
c
c
c
d
d
d
d
d
e,h
e,h
e,h
g,[m],s,t
g,s,t
g,z51
i
i
i
i
k
k
k
k
k
k
k
k
l,v
l,v
l,v
l,v
l,v
l,v
l,w
l,z13,z28
l,z13,z28
l,z28
m,t
m,t
r
r
r
r,i
y
y
y
z
z
z
e,n,z15
1,5
e,n,x
e,n,z15
1,2
1,5
e,n,x:[r]
e,n,z15
z6
1,2
1,6
l,z13,z28
[z39]
1,2
1,5
1,6
e,n,x
1,2
1,5
1,6
1,7
e,n,x,[z15]
l,w
l,z13,z28
z53
1,2
1,5
e,n,x
e,n,z15
z53
z
1,5
1,5
e,n,x
e,n,x
e,n,x
1,5
[e,n,x]
l,z13,z28
e,n,x,z15
1,5
1,7
e,n,x
1,5
1,7
e,n,x
NOTE
(Ar. 17:29:25)
(Ar. 17:23:25)
(Ar. 17:23:31). May possess H phase Rz56 (Ar. 38).
The Difco Manual
Section V
II
I
I
I
I
I
IIIa
IV
I
I
I
IV
I
II
I
II
I
II
I
I
II
I
F
F
F
F
F
F
F
F
F
F
F
F
F
G
G
G
G
G
G
G
G
G
I
I
II
I
I
I
I
I
I
I
II
I
I
I
I
I
I
II
I
I
II
I
I
I
I
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
The Difco Manual
SEROTYPE
Soutpan
Nyanza
Wentworth
Straengnaes
Telhashomer
Lene
Parera
Remete
Etterbeek
Yehuda
Maastricht
Chagoua
Mim
Wyldegreen
Marshall
Tygerberg
Atlanta
Ibadan
Mississippi
Acres
Bracknell
Oudwijk
Rottnest
Ullevi
Vaertan
Bahati
Durham
Haouaria
Handen
Mishmarhaemek
Friedenau
Wichita
Grumpensis
Diguel
Telelkebir
Putten
Isuge
Tschangu
Willemstad
O ANTIGENS
PHASE 1
PHASE 2
11
11
11
11
11
11
11
11
11
11
11
11
11
13,23
1,13,23
1,13,23
13,22
13,22
1,13,23
13,22
1,13,23
13,23
z
z
z10
z10
z10
z38
z4,z23
z4,z23
z4,z23
z4,z23
z4,z24
z4,z32
z41
a
a
a
a
a
a
a
b
z39
z6
1,2
1,5
e,n,x
1,6
e,n,z15
1,2
1,6
1,5
1,5
1,6
e,n,x
l,w
l,z13,z28
z42
13,22
1,13,23
1,13,23
13,23
13,22
1,13,22
1,13,23
13,22
13,22
13,23
1,13,22
13,22
1,13,23
1,13,23
13,22
1,13,23
1,13,23
13,23
1,13,22
13,23
1,13,23
13,23
13,23
1,13,23
1,13,22
b
b
b
b
b
b
b
b
b
b
b
c
d
d
d
d
d
d
d
d
d
d
d
e,h
e,h
1,5
[1,5]
[1,5]:z42
1,6
1,6
1,7
e,n,x
e,n,x
e,n,z15
e,n,z15
z42
e,n,x,z15
1,2
1,5
1,6
1,6
1,7
e,n,x
e,n,z15
e,n,z15
e,n,z15
l,w
z6
1,5
1,6
NOTE
(Ar. 17:1,2,5:-)
Atlanta was combined with Mississippi

(1,13,23:b:1,5). The name Atlanta has
been dropped.
Wichita may possess H phase Rz37.
699

Section V
SEROTYPE
PHASE 1
PHASE 2
e,h
e,n,x
f,g
f,g,[s]
g,51
g,m
g,m,s,t
g,m,s,t
l,w
1,[5],7
e,n,x
[e,n,z15]
1,5
[e,n,x]
I
II
I
I
IIIa
I
II
II
G
G
G
G
G
G
G
G
Luanshya
1,13,23
1,13,23
13,22
1,13,23
1,13,23
13,22
1,13,23
1,13,23
II
II
II
G
G
G
Limbe
Kraaifontein
1,13,23
1,13,22
1,13,23
g,[s],t
g,m,t
g,m,t
z42
[1,5]
[e,n,x]
I
I
I
I
II
II
II
V
I
I
I
I
II
II
I
IIIb
I
I
II
II
II
II
II
I
I
II
II
II
II
V
I
I
I
I
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
1,13,23
13,23
13,22
13,23
1,13,23
1,13,23
1,13,22
1,13,22
1,13,23
13,23
1,13,23
13,23
13,22
13,23
13,22
13,22
13,22
1,13,23
13,23
13,22
13,23
1,13,23
13,23
1,13,23
13,22
1,13,23
1,13,23
1,13,23
13,22
13,22
13,23
13,23
1,13,22
13,23
g,m,[t]
g,[m],[s],t
g,s,t
g,[s],t
g,m,s,t
g,t
g,t
i
i
i
i
k
k
k
l,v
l,v
l,v
l,v
l,w
l,z28
l,z28
l,z28
l,z28
m,t
m,t
m,t
m,t
m,t
m,t
r
r
r
y
y
z42
1,5
1,5
1,5
e,n,z15
l,w
1,5:z42
z41
1,5
1,5,7
1,6
e,n,z15
e,n,x
1,5
1,5
z42
z6
1,5
e,n,x
z42
z42:z39
1,6
e,n,z15
1,6
1,7
700
Vridi
Epping
Raus
Havana
O ANTIGENS
Bron
Agbeni
Congo
Newyork
Okatie
Gojenberg
Rotterdam
Idikan
Jukestown
Kedougou
Marburg
Lovelace
Borbeck
Nanga
Vredelust
Kintambo
Washington
Katesgrove
Worcester
Boulders
Adjame
Linton
Tanger
Yarrabah
NOTE
Havana may possess H phase Rz45 or Rz79.

(Ar. 18:13,14:-)
IP combined Luanshya with Kraaifontein

(1,13,23:g,m,t:[e,n,x]) to form Luanshya
1,13,23:g,m,[s],t:[e,n,x].
IP combined Kraaifontein with Luanshya

(1,13,23:g,m,s,t:[e,n,x]) to form Luanshya
1,13,23:g,m,[s],t:[e,n,x]. The name
Kraaifontein has been dropped.
IP calls Congo 13,23:g,m,s,t:-.
(Ar. 18:23:30)
The Difco Manual
Section V
I
I
II
II
I
I
I
I
I
II
I
I
II
I
I
II
II
II
II
I
I
I
I
I
II
II
I
IIIa
I
IIIa
I
IIIa
II
I
VI
VI
I
I
I
I
IIIb
I
I
I
I
I
I
I
I
The Difco Manual
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
Ordonez
Tunis
1,13,23
1,13,23
13,22
1,13,23
13,23
1,13,22
13,22
1,13,22
1,13,23
1,13,23
1,13,22
13,23
1,13,22
13,22
1,13,23
13,22
1,13,23
13,22
1,13,23
13,22
13,23
13,22
13,23
13,22
1,13,23
13,22
13,23
13,22
1,13,22
13,23
1,13,23
1,13,23
1,13,23
1,6,14,25
[1],6,14
1,6,14,25
1,6,14,25
1,6,14,25
1,6,14,25
[1],6,14,[25]
(6),14
1,6,14,25
1,6,14,25
1,6,14,25
1,6,14,25
[1],6,14,[25]
6,14,[24]
1,6,14,25
[1],6,14,[25]
y
y
z
z
z
z
z
z
z
z
z10
z10
z10
z29
z29
z29
z29
z29
z29
z35
z35
z35
z38
z38
z39
z39
z4,z23
z4,z23
z4,z23
z4,z23,z32
z4,z24
z4,z24
[z42]
a
a
a
a
b
b
b
b
c
c
c
c
d
d
d
d
l,w
z6
1,5
1,6
1,6
1,7
e,n,z15
l,w
z42
1,5
l,w
z6
1,5
1,5
e,n,x
e,n,x
1,6
e,n,z15
e,n,z15
1,5,7
1,7
[e,n,z15]
1,[5],7
1,5
1,5
e,n,x
e,n,z15
1,2
1,5
[1,7]
e,n,x,z15
1,5
1,6
e,n,x
l,w
1,5
1,5
1,6
1,7
Nachshonim
Farmsen
Poona
Bristol
Tanzania
Worthington
Roodepoort
Demerara
Agoueve
Cubana
Clifton
Goodwood
Mampong
Anna
Nimes
Fanti
Leiden
Ajiobo
Ried
Romanby
Stevenage
Garba
Ferlac
Banjul
Ndjamena
Kuntair
Tucson
Blijdorp
Kassberg
Runby
Minna
Finkenwerder
Heves
Woodhull
Florida
NOTE
Poona may possess H phase Rz59.
Worthington may possess H phase Rz45.
Cubana may possess H phase Rz37 or Rz43.
(Ar. 18:1,2,5:-)
(Ar. 18:1,6,7:-). CDC would call this 1,6,7,9.
(Ar. 18:1,3,11:-)
(Ar. 7a,7c:43:28)
701

I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
II
II
IIIb
IIIb
II
I
I
IIIb
IIIb
IIIb
I
I
II
II
IIIb
I
I
I
I
I
I
I
I
I
I
I
I
I
VI
II
702
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Section V
SEROTYPE
Midway
Charity
Lindern
Teko
Encino
Albuquerque
Bahrenfeld
Onderstepoort
Magumeri
Beaudesert
Warragul
Caracas
Sylvania
Catanzaro
Mampeza
Buzu
Schalkwijk
Moussoro
Harburg
Boecker
Horsham
Aflao
Kaitaan
Rooikrantz
Emmerich
Istoria
Surat
Carrau
Madelia
Fischerkietz
Mornington
Homosassa
Kanifing
Soahanina
Sundsvall
Royan
Poano
Nessa
Bornheim
Simonstown
O ANTIGENS
PHASE 1
PHASE 2
6,14,24
[1],6,14,[25]
6,14,[24]
1,6,14,25
1,6,14,25
1,6,14,24
6,14,24
1,6,14,[25]
1,6,14,25
[1],6,14,[25]
[1],6,14,[25]
[1],6,14,[25]
[1],6,14,[25]
6,14
1,6,14,25
[1],6,14,[25]
6,14,[24]
1,6,14,25
[1],6,14,[25]
6,14,[24]
6,14
(6),14
(6),14
1,6,14
[1],6,14,[25]
1,6,14,[25]
(6),14
(6),14
(6),14
1,6,14,25
1,6,14,25
1,6,14
6,14
(6),14
1,6,14,25
[1],6,14,[25]
6,14,[24]
1,6,14,25
1,6,14,25
1,6,14,25
1,6,14,25
1,6,14,25
6,14,24
[1],6,14,[25]
1,6,14,25
1,6,14,25
1,6,14,25
1,6,14,25
1,6,14
d
d
d
d
d
d
e,h
e,h
e,h
e,h
g,m
g,m,s
g,p
g,s,t
i
i
i
i
k
k
k
k
k
k
l,v
l,v
l,v
l,v
l,v
l,z28
m,t
m,t
[m,t]
r
r,i
[r],[i]
y
y
y
y
z
z
z
z
z
z
z10
z10
z10
1,7
e,n,x
e,n,x
e,n,z15
l,z13,z28
z6
1,5
1,5
1,6
1,7
1,5
1,7
e,n,..
e,n,z15
1,5
1,6
[e,n,x]
z
z53
z6:z42
1,7
e,n,x
z
z35
z53
e,n,x
1,5
e,n,x
z
1,5
e,n,z15
1,7
1,7
e,n,x
e,n,z15
1,5
1,6
e,n,x
e,n,x
e,n,z15
l,z13,z28
1,2
1,(2),7
1,5
NOTE
(Ar. 7a,7c:29:31)
(Ar. 7a,7c:29:25)
(Ar. 7a,7c:23:31)
(Ar. 7a,7c:23:21)
(Ar. 7a,7c:23:25)
(Ar. 7a,7c:24:31)
Bornheim was formerly in Subspecies II.

The Difco Manual
Section V
IIIb
IIIb
II
IIIb
I
I
IV
I
I
I
I
II
IIIb
IIIb
I
I
I
I
I
I
I
I
I
I
I
II
I
I
II
II
I
I
I
I
I
I
I
I
II
I
I
I
I
I
I
I
I
I
I
The Difco Manual
H
H
H
H
H
H
H
H
H
H
H
H
H
H
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
SEROTYPE
Slangkop
Potosi
Sara
Arapahoe
Bousso
Chichiri
Uzaramo
Hannover
Brazil
Amunigun
Nyeko
Togba
Fischerhuette
Heron
Hull
Wa
Glasgow
Hvittingfoss
Sangera
Vegesack
Malstatt
Vancouver
Gafsa
Shamba
Hithergreen
Yoruba
Oldenburg
Sculcoates
Sherbrooke
Gaminara
Barranquilla
Nottingham
Caen
Barmbek
Malakal
Saboya
Rhydyfelin
Weston
O ANTIGENS
PHASE 1
PHASE 2
(6),14
(6),14
1,6,14
(6),14
6,14
1,6,14,25
6,14
1,6,14
1,6,14,25
6,14,24
1,6,14,25
1,6,14
(6),14
(6),14
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
z10
z10
z10
z10
z36
z38
z4,z23
z4,z23
z4,z23
z4,z24
z4,z24
z42
z52
z52
a
a
a
a
a
a
a
b
b
b
b
b
b
b
b
b
b
c
c
c
c
c
d
d
d
d
d
d
d
d
d
e,h
e,h
e,h
e,h
e,n,x,z15
z53
z6:z42
z:[z53]
1,5
[e,n,x]
1,5
[e,n,z15]
1,6
e,n,x,z15
z35
1,2
1,5
1,6
1,7
e,n,x
e,n,z15
z6
1,2
1,5
1,6
e,n,x
e,n,x
e,n,z15
l,w
z39
z42
z6
1,5
1,6
e,n,x
e,n,z15
l,w
1,2
1,5
1,5
1,6
1,7
e,n,x
e,n,z15
l,w
z6
1,2
1,5
e,n,x
z6
NOTE
(Ar. 7a,7c:27:28)
(Ar. 7a,7c:27:25)
(Ar. 7a,7c:27:31:[25])
(Ar. 7a,7c:26:28)
(Ar. 7a,7c:26:21)
703

I
II
I
I
I
II
II
I
II
I
I
I
I
I
IIIb
I
I
I
I
IIIb
IIIb
IIIb
IIIb
I
I
I
I
IIIb
IIIb
I
IIIb
I
I
I
I
II
I
I
I
I
II
I
II
II
I
I
I
I
I
704
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
Section V
SEROTYPE
Bellville
Elsiesrivier
Tees
Nikolaifleet
Adeoyo
Mobeni
Cardoner
Merseyside
Amina
Agbara
Wisbech
Frankfurt
Pisa
Abobo
Szentes
Nuatja
Orientalis
Shanghai
Welikade
Salford
Burgas
Losangeles
Zigong
Westeinde
Brooklyn
Lomnava
Noordhoek
Mandera
Battle
Ablogame
Enugu
Sarepta
Mpouto
Rowburton
Ivory
Brunflo
Annedal
Zwickau
Rovaniemi
O ANTIGENS
PHASE 1
PHASE 2
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
e,n,x
[e,n,x]
f,g
g,m,s
g,m,[t]
g,[m],[s],t
g,[m],[s],t
g,s,t
g,t
i
i
i
i
i
i
i
k
k
k
k
k
(k)
l,v
l,v
l,v
l,v
l,v
l,v
l,v
l,v
l,v
l,w
l,w
l,w
l,w
l,w
l,z13
l,z13,z28
l,z13,z28
l,[z13],z28
l,z28
m,t
m,t
m,t
r
r
r,i
r,i
r,[i]
1,(5),7
1,6:z42
[e,n,x]
z42
[1,5]
1,5
1,6
1,7
e,n,z15
l,w
z35
z6
1,2
e,n,x
e,n,z15
z
z53
z35
1,5,7
1,6
1,7
e,n,x
e,n,z15
z35
z53
z6
z:[z61]
1,5
1,6
e,n,x
e,n,z15
z6
e,n,z15
1,6
z6
[1,5]
z42
e,n,x
[z42]
1,6
1,7
e,n,x
e,n,z15
1,5
NOTE
(Ar. 25:33:21)
(Ar. 25:29:31)
(Ar. 25:29:25)
(Ar. 25:22:21)
(Ar. 25:23:30)
(Ar. 25:23:21)
(Ar. 25:23:25)
(Ar. 25:23:31:[41])
The Difco Manual
Section V
SEROTYPE
I
I
I
I
I
I
II
I
II
I
I
IIIb
I
IIIb
I
I
I
II
II
I
I
IV
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
Saphra
Akuafo
Kikoma
Avignon
Fortlamy
Lingwala
Louwbester
Brevik
II
IV
I
II
IV
II
IIIb
II
I
I
I
I
I
II
I
II
I
II
I
I
I
II
II
II
I
I
I
I
I
I
I
I
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
Haddon
Ochsenzoll
Kibi
The Difco Manual
Bouake
Badagry
Lisboa
Redlands
Angouleme
Saloniki
Jacksonville
Trier
Dakota
Naware
Grancanaria
Chameleon
Woodstock
Bonames
Jangwani
Kinondoni
Kirkee
Dahra
Hillbrow
Bignona
Victoriaborg
Woerden
Berlin
Niamey
Jubilee
Verity
Bleadon
O ANTIGENS
PHASE 1
PHASE 2
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
y
y
y
y
z
z
z
z
z
z
z10
z10
z10
z10
z10
z10
z29
z29
z29
z35
z35
z36
z38
z39
1,5
1,6
e,n,x
e,n,z15
1,6
1,7
[e,n,x]
e,n,[x],z15
z42
z6
1,5
1,5,7
1,6
e,n,x,z15
e,n,z15
z6
1,5
[e,n,x]
1,6
e,n,z15
[1,6]
16
16
16
16
16
16
16
16
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
z4,z23
z4,z23
z4,z23
z4,z24
z4,z32
z42
z52
z6
a
a
a
b
b
b
b
b
c
c
d
d
e,h
e,n,x,z15
e,n,x,z15
(f),g,t
[1,6]
1,[5],7
z35
1,6
1,2
1,5
e,n,x
1,2
1,5
e,n,x,z15
e,n,z15
z6
1,6
z39
1,5
l,w
1,2
1,[5],7
1,6
[e,n,x,z15]
NOTE
(Ar. 25:27:30)
(Ar. 25:27:28)
Grancanaria can be d-tartrate neg., dulcitol

neg., ONPG pos., and anaerogenic.
(Ar. 25:26:21)
IP has dropped f.
705

II
I
II
I
IIIb
II
I
I
I
I
IIIb
I
I
I
I
IIIb
IIIb
I
I
II
I
IIIb
II
I
I
I
I
II
I
IIIb
IIIb
I
IIIa
IV
IIIa
IV
IIIa
IIIa
IIIa
IIIa
I
I
I
I
I
I
I
IIIa
I
706
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
K
K
K
K
K
K
K
K
K
Section V
SEROTYPE
Lowestoft
Ahanou
Irenea
Warri
Matadi
Zaria
Morotai
Michigan
Lancaster
Carmel
Granlo
Bama
Lode
Hadejia
Gori
Warengo
Tchamba
Constantia
Djibouti
Kandla
Cotia
Brazos
Fluntern
Rawash
Groenekan
Usumbura
Pontypridd
Memphis
O ANTIGENS
PHASE 1
PHASE 2
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
17
18
6,14,18
6,14,18
6,14,18
18
6,14,18
18
18
18
g,m,s,t
g,s,t
g,t
i
i
k
k
k
k
k
k
l,v
l,v
l,v
l,v
l,v
l,v
l,z28
m,t
m,t
r
r
y
y
z
z
z
z
z10
z10
z10
z29
z29
z29
z36
z36
z4,z23
z4,z23,z32
z4,z24
z4,z32
a
b
c
d
d
g,m
g,z51
k
z39
1,7
z35
1,5
1,7
e,n,x
e,n,z15
z
1,2
1,5
1,7
e,n,x
e,n,x,z15
z35
e,n,x
1,2
z
e,n,z15
1,2
1,5
e,n,z15
l,w:z42
e,n,x
e,n,x,z15
z
1,6
e,n,z15
1,5
e,n,x
1,5
1,7
1,5
NOTE
(Ar. 12:33:21)
(Ar. 12:29:32)
(Ar. 12:23:28)
(Ar. 12:23:21)
(Ar. 12:24:31)
(Ar.12:27:28). May possess H phase Rz56 (Ar. 38).

(Ar. 12:27:31)
(Ar. 12:16,17,18:-)
(Ar. 12:17,20:-)
(Ar. 12:1,2,5:- and 12:1,2,6:-)
(Ar. 12:1,6,7,9:-)
(Ar. 12:1,3,11:-)
(Ar. 12:1,6,7:- and 12:1,7,8:-)
(Ar. 7a,7b:13,14:-)
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
(k)
(k)
l,v
l,v
l,v
l,v
l,w
m,t
m,t
r
y
z
z10
z10
z10
z10
z36
z36,z38
z38
z4,z23
z4,z23
z4,z23
z53
z54
e,n,x,z15
e,n,z15
z
z53
e,n,z15
1,5
z
e,n,x,z15
1,5
e,n,x,z15
z6
z6
[1,5]
IIIb
IIIb
IIIb
I
IIIb
IIIb
I
I
II
IIIb
II
I
I
IIIb
I
II
II
IV
I
II
IIIa
I
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
Siegburg
6,14,18
z4,z23
[1,5]
I
I
II
IIIa
K
K
K
K
Aarhus
Blukwa
18
18
18
18
z4,z23
z4,z24
z4,z24
z4,z32
z64
IIIa
I
I
II
I
I
I
I
II
IIIb
I
I
I
I
II
IIIa
K
K
L
L
L
L
L
L
L
L
L
L
L
L
L
L
18
18
21
21
21
21
21
21
21
21
21
21
21
21
21
21
z4,z32
z71
a
b
b
b
c
c
c
c
d
d
d
f,g
g,[m],[s],t
g,z51
[1,5]
1,5
1,6
e,n,x
1,6
e,n,x
e,n,x
e,n,x,z15
1,5
e,n,x
z6
e,n,x
The Difco Manual
Orlando
Toulon
Langenhorn
Potengi
Leer
Carnac
Zeist
Beloha
Sinthia
Cerro
Shomron
Delmenhorst
Assen
Ghana
Minnesota
Hydra
Rhone
Spartel
Magwa
Madison
Good
NOTE
(Ar. 7a,7b:22:25)
(Ar. 7a,7b:22:34)
(Ar. 7a,7b:23:28)
(Ar. 7a,7b:23:31)
(Ar. 7a,7b:23:25)
(Ar. 7a,7b:24,31)
(Ar. 7a,7b:27:28)
(Ar. 7a,7b:1,2,5:- and 7a,7b:1,2,6:-)

Cerro was combined with Siegburg
(6,14,18:z4,z23:[1,5]) and called Cerro.
Cerro may possess H phase Rz45.
IP combined Siegburg with Cerro (18:z4,z23:[1,5])
to form Cerro 6,14,18:z4,z23:[1,5]. Siegburg
is now called Cerro var. O 14+. The name
Siegburg has been dropped.
Shomron was formerly in Subspecies II, but

is now combined with Arizona 7a,7b:1,7,8:-.
The name Shomron has been dropped.
(Ar. 7a,7b:1,6,7:- and 7a,7b:1,7,8:-)
Minnesota may possess H phase Rz33 or Rz49.
(Ar. 22:32:28)
(Ar. 22:13,14:-)
707

IV
I
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
I
I
II
I
IIIb
I
I
II
IIIb
IIIb
IIIb
II
IIIa
I
IV
I
IIIa
IV
II
IIIa
IV
IIIb
I
I
I
I
II
I
I
I
I
I
I
II
I
II
I
I
I
I
I
708
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
Section V
SEROTYPE
Diourbel
Keve
Jambur
Mountmagnet
Ibaragi
Ruiru
Wandsbek
Gambaga
Baguida
Soesterberg
Gwaai
Solna
Dakar
Bakau
Seattle
Honelis
Dibra
Moero
Ashanti
Bokanjac
Soumbedioune
Langford
Kaltenhausen
Hermannswerder
Eberswalde
Halle
Dresden
Wedding
O ANTIGENS
PHASE 1
PHASE 2
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
g,z51
i
i
i
k
k
l,v
l,v
l,w
l,z28
m,t
r
r
y
y
z
z10
z10
z10
z10
z29
z35
z36
z4,z23
z4,z23
z4,z23
z4,z24
z4,z24
z4,z32
z65
a
a
a
a
a
a
a
b
b
b
b
b
b
b
c
c
c
c
c
1,2
1,5,7
e,n,x,z15
e,n,x,z15
z
z
z57
e,n,z15
z
1,2
e,n,x
e,n,x,z15
z
z53
[z6]
e,n,z15
e,n,x,z15
1,5
1,6
1,7
e,n,x
e,n,x
e,n,z15
z6
1,5
1,6
1,7
e,n,x
e,n,x
e,n,z15
z6
1,5
1,6
1,7
e,n,x
e,n,z15
NOTE
(Ar. 22:33:30)
(Ar. 22:33:28)
(Ar. 22:29:28)
(Ar. 22:29:31)
(Ar. 22:23:31)
(Ar. 22:23:40)
(Ar. 22:24:31)
CDC does not have this.

(Ar. 22:27:28)
(Ar. 22:27:31)
(Ar. 22:27:25)
(Ar. 22:16,17,18:-)
(Ar. 22:1,2,5:- and 22:1,2,6:-)
(Ar. 22:1,3,11:-)
(Ar. 22:32:28)
The Difco Manual
Section V
I
I
I
I
I
I
I
I
I
II
I
I
I
I
II
II
II
I
II
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
II
I
I
II
I
I
I
II
I
I
I
The Difco Manual
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
SEROTYPE
Techimani
Amoutive
Hatfield
Mundonobo
Mocamedes
Patience
Cullingworth
Kpeme
Gozo
Friedrichsfelde
Yardley
Abadina
Croft
Llandudno
Ona
Doorn
Cotham
Volksmarsdorf
Dieuppeul
Warnemuende
Kuessel
Douala
Guildford
Ilala
Adamstown
Ikeja
Taunton
Ank
Leoben
Vitkin
Nashua
Ramsey
Catalunia
Penilla
Fajara
Morillons
Vinohrady
Bassadji
Kibusi
Oevelgoenne
Fairfield
Banco
Chicago
O ANTIGENS
PHASE 1
PHASE 2
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
c
d
d
d
d
d
d
e,h
e,h
e,n,x
f,g
g,m
g,m
g,m,s
g,m,t
g,m,t
g,[m],[s],t
g,s,t
g,s,t
i
i
i
i
i
i
i
k
k
k
k
k
k
l,v
l,v
l,v
l,w
l,z13,z28
l,z13,z28
l,z28
l,z28
m,t
m,t
m,t
r
r
r
r
r,i
r,[i]
z6
1,5
1,6
1,7
e,n,x
e,n,z15
l,w
1,7
e,n,z15
1,7
1,6
[e,n,z15]
[e,n,z15]
e,n,x
z39
1,5
e,n,x
1,2
1,5
1,6
1,7
e,n,x
e,n,z15
l,w
1,2
1,5
1,6
1,7
e,n,x
e,n,z15
1,5
e,n,x
e,n,z15
1,6
1,5
e,n,z15
1,5
e,n,x
1,6
[e,n,x]
[e,n,z15]
1,6
e,n,x
e,n,z15
l,w
1,7
1,5
NOTE
709

Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
NOTE
Pomona may possess H phases Rz60, Rz70 or Rz80.
I
I
I
I
M
M
M
M
Sanktgeorg
Oskarshamn
Nima
Pomona
28
28
28
28
r,[i]
y
y
y
e,n,z15
1,2
1,5
1,7
I
I
I
I
I
II
I
I
I
II
I
I
I
I
I
I
I
IIIb
IIIb
I
II
II
I
I
I
I
I
I
I
I
I
I
I
II
I
I
I
I
II
I
I
II
I
I
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Kitenge
Telaviv
Shomolu
Selby
Vanier
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
28
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
y
y
y
y
z
z
z
z
z
z
z10
z10
z10
z10
z10
z10
z10
z10
z10
z29
z29
z29
z35
z35
z35
z38
z4,z23
z4,z23
z4,z23
a
a
a
a
a
b
b
b
b
b
c
c
c
d
e,h
e,n,x
e,n,z15
l,w
z6
1,5
1,5
1,6
1,7
e,n,z15
z39
1,2
1,5
1,6
1,7
e,n,x
e,n,z15
l,w
z
z:[z57]
1,5
e,n,x
1,6
1,7
e,n,z15
[e,n,z15]
1,5
1,6
[e,n,z15]
1,2
1,5
e,n,x
e,n,z15
z39
1,2
1,5
e,n,x
e,n,z15
z6
1,7
e,n,z15
z39
1,5
1,2
710
Doel
Ezra
Brisbane
Ceres
Rogy
Farakan
Libreville
Malaysia
Umbilo
Luckenwalde
Moroto
Djermaia
Konolfingen
Babili
Santander
Aderike
Cannobio
Teltow
Babelsberg
Overvecht
Zehlendorf
Guarapiranga
Doulassame
Odijk
Louga
Aschersleben
Urbana
Neudorf
Zaire
Morningside
Messina
Livulu
(Ar. 35:27:31)
(Ar. 35:27:31:[40])
The Difco Manual
Section V
I
I
II
I
I
II
I
I
I
I
I
I
I
I
I
II
I
I
I
I
I
II
II
I
I
I
I
I
I
I
I
I
I
I
I
I
I
II
I
I
II
I
I
I
I
II
I
I
I
The Difco Manual
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
O
O
O
O
O
O
O
O
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
Torhout
Giessen
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
30
35
35
35
35
35
35
35
35
e,h
g,m,s
g,m,s
g,m,[t]
g,s,t
g,t
g,z51
i
i
i
i
k
k
k
k
k
k
l,v
l,v
l,z13,z28
l,z13,z28
l,z28
m,t
r
r
y
y
y
y
y
z10
z10
z10
z29
z35
z35
z38
z39
z4,z23
z4,z24
z6
a
b
c
c
d
d
e,h
f,g
1,5
e,n,x
1,2
1,5
e,n,z15
l,w
1,2
1,5
1,6
e,n,x
e,n,x,z15
e,n,[x],z15
1,2
1,5
1,6
e,n,z15
z6
1,2
1,5
1,2
1,5
1,6
e,n,x
e,n,z15
1,2
1,5
e,n,z15
1,6
e,n,z15
1,7
1,6
e,n,z15
1,5
[e,n,z15]
1,5
l,w
z6
Godesberg
Sternschanze
Slatograd
Wayne
Landau
Morehead
Mjordan
Soerenga
Hilversum
Ramatgan
Aqua
Angoda
Odozi
Ligeo
Donna
Ockenheim
Morocco
Grandhaven
Gege
Matopeni
Bietri
Steinplatz
Baguirmi
Nijmegen
Sada
Senneville
Kumasi
Aragua
Kokoli
Wuiti
Ago
Stoneferry
Bodjonegoro
Umhlatazana
Tchad
Gouloumbo
Yolo
Dembe
Gassi
Adelaide
NOTE
Sternschanze may possess H phase Rz59.
Adelaide may possess H phase Rz27.
711

I
II
I
I
I
II
II
IIIa
IIIb
I
I
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
IIIb
IIIb
IIIb
IIIb
IIIb
II
I
II
IIIb
I
IIIb
IIIb
IIIb
I
I
IIIb
I
I
IIIa
II
IIIa
I
I
IIIa
I
IIIa
IIIb
IIIb
IIIb
IIIb
II
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
P
712
Section V
SEROTYPE
Ealing
Ebrie
Anecho
Agodi
Gambia
Bandia
Monschaui
Massakory
Camberene
Enschede
Ligna
Widemarsh
Utbremen
Haga
Alachua
Westphalia
O ANTIGENS
PHASE 1
PHASE 2
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
g,m,s
g,m,s,t
g,m,t
g,s,t
g,t
g,t
g,t
g,z51
i
i
i
i
i
i
k
k
k
1,5
z42
e,n,x,z15
e,n,z15
l,w
z
z35
z53
e,n,x,z15
z
z53
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
38
(k)
(k)
l,v
l,v
l,v
l,z28
m,t
m,t
r
r
r
r
r
z10
z10
z10
z10
z29
z29
z29
z36
z38
z4,z23
z4,z23
z4,z24
z4,z32
z52
z52
z52
z52
b
z
z35
1,5,7
e,n,x,z15
z35
e,n,x,z15
l,w
z
z35
z61
1,5
l,w
z35
z6
e,n,x
1,5,7
e,n,x,z15
z
z35
1,2
NOTE
(Ar. 20:13,14:-)
(Ar. 20:33:28)
(Ar. 20:33:31)
(Ar. 20:33:21)
(Ar. 20:33:25)
(Ar. 20:29:28)
(Ar. 20:29:31)
(Ar. 20:29:25). May possess H phase
Rz50 (Ar.42).
(Ar. 20:22:31)
(Ar. 20:22:21)
(Ar. 20:23:30)
(Ar. 20:23:28)
(Ar. 20:23:21)
(Ar. 20:24:28)
(Ar. 20:24:31)
(Ar. 20:24:21)
(Ar. 20:24:41)
(Ar. 20:27:21)
(Ar. 20:16,17,18:-)
(Ar. 20:17,20:-)
Alachua may possess H phase Rz37 or Rz45.
(Ar. 20:1,2,6:-)
(Ar. 20:1,7,8:-)
(Ar. 20:26:30)
(Ar. 20:26:28)
(Ar. 20:26:31)
(Ar. 20:26:21)
The Difco Manual
Section V
I
I
I
II
I
I
I
II
IIIa
IV
I
I
IIIb
IIIb
I
I
I
I
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
I
I
I
IIIb
IIIb
IIIb
I
I
IIIb
I
IIIb
IIIb
IIIb
I
I
I
I
I
IIIb
IIIb
I
IIIa
IV
The Difco Manual
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
SEROTYPE
Rittersbach
Sheffield
Kidderminster
Carletonville
Thiaroye
Kasenyi
Korovi
Foulpointe
Mgulani
Lansing
Echa
Mango
Inverness
Njala
Alger
Kimberley
Roan
Rothenburgsort
Lindi
Emmastad
Freetown
Colombo
Perth
Stachus
Neunkirchen
Klouto
O ANTIGENS
PHASE 1
PHASE 2
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
b
c
c
d
e,h
e,h
g,m,[s]
g,t
g,z51
g,z51
i
i
i
i
k
k
k
k
k
k
k
(k)
(k)
(k)
(k)
(k)
l,v
l,v
l,v
l,v
l,v
l,v
m,t
r
r
r
r
r
r
y
y
y
z
z10
z10
z10
z38
z4,z23
z4,z23
e,n,z15
1,5
1,6
[1,5]
1,2
1,5
1,2
1,5
z
z53
1,2
1,5
1,6
e,n,x
e,n,x,z15
z
z53
1,5,7
z
z35
z54
z55
1,2
1,5
e,n,x
z
z35
z53:[z54]
1,5
1,5,7
1,6
e,n,x,z15
z:[z57]
z35
1,5
1,6
e,n,x
z
z53
NOTE
(Ar. 16:13,14:-)
(Ar. 16:33:31)
(Ar. 16:33:25)
(Ar. 16:29:28)
(Ar. 16:29:31)
(Ar. 16:29:25)
(Ar. 16:22:30)
(Ar. 16:22:31)
(Ar. 16:22:34)
(Ar. 16:22:37)
(Ar. 16:23:31)
(Ar. 16:23:21)
(Ar. 16:23:25:[34])
(Ar. 16:24:30)
(Ar. 16:24:28)
(Ar. 16:24:31:[40])
(Ar. 16:24:21)
(Ar. 16:27:31)
(Ar. 16:27:25)
(Ar. 16:1,2,6:-)
713

I
I
IIIb
IIIb
IIIb
IIIb
P
P
P
P
P
P
IIIa
IIIb
II
II
I
I
II
I
I
II
II
I
I
I
II
I
I
II
II
II
I
P
P
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
I
I
I
I
II
II
I
II
II
I
I
I
I
I
II
II
II
I
I
Q
Q
Q
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
714
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
Yoff
Bangkok
38
38
38
38
38
38
z4,z23
z4,z24
z47
z52
z52
z53
1,2
z53
z35
z53
38
38
39
39
39
39
39
39
39
39
39
39
39
39
39
39
39
39
39
39
39
z61
z61
a
b
b
c
d
e,h
e,n,x
g,m,t
i
i
k
l,v
l,v
l,v
l,z28
l,z28
m,t
Rz48
z53
1,7
z39
1,2
l,w
e,n,x
1,5
[1,5]
1,7
1,5
e,n,x
1,5
1,5
e,n,x
e,n,z15
e,n,x
z39
e,n,x
1,5
39
39
39
40
1,40
40
40
1,40
40
40
40
1,40
1,40
40
1,40
1,40
40
1,40
1,40
y
y
z10
a
a
a
a
a
b
b
b
b
b
b
c
c
d
d
e,h
1,2
1,5
1,5
1,5
z39
z6
z6
1,5
1,7
e,n,x
e,n,z15
z6
e,n,x,z15
z39
1,5
1,2
Wandsworth
Abidjan
Logone
Mara
Hofit
Cumberland
Champaign
Kokomlemle
Oerlikon
Mondeor
Cook
Anfo
Windermere
Hegau
Shikmonah
Springs
Greiz
Riogrande
Saugus
Johannesburg
Duval
Benguella
Suarez
Ottershaw
Driffield
Tilene
NOTE
(Ar. 16:39:25)
(Ar. 16:26:21)
(Ar. 16:26:25)
(Ar. 16:25:-). May possess H phase Rz50
(Ar. 42) or Rz76 (Ar. Rz76). CDC does not
have monophasic.
(Ar. 16:41:-)
(Ar. 16:41:25)
Champaign may possess H phase Rz48
IP combined Cook with Champaign (39:k:1,5).

The name Cook has been dropped.
The Difco Manual
Section V
II
II
I
II
II
II
II
II
II
II
IV
IIIb
IIIb
I
I
I
II
IIIb
II
IIIb
I
I
IIIb
IIIb
I
I
I
II
I
II
IV
II
II
I
II
I
II
II
I
II
IIIb
I
I
IIIa
V
II
I
IIIa
II
The Difco Manual
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
SEROTYPE
Bijlmer
Alsterdorf
Boksburg
Seminole
Goulfey
Allandale
Hann
Sunnydale
Millesi
Canary
Overchurch
Tiko
Bukavu
Santhiaba
Odienne
Bulawayo
Casamance
Nowawes
Trotha
Omifisan
Fandran
Yekepa
O ANTIGENS
PHASE 1
PHASE 2
1,40
1,40
1,40
1,40
40
1,40
1,40
1,40
1,40
40
1,40
40
40
1,40
1,40
40
1,40
40
40
40
1,40
40
40
40
1,40
40
1,40
1,40
40
1,40
40
40
1,40
40
1,40
40
1,40
40
40
1,40
40
40
40
40
1,40
1,40
1,40
40
40
e,n,x
e,n,x,z15
g,m
g,m,[s],t
g,[m],s,[t]
g,[m],[s],t
g,t
g,t
g,t
g,t
g,z51
g,z51
i
k
k
k
k
k
k
k
l,v
l,v
l,v
l,v
l,w
l,z13,z28
l,z28
l,z28
l,z28
l,z28
m,t
m,t
m,t
y
z
z
z
z
z
z
z10
z10
z29
z29
z35
z35
z35
z35
z39
1,[5],7
1,6
[1,5]
e,n,x
z42
[e,n,x]
1,5
e,n,x,z15
z39
[e,n,x,z15]
1,5,7
1,5
1,6
e,n,x
e,n,x,z15
z53
z6
z:z57
1,2
1,6
z
z53
[1,2]
1,2
1,5
1,5:z42
1,6
z39
z39
z42
1,5
1,5
e,n,x
z39
z42
z6
z6
z35
z6
e,n,x,z15
e,n,z15
1,5:z42
NOTE
(Ar. 10a,10b:13,14:[28])
(Ar. 10a,10b:33:30)
(Ar. 10a,10b:29:25)
(Ar. 10a,10b:29:31:40)
(Ar. 10a,10b,(10c):23:31)
(Ar. 10a,10b:23:25)
(Ar. 10a,10b:27:21)
(Ar. 10a,10b:16,18:-)
(Ar. 10a,10b:17,20:-)
715

Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
1,40
40
40
z39
z39
z4,z23
1,6
1,7
II
II
IIIa
R
R
R
Grunty
IV
IIIa
IV
II
IIIa
R
R
R
R
R
Sachsenwald
1,40
40
Degania var. subsp. IV 40
Degania
40
40
z4,z23
z4,z24
z4,z24
z4,z24
z4,z32
[z39]
IIIa
IV
I
II
II
II
V
II
I
II
II
I
VI
I
I
IIIb
II
I
II
II
II
IIIa
I
I
I
II
II
IIIb
II
I
I
II
II
I
I
I
II
II
I
R
R
R
R
R
R
R
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
40
1,40
40
1,40
1,40
1,40
40
41
Burundi
41
Vietnam var. subsp. II 41
41
Vaugirard
41
41
Sica
41
Vietnam
41
41
41
Egusi
41
Hennepin
41
41
Lethe
41
41
Samaru
41
Verona
41
Ferlo
41
41
41
41
41
Leatherhead
41
Lubumbashi
41
Dubrovnik
41
Negev
41
Leipzig
41
Landala
41
Inpraw
41
Lurup
41
Lichtenberg
41
Lodz
41
z4,z32
z4,z32
z41
z42
[z42]
z6
z81
a
b
b
b
b
b
b
c
c
d
d
g,m,s,t
g,t
g,z51
i
i
k
k
k
(k)
l,z13,z28
m,t
r
z
z10
z10
z10
z10
z10
z10
z29
1,2
1,6
1,(5),7
1,5
1,6
[1,5]
1,6
1,7
e,n,z15
z6
e,n,x,z15
[z6]
[1,5]
z6
z6
1,5
1,6
1,6
1,6
[z6]
[z35]
e,n,x,z15
1,6
1,5
1,5
1,2
1,5
1,6
e,n,x
e,n,x,z15
[z6]
716
Bern
Karamoja
NOTE
(Ar. 10a,10b:1,2,5:-; 10a,10b:1,2,5,6:-;

and 10a,10b:1,2,6:-)
(Ar. 10a,10b:1,3,11:-)
(Ar. 10a,10b:1,2,10:-; 10a,10c:1,2,10:-;

and 10a,10b:1,7,8:-)
H z81 was formerly H a in S. bongori.
(Ar. 13:32:28)
(Ar. 13:13,14:-)
(Ar. 13:22:[21])
The Difco Manual
Section V
IIIa
IV
I
IIIa
I
IIIa
IV
I
IIIa
IIIa
I
IIIa
IV
I
II
I
II
I
I
II
I
I
II
II
II
IIIa
IV
I
I
I
I
IIIb
I
IIIb
IIIb
IIIb
I
IIIb
IIIb
II
IIIb
I
IIIb
IIIb
II
II
I
II
II
The Difco Manual
S
S
S
S
S
S
S
S
S
S
S
S
S
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
SEROTYPE
O ANTIGENS
41
41
Ahoutoue
41
41
Offa
41
41
Waycross var. subsp. IV41
Waycross
41
41
41
Ipswich
41
41
41
Faji
1,42
Chinovum
42
Orbe
42
Uphill
42
Tomegbe
1,42
Egusitoo
1,42
42
Antwerpen
1,42
Kampala
1,42
42
42
Fremantle
42
42
1,42
Maricopa
1,42
Borromea
42
Kaneshie
1,42
Middlesbrough
1,42
42
Haferbreite
42
42
42
42
Gwale
1,42
42
42
Portbech
42
42
Coogee
42
42
42
1,42
42
Waral
1,42
42
Nairobi
42
PHASE 1
PHASE 2
z29
z29
z35
z36
z38
z4,z23
z4,z23
z4,z23
z4,z23,z32
z4,z24
z4,z24
z4,z32
z52
a
b
b
b
b
b
b
c
c
d
[e,n,x]
(f),g,t
g,z51
g,z51
g,z51
i
i
i
k
k
k
k
k
k
(k)
l,v
l,v
l,v
l,v
l,v
l,v
l,w
l,[z13],z28
m,t
m,t
r
1,6
[e,n,z15]
[1,5]
e,n,z15
1,5
1,6
e,n,x,z15
e,n,z15
z6
z6
e,n,z15
z6
z6
1,6
1,5
1,6
l,w
z6
[1,6]
e,n,x,z15
z
z35
z6
z35
1,5,7
e,n,x,z15
e,n,x,z15
e,n,z15
z
z53
e,n,x
[z6]
[e,n,x,z15]
NOTE
(Ar. 13:16,17,18:-)
(Ar. 13:17,20:-)
(Ar. 13:1,2,5:- and 13:1,2,6:-)
(Ar. 13:1,6,7,9:-)
(Ar. 13:1,3,11:-)
(Ar. 13:1,6,7:- and 13:1,7,8:-)
(Ar. 15:13,14:-)
(Ar. 15:29:-)
(Ar. 15:29:28)
(Ar. 15:29:31)
(Ar. 15:29:21)
(Ar. 15:22:21)
(Ar. 15:23:30)
(Ar. 15:23:28)
(Ar. 15:23:31)
(Ar. 15:23:25)
717

IIIb
I
I
IIIb
IIIb
I
I
II
I
II
I
II
IIIb
T
T
T
T
T
T
T
T
T
T
T
T
T
II
II
IIIb
IIIb
IIIb
I
II
IIIb
I
I
I
I
IV
I
IIIa
I
I
I
IIIa
IV
I
IIIb
II
I
I
II
II
II
I
I
II
II
II
II
II
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
U
U
U
U
U
U
U
U
U
U
U
U
718
Section V
SEROTYPE
Sipane
Brive
Spalentor
Harvestehude
Detroit
Ursenbach
Rand
Melbourne
Nuernberg
Loenga
Djama
Kahla
Tema
Weslaco
Vogan
Gera
Broc
Toricada
Taset
Graz
Berkeley
Kommetje
Montreal
Orleans
O ANTIGENS
PHASE 1
PHASE 2
42
1,42
1,42
42
42
1,42
1,42
42
1,42
42
42
42
42
r
r
r
r
r
y
y
z
z
z
z
z
z10
e,n,z15
l,w
z
z53
e,n,z15
z6
1,5
1,6
e,n,x,z15
e,n,z15
z6
42
42
42
42
42
1,42
42
42
1,42
1,42
1,42
42
42
1,42
42
1,42
42
1,42
42
1,42
1,42
42
42
43
43
43
43
43
43
43
43
43
43
43
43
z10
z10
z10
z10
z10
z10
z10
z10
z29
z35
z35
z36
z36
z38
z4,z23
z4,z23
z4,z23
z4,z24
z4,z24
z4,z24
z41
z52
z6
a
a
a
a
b
c
d
d
d
d
e,n,x,z15
e,n,x,z15
1,2
e,n,x,z15
e,n,x,z15
z
z35
z6
z6
z67
[1,5]
1,6
z6
z6
[1,6]
e,n,z15
z
1,6
1,2
1,5
1,5
z6
z42
1,5
1,5
e,n,x,z15
z39
z42
1,(5),7
1,6
NOTE
(Ar. 15:24:-). May possess H phase Rz50 (Ar. 42).
(Ar. 15:24:31)
(Ar. 15:24:25)

(Ar. 38) and Rz50 (Ar. 42).
(Ar. 15:27:28)
(Ar. 15:27:31)
(Ar. 15:27:21)
(Ar. 15:27:46)
(Ar. 15:1,2,5:- and 15:1,2,6:-)
(Ar. 15:1,3,11:-)
(Ar. 15:26:31)
The Difco Manual
Section V
I
II
II
II
IIIa
IV
II
I
I
I
I
IIIb
IIIb
I
II
IIIb
IIIb
IIIb
I
I
I
II
I
I
I
IV
II
II
I
IIIa
IV
I
IIIa
IV
IV
IIIa
IV
IV
II
IIIb
I
I
I
I
I
I
I
I
I
The Difco Manual
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
V
V
V
V
V
V
V
V
V
SEROTYPE
Milwaukee
Mosselbay
Veddel
Mbao
Voulte
Thetford
Ahuza
Sudan
Farcha
Kingabwa
Ogbete
Arusha
Adana
Makiling
Ahepe
Volksdorf
Irigny
Houten
Tuindorp
Bunnik
Niakhar
Tiergarten
Niarembe
Sedgwick
Madigan
Quebec
Bobo
Kermel
Fischerstrasse
O ANTIGENS
PHASE 1
PHASE 2
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
44
44
44
44
44
44
44
44
44
f,g,[t]
g,m,[s],t
g,t
g,t
g,z51
g,z51
g,z62
i
i
k
k
k
l,v
l,z13
l,z13,z28
r
r
r
y
y
z
z
z
z10
z29
z29
z29
z29
z35
z36
z36,z38
z38
z4,z23
z4,z23
z4,z23
z4,z24
z4,z24
z4,z32
z42
z52
a
a
a
b
c
c
d
d
d
[z42]
1,5
e,n,x
1,2
e,n,x
1,2
1,5
z
z53:[Rz56]
1,5
e,n,x,z15
z
z53
1,2
1,5
1,5
1,5
e,n,z15
1,5
e,n,x
z42
1,6
[1,5,7]
z53
1,5
e,n,x
l,w
e,n,z15
1,5
e,n,z15
1,5
e,n,x
e,n,z15
NOTE
(Ar. 21:13,14:-)
(Ar. 21:29:31)
(Ar. 21:23:25:[38])
(Ar. 21:24:28)
(Ar. 21:24:31)
(Ar. 21:24:25)
(Ar. 21:17,20:-)
(Ar. 21:1,2,5:- and 21:1,2,6:-)
(Ar. 21:1,3,11:-)
(Ar. 21:26:25)
719

I
II
I
I
I
II
I
IV
I
I
I
I
V
I
I
I
I
I
I
IV
II
IV
I
V
II
I
II
IIIa
IV
I
IIIa
I
IIIa
IV
IIIa
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
V
IV
VI
I
II
I
I
I
I
I
I
I
II
II
V
W
W
W
W
W
W
W
W
W
W
W
W
720
Section V
SEROTYPE
Palamaner
Vleuten
Gamaba
Splott
Carswell
Maritzburg
Lawra
Malika
Muguga
Camdeni
Brefet
Bolama
Uhlenhorst
Guinea
Llobregat
Zinder
Koketime
Clovelly
Kua
Ploufragan
Christiansborg
Lohbruegge
Vrindaban
Meekatharra
Ejeda
Riverside
Fomeco
Deversoir
Dugbe
Karachi
Warmsen
Suelldorf
Windhoek
Bremen
O ANTIGENS
PHASE 1
PHASE 2
1,44
1,44
44
1,44
44
44
44
44
1,44
44
44
44
44
44
44
44
1,44
1,44
44
44
44
44
44
44
1,44
44
44
44
44
1,44
44
44
44
44
44
d
e,n,x
f,g
g,m,[s]
g,s,t
g,t
g,z51
g,z51
i
k
l,z28
m,t
r
r
z
z
z10
z10
z29
z29
z29
z36,[z38]
z38
z39
z39
z4,z23
z4,z23
z4,z23
z4,z23
z4,z23
z4,z23,z32
z4,z24
z4,z24
z4,z24
z4,z32
z35
1,6
z42
e,n,z15
e,n,z15
1,5
e,n,z15
e,n,x
l,w
[1,7]
e,n,x
e,n,x:z42
[e,n,x,z15]
e,n,z15
44
45
45
45
45
45
45
45
45
45
45
45
45
z4,z32
a
a
a
b
b
c
d
d
d
f,g
g,m,s,t
g,m,s,t
e,n,x
e,n,z15
z10
1,5
e,n,z15
e,n,x
1,6
e,n,x
e,n,z15
1,5
e,n,x
NOTE
(Ar. 1,3:1,2,5:- and 1,3:1,2,6:-)
(Ar. 1,3:1,6,7,9:-)
(Ar. 1,3:1,3,11:-)
(Ar. 1,3:1,2,10:- and 1,3:1,7,8:-). IP calls
z4,z23,z32, Ar. 1,2,10.
The Difco Manual
Section V
I
II
I
IIIa
IV
I
I
I
I
I
II
I
II
I
II
I
IIIa
II
II
II
I
I
IV
IIIa
IV
I
IIIa
IIIa
II
II
I
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
W
X
X
X
I
II
II
I
II
IIIb
I
IIIb
IIIb
IIIb
II
I
II
II
I
I
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
The Difco Manual
SEROTYPE
Tornow
Perinet
Binningen
Verviers
Casablanca
Cairns
Imo
Apapa
Kofandoka
Yopougon
Klapmuts
Jodhpur
Lattenkamp
Balcones
Transvaal
Bilthoven
Saka
Wenatchee
Phoenix
Khami
Sya
Kodjovi
Stellingen
Quimbamba
Sljeme
Anie
O ANTIGENS
PHASE 1
PHASE 2
45
45
45
45
45
45
45
45
45
45
45
45
45
45
45
45
45
45
45
45
45
45
45
45
45
45
45
45
47
47
47
g,m,[s],[t]
g,m,t
g,s,t
g,z51
g,z51
k
k
k
l,v
m,t
m,t
r
z
z
z
z29
z29
z29
z29
z29
z35
z36
z36,z38
z4,z23
z4,z23
z4,z24
z4,z24
z4,z32
a
a
b
e,n,x,z15
1,5
1,7
e,n,z15
[e,n,z15]
1,5
e,n,z15
1,5
e,n,z15
z39
1,5
e,n,x
z42
1,5
[1,5]
e,n,x,z15
47
47
47
47
47
47
47
47
47
47
47
47
47
47
1,47
47
b
b
b
b
b
c
c
c
c
c
d
d
d
e,n,x,z15
f,g
(g),m,t
1,2
1,5
[e,n,x,z15]
z6
z6
1,5,7
[1,6]
e,n,x,z15:[z57]
z
z35
e,n,x,z15
[e,n,x]
z39
1,6
NOTE
(Ar. 11:13,14:-)
(Ar. 11:16,18:-)
(Ar. 11:1,2,5:-)
(Ar. 11:1,3,11:-)
(Ar. 11:1,7,8:-)
IP combined Saka with Sya (47:b:z6) and

called it Sya.
(Ar. 28:32:30)
Kodjovi may possess H phase Rz78.
(Ar. 23:32:28 and 28:32:28:[40])
(Ar. 28:32:31)
(Ar. 28:32:21)
IP combined Anie with Mesbit

(47:m,t:[e,n,z15]) and called it Mesbit.
721

Section V
SEROTYPE
I
II
IIIa
IIIb
X
X
X
X
Luke
I
IIIb
IIIb
IIIb
I
I
IIIb
I
IIIb
I
IIIb
IIIb
IIIb
IV
IIIb
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Bergen
IIIa
IIIb
IIIb
IIIb
IIIb
I
I
IIIa
I
IIIb
IIIb
IIIb
IIIb
X
X
X
X
X
X
X
X
X
X
X
X
X
IIIb
IIIb
X
X
I
I
I
I
II
II
IIIb
IIIb
IIIb
I
IIIa
X
X
X
X
X
X
X
X
X
X
X
722
Staoueli
Bootle
Dahomey
Lyon
Teshie
Mesbit
Dapango
Moualine
Blitta
Mountpleasant
Kaolack
Chersina
Ekpoui
O ANTIGENS
PHASE 1
PHASE 2
1,47
47
47
47
g,m
g,t
g,z51
i
e,n,x
e,n,x,z15
47
47
47
47
47
47
47
47
47
47
47
47
47
47
47
i
i
i
i
k
k
k
k
k
k
k
k
k
l,v
l,v
e,n,z15
z
z35
z53:[z57]
1,2
1,5
1,5,7
1,6
e,n,x,z15
e,n,z15
z
z35
z53
1,5,(7)
47
47
47
47
47
1,47
47
47
47
47
47
47
47
l,v
l,v
l,v
l,v
l,v
l,z13,z28
m,t
r
r
r
r
r
r
e,n,x,z15
z
z35
z53
z57
e,n,z15
[e,n,z15]
1,2
1,5,7
z
z35
z53
47
47
r
r
z53:Rz50:z60
z53:[z60]
47
47
47
47
47
47
47
47
47
47
47
y
y
z
z
z
z
z10
z10
z10
z29
z29
1,6
e,n,x
1,5
1,6
e,n,x,z15
z6
1,5,7
z
z35
NOTE
(Ar. 28:13,14)
(Ar. 28:33:31)
(Ar. 23:33:21 and 28:33:21)
(Ar. 23:33:25 and 28:33:25:[40])
(Ar. 28:29:30)
Dahomey may possess H phase Rz58.
(Ar. 28:29:28)
(Ar. 28:29:31)
(Ar. 23:29:21)
(Ar. 23:29:25)
(Ar. 28:23:28)
(Ar. 23:23:31)
(Ar. 28:23:21)
(Ar. 28:23:25)
(Ar. 28:23:40)
(Ar. 23:24:-). CDC does not have this.

(Ar. 23:24:30)
(Ar. 23:24:31)
(Ar. 23:24:21 and 28:24:21)
(Ar. 23:24:25). May possess H phase Rz74 (Ar. Rz74)
(Ar. 28:24:25:42:44). Not in IP book.
(Ar. 23:24:25:[44]). May possess H phase Rz70
and Rz72 (Ar. Rz70 or Rz72).
(Ar. 28:27:30)
(Ar. 28:27:31)
(Ar. 28:27:21)
(Ar. 28:16,18:-)
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
47
47
47
47
47
47
47
47
47
47
47
47
47
47
47
48
48
48
48
48
48
48
48
z29
z35
z36
z38
z4,z23
z4,z23
z4,z23
z4,z23
z4,z24
z44
z52
z52
z52
z52
z6
a
a
a
a
b
b
c
d
e,n,x,z15
e,n,z15
[e,n,z15]
l,w
[z6]
1,5,7
e,n,x,z15
z
z35
1,6
1,5,7
z35
z39
z6
[z6]
z
1,11
48
48
48
48
48
48
48
48
d
d
e,h
e,n,x,z15
g,m,t
g,z51
g,z51
i
1,2
z6
1,5
z6
II
I
IV
I
IIIa
I
I
I
I
I
IIIb
IIIb
IIIb
IIIb
II
I
IIIb
II
II
V
II
IIIb
II
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Y
Y
Y
Y
Y
Y
Y
Y
II
II
I
II
II
IIIa
IV
IIIb
Y
Y
Y
Y
Y
Y
Y
Y
IIIb
IIIb
IIIb
IIIb
Y
Y
Y
Y
48
48
48
48
i
i
i
i
z35:[z57]
z53
z61
z:[z72]
IIIb
II
IIIb
I
IIIb
IIIb
IIIb
IIIb
Y
Y
Y
Y
Y
Y
Y
Y
48
48
48
48
48
48
48
48
k
k
k
k
k
k
k
(k)
1,5,(7)
e,n,x,z15
e,n,x,z15
e,n,z15
z
z35:[Rz75]
z53
z53
II
I
Y
Y
48
48
[k]
l,v
z39
1,5
The Difco Manual
Bingerville
Alexanderplatz
Tabligbo
Binche
Bere
Tamberma
Quinhon
Hisingen
Etosha
Hagenbeck
Fitzroy
Hammonia
Erlangen
Marina
Sydney
Dahlem
Sakaraha
Australia
NOTE
(Ar. 28:1,2,5:-)
Bere may possess H phase Rz45.
(Ar. 28:26:30)
(Ar. 28:26:28)
(Ar. 28:26:31)
(Ar. 28:26:21)
(Ar. 5:35:21). Not in 1992 IP, but is in Bergey.
(Ar. 29:32:31)
Etosha was not considered a new serotype by
Kauffmann and is not used.
(Ar. 5:13,14:-)
Sydney was formerly in subspecies II, but it
it now combined with Arizona 5:33:31. The
name Sydney has been dropped.
(Ar. 29:33:21:[40])
(Ar. 5:33:25)
(Ar. 5,29:33:41)
(Ar. 5,29:33:31:[z72]). CDC does not have
z72 strain.
(Ar. 5:29:30)
(Ar. 5:29:28)
(Ar. 5,29:29:31)
(Ar. [5:29:21:Rz75]). CDC does not have Rz75.
(Ar. 5,29:29:25)
(Ar. 5:22:25 and Ar. 5,29:22:25).
Called 5:22:25 by IP.
723

Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
NOTE
(Ar. 5:23:30). May possess H phase Rz47

or Rz50 (Ar. 39 or 42).
(Ar. 5,29:23:31)
(Ar. 5:24:28)
(Ar. 5,29:24:31)
Toucra may possess H phase Rz58.
IIIb
48
l,v
1,5,(7)
IIIb
IIIb
IIIb
I
II
VI
II
I
IIIb
IIIb
II
IIIa
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
48
48
48
48
48
48
48
48
48
48
48
48
l,v
r
r
z
z
z10
z10
z10
z10
z10
z29
z29
z
e,n,x,z15
z
1,5
1,5
1,5
[1,5]
e,n,x
e,n,x,z15
z
IV
V
IIIb
IIIa
IV
V
IIIa
IV
IIIa
I
IIIa
IIIa
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
48
48
48
48
48
48
48
48
48
48
48
48
z29
z35
z35
z36
z36,[z38]
z39
z4,z23
z4,z23
z4,z23,z32
z4,z24
z4,z24
z4,z32
z52
IV
V
IIIb
IIIb
V
V
IV
IV
I
II
IV
I
II
II
IV
II
IIIb
IIIb
IIIb
IIIb
IIIb
Y
Y
Y
Y
Y
Y
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
48
48
48
48
48
48
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
z4,z32
z41
z52
z52
z65
z81
a
b
b
b
d
d
e,n,x
g,[m],s,t
g,z51
g,z62
i
i
i
k
k
e,n,x,z15
z
e,n,x
z6
1,5
1,7
[1,5]
e,n,x
1,5,7
e,n,x,z15
z
1,5,7
e,n,x,z15
724
Toucra
Ngozi
Isaszeg
Bongor
Djakarta
Balboa
Rochdale
Hemingford
Krugersdorp
Namib
Wassenaar
(Ar. 5:27:28)
(Ar. 5,29:27:31)
(Ar. 5:16,18). This is not in IP book, but is
on Rohdes list.
(Ar. 5:21:26)
(Ar. 5,29:17,20:-)
(Ar. 5:1,2,5:-; 5:1,2,5,6:-; and 5:1,6:-)

(Ar. 5:1,6,7,9:-). IP calls this 5:1,6,7:-.
(Ar. 5:1,3,11:-)
(Ar. 5:1,6,7:-; 5:1,7,8:-; and Ar. 5:1,2,10:-). IP calls
z4,z32, Ar. 1,7,8; and would call z4,z23,z32, Ar. 1,2,10.
(Ar. 29:26:28)
(Ar. 5:26:31)
Hemingford may possess H phase Rz82.
(Ar. 9a,9c:33:30)
(Ar. 9a,9c:33:28)
(Ar. 9a,9c:33:31)
(Ar. 9a,9c:29:30)
(Ar. 9a,9c:29:28)
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
II
IIIb
Z
Z
50
50
k
k
e,n,x:z42
z
IIIb
IIIb
Z
Z
50
50
k
k
z35
z53
II
IIIb
IIIb
I
IIIb
IIIb
Z
Z
Z
Z
Z
Z
50
50
50
50
50
50
k
(k)
(k)
l,v
l,v
l,v
z6
z
z35
1,2
e,n,x,z15
z
IIIb
II
II
II
IIIb
IIIb
IIIb
IIIb
Z
Z
Z
Z
Z
Z
Z
Z
50
50
50
50
50
50
50
50
l,v
l,w
l,z28
m,t
r
r
r
r
z35
e,n,x,z15:z42
z42
z6:z42
1,5,(7)
e,n,x,z15
z
z35
IIIb
50
z53
I
II
IIIb
Z
Z
Z
50
50
50
y
z
z10
1,6
e,n,x
z
IIIb
II
I
IIIa
IIIa
IIIa
IV
IIIa
Z
Z
Z
Z
Z
Z
Z
Z
50
50
50
50
50
50
50
50
z10
z10
z29
z29
z36
z4,z23
z4,z23
z4,z23,z32
z53
z6:z42
IIIa
IV
IIIa
Z
Z
Z
50
50
50
z4,z24
z4,z24
z4,z32
IV
II
IIIb
IIIb
IIIb
IIIb
II
I
Z
Z
Z
Z
Z
Z
51
51
50
50
50
50
50
50
51
51
z4,z32
z42
z52
z52
z52
z52
1,7
1,5,7
z
z35
z53
1,7
e,n,x
The Difco Manual
Seaforth
Fass
Atra
Dougi
Greenside
Hooggraven
Ivorycoast
Flint
Bonaire
Faure
Roggeveld
Tione
NOTE
(Ar. 9a,9b:29:31 and 9a,9c:29:31). Ar. 9a,9b

may possess H phase Rz50 (Ar. 42).
(Ar. 9a,9b:29:21)
(Ar. 9a,9b:29:25 and 9a,9c:29:25). IP and
Rohde only list the 9a,9c.
(Ar. 9a,9b:22:31)
(Ar. 9a,9b:22:21)
(Ar. 9a,9b:23:28)
(Ar. 9a,9b:23:31 and 9a,9c:23:31). IP only
lists 9a,9c.
(Ar. 9a,9c:23:21)
(Ar. 9a,9b:24:30)
(Ar. 9a,9c:24:28)
(Ar. 9a,9b:24:31 and 9a,9c:24:31).
(Ar. 9a,9b:24:21). May possess H phase Rz58
(Ar. Rz58). This is not in IP book, but is on
Rohdes list.
(Ar. 42). This is not in IP book, but is on
Rohdes list.
(Ar. 9a,9c:27:31). May possess H phase Rz56

(Ar. 38).
(Ar. 9a,9c:27:25)
(Ar. 9a,9b:16,18:-)
(Ar. 9a,9b:17,20:-)
(Ar. 9a,9b:1,2,5:- and 9a,9b:1,2,6:-)
(Ar. 9a,9b:1,2,10:-). Called 9a,9b:1,6,7:- by
IP and Rohde.
(Ar. 9a,9b:1,3,11:-)
(Ar. 9a,9b:1,2,10; 9a,9b:1,6,7:-; and
9a,9b:1,7,8:-). 9a,9b:1,2,10:- and 9a,9b:1,7,8:used by IP and Rohde.
(Ar. 9a,9b:26:30 and 9a,9c:26:30)

(Ar. 9a,9b:26:31 and 9a,9c:26:31)
(Ar. 9a,9b:26:21 and 9a,9c:26:21)
(Ar. 9a,9b:26:25 and 9a,9c:26:25)
725

IV
I
II
I
I
II
IIIa
I
I
IIIb
I
I
I
IIIb
I
II
I
I
I
I
I
II
IIIa
IV
IIIa
IIIa
I
I
I
I
II
IIIb
51
51
51
51
51
51
51
51
51
51
51
51
51
51
51
51
51
51
51
51
51
51
51
51
51
51
52
52
52
51
52
52
I
II
II
I
I
I
II
I
IIIb
IIIb
IIIb
IIIb
II
II
52
52
52
52
52
52
52
52
52
52
52
52
52
52
726
Section V
SEROTYPE
Karaya
Gokul
Meskin
Kabete
Dan
Harcourt
Overschie
Dadzie
Moundou
Askraal
Lutetia
Antsalova
Treforest
Lechler
Bergues
Harmelen
Uithof
Ord
Molesey
Flottbek
Utrecht
Butare
Derkle
Saintemarie
Bordeaux
Wilhemstrasse
O ANTIGENS
PHASE 1
PHASE 2
51
51
51
1,51
51
51
51
51
51
51
51
51
51
51
51
51
51
51
1,51
51
51
51
51
51
51
51
52
52
52
52
52
52
b
b
c
d
e,h
g,s,t
g,z51
i
k
k
l,v
l,v
l,v
l,v
l,z28
l,z28
r,i
z
z
z
z10
z29
z4,z23
z4,z23
z4,z24
z4,z32
a
a
b
b
c
c
1,5
[1,5]
1,2
e,n,x
1,5
e,n,z15
z35
1,2
1,5
e,n,x
z
1,5
[z6]
l,z13,z28
1,5
1,6
e,n,z15
1,5
e,n,x,z15
1,5
e,n,z15
1,5
[e,n,x]
k
k
52
52
52
52
52
52
52
52
52
52
52
52
52
52
d
d
d
e,h
e,h
g,t
g,t
k
k
k
(k)
l,v
z
z44
1,5
e,n,x,z15
z39
1,6
1,7
1,5
z35
z53
z35
z53
z39
1,5
NOTE
(Ar. 1,2:13,14:-)
(Ar. 1,2:29:21)
(Ar. 1,2:23:31)
(Ar. 1,2:1,2,5:- and 1,2:1,2,6:-)

(Ar. 1,2:1,3,11:-)
(Ar. 1,2:1,7,8:-)
(Ar. 31:32:29). This is not in IP book, but is

on Rohdes list.
(Ar. 31:29:21)
(Ar. 31:29:25)
(Ar. 31:22:21)
(Ar. 31:23:25)
IP combined Wilhemstrasse with Lobatsi
(52:z44:1,5,7). The name Wilhemstrasse has
been dropped.
The Difco Manual
Section V
II
IIIb
II
II
II
II
IIIa
IV
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
II
II
II
IIIb
IIIb
IIIb
52
52
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
II
IIIb
II
IIIb
IIIa
IV
IIIa
IV
IIIa
II
IIIa
IIIa
53
53
53
53
53
53
53
53
53
53
53
53
IIIb
IIIb
I
I
I
I
I
I
I
I
I
I
I
I
53
53
54
54
54
54
54
54
54
54
54
54
54
54
The Difco Manual
SEROTYPE
Lobatsi
Midhurst
Bockenheim
Humber
Tonev
Winnipeg
Rossleben
Borreze
Uccle
Poeseldorf
Ochsenwerder
Newholland
Czernyring
Steinwerder
Canton
Barry
O ANTIGENS
PHASE 1
PHASE 2
52
52
53
53
1,53
53
53
1,53
53
53
53
53
53
53
53
53
53
53
53
53
53
z44
z52
c
d
d
d
g,z51
g,z51
i
k
k
(k)
(k)
l,v
l,v
l,z28
l,z28
l,z28
r
r
r
1,5,7
z
1,5
1,5
z39
z42
z
e,n,x,z15
z
z
z35
e,n,x,z15
z35
e,n,x
z39
z6
z
z35
z68
53
53
53
53
53
1,53
53
53
53
53
53
53
z
z
z
z10
z29
z36,z38
z4,z23
z4,z23
z4,z23,z32
z4,z24
z4,z24
z4,z32
1,5
1,5,(7)
z6
z35
53
53
21,54
54
54
54
3,54
8,20,54
6,7,54
4,12,54
54
3,15,54
54
54
z52
z52
b
e,h
e,h
f,g,s
g,s,t
i
k
m,t
r
y
z10
z10
z35
z53
e,n,x
1,5
1,6
z6
1,5
1,5
1,5
e,n,x
e,n,z15
NOTE
(Ar. 31:26:31)
(Ar. 1,4:13,14:-)
(Ar. 1,4:33:31)
(Ar. 1,4:29:28)
(Ar. 1,4:29:31)
(Ar. 1,4:22:31)
(Ar. 1,4:22:21)
(Ar. 1,4:23:28)
(Ar. 1,4:23:21)
(Ar. 1,4:24:31)
(Ar. 1,4:24:21)
(Ar. 1,4:24:47). This was formerly called z56
(Ar. 38), but was changed to z68 (Ar. 47).
(Ar. 1,4:30:31)
(Ar. 1,4:27:21)
(Ar. 1,4:16,18:-)
(Ar. 1,4:1,2,5:- and 1,4:1,2,6:-)
(Ar. 1,4:1,6,7:- and 1,4:1,6,7,9:-)
(Ar. 1,4:1,3,11:-)
(Ar. 1,4:1,6,7:-). IP combined this with
53:z4,z23,z32:- (Ar. 1,4:1,6,7,9:-).
(Ar. 1,4:26:21)
(Ar. 1,4:26:25)
727

Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
Yerba
Tranoroa
Artis
54
55
56
56
56
56
56
56
56
56
56
56
57
57
57
57
57
57
57
57
57
57
57
z4,z23
k
b
d
e,n,x
l,v
l,z28
z
z10
z29
z4,z23
z4,z23,z32
a
a
b
b
c
d
g,[m],s,t
g,t
i
i
k
z39
1,7
z39
z6
e,n,x
z42
z6
1,7
e,n,z15
z:[z60]
1,5
z42
e,n,x,z15
z
e,n,x,z15
57
57
57
57
57
58
58
58
58
58
58
58
58
58
58
58
58
58
z10
z29
z39
z4,z23
z42
a
b
c
d
i
k
l,v
l,v
l,z13,z28
l,z13,z28
r
r
r
z
z42
e,n,x,z15
1,6:z53
z6
1,5
z6
z6
e,n,x,z15
z
e,n,x,z15
z35
1,5
z6
e,n,x,z15
z
z53
I
II
II
II
II
II
II
II
II
IIIa
IIIa
IIIa
II
I
I
I
IIIb
II
II
II
IIIb
IIIb
IIIb
54
55
56
56
56
56
56
56
56
56
56
56
57
57
57
57
57
57
57
57
57
57
57
IIIb
II
II
IV
II
II
II
II
II
IIIb
IIIb
IIIb
IIIb
II
II
IIIb
IIIb
IIIb
57
57
57
57
57
58
58
58
58
58
58
58
58
58
58
58
58
58
II
IIIb
IIIb
58
58
58
58
58
58
z10
z10
z10
1,6
e,n,x,z15
z53
II
II
58
58
58
58
z10
z39
z6
e,n,x,z15
728
Antonio
Maryland
Batonrouge
Locarno
Manombo
Tokai
Basel
NOTE
(Ar. 14:16,18:-)
(Ar. 14:1,2,5:- and 14:1,2,6:-)
(Ar. 14:1,6,7,9:-)
(Ar. 34:32:31:[44])
(Ar. 34:33:28)
(Ar. 34:33:31)
(Ar. 34:29:28). CDC does not have this
and not on Rohdes list.
(Ar. 34:27:31)
(Ar. 1,33:33:28)
(Ar. 1,33:29:31)
(Ar. 1,33:23:28)
(Ar. 1,33:23:21)
(Ar. 1,33:24:28)
(Ar. 1,33:24:31)
(Ar. 1,33:24:25). May possess H phase Rz47
(Ar. 39) or Rz57 (Ar. 40) or Rz70 (Ar. Rz70).
(Ar. 1,33:27:28)
(Ar. 1,33:27:25). May possess H phase
Rz50 (Ar. 42).
The Difco Manual
Section V
IIIb
IIIb
II
IIIb
IIIb
IIIb
IIIb
II
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
II
IIIb
IIIb
IIIa
IIIa
IIIa
IIIb
II
II
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
II
IIIb
IIIb
IIIb
II
V
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
The Difco Manual
58
58
58
59
59
59
59
59
59
59
59
59
59
59
59
59
59
59
59
59
59
59
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
61
61
61
SEROTYPE
Betioky
Setubal
Luton
O ANTIGENS
PHASE 1
PHASE 2
58
58
58
59
59
59
59
59
59
59
59
59
59
59
59
1,59
59
59
59
59
59
59
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
61
61
61
z52
z52
z6
c
i
i
i
k
k
(k)
(k)
(k)
l,v
l,v
r
z
z10
z10
z29
z36
z4,z23
z52
b
g,m,t
i
i
i
k
k
(k)
l,v
r
r
r
r
z
z10
z10
z10
z29
z41
z52
z52
z52
z52
c
c
i
z
z35
1,6
e,n,x,z15
e,n,x,z15
z
z35
(z)
z53
e,n,x,z15
z
z35
z
z53
z35
z6
z53
z57
[z53]
[1,16]
z6
e,n,x,z15
z35
z
z35
z53
z
e,n,x,z15
z
z35
z53
e,n,x
z
z35
z53
e,n,x
1,5,[7]
z
z35
z53
1,5,(7)
z35
e,n,x,z15
NOTE
(Ar. 1,33:26:31)
(Ar. 1,33:26:21)
(Ar. 19:32:28)
(Ar. 19:33:28)
(Ar. 19:33:31)
(Ar. 19:33:21)
(Ar. 19:29:25)
(Ar. 19:22:28)
(Ar. 19:22:31)
(Ar. 19:22:21)
(Ar. 19:23:31)
(Ar. 19:23:25)
(Ar. 19:24:21)
(Ar. 19:27:25)
(Ar. 19:27:40)
(Ar. 19:16,18:-)
(Ar. 19:17,20:-)
(Ar. 19:1,2,5:- and 19:1,2,6:-)
(Ar. 19:26:[25])

(Ar. 24:33:28)
(Ar. 24:33:21)
(Ar. 24:29:31)
(Ar. 24:29:21)
(Ar. 24:22:25)
(Ar. 24:23:31)
(Ar. 24:24:28)
(Ar. 24:24:31)
(Ar. 24:24:21)
(Ar. 24:24:25)
(Ar. 24:27:31)
(Ar. 24:27:21)
(Ar. 24:27:25)
(Ar. 24:26:30)
(Ar. 24:26:31)
(Ar. 24:26:21)
(Ar. 24:26:25)
(Ar. 26:32:30)
(Ar. 26:32:21)
(Ar. 26:33:28)
729

Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
Eilbeck
61
IIIb
61
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
i
i
i
k
k
(k)
l,v
l,v
l,v
r
r
r
r
z
z35
z53
1,5,(7)
z35
z53
1,5,7:[z57]
z
z35
1,5,7
z
z35
z53
IIIb
V
IIIb
IIIb
IIIb
IIIb
IIIa
IIIa
IIIa
IIIa
IIIa
IIIa
IIIa
IIIa
IIIa
II
IIIb
IIIb
IIIb
II
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
61
61
61
61
61
61
62
62
62
62
62
63
63
63
63
65
65
65
65
65
65
65
65
65
65
65
65
65
65
65
65
65
61
61
61
61
61
61
62
62
62
62
62
63
63
63
63
65
65
65
65
65
65
65
65
65
65
65
65
65
65
65
65
65
z10
z35
z52
z52
z52
z52
g,z51
z29
z36
z4,z23
z4,z32
g,z51
z36
z4,z23
z4,z32
c
c
c
g,t
i
(k)
(k)
(k)
l,v
l,v
l,v
l,v
r
z10
z10
z52
z35
1,5,7
z
z35
z53
1,6
1,5,7
z
z53
e,n,x,z15
z
z35
z53
e,n,x,z15
z
z35
z53
z35
e,n,x,z15
z
e,n,x,z15
730
NOTE
Eilbeck was formerly in subspecies II, but is

now combined with Arizona 26:33:31. The
name Eilbeck has been dropped.
(Ar. 26:33:31)
(Ar. 26:33:21)
(Ar. 26:33:25)
(Ar. 26:29:30)
(Ar. 26:29:21). CDC does not have this.
(Ar. 26:22:25)
(Ar. 26:23:30:[40])
(Ar. 26:23:31)
(Ar. 26:23:21)
(Ar. 26:24:30)
(Ar. 26:24:31)
(Ar. 26:24:21)
(Ar. 39).
(Ar. 26:27:21)
(Ar. 26:26:30)
(Ar. 26:26:31)
(Ar. 26:26:21)
(Ar. 26:26:25)
(Ar. 6:13,14:-)
(Ar. 6:17,18:-)
(Ar. 6:17,20:-)
(Ar. 6:1,2,5:-)
(Ar. 6:1,7,8:-)
(Ar. 8:13,14:-)
(Ar. 8:17,20:-)
(Ar. 8:1,2,5:-)
(Ar. 8:1,7,8:-)
(Ar. 30:32:30)
(Ar. 30:32:31)
(Ar. 30:32:25)
(Ar. 30:33:28)
(Ar. 30:22:31)
(Ar. 30:22:21)
(Ar. 30:22:25)
(Ar. 30:23:28)
(Ar. 30:23:31)
(Ar. 30:23:21)
(Ar. 30:23:25)
(Ar. 30:24:21)
(Ar. 30:27:28)
(Ar. 30:27:31)
(Ar. 30:26:28)
The Difco Manual
Section V
IIIb
IIIb
IIIb
V
V
V
V
V
I
65
65
65
66
66
66
66
66
67
SEROTYPE
Maregrosso
Brookfield
Malawi
Crossness
O ANTIGENS
PHASE 1
PHASE 2
65
65
65
66
66
66
66
66
67
z52
z52
z52
z35
z39
z41
z65
z81
r
z
z35
z53
1,2
O ANTIGENS
PHASE 1
PHASE 2
NOTE
(Ar. 30:26:31)
(Ar. 30:26:21)
(Ar. 30:26:25)
Appendix B
SEROTYPE
I
I
I
I
I
I
I
I
I
K
C2
M
F
F
Q
I
I
B
Aarhus
Aba
Abadina
Abaetetuba
Aberdeen
Abidjan
Ablogame
Abobo
Abony
18
6,8
28
11
11
39
16
16
1,4,5,12
z4,z23
i
g,m
k
i
b
l,z13,z28
i
b
z64
e,n,z15
[e,n,z15]
1,5
1,2
l,w
z6
z6
e,n,x
Abortusbovis
1,4,12,27
e,n,x
Abortuscanis
4,5,12
Rz5
I
I
I
I
II
I
I
I
I
I
I
I
I
I
I
I
I
I
B
B
E4
D2
G
E1
M
F
U
O
I
M
C1
G
C1
C2
H
B
Abortusequi
Abortusovis
Accra
Ackwepe
Acres
Adabraka
Adamstown
Adamstua
Adana
Adelaide
Adeoyo
Aderike
Adime
Adjame
Aequatoria
Aesch
Aflao
Africana
4,12
4,12
1,3,19
9,46
1,13,23
3,10
28
11
43
35
16
28
6,7
13,23
6,7
6,8
1,6,14,25
4,12
c
b
l,w
b
z4,z23
k
e,h
z10
f,g
g,m,[t]
z38
b
r
z4,z23
z60
l,z28
r,i
e,n,x
1,6
z6
[1,5]:z42
[1,7]
1,6
1,6
1,5
[e,n,z15]
1,6
1,6
[e,n,z15]
1,2
e,n,x
l,w
The Difco Manual
NOTE
IP combined Sladun (1,4,12,27:b:e,n,x) with

Abony to form Abony 1,4,[5],12,27:b:e,n,x
(gelatin neg.).
Gelatin pos., mucate pos.1-4 days. Abortusbovis
was combined with Abony (1,4,5,12:b:e,n,x),
gelatin neg. The name Abortusbovis was dropped.
Abortuscanis was combined with Paratyphi B
(1,4,[5],12:b:1,2) in 1938 and the name
Abortuscanis was dropped.
Adelaide may possess H phase Rz27.
731

732
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
6,7
4,12
16
1,13,23
3,10
30
35
1,4,[5],12
13,22
17
43
1,3,19
41
43
13,23
6,8
16
9,12
35
8
6,7
8,20
4,12
1,6,14,24
3,10
47
8
3,10
38
1,3,19
1,40
3,10
8,20
1,40
4,12
f,g,t
i
i
g,m,[t]
c
z38
g,t
f,g,s
z29
i
z35
d
z35
k
z4,z23
r
y
c
z4,z23
y
g,z51
z4,z24
z10
d
z
z38
c
f,g
l,v
a
k
b
g,s,t
g,m,[s],t
c
e,n,x
1,6
1,6
e,n,z15
[1,2]
1,7
1,6
1,5
1,6
1,5
1,7
1,6
e,n,z15
1,7
1,5
e,n,x
z6
1,5
l,w
e,n,x
1,2
l,w
1,6
1,6
[1,5]
1,7
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
II
I
I
I
I
I
I
I
I
II
I
C1
B
I
G
E1
N
O
B
G
J
U
E4
S
U
G
C2
I
D1
O
C2
C1
C2
B
H
E1
X
C2
E1
P
E4
R
E1
C2
R
B
Afula
Agama
Agbara
Agbeni
Agege
Ago
Agodi
Agona
Agoueve
Ahanou
Ahepe
Ahmadi
Ahoutoue
Ahuza
Ajiobo
Akanji
Akuafo
Alabama
Alachua
Alagbon
Alamo
Albany
Albert
Albuquerque
Alexander
Alexanderplatz
Alexanderpolder
Alfort
Alger
Alkmaar
Allandale
Allerton
Alminko
Alsterdorf
Altendorf
C2
Altona
8,20
r,[i]
z6
E1
Amager
3,10
1,2
I
I
F
C1
Amba
Amersfoort
11
6,7
k
d
l,z13,z28
e,n,x
I
I
I
I
I
C2
I
E1
E1
M
Amherstiana
Amina
Aminatu
Amounderness
Amoutive
8
16
3,10
3,10
28
l,(v)
i
a
i
d
1,6
1,5
1,2
1,5
1,5
NOTE
Alachua may possess H phase Rz37 or Rz45.
Albany may possess H phase Rz45.
IP combined Womba (4,12,27:c:1,7) with

Altendorf to form Altendorf 4,12,27:c:1,7.
Pikine (8,20:r:z6) was combined with Altona
and called Altona.
IP combined Tuebingen (3,15:y:1,2) with
Amager to form Amager 3,10,[15]:y:1,2.
Amager may possess H phase Rz45.
IP combined Omderman (6,7,14:d:e,n,x) with
Amersfoort to form Amersfoort 6,7,14:d:e,n,x.
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
E1
Amsterdam
3,10
g,m,s
I
I
I
E1
Amunigun
Anatum
16
3,10
a
e,h
1,6
1,6
I
I
I
I
I
II
I
I
E1
O
Q
C2
N
D1
I
X
Anderlecht
Anecho
Anfo
Angers
Angoda
Angola
Angouleme
Anie
3,10
35
39
8,20
30
1,9,12
16
47
c
g,s,t
y
z35
k
z
z10
(g),m,t
l,w
1,2
z6
e,n,x
z6
z6
I
I
I
I
I
I
I
I
I
I
I
I
I
M
G
I
D1
57
51
T
W
C2
N
N
H
C1
Ank
Anna
Annedal
Antarctica
Antonio
Antsalova
Antwerpen
Apapa
Apeyeme
Aqua
Aragua
Arapahoe
Ardwick
28
13,23
16
9,12
57
51
1,42
45
8,20
30
30
1,6,14
6,7,14
k
z35
r,i
g,z63
a
z
c
m,t
z38
k
z29
z4,z23
f,g
e,n,z15
e,n,z15
e,n,x
z6
1,5
e,n,z15
1,6
1,5
I
IV
I
B
C1
E3
Arechavaleta
Argentina
Arkansas
4,[5],12
6,7
3,15,34
a
z36
e,h
[1,7]
1,5
II
I
I
I
II
I
I
I
I
I
I
I
I
56
U
N
M
51
L
E1
E1
C2
F
C1
F
G
Artis
Arusha
Aschersleben
Ashanti
Askraal
Assen
Assinie
Asylanta
Atakpame
Atento
Athinai
Ati
Atlanta
56
43
30
28
51
21
3,10
3,10
8,20
11
6,7
11
13,23
b
z
b
b
l,z28
a
l,w
c
e,h
b
i
d
b
e,n,z15
1,5
1,6
[z6]
[1,5]
z6
1,2
1,7
1,2
e,n,z15
1,2
The Difco Manual
NOTE
IP combined Drypool (3,15:g,m,s) and Drypool

var. O 34+ with Amsterdam to form
Amsterdam 3,10,[15],[15,34]:g,m,s:-.
IP combined Newington (3,15:e,h:1,6) and
Minneapolis (3,15,34:e,h:1,6) with Anatum to
form Anatum 3,10,[15],[15,34]:e,h:1,6.
IP combined Anie with Mesbit

(47:m,t:[e,n,z15]) and called it Mesbit.
IP combined Ardwick with Rissen (6,7:f,g:-)

to form Rissen 6,7,14:f,g:-. Ardwick is now
called Rissen var. O 14+ by IP.
IP combined Arkansas and Newhaw (3,15:e,h:1,5)

with Muenster (3,10:e,h:1,5) to form
Muenster 3,10,[15],[15,34]:e,h:1,5. Arkansas
is now callled Muenster var. O 15+, 34+ by IP.
Assinie may possess H phase Rz45.
Atlanta was combined with Mississippi

(1,13,23:b:1,5). The name Atlanta has been
dropped.
733

734
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
II
I
I
I
I
I
I
I
I
I
I
II
I
I
I
I
I
I
I
I
V
I
I
Z
C1
C1
Y
I
E4
B
B
B
M
M
C1
I
L
N
G
H
C1
D2
M
Y
W
B
Atra
Augustenborg
Austin
Australia
Avignon
Avonmouth
Ayinde
Ayton
Azteca
Babelsberg
Babili
Bacongo
Badagry
Baguida
Baguirmi
Bahati
Bahrenfeld
Baiboukoum
Baildon
Bakau
Balboa
Balcones
Ball
50
6,7,14
6,7
48
16
1,3,19
1,4,12,27
1,4,12,27
4,[5],12,27
28
28
6,7
16
21
30
13,22
6,14,24
6,7
9,46
28
48
45
1,4,12,27
m,t
i
a
l,v
y
i
d
l,w
l,v
z4,z23
z35
z36
z10
z4,z23
y
b
e,h
k
a
a
z41
z36
y
z6:z42
1,2
1,7
1,5
e,n,z15
e,n,z15
z6
z6
1,5
[e,n,z15]
1,7
z42
1,5
e,n,x
e,n,z15
1,5
1,7
e,n,x
1,7
e,n,x
I
I
I
I
I
I
I
I
I
I
II
I
I
I
I
I
I
II
I
I
I
I
I
I
J
D2
D2
C2
B
M
O
P
D1
H
C2
C2
C1
C2
I
I
54
58
D
C2
M
57
I
C2
Bama
Bamboye
Bambylor
Banalia
Banana
Banco
Bandia
Bangkok
Bangui
Banjul
Baragwanath
Bardo
Bareilly
Bargny
Barmbek
Barranquilla
Barry
Basel
Basingstoke
Bassa
Bassadji
Batonrouge
Battle
Bazenheid
17
9,46
9,46
6,8
1,4,[5],12
28
35
38
9,12
1,6,14,25
6,8
8
6,7,14
8,20
16
16
54
58
9,46
6,8
28
57
16
8,20
m,t
b
z
b
m,t
r,i
i
z4,z24
d
a
m,t
e,h
y
i
d
d
z10
l,z13,z28
z35
m,t
r
b
l,z13,z28
z10
l,w
e,n,z15
z6
[1,5]
1,7
l,w
e,n,z15
e,n,z15
1,5
1,2
1,5
1,5
z6
e,n,x
e,n,z15
1,5
e,n,z15
1,6
e,n,z15
1,6
1,2
NOTE
IP combined Ball with Ruki (4,5,12:y:e,n,x)

1,4,[5],12,27:y:e,n,x.
Banana may possess H phase Rz45.
The Difco Manual
Section V
SEROTYPE
I
I
II
I
I
I
I
I
II
I
I
I
I
I
I
I
I
I
I
IV
I
I
I
II
I
I
I
I
I
II
I
I
I
I
I
C2
H
B
E4
C2
C2
C2
I
K
E1
R
D2
C2
X
D2
X
51
U
J
R
D1
E1
E4
59
E1
E4
N
J
R
X
E4
X
X
W
E2
Be
Beaudesert
Bechuana
Bedford
Belem
Belfast
Bellevue
Bellville
Beloha
Benfica
Benguella
Benin
Benue
Bere
Bergedorf
Bergen
Bergues
Berkeley
Berlin
Bern
Berta
Bessi
Bethune
Betioky
Biafra
Bida
Bietri
Bignona
Bijlmer
Bilthoven
Bilu
Binche
Bingerville
Binningen
Binza
I
I
I
I
II
II
I
I
I
I
II
C1
E1
B
B
D1
J
D1
H
X
C2
C1
Birkenhead
Birmingham
Bispebjerg
Bissau
Blankenese
Bleadon
Blegdam
Blijdorp
Blitta
Blockley
Bloemfontein
The Difco Manual
O ANTIGENS
PHASE 1
PHASE 2
8,20
[1],6,14,[25]
1,4,12,27
1,3,19
6,8
6,8
8
16
18
3,10
40
9,46
6,8
47
9,46
47
51
43
17
1,40
1,9,12
3,10
1,3,19
59
3,10
1,3,19
30
17
1,40
47
(1),3,10,(19)
47
47
45
3,15
a
e,h
g,[m],t
l,z13,z28
c
c
z4,z23
e,n,x
z36
b
b
y
y
z4,z23
e,h
i
z10
a
d
z4,z32
[f],g,t
i
k
k
z10
c
y
b
g,m
a
f,g,t
z4,z23
z35
g,s,t
y
[z6]
1,7
[1,5]
e,n,z15
e,n,x
1,7
1,7
1,(5),7
e,n,x
z6
1,7
l,w
[z6]
1,2
e,n,z15
1,5
1,5
1,5
e,n,x
1,7
(z)
z6
1,6
1,5
e,n,z15
[1,5]
1,(2),7
l,w
e,n,z15
1,5
6,7
3,10
1,4,[5],12
4,12
1,9,12
17
9,12
1,6,14,25
47
6,8
6,7
c
d
a
c
b
(f),g,t
g,m,q
c
y
k
b
1,6
l,w
e,n,x
e,n,x
z6
[e,n,x,z15]
1,5
e,n,x
1,5
[e,n,x]:z42
NOTE
Bere may possess H phase Rz45.
IP combined Binza and Thomasville

(3,15,34:y:1,5) with Orion (3,10:y:1,5) to
form Orion 3,10,[15],[15,34]:y:1,5. Binza is
now called Orion var. O 15+ by IP.
IP has dropped f.
Blockley may possess H phase Rz58
735

736
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
I
I
I
I
IV
I
I
I
II
I
I
I
IV
I
I
V
I
I
I
I
VI
I
E1
K
V
B
53
N
H
M
R
V
E1
E1
Z
J
C2
Y
C1
X
G
52
H
C1
Bloomsbury
Blukwa
Bobo
Bochum
Bockenheim
Bodjonegoro
Boecker
Bokanjac
Boksburg
Bolama
Bolombo
Bolton
Bonaire
Bonames
Bonariensis
Bongor
Bonn
Bootle
Borbeck
Bordeaux
Bornheim
Bornum
3,10
18
44
4,[5],12
1,53
30
[1],6,14,[25]
28
40
44
3,10
3,10
50
17
6,8
48
6,7
47
13,22
52
1,6,14,25
6,7,14
g,t
z4,z24
d
r
z36,z38
z4,z24
l,v
b
g,[m],s,[t]
z
z38
y
z4,z32
a
i
z35
l,v
k
l,v
k
z10
z38
1,5
1,5
l,w
1,7
1,7
e,n,x
e,n,x
[z6]
e,n,z15
1,2
e,n,x
e,n,x
1,5
1,6
1,5
1,(2),7
I
I
I
II
I
I
I
I
I
I
I
I
I
I
I
I
I
I
II
I
I
I
I
I
I
54
T
I
G
D1
H
C2
G
B
C1
B
B
I
K
C1
C2
B
V
W
C2
I
B
F
C2
M
Borreze
54
Borromea
42
Bouake
16
Boulders
1,13,23
Bournemouth
9,12
Bousso
1,6,14,25
Bovismorbificans
6,8
Bracknell
13,23
Bradford
4,12,27
Braenderup
6,7,14
Brancaster
1,4,12,27
Brandenburg
1,4,[5],12,27
Brazil
16
Brazos
6,14,18
Brazzaville
6,7
Breda
6,8
Bredeney
1,4,12,27
Brefet
44
Bremen
45
Breukelen
6,8
Brevik
16
Brezany
1,4,12,27
Brijbhumi
11
Brikama
8,20
Brisbane
28
f,g,s
i
z
m,t
e,h
z4,z23
r,[i]
b
r
e,h
z29
l,v
a
a
b
z4,z23
l,v
r
g,m,s,t
l,z13,[z28]
z
d
i
r,[i]
z
1,6
z6
z42
1,2
[e,n,z15]
1,5
1,6
1,5
e,n,z15
e,n,z15
1,5
e,n,z15
1,2
e,n,x
1,7
e,n,z15
e,n,x
e,n,z15
e,n,[x],z15
1,6
1,5
l,w
e,n,z15
NOTE
Bornheim was formerly in Subspecies II.

IP combined Bornum with Lille (6,7:z38:-) to
form Lille 6,7,14:z38:-. Bornum is now called
Lille var. O 14+ by IP.
Bredeney may possess H phase Rl,z40 instead of l,v.
The Difco Manual
Section V
SEROTYPE
I
I
I
I
I
V
I
I
I
G
T
T
G
C2
66
I
E4
I
I
I
I
I
I
I
II
I
I
I
II
I
I
I
I
I
C1
C2
I
C2
B
R
C2
R
C2
F
C1
U
I
S
B
C1
E1
Bruck
Brunei
Brunflo
Bsilla
Budapest
Bukavu
Bukuru
Bulawayo
Bulgaria
Bullbay
Bulovka
Bunnik
Burgas
Burundi
Bury
Businga
Butantan
I
I
I
I
I
I
52
H
I
E1
W
B
Butare
Buzu
Caen
Cairina
Cairns
Cairo
I
II
I
II
I
I
I
E4
B
B
C1
O
D1
E2
V
I
I
V
D1
B
The Difco Manual
Bristol
Brive
Broc
Bron
Bronx
Brookfield
Brooklyn
Broughton
Broxbourne
O ANTIGENS
PHASE 1
PHASE 2
13,22
1,42
42
13,22
6,8
66
16
1,3,19
z
r
z4,z23
g,m
c
z41
l,w
b
1,7
l,w
e,n,z15
[e,n,z15]
1,6
e,n,x
l,w
NOTE
Combined with Wien. The name Broxbourne

has been dropped.
6,7
8,20
16
6,8
1,4,12,27
1,40
6,8
1,40
6,8
11
6,7
43
16
41
4,12,27
6,7
3,10
z
y
r
r
g,t
l,z28
b
z
y
l,v
z44
z42
l,v
a
c
z
b
l,w
1,5
1,7
1,2
1,5
l,w
1,5
1,6
e,n,z15
[1,5,7]
e,n,z15
z6
e,n,z15
1,5
52
[1],6,14,[25]
16
3,10
45
1,4,12,27
e,h
i
d
z35
k
d
1,6
1,7
l,w
z6
e,n,z15
1,2
Calabar
Caledon
California
Calvinia
Camberene
Camberwell
Cambridge
1,3,19
1,4,12,27
4,12
6,7
35
9,12
3,15
e,h
g,[m],[s],t
g,m,t
a
z10
r
e,h
l,w
e,n,x
z42
1,5
1,7
l,w
Camdeni
Campinense
Canada
44
9,12
4,12,27
r
r
b
e,n,z15
1,6
IP combined Rosenthal (3,15:b:1,5) and

unnamed 3,15,34:b:1,5 with Butantan to
form Butantan 3,10,[15],[15,34]:b:1,5.
IP combined Cairo with Stanley (4,5,12:d:1,2)

to form Stanley 1,4,[5],12,27:d:1,2. The name
Cairo has been dropped.
IP combined Cambridge and Wildwood

(3,15,34:e,h:l,w) with Meleagridis (3,10:e,h:l,w)
Cambridge is now called Meleagridis var. O
15+ by IP.
737

738
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
40
9,12
28
1,3,15,19
1,3,19
3,15,34
l,v
z29
z4,z23
y
m,t
g,s,t
1,6
[1,5]
1,5
e,n,x
54
6,7
[1],6,14,[25]
6,7
z10
z6
g,m,s
k
e,n,x
1,7
R1,10
16
38
17
18
1,3,19
6,14,[24]
44
45
40
28
6,14
6,7
28
18
g,s,t
d
l,v
z10
z
y
g,z51
k
z
l,z13,z28
g,s,t
z
z
z4,z23
[1,5]
e,n,x
z6
l,w
1,7
1,7
e,n,x
1,5
e,n,x
z39
[1,5]
9,46
1,13,23
6,8
16
39
11
[1],6,14,[25]
6,8
9,46
47
1,4,[5],12
28
1,3,19
6,14,24
6,7
6,8
11
42
11
1,3,10,19
k
a
z4,z23
z4,z32
k
d
d
k
b
z
e,h
r,[i]
i
z4,z24
z
g,m,[s]
e,h
b
c
b
z35
1,5
[e,n,z15]
1,5
e,n,x:[r]
e,n,x
e,n,z15
1,5
z6
e,n,x
1,5
1,6
1,2
[e,n,x]
1,2
1,5
1,5
z35
I
II
I
I
I
I
R
D1
M
E4
E4
E3
Canary
Canastel
Cannobio
Cannonhill
Cannstatt
Canoga
I
II
I
I
54
C1
H
C1
Canton
Cape
Caracas
Cardiff
I
II
I
I
I
I
I
I
I
I
I
I
II
I
I
P
J
K
E4
H
V
W
R
M
H
C1
M
K
Cardoner
Carletonville
Carmel
Carnac
Carno
Carrau
Carswell
Casablanca
Casamance
Catalunia
Catanzaro
Cayar
Ceres
Cerro
I
I
I
IV
I
I
I
I
I
II
I
I
I
I
I
I
I
II
I
I
D2
G
C2
I
Q
F
H
C2
D2
X
B
M
E4
H
C1
C2
F
T
F
E4
Ceyco
Chagoua
Chailey
Chameleon
Champaign
Chandans
Charity
Charlottenburg
Cheltenham
Chersina
Chester
Chicago
Chichester
Chichiri
Chile
Chincol
Chingola
Chinovum
Chiredzi
Chittagong
NOTE
IP combined Canoga and Halmstad (3,15:g,s,t:-)

Westhampton 3,10,[15],[15,34]:g,s,t:-. Canoga
is now called Westhampton var. O 15+, 34+ by IP.

R1,10 with Thompson (6,7,14:k:1,5).
Cerro was combined with Siegburg

(6,14,18:z4,z23:[1,5]) and called Cerro.
Cerro may possess H phase Rz45.
Champaign may possess H phase Rz48
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
NOTE
c
6,7
z10
z4,z24
e,n,x
l,v,[z13]
k
z
z10
a
1,5
c
e,n,z15
1,7
1,6
1,5
l,w
1,7
1,5
H2S negative
[1,5] H2S positive
I
I
I
I
II
I
I
I
I
I
C1
C1
C2
V
E1
B
D1
E1
C2
E2
Choleraesuis
6,7
Choleraesuis var. Kunzendorf
Chomedey
8,20
Christiansborg
44
Chudleigh
3,10
Clackamas
4,12
Claibornei
1,9,12
Clerkenwell
3,10
Cleveland
6,8
Clichy
3,15
II
II
I
I
I
I
I
I
I
I
I
I
I
II
I
I
G
V
D2
C2
B
C1
C1
F
P
C1
C1
G
F
J
T
Q
Clifton
Clovelly
Cochin
Cocody
Coeln
Coleypark
Colindale
Colobane
Colombo
Colorado
Concord
Congo
Connecticut
Constantia
Coogee
Cook
13,22
1,44
9,46
8,20
4,[5],12
6,7,14
6,7
11
38
6,7
6,7
13,23
11
17
42
39
z29
z39
k
r,i
y
a
r
k
y
l,w
l,v
g,[m],[s],t
l,z13,z28
z
l,v
Rz48
1,5
[e,n,x,z15]
1,5
e,n,z15
1,2
l,w
1,7
1,7
1,6
1,5
1,2
1,5
l,w:z42
e,n,z15
1,5
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
E1
C1
C2
M
K
C2
F
M
67
G
E1
M
Q
C2
C2
54
C2
C2
51
Y
Coquilhatville
Coromandel
Corvallis
Cotham
Cotia
Cremieu
Crewe
Croft
Crossness
Cubana
Cuckmere
Cullingworth
Cumberland
Curacao
Cyprus
Czernyring
Daarle
Dabou
Dadzie
Dahlem
3,10
6,7
8,20
28
18
6,8
11
28
67
1,13,23
3,10
28
39
6,8
6,8
54
6,8
8,20
51
48
z10
l,v
z4,z23
i
e,h
z
g,m,s
r
z29
i
d
i
a
i
r
y
z4,z23
l,v
k
1,7
z35
[z6]
1,5
1,6
1,[6]
1,5
[e,n,z15]
1,2
1,2
l,w
e,n,x
1,6
l,w
1,5
e,n,x
l,w
e,n,x
e,n,z15
The Difco Manual
IP does not get z13
IP combined Clichy with Goelzau (3,10:a:1,5)

to form Goelzau 3,10,[15]:a:1,5. Clichy is
now called Goelzau var. O 15+ by IP.
IP calls Congo 13,23:g,m,s,t:-.
IP combined Cook with Champaign (39:k:1,5).

The name Cook has been dropped.
Cubana may possess H phase Rz37 or Rz43.
739

740
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
NOTE
Dahomey may possess H phase Rz58.
I
I
I
I
I
X
J
M
I
B
Dahomey
Dahra
Dakar
Dakota
Dalat
47
17
28
16
4,5,27
k
b
a
z35
y
1,6
1,5
1,6
e,n,z15
e,n,x
I
I
I
I
II
I
I
I
E4
C1
51
X
D1
C2
C1
C1
Dallgow
Damman
Dan
Dapango
Daressalaam
Daula
Daytona
Decatur
1,3,19
6,7
51
47
1,9,12
8,20
6,7
6,7
z10
a
k
r
l,w
z
k
c
e,n,z15
z6
e,n,z15
1,2
e,n,x
z6
1,6
1,5
I
II
IV
I
I
I
I
I
I
I
II
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
D2
R
R
K
O
G
C1
B
52
E4
T
W
M
M
G
C2
L
Y
T
C2
M
J
C1
D2
M
C2
N
M
M
Z
N
M
R
B
Deckstein
9,46
Degania
40
Degania var. subsp. IV 40
Delmenhorst
18
Dembe
35
Demerara
13,23
Denver
6,7
Derby
1,4,[5],12
Derkle
52
Dessau
1,3,15,19
Detroit
42
Deversoir
45
Dibra
28
Dieuppeul
28
Diguel
1,13,22
Diogoye
8,20
Diourbel
21
Djakarta
48
Djama
1,42
Djelfa
8
Djermaia
28
Djibouti
17
Djugu
6,7
Doba
9,46
Doel
28
Doncaster
6,8
Donna
30
Doorn
28
Douala
28
Dougi
50
Doulassame
30
Dresden
28
Driffield
1,40
Drogana
1,4,12,27
r
z4,z24
z4,z24
z71
d
z10
a
f,g
e,h
g,s,t
z
c
a
i
d
z41
i
z4,z24
z29
b
z29
z10
z10
a
z
a
l,v
i
i
y
a
c
d
r,(i)
1,7
[z39]
l,w
l,w
e,n,z15
[1,2]
1,7
1,5
e,n,x
z6
1,7
e,n,z15
z6
1,2
[1,5]
1,2
e,n,x
e,n,x
e,n,z15
1,6
1,5
1,5
1,2
l,w
1,6
e,n,z15
e,n,x
1,5
e,n,z15
Dalat was combined with Ball. The name

Dalat has been dropped.
IP has dropped Decatur and calls it dulcitol

positive, mucate positive variant of Choleraesuis.
IP calls Drogana r,i.

The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
3,[15],[15,34]
g,m,s
E2
Drypool
I
II
I
I
I
II
I
I
I
II
I
I
I
I
I
I
I
I
I
I
I
I
I
IIIb
D1
S
C2
W
B
D2
E1
C2
D1
B
G
R
O
D1
E4
M
C2
O
P
C1
C2
S
T
61
Dublin
Dubrovnik
Duesseldorf
Dugbe
Duisburg
Duivenhoks
Dumfries
Dunkwa
Durban
Durbanville
Durham
Duval
Ealing
Eastbourne
Eastglam
Eberswalde
Eboko
Ebrie
Echa
Edinburg
Edmonton
Egusi
Egusitoo
Eilbeck
1,9,12,[Vi]
41
6,8
45
1,4,12,27
9,46
3,10
6,8
9,12
1,4,12,27
13,23
1,40
35
1,9,12
1,3,19
28
6,8
35
38
6,7
6,8
41
1,42
61
g,p
z
z4,z24
d
d
g,[m],[s],t
r,[i]
d
a
[z39]
b
b
g,m,s
e,h
c
c
b
g,m,t
k
b
l,v
d
b
i
1,5
1,6
e,n,z15
[e,n,x]
1,6
1,7
e,n,z15
1,[5],7
e,n,z15
e,n,z15
1,5
1,5
1,6
1,7
1,2
1,5
e,n,z15
[1,5]
z6
z
C1
Eimsbuettel
6,7,14
l,w
I
II
I
I
I
I
I
I
II
I
I
II
C1
W
B
D2
X
E1
D1
D1
I
C2
P
H
I
O
B
D1
Eingedi
Ejeda
Eko
Ekotedo
Ekpoui
Elisabethville
Elokate
Elomrane
Elsiesrivier
Emek
Emmastad
Emmerich
H
Enschede
Entebbe
Enteritidis
6,7
45
4,12
9,46
47
3,10
9,12
1,9,12
16
8,20
38
6,14
Encino
35
1,4,12,27
1,9,12
f,g,t
a
e,h
z4,z23
z29
r
c
z38
[e,n,x]
g,m,s
r
[m,t]
1,6,14,25
z10
z
[f],g,m,[p],[t]
1,2,7
z10
1,6
1,7
1,7
1,6:z42
1,6
e,n,x
d
l,w
z6
[1,7]
I
I
I
The Difco Manual
NOTE
IP combined Drypool 3,15:g,m,s:- and Drypool

var. O34+ with Amsterdam (3,10:g,m,s:-) to
or O 15+, 34+ by IP.
Eilbeck was formerly in subspecies II, but is

now combined with Arizona 26:33:31. The
name Eilbeck has been dropped.
IP combined Eimsbuettel with Livingstone
(6,7:d:l,w) to form Livingstone 6,7,14:d:l,w.
Eimbuettel is now called Livingstone var. O
14+ by IP.
l,z13,z28
741

742
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
I
I
I
I
II
II
I
I
I
I
E1
F
B
G
Y
C1
D1
E2
Enugu
Epicrates
Epinay
Eppendorf
Epping
Erlangen
Escanaba
Eschberg
Eschersheim
16
3,10
11
1,4,12,27
1,13,23
48
6,7
9,12
3,15
l,[z13],z28
b
a
d
e,n,x
g,m,t
k
d
d
[1,5]
l,w
l,z13,z28
1,5
1,[5],7
e,n,z15
1,7
e,n,x
I
I
II
C1
B
Y
Eschweiler
Essen
Etosha
6,7
4,12
48
z10
g,m
d
1,6
1,11
I
I
I
I
I
I
I
I
I
II
I
I
I
I
I
I
I
I
II
I
VI
I
I
I
II
I
I
I
I
I
I
IV
I
I
F
F
E1
M
M
M
T
E1
E1
R
F
G
M
U
E4
G
B
Z
Z
C2
H
S
C2
B
E1
F
H
I
H
V
Y
Z
E1
H
Etterbeek
Euston
Everleigh
Ezra
Fairfield
Fajara
Faji
Falkensee
Fallowfield
Fandran
Fann
Fanti
Farakan
Farcha
Fareham
Farmsen
Farsta
Fass
Faure
Fayed
Ferlac
Ferlo
Ferruch
Finaghy
Finchley
Findorff
Finkenwerder
Fischerhuette
Fischerkietz
Fischerstrasse
Fitzroy
Flint
Florian
Florida
11
11
3,10
28
28
28
1,42
3,10
3,10
1,40
11
13,23
28
43
1,3,19
13,23
4,12
50
50
6,8
1,6,14,25
41
8
4,12
3,10
11
[1],6,14,[25]
16
1,6,14,25
44
48
50
3,10,[15]
[1],6,14,[25]
z4,z23
r,i
z29
z
r
l,z28
a
i
l,z13,z28
z35
l,v
z38
z10
y
r,i
z
i
l,v
z42
l,w
a
k
e,h
y
z
d
d
a
y
d
e,h
z4,z23
z4,z24
d
e,n,z15
e,n,x,z15
e,n,x
1,7
l,w
e,n,x
e,n,z15
e,n,z15
e,n,z15
e,n,x,z15
e,n,x
1,5
1,2
l,w
1,6
e,n,x
1,2
1,7
1,2
e,n,x
1,6
1,5
1,6
e,n,x
z6
1,5
e,n,z15
e,n,x
e,n,z15
1,5
1,7
NOTE
IP combined Eschersheim with Souza

(3,10:d:e,n,x) to form Souza 3,10,[15]:d:e,n,x.
Eschersheim is now called Souza
var. O 15+ by IP.
Etosha was not considered a new serotype by

Kauffmann and is not used.
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
I
I
I
I
I
II
I
I
I
I
II
I
I
I
I
I
II
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
51
K
W
I
B
P
D1
I
P
E1
T
D2
G
M
D1
E1
E1
E4
B
B
C1
I
C1
E1
F
D1
V
L
O
I
H
C1
O
D2
E4
C1
C2
E1
C1
Flottbek
Fluntern
Fomeco
Fortlamy
Fortune
Foulpointe
Franken
Frankfurt
Freetown
Freiburg
Fremantle
Fresno
Friedenau
Friedrichsfelde
Frintrop
Fufu
Fuhlsbuettel
Fulda
Fulica
Fyris
Gabon
Gafsa
Galiema
Galil
Gallen
Gallinarum
Gamaba
Gambaga
Gambia
Gaminara
Garba
Garoli
Gassi
Gateshead
Gatineau
Gatow
Gatuni
Gbadago
Gdansk
52
6,14,18
45
16
1,4,12,27
38
1,9,12
16
38
3,10
42
9,46
13,22
28
1,9,12
3,10
3,10
1,3,19
4,[5],12
4,[5],12
6,7
16
6,7,14
3,10
11
1,9,12
1,44
21
35
16
1,6,14,25
6,7
35
9,46
1,3,19
6,7
6,8
3,10,[15]
6,7
b
b
b
z
z10
g,t
z60
i
y
l,z13
(f),g,t
z38
d
f,g
b
z
l,v
l,w
a
l,v
l,w
c
k
a
a
g,m,[s]
z35
i
d
a
i
e,h
g,s,t
y
y
b
c
l,v
[e,n,x]
1,5
e,n,z15
1,6
z6
z67
e,n,z15
1,5
1,2
1,6
1,5
1,5
z6
1,5
1,5
1,2
1,2
1,6
1,2
e,n,z15
1,2
e,n,z15
e,n,z15
1,7
1,5
1,6
z6
1,5
1,7
e,n,x
1,5
z6
I
I
N
C1
Gege
Gelsenkirchen
30
6,7,14
r
l,v
1,5
z6
I
I
I
II
I
C1
T
D2
C2
L
Georgia
Gera
Geraldton
Germiston
Ghana
6,7
1,42
9,46
6,8
21
b
z4,z23
l,v
m,t
b
e,n,z15
[1,6]
1,6
e,n,x
1,6
The Difco Manual
NOTE
Gallinarum must be identified biochemically.
IP combined Gelsenkirchen (6,7,14:l,v:z6)

with Gdansk to form Gdansk 6,7,14:l,v:z6.
IP combined Gelsenkirchen with Gdansk
(6,7:l,v:z6) to form Gdansk 6,7,14:l,v:z6.
Gelsenkirchen is now called Gdansk var.
O 14+ by IP.
743

744
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
30
6,7
3,10
g,m,s
z39
l,v
1,5,7
1,7
8,20
16
11
11
6,8
1,4,12,27
1,3,19
30
3,10
y
b
a
l,w
z10
i
b
g,m,[t]
a
1,2
1,6
z6:z42
1,5
e,n,z15
l,w
1,5
1,5
3,15
e,h
1,2
9,12
9,12
1,13,23
1,51
6,8
6,7
6,7,14
21
13,22
17
1,40
35
9,12
28
11
6,7
16
c
l,v
g,t
d
r
z4,z23
d
f,g
z29
z
k
c
k
e,h
g,[m],s,t
r
z39
1,5
e,n,z15
1,5
[1,5]
l,w
z6
e,n,z15
e,n,x
e,n,x
1,2
1,5
1,5
1,6
e,n,z15
[z39]
l,w
[1,6]
I
II
I
N
C1
E1
Giessen
Gilbert
Give
I
I
II
I
I
I
I
I
I
C2
I
F
F
C2
B
E4
N
E1
Giza
Glasgow
Glencairn
Glidji
Glostrup
Gloucester
Gnesta
Godesberg
Goelzau
E2
Goerlitz
I
I
II
I
I
I
I
I
II
I
I
I
I
I
II
I
I
D1
D1
G
51
C2
C1
C1
M
G
J
R
O
D1
M
F
C1
I
Goeteborg
Goettingen
Gojenberg
Gokul
Goldcoast
Goma
Gombe
Good
Goodwood
Gori
Goulfey
Gouloumbo
Goverdhan
Gozo
Grabouw
Grampian
Grancanaria
I
I
I
II
I
I
N
J
U
Z
R
K
Grandhaven
30
r
Granlo
17
l,z28
Graz
43
a
Greenside
50
z
Greiz
40
a
Groenekan
18
d
Group A
1,2,12
Group B 4,12; 1,4,5,12; or 1,4,12,27
Group C1
6,7,[Vi] or 6,7,14
Group C2
6,8
Group C3
8; or 8,20
Group D1
1,9,12
Group D2
9,46
NOTE
IP combined Newbrunswick (3,15:l,v:1,7) and

Menhaden (3,15,34:l,v:1,7) with Give to form
Give 3,10,[15],[15,34]:[d],l,v:[d],1,7. Give may
possess H phase d; Rl,z40; or Rz77.
IP combined Clichy (3,15:a:1,5) with Goelzau

to form Goelzau 3,10,[15]:a:1,5.
IP combined Goerlitz with Vejle (3,10:e,h:1,2)
to form Vejle 3,10,15:e,h:1,2. Goerlitz is now
called Vejle var. O 15+ by IP.
Grancanaria can be d-tartrate neg., dulcitol

neg., ONPG pos., and anaerogenic.
1,2
e,n,x
1,2
e,n,x
z6
1,5
IP combined C2 and C3.

IP combined with C2.
The Difco Manual
Section V
I
II
I
I
I
I
I
The Difco Manual
G
R
N
D2
M
V
F
SEROTYPE
O ANTIGENS
PHASE 1
Group D3
1,9,12,46,27
Group E1
3,10
Group E2
3,15
Group E3
3,15,34
Group E4
1,3,19
Group F
11
Group G
13,22 or 13,23
Group H 6,14; 6,14,24; or 1,6,14,25
Group I
16
Group J
17
Group K
18
Group L
21
Group M
28
Group N
30
Group O
35
Group P
38
Group Q
39
Group R
40
Group S
41
Group T
42
Group U
43
Group V
44
Group W
45
Group X
47
Group Y
48
Group Z
50
Group 51
51
Group 52
52
Group 53
53
Group 54
54
Group 55
55
Group 56
56
Group 57
57
Group 58
58
Group 59
59
Group 60
60
Group 61
61
Group 62
62
Group 63
63
Group 65
65
Group 66
66
Group 67
67
Grumpensis
1,13,23
d
Grunty
1,40
z39
Guarapiranga
30
a
Guerin
9,46
e,h
Guildford
28
k
Guinea
1,44
z10
Gustavia
11
d
PHASE 2
NOTE
IP combined E2 and E3 with E1.

IP combined E2 and E3 with E1.
1,7
1,6
e,n,x
z6
1,2
[1,7]
1,5
745

746
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
21
1,42
1,3,19
8
9,46
6,8
16
17
4,12
6,7
42
35
48
1,4,[5],12
28
1,4,12,27
3,15
z4,z24
k
a
k
z
z10
z4,z23
y
l,z13,[z28]
m,p,t,[u]
k
z38
d
z10
c
c
g,s,t
z6
e,n,z15
1,5
e,n,x
e,n,x
e,n,z15
1,6
[1,6]
z6
1,2
1,7
l,w
II
I
I
I
II
I
II
I
I
I
I
I
II
I
I
I
I
L
T
E4
C2
D2
C2
I
J
B
C1
T
O
Y
B
M
B
E2
Gwaai
Gwale
Gwoza
Haardt
Haarlem
Hadar
Haddon
Hadejia
Haduna
Haelsingborg
Haferbreite
Haga
Hagenbeck
Haifa
Halle
Hallfold
Halmstad
II
D1
Hamburg
1,9,12
g,t
E2
Hamilton
3,15
Rz27
II
I
I
I
I
I
I
I
IV
I
I
I
I
I
I
I
I
I
I
II
II
I
Y
G
R
I
G
H
51
E1
51
E1
C1
T
M
B
G
E4
F
Q
B
C1
B
Z
Hammonia
Handen
Hann
Hannover
Haouaria
Harburg
Harcourt
Harleystreet
Harmelen
Harrisonburg
Hartford
Harvestehude
Hatfield
Hato
Havana
Hayindogo
Heerlen
Hegau
Heidelberg
Heilbron
Helsinki
Hemingford
48
e,n,x,z15
1,13,23
d
40
k
16
a
13,22
c
[1],6,14,[25]
k
51
l,v
3,10
z
51
z4,z23
3,10,[15],[15,34]
z10
6,7
y
1,42
y
28
d
4,[5],12
g,m,s
1,13,23
f,g,[s]
1,3,19
e,h
11
i
39
z10
1,4,[5],12
r
6,7
l,z28
1,4,12
z29
50
d
z6
1,2
e,n,x
1,2
e,n,x,z15
1,5
1,2
1,6
1,6
e,n,x
z6
1,6
1,6
1,6
1,2
1,5:[z42]
[e,n,x]
1,5
NOTE
IP combined Halmstad and Canoga

(3,15,34:g,s,t:-) with Westhampton (3,10:g,s,t:-)
to form Westhampton 3,10,[15],[15,34]:g,s,t:-.
Halmstad is now called Westhampton
var. O 15+ by IP.
IP combined Hamburg with Manica
(1,9,12:g,m,s,t:z42) and Muizenberg
(9,12:g,m,s,t:1,5) to form S. II
1,9,12:g,m,[s],t:[1,5,7]:[z42].
IP combined Hamilton with Goerlitz
(3,15:e,h:1,2) and Vejle (3,10:e,h:1,2) to form
Vejle 3,10,15:e,h:1,2:Rz27. Hamilton is now
called Vejle var. Rz27+. The name Hamilton
has been dropped.
Hartford may possess H phase Rz50 or Rz67.
Havana may possess H phase Rz45 or Rz79.
Hemingford may possess H phase Rz82.

The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
d
c
a
d
y
a
d
r,i
l,z13,z28
b
z35
g,m
z41
k
r
a
c
c
i
i
l,z13,z28
l,v
z
a
z
z10
l,v
z4,z23
z
z
a
l,z28
b
z4,z24
b
b
c
b
y
i
c
z6
1,5
z6
e,n,z15
e,n,x
1,5
1,5
e,n,z15
1,5
e,n,x,z15
1,5
l,w
1,2
1,5
1,5,7
1,2
e,n,z15
l,w
1,5
z6
e,n,x
1,5
e,n,z15
z6
z6:z42
e,n,x
1,7
z39
l,w
e,n,x
1,2
1,7
e,n,x
1,6
1,5
1,2
1,5
1,6
II
I
I
I
I
I
I
I
I
II
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
II
I
IV
I
II
I
II
I
II
I
I
I
I
I
I
I
S
M
I
C2
F
B
H
C2
C2
J
D2
D2
C1
N
C2
Y
C1
I
E1
Q
E1
C2
H
M
E4
Z
H
U
E1
D1
B
F
I
53
E1
I
L
G
L
G
E1
Hennepin
41
Hermannswerder
28
Heron
16
Herston
6,8
Herzliya
11
Hessarek
4,12,27
Heves
6,14,[24]
Hidalgo
6,8
Hiduddify
6,8
Hillbrow
17
Hillegersberg
9,46
Hillingdon
9,46
Hillsborough
6,7
Hilversum
30
Hindmarsh
8,20
Hisingen
48
Hissar
6,7,14
Hithergreen
16
Hoboken
3,10
Hofit
39
Hoghton
3,10
Holcomb
6,8
Homosassa
1,6,14,25
Honelis
28
Hongkong
1,3,19
Hooggraven
50
Horsham
1,6,14,[25]
Houten
43
Huddinge
3,10
Hueningen
9,12
Huettwilen
1,4,12
Huila
11
Hull
16
Humber
53
Huvudsta
3,10
Hvittingfoss
16
Hydra
21
Ibadan
13,22
Ibaragi
21
Idikan
1,13,23
Ikayi
3,10,[15]
I
I
I
M
M
E3
Ikeja
Ilala
Illinois
28
28
3,15,34
k
k
z10
1,7
1,5
1,5
E4
Ilugun
1,3,10,19
z4,z23
z6
The Difco Manual
NOTE
Ikayi Var. O 15+ was described after E1 and

E2 were combined.
IP combined Illinois and Manila (3,15:z10:1,5)

with Lexington (3,10:z10:1,5) to form
Lexington 3,10,[15],[15,34]:z10:1,5. Illinois is
now called Lexington var. O 15+, 34+ by IP.
747

748
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
45
6,8
9,46
1,4,12
6,7,14
6,7
9,46
41
38
9,12
41
9,46
17
43
6,7
6,7,14
48
3,10
9,12
8
1,6,14,25
13,23
9,12
l,v
y
l,v
z
r
z10
z10
z10
k
c
z4,z24
y
k
z38
l,v
d
z10
g,t
e,h
z10
r,i
d
l,v
[e,n,z15]
1,7
1,5
1,7
1,5
1,5
e,n,x
e,n,x
1,6
1,6
[1,5]
e,n,x
1,5
1,5
1,5
e,n,x
e,n,z15
e,n,x
1,5
z6
R1,11
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
II
I
I
I
I
I
W
C2
D2
B
C1
C1
D2
S
P
D1
S
D2
J
U
C1
C1
Y
E1
D1
C2
H
G
D1
Imo
Inchpark
India
Indiana
Infantis
Inganda
Inglis
Inpraw
Inverness
Ipeko
Ipswich
Irchel
Irenea
Irigny
Irumu
Isangi
Isaszeg
Islington
Israel
Istanbul
Istoria
Isuge
Italiana
I
I
I
I
I
II
I
I
D1
B
D2
I
Z
I
D1
B
Itami
Ituri
Itutaba
Ivory
Ivorycoast
Jacksonville
Jaffna
Jaja
9,12
1,4,12
9,46
16
50
16
1,9,12
4,12,27
l,z13
z10
c
r
z29
z29
d
z4,z23
1,5
1,5
z6
1,6
[e,n,x]
z35
I
I
I
I
I
F
D1
L
J
B
Jalisco
Jamaica
Jambur
Jangwani
Java
11
9,12
21
17
1,4,5,12
y
r
l,z28
a
b
1,7
1,5
e,n,z15
1,5
[1,2],
(tartrate +)
I
I
I
I
I
I
D1
E1
B
C1
E1
W
Javiana
Jedburgh
Jericho
Jerusalem
Joal
Jodhpur
1,9,12
3,10,[15]
1,4,12,27
6,7,14
3,10
45
l,z28
z29
c
z10
l,z28
z29
1,5
e,n,z15
l,w
1,7
NOTE
Infantis may possess H phase Rz49.
IP combined Italiana that contains H phase

R1,11 with Panama (1,9,12:l,v:1,5). The
name Italiana has been dropped.
IP combined Jaja with Stanleyville

(1,4,[5],12:z4,z23:[1,5]) to form Stanleyville
1,4,[5],12,27:z4,z23:[1,5]. Jaja is now called
Stanleyville var. O 27+. The name Jaja has
been dropped.
IP calls Java, Paratyphi B var. Java. Java is

often monophasic in the U.S. May possess
H phase Rz33.
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
Joenkoeping
4,5,12
g,s,t
I
I
I
I
I
I
I
I
I
I
I
I
I
I
II
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
II
I
I
I
I
I
II
I
R
B
E4
J
G
B
51
C1
T
E4
H
B
E1
C2
M
C2
C1
B
T
E4
J
T
H
B
X
D1
W
R
51
P
H
G
G
C2
C1
V
L
X
E3
Johannesburg
Jos
Juba
Jubilee
Jukestown
Kaapstad
Kabete
Kaduna
Kahla
Kainji
Kaitaan
Kalamu
Kalina
Kallo
Kaltenhausen
Kalumburu
Kambole
Kamoru
Kampala
Kande
Kandla
Kaneshie
Kanifing
Kano
Kaolack
Kapemba
Karachi
Karamoja
Karaya
Kasenyi
Kassberg
Katesgrove
Kedougou
Kentucky
Kenya
Kermel
Keve
Khami
Khartoum
1,40
1,4,12,27
1,3,19
17
13,23
4,12
51
6,7,14
1,42
1,3,19
1,6,14,25
4,[5],12
3,10
6,8
28
6,8
6,7
4,12,27
1,42
1,3,19
17
1,42
1,6,14,25
1,4,12,27
47
9,12
45
40
51
38
1,6,14,25
1,13,23
1,13,23
8,20
6,7
44
21
47
3,15,34
b
y
a
e,h
i
e,h
i
c
z35
z
m,t
z4,z24
b
k
b
z
d
y
c
b
z29
i
z
l,z13,z28
z
l,v
d
z41
b
e,h
c
m,t
i
i
l,z13
d
l,w
b
a
e,n,x
e,n,z15
1,7
1,2
e,n,z15
1,7
1,5
e,n,z15
1,6
1,6
[1,5]
1,2
1,2
z6
e,n,z15
1,[2],7
z6
z6
e,n,z15
l,w
1,6
e,n,x
1,6
1,7
e,n,x
1,2
1,5
1,5
1,6
1,5
l,w
z6
e,n,x
e,n,x
[e,n,x,z15]
1,7
I
I
I
B
I
M
Kiambu
Kibi
Kibusi
4,12
16
28
z
z4,z23
r
1,5
[1,6]
e,n,x
The Difco Manual
NOTE
IP combined Joenkoeping with Kingston

(1,4,12,27:g,s,t:-) to form Kingston
1,4,[5],12,27:g,s,t:-. The name Joenkoeping
has been dropped.
IP combined Khartoum with Oxford (3,10:a:1,7)

to form Oxford 3,10,[15],[15,34]:a:1,7.
Khartoum is now called Oxford var. O 15+ by
IP. CDC has no 3,15:a:1,7. Khartoum was
found by IP with colonies containing O 3,15.
749

750
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
38
1,2,12
16
4,12
38
9,12
1,4,12,27
1,3,19
43
1,4,12,27
c
g,p
y
l,w
l,v
z
l,v
l,z28
y
g,s,t
1,6
e,n,x
e,n,x
1,5
1,6
e,n,x
e,n,x
1,5
[1,2]
17
3,15
a
l,z13
e,n,x
1,5
1,3,19
1,13,23
17
1,4,[5],12
11
6,7
28
6,7
45
38
4,12
47
1,4,[5],12
2,12
45
44
30
39
9,46
8,20
43
28
8
8,20
1,3,19
38
6,7
6,8
6,7
9,12
1,3,19
6,7
28
y
m,t
b
a
k
d
y
d
z
z38
d
c
z
l,v
r
z38
z35
l,v
b
z35
b
z35
b
b
z
g,m,[s]
l,v
e,h
b
l,z28
g,m,[t]
b
e,h
e,n,x
1,2
1,2
e,n,x,[z15]
1,2
e,n,x
1,6
z39
e,n,x
[1,6]
e,n,z15
1,5
e,n,z15
1,6
e,n,x
z35
1,2
z42
1,6
e,n,x
1,5
1,5
1,7
1,5
z35
1,6
1,7
1,7
I
I
I
II
I
I
I
I
I
I
P
A
I
B
P
D1
B
E4
U
B
Kidderminster
Kiel
Kikoma
Kilwa
Kimberley
Kimpese
Kimuenza
Kindia
Kingabwa
Kingston
I
I
J
E2
Kinondoni
Kinshasa
I
I
I
I
I
I
I
I
II
I
II
I
I
I
I
I
I
I
I
I
II
I
I
I
I
I
I
I
I
I
I
I
I
E4
G
J
B
F
C1
M
C1
W
P
B
X
B
A
W
V
N
Q
D2
C2
U
M
C2
C2
E4
P
C1
C2
C1
D1
E4
C1
M
Kinson
Kintambo
Kirkee
Kisangani
Kisarawe
Kisii
Kitenge
Kivu
Klapmuts
Klouto
Kluetjenfelde
Kodjovi
Koenigstuhl
Koessen
Kofandoka
Koketime
Kokoli
Kokomlemle
Kolar
Kolda
Kommetje
Konolfingen
Konstanz
Korbol
Korlebu
Korovi
Kortrijk
Kottbus
Kotte
Kotu
Kouka
Koumra
Kpeme
NOTE
IP combined Kingston with Joenkoeping

(4,5,12:g,s,t:-) to form Kingston
1,4,[5],12,27:g,s,t:[1,2]. Kingston may
possess H phase Rz27 or Rz43.
IP combined Kinshasa with Uganda
(3,10:l,z13:1,5) to form Uganda 3,10,[15]:l,z13:1,5.
Kinshasa is now called Uganda var. O 15+ by IP.
Kodjovi may possess H phase Rz78.
The Difco Manual
Section V
SEROTYPE
II
Kraaifontein
IV
I
I
I
II
I
I
I
II
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
C1
C2
E4
E1
Z
V
B
M
D1
N
B
H
C2
C2
B
E1
E1
J
S
N
E1
K
E1
M
E2
Kralendyk
Kralingen
Krefeld
Kristianstad
Krugersdorp
Kua
Kubacha
Kuessel
Kuilsrivier
Kumasi
Kunduchi
Kuntair
Kuru
Labadi
Lagos
Lamberhurst
Lamin
Lancaster
Landala
Landau
Landwasser
Langenhorn
Langensalza
Langford
Lanka
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
II
P
C1
W
D1
V
S
51
K
F
B
G
S
C2
E1
F
M
C1
E4
S
Lansing
Larochelle
Lattenkamp
Lawndale
Lawra
Leatherhead
Lechler
Leer
Leeuwarden
Legon
Leiden
Leipzig
Leith
Lekke
Lene
Leoben
Leopoldville
Lerum
Lethe
The Difco Manual
O ANTIGENS
PHASE 1
PHASE 2
NOTE
1,13,23
g,m,t
[e,n,x]
IP combined Kraaifontein with Luanshya

(1,13,23:g,m,s,t:[e,n,x]) to form Luanshya
1,13,23:g,m,[s],t:[e,n,x]. The name
Kraaifontein has been dropped.
6,7
z4,z24
8,20
y
1,3,19
y
3,10
z10
50
e,n,x
44
z4,z23
1,4,12,27
l,z13,z28
28
i
1,9,12
g,m,s,t
30
z10
1,4,[5],12,27 l,[z13],[z28]
1,6,14,25
b
6,8
z
8,20
d
1,4,[5],12
i
3,10
e,h
3,10
l,z28
17
l,v
41
z10
30
i
3,10
z
18
m,t
3,10
y
28
b
3,15
r
38
6,7
45
1,9,12
44
41
51
18
11
1,4,12,27
13,22
41
6,8
3,10
11
28
6,7,14
1,3,19
41
i
e,h
z35
z
k
m,t
z
z10
b
c
z38
z10
a
d
z38
l,v
b
z
g,t
z6
l,w
e,n,z15
1,7
1,7
e,n,z15
e,n,x
e,n,z15
[1,2]
1,5
l,w
z6
1,5
e,n,z15
e,n,x
1,7
1,6
1,2
z6
l,w
e,n,z15
z6
IP combined Lanka with Weltevreden (3,10:r:z6)

to form Weltevreden 3,10,[15]:r:z6. Lanka is
now called Weltevreden var. O 15+ by IP.
1,5
1,2
1,5
1,5
e,n,z15
1,6
e,n,z15
1,5
1,5
1,5
1,5
e,n,z15
1,6
1,5
z6
1,7
751

752
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
E1
Lexington
3,10
z10
1,5
I
I
II
I
I
I
I
C2
M
S
N
O
C1
C1
Lezennes
Libreville
Lichtenberg
Ligeo
Ligna
Lika
Lille
6,8
28
41
30
35
6,7
6,7
z4,z23
z10
z10
l,v
z10
i
z38
1,7
1,6
[z6]
1,2
z6
1,7
II
I
II
I
I
I
II
I
I
I
I
I
I
I
I
G
B
F
C2
H
P
D1
D2
I
G
I
D2
C2
E4
C1
Limbe
Limete
Lincoln
Lindenburg
Lindern
Lindi
Lindrick
Linguere
Lingwala
Linton
Lisboa
Lishabi
Litchfield
Liverpool
Livingstone
1,13,22
1,4,12,27
11
6,8
6,14,[24]
38
9,12
9,46
16
13,23
16
9,46
6,8
1,3,19
6,7
g,m,t
b
m,t
i
d
r
e,n,x
b
z
r
z10
z10
l,v
d
d
[1,5]
1,5
e,n,x
1,2
e,n,x
1,5
1,[5],7
z6
1,7
e,n,z15
1,6
1,7
1,2
e,n,z15
l,w
I
I
I
II
I
I
II
II
I
I
I
I
I
IV
I
I
I
I
I
I
N
B
E4
M
V
C2
52
57
C1
J
S
T
Q
V
E4
D1
D1
C1
I
E1
Livulu
Ljubljana
Llandoff
Llandudno
Llobregat
Loanda
Lobatsi
Locarno
Lockleaze
Lode
Lodz
Loenga
Logone
Lohbruegge
Lokstedt
Lomalinda
Lome
Lomita
Lomnava
London
30
4,12,27
1,3,19
28
1,44
6,8
52
57
6,7,14
17
41
1,42
39
44
1,3,19
1,9,12
9,12
6,7
16
3,10
e,h
k
z29
g,[m],[s],t
z10
l,v
z44
z29
b
r
z29
z10
d
z4,z32
l,z13,z28
a
r
e,h
l,w
l,v
1,2
e,n,x
[z6]
1,5
e,n,x
1,5
1,5,7
z42
e,n,x
1,2
z6
1,5
1,2
e,n,x
z6
1,5
e,n,z15
1,6
Losangeles
16
l,v
z6
NOTE
IP combined Lexington with Manila (3,15:z10:1,5)

and Illinois (3,15,34:z10:1,5) to form Lexington
3,10,[15],[15,34]:z10,1,5. Lexington may
IP combined Lille with Bornum (6,7,14:z38:-)

to form Lille 6,7,14:z38:-.
IP combined Eimsbuettel (6,7,14:d:l,w) with

Livingstone to form Livingstone 6,7,14:d:l,w.
IP combined London with Portsmouth

The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
I
I
I
II
I
I
II
B
N
D2
I
G
J
G
Loubomo
Louga
Louisiana
Louwbester
Lovelace
Lowestoft
Luanshya
4,12
30
9,46
16
13,22
17
1,13,23
z
b
z10
z
l,v
g,s,t
g,m,s,t
1,6
1,2
z6
[e,n,x]
1,5
[e,n,x]
I
I
I
I
II
II
I
II
I
II
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
II
II
I
V
I
I
I
I
I
I
I
I
I
S
F
M
X
D2
S
51
60
X
D2
F
E1
D2
E4
H
E4
V
L
E1
B
C2
H
L
D2
E4
U
C1
B
B
I
66
M
V
C2
I
H
G
C2
I
P
Lubumbashi
Luciana
Luckenwalde
Luke
Lundby
Lurup
Lutetia
Luton
Lyon
Maarssen
Maastricht
Macallen
Macclesfield
Machaga
Madelia
Madiago
Madigan
Madison
Madjorio
Madras
Magherafelt
Magumeri
Magwa
Mahina
Maiduguri
Makiling
Makiso
Makoma
Makumira
Malakal
Malawi
Malaysia
Malika
Malmoe
Malstatt
Mampeza
Mampong
Manchester
Mandera
Mango
41
11
28
1,47
9,46
41
51
60
47
9,46
11
3,10
9,46
1,3,19
1,6,14,25
1,3,19
44
21
3,10
4,[5],12
8,20
1,6,14,25
21
9,46
1,3,19
43
6,7
1,4,[5],12,27
1,4,12,27
16
66
28
44
6,8
16
1,6,14,25
13,22
6,8
16
38
r
a
z10
g,m
b
z10
r,i
z
k
z4,z24
z41
z36
g,m,s,t
i
y
c
c
d
d
m,t
i
e,h
d
z10
f,g,t
z29
l,z13,z28
a
e,n,x
e,h
z65
z10
l,z28
i
b
i
z35
l,v
l,z13
k
1,5
e,n,z15
e,n,z15
e,n,x
e,n,x,z15
l,z13,z28
e,n,x
e,n,z15
z39:z42
1,2
1,(2),7
e,n,x
1,7
1,7
1,5
z6
e,n,z15
e,n,z15
l,w
1,6
e,n,x
e,n,z15
e,n,z15
z6
[e,n,x]
1,[5],7
1,2
1,7
1,5
1,7
z6
1,5
1,6
1,7
e,n,z15
1,5
The Difco Manual
NOTE
IP combined Luanshya with Kraaifontein

(1,13,23:g,m,t:[e,n,x]) to form Luanshya
1,13,23:g,m,[s],t:[e,n,x].
753

754
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
6,8
1,9,12
d
g,m,s,t
1,5
z42
3,15
z10
1,5
11
57
6,8
39
11
13,23
66
1,42
3,10
48
1,44
3,10
11
11
13,22
57
9,46
3,10
1,4,12,27
35
1,4,12,27
17
9,46
30
3,10
9,46
6,7,14
43
45
42
3,10
k
z39
z10
e,h
l,v
k
z35
g,z51
k
g,z51
i
d
z
a
a
b
k
a
z41
r
k
k
i
y
a
y
z10
i
a
z
e,h
l,w
e,n,x,z15
1,5
[1,5]
1,5
1,5
e,n,z15
e,n,z15
z35
1,7
1,5
l,z13,z28
1,7
1,2
e,n,x
e,n,z15
l,w
1,5
e,n,x
e,n,z15
1,2
e,n,x
z6
e,n,z15
1,2
e,n,z15
e,n,z15
l,w
I
II
C2
D1
Manhattan
Manica
E2
Manila
I
II
I
I
I
I
V
I
I
IV
I
I
I
I
I
I
I
I
I
I
I
I
I
I
II
I
I
I
I
I
I
F
57
C2
Q
F
G
66
T
E1
Y
V
E1
F
F
G
57
D2
E1
B
O
B
J
D2
N
E1
D2
C1
U
W
T
E1
Mannheim
Manombo
Mapo
Mara
Maracaibo
Marburg
Maregrosso
Maricopa
Marienthal
Marina
Maritzburg
Maron
Maroua
Marseille
Marshall
Maryland
Marylebone
Masembe
Maska
Massakory
Massenya
Matadi
Mathura
Matopeni
Matroosfontein
Mayday
Mbandaka
Mbao
Meekatharra
Melbourne
Meleagridis
I
I
I
I
K
C1
D1
E3
Memphis
Menden
Mendoza
Menhaden
18
6,7
9,12
3,15,34
k
z10
l,v
l,v
1,5
1,2
1,2
1,7
I
II
I
C1
I
X
Menston
Merseyside
Mesbit
6,7
16
47
g,s,[t]
g,t
m,t
[1,6]
[1,5]
[e,n,z15]
NOTE
IP combined Manica with Hamburg (1,9,12:g,t:-)

and Muizenberg (9,12:g,m,s,t:1,5) to form
S. II 1,9,12:g,[m],[s],t:[1,5,7]:[z42].
IP combined Manila and Illinois (3,15,34:z10:1,5)
with Lexington (3,10:z10:1,5) to form Lexington
3,10,[15],[15,34]:z10:1,5. Manila is now
called Lexington var. O 15+ by IP.
Masembe may possess H phase Rz5.
IP combined Meleagridis with Cambridge

(3,15:e,h:l,w) and Wildwood (3,15,34:e,h:l,w)
IP combined Menhaden with Give (3,10:l,v:1,7)

and Newbrunswick (3,15:l,v:1,7) to form Give
3,10,[15],[15,34]:[d],l,v:1,7. Menhaden is now
called Give var. O 15+, 34+ by IP.
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
I
I
I
51
N
C2
Meskin
Messina
Mexicana
51
30
6,8
e,h
d
d
1,2
1,5
1,2
I
I
P
D1
Mgulani
Miami
38
1,9,12
i
a
1,2
1,5
I
I
II
I
I
I
I
I
I
I
J
T
53
H
C1
R
U
G
H
E3
Michigan
Middlesbrough
Midhurst
Midway
Mikawasima
Millesi
Milwaukee
Mim
Minna
Minneapolis
17
1,42
53
6,14,24
6,7,14
1,40
43
13,22
1,6,14,25
3,15,34
l,v
i
l,z28
d
y
l,v
f,g,[t]
a
c
e,h
1,5
z6
z39
1,7
e,n,z15
1,2
1,6
l,w
1,6
I
I
I
L
G
C1
Minnesota
Mishmarhaemek
Mission
21
1,13,23
6,7
b
d
d
e,n,x
1,5
1,5
I
I
I
II
I
I
II
I
I
I
I
I
I
II
I
I
I
I
II
I
I
I
I
I
I
I
G
F
D1
D1
N
C1
I
M
M
F
E1
C2
52
Q
B
B
O
C1
F
U
N
M
N
H
N
J
Mississippi
Missouri
Miyazaki
Mjimwema
Mjordan
Mkamba
Mobeni
Mocamedes
Moero
Moers
Mokola
Molade
Molesey
Mondeor
Mono
Mons
Monschaui
Montevideo
Montgomery
Montreal
Morehead
Morillons
Morningside
Mornington
Morocco
Morotai
1,13,23
11
9,12
1,9,12
30
6,7
16
28
28
11
3,10
8,20
52
39
4,12
1,4,12,27
35
6,7,14
11
43
30
28
30
1,6,14,25
30
17
b
g,s,t
l,z13
b
i
l,v
g,[m],[s],t
d
b
m,t
y
z10
b
l,z28
l,w
d
m,t
g,m,[p],s
a,[d]
c
i
m,t
c
y
l,z13,z28
l,v
[1,5]
1,7
e,n,x
e,n,z15
1,6
[e,n,x]
e,n,x
1,5
1,7
z6
1,5
e,n,x
1,5
l,w
[1,2,7]
[d]:e,n,z15
1,5
1,5
1,6
e,n,z15
e,n,z15
e,n,z15
1,2
The Difco Manual
NOTE
Mexicana was combined with Muenchen. The

name Mexicana has been dropped.
Miami must be differentiated from Sendai
with biochemical tests. Miami is pos. for H2S,
citrate, and tartrate; Sendai is neg.
Mikawasima may possess H phase Rz47 or Rz50.
IP combined Minneapolis and Newington

(3,15:e,h:1,6) with Anatum (3,10:e,h:1,6) to form
Anatum 3,10,[15],[15,34]:e,h:1,6. Minneapolis
is now called Anatum var. O 15+ by IP.
Minnesota may possess H phase Rz33 or Rz49.
Mission was combined with Isangi 6,7,14:d:1,5.
The name Mission has been dropped.
755

756
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
28
9,12
43
47
51
21
47
1,6,14,25
6,8
3,10
16
6,8
3,10
z10
g,q
g,m,[s],t
y
l,z28
r
z
i
z
z38
m,t
d
e,h
l,w
[z42]
1,6
1,5
1,5
e,n,z15
1,5
z42
1,2
1,5
I
I
II
I
I
I
I
I
I
II
I
I
I
M
D1
U
X
51
L
X
H
C2
E1
I
C2
E1
Moroto
Moscow
Mosselbay
Moualine
Moundou
Mountmagnet
Mountpleasant
Moussoro
Mowanjum
Mpila
Mpouto
Muenchen
Muenster
I
II
V
D1
Muguga
Muizenberg
44
9,12
m,t
g,m,s,t
1,5
I
IV
I
II
I
I
II
I
II
I
I
M
F
B
G
D1
C2
T
B
Z
C1
E2
Mundonobo
Mundsburg
Mura
Nachshonim
Naestved
Nagoya
Nairobi
Nakuru
Namib
Namibia
Nancy
28
11
1,4,12
1,13,23
1,9,12
6,8
42
1,4,12,27
50
6,7
3,15
d
g,z51
z10
z
g,p,s
b
r
a
g,[m],s,t
c
l,v
1,7
l,w
1,5
1,5
z6
[1,5]
e,n,x
1,2
I
I
I
I
I
I
I
I
I
C2
G
D2
D1
C2
M
D1
I
E1
Nanergou
Nanga
Nantes
Napoli
Narashino
Nashua
Natal
Naware
Nchanga
6,8
1,13,23
9,46
1,9,12
6,8
28
9,12
16
3,10
g,s,t
l,v
y
l,z13
a
l,v
z4,z24
z38
l,v
e,n,z15
l,w
e,n,x
e,n,x
e,n,z15
1,2
I
I
II
I
II
I
I
I
I
H
D1
D1
B
S
H
C1
N
C1
Ndjamena
Ndolo
Neasden
Neftenbach
Negev
Nessa
Nessziona
Neudorf
Neukoelin
1,6,14,25
1,9,12
9,12
4,12
41
1,6,14,25
6,7
30
6,7
b
d
g,s,t
z
z10
z10
l,z13
b
l,z13,[z28]
1,2
1,5
e,n,x
e,n,x
1,2
1,2
1,5
e,n,z15
e,n,z15
NOTE
IP combined Muenster with Newhaw

(3,15:e,h:1,5) and Arkansas (3,15,34:e,h:1,5)
IP combined Muizenberg with Hamburg
(1,9,12:g,t:-) and Manica (1,9,12:g,m,s,t:z42)
to form S. II 1,9,12:g,[m],[s],t:[1,5,7]:[z42].
IP combined Nancy with Nchanga (3,10:l,v:1,2)

to form Nchanga 3,10,[15]:l,v:1,2. Nancy is
now called Nchanga var. O 15+ by IP.
IP combined Nchanga with Nancy (3,15:l,v:1,2)

to form Nchanga 3,10,[15]:l,v:1,2.
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
1,4,12,27
38
3,15
k
z10
l,v
1,6
1,7
3,15
e,h
1,5
I
I
I
B
P
E2
Neumuenster
Neunkirchen
Newbrunswick
E2
Newhaw
I
I
54
E2
Newholland
Newington
4,12,54
3,15
m,t
e,h
1,6
I
I
I
E1
D1
C2
Newlands
Newmexico
Newport
3,10,[15,34]
9,12
6,8,20
e,h
g,z51
e,h
e,n,x
1,5
1,2
C2
6,8
1,2
I
I
I
I
I
II
I
I
I
I
E1
G
D2
C1
E4
Y
V
J
V
C1
Newport var.
Puerto Rico
Newrochelle
Newyork
Ngaparou
Ngili
Ngor
Ngozi
Niakhar
Niamey
Niarembe
Nienstedten
3,10
13,22
9,46
6,7
1,3,19
48
44
17
44
6,7,14
k
g,s,t
z4,z24
z10
l,v
z10
a
d
a
b
l,w
1,7
1,5
[1,5]
1,5
l,w
l,w
[l,w]
I
I
I
I
I
I
I
I
C1
C1
N
I
E4
M
G
C1
Nieukerk
Nigeria
Nijmegen
Nikolaifleet
Niloese
Nima
Nimes
Nissii
6,7,14
6,7
30
16
1,3,19
28
13,22
6,7,14
d
r
y
g,m,s
d
y
z35
b
z6
1,6
e,n,z15
z6
1,5
e,n,z15
I
I
I
I
II
II
A
E4
P
C1
I
B
Nitra
Niumi
Njala
Nola
Noordhoek
Nordenham
2,12
1,3,19
38
6,7
16
1,4,12,27
g,m
a
k
e,h
l,w
z
1,5
e,n,x
1,7
z6
e,n,x
The Difco Manual
NOTE
IP combined Newbrunswick and Menhaden

(3,15,34:l,v:1,7) with Give (3,10:l,v:1,7) to
form Give 3,10,[15],[15,34]:[d],l,v:[d],1,7.
Newbrunswick is now called Give
var. O 15+ by IP.
IP combined Newhaw and Arkansas
(3,15,34:e,h:1,5) with Muenster (3,10:e,h:1,5)
Newhaw is now called Muenster var. O 15+ by IP.
IP combined Newington and Minneapolis
(3,15,34:e,h:1,6) with Anatum (3,10:e,h:1,6)
to form Anatum 3,10,[15],[15,34]:e,h:1,6.
Newington is now called Anatum
var. O 15+ by IP.
Newport may possess H phase Rz50 or Rz58

or Rz78 or R1,12
Nienstedten was combined with Nissii

(6,7,14:b:-) and called Nienstedten; then IP
combined Nienstedten with Ohio (6,7:b:l,w)
to form Ohio (6,7,14:b:[l,w]). Nienstedten is
now called Ohio var.O 14+ by IP.
Nissii was combined with Nienstedten

(6,7,14:b:l,w) as a monophasic variant of
Nienstedten. Nienstedten is now called a variant
of Ohio by IP. The name Nissii has been dropped.
757

758
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
6,8
6,7
6,7
16
40
8
16
42
11
3,10
a
i
e,h
d
z
r
k
z
z
e,h
1,7
l,w
1,6
e,n,z15
z6
1,7
e,n,x
z6
z6
1,7
I
I
I
I
I
I
I
II
I
I
C2
C1
C1
I
R
C2
I
T
F
E1
Nordufer
Norton
Norwich
Nottingham
Nowawes
Noya
Nuatja
Nuernberg
Nyanza
Nyborg
I
I
I
I
I
I
IV
I
I
II
I
I
II
I
I
I
I
C1
C1
C1
E4
54
I
N
R
N
N
Q
M
S
U
C1
Nyeko
Oakey
Oakland
Obogu
Ochiogu
Ochsenwerder
Ochsenzoll
Ockenheim
Odienne
Odijk
Odozi
Oerlikon
Oevelgoenne
Offa
Ogbete
Ohio
16
6,7
6,7
6,7
1,3,19
6,7,54
16
30
40
30
30
39
28
41
43
6,7
a
m,t
z
z4,z23
z38
k
z4,z23
l,z13,z28
y
a
k
l,v
r
z38
z
b
1,7
z64
1,6,[7]
1,5
[e,n,z15]
1,5
1,6
1,5
z39
e,n,[x],z15
e,n,z15
e,n,z15
1,5
l,w
I
I
I
I
I
I
I
E1
G
E1
E1
I
D2
C1
Ohlstedt
Okatie
Okefoko
Okerara
Oldenburg
Olten
Omderman
3,10
13,23
3,10
3,10
16
9,46
6,7,14
y
g,[s],t
c
z10
d
d
d
e,n,x
z6
1,2
1,2
e,n,z15
e,n,x
I
I
I
I
I
I
I
I
R
C1
M
D1
H
E1
D2
C1
Omifisan
Omuna
Ona
Onarimon
Onderstepoort
Onireke
Ontario
Oranienburg
40
6,7
28
1,9,12
1,6,14,[25]
3,10
9,46
6,7
z29
z10
g,s,t
b
e,h
d
d
m,t
z35
1,2
1,5
1,7
1,5
I
I
T
52
Orbe
Ord
42
52
b
a
1,6
e,n,z15
NOTE
IP combined Selandia (3,15:e,h:1,5) with

Nyborg to form Nyborg 3,10,[15]:e,h:1,7.
IP combined Nienstedten (6,7,14:b:[l,w]) with

Ohio to form Ohio 6,7,14:b:[l,w}. Ohio may
IP combined Omderman with Amersfoort

(6,7:d:e,n,x) to form Amersfoort 6,7,14:d:e,n,x.
Omderman is now called Amersfoort
var. O 14+ by IP.
IP combined Theilallee (6,7,14:m,t:-) with

Oranienburg to form Oranienburg 6,7,14:m,t:-.
Oranienburg may possess H phase Rz57.
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
I
I
I
G
I
E1
Ordonez
Orientalis
Orion
1,13,23
16
3,10
y
k
y
l,w
e,n,z15
1,5
I
I
I
I
I
I
I
I
I
II
I
I
I
I
I
I
C1
K
U
D1
M
C1
F
C1
D1
R
D2
G
R
51
N
E1
Oritamerin
Orlando
Orleans
Os
Oskarshamn
Oslo
Osnabrueck
Othmarschen
Ottawa
Ottershaw
Ouakam
Oudwijk
Overchurch
Overschie
Overvecht
Oxford
6,7
18
43
9,12
28
6,7,14
11
6,7,14
1,9,12
40
9,46
13,22
1,40
51
30
3,10
i
l,v
d
a
y
a
l,z13,z28
g,m,[t]
z41
d
z29
b
l,w
l,v
a
a
1,5
e,n,z15
1,5
1,6
1,2
e,n,x
e,n,x
1,5
1,6
[1,2]
1,5
1,2
1,7
I
II
I
I
I
I
I
C1
C1
C2
V
C1
D1
E2
Oyonnax
Oysterbeds
Pakistan
Palamaner
Palime
Panama
Pankow
6,7
6,7
8
1,44
6,7
1,9,12
3,15
y
z
l,v
d
z35
l,v
d
1,6
z42
1,2
z35
e,n,z15
1,5
1,5
I
I
I
C1
A
B
Papuana
Paratyphi A
Paratyphi B
6,7
1,2,12
1,4,[5],12
r
a
[b]
e,n,z15
[1,5]
[1,2]
I
IV
I
I
II
I
I
I
I
I
I
II
I
I
C1
C2
C2
E4
E1
B
M
D1
M
F
D1
W
P
E4
Paratyphi C
Parera
Paris
Parkroyal
Parow
Pasing
Patience
Penarth
Penilla
Pennsylvania
Pensacola
Perinet
Perth
Petahtikva
6,7,[Vi]
11
8,20
1,3,19
3,10,[15]
4,12
28
9,12
28
11
1,9,12
45
38
1,3,19
c
z4,z23
z10
l,v
g,m,s,t
z35
d
z35
l,z13,z28
d
m,t
g,m,t
y
f,g,t
1,5
1,5
1,7
1,5
e,n,z15
z6
e,n,z15
e,n,z15
[1,2]
e,n,x,z15
e,n,x
1,7
The Difco Manual
NOTE
IP combined Binza (3,15:y:1,5) and Thomasville

(3,15,34:y:1,5) with Orion to form Orion
3,10,[15],[15,34]:y:1,5.
IP combined Khartoum (3,15,34:a:1,7) with

Oxford to form Oxford 3,10,[15],[15,34]:a:1,7.
Panama may possess H phase R1,11

IP combined Pankow with Shangani (3,10:d:1,5)
to form Shangani 3,10,15:d:1,5. Pankow is
now called Shangani var. O 15+ by IP.
Paratyphi B is tartrate neg.; Paratyphi B var.

Java (CDC calls this S. ser. Java) is often
monophasic (1,4,5,12:b:-) and is tartrate pos.
Paratyphi B and Java may possess H phase Rz33.
759

760
Section V
SEROTYPE
I
I
II
I
I
C2
F
X
E1
C2
Phaliron
Pharr
Phoenix
Pietersburg
Pikine
I
I
I
I
I
I
I
I
I
I
I
II
I
I
I
C1
V
D2
H
54
C1
M
K
G
C2
T
D1
E2
Pisa
Planckendael
Ploufragan
Plymouth
Poano
Poeseldorf
Poitiers
Pomona
Pontypridd
Poona
Portanigra
Portbech
Portland
Portsmouth
I
I
I
I
I
I
I
I
I
I
I
K
H
C1
D2
D1
C2
E1
C2
B
F
D1
Potengi
Potosi
Potsdam
Potto
Powell
Praha
Pramiso
Presov
Preston
Pretoria
Pullorum
I
I
I
II
I
I
I
I
II
I
I
I
I
I
G
V
D2
X
X
C2
N
M
T
G
K
B
C2
C1
Putten
Quebec
Quentin
Quimbamba
Quinhon
Quiniela
Ramatgan
Ramsey
Rand
Raus
Rawash
Reading
Rechovot
Redba
O ANTIGENS
PHASE 1
PHASE 2
8
11
47
3,10,[15,34]
8,20
z
b
b
z69
r
e,n,z15
e,n,z15
1,5
1,7
z6
16
6,7
1,44
9,46
1,6,14,25
8,20,54
6,7
28
18
1,13,22
8,20
42
9,12
3,15
i
z4,z23
z4,z23
d
z
i
z
y
g,m
z
d
l,v
z10
l,v
l,w
1,6
e,n,z15
z6
l,z13,z28
z6
1,5
1,7
1,6
1,7
e,n,x,z15
1,5
1,6
18
6,14
6,7,14
9,46
9,12
6,8
3,10
6,8
1,4,12
11
1,9,12
z
z36
l,v
i
y
y
c
b
z
k
1,5
e,n,z15
z6
1,7
e,n,z15
1,7
e,n,z15
l,w
1,2
13,23
44
9,46
47
47
6,8
30
28
42
13,22
6,14,18
1,4,[5],12
8,20
6,7
d
c
d
d
z44
c
k
l,w
z
f,g
c
e,h
e,h
z10
l,w
e,n,z15
1,6
z39
e,n,z15
1,5
1,6
e,n,x,z15
e,n,x
e,n,x
[1,5]
z6
z6
NOTE
Pikine was combined with Altona (8,20:r,[i]:z6).

The name Pikine has been dropped.
Pomona may possess H phases Rz60, Rz70 or Rz80.

Poona may possess H phase Rz59.
IP combined Portsmouth with London

Portsmouth is now called London
var. O 15+ by IP.
IP combined Pullorum with Gallinarum

(1,9,12:-:-). They must be identified
biochemically.
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
11
16
3,10
4,12
11
8,20
1,4,12,27
8,20
9,12
21
16
6,7
1,3,19
9,12
1,13,22
6,7
40
6,7
e,h
z10
f,g,[s]
l,z28
z4,z23
z10
r
g,m,t
d
c
e,h
y
f,g
c
z4,z23
g,t
b
f,g
l,z13,z28
e,n,z15
[1,6]
e,n,x
1,6
l,w
1,7
e,n,x
e,n,x
e,n,x
1,2
z6
[e,n,z15]
1,5
38
45
38
50
51
28
1,13,23
1,13,22
1,6,14
3,15
b
b
l,v
b
z10
z4,z24
z10
m,t
b
e,n,z15
1,5
e,n,x
e,n,x
1,7
1,2
1,5
1,5
1,5
I
I
I
I
I
I
I
I
II
I
I
I
I
I
I
I
I
I
F
I
E1
B
F
C2
B
C2
D1
L
I
C1
E4
D1
G
C1
R
C1
Redhill
Redlands
Regent
Reinickendorf
Remete
Remiremont
Remo
Reubeuss
Rhodesiense
Rhone
Rhydyfelin
Richmond
Rideau
Ridge
Ried
Riggil
Riogrande
Rissen
I
I
I
I
II
I
I
I
II
I
P
W
P
Z
51
M
G
G
H
E2
Rittersbach
Riverside
Roan
Rochdale
Roggeveld
Rogy
Romanby
Roodepoort
Rooikrantz
Rosenthal
I
I
IV
I
II
I
I
II
I
I
I
I
I
54
D1
C1
P
G
G
I
I
H
D1
F
L
B
Rossleben
Rostock
Roterberg
Rothenburgsort
Rotterdam
Rottnest
Rovaniemi
Rowburton
Royan
Ruanda
Rubislaw
Ruiru
Ruki
54
1,9,12
6,7
38
1,13,22
1,13,22
16
16
1,6,14,25
9,12
11
21
4,5,12
e,h
g,p,u
z4,z23
m,t
g,t
b
r,[i]
m,t
z
z10
r
y
y
1,6
1,5
1,7
1,5
[z42]
e,n,z15
e,n,z15
[e,n,x]
e,n,x
e,n,x
I
I
C1
H
Rumford
Runby
6,7
1,6,14,25
z38
c
1,2
e,n,x
The Difco Manual
NOTE
IP combined Ardwick (6,7,14:f,g:-) with

Rissen for form Rissen 6,7,14:f,g:-.
IP combined Rosenthal and unnamed

3,15,34:b:1,5 with Butantan (3,10:b:1,5) to form
Butantan 3,10,[15],[15,34]:b:1,5. Rosenthal is
now called Butantan var. O 15+ by IP.
IP combined Ruki with Ball (1,4,12,27:y:e,n,x)

1,4,[5],12,27:y:e,n,x. The name Ruki has
been dropped.
761

762
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
3,10
Rl,z40
1,7
3,10
1,9,12
16
1,40
30
52
1,4,[5],12
47
l,v
a
e,h
z4,z23
z10
g,t
e,h
b
e,n,z15
1,7
1,5
1,2
1,2
48
16
4,12
[k]
l,v
d,e,h
z39
e,n,x
d,e,n,z15
z29
i
z4,z24
[e,h]
f,g
b
m,t
b
a
r,[i]
e,h
z35
l,z28
c
e,h
y
z38
d
l,z28
b
i
b
z
c
d
k
d
k
a
b
1,5
e,n,z15
e,n,z15
1,7
e,n,z15
1,5
e,n,z15
1,7
e,n,z15
1,6
e,n,x
e,n,z15
1,5
[e,n,x]
e,n,x
z42
1,7
e,n,..
e,n,z15
1,7
1,7
e,n,x
1,5
z6
e,n,x
e,n,z15
E1
Rutgers
I
I
I
IV
I
I
I
I
E1
D1
I
R
N
52
B
X
Ruzizi
Saarbruecken
Saboya
Sachsenwald
Sada
Saintemarie
Saintpaul
Saka
II
I
I
Y
I
B
Sakaraha
Salford
Salinatis
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
II
I
I
I
I
I
I
I
I
II
I
I
I
S
E4
B
C2
C2
D2
I
C1
M
E4
M
R
C2
E4
I
H
B
I
R
H
B
E4
C1
B
C2
I
Z
M
V
Saloniki
16
Samaru
41
Sambre
1,3,19
Sandiego
4,[5],12
Sandow
6,8
Sanga
8
Sangalkam
9,46
Sangera
16
Sanjuan
6,7
Sanktgeorg
28
Sanktmarx
1,3,19
Santander
28
Santhiaba
40
Santiago
8,20
Sao
1,3,19
Saphra
16
Sara
1,6,14,25
Sarajane
1,4,[5],12,27
Sarepta
16
Saugus
40
Schalkwijk
6,14,[24]
Schleissheim
4,12,27
Schoeneberg
1,3,19
Schwabach
6,7
Schwarzengrund 1,4,12,27
Schwerin
6,8
Sculcoates
16
Seaforth
50
Seattle
28
Sedgwick
44
NOTE
Rutgers has been dropped from the scheme

and the H phase Rl,z40 is now considered an
R phase of Give.
IP combined Saka with Sya (47:b:z6) and

called it Sya.
IP states that Salinatis was combined with

Duisburg (1,4,12,27:d:e,n,z15). This is
incorrect; IP should have stated that it was
combined with Sandiego (4,[5],12:e,h:e,n,z15),
because Salinatis loses the d and becomes
Sandiego.
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
I
I
I
E1
E1
E2
Seegefeld
Sekondi
Selandia
3,10
3,10
3,15
r,[i]
e,h
e,h
1,2
z6
1,7
I
IV
I
M
R
D1
Selby
Seminole
Sendai
28
1,40
1,9,12
y
g,z51
a
z6
1,5
I
I
F
E4
Senegal
Senftenberg
11
1,3,19
r
g,[s],t
1,5
I
I
I
II
I
I
N
D1
E1
60
I
E1
Senneville
Seremban
Serrekunda
Setubal
Shamba
Shangani
30
9,12
3,10
60
16
3,10
z10
i
k
g,m,t
c
d
1,5
1,5
1,7
z6
e,n,x
1,5
I
I
I
I
I
I
I
I
IIIa
I
E1
F
P
I
R
C2
M
K
Shanghai
Shannon
Sharon
Sheffield
Sherbrooke
Shikmonah
Shipley
Shomolu
Shomron
16
3,10
11
38
16
40
8,20
28
18
l,v
z35
k
c
d
a
b
y
z4,z32
1,6
l,w
1,6
1,5
1,6
1,5
e,n,z15
l,w
I
I
I
I
D2
B
S
K
Shoreditch
Shubra
Sica
Siegburg
9,46
4,[5],12
41
6,14,18
r
z
b
z4,z23
e,n,z15
1,2
e,n,z15
[1,5]
I
II
I
E1
H
E4
Simi
Simonstown
Simsbury
3,10
1,6,14
1,3,19
r
z10
Rz27
e,n,z15
1,5
I
I
I
I
I
I
I
E1
C2
C1
E1
K
T
C2
Sinchew
Sindelfingen
Singapore
Sinstorf
Sinthia
Sipane
Skansen
3,10
8,20
6,7
3,10
18
1,42
6,8
l,v
y
k
l,v
z38
r
b
z35
l,w
e,n,x
1,5
e,n,z15
1,2
The Difco Manual
NOTE
IP combined Selandia with Nyborg (3,10:e,h:1,7)

to form Nyborg 3,10,[15]:e,h:1,7. Selandia is
now called Nyborg var. O 15+ by IP.
Sendai must be differentiated from Miami with

biochemical tests. Sendai is neg. for H2S,
citrate, and tartrate; Miami is pos.
Senftenberg may possess H phase Rz37 or Rz43
or Rz45 or Rz46. Simsbury (1,3,19:Rz27:-) is now
considered an H phase Rz27 of Senftenberg.
IP combined Pankow (3,15:d:1,5) with

Shangani to form Shangani 3,10,[15]:d:1,5.
Shomron was formerly in Subspecies II, but is

now combined with Arizona 7a,7b:1,7,8:-.
The name Shomron has been dropped.
IP combined Siegburg with Cerro (18:z4,z23:[1,5])

to form Cerro 6,14,18:z4,z23:[1,5]. Siegburg is
now called Cerro var. O 14+. The name
Siegburg has been dropped.
IP combined Simsbury with Senftenberg

1,3,19:g,[s],t:-. Simsbury is now considered
an R phase of Senftenberg. The name
Simsbury has been dropped.
763

764
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
I
I
E4
B
Slade
Sladun
1,3,19
1,4,12,27
y
b
e,n,z15
e,n,x
II
II
I
I
I
I
IV
II
I
I
I
I
I
I
I
II
I
H
N
X
B
H
N
L
B
D2
M
F
C1
M
B
E1
F
E1
Slangkop
Slatograd
Sljeme
Sloterdijk
Soahanina
Soerenga
Soesterberg
Sofia
Sokode
Solna
Solt
Somone
Soumbedioune
Southampton
Southbank
Soutpan
Souza
1,6,14
30
1,47
1,4,12,27
6,14,24
30
21
1,4,12,27
9,46
28
11
6,7
28
1,4,12,27
3,10,15,34
11
3,10
z10
g,t
f,g
z35
z
i
z4,z23
b
r
a
y
z4,z24
b
r
m,t
z
d
z6:z42
z6
e,n,x
l,w
[e,n,x]
z6
1,5
1,5
e,n,x
z6
[1,6]
z39
e,n,x
I
I
I
II
VI
I
I
T
L
V
R
F
P
B
Spalentor
Spartel
Splott
Springs
Srinagar
Stachus
Stanley
1,42
21
44
40
11
38
4,5,12
y
d
g,s,t
a
b
z
d
e,n,z15
1,5
z39
e,n,x
1,2
Stanleyville
1,4,[5],12
z4,z23
[1,2]
I
I
I
II
I
I
I
I
II
II
I
X
N
54
D1
X
F
N
C2
G
E1
E1
Staoueli
Steinplatz
Steinwerder
Stellenbosch
Stellingen
Stendal
Sternschanze
Sterrenbos
Stevenage
Stikland
Stockholm
47
30
3,15,54
1,9,12
47
11
30
6,8
1,13,23
3,10
3,10
k
y
y
z
d
l,v
g,s,t
d
[z42]
m,t
y
1,2
1,6
1,5
1,7
[e,n,x]
1,2
e,n,x
1,[5],7
e,n,x
z6
I
I
I
I
I
N
E1
C2
F
D2
Stoneferry
Stormont
Stourbridge
Straengnaes
Strasbourg
30
3,10
6,8
11
9,46
z4,z23
d
b
z10
d
1,2
1,6
1,5
1,7
NOTE
IP combined Sladun with Abony (1,4,5,12:b:e,n,x)

to form Abony 1,4,[5],12,27:b:e,n,x. Sladun
is now called Abony var. O 27+. The name
Sladun has been
IP combined Eschersheim (3,15:d:e,n,x) with

Souza to form Souza 3,10,[15]:d:e,n,x.
IP combined Cairo (1,4,12,27:d:1,2) with

Stanley to form Stanley 1,4,[5],12,27:d:1,2
IP combined Jaja (4,12,27:z4,z23:-)
with Stanleyville to form Stanleyville
1,4,[5],12,27:z4,z23:[1,2].
Sternschanze may possess H phase Rz59.
IP combined Tournai (3,15:y:z6) with

Stockholm to form Stockholm 3,10,[15]:y:z6.
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
I
I
I
I
II
I
I
II
I
II
I
I
II
I
I
I
IIIb
E4
C1
E4
C1
R
E1
U
D1
W
C1
H
C2
R
H
E4
X
Y
Stratford
Strathcona
Stuivenberg
Stuttgart
Suarez
Suberu
Sudan
Suederelbe
Suelldorf
Sullivan
Sundsvall
Sunnycove
Sunnydale
Surat
Svedvi
Sya
Sydney
1,3,19
6,7
1,3,19
6,7,14
1,40
3,10
43
1,9,12
45
6,7
[1],6,14,[25]
8
1,40
[1],6,14,[25]
1,3,19
47
48
i
l,z13,z28
l,z13,z28
i
c
g,m
l,z13
b
f,g
z42
z
y
k
[r],[i]
l,v
b
i
1,2
1,7
1,5
z6
e,n,x,z15
z39
1,7
e,n,x
e,n,x
e,n,x,z15
e,n,z15
e,n,z15
z6
z
I
I
I
I
II
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
H
I
X
C2
E1
B
C2
E4
C2
C2
E4
X
C1
C1
C2
G
G
D1
T
M
O
J
M
B
I
B
H
M
G
F
Sylvania
Szentes
Tabligbo
Tado
Tafelbaai
Tafo
Takoradi
Taksony
Tallahassee
Tamale
Tambacounda
Tamberma
Tamilnadu
Tampico
Tananarive
Tanger
Tanzania
Tarshyne
Taset
Taunton
Tchad
Tchamba
Techimani
Teddington
Tees
Tejas
Teko
Telaviv
Telelkebir
Telhashomer
[1],6,14,[25]
16
47
8,20
3,10
1,4,12,27
6,8
1,3,19
6,8
8,20
1,3,19
47
6,7
6,7
6,8
1,13,22
1,13,22
9,12
1,42
28
35
17
28
1,4,12,27
16
4,12
1,6,14,25
28
13,23
11
g,p
k
z4,z23
c
z
z35
i
[i]
z4,z32
z29
b
z4,z24
z41
z36
y
y
z
d
z41
k
b
z
c
y
f,g
z36
d
y
d
z10
1,2
[e,n,z15]
z6
z39
1,7
1,5
z6
[e,n,z15]
e,n,x
z35
e,n,z15
1,5
1,6
e,n,z15
1,6
e,n,x
e,n,z15
z6
1,7
e,n,z15
e,n,z15
e,n,z15
e,n,x
The Difco Manual
NOTE
Sydney was formerly in subspecies II, but it

is now combined with Arizona 5:33:31. The
name Sydney has been dropped.
765

766
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
28
1,42
6,7,14
4,5,12
1,47
4,[5],12
1,4,12,27
43
38
6,7,14
z4,z23
z35
z29
g,z51
l,z13,z28
k
z41
k
e,h
m,t
1,6
z6
[1,2,7]
e,n,z15
e,n,z15
e,n,z15
1,(2),5
1,2
1,2
I
I
I
I
I
I
I
I
I
I
M
T
C1
B
X
B
B
U
P
C1
Teltow
Tema
Tennessee
Tennyson
Teshie
Texas
Thayngen
Thetford
Thiaroye
Thielallee
I
I
E4
E3
Thies
Thomasville
1,3,19
3,15,34
y
y
1,7
1,5
C1
Thompson
6,7,14
[k]
[1,5]
I
I
I
I
I
I
I
I
I
II
I
I
I
I
I
I
I
I
I
II
I
I
I
I
C1
V
R
E4
R
B
51
I
B
57
B
T
E4
54
F
N
T
W
D2
C1
Y
K
C2
E2
Tienba
Tiergarten
Tiko
Tilburg
Tilene
Tinda
Tione
Togba
Togo
Tokai
Tokoin
Tomegbe
Tomelilla
Tonev
Toowong
Torhout
Toricada
Tornow
Toronto
Tosamanga
Toucra
Toulon
Tounouma
Tournai
6,7
44
40
1,3,19
1,40
1,4,5,27
51
16
4,12
57
4,12
1,42
1,3,19
21,54
11
30
1,42
45
9,46
6,7
48
18
8,20
3,15
z35
a
l,z13,z28
d
e,h
a
a
a
l,w
z42
z10
b
l,z28
b
a
e,h
z4,z24
g,m,[s],[t]
l,v
z
z
l,w
b
y
1,6
e,n,x
1,2
l,w
1,2
e,n,z15
e,n,x
e,n,x
1,6
1,6:z53
e,n,z15
e,n,z15
1,7
e,n,x
1,7
1,5
e,n,x;[z44]
1,5
1,5
e,n,z15
z6
z6
I
II
I
I
I
B
55
W
B
51
Trachau
Tranoroa
Transvaal
Travis
Treforest
4,12,27
55
45
4,[5],12
1,51
y
k
z4,z24
g,z51
z
1,5
z39
1,7
1,6
NOTE
IP combined Thielallee with Oranienburg

(6,7:m,t:-) to form Oranienburg 6,7,14:m,t:-.
Theilallee is now called Oranienburg
var. O 14+ by IP.
IP combined Thomasville and Binza (3,15:y:1,5)
with Orion (3,10:y:1,5) to form Orion
3,10,[15],[15,34]:y:1,5. Thomasville is now
called Orion var. O 15+ by IP.
R1,10 (6,7:k:R1,10) with Thompson.
Tilburg may possess H phase Rz49.
Toucra may possess H phase Rz58.
IP combined Tournai with Stockholm (3,10:y:z6)

to form Stockholm 3,10,[15]:y:z6. Tournai is
now called Stockholm var. O 15+ by IP.
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
I
I
I
I
I
I
I
I
I
I
I
I
D1
I
D2
B
R
E1
G
B
C2
H
B
E2
Treguier
Trier
Trimdon
Tripoli
Trotha
Truro
Tschangu
Tsevie
Tshiongwe
Tucson
Tudu
Tuebingen
1,9,12
16
9,46
1,4,12,27
40
3,10
1,13,23
4,12
6,8
[1],6,14,[25]
4,12
3,15
z10
z35
z35
b
z10
i
e,h
i
e,h
b
z10
y
z6
1,6
z6
z6
z6
1,7
1,5
e,n,z15
e,n,z15
[1,7]
1,6
1,2
IV
II
I
I
II
I
U
C2
B
G
G
D1
Tuindorp
Tulear
Tumodi
Tunis
Tygerberg
Typhi
43
6,8
1,4,12
1,13,23
1,13,23
9,12,[Vi]
z4,z32
a
i
y
a
d
z52
z6
z6
z42
I
I
I
B
B
C1
Typhimurium
1,4,5,12
Typhimurium var. Copenhagen
Typhisuis
6,7
i
1,4,12
c
1,2,[7]
i
1,5
l,[z13],z28
g,s,t
l,z13
r
z
a
b
z10
a
a
z29
b
b
b
z
d
c
z29
d
z4,z24
b
a
c
z
b
1,5
1,5
1,5
l,w
1,5
e,n,x
e,n,x
1,6
e,n,z15
[e,n,z15]
e,n,x,z15
1,7
e,n,x
1,6
1,7
1,5
e,n,x
1,5
e,n,x
1,2
1,5
1,5
1,6
I
B
I
54
I
E1
I
E1
I
V
I
52
I
G
I
M
I
C1
I
O
I
C2
II
T
I
B
I
N
I
T
I
K
I
C2
II
O
I
52
I
H
I
G
I
C2
I
I
I
M
I
S
The Difco Manual
Tyresoe
Uccle
Uganda
Ughelli
Uhlenhorst
Uithof
Ullevi
Umbilo
Umhlali
Umhlatazana
Uno
Uphill
Uppsala
Urbana
Ursenbach
Usumbura
Utah
Utbremen
Utrecht
Uzaramo
Vaertan
Valdosta
Vancouver
Vanier
Vaugirard
4,12
3,54
3,10,15
3,10
44
52
1,13,23
28
6,7
35
6,8
42
4,12,27
30
1,42
6,14,18
6,8
35
52
1,6,14,25
13,22
6,8
16
28
41
NOTE
IP combined Tuebingen with Amager (e,10:y:1,2)

to form Amager 3,10,[15]:y:1,2. Tuebingen is
now called Amager var. O 15+ by IP.
Typhi may possess H phase Rj or Rz66.
1,2
Typhisuis is a bioserotype found in pigs. It is
like Choleraesuis except tartrate negative.
767

768
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
43
16
3,10
g,t
b
e,h
l,w
1,2
z10
i
e,n,x,z15
i
k
l,w
c
b
b
e,h
m,t
r
d
b
l,v
f,g
z38
z36,z38
i
r
l,[z13],[z28]
i
l,z28
e,h
a
z4,z23
b
e,h
z4,z23
b
z10
b
z4,z23
m,t
z
d
i
i
g,m
k
m,t
g,z51
z4,z23
z35
e,n,x
1,6
1,6
1,5
1,5
1,6
z6
1,5
[e,n,z15]
1,2
[1,2]
1,6
e,n,x
z6
1,6
l,z13,z28
e,n,z15
e,n,x
z42
l,w
e,n,x
z6
1,5
1,5
z6
e,n,z15
[z6]
1,2
[1,7]
1,5
e,n,z15
e,n,x
1,6
1,7
[e,n,z15]
z4,z23
II
I
I
U
I
E1
Veddel
Vegesack
Vejle
I
I
II
I
I
I
I
I
II
I
I
I
I
I
I
I
I
IV
I
I
I
I
II
I
VI
I
I
I
I
I
II
I
I
I
I
I
I
I
I
I
I
IV
I
IV
B
F
J
S
W
D1
J
S
S
E4
M
C1
C2
E4
M
V
T
U
M
F
B
U
G
G
W
B
I
D2
E1
B
L
Q
D1
T
J
W
M
C2
H
J
G
Z
S
S
Vellore
1,4,12,27
Veneziana
11
Verity
17
Verona
41
Verviers
45
Victoria
1,9,12
Victoriaborg
17
Vietnam
41
Vietnam var. subsp. II 41
Vilvoorde
1,3,19
Vinohrady
28
Virchow
6,7
Virginia
8
Visby
1,3,19
Vitkin
28
Vleuten
44
Vogan
1,42
Volksdorf
43
Volksmarsdorf
28
Volta
11
Vom
1,4,12,27
Voulte
43
Vredelust
1,13,23
Vridi
1,13,23
Vrindaban
45
Vuadens
4,12,27
Wa
16
Waedenswil
9,46
Wagadugu
3,10
Wagenia
1,4,12,27
Wandsbek
21
Wandsworth
39
Wangata
1,9,12
Waral
1,42
Warengo
17
Warmsen
45
Warnemuende
28
Warnow
6,8
Warragul
[1],6,14,[25]
Warri
17
Washington
13,22
Wassenaar
50
Waycross
41
Waycross var.
subsp.IV
41
NOTE
IP combined Goerlitz (3,15:e,h:1,2) with

Vejle to form Vejle 3,10,[15]:e,h:1,2.
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
I
I
I
I
N
M
I
E1
Wayne
Wedding
Welikade
Weltevreden
30
28
16
3,10
g,z51
c
l,v
r
e,n,z15
1,7
z6
I
I
I
I
I
I
I
I
X
F
D2
T
D1
I
E4
E1
Wenatchee
Wentworth
Wernigerode
Weslaco
Westafrica
Westeinde
Westerstede
Westhampton
47
11
9,46
42
9,12
16
1,3,19
3,10
b
z10
f,g
z36
e,h
l,w
l,z13
g,s,t
1,2
1,2
1,7
1,6
[1,2]
I
I
II
I
I
I
I
I
I
I
E1
I
E1
O
E1
G
O
B
C1
E3
Westminster
Weston
Westpark
Westphalia
Weybridge
Wichita
Widemarsh
Wien
Wil
Wildwood
3,10,[15]
16
3,10
35
3,10
1,13,23
35
1,4,12,27
6,7
3,15,34
b
e,h
l,z28
z4,z24
d
d
z29
b
d
e,h
z35
z6
e,n,x
z6
1,6
l,w
l,z13,z28
l,w
I
II
B
52
Wilhelmsburg
Wilhemstrasse
1,4,[5],12,27
52
z38
z44
[e,n,z15]
1,5
I
I
I
II
I
II
I
I
I
I
I
I
I
II
I
G
E1
E1
E1
Q
W
C2
B
54
C1
E4
C2
I
J
F
Willemstad
Wilmington
Wimborne
Winchester
Windermere
Windhoek
Wingrove
Winneba
Winnipeg
Winston
Winterthur
Wippra
Wisbech
Woerden
Wohlen
1,13,22
3,10
3,10
3,10
39
45
6,8
4,12
54
6,7
1,3,19
6,8
16
17
11
e,h
b
k
z39
y
g,m,s,t
c
r
e,h
m,t
l,z13
z10
i
c
b
1,6
z6
1,2
1,[5],7
1,5
1,5
1,2
1,6
1,5
1,6
1,6
z6
1,7
z39
1,6
The Difco Manual
NOTE
IP combined Lanka 3,15:r:z6 with Weltevreden

to form Weltevreden 3,10,[15]:r:z6.
IP combined Halmstad (3,15:g,s,t:-) and

Canoga (3,15,34:g,s,t:-) with Westhampton to
form Westhampton 3,10,[15],[15,34]:g,s,t:-.
Westhampton may possess H phase Rz37
or Rz43 or Rz45.
CDC has no 3,10:b:z35.
Wichita may possess H phase Rz37.
IP combined Wildwood and Cambridge

(3,15:e,h:l,w) with Meleagridis (3,10:e,h:l,w)
Wildwood is now called Meleagridis
var. O 15+, 34+ by IP.
IP combined Wilhemstrasse with Lobatsi
(52:z44:1,5,7). The name Wilhemstrasse has
been dropped.
769

770
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
4,12,27
1,7
Womba
I
I
II
I
II
I
H
F
I
D2
G
G
Woodhull
Woodinville
Woodstock
Worb
Worcester
Worthington
1,6,14,25
11
16
9,46
1,13,23
1,13,23
d
c
z42
b
m,t
z
1,6
e,n,x
1,[5],7
e,n,x
e,n,x
l,w
I
I
I
II
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
II
I
I
I
I
II
I
I
II
II
N
D2
G
D1
E1
E4
B
M
C2
G
E1
F
R
54
P
C2
O
W
I
C2
E1
D2
D1
N
E1
J
D1
N
K
C2
I
V
E1
D3
E4
I
B
B
Wuiti
Wuppertal
Wyldegreen
Wynberg
Yaba
Yalding
Yaounde
Yardley
Yarm
Yarrabah
Yeerongpilly
Yehuda
Yekepa
Yerba
Yoff
Yokoe
Yolo
Yopougon
Yoruba
Yovokome
Yundum
Zadar
Zaiman
Zaire
Zanzibar
Zaria
Zega
Zehlendorf
Zeist
Zerifin
Zigong
Zinder
Zongo
Zuerich
Zuilen
Zwickau
30
9,46
1,13,23
1,9,12
3,10,[15]
1,3,19
1,4,12,27
28
6,8
13,23
3,10
11
1,40
54
38
8,20
35
45
16
8,20
3,10
9,46
9,12
30
3,10,[15]
17
9,12
30
18
6,8
16
44
3,10
1,9,12,46,27
1,3,19
16
1,4,[5],12,27
1,4,12,27
z35
z41
a
z39
b
r
z35
g,m
z35
y
i
z4,z24
z35
z4,z23
z4,z23
m,t
c
z
c
d
k
b
l,v
c
k
k
d
a
z10
z10
l,w
z29
z35
c
i
r,i
a
a
e,n,z15
l,w
1,7
e,n,z15
e,n,z15
e,n,z15
1,6
1,2
1,7
z6
e,n,z15
1,2
[e,n,z15]
e,n,z15
l,w
1,5
e,n,x
1,6
e,n,x
1,7
1,5
e,n,z15
z6
1,5
z6
1,2
1,5
1,7
z39
l,w
e,n,z15
e,n,x
z39
NOTE
IP combined Womba with Altendorf (4,12:c:1,7)

to form Altendorf 4,12,27:c:1,7. Womba is
now called Altendorf var. O 27+. The name
Womba has been dropped.
Worthington may possess H phase Rz45.
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
II
II
II
II
II
II
B
B
B
B
B
B
4,12
4,12
4,[5],12
4,12
4,12
4,12
b
e,n,x
f,g,t
(f),g
g,m,t
g,m,t
1,5
1,2,7
z6,z42
z39
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
IV
II
IIIa
IIIb
II
II
II
II
II
II
II
II
II
II
VI
II
II
II
II
II
II
II
II
B
B
B
B
B
B
B
B
B
B
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C1
C2
C2
C2
C2
C2
C2
4,12
4,12,27
1,4,12,27
1,4,12,27
1,4,12,27
4,12
1,4,12,27
4,12
4,12
4,12
6,7,14
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,7
6,8
6,8
6,8
6,8
6,8
6,8
g,z62
i
k
l,v
l,v
l,z28
z
z
z
a
a
b
d
g,m,[s],t
(g),m,[s],t
g,[m],s,t
g,t
g,z51
k
(k)
l,v
l,w
l,w
l,z28
l,z28
m,t
z
z
z4,z24
z10
z29
z41
z42
a
a
b
d
f,g,m,t
g,m,t
z35
1,6
e,n,x
z39
1,5
1,7
z39
1,6
1,5
z6
z39
z42
e,n,x
[1,5]
[z42]
e,n,x:z42
[z6]
z:[z55]
z53
1,5,7
z42
e,n,x
z6
z6
z39
z42
z35
1,7
e,n,x:1,6
1,6
e,n,x
z39
1,5
z6:z42
[e,n,x]
1,7
The Difco Manual
NOTE
Not in IP book
IP calls this monophasic var. of Bechuana.
(Ar. 27:22:31:[37])
(Ar. 27:23:25)
771

II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
II
772
C2
C2
C2
C2
C2
C2
C2
C2
C2
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D1
D2
D2
D2
D2
D2
D2
D2
D3
D3
D3
D3
D3
D3
D3
E1
E1
E1
E1
E1
E1
E1
E1
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
6,8
6,8
6,8
6,8
6,8
6,8
8
6,8
6,8
9,12
1,9,12
9,12
1,9,12
9,12
9,12
1,9,12
1,9,12
9,12
9,12
9,12
9,12
1,9,12
1,9,12
9,12
1,9,12
1,9,12
9,46
9,46
9,46
9,46
9,46
9,46
9,46
9,12,46,27
1,9,12,46,27
1,9,12,46,27
1,9,12,46,27
1,9,12,46,27
1,9,12,46,27
1,9,12,46,27
3,10
3,10
3,10
3,10
3,10
3,10
3,10
3,10
l,v
l,w
l,z28
y
z
z29
z29
z29
a
a
a
a
d
e,n,x
g,m,[s],t
g,z62
l,v
l,v
l,z28
l,z28
m,t
m,t
m,t
z29
z42
e,n,x
g,z62
m,t
z
z10
z10
z39
g,t
l,z13,z28
y
z10
z10
z10
z4,z24
a
b
b
d
l,v
l,z28
l,z28
m,t
e,n,x
z6:z42
e,n,x
1,6:z42
1,5
1,5
e,n,x:z42
e,n,x
1,5,7
1,5
e,n,x
z39
z42
z39
1,6
[1,5,7]:[z42]
e,n,x
z39
1,5:[z42]
e,n,x
1,5
z39
e,n,x
1,[5],7
1,5,7
e,n,x
1,5
z39
z6
1,7
e,n,x
z39
z39
1,5
e,n,x
z39
[1,5]
z39
e,n,x
z39
e,n,x
e,n,x
1,5
z39
1,5
NOTE
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
II
II
II
VI
VI
II
IIIb
IIIb
E1
E1
E1
F
F
F
F
F
3,10
3,10
3,10
11
11
11
11
11
z4,z24
z29
z29
a
b
c
k
l,v
e,n,x
1,5
1,7
e,n,z15
z53
z
IIIb
II
IIIa
IV
II
II
II
II
II
II
II
II
II
IIIa
V
II
II
IIIb
II
II
II
II
II
V
II
II
IIIa
IIIa
F
F
F
F
F
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
11
11
11
11
11
1,13,23
13,22
1,13,22
13,23
1,13,23
1,13,23
1,13,23
1,13,23
1,13,23
1,13,22
13,22
13,23
13,22
13,23
13,22
13,23
13,23
13,22
13,22
13,22
1,13,23
13,22
13,23
l,v
z
z4,z23
z4,z32
a
a
b
d
d
g,m,s,t
g,m,s,t
g,[s],t
g,51
i
k
k
l,v
l,w
l,z28
l,z28
l,z28
m,t
r
z
z
z4,z23
z4,z23,z32
z53
e,n,x
1,5
1,5
e,n,x
z42
e,n,x
e,n,z15
1,5
z42
z42
1,5:z42
z41
1,5,7
e,n,x
1,5
1,5
z6
z42:z39
z42
IIIa
II
II
II
II
II
II
VI
IIIb
II
II
G
G
G
G
G
G
G
H
H
H
H
1,13,23
1,13,22
1,13,23
1,13,23
1,13,23
13,22
13,23
[1],6,14
(6),14
6,14,[24]
6,14
z4,z24
z10
z29
z29
z39
z39
a
b
k
k
z6
1,5
e,n,x
1,5,7
1,7
1,6
1,5
e,n,x,z15
1,6
[e,n,x]
The Difco Manual
NOTE
(Ar. 17:29:25)
(Ar. 38).
(Ar. 17:23:25)
(Ar. 17:1,2,5:-)
(Ar. 18:13,14:-)
(Ar. 18:23:30)
(Ar. 18:1,2,5:-)
(Ar. 18:1,6,7:-). CDC would call this 1,6,7,9.
(Ar. 18:1,3,11:-)
(Ar. 7a,7c:43:28)
773

IIIb
II
IIIb
IIIb
IIIb
IIIb
IIIb
IV
IIIb
IIIb
IIIb
II
IIIb
IIIb
II
II
II
II
II
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
II
II
II
II
IIIb
IIIb
II
IV
IIIb
II
II
II
II
IIIb
IIIb
II
IIIb
IIIb
II
IIIb
II
774
H
H
H
H
H
H
H
H
H
H
H
H
H
H
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
J
J
J
J
J
J
J
J
J
J
J
J
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
(6),14
1,6,14
(6),14
(6),14
(6),14
(6),14
(6),14
6,14
(6),14
(6),14
(6),14
1,6,14
(6),14
(6),14
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
16
17
17
17
17
17
17
17
17
17
17
17
17
k
k
k
l,v
l,v
l,v
r
z4,z23
z10
z10
z10
z42
z52
z52
b
b
b
d
g,[m],[s],t
i
k
k
(k)
l,v
l,v
l,v
l,v
m,t
z
z4,z24
z6
z10
z10
z29
z36
z52
b
e,n,x,z15
g,m,s,t
g,t
i
k
k
l,v
l,v
m,t
r
y
z
z6:z42
z53
z
z35
z53
z
e,n,x,z15
z:[z53]
z53
1,6
e,n,x,z15
z35
e,n,x
z39
z42
1,5
z42
z35
z
z53
z35
1,5,7
z:[z61]
z35
z53
e,n,x
z42
1,6
1,5,7
e,n,x,z15
1,5
z35
z6
1,[5],7
z39
z35
z
e,n,x,z15
z35
NOTE
(Ar. 7a,7c:29:31)
(Ar. 7a,7c:29:25)
(Ar. 7a,7c:23:31)
(Ar. 7a,7c:23:21)
(Ar. 7a,7c:23:25)
(Ar. 7a,7c:24:31)
(Ar. 7a,7c:27:28)
(Ar. 7a,7c:27:31:[25])
(Ar. 7a,7c:27:25)
(Ar. 7a,7c:26:28)
(Ar. 7a,7c:26:21)
(Ar. 25:33:21)
(Ar. 25:29:31)
(Ar. 25:29:25)
(Ar. 25:22:21)
(Ar. 25:23:30)
(Ar. 25:23:31:[41])
(Ar. 25:23:21)
(Ar. 25:23:25)
(Ar. 25:27:30)
(Ar. 25:27:28)
(Ar. 25:26:21)
(Ar. 12:33:21)
(Ar. 12:29:32)
(Ar. 12:23:28)
(Ar. 12:23:21)
(Ar. 12:24:31)
The Difco Manual
Section V
IIIa
IIIa
IIIa
IIIa
IIIb
IIIb
IIIa
IV
IIIa
IV
IIIa
IIIb
IIIb
IIIb
IIIb
IIIb
II
IIIb
II
II
IIIa
II
IIIa
IIIb
IV
II
II
IIIb
II
IIIa
IV
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
II
IIIb
II
IIIa
IIIa
IV
IIIb
IIIb
IIIb
IIIa
IV
The Difco Manual
J
J
J
J
J
J
J
J
J
J
K
K
K
K
K
K
K
K
K
K
K
K
K
K
K
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
17
17
17
17
17
17
17
17
17
17
18
18
18
18
18
18
18
18
18
18
18
18
18
18
18
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
z4,z23
z4,z23,z32
z4,z24
z4,z32
z10
z10
z29
z29
z36
z36
g,z51
(k)
(k)
l,v
l,v
l,v
m,t
r
y
z4,z23
z4,z23
z4,z24
z4,z32
z10
z36,z38
b
c
c
g,[m],[s],t
g,z51
g,z51
i
i
k
k
l,v
l,v
m,t
r
z
z4,z23
z4,z24
z4,z32
z10
z10
z10
z29
z36
e,n,x,z15
z
z53
z54
e,n,x,z15
z
z53
1,5
z
e,n,x,z15
e,n,x,z15
1,5
e,n,x
e,n,x,z15
1,5,7
e,n,x,z15
e,n,x,z15
z
z
z57
e,n,x,z15
z
z53
NOTE
(Ar. 12:1,2,5:- and 12:1,2,6:-)

(Ar. 12:1,6,7,9:-)
(Ar. 12:1,3,11:-)
(Ar. 12:1,6,7:- and 12:1,7,8:-)
(Ar.12:27:28). May possess H phase Rz56 (Ar. 38).
(Ar. 12:27:31)
(Ar. 12:16,17,18:-)
(Ar. 12:17,20:-)
(Ar. 7a,7b:13,14:-)
(Ar. 7a,7b:22:25)
(Ar. 7a,7b:22:34)
(Ar. 7a,7b:23:28)
(Ar. 7a,7b:23:31)
(Ar. 7a,7b:23:25)
(Ar. 7a,7b:24,31)
(Ar. 7a,7b:1,2,5:- and 7a,7b:1,2,6:-)

(Ar. 7a,7b:1,6,7:- and 7a,7b:1,7,8:-)
(Ar. 7a,7b:27:28)
(Ar. 22:32:28)
(Ar. 22:13,14:-)
(Ar. 22:33:30)
(Ar. 22:33:28)
(Ar. 22:29:28)
(Ar. 22:29:31)
(Ar. 22:23:31)
(Ar. 22:23:40)
(Ar. 22:24:31)
(Ar. 22:1,2,5:- and 22:1,2,6:-)
(Ar. 22:1,3,11:-)
(Ar. 22:27:28)
(Ar. 22:27:31)
(Ar. 22:27:25)
(Ar. 22:16,17,18:-)
775

IIIb
II
II
II
II
II
II
II
II
II
IIIb
IIIb
II
II
II
II
II
II
II
II
II
II
II
II
II
II
IIIa
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
II
II
IIIb
IIIb
IIIb
IIIb
IIIa
IIIa
IIIb
776
L
M
M
M
M
M
M
M
M
M
M
M
M
M
N
N
N
N
N
N
N
N
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
21
28
28
28
28
28
28
28
28
28
28
28
28
28
30
30
30
30
30
30
30
30
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
z65
a
b
e,n,x
g,m,t
g,m,t
g,s,t
l,z28
m,t
z
z10
z10
z29
z29
b
c
g,m,s
k
l,z28
m,t
z6
z39
d
g,m,s,t
g,t
g,t
g,z51
i
i
i
i
k
k
k
(k)
(k)
l,v
l,v
l,v
l,z28
m,t
r
r
r
r
z4,z23
z4,z32
z10
e,n,x,z15
e,n,x
e,n,x
1,7
e,n,x
z39
e,n,x
1,5
[e,n,x]
1,5
z
z:[z57]
1,5
e,n,x
z6
z39
e,n,x
e,n,x,z15
z6
1,6
1,7
1,5
1,5
z42
e,n,x,z15
z
z35
z53
e,n,x,z15
z
z53
z
z35
1,5,7
e,n,x,z15
z35
e,n,x,z15
z
z35
z61
z35
NOTE
(Ar. 22:32:28)
(Ar. 35:27:31)
(Ar. 35:27:31:[40])
(Ar. 20:13,14:-)
(Ar. 20:33:28)
(Ar. 20:33:31)
(Ar. 20:33:21)
(Ar. 20:33:25)
(Ar. 20:29:28)
(Ar. 20:29:31)
(Ar. 20:29:25). May possess H phase Rz50 (Ar.42).
(Ar. 20:22:31)
(Ar. 20:22:21)
(Ar. 20:23:30)
(Ar. 20:23:28)
(Ar. 20:23:21)
(Ar. 20:24:28)
(Ar. 20:24:31)
(Ar. 20:24:21)
(Ar. 20:24:41)
(Ar. 20:1,2,6:-)
(Ar. 20:1,7,8:-)
(Ar. 20:27:21)
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
IIIa
IIIa
IIIb
IIIb
IIIb
IIIb
II
IIIa
IV
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
O
O
O
O
O
O
P
P
P
P
P
P
P
P
P
P
P
35
35
35
35
35
35
38
38
38
38
38
38
38
38
38
38
38
z29
z36
z52
z52
z52
z52
b
g,z51
g,z51
i
i
k
k
k
(k)
(k)
(k)
1,5,7
e,n,x,z15
z
z35
1,2
z
z53
e,n,x,z15
z
z53
1,5,7
z
z35
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIa
IV
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
P
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
38
(k)
(k)
l,v
l,v
l,v
r
r
r
r
z4,z23
z4,z23
z10
z10
z47
z52
z52
z53
z54
z55
z
z35
z53:[z54]
1,5,7
e,n,x,z15
z:[z57]
z35
z
z53
z53
z35
z53
IIIa
IIIb
II
II
II
II
II
II
II
II
II
II
P
P
Q
Q
Q
Q
Q
Q
Q
Q
R
R
38
38
39
39
39
39
39
39
39
39
1,40
1,40
z61
z61
a
c
e,n,x
g,m,t
l,v
l,z28
m,t
a
a
z53
z39
e,n,x
1,7
1,5
z39
e,n,x
1,7
1,5
z6
The Difco Manual
NOTE
(Ar. 20:16,17,18:-)
(Ar. 20:17,20:-)
(Ar. 20:26:30)
(Ar. 20:26:28)
(Ar. 20:26:31)
(Ar. 20:26:21)
(Ar. 16:13,14:-)
(Ar. 16:33:31)
(Ar. 16:33:25)
(Ar. 16:29:28)
(Ar. 16:29:31)
(Ar. 16:29:25)
(Ar. 16:22:30)
(Ar. 16:22:31)
(Ar. 38).
(Ar. 16:22:34)
(Ar. 16:22:37)
(Ar. 16:23:31)
(Ar. 16:23:21)
(Ar. 16:23:25:[34])
(Ar. 16:24:30)
(Ar. 16:24:28)
(Ar. 16:24:31:[40])
(Ar. 16:24:21)
(Ar. 16:1,2,6:-)
(Ar. 16:27:31)
(Ar. 16:27:25)
(Ar. 16:39:25)
(Ar. 16:26:21)
(Ar. 16:26:25)
(Ar. 42) or Rz76 (Ar. Rz76). CDC does not
have monophasic.
(Ar. 16:41:-)
(Ar. 16:41:25)
777

Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
II
II
II
II
II
II
II
II
II
IIIb
IIIb
IIIb
II
IIIb
IIIb
IIIb
II
II
II
II
IV
II
II
II
IIIa
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
40
1,40
1,40
1,40
1,40
1,40
1,40
40
1,40
40
40
40
40
40
40
40
1,40
1,40
40
1,40
40
1,40
1,40
40
40
b
c
e,n,x
e,n,x,z15
g,t
g,t
g,t
g,t
g,[m],[s],t
g,z51
i
k
k
k
l,v
l,v
l,z28
l,z28
m,t
m,t
m,t
z
z
z
z4,z23
z39
1,[5],7
1,6
1,5
[e,n,x]
e,n,x,z15
z39
z42
[e,n,x,z15]
1,5,7
z:z57
z6
z53
z
z53
1,5:z42
z39
z39
z42
z6
z39
z42
IIIa
IV
IIIa
R
R
R
40
40
40
z4,z24
z4,z24
z4,z32
IIIa
II
IIIb
IIIa
V
IIIa
II
II
II
II
V
II
VI
IIIb
II
II
IIIa
II
II
R
R
R
R
R
R
R
R
R
R
R
S
S
S
S
S
S
S
S
40
1,40
40
40
1,40
40
40
40
1,40
1,40
40
41
41
41
41
41
41
41
41
z4,z32
z6
z10
z29
z35
z35
z39
z39
z42
[z42]
z81
b
b
c
c
g,m,s,t
g,z51
k
k
1,5
z35
1,5:z42
1,7
1,6
1,(5),7
[1,5]
1,7
e,n,x,z15
[z6]
z6
1,6
[z6]
778
NOTE
(Ar. 10a,10b:13,14:[28])
(Ar. 10a,10b:33:30)
(Ar. 10a,10b:29:31:40)
(Ar. 10a,10b:29:25)
(Ar. 10a,10b,(10c):23:31)
(Ar. 10a,10b:23:25)
(Ar. 10a,10b:1,2,5:-; 10a,10b:1,2,5,6:-; and

10a,10b:1,2,6:-)
(Ar. 10a,10b:1,3,11:-)
Also called Degania var. subsp. IV.
(Ar. 10a,10b:1,2,10:-; 10a,10c:1,2,10:-;
and 10a,10b:1,7,8:-)
(Ar. 10a,10b:27:21)
(Ar. 10a,10b:16,18:-)
(Ar. 10a,10b:17,20:-)
H z81 was formerly H a in S. bongori.
(Ar. 13:32:28)
(Ar. 13:13,14:-)
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
IIIb
II
IIIa
IV
IIIa
IIIa
IIIa
IIIa
IV
IIIa
IV
II
II
II
II
IIIa
IV
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
II
II
II
IIIb
IIIb
IIIb
IIIa
IIIa
IV
II
IIIb
S
S
S
S
S
S
S
S
S
S
S
S
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
41
41
41
41
41
41
41
41
41
41
41
41
42
42
42
42
1,42
42
42
42
42
42
42
42
42
42
1,42
42
42
42
42
42
42
42
1,42
42
42
(k)
l,z13,z28
z4,z23
z4,z23
z4,z23,z32
z4,z24
z4,z32
z29
z29
z36
z52
b
d
[e,n,x]
g,z51
g,z51
k
k
k
k
(k)
l,v
l,v
l,v
l,v
l,w
l,[z13],z28
m,t
r
r
r
z4,z23
z4,z24
z4,z24
z6
z10
[z35]
e,n,x,z15
1,6
z6
z6
1,6
e,n,x,z15
z
z35
z35
1,5,7
e,n,x,z15
z
z53
e,n,x
[z6]
[e,n,x,z15]
z
z53
1,6
II
II
IIIb
IIIb
II
IIIb
IIIb
IV
IIIb
II
T
T
T
T
T
T
T
T
T
U
42
42
42
42
42
42
42
42
42
43
z10
z10
z10
z10
z10
z10
z10
z36
z52
a
1,2
e,n,x,z15
e,n,x,z15
z
z6
z35
z67
z
1,5
The Difco Manual
NOTE
(Ar. 13:22:[21])
(Ar. 13:1,2,5:- and 13:1,2,6:-)
Also called Waycross var. subsp. IV.
(Ar. 13:1,6,7,9:-)
(Ar. 13:1,3,11:-)
(Ar. 13:1,6,7:- and 13:1,7,8:-)
(Ar. 13:16,17,18:-)
(Ar. 13:17,20:-)
(Ar. 15:13,14:-)
(Ar. 15:29:-)
(Ar. 15:29:28)
(Ar. 15:29:31)
(Ar. 15:29:21)
(Ar. 15:22:21)
(Ar. 15:23:30)
(Ar. 15:23:28)
(Ar. 15:23:31)
(Ar. 15:23:25)

(Ar. 15:24:31)
(Ar. 15:24:25)
(Ar. 15:1,2,5:- and 15:1,2,6:-)
(Ar. 15:1,3,11:-)

(Ar. 38) and Rz50 (Ar. 42).
(Ar. 15:27:28)
(Ar. 15:27:31)
(Ar. 15:27:21)
(Ar. 15:27:46)
(Ar. 15:26:31)
779

Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
II
II
II
II
II
II
II
IIIa
IV
II
IIIb
IIIb
II
IIIb
IIIb
IIIb
II
IV
IIIa
IIIa
IV
IV
II
II
IIIa
IIIb
II
II
IV
II
IIIa
IV
IIIa
IIIa
IV
IIIa
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
U
V
V
V
V
V
V
V
V
V
V
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
43
1,44
44
44
44
44
44
44
44
44
44
a
d
d
d
e,n,x,z15
e,n,x,z15
g,t
g,z51
g,z51
g,z62
k
l,v
l,z13,z28
r
r
r
z
z4,z23
z4,z23
z4,z24
z4,z24
z29
z29
z29
z36
z52
e,n,x
g,t
g,z51
z4,z23
z4,z23
z4,z23
z4,z23,z32
z4,z24
z4,z24
z4,z32
z6
e,n,x,z15
z39
z42
1,(5),7
1,6
1,5
e,n,x
z
z53:[Rz56]
1,5
e,n,x,z15
z
z53
1,5
e,n,x
z42
z53
1,6
z42
IV
II
IV
V
IIIa
IV
II
II
IIIa
IV
IIIa
V
V
V
V
W
W
W
W
W
W
W
44
44
44
44
45
45
45
45
45
45
45
z29
z29
z36,[z38]
z39
g,z51
g,z51
m,t
z
z4,z23
z4,z23
z4,z24
e,n,x:z42
1,5
1,5
780
NOTE
(Ar. 21:13,14:-)
(Ar. 21:29:31)
(Ar. 21:23:25:[38])
(Ar. 21:24:28)
(Ar. 21:24:31)
(Ar. 21:24:25)
(Ar. 21:1,2,5:- and 21:1,2,6:-)

(Ar. 21:1,3,11:-)
(Ar. 21:17,20:-)
(Ar. 21:26:25)
(Ar. 1,3:1,2,5:- and 1,3:1,2,6:-)

(Ar. 1,3:1,6,7,9:-)
(Ar. 1,3:1,3,11:-)
(Ar. 1,3:1,2,10:- and 1,3:1,7,8:-). IP calls
z4,z23,z32, Ar. 1,2,10.
(Ar. 11:13,14:-)
(Ar. 11:1,2,5:-)
(Ar. 11:1,3,11:-)
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
IIIa
IIIa
II
II
II
IV
II
II
IIIb
IIIb
IIIb
IIIb
II
II
II
IIIa
IIIb
W
W
W
W
W
W
X
X
X
X
X
X
X
X
X
X
X
45
45
45
45
45
45
47
47
47
47
47
47
47
47
47
47
47
z4,z32
z29
z29
z29
z29
z36,z38
a
b
c
c
c
c
d
e,n,x,z15
g,t
g,z51
i
1,5
e,n,x
z42
e,n,x,z15
z6
1,5,7
e,n,x,z15:[z57]
z
z35
e,n,x,z15
1,6
e,n,x
e,n,x,z15
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IV
IIIb
X
X
X
X
X
X
X
X
X
X
47
47
47
47
47
47
47
47
47
47
i
i
i
k
k
k
k
k
l,v
l,v
z
z35
z53:[z57]
1,5,7
e,n,x,z15
z
z35
z53
1,5,(7)
IIIa
IIIb
IIIb
IIIb
IIIb
IIIa
IIIb
IIIb
IIIb
IIIb
X
X
X
X
X
X
X
X
X
X
47
47
47
47
47
47
47
47
47
47
l,v
l,v
l,v
l,v
l,v
r
r
r
r
r
e,n,x,z15
z
z35
z53
z57
1,5,7
z
z35
z53
IIIb
47
z53:[z60]
IIIb
II
IIIa
II
IIIb
IIIb
IIIb
X
X
X
X
X
X
X
47
47
47
47
47
47
47
r
z
z4,z23
z6
z10
z10
z10
z53:Rz50:z60
e,n,x,z15
1,6
1,5,7
z
z35
The Difco Manual
NOTE
(Ar. 11:1,7,8:-)
(Ar. 11:16,18:-)
(Ar. 28:32:30)
(Ar. 23:32:28 and 28:32:28:[40])
(Ar. 28:32:31)
(Ar. 28:32:21)
(Ar. 28:13,14)
(Ar. 42).
(Ar. 28:33:31)
(Ar. 23:33:21 and 28:33:21)
(Ar. 23:33:25 and 28:33:25:[40])
(Ar. 28:29:30)
(Ar. 28:29:28)
(Ar. 28:29:31)
(Ar. 23:29:21)
(Ar. 23:29:25)
(Ar. 42).
(Ar. 28:23:28)
(Ar. 23:23:31)
(Ar. 28:23:21)
(Ar. 28:23:25)
(Ar. 28:23:40)
(Ar. 23:24:-). CDC does not have this.
(Ar. 23:24:30)
(Ar. 23:24:31)
(Ar. 23:24:21 and 28:24:21)
(Ar. Rz74)
(Ar. 23:24:25:[44]). May possess H phase
Rz70 and Rz72 (Ar. Rz70 or Rz72).
(Ar. 28:24:25:42:44). Not in IP book.
(Ar. 28:1,2,5:-)
(Ar. 28:27:30)
(Ar. 28:27:31)
(Ar. 28:27:21)
781

Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
IIIa
II
IV
IIIb
IIIb
IIIb
IIIb
II
IIIb
II
V
II
IIIb
II
IIIa
IIIb
X
X
X
X
X
X
X
Y
Y
Y
Y
Y
Y
Y
Y
Y
47
47
47
47
47
47
47
48
48
48
48
48
48
48
48
48
z29
z29
z36
z52
z52
z52
z52
a
a
a
b
b
c
d
g,z51
i
e,n,x,z15
1,5,7
e,n,x,z15
z
z35
z6
z35
z39
[z6]
z
1,2
z:[z72]
IIIb
IIIb
IIIb
IIIb
II
IIIb
IIIb
IIIb
IIIb
IIIb
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
48
48
48
48
48
48
48
48
48
48
i
i
i
k
k
k
k
k
k
(k)
z35:[z57]
z53
z61
1,5,(7)
e,n,x,z15
e,n,x,z15
z
z35:[Rz75]
z53
z53
IIIb
48
l,v
1,5,(7)
IIIb
IIIb
IIIb
II
IIIa
IV
IIIa
IIIa
IIIa
Y
Y
Y
Y
Y
Y
Y
Y
Y
48
48
48
48
48
48
48
48
48
l,v
r
r
z
z4,z23
z4,z23
z4,z23,z32
z4,z24
z4,z32
z
e,n,x,z15
z
1,5
IV
VI
IIIb
IIIb
II
IIIa
Y
Y
Y
Y
Y
Y
48
48
48
48
48
48
z4,z32
z10
z10
z10
z29
z29
1,5
e,n,x,z15
z
IV
IIIb
Y
Y
48
48
z29
z35
z52
782
NOTE
(Ar. 28:16,18:-)
(Ar. 28:26:30)
(Ar. 28:26:28)
(Ar. 28:26:31)
(Ar. 28:26:21)
(Ar. 5:35:21). Not in 1992 IP, but is in Bergey.
(Ar. 29:32:31)
(Ar. 5:13,14:-)
(Ar. 5,29:33:31:[z72]). CDC does not have
z72 strain.
(Ar. 29:33:21:[40])
(Ar. 5:33:25)
(Ar. 5,29:33:41)
(Ar. 5:29:30)
(Ar. 5:29:28)
(Ar. 5,29:29:31)
(Ar. [5:29:21:Rz75]). CDC does not have Rz75.
(Ar. 5,29:29:25)
(Ar. 5:22:25 and Ar. 5,29:22:25). Called
5:22:25 by IP.
or Rz50 (Ar. 39 or 42).
(Ar. 5,29:23:31)
(Ar. 5:24:28)
(Ar. 5,29:24:31)
(Ar. 5:1,2,5:-; 5:1,2,5,6:-; and 5:1,6:-)
(Ar. 5:1,6,7,9:-). IP calls this 5:1,6,7:-.
(Ar. 5:1,3,11:-)
(Ar. 5:1,6,7:-; 5:1,7,8:-; and Ar. 5:1,2,10:-).
IP calls z4,z32, Ar. 1,7,8; and would call
z4,z23,z32, Ar. 1,2,10.
(Ar. 5:27:28)
(Ar. 5,29:27:31)
(Ar. 5:16,18). This is not in IP book, but is
on Rohdes list.
(Ar. 5:21:26)
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
IIIa
IV
V
IIIb
IIIb
V
V
IV
IV
II
IV
II
IIIb
IIIb
IIIb
IIIb
IIIb
II
IIIb
Y
Y
Y
Y
Y
Y
Y
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
Z
48
48
48
48
48
48
48
50
50
50
50
50
50
50
50
50
50
50
50
z36
z36,[z38]
z39
z52
z52
z65
z81
a
b
b
d
g,z62
i
i
i
k
k
k
k
e,n,x,z15
z
z6
e,n,x
1,5,7
e,n,x,z15
z
1,5,7
e,n,x,z15
e,n,x:z42
z
IIIb
IIIb
Z
Z
50
50
k
k
z35
z53
IIIb
IIIb
IIIb
IIIb
Z
Z
Z
Z
50
50
50
50
(k)
(k)
l,v
l,v
z
z35
e,n,x,z15
z
IIIb
II
II
IIIb
IIIb
IIIb
IIIb
Z
Z
Z
Z
Z
Z
Z
50
50
50
50
50
50
50
l,v
l,w
l,z28
r
r
r
r
z35
e,n,x,z15:z42
z42
1,5,(7)
e,n,x,z15
z
z35
IIIb
50
z53
IIIa
IIIa
Z
Z
50
50
z4,z23
z4,z23,z32
IIIa
IV
IIIa
Z
Z
Z
50
50
50
z4,z24
z4,z24
z4,z32
IIIb
50
z10
IIIb
50
z10
z53
The Difco Manual
NOTE
(Ar. 5,29:17,20:-)
(Ar. 29:26:28)
(Ar. 5:26:31)
(Ar. 9a,9c:33:30)
(Ar. 9a,9c:33:28)
(Ar. 9a,9c:33:31)
(Ar. 9a,9c:29:30)
(Ar. 9a,9c:29:28)
(Ar. 9a,9b:29:31 and 9a,9c:29:31). Ar. 9a,9b
may possess H phase Rz50 (Ar. 42).
(Ar. 9a,9b:29:21)
(Ar. 9a,9b:29:25 and 9a,9c:29:25). IP and
Rohde only list the 9a,9c.
(Ar. 9a,9b:22:31)
(Ar. 9a,9b:22:21)
(Ar. 9a,9b:23:28)
(Ar. 9a,9b:23:31 and 9a,9c:23:31).
IP only lists 9a,9c.
(Ar. 9a,9c:23:21)
(Ar. 9a,9b:24:30)
(Ar. 9a,9c:24:28)
(Ar. 9a,9b:24:31 and 9a,9c:24:31).
(Ar. Rz58). This is not in IP book, but is on
Rohdes list.
(Ar. 42). This is not in IP book, but is on
Rohdes list.
(Ar. 9a,9b:1,2,5:- and 9a,9b:1,2,6:-)
(Ar. 9a,9b:1,2,10:-). Called 9a,9b:1,6,7:- by IP
and Rohde.
(Ar. 9a,9b:1,3,11:-)
(Ar. 9a,9b:1,2,10; 9a,9b:1,6,7:-; and
9a,9b:1,7,8:-). 9a,9b:1,2,10:- and 9a,9b:1,7,8:used by IP and Rohde.
(Ar. 9a,9c:27:31). May possess H phase Rz56
(Ar. 38).
(Ar. 9a,9c:27:25)
783

Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
IIIa
IIIa
IIIb
IIIb
IIIb
IIIb
IV
II
II
IIIa
IIIb
IIIb
IIIa
IIIa
IIIa
II
II
IIIb
Z
Z
Z
Z
Z
Z
51
51
51
51
51
51
51
51
51
51
52
52
50
50
50
50
50
50
51
51
51
51
51
51
51
51
51
51
52
52
z29
z36
z52
z52
z52
z52
b
c
g,s,t
g,z51
k
l,v
z4,z23
z4,z24
z4,z32
z29
c
c
1,5,7
z
z35
z53
e,n,x
z35
z
e,n,x,z15
k
k
II
II
II
IIIb
IIIb
IIIb
IIIb
II
IIIb
II
II
II
II
IIIa
IV
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
II
II
IIIb
IIIb
IIIb
52
52
52
52
52
52
52
52
52
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
53
52
52
52
52
52
52
52
52
52
53
53
1,53
53
53
1,53
53
53
53
53
53
53
53
53
53
53
53
53
d
d
g,t
k
k
(k)
l,v
z
z52
c
d
d
d
g,z51
g,z51
i
k
k
(k)
(k)
l,v
l,v
l,z28
l,z28
r
r
r
e,n,x,z15
z39
z35
z53
z35
z53
z39
z
1,5
1,5
z39
z42
z
e,n,x,z15
z
z
z35
e,n,x,z15
z35
e,n,x
z6
z
z35
z68
II
IIIb
53
53
53
53
z
z
1,5
1,5,(7)
784
NOTE
(Ar. 9a,9b:16,18:-)
(Ar. 9a,9b:17,20:-)
(Ar. 9a,9b:26:30 and 9a,9c:26:30)
(Ar. 9a,9b:26:31 and 9a,9c:26:31)
(Ar. 9a,9b:26:21 and 9a,9c:26:21)
(Ar. 9a,9b:26:25 and 9a,9c:26:25)
(Ar. 1,2:13,14:-)
(Ar. 1,2:29:21)
(Ar. 1,2:23:31)
(Ar. 1,2:1,2,5:- and 1,2:1,2,6:-)
(Ar. 1,2:1,3,11:-)
(Ar. 1,2:1,7,8:-)
(Ar. 31:32:29). This is not in IP book,

but is on Rohdes list.
(Ar. 31:29:21)
(Ar. 31:29:25)
(Ar. 31:22:21)
(Ar. 31:23:25)
(Ar. 31:26:31)
(Ar. 1,4:13,14:-)
(Ar. 1,4:33:31)
(Ar. 1,4:29:28)
(Ar. 1,4:29:31)
(Ar. 1,4:22:31)
(Ar. 1,4:22:21)
(Ar. 1,4:23:28)
(Ar. 1,4:23:21)
(Ar. 1,4:24:31)
(Ar. 1,4:24:21)
(Ar. 1,4:24:47). This was formerly called z56
(Ar. 38), but was changed to z68 (Ar. 47).
(Ar. 1,4:30:31)
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
II
IIIa
IV
IIIa
IIIa
IIIa
53
53
53
53
53
53
53
53
53
53
53
53
z
z4,z23
z4,z23
z4,z23,z32
z4,z24
z4,z32
z6
IIIb
IIIa
IIIb
IIIb
II
II
II
II
II
IIIa
IIIa
II
IIIa
II
IIIb
II
II
II
IIIb
IIIb
IIIb
53
53
53
53
56
56
56
56
56
56
56
56
56
57
57
57
57
57
57
57
57
53
53
53
53
56
56
56
56
56
56
56
56
56
57
57
57
57
57
57
57
57
z10
z29
z52
z52
d
e,n,x
l,v
l,z28
z
z4,z23
z4,z23,z32
z10
z29
a
c
d
g,[m],s,t
g,t
i
i
k
z35
z35
z53
1,7
z39
z6
e,n,x
z42
z:[z60]
1,5
z42
e,n,x,z15
z
e,n,x,z15
IV
IIIb
II
II
II
II
IIIb
IIIb
IIIb
IIIb
II
IIIb
IIIb
IIIb
57
57
58
58
58
58
58
58
58
58
58
58
58
58
57
57
58
58
58
58
58
58
58
58
58
58
58
58
z4,z23
z10
a
b
c
d
i
k
l,v
l,v
l,z13,z28
r
r
r
z
z6
1,5
z6
z6
e,n,x,z15
z
e,n,x,z15
z35
z6
e,n,x,z15
z
z53
II
II
IIIb
II
IIIb
58
58
58
58
58
58
58
58
58
58
z6
z10
z10
z10
z10
1,6
1,6
e,n,x,z15
z6
z53
The Difco Manual
NOTE
(Ar. 1,4:1,2,5:- and 1,4:1,2,6:-)

(Ar. 1,4:1,6,7,9:-)
(Ar. 1,4:1,3,11:-)
(Ar. 1,4:1,6,7:-). IP combined this with
53:z4,z23,z32:- (Ar. 1,4:1,6,7,9:-).
(Ar. 1,4:27:21)
(Ar. 1,4:16,18:-)
(Ar. 1,4:26:21)
(Ar. 1,4:26:25)
(Ar. 14:1,2,5:- and 14:1,2,6:-)

(Ar. 14:1,6,7,9:-)
(Ar. 14:16,18:-)
(Ar. 34:32:31:[44])
(Ar. 34:33:28)
(Ar. 34:33:31)
(Ar. 34:29:28). CDC does not have this
and not on Rohdes list.
(Ar. 34:27:31)
(Ar. 1,33:33:28)
(Ar. 1,33:29:31)
(Ar. 1,33:23:28)
(Ar. 1,33:23:21)
(Ar. 1,33:24:28)
(Ar. 1,33:24:31)
(Ar. 39) or Rz57 (Ar. 40) or Rz70 (Ar. Rz70).
(Ar. 1,33:27:28)
(Ar. 42).
785

II
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
II
IIIa
IIIb
IIIb
IIIa
IIIa
IIIb
II
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
II
V
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
786
58
58
58
59
59
59
59
59
59
59
59
59
59
59
59
59
59
59
59
59
59
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
61
61
61
61
61
61
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
58
58
58
59
59
59
59
59
59
59
59
59
59
59
1,59
59
59
59
59
59
59
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
61
61
61
61
61
61
z39
z52
z52
c
i
i
i
k
(k)
(k)
(k)
l,v
l,v
r
z
z4,z23
z10
z10
z29
z36
z52
b
i
i
i
k
k
(k)
l,v
r
r
r
r
z10
z10
z10
z29
z41
z52
z52
z52
z52
c
c
i
i
i
i
e,n,x,z15
z
z35
e,n,x,z15
e,n,x,z15
z
z35
z53
e,n,x,z15
z
z35
z
z53
z35
z6
z53
z57
[z53]
[1,16]
e,n,x,z15
z35
z
z35
z53
z
e,n,x,z15
z
z35
z53
z
z35
z53
e,n,x
1,5,[7]
z
z35
z53
1,5,(7)
z35
e,n,x,z15
z
z35
z53
NOTE
(Ar. 1,33:26:31)
(Ar. 1,33:26:21)
(Ar. 19:32:28)
(Ar. 19:33:28)
(Ar. 19:33:31)
(Ar. 19:33:21)
(Ar. 19:29:25)
(Ar. 19:22:28)
(Ar. 19:22:31)
(Ar. 19:22:21)
(Ar. 19:23:31)
(Ar. 19:23:25)
(Ar. 19:24:21)
(Ar. 19:1,2,5:- and 19:1,2,6:-)
(Ar. 19:27:25)
(Ar. 19:27:40)
(Ar. 19:16,18:-)
(Ar. 19:17,20:-)
(Ar. 19:26:[25])
(Ar. 24:33:28)
(Ar. 24:33:21)
(Ar. 24:29:31)
(Ar. 24:29:21)
(Ar. 24:22:25)
(Ar. 24:23:31)
(Ar. 24:24:28)
(Ar. 24:24:31)
(Ar. 24:24:21)
(Ar. 24:24:25)
(Ar. 24:27:31)
(Ar. 24:27:21)
(Ar. 24:27:25)
(Ar. 24:26:30)
(Ar. 24:26:31)
(Ar. 24:26:21)
(Ar. 24:26:25)
(Ar. 26:32:30)
(Ar. 26:32:21)
(Ar. 26:33:28)
(Ar. 26:33:31)
(Ar. 26:33:21)
(Ar. 26:33:25)
The Difco Manual
Section V
SEROTYPE
O ANTIGENS
PHASE 1
PHASE 2
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
61
k
k
(k)
l,v
l,v
l,v
r
r
r
r
1,5,(7)
z35
z53
1,5,7:[z57]
z
z35
1,5,7
z
z35
z53
IIIb
V
IIIb
IIIb
IIIb
IIIb
IIIa
IIIa
IIIa
IIIa
IIIa
IIIa
IIIa
IIIa
IIIa
IIIb
IIIb
IIIb
II
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
IIIb
II
V
V
61
61
61
61
61
61
62
62
62
62
62
63
63
63
63
65
65
65
65
65
65
65
65
65
65
65
65
65
65
65
65
65
65
65
65
66
66
61
61
61
61
61
61
62
62
62
62
62
63
63
63
63
65
65
65
65
65
65
65
65
65
65
65
65
65
65
65
65
65
65
65
65
66
66
z10
z35
z52
z52
z52
z52
g,z51
z4,z23
z4,z32
z29
z36
g,z51
z4,z23
z4,z32
z36
c
c
c
g,t
i
(k)
(k)
(k)
l,v
l,v
l,v
l,v
r
z10
z10
z52
z52
z52
z52
z39
z81
z35
1,5,7
z
z35
z53
1,5,7
z
z53
e,n,x,z15
z
z35
z53
e,n,x,z15
z
z35
z53
z35
e,n,x,z15
z
e,n,x,z15
z
z35
z53
1,6
The Difco Manual
NOTE
(Ar. 26:29:30)
(Ar. 26:29:21). CDC does not have this.
(Ar. 26:22:25)
(Ar. 26:23:30:[40])
(Ar. 26:23:31)
(Ar. 26:23:21)
(Ar. 26:24:30)
(Ar. 26:24:31)
(Ar. 26:24:21)
(Ar. 39).
(Ar. 26:27:21)
(Ar. 26:26:30)
(Ar. 26:26:31)
(Ar. 26:26:21)
(Ar. 26:26:25)
(Ar. 6:13,14:-)
(Ar. 6:1,2,5:-)
(Ar. 6:1,7,8:-)
(Ar. 6:17,18:-)
(Ar. 6:17,20:-)
(Ar. 8:13,14:-)
(Ar. 8:1,2,5:-)
(Ar. 8:1,7,8:-)
(Ar. 8:17,20:-)
(Ar. 30:32:30)
(Ar. 30:32:31)
(Ar. 30:32:25)
(Ar. 30:33:28)
(Ar. 30:22:31)
(Ar. 30:22:21)
(Ar. 30:22:25)
(Ar. 30:23:28)
(Ar. 30:23:31)
(Ar. 30:23:21)
(Ar. 30:23:25)
(Ar. 30:24:21)
(Ar. 30:27:28)
(Ar. 30:27:31)
(Ar. 30:26:28)
(Ar. 30:26:31)
(Ar. 30:26:21)
(Ar. 30:26:25)
787
Shigella Antisera
Section V
Bacto Shigella Antisera

Shigella Antiserum Poly Group A . Shigella Antiserum Poly
Group A1 . Shigella Antiserum Poly Group B . Shigella
Antiserum Poly Group C . Shigella Antiserum Poly Group C1
Shigella Antiserum Poly Group C2 . Shigella Antiserum Poly
Group D . Alkalescens-Dispar Antiserum Poly
Intended Use
Bacto Shigella Antisera are used for identifying Shigella species by
the slide agglutination test. Bacto Alkalescens-Dispar Antiserum Poly
is used for identifying the Alkalescens-Dispar Group of microorganisms
by the slide agglutination test.

Shigella species cause the human diarrheal disease shigellosis (classic
bacillary dysentery). The range of illness is from mild diarrhea to severe
dysentery characterized by abdominal cramps and frequent passage of
bloody, mucoid stools. While the disease is usually self-limiting, it can

Shigella Poly Antisera Group A-D
powdered cake.
powdered cake.
Culture Response
Rehydrate Shigella Antiserum Poly Groups A-D and
Alkalescens-Dispar Antiserum Poly per label directions.
Perform the slide agglutination test using appropriate QC
Antigens Shigella Group A-D or Alkalescens-Dispar.
SHIGELLA ANTISERUM
QC ANTIGEN
Poly Group A
Poly Group A1
Poly Group B
Poly Group C
Poly Group C1
Poly Group C2
Poly Group D
Alkalescens-Dispar
Antiserum Poly
Shigella Group A
Shigella Group A1
Shigella Group B
Shigella Group C
Shigella Group C1
Shigella Group C2
Shigella Group D
788
REACTION
3+
3+
3+
3+
3+
3+
3+
3+
be life threatening to the young, the elderly and malnourished persons.

Shigella species are carried primarily in humans and are not generally
distributed in nature. While transmission is usually direct person-toperson and through contaminated water supplies, foodborne outbreaks
do occur.
The genus Shigella belongs to the family Enterobacteriaceae. Shigella
species are facultatively anaerobic, gram-negative bacilli that typically
are oxidase negative, lactose negative, H2S negative and non-gas
producing. Shigella and Escherichia are genetically related. Certain
strains of E. coli may resemble Shigella biochemically because both
can be lactose-negative, nonmotile or non-gas-producing. These
anaerogenic, nonmotile types have historically been called the
Alkalescens-Dispar group and are presently classified as E. coli.
Serological testing with polyvalent and group specific antisera should
be used to confirm the identification of isolates that are morphologically
and biochemically identified as Shigella species. Shigella species are
nonmotile, so serological identification is based on somatic (O)
antigens. However, some strains have envelope antigens that prevent
agglutination in somatic antisera. Heating the suspension at 100C for
15-60 minutes destroys these interfering antigens. The four named
species or serotypes of Shigella are S. dysenteriae (10 serovars),
S. flexneri (six serovars), S. boydii (15 serovars) and S. sonnei. For a
complete and current explanation of the classification of Shigella,
consult appropriate references.1
The Alkalescens-Dispar group of microorganisms is currently recognized
as anaerogenic, nonmotile biotypes of E. coli. Consult appropriate
references for biochemical tests specific for differentiating these strains
from Shigella.1-6

Identification of Shigella species includes the isolation of the microorganism, biochemical identification and serological confirmation.
desired homologous reaction is rapid, does not dissociate (high avidity)
and binds strongly (high affinity).
Because a microorganism (antigen) may agglutinate with antibodies
produced in response to other species, heterologous reactions are possible. These are characterized as weak in strength or slow in formation.
The Difco Manual
Section V
Shigella Antisera
Such unexpected and, perhaps, unpredictable reactions may lead to

some confusion in serological identification. Therefore, a positive
homologous agglutination reaction should support the morphological
and biochemical identification of the microorganism. Homologous
reactions occur rapidly and are strong. Heterologous reactions form
slowly and are weak.
Expiration Date
Reagents
Rehydrated Shigella Antisera Poly and Alkalescens-Dispar Antiserum

Poly that are cloudy or have a precipitate at any time during the period
of use should be discarded.
Shigella Antisera Poly and Alkalescens-Dispar Antiserum Poly are

lyophilized, polyclonal rabbit antisera containing approximately
0.04% Thimerosal as a preservative.
Shigella Antisera Poly are absorbed when necessary to render each lot
of serum as specific as practical. Antisera are absorbed to a certain
point without reducing homologous reactions to an unsatisfactory level.
They have been absorbed inter- and intra-specifically except that
Shigella antisera are not prepared from or tested for:
S. dysenteriae provisional serotypes,
S. flexneri X and Y variants, or
Alkalescens-Dispar Groups other than types 1-4.
ANTISERUM
REACTS WITH

S. dysenteriae types 1-7

S. dysenteriae types 8ab, 8ac, 9, 10
S. flexneri types 1-6
S. boydii types 1-7
S. boydii types 8-11
S. boydii types 12-15
S. sonnei I and II
Alkalescens-Dispar
Groups 1,2,3 and 4
When rehydrated and used as described, each vial of Shigella Antisera

Poly and Alkalescens-Dispar Antiserum Poly contains sufficient
Precautions
2. Shigella Antiserum Poly Group A
Storage
Store lyophilized and rehydrated Shigella Antisera Poly and
Alkalescens-Dispar Antiserum Poly at 2-8C. Prolonged exposure of
reagents to temperatures other than those specified is detrimental to
the products.
The Difco Manual
Lyophilized Shigella Antisera Poly and Alkalescens-Dispar Poly are
stable through the expiration date on the label when stored as described.
Procedure
Materials Provided
Shigella Antisera Poly Group A
Shigella Antisera Poly Group A1
Shigella Antisera Poly Group B
Shigella Antisera Poly Group C
Shigella Antisera Poly Group C1
Shigella Antisera Poly Group C2
Shigella Antisera Poly Group D

Agglutination slides with 1 inch squares
Applicator sticks
Waterbath, boiling
QC Antigen Shigella Groups
Reagent Preparation
Shigella Antisera Poly and Alkalescens-Dispar Antiserum Poly: To
rehydrate, add 3 ml of sterile 0.85% NaCl solution and rotate gently to
completely dissolve the contents. The rehydrated antiserum is considered a 1:2 working dilution. Subsequent dilutions are based on
this as a starting dilution.

From clinical specimens, Shigella can be recovered on selective differential media such as Hektoen Enteric Agar or XLD Agar. For specific
recommendations, consult appropriate references.2,3,4 Determine that
a pure culture of the microorganism has been obtained and that
biochemical test reactions are consistent with the identification of the
organism as a Shigella species. After these criteria are met, serological
Shigella can be recovered from various types of foods when samples
are processed to recover injured microorganisms and to prevent
overgrowth of competing microorganisms. Consult appropriate
references for recommended procedures when testing food samples.5,6
After following an established protocol, determine that a pure culture
of the microorganism has been obtained. Biochemical test reactions
789
Shigella Antisera
should be consistent with the identification of the organism as a

Shigella species. After these criteria are met, serological identification
can be performed.
Test Procedure
Use this procedure to test the isolate with each selected Shigella
Antisera Poly or Alkalescens-Dispar Antiserum Poly.
1. Shigella Antiserum: Dispense 1 drop (35 l) of the antiserum to
be tested on an agglutination slide.
2. Negative control: Dispense 1 drop of sterile 0.85% NaCl solution
on an agglutination slide.
3. Test isolate: Transfer a loopful of growth of the test organism to
the drops of antisera and NaCl solution and mix thoroughly.
4. Positive control: Dispense 1 drop of the Shigella Antiserum to
be tested on an agglutination slide. Add 1 drop of the appropriate
QC Antigen.
5. Mix each reaction area with a separate applicator stick and rock
for 1 minute. Read for agglutination.
Results
No agglutination.
3. Negative control: Should show no agglutination. If autoagglutination
occurs, tests results cannot be reported. To test for autoagglutination,
transfer the isolate to selective medium.
4. Test isolates: 3+ or greater agglutination within 1-2 minutes is a
positive result.
5. If no agglutination occurs or agglutination is weak, follow this
procedure to remove blocking envelope antigens:
Prepare a dense suspension of the isolate from an agar medium
in 3-5 ml of sterile 0.85% NaCl solution.
Heat in a boiling waterbath for 30-60 minutes and cool. The
suspension should not show precipitation after heating. If this
occurs, select another colony for testing.
Centrifuge at 1,000 rpm for 10-15 minutes.
Aspirate and discard the supernatant.
Resuspend the sediment in 0.5 ml sterile 0.85% NaCl solution.
Use a drop of the suspension and perform the slide agglutination
test as outlined above.
6. A partial (less than 3+) or delayed agglutination reaction should be
considered negative.
7. If test results for either the positive control or negative control
are not as described, the test is invalid and results cannot be
reported.
790
Section V

purity, morphological characteristics and biochemical reactions that
are consistent with identification of the microorganism as a
Shigella species.
2. Serological methods alone cannot identify the isolate as a Shigella
species.
5. Shigella Antisera Poly and Alkalescens-Dispar Poly have been
tested using cultures taken directly from agar media. These antisera
have not been tested using antigen suspensions in NaCl solution or
other diluents. If the user applies variations in the recommended
steps, each lot of antiserum must be tested with known control
cultures to verify expected reactions under the modified procedure.
References
1. Ewing, W.H. (ed.). 1986. Edwards and Ewings identification of
New York, NY.
St. Louis, MO.
p. 6.01-6.06. In FDA Bacteriological Analytical Manual, 8th ed.
6. Vanderzant, C., and D. F. Splittstoesser (eds.). 1992.
of foods, 3rd edition. American Public Health Association,
Washington, D.C.
Packaging
3 ml
2834-47
3 ml
2776-47
3 ml
2835-47
3 ml
2836-47
3 ml
2777-47
3 ml
2778-47
3 ml
2837-47
3 ml
2838-47
The Difco Manual
Section V
Streptococcus Antigens and Antisera
Bacto Streptococcus Antigens and Antisera

Streptococcus Antiserum Group A . Streptococcus Antiserum
Group B . Streptococcus Antigen Group A . Streptococcus
Antigen Group B
Intended Use
Bacto Streptococcus Antisera are used in the serological grouping of
Group A and Group B streptococci by the capillary tube precipitin
technique.
Streptococcus agalactiae (Group B streptococci) causes neonatal

sepsis and meningitis. Other infections in children and adults include
bacteremia, endocarditis and pneumonia.
Identification of Group A and Group B streptococci includes isolation

of the microorganism and biochemical and serological identification.
Serological identification involves the reaction in which the microorganism (antigen) reacts with its corresponding antibody. This in vitro
reaction produces fine particles called precipitation.
Streptococci are gram-positive cocci that are facultative anaerobes.

They are catalase negative and may be alpha-, beta- or non-hemolytic.
Streptococcus pyogenes (Group A) is the most common cause of

bacterial pharyngitis in children. Symptoms include fever, pharyngeal erythema and edema, tonsillar exudate and enlarged cervical
lymph nodes. Physical findings alone cannot distinguish between
Group A streptococcal pharyngitis and pharyngitis caused by other
agents such as viruses or mycoplasma. Other infections caused
by Group A streptococci include scarlet fever, impetigo and skin
infections that range from mild to severe with toxic shock symptoms
and tissue necrosis.
Reagents
Bacto Streptococcus Antigens are used in the quality control testing

of Bacto Streptococcus Antisera Groups A and B.

Streptococcus Antisera Groups A and B
powdered cake.
Streptococcus Antigens Groups A and B
Solution Appearance: Colorless to light yellow, clear liquid.
Quality Control Results

Rehydrate Streptococcus Antisera per label directions.
Perform the capillary tube precipitin technique using
appropriate Streptococcus Antigens.
ANTISERUM
ANTIGEN
REACTION
Streptococcus Antiserum
Group A
Group A
Group B
Group B
Streptococcus Antigen
Group A
Group B
Group B
Group A
Positive
The Difco Manual
Beta-hemolytic streptococci Group A and Group B have carbohydrate

group-specific antigens that can be extracted. Streptococcus Antisera
and Streptococcus Antigens are used together in the capillary precipitin
test to serologically identify the microorganisms.
Streptococcus Antisera Groups A and B are lyophilized,

polyclonal rabbit antisera containing approximately 0.02%
Thimerosal as a preservative. When rehydrated and used as described,
each vial of Streptococcus Antisera contains sufficient reagent for
50 precipitin tests.
Streptococcus Antigens Groups A and B are ready-to-use cellular
extracts of S. pyogenes and S. agalactiae, respectively, containing
Thimerosal as a preservative. When used as described, each vial
of Streptococcus Antigens contains sufficient reagent for 50
precipitin tests.
Precautions
2. Streptococcus Antiserum Group A
Streptococcus Antiserum Group B
Streptococcus Antigen Group A
Streptococcus Antigen Group B
4. Streptococcus Antigens are not intended for use in the immunization
of humans or animals.
Negative
Storage
Positive
Store lyophilized and rehydrated Streptococcus Antisera at 2-8C.
Negative
Store Streptococcus Antigens at 2-8C.

Prolonged exposure of reagents to temperatures other than those
791
Streptococcus Antigens and Antisera
Section V
Expiration Date
Rehydrated Streptococcus Antiserum that is cloudy or has a precipitate anytime during use should be discarded.
4.
5.
Antigens must be smooth uniform suspensions. Examine antigen vials

for precipitation before use. Suspensions with precipitation are not
usable and should be discarded.
Procedure
6.
Materials Provided
Streptococcus Antiserum Group A

Capillary tubes
Plasticine block
Reagent Preparation
tests. Ensure that glassware and pipettes are clean and free of residues
such as detergent.
Streptococcus Antisera: To rehydrate, add 1 ml sterile distilled or
Streptococcus Antigens are ready to use.

Group A and Group B streptococci can be recovered on routine culture
media such as sheep blood agar. For specific recommendations on
isolation and presumptive identification, consult appropriate references.1,2
of the organism as a Group A or Group B Streptococcus. After these
criteria are met, serological identification can be performed.
Test antigen extract: To prepare, extract the carbohydrate, group-specific
antigen from a pure culture of the microorganism by the Lancefield hot
HCl, autoclave, enzyme or other such method. For specific information
on these methods, consult appropriate references.1,2
Test Procedure
Add the antiserum to the capillary tube first so that it will be layered
above the extract.
1. Streptococcus Antiserum: Dip a capillary tube into the antiserum
and allow a column of 2-3 cm to rise into the tube.
2. Holding the forefinger on the top end of the capillary tube, remove
the tube from the antiserum vial. Clean the tip with a lint-free
tissue to remove excess antiserum. Do not allow air into the tube.
If this occurs, discard the tube and begin again.
3. Test antigen extract: Dip the capillary tube into the prepared
extract until the antiserum and the antigen come in contact with
792
7.
8.
each other. If an air bubble separates them, discard the tube and
repeat steps 1-3.
Remove the tube from the extract and invert slightly to allow the
column to move to the center of the tube.
Wipe excess fluid from the tube and insert in a plasticine block,
antiserum end upward. Wipe the capillary tube so that it is free
of fingerprints or any material that might interfere with a clear
reading.
Positive control: Repeat steps 1-5, using (in step 3) a Streptococcus
Antigen (Group A or B) that is homologous to the antiserum used
in step 1.
Negative control: Repeat steps 1-5, using (in step 3) a Streptococcus
Antigen (Group A or B) that is not homologous to the antiserum
used in step 1.
Incubate all capillary tubes at 22 2C for 5 minutes. Examine
for the formation of a white precipitate at the interface of the
antiserum and the antigen. Observe at 5 minute intervals for up to
30 minutes.
Results
1. A strongly positive reaction develops within 5 minutes, a weaker
reaction develops within 30 minutes.
2. Disregard any precipitate that appears after 30 minutes.
3. Precipitation in a tube indicates that the test antigen extract is
homologous to the Streptococcus Antiserum Group A or Group B
used.
4. Observe at 5 minute intervals within the 30 minute period because
the precipitate may dissolve (prozone phenomenon).

purity as well as on morphological characteristics and biochemical
reactions that are consistent with identification of the microorganism as S. pyogenes or S. agalactiae.
2. Serological methods alone cannot identify the isolate as S. pyogenes
or S. agalactiae.
References
Washington, D.C.
Packaging
Streptococcus Antiserum Group A
1 ml
2672-50
1 ml
2741-50
1 ml
2978-50
1 ml
2979-50
The Difco Manual
Section V
USR Antigen & USR Test Control Serum Set
Bacto USR Antigen

Bacto USR Test Control Serum Set
Intended Use
Bacto USR Antigen is nontreponemal antigen used in the Unheated
Serum Reagin (USR) Test.1
Bacto USR Test Control Serum Set is standardized human sera used
for controlling the USR Test.

Treponema pallidum is the causative agent of syphilis. Syphilis is a
chronic infection with clinical manifestations that occur in distinct
stages. Specific laboratory tests are recommended for the detection of
each stage of the disease.
During the primary stage, treponemes present in the characteristic
lesion, a chancre, are detectable by dark-field microscopy2 or by the
Direct Fluorescent Antibody Test for T. pallidum (DFA-TP). During
the secondary stage, most serological tests for syphilis are reactive and
treponemes may be found in the lesions by using dark-field microscopy.
The latent period, which is asymptomatic, may last for years. Serological
tests are usually reactive in the early latent period, but the reactivity of

USR Antigen
Appearance:
Milky white, opaque suspension

after gentle mixing.
Nontreponemal Antigen Reactive Serum

Lyophilized Appearance: White to cream colored, button to
powdered cake.
Rehydrated Appearance: Light gold to light amber, clear to
USR Weakly Reactive Serum
powdered cake.
Nontreponemal Antigen Nonreactive Serum
powdered cake.
Rehydrate the sera contained in the USR Test Control Serum
Set per label directions. Perform the USR Test according to
the Test Procedure. Each serum in the USR Test Control
Serum Set should yield appropriate reactions when tested with
the USR Antigen.
Use the USR Antigen suspension only if it produces the
expected reactivity with the control sera.
The Difco Manual
non-treponemal tests decreases during the late latent period. Symptoms

of the tertiary or late stage of syphilis may occur 10-20 years after
initial infection. Approximately 71% of patients in the tertiary stage
of syphilis have reactive non-treponemal tests.3,4 In the tertiary stage,
treponemal tests will usually be reactive and are the only basis for
diagnosis. The lesions in tertiary syphilis will have few treponemes.
Neurosyphilis and late cardiovascular syphilis are complications of
tertiary syphilis.
Since the clinical manifestations of syphilis can be confused with other
infectious or noninfectious conditions, proper diagnosis must include
microscopic examination of lesion material and serological results.3
The USR Antigen is a nontreponemal antigen composed of cardiolipin,
cholesterol and lecithin. The antigen is a modification of the VDRL
Antigen emulsion in a USR suspending solution. The antigen suspension
contains choline chloride, which enhances the reactivity of reagin in
unheated serum.5 The USR Test is suitable for qualitative as well as
quantitative determinations.
Nontreponemal tests measure reagin, an antibody-like substance that
can be detected in syphilitic serum. Reagin is also occasionally found
in the serum of persons with other acute or chronic diseases. Reactive
nontreponemal tests aid in the diagnosis of latent subclinical syphilis
and are effective tools for detecting cases in epidemiological investigations. Nontreponemal tests are superior to treponemal tests for
following the response to therapy.3
Nontreponemal antigen tests are not entirely specific for syphilis,
nor do they have satisfactory sensitivity in all stages of syphilis.
Whenever the results of a nontreponemal antigen test disagree with the
clinical impression, a treponemal antigen test such as the Fluorescent
Treponemal Antibody-Absorption (FTA-ABS)2,3 should be performed.
Nontreponemal tests such as the USR, RPR and VDRL tests are used
to screen patient serum. Treponemal tests such as the FTA-ABS are
used for confirmation.
The likelihood of obtaining a reactive USR Test result in various stages
of untreated syphilis has been reported as follows:3
STAGES OF UNTREATED SYPHILIS
% REACTIVE
Primary
Secondary
Latent
80
100
95

In the USR Test procedure, the patients unheated serum is mixed with
a buffered saline suspension of USR Antigen containing cardiolipin,
lecithin and cholesterol. The combination of reagin and USR Antigen
forms microscopic clumping called flocculation.
Reagents
USR Antigen is 0.03% cardiolipin and 0.9% cholesterol dissolved in
absolute alcohol with sufficient lecithin (approximately 0.2%) to
produce standard reactivity. The antigen is suspended in a solution
793
containing EDTA, choline chloride and phosphate with 0.2%

Thimerosal as a preservative.7,8
USR Test Control Serum Set contains 3 ml, each, of the following
lyophilized human sera: Nontreponemal Antigen Reactive Serum,
USR Weakly Reactive Serum and Nontreponemal Antigen Nonreactive
Serum. These reagents are standardized to provide reactive, weakly
reactive and nonreactive readings, respectively, when tested according
to the USR Test procedure.
Precautions
2. WARNING! POTENTIAL BIOHAZARDOUS REAGENTS. Each
donor unit used in preparation of USR Antigen and USR Test
Control Serum Set was tested by an FDA approved method for the
presence of the antibody to human immunodeficiency virus (HIV)
and for hepatitis B surface antigen and found negative (were not
repeatedly reactive).
hepatitis B virus or other infectious agents are absent, these reagents should be handled at the Biosafety Level 2 as recommended
for any potentially infectious human serum or blood specimen.9
3. USR Test Control Serum Set
Storage
Store USR Antigen at 2-8C. If the original 3 ml quantity exceeds what
is needed for one testing period, transfer the remainder from the first
days use to one or more aliquot vials and store at 2-8C.
Store the lyophilized sera in the USR Test Control Serum Set at 2-8C.
Store the rehydrated control sera at 2-8C or divide into aliquots sufficient for one day of testing and store at -20C. Do not thaw and refreeze.
Expiration Date
Procedure
Materials Provided
USR Antigen
USR Test Control Serum Set

0.9% saline
Nondisposable glass syringe, 1-2 cc
Nondisposable calibrated 18-gauge needles without bevel
Micropipettor, 50 l
Pipettes, serological, graduated to tip:
1.0 ml, graduated in 1/100 ml
794
Section V
Slides, 2 x 3 inches with paraffin or ceramic rings approximately 14 mm

in diameter and high enough to prevent spillage during rotation.
Slide holder for 2 x 3 inch slides
Mechanical rotator, adjustable to 180 2 rpm circumscribing
a circle 19 mm in diameter on a horizontal plane.
Light microscope with 10X ocular and 10X objective
Absolute alcohol
Acetone
Timer
Reagent Preparation
USR Antigen is ready to use.
Equilibrate all materials to room temperature (23-29C) before
performing the tests. Ensure that all glassware and pipettes are clean
and free of detergent residues.
USR Test Control Serum Set: To rehydrate the control sera, add 3 ml
sterile distilled or deionized water and rotate gently to completely
dissolve the contents.

Collect a blood specimen by aseptic venipuncture into a clean, dry
tube without anticoagulant. After the specimen has clotted, centrifuge
the specimen at 1,500-2,000 rpm for five minutes to obtain test serum.
for prolonged storage, keep at 2-8C for up to 5 days or maintain
below -20C. Serum specimens must be clear, free of hemolysis and
show no visible evidence of bacterial contamination, such as turbidity
or particulate matter. Refer to appropriate references for more
information on collection of specimens.1,3,12
Test Procedure
Preparation of Specific Glassware
Syringes with needles:
1. Prerinse with tap water.
2. Soak and hand wash thoroughly in a glassware detergent solution.
3. Rinse with tap water 6-8 times.
4. Rinse with unused distilled or deionized water.
5. Rinse with absolute alcohol.
6. Rinse with acetone.
7. Air dry until the acetone odor is completely eliminated.
8. Remove needles from syringes for storage.
Ceramic-ringed slides:
1. Prerinse with tap water.
2. Wash with a glassware detergent solution. Avoid prolonged
soaking of ceramic-ringed slides in detergent solution because the
ceramic rings will become brittle and flake off.
5. Wipe dry with a clean lint-free cloth. If cleaned slides do not allow
serum to spread evenly within the inner surface of the circle,
proceed as follows.
6. Scrub the slides with a nonscratching cleanser.
7. Rinse, dry and polish with a clean, lint-free cloth.
The Difco Manual
Section V
Testing the Accuracy of the Antigen Suspension Needle

1. The accuracy of the test depends on the amount of antigen suspension used. Check the calibration of the needle periodically to
ensure delivery of the correct volume of USR Antigen suspension.
2. For the qualitative and quantitative tests on serum, dispense the
antigen suspension from a syringe fitted with an 18-gauge needle
without bevel that will deliver 45 1 drops (22 ml) of antigen
suspension per ml when held vertically.
3. Place the needle on a 1-2 ml syringe. Fill the syringe with 1 ml
of USR Antigen suspension. Holding the syringe in a vertical
position, count the number of drops delivered in 1.0 ml. The needle
is correctly calibrated if 45 1 drops are delivered in 1.0 ml.
4. Adjust or replace the needle if it does not meet this specification.
Repeat calibration on the new or adjusted needle.
Testing and Storing the USR Antigen Suspension

1. Store the antigen suspension at 2-8C.
2. For daily use, withdraw a sufficient amount of suspension for
1 days testing and store the remainder at 2-8C. Antigen suspensions
must be at room temperature (23-29C) before use.
2. Test antigen suspension reactivity with the Reactive, Weakly
Reactive and Nonreactive control sera. Use the antigen suspension
only if it produces the expected reactivity with the control sera.
3. After each day of use, clean the dispensing needle, bottle and
syringe by rinsing with water, alcohol and acetone, in that order.
Remove the needle from the syringe after cleaning.
USR Qualitative Slide Test on Serum1,13

For reliable and reproducible test results, the USR Antigen suspension,
controls and test specimens must be at 23-29C when tests are performed.
1. Pipette 50 l of unheated serum into one ring of a paraffin- or
ceramic-ringed slide using a safety pipetting device. Do not use a
glass slide with concavities, wells or glass rings. Spread the serum
with a circular motion of the pipette tip so that the serum covers
the entire inner surface of the paraffin or ceramic ring. Include
control sera when performing the test.
2. Gently resuspend the USR Antigen and withdraw the desired
quantity with a syringe and needle.
3. Hold the syringe and needle containing the USR Antigen suspension
in a vertical position. Dispense several drops to clear the needle of
air. Add exactly 1 free-falling drop (22 l) of antigen suspension to
each circle containing serum. Do not allow the needle to touch the
serum.
4. Place the slide on the mechanical rotator. Rotate the slide for
4 minutes at 180 2 rpm. If the environment is dry, cover the slides
with a moist, humidifying cover during rotation to prevent
excessive evaporation.
5. Immediately after rotating the slide, remove the slide from the
rotator and read the test results microscopically, using a 10X
ocular and a 10X objective.
Results Qualitative Test

Medium to large clumps - Reactive (R)
Small clumps - Weakly reactive (WR)
No clumping or very slight roughness - Nonreactive (N)
The Difco Manual
2. Verify that the control sera results are as expected. If reactions are
not as expected, the test is invalid and results cannot be reported.
3. Perform a quantitative test on all serum specimens that produce
Reactive, Weakly Reactive or rough Nonreactive results, since
prozone reactions are occasionally encountered.
USR Quantitative Test1,13 on Serum

1. To quantitate serum samples to an endpoint titer, prepare serum
dilutions on the slide at 1:1, 1:2, 1:4 and 1:8, as follows.
2. Dispense 50 l of 0.9% saline in circles 2-4. Do not spread the saline.
3. Dispense 50 l of serum in circles 1 and 2.
4. Mix the saline and the serum in circle 2 by drawing the mixture up
and down in the pipette 8 times. Mix gently to prevent bubbles.
5. Transfer 50 l from circle 2 (1:2) to circle 3 and mix.
6. Transfer 50 l from circle 3 (1:4) to circle 4 (1:8), mix, and then
discard 50 l from circle 4.
7. Holding the syringe and needle containing the USR Antigen
suspension in a vertical position, dispense several drops to clear
the needle of air. Then, add exactly 1 free-falling drop (22 l) of
antigen suspension to each circle containing serum. Do not allow
the needle to touch the serum.
4 minutes at 180 2 rpm. If the environment is dry, cover the slides
with a moist, humidifying cover during rotation to prevent
excessive evaporation.
9. Immediately after rotating the slide, remove the slide from the rotator and read the test results microscopically using a 10X ocular
and a 10X objective.
10. If the highest dilution tested (1:8) is reactive, prepare a 1:8 dilution
of the test specimen by adding 0.1 ml of serum to 0.7 ml of 0.9%
saline. Mix thoroughly. Retest as in steps 1-9, above.
Results Quantitative Test

Report the titer as the highest dilution that produces a Reactive result.
Table 1. Sample quantitative USR Test results.
Undiluted
(1:1)
1:2
1:4
1:8
1:16
1:32
N
(rough)
W
Reactive,
undiluted
Reactive,
1:2 dilution
Reactive,
1:4 dilution
Reactive,
1:8 dilution
Reactive,
1:16 dilution
Weakly
reactive,
undiluted
If reactive results are obtained through dilution 1:32, prepare further

twofold serial dilutions in 0.9% saline (1:64, 1:128 and 1:256) and
retest using the quantitative test procedure.
795
Interpretation
1. The results of the serum USR Test must be confirmed by a
treponemal test.
2. The diagnosis of syphilis depends on the results of the USR Test,
treponemal confirmatory test, clinical signs and symptoms, and risk
factors.
3. A Reactive USR Test may indicate past or present infection with a
pathogenic treponeme. However, it may be a false-positive reaction.
A false positive is determined if the confirmatory treponemal test
is negative.
4. A Nonreactive USR Test with clinical evidence of syphilis may
indicate early, primary syphilis, a prozone reaction in secondary
syphilis, or late syphilis.
5. A Nonreactive USR Test with no clinical evidence of syphilis
indicates no current infection or an effectively treated infection.
6. A quantitative USR Test detects changes in reagin titer. Therefore,
a serum specimen showing a fourfold increase in titer on a repeat
specimen may indicate an infection, a reinfection or a treatment
failure. Likewise, a fourfold decrease during treatment indicates
adequate syphilis therapy.

1. A prozone reaction may occur in which reactivity with true positive
undiluted serum is inhibited. The prozone phenomenon often gives
Weakly Reactive or rough Nonreactive results in the qualitative
test. Specimens with such nonreactive results must be quantitatively
tested.
2. Biological false-positive reactions can occur with nontreponemal
tests in persons who abuse drugs, have diseases such as lupus
erythematosus, mononucleosis, malaria, leprosy or viral pneumonia,
or who have recently been immunized.1
4. If the temperature of the testing area, specimens, or reagents is less
than 23C, test, reactivity is decreased. If the temperature is greater
than 29C, test reactivity is increased.1
5. Test results are unpredictable when testing hemolyzed, contaminated
or extremely turbid serum specimens.
6. Test results may be erroneous if the speed and time of rotation are
not correct.
7. Positive results obtained by using USR Antigen should not be
considered as conclusive evidence that the patient is syphilitic.
Conversely, a nonreactive USR Test, by itself, does not rule out the
diagnosis of syphilis.
References
1. Larsen, S. A., E. F. Hunter, and S. J. Kraus. 1990. A manual of
tests for syphilis. American Public Health Association.
796
Section V

4. Norris, S. J., and S. A. Larsen. 1995. Treponema and other hostassociated spirochetes, p. 636-651. In P. R. Murray, E. J. Baron,
Washington, D.C.
5. Perine, P. L., A. L Wallace, J. H. Blount, and S. T. Brown. 1981.
Syphilis, p. 631-673. In A. Ballows and W. J. Hausler, Diagnostic
procedures for bacterial, mycotic and parasitic infections. American
6. Matthews, H. M., T. K. Yang, and H. M. Jenkin. 1979. Unique
lipid composition of Treponema pallidum (Nichols virulent strain).
Infect. Immun. 24:713-719.
7. Portnoy, J., and W. Garson. 1960. New and improved antigen
suspension for rapid reagin test for syphilis. Public Health Rep.
75:985-988.
8. Portnoy, J., H. W. Bossak, V. H. Falcone, and A. Harris. 1961. A
rapid reagin test with unheated serum and new improved antigen
suspension. Public Health Rep. 76:933-935.
Biosafety in microbiological and biomedical laboratories, 2nd ed.
U. S. Department of Health and Human Services publication no.
88-8395. U. S. Government Printing Office, Washington, D.C.
387-388.
exposure to bloodborne pathogens, final rule. Federal Register
56:64175-64182.
Washington, D.C.
13. Pettit, D. E., S. A. Larsen, V. Pope, M. D. Perryman, and
M. R. Adams. 1982. Unheated serum reagin test as a quantitative
test for syphilis. J. Clin Microbiol. 15:238-242.
Packaging
USR Antigen
USR Test Control Serum Set
Contains:
Nontreponemal Antigen
Reactive Serum
USR Weakly Reactive Serum
Nonreactive Serum
Aliquant Vials
6 x 3 ml
2405-46
1 set
3516-32
3 ml
3 ml
3 ml
3 vials
The Difco Manual
Section V
VDRL Antigen & VDRL Test Control Serum Set
Bacto VDRL Antigen

Bacto VDRL Test Control Serum Set
Intended Use
Bacto VDRL Antigen with Bacto VDRL Buffered Saline is used in the
Venereal Disease Research Laboratory (VDRL)1 Test for detecting
reagin, an antibody-like substance, by the qualitative and quantitative
slide procedures.
Bacto VDRL Test Control Serum Set is used for controlling the VDRL Test.

Treponema pallidum is the causative agent of syphilis. Syphilis is a
chronic infection with many clinical manifestations which occur in
distinct stages. Specific laboratory tests are recommended for the
detection of each stage of the disease.2-4
The VDRL Antigen is a nontreponemal antigen composed of cardiolipin,
cholesterol, and lecithin. The nontreponemal tests measure antilipid
antibodies (IgG and IgM, which are formed by the host in response to
lipoidal material released from damaged host cells early in infection
with T. pallidum) and lipid like material from the treponemal cell
surface.5 During infection with syphilis, an antibody-like substance

called reagin can be detected in the patients serum. In syphilis infection
of the central nervous system, reagin can be detected in the cerebrospinal
fluid (CSF).
Reactive nontreponemal tests confirm the diagnosis in the presence of
early or late lesion syphilis. They offer a clue in latent subclinical syphilis,
and are effective tools for detecting cases in epidemiological investigations. Nontreponemal tests are superior to the treponemal test for
following the response to therapy.3
Nontreponemal antigen tests are not entirely specific for syphilis, nor
do they have satisfactory sensitivity in all stages of syphilis. Whenever
the results of a nontreponemal antigen test disagree with the clinical
impression, a treponemal antigen test such as the FTA-ABS2,3 should
be performed. Nontreponemal tests such as the VDRL are used to
screen patient serum, while treponemal tests such as the FTA-ABS are
used for confirmation. The likelihood of obtaining a reactive VDRL
test result in various stages of untreated syphilis has been reported as
follows3:
STAGES OF UNTREATED SYPHILIS

VDRL Antigen
Appearance:
Clear, colorless solution.
VDRL Buffered Saline

Appearance:
Clear, colorless solution.
Nontreponemal Antigen Reactive Serum
powdered cake.
VDRL Weakly Reactive Serum
powdered cake.
Nontreponemal Antigen Nonreactive Serum
powdered cake.
Rehydrate the sera contained in the VDRL Test Control Serum
Set per label directions. Perform the VDRL Slide Test
according to the Test Procedure. Each serum in the VDRL Test
Control Serum Set should yield appropriate reactions when
tested with the VDRL Antigen.
Use an antigen suspension only if it produces the expected
reactivity with the control sera.
The Difco Manual
% REACTIVE VDRL TEST
Primary
Secondary
Latent
Late
78
100
96
71

In the VDRL Test procedure, the patients serum is heat-inactivated
and then mixed with a buffered saline suspension of VDRL Antigen
containing cardiolipin, lecithin and cholesterol. The combination
of reagin and VDRL Antigen forms microscopic clumping called
flocculation. With certain modification, the serum test procedure can
be used for testing CSF.
Reagents
VDRL Antigen is 0.03% cardiolipin and 0.9% cholesterol dissolved
in absolute alcohol with sufficient lecithin (approximately 0.18-0.2%)
to produce standard reactivity.
It is prepared according to the modifications of Harris, Rosenberg and
Riedel. 6 Cardiolipin and lecithin are prepared according to the
directions of Pangborn.7,8,9
VDRL Buffered Saline is a 1% NaCl solution at pH 6.0 0.1. It is
packaged with VDRL Antigen and used to prepare the VDRL Antigen
suspension.
Nontreponemal Antigen Reactive Serum is a lyophilized human serum
standardized to provide a reactive reading when tested according to
the USR or VDRL test procedure.
VDRL Weakly Reactive Serum is a lyophilized human serum
standardized to provide a weakly reactive reading when tested according
to the VDRL test procedure.
Nontreponemal Antigen Nonreactive Serum is a lyophilized human
serum standardized to provide a nonreactive reading when tested
according to the USR or VDRL test procedure.
797
Precautions
2. WARNING! POTENTIALLY BIOHAZARDOUS REAGENTS.
Each donor unit used in preparation of VDRL Antigen and VDRL
Test Control Serum Set was tested by an FDA approved method
for the presence of the antibody to human immunodeficiency virus
(HIV) and for hepatitis B surface antigen and found negative (were
not repeatedly reactive).
hepatitis B virus or other infectious agents are absent, these
reagents should be handled at the Biosafety Level 2 as recommended
for any potentially infectious human serum or blood specimen.10
4. VDRL Antigen
HIGHLY FLAMMABLE. IRRITATING TO EYES, RESPIRATORY SYSTEM AND SKIN. us POSSIBLE RISK OF IRREVERSIBLE EFFECTS.us POSSIBLE RISK OF HARM TO THE
UNBORN CHILD.us Avoid contact with skin and eyes. Do not
breathe mist. Wear suitable protective clothing. Keep container
tightly closed. Keep away from sources of ignition. No smoking.
Target Organs: Blood, Intestines, Liver, Muscles, Nerves.
5. VDRL Test Control Serum Set
Storage
Store VDRL Antigen at 15-30C in the dark.
Store VDRL Buffered Saline at 15-30C. After the bottle is opened,
store at 2-8C.
Store the lyophilized control sera in the VDRL Test Control Serum Set
at 2-8C. Store the rehydrated control sera at 2-8C or divide into
aliquots sufficient for one day of testing and store at -20C. Do not
thaw and refreeze.
Section V
Nondisposable calibrated needles without bevel:

Serum test: 18 gauge
CSF test: 21 or 22 gauge
Bottles, 30 ml, round, narrow-mouthed, 35 mm in diameter with
glass stoppers and a flat inner-bottom surface
Micropipettor, 50 l
Pipettes, serological, graduated to tip:
Slides:
Serum test: 2 x 3 inches with paraffin or ceramic rings
approximately 14 mm in diameter and high enough to prevent
spillage during rotation
CSF test: Kline concavity slides, 3 x 2 3 inches x 3 mm thick,
12 concavities measuring 16 mm in diameter and 1.75 mm
in depth
Slide holder for 2 x 4 inch slides
Mechanical rotator adjustable to 180 2 rpm, circumscribing a
circle 19 mm in diameter on a horizontal plane
Waterbath, 56C
Light microscope with 10X ocular and 10X objective
Absolute alcohol
Acetone
Timer
Reagent Preparation
VDRL Antigen and VDRL Buffered Saline are ready to use in preparing
VDRL Antigen suspension.
Equilibrate all materials to room temperature before performing
detergent residues.
VDRL Test Control Serum Set: To rehydrate the control sera, add
3 ml sterile distilled or deionized water each and rotate gently to
completely dissolve the contents.

has clotted, centrifuge to obtain serum. Store serum specimens at room
temperature for no longer than 4 hours; for prolonged storage, keep at
2-8C for up to 5 days or maintain below -20C. Serum specimens
must be clear, free of hemolysis and show no visible evidence of bacterial contamination such as turbidity, hemolysis or particulate matter.
Consult appropriate references for more information on collection of
specimens.1,3,13
Before testing, heat the test sera at 56C for 30 minutes. Specimens
that are not tested within four hours must be reheated for 10 minutes
at 56C.
Procedure
Preparation of Specific Glassware
Materials Provided
Syringes with needles and emulsion bottles:

1. Pre-rinse with tap water.
2. Soak and hand wash thoroughly in a glassware detergent solution.
5. Rinse with absolute alcohol.
6. Rinse with acetone.
Expiration Date
VDRL Antigen with VDRL Buffered Saline

VDRL Test Control Serum Set

0.9% saline
Nondisposable syringe, 1 cc
798
The Difco Manual
Section V
7. Air dry until the acetone odor is completely eliminated.

8. Remove needles from syringes for storage.
Ceramic-ringed slides:
1. Pre-rinse with tap water.
2. Wash with a glassware detergent solution. Avoid prolonged soaking
in detergent solution because the ceramic rings will become brittle
and flake off.
5. Wipe dry with a clean lint-free cloth. If, after cleaning, the slides
do not allow serum to spread evenly within the inner surface of the
circle, proceed as follows.
6. Scrub the slides with a nonscratching cleanser.
7. Rinse, dry and polish with a clean lint-free cloth.
Prepare the Antigen Suspension

Check the pH of VDRL Buffered Saline before preparing the
VDRL Antigen emulsion. VDRL Buffered Saline outside the range of
pH 6.0 0.1 should be discarded.
Allow VDRL Antigen and VDR Buffered Saline to reach 23-29C
before preparing the VDRL Antigen emulsion.
Use only emulsion bottles with flat inner-bottom surfaces that allow
the initial VDR Buffered Saline to evenly cover the inner-bottom
surface of the bottle. If the VDRL Buffered Saline beads or does not
spread evenly to cover the bottom of the bottle, rewash the bottle.
For reproducible results, the VDRL Antigen emulsion must be checked
daily for proper reactivity by testing with VDRL Test Control Serum
Set. Only those VDRL emulsions producing the established reactivity
pattern of the control serum should be used.
1. Prepare a fresh VDRL Antigen suspension each testing day. The
temperature of the VDRL Buffered Saline, VDRL Antigen and
equipment should be between 23-29C at the time the antigen
suspension is prepared.
2. Pipette 0.4 ml of VDRL Buffered Saline to the bottom of a round,
30 ml glass-stoppered bottle with a flat inner-bottom surface.
Gently tilt the bottle so that the VDR Buffered Saline will cover
the entire inner-bottom surface of the bottle.
3. Add 0.5 ml of VDRL Antigen (from the lower half of a 1.0 ml
pipette graduated to the tip) directly into the saline while continuously
but gently rotating the bottle on a flat surface. Add antigen drop by
drop at a rate allowing approximately 6 seconds for each 0.5 ml of
antigen. Keep the pipette tip in the upper third of the bottle. Do not
splash saline onto the pipette. The proper speed of rotation is
obtained when the center of the bottle circumscribes a 2-inch
diameter circle approximately three times per second.
4. Expel the last drop of antigen from the pipette without touching the
pipette to the saline and continue rotating the bottle for 10 seconds.
5. Add 4.1 ml of buffered saline from a 5 ml pipette. Do not drop the saline
directly onto the antigen; allow it to flow down the side of the bottle.
6. Cap the bottle and shake it from bottom to top and back approximately 30 times in 10 seconds. Let the VDRL Antigen emulsion
stand without further disturbance for 10 minutes. The antigen
suspension is ready for use and may be used during 1 day (8 hours).
7. Mix the VDRL Antigen suspension by gently swirling it each time
it is used. Do not mix the suspension by forcing it back and forth
The Difco Manual
through the syringe and needle, since this may cause breakdown of
particles and loss of reactivity.
Testing the Accuracy of the Antigen Suspension Needle

1. The accuracy of the test depends on the amount of antigen suspension
used. Check the calibration of the needle periodically to ensure
delivery of the correct volume of VDRL Antigen suspension.
2. For the qualitative and quantitative tests on serum, dispense the
antigen suspension from a syringe fitted with an 18-gauge needle
without bevel that will deliver 60 2 drops of antigen suspension
per ml when held vertically.
3. Place the needle on a 1 ml syringe. Fill the syringe with VDRL
Antigen suspension. Holding the syringe in a vertical position,
count the number of drops delivered in 0.5 ml. The needle is
correctly calibrated if 30 1 drops are delivered in 0.5 ml.
4. Adjust or replace the needle if it does not meet this specification.
Repeat calibration of the new needle.
Testing and Storing the VDRL Antigen Suspension

1. Prepare a fresh antigen suspension each testing day. Once prepared,
it should be used within 8 hours.
2. Store the antigen suspension at 23-29C.
3. Test the reactivity of the antigen suspension with the Reactive,
Weakly Reactive and Nonreactive control sera. Test the serum
dilutions within 1 hour after inactivation.
4. Use the antigen suspension only if it produces the expected
reactivity with the control sera (Reactive, Weakly Reactive and
Nonreactive).
5. After each day of use, clean the dispensing needle, bottle and
syringe by rinsing with water, alcohol and acetone, as described
above. Remove the needle from the syringe after cleaning.
VDRL Qualitative Slide Test on Serum

1. Slide flocculation tests for syphilis are affected by the temperature
of the room. For reliable and reproducible test results, the VDRL
Antigen suspension, controls and test specimens must be at
23-29C when tests are performed.
2. Pipette 50 l of serum into one ring of a paraffin or ceramic-ringed
slide using a safety pipetting device. Do not use a glass slide with
concavities, wells or glass rings. Spread the serum with a circular
motion of the pipette tip so that the serum covers the entire inner
surface of the paraffin or ceramic ring. Use only clean slides that
allow the serum to evenly cover the entire surface within the
ceramic or paraffin ring.
3. Gently Resuspend the VDRL Antigen suspension.
4. Holding the VDRL Antigen suspension dispensing needle and
syringe in a vertical position, dispense several drops to clear the
needle of air. Then add exactly 1 free-falling drop (17 l) of antigen
suspension to each circle containing serum. Do not allow the needle
to touch the serum.
4 minutes at 180 2 rpm. When performing the test in a dry
climate, cover the slides with a moist, humidifying cover during
rotation to prevent excessive evaporation.
6. Immediately after rotating the slide, remove it from the rotator and
read the test results microscopically using a 10X ocular and a 10X
objective.
799
Section V
Results - Qualitative Slide Test

Reactive (R) - Medium to large clumps
Weakly reactive (WR) - Small clumps
Nonreactive (N) - No clumping or very slight roughness
2. Verify that the control sera results are as expected. If reactions are
not as expected, the test is invalid and results cannot be reported.
3. Perform a quantitative test on all serum specimens that produce
Reactive, Weakly Reactive or rough Nonreactive results, since
prozone reactions are encountered occasionally.
VDRL Quantitative Test on Serum

1. To quantitate serum samples to an endpoint titer, prepare serum
dilutions on the slide at 1:1, 1:2, 1:4 and 1:8, as follows.
2. Dispense 50 l of 0.9% saline in circles 2-4. Do not spread the saline.
3. Dispense 50 l of serum in circles 1 and 2.
4. Mix the saline and the serum in circle 2 by drawing the mixture up
and down in the pipette 8 times. Avoid forming bubbles.
5. Transfer 50 l from circle 2 (1:2) to circle 3 (1:4) and mix.
6. Transfer 50 l from circle 3 (1:4) to circle 4 (1:8), mix, and then
discard 50 l from circle 4.
7. Gently resuspend the antigen suspension.
8. Holding the antigen suspension dispensing needle and syringe in a
vertical position, dispense several drops to clear the needle of air.
Then add exactly 1 free-falling drop (17 l) of antigen suspension
to each circle.
4 minutes at 180 2 rpm. When performing the test in a dry climate,
place the slides under a moist, humidifying cover during rotation
to prevent excessive evaporation.
10. Immediately after rotation, read the test results microscopically
using a 10X ocular and a 10X objective.
11. If the highest dilution tested (1:8) is reactive, prepare a 1:8 dilution
of the test specimen by adding 0.1 ml of serum to 0.7 ml of 0.9%
saline. Mix thoroughly. Retest as in steps 1-10, above.
Results - Quantitative Test

Report the titer as the highest dilution that produces a Reactive (not
Weakly Reactive) result.
Table 1. Sample quantitative VDRL Test results.
SERUM DILUTIONS
REPORT
Undiluted
(1:1)
1:2
1:4
1:8
1:16
1:32
N
(rough)
W
800
Reactive,
undiluted
Reactive,
1:2 dilution
Reactive,
1:4 dilution
Reactive,
1:8 dilution
Reactive,
1:16 dilution
Weakly
reactive,
undiluted
If reactive results are obtained through dilution 1:32, prepare further

twofold serial dilutions in 0.9% saline (1:64, 1:128 and 1:256) and
retest using the quantitative test procedure.
Interpretation
1. The results of the serum VDRL Test must be confirmed by a
treponemal test.
2. The diagnosis of syphilis depends on the results of the VDRL test,
the treponemal confirmatory test, clinical signs and symptoms,
and risk factors.
3. A reactive VDRL Test may indicate past or present infection with a
pathogenic treponeme. However, it may be a false-positive reaction.
A false positive is determined if the confirmatory treponemal test
is negative.
4. A nonreactive VDRL Test with clinical evidence of syphilis may
indicate early, primary syphilis, a prozone reaction in secondary
syphilis, or late syphilis.
5. A nonreactive VDRL Test with no clinical evidence of syphilis
indicates no current infection or an effectively treated infection.
6. A quantitative VDRL Test detects changes in reagin titer. Therefore
a serum specimen showing a fourfold increase in titer on a repeat
specimen may indicate an infection, a reinfection or a treatment
failure. Likewise, a fourfold decrease during treatment indicates
adequate syphilis therapy.
VDRL Test on Spinal Fluid

Consult an appropriate reference for the procedure to use when testing
spinal fluids by the VDRL Test.1

1. A prozone reaction may occur in which reactivity with undiluted
serum is inhibited. The prozone phenomenon often gives Weakly
Reactive or rough Nonreactive results in the qualitative test.
Specimens with such results must be quantitatively tested.
2. Biological false-positive reactions can occur with nontreponemal
tests in persons who abuse drugs, have diseases such as lupus
erythematosus, mononucleosis, malaria, leprosy or viral pneumonia,
or who have recently been immunized.1
3. During manufacturing, VDRL Antigen with VDRL Buffered Saline
is tested only with serum. To modify the serum test products and
procedures for testing CSF, consult the appropriate reference.1 The
user is responsible for modifying the products and procedures and
for the required quality control standards according to this manual.
5. VDRL Buffered Saline showing turbidity or mold growth should
be discarded.
6. If the temperature of the testing area, specimens or reagents is less
than 23C, test reactivity is decreased. If the temperature is greater
than 29C, test reactivity is increased.1
7. Test results are unpredictable when testing hemolyzed, contaminated
or extremely turbid serum specimens.
8. For correct test results, adhere strictly to the correct speed and
length of time for rotating the specimens and antigen.
The Difco Manual
Section V
Vibrio Cholerae Antisera
References
1. Larsen, S. A., E. F. Hunter, and S. J. Kraus. 1990. A manual of
tests for syphilis. American Public Health Association.
4. Norris, S. J., and S. A. Larsen. 1995. Treponema and other hostassociated spirochetes, p. 636-651. In P. R. Murray, E. J. Baron,
Washington, D.C.
5. Matthews, H. M., T. K. Yang, and H. M. Jenkin. 1979. Unique
lipid composition of Treponema pallidum (Nichols virulent strain).
Infect. Immun. 24:713-719.
6. Harris, A., A. A. Rosenberg, and L. M. Riedel. 1946.
A microflocculation test for syphilis using cardiolipin antigen.
J. Ven. Dis. Infor. 27:169-174.
7. Pangborn, M. C. 1941. A new serologically active phospholipid
from beef heart. Proc. Soc. Exp. Biol. and Med. 48:484-486.
8. Pangborn, M. C. 1944. Acid cardiolipin and an improved method
for the preparation of cardiolipin from beef heart. J. Biol. Chem.
153:343-348.
9. Pangborn, M. C. 1945. A simplified preparation of cardiolipin,
with a note on purification of lecithin for serologic use. J. Biol.
Chem. 161:71-82.

Biosafety in microbiological and biomedical laboratories, 2nd ed.
U. S. Department of Health and Human Services publication no.
88-8395. U. S. Government Printing Office, Washington, D.C.
387-388.
exposure to bloodborne pathogens, final rule. Federal Register
56:64175-64182.
13. Miller, J. M., and H. T. Holmes. 1995. Specimen collection, transport and storage. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C.
Packaging
VDRL Antigen w/Buffered Saline
VDRL Test Control Serum Set
Contains:
Reactive Serum
VDRL Weakly Reactive Serum
Nonreactive Serum
Aliquant Vials
5 ml
10 x 0.5 ml
0388-56
0388-49
1 set
3520-32
3 ml
3 ml
3 ml
3 vials
Bacto Vibrio Cholerae Antisera

Vibrio Cholerae Antiserum Inaba . Vibrio Cholerae Antiserum
Ogawa . Vibrio Cholerae Antiserum Poly
Intended Use
Vibrio Cholerae Antiserum Inaba
Vibrio Cholerae Antiserum Ogawa
Vibrio Cholerae Antiserum Poly
Lyophilized appearance: Light gold to amber button to
powdered cake
Rehydrated appearance: Light gold to amber, clear liquid
Rehydrate Vibrio Cholerae Antisera per label directions.
Perform the slide agglutination test using appropriate known
cultures of Vibrio cholerae as positive and negative controls.
The Difco Manual
Bacto Vibrio Cholerae Antisera are used for serotyping Vibrio cholerae
in slide agglutination tests.

V. cholerae are facultative anaerobes that in microscopic morphology
are gram-negative, curved or straight bacilli. The microorganisms
either require sodium chloride or grow best in its presence. Various
media designed to select and cultivate the growth of this microorganism
are used in the clinical laboratory, the food industry and environmental
testing.
V. cholerae is the causative agent of a secretory diarrhea known as
cholera. Two biotypes, El Tor and Classical, are associated with
human disease.1 These two biotypes cannot be differentiated serologically. The spread of the disease is primarily through contaminated
801
Section V
water and by the fecal-oral route. Infections with V. cholerae may be

asymptomatic, mild or severe. If not treated, patients with severe
cholera may die within five hours from massive fluid and electrolyte
loss. 1 Because Vibrio species are natural inhabitants of aquatic
environments, food products such as uncooked or incorrectly handled
seafood can spread infection.
Gastrointestinal symptoms of cholera include rice water stools
caused by a potent enterotoxin. The primary human specimen is feces.
Seafood products are frequently tested as vehicles of human infection.
In 1935, Gardner and Ventkatraman2 published a classification scheme
for Vibrio cholerae. V. cholerae isolated from cholera patients was
classified as O1. All other strains were designated non-O1. Non-O1
V. cholerae causes both gastroenteritis and systemic infections. Some
strains produce cholera enterotoxin. Non-O1 V. cholerae has been
isolated from blood, wounds, ears, sputum, cerebro-spinal fluid and urine.3,4
V. cholerae of the O1 serogroup are divided into the serotypes Ogawa,
Inaba and Hikojima.5,6 The antigenic factors are:
SEROTYPE
O ANTIGEN FACTORS
Precautions
2. Vibrio Cholerae Antiserum Inaba
Storage
Store lyophilized and rehydrated Vibrio Cholerae Antisera at 2-8C.
Expiration Date
The expiration date applies to product in its intact container when stored
Procedure
Ogawa
AB
Inaba
AC
Hikojima
ABC
Cholera can be diagnosed retrospectively. A fourfold rise in titer
between acute-phase serum and that collected 10-14 days later is
considered diagnostic.7
Materials Provided
Applicator sticks
Identification of V. cholerae includes the isolation of the microorganism

as well as biochemical identification and serological confirmation.
Serological confirmation requires that the microorganism (antigen)
react with its corresponding antibody. This in vitro reaction produces
macroscopic clumping called agglutination. The desired homologous
reaction is rapid, does not dissociate (high avidity) and binds (high
affinity).
Because a microorganism may agglutinate with an antibody produced
in response to another species, heterologous reactions are possible.
These are characterized as weak in strength or slow in formation. Such
unexpected and unpredictable reactions may lead to some confusion in
serological identification. A positive homologous agglutination reaction
should support the morphological and biochemical identification of
the microorganism.
Homologous reactions occur rapidly and are strong. Heterologous
reactions form slowly and are weak.
Reagents
Vibrio Cholerae Antisera are lyophilized, polyclonal rabbit Vibrio
cholerae O1 antisera containing approximately 0.04%Thimerosal as
a preservative. Vibrio Cholerae Antisera Inaba and Ogawa are
monospecific absorbed antisera.
Vibrio Cholerae Antisera possess the following antibodies:
ANTISERUM

O ANTIBODIES
C
B
ABC
Each vial of Vibrio Cholerae Antiserum contains sufficient reagent for

20 slide tests.
802

Reagent Preparation
Vibrio Cholerae Antisera: To rehydrate, add 3 ml sterile 0.85% NaCl
solution and rotate gently to completely dissolve the contents. The
rehydrated antiserum is considered a 1:2 working dilution.

V. cholerae can be recovered from clinical specimens on selective
media such as thiosulfate-citrate-bile salts-sucrose (TCBS) agar. For
specific recommendations, consult appropriate references.1,8,9
V. cholerae can be recovered from various types of foods, particularly
seafood. Samples are processed to prevent overgrowth of competing
microorganisms and selective media are used to enhance the growth of
the microorganism. Some isolation media have been specifically
developed for the food industry. Consult appropriate references for
recommended procedures for isolating V. cholerae from food.10,11
The isolate for serological testing should be subcultured from
selective media to a nonselective agar such as Nutrient Agar. Consult
standard protocols in appropriate references for the type of specimen
and the appropriate medium.1, 8-11
Having followed an established protocol, determine that a pure culture
of the microorganism has been obtained and that biochemical test
reactions are consistent with the identification of the organism as
V. cholerae. After these criteria are met, serological identification can
be performed.
The Difco Manual
Section V
Testing the isolate for autoagglutination:

1. From the test culture, transfer a loopful of growth to a drop (35 l)
of sterile 0.85% NaCl solution on a clean slide and emulsify the
organism.
2. Rotate the slide for one minute, then observe for agglutination.
3. If autoagglutination occurs, the culture is rough and cannot be
tested. Subculture to a nonselective medium, incubate and test the
organism again as described in steps 1 and 2.
If no agglutination occurs, proceed with testing the organism as
described below.
Test Procedure
Use Vibrio Cholerae Antiserum Poly to screen possible V. cholerae
isolates. Continue testing with Vibrio Cholerae Antisera Inaba and
Ogawa. Include known positive and negative control cultures.
1. Vibrio Cholerae Antiserum: Dispense a drop of the antiserum to
be tested on an agglutination slide.
2. Test organism: Transfer a loopful of growth to the drop of antiserum
and mix thoroughly.
Results
No agglutination.
3. Test isolates: 3+ or greater agglutination within one minute is a
positive result.
Interpretation
Agglutination of the monospecific antiserum, when used, provides
preliminary presumptive identification of the serotype.
0.85% NACL
SOLUTION
CONTROL
POLY
+
+
+
+
Any
OGAWA
+
Any
INABA
+
+
Any

as V. cholerae.
2. Serological methods alone cannot identify the isolate as V. cholerae.
causing agglutination of the negative control reaction (autoagglutination). Smooth colonies must be selected and tested in serological
procedures.
5. Vibrio Cholerae Antisera have been tested using undiluted cultures
taken from agar media. These antisera have not been tested using
antigen suspensions in NaCl solution or other diluents. If the user
applies variations to the recommended procedure, each lot of
antiserum must be tested with known control cultures to verify
expected reactions under the modified procedure.
7. Rehydrated Vibrio Cholerae Antiserum that is cloudy or has a
precipitate at any time during its use should be discarded.
INTERPRETATION
References
Not V. cholerae
Presumptively V. cholerae
V. cholerae serotype Ogawa
V. cholerae serotype Inaba
V. cholerae serotype Hikojima
Autoagglutination.
Unsuitable test culture.
1. McLaughlin, J. C. 1995. Vibrio, p. 465-476. In P. R. Murray, E. J.

2. Gardner, A. D., and K. V. Venkatraman. 1935. The antigens of
the cholera group of vibrios. J. Hyg. 35:262-282.
3. Florman, A. L., A. H. Cushing, T. Byers, and S. Popejoy. 1990.
Vibrio cholerae bacteremia in a 22-month-old New Mexican child.
Pediatr. Infect. Dis. J. 9:63-65. (Letter).
4. Morris, J. G., Jr. 1990. Non-O group 1 Vibrio cholerae: a look at
the epidemiology of an occasional pathogen. Epidemiol. Rev.
12:179-191.
5. Nobechi, K. 1923. Contributions to the knowledge of V. cholerae.
3. Immunological studies upon the types of V. cholerae. Sci. Repr.
Govt. Inst. Infect. Dis. 2:43-87.
+ agglutination
no agglutination
1. Positive agglutination using Vibrio Cholerae Antiserum Poly with

typical biochemical test results gives presumptive identification of
V. cholerae O1.
2. Cultures with positive agglutination in Vibrio Cholerae Antiserum
Poly may be serotyped using the Vibrio Cholerae Antiserum Ogawa
and Vibrio Cholerae Antiserum Inaba. Positive agglutination in
both antisera is rare and, when it occurs, is usually interpreted as
The Difco Manual
identifying V. cholerae serotype Hikojima.6 V. cholerae serotype

Hikojima is a rare serotype and should be sent to a reference
laboratory for further study.
3. Positive agglutination will be immediate and strong. The strongest
and most rapid reaction should be used to identify the serotype.
V. cholerae O1 strains frequently cross-react slowly or weakly in
monospecific antiserum for the other serotype.
4. Isolates that weakly or slowly agglutinate with Vibrio Cholerae
Antiserum Poly and not with Vibrio Cholerae Antiserum Inaba or
Vibrio Cholerae Antiserum Ogawa are usually considered
V. cholerae non-O1. The isolate may be sent to a reference laboratory
for further study.
803
Widal Antigen Set
6. Kelly, M. T., F. W. Hickman-Brenner, and J. J. Farmer III.

1991. Vibrio, p. 384-395. In A. Balows, W. J. Hausler, K. L.
Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.), Manual of
Washington, D.C.
7. Smith, H. L. 1981. Vibrio Infections, p. 715-722. In A. Balows,
and W. J. Hausler (ed.), Bacterial, mycotic and parasitic infections,
8. Pezzlo, M. 1992. Aerobic bacteriology, p. 1.0.1.-1.20.47. In H. D.
Isenberg (ed.). Clinical microbiology procedures handbook, vol.
St. Louis, MO.
Section V
10. Kaysner, C. A., M. L. Tamplin, and R. M. Twedt. 1992. Vibrio,

p. 451-473. In C. Vanderzant and D. F. Splittstoesser, (ed.), Compendium of methods for the microbiological examination of foods,
11. Elliot, E. L., C. A. Kaysner, L. Jackson, and M. L. Tamplin.
1995. Vibrio cholerae, V. parahaemolyticus, V. vulnificus, and
other Vibrio sp., p. 9.01-9.27. In Food and Drug Administration,
Gaithersburg, MD.
Packaging
Vibrio Cholera Antiserum Inaba
3 ml
2430-47
Vibrio Cholera Antiserum Ogawa
3 ml
2431-47
Vibrio Cholera Antiserum Poly
3 ml
2432-47
Bacto Widal Antigen Set

CONTAINS: Salmonella O Antigen . Salmonella H Antigens
Salmonella Vi Antigen . Febrile Positive Control Polyvalent
ALSO AVAILABLE: Salmonella O Antigens . Salmonella H Antigen
Intended Use
Bacto Widal Antigen Set, which contains four Salmonella antigens,
is used in detecting Salmonella antibodies by slide and tube agglutination tests.
Bacto Salmonella H, O and Vi Antigens, available individually, are
used in detecting Salmonella antibodies by slide and tube agglutination tests.

Salmonellae represent many species of pathogenic microorganisms
that, upon invasion, produce a fever in their host. Consequently, they
are often called Febrile Antigens. Salmonella species cause a variety
of human diseases called salmonelloses. The range of disease is from
mild self-limiting gastroenteritis to a more severe form, possibly with
bacteremia or typhoid fever, which can be severe and life-threatening.
Severe disease and bacteremia are associated primarily with
S. Choleraesuis, S. Paratyphoid A and S. Typhi, while most of the other
2,300 or more strains are associated with gastroenteritis. The severity
of the diarrheal disease depends on the virulence of the strain and the
condition of the human host.
Salmonella is found in nature and occurs in the intestinal tract of many
animals, both wild and domestic. The microorganism can spread to
man from contact with the environment or from eating contaminated
meat or vegetable food products.
The genus Salmonella is in the family Enterobacteriaceae. Salmonellae
804
oxidase negative, non-lactose fermenting, H2S positive and produce

gas. Serotypes of Salmonella are defined based on antigenic structure,
both somatic or cell wall (O) antigens and flagellar (H) antigens. The
antigenic formula lists the O antigen(s) followed by the H antigen(s).
In 1896, Widal introduced techniques for testing patient serum for
antibodies in cases of typhoid fever.1 The Widal test was used diagnostically in two ways. First, it was considered diagnostic when a single
high antibody titer occurred during the first week of illness. Further, it
was diagnostic if a greater than fourfold rise in titer existed in serum
samples taken 1 to 2 weeks apart.2,3,4 The Widal test was developed to
include Salmonella Typhi and other species of Salmonella detected by
a variety of O and H antigens. S. Typhi and S. Paratyphi A and B are
the major pathogens in this group that can produce clinically distinct
systemic illness. In areas such as developing countries where typhoid
is highly endemic, the diagnostic value of the Widal test has been well
documented.5,6,7 The Widal test for antibodies to the O antigens of
Salmonella serotypes most likely to cause typhoid fever (usually
S. Typhi and S. Paratyphi A and B) can be useful in helping diagnose
typhoid fever when other methods have failed.8
and previous immunizations. Patients with known typhoid fever have
developed diagnostic titers of antibodies that are low.9,10 Also, patients
treated with antibiotics early in their disease may not develop a
significant titer rise.2
The Difco Manual
Section V
The various species of Salmonella contain multiple antigens that are

cross-reactive. This prevents using increased antibody levels, alone, to
identify infecting organisms by species or serotype. Other nontyphoidal febrile illness or unrelated immunological disorders may
produce significant elevations of antibody titers.2
may react with one or more antigens in an antibody test procedure,
resulting in low-level antibody titers that may not, singly, suggest disease.
The rapid slide procedure is a screening test designed to detect agglutinins. The macroscopic tube test11 is a confirmatory procedure
designed to quantitate agglutinin compositions. Any positive results
obtained in the screening (slide) test of specimens must be confirmed
by a tube test.
The rapid slide test is the most widely used procedure employing the
Widal antigens because of the simplicity with which the results may be
reported. Negative slide test reactions can usually be reported as such
if all five serum dilutions have been used. Although the slide test is not
quantitative, running the series of dilutions is necessary to detect
agglutinins that might be overlooked with a prozone phenomenon. This
often occurs in serum containing a high titer of typhoid agglutinins where
higher concentrations of the serum may yield negative results but a
dilution of the serum is positive.

Agglutination tests involving the use of Salmonella antigens determine
the presence of antibodies that react with the test antigen. The serological

Salmonella O Antigens
Salmonella H Antigens
Salmonella Vi Antigen
Appearance:
Turquiose-blue-violet suspension.
powdered cake.
powdered cake.
Rehydrate Febrile Positive Control Polyvalent and Febrile
Negative Control per label directions. Perform the slide or
tube agglutination test using Salmonella O, H or Vi Antigens
and positive and negative controls diluted in the same
proportion as a patient serum.
A Salmonella Antigen is considered satisfactory if it does not
agglutinate with the negative control and gives a 2+ or greater
reaction at a 1:80 dilution with the positive control.
The Difco Manual
Widal Antigen Set
a standard volume of antigen. The end point of the test is the last
dilution of the serum that shows a specific amount of agglutination.
The end point, reported as a dilution of the serum, is called the patients
antibody titer.
Reagents
Antigens
1. Salmonella Antigens are ready-to-use suspensions of the
Salmonella organisms listed below. Salmonella O Antigens
contain 20% glycerin.
Widal Antigen Set contents:
Salmonella O Antigen Group D - Salmonella Typhi O901,
factors 9,12 (selected strain)
Salmonella H Antigen a - Salmonella Paratyphi A.
Salmonella H Antigen b - Salmonella Paratyphi B.
Salmonella H Antigen d - Salmonella Typhi H901.
Salmonella O and H Antigens, available individually, are
prepared from selected strains containing the following groupspecific antigens:
Salmonella H Antigen c - flagellar antigen c
Salmonella O Antigen Group A - factors 1, 2, 12
Salmonella O Antigen Group B - factors 1, 4, 5, 12
Salmonella O Antigen Group C - factors 6, 7, (8), 20
When used as described, each vial contains sufficient reagent for
20 slide tests or 25 tube tests.
2. Concentration of Antigen: Salmonella O, H and Vi Antigens are
used undiluted for the slide test and diluted 1:20 for the tube test.
3. Antigen Density: Salmonella O, H and Vi Antigens are adjusted to
a density approximating 20 times a McFarland Barium Sulfate
Standard No. 3 (1.8 x 1010 organisms per ml).
Because antigen density may vary, it is adjusted for optimum
performance when standardized with hyperimmune sera obtained
from laboratory animals.
Variation in color intensity of the antigen is normal and will not
affect the outcome of the test.
4. Salmonella Antigens contain the following preservatives:
Salmonella O Antigens: 0.5% phenol, and approximately 0.002%
crystal violet and 0.005% brilliant green.
Salmonella H Antigens: 0.5% formaldehyde, and approximately
Salmonella Vi Antigen: 0.5% phenol, and approximately 0.002%
crystal violet and 0.005% brilliant green.
Antisera:
1. Febrile Positive Control Polyvalent is lyophilized, polyclonal,
polyvalent goat antisera containing approximately 0.04% Thimerosal as a preservative. This reagent contains antibodies at a titer
of 1:80 or greater for the Salmonella O and H Antigens in the
Widal Antigen Set.
Each vial of Febrile Positive Control Polyvalent contains sufficient
reagent for 32 slide tests or 50 tube tests using the four antigens
contained in the Widal Antigen Set. When using the Salmonella O,
H and Vi Antigens separately, there is sufficient reagent for 20 slide
or 25 tube tests.
805
Widal Antigen Set
2. Febrile Negative Control is a lyophilized, standard protein solution

Each vial of Febrile Negative Control contains sufficient reagent
for 32 slide tests when using the four antigens contained in the Widal
Antigen Set. When using the Salmonella O, H and Vi Antigens
separately, there is sufficient reagent for 20 slide tests.
Precautions
3. Salmonella H Antigen a
Salmonella H Antigen c
5. Salmonella O, H and Vi Antigens are not intended for use in the
immunization of humans or animals.
Storage
Store Salmonella O, H and Vi Antigens at 2-8C.
Store lyophilized and rehydrated Febrile Positive Control Polyvalent
at 2-8C.
Expiration Date
Procedure
Materials Provided
Widal Antigen Set:
Salmonella H Antigen Group a
Salmonella H Antigen Group b
Salmonella H Antigen Group d
Available separately:
Salmonella O Antigens
Salmonella H Antigens
806
Section V

Droppers, 0.03 ml per drop (supplied)

Slide test
Applicator sticks
Tube Test
Waterbath, 50 2C
Reagent Preparation
Salmonella O, H and Vi Antigens are ready to use in the slide test.
Salmonella O and H antigens must be diluted 1:20 for the tube tests.
(See Test Procedure for preparation instructions).
Febrile Positive Control Polyvalent: To rehydrate, add 5 ml sterile
distilled or deionized water and rotate gently to completely dissolve
the contents.

maintain at or below -20C. Serum specimens must be clear, free of
hemolysis and show no visible evidence of bacterial contamination (turbidity, hemolysis or particulate matter). Refer to appropriate references
for more information on collection of specimens.14,15 Serum specimens
must not be heated. Heat may inactivate or destroy certain antibodies.
Because changes in titer over a period of time are the best indicators of
active infection, and because the accuracy and precision of the tests
can be affected not only by test conditions but also by the subjectivity of
the person reading the endpoint, the following protocol is recommended.
A preliminary test using either the rapid slide test and/or the macroscopic tube test may be performed on the initial serum specimen and
reported to the physician at that time. An aliquot of the serum should
be transferred to a sterile test tube, sealed tightly, and kept in the freezer.
When the second serum is obtained, it should be run in parallel with
the original specimen. In this manner, the original serum will serve as
a control. Any difference in titer will be more credible, since the bias
associated with the performance of the test and determining the
endpoint will be reduced.
Test Procedure
Rapid Slide Test
the tube test.
The Difco Manual
Section V
Widal Antigen Set

an agglutination slide.
0.04, 0.02, 0.01 and 0.005 ml of Febrile Positive Control Polyvalent into a row of squares on the agglutination slide.
4. Salmonella Antigen: Shake the vial of antigen well to ensure a
smooth, uniform suspension. Place one drop (35 l) of antigen
suspension in each drop of test serum, positive control and
negative control.
5. Mix each row of test sera and control sera, using a separate
7. The final dilutions in squares 1-5 correspond with tube dilutions
of 1:20, 1:40, 1:80, 1:160, 1:320, respectively.
Results
4+
3+
2+
1+
100% agglutination; background is clear to slightly hazy.

75% agglutination; background is slightly cloudy.
50% agglutination; background is moderately cloudy.
25% agglutination; background is cloudy.
No agglutination.

1:80 dilution.
4. If results for either the positive or negative control are not as
described, the test is invalid and results cannot be reported.
5. Test specimens: The serum titer is that dilution that shows a 2+
or greater agglutination. See Table 1.
6. The slide test is a screening test, only, and results must be
confirmed with the tube test.
ml SERUM
CORRELATED
DILUTION
0.08
1:20
0.04
1:40
0.02
1:80
0.01
1:160
0.005
1:320
Serum titer
Results
1. Tube agglutination reactions detect antibodies to either somatic
(O) antigens or flagellar (H) antigens and these antibodies give
two different reactions. An O antigen and the corresponding antibody
give a coarse, compact agglutination which may be difficult to
disperse. An H antigen and its corresponding antibody give a loose,
flocculent agglutination. Do not vigorously shake tubes containing
H antigens. Characteristic O and H agglutination is shown in the
following diagrams.
Somatic O Agglutination
REACTIONS
SPECIMEN 1
SPECIMEN 2
SPECIMEN 3
3+
2+
1+
1:40
4+
3+
3+
2+
1+
1:160
4+
3+
2+
+
1:80
Macro Tube Test

Prepare a 1:20 dilution of the Salmonella O and H Antigens by adding
1 part of the antigen to 19 parts of sterile 0.85% NaCl solution.
1. Prepare a row of eight culture tubes (12 x 75 ml) for each test
serum, including a row for the Febrile Positive Control Polyvalent.
The Difco Manual
2. Sterile 0.85% NaCl solution: Dispense 0.9 ml in the first tube of

test serum in the first tube in the row and mix thoroughly. Transfer
0.5 ml from tube 1 to tube 2 and mix thoroughly. Similarly, continue transferring 0.5 ml through tube 7, discarding 0.5 ml from
tube 7 after mixing. Tube 8 is the antigen control tube and contains
of Febrile Positive Control Polyvalent in the first tube in the row
and mix thoroughly. Transfer 0.5 ml from tube 1 to tube 2 and mix
5. Salmonella Antigen: Add 0.5 ml of the diluted antigen to all 8
tubes in each row and shake the rack to mix the suspensions.
6. The final dilutions in tubes 1-7 are 1:20, 1:40, 1:80, 1:160, 1:320,
1:640 and 1:1280, respectively.
7. H antigen tests: Incubate in a waterbath at 50 2C for 1 hour.
O antigen tests: Incubate in a waterbath at 50 2C for 17 1 hours.
8. Remove from the waterbath. Avoid excessive shaking before
reading the reactions, either when the tubes are in the waterbath or
when removing them from the waterbath.
9. Read and record the results.
Flagellar H Agglutination
No agglutination.
1:80 dilution.
807
Widal Antigen Set
Section V
4. Antigen control: Tube #8 of each row should show no agglutination.

5. If results of the positive control and antigen control are not as
described, the test is invalid and results cannot be reported.
6. Test serum: The serum titer is that dilution which shows 2+ or
greater agglutination. See Table 2.
REACTIONS
SERUM DILUTION
SPECIMEN 1
SPECIMEN 2
SPECIMEN 3
1:20
1:40
1:80
1:160
1:320
1:640
1:1280
Serum Titer
4+
4+
3+
2+
1+
1:160
3+
2+
1+
1:40
4+
4+
4+
4+
3+
2+
1+
1:640
Interpretation
For a single serum specimen, a titer of 1:80 suggests infection.
A pair of serum specimens (acute and convalescent) showing a twodilution increase in titer is significant and suggests infection. A one
dilution difference is within the limits of laboratory error.
Table 3 presents data that will be helpful in interpreting serological
tests with the Salmonella antigens. The values tabulated will vary in
certain cases.
Table 3. Disease states and associated Salmonella Antigens.
SALMONELLA ANTIGEN
SUGGESTED
PATHOLOGY
Salmonella H Antigen d Typhoid Fever

(Typhoid H)
Salmonella O Antigen
Typhoid Fever
Group D (Typhoid O)
Salmonella H Antigen a Paratyphoid Fever
(Paratyphoid A)
Salmonella H Antigen b Paratyphoid Fever
(Paratyphoid B)
TIME TO
MAXIMUM TITER
SIGNIGICANT
TITER
4-5 weeks
1:80
3-5 weeks
1:80
3-5 weeks
1:80
3-5 weeks
1:80

1. The slide test is intended for screening only and should be confirmed
by the tube test. Slide test dilutions are made to detect a prozone
reaction and do not represent true quantitation of the antibody. A
serum specimen with a prozone reaction shows no agglutination
because of excessively high antibody concentrations. To avoid this
occurrence, all five serum dilutions of the slide test should be run.
2. Detection of antibodies in serum specimens may complete the clinical
picture of a patient having infection. However, isolation of the causative agent from patient specimens may be required. A definitive
diagnosis must be made by a physician and must be based on patient
history, physical examination and data from all laboratory tests.
808
3. In some cases of typhoid fever, sera may show a prozone reaction,

the inability of an antigen to react in higher serum antibody concentrations. It is advised that all five serum dilutions be run in the rapid
slide test, rather than just one dilution, to eliminate the possibility of
missing positive reactions due to the prozone phenomenon.
low titer reactions. Infections with other organisms, vaccinations
and a history of disease may result in a low level of antibody titer.
5. Previous immunization with typhoid vaccine or previous infection
with Salmonella species sharing common antigens with S. Typhi
can cause elevated antibody titers for prolonged periods. Other
non-typhoid febrile illnesses may cause elevation of cross-reacting
antibodies.
7. Nonspecific agglutination has been noted with Salmonella O
Group D antigen in the sera of patients with influenza.
8. Sera from narcotic addicts appear to contain broad nonspecific
activity to the Widal antigen.16
9. Sera from patients with chronic active liver disease may show high
agglutinin titers.17
are necessary: an acute specimen (obtained at the time of initial
symptoms) and a convalescent specimen (obtained 7 to 14 days later).
A two-dilution difference in the titer is a significant increase in
antibody level and suggests infection.
11. Antibody titers may be clinically useful in detecting chronic carriers
of S. Typhi and in diagnosing typhoid fever in endemic areas.
Detection of antibodies to heat-labile Salmonella envelope antigen
(Vi) may be useful for the detection of a chronic carrier state for
S. Typhi. The presence of capsular Vi antigen may mask somatic
(O) antigen activity.
12. In Salmonella infections, the stage of the patients disease is important
and may affect test results. The optimum time for detecting peak
titers may be missed if symptoms do not correlate with increased
antibody levels. Antibodies to O antigens appear earlier and disappear
earlier than antibodies to H antigens.
Antigens must be smooth uniform suspensions; before use, examine
antigen vials for agglutination. Suspensions with agglutination are
14. Discard rehydrated Febrile Positive Control Polyvalent or Febrile
Negative Control that is cloudy or has a precipitate anytime during
its period of use.
References
1. Widal, F. 1896. Serodiagnostic de la fivre typhoide. Sem. Med.
16:259.
2. Sack, B. R. 1986. Serologic tests for the diagnosis of enterobacterial
infections, p. 359-362. In N. R. Rose, H. Friedman, and J. L. Fahey
(eds.), Manual of clinical laboratory immunology, 3rd ed. American
The Difco Manual
Section V
3. Miller, L. E., H.R. Ludke, J. E. Peacock, and R. H. Tomar. 1991.

Manual of laboratory immunology, 2nd ed. Lea & Febiger.
5. Senewiratne, B., B. Chir, and K. Senewiratne. 1977. Reassessment
of the Widal test in the diagnosis of typhoid. Gastroenterology
73:233-236.
6. Sangpetchsong, V., and S. Tharavanij. 1983. Diagnosis of
typhoid fever by indirect hemagglutination with lyophilized cells.
Southeast Asian J. Trop. Med. Public Health 14:374-379.
7. Pang, T., and S. D. Puthucheary. 1983. Significance and value of
the Widal test in the diagnosis of typhoid fever in an endemic area.
J. Clin. Pathol. 36:471-475.
8. Sack, R. B., and D. A. Sack. 1992. Immunologic methods for the
diagnosis of infections by Enterobacteriaceae and Vibrionaceae,
p. 482-488. In N. R. Rose, E. C. De Macario, J. L. Fahey, H.
Friedman, and G. M. Penn (eds.), Manual of clinical laboratory
immunology, 4th ed. American Society for Microbiology,
Washington, D.C.
9. Brodie, J. 1977. Antibodies and the Aberdeen typhoid outbreak of
1964. I. The Widal reaction. J. Hyg. 79:161-180.
10. Hoffman, T. A., C. J. Ruiz, G. W. Counts, J. M. Sachs, and J. L.
Nitzkin. 1975. Waterborne typhoid fever in Dade County, Florida.
Clinical and therapeutic evaluation of 105 bacteremic patients. Am.
J. Med. 59:481-487.
11. Spink, W. W., N. D. McCullough, L. M. Hutchings, and C. K.
Mingle. 1954. A standardized antigen for agglutination technique
for human brucellosis. Report no. 3 of the National Research
Council, Committee on Public Health Aspects of Brucellosis.
Am. J. Pathol. 24:496-498.
387-388.
The Difco Manual
Widal Antigen Set

56:64175-64182.
15. Miller, J. M., and H. T. Holmes. 1995. Specimen collection, transport and storage. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C.
16. Vogel, H., C. E. Cherubin, and S. J. Millian. 1970. Amer. J. Clin.
Path. 53:932.
17. Protell, R. L., R. D. Soloway, W. J. Martin, L. J. Schoenfield,
and W. H. J. Summerskill. 1971. Lancet ii:330.
Packaging
Widal Antigen Set
6 x 5 ml
Contains:
AVAILABLE SEPARATELY:
5 ml
2642-32
5 ml
2845-56
Salmonella H Antigen c
5 ml
2846-56
5 ml
2847-56
Salmonella O Antigen Group A
5 ml
2839-56
Salmonella O Antigen Group B
5 ml
2840-56
Salmonella O Antigen Group C
5 ml
2841-56
5 ml
2842-56
5 ml
2953-56
2844-56
809
Section VI
Color Cover Page
The Difco Manual
811
Section VI
Agar Selection Guide
Agar Selection Guide
Ag
ar,
ar,
Ag
++
++
++
++
+
+
++
++
++
++
+
++
+
++
++
ica
Te
ch
n
ula
Gr
an
ar
Ag
cto
Ba
APPLICATIONS
Auxotrophic studies
Bacteriology, research
Bacteriology, general purpose
Bacteriophage studies
Biotechnology production
General microbial production
Growth of fastidious organisms
Identification of pathogenic organisms
Microaerophilic studies
Molecular genetics
Prepared plate manufacture
Quality control, production
Quality control, environmental
Transformation of bacteria
Transformation of yeast
ted
BACTERIOLOGICAL USES OF AGARS
+
+
+
++
++
+/
+/
+/
+
++
+
++
+
+/
+/
++
+
++
Ag
Ag
ar
ar
,T
,G
ra
ec
nu
ar
Ag
cto
Ba
APPLICATIONS
Immunodiffusion
Electrophoresis
Tissue Culture, mammalian
Tissue Culture, plant
Histology, tissue embedding
Histology, bone marrow embedding
Insect growth substrate
hn
lat
ica
ed
NON-BACTERIOLOGICAL USES OF AGARS
++
++
++
+
++
+
++
Key
++
+
+/
Recommended
Suitable
Marginal
The Difco Manual
813
Anaerobes - General
Section VI
Anaerobes - General
ANAEROBES - GENERAL
Anaerobe Broth MIC
Anaerobic Agar
Blood Culture Bottles Columbia Broth w/CO2
Blood Culture Bottles Columbia Broth w/SPS and CO2
Blood Culture Bottles Fluid Thioglycollate Medium w/SPS and CO2
Blood Culture Bottles Thioglycollate w/CO2
Blood Culture Bottles Thioglycollate w/SPS and CO2
CHO Medium Base
Cooked Meat Medium
ESP Anaerobic Broth
Liver Veal Agar
Schaedler Agar
Schaedler Broth
SFP Agar Base
Sterility Bottles with Septum Fluid Thioglycollate Medium
Sterility Bottles with Screw Cap Fluid Thioglycollate Medium
SPS Agar
Sulfite Agar
Thioglycollate Medium USP Alternative
Wilkens-Chalgren Agar
814
The Difco Manual
Section VI
Antimicrobic Selective Agents For Culture Media
Antimicrobic Selective Agents For Culture Media

AGENT
CONCENTRATION PER VIAL
PRODUCT NAME
Cefsulodin-novobiocin
4 mg/2.5 mg
Ceftozidime
40 mg
Palcam Antimicrobic Supplement
0.05 g
Chloramphenicol
Chlortetracycline (Aureomycin )
25 mg
Antimicrobic Vial A
Colistin Sulfate - Nystatin - Vancomycin
7,500 mcg/12,500 units/

3,000 mcg
Colistin Sulfate - Nystatin - Vancomycin

Trimethoprim
7,500 mcg/12,500 units/

3,000 mcg/5,000 mcg
(10 ml vial)
Cyclohexamide - Colistin Sulfate Acriflavine - Cefotetan - Fosfomycin
400 mg/20 mg/5 mg/2 mg/

10 mg
Cycloserine - Cefoxitin
125 mg/ 5 mg
Clostridium Difficile Antimicrobic

Supplement CC
Kanamycin
25,000 mcg
Antimicrobic Vial K
Moxalactam
20 mg
Moxalactam - Colistin Sulfate
20 mg/10 mg

Supplement
Novobiocin
20 mg
Oxytetracycline
100 mg
Polymyxin B
30,000 units
Antimicrobic Vial P
Potassium Tellurite Solution 1%
1%
Chapman Tellurite Solution
3.5%
Sodium 7- ethyl - 2 - methyl 4 - undecyl sulfate
The Difco Manual
XLT4 Supplement
815
Cosmetic Testing
Section VI
Cosmetic Testing
AC Broth
AC Broth Medium w/o Dextrose
Cetrimide Agar Base/PSEUDOSEL Agar
RODAC Plates
EMB Agar/Eosin Methylene Blue Agar Modified
HC Agar Base
HYcheck
Letheen Agar
Letheen Broth/Letheen Broth AOAC
MacConkey Agar
Malt Agar
Malt Extract Agar
Malt Extract Broth
Mannitol Salt Agar
Microbial Content Test Agar/
TRYPTICASE Soy w/Lec. poly.
Mycological Agar/MYCOPHIL Agar
Mycobiotic Agar/MYCOPHIL Agar
Neutralizing Buffer
Phenylethanol Agar/Phenylethyl Alcohol Agar
Pseudomonas Agar F/Flo Agar
Pseudomonas Agar P/Tech Agar
Pseudomonas Isolation Agar/Pseudomonas ISO
Staph Latex Test Kit/STAPHYLOSLIDE Test Kit
Staphylococcus Medium 110/Staphylococcus Agar #10
Sterility Test Bottles, Prepared
TAT Broth Base
Tryptic Soy Broth/ TRYPTICASE Soy Broth
VJ Agar/Vogel & Johnson Agar
Iso
old
tin
Te
s
st
&
ity
us
Ye
a
Sta
ylo
ph
c
oc
lat
ion
ion
ril
a
on
Ste
Ps
o
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lat
Iso
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ga
Gr
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-N
e
En
PRODUCTS
vir
on
me
nt
al
(Se
e
and also
Di Env
sin iro
fec nm
tan en
t T tal
est Sa
ing mp
) lin
cre
en
ing
APPLICATIONS
Available from Difco & Becton Dickinson Microbiology Systems.
816
The Difco Manual
Section VI
Dairy Product Testing - Products and Applications
Lip
nt
al
oly
tic
Mi
cro
or
ga
nis
An
ms
aly
Sta
sis
nd
ard
Pla
te
Co
un
t
Ye
as
De
t&
tec
M
tio
old
n
An
aly
sis
me
aly
An
vir
on
m
or
lif
ep
Str
Concentration Disks 1/2" Penase
Concentration Disks 1/2"

Penicillin 0.05 Unit/PM Discs, 0.05, 1/4" Taxo
Concentration Disks 1/2" Sterile Blanks,

Antibiotic Detect Disc 1/2" Taxo
EC Medium w/MUG/EC Broth w/ MUG

m FC Agar/M-FC Agar
Fraser Broth Base and/Fraser Broth Base Modified
Fraser Broth Supplement/Fraser Broth Additive
LPM Agar Base

Lactose Broth
Lauryl Tryptose Broth w/MUG/

Lauryl Sulfate Broth w/ MUG
Letheen Agar
Levine EMB Agar/Levine Eosin Methylene Blue Agar
Listeria Antisera
Malt Extract Agar
Malt Extract Broth

Trypticase Soy Agar w/Lec & Polysorbate
Milk Agar IDF Formulation

ia
us
M17 Broth
er
Brilliant Green Bile 2%/Brilliant Green Bile Broth
HYcheck
t
Lis
cc
o
toc
Antibiotic Medium 1
Antibiotic Medium 4
os
Cl
En
Be
PRODUCTS
iu
rid
Co
ta-
La
cta
ms
in
sis
ilk
APPLICATIONS
The Difco Manual
817
Section VI
Lip
nt
al
oly
tic
Mi
cro
or
ga
nis
An
ms
aly
Sta
sis
nd
ard
Pla
te
Co
un
t
Ye
as
D
t&
ete
cti
M
old
on
An
aly
sis
me
aly
An
vir
on
m
or
lif
os
Cl
En
Be
PRODUCTS
Supplement
iu
rid
Co
ta-
La
cta
ms
in
sis
ilk
APPLICATIONS
Nutrient Agar
ia
Oxford Medium Base/ Oxford Agar Base Modified
PALCAM Medium Base and

PM Indicator Agar
PM Negative Control
PM Positive Control
Penase Concentrate/Penicillinase Concentrate
Phenylethanol Agar/Phenylethanol Alchol Agar

Plate Count Agar/Standard Methods Agar
Spirit Blue Agar w/Lipase Reagent

Strep Grouping Kit
Subtilis Spore Suspension
Thermospore Suspension PM
Tryptic Soy Broth/TRYPTICASE Soy Broth
Tryptone Glucose Extract Agar/

TRYPTICASE Glucose Extract Agar
UVM Modified Enrichment Broth/

UVM Mod. Listeria Enrichment Broth
Violet Red Bile Agar w/MUG

ep
Str
us
Nutrient Broth
er
cc

Neutralizing Buffer
t
Lis
o
toc
818
The Difco Manual
Section VI
Environmental Sampling and Disinfectant Testing & Fermentation Products
Environmental Sampling and Disinfectant Testing

CULTURE MEDIA
HYGIENE CONTACT SLIDES

Bushnell-Haas Broth
Disinfectant Test Broth AOAC
Egg Meat Medium
Letheen Agar
Microbial Content Test Agar /Trypticase Soy Agar
w/ Lec. & Polysorbate
Neutralizing Buffer

HYcheck for Plate Count Agar with TTC
CONTACT PLATES
SETTLING PLATES (TRIPLE WRAPPED)
DOUBLE WRAPPED (IRRADIATED)

Sabouraud Dextrose Agar w/Lecithin and
Polysorbate 80
Tryptic Soy Agar w/Lecithin and Polysorbate 80
STERILE 100mm OR 150mm

Tryptic Soy Agar
Tryptic Soy Agar w/Lecithin and Polysorbate 80
Fermentation Products
FERMENTATION*
Brucella Broth
Casamino Acids/Select
Casamino Acids
Casitone
Eugon Broth/EUGONBROTH
Gelatone/Gelysate Peptone
M Broth
Malt Extract
Neopeptone
Peptamin
Peptone, Bacto
Proteose Peptone/Meat Peptone
Soytone/PHYTONE Peptone
TC Yeastolate/Yeastolate, TC
Todd Hewitt Broth

Tryptic Soy Broth/TRYPTICASE
Soy Broth
Tryptic Soy Broth w/o Dextrose/
TRYPTICASE Soy Broth w/o Dextrose
Tryptone/Select TRYPTICASE
Peptone
Tryptose/ POLYPEPTONE Peptone
Yeast Extract/Select Yeast
Extract
*Custom formulations and packaging are also available.
The Difco Manual
819
Escherichia coli-Products and Applications
Section VI
Escherichia coli - Products and Applications
lat
rit
PRODUCTS
BAGG Broth
CULTURESWAB Transport System Amies Medium
CULTURESWAB Transport System Amies Medium
w/o Charcoal/CULTURETTE Amies w/o Charcoal
CULTURESWAB Transport System Cary-Blair Medium/
Anaerobic CULTURETTE Cary-Blair Single
CULTURESWAB Transport System Stuarts Medium
Modified/CULTURETTE Modified Stuart's Medium
EC Medium w/MUG
E. coli H Antiserum H7
E. coli O Antiserum O157
m Endo Agar LES/M-Enda Agar LES
m Endo Broth MF/M-Endo Broth
EZ Coli Rapid Detection System for E. coli 0157
Lauryl Tryptose Broth w/MUG/Lauryl Sulfate Broth w/MUG
MacConkey Agar
MacConkey Sorbitol Agar/MacConkey II Agar w/Sorbitol
Nutrient Agar
Nutrient Agar w/MUG
Tryptic Soy Agar/TRYPTICASE Soy Agar
TSA Blood Agar Base
Veal Infusion Agar
Pu
No
ns
yP
ele
cti
ve
M
ed
ia
e
Se
le
M ctive
ed - D
ia i f
fer
Se
en
ro
tia
log
l
y
Sp
e
an cime
d n
Sh Co
ipm lle
Ra
c
pid ent tion
Te
st
APPLICATIONS
BIOCHEMICAL TESTS
Purple Agar/Broth Base
Adonitol
Dulcitol
Glucose
Inositol
Lactose
Mannitol
Salicin
Sucrose
V
+
V
+
V
V
Ammonium Citrate
Gelatin
H2 S
Indole
KCN
Methyl Red
Phenylalanine
Sodium Malonate
Tryptone Water
Urease
Voges-Proskauer
Key
Negative
+ Positive
d Delayed
V Variable +, d or
820
The Difco Manual
Section VI
Food and Beverage Testing - Products and Applications

aly
sp
old
Ye
a
st
&
te
Pla
al
r
Ye
p.
An
un
Co
sis
aly
To
t
En
lif
Co
lus
An
An
ge
sis
sp
p.
lla
ige cus
h
oc
a/S
il
ell yloc
a c ia
n
b
o
r
h
io
cto te lm tap
br
S
La Lis Sa
Vi
or
m
v i r Ana
on lys
m e is
nt
al
r
cte m
a
u
lob di
py stri
m
o
Ca Cl
aly
lus
Be
ve
ra
PRODUCTS
A-1 Medium/A-1 Broth
APT Agar
APT Broth
Baird-Parker Agar Base w/EY Tellurite Enrichment
Brilliant Green Agar Modified (Edel-Kampelmacher)
Brilliant Green Bile 2%/Brilliant Green Bile Broth 2%
Brucella Agar
Brucella Broth
Campylobacter Agar Kit Blaser/Campylobacter Agar
w/5 Antimicrobics and 10% Sheep Blood
Coagulase Plasma (Rabbit)
Coagulase Plasma EDTA (Rabbit)/
Coagulase Plasma, Rabbit w/ EDTA
Cooked Meat Medium
DNAse Test Agar
DNAse Test Agar w/Methyl Green/
DNAse Test Agar w/Toluidine Blue
DRBC Agar
Desoxycholate Citrate Agar Hynes
EC Medium/EC Broth
EC Medium with MUG/EC Broth w/MUG
Elliker Broth
m Endo Agar LES
m Endo Broth MF/ mEndo Broth, ALPHA
EZ Coli Rapid Detection System
Fraser Broth Base/Fraser Broth Base Modified
Fraser Broth Supplement/Fraser Broth Base Supplement
HYcheck for Yeasts and Molds w/TTC
LPM Agar Base
il
ac
An
aly
sis
sis
APPLICATIONS
sin
ia
The Difco Manual
821
Section VI

APPLICATIONS
is
lys
a
An
lys
lla
is
na
sis l
nt
i s hige u s
y
u
s
la ys er
A
l
a
n ct
na nt lus aly /S occ Co
old sp.
e A loba dium m A nme acil a An nella loc late
M
s
g
ia
r
i
illu vera mpy ostr lifo viro ctob steri lmo aphy tal P brio ast & rsin
c
Sa
St To Vi Ye
Ba Be Ca Cl Co En La Li
Ye
PRODUCTS
Lactobacilli MRS Agar/MRS Agar
Lactobacilli MRS Broth/MRS Broth
Lactose Broth
Lauryl Tryptose Broth/Lauryl Sulfate Broth
Lauryl Tryptose Broth with MUG/Lauryl Sulfate Broth w/MUG
Letheen Agar
Listeria O Antisera
Liver Veal Agar
Lysine Medium
MYP Agar
M Broth
MacConkey Agar
MacConkey Sorbitol Agar/MacConkey II Agar w/Sorbitol
Malt Agar
Malt Extract Agar
Malt Extract Broth
Mannitol Salt Agar
TRYPTICASE Soy Agar w/Lec. & Polysorbate 80
Minerals Modified Glutamate Agar
Modified EC Medium w/Indicator and
Neutralizing Buffer
OGYE Agar Base w/Antimicrobic Vial Oxytetracycline
Orange Serum Agar
Oxford Medium Base/Oxford Agar Base Modified
PALCAM Medium Base with
Rappaport-Vassiliadis (MSRV) Medium
Semisolid Modification
is
822
The Difco Manual
Section VI

APPLICATIONS
PRODUCTS
Reinforced Clostridial Agar
Rogosa SL Agar
Rogosa SL Broth
Rose Bengal Antimicrobic Supplement C/
Chloramphenicol Selective
SFP Agar Base/TSN Agar
SS Agar/Salmonella Shigella Agar
Salmonella H Antiserum Poly a-z
Selenite Broth/Selenite F Broth
Special Yeast and Mold Medium
Staphylococcus Medium 110/Staphylococcus Agar 110
Staph Latex Test/STAPHYLOSLIDE Test
Sulfite Agar
TCBS Agar
TT Broth Base, Hajna/Tetrathionate H
Tomato Juice Agar
Tryptone Water
UBA Medium/Universal Beer Agar
WL Differential Medium/WL Differential Agar
WL Nutrient Broth
WL Nutrient Medium
Wort Agar
XLD Agar
XLT4 Agar Base and XLT4 Supplement
YM Agar
YM Broth
Yersinia Selective Agar Base/CIN Agar Base
Yersinia Antimicrobic Supplement CN/CN Inhibitor
.
is
is
sp
lys
a
l
lys
a
s
l
s
i An
a
t
i
e
s
s
n
s
s
n
l
ig
y
u
u
si
ly r
A
na acte m Anal enta illus naly a/Sh occ Co
ld p.
o
A
e
l
c
u
l
t
M ia s
s
l o la
ge lob idi rm
n m a c a A ne
ic llu vera mpy ostr lifo viro ctob steri lmo aphy tal P brio ast & rsin
Sa
St To Vi Ye
Ba Be Ca Cl Co En La Li
Ye
The Difco Manual
823
McFarland Standard Preparation & Molecular Genetics - Media and Ingredients
Section VI
McFarland Standard Preparation

"MCFARLAND"
SULFURIC ACID, 1%
AQUEOUS SOLUTION
ML
BARIUM CHLORIDE, 1%
AQUEOUS SOLUTION
ML
CORRESPONDING
DENSITY OF
BACTERIA -106
INTERNATIONAL
UNITS (IU)
OF OPACITY
9.9
0.1
300
9.8
0.2
600
9.7
0.3
900
10
9.6
0.4
1200
12
9.5
0.5
1500
15
9.4
0.6
1800
9.3
0.7
2100
20
9.2
0.8
2400
9.1
0.9
2700
10
9.0
1.0
3000
30
1. Prepare the tubes by mixing 1% sulfuric acid with 1% barium chloride according to the table.
2. Make sure the tubes are uniform in size and made of chemically resistant glass.
3. Plug the tubes with rubber stoppers and carefully seal with paraffin. Store the tubes upright.
4. To estimate bacterial cell density, compare the bacterial suspension with the standards.
5. The above set is used to determine bacterial density in saline suspension. To estimate bacterial density
in broth, make the set by dissolving the sulfuric acid and barium chloride in sterile broth.
REFERENCE IN: Gradwohl's Clinical Laboratory Methods and Diagnosis. In. A.C. Sonnenwirth and
L. Jarett (ed.). C.V. Mosby Company, 1980 p. 1363.
Molecular Genetics - Media and Ingredients

LB MEDIA
NZ MEDIA AND INGREDIENTS
LB Agar, Lennox/LB Agar

(Lennox LAgar)
Casein Digest/Casein Digest

Peptone
LB Agar, Miller (Luria-Bertani)/

Luria Agar
NZCYM Broth
LB Broth, Lennox/LB Broth
NZYM Broth/NZY Broth
NZM Broth
LB Broth, Miller/Luria Broth

GENERAL MEDIA AND INGREDIENTS

M9CA Medium
M9 Minimal Salts, 5x/
M9 Minimal Salt
Minimal Agar Davis
SOB Medium
Terrific Broth
2xYT
YPD Agar/YEPD Agar
YPD Broth/YEPD Broth
Yeast Nitrogen Base
Yeast Nitrogen Base w/o Amino
Acids & Ammonium Sulfate
824
The Difco Manual
Section VI
Mycobacteria
Mycobacteria
MYCOBACTERIA
ATS Medium
Dubos Albumin Broth
Dubos Broth Base
Dubos Oleic Albumin Complex/
Oleic Albumin Complex
ESP Myco
ESP Myco GS
ESP Myco PVNA
Lowenstein Medium Base/
Lowenstein Jensen Medium Base
Lowenstein Medium Gruft
Middlebrook 7H9 Broth/Middlebrook 7H9 Broth Base

Middlebrook 7H10 Agar/Middlebrook & Cohn 7H10
Agar Base
w/WR 1339
Mycobacteria 7H11 Agar/Seven H11 Agar Base
Petragnani Medium
The Difco Manual
825
Mycology
Section VI
Mycology
CLINICAL REAGENTS FOR DETECTION
OF CANDIDA ALBICANS
MEDIA FOR CLASSIFICATION OF YEASTS

Yeast Carbon Base
BiGGY Agar
Yeast Nitrogen Base

Yeast Nitrogen Base w/o Amino Acids and
Ammonium Sulfate
rm a
ato nd/
ph or
yte
s
nA
mi
n
Ca
did
De
Vi
ta
MEDIA
Blood Agar Base
Brain Heart Infusion w/PAB
Corn Meal Agar
Malt Agar
Malt Extract Agar
Malt Extract Broth
Mycological Agar w/Low pH/MYCOPHIL w/ Low pH
Oatmeal Agar
Pagano Levin Base and TTC
Rice Extract Agar
SABHI Agar Base/SABHI Agar
Sabouraud Agar Modified/
Sabouraud Dextrose Agar Emmons
Special Yeast and Mold Medium
YM Agar
YM Broth
Ge
ne
ral
Us
ssa
y
NON-INHIBITORY MEDIA FOR FUNGI
826
The Difco Manual
Section VI
Mycology
Mycology
Reagents for Direct Microscopic Detection of Fungi
PRODUCT

3-Step Gram Stain Sets and Reagents
SpotTest Calcofluor White Reagent/Calcofluor White Droppers
USES
General screening
General screening
Screening of cultures for presence of fungi
A nonspecific fluorochrome stain which allows the rapid

examination of clinical specimens for the presence of fungi
under an FA microscope
SpotTest India Ink/India Ink Droppers

For use in the direct microscopic examination of clinical material
for the presence of encapsulated yeast cells
SpotTest KOH 10%/Potassium Hydroxide 10% Droppers

A 10% solution of potassium hydroxide
SpotTest Lactophenol Cotton Blue Stain/

Lactophenol Cotton Blue Droppers
Screening CSF for Cryptococcus neoformans

Negative staining
Used in wet mounts of clinical specimens to
examine for the presence of fungi
Teased mount method
Slide culture method
For use in the direct mounting and staining of yeast and molds
Selective and/or Differential Media for Fungi

GENERAL USE
Brain Heart CC Agar

Cooke Rose Bengal Agar and Antimicrobic Vial A
DRBC Agar
HC Agar Base
Mycobiotic Agar/Mycosel Agar
Mycological Agar w/Low pH/Mycophil Agar w/Low pH
Rose Bengal Agar Base and Rose Bengal Antimicrobic
Supplement C
CANDIDA AND/OR DERMATOPHYTES
BiGGY Agar
DTM Agar
Littman Oxgall Agar
Trichophyton Agars 1-7
The Difco Manual
827
Pasco Panel Contents - Selection Guide
Section VI
Pasco Panel Contents - Selection Guide
ANTIMICROBIAL AGENT
(MCG/ML)
AK
A/C
AM
A/S
AZT
CB
CCL
CFZ
FIX
CPZ
CTX
CTN
CX
TAZ
CTZ
FRX
CFX
CF
C
CIP
CLM
CD
EN
E
GM
IMI
LOM
MZ
NET
FD
NOR
OFX
OX
P
PIP
P/T
RI
STR
SFX
TE
TC
T/C
TO
T/S
VA
MIC
GRAM-NEG.
MIC/ID
MIC/ID GRAMGRAM-NEG. NEG. SPECIAL
BREAKPOINT/ID
GRAM-NEG.
MIC
ORAL
MIC
SUPP.
MIC
MIC/ID
GRAM-POS. GRAM-POS.
Amikacin
Amoxicillin/
Clavulanic Acid
Ampicillin
Ampicillin/
Sulbactam
Aztreonam
Carbenicillin
Cefaclor
Cefazolin
Cefixime
Cefoperazone
Cefotaxime
Cefotetan
Cefoxitin
Ceftazidime
Ceftizoxime
Ceftriaxone
Cefuroxime
Cephalothin
Chloramphenicol
Ciprofloxacin
Clarithromycin
Clindamycin
Enoxacin
Erythromycin
Gentamicin
Imipenem
Lomefloxacin
Mezlocillin
Netilmicin
Nitrofurantoin
Norfloxacin
Ofloxacin
Oxacillin
Penicillin
Piperacillin
Piperacillin/
Tazobactam
Rifampin
Streptomycin
Sulfisoxasole
Tetracycline
32,24,16-1
32-4
32-4
16-4/8-2
32,24,16-4
16-0.5/8-0.25
16-0.25/8-0.12
16-2
16-2/8-1
16-2
16-8/8-4
16-8
16-8/8-4
16-8
16-8/8-4
16-0.12
8-0.12
16-2/8-1
8-0.12
16-2/8-1
16-4
16-2
2-1
32-4
32-8
32-8
16-2
16-2
32-4
32-4
16-2
16-2
2-0.25
8,6,4-0.25
4-2
64-8
16-8
64-32
8-4
4-0.5
64-8
64-8/4
16-8
16-2
2-1
32-16
32-8
16-4
16-4
32-8
32-8
16-2
2-0.25
8,6,4-0.5
4-2
64-8
64-32
______
4-0.5
______
64-8/4
16-8
16-8
32-16
32-8
32-16
16-8
16-4
32-8
16-8
16-8
16-8
2-1
8,6,4-0.5
8-4
64-16
64-32
8-4
4-2
64-16
64-16/4
16-8
16-8
2-1
32-16
32-8
32-16
16-8
16-8
32-8
32-8
16-8
16-8
16-8
2-1
4-2
8,6,4-1
4-2
64-32
8-4
4-2
64-8
64-16/4
256-128
16-1
2-0.25
16-0.5
16-1
16-0.5
2-0.06
4-0.25
2-0.25
4-0.25
4-2
64-32
8-4
4-0.12
6,4-0.5
8-0.03
256-128
16-1
16-0.12
8-0.25
4-0.25
16-8
8-1
4-0.12
2-0.06
4-0.25
8-0.06
64-32
8-2
8-1
2-0.03
16-2
2-0.5
32-4
32-4
16-4
16-4
2-0.25
4-0.5
2-0.25
4-0.5
500,8,6,4-0.25
16-8
2-1
32-8
32-8
16-8
16-4
2-0.25
4-0.5
2-0.25
4-0.5
500,8,6,4-1
4-2
4-0.25
6.4-0.5
8-0.03
16-2/4
4-2
64-32
4-1
6,4-1
8-0.03
16-4/4
8-4
8-4
2-1
256
8-0.5
4-0.5
8-1
2-1
1000
8-2
2-1
1000
Ticarcillin
Ticarcillin/
Clavulanic Acid
Tobramycin
Trimethoprim/
Sulfamethoxazole
Vancomycin
64-16/2
64-8
64-16/2
8,6,4-0.25
2-1/38-19
8,6,4,-0.5
2-1/38-19
8,6,4,-0.5
2-38
8,6,4-1
2/38
2-0.5/38-9.5
2-0.5/38-9.5
8,6,4-0.25
2-0.5/38-9.5
8,6,4-1
2/38
16-4
4-0.5
16-1
16-2
30*
30*
30*
Biochemical Substrates
18**
* Gram-Negative Panels
** Gram-Positive Panels
828
The Difco Manual
Section VI
Peptones & Hydrolysates Selection Guide

Typical Analyses
6.01
ine
e
Th
re
on
ine
Try
pt
op
ha
n
Ty
ro
sin
e
Va
lin
e
lan
rin
ne
Se
Pr
oli
ine
en
yla
Ph
Le
Ly
s
eth
ine
ion
e
uc
leu
ine
cin
e
din
Iso
sti
Hi
Gl
ut
am
i
G l c Ac
yc
i
ine d
sti
ne
cA
As
Cy
pa
rti
ine
gin
Ar
Al
PRODUCT
an
ine
cid
AMINO ACIDS - %
Beef Extract
2.54
1.39
1.67
0.18
4.14
4.94
0.53
1.00
1.45
0.30 <0.01
2.16
0.90
0.67
0.05
1.99
0.86
Beef Extract, Dessicated
8.96
5.66
4.30
0.17 12.55 16.25
2.50
1.45
3.63
3.27
1.08
2.00
9.58
2.10
1.42
0.32
1.03
2.62
Casamino Acids
3.26
2.20
4.76
0.16 15.30
1.31
1.66
3.34
5.47
5.71
1.28
2.11
6.17
2.19
2.41
<0.01
0.47
4.30
1.64
1.77
3.42
0.34 10.97
1.09
1.31
0.13
2.54
2.14
1.19
5.23
4.44
2.64
1.99
0.01
1.33
3.24
Casein Digest
2.92
3.02
6.75
0.17 23.10
1.93
2.52
4.66
8.29
7.70
2.66
4.27 11.04
5.55
4.33
1.16
2.54
6.51
Casitone
3.01
3.76
6.61
0.02 20.03
1.97
2.17
4.16
8.74 13.62
1.71
4.02
8.57
4.82
3.74
0.14
2.09
4.06
Neopeptone
4.03
4.14
6.19
0.26 13.22
7.02 <0.01
0.36
3.65
5.16
2.00
8.67
6.73
4.22
3.69
0.96
4.21
4.96
Bacto Peptone
8.67
6.76
5.60
0.20 10.21 15.59
0.58
1.45
3.01
3.42
1.19
1.81
8.80
2.87
1.81
0.36
0.64
2.35
Proteose Peptone
6.50
5.12
7.28
0.87 11.95
2.01
3.04
5.66
5.33
1.97
2.86
5.93
3.49
3.14
0.60
2.35
3.76
6.08
5.47
7.45
0.40 10.57 10.84 <0.01
1.00
3.57
5.22
1.51
7.94
5.31
4.64
3.90
0.94
1.92
4.73
9.68
5.99
5.49
6.92
1.12 12.38
9.26
1.74
2.65
5.70
5.02
1.86
2.72
4.94
3.65
3.32
0.59
1.96
3.62
Soytone
2.46
3.82
7.27
1.45 12.76
2.51
1.24
2.37
4.03
3.45
0.86
2.46
2.92
2.87
2.17
0.47
1.93
2.65
3.70
2.67
7.13
0.55 16.30
2.02
1.83
1.18
5.43
4.11
2.11 10.81
6.62
4.62
4.76
1.75
1.22
5.82
TC Yeastolate
4.84
2.99
5.58
0.45 10.53
4.02 <0.01
0.56
3.23
3.82
0.96
5.59
2.59
2.81
2.80
0.79
1.21
3.80
Tryptone
2.86
3.03
6.11
0.42 17.05
1.75
2.02
4.40
7.11
6.70
2.57
3.71
7.45
4.29
3.58
0.71
1.42
5.00
Tryptose
4.45
4.65
6.34
0.44 13.92
2.84 <0.01
0.34
3.67
4.64
1.92
7.52
6.33
4.09
3.55
0.62
2.21
1.93
Yeast Extract
5.36
3.02
6.69
0.74 14.20
3.25
3.23
4.69
5.15
1.05
2.53
2.60
2.84
2.95
1.36
1.20
3.79
1.20
CARBOHYDRATE
)
(%
Beef Extract
0.068
1.284 <0.001 <0.001 <0.001 <0.001 0.239 <0.001 5.458
5.477
2.315
0.629
0.707 <0.001 <0.001
0.018
1.576 <0.001
0.011 <0.001 0.022 <0.001 0.345
1.994
2.774
0.829
0.661 <0.001
7.400 <0.001 <0.001 <0.001 <0.001 0.002 <0.001 3.325
0.410
0.420 <0.001 <0.001
Casamino Acids
<0.001
0.001
0.002
al
To
t
Zi
nc
r
Tin
lfu
Su
Su
lfa
te
m
diu
So
as
siu
ate
ph
os
Ph
Po
t
ne
an
ga
siu
ne
ag
M
ad
Le
er
Iro
pp
Co
Co
ba
lt
ide
lor
Ch
Ca
PRODUCT
lci
um
se
INORGANICS - %
0.2
<0.1
8.710
0.045
0.019 21.212 <0.001 <0.001 <0.001 <0.001 0.008 <0.001 1.358
0.273 13.721
0.167
0.424 <0.001
0.002
3.4
Casein Digest
0.019
0.059 <0.001 <0.001
0.002 <0.001 0.018 <0.001 2.670
0.078
0.167
0.621 <0.001
0.002
4.2
Casitone
0.010
0.110 <0.001 <0.001
0.003 <0.001 0.019 <0.001 2.604
0.162
3.073
0.339
0.676 <0.001
0.004
0.2
Neopeptone
0.012
0.344 <0.001 <0.001 <0.001 <0.001 0.006 <0.001 2.209
0.149
2.057
0.340
0.657 <0.001 <0.001
0.8
Bacto Peptone
0.008
1.086 <0.001 <0.001
0.004 <0.001 0.007 <0.001 0.445
0.203
1.759
0.244
0.410 <0.001
0.001
6.9
Proteose Peptone
0.021
4.510 <0.001 <0.001
0.002 <0.001 0.027 <0.001 0.872
0.685
3.677
0.162
0.812 <0.001
0.002
<0.1
0.024
3.644 <0.001 <0.001 <0.001 <0.001 0.024 <0.001 1.674
0.815
3.956
0.232
0.698 <0.001
0.003
1.3
2.937
0.023
3.581 <0.001 <0.001
0.002 <0.001 0.027 <0.001 1.447
0.982
3.815
0.232
0.975 <0.001
0.007
1.4
Soytone
0.055
0.165 <0.001 <0.001
0.008 <0.001 0.161 <0.001 0.820
2.220
3.404
2.334
1.660 <0.001
0.001
24.0
0.095 <0.010 <0.001 <0.001
TC Yeastolate
0.002
Tryptone
Tryptose
Yeast Extract
The Difco Manual
0.002 <0.001 0.026 <0.001 1.309
0.932
1.357
0.379
0.750 <0.001
0.002
9.2
0.299 <0.001 <0.001 <0.001 <0.001 0.025 <0.001 2.633
5.085
0.819
0.488
0.528 <0.001
0.007
10.3
0.013
0.186 <0.001 <0.001 <0.001 <0.001 0.017 <0.001 2.669
0.229
2.631
0.241
0.740 <0.001
0.003
7.7
0.001
1.886 <0.001 <0.001
0.002 <0.001 0.022 <0.001 2.144
0.679
3.410
0.308
0.737 <0.001
0.005
7.1
0.013
0.380 <0.001 <0.001 <0.001 <0.001 0.075 <0.001 3.270
3.195
1.490
0.091
0.634 <0.001
0.011
17.5
829
Section VI

Typical Analyses
or
eC
Co
As
ell
Sp
or
lif
h(
Sa
n
mo
NITROGEN CONTENT
ou
nt
Sta
nd
ard
Pla
Th
te
erm
Co
un
op
t
hil
eC
ou
nt
To
tal
Ni
tro
ge
Am
n(
ino
%
)
Ni
tro
AN
g
e
/T
n
N
(%
)
BIOLOGICAL TESTING - CFU/g
%
)
Cla
rity
1%
So
Fil
ln
ter
(N
TU
ab
ilit
)
y
Lo
(g/
ss
cm 2
on
)
Dr
y
ing
pH
,1
(%
%
)
So
ln
PHYSICAL CHARACTERISTICS
PRODUCT
Beef Extract
24.1 116.8
0.1
77.2*
5.4
neg
neg
299
117
33
11.2
3.8
10.2
1.7
0.6
2.5
6.9
neg
neg
585
690
28
14.0
2.2
15.7
Casamino Acids
24.4
0.5
2.9
4.5
6.4
neg
neg
390
950
25
10.5
8.8
83.8
38.3
0.3
2.6
4.5
6.7
neg
neg
2375
2250
<50
8.1
6.4
79.0
33.8
Casein Digest
6.4
0.4
2.6
4.7
7.2
neg
neg
235
250
178
13.4
7.2
53.9
Casitone
7.0
0.6
1.7
3.7
7.2
neg
neg
300
1850
100
13.3
4.7
35.3
Neopeptone
7.0
1.2
0.3
3.2
7.4
neg
neg
175
400
75
13.7
3.3
23.8
Bacto Peptone
4.4
0.5
0.5
3.0
7.0
neg
neg
90
273
13
15.5
3.1
20.0
Proteose Peptone
11.1
1.4
0.9
3.1
7.2
neg
neg
393
443
73
14.0
2.9
20.7
12.7
1.5
0.6
3.5
7.2
neg
neg
75
1450
<50
12.6
5.0
39.7
11.4
2.2
0.5
4.0
7.2
neg
neg
890
915
25
13.2
3.5
26.5
Soytone
12.0
1.0
1.2
4.6
7.2
neg
neg
10
38
<3
9.4
3.1
33.0
7.2
0.4
7.3
4.6
7.1
neg
neg
<50
300
<50
13.0
6.3
48.3
13.0
1.4
4.5
3.6
6.9
neg
neg
175
175
<50
10.8
6.5
59.8
Tryptone
6.8
0.5
1.3
3.7
7.2
neg
neg
73
870
13.0
5.2
40.0
Tryptose
9.7
0.8
2.3
3.2
7.4
neg
neg
875
825
100
13.4
4.4
32.5
11.2
1.5
2.7
3.1
6.7
neg
neg
60
<5
10.9
6.0
55.0
TC Yeastolate
Yeast Extract
Beef Extract
0.1 1171.5
0.5
3.3
4113.2
774.7
20.0
91.0
7.3
0.4
0.1 1300.0
<0.1
0.6
2100.0
138.1
40.5
8.7
2.8
<0.1
<0.1
111.3
<100.0 <20.0
<5.0
<0.1
<0.1
1.8
1.2
<30.0
0.2
<14.0
<0.1
160.0
<0.1
<0.1
<0.1
<50.0
<0.1
<0.1
<38.0
<0.1
9.8
<0.6
<0.1
0.2
0.1
<40.0
<0.1
1.0
490.0
14.1
6.1
6.7
0.4
<0.1
Casitone
0.2
550.0
<0.1
0.8
980.0
20.3
15.9
7.7
1.3
0.4
<0.1
342.9
Neopeptone
0.2 3100.0
<0.1
0.4
3600.0
52.2
2.9
16.0
2.3
1.3
<0.1
<14.0
Bacto Peptone
0.2 2000.0
<0.1
0.3
2400.0
21.9
<0.5
5.9
1.7
3.9
<0.1
413.0
Proteose Peptone
0.1 2300.0
<0.1
0.4
5000.0
79.9
4.2
20.0
1.1
<0.1
1.2
99.7
0.3 4500.0
<0.1
0.5
4700.0
157.1
1.2
47.0
4.0
6.4
0.4 3700.0
<0.1
0.3
8900.0
124.2
<0.5
20.0
1.3
6.8
0.1
659.6
Soytone
0.2 2200.0
<0.1
3.0
2100.0
19.1
9.0
13.0
11.0
<0.1
1.2
113.2
0.5
<14.0
<0.1
1.5 8297.0
1.6 1319.0
280.0
<0.1
0.2
360.0
<0.1
11.3
4.3
1.0
8.5
TC Yeastolate
6.7 3400.0
<0.1
25.2
1900.0
945.0
96.6
300.0
77.5
21.8
Tryptone
0.1
350.0
<0.1
0.3
1400.0
97.8
3.7
5.3
0.6
<0.1
0.4
93.4
Tryptose
0.2 2700.0
<0.1
0.4
5400.0
47.4
11.4
16.0
1.4
4.3
0.1
769.0
Yeast Extract
3.3
<0.1
1.5
1400.0
597.9 763.0
273.7
43.2
116.5
529.9
217.5
300.0
ne
<0.1 1093.4
Casamino Acids
Casein Digest
idi
ne
mi
Th
ym
bo
Ri
Th
ia
ic
Py
rid
ox
in
en
th
to
Pa
n
fla
vin
Ac
id
Ac
ic
tin
PA
BA
co
Ni
ito
os
In
Fo
li
cA
cid
ob
oc
ne
Cy
an
oli
Ch
PRODUCT
Bi
ot
in
ala
mi
id
VITAMINS - g/g
54.3 2975.0
*Represents Total Solids value rather than loss on drying.
830
The Difco Manual
Section VI
Pharmaceutical Testing - Products and Applications
Am
As
n/
ic
Vi
ta
mi
iot
tib
Antibiotic Medium 1
Antibiotic Medium 2/Base Agar, Penicillin Assay
Antibiotic Medium 3
Antibiotic Medium 4/Yeast Beef Agar
Antibiotic Medium 5/
Streptomycin Assay Agar w/YE
Antibiotic Medium 8/Base Agr w/Low pH

Antibiotic Medium 9/Polymixin Base Agar
Antibiotic Medium 10/Polymixin Seed Agar
Antibiotic Medium 19/Nystatin Assay Agar
Antibiotic Medium 11/Neomycin Assay Agar
APT Agar
APT Broth
Biotin Assay Medium
An
Agar Medium No. F
ino
say
g
tin
Te
s
ity
ril
Ste
PRODUCTS
Ac
id
As
say
APPLICATIONS
Folic AOAC Medium
Lysine Assay Medium
Methionine Assay Medium
The Difco Manual
831
Section VI
Am
As
n/
ic
Vi
ta
mi
iot
tib
Neurospora Culture Agar
Niacin Assay Medium
Pyridoxine Y Medium
Sterility Bottles w/ Screw Cap Fluid

Thioglycollate Medium/
Sterility Bottles w/ Screw Cap Tryptic

Soy Broth/Trypticase Soy Broth
Sterility Bottles w/ Septum Fluid A/

Peptone Water (0.1%)
Sterility Bottles w/ Septum Fluid D/

Peptone Water (0.1%) w/Polysorbate
Sterility Bottles w/ Septum Fluid

Thioglycollate Medium
Sterility Bottles w/ Septum Tryptic Soy Broth/

Septum Fluid Thioglycollate Medium

Tryptic Soy Broth/Trypticase Soy Broth
An
NIH Thioglycollate Medium
ino
say
g
tin
Te
s
ity
ril
Ste
PRODUCTS
Ac
id
As
say
APPLICATIONS
832
The Difco Manual
Section VI
Salmonella- Products and Applications
Salmonella - Products and Applications
Adonitol/Adonitol CP
Arginine
+ or (+)
ed
ia
Str
en
ea
tif
ica
co k la
lon ct
tio
n
ies ose
on ne
ga
tiv
e
al
Id
ial
re
ffe
gic
Di
ro
lo
Se
im
Pr

BG Sulfa Agar
Brilliant Green Agar Modified (Edel-Kampelmacher)

Dulcitol
ary
ly
S
gh
Hi
En
Alginate
Desoxycholate Agar
nt
cti
ve
M
ele
t
en
hm
ric
re
ffe
Di
Bi
PRODUCTS
oc
he
mi
nt
ca
ial
Ag
lT
es
ar
ts
ed
ia
APPLICATIONS
(+) d
EMB Agar
Erythritol
Esculin
Gelatin
Glucose
GN Broth Hajna

H2 S
+()
Individual O and H Antisera

Indole
Inositol
KCN
Lactose
Lysine
Lysine Iron Agar

MacConkey Agar
Methyl Red
MacConkey Agar CS
+
Key
Negative
+ Positive
d Delayed
(+) Variable
() Variable
The Difco Manual
833
Section VI

ed
ia
Str
en
ea
tif
ica
co k la
lon ct
tio
n
ies ose
on ne
ga
tiv
e
al
gic
ro
lo
Se
im
Pr
Id
ial
re
ffe
Di
ary
ly
S
gh
Hi
En
nt
cti
ve
M
ele
t
en
hm
ric
re
ffe
Di
Bi
PRODUCTS
oc
he
mi
nt
ca
ial
Ag
lT
es
ar
ts
ed
ia
APPLICATIONS
MIO Medium

Nitrate Reduction
Ornithine
+ or (+)
Oxidase
Phenylalanine
Polyvalent Antisera
Raffinose
Salicin
SBG Enrichment
Selenite Broth
SIM Medium
Simmons Citrate
(+) d
Sodium Malonate
SS Agar
Sucrose

Urea Agar Base

Urease
VP
XLD Agar
Key
Negative
+ Positive
d Delayed
(+) Variable
() Variable
834
The Difco Manual
Shigella and Alkalescens-Dispar Group - Products and Applications
Shigella and Alkalescens-Dispar Group Products and Applications
APPLICATIONS
en
t
Pr
im
a
M ry
ed D
ia iff
e
Pr
im
ary
hm
ric
En
PRODUCTS
ell
A-
ig
Sh
Gr
ou
re
nt
ial
Biochemical
Tests
Adonitol
Arabinose
Arginine Dihydrolase
Christensen Citrate
Desoxycholate Agar
Dulcitol
EMB Agar
Gas from Glucose
Gelatin (22C)
(1)
GN Broth Hajna

H2 S
Indole
or+
Inositol
KCN
Lactose
Lysine Decarboxylase
(1)
Lysine Iron Agar

MacConkey Agar
Pla
tin
Str
gM
e
ed
ne ak la
ia
ga ct
tiv os
ec e
olo
nie
so
n
Section VI
MacConkey Agar CS
Malonate
Maltose
Mannitol
+ or
Methyl Red
Motility
Key
Negative
+ Positive
d Different reactions
(1) Certain biotypes of S. flexneri produce gas; cultures of S. sonnei ferment lactose and sucrose slowly and decarboxylate ornithine.
() Variable
The Difco Manual
835
Shigella and Alkalescens-Dispar Group - Products and Applications
Section VI
Mucate
Ornithine Decarboxylase
Phenylalanine
Raffinose
Rhamnose
Salicin
Sodium Acetate
Sucrose
+(+)
(1)
en
t
Pr
im
a
M ry
ed D
ia iff
e
Pr
im
ary
hm
ric

Urease
Voges-Proskauer
XLD Agar
Xylose
En
PRODUCTS
ell
A-
ig
Sh
Gr
ou
re
nt
ial
Biochemical
Tests
Pla
tin
Str
gM
ea
k
ed
ne la
ia
ga ct
tiv os
ec e
olo
nie
so
n
Shigella and Alkalescens-Dispar Group Products and Applications

APPLICATIONS
Key
Negative
+ Positive
d Different reactions
(1) Certain biotypes of S. flexneri produce gas; cultures of S. sonnei ferment lactose and sucrose slowly and decarboxylate ornithine.
() Variable
836
The Difco Manual
Section VI
Sterility Testing
Sterility Testing
STERILITY TESTING, MANUAL
AC Broth
Sterility Bottles w/Screw Cap Tryptic Soy Broth
AC Medium
Sterility Bottles w/Septum Fluid A
Agar Medium No. F
Sterility Bottles w/Septum Fluid D

Sterility Bottles w/Septum Fluid Thioglycollate

Medium
Sterility Bottles w/Septum Tryptic Soy Broth
NIH Thioglycollate Medium
Sterility Bottles w/Screw Cap Fluid

Thioglycollate Medium

Tryptic Soy Broth
See also: Environmental Sampling Section of the Reference Guide
STERILITY TESTING, AUTOMATED
See ESP, Industrial Applications
The Difco Manual
837
Veterinary Testing - Products and Applications
Section VI

cis
ell
a
Gr
a
Ba m N
cte eg
ria ativ
(G e E
Le
en nte
pt
era ri
os
l) c
pir
a
Ps
eu
do
mo
na
s
m
tri
diu
lla
os
PRODUCTS
ce
Cl
u
Br
Fra
n
Co
ll
Ba e c t i
cte o n
ria /Tr
l ans
po
Co
rt
lle
cti
on
/T
ran
Ge
sp
n
or
Cu era
t, V
ltu l P
ira
re ur
l
M po
ed se
ia Ba
cte
ria
APPLICATIONS

Brain Heart Infusion Agar and Clostridium

Difficile Antimicrobic Supplement CC
Brucella Agar
Brucella Broth
Brucella Antisera
Cellmatics Viral Transport Pack/

CULTURETTE Viral Single Swab
Cetrimide Agar Base/PSEUDOSEL Agar

Cooked Meat Medium
CULTURESWAB Amies Medium
CULTURESWAB Amies Mediumw/o Charcoal/

CULTURETTE Amies w/o Charcoal
CULTURESWAB Cary-Blair Medium/Anaeno

CULTURETTE Cary-Blair Single
CULTURESWAB Perinasal Swab w/Amies Medium
CULTURESWAB Urethral Swab w/Amies Medium
CULTURESWAB Stuarts Medium Modified/

CULTURETTE Modified Stuart's Medium
Cystine Heart Agar

Eugon Agar/EUGONAGAR
Eugon Broth/EUGONBROTH

Francisella Tularensis Antigen

MacConkey Agar
Pseudomonas Agar F/Flo Agar
Pseudomonas Agar P/Tech Agar

Pseudomonas Isolation Agar/Pseudomonas ISO
Reinforced Clostridium Medium/
Reinforced Clostridum Agar
838
The Difco Manual
Section VI

cis
ell
a
Gr
am
Ba N
cte eg
ria ativ
(G e E
Le
en nte
pt
era ri
os
l) c
pir
a
Ps
eu
do
mo
na
s
Fra
n
tri
diu
lla
os
PRODUCTS
ce
Cl
u
Br
Co
ll
Ba e c t i
cte o n
ria /Tr
l ans
po
Co
rt
lle
cti
on
/T
ran
Ge
sp
n
or
Cu era
t, V
ltu l P
ira
re ur
l
M po
ed se
ia Ba
cte
ria
APPLICATIONS
SFP Agar Base/TSN Agar
SPS Agar
Sterile Swab/Aerobic Collection & Trans. System w/o Agar
Tryptic Soy Agar/TRYPTICASE Soy Agar

Blood Culture Bottles Brain Heart

Infusion w/ PAB, SPS + CO2
Blood Culture Bottles Columbia

Broth w/CO2
Blood Culture Bottles Columbia

Broth w/SPS + CO2
Blood Culture Bottles Fluid

Thioglycollate Medium w/SPS + CO2
Blood Culture BottlesThiol

Broth w/CO2
Blood Culture BottlesThiol

Broth w/SPS + CO2
Blood Culture Bottles

Tryptic Soy Broth w/CO2
Blood Culture Bottles Tryptic

Soy Broth w/SPS + CO2/
SEPTIC-CHEK TSB w/ SPS + CO2
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Coagulase Plasma (Rabbit)/ Coagulase

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w/ EY Tellurite Enrichment
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APPLICATIONS
The Difco Manual
839
Veterinary Testing-Products and Applications
Section VI
Coagulase Plasma EDTA (Rabbit)/ Coagulase

Plasma, Rabbit w/EDTA
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Dispens-O-Disc Susceptilbility Disks: Apramycin 15 mcg
Ceftiofur 30 mcg
Enrofloxacin 5 mcg
Tilmicosin 15 mcg
DNase Test Agar
DNase Test Agar w/ Methyl Green/DNase

Test Agar w/Toluidine Blue
DTM Agar
Lysine Iron Agar
Mannitol Salt Agar

MIO Medium
MR-VP Medium/MR-VP Broth
Mueller Hinton Medium/Mueller Hinton II Agar
Mycological Agar/MYCOPHIl Agar
OF Basal Medium

Phenylethanol Agar/Phenylethyl Alcohol Agar
Purple Agar Base
Purple Broth Base
SS Agar/Salmonella Shigella Agar

Staph Latex Test/STAPHYLOSLIDE Test Kit
Strep Grouping Kit
TCBS Agar
Urea Agar Base

VJ Agar (Vogel-Johnson Medium)
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Columbia CNA Agar

Corn Meal Agar
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APPLICATIONS
XLD Agar
840
The Difco Manual
Section VI
Water/Wastewater Testing-Products and Applications
Water/Wastewater Testing - Products and Applications
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A-1 Medium/A-1 Broth

BAGG Broth
Baird-Parker Agar Base w/
EY Tellurite Enrichment/Egg Yolk Tellurite Solution
Bile Esculin Agar
Bile Esculin Azide Agar/ENTEROCOCCOSEL Agar
(Edel-Kempelmacher)
Brilliant Green Bile 2%/Brilliant Green Bile Broth 2%
m Brilliant Green Broth
m E Agar/M-E Agar Base
EC Medium/EC Broth
EC Medium with MUG/EC Broth w/ MUG
m Endo Agar LES/m Endo Agar LES
m Endo Broth MF/m Endo Broth
m Enterococcus Agar/m Enterococcus Agar
Esculin Iron Agar
EVA Broth/Ethyl Violet Azide Broth
m FC Agar/ m FC Agar
m FC Basal Medium
m FC Broth Base/m FC Broth
m HPC Agar/m HPC Agar Base
Lauryl Tryptose Broth/Lauryl Sulfate Broth
Lauryl Tryptose Broth with MUG/
Lauryl Sulfate Broth w/ MUG
Muller Kauffmann Tetrathionate
Broth Base
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The Difco Manual
841
Water/Wastewater Testing-Products and Applications
Section VI
Water/Wastewater Testing - Products and Applications
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Rosolic Acid
m T7 Agar
m TEC Agar
Tryptone Water
XLD Agar
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842
The Difco Manual
Section VII
(insert color page here)
Section VII
Alphabetical Index
Alphabetical Index
Azide Blood Agar Base . . . . . . . . . . . . . . . . . . . . . 0409 . . . . . . . . . . . . . 46

Azide Dextrose Broth . . . . . . . . . . . . . . . . . . . . . . 0387 . . . . . . . . . . . . . 48
Product #
Pg. #
2xYT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0440 . . . . . . . . . . . . . 23
Product #
Pg. #
A-1 Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1823 . . . . . . . . . . . . . 24
B12 Assay Medium USP . . . . . . . . . . . . . . . . . . . . . 0457 . . . . . . . . . . . . . 49
AC Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0317 . . . . . . . . . . . . . 26
B12 Culture Agar USP . . . . . . . . . . . . . . . . . . . . . . 0541 . . . . . . . . . . . . . 51
AC Broth w/o Dextrose . . . . . . . . . . . . . . . . . . . . . 0599 . . . . . . . . . . . . . 26
B12 Inoculum Broth USP . . . . . . . . . . . . . . . . . . . . 0542 . . . . . . . . . . . . . 51
Acetate Differential Agar . . . . . . . . . . . . . . . . . . . 0742 . . . . . . . . . . . . . 29
BAGG Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0442 . . . . . . . . . . . . . 53
Acridine Orange Stain . . . . . . . . . . . . . . . . . . . . . . 3336 . . . . . . . . . . . . 597
BG Sulfa Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0717 . . . . . . . . . . . . . 55
Actinomycete Isolation Agar . . . . . . . . . . . . . . . . . 0957 . . . . . . . . . . . . . 31
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0140 . . . . . . . . . . . . . 21
Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Bactrol Gram Silde . . . . . . . . . . . . . . . . . . . . . . . 3140 . . . . . . . . . . . . 601
Agar Bacteriological Technical . . . . . . . . . . . . . . . 0812 . . . . . . . . . . . . . 21
Bactrol TB Slides . . . . . . . . . . . . . . . . . . . . . . . . 3139 . . . . . . . . . . . . 603
Agar Flake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0970 . . . . . . . . . . . . . 21
Baird-Parker Agar Base . . . . . . . . . . . . . . . . . . . . . 0768 . . . . . . . . . . . . . 58
Agar Medium No. F . . . . . . . . . . . . . . . . . . . . . . . . 0666 . . . . . . . . . . . . . 32
Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0126 . . . . . . . . . . . . . 60
Agar Noble . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0142 . . . . . . . . . . . . . 21
Beef Extract, Desiccated . . . . . . . . . . . . . . . . . . . . 0115 . . . . . . . . . . . . . 60
Agar Selection Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 813
BiGGY Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0635 . . . . . . . . . . . . . 62
Agar, Granulated . . . . . . . . . . . . . . . . . . . . . . . . . . 0145 . . . . . . . . . . . . . 21
Bile Esculin Agar . . . . . . . . . . . . . . . . . . . . . . . . . . 0879 . . . . . . . . . . . . . 64
Alkalescens-Dispar Antiserum Poly . . . . . . . . . . . 2838 . . . . . . . . . . . . 788
Bile Esculin Agar Base . . . . . . . . . . . . . . . . . . . . . 0878 . . . . . . . . . . . . . 64
Amino Acid Assay Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Bile Esculin Azide Agar . . . . . . . . . . . . . . . . . . . . 0525 . . . . . . . . . . . . . 67
Anaerobes - General Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 814
Biotin Assay Medium . . . . . . . . . . . . . . . . . . . . . . 0419 . . . . . . . . . . . . . 68
Anaerobic Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . 0536 . . . . . . . . . . . . . 36
Bismuth Sufite Agar . . . . . . . . . . . . . . . . . . . . . . . 0073 . . . . . . . . . . . . . 70
Antibiotic Assay Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Blood Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . 0045 . . . . . . . . . . . . . 73
Antibiotic Medium 1 . . . . . . . . . . . . . . . . . . . . . . . 0263 . . . . . . . . . . . . . 38
Blood Agar Base No. 2 . . . . . . . . . . . . . . . . . . . . . 0696 . . . . . . . . . . . . . 73
Bordet Gengou Agar Base . . . . . . . . . . . . . . . . . . . 0048 . . . . . . . . . . . . . 76
Bordetella Antigens and Antiserum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609
Bordetella Parapertussis Antiserum . . . . . . . . . . . 2310 . . . . . . . . . . . . 609
Bordetella Pertussis Antigen . . . . . . . . . . . . . . . . . 2585 . . . . . . . . . . . . 609
Bordetella Pertussis Antiserum . . . . . . . . . . . . . . . 2309 . . . . . . . . . . . . 609
Bovine Albumin 5% . . . . . . . . . . . . . . . . . . . . . . . . 0668 . . . . . . . . . . . . . 78
Antibiotic Medium 10 . . . . . . . . . . . . . . . . . . . . . . 0463 . . . . . . . . . . . . . 38
Brain Heart CC Agar . . . . . . . . . . . . . . . . . . . . . . . 0483 . . . . . . . . . . . . . 79
Brain Heart Infusion . . . . . . . . . . . . . . . . . . . . . . . 0037 . . . . . . . . . . . . . 79
Brain Heart Infusion Agar . . . . . . . . . . . . . . . . . . . 0418 . . . . . . . . . . . . . 79
Brain Heart Infusion Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Antimicrobic Selective Agents

For Culture Media Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 815
Brain Heart Infusion, Porcine . . . . . . . . . . . . . . . . 0561 . . . . . . . . . . . . . 84
Antimicrobic Vial A . . . . . . . . . . . . . . . . . . . . . . . . 3333 . . . . . . . . . . . . 129
Brain Heart Infusion w/o Dextrose . . . . . . . . . . . . 0502 . . . . . . . . . . . . . 79
Antimicrobic Vial CNV . . . . . . . . . . . . . . . . . . . . . 3260 . . . . . . . . . . . . 207
Brewer Anaerobic Agar . . . . . . . . . . . . . . . . . . . . . 0279 . . . . . . . . . . . . . 85
Antimicrobic Vial CNVT . . . . . . . . . . . . . . . . . . . 3198 . . . . . . . . . . . . 207
Brewer Thioglycollate Medium . . . . . . . . . . . . . . 0236 . . . . . . . . . . . . 502
Antimicrobic Vial K . . . . . . . . . . . . . . . . . . . . . . . . 3339 . . . . . . . . . . . . 440
Brilliant Green Agar . . . . . . . . . . . . . . . . . . . . . . . 0285 . . . . . . . . . . . . . 87
Antimicrobic Vial Oxytetracycline . . . . . . . . . . . . 3267 . . . . . . . . . . . . 360
Brilliant Green Agar Modified . . . . . . . . . . . . . . . 1880 . . . . . . . . . . . . . 89
Antimicrobic Vial P . . . . . . . . . . . . . . . . . . . . . . . . 3268 . . . . . . . 284, 440
Brilliant Green Bile 2% . . . . . . . . . . . . . . . . . . . . . 0007 . . . . . . . . . . . . . 93
APT Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0654 . . . . . . . . . . . . . 27
Brilliant Green Bile Agar . . . . . . . . . . . . . . . . . . . 0014 . . . . . . . . . . . . . 91
APT Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0655 . . . . . . . . . . . . . 27
mBrilliant Green Broth . . . . . . . . . . . . . . . . . . . . . 0494 . . . . . . . . . . . . . 94
Aseptic Commissioning Medium . . . . . . . . . . . . . 1862 . . . . . . . . . . . . . 45
Brucella Abortus Antigen (Slide) . . . . . . . . . . . . . 2909 . . . . . . . 611, 637
ATS Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1019 . . . . . . . . . . . . 587
Brucella Abortus Antigen (Tube) . . . . . . . . . . . . . 2466 . . . . . . . 611, 640
Autolyzed Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . 0229 . . . . . . . . . . . . 572
Brucella Abortus Antiserum . . . . . . . . . . . . . . . . . 2871 . . . . . . . . . . . . 611
The Difco Manual
Brain Heart Infusion w/PAB and Agar . . . . . . . . . 0499 . . . . . . . . . . . . . 79
845
Alphabetical Index
Section VII
Brucella Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0964 . . . . . . . . . . . . . 96

Brucella Antigens and Antisera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
Brucella Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0495 . . . . . . . . . . . . . 96
Product #
Pg. #
DCLS Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0759 . . . . . . . . . . . . 139

D/E Neutralizing Agar . . . . . . . . . . . . . . . . . . . . . . 0686 . . . . . . . . . . . . 140
Brucella Melitensis Antigen (Slide) . . . . . . . . . . . 2916 . . . . . . . . . . . . 611
D/E Neutralizing Broth . . . . . . . . . . . . . . . . . . . . . 0819 . . . . . . . . . . . . 140
Brucella Suis Antigen (Slide) . . . . . . . . . . . . . . . . 2915 . . . . . . . . . . . . 611
DNase Test Agar . . . . . . . . . . . . . . . . . . . . . . . . . . 0632 . . . . . . . . . . . . 143
Bryant and Burkey Medium . . . . . . . . . . . . . . . . . 0645 . . . . . . . . . . . . . 97
DNase Test Agar w/Methyl Green . . . . . . . . . . . . 0220 . . . . . . . . . . . . 143
Buffered Peptone Water . . . . . . . . . . . . . . . . . . . . . 1810 . . . . . . . . . . . . . 99
DRBC Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0587 . . . . . . . . . . . . 145
Bushnell-Haas Broth . . . . . . . . . . . . . . . . . . . . . . . 0578 . . . . . . . . . . . . 100
Dairy Product Testing - Products

and Applications Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 817
Decarboxylase Base Moeller . . . . . . . . . . . . . . . . . 0890 . . . . . . . . . . . . 147
Product #
Pg. #
CLED Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0971 . . . . . . . . . . . . 102

Campylobacter Agar Base . . . . . . . . . . . . . . . . . . . 1820 . . . . . . . . . . . . 103
Campylobacter Agar Kit Blaser . . . . . . . . . . . . . . 3279 . . . . . . . . . . . . 103
Campylobacter Agar Kit Skirrow . . . . . . . . . . . . . 3280 . . . . . . . . . . . . 103
Candida Albicans Antiserum . . . . . . . . . . . . . . . . . 2281 . . . . . . . . . . . . 615
Candida BCG Agar Base . . . . . . . . . . . . . . . . . . . . 0835 . . . . . . . . . . . . 106
Candida Isolation Agar . . . . . . . . . . . . . . . . . . . . . 0507 . . . . . . . . . . . . 108
Cary-Blair Transport Medium . . . . . . . . . . . . . . . . 0505 . . . . . . . . . . . . 515
Casamino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . 0230 . . . . . . . . . . . . 110
Casamino Acids, Technical . . . . . . . . . . . . . . . . . . 0231 . . . . . . . . . . . . 110
Decarboxylase Differential Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

Decarboxylase Medium Base . . . . . . . . . . . . . . . . 0872 . . . . . . . . . . . . 147
Demi-Fraser Broth Base . . . . . . . . . . . . . . . . . . . . 0653 . . . . . . . . . . . . 150
Desoxycholate Agar . . . . . . . . . . . . . . . . . . . . . . . . 0273 . . . . . . . . . . . . 152
Desoxycholate Citrate Agar . . . . . . . . . . . . . . . . . 0274 . . . . . . . . . . . . 154
Desoxycholate Lactose Agar . . . . . . . . . . . . . . . . . 0420 . . . . . . . . . . . . 155
Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0155 . . . . . . . . . . . . 276
Dextrose Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0067 . . . . . . . . . . . . 157
Dextrose Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0063 . . . . . . . . . . . . 157
Dextrose Starch Agar . . . . . . . . . . . . . . . . . . . . . . . 0066 . . . . . . . . . . . . 158
Dextrose Tryptone Agar . . . . . . . . . . . . . . . . . . . . 0080 . . . . . . . . . . . . 160
Casein Digest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0116 . . . . . . . . . . . . 112
Differential Reinforced Clostridial Agar . . . . . . . 0641 . . . . . . . . . . . . 161
Casitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0259 . . . . . . . . . . . . 113
Dubos Albumin Broth . . . . . . . . . . . . . . . . . . . . . . 1022 . . . . . . . . . . . . 163
Casman Medium Base . . . . . . . . . . . . . . . . . . . . . . 0290 . . . . . . . . . . . . 114
Dubos Broth Base . . . . . . . . . . . . . . . . . . . . . . . . . 0385 . . . . . . . . . . . . 163
Cetrimide Agar Base . . . . . . . . . . . . . . . . . . . . . . . 0854 . . . . . . . . . . . . 116
Dubos Medium Albumin . . . . . . . . . . . . . . . . . . . . 0309 . . . . . . . . . . . . 163
Chapman Stone Medium . . . . . . . . . . . . . . . . . . . . 0313 . . . . . . . . . . . . 118
Dubos Oleic Agar Base . . . . . . . . . . . . . . . . . . . . . 0373 . . . . . . . . . . . . 163
Chapman Tellurite Solution 1% . . . . . . . . . . . . . . 0299 . . . 232, 324, 489
Dubos Oleic Albumin Complex . . . . . . . . . . . . . . 0375 . . . . . . . . . . . . 163
Charcoal Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0894 . . . . . . . . . . . . 119

Choline Assay Medium . . . . . . . . . . . . . . . . . . . . . 0460 . . . . . . . . . . . . 121
Clostridium Difficile
Antimicrobic Supplement CC . . . . . . . . . . . . . . 3194 . . . . . . . . . . . . . 79
Product #
Pg. #
m E Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0333 . . . . . . . . . . . . 165
Coagulase Plasma . . . . . . . . . . . . . . . . . . . . . . . . . 0286 . . . . . . . . . . . . 617
E. Coli Antisera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 621
Coagulase Plasma EDTA . . . . . . . . . . . . . . . . . . . . 0803 . . . . . . . . . . . . 617
E. Coli H Antiserum H7 . . . . . . . . . . . . . . . . . . . . 2159 . . . . . . . . . . . . 621
Columbia Blood Agar Base . . . . . . . . . . . . . . . . . . 0792 . . . . . . . . . . . . 123
E. Coli O Antiserum O157 . . . . . . . . . . . . . . . . . . 2970 . . . . . . . . . . . . 621
Columbia Blood Agar Base EH . . . . . . . . . . . . . . 0790 . . . . . . . . . . . . 123
EC Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0314 . . . . . . . . . . . . 168
Columbia Blood Agar Base No. 2 . . . . . . . . . . . . . 0793 . . . . . . . . . . . . 123
EC Medium with MUG . . . . . . . . . . . . . . . . . . . . . 0022 . . . . . . . . . . . . 170
Columbia Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . 0944 . . . . . . . . . . . . 125
EE Broth Mossel . . . . . . . . . . . . . . . . . . . . . . . . . . 0566 . . . . . . . . . . . . 171
Columbia CNA Agar . . . . . . . . . . . . . . . . . . . . . . . 0867 . . . . . . . . . . . . 127
EMB Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0076 . . . . . . . . . . . . 173
Cooke Rose Bengal Agar . . . . . . . . . . . . . . . . . . . . 0703 . . . . . . . . . . . . 129
EVA Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0606 . . . . . . . . . . . . 175
Cooked Meat Medium . . . . . . . . . . . . . . . . . . . . . . 0267 . . . . . . . . . . . . 130
EY Tellurite Enrichment . . . . . . . . . . . . . . . . . . . . 0779 . . . . . . . . . . . . . 58
Corn Meal Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . 0386 . . . . . . . . . . . . 132
Egg Meat Medium . . . . . . . . . . . . . . . . . . . . . . . . . 0042 . . . . . . . . . . . . 176
Cosmetic Testing Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 816
Egg Yolk Enrichment 50% . . . . . . . . . . . . . . . . . . 3347 . . . . . . . 307, 440
Cystine Assay Medium . . . . . . . . . . . . . . . . . . . . . 0467 . . . . . . . . . . . . . 33
Elliker Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0974 . . . . . . . . . . . . 177
Cystine Heart Agar . . . . . . . . . . . . . . . . . . . . . . . . 0047 . . . . . . . . . . . . 134
Emerson YpSs Agar . . . . . . . . . . . . . . . . . . . . . . . . 0739 . . . . . . . . . . . . 179
Cystine Tryptic Agar . . . . . . . . . . . . . . . . . . . . . . . 0523 . . . . . . . . . . . . 135
Endo Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0006 . . . . . . . . . . . . 180
Czapek-Dox Broth . . . . . . . . . . . . . . . . . . . . . . . . . 0338 . . . . . . . . . . . . 137
m Endo Agar LES . . . . . . . . . . . . . . . . . . . . . . . . . 0736 . . . . . . . . . . . . 181
Czapek Solution Agar . . . . . . . . . . . . . . . . . . . . . . 0339 . . . . . . . . . . . . 137
m Endo Broth MF . . . . . . . . . . . . . . . . . . . . . . . . 0749 . . . . . . . . . . . . 183

Enteric Fermentation Base . . . . . . . . . . . . . . . . . . 1828 . . . . . . . . . . . . 185
846
The Difco Manual
Section VII
Alphabetical Index
m Enterococcus Agar . . . . . . . . . . . . . . . . . . . . . . . 0746 . . . . . . . . . . . . 187
Francisella Tularensis Antiserum . . . . . . . . . . . . . 2241 . . . . . . . . . . . . 642
Environmental Sampling and

Disinfectant Testing Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 819
Fraser Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
Escherichia coli - Products

Fraser Broth Supplement . . . . . . . . . . . . . . . . . . . . 0211 . . . . . . . 150, 204
Esculin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0158 . . . . . . . . . . . . . 65
Fraser Broth Base . . . . . . . . . . . . . . . . . . . . . . . . . 0219 . . . . . . . . . . . . 204
Esculin Iron Agar . . . . . . . . . . . . . . . . . . . . . . . . . . 0488 . . . . . . . . . . . . 165
Eugon Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0589 . . . . . . . . . . . . 189
GC Medium Base . . . . . . . . . . . . . . . . . . . . . . . . . . 0289 . . . . . . . . . . . . 207
Eugon Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0590 . . . . . . . . . . . . 189
GN Broth, Hajna . . . . . . . . . . . . . . . . . . . . . . . . . . 0486 . . . . . . . . . . . . 211
Product #
Pg. #
Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0143 . . . . . . . . . . . . 212
Product #
Pg. #
FA Bordetella Parapertussis . . . . . . . . . . . . . . . . . 2378 . . . . . . . . . . . . 623
Gelatone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0657 . . . . . . . . . . . . 212

Giolitti-Cantoni Broth Base . . . . . . . . . . . . . . . . . 1809 . . . . . . . . . . . . 214
FA Bordetella Pertussis . . . . . . . . . . . . . . . . . . . . . 2359 . . . . . . . . . . . . 623
Glycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0282 . . . . 31, 117, 218

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315, 414, 493
FA Buffer, Dried . . . . . . . . . . . . . . . . . . . . . . . . . . 2314 . . . . . . . 626, 630
3-Step Gram Safranin-S . . . . . . . . . . . . . . . . . . . . 3335 . . . . . . . . . . . . 599
FA Human Globulin Antiglobulin (Rabbit) . . . . . 2449 . . . . . . . . . . . . 630
3-Step Gram Safranin-T . . . . . . . . . . . . . . . . . . . . 3341 . . . . . . . . . . . . 599
FA Mounting Fluid pH 7.2 . . . . . . . . . . . . . . . . . . 2329 . . . . . . . 626, 630
3-Step Gram Stain Set-S . . . . . . . . . . . . . . . . . . . . 3334 . . . . . . . . . . . . 598
FA Mounting Fluid pH 9 . . . . . . . . . . . . . . . . . . . . 3340 . . . . . . . 626, 628
3-Step Gram Stain Set-T . . . . . . . . . . . . . . . . . . . . 3337 . . . . . . . . . . . . 598
FA Product Accessories and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . 626
Gram Basic Fuchsin . . . . . . . . . . . . . . . . . . . . . . . . 3343 . . . . . . . . . . . . 599
FA Streptococcus Group A . . . . . . . . . . . . . . . . . . 2318 . . . . . . . . . . . . 628
Gram Crystal Violet . . . . . . . . . . . . . . . . . . . . . . . . 3329 . . . . . . . . . . . . 599
m FC Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0677 . . . . . . . . . . . . 190
Gram Decolorizer . . . . . . . . . . . . . . . . . . . . . . . . . 3330 . . . . . . . . . . . . 599
m FC Basal Medium . . . . . . . . . . . . . . . . . . . . . . . 0698 . . . . . . . . . . . . 193
Gram Iodine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3331 . . . . . . . . . . . . 599
m FC Broth Base . . . . . . . . . . . . . . . . . . . . . . . . . . 0883 . . . . . . . . . . . . 190
Gram Safranin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3332 . . . . . . . . . . . . 599
FTA-ABS Test Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 630
Gram Stain Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3328 . . . . . . . . . . . . 598
FTA Serum Non-Reactive . . . . . . . . . . . . . . . . . . . 2440 . . . . . . . . . . . . 630
Gram Stain Set (with Stabilized Iodine) . . . . . . . . 3338 . . . . . . . . . . . . 598
FTA Serum Reactive . . . . . . . . . . . . . . . . . . . . . . . 2439 . . . . . . . . . . . . 630
Gram Stain Sets and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 598
FTA Sorbent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3259 . . . . . . . . . . . . 630

FTA Sorbent Control . . . . . . . . . . . . . . . . . . . . . . . 3266 . . . . . . . . . . . . 630
Febrile Antigen Set . . . . . . . . . . . . . . . . . . . . . . . . 2407 . . . . . . . . . . . . 637
Febrile Negative Control . . . . . . . . . . . . . . . . . . . . 3239 . . . 611, 637, 642
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 658, 659, 663, 804
Product #
Pg. #
HC Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0685 . . . . . . . . . . . . 215

m HPC Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0752 . . . . . . . . . . . . 217
Febrile Positive Control Polyvalent . . . . . . . . . . . 3238 . . . . . . . 637, 804
Haemophilus Influenzae Antisera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 646
Fermentation Products Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 819
Haemophilus Influenzae Antiserum Poly . . . . . . . 2237 . . . . . . . . . . . . 646
Fildes Enrichment . . . . . . . . . . . . . . . . . . . . . . . . . 0349 . . . . . . . . . . . . 194
Haemophilus Influenzae Antiserum Type a . . . . . 2250 . . . . . . . . . . . . 646
Fish Peptone No. 1 . . . . . . . . . . . . . . . . . . . . . . . . . 0551 . . . . . . . . . . . . 195
Haemophilus Influenzae Antiserum Type b . . . . . 2236 . . . . . . . . . . . . 646
Fletcher Medium Base . . . . . . . . . . . . . . . . . . . . . . 0987 . . . . . . . . . . . . 197
Haemophilus Influenzae Antiserum Type c . . . . . 2789 . . . . . . . . . . . . 646
Fluid Sabouraud Medium . . . . . . . . . . . . . . . . . . . 0642 . . . . . . . . . . . . 448
Haemophilus Influenzae Antiserum Type d . . . . . 2790 . . . . . . . . . . . . 646
Fluid Thioglycollate Medium . . . . . . . . . . . . . . . . 0256 . . . . . . . . . . . . 502
Haemophilus Influenzae Antiserum Type e . . . . . 2791 . . . . . . . . . . . . 646

w/Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . 0697 . . . . . . . . . . . . 502
Haemophilus Influenzae Antiserum Type f . . . . . 2792 . . . . . . . . . . . . 646
Fluid Thioglycollate Medium w/K Agar . . . . . . . . 0607 . . . . . . . . . . . . 502
Heart Infusion Broth . . . . . . . . . . . . . . . . . . . . . . . 0038 . . . . . . . . . . . . 219
Folic Acid Assay Medium . . . . . . . . . . . . . . . . . . . 0318 . . . . . . . . . . . . 200
Hektoen Enteric Agar . . . . . . . . . . . . . . . . . . . . . . 0853 . . . . . . . . . . . . 221
Folic Acid Casei Medium . . . . . . . . . . . . . . . . . . . 0822 . . . . . . . . . . . . 202
Hemoglobin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0136 . . . 134, 207, 223
Folic AOAC Medium . . . . . . . . . . . . . . . . . . . . . . . 0967 . . . . . . . . . . . . 198
Hemoglobin Solution 2% . . . . . . . . . . . . . . . . . . . 3248 . . . 135, 208, 408
Folic Buffer A, Dried . . . . . . . . . . . . . . . . . . . . . . . 3246 . . . . . . . . . . . . 202
Horse Serum, Desiccated . . . . . . . . . . . . . . . . . . . 0261 . . . . . . . . . . . . 224
Food and Beverage Testing - Products

HYcheck . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588
Francisella Tularensis Antigen (Slide) . . . . . . . . . 2240 . . . . . . . . . . . . 642
HYcheck for Disinfection Control . . . . . . . . . . . . 9039 . . . . . . . . . . . . 588
Francisella Tularensis Antigen (Tube) . . . . . . . . . 2251 . . . . . . . . . . . . 642
HYcheck for Enterobacteriaceae . . . . . . . . . . . . . . 9037 . . . . . . . . . . . . 588
The Difco Manual
Heart Infusion Agar . . . . . . . . . . . . . . . . . . . . . . . . 0044 . . . . . . . . . . . . 219
HYcheck D/E Neutralizing Agar . . . . . . . . . . . . . 9041 . . . . . . . . . . . . 588
847
Alphabetical Index
Section VII
HYcheck for Total Count . . . . . . . . . . . . . . . . . . . 9053 . . . . . . . . . . . . 588
Listeria O Antigen Type 4 (Slide) . . . . . . . . . . . . . 2304 . . . . . . . . . . . . 648
HYcheck for Yeasts and Molds . . . . . . . . . . . . . . . 9038 . . . . . . . . . . . . 588
Listeria O Antigen Type 4 (Tube) . . . . . . . . . . . . . 2306 . . . . . . . . . . . . 648
HYcheck for Yeasts and Molds with TTC . . . . . . 9046 . . . . . . . . . . . . 588
Listeria O Antiserum Poly . . . . . . . . . . . . . . . . . . . 2302 . . . . . . . . . . . . 648
HYcheck Plate Count Agar with TTC . . . . . . . . . 9045 . . . . . . . . . . . . 588
Listeria O Antiserum Type 1 . . . . . . . . . . . . . . . . . 2300 . . . . . . . . . . . . 648

Listeria O Antiserum Type 4 . . . . . . . . . . . . . . . . . 2301 . . . . . . . . . . . . 648
Product #
Pg. #
ISP Medium 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0769 . . . . . . . . . . . . 225

ISP Medium 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0770 . . . . . . . . . . . . 225
ISP Medium 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0772 . . . . . . . . . . . . 225
Inositol Assay Medium . . . . . . . . . . . . . . . . . . . . . 0995 . . . . . . . . . . . . 226
Litmus Milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0107 . . . . . . . . . . . . 261

Littman Oxgall Agar . . . . . . . . . . . . . . . . . . . . . . . 0294 . . . . . . . . . . . . 262
Liver Infusion Agar . . . . . . . . . . . . . . . . . . . . . . . . 0052 . . . . . . . . . . . . 264
Liver Infusion Broth . . . . . . . . . . . . . . . . . . . . . . . 0269 . . . . . . . . . . . . 264
Liver Veal Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . 0059 . . . . . . . . . . . . 265
Loeffler Blood Serum . . . . . . . . . . . . . . . . . . . . . . 0070 . . . . . . . . . . . . 266
Lowenstein Medium Base . . . . . . . . . . . . . . . . . . . 0444 . . . . . . . . . . . . 268
Product #
Pg. #
Lowenstein Medium w/5% NaCl . . . . . . . . . . . . . 1423 . . . . . . . . . . . . 268
KF Streptococcus Agar . . . . . . . . . . . . . . . . . . . . . 0496 . . . . . . . . . . . . 228
Lowenstein Medium, Gruft . . . . . . . . . . . . . . . . . . 1417 . . . . . . . . . . . . 268
KF Streptococcus Broth . . . . . . . . . . . . . . . . . . . . 0997 . . . . . . . . . . . . 230
Lowenstein Medium, Jensen . . . . . . . . . . . . . . . . . 1017 . . . . . . . . . . . . 268
KL Antitoxin Strips . . . . . . . . . . . . . . . . . . . . . . . . 3101 . . . . . . . . . . . . 232
Lowenstein Medium, Jensen Deeps . . . . . . . . . . . 1289 . . . . . . . . . . . . 268
KL Virulence Agar . . . . . . . . . . . . . . . . . . . . . . . . . 0985 . . . . . . . . . . . . 232
Luria Agar Base, Miller . . . . . . . . . . . . . . . . . . . . . 0413 . . . . . . . . . . . . 271
KL Virulence Enrichment . . . . . . . . . . . . . . . . . . . 0986 . . . . . . . . . . . . 232
Luria Broth Base, Miller . . . . . . . . . . . . . . . . . . . . 0414 . . . . . . . . . . . . 272
Kligler Iron Agar . . . . . . . . . . . . . . . . . . . . . . . . . . 0086 . . . . . . . . . . . . 234
Lysine Assay Medium . . . . . . . . . . . . . . . . . . . . . . 0422 . . . . . . . . . . . . . 33
Koser Citrate Medium . . . . . . . . . . . . . . . . . . . . . . 0015 . . . . . . . . . . . . 236
Lysine Decarboxylase Broth . . . . . . . . . . . . . . . . . 0215 . . . . . . . . . . . . 147

Lysine Iron Agar . . . . . . . . . . . . . . . . . . . . . . . . . . 0849 . . . . . . . . . . . . 273
Product #
Pg. #
LB Agar, Lennox . . . . . . . . . . . . . . . . . . . . . . . . . . 0401 . . . . . . . . . . . . 238
Lysine Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . 1894 . . . . . . . . . . . . 274
LB Agar, Miller . . . . . . . . . . . . . . . . . . . . . . . . . . . 0445 . . . . . . . . . . . . 239
LB Broth, Lennox . . . . . . . . . . . . . . . . . . . . . . . . . 0402 . . . . . . . . . . . . 240
M Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0940 . . . . . . . . . . . . 279
LB Broth, Miller . . . . . . . . . . . . . . . . . . . . . . . . . . 0446 . . . . . . . . . . . . 241
M17 Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1857 . . . . . . . . . . . . 278
LPM Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . 0221 . . . . . . . . . . . . 242
M17 Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1856 . . . . . . . . . . . . 278
Lactobacilli Agar AOAC . . . . . . . . . . . . . . . . . . . . 0900 . . . . . . . . . . . . 244
M9 Minimal Salts, 5x . . . . . . . . . . . . . . . . . . . . . . 0485 . . . . . . . . . . . . 277
Lactobacilli Broth AOAC . . . . . . . . . . . . . . . . . . . 0901 . . . . . . . . . . . . 244
M9CA Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . 1454 . . . . . . . . . . . . 276
Lactobacilli MRS Agar . . . . . . . . . . . . . . . . . . . . . 0882 . . . . . . . . . . . . 246
MIL Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1804 . . . . . . . . . . . . 281
Lactobacilli MRS Broth . . . . . . . . . . . . . . . . . . . . 0881 . . . . . . . . . . . . 246
MIO Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0735 . . . . . . . . . . . . 283
Lactose Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0004 . . . . . . . . . . . . 248

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9070 . . . . . . . . . . . . 248
MR-VP Medium . . . . . . . . . . . . . . . . . . . . . . . . . . 0016 . . . . . . . . . . . . 308
Lactose Peptone Broth . . . . . . . . . . . . . . . . . . . . . . 0665 . . . . . . . . . . . . 250
MacConkey Agar . . . . . . . . . . . . . . . . . . . . . . . . . . 0075 . . . . . . . . . . . . 288
Lauryl Tryptose Broth . . . . . . . . . . . . . . . . . . . . . . 0241 . . . . . . . 181, 251
MacConkey Agar Base . . . . . . . . . . . . . . . . . . . . . 0818 . . . . . . . . . . . . 288
Leptospira Enrichment EMJH . . . . . . . . . . . . . . . . 0795 . . . . . . . . . . . . 253
MacConkey Agar CS . . . . . . . . . . . . . . . . . . . . . . . 1818 . . . . . . . . . . . . 288
Leptospira Medium Base EMJH . . . . . . . . . . . . . . 0794 . . . . . . . . . . . . 253
MacConkey Agar w/o CV . . . . . . . . . . . . . . . . . . . 0470 . . . . . . . . . . . . 288
Leptospira Medium EMJH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
MacConkey Agar w/o Salt . . . . . . . . . . . . . . . . . . . 0331 . . . . . . . . . . . . 288
Letheen Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0680 . . . . . . . . . . . . 255
MacConkey Broth . . . . . . . . . . . . . . . . . . . . . . . . . 0020 . . . . . . . . . . . . 286
Letheen Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0681 . . . . . . . . . . . . 255
MacConkey Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
Levine EMB Agar . . . . . . . . . . . . . . . . . . . . . . . . . 0005 . . . . . . . . . . . . 257
MacConkey Sorbitol Agar . . . . . . . . . . . . . . . . . . . 0079 . . . . . . . . . . . . 292
Lima Bean Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . 0117 . . . . . . . . . . . . 258
Malonate Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . 0395 . . . . . . . . . . . . 294
Lipase Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0431 . . . . . . . . . . . . 463
Malonate Broth Modified . . . . . . . . . . . . . . . . . . . 0569 . . . . . . . . . . . . 295
Listeria Antigens and Antisera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 648
Malt Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0024 . . . . . . . . . . . . 297
Listeria Enrichment Broth . . . . . . . . . . . . . . . . . . . 0222 . . . . . . . . . . . . 259
Malt Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0186 . . . . . . . . . . . . 298
Listeria O Antigen Type 1 (Slide) . . . . . . . . . . . . . 2303 . . . . . . . . . . . . 648
Malt Extract Agar . . . . . . . . . . . . . . . . . . . . . . . . . 0112 . . . . . . . . . . . . 299
Listeria O Antigen Type 1 (Tube) . . . . . . . . . . . . . 2305 . . . . . . . . . . . . 648
Malt Extract Broth . . . . . . . . . . . . . . . . . . . . . . . . . 0113 . . . . . . . . . . . . 299
848
Product #
Pg. #
MYP Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0810 . . . . . . . . . . . . 284
The Difco Manual
Section VII
Mannitol Salt Agar . . . . . . . . . . . . . . . . . . . . . . . . . 0306 . . . . . . . . . . . . 300

Marine Agar 2216 . . . . . . . . . . . . . . . . . . . . . . . . . 0979 . . . . . . . . . . . . 302
Marine Broth 2216 . . . . . . . . . . . . . . . . . . . . . . . . . 0791 . . . . . . . . . . . . 302
Maximum Recovey Diluent . . . . . . . . . . . . . . . . . . 1897 . . . . . . . . . . . . 304
McBride Listeria Agar . . . . . . . . . . . . . . . . . . . . . . 0922 . . . . . . . . . . . . 305
McClung Toabe Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
McClung Toabe Agar Base . . . . . . . . . . . . . . . . . . 0941 . . . . . . . . . . . . 307
McFarland Standard Preparation Guide . . . . . . . . . . . . . . . . . . . . . . . . . . 824
Methionine Assay Medium . . . . . . . . . . . . . . . . . . 0423 . . . . . . . . . . . . . 33
Methyl Red and Voges-Proskauer Tests . . . . . . . . . . . . . . . . . . . . . . . . . . 308
Micro Assay Culture Agar . . . . . . . . . . . . . . . . . . . 0319 . . . . . . . . . . . . 312
Micro Inoculum Broth . . . . . . . . . . . . . . . . . . . . . . 0320 . . . . . . . . . . . . 312
Microbial Content Test Agar . . . . . . . . . . . . . . . . . 0553 . . . . . . . . . . . . 313
Middlebrook 7H10 Agar . . . . . . . . . . . . . . . . . . . . 0627 . . . . . . . . . . . . 315
Middlebrook 7H9 Broth . . . . . . . . . . . . . . . . . . . . 0713 . . . . . . . . . . . . 315
Alphabetical Index
Product #
Pg. #
NIH Thioglycollate Broth . . . . . . . . . . . . . . . . . . . 0257 . . . . . . . . . . . . 502

Neisseria Meningitidis Antisera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 652
Neisseria Meningitidis Antiserum Group A . . . . . 2228 . . . . . . . . . . . . 652
Neisseria Meningitidis Antiserum Group B . . . . . 2229 . . . . . . . . . . . . 652
Neisseria Meningitidis Antiserum Group C . . . . . 2230 . . . . . . . . . . . . 652
Neisseria Meningitidis Antiserum Group D . . . . . 2231 . . . . . . . . . . . . 652
Neisseria Meningitidis Antiserum Group W135 . 2253 . . . . . . . . . . . . 652
Neisseria Meningitidis Antiserum Group X . . . . . 2880 . . . . . . . . . . . . 652
Neisseria Meningitidis Antiserum Group Y . . . . . 2881 . . . . . . . . . . . . 652
Neisseria Meningitidis Antiserum Group Z . . . . . 2891 . . . . . . . . . . . . 652
Neisseria Meningitidis Antiserum Group Z . . . . 2252 . . . . . . . . . . . . 652
(Groups A, B, C, D) . . . . . . . . . . . . . . . . . . . . . . 2232 . . . . . . . . . . . . 652
Middlebrook ADC Enrichment . . . . . . . . . . . . . . . 0714 . . . . . . . . . . . . 315

(Groups X, Y, Z) . . . . . . . . . . . . . . . . . . . . . . . . . 2910 . . . . . . . . . . . . 652
Middlebrook OADC Enrichment . . . . . . . . . . . . . 0722 . . . . . . . . . . . . 315
Neopeptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0119 . . . . . . . . . . . . 344
Middlebrook OADC Enrichment w/WR 1339 . . . 0801 . . . . . . . . . . . . 315
Neutralizing Buffer . . . . . . . . . . . . . . . . . . . . . . . . 0362 . . . . . . . . . . . . 345
Milk Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1859 . . . . . . . . . . . . 318
Niacin Assay Medium . . . . . . . . . . . . . . . . . . . . . . 0322 . . . . . . . . . . . . 346
Mineral Oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6663 . . . . . . . . . . . . 359
Nitrate Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0268 . . . . . . . . . . . . 348
Minerals Modified Glutamate Broth . . . . . . . . . . . 1850 . . . . . . . . . . . . 320
Novobiocin Antimicrobic Supplement . . . . . . . . . 3197 . . . . . . . 326, 424
Minimal Agar Davis . . . . . . . . . . . . . . . . . . . . . . . 0544 . . . . . . . . . . . . 322
Nutrient Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0001 . . . . . . . . . . . . 349
Minimal Broth Davis w/o Dextrose . . . . . . . . . . . 0756 . . . . . . . . . . . . 322
Nutrient Agar 1.5% . . . . . . . . . . . . . . . . . . . . . . . . 0069 . . . . . . . . . . . . 351
Mitis Salivarius Agar . . . . . . . . . . . . . . . . . . . . . . . 0298 . . . . . . . . . . . . 324
Nutrient Agar with MUG . . . . . . . . . . . . . . . . . . . . 0023 . . . . . . . . . . . . 352
Modified Buffered Peptone Water . . . . . . . . . . . . . 1833 . . . . . . . . . . . . . 99
Nutrient Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0003 . . . . . . . . . . . . 354
Modified EC Medium . . . . . . . . . . . . . . . . . . . . . . 0340 . . . . . . . . . . . . 326
Nutrient Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . 0011 . . . . . . . . . . . . 355
Modified Letheen Agar . . . . . . . . . . . . . . . . . . . . . 0631 . . . . . . . . . . . . 328
NZCYM Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0404 . . . . . . . . . . . . 356
Modified Letheen Broth . . . . . . . . . . . . . . . . . . . . 0630 . . . . . . . . . . . . 328
NZM Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0435 . . . . . . . . . . . . 356
Modified Listeria Enrichment Broth . . . . . . . . . . . 0205 . . . . . . . . . . . . 330
NZYM Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0415 . . . . . . . . . . . . 356
Modified Oxford Antimicrobic Supplement . . . . . 0218 . . . . . . . . . . . . 364

Molecular Genetics - Media and
Ingredients Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 824
Motility GI Medium . . . . . . . . . . . . . . . . . . . . . . . 0869 . . . . . . . . . . . . 332
OF Basal Medium . . . . . . . . . . . . . . . . . . . . . . . . . 0688 . . . . . . . . . . . . 358
Motility Medium S . . . . . . . . . . . . . . . . . . . . . . . . 0761 . . . . . . . . . . . . 333
OGYE Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360
Motility Test Medium . . . . . . . . . . . . . . . . . . . . . . 0105 . . . . . . . . . . . . 335
OGYE Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . 1811 . . . . . . . . . . . . 360
Moxalactam Antimicrobic Supplement . . . . . . . . 0216 . . . . . . . . . . . . 242
Oatmeal Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0552 . . . . . . . . . . . . 361
Mueller Hinton Broth . . . . . . . . . . . . . . . . . . . . . . 0757 . . . . . . . . . . . . 336
Orange Serum Agar . . . . . . . . . . . . . . . . . . . . . . . . 0521 . . . . . . . . . . . . 362
Mueller Hinton Medium . . . . . . . . . . . . . . . . . . . . 0252 . . . . . . . . . . . . 336
Orange Serum Broth Concentrate 10X . . . . . . . . . 0518 . . . . . . . . . . . . 362
Muller Kauffmann Tetrathionate Broth Base . . . . 1853 . . . . . . . . . . . . 339
Oxford Antimicrobic Supplement . . . . . . . . . . . . . 0214 . . . . . . . . . . . . 364
Mycobacteria 7H11 Agar . . . . . . . . . . . . . . . . . . . 0838 . . . . . . . . . . . . 315
Oxford Medium Base . . . . . . . . . . . . . . . . . . . . . . 0225 . . . . . . . . . . . . 364
Product #
Pg. #
Mycobacteria Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 825

Mycobiotic Agar . . . . . . . . . . . . . . . . . . . . . . . . . . 0689 . . . . . . . . . . . . 341
Mycological Agar . . . . . . . . . . . . . . . . . . . . . . . . . 0405 . . . . . . . . . . . . 342
Product #
Pg. #
Mycological Agar w/Low pH . . . . . . . . . . . . . . . . 0305 . . . . . . . . . . . . 342
PALCAM Antimicrobic Supplement . . . . . . . . . . 0637 . . . . . . . . . . . . 367
Mycological Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
PALCAM Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
Mycology Guides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 826, 827
PALCAM Medium Base . . . . . . . . . . . . . . . . . . . . 0636 . . . . . . . . . . . . 367
Mycoplasma Supplement . . . . . . . . . . . . . . . . . . . 0836 . . . . . . . . . . . . 372
PKU Test Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0980 . . . . . . . . . . . . 369
Mycoplasma Supplement S . . . . . . . . . . . . . . . . . . 0837 . . . . . . . . . . . . 372
PKU Test Agar w/o Thienylalanine . . . . . . . . . . . 0474 . . . . . . . . . . . . 369
The Difco Manual
849
Alphabetical Index
Section VII
PPLO Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0412 . . . . . . . . . . . . 372
Proteus OX19 Antiserum . . . . . . . . . . . . . . . . . . . . 2235 . . . . . . . . . . . . 655
PPLO Broth w/o CV . . . . . . . . . . . . . . . . . . . . . . . 0554 . . . . . . . . . . . . 372
Proteus OX2 Antigen (Slide) . . . . . . . . . . . . . . . . . 2243 . . . . . . . . . . . . 655
PPLO Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
Proteus OX2 Antigen (Tube) . . . . . . . . . . . . . . . . . 2248 . . . . . . . . . . . . 655
Pagano Levin Base . . . . . . . . . . . . . . . . . . . . . . . . . 0141 . . . . . . . . . . . . 374
Proteus OX2 Antiserum . . . . . . . . . . . . . . . . . . . . . 2245 . . . . . . . . . . . . 655
Panthenol Assay Medium . . . . . . . . . . . . . . . . . . . 0994 . . . . . . . . . . . . 376
Proteus OXK Antigen (Slide) . . . . . . . . . . . . . . . . 2244 . . . . . . . . . . . . 655
Panthenol Supplement . . . . . . . . . . . . . . . . . . . . . . 0212 . . . . . . . . . . . . 376
Proteus OXK Antigen (Tube) . . . . . . . . . . . . . . . . 2249 . . . . . . . . . . . . 655
Pantothenate Assay Medium . . . . . . . . . . . . . . . . . 0604 . . . . . . . . . . . . 378
Proteus OXK Antiserum . . . . . . . . . . . . . . . . . . . . 2246 . . . . . . . . . . . . 655
Pantothenate Medium AOAC USP . . . . . . . . . . . . 0816 . . . . . . . . . . . . 380
Pseudomonas Agar F . . . . . . . . . . . . . . . . . . . . . . . 0448 . . . . . . . . . . . . 412
Pasco Panel Contents - Selection Guide . . . . . . . . . . . . . . . . . . . . . . . . . . 828
Pseudomonas Agar Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
Peptamin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0905 . . . . . . . . . . . . 382
Pseudomonas Agar P . . . . . . . . . . . . . . . . . . . . . . . 0449 . . . . . . . . . . . . 412
Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0118 . . . . . . . . . . . . 383
Pseudomonas Isolation Agar . . . . . . . . . . . . . . . . . 0927 . . . . . . . . . . . . 414
Peptone Bacteriological Technical . . . . . . . . . . . . 0885 . . . . . . . . . . . . 383
Purple Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . 0228 . . . . . . . . . . . . 415
Peptone Iron Agar . . . . . . . . . . . . . . . . . . . . . . . . . 0089 . . . . . . . . . . . . 385
Purple Broth Base . . . . . . . . . . . . . . . . . . . . . . . . . 0227 . . . . . . . . . . . . 415
Peptone Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1807 . . . . . . . . . . . . 386
Purple Lactose Agar . . . . . . . . . . . . . . . . . . . . . . . . 0082 . . . . . . . . . . . . 418
Peptones & Hydrolysates Selection Guide . . . . . . . . . . . . . . . . . . . . . . . . 829
Pyridoxine Y Medium . . . . . . . . . . . . . . . . . . . . . . 0951 . . . . . . . . . . . . 419
Petragnani Medium . . . . . . . . . . . . . . . . . . . . . . . . 1010 . . . . . . . . . . . . 592

Pharmaceutical Testing - Products and
Applications Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 831
Phenol Red Agar Base . . . . . . . . . . . . . . . . . . . . . . 0098 . . . . . . . . . . . . 388
Phenol Red Agar Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
Phenol Red Broth Base . . . . . . . . . . . . . . . . . . . . . 0092 . . . . . . . . . . . . 390
Product #
Pg. #
QC Antigen Alkalescens-Dispar Group 1 . . . . . . . 2116 . . . . . . . . . . . . 662

QC Antigen Salmonella O Group A . . . . . . . . . . . 2130 . . . . . . . . . . . . 659
QC Antigen Salmonella O Group B . . . . . . . . . . . 2131 . . . . . . . . . . . . 659
Phenol Red Carbohydrate Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
QC Antigen Salmonella O Group C1 . . . . . . . . . . 2132 . . . . . . . . . . . . 659
Phenol Red Dextrose Broth . . . . . . . . . . . . . . . . . . 0093 . . . . . . . . . . . . 390
QC Antigen Salmonella O Group C2 . . . . . . . . . . 2133 . . . . . . . . . . . . 659
Phenol Red Lactose Agar . . . . . . . . . . . . . . . . . . . 0100 . . . . . . . . . . . . 388

Phenol Red Lactose Broth . . . . . . . . . . . . . . . . . . . 0094 . . . . . . . . . . . . 390
Phenol Red Mannitol Agar . . . . . . . . . . . . . . . . . . 0103 . . . . . . . . . . . . 388
Phenol Red Mannitol Broth . . . . . . . . . . . . . . . . . . 0097 . . . . . . . . . . . . 390
Phenol Red Saccharose Broth . . . . . . . . . . . . . . . . 0095 . . . . . . . . . . . . 390
Phenylalanine Agar . . . . . . . . . . . . . . . . . . . . . . . . 0745 . . . . . . . . . . . . 393
Phenylethanol Agar . . . . . . . . . . . . . . . . . . . . . . . . 0504 . . . . . . . . . . . . 395
Phytohemagglutinin M . . . . . . . . . . . . . . . . . . . . . 0528 . . . . . . . . . . . . 396
Phytohemagglutinin P . . . . . . . . . . . . . . . . . . . . . . 3110 . . . . . . . . . . . . 396
Plate Count Agar . . . . . . . . . . . . . . . . . . . . . . . . . . 0479 . . . . . . . . . . . . 399
m Plate Count Broth . . . . . . . . . . . . . . . . . . . . . . . 0751 . . . . . . . . . . . . 401
Potassium Tellurite Solution 3.5% . . . . . . . . . . . . 1814 . . . . . . . . . . . . 214
Potato Dextrose Agar . . . . . . . . . . . . . . . . . . . . . . . 0013 . . . . . . . . . . . . 402
Potato Dextrose Broth . . . . . . . . . . . . . . . . . . . . . . 0549 . . . . . . . . . . . . 402
Potato Infusion Agar . . . . . . . . . . . . . . . . . . . . . . . 0051 . . . . . . . . . . . . 404
Presence-Absence Broth . . . . . . . . . . . . . . . . . . . . 0019 . . . . . . . . . . . . 405
Proteose No. 3 Agar . . . . . . . . . . . . . . . . . . . . . . . . 0065 . . . . . . . . . . . . 407
QC Antigen Salmonella O Group D . . . . . . . . . . . 2134 . . . . . . . . . . . . 659

QC Antigen Salmonella O Group E1 . . . . . . . . . . . 2135 . . . . . . . . . . . . 659
QC Antigen Salmonella O Group F . . . . . . . . . . . 2138 . . . . . . . . . . . . 659
QC Antigen Salmonella O Group G1 . . . . . . . . . . 2139 . . . . . . . . . . . . 659
QC Antigen Salmonella O Group H . . . . . . . . . . . 2140 . . . . . . . . . . . . 659
QC Antigen Salmonella O Group I . . . . . . . . . . . . 2141 . . . . . . . . . . . . 659
QC Antigen Salmonella O Group Vi . . . . . . . . . . . 2142 . . . . . . . . . . . . 659
QC Antigen Shigella Group A . . . . . . . . . . . . . . . . 2100 . . . . . . . . . . . . 662
QC Antigen Shigella Group A1 . . . . . . . . . . . . . . . 2101 . . . . . . . . . . . . 662
QC Antigen Shigella Group B . . . . . . . . . . . . . . . . 2102 . . . . . . . . . . . . 662
QC Antigen Shigella Group C . . . . . . . . . . . . . . . . 2103 . . . . . . . . . . . . 662
QC Antigen Shigella Group C1 . . . . . . . . . . . . . . . 2104 . . . . . . . . . . . . 662
QC Antigen Shigella Group C2 . . . . . . . . . . . . . . . 2105 . . . . . . . . . . . . 662
QC Antigen Shigella Group D . . . . . . . . . . . . . . . 2106 . . . . . . . . . . . . 662
Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . 0120 . . . . . . . . . . . . 409
Proteose Peptone No. 2 . . . . . . . . . . . . . . . . . . . . . 0121 . . . . . . . . . . . . 409
R2A Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1826 . . . . . . . . . . . . 421
Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . 0122 . . . . . . . . . . . . 409
Raka-Ray No. 3 Broth . . . . . . . . . . . . . . . . . . . . . . 1865 . . . . . . . . . . . . 423
Proteose Peptones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
Raka-Ray No. 3 Medium . . . . . . . . . . . . . . . . . . . . 1867 . . . . . . . . . . . . 423

(The Weil-Felix Test) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 655
Rappaport-Vassiliadis Medium Semisolid . . . . . . . . . . . . . . . . . . . . . . . . 424
Product #
Pg. #
Proteus OX19 Antigen (Slide) . . . . . . . . . . . . . . . . 2234 . . . . . . . 637, 655

Semisolid Modification . . . . . . . . . . . . . . . . . . . 1868 . . . . . . . . . . . . 424
Proteus OX19 Antigen (Tube) . . . . . . . . . . . . . . . . 2247 . . . . . . . 640, 655
Rappaport-Vassiliadis R10 Broth . . . . . . . . . . . . . 1858 . . . . . . . . . . . . 427
850
The Difco Manual
Section VII
Alphabetical Index
Reinforced Clostridial Medium . . . . . . . . . . . . . . . 1808 . . . . . . . . . . . . 428
Salmonella H Antiserum h . . . . . . . . . . . . . . . . . . 2545 . . . . . . . . . . . . 667
Riboflavin Assay Medium . . . . . . . . . . . . . . . . . . . 0325 . . . . . . . . . . . . 430
Salmonella H Antiserum I . . . . . . . . . . . . . . . . . . . 2824 . . . . . . . . . . . . 667
Rice Extract Agar . . . . . . . . . . . . . . . . . . . . . . . . . . 0899 . . . . . . . . . . . . 431
Salmonella H Antiserum k . . . . . . . . . . . . . . . . . . 2274 . . . . . . . . . . . . 667
Rogosa SL Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . 0480 . . . . . . . . . . . . 433
Salmonella H Antiserum L Complex . . . . . . . . . . 2271 . . . . . . . . . . . . 667
Rogosa SL Broth . . . . . . . . . . . . . . . . . . . . . . . . . . 0478 . . . . . . . . . . . . 433
Salmonella H Antiserum m . . . . . . . . . . . . . . . . . . 2546 . . . . . . . . . . . . 667
Rose Bengal Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434
Salmonella H Antiserum p . . . . . . . . . . . . . . . . . . 2548 . . . . . . . . . . . . 667
Rose Bengal Agar Base . . . . . . . . . . . . . . . . . . . . . 1831 . . . . . . . . . . . . 434

Rose Bengal Antimicrobic Supplement C . . . . . . 3352 . . . . . . . . . . . . 434
Salmonella H Antiserum Poly A

(a,b,c,d,i,z10,z29) . . . . . . . . . . . . . . . . . . . . . . . . . 2539 . . . . . . . . . . . . 667
Rosolic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3228 . . . . . . . . . . . . 190
Salmonella H Antiserum Poly a-z . . . . . . . . . . . . . 2406 . . . . . . . . . . . . 667
Salmonella H Antiserum Poly B

(eh,en,enx,enz15, and G Complex) . . . . . . . . . . . 2540 . . . . . . . . . . . . 667
Product #
Pg. #
Salmonella H Antiserum Poly C (k,l,r,y,z1,z4) . . . 2541 . . . . . . . . . . . . 667
SABHI Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . 0797 . . . . . . . . . . . . 437
Salmonella H Antiserum Poly D

(z35,z36,z37,z38,z39,z41,z42) . . . . . . . . . . . . . . . . . . . 2542 . . . . . . . . . . . . 667
SBG Enrichment . . . . . . . . . . . . . . . . . . . . . . . . . . 0661 . . . . . . . . . . . . . 55

SBG Sulfa Enrichment . . . . . . . . . . . . . . . . . . . . . 0715 . . . . . . . . . . . . . 55
SF Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0315 . . . . . . . . . . . . 438
SFP Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440
SFP Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0811 . . . . . . . . . . . . 440
SIM Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0271 . . . . . . . . . . . . 442
SOB Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0443 . . . . . . . . . . . . 444
SPS Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0845 . . . . . . . . . . . . 445
SS Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0074 . . . . . . . . . . . . 446
Sabouraud Agar Modified . . . . . . . . . . . . . . . . . . . 0747 . . . . . . . . . . . . 448
Sabouraud Dextrose Agar . . . . . . . . . . . . . . . . . . . 0109 . . . . . . . . . . . . 448
Sabouraud Dextrose Broth . . . . . . . . . . . . . . . . . . 0382 . . . . . . . . . . . . 448
Sabouraud Maltose Agar . . . . . . . . . . . . . . . . . . . . 0110 . . . . . . . . . . . . 448
Sabouraud Maltose Broth . . . . . . . . . . . . . . . . . . . 0429 . . . . . . . . . . . . 448
Sabouraud Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448
Salmonella, Antigenic Scheme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 674
Appendix A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 675
Appendix B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 731
Salmonella H Antiserum Poly E

(1 Complex, z6) . . . . . . . . . . . . . . . . . . . . . . . . . . 2543 . . . . . . . . . . . . 667
Salmonella H Antiserum r . . . . . . . . . . . . . . . . . . . 2275 . . . . . . . . . . . . 667
Salmonella H Antiserum s . . . . . . . . . . . . . . . . . . . 2550 . . . . . . . . . . . . 667
Salmonella H Antiserum Single Factor 2 . . . . . . . 2474 . . . . . . . . . . . . 667
Salmonella H Antiserum Spicer-Edwards 1 . . . . . 2265 . . . . . . . . . . . . 667
Salmonella H Antiserum t . . . . . . . . . . . . . . . . . . . 2551 . . . . . . . . . . . . 667
Salmonella H Antiserum w . . . . . . . . . . . . . . . . . . 2554 . . . . . . . . . . . . 667
Salmonella H Antiserum x . . . . . . . . . . . . . . . . . . 2555 . . . . . . . . . . . . 667
Salmonella H Antiserum y . . . . . . . . . . . . . . . . . . 2276 . . . . . . . . . . . . 667
Salmonella H Antiserum z . . . . . . . . . . . . . . . . . . . 2277 . . . . . . . . . . . . 667
Salmonella - Products and

Salmonella H Antiserum z6 . . . . . . . . . . . . . . . . . . 2473 . . . . . . . . . . . . 667
Salmonella Antisera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 664
Salmonella H Antiserum z10 . . . . . . . . . . . . . . . . . 2279 . . . . . . . . . . . . 667
Salmonella H Antigen a . . . . . . . . . . . . . . . . . . . . . 2844 . . . . . . . 637, 806
Salmonella H Antigen b . . . . . . . . . . . . . . . . . . . . . 2845 . . . . . . . 637, 806
Salmonella H Antigen c . . . . . . . . . . . . . . . . . . . . . 2846 . . . . . . . . . . . . 806
Salmonella H Antigen d . . . . . . . . . . . . . . . . . . . . . 2847 . . . . . . . 637, 806
Salmonella H Antisera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 664
Salmonella H Antisera Spicer-Edwards . . . . . . . . . . . . . . . . . . . . . . . . . . 664
Salmonella H Antiserum 1 Complex . . . . . . . . . . 2272 . . . . . . . . . . . . 667
Salmonella O Antigen Group A . . . . . . . . . . . . . . 2839 . . . . . . . . . . . . 806
Salmonella H Antiserum a . . . . . . . . . . . . . . . . . . . 2820 . . . . . . . . . . . . 667
Salmonella O Antigen Group B . . . . . . . . . . . . . . 2840 . . . . . . . . . . . . 806
Salmonella H Antiserum b . . . . . . . . . . . . . . . . . . 2821 . . . . . . . . . . . . 667
Salmonella O Antigen Group C . . . . . . . . . . . . . . 2841 . . . . . . . . . . . . 806
Salmonella H Antiserum c . . . . . . . . . . . . . . . . . . . 2822 . . . . . . . . . . . . 667
Salmonella O Antigen Group D . . . . . . . . . . . . . . 2842 . . . . . . . 637, 806
Salmonella H Antiserum d . . . . . . . . . . . . . . . . . . 2823 . . . . . . . . . . . . 667
Salmonella Vi Antigen . . . . . . . . . . . . . . . . . . . . . . 2953 . . . . . . . . . . . . 804
Salmonella H Antiserum eh . . . . . . . . . . . . . . . . . . 2273 . . . . . . . . . . . . 667
Salmonella O Antisera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 664
Salmonella H Antiserum EN Complex . . . . . . . . . 2270 . . . . . . . . . . . . 667
Salmonella O Antiserum Factor 2 . . . . . . . . . . . . . 2814 . . . . . . . . . . . . 667
Salmonella H Antiserum f . . . . . . . . . . . . . . . . . . . 2544 . . . . . . . . . . . . 667
Salmonella H Antiserum Z4 Complex . . . . . . . . . . 2278 . . . . . . . . . . . . 667
Salmonella H Antiserum G Complex . . . . . . . . . . 2269 . . . . . . . . . . . . 667
The Difco Manual
851
Alphabetical Index
Section VII
Salmonella O Antiserum Factor 4,5 . . . . . . . . . . . 2815 . . . . . . . . . . . . 667
Salmonella O Antiserum Group I Factor 16 . . . . . 2263 . . . . . . . . . . . . 667
Salmonella O Antiserum Group J Factor 17 . . . . . 2517 . . . . . . . . . . . . 667
Salmonella O Antiserum Group K Factor 18 . . . . 2518 . . . . . . . . . . . . 667
Salmonella O Antiserum Group L Factor 21 . . . . 2519 . . . . . . . . . . . . 667
Salmonella O Antiserum Group M Factor 28 . . . . 2520 . . . . . . . . . . . . 667
Salmonella O Antiserum Factor 10 . . . . . . . . . . . . 2257 . . . . . . . . . . . . 667
Salmonella O Antiserum Group N Factor 30 . . . . 2521 . . . . . . . . . . . . 667
Salmonella O Antiserum Group O Factor 35 . . . . 2522 . . . . . . . . . . . . 667

Salmonella O Antiserum Poly A

(A,B,D,E1,E2,E3,E4,& L) . . . . . . . . . . . . . . . . . . . 2534 . . . . . . . . . . . . 667
Salmonella O Antiserum Poly A-I & Vi . . . . . . . . 2264 . . . . . . . . . . . . 667
Salmonella O Antiserum Poly B

(C1,C2,F,G, & H) . . . . . . . . . . . . . . . . . . . . . . . . . 2535 . . . . . . . . . . . . 667

Salmonella O Antiserum Group A
Factors 1,4,5,12 . . . . . . . . . . . . . . . . . . . . . . . . . 2947 . . . . . . . . . . . . 667
Factors 1,4,12,27 . . . . . . . . . . . . . . . . . . . . . . . . 2973 . . . . . . . . . . . . 667
Factors 1,4,5,12 . . . . . . . . . . . . . . . . . . . . . . . . . 2948 . . . . . . . . . . . . 667
Salmonella O Antiserum Poly C

(I,J,K,M,N,& O) . . . . . . . . . . . . . . . . . . . . . . . . . 2536 . . . . . . . . . . . . 667
Salmonella O Antiserum Poly D
(P,Q,R,S,T,& U) . . . . . . . . . . . . . . . . . . . . . . . . . 2537 . . . . . . . . . . . . 667
Salmonella O Antiserum Poly E
(V,W,X,Y,& Z) . . . . . . . . . . . . . . . . . . . . . . . . . . 2538 . . . . . . . . . . . . 667
Salmonella O Antiserum Poly F
(Groups 51-55) . . . . . . . . . . . . . . . . . . . . . . . . . . 2645 . . . . . . . . . . . . 667
Salmonella O Antiserum Poly G
(Groups 56-61) . . . . . . . . . . . . . . . . . . . . . . . . . . 2646 . . . . . . . . . . . . 667
Salmonella O Antiserum Vi . . . . . . . . . . . . . . . . . . 2827 . . . . . . . . . . . . 667

Factors 6,7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2949 . . . . . . . . . . . . 667
Schaedler Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0403 . . . . . . . . . . . . 452

Factors 6,8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2950 . . . . . . . . . . . . 667
Selenite Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0275 . . . . . . . . . . . . 454

Factors (8),20 . . . . . . . . . . . . . . . . . . . . . . . . . . . 3016 . . . . . . . . . . . . 667
Factors 1,9,12 . . . . . . . . . . . . . . . . . . . . . . . . . . . 2951 . . . . . . . . . . . . 667
Schaedler Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . 0534 . . . . . . . . . . . . 452

Selenite Cystine Broth . . . . . . . . . . . . . . . . . . . . . . 0687 . . . . . . . . . . . . 455
Shigella and Alkalescens-Dispar Group Products and Applications Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 835
Shigella Antisera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 788

Factors (9),46 . . . . . . . . . . . . . . . . . . . . . . . . . . . 3017 . . . . . . . . . . . . 667
Shigella Antiserum Poly Group A . . . . . . . . . . . . . 2834 . . . . . . . . . . . . 788

Factors 1,3,10,15,19,34 . . . . . . . . . . . . . . . . . . . 2819 . . . . . . . . . . . . 667
Shigella Antiserum Poly Group B . . . . . . . . . . . . . 2835 . . . . . . . . . . . . 788

Factors 3,10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2952 . . . . . . . . . . . . 667
Shigella Antiserum Poly Group C1 . . . . . . . . . . . . 2777 . . . . . . . . . . . . 788
Shigella Antiserum Poly Group A1 . . . . . . . . . . . . 2776 . . . . . . . . . . . . 788

Shigella Antiserum Poly Group C . . . . . . . . . . . . . 2836 . . . . . . . . . . . . 788

Factors 3,15 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2954 . . . . . . . . . . . . 667
Shigella Antiserum Poly Group C2 . . . . . . . . . . . . 2778 . . . . . . . . . . . . 788

Factors (3),(15),34 . . . . . . . . . . . . . . . . . . . . . . . 3018 . . . . . . . . . . . . 667
Simmons Citrate Agar . . . . . . . . . . . . . . . . . . . . . . 0091 . . . . . . . . . . . . 457

Factors 1,3,19 . . . . . . . . . . . . . . . . . . . . . . . . . . . 3019 . . . . . . . . . . . . 667
Snyder Test Agar . . . . . . . . . . . . . . . . . . . . . . . . . . 0247 . . . . . . . . . . . . 460
Salmonella O Antiserum Group F

Factor 11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2260 . . . . . . . . . . . . 667
Soytone No. 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0508 . . . . . . . . . . . . 462
Shigella Antiserum Poly Group D . . . . . . . . . . . . 2837 . . . . . . . . . . . . 788

Skim Milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0032 . . . . . . . . . . . . 459
Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0436 . . . . . . . . . . . . 462

Factors 13,22,23,(36),(37) . . . . . . . . . . . . . . . . . 3029 . . . . . . . . . . . . 667
Spirit Blue Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . 0950 . . . . . . . . . . . . 463

Factors 13,22,(36) . . . . . . . . . . . . . . . . . . . . . . . . 2216 . . . . . . . . . . . . 667
SpotTest Nitrate Reagent A . . . . . . . . . . . . . . . . 3554 . . . . . . . . . . . . 524

Factors 1,13,23,(37) . . . . . . . . . . . . . . . . . . . . . . 3020 . . . . . . . . . . . . 667
SpotTest Nitrate Reagent C . . . . . . . . . . . . . . . . 3556 . . . . . . . . . . . . 524

Factors 1,6,14,24,25,47 . . . . . . . . . . . . . . . . . . . 2262 . . . . . . . . . . . . 667
SpotTest Voges-Proskauer Reagent B . . . . . . . . 3559 . . . . . . . . . . . . 308
852
SpotTest Acridine Orange Stain . . . . . . . . . . . . 3561 . . . . . . . . . . . . 597

SpotTest Nitrate Reagent B . . . . . . . . . . . . . . . . 3555 . . . . . . . . . . . . 524
SpotTest Voges-Proskauer Reagent A . . . . . . . . 3558 . . . . . . . . . . . . 308
The Difco Manual
Section VII
Alphabetical Index
Stabilized Gram Iodine . . . . . . . . . . . . . . . . . . . . . 3342 . . . . . . . . . . . . 599
Tellurite Glycine Agar . . . . . . . . . . . . . . . . . . . . . . 0617 . . . . . . . . . . . . 489
Staining Tray . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5251 . . . . . . . 626, 629
Tergitol 7 Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0455 . . . . . . . . . . . . 491
Standard Methods Agar . . . . . . . . . . . . . . . . . . . . . 9081 . . . . . . . . . . . . 399
Tergitol 7 Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . 0912 . . . . . . . . . . . . 491
m Staphylococcus Broth . . . . . . . . . . . . . . . . . . . . 0649 . . . . . . . . . . . . 465
Terrific Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0438 . . . . . . . . . . . . 493
Staphylococcus Medium 110 . . . . . . . . . . . . . . . . 0297 . . . . . . . . . . . . 466
Tetrathionate Broth Base . . . . . . . . . . . . . . . . . . . . 0104 . . . . . . . . . . . . 494
Starch Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0072 . . . . . . . . . . . . 468
m Tetrathionate Broth Base . . . . . . . . . . . . . . . . . . 0580 . . . . . . . . . . . . 496
Sterility Testing Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 837
Thermoacidurans Agar . . . . . . . . . . . . . . . . . . . . . 0303 . . . . . . . . . . . . 498
Stock Culture Agar . . . . . . . . . . . . . . . . . . . . . . . . 0054 . . . . . . . . . . . . 469
Thiamine Assay Medium . . . . . . . . . . . . . . . . . . . . 0326 . . . . . . . . . . . . 499
Streptococcus Antigen Group A . . . . . . . . . . . . . . 2978 . . . . . . . . . . . . 791
Thiamine Assay Medium LV . . . . . . . . . . . . . . . . . 0808 . . . . . . . . . . . . 499
Streptococcus Antigen Group B . . . . . . . . . . . . . . 2979 . . . . . . . . . . . . 791
Thioglycollate Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 502
Streptococcus Antigens and Antisera . . . . . . . . . . . . . . . . . . . . . . . . . . . . 791
Thioglycollate Medium w/o Dextrose . . . . . . . . . 0363 . . . . . . . . . . . . 502
Streptococcus Antiserum Group A . . . . . . . . . . . . 2672 . . . . . . . . . . . . 791

or Indicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0432 . . . . . . . . . . . . 502
Streptococcus Antiserum Group B . . . . . . . . . . . . 2741 . . . . . . . . . . . . 791

Subtilis Spore Suspension No. 2 . . . . . . . . . . . . . . 0981 . . . . . . . . . . . . 369
Sulfite Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0972 . . . . . . . . . . . . 470
Supplement B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0276 . . . . . . . 207, 408
Supplement VX . . . . . . . . . . . . . . . . . . . . . . . . . . . 3354 . . . . . . . 207, 408
Synthetic Broth AOAC . . . . . . . . . . . . . . . . . . . . . 0352 . . . . . . . . . . . . 472
Thioglycollate Medium w/o Indicator . . . . . . . . . 0430 . . . . . . . . . . . . 502

Thiol Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0434 . . . . . . . . . . . . 507
Thiol Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0307 . . . . . . . . . . . . 507
Tinsdale Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509
Tinsdale Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0786 . . . . . . . . . . . . 509
Tinsdale Enrichment Desiccated . . . . . . . . . . . . . . 0342 . . . . . . . . . . . . 509
Todd Hewitt Broth . . . . . . . . . . . . . . . . . . . . . . . . . 0492 . . . . . . . . . . . . 511

Pg. #
Tomato Juice Agar . . . . . . . . . . . . . . . . . . . . . . . . . 0031 . . . . . . . . . . . . 512
m T7 Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0018 . . . . . . . . . . . . 474
Tomato Juice Agar Special . . . . . . . . . . . . . . . . . . 0389 . . . . . . . . . . . . 512
TAT Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9072 . . . . . . . . . . . . 475
Tomato Juice Broth . . . . . . . . . . . . . . . . . . . . . . . . 0517 . . . . . . . . . . . . 512
TAT Broth Base . . . . . . . . . . . . . . . . . . . . . . . . . . . 0984 . . . . . . . . . . . . 475
Tomato Juice Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
TB Auramine M . . . . . . . . . . . . . . . . . . . . . . . . . . . 3316 . . . . . . . . . . . . 603
Transport Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515
TB Auramine-Rhodamine T . . . . . . . . . . . . . . . . . 3317 . . . . . . . . . . . . 603
Transport Medium Amies . . . . . . . . . . . . . . . . . . . 0996 . . . . . . . . . . . . 515
TB Brilliant Green K . . . . . . . . . . . . . . . . . . . . . . . 3327 . . . . . . . . . . . . 603
Transport Medium Amies w/o Charcoal . . . . . . . . 0832 . . . . . . . . . . . . 515
TB Carbolfuchsin KF . . . . . . . . . . . . . . . . . . . . . . 3321 . . . . . . . . . . . . 602
Transport Medium Stuart . . . . . . . . . . . . . . . . . . . 0621 . . . . . . . . . . . . 515
TB Carbolfuchsin ZN . . . . . . . . . . . . . . . . . . . . . . 3313 . . . . . . . . . . . . 603
Trichophyton Agar 1 . . . . . . . . . . . . . . . . . . . . . . . 0877 . . . . . . . . . . . . 518
TB Decolorizer . . . . . . . . . . . . . . . . . . . . . . . . . . . 3318 . . . . . . . . . . . . 603
TB Decolorizer TM . . . . . . . . . . . . . . . . . . . . . . . . 3314 . . . . . . . . . . . . 603
TB Fluorescent Stain Set M . . . . . . . . . . . . . . . . . 3323 . . . . . . . . . . . . 602
TB Fluorescent Stain Set T . . . . . . . . . . . . . . . . . . 3325 . . . . . . . . . . . . 602
TB Hydrolysis Reagent . . . . . . . . . . . . . . . . . . . . . 3192 . . . . . . . . . . . . 477
TB Methylene Blue . . . . . . . . . . . . . . . . . . . . . . . . 3319 . . . . . . . . . . . . 603
Triple Sugar Iron Agar . . . . . . . . . . . . . . . . . . . . . . 0265 . . . . . . . . . . . . 521
TB Potassium Permanganate . . . . . . . . . . . . . . . . . 3315 . . . . . . . . . . . . 603
Tryptic Nitrate Medium . . . . . . . . . . . . . . . . . . . . . 0367 . . . . . . . . . . . . 523
TB Stain Set K . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3326 . . . . . . . . . . . . 602
Tryptic Soy Agar . . . . . . . . . . . . . . . . . . . . . . . . . . 0369 . . . . . . . . . . . . 525
TB Stain Set ZN . . . . . . . . . . . . . . . . . . . . . . . . . . . 3324 . . . . . . . . . . . . 602
Tryptic Soy Blood Agar Base EH . . . . . . . . . . . . . 0028 . . . . . . . . . . . . 484
TB Stain Sets and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602
Tryptic Soy Blood Agar Base No. 2 . . . . . . . . . . . 0027 . . . . . . . . . . . . 484
TCBS Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0650 . . . . . . . . . . . . 478
Tryptic Soy Broth . . . . . . . . . . . . . . . . . . . . . . . . . 0370 . . . . . . . . . . . . 527
m TEC Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0334 . . . . . . . . . . . . 480
Tryptic Soy Broth w/o Dextrose . . . . . . . . . . . . . . 0862 . . . . . . . . . . . . 527
m TGE Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0750 . . . . . . . . . . . . 531
Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0123 . . . . . . . . . . . . 529
TPEY Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . 0556 . . . . . . . . . . . . 482
Tryptone Glucose Extract Agar . . . . . . . . . . . . . . . 0002 . . . . . . . . . . . . 531
TSA Blood Agar Base . . . . . . . . . . . . . . . . . . . . . . 0026 . . . . . . . . . . . . 484
Tryptone Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0644 . . . . . . . . . . . . 533
TT Broth Base Hajna . . . . . . . . . . . . . . . . . . . . . . . 0491 . . . . . . . . . . . . 486
Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0124 . . . . . . . . . . . . 534
TTC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0643 . . . . . . . . . . . . 334
Tryptose Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0064 . . . . . . . . . . . . 536
TTC Solution 1% . . . . . . . . . . . . . . . . . . . . . . . . . . 3112 . . . . . . . 333, 491
Tryptose Blood Agar Base . . . . . . . . . . . . . . . . . . . 0232 . . . . . . . . . . . . 538
Tellurite Blood Solution . . . . . . . . . . . . . . . . . . . . 0139 . . . . . . . . . . . . 488
Tryptose Blood Agar Base w/Yeast Extract . . . . . 0662 . . . . . . . . . . . . 538
The Difco Manual
Product #
853
Alphabetical Index
Section VII
Tryptose Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0062 . . . . . . . . . . . . 536

Tryptose Phosphate Broth . . . . . . . . . . . . . . . . . . . 0060 . . . . . . . . . . . . 540
Tween 80 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3118 . . . . . . . . . . . . 630
Product #
Pg. #
YM Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0712 . . . . . . . . . . . . 569

YM Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0711 . . . . . . . . . . . . 569
YPD Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0427 . . . . . . . . . . . . 571
Product #
Pg. #
YPD Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0428 . . . . . . . . . . . . 571
UBA Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0856 . . . . . . . . . . . . 541
Yeast Carbon Base . . . . . . . . . . . . . . . . . . . . . . . . . 0391 . . . . . . . . . . . . 576
USR Antigen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2405 . . . . . . . . . . . . 793
Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0127 . . . . . . . . . . . . 572
USR Test Control Serum Set . . . . . . . . . . . . . . . . . 3516 . . . . . . . . . . . . 793
Yeast Extract Glucose Chloramphenicol Agar . . . 1900 . . . . . . . . . . . . 574
UVM Modified Listeria Enrichment Broth . . . . . 0223 . . . . . . . . . . . . 543
Yeast Extract, Technical . . . . . . . . . . . . . . . . . . . . 0886 . . . . . . . . . . . . 572
Universal Preenrichment Broth . . . . . . . . . . . . . . . 0235 . . . . . . . . . . . . 544
Yeast Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576
Urea Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . 0283 . . . . . . . . . . . . 546
Yeast Morphology Agar . . . . . . . . . . . . . . . . . . . . . 0393 . . . . . . . . . . . . 576
Urea Agar Base Concentrate . . . . . . . . . . . . . . . . . 0284 . . . . . . . . . . . . 546
Yeast Nitrogen Base . . . . . . . . . . . . . . . . . . . . . . . 0392 . . . . . . . . . . . . 576
Urea Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0272 . . . . . . . . . . . . 546
Yeast Nitrogen Base w/o Amino Acids . . . . . . . . . 0919 . . . . . . . . . . . . 576
Urea Broth Concentrate . . . . . . . . . . . . . . . . . . . . . 0280 . . . . . . . . . . . . 546

and Ammonium Sulfate . . . . . . . . . . . . . . . . . . . 0335 . . . . . . . . . . . . 576
Yersinia Antimicrobic Supplement CN . . . . . . . . . 3196 . . . . . . . . . . . . 581
Product #
Pg. #
VDRL Antigen . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0388 . . . . . . . . . . . . 797

VDRL Test Control Serum Set . . . . . . . . . . . . . . . 3520 . . . . . . . . . . . . 797
Yersinia Selective Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581

Yersinia Selective Agar Base . . . . . . . . . . . . . . . . . 1817 . . . . . . . . . . . . 581
VJ Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0562 . . . . . . . . . . . . 550

Veal Infusion Agar . . . . . . . . . . . . . . . . . . . . . . . . . 0343 . . . . . . . . . . . . 551
Veal Infusion Broth . . . . . . . . . . . . . . . . . . . . . . . . 0344 . . . . . . . . . . . . 551
Veillonella Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . 0917 . . . . . . . . . . . . 553
Veterinary Testing - Products and
Vibrio Cholerae Antisera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 801
Vibrio Cholerae Antiserum Inaba . . . . . . . . . . . . . 2430 . . . . . . . . . . . . 801
Vibrio Cholerae Antiserum Ogawa . . . . . . . . . . . . 2431 . . . . . . . . . . . . 801
Vibrio Cholerae Antiserum Poly . . . . . . . . . . . . . . 2432 . . . . . . . . . . . . 801
Violet Red Bile Agar . . . . . . . . . . . . . . . . . . . . . . . 0012 . . . . . . . . . . . . 554
Violet Red Bile Agar with MUG . . . . . . . . . . . . . . 0029 . . . . . . . . . . . . 556
Violet Red Bile Glucose Agar . . . . . . . . . . . . . . . . 1866 . . . . . . . . . . . . 558
Vitamin Assay Casamino Acids . . . . . . . . . . . . . . 0288 . . . . . . . . . . . . 110
Vitamin B12 Assay Medium . . . . . . . . . . . . . . . . . . 0360 . . . . . . . . . . . . 560
Product #
Pg. #
WL Differential Medium . . . . . . . . . . . . . . . . . . . . 0425 . . . . . . . . . . . . 562

WL Nutrient Broth . . . . . . . . . . . . . . . . . . . . . . . . . 0471 . . . . . . . . . . . . 562
WL Nutrient Medium . . . . . . . . . . . . . . . . . . . . . . 0424 . . . . . . . . . . . . 562
Water/Wastewater Testing - Products
Widal Antigen Set . . . . . . . . . . . . . . . . . . . . . . . . . 2642 . . . . . . . . . . . . 804
Product #
Pg. #
XL Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0555 . . . . . . . . . . . . 564

XLD Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0788 . . . . . . . . . . . . 564
XLT4 Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . 0234 . . . . . . . . . . . . 567
XLT4 Agar Supplement . . . . . . . . . . . . . . . . . . . . . 0353 . . . . . . . . . . . . 567
854
The Difco Manual
Section VII
Numerical Index
Numerical Index
Product #
0001
0002
0003
0004
0005
0006
0007
0011
0012
0013
0014
0015
0016
0018
0019
0020
0022
0023
0024
0026
0027
0028
0029
0031
0032
0037
0038
0042
0043
0044
0045
0047
0048
0051
0052
0054
0059
0060
0062
0063
0064
0065
0066
0067
0069
0070
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Product #
Pg. #
Nutrient Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349

Tryptone Glucose Extract Agar . . . . . . . . . . . . . . . . . . . . . . . . 531
Nutrient Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
Lactose Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
Levine EMB Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Endo Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
Brilliant Green Bile 2% . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Nutrient Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
Violet Red Bile Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 554
Potato Dextrose Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402
Brilliant Green Bile Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
Koser Citrate Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236
MR-VP Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
m T7 Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474
Presence-Absence Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405
MacConkey Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286
EC Medium with MUG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
Nutrient Agar with MUG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352
Malt Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
TSA Blood Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484
Tryptic Soy Blood Agar Base No. 2 . . . . . . . . . . . . . . . . . . . . . 484
Tryptic Soy Blood Agar Base EH . . . . . . . . . . . . . . . . . . . . . . . 484
Violet Red Bile Agar with MUG . . . . . . . . . . . . . . . . . . . . . . . 556
Tomato Juice Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
Skim Milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459
Brain Heart Infusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Heart Infusion Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
Egg Meat Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
Antibiotic Medium 19 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Heart Infusion Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
Blood Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Cystine Heart Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
Bordet Gengou Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Potato Infusion Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 404
Liver Infusion Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264
Stock Culture Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469
Liver Veal Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
Tryptose Phosphate Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 540
Tryptose Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 536
Dextrose Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Tryptose Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 536
Proteose No. 3 Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407
Dextrose Starch Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
Dextrose Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Nutrient Agar 1.5% . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
Loeffler Blood Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266
The Difco Manual
0072
0073
0074
0075
0076
0079
0080
0082
0086
0089
0091
0092
0093
0094
0095
0097
0098
0100
0103
0104
0105
0107
0109
0110
0112
0113
0115
0116
0117
0118
0119
0120
0121
0122
0123
0124
0126
0127
0136
0139
0140
0141
0142
0143
0145
0155
0158
0186
0197
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Pg. #
Starch Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468

Bismuth Sufite Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
SS Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446
MacConkey Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
EMB Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
MacConkey Sorbitol Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
Dextrose Tryptone Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
Purple Lactose Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418
Kligler Iron Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
Peptone Iron Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
Simmons Citrate Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457
Phenol Red Broth Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
Phenol Red Dextrose Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
Phenol Red Lactose Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
Phenol Red Saccharose Broth . . . . . . . . . . . . . . . . . . . . . . . . . . 390
Phenol Red Mannitol Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
Phenol Red Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
Phenol Red Lactose Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
Phenol Red Mannitol Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
Tetrathionate Broth Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494
Motility Test Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
Litmus Milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
Sabouraud Dextrose Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448
Sabouraud Maltose Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448
Malt Extract Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
Malt Extract Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
Beef Extract, Desiccated . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Casein Digest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Lima Bean Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
Neopeptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344
Proteose Peptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
Proteose Peptone No. 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
Proteose Peptone No. 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409
Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529
Tryptose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 534
Beef Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Yeast Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572
Hemoglobin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134, 207, 223
Tellurite Blood Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488
Bacto Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Pagano Levin Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374
Agar Noble . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
Agar, Granulated . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
Esculin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Malt Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298
Trichophyton Agar 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518
855
Numerical Index
Product #
0205
0211
0212
0214
0215
0216
0218
0219
0220
0221
0222
0223
0225
0227
0228
0229
0230
0231
0232
0234
0235
0236
0241
0243
0244
0247
0252
0256
0257
0259
0261
0263
0265
0267
0268
0269
0270
0271
0272
0273
0274
0275
0276
0277
0279
0280
0282
Pg. #
Modified Listeria Enrichment Broth . . . . . . . . . . . . . . . . . . . . 330

Fraser Broth Supplement . . . . . . . . . . . . . . . . . . . . . . . . . 150, 204
Panthenol Supplement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376
Oxford Antimicrobic Supplement . . . . . . . . . . . . . . . . . . . . . . 364
Lysine Decarboxylase Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Moxalactam Antimicrobic Supplement . . . . . . . . . . . . . . . . . . 242
Modified Oxford Antimicrobic Supplement . . . . . . . . . . . . . . 364
Fraser Broth Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
DNase Test Agar w/Methyl Green . . . . . . . . . . . . . . . . . . . . . . 143
LPM Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
Listeria Enrichment Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
UVM Modified Listeria Enrichment Broth . . . . . . . . . . . . . . . 543
Oxford Medium Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
Purple Broth Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415
Purple Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 415
Autolyzed Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572
Casamino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Casamino Acids, Technical . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Tryptose Blood Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . 538
XLT4 Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567
Universal Preenrichment Broth . . . . . . . . . . . . . . . . . . . . . . . . 544
Brewer Thioglycollate Medium . . . . . . . . . . . . . . . . . . . . . . . . 502
Lauryl Tryptose Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181, 251
Antibiotic Medium 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Snyder Test Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460
Mueller Hinton Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336
Fluid Thioglycollate Medium . . . . . . . . . . . . . . . . . . . . . . . . . . 502
NIH Thioglycollate Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 502
Casitone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Horse Serum, Desiccated . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
Triple Sugar Iron Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 521
Cooked Meat Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
Nitrate Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348
Liver Infusion Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264
SIM Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 442
Urea Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 546
Desoxycholate Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
Desoxycholate Citrate Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
Selenite Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454
Supplement B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207, 408
Brewer Anaerobic Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Urea Broth Concentrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 546
Glycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31, 117, 218
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315, 414, 493
0283 . . Urea Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 546
856
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Section VII
Product #
0284 . .
0285 . .
0286 . .
0288 . .
0289 . .
0290 . .
0294 . .
0297 . .
0298 . .
0299 . .
0303 . .
0305 . .
0306 . .
0307 . .
0309 . .
0313 . .
0314 . .
0315 . .
0317 . .
0318 . .
0319 . .
0320 . .
0322 . .
0325 . .
0326 . .
0331 . .
0333 . .
0334 . .
0335 . .
...
0338 . .
0339 . .
0340 . .
0342 . .
0343 . .
0344 . .
0349 . .
0352 . .
0353 . .
0360 . .
0362 . .
0363 . .
0367 . .
0369 . .
0370 . .
0373 . .
0375 . .
0382 . .
0385 . .
Pg. #
Urea Agar Base Concentrate . . . . . . . . . . . . . . . . . . . . . . . . . . . 546

Brilliant Green Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Coagulase Plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 617
Vitamin Assay Casamino Acids . . . . . . . . . . . . . . . . . . . . . . . . 110
GC Medium Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Casman Medium Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
Littman Oxgall Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
Staphylococcus Medium 110 . . . . . . . . . . . . . . . . . . . . . . . . . . 466
Mitis Salivarius Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324
Chapman Tellurite Solution 1% . . . . . . . . . . . . . . . . 232, 324, 489
Thermoacidurans Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498
Mycological Agar w/Low pH . . . . . . . . . . . . . . . . . . . . . . . . . . 342
Mannitol Salt Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
Thiol Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 507
Dubos Medium Albumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Chapman Stone Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
EC Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
SF Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438
AC Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Folic Acid Assay Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Micro Assay Culture Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
Micro Inoculum Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
Niacin Assay Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
Riboflavin Assay Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430
Thiamine Assay Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499
MacConkey Agar w/o Salt . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
m E Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
m TEC Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 480
and Ammonium Sulfate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576
Czapek-Dox Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Czapek Solution Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
Modified EC Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326
Tinsdale Enrichment Desiccated . . . . . . . . . . . . . . . . . . . . . . . 509
Veal Infusion Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551
Veal Infusion Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551
Fildes Enrichment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
Synthetic Broth AOAC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472
XLT4 Agar Supplement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567
Vitamin B12 Assay Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560
Neutralizing Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
Thioglycollate Medium w/o Dextrose . . . . . . . . . . . . . . . . . . . 502
Tryptic Nitrate Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 523
Tryptic Soy Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525
Tryptic Soy Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 527
Dubos Oleic Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Dubos Oleic Albumin Complex . . . . . . . . . . . . . . . . . . . . . . . . 163
Sabouraud Dextrose Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448
Dubos Broth Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
The Difco Manual
Section VII
Product #
0386
0387
0388
0389
0391
0392
0393
0395
0401
0402
0403
0404
0405
0409
0412
0413
0414
0415
0418
0419
0420
0422
0423
0424
0425
0427
0428
0429
0430
0431
0432
0434
0435
0436
0438
0440
0442
0443
0444
0445
0446
0448
0449
0455
0457
0460
0462
0463
0467
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Numerical Index
Pg. #
Corn Meal Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

Azide Dextrose Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
VDRL Antigen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797
Tomato Juice Agar Special . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
Yeast Carbon Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576
Yeast Nitrogen Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576
Yeast Morphology Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576
Malonate Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294
LB Agar, Lennox . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
LB Broth, Lennox . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
Schaedler Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
NZCYM Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
Mycological Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
Azide Blood Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
PPLO Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
Luria Agar Base, Miller . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
Luria Broth Base, Miller . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
NZYM Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
Brain Heart Infusion Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Biotin Assay Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Desoxycholate Lactose Agar . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Lysine Assay Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Methionine Assay Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
WL Nutrient Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562
WL Differential Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562
YPD Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571
YPD Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571
Sabouraud Maltose Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448
Thioglycollate Medium w/o Indicator . . . . . . . . . . . . . . . . . . . 502
Lipase Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463
Thioglycollate Medium w/o Dextrose or Indicator . . . . . . . . . 502
Thiol Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 507
NZM Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
Soytone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
Terrific Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493
2xYT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
BAGG Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
SOB Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444
Lowenstein Medium Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
LB Agar, Miller . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
LB Broth, Miller . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
Pseudomonas Agar F . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
Pseudomonas Agar P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 412
Tergitol 7 Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491
B12 Assay Medium USP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Choline Assay Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Cystine Assay Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
The Difco Manual
Product #
0470
0471
0474
0478
0479
0480
0483
0485
0486
0488
0491
0492
0494
0495
0496
0499
0502
0504
0505
0507
0508
0517
0518
0521
0523
0524
0525
0528
0534
0536
0541
0542
0544
0549
0551
0552
0553
0554
0555
0556
0561
0562
0566
0569
0578
0580
0587
0589
0590
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
Pg. #
MacConkey Agar w/o CV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288

WL Nutrient Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562
PKU Test Agar w/o Thienylalanine . . . . . . . . . . . . . . . . . . . . . 369
Rogosa SL Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433
Plate Count Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
Rogosa SL Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433
Brain Heart CC Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
M9 Minimal Salts, 5x . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
GN Broth, Hajna . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Esculin Iron Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
TT Broth Base Hajna . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486
Todd Hewitt Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511
mBrilliant Green Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
Brucella Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
KF Streptococcus Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
Brain Heart Infusion w/PAB and Agar . . . . . . . . . . . . . . . . . . . . 79
Brain Heart Infusion w/o Dextrose . . . . . . . . . . . . . . . . . . . . . . . 79
Phenylethanol Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
Cary-Blair Transport Medium . . . . . . . . . . . . . . . . . . . . . . . . . 515
Candida Isolation Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Soytone No. 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
Tomato Juice Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 512
Orange Serum Broth Concentrate 10X . . . . . . . . . . . . . . . . . . . 362
Orange Serum Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
Cystine Tryptic Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
Bile Esculin Azide Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Phytohemagglutinin M . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396
Schaedler Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
Anaerobic Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
B12 Culture Agar USP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
B12 Inoculum Broth USP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Minimal Agar Davis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
Potato Dextrose Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402
Fish Peptone No. 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Oatmeal Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
Microbial Content Test Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
PPLO Broth w/o CV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
XL Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 564
TPEY Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 482
Brain Heart Infusion, Porcine . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
VJ Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 550
EE Broth Mossel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Malonate Broth Modified . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Bushnell-Haas Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
m Tetrathionate Broth Base . . . . . . . . . . . . . . . . . . . . . . . . . . . 496
DRBC Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Eugon Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Eugon Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
857
Numerical Index
Product #
0593
0599
0604
0606
0607
0617
0621
0627
0630
0631
0632
0635
0636
0637
0641
0642
0643
0644
0645
0649
0650
0653
0654
0655
0657
0661
0662
0665
0666
0667
0668
0669
0677
0680
0681
0685
0686
0687
0688
0689
0696
0697
0698
0703
0711
0712
0713
0714
0715
858
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
Section VII
Pg. #
AC Broth w/o Dextrose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Pantothenate Assay Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . 378
EVA Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Fluid Thioglycollate Medium w/K Agar . . . . . . . . . . . . . . . . . 502
Tellurite Glycine Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489
Transport Medium Stuart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515
Middlebrook 7H10 Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
Modified Letheen Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328
Modified Letheen Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328
DNase Test Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
BiGGY Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
PALCAM Medium Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
PALCAM Antimicrobic Supplement . . . . . . . . . . . . . . . . . . . . 367
Differential Reinforced Clostridial Agar . . . . . . . . . . . . . . . . . 161
Fluid Sabouraud Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448
TTC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
Tryptone Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533
Bryant and Burkey Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
m Staphylococcus Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465
TCBS Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 478
Demi-Fraser Broth Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
APT Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
APT Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Gelatone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212
SBG Enrichment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Tryptose Blood Agar Base w/Yeast Extract . . . . . . . . . . . . . . . 538
Lactose Peptone Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
Agar Medium No. F . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Bovine Albumin 5% . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
m FC Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
Letheen Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Letheen Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
HC Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
D/E Neutralizing Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
Selenite Cystine Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455
OF Basal Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
Mycobiotic Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
Blood Agar Base No. 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Fluid Thioglycollate Medium w/Beef Extract . . . . . . . . . . . . . 502
m FC Basal Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Cooke Rose Bengal Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
YM Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 569
YM Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 569
Middlebrook 7H9 Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
Middlebrook ADC Enrichment . . . . . . . . . . . . . . . . . . . . . . . . . 315
SBG Sulfa Enrichment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Product #
0717
0722
0735
0736
0739
0742
0745
0746
0747
0749
0750
0751
0752
0756
0757
0759
0761
0768
0769
0770
0772
0779
0786
0788
0790
0791
0792
0793
0794
0795
0797
0801
0803
0808
0810
0811
0812
0816
0818
0819
0822
0832
0835
0836
0837
0838
0845
0849
0853
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
Pg. #
BG Sulfa Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Middlebrook OADC Enrichment . . . . . . . . . . . . . . . . . . . . . . . 315
MIO Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
m Endo Agar LES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Emerson YpSs Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Acetate Differential Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Phenylalanine Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393
m Enterococcus Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Sabouraud Agar Modified . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448
m Endo Broth MF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
m TGE Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 531
m Plate Count Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401
m HPC Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
Minimal Broth Davis w/o Dextrose . . . . . . . . . . . . . . . . . . . . . 322
Mueller Hinton Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336
DCLS Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
Motility Medium S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
Baird-Parker Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
ISP Medium 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
ISP Medium 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
ISP Medium 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
EY Tellurite Enrichment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Tinsdale Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509
XLD Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 564
Columbia Blood Agar Base EH . . . . . . . . . . . . . . . . . . . . . . . . 123
Marine Broth 2216 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
Columbia Blood Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Columbia Blood Agar Base No. 2 . . . . . . . . . . . . . . . . . . . . . . 123
Leptospira Medium Base EMJH . . . . . . . . . . . . . . . . . . . . . . . 253
Leptospira Enrichment EMJH . . . . . . . . . . . . . . . . . . . . . . . . . 253
SABHI Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437
Middlebrook OADC Enrichment w/WR 1339 . . . . . . . . . . . . . 315
Coagulase Plasma EDTA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 617
Thiamine Assay Medium LV . . . . . . . . . . . . . . . . . . . . . . . . . . 499
MYP Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
SFP Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440
Agar Bacteriological Technical . . . . . . . . . . . . . . . . . . . . . . . . . 21
Pantothenate Medium AOAC USP . . . . . . . . . . . . . . . . . . . . . . 380
MacConkey Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
D/E Neutralizing Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
Folic Acid Casei Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
Transport Medium Amies w/o Charcoal . . . . . . . . . . . . . . . . . 515
Candida BCG Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
Mycoplasma Supplement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
Mycoplasma Supplement S . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
Mycobacteria 7H11 Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
SPS Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445
Lysine Iron Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
Hektoen Enteric Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
The Difco Manual
Section VII
Product #
0854
0856
0862
0867
0869
0872
0874
0877
0878
0879
0881
0882
0883
0885
0886
0890
0894
0899
0900
0901
0905
0912
0917
0919
0922
0927
0940
0941
0944
0950
0951
0955
0957
0964
0965
0967
0970
0971
0972
0974
0979
0980
0981
0984
0985
0986
0987
0994
0995
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
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..
..
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..
..
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..
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..
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..
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..
..
..
..
Numerical Index
Pg. #
Cetrimide Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

UBA Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 541
Tryptic Soy Broth w/o Dextrose . . . . . . . . . . . . . . . . . . . . . . . . 527
Columbia CNA Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
Motility GI Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332
Decarboxylase Medium Base . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Bile Esculin Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Bile Esculin Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Lactobacilli MRS Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
Lactobacilli MRS Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
m FC Broth Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
Peptone Bacteriological Technical . . . . . . . . . . . . . . . . . . . . . . 383
Yeast Extract, Technical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572
Decarboxylase Base Moeller . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Charcoal Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Rice Extract Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431
Lactobacilli Agar AOAC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
Lactobacilli Broth AOAC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
Peptamin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382
Tergitol 7 Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491
Veillonella Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 553
Yeast Nitrogen Base w/o Amino Acids . . . . . . . . . . . . . . . . . . 576
McBride Listeria Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
Pseudomonas Isolation Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . 414
M Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
McClung Toabe Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
Columbia Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
Spirit Blue Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463
Pyridoxine Y Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419
Actinomycete Isolation Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Brucella Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
Folic AOAC Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
Agar Flake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
CLED Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Sulfite Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 470
Elliker Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Marine Agar 2216 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302
PKU Test Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
Subtilis Spore Suspension No. 2 . . . . . . . . . . . . . . . . . . . . . . . . 369
TAT Broth Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 475
KL Virulence Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
KL Virulence Enrichment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
Fletcher Medium Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Panthenol Assay Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376
Inositol Assay Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
The Difco Manual
Product #
0996 . .
0997 . .
1010 . .
1017 . .
1019 . .
1022 . .
1289 . .
1417 . .
1423 . .
1454 . .
1804 . .
1807 . .
1808 . .
1809 . .
1810 . .
1811 . .
1814 . .
1817 . .
1818 . .
1820 . .
1823 . .
1826 . .
1828 . .
1831 . .
1833 . .
1850 . .
1853 . .
1856 . .
1857 . .
1858 . .
1859 . .
1862 . .
1865 . .
1866 . .
1867 . .
1868 . .
...
1880 . .
1894 . .
1897 . .
1900 . .
2100 . .
2101 . .
2102 . .
2103 . .
2104 . .
2105 . .
2106 . .
2116 . .
Pg. #
Transport Medium Amies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 515

KF Streptococcus Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
Petragnani Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592
Lowenstein Medium, Jensen . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
ATS Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587
Dubos Albumin Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Lowenstein Medium, Jensen Deeps . . . . . . . . . . . . . . . . . . . . . 268
Lowenstein Medium, Gruft . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268
Lowenstein Medium w/5% NaCl . . . . . . . . . . . . . . . . . . . . . . . 268
M9CA Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
MIL Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
Peptone Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
Reinforced Clostridial Medium . . . . . . . . . . . . . . . . . . . . . . . . 428
Giolitti-Cantoni Broth Base . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
Buffered Peptone Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
OGYE Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360
Potassium Tellurite Solution 3.5% . . . . . . . . . . . . . . . . . . . . . . 214
Yersinia Selective Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . 581
MacConkey Agar CS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288
Campylobacter Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
A-1 Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
R2A Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421
Enteric Fermentation Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Rose Bengal Agar Base . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434
Modified Buffered Peptone Water . . . . . . . . . . . . . . . . . . . . . . . 99
Minerals Modified Glutamate Broth . . . . . . . . . . . . . . . . . . . . 320
Muller Kauffmann Tetrathionate Broth Base . . . . . . . . . . . . . . 339
M17 Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
M17 Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
Rappaport-Vassiliadis R10 Broth . . . . . . . . . . . . . . . . . . . . . . . 427
Milk Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
Aseptic Commissioning Medium . . . . . . . . . . . . . . . . . . . . . . . . 45
Raka-Ray No. 3 Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423
Violet Red Bile Glucose Agar . . . . . . . . . . . . . . . . . . . . . . . . . . 558
Raka-Ray No. 3 Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423
Semisolid Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 424
Brilliant Green Agar Modified . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Lysine Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
Maximum Recovey Diluent . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
Yeast Extract Glucose Chloramphenicol Agar . . . . . . . . . . . . . 574
QC Antigen Shigella Group A . . . . . . . . . . . . . . . . . . . . . . . . . 662
QC Antigen Shigella Group A1 . . . . . . . . . . . . . . . . . . . . . . . . . 662
QC Antigen Shigella Group B . . . . . . . . . . . . . . . . . . . . . . . . . 662
QC Antigen Shigella Group C . . . . . . . . . . . . . . . . . . . . . . . . . 662
QC Antigen Shigella Group C1 . . . . . . . . . . . . . . . . . . . . . . . . . 662
QC Antigen Shigella Group C2 . . . . . . . . . . . . . . . . . . . . . . . . . 662
QC Antigen Shigella Group D . . . . . . . . . . . . . . . . . . . . . . . . . 662
QC Antigen Alkalescens-Dispar Group 1 . . . . . . . . . . . . . . . . 662
859
Numerical Index
Product #
2130 . .
2131 . .
2132 . .
2133 . .
2134 . .
2135 . .
2136 . .
2137 . .
2138 . .
2139 . .
2140 . .
2141 . .
2142 . .
2159 . .
2216 . .
...
2228 . .
2229 . .
2230 . .
2231 . .
2232 . .
...
2234 . .
2235 . .
2236 . .
2237 . .
2240 . .
2241 . .
2243 . .
2244 . .
2245 . .
2246 . .
2247 . .
2248 . .
2249 . .
2250 . .
2251 . .
2252 . .
2253 . .
2257 . .
2258 . .
2259 . .
2260 . .
2262 . .
...
2263 . .
2264 . .
2265 . .
2266 . .
860
Section VII
Pg. #
QC Antigen Salmonella O Group A . . . . . . . . . . . . . . . . . . . . . 659

QC Antigen Salmonella O Group B . . . . . . . . . . . . . . . . . . . . . 659
QC Antigen Salmonella O Group C1 . . . . . . . . . . . . . . . . . . . . 659
QC Antigen Salmonella O Group C2 . . . . . . . . . . . . . . . . . . . . 659
QC Antigen Salmonella O Group D . . . . . . . . . . . . . . . . . . . . . 659
QC Antigen Salmonella O Group E1 . . . . . . . . . . . . . . . . . . . . 659
QC Antigen Salmonella O Group F . . . . . . . . . . . . . . . . . . . . . 659
QC Antigen Salmonella O Group G1 . . . . . . . . . . . . . . . . . . . . 659
QC Antigen Salmonella O Group H . . . . . . . . . . . . . . . . . . . . . 659
QC Antigen Salmonella O Group I . . . . . . . . . . . . . . . . . . . . . . 659
QC Antigen Salmonella O Group Vi . . . . . . . . . . . . . . . . . . . . 659
E. Coli H Antiserum H7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 621
Factors 13,22,(36) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
Neisseria Meningitidis Antiserum Group A . . . . . . . . . . . . . . . 652
Neisseria Meningitidis Antiserum Group B . . . . . . . . . . . . . . . 652
Neisseria Meningitidis Antiserum Group C . . . . . . . . . . . . . . . 652
Neisseria Meningitidis Antiserum Group D . . . . . . . . . . . . . . . 652
(Groups A, B, C, D) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 652
Proteus OX19 Antigen (Slide) . . . . . . . . . . . . . . . . . . . . . 637, 655
Proteus OX19 Antiserum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 655
Haemophilus Influenzae Antiserum Type b . . . . . . . . . . . . . . . 646
Haemophilus Influenzae Antiserum Poly . . . . . . . . . . . . . . . . . 646
Francisella Tularensis Antigen (Slide) . . . . . . . . . . . . . . . . . . . 642
Francisella Tularensis Antiserum . . . . . . . . . . . . . . . . . . . . . . . 642
Proteus OX2 Antigen (Slide) . . . . . . . . . . . . . . . . . . . . . . . . . . 655
Proteus OXK Antigen (Slide) . . . . . . . . . . . . . . . . . . . . . . . . . . 655
Proteus OX2 Antiserum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 655
Proteus OXK Antiserum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 655
Proteus OX19 Antigen (Tube) . . . . . . . . . . . . . . . . . . . . . 640, 655
Proteus OX2 Antigen (Tube) . . . . . . . . . . . . . . . . . . . . . . . . . . 655
Proteus OXK Antigen (Tube) . . . . . . . . . . . . . . . . . . . . . . . . . . 655
Haemophilus Influenzae Antiserum Type a . . . . . . . . . . . . . . . 646
Francisella Tularensis Antigen (Tube) . . . . . . . . . . . . . . . . . . . 642
Neisseria Meningitidis Antiserum Group Z . . . . . . . . . . . . . . 652
Neisseria Meningitidis Antiserum Group W135 . . . . . . . . . . . 652
Salmonella O Antiserum Factor 10 . . . . . . . . . . . . . . . . . . . . . . 667
Salmonella O Antiserum Group F Factor 11 . . . . . . . . . . . . . . 667
Factors 1,6,14,24,25,47 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
Salmonella O Antiserum Group I Factor 16 . . . . . . . . . . . . . . . 667
Salmonella O Antiserum Poly A-I & Vi . . . . . . . . . . . . . . . . . . 667
Salmonella H Antiserum Spicer-Edwards 1 . . . . . . . . . . . . . . . 667
Product #
2267
2268
2269
2270
2271
2272
2273
2274
2275
2276
2277
2278
2279
2280
2281
2300
2301
2302
2303
2304
2305
2306
2309
2310
2314
2318
2329
2359
2378
2405
2406
2407
2430
2431
2432
2439
2440
2449
2466
2473
2474
2475
2476
2477
2512
2517
2518
2519
2520
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
Pg. #

Salmonella H Antiserum G Complex . . . . . . . . . . . . . . . . . . . . 667
Salmonella H Antiserum EN Complex . . . . . . . . . . . . . . . . . . . 667
Salmonella H Antiserum L Complex . . . . . . . . . . . . . . . . . . . . 667
Salmonella H Antiserum 1 Complex . . . . . . . . . . . . . . . . . . . . 667
Salmonella H Antiserum eh . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
Salmonella H Antiserum k . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
Salmonella H Antiserum r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
Salmonella H Antiserum y . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
Salmonella H Antiserum z . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
Salmonella H Antiserum Z4 Complex . . . . . . . . . . . . . . . . . . . 667
Salmonella H Antiserum z10 . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
Salmonella H Antiserum z29 . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
Candida Albicans Antiserum . . . . . . . . . . . . . . . . . . . . . . . . . . 615
Listeria O Antiserum Type 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . 648
Listeria O Antiserum Type 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . 648
Listeria O Antiserum Poly . . . . . . . . . . . . . . . . . . . . . . . . . . . . 648
Listeria O Antigen Type 1 (Slide) . . . . . . . . . . . . . . . . . . . . . . . 648
Listeria O Antigen Type 4 (Slide) . . . . . . . . . . . . . . . . . . . . . . . 648
Listeria O Antigen Type 1 (Tube) . . . . . . . . . . . . . . . . . . . . . . . 648
Listeria O Antigen Type 4 (Tube) . . . . . . . . . . . . . . . . . . . . . . . 648
Bordetella Pertussis Antiserum . . . . . . . . . . . . . . . . . . . . . . . . . 609
Bordetella Parapertussis Antiserum . . . . . . . . . . . . . . . . . . . . . 609
FA Buffer, Dried . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 626, 630
FA Streptococcus Group A . . . . . . . . . . . . . . . . . . . . . . . . . . . . 628
FA Mounting Fluid pH 7.2 . . . . . . . . . . . . . . . . . . . . . . . . 626, 630
FA Bordetella Pertussis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623
FA Bordetella Parapertussis . . . . . . . . . . . . . . . . . . . . . . . . . . . 623
USR Antigen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 793
Salmonella H Antiserum Poly a-z . . . . . . . . . . . . . . . . . . . . . . 667
Febrile Antigen Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 637
Vibrio Cholerae Antiserum Inaba . . . . . . . . . . . . . . . . . . . . . . . 801
Vibrio Cholerae Antiserum Ogawa . . . . . . . . . . . . . . . . . . . . . . 801
Vibrio Cholerae Antiserum Poly . . . . . . . . . . . . . . . . . . . . . . . . 801
FTA Serum Reactive . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 630
FTA Serum Non-Reactive . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 630
FA Human Globulin Antiglobulin (Rabbit) . . . . . . . . . . . . . . . 630
Brucella Abortus Antigen (Tube) . . . . . . . . . . . . . . . . . . . 611, 640
Salmonella H Antiserum z6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
Salmonella H Antiserum Single Factor 2 . . . . . . . . . . . . . . . . . 667
Salmonella O Antiserum Factor 34 . . . . . . . . . . . . . . . . . . . . . 667
Salmonella O Antiserum Group J Factor 17 . . . . . . . . . . . . . . 667
Salmonella O Antiserum Group K Factor 18 . . . . . . . . . . . . . . 667
Salmonella O Antiserum Group L Factor 21 . . . . . . . . . . . . . . 667
Salmonella O Antiserum Group M Factor 28 . . . . . . . . . . . . . 667
The Difco Manual
Section VII
Product #
2521 . . Salmonella O Antiserum Group N Factor 30 . . . . . . . . . . . . . .

2522 . . Salmonella O Antiserum Group O Factor 35 . . . . . . . . . . . . . .
2534 . . Salmonella O Antiserum Poly A
(A,B,D,E1,E2,E3,E4,& L) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2535 . . Salmonella O Antiserum Poly B
(C1,C2,F,G, & H) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2536 . . Salmonella O Antiserum Poly C
(I,J,K,M,N,& O) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2537 . . Salmonella O Antiserum Poly D
(P,Q,R,S,T,& U) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2538 . . Salmonella O Antiserum Poly E
(V,W,X,Y,& Z) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2539 . . Salmonella H Antiserum Poly A (a,b,c,d,i,z10,z29) . . . . . . . . . .
2540 . . Salmonella H Antiserum Poly B
(eh,en,enx,enz15, and G Complex) . . . . . . . . . . . . . . . . . . . . . .
2541 . . Salmonella H Antiserum Poly C (k,l,r,y,z1,z4) . . . . . . . . . . . . .
2542 . . Salmonella H Antiserum Poly D
(z35,z36,z37,z38,z39,z41,z42) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2543 . . Salmonella H Antiserum Poly E
(1 Complex, z6) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2544 . . Salmonella H Antiserum f . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2545 . . Salmonella H Antiserum h . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2546 . . Salmonella H Antiserum m . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2548 . . Salmonella H Antiserum p . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2550 . . Salmonella H Antiserum s . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2551 . . Salmonella H Antiserum t . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2554 . . Salmonella H Antiserum w . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2555 . . Salmonella H Antiserum x . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2556 . . Salmonella H Antiserum z13 . . . . . . . . . . . . . . . . . . . . . . . . . . .
2585 . . Bordetella Pertussis Antigen . . . . . . . . . . . . . . . . . . . . . . . . . . .
2642 . . Widal Antigen Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2645 . . Salmonella O Antiserum Poly F
(Groups 51-55) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2646 . . Salmonella O Antiserum Poly G
(Groups 56-61) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2659 . . Salmonella O Antiserum Factor 4 . . . . . . . . . . . . . . . . . . . . . .
2662 . . Salmonella O Antiserum Factor 20 . . . . . . . . . . . . . . . . . . . . .
2672 . . Streptococcus Antiserum Group A . . . . . . . . . . . . . . . . . . . . . .
2741 . . Streptococcus Antiserum Group B . . . . . . . . . . . . . . . . . . . . . .
2776 . . Shigella Antiserum Poly Group A1 . . . . . . . . . . . . . . . . . . . . . .
2777 . . Shigella Antiserum Poly Group C1 . . . . . . . . . . . . . . . . . . . . . .
The Difco Manual
Numerical Index
Pg. #
667
667
667
667
667
667
667
667
667
667
667
667
667
667
667
667
667
667
667
667
667
667
667
667
667
609
804
667
667
667
667
667
667
667
667
667
667
791
791
788
788
Product #
2778 . .
2779 . .
2789 . .
2790 . .
2791 . .
2792 . .
2814 . .
2815 . .
2816 . .
2817 . .
2818 . .
2819 . .
...
2820 . .
2821 . .
2822 . .
2823 . .
2824 . .
2827 . .
2834 . .
2835 . .
2836 . .
2837 . .
2838 . .
2839 . .
2840 . .
2841 . .
2842 . .
2844 . .
2844 . .
2845 . .
2846 . .
2847 . .
2871 . .
2880 . .
2881 . .
2891 . .
2909 . .
2910 . .
2915
2916
2947
2948
2949
2950
2951
2952
2953
..
..
..
..
..
..
..
..
..
Pg. #
Shigella Antiserum Poly Group C2 . . . . . . . . . . . . . . . . . . . . . . 788

Haemophilus Influenzae Antiserum Type c . . . . . . . . . . . . . . . 646
Haemophilus Influenzae Antiserum Type d . . . . . . . . . . . . . . . 646
Haemophilus Influenzae Antiserum Type e . . . . . . . . . . . . . . . 646
Haemophilus Influenzae Antiserum Type f . . . . . . . . . . . . . . . 646
Salmonella O Antiserum Factor 4,5 . . . . . . . . . . . . . . . . . . . . . 667
Factors 1,3,10,15,19,34 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
Salmonella H Antiserum a . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
Salmonella H Antiserum b . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
Salmonella H Antiserum c . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
Salmonella H Antiserum d . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
Salmonella H Antiserum I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
Salmonella O Antiserum Vi . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
Shigella Antiserum Poly Group A . . . . . . . . . . . . . . . . . . . . . . 788
Shigella Antiserum Poly Group B . . . . . . . . . . . . . . . . . . . . . . 788
Shigella Antiserum Poly Group C . . . . . . . . . . . . . . . . . . . . . . 788
Shigella Antiserum Poly Group D . . . . . . . . . . . . . . . . . . . . . . 788
Alkalescens-Dispar Antiserum Poly . . . . . . . . . . . . . . . . . . . . . 788
Salmonella O Antigen Group A . . . . . . . . . . . . . . . . . . . . . . . . 806
Salmonella O Antigen Group B . . . . . . . . . . . . . . . . . . . . . . . . 806
Salmonella O Antigen Group C . . . . . . . . . . . . . . . . . . . . . . . . 806
Salmonella O Antigen Group D . . . . . . . . . . . . . . . . . . . . 637, 806
Salmonella H Antigen a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 637
Salmonella H Antigen a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 806
Salmonella H Antigen b . . . . . . . . . . . . . . . . . . . . . . . . . . 637, 806
Salmonella H Antigen c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 806
Salmonella H Antigen d . . . . . . . . . . . . . . . . . . . . . . . . . . 637, 806
Brucella Abortus Antiserum . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
Neisseria Meningitidis Antiserum Group X . . . . . . . . . . . . . . . 652
Neisseria Meningitidis Antiserum Group Y . . . . . . . . . . . . . . . 652
Neisseria Meningitidis Antiserum Group Z . . . . . . . . . . . . . . . 652
Brucella Abortus Antigen (Slide) . . . . . . . . . . . . . . . . . . . 611, 637
(Groups X, Y, Z) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 652
Brucella Suis Antigen (Slide) . . . . . . . . . . . . . . . . . . . . . . . . . . 611
Brucella Melitensis Antigen (Slide) . . . . . . . . . . . . . . . . . . . . . 611
Salmonella O Antiserum Group A Factors 1,4,5,12 . . . . . . . . 667
Salmonella O Antiserum Group B Factors 1,4,5,12 . . . . . . . . 667
Salmonella O Antiserum Group C1 Factors 6,7 . . . . . . . . . . . . 667
Salmonella O Antiserum Group C2 Factors 6,8 . . . . . . . . . . . . 667
Salmonella O Antiserum Group D1 Factors 1,9,12 . . . . . . . . . 667
Salmonella O Antiserum Group E1 Factors 3,10 . . . . . . . . . . . 667
Salmonella Vi Antigen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 804
861
Numerical Index
Product #
2954 . .
2970 . .
2973 . .
2978 . .
2979 . .
3016 . .
3017 . .
3018 . .
...
3019 . .
3020 . .
...
3029 . .
...
3101 . .
3110 . .
3112 . .
3118 . .
3139 . .
3140 . .
3192 . .
3194 . .
3196 . .
3197 . .
3198 . .
3228 . .
3238 . .
3239 . .
3246
3248
3259
3260
3266
3267
3268
3279
3280
3313
3314
3315
3316
3317
3318
3319
3321
3323
3324
3325
862
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
Section VII
Pg. #
Salmonella O Antiserum Group E2 Factors 3,15 . . . . . . . . . . . 667

E. Coli O Antiserum O157 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 621
Salmonella O Antiserum Group B Factors 1,4,12,27 . . . . . . . 667
Streptococcus Antigen Group A . . . . . . . . . . . . . . . . . . . . . . . . 791
Streptococcus Antigen Group B . . . . . . . . . . . . . . . . . . . . . . . . 791
Salmonella O Antiserum Group C3 Factors (8),20 . . . . . . . . . . 667
Salmonella O Antiserum Group D2 Factors (9),46 . . . . . . . . . 667
Factors (3),(15),34 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
Salmonella O Antiserum Group E4 Factors 1,3,19 . . . . . . . . . 667
Factors 1,13,23,(37) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
Factors 13,22,23,(36),(37) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 667
KL Antitoxin Strips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
Phytohemagglutinin P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396
TTC Solution 1% . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333, 491
Tween 80 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 630
Bactrol TB Slides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603
Bactrol Gram Silde . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 601
TB Hydrolysis Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477
Clostridium Difficile Antimicrobic Supplement CC . . . . . . . . . 79
Yersinia Antimicrobic Supplement CN . . . . . . . . . . . . . . . . . . 581
Novobiocin Antimicrobic Supplement . . . . . . . . . . . . . . . 326, 424
Antimicrobic Vial CNVT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Rosolic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
Febrile Positive Control Polyvalent . . . . . . . . . . . . . . . . . 637, 804
Febrile Negative Control . . . . . . . . . . . . . . . . . . . . . 611, 637, 642,
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 658, 659, 663, 804
Folic Buffer A, Dried . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
Hemoglobin Solution 2% . . . . . . . . . . . . . . . . . . . . . 135, 208, 408
FTA Sorbent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 630
Antimicrobic Vial CNV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
FTA Sorbent Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 630
Antimicrobic Vial Oxytetracycline . . . . . . . . . . . . . . . . . . . . . . 360
Antimicrobic Vial P . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284, 440
Campylobacter Agar Kit Blaser . . . . . . . . . . . . . . . . . . . . . . . . 103
Campylobacter Agar Kit Skirrow . . . . . . . . . . . . . . . . . . . . . . . 103
TB Carbolfuchsin ZN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603
TB Decolorizer TM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603
TB Potassium Permanganate . . . . . . . . . . . . . . . . . . . . . . . . . . 603
TB Auramine M . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603
TB Auramine-Rhodamine T . . . . . . . . . . . . . . . . . . . . . . . . . . . 603
TB Decolorizer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603
TB Methylene Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603
TB Carbolfuchsin KF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602
TB Fluorescent Stain Set M . . . . . . . . . . . . . . . . . . . . . . . . . . . 602
TB Stain Set ZN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602
TB Fluorescent Stain Set T . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602
Product #
3326
3327
3328
3329
3330
3331
3332
3333
3334
3335
3336
3337
3338
3339
3340
3341
3342
3343
3347
3352
3354
3516
3520
3554
3555
3556
3558
3559
3561
5251
6663
9037
9038
9039
9041
9045
9046
9053
9070
9072
9081
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
..
Pg. #
TB Stain Set K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602

TB Brilliant Green K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603
Gram Stain Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 598
Gram Crystal Violet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599
Gram Decolorizer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599
Gram Iodine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599
Gram Safranin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599
Antimicrobic Vial A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
3-Step Gram Stain Set-S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 598
3-Step Gram Safranin-S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599
Acridine Orange Stain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 597
3-Step Gram Stain Set-T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 598
Gram Stain Set (with Stabilized Iodine) . . . . . . . . . . . . . . . . . 598
Antimicrobic Vial K . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440
FA Mounting Fluid pH 9 . . . . . . . . . . . . . . . . . . . . . . . . . . 626, 628
3-Step Gram Safranin-T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599
Stabilized Gram Iodine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599
Gram Basic Fuchsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599
Egg Yolk Enrichment 50% . . . . . . . . . . . . . . . . . . . . . . . . 307, 440
Rose Bengal Antimicrobic Supplement C . . . . . . . . . . . . . . . . 434
Supplement VX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207, 408
USR Test Control Serum Set . . . . . . . . . . . . . . . . . . . . . . . . . . 793
VDRL Test Control Serum Set . . . . . . . . . . . . . . . . . . . . . . . . . 797
SpotTest Nitrate Reagent A . . . . . . . . . . . . . . . . . . . . . . . . . . 524
SpotTest Nitrate Reagent B . . . . . . . . . . . . . . . . . . . . . . . . . . 524
SpotTest Nitrate Reagent C . . . . . . . . . . . . . . . . . . . . . . . . . . 524
SpotTest Voges-Proskauer Reagent A . . . . . . . . . . . . . . . . . . 308
SpotTest Voges-Proskauer Reagent B . . . . . . . . . . . . . . . . . . 308
SpotTest Acridine Orange Stain . . . . . . . . . . . . . . . . . . . . . . . 597
Staining Tray . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 626, 629
Mineral Oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
HYcheck for Enterobacteriaceae . . . . . . . . . . . . . . . . . . . . . . . 588
HYcheck for Yeasts and Molds . . . . . . . . . . . . . . . . . . . . . . . . . 588
HYcheck for Disinfection Control . . . . . . . . . . . . . . . . . . . . . . 588
HYcheck D/E Neutralizing Agar . . . . . . . . . . . . . . . . . . . . . . . 588
HYcheck Plate Count Agar with TTC . . . . . . . . . . . . . . . . . . . 588
HYcheck for Yeasts and Molds with TTC . . . . . . . . . . . . . . . . 588
HYcheck for Total Count . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588
Lactose Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
TAT Broth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 475
Standard Methods Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
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11th Edition

The Difco Manual

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The Difco Manual

dependent upon nutritional requirements which are known to vary

products of protein decomposition are equally utilizable by all

The Difco Manual

History of Difco Laboratories

Difco pursued the challenging task of producing bacterial antisera

Bactrol Disks were introduced by Difco Laboratories in 1972. Bactrol

The development of Proteose Peptone, Proteose Peptone No. 2 and

The Difco Manual

Throughout the 1950s and 1960s, Difco continued to add products

In 1983, Difco purchased the Paul A. Smith Company, later to be known

History of Microbiology and Culture Media

Early years at Difco Laboratories.

The study of immunity began after the discovery of the tubercle

The Difco Manual

demonstrated that a virus could be grown in chick embryos and would

With rapid advances in technologies and instrumentation, the basic

One organism that has made a great contribution to molecular

In the 1980s, instrumentation entered the microbiology laboratory.

1. Marti-Ibanez, F. 1962. Baroque medicine, p. 185-195. In

Microorganism Growth Requirements

The Difco Manual

Common Media Constituents

Peptone, protein hydrolysate,

Blood, serum, yeast extract or

Sugar, alcohols and carbohydrates

Phosphates, acetates and citrates

Mineral Salts and Metals

Phosphate, sulfate, magnesium,

Chemicals, antimicrobials and dyes

Phenol red, neutral red

Agar, gelatin, alginate, silica gel

Selective Agents include Bile Salts, dyes and antimicrobial agents.

Environmental Factors in Culture Media

Absorption of the oxygen by chemicals;

Protective Agents and Growth Factors

Culture Media Ingredients Agars

the temperatures required for growth of human pathogens and was

The Difco Manual

The Manufacturing Process

The Difco Manual

Agar Granulated is qualified to grow recombinant strains of

Culture Media Ingredients Peptones and Hydrolysates

Typical fermentation process.

Meat (fresh, frozen or dried)

Infusions and Extracts

Other non-chemically defined ingredients, including Bacto Liver, Bacto

The water-soluble fractions of materials such as muscle, liver, yeast

Proteins consist of amino acids joined together by means of the

The quality and performance of peptones, infusions and extracts are

The Difco Manual

A typical analysis was performed on Difco peptones and hydrolysates

The Difco Manual

The Difco Manual

Quantities of media in excess of two liters may require an

Use vessels at least 2-3 times the volume of medium.

For filter sterilization, adjust the pH, if necessary, prior to filtering.