Medios Salmonella

Bismuth Sulfite Agar
3. The use of altered or deficient media may cause mutants Reference

having different nutritional requirements that will not give 1. United States Pharmacopeial Convention, Inc. 2008. The United States pharmacopeia 31/The
national formulary 26, Supp. 1, 8-1-08, online. United States Pharmacopeial Convention, Inc.,
a satisfactory response. Rockville, Md.
4. For successful results to these procedures, all conditions of

Availability
the assay must be followed precisely.
Difco™ Biotin Assay Medium B
Cat. No. 241910 Dehydrated – 100 g*
*Store at 2-8˚C.
Bird Seed Agar

Intended Use The addition of the antimicrobial agent chloramphenicol
Bird Seed Agar is a selective and differential medium used in improves the recovery of Cryptococcus from specimens
the identification of Cryptococcus neoformans. containing mixed flora by suppressing bacterial growth.
Summary and Explanation Procedure

Bird Seed Agar was initially described by Staib. He found
1 To prepare plates from agar deeps, liquefy medium in boiling
that Cryptococcus neoformans produced characteristic brown water bath and pour molten medium into a sterile Petri dish;
colonies when cultivated on a growth medium containing an allow medium to solidify and dry before use.
extract prepared from the seeds of the Indian thistle plant Using a sterile inoculating loop or needle, pick two or three
Guizotia abyssinica. C. neoformans is the only yeast known to isolated colonies from the subculture medium and streak over slant
produce this pigmentation.2 or plate surface. Incubate media at 25-30°C for up to 7 days.
Shields and Ajello later modified the original formulation by
the addition of the antimicrobial agent chloramphenicol.3 The Expected Results
concentration of chloramphenicol has been doubled in this Yeast-like organisms that produce tan to brown colonies on
medium to improve the inhibition of bacteria. this medium within 4-7 days may be presumptively identified
as C. neoformans.
Principles of the Procedure
The seed extract contains caffeic acid, which serves as a References
1. Staib. 1962. Z. Hyg. Infekt. Med. Mikrobiol. Immunol. 148:466.
substrate for phenol oxidase, an enzyme present in the cell 2. Warren and Hazen. 1999. In Murray, Baron, Pfaller, Tenover and Yolken (ed.), Manual of clinical
microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
wall of C. neoformans. The subsequent enzymatic reaction 3. Shields and Ajello. 1966. Science 151:208.
produces the brown pigment melanin, resulting in tan to
brown pigmentation of the yeast colonies. C. neoformans is Availability
the only species known to produce this enzyme, although with BBL™ Bird Seed Agar
occasional isolates (particularly serotype C) the production of Cat. No. 297875 Prepared Plates – Pkg. of 10*
phenol oxidase may have to be induced.2 297096 Prepared Pour Tubes (20 mL) – Pkg. of 10
*Store at 2-8°C.
Bismuth Sulfite Agar

Intended Use illnesses result from consumption of raw, undercooked or
Bismuth Sulfite Agar is a highly selective medium used for improperly processed foods contaminated with Salmonella.
isolating Salmonella spp., particularly Salmonella Typhi, from Many cases of Salmonella-related gastroenteritis are due to
food and clinical specimens. improper handling of poultry products. United States federal
guidelines require various poultry products to be routinely
Summary and Explanation monitored before distribution for human consumption but
Salmonellosis continues to be an important public health contaminated food samples often elude monitoring.
problem worldwide, despite efforts to control the prevalence of Bismuth Sulfite Agar is a modification of the Wilson and Blair2-4
Salmonella in domesticated animals. Infection with nontyphi formula. Wilson5,6 and Wilson and Blair2-4 clearly showed
Salmonella often causes mild, self-limiting illness.1 Typhoid the superiority of Bismuth Sulfite medium for isolation of
fever, caused by S. Typhi, is characterized by fever, headache, S. Typhi. Cope and Kasper7 increased their positive findings
diarrhea and abdominal pain, and can produce fatal respira- of typhoid from 1.2 to 16.8% among food handlers and from
tory, hepatic, splenic and/or neurological damage. These 8.4 to 17.5% among contacts with Bismuth Sulfite Agar.
79
Difco Manual Sect III Ba.indd 79 3/16/09 3:46:30 PM

Section III
B Bismuth Sulfite Agar, cont.
Employing this medium in the routine laboratory examination Bismuth Sulfite Agar is valuable when investigating outbreaks of
of fecal and urine specimens, these same authors8 obtained Salmonella spp., especially S. Typhi.17-19
40% more positive isolations of S. Typhi than were obtained
Bismuth Sulfite Agar is used for the isolation of S. Typhi and
on Endo medium. Gunther and Tuft,9 employing various
other Salmonella from food, feces, urine, sewage and other
media in a comparative way for the isolation of typhoid from
infectious materials. The typhoid organism grows luxuriantly
stool and urine specimens, found Bismuth Sulfite Agar most
on the medium, forming characteristic black colonies, while
productive. On Bismuth Sulfite Agar, they obtained 38.4%
gram-positive bacteria and members of the coliform group
more positives than on Endo Agar, 33% more positives than
are inhibited. This inhibitory action of Bismuth Sulfite Agar
on Eosin Methylene Blue Agar, and 80% more positives than
toward gram-positive and coliform organisms permits the use
on the Desoxycholate media. These workers found Bismuth
of a much larger inoculum than possible with other media
Sulfite Agar to be superior to Wilson’s original medium.
employed for similar purposes in the past. The use of larger
Bismuth Sulfite Agar was stable, sensitive and easier to
inocula greatly increases the possibility of recovering the
prepare. Green and Beard,10 using Bismuth Sulfite Agar, claimed
pathogens, especially when they are present in relatively small
that this medium successfully inhibited sewage organisms.
numbers. Small numbers of organisms may be encountered in
The value of Bismuth Sulfite Agar as a plating medium after
the early course of the disease or in the checking of carriers
enrichment has been demonstrated by Hajna and Perry.11
and releases.
Since these earlier references to the use of Bismuth Sulfite
Agar, this medium has been generally accepted as routine for Principles of the Procedure
the detection of most Salmonella. The value of the medium is In Bismuth Sulfite Agar, beef extract and peptone provide
demonstrated by the many references to the use of Bismuth Sulfite nitrogen, vitamins and minerals. Dextrose is an energy source.
Agar in scientific publications, laboratory manuals and texts. Disodium phosphate is a buffering agent. Bismuth sulfite
indicator and brilliant green are complementary in inhibiting
For food testing, the use of Bismuth Sulfite Agar is specified
gram-positive bacteria and members of the coliform group,
for the isolation of pathogenic bacteria from raw and pasteur-
while allowing Salmonella to grow luxuriantly. Ferrous
ized milk, cheese products, dry dairy products, cultured milks
sulfate is included for detection of H2S production. When
and butter.1,12-14 The use of Bismuth Sulfite Agar is also recom-
H2S is present, the iron in the formula is precipitated, giving
mended for use in testing clinical specimens.15,16 In addition,
Uninoculated Salmonella Typhi

User Quality Control Plate ATCC™ 19430
Identity Specifications
Difco™ Bismuth Sulfite Agar
Dehydrated Appearance: Light beige to light green, free-flowing, homo-
geneous.
Solution: 5.2% solution, soluble in purified water upon
boiling. Solution is light green, opaque with a
flocculent precipitate that can be dispersed by
swirling contents of flask.
Prepared Appearance: Light gray-green to medium green, opaque with
a flocculent precipitate.
Reaction of 5.2%
Solution at 25°C: pH 7.7 ± 0.2
Cultural Response
Prepare the medium per label directions. Inoculate and incubate at
35 ± 2°C for 40-48 hours.
INOCULUM COLONY
ORGANISM ATCC™ CFU RECOVERY COLOR
Enterococcus faecalis 29212 103 Marked to –
complete inhibition Salmonella
Escherichia coli 25922 103 Partial inhibition Brown to green Typhimurium
ATCC™ 14028
Salmonella enterica
subsp. enterica
serotype Typhi 19430 102-103 Good Black with sheen
Salmonella enterica Black or greenish-gray,
subsp. enterica may or may not
serotype Typhimurium 14028 102-103 Good have sheen
80

Bismuth Sulfite Agar, cont.
positive cultures the characteristic brown to black color with Generally, Shigella spp. other than S. flexneri and S. sonnei are
metallic sheen. Agar is the solidifying agent. inhibited. S. flexneri and S. sonnei strains that do grow on this
medium produce brown to green, raised colonies with depressed
Formula centers and exhibit a crater-like appearance.
Approximate Formula* Per Liter
Escherichia coli is partially inhibited. Occasionally a strain
will be encountered that will grow as small brown or greenish
B
Beef Extract.................................................................. 5.0 g
Peptone..................................................................... 10.0 g glistening colonies. This color is confined entirely to the colony
Dextrose...................................................................... 5.0 g itself and shows no metallic sheen. A few strains of Enterobacter
Disodium Phosphate.................................................... 4.0 g
Ferrous Sulfate............................................................. 0.3 g aerogenes may develop on this medium, forming raised,
Bismuth Sulfite Indicator.............................................. 8.0 g mucoid colonies. Enterobacter colonies may exhibit a silvery
Agar.......................................................................... 20.0 g sheen, appreciably lighter in color than that produced by
Brilliant Green............................................................ 25.0 mg
*Adjusted and/or supplemented as required to meet performance criteria.
S. Typhi. Some members of the coliform group that produce
hydrogen sulfide may grow on the medium, giving colonies
Directions for Preparation from similar in appearance to S. Typhi. These coliforms may be readily
Dehydrated Product differentiated because they produce gas from lactose in dif-
1. Suspend 52 g of the powder in 1 L of purified water. Mix ferential media, for example, Kligler Iron Agar or Triple Sugar
thoroughly. Iron Agar. The hydrolysis of urea, demonstrated in Urea Broth
2. Heat with frequent agitation and boil for 1 minute to or on Urea Agar Base, may be used to identify Proteus sp.
completely dissolve the powder. DO NOT AUTOCLAVE. To isolate S. Typhi for agglutination or fermentation studies,
3. Evenly disperse the precipitate when dispensing. Use the pick characteristic black colonies from Bismuth Sulfite Agar and
medium the same day it is prepared. subculture them on MacConkey Agar. The purified colonies
4. Test samples of the finished product for performance using from MacConkey Agar may then be picked to differential tube
stable, typical control cultures. media such as Kligler Iron Agar, Triple Sugar Iron Agar or
other satisfactory differential media for partial identification. All
Procedure cultures that give reactions consistent with Salmonella spp. on
For isolation of Salmonella spp. from food, samples are these media should be confirmed biochemically as Salmonella
enriched and selectively enriched. Streak 10 µL of selective spp. before any serological testing is performed. Agglutination
enrichment broth onto Bismuth Sulfite Agar. Incubate plates tests may be performed from the fresh growth on the differential
for 24-48 hours at 35°C. Examine plates for the presence tube media or from the growth on nutrient agar slants inoculated
of Salmonella spp. Refer to appropriate references for the from the differential media. The growth on the differential tube
complete procedure when testing food samples.1,12-14 media may also be used for inoculating carbohydrate media for
For isolation of Salmonella spp. from clinical specimens, fermentation studies.
inoculate fecal specimens and rectal swabs onto a small area
of one quadrant of the Bismuth Sulfite Agar plate and streak Limitations of the Procedure
for isolation. This will permit the development of discrete 1. It is important to streak for well-isolated colonies. In heavy
colonies. Incubate plates at 35°C. Examine at 24 hours and again growth areas, S. Typhi appears light green and may be
at 48 hours for colonies resembling Salmonella spp. misinterpreted as negative growth for S. Typhi.20
2. S. Typhi and S. arizonae are the only enteric organisms to
For additional information about specimen preparation exhibit typical brown zones on the medium. Brown zones are
and inoculation of clinical specimens, consult appropriate not produced by other members of the Enterobacteriaceae.
references.15-19 However, S. arizonae is usually inhibited.20
3. Colonies on Bismuth Sulfite Agar may be contaminated with
Expected Results other viable organisms; therefore, isolated colonies should
The typical discrete S. Typhi surface colony is black and
be subcultured to a less selective medium (e.g., MacConkey
surrounded by a black or brownish-black zone which may be
Agar).20
several times the size of the colony. By reflected light, preferably
4. Typical S. Typhi colonies usually develop within 24 hours;
daylight, this zone exhibits a distinctly characteristic metallic
however, all plates should be incubated for a total of
sheen. Plates heavily seeded with S. Typhi may not show this
48 hours to allow growth of all typhoid strains.20
reaction except near the margin of the mass inoculation. In
5. DO NOT AUTOCLAVE. Heating this medium for a period
these heavy growth areas, this organism frequently appears as
longer than necessary to just dissolve the ingredients destroys
small light green colonies. This fact emphasizes the importance
its selectivity.
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.
81

Section III
B Brewer Anaerobic Agar, cont.
Directions for Preparation from the surface of the medium, and the oxygen in this space
Dehydrated Product reacts with the reducing agents to form an anaerobic
1. Suspend 58 g of the powder in 1 L of purified water. Mix environment.
thoroughly. 4. Incubate aerobically as desired.
2. Heat with frequent agitation and boil for 1 minute to com- For a complete discussion on anaerobic and microaerophilic
pletely dissolve the powder. bacteria from clinical specimens, refer to the appropriate
3. Autoclave at 121°C for 15 minutes. procedures outlined in the references.2-4 For the examination of
4. Test samples of the finished product for performance using anaerobic bacteria in food refer to standard methods.6-8
stable, typical control cultures.
Expected Results
Procedure Refer to appropriate references and procedures for results.
Standard Petri Dishes2
1. Inoculate a properly obtained specimen onto the medium, Limitations of the Procedure
and streak to obtain isolated colonies. 1. Clinical specimens must be obtained properly and trans-
2. Immediately incubate anaerobically at 35 ± 2°C. ported to the laboratory in a suitable anaerobic transport
3. Examine at 24 hours if incubating plates in an anaerobic container.2
chamber. Examine at 48 hours if incubating plates in an 2. The microbiologist must be able to verify quality control
anaerobic jar or pouch, or if using Brewer anaerobic dish of the medium and determine whether the environment is
cover. anaerobic.2
4. Extended incubation may be necessary to recover some 3. The microbiologist must perform aerotolerance testing on each
anaerobes. isolate recovered to ensure the organism is an anaerobe.2
Brewer Anaerobic Agar Plates
References
1. Dispense 50-60 mL of Brewer Anaerobic Agar into a 1. Brewer. 1942. Science 95:587.
2. Isenberg and Garcia (ed.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd ed.
standard Petri dish. For best results use porous tops to American Society for Microbiology, Washington, D.C.
obtain a dry surface. 3. Baron, Peterson and Finegold. 1994. Bailey & Scott’s diagnostic microbiology, 9th ed. Mosby-Year
Book, Inc., St. Louis, Mo.
2. Inoculate the surface of the medium by streaking; avoid the 4. Murray, Baron, Jorgensen, Landry and Pfaller, (ed.). 2007. Manual of clinical microbiology, 9th ed.
American Society for Microbiology, Washington, D.C.
edges of the plates. 5. Smith. 1975. The pathogenic anaerobic bacteria, 2nd ed. Charles C. Thomas, Springfield, Ill.
6. Wehr and Frank (ed.). 2004. Standard methods for the microbiological examination of dairy products,
3. Replace the standard Petri dish lid with a sterile Brewer 17th ed. American Public Health Association, Washington, D.C.
7. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna-
anaerobic dish cover. The cover should not rest on the tional, Gaithersburg, Md.
Petri dish bottom. The inner glass ridge should seal against 8. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
4th ed. American Public Health Association, Washington, D.C.
the uninoculated periphery of the agar. It is essential that
the sealing ring inside the cover is in contact with the Availability
medium. This seal must not be broken before the end of Difco™ Brewer Anaerobic Agar
the incubation period. A small amount of air is caught over Cat. No. 227920 Dehydrated – 500 g
Brilliant Green Agar

Intended Use Formula
Brilliant Green Agar is a highly selective medium for the isola- Difco™ Brilliant Green Agar
tion of Salmonella other than S. Typhi from feces and other Approximate Formula* Per Liter
materials. Proteose Peptone No. 3.............................................. 10.0 g
Yeast Extract................................................................ 3.0 g
Lactose...................................................................... 10.0 g
Summary and Explanation Saccharose................................................................. 10.0 g
Brilliant Green Agar was first described by Kristensen et al. in Sodium Chloride.......................................................... 5.0 g
Agar.......................................................................... 20.0 g
1925.1 Their formulation was modified slightly by Kauffmann in Brilliant Green............................................................ 12.5 mg
1935.2 The medium is included in procedures for the examination Phenol Red.................................................................. 0.08 g
of water and wastewater.3 *Adjusted and/or supplemented as required to meet performance criteria.

Brilliant green dye inhibits gram-positive bacteria and a major-
ity of gram-negative bacilli. Phenol red serves as a pH indicator
and yields a yellow color as a result of acid production in the
fermentation of the lactose and/or sucrose in the medium.
96
Difco Manual Sect III Bb.indd 96 3/16/09 3:44:19 PM

Brilliant Green Agar Modified
Directions for Preparation from User Quality Control

Dehydrated Product
1. Suspend 58 g of the powder in 1 L of purified water. Mix Identity Specifications
thoroughly. Difco™ Brilliant Green Agar
B
Dehydrated Appearance: Pink, free-flowing, homogeneous.
2. Heat with frequent agitation and boil for 1 minute to
completely dissolve the powder. boiling. Solution is brownish-green, clear to very
3. Autoclave at 121°C for 15 minutes. slightly opalescent.
4. Test samples of the finished product for performance using Prepared Appearance: Orange-brown, very slightly to slightly opales-
stable, typical control cultures. cent.
Reaction of 5.8%
Procedure
Use standard procedures to obtain isolated colonies from Cultural Response
specimens. A less selective medium and a nonselective medium Difco™ Brilliant Green Agar
should also be streaked to increase the chance of recovery Prepare the medium per label directions. Inoculate and incubate at
when the population of gram-negative organisms is low and 35 ± 2°C for 18-24 hours.
to provide an indication of other organisms present in the INOCULUM COLONY
specimen. Incubate plates, protected from light, at 35 ± 2°C
Escherichia coli 25922 ~104 Poor Yellow-green
for 18-24 hours. If negative after 24 hours, reincubate an
Salmonella enterica
additional 24 hours. subsp. enterica
serotype Enteritidis 13076 30-300 Good Red
Expected Results Salmonella enterica
Typical colonial morphology on Brilliant Green Agar is as subsp. enterica None to
serotype Typhi 19430 30-300 poor Red
follows:
Salmonella enterica
Salmonella (other than subsp. enterica
S. Typhi and S. Paratyphi)....... White to red, opaque colonies serotype Typhimurium 14028 30-300 Good Red
. ............................................ surrounded by red zones in the
. ............................................ medium Staphylococcus Marked
aureus 25923 ~104 inhibition –
S. Typhi and S. Paratyphi........ No growth to trace growth
Shigella.................................. No growth to trace growth
Escherichia coli and
Availability
Enterobacter/Klebsiella........... Yellow to greenish-yellow Difco™ Brilliant Green Agar
. ............................................ colonies surrounded by intense EP SMWW
. ............................................ yellow-green zones in medium Cat. No. 228530 Dehydrated – 500 g
Proteus.................................. No growth to trace growth BBL™ Brilliant Green Agar
Pseudomonas......................... Pink to red colonies EP SMWW
Gram-positive bacteria........... No growth to trace growth Cat. No. 295963 Prepared Plates – Pkg. of 20*
*Store at 2-8°C.
References
1. Kristensen, Lester and Jurgens. 1925. Br. J. Exp. Pathol. 6:291.
2. Kauffmann. 1935. Z. Hyg. Infektionskr. 117:26.
3. Eaton, Rice and Baird (ed.). 2005. Standard methods for the examination of water and wastewater,
21st ed., online. American Public Health Association, Washington, D.C.
Brilliant Green Agar Modified

Intended Use Salmonella enterica grows well on Brilliant Green Agar Modified
Brilliant Green Agar Modified is used for isolating Salmonella compared to Desoxycholate Citrate Agar.2
from water, sewage and foodstuffs. Brilliant Green Agar Modified is recommended for the isolation
of Salmonella, other than Salmonella Typhi, from water and
Summary and Explanation associated materials3 and meat and meat products.4 It is
Kampelmacher1 proposed the formula for a selective medium recommended by the British Poultry Meat Society5 for the
to isolate Salmonella from pig feces and minced meat. Brilliant examination of poultry and poultry products. The recom-
Green Agar Modified is more selective than Desoxycholate mended procedures include using complementary selective
Citrate Agar and other brilliant green media, and inhibits the culture media and techniques to increase the likelihood of
growth of Pseudomonas aeruginosa and partially inhibits isolating multiple serotypes of Salmonella from samples.6
the growth of Proteus spp. which may resemble Salmonella.
97
Difco Manual Sect III Bb.indd 97 3/16/09 3:44:21 PM

Hektoen Enteric Agar
Hektoen Enteric Agar

Intended Use HE Agar is currently recommended as one of several plating
Hektoen Enteric (HE) Agar is a modrately selective medium media for the culture of Enterobacteriaceae from stool speci-
used in qualitative procedures for the isolation and cultivation mens.5 Foods containing poultry, eggs or dairy products are the
of gram-negative enteric microorganisms, especially Shigella, most frequent vehicles for foodborne salmonellosis, and a variety
from a variety of clinical and nonclinical specimens. of procedures have been developed using Hektoen Enteric Agar
as part of the multi-step procedure to isolate Salmonella.6-9
Summary and Explanation
Through the years many media have been devised for the Principles of the Procedure
isolation of enteric pathogens. These various formulations have The selective nature of Hektoen Enteric Agar is due to the
differed in their degree of selectivity for the pathogenic species. incorporation of bile salts in the formulation. These substances
Some were designed to isolate and differentiate Shigella species inhibit gram-positive organisms but also can be toxic for some
whereas others were formulated for the selective isolation of the gram-negative strains.
salmonellae. Media that isolated a broader spectrum of enteric This medium contains three carbohydrates, lactose, sucrose
pathogens were less inhibitory to members of the nonpathogenic (saccharose) and salicin, for optimal differentiation of en-
intestinal flora. teric pathogens by the color of the colonies and of the medium
Hektoen Enteric Agar was developed in 1967 by King and adjacent to the colonies. The lactose concentration is higher
Metzger of the Hektoen Institute in order to increase the than in many other media used for enterics in order to aid
frequencies of isolation of Shigella and Salmonella organisms in the visualization of enteric pathogens and minimize the
when compared with their recovery on other media frequently problem of delayed lactose fermentation. Ferric ammo-
utilized in clinical laboratories at that time.1-3 This medium nium citrate and sodium thiosulfate in the medium enable the
is considered to be moderately selective, and is particularly detection of hydrogen sulfide production, thereby aiding in
useful in the isolation of Shigella species. The present formulation the differentiation process due to the production of black-
differs from that of the original in that sodium desoxycholate has centered colonies. The indicator system, consisting of acid
been eliminated and the concentration of bile salts is reduced. fuchsin and bromthymol blue, has a lower toxicity than that
Additionally, the peptone concentrations have been increased in of many other enteric media, resulting in improved recovery of
order to offset the inhibitory effects of the bile salts.4 enteric pathogens.
User Quality Control Salmonella Typhimurium

ATCC™ 14028
Shigella flexneri
ATCC™ 12022
Identity Specifications H
Difco™ Hektoen Enteric Agar -
Dehydrated Appearance: Light beige, may have a slight green cast, free-
flowing, homogeneous.
K
boiling. Solution is brown with greenish cast,
slightly opalescent.
Prepared Appearance: Green with yellowish cast, slightly opalescent.
Reaction of 7.6%
Cultural Response
Difco™ Hektoen Enteric Agar
Prepare the medium per label directions. Inoculate and incubate at
35 ± 2°C for 18-24 hours.
INOCULUM COLONY
ORGANISM ATCC™ CFU RECOVERY color
Enterococcus Marked to
faecalis 29212 103 complete inhibition –
Escherichia coli 25922 10 -3×10 2
Partial
2
Salmon-orange,
inhibition may have bile
precipitate
Salmonella enterica
subsp. enterica Greenish blue,
serotype Typhimurium 14028 102-3×102 Good w/black centers
Shigella flexneri 12022 102-3×102 Good Greenish blue
265
Difco Manual Sect III HIJK.indd 265 3/16/09 4:16:51 PM

Section III
H-K Hektoen Enteric Agar, cont.
Formula Limitation of the Procedure

Difco™ Hektoen Enteric Agar Proteus species may resemble salmonellae or shigellae. Further
Approximate Formula* Per Liter testing should be conducted to confirm the presumptive identi-
Proteose Peptone....................................................... 12.0 g fication of organisms isolated on this medium.
Yeast Extract................................................................ 3.0 g
Bile Salts No. 3............................................................. 9.0 g
Lactose...................................................................... 12.0 g References
Saccharose................................................................. 12.0 g 1. King and Metzger. 1967. Abstr. M99, p. 77. Bacteriol. Proc. Am. Soc. Microbiol. 1967.
2. King and Metzger. 1968. Appl. Microbiol. 16:577.
Salicin.......................................................................... 2.0 g 3. King and Metzger. 1968. Appl. Microbiol. 16:579.
Sodium Chloride.......................................................... 5.0 g 4. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria,
Sodium Thiosulfate...................................................... 5.0 g vol. l. Williams & Wilkins, Baltimore, Md.
5. Murray, Baron, Jorgensen, Landry and Pfaller, (ed.). 2007. Manual of clinical microbiology, 9th ed.
Ferric Ammonium Citrate............................................. 1.5 g American Society for Microbiology, Washington, D.C.
Agar.......................................................................... 14.0 g 6. Wehr and Frank. (ed.). 2004. Standard Methods for the examination of dairy products, 17th ed.
Bromthymol Blue....................................................... 65.0 mg American Public Health Association, Washington, D.C.
Acid Fuchsin................................................................. 0.1 g tional, Gaithersburg, Md.
*Adjusted and/or supplemented as required to meet performance criteria. 8. Horwitz (ed.). 2007. Official methods of analysis of AOAC International, 18th ed., online. AOAC
International, Gaithersburg, Md.
9. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
Directions for Preparation from 4th ed. American Public Health Association, Washington, D.C.
Dehydrated Product
1. Suspend 76 g of the powder in 1 L of purified water. Mix Availability
thoroughly. Difco™ Hektoen Enteric Agar
AOAC BAM BS12 CCAM CMPH2 COMPF MCM9 SMD
2. Heat to boiling with frequent agitation to dissolve
Cat. No. 285340 Dehydrated – 500 g
completely. Do not overheat. DO NOT AUTOCLAVE. 285310 Dehydrated – 2 kg
3. Cool to 45-50°C and use immediately. 285320 Dehydrated – 10 kg
4. Test samples of the finished product for performance using BBL™ Hektoen Enteric Agar
stable, typical control cultures. AOAC BAM BS12 CCAM CMPH2 COMPF MCM9 SMD
United States and Canada
Procedure Cat. No. 221365 Prepared Plates – Pkg. of 20*
Use standard procedures to obtain isolated colonies from speci- 221366 Prepared Plates – Ctn. of 100*
mens. A nonselective medium should also be streaked to increase Europe
Cat. No. 254009 Prepared Plates – Pkg. of 20*
the chance of recovery when the population of gram-negative
254075 Prepared Plates – Ctn. of 120*
organisms is low and to provide an indication of other organisms
Mexico
present in the specimen. Cat. No. 224450 Prepared Plates – Pkg. of 10*
Incubate plates, protected from light, at 35 ± 2°C for 18-24 BBL™ Hektoen Enteric Agar//Salmonella Shigella Agar
hours. Cat. No. 297426 Prepared I Plate™ Dishes – Pkg. of 20*
BBL™ Hektoen Enteric Agar//XLD Agar
Expected Results Cat. No. 295646 Prepared I Plate™ Dishes – Pkg. of 20*
After incubation most plates will show an area of confluent *Store at 2-8°C.
growth. Because the streaking procedure is, in effect, a “dilution”
technique, diminishing numbers of microorganisms are deposited
on the streaked areas. Consequently, one or more of these areas
should exhibit isolated colonies of the organisms contained in
the specimen. Better isolation is obtained due to the inhibitory
action of the medium.
Hemo (Haemophilus) Identification (ID) QUAD Plate

Intended Use ship with Staphylococcus aureus.1 In 1921, Thjotta and Avery
The Hemo (Haemophilus) Identification (ID) Quad Plate (with described the growth factor from blood as X factor and the other
growth factors) is used for the differentiation and identification as V factor.2 The X factor was found in hemin and later identified
of Haemophilus species based on requirements for growth fac- as protoporphyrin IX, or protoheme, and V factor was identified
tors X and/or V and hemolytic reactions. as nicotinamide adenine dinucleotide (NAD).3,4
Pathogenic Haemophilus species may be differentiated and
Summary and Explanation presumptively identified by determining in vitro growth
In 1917, Davis reported that bacteria causing influenza required requirements for X and/or V factors and by determining
two factors for in vitro growth, one from hemoglobin and hemolytic reactions.5
another that the organism derived through a satellite relation-
266
Difco Manual Sect III HIJK.indd 266 3/16/09 4:16:53 PM

Section III
M MYP Agar, cont.
Formulae Procedure
Difco™ MYP Agar Consult appropriate references.4-6
Approximate Formula* Per 900 mL
Beef Extract.................................................................. 1.0 g Expected Results
Peptone..................................................................... 10.0 g
D-Mannitol................................................................ 10.0 g Consult appropriate references.4-6
Sodium Chloride........................................................ 10.0 g
Phenol Red................................................................ 25.0 mg References
Agar.......................................................................... 15.0 g 1. Mossel, Koopman and Jongerius. 1967. Appl. Microbiol. 15:650.
2. Donovan. 1958. J. Appl. Bacteriol. 21:100.
Difco™ Antimicrobic Vial P 3. Coliner. 1948. J. Bacteriol. 55:777.
Approximately 30,000 units polymyxin B per vial. tional, Gaithersburg, Md.
*Adjusted and/or supplemented as required to meet performance criteria. 5. Bennett and Belay. 2001. In Downes and Ito (ed.), Compendium of methods for the microbiological
examination of foods, 4th ed. American Public Health Association, Washington, D.C.
6. Horwitz (ed.). 2007. Official methods of analysis of AOAC International, 18th ed., online. AOAC
Directions for Preparation from International, Gaithersburg, Md.
Dehydrated Product
Difco™ MYP Agar Availability
Difco™ MYP Agar
1. Suspend 46 g of the powder in 900 mL of purified water.
AOAC BAM COMPF ISO USDA
Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to com-
Europe
pletely dissolve the powder. Cat. No. 257004 Prepared Plates – Pkg. of 20*
3. Dispense 225 mL into 500 mL flasks. Japan
4. Autoclave at 121°C for 15 minutes. Cool to 45-50°C. Cat. No. 251264 Prepared Plates – Pkg. of 20*
5. Aseptically add 12.5 mL Egg Yolk Enrichment 50% and Difco™ Antimicrobic Vial P
4.1 mL Antimicrobic Vial P rehydrated with 5 mL sterile AOAC BAM COMPF ISO USDA
water (25,000 units of polymyxin B). Mix thoroughly. Cat. No. 232681 Vial – 6 × 10 mL*
6. Test samples of the finished product for performance using
Difco™ Egg Yolk Enrichment 50%
stable, typical control cultures.
AOAC BAM COMPF ISO USDA
Difco™ Antimicrobic Vial P (Polymyxin B) Cat. No. 233471 Tube – 12 × 10 mL*
233472 Bottle – 6 × 100 mL*
1. To rehydrate, aseptically add 5 mL sterile purified water
*Store at 2-8°C.
(to achieve the desired concentration for MYP Agar).
2. Rotate in an end-over-end motion to dissolve the contents
completely.
MacConkey Agars
MacConkey Agar • MacConkey Agar Base
MacConkey Agar without Crystal Violet
MacConkey Agar without Crystal Violet or Salt
MacConkey Agar without Salt
Intended Use of staphylococci and enterococci. The medium can be used also
MacConkey agars are slightly selective and differential plating to separate Mycobacterium fortuitum and M. chelonae from
media mainly used for the detection and isolation of gram-nega- other rapidly growing mycobacteria.
tive organisms from clinical,1-3 dairy,4 food,5-7 water,8 pharma- MacConkey Agar without Crystal Violet or Salt and MacConkey
ceutical,9-11 cosmetic,6,7 and other industrial sources. Agar without Salt are used for isolating and differentiating
MacConkey Agar is used for isolating and differentiating gram-negative bacilli while suppressing the swarming of most
lactose-fermenting from lactose-nonfermenting gram-negative Proteus species.
enteric bacilli. MacConkey Agar meets United States Pharmacopeia (USP),
MacConkey Agar Base is used with added carbohydrate in European Pharmacopoeia (EP) and Japanese Pharmacopoeia
differentiating coliforms based on fermentation reactions. (JP)9-11 performance specifications, where applicable.
MacConkey Agar without Crystal Violet is used for isolating and
differentiating enteric microorganisms while permitting growth
328
Difco Manual Sect III Ma.indd 328 3/16/09 4:41:34 PM

MacConkey Agars, cont. M
User Quality Control
NOTE: Differences in the Identity Specifications and Cultural Response testing for media offered as both Difco™ and BBL™ brands may reflect differences in the
development and testing of media for industrial and clinical applications, per the referenced publications.
Difco™ MacConkey Agar Difco™ MacConkey Agar without Crystal Violet
Dehydrated Appearance: Pink to pinkish beige, free-flowing, homoge- Dehydrated Appearance: Pinkish beige, free-flowing, homogeneous.
neous. Solution: 5.2% solution, soluble in purified water upon
Solution: 5.0% solution, soluble in purified water upon boiling. Solution is reddish orange, clear to very
boiling. Solution is reddish-purple, slightly opal- slightly opalescent.
escent. Prepared Appearance: Reddish orange, clear to very slightly opales-
Prepared Appearance: Pinkish red, slightly opalescent. cent.
Reaction of 5.0% Reaction of 5.2%
Solution at 25°C: pH 7.1 ± 0.2 Solution at 25°C: pH 7.4 ± 0.2
Difco MacConkey Agar Base
™
Difco MacConkey Agar without Salt
™
Dehydrated Appearance: Pinkish beige, free-flowing, homogeneous. Dehydrated Appearance: Beige to pinkish beige, free-flowing, homoge-
Solution: 4.0% solution, soluble in purified water upon neous.
boiling. Solution is red, very slightly to slightly Solution: 4.7% solution, soluble in purified water upon
opalescent. boiling. Solution is reddish orange, slightly
Prepared Appearance: Red, slightly opalescent. opalescent.
Reaction of 4.0% Prepared Appearance: Reddish orange, slightly opalescent.
Solution at 25°C: pH 7.1 ± 0.2 Reaction of 4.7%
Cultural Response
Difco™ MacConkey Agar Difco™ MacConkey Agar without Crystal Violet
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C Prepare the medium per label directions. Inoculate and incubate at
for 18-24 hours (incubate E. coli ATCC 25922 for 40-48 hours). For E. coli 35 ± 2°C for 18-48 hours.
ATCC 8739, inoculate in duplicate and incubate one plate at 30-35°C for
INOCULUM COLOny BILE
18-24 hours and the other plate at 35-37°C for 18-72 hours. ORGANISM ATCC™ CFU RECOVERY COLOr PPT.
INOCULUM Colony BILE Enterococcus faecalis 29212 30-300 Good Red –
ORGANISM ATCC™ CFU RECOVERY COLOR PPT.
Escherichia coli 25922 30-300 Good Pink to red –
Enterococcus faecalis 29212 103 Marked to – –
complete inhibition Proteus mirabilis 12453 30-300 Good Colorless –
Escherichia coli 25922 30-300 Good Pink to red + Salmonella enterica
subsp. enterica
Proteus mirabilis 12453 30-300 Good Colorless – serotype Typhimurium 14028 30-300 Good Colorless –
Salmonella enterica Staphylococcus aureus 25923 30-300 Good Pink to red –
subsp. enterica
serotype Typhimurium 14028 30-300 Good Colorless – Difco™ MacConkey Agar without Salt
Escherichia coli 8739 <100 Growth Pink + Prepare the medium per label directions. Inoculate and incubate at
(18-24 hours to red 35 ± 2°C for 18-48 hours.
at 30-35°C)
INOCULUM Colony BILE
Escherichia coli 8739 <100 Growth Pink + ORGANISM ATCC™ CFU RECOVERY COLOR PPT.
(18-72 hours to red
Enterococcus faecalis 33186 30-300 Good Red –
at 35-37°C)
Escherichia coli 25922 30-300 Good Pink to red –
Difco™ MacConkey Agar Base Proteus mirabilis 12453 30-300 Good Colorless, –
Prepare the medium per label directions without and with 1% added no swarming
lactose. Inoculate and incubate at 35 ± 2°C for 18-24 hours.
Salmonella enterica
INOCULUM Colony Color BILE subsp. enterica
ORGANISM ATCC™ CFU RECOVERY Plain w/Lactose PPT. serotype Typhimurium 14028 30-300 Good Colorless –
Enterococcus 29212 103 Marked to – – – Shigella flexneri 12022 30-300 Good Colorless –
faecalis complete
inhibition
Continued
Escherichia coli 25922 30-300 Good Colorless Pink +
to red (w/lactose)
Proteus
mirabilis 12453 30-300 Good Colorless Colorless –
Salmonella
enterica subsp.
enterica serotype
Typhimurium 14028 30-300 Good Colorless Colorless –
329

Section III
M MacConkey Agars, cont.
BBL™ MacConkey Agar BBL™ MacConkey Agar without Crystal Violet
Dehydrated Appearance: Fine, homogenous, may contain dark Dehydrated Appearance: Fine, homogeneous, free of extraneous
particles. material.
Solution: 5.0% solution, soluble in purified water Solution: 5.2% solution, soluble in purified water
upon boing. Solution is medium to dark, upon boiling. Solution is medium, red-
rose to brown-rose with or without a trace orange to red-rose, slightly hazy to hazy.
orange tint; clear to slightly hazy. Prepared Appearance: Medium, red-orange to red-rose, slightly
Prepared Appearance: Medium to dark, rose to brown-rose with or hazy to hazy.
without a trace orange tint; clear to slightly Reaction of 5.2%
hazy. Solution at 25°C: pH 7.4 ± 0.2
Reaction of 5.0%
BBL MacConkey Agar without Crystal Violet or Salt
™
Dehydrated Appearance: Fine, homogeneous, free of extraneous
BBL™ MacConkey Agar (prepared) material.
Appearance: Medium-dark, rose-tan and trace hazy. Solution: 4.37% solution, soluble in purified water
Reaction at 25°C: pH 7.1 ± 0.2 upon boiling. Solution is medium, red-
orange to red-rose, slightly hazy to hazy.
Prepared Appearance: Medium, red-orange to red-rose, slightly
hazy to hazy.
Reaction of 4.37%
Cultural Response
BBL™ MacConkey Agar BBL™ MacConkey Agar without Crystal Violet
Prepare the medium per label directions. Inoculate and incubate at Prepare the medium per label directions. Inoculate and incubate at
35 ± 2°C for 48 hours. For E. coli ATCC 8739, inoculate in duplicate and 35 ± 2°C for 18-24 hours and up to 48 hours if necessary (up to 11 days
incubate one plate at 30-35°C for 18-24 hours and the other plate at for M. fortuitum).
35-37°C for 18-72 hours.
INOCULUM Colony BILE
INOCULUM COLOny BILE ORGANISM ATCC™ CFU RECOVERY Color PPT.
ORGANISM ATCC™ CFU RECOVERY COLOr PPT.
Enterococcus faecalis 29212 103-104 Good Rose red –
Enterococcus faecalis 29212 104-105 Partial to – – Escherichia coli 25922 103-104 Good Pink to –
complete inhibition rose red
Escherichia coli 25922 103-104 Good Red to + Mycobacterium
rose-red fortuitum 6841 103-104 Good Rose red –
Proteus mirabilis 12453 103-104 Good Colorless – Salmonella enterica
Salmonella enterica subsp. enterica
subsp. enterica serotype Typhimurium 14028 103-104 Good Colorless –
serotype Typhimurium 14028 103-104 Good Colorless – Staphylococcus aureus 25923 103-104 Good Pink to –
Shigella flexneri 12022 103-104 Good Colorless – rose red
Escherichia coli 8739 <100 Growth Red to +
(18-24 hours rose-red
BBL™ MacConkey Agar without Crystal Violet or Salt
at 30-35°C) Prepare the medium per label directions. Inoculate and incubate at
35 ± 2°C for 18-24 hours and up to 48 hours if necessary (up to 11 days
Escherichia coli 8739 <100 Growth Red to + for M. fortuitum).
(18-72 hours rose-red
at 35-37°C) INOCULUM COLOny BILE
BBL™ MacConkey I Agar (prepared) Enterococcus faecalis 29212 103-104 Good Rose red –
Inoculate and incubate at 35 ± 2°C for 18-24 hours. Incubate E. coli Escherichia coli 25922 103-104 Good Pink to –
ATCC 8739 at 30-35°C for 18-72 hours. rose red
INOCULUM COLOny BILE Proteus mirabilis 12453 103-104 Good Colorless, –
no swarming
Enterococcus faecalis 29212 104-105 Partial to – – Salmonella enterica
complete inhibition subsp. enterica
Escherichia coli 25922 103-104 Good Red to + serotype Typhimurium 14028 103-104 Good Colorless –
rose-red
Pseudomonas Greenish
aeruginosa 10145 103-104 Good yellow –
Salmonella enterica
subsp. enterica
serotype Typhimurium 14028 103-104 Good No reaction –
Shigella dysenteriae 9361 10 -10
3 4
Good No reaction –
Escherichia coli 8739 10-100 Growth Red to +
rose-red
330

MacConkey Agars, cont. M
MacConkey Agar w/o CV Staphylococci produce pale pink to red colonies and enterococci
Uninoculated Escherichia coli
Plate ATCC™ 25922
produce compact tiny red colonies either on or beneath the
surface of the medium. The medium is used also to separate
Mycobacterium fortuitum and M. chelonae from other rapidly
growing mycobacteria.13,14
MacConkey Agar without Crystal Violet or Salt and MacConkey
Agar without Salt (which also lacks crystal violet) are differential
media used for isolating and cultivating gram-negative enteric
organisms and gram-positive cocci from waters, feces and other
sources suspected of containing these organisms, as well as limit-
ing the swarming of Proteus species.

Peptones are sources of nitrogen and other nutrients. Yeast
extract is a source of trace elements, vitamins, amino acids and
carbon. 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, oxgall and crystal violet are selective
Proteus Salmonella agents that inhibit growth of gram-positive organisms. Sodium
mirabilis Typhimurium
ATCC™ 12453 ATCC™ 14028 chloride maintains osmotic balance in the medium. Magnesium
sulfate is a source of divalent cations. Agar is the solidifying
agent.
MacConkey Agar is based on the bile salt-neutral red-lactose
Formulae
agar of MacConkey.12
Difco™ MacConkey Agar
The original MacConkey medium was used to differentiate Approximate Formula* Per Liter
strains of Salmonella typhosa from members of the coliform Pancreatic Digest of Gelatin....................................... 17.0 g
Peptones (meat and casein).......................................... 3.0 g
group. Formula modifications improved the growth of Shigella Lactose...................................................................... 10.0 g
and Salmonella strains. These modifications included the ad- Bile Salts No. 3............................................................. 1.5 g
dition of 0.5% sodium chloride, decreased agar content, and Sodium Chloride.......................................................... 5.0 g
Agar.......................................................................... 13.5 g
altered bile salts and neutral red concentrations. The formula Neutral Red.................................................................. 0.03 g
improvements gave improved differential reactions between Crystal Violet............................................................... 1.0 mg
these enteric pathogens and the coliform group. Difco™ MacConkey Agar Base
MacConkey Agar contains crystal violet and bile salts that inhibit Consists of the same ingredients without the lactose.
gram-positive organisms and allow gram-negative organisms BBL™ MacConkey Agar
to grow. Isolated colonies of coliform bacteria are brick red in Approximate Formula* Per Liter
Pancreatic Digest of Gelatin....................................... 17.0 g
color and may be surrounded by a zone of precipitated bile. This Peptones (meat and casein).......................................... 3.0 g
bile precipitate is due to a local pH drop around the colony due Lactose...................................................................... 10.0 g
to lactose fermentation. Colonies that do not ferment lactose Bile Salts...................................................................... 1.5 g
Sodium Chloride.......................................................... 5.0 g
(such as typhoid, paratyphoid and dysentery bacilli) remain Agar.......................................................................... 13.5 g
colorless. When lactose nonfermenters grow in proximity to Neutral Red.................................................................. 0.03 g
coliform colonies, the surrounding medium appears as cleared Crystal Violet............................................................... 1.0 mg
areas. MacConkey Agar is listed as one of the recommended Difco™ MacConkey Agar without Crystal Violet
media for the isolation of E. coli from nonsterile pharmaceutical Approximate Formula* Per Liter
products.9 Peptone..................................................................... 20.0 g
Lactose...................................................................... 10.0 g
MacConkey Agar Base is prepared without added carbohy- Bile Salts...................................................................... 5.0 g
drates, which permits their addition either individually or in Agar.......................................................................... 12.0 g
combination. It is recommended that carbohydrates such as Neutral Red.................................................................. 0.05 g
sucrose or lactose be added in a concentration of 1% to the
basal medium.
MacConkey Agar without 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.
331

Section III
M MacConkey Agars, cont.
BBL™ MacConkey Agar without Crystal Violet For pharmaceutical samples, refer to USP General Chapter <62>
Approximate Formula* Per Liter for details on the examination of nonsterile products and tests
Pancreatic Digest of Casein........................................ 10.0 g
Peptic Digest of Animal Tissue.................................... 10.0 g
for isolating E. coli using MacConkey Agar.9
Lactose...................................................................... 10.0 g
Bile Salts...................................................................... 5.0 g Procedure
Sodium Chloride.......................................................... 5.0 g Refer to appropriate standard references for details on test
Agar.......................................................................... 12.0 g
Neutral Red.................................................................. 0.05 g methods to obtain isolated colonies from specimens or samples
Difco™ MacConkey Agar without Salt
using MacConkey Agar.1-11 Incubate plates for 18-72 hours
Approximate Formula* Per Liter at 35 ± 2°C under appropriate atmospheric conditions, or as
Peptone..................................................................... 20.0 g instructed in the standard reference.1-11
Lactose...................................................................... 10.0 g
Bile Salts...................................................................... 5.0 g Expected Results
Agar.......................................................................... 12.0 g
Neutral Red................................................................ 75.0 mg Lactose-fermenting organisms grow as pink to brick-red colonies
BBL MacConkey Agar without Crystal Violet or Salt
™ with or without a zone of precipitated bile. Lactose-nonferment-
Approximate Formula* Per Liter ing organisms grow as colorless or clear colonies.
Pancreatic Digest of Gelatin....................................... 10.0 g
Yeast Extract.............................................................. 10.0 g
Swarming by Proteus spp. is reduced on MacConkey agars
Lactose...................................................................... 10.0 g without salt.
Oxgall.......................................................................... 5.0 g
Magnesium Sulfate...................................................... 0.2 g On MacConkey Agar without Crystal Violet and MacConkey
Agar.......................................................................... 12.0 g agars without salt, staphylococci produce pale pink to red
Neutral Red................................................................ 75.0 mg colonies and enterococci produce tiny red colonies; these
organisms are inhibited on MacConkey Agar.
Directions for Preparation from On MacConkey Agar without Crystal Violet, potentially
Dehydrated Product pathogenic rapid growers of the M. fortuitum complex usually
1. Suspend the powder in 1 L of purified water: grow in 5-11 days, while the commonly saprophytic species
Difco™ MacConkey Agar – 50 g; are inhibited.3,13
BBL™ MacConkey Agar – 50 g; On MacConkey agars without salt, the swarming of Proteus
Difco™ MacConkey Agar Base – 40 g; is reduced.
Difco™ MacConkey Agar without Crystal Violet – 52 g;
BBL™ MacConkey Agar without Crystal Violet – 52 g; Limitations of the Procedure
BBL™ MacConkey Agar without Crystal Violet or Salt – 47.3 g; 1. Although MacConkey media are selective primarily for
Difco™ MacConkey Agar without Salt – 47 g. gram-negative enteric bacilli, biochemical and, if indicated,
Mix thoroughly. serological testing using pure cultures are recommended for
2. Heat with frequent agitation and boil for 1 minute to com- complete identification. Consult appropriate references for
pletely dissolve the powder. further information.1,3
3. Autoclave at 121°C for 15 minutes. 2. Incubation of MacConkey Agar plates under increased CO2
NOTE: If MacConkey Agar Base is to be used within 12 hours, has been reported to reduce the growth and recovery of a
omit autoclaving and gently boil medium for 5 minutes. Add 1% number of strains of gram-negative bacilli.14
carbohydrate before or after autoclaving, depending upon heat 3. Some strains of M. smegmatis from humans may grow on
lability. The surface of MacConkey agars without salt should MacConkey Agar without Crystal Violet, but these strains
be thoroughly air-dried prior to inoculation. can be differentiated from M. fortuitum complex by the
3-day arylsulfatase test.9
stable, typical control cultures. References
1. Murray, Baron, Jorgensen, Landry and Pfaller (eds.). 2007. Manual of clinical microbiology, 9th ed.
Sample Collection and Handling American Society for Microbiology, Washington, D.C.
2. Forbes, Sahm and Weissfeld. 2007. Bailey & Scott’s diagnostic microbiology, 12th ed. Mosby Elsevier,
For clinical specimens, refer to laboratory procedures for details St. Louis, Mo.
3. Isenberg and Garcia (eds.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd
on specimen collection and handling.1-3 ed., American Society for Microbiology, Washington, D.C.
4. Wehr and Frank (eds.). 2004. Standard methods for the examination of dairy products, 17th ed.
For food or dairy samples, follow appropriate standard methods American Public Health Association, Washington, D.C.
5. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
for details on sample collection and preparation according to 4th ed. American Public Health Association, Washington. D.C.
6. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online (25 Sept 2008).
sample type and geographic location.4-7 AOAC International, Gaithersburg, Md.
7. Horwitz (ed.). 2007. Official methods of analysis of AOAC International, 18th ed., online. AOAC
For cosmetics, water, or other industrial samples, follow 8. Eaton, Rice and Baird (eds.). 2005. Standard methods for the examination of water and wastewater,
appropriate standard methods for details on sample collection 21st ed., online. American Public Health Association, Washington, D.C.
9. United States Pharmacopeial Convention, Inc. 2008. The United States pharmacopeia 31/The national
and preparation according to sample type and geographic formulary 26, Supp. 1, 8-1-08, online. United States Pharmacopeial Convention, Inc., Rockville, Md.
location.6-11
332

MacConkey II Agar M
10. European Directorate for the Quality of Medicines and Healthcare. 2008. The European pharma- BBL MacConkey I Agar
™
copoeia, 6th ed., Supp. 1, 4-1-2008, online. European Directorate for the Quality of Medicines
and Healthcare, Council of Europe, 226 Avenue de Colmar BP907-, F-67029 Strasbourg Cedex 1, AOAC BAM BS12 CCAM CMPH2 COMPF EP JP MCM9
France.
11. Japanese Ministry of Health, Labour and Welfare. 2006. The Japanese pharmacopoeia, 15th ed., SMD SMWW USP
online. Japanese Ministry of Health, Labour and Welfare. United States and Canada
12. MacConkey. 1905. J. Hyg. 5:333.
13. Kent and Kubica. 1985. Public health mycobacteriology: a guide for the level III laboratory. USDHHS, Cat. No. 215197 Prepared Plates – Pkg. of 20*†
Centers for Disease Control, Atlanta, Ga. 297064 Prepared Plates – Ctn. of 100*†
14. Mazura-Reetz, Neblett and Galperin. 1979. Abstr. C179, p. 339. Abstr. Annu. Meet. American Society
for Microbiology, 1979. Difco™ MacConkey Agar Base
Availability Difco™ MacConkey Agar without Crystal Violet
Difco™ MacConkey Agar
AOAC BAM BS12 CCAM CMPH2 COMPF EP JP MCM9
SMD SMWW USP
BBL™ MacConkey Agar without Crystal Violet
Cat. No. 212123 Dehydrated – 500 g† Cat. No. 211393 Dehydrated – 500 g
212122 Dehydrated – 2 kg† Europe
275300 Dehydrated – 10 kg† Cat. No. 256008 Prepared Plates – Pkg. of 20*
BBL™ MacConkey Agar BBL™ MacConkey Agar without Crystal Violet or Salt
AOAC BAM BS12 CCAM CMPH2 COMPF EP JP MCM9 Cat. No. 294584 Dehydrated – 500 g
SMD SMWW USP
Difco™ MacConkey Agar without Salt
Cat. No. 211387 Dehydrated – 500 g†
211390 Dehydrated – 5 lb (2.3 kg)†
233110 Dehydrated – 10 kg
211391 Dehydrated – 25 lb (11.3 kg)†
Europe
* Store at 2-8°C.
† QC testing performed according to USP/EP/JP performance specifications.
MacConkey II Agar • MacConkey II Agar with MUG

Intended Use Differentiation of enteric microorganisms is achieved by the
MacConkey II Agar is a slightly selective and differential medium combination of lactose and the neutral red indicator. Colorless
for the detection of coliform organisms and enteric pathogens. or pink to red colonies are produced depending upon the ability
of the isolate to ferment the carbohydrate.
MacConkey II Agar with MUG is used for the presumptive
identification of Escherichia coli. Most strains (96-97%) of E. coli produce β-D-glucuronidase.2
The enzyme hydrolyzes MUG to yield 4-methylumbelliferone,
MacConkey II Agar meets United States Pharmacopeia (USP)
a compound that fluoresces under long-wave (366 nm)
performance specifications.
UV light. The addition of MUG to the formulation allows
β-D-glucuronidase-positive strains of E. coli to fluoresce
blue-green when examined under UV light.
The BBL™ MacConkey II Agar formulation was made available
in 1983. It was specially designed to improve the inhibition BBL MacConkey II Agar with MUG contains 0.1 g of MUG
of swarming Proteus species, to achieve more definitive differ- per liter of MacConkey II Agar.
entiation of lactose fermenters and nonfermenters, and for the
promotion of superior growth of enteric pathogens. Formula
BBL™ MacConkey II Agar
Trepeta and Edberg1 modified MacConkey Agar by the incor- Approximate Formula* Per Liter
poration of MUG (4-methylumbelliferyl-β-D-glucuronide). Pancreatic Digest of Gelatin....................................... 17.0 g
The resulting medium allowed the authors to presumptively Pancreatic Digest of Casein.......................................... 1.5 g
identify E. coli from the primary plating medium within Peptic Digest of Animal Tissue...................................... 1.5 g
Lactose...................................................................... 10.0 g
5 minutes. Bile Salts...................................................................... 1.5 g
Principles of the Procedure Agar.......................................................................... 13.5 g
Neutral Red.................................................................. 0.03 g
MacConkey II Agar is a selective and differential medium. It is Crystal Violet............................................................... 1.0 mg
only slightly selective since the concentration of bile salts, which *Adjusted and/or supplemented as required to meet performance criteria.
inhibit gram-positive microorganisms, is low in comparison

with other enteric plating media. Crystal violet also is included
in the medium to inhibit the growth of gram-positive bacteria,
especially enterococci and staphylococci.
333

SS Agar
User Quality Control Uninoculated

Plate
Clostridium perfringens
ATCC™ 12919
Difco™ SPS Agar
Dehydrated Appearance: Beige, free-flowing, homogeneous.
boiling. Solution is light to medium amber, very
slightly to slightly opalescent.
Prepared Appearance: Light to medium amber, slightly opalescent.
Reaction of 4.1%
Cultural Response
Difco™ SPS Agar
Prepare the medium per label directions. Inoculate using the pour plate
technique and incubate anaerobically at 35 ± 2°C for 24-48 hours.
INOCULUM COLONY
Organism ATCC™ CFU RECOVERY COLOR
Clostridium perfringens 12919 102-103 Good Black
Clostridium sporogenes 11437 102-103 None to fair Black
Escherichia coli 25922 102-103 Marked to –
complete inhibition
Salmonella enterica Staphylococcus
aureus
subsp. enterica Marked to
S
ATCC™ 25923
serotype Typhimurium 14028 102-103 complete inhibition –
Staphylococcus aureus 25923 102-103 Fair to good White
Limitation of the Procedure Availability

The high degree of selectivity of SPS Agar may inhibit some Difco™ SPS Agar
strains of C. perfringens while other strains that grow may fail Cat. No. 284530 Dehydrated – 500 g*
to produce distinguishing black colonies.4 *Store at 2-8°C.
References
1. Mossel. 1959. J. Sci. Food Agric. 19:662.
2. Mossel, DeBruin, van Diepen, Vendrig and Zoutewelle. 1956. J. Appl. Microbiol. 19:142.
3. Angelotti, Hall, Foster and Lewis. 1962. Appl. Microbiol. 10:193.
4. Labbe. 2001. In Downes and Ito (ed.), Compendium of methods for the microbiological examination
of foods, 4th ed. American Public Health Association, Washington, D.C.
tional, Gaithersburg, Md.
SS Agar • Salmonella Shigella Agar

Intended Use Principles of the Procedure
SS Agar and Salmonella Shigella Agar are moderately selective and SS Agar and Salmonella Shigella Agar are designated as
differential media for the isolation of pathogenic enteric bacilli, moderately selective media based upon the degree of inhibition
especially those belonging to the genus Salmonella. This formula- of gram-positive microorganisms that they inhibit due to their
tion is not recommended for the primary isolation of Shigella. content of bile salts, brilliant green and citrates. Differentiation
of enteric organisms is achieved by the incorporation of lactose
Summary and Explanation in the medium. Organisms that ferment lactose produce acid
The culture media that have been developed for the selection which, in the presence of the neutral red indicator, results in
and differentiation of enteric microorganisms from clinical the formation of red colonies. Lactose nonfermenters form
and nonclinical materials inhibit the growth of gram-positive colorless colonies. The latter group contains the majority of the
species to a varying degree due to the presence of either pure intestinal pathogens, including Salmonella and Shigella.
bile salts, mixtures of bile salts or dyes. SS Agar and Salmonella
The sodium thiosulfate and ferric citrate enable the detection
Shigella Agar are examples of media used in the plating of
of hydrogen sulfide production as evidenced by colonies with
samples for the detection of enteric pathogens that contain bile
black centers.
salt mixtures. This formulation is essentially a modification of
the Desoxycholate-Citrate Agar described by Leifson.1
485
Difco Manual Sect III S new.indd485 485 3/16/09 4:53:46 PM

Section III
S SS Agar, cont.

Identity Specifications Identity Specifications

Difco™ SS Agar BBL™ Salmonella Shigella Agar
Dehydrated Appearance: Very light buff to pink, free-flowing, homo- Dehydrated Appearance: Fine, homogeneous, free of extraneous material,
geneous. may contain many small tan flecks.
Solution: 6.0% solution, soluble in purified water upon Solution: 6.0% solution, soluble in purified water upon
boiling. Solution is red-orange, very slightly to boiling. Solution is medium, tan-orange to tan-red,
slightly opalescent. clear to moderately hazy.
Prepared Appearance: Red-orange, slightly opalescent. Prepared Appearance: Medium, tan-orange to tan-red, clear to mod-
Reaction of 6.0% erately hazy.
Solution at 25°C: pH 7.0 ± 0.2 Reaction of 6.0%
Cultural Response
Difco™ SS Agar Cultural Response
Prepare the medium per label directions. Inoculate and incubate at BBL™ Salmonella Shigella Agar
35 ± 2°C for 18-24 hours. Prepare the medium per label directions. Inoculate and incubate at
35 ± 2°C for 24 hours.
inoculum COLONY
ORGANISM ATCC™ CFU RECOVERY COLOR H2S inoculum COLONY
ORGANISM ATCC™ CFU RECOVERY COLOR H2S
Enterococcus faecalis 29212 103-2×103 Partial Colorless –
inhibition Enterococcus faecalis 29212 104-105 Complete – –
Escherichia coli 25922 10 -2×10 Partial 3 3
Pink – inhibition
inhibition to red Escherichia coli 25922 104-105 Partial Pink to –
Salmonella enterica to complete red
subsp. enterica inhibition
serotype Typhimurium 14028 102-103 Good Colorless + Salmonella enterica
Shigella flexneri 12022 102-103 Fair to Colorless – subsp. enterica
good serotype Typhimurium 14028 103-104 Good Colorless +
Shigella flexneri 12022 103-104 Good Colorless –
Formulae 2. Heat with frequent agitation and boil for 1 minute to com-
Difco SS Agar
™ pletely dissolve the powder. DO NOT AUTOCLAVE.
Approximate Formula* Per Liter 3. Cool the medium to approximately 45-50°C and pour into
Beef Extract................................................................. 5.0 g Petri dishes.
Proteose Peptone........................................................ 5.0 g
Lactose..................................................................... 10.0 g 4. Allow the plates to dry for approximately 2 hours with the
Bile Salts No. 3............................................................ 8.5 g covers partially removed.
Sodium Citrate............................................................ 8.5 g 5. Test samples of the finished product for performance using
Sodium Thiosulfate.................................................... 8.5 g
Ferric Citrate............................................................... 1.0 g stable, typical control cultures.
Agar......................................................................... 13.5 g
Brilliant Green............................................................. 0.33 mg Procedure
Neutral Red............................................................... 25.0 mg Use standard procedures to obtain isolated colonies from speci-
BBL™ Salmonella Shigella Agar mens. A nonselective medium should also be streaked to increase
Approximate Formula* Per Liter the chance of recovery when the population of gram-negative
Beef Extract................................................................. 5.0 g
Pancreatic Digest of Casein......................................... 2.5 g organisms is low and to provide an indication of other organisms
Peptic Digest of Animal Tissue..................................... 2.5 g present in the specimen. Incubate plates, protected from light, at
Lactose..................................................................... 10.0 g 35 ± 2°C for 18-24 hours. If negative after 24 hours, reincubate
Bile Salts..................................................................... 8.5 g
Sodium Citrate............................................................ 8.5 g an additional 24 hours.
Sodium Thiosulfate..................................................... 8.5 g
Ferric Citrate............................................................... 1.0 g Expected Results
Agar......................................................................... 13.5 g Typical colonial morphology on Salmonella Shigella Agar is
Brilliant Green............................................................. 0.33 mg
Neutral Red............................................................... 25.0 mg as follows:
*Adjusted and/or supplemented as required to meet performance criteria. Escherichia coli................... Slight growth, pink or red
Enterobacter/Klebsiella....... Slight growth, pink
Directions for Preparation from Proteus.............................. Colorless, usually with black center
Salmonella......................... Colorless, usually with black center
Dehydrated Product Shigella.............................. Colorless
1. Suspend 60 g of the powder in 1 L of purified water. Mix Pseudomonas..................... Irregular, slight growth
thoroughly. Gram-positive bacteria....... No growth
486

SXT Blood Agar
Shigella flexneri
ATCC™ 12022
Salmonella Typhimurium
ATCC™ 14028 References
1. Leifson. 1935. J. Pathol. Bacteriol. 40:581.
2. Taylor and Harris. 1965. Am. J. Clin. Pathol. 44:476.
3. Pollock and Dahlgren. 1974. Appl. Microbiol. 27:197.
Availability
Difco™ SS Agar
BS12 CMPH2 COMPF MCM9
BBL™ Salmonella Shigella Agar
BS12 CMPH2 COMPF MCM9
211597 Dehydrated – 500 g
211600 Dehydrated – 5 lb (2.3 kg)
293306 Dehydrated – 25 lb (11.3 kg)
United States and Canada
Europe
Japan
Limitation of the Procedure

251279
251134
Prepared Plates – Ctn. of 100*
Prepared Plates – Ctn. of 200* S
Due to the relatively high level of selectivity, some Shigella strains 251826 Prepared I Plate™ Dishes – Ctn. of 200*
may not grow on SS Agar and Salmonella Shigella Agar and, BBL™ Salmonella Shigella Agar//Hektoen Enteric Agar
therefore, these media are not recommended for the primary Cat. No. 297426 Prepared I Plate™ Dishes – Pkg. of 20*
isolation of Shigella.1,2 Media recommended for the isolation *Store at 2-8°C.
of Shigella are Hektoen Enteric and XLD agars.3
SXT Blood Agar

Intended Use tion of group A and B streptococci from throat cultures.5 They
SXT Blood Agar is used in the isolation of Lancefield groups A reported that most normal flora and beta-hemolytic streptococci
and B streptococci from throat cultures and other specimens. other than groups A and B were inhibited on the SXT agar,
The growth of viridans streptococci, other beta-hemolytic and resulting in the recovery of 42% more group A and 49% more
nonhemolytic streptococci, most Enterobacteriaceae, Neisseria group B streptococci than with sheep blood agar alone.
species and some Pseudomonas species is inhibited. In similar studies, Mirrett and Reller and others have reported
that SXT agar is more sensitive and more specific in the recovery
Summary and Explanation of group A streptococci than sheep blood agar alone.6-9
Groups A and B streptococcal infections may cause serious medi-
cal complications. Group A streptococcal infections may result in Principles of the Procedure
scarlet fever, rheumatic fever or acute glomerulonephritis. Group SXT Blood Agar is a primary plating medium suitable for isolat-
B infections may produce neonatal sepsis and meningitis.1 ing group A streptococci (S. pyogenes) and group B streptococci
To aid the detection of group A and B streptococci, chemicals (S. agalactiae) from clinical specimens. Sulfamethoxazole and
(such as crystal violet and sodium azide) and antimicrobial trimethoprim act synergistically in this medium to suppress the
agents (such as neomycin and gentamicin) have been incorporated growth of normal flora.5 Defibrinated sheep blood supplies the
into sheep blood agar.2-4 These inhibitory agents suppress the nutrients necessary to support the growth of streptococci and,
growth of normal flora and other organisms that could mask the simultaneously, it allows detection of hemolytic reactions. Sheep
presence of group A and B streptococci. blood also inhibits the growth of Haemophilus haemolyticus, a
bacterium commonly found in nose and throat cultures that is
Gunn et al. introduced SXT Sheep Blood Agar, consisting of
indistinguishable from beta-hemolytic streptococci.10
Trypticase™ Soy Agar with 5% Sheep Blood and two antimicro-
bial agents, sulfamethoxazole and trimethoprim, for the isola-
487

Section III
U-Z Wilkins-Chalgren Agar, cont.
Formulae Expected Results

Difco™ Wilkins-Chalgren Agar Refer to appropriate references for acceptable ranges.
Pancreatic Digest of Casein........................................ 10.0 g Limitation of the Procedure
Peptone..................................................................... 10.0 g
Yeast Extract................................................................ 5.0 g Anaerobe Broth MIC is supplemented to a final concentration
Dextrose...................................................................... 1.0 g of 0.5 µg per mL of vitamin K1 and 5.0 µg of hemin per mL.
Sodium Chloride.......................................................... 5.0 g CLSI changed their recommendations to include use of broth
L-Arginine.................................................................... 1.0 g
Sodium Pyruvate.......................................................... 1.0 g with a final concentration of 1 µg of vitamin K1 per mL.2
Hemin......................................................................... 5.0 mg To follow CLSI recommendations, the concentration of vitamin
Vitamin K1 .................................................................. 0.5 mg K1 should be increased accordingly. A final concentration of
Agar.......................................................................... 15.0 g
0.5 µg of vitamin K1 per mL is sufficient, but some fastidious
Difco™ Anaerobe Broth MIC anaerobes may need a higher concentration of vitamin K1.5
Consists of the same ingredients without the agar.
References
1. Wilkins and Chalgren. 1976. Antimicrob. Agents Chemother. 10:926.
Directions for Preparation from 2. Clinical and Laboratory Standards Institute. 1993. Methods for antimicrobial susceptibility testing of
anaerobic bacteria. Approved standard M11-A3. CLSI, Wayne, Pa.
Dehydrated Product 3. Clinical and Laboratory Standards Institute. 2001. Methods for antimicrobial susceptibility testing of
anaerobic bacteria. Approved standard M11-A5. CLSI, Wayne, Pa.
1. Suspend the powder in 1 L of purified water: 4. Wexler and Doern. 1995. In Murray, Baron, Pfaller, Tenover and Yolken (ed.). Manual of clinical
Difco™ Wilkins-Chalgren Agar – 48 g; microbiology, 6th ed. American Society for Microbiology, Washington, D.C.
5. Isenberg (ed.). 1995. Clinical microbiology procedures handbook, vol 1. American Society for
Difco™ Anaerobe Broth MIC – 33 g. Microbiology, Washington, D.C.
Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to Availability
completely dissolve the powder. Difco™ Wilkins-Chalgren Agar
3. Autoclave at 121°C for 15 minutes. CCAM
stable, typical control cultures. Difco™ Anaerobe Broth MIC
CCAM
Procedure Cat. No. 218151 Dehydrated – 500 g
For a complete discussion on susceptibility testing of an-
aerobic bacteria refer to appropriate procedures outlined in the
references.2-5
XL Agar Base • XLD Agar

Intended Use XL Agar Base was developed by Taylor4 for the nonselective
XL (Xylose Lysine) Agar Base is used for the isolation and dif- isolation and differentiation of gram-negative enteric bacilli.
ferentiation of enteric pathogens and, when supplemented with It is particularly recommended for obtaining counts of enteric
appropriate additives, as a base for selective enteric media. organisms. This medium can be rendered moderately selective
for enteric pathogens, particularly Shigella, by the addition of
XLD Agar is the complete Xylose Lysine Desoxycholate Agar,
sodium desoxycholate (2.5 g/L) to make XLD Agar.4
a moderately selective medium recommended for isolation and
differentiation of enteric pathogens, especially Shigella species. XL Agar Base can be made selective for Salmonella by adding
1.25 mL/L of 1% aqueous brilliant green to the base prior to
XLD Agar meets United States Pharmacopeia (USP), European
autoclaving. Its use is recommended for Salmonella isolation
Pharmacopoeia (EP) and Japanese Pharmacopoeia (JP)1-3
after selenite or tetrathionate enrichment in food analysis; both
performance specifications, where applicable.
coliforms and Shigella are inhibited.4
Summary and Explanation XLD Agar was developed by Taylor in order to increase the
A wide variety of media have been developed to aid in the efficiency of the isolation and identification of enteric pathogens,
selective isolation and differentiation of enteric pathogens. Due particularly Shigella.4 The pathogens are differentiated not only
to the large numbers of different microbial species and strains from the nonpathogenic lactose fermenters but also from many
with varying nutritional requirements and chemical resistance nonpathogens which do not ferment lactose or sucrose. Addi-
patterns, investigators have developed various formulae to tionally, the medium was formulated to increase the frequency
meet general as well as specific needs relative to isolation and of growth of the more fastidious pathogens,4 which in other
identification of the microorganisms. formulations have often failed to grow due to the inclusion of
excessively toxic inhibitors. The results obtained in a number of
616
Difco Manual Sect III UVWXYZ.ind616 616 3/16/09 5:08:37 PM

XL Agar Base, cont.

Identity Specifications Identity Specifications

Difco™ XLD Agar BBL™ XL Agar Base
Dehydrated Appearance: Pink, free-flowing, homogeneous. Dehydrated Appearance: Fine, homogeneous, free of extraneous
Solution: 5.5% solution, soluble in purified water upon material.
boiling. Solution is red, very slightly to slightly Solution: 4.5% solution, soluble in purified water
opalescent. upon boiling. Solution is dark medium
Prepared Appearance: Red, slightly opalescent. to dark, red to rose-red, clear to slightly
hazy.
Reaction of 5.5%
Solution at 25°C: pH 7.4 ± 0.2 Prepared Appearance: Dark medium to dark, red to rose-red, clear
to slightly hazy.
Cultural Response Reaction of 4.5%
Difco™ XLD Agar
Prepare the medium per label directions. Inoculate and incubate at BBL XLD Agar (prepared)
™
35 ± 2°C for 18-24 hours. Incubate (*) cultures at 30-35°C for 18-48 hours Appearance: Medium orange red to red, trace hazy.
and (**) culture at 35-37°C for 18-72 hours. Reaction at 25°C: pH 7.4 ± 0.2
INOCULUM COLONY
ORGANISM ATCC™ CFU RECOVERY COLOR Cultural Response
Enterococcus BBL™ XL Agar Base
faecalis 29212 ~103 Partial inhibition – Prepare the medium per label directions. Inoculate and incubate at
Escherichia coli 25922 ~103 Partial inhibition Yellow 35 ± 2°C for 18-24 hours (up to 48 hours if necessary).
Providencia INOCULUM COLONY
alcalifaciens 9886 100-300 Good Red ORGANISM ATCC™ CFU RECOVERY COLOR
Shigella flexneri 12022 100-300 Good Red Escherichia coli 25922 103-104 Good Yellow
Escherichia coli* 8739 >100 Partial to Yellow Salmonella enterica Red to yellow with
complete inhibition subsp. enterica black centers to
(30-35°C) serotype Typhimurium 14028 103-104 Good predominantly black
Salmonella enterica Shigella flexneri 12022 103-104 Good Red
subsp. enterica Growth Red with
serotype Typhimurium* 14028 <100 (30-35°C) black centers BBL™ XLD Agar (prepared)
Salmonella enterica Inoculate and incubate at 35 ± 2°C for 24 hours. Incubate (*) cultures at
subsp. enterica Growth Red with 30-35°C for 18-48 hours and (**) culture at 35-37°C for 18-48 hours.
serotype Typhimurium** 14028 <100 (35-37°C) black centers INOCULUM COLONY
U
Enterococcus
faecalis 29212 104- 105 Partial inhibition – -
clinical evaluations have supported the claim for the relatively
high efficiency of XLD Agar in the primary isolation of Shigella Escherichia coli 25922 104- 105
complete inhibition yellow-red
Partial to Yellow to Z
and Salmonella.5-9
Salmonella enterica
XLD Agar is a selective and differential medium used for the subsp. enterica Red with
serotype Typhimurium 14028 103-104 Good black centers
isolation and differentiation of enteric pathogens from clinical
Shigella flexneri 12022 103-104 Good Red
specimens.10-12 The value of XLD Agar in the clinical laboratory
Escherichia coli* 8739 102-103 Partial to Yellow
is that the medium is more supportive of fastidious enteric organ- complete inhibition to red
isms such as Shigella.12 XLD Agar is also recommended for the (30-35°C)
testing of food, dairy products and water in various industrial Salmonella enterica Red with
standard test methods.13-17 General Chapter <62> of the USP subsp. enterica Growth black
serotype Typhimurium* 14028 <100 (30-35°C) centers
describes the test method for the isolation of Salmonella from
Salmonella enterica Red with
nonsterile pharmaceutical products using XLD Agar as the solid subsp. enterica Growth black
culture medium.1 serotype Typhimurium** 14028 <100 (35-37°C) centers

Xylose is incorporated into the medium because it is fermented
by practically all enterics except for the shigellae. This property
enables the differentiation of Shigella species. Lysine is included
to enable the Salmonella group to be differentiated from the non-
pathogens. Without lysine, salmonellae rapidly would ferment
617

Section III
U-Z XL Agar Base, cont.
XL Agar Base To add to the differentiating ability of the formulation, an H2S

Uninoculated Escherichia coli
Plate ATCC™ 25922 indicator system, consisting of sodium thiosulfate and ferric
ammonium citrate, is included for the visualization of the hy-
drogen sulfide produced, resulting in the formation of colonies
with black centers. The nonpathogenic H2S producers do not
decarboxylate lysine; therefore, the acid reaction produced by
them prevents the blackening of the colonies.4 Sodium chloride
maintains the osmotic balance. Yeast extract supplies B-complex
vitamins which stimulate bacterial growth. Agar is the solidify-
ing agent.
XLD Agar is both a selective and differential medium. It utilizes
sodium desoxycholate as the selective agent and, therefore, it is
inhibitory to gram-positive microorganisms.
Formulae
BBL™ XL Agar Base
Xylose.......................................................................... 3.5 g
L-Lysine........................................................................ 5.0 g
Lactose........................................................................ 7.5 g
Sucrose........................................................................ 7.5 g
Yeast Extract................................................................ 3.0 g
XLD Agar Phenol Red.................................................................. 0.08 g
Salmonella Typhimurium Shigella flexneri Agar.......................................................................... 13.5 g
ATCC™ 14028 ATCC™12022
Difco™ XLD Agar
Xylose.......................................................................... 3.5 g
L-Lysine........................................................................ 5.0 g
Lactose........................................................................ 7.5 g
Saccharose................................................................... 7.5 g
Yeast Extract................................................................ 3.0 g
Phenol Red.................................................................. 0.08 g
Sodium Desoxycholate................................................. 2.5 g
Ferric Ammonium Citrate............................................. 0.8 g
Sodium Thiosulfate...................................................... 6.8 g
Agar.......................................................................... 13.5 g
Directions for Preparation from

Dehydrated Product
BBL™ XL Agar Base
1. Suspend 45 g of the powder in 1 L of purified water. Mix
thoroughly.
2. Heat with frequent agitation and boil for 1 minute to com-
pletely dissolve the powder. Add brilliant green, if desired.
3. Autoclave at 118°C for 10 minutes. Cool to 55-60°C.
4. Add 20 mL of an aqueous solution containing 34% sodium
the xylose and be indistinguishable from nonpathogenic species. thiosulfate and 4% ferric ammonium citrate. For XLD agar,
After the salmonellae exhaust the supply of xylose, the lysine is add 25 mL of 10% aqueous sodium desoxycholate. Pour into
attacked via the enzyme lysine decarboxylase, with reversion to plates.
an alkaline pH, which mimics the Shigella reaction. To prevent 5. Test samples of the finished product for performance using
similar reversion by lysine-positive coliforms, lactose and sucrose stable, typical control cultures.
(saccharose) are added to produce acid in excess.4 Degradation
Difco™ XLD Agar
of xylose, lactose and sucrose generates acid products, which
in the presence of the pH indicator phenol red, causes a color 1. Suspend 55 g of the powder in 1 L of purified water. Mix
change in the medium from red to yellow. thoroughly.
2. Heat with agitation just until the medium boils. DO NOT
OVERHEAT. DO NOT AUTOCLAVE.
618

XL Agar Base, cont.
3. Cool to 45-50°C in a water bath and use immediately. Limitations of the Procedure
Overheating causes precipitation. 1. Red, false-positive colonies may occur with some Proteus
4. Test samples of the finished product for performance using and Pseudomonas species.
stable, typical control cultures. 2. Incubation in excess of 48 hours may lead to false-positive
results.
Sample Collection and Handling 3. S. Paratyphi A, S. Choleraesuis, S. pullorum and S. gallinarum
For clinical specimens, refer to laboratory procedures for details may form red colonies without black centers, thus resembling
on specimen collection and handling.10-12 Shigella species.
For food, dairy or water samples, follow appropriate standard 4. Some Proteus strains will give black-centered colonies on
methods for details on sample collection and preparation ac- XLD Agar.
cording to sample type and geographic location.13-17
References
For pharmaceutical samples, refer to the USP for details on 1. United States Pharmacopeial Convention, Inc. 2008. The United States pharmacopeia 31/The national
formulary 26, Supp. 1, 8-1-08, online. United States Pharmacopeial Convention, Inc., Rockville,
sample collection and preparation for testing of nonsterile Md.
products.1 2. European Directorate for the Quality of Medicines and Healthcare. 2008. The European pharma-
copoeia, 6th ed., Supp. 1, 4-1-2008, online. European Directorate for the Quality of Medicines
and Healthcare, Council of Europe, 226 Avenue de Colmar BP907-, F-67029 Strasbourg Cedex 1,
France.
Procedure 3. Japanese Ministry of Health, Labour and Welfare. 2006. The Japanese pharmacopoeia, 15th ed.,
online. Japanese Ministry of Health, Labour and Welfare.
For clinical specimens, refer to appropriate standard references 4. Taylor. 1965. Am. J. Clin. Pathol. 44:471.
for details on testing protocol to obtain isolated colonies from 5. Taylor and Harris. 1965. Am. J. Clin. Pathol. 44:476.
6. Taylor and Harris. 1967. Am. J. Clin. Pathol. 48:350.
specimens using XLD Agar.10-12 7. Taylor and Schelhart. 1967. Am. J. Clin. Pathol. 48:356.
8. Taylor and Schelhart. 1968. Appl. Microbiol. 16:1387.
9. Pollock and Dahlgren. 1974. Appl. Microbiol. 27:197.
For food, dairy and water samples, refer to appropriate standard 10. Forbes, Sahm and Weissfeld. 2007. Bailey & Scott’s diagnostic microbiology, 12th ed. Mosby, Inc.,
St. Louis, Mo.
references for details on test methods using XLD Agar.13-17 11. Isenberg and Garcia (ed.). 2004 (update, 2007). Clinical microbiology procedures handbook, 2nd ed.
For pharmaceutical samples, refer to USP General Chapter <62> 12. Murray, Baron, Jorgensen, Landry and Pfaller (ed.). 2007. Manual of clinical microbiology, 9th ed.
for details on the examination of nonsterile products and the 13. U.S. Food and Drug Administration. 2001. Bacteriological analytical manual, online. AOAC Interna-
tional, Gaithersburg, Md.
isolation of Salmonella using XLD Agar.1 14. Horwitz (ed.). 2007. Official methods of analysis of AOAC International, 18th ed., online. AOAC
A nonselective medium should also be streaked to increase the 15. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
4th ed. American Public Health Association, Washington, D.C.
chance of recovery when the population of gram-negative or- 16. Wehr and Frank (ed.). 2004. Standard methods for the examination of dairy products, 17th ed.
American Public Health Association, Washington, D.C.
ganisms is low and to provide an indication of other organisms 17. Eaton, Rice and Baird (ed.). 2005. Standard methods for the examination of water and wastewater,
21st ed., online. American Public Health Association, Washington, D.C.
present in the specimen. Incubate plates, protected from light, at
35 ± 2°C for 18-24 hours. Colonies on XLD agar may require Availability
48 hours incubation for full color development. BBL™ XL Agar Base
SMWW
U
Expected Results Cat. No. 211836 Dehydrated – 500 g
-
Degradation of xylose, lactose and sucrose generates acid
products, causing a color change in the medium from red to
Difco™ XLD Agar
AOAC BAM BS12 CCAM CMPH2 COMPF EP ISO JP
Z
yellow. MCM9 SMD SMWW USP
Hydrogen sulfide production under alkaline conditions causes Cat. No. 278850 Dehydrated – 500 g†
278820 Dehydrated – 2 kg†
colonies to develop black centers. This reaction is inhibited by the 278830 Dehydrated – 10 kg†
acid conditions that accompany carbohydrate fermentation.
BBL™ XLD Agar
Lysine decarboxylation in the absence of lactose and sucrose AOAC BAM BS12 CCAM CMPH2 COMPF EP ISO JP
fermentation causes reversion to an alkaline condition and the MCM9 SMD SMWW USP
color of the medium changes back to red. United States and Canada
Cat. No. 221192 Prepared Plates – Pkg. of 20*†
Typical colonial morphology and reactions on XLD Agar are 221284 Prepared Plates – Ctn. of 100*†
as follows: Europe
E.coli............................................... Large, flat, yellow; some Cat. No. 254055 Prepared Plates – Pkg. of 20*
strains may be inhibited 254090 Prepared Plates – Ctn. of 120*
Enterobacter / Klebsiella.................. Mucoid, yellow Japan
Proteus............................................ Red to yellow; most Cat. No. 252020 Prepared Plates – Pkg. of 20*
strains have black centers 251159 Prepared Plates – Ctn. of 100*
Salmonella...................................... Red-yellow with black
centers BBL™ XLD Agar//Hektoen Enteric Agar
Shigella, Salmonella H2S-negative.... Red Cat. No. 295646 Prepared I Plate™ Dishes – Pkg. of 20*
Pseudomonas.................................. Red *Store at 2-8°C.
Gram-positive bacteria.................... No growth to slight †QC testing performed according to USP/EP/JP performance specifications.
growth
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Medios Salmonella

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Bismuth Sulfite Agar

3. The use of altered or deficient media may cause mutants Reference

4. For successful results to these procedures, all conditions of

Bird Seed Agar

Summary and Explanation Procedure

Bismuth Sulfite Agar

Difco Manual Sect III Ba.indd 79 3/16/09 3:46:30 PM

Uninoculated Salmonella Typhi

Difco Manual Sect III Ba.indd 80 3/16/09 3:46:32 PM

Difco Manual Sect III Ba.indd 81 3/16/09 3:46:33 PM

Brilliant Green Agar

Principles of the Procedure

Difco Manual Sect III Bb.indd 96 3/16/09 3:44:19 PM

Directions for Preparation from User Quality Control

Brilliant Green Agar Modified

Difco Manual Sect III Bb.indd 97 3/16/09 3:44:21 PM

Hektoen Enteric Agar

User Quality Control Salmonella Typhimurium

Difco Manual Sect III HIJK.indd 265 3/16/09 4:16:51 PM

Formula Limitation of the Procedure

Hemo (Haemophilus) Identification (ID) QUAD Plate

Difco Manual Sect III HIJK.indd 266 3/16/09 4:16:53 PM

Difco Manual Sect III Ma.indd 328 3/16/09 4:41:34 PM

Difco Manual Sect III Ma.indd 329 3/16/09 4:41:35 PM

Difco Manual Sect III Ma.indd 330 3/16/09 4:41:37 PM

Principles of the Procedure

Difco Manual Sect III Ma.indd 331 3/16/09 4:41:39 PM

Difco Manual Sect III Ma.indd 332 3/16/09 4:41:40 PM

MacConkey II Agar • MacConkey II Agar with MUG

inhibit gram-positive microorganisms, is low in comparison

Difco Manual Sect III Ma.indd 333 3/16/09 4:41:42 PM

User Quality Control Uninoculated

Limitation of the Procedure Availability

SS Agar • Salmonella Shigella Agar

Difco Manual Sect III S new.indd485 485 3/16/09 4:53:46 PM

User Quality Control

Identity Specifications Identity Specifications

Difco Manual Sect III S new.indd486 486 3/16/09 4:53:47 PM

Limitation of the Procedure

of Shigella are Hektoen Enteric and XLD agars.3

SXT Blood Agar

Difco Manual Sect III S new.indd487 487 3/16/09 4:53:50 PM

Formulae Expected Results

XL Agar Base • XLD Agar

Difco Manual Sect III UVWXYZ.ind616 616 3/16/09 5:08:37 PM

User Quality Control

Identity Specifications Identity Specifications

Principles of the Procedure

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XL Agar Base To add to the differentiating ability of the formulation, an H2S

Directions for Preparation from

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