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FUNDAMENTALS OF
Microbiology
FUNDAMENTALS OF
icrobiology /
SIXTH EDITION, ILLUSTRATED
MARTIN FROBISHER, Se.D.

Special Consultant, Laboratory Branch, Communicable Disease Center,
United States Public Health Service; Associate Professor of Bacteriology,
Emory University Medical School, Atlanta, Georgia. Formerly, Special
Member, International Health Division, Rockefeller Foundation; Associate
Professor of Bacteriology, Johns Hopkins University; Chief, Bacteriology
Section, Communicable Disease Center, United States Public Health
Service; Professor and Head, Department of Bacteriology, University of
Georgia.
W. B. SAUNDERS COMPANY, Philadelphia and London
CHARLES E. TUTTLE COMPANY, Tokyo

A millipore- or membrane-filter disk showing colonies of coliform bacteria. This paper-thin
disk is made of cellulose acetate or similar material. It is traversed from surface to surface
by tiny pores or tubes so fine that bacteria cannot pass through them. When bacteria-con-
taining fluid is passed through the porous disk tlie bacteria are held on the surface of the
disk. (See description of apparatus, Fig. 13-4.)
After passing a sample of well water (suspected of being polluted) through the filter disk
shown above, the disk, with bacteria on its upper surface, was placed on a pad saturated
with liquid Endo medium (from which the disk absorbed the medium) and incubated over-
night at 35° C. Endo medium contains lactose, and dyes which change color when the grow-
ing colonies of coliform bacteria produce acid by fermenting the lactose. The rapid growth
and distinctive color of the colonies on the surface of the medium-saturated filter disk
permit quick detection and (aided by the grid-like markings) easy enumeration of the coli-
form organisms, each represented by a colony. These bacteria are indicative of sewage
pollution in well water.
For other purposes appropriate special media other than Endo's may be substituted to
cultivate, selectively, organisms from blood, fruit juices, and many other fluids of medical, _
industrial and research interest. The possibilities of fine-pored filter membranes, like the
disk shown here, made of cellulosic or "plastic" materials, are only beginning 10 be realized.
(Color plates courtesy of The Millipore Filter Corporation, Watertown 72, Massachusetts.)
This Modern Asia Edition is a complete and unabridged photo-
offset reproduction of the latest American edition, specially pub-
lished for sale in the Far East. It is the only authorized edition
so published and is offered in the public interest at a great re-
duction in price.
Inquiries or suggestions concerning Modern Asia Editions will
be welcomed by the Ch~rles E. Tuttle Company,15 Edogawa-cho,
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© 1957, by W. B. Saunders Company
All rights reserved
No part of this book may be reproduced in any

form, by mimeograph or any other means, with-
out permission in ~riting from the publishers.
First printing (Modern Asia Edition), 1961
PRINTED IN JAPAN
Preface to the Sixth Edition
THE AIM in this edition has been to present current information about funda-
mental principles of microbiology, with modern exemplifications, in a simple,
direct and concise form, yet, having in mind the burdens of the college student
of today, in as readable and interesting a manner as possible. Because of
enormous expansion of research in the field, exhaustive coverage is no longer
feasible in an elementary textbook. For this reason the material included has
been very carefully selected. It is intended for the student interested in acquir-
ing an introduction to the field of microbiology; whether for future profes-
sional specialization or as part of a well-rounded general education-for the
Liberal Arts student, students in Home Economics, Pharmacy, Agriculture,
Industry, Premedical and Veterinary courses. It is assumed that the reader has
at least an elementary knowledge of chemistry, physics and biology.
This edition represents almost a complete rewriting. The chemistry and
physics of metabolism and enzymes have been much simplified. The chapters
on viruses have been completely rewritten in line with new developments, both
in concept and technique. So, also, have the sections on growth, structure,
methodology, mutation, variation and sex, antibiotics, disinfection, micro-
biology of soil, of water and sewage, of industrial spoilage, the lactic acid
bacteria, the anaerobes and n~merous others. The emphasis on infectious dis-
eases and public health, while still present in appropriate aspects, has been
lessened and more has been placed on other important aspects of microbi-
ology. Much information has been tabulated for conciseness and the con-
venience of the student.
A special effort has been made to improve the illustrative material and to -
make the explanatory legends carry as much of a story as possible. Scientists
at home and abroad have contributed generously of superb electron microg-
raphy and microphotography and the author feels a special gratitude to them.
All are thankfully acknowledged, as well as the courtesy of publishers, in
legends accompanying the pictures.
It is neither practicable nor desirable to include complete bibliographies.
The references listed after each chapter have been selected with care as to con-
tent, date and inclusive bibliography as well as to pertinence. Recent review
articles bave been included so far as possible. It is hoped these lists may afford
keys to the literature for the interested student and useful material for in-
structors in fields other than their specialties.
It is a pleasure to acknowledge the informed direction of Dr. Mauricf'
v
vi Preface to the 6th Edition
Green, Assistant Professor of Microbiology, St. Louis University, in the
preparation of Chapter 13 (Nutrition, Metabolism and Cultivation of Micro-'
. organisms) and the wise counsel of Dr. Libero Ajello and of Dr. Lucille
Georg, both of the Co~municable Disease Center, U. S. Public Health
Service, Atlanta, Georgia, in revising Chapters 3 and 4 (Yeasts and Molds).
Their efforts are evident in the good there is in these sections; the author is
responsible for any defects.
Finally, only the expert guidance of the publisher's staff and the unflagging
assistance of the author's long-suffering helpmate, Amy W. Frobisher, have
made possible the transformation of rough manuscript into a beautifully
crafted volume. To both the author expresses his undying thankfulness.
MARTIN FROBISHER, JR.

Contents
SECTION 1. THE RELATIONSHIPS OF MICROORGANISMS TO

EACH OTHER AND TQ THE LIVING WORLD
CHAPTER 1
THE MICROSCOPIC WORLD 1. PROTOZOA, ALGAE, FUNGI AND BACTERIA 3
Animal or Vegetable?, 3.
Bacteri~ and the Animal Kingdom. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Bacteria and Protozoa, 5. Classification of Protozoa, 8. Reproduc-
duction and Life Cycles of Protozoa, 9. Mode of Nutrition, 9.
Bacteria and the Vegetable' Kingdom ..... " .................... ,. . 10
Bacteria and Algae, 11. Bacteria and Fungi, 12. Recapitulation, 12.
Definition of Terms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
S~ Variation, 13. Aerobic and Anaerobic Growth, 13.
. /
CHAPTER 2
THE MICROSCOPIC WORLD 2. GROWTH OF BACTERIOLOGY. . . .. .. . .. 15
The Primitive World, 16. The First Microscopes. Leeuwenhoek, 16.
Ancient Theories Concerning the Origin of Life, 19.
The Dispute Over Spontaneous Generation. . . . . . . . . . . . . . . . . . . . . . . .. 20
Francesco Redi (1626-1679), 20. Louis Joblot (1645-1723), 20. John
Needham (1713-1780, 20. Lazzaro Spallanzani (1729-1799), 21.
Schulze ,and Schwann. 21. Louis Pasteur (1822-1895), 21. Lord
Lister (1'827-1912), 25.
The Beginnings of Precise Bacteriology. . . . . . . . . . . . . . . . . . . . . . . . . . .. 25
Robert Koch (1843-1910), 26.
Development of Staining Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 27
Weigert and Eqrlich, 27. Fluorescence Microscopy, 27. Develop-
ment or:Gram's
, Stain, 28. Other Developments, 29.
CHAPTER 3
THE MICROSCO),IC WORLD 3. YEASTS AND MOLDS. . . . . . . . . . . . . . . .. 32
General Characteristics .................... , . ... . . . . .. . . . . .... . .. 32
Oassification of Fungi, 33.
vii
viii Contents
The Yeasts ...................... " ........................ '" " 34
Dimorphism, 35. Structure of Yeast Cells, 35. Multiplication of
Yeasts, 35. Habitat of Yeasts, 37. Activities of Yeasts,,37.
Classification of Yeasts ...................................... " 38
Family Saccharomycetaceae 40. The Non-spore-forming Yeasts. 41.
CHAPTER 4
THE MICROSCOPIC WORLD 4. THE MOLDS ....... , . . . . . . . . . . . . . . . . 43
Structure of Molds ............................................ " 43
Reproduction of Molds .......................... , . . . . . . . . . . . . . 44
Formation of oidia or arthrospores, 44. Blastospores, 44. Chlamy.
dospores, 44. Sporangiospores, 45. Conidiospores, 45.
Culture Media for Molds........................................ 4S
Giant Colonies, 46. Slide Cultures, 47.
Habitat and General Properties of Molds ........ , ......... , . , . . . .. 48
The Phycomycetes ............. '.......... ' ..... , .... , . , .. , . . . . .. 49
Genus Mucor, 49. Genus Rhizopus, 50.
The Filamentous Ascomycetes ..... '.................... '. . . . . . . . . .. 51
Genus Aspergillus. 52. Genus Penicillium, 53.
Fungi lmperfecti................................................ 54
Genus Trichoderma, 54. Genera Candida. Trichosporon, Geo-
trichum, 55. Genus Alternaria, 55. Genus Hormodendrum or Clado-
sporium, 56.
Some Pathogenic Fungi .....................................'. . . . . 56
Blastomyces cermatitidis, 56. Coccidioides immitis, 57. Histo-
plasma capsulatum, 57.
The Dermatophytes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 59
CHAPTER 5
THE MICROSCOPIC. WORLD 5. THE VIRUSES. A. GENERAL DISCUSSSION 61
First Demonstration of Filtrable Infectious Agents.... . . . . . . ...... .. 61
Plant Viruses, 61. Bacterial Viruses, 61. Animal Viruses, 62.
General Properties............... ................................ 62
Methods of Virology ............................... , ......~. . . . . . 62
Microscopy of Viruses, 62. Chemistry and Physics of Viruses,
63. Viruses and Nucleoproteins, 64.
Tissue Cultures and Viruses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Multiplication of Viruses,...................................... " 68
Cell Receptors, 68. The Latent Period, 69. 'Formation of New
'Phage, 69. Cell Lysis, 70. Burst Size, 71. Resistance, 71. Virulent
and TeJ,llperate 'Phage, 71. Prophage, 71. Lysogenicity and Induc-
tion, 71. Provirus. 72. Resistance of Viruses, 72. Viruses and Anti-
biotics, 72. Parasitic Status of Viruses, 73. Classification of Viruses,
74. Mutual Interference by Viruses, 74.
CHAPTER 6
THE MICROSCOPIC WORLD 6. THE VIRUSES. B. BACTERIOPHAGE ... " 78
Isolation of 'Phage, 78. Transduction, 79. Plaque Formation, 79.
Enumeration of Bacteriophage, 80. Effect of Colloidal Matter on
Contents ix
Bacteriophage, 81. Varieties of Bacteriophage, 81. "Typing" of
Bacteria with Bacteriophage, 83.
CHAPTER 7
THE MICROSCOPIC WORLD 7. THE RICKETISIAE ........ '" . .. .. .... 85
Discovery. . • . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 85
Characteristics of Rickettsiae.. ........ ........ . ..... . ............ 85
Morphology, 85. Growth, 87. Rickettsiae and Antibiotics, 87.
Habitat, 88.
Rickettsial Diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
CHAPTER 8
THE MICROSCOPIC WORLD 8. THE PLEUROPNEUMONIA AND PLEURO-
PNEUMONIA-LIKE ORGANISMS......................................... 89
Principal Groups. • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 89
Pleuropneumonia Organisms, 89. Pleuropneumonia-like Organ-
isms, 89. L Forms, 89. General Properties, 90. Cultivation, 91.
Colonies (All Forms) ....................................... '" 93
Staining and Morphology, 94. Life Cycle, 94. Occurrence of L
Bodies, 95. Relations of PPO, PPLO and L Forms, 96. Streptoba-
cillus~ Moniliformis, 96.
SECTION 2. METHODS AND PHENOMENA OF MICROBIOLOGY
CHAPTER 9 /
OPTICAL METHODS IN MICROBIOLOGY.......................... 101

Light Microscopy .•...•...•..................................... 101
The Compound Microscope ..........................•............ 101
General Description, 101. Oil Immersion, 102. Real and Virtual
Image, 102. Resolving Power, 104. "Hanging Drop" Preparations,
104. Smears for Staining, 105. Nature of Dyes, 105.
Staining Bacteria ............................................... 105
Simple Stains, 105. Gram's Stain, 106. Ziehl-Neelsen Stain, 107.
Negative Staining, 109. Darkfield Method, 110.
Electron Microscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 110
Shadowing Technique, 114.
Fluorescence Microscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 117
Phase Microscopy .............................................. 118
I
CHAPTER 10 I
MORPHOLOGY AND STRUCTURE OF BACTERIA. . . . . . . . . . . . . . . .. 120

Morphologic~l Types of Bacteria ...................•...•..•.. " .. 120
Spherical Bacteria, 120. Cylindrical or Rod-like Bacteria, 120.
Helicoidal or Spiral Bacteria, 120.
x Contents
Size of Bacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 121
Size and Fission, 122.
Bacterial Structure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 122
Flagella, 122. Capsule (Slime Layer, or Sheath), 125. Cell Wall, 127.
Protoplast, 128. Mitochondria-like Particles, 131. Spores, 131.
Granules, 134. Chemical Composition of Bacteria, 136. Pigments
of Microorganisms, 138.
CHAPTER 11
CLASSIFICATION AND TAXONOMY OF BACTERIA ............... 140

Development of Bacterial Classification, 140. Classification schemes,
141. Species and Genus, 146. Strains, 147. Clones, 147. Bacterial
Nomenclature, 147. Type Species, 148. Biochemistry and Classifica-
tion, 148. Antigenic Structure and Classification, 148.
CHAPTER 12
EFFECT OF CHEMICAL AND PHYSICAL AGENTS ON MICROOR-
GANISMS ....................................................... 150
Temperature ................................................... 150
Temperature and Growth, 150. Thermal Resistance, 151. Rate of
Death, 152. Extreme Cold, 153.
Hydrogen Ion Concentration ........... : ......... : .... .....J • • • • • • 153
pH in Microbiology ........................................... 154
Buffers and Buffer Action ...................................... 155
Other Ions ................................................... 156
Cations, 156. Anions, 158. Complete Molecules, 158.
Magnetism .................................. '. . . . . . . . . . . . . . . . . .. 158
Electricity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 158
Electrophoresis, 158.
Moisture and Desiccation ........' ............................ : . .. 159
Desiccation and Vacuum, 160.
Preservation of Bacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 160
Desiccation in Vacuo, 160. Freezing, 160. Freeze-Drying; 161.
Effects of Autolysis, 161.
Osmotic Pressure ............................................... 162
Halophilic Organisms; 162. Effects of Evaporation, 162. Altera-
tions in Permeability; 162. Involution Forms, 163.
Radiant Energy ................................................... 163
The Electromagnetic Spectrum, 1.63.
Biological Effects ofIrradiations .................................. 163
Effects'of Irradiations, 163. Mechanisms of Irradiation Effects, 165.
Recovery from Irradiation, 165. Special Uses of Ultraviolet Light,
165. Ionizing Radiations and Food Preservation, 166. Genetic
Effects of Irradiations, 166. Photodynamic Sensitization, 166. Sun-
light, 167.
Hydrostatic Pressures ........................................... 167
Pressure and Temperature, 167.
Surface Forces .................................................. 167
Surface Tension, 168. Adsorption, 168. Surface Tension and Wet-
ness, 169.
C:ontents xi
Rapid Vibrations ............................................... 169
Production and'-Effects of Viprations, 169.
Natural Environments ••........................................ , 170
Germ-free Life .•............................................... 171
OiAPTER 13
NUTRITION, METABOLlSM, AND CULTIVATION OF MICROOR-

GANISMS •..................................................... , 176
Nutrition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 176
Food, 176. Holozoic Nutrition, 176. Holophytic Nutrition, 177.
Nutritional Types .........•.....•............................• 178
General Requirements, 178. Elemental Requirements, 178. Auto-
trophs, 178. Heterotrophs, 179. Saprophytes, 179. Parasites, 180.
Recapitulation ..........•.................................... 180
Nutritional Requirements and Culture Media. . . . . . . . . . . . . . . . . . .. 180
Metabolism .................................................... 180
1. Nutrients as Sources of Energy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 180
Dehydrogenation, 181. Aerobic Respiration, 181. Anaerobic Respi-
ration, 182. Stages and Products of Energy Metabolism, 185. Facul-
tativeRespiration, 185. Direct Oxidation, 186. Oxidation-Reduction,
186.
2. Nutrients as Sources of Cell Building Materials. . . . . . . . . . . . . . . . .. 186
Assimilation, 187.
3.. Nutrients as AcCessory Substances ............................ 187
Culture Media.-................................................ 188
Natural and Empirical Culture Media, 188. Synthetic Culture
Media, '188. Living Culture Media, 189.
Cultural Methods ••........................................... 189
Synthetic Inorganic Media, 189. Synthetic Organic Media, 190.
Empirical, Complex, Organic Media, 191. Special Media, 191.
Pure Culture Methods ..................... : ..................... 192
Difficulties with Mixed Cultures, 192. Origin of Pure Culture Tech-
nique, 192. The Use of Gelatin, 193. First Use of Agar-Agar, 193.
Origin of the Petri Plate, 193. Preparation of Solid Media, 193.
Silica Gels, 194. Cultivation on Fine Pore Filters, 194. Selective
Cultivation, 195. Enrichment, 196.
Use of Living Cells for Cultivation of Microorganisms. . . . . . . . . . . . . .. 196
Propagation and Cultivation of Viruses and Rickettsiae ............. , 196
Tissue Cultures, 196.
Cultivation in Chick Embryos ................................... , 199
CltAPTER 14
!HF, G~OWTH '-O!_ B~~ ..................................... 204
...:...._Reproductio_n_.__,__._.. '_'_~'_'_"""_"'" ................................... 204
Binary Fission, 204. Bacterial Multiplication by Means Other than
Binary Fission, 204. Sexual Multiplication of Bacteria, 204.
Bacterial "Populations" . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 20S
The Enumeration of Bacteria, 206. The Colony Count, 207. Deter-
mination of Growth Curve, 207. Phases of the Growth Curve, 208.
Factors Affecting Growth Phases, 211. Colony Growth, 212.
xii Contents
CHAPTER 15
VARIATION OF MICROORGANISMS ••............................. 214
Hereditary Mechanisms ......................................... 214
Genes, 214. Plasmagenes, 216.
Mutagenic Agents .............................................. 216
A. Extrinsic Mutagens ......................................... 216
Microbiological Agents, 216. Fertilization as a Mutagenic Agent,
220. Disease and Mutation, 220. Genetic Recombination, 220.
Summary, 221.
CHAPTER 16
SOME METHODS OF STUDYING VARIATIONS; COMMON VARIA~
TIONAL TYPES ..•............................................... 223
Rates of Mutation, 223. Detection and Isolation of Mutants, 223.
Adaptation, 226. Mutations Affecting Whole Populations, 227.
Sectors in Colonies, 228.
Variations in Colony Form. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 228
Rough and Smooth Colonies, 228. Mucoid Colonies, 231. Minute
or Small Colonies, 231. Hand 0 Forms, 231. Secondary Colonies,
231.
CHAPTER 17
THE SYSTEMATIC STUDY OF BACTERIA ......................... 233
Procedures in Identification. . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . .. 233
Purification of Culture, 234. Arrangement and Motility, 235. Stain-
ing Reaction and Morphology, 235. Biochemical Tests, 236.·Rapid
Microtechniques, 242. Identification of the Unknown .organism,
243. Need for Keys, 243.
CHAPTER 18
DESTRUCTION, REMOVAL AND INHIBITION OF MICROORGAN-
ISMS 1. BASIC PRINCIPLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
Definition of Terms ............................................. 247
Sterilizat_ion, 247. Bactericide, 247. Disinfection, 247. Sepsis, 248.
=Asepsis', '248~Antiseptic, 248. Bacteriostasis; Bacteriostatic Agents,
248.
Principal Methods ......................... " . . . . . . . . . . . . . . . . . . .. 249
Destruction of Physical Structure, 249. Non-specific Chemical Com-
binations, 249. Specific Chemical Combinations and Bacteriostatic
Agents, 249. Non-specific Bacteriostatic Methods, 249. Combined
and Variable Effects, 250.
The Role of Hydration in Disinfection.. .. . . . . . . . . . . . . . . . . . . . . . . . .. 250
Factors Affecting Chemical Disinfection ........................... 251
Contact, 251. Synthetic Detergent-Disinfectants, 252. Chemical
Structure and Activity, 252. Concentration, Time, Temperature and
pH or pOH, 253. Inactivation of Antimicrobial Agents, 256.
Contents xiii
The Evaluation of Disinfectants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 260
The Phenol Coefficient, 261. Inactivators, 262. The Use-dilution
Test, 263. Toxicity of Disinfectants, 263.
Factors Affecting Sterilization by Heat. . . . . . . . . . . . . . . . . . . . . . . . . . .. 264
Time and Temperature, 264. Numbers of Organisms, 264. pH, Hy-
dration, etc., 264.
CHAPTER 19

ISMS 2. PRACTICAL APpuCATIONS ••••••••••••.•••..•••••••••••••••• 267
Uses of Heat ..•...................................... ~. . . . . . . . .. 267
A. Moist Heat, 267. B. Dry Heat, 270. Thorough Heating Neces-
sary,270.
Sterilization by Filtration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 271
. Clay and Paper Filters, 271. Membrane Or Ultra-Filters, 271.
Some Useful Disinfectants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 272
KMn04 and H 20 2, 272. Halogens, 272. Inorganic Acids and Alka-
lies, 272. Heavy Metal Salts, 272. Phenolic Compounds, 275. Radia-
tions, 276. Alcohols, 276. Quaternaries, 277. Ethylene Oxide, 277.
CHAPTER 20
ISMS 3. ANTmIOTICS.· ••.•.•...••.•••••..•.. , • • • • • • • • • • • • • • . • . . • • •• 279
Penicillin. . ......-.............. '.......... , . . . . . . . . . . . . . . . . . . . . .. 279
Production, 281. Properties, 282. Mode of Action, 283. Chemistry
and Varieties of .Penicillin, 283. Biosynthesis of Penicillins, 283.
Various Strains of Penicillium Notatum, 284. Factors Inhibiting
Penicillin, 284. Development of Resistance or Drug-Fastness; De-
pendence, 284. Laboratory Uses ofPeniciUiI), 284. The Clinical Uses
of Penicillin, 284. Standardization, 285. Methods of Assay and Sen-
sitivity Testing, 285.
Antibiotics·from Streptomyces .................................... 290
Streptomycin, 291. Chloramphenicol, 293. The Tetracyclines, 294.
Antibiotics from the Genus Bacillus ............................... 294
Bacitracin, 294.
Non-Medical Uses of Antibiotics ................................. 295
Industrial Uses, 295. Antibiotics as Growth Stimulants, 295. Anti-
biotics for Plant Diseases, 295.
SECTION 3. IMMUNOLOGY: REACTION OF THE ORGANISM TO

SUBSTANCES OF EXTRANEOus ORIGIN
CHAPTER 21
IMMUNOLOGY AND MICROBIOLOGY 1. I>ruNCIPLES AND METIIODs. 301
I
Blood ............. : ........................................... 301
Important Blood COnstituents .................................. 301
xiv Contenu
Plasma, 301. Fibrin Comoonents, 301. Platelets, 302. Serum, 302.
Lymph, 302. Erythrocytes, 302. Leukocytes, 303.
Immunology in Relation to Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 303
Defensive Mechanisms, 303.
l. Non-Specific Resistance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 304
A. Genetic, 304. B. Physiological, 305.
II. Specific Resistance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 309
Natural Active Immunity, 309. Nature of Antigens, 309. Antibodies
and Adaptive Enzymes, 310.
Antigen-Antibody Reactions ..................................... 310
Stability of Antigen-Antibody Combinations, 311. Factors Influ-
encing Development of Active Immunity, 312. Antigens in Nature
Are Usually Mixed, 312. Antibody Adsorption, 313. Labeled Anti-
bodies, 314.
Antigenic Structure of Bacterial Cells... . . . . . . . . . . . . . . . . . . . . • • . . . .. 314
Somatic (0) Antigens, 314. Flagella (H) Antigens, 314. Capsular
(K) Antigens, 316. Extracellular Antigens, 316.
Types of Antibody Response"and Reaction ......................... 316
Antitoxins, 316. Cytolysins and Complement, 318. Complement,
318. Immobilizing Antibodies, 319. The Immune-Adherence Phe-
nomenon, 320. Agglutinins, 320. Precipitins, 322. Antigen-Antibody
(Precipitin) Reactions in Gels, 323. Protective Antibodies, 325.
CHAPTER 22
IMMUNOLOGY AND MICROBIOLOGY 2. ARTIFICIAL IMMUNITY •.... 327
Active Artificial Immunity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 328
Primary and Secondary Stimulus ............... " . . . . . . . . . . . . . .. 328
"Booster Doses," 328. (A) Immunization by Means of Exotoxins.
329. (B) Immunization with Dead Microorganisms, 330. (C) Im-
munization with Attenuated, Living. Infectious Agents, 331.
Passive Immunity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 334
Artificial Passive Immunity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 334
Passive Immunity in the Prevention of Disease, 334. Passive Im-
munity and Serum Jaundice, 335.
Natural Passive Immunity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 335
Transitory Nature of Passive Imm,unity, 335.
CHAPTER 23
lMUNOLOGY AND MICROBIOLOGY 3. THE TISSUES IN RELATION TO
IMMUNITY ...... '...................... " ............................ 337
The Allergic State ................... ,...... : ..................... 337
The Induction Period, 337. Passive Allergy, 337. Allergens and Re-
agins.337.
Types of Allergic Reaction •......•.......... '..................... 338
Manifestations of Immediate Allergy. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 338
Anaphylaxis, 338.
Other Reactions of Immediate Allergy. . . . . . . . . . . . . . . . . . . . . . . . . .. 339
Dermal Reactions, 339.
Manifestations of Delayed Allergy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 340
Bacterial Allergy, 340. Allergy and Disease, 340.
.
Contents, xv
Harmful Effects of Allergy ....................................... 341
Allergy as a Defensive Mechanism ................................ 341
Non-antitoxic Immunity to Toxin, 341. The Koch Phenomenon,
341.
CHAPTER 24
MICROORGANISMS AND DISEASE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 343

Obstacles to Parasitism, 343. What Is Disease?, 343. Parasitism and
Pathogenicity, 343. Pathogeilic Saprophytes, 344. Pathogenicity Is
Fortuitous, 344. Mutual Adaptation, 344.
Factors in the Occurrence of Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 345
Portal of Entry, 345. Vegetative Vigor (or Aggressiveness), 345.
Toxicity, 346. Dosage, 347. Infectiousness, Pathogenicity and Viru-
lence. 347. Koch's Postulates, 347. Rivers' Postulates in Viral Dis-
eases, 348.
CHAPTER 25
TRANSMISSION OF DISEASE ...................................... 350
Vectors... . . .. . ............................................... 350
Types of Di~ease Vector .................... '" .................. 350
1. Mechanical Jr!lnsmission .................................... 351
Fomites, 351. Transmission by Foods, 354. Transmission by Hands,
355. Transmission by Droplets of Saliva, Mucus, etc., 355. Trans-
mission by Dust, 357. Transmission by Direct Contact, 358. Domes-
tic Environments and Diseases, 358.
2. BioJogicili Transmission of Disease. . . . . . . . . . . . . . . . . . . . . . . . . . .. 358
Human Blood and Blood Derivatives. 359. Bites of Vertebrates, 359.
Bites of Arthropods, 360. Insect Feces, 360. Bodies of Insects, 360.
Vertebrate Animals as Vectors of Disease, 3,510.
SEcrION 4. THE BACTERIA. (CLASS SCHIZOMYCETES)
CHAPTER 26
THE "SLIME BACTERIA" (ORDER MYXOBACTERIALES).......... 367
The Swarm Stage. 367. The Encystment Stage, 367. Enzymic Activi-
ties of Myxobacteriales, 368. Genus Cytophaga, 368. Cultivation of
Saprophytic Myxobacteriales, 369. Relationship to Other Forms of
Life, 370. The Mycetozoa, 371.
CHAPTER 27 '
THE "SHEA THEq" BACTERIA (ORDER CHLAMYDOBACTERIALES)
AND "STALK-FORMING" BACTERIA (SUBORDER CAULOBAC-
TERIINEAE). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 372
Structure, 372. Growth, 372. Sphaerotilus (or Leptothrix) and the
Iron Bacteria, 372, Genus Crenothrix, 373. Systematic Relation-
ships of: the "Sheath-Formers," 374.
xvi Contents
The Caulobacteriineae ........................................... 374
Gallionella Ferruginea. 374. Siderocapsa Treubii, 375. Caulobacter
Vibrioides, 376.
CHAPTER 28
THE SULFUR BACTERIA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 378
Groups of Sulfur Bacteria, 378.
A. Sulfur Oxidizers ............................................. 378
B. Sulfate Reducers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 379
Habitat of Sulfur Bacteria, 379.
Sulfur Oxidation ................................................ 380
Beggiatoacea and Achromatiaceae, 380. The Genus Thiobacillus,
380.
Functions of Sulfur Oxidizers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 382
Sulfate Reduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 383
Distribution and Structure, 383.
CHAPTER 29
THE PHOTOSYNTHETIC BACTERIA ...•.•.......................... 385
Cblorophylls, 385. Habitat and General Properties, 385. Use of
Photosynthesis. 385.
Photosynthesis as a Type of Reaction ........................... :. 386
Green Plants, 386. Chlorobacteriaceae and Thiorhodaceae, 386.
Athiorhodaceae. 387. General Type Reaction, 387. Sulfur Utiliza-
tion by Bacteria. 387. Other Relations of Light to Photosynthetic
Bacteria, 387.
CHAPTER. 30
THE MOLD-LIKE BACTERIA (ORDER ACTINOMYCETALES) ....... 389
Three Families of Actinomycetales ................................. 389
The Family Mycobacteriaceae, 389. The Family Actinomycetaceae,
389. The Family Streptomycetaceae, 389. Molds and Actinomy-
cetes, 390.
Family Actinomycetaceae ........................................ 392
Genus Nocardia. 392. Genus Actinomyces, 392. "Ray-fungi," 392.
Family Streptomycetaceae ......... : . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 393
Streptomyces and Antibiotics, 394. .
Family Mycobacteriaceae ........................................ 394
Non-pathogenic Species of Mycobacterium, 394. Mycobacterium
Tuberculosis, 395.
Tuberculosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 395
Tubercles and Tuberculosis, 395. Tuberculin Reaction, 396. Im-
munization Against Tuberculosis, 396. Laboratory Diagnosis of
Human Tuberculosis, 396. Mycobacteria and Wetting Agents, 397.
Leprosy. . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 397
Etiology, 397. The Method of l'ransmission, 398. History, 398.
Contents xvii
CHAPTER 31
THE SPIRAL, FLEXIBLE BACTERIA (ORDER SPIROCHAETALES) .. 400
General Characters and Structure of the Spirochaetales, 400.
Genus Spirochaeta .............................................. 403
Genus Saprospira ............................................... 403
Genus Cristispira. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 403
Genus Treponema. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 4Q3
Resistance- and Cultivation, 404. Syphilis, 404.
Genus Borrelia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .• 405
Trench Mouth, 406.
Genus Leptospira .•..........................................•.. 406
Leptospirosis, 407.
CHAPTER 32
THE SPIRAL, RIGID BACTERIA ................................... 409
Genus Vibrio ................................................... 409
V. comma et aI., 409. Isolation of Intestinal Vibrios, 410. Asiatic
Cholera, 410. V. fetus, 411. Anaerobic Vibrios, 411.
Genus Spirillum ................................................ 412
Spirillum Volutans, 412. Spirillum minus and Rat-Bite Fever, 412.
CHAPTER 33
THE AEROBIC SPORE-FORMING RODS (GENUS BACILLUS) ....... 413
Genus Bacillus ................................................. 413
Variability, 414. Thermophilic Species, 414. Types of Sporulation
and Classification, 414. Spore Germination, 415. Biocheffi1cal Char-
acters, 415. Structure of Bacillus, 415. Distribution, 415.
Bacillus Anthracis ...................... . 1 ...............••••• 416
Anthrax, 416 ..
Bacillus Subtilis .............................................. 417
Bacillus Cereus, B. Mycoides, B. Vulgatus, B. Mesentericus, Etc..... 417
Bacillus Coagulans ......................................... : .. 417
Bacillus Popilliae and B. Lentimorbus. . . . . . . . . . . . . . . . . . . . . . . . . .. 418
Bacillus Rotans and B. Alvei. .................................. 418
Foulbrood,418.
CHAPTER 34
ANAEROBIOSIS. THE GENUS CLOSTRIDIUM ..................... 420
I
AnaerobiOSIS ..................................................... 420

Relations to Oxygen, 420.
Cultivation 'of Anaerobic Bacteria.. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 422
Oxidation-Reduction Potentials, 422. O-R Requirements of Micro-
organisms, 422.
Anaerobic Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 422
Chemical, 422. Replacement Methods, 424. Oxygen Exclusion
Methods, 424. Cultivation of Anaerobes in Media Freely Exposed
to Air, 425.
Anaerobic :Bacteria. . . . . . . . . . . . . . . . . . . . . . . . •. . . . . . . . . . . . . . . . . . . .. 426
xviii Contents
Tribe Bacteroideae •..... '" ................................... 426
Genus Clostridium .•........ '" ............................... 427
Clostridium Butyricum, 427. Anaerobic Nitrogen Fixation, 427.
Clostridium Tetani and "Lockjaw," 428. Clostridium Perfringens,
428. The Gas Gangrene Organisms, 430. Clostridium Botulinum,
430.
CliAPTI!R 35
FAMILYCORYNEBACTERIACEAE;FAMILYLACfOBACTERIACEAE 432
The Corynebacteriaceae ......................................... 432
The Genus Corynebacterium. . . . . . . . . . .. .. . . . . . . . . . .. . .. . . . . . .. 432
Diphtheroids, 433. Toxin Production by Corynebacterium Diph-
theriae, 434. Immunity to Diphtheria, 434. Laboratory Methods for
the Study of Diphtheria, 436. Alterations in Virulence of C. Diph-
theriae, 437.
Genus Listeria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 437
Genus Erysipelothrix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 439
Swine Erysipelas, 439.
The Family Lactobacteriaceae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 439
Tribe Lactobacilleae .................................... : ...... 439
The "Lactic Bacteria" ... " . '" ................................ 441
CHAPTER 36
SPHERICAL BACTERIA ............................................ 443
1. The Family Micrococcaceae ................................. " 443
General Characters, 443. Micrococcaceae in Nature, 444.
Pathpgenic Micrococci ........................................ " 445
Gaffkya, 445.
Tribe Streptococceae .......................................... " 445
Bloo:~~~!:i~~~e~: ~~: ............ : ....................... ..\ 446

Alpha-Type Hemolytlc StreptococcI, 446. Beta-Type HemolytI~\
Streptococci, 447. Double-Zone Beta-Type Streptococc, 447. Gam-
ma Type, 447.
1. The Lactic Streptococci .............................. ;-. ...... 447
Streptococci Related to S. Lactis: 448.
2. The EnterococCi. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 448
3. The Pyogenic Streptococci ............... :. . . . . . . . . . . . . . . . . .. 448
The Beta Hemolytic Species, 448. The Alpha Hemolytic ("Viri-
dans"),Streptococci,451.
Genus Diplococcus ........... '.. ~ ... ' ....... , .. , .. '., ......... 451
Serological Types of Pneumococci, 452, Pathogenic Action of Pneu-
mococci, 453.
Genus Leuconostoc ................ , .. , .......... , ............ 453
Leuconostoc Citrovorum, 454.
The Lactic Bacteria .................. , .. , .......... , . . . . . . . . . . .. .454
Recapitulation .......... , ....................... , ........... , 454
The Family Neisseriaceae ... , .. , .... , .. , , ..... , ......... , ........ 455
Genus Neisseria ......................... , . . . . . . . . . . . . . . . . . . .. 455
The Oxidase Test, 457. The Catalase Test, 457. Gonorrhea, 457.
Gonorrheal Ophthalmia, 458. Meningitis, 458.
Contents xix
SECTION 5. THE MICROBIOLOGY OF SPECIAL ENVIRONMENTS
CHAPTER 37
THE MICROBIOLOGY OF WATER ................................. 463
Controlling Factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 463
Aqueous Environments .......................................... 463
Bacteria in Fresh Waters, 463.
The Pseudomonadaceae and Enterobacteriaceae. . . . . . . . . . . . . . . . . . .. 465
FamilyPseudomonadaceae ....................................... 465
General Properties, 465. Pseudomonas as Pathogen, 466.
Family Enterobacteriaceae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 466
General Properties, 466. Enteric Genera, 468.
Genus Proteus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 468
Proteus Colonies, 4
The Coliform Group. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 468
Genus Escherichia, 468. Genera Aerobacter and Klebsiella, 468.
Sanitary Relationships of the Coliform Group. . . . . . . . . . . . . . . . . . . . .. 469
Pollution of Water with Fecal Material, 469. Indices of Fecal Pollu-
tion, 469. The "Membrane" or "Millipore" Filter (M.F.) Method,
470.
Differentiation of Fecal and Non-Fecal Types .................... 472
Imvic Formula, 472.
Genus Paracolobactrum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 473
Marine Bacteria .... -. ........................................... 473
Marine Zones, 473. Photogenic Bacteria, 474. Marine Bacteria and
Petroleum, 474.
CHAPTER 38
SANITATION OF DRINKING WATER AND SEWAGE DISPOSAL. .. 476
-._
Filter Plants ....................................................
- /
476
The Slow Sand Filter, 477. The Rapid Sand Filter, 477. Combina-
tion Process, 478.
Sewage Purification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 479
Composition of Sewage, 479. Microorganisms in Sewage, 479.
Changes in Sewage, 480. Biological Actions, 480. Importance of
Oxygen, 481.
Sewage Disposal Plants ........................................ 481
"Two-Story" Tanks, 482. Aeration and Disposal of Fluid, 482. Ac-
tivated Sludge, 484. Rapid Methods of Sewage Purification, 485.
Prevention of Water-Borne Disease in Absence of Filtration ........ 485
CHAPTER 39
SOME PATHOGENIC GRAM-NEGATIVE RODS: ENTEROBACTERIA-
CEAE; PARVOBACTERIACEAE .................................. 487
The Pathogenic Enterobacteriaceae ................................ 487
Genus SalInonella. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • .. 487
Isolatioh of Salmonella, 489. Antigenic Analysis, 489. Names of
Salmonella, 492. Variation of Species, 492. Salmonellosis, 492.
xx Contents
Genus Shigella and Bacillary Dysentery. . . . . . . . . . . . . . . . . . . . . . . . .. 493
Bacillary Dysentery, 493.
Pathogenic Escherichia. . . . . . . . . . . . . . . . . . . . . • . . . . . . . . . . . . . . . . .. 493
Paracolons as Pathogens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 493
The Parvobacteriaceae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 494
Genus Pasteurella.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 494
Pasteurella Pestis, 494. Pasteurella Tularensis, 495.
Genus Brucella. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • .. 496
Isolation, 496. Survival and Distribution, 496.
Genera Hemophilus, Bordetella, Moraxella..... . . . . . . . . . . . . . . . . .. 497
Genus Hemophilus, 497. Bordetella, 497.
Summary of Rod-Shaped Bacteria. . . . . . . . . . . . . . . . . . . . . . . .. .. .. ... 497
CHAPTER 40
THE SOIL AS AN ENVIRONMENT FOR MICROORGANISMS ...... 500
Composition of Soils, 500. Soil as a Culture Medium, 500. Varia-
tions in Soil, 500.
Soil Populations .................................................... 501
Bacterial, 501. The Soil a Microbial Universe, 502.
Syntropism in the Soil ........................................... 502
Cellulose Decomposition, 502. Aerobe-Anaerobe Relationships,
502. Formation of Humus, 503.
Bacteriological Examination of Soil. . . . . . . . . . . . . . . . . . .. . . . . . . . . . .. 503
Plating Methods, 503. Selective Methods, 503. Enrichment Meth-
ods 504. Microscopic Examination, 504.
Cycles of the Elements. . . . .. . . . . . . . . . .. .. . . .. . . . . . .. .. .. . . . . . ... 504
The Nitrogen Cycle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 5Qi
Processes in the Nitrogen Cycle, 504.
I. Nitrogen Fixation... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 505
A. Non-Symbiotic Nitrogen-Fixing Organisms. . . . . . . . . . . . . . . . . .. 505
Genus Azotobacter, 506. .
B. Symbiotic Nitrogen-Fixing Organisms ........................ 507
The Rhizosphere, 507. Genus Rhizobium, 507. Nitrogen Fixation,
508. Soil Inoculation, 509.
Other Rhizobiaceae .......................................... : 510
Genus Agrobacterium, 510.
The Rhizosphere .............. ,...................... : ......... 510
Antibiotics and Plant Diseases, 511.
n Nitrogen Oxidation ........................................ ~ .. 512
Tribe Nitrobacterieae .................... ' ..................... 5.12
Ammonium Salts to NaN0 2• 512. NaN0 2 to NaN0 3• 512.
III. Nitrogen Reduction ........................................... 513
Importance of Nitrogen Redtiction, 513. Dentrification, 513. Am-
monification, 513.
The Sulfur Cycle................................................; 514
Forms of Sulfur in Soil, 514. Reduction and Oxidation of Sulfur,
515.
The Carbon Cycle .............................................. 515
Carbon Oxidation, 515. Carbon Reduction, 515.
The Phosphorus Cycle ........................................... 515
Microbiology of Petroleum ....................................... 516
Destruction of Petroleum, 516.
Contents xxi
CHAPTER 41
THE MICROBIOLOGY OF THE ATMOSPHERE., ................... 519
Collection and Enumeration of Aerial Microorganisms. . . . . . . . . . . . .. 520
Dust, Droplets and Droplet Nuclei ............ , ................... 520
Air Pollution •.............................. , . . . . . . . . . . . . . . . . . .. 523
Control of Air-Borne Infection, 523, Effectiveness of Methods, 524.
CHAPTER 42
I
THE MICROBIOLOGY OF MILK ............... , ................... 526
Types, Numbers and Significance of Bacteria Normally in Milk,
526. Changes in Flora of Milk, 526. Significance of Coliform Organ-
isms in Milk, 529. Factors Affecting Bacteria in Milk, 529. The
Phosphatase Test, 529. Counting the Bacteria in Milk, 530. Enumer-
ation of Bacteria in Milk, 530. The Reductase Test, 531. Grades of
Milk, 531. Criteria of Good Milk, 532. Certified Milk, 532. Lacto-
bacilli in Milk, 533. Fermented Milk Beverages, 533. Milk as a Dis-
ease Vector, 533.
CHAPTER 43
THE MICROBIOLOGY 9F FOODS.... . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 535
Definitions and Classifications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 535
Autolytic Enzymes, 536.
Fresh Foods ...•............................. , ................• 536
Meat, 536. Microorganisms in Meat, 537. Ground Meat, 538. Bac-
teria in Comminuted Foods, 538. Fish, 539. Oysters, 539. Fruits and
and Vegetables, 540. Eggs, 540. Bread, 541.
Some Fermented Foods ....................../ . . . . . . . . . . . . . . . . .• 543
Ensilage, 543. Sauerkraut, 544. Pickles, 544.
Preservation of Foods ......................... , ................. 545
Canned Foods, 545. Freezing, 548. Refrigeration, 549. Chemical
Preservatives, 549. Smoking, 549. Drying, 549. Preservatives Com-
monly Classed as Foods, 550. Foods in the Kitchen, 551.
Bacteriological Examination of Foods. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 552
Quantitative Microbiology of Food, 552. Qualitative Microbiology
of Food, 552. Opening Containers, 552.
Food Spoilage ........................................... '....... 553
Specific Causes of Spoilage or "Diseases" of Foods, 553.
CHAPTER 44 I
MICROBIOLOGY;AND INDUSTRY ................................. 556

Kinds of Indpstrial Processes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 556
Factors of Importance in Developing an Industrial Process. . . . . . . . . .. 557
Purity and Nature of Cultures, 557. Medium or Raw Material, 557.
Nature of the Process, 557. Preliminary Experimentation, 557.
Some Manufactured Dairy Products ............................... 558
Maintenance of Starter Cultures, 558. Use of Starters, 558. Interfer-
ence with Starter, 558. Butter, 558. Cheese, 559.
xxii Contents
Curing or Ripening. . . . .. . .. .. . .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 560
Hard Cheeses, 560. Semi-soft Cheese, 562. The Curing of Soft
Cheeses, 562.
Vinegar and Acetic Acid; The Acetobacter ......................... 562
Beer .......................................................... 564
Wine .......................................................... 564
Industrial Fermentations ......................................... 566
Types of Process. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 566
Batch Fermentations, 566. The Continuous Process, 566. Sub-
merged Cultures, 566.
Industrial Ethyl Alcohol Manufacture ............................. 567
Wood as a Source of Alcohol and Food, 567. Distilled Beverage In-
dustries, 568.
Production of Butanol. .............................. '. . . . . . . . . . .. 570
Relation of Sporulation, 570.
Other Processes: Use of "By-Products" ............... '" . '" ...... 571
Enzymes of Microorganisms in Industry. . . . . . . . . . . . . . . . . . . . . . . . . .. 571
Enzymes from Bacteria ........................................ 571
Amylases and Proteases, 571.
Microbiological Assays. . . . . . . . . . . . . . . . . . . .. . . . .. . . . . . . . . . . . . . . .. 572
Assay Methods ............................................... 572
Measurement of Gas, 572. Measurement of Acid Production by
Bacteria, 572. Measurement of Turbidity, 572. Gravimetric Meas-
urement of the Mycelia of Molds, 572
Industrial Spoilage ................... , .......................... 573
Lactobacilli and Leuconostoc, 573. Prevention of Spoilage, 573.
CHAPTER 45
SOME VIRAL DISEASES .................................. , ......... 575

Virus Groups, 575.
A Viscerotropic Virus ......................................... '" 575
Yellow Fever. 575. .
Respiratory Viruses ........................................... " 579
Human Influenza, 580. .
Dermotropic Viruses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 581
Measles, 581.
Prenatal Injury by. I nfectious Agents .......................... " 581
Rubella and Pre-natal Injury, 581.
Neurotropic Viruses. ,,' . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 581
Poliomyelitis, 581. Rabies, 584.
Enteric Viruses .......................... ',' ... , ............. , ... 585
Neoplastic Diseases ........•... l . . . . . . . • . . . . . . . . . . . . . . • . . . . . . . " 586
Neoplasms, Viruses and Mutation. ,586. Antagonisms of Viruses'
and Neoplasms, 587 ..
INDEX ............................................................. 589

SECTION 1
The Relationships of Micro-
organisms to Each Other
and to the Living World
THE FIELD of microbiology is a large one. There are many species, genera,
tribes, orders and classes of microorganisms. One may devote one's life to any
of a vast number of highly specialized problems. Some of these sound utterly
fantastic: the role and exploitation of certain protozoa in the purification of
sewage; how to make poultry and livestock out of sawdust; what microscopic
fungi interest the telephone companies and why; how to find (and manufac-
ture?) petroleum with bacteria; how to control the fertility of the soil; develop-
ment of an antibiotic, an antimetabolite or a viral vaccine to combat cancer-
and so on. Before becoming an expert in anyone of these always fascinating
and sometimes lucrative activities one must have a good basic idea of what
microorganisms-are: their form, structure and size; what they do; how they
act; how they are controlled, classified, identified; and several other useful
facts about them. The.first section of this textbook introduces the beginning
student to the eight great biological groups in which microorganisms occur:
algae, protozoa, yeasts, molds, bacteria, viruses, rickettsiae and pleuropneu-
monia-like organisms. In this section each group is described in sufficient de-
tail, and in comparison with the other groups, so that the beginning student
may define and distinguish each and identify any microorganism with respect
to its major group. IThis section opens the door, as it were, to that beckoning
enigma-the living ,microscopic universe.
r
The Microscopic World
1. PROTOZOA, ALGAE, FUNGI AND BACTERIA
THE WORLD of microorganisms must not be thought of as sharply differ-

entiated from the world of macroscopic life. If we begin our survey of life
with the largest of living things, such as whales or giant redwood trees, and
move downwar:d in the scale of size to those ofless and less magnitude, it im-
mediately becomes clear that there is a continuous gradation in size, from the
very large to the very small. As we proceed downward in the scale we reach
a point where, be,cause of diminishing size, the objects of our survey disappear,
as it were, behind a curtain representing the limit of our unaided eyes to see
(resolve) minute objects. They have entered the realm of the microscopic.
We may follow with our eyes into the microscopic world for a short distance
by poking a hole through,the curtain with a microscope. On the other side
of the curtain we find, as did Alice when she passed "Through the Looking
Glass," many more strange and beautiful creatures. In a still-diminishing
scale of size these extend far back into the secret shadows of the seeable
cosmos to some inconceivably small vanishing point among the molecules,
atoms, electrons, neutrons, protons, and the unknown.
Animal or Vegetable? We generally think of the living world as divided
into two well-defined kingdoms: animal and vegetable. Usually in the macro-
scopic world the properties of p!ants distinguish them clearly from arumals.
One could never confuse a rabbit with a rose. But when one cannot see the
creatures he wishes to distinguish, when one cannot even accurately determine
form, color, chemiCal properties, food, mode of multiplication, waste prod-
ucts, and other matters of vital importance to the classifier, how is he to know
in which kingdom to place them, especially when he finds creatures possessing
some of the properties of both plants and animals'? This problem has led
some workers to suggest a third kingdom, the Protista, to include all micro-
scopic creatures nO,t readily classified as plant or animal. This suggestion has
been discussed but! never generally adopted.
The places of creatures which are pretty clearly plants, and of those which
have definitely animal characters, are well established in their respective king-
doms, as shown in, the accompanying diagram (Fig. 1-1). In the diagram it is
3
4 The Relationships of Microorganisms to Each Other and the Living World
Fig. 1-1. A suggested relationship of various divisions of 8rumate nature. With a few
borderline exceptions microscopic forms are found m all groups except multicellular ani-
mals and vascular plants. Of the bacteria, Spirochaetales are shown as most like protozoa;
Myxobacteriales are perhaps more like spirochaetes than they are like other bacteria because
of their flexing movements. The Eubacteriales (Eubacteriineae) have least resemblance to
protozoa or algae. The Rhodobacteriioeae are seen as most like the algae because of their
photosynthetic pigment. True chlorophyll is shown only for algae <,-nd vascuiar plants. The
Actinomycetales are most like the molds, while yeasts, perhaps, are more like true bacteria'
than are the molds although certainly this arrangement is open to debate. The position o~
the Chlarnydobacteriales is doubtful and is not shown. Probably it should be next to Rhoao-
bacteriineae since the Chlamydobacteriales resemble algae in some ways but are without
photosynthetic pigment. The relationships of the viruses, rickettsiae and pleuropneumonia~
like organisms (PPLO) are entirely speculative.
seen that the' field of the microbiologist embraces one phylum of the animal
kingdom (Protozoa-microscopic animals) and parts of one phylum of the
vegetable kingdom (Thallophyta-microscopic fungi and some minute algae).
Some of the Schizomycetes, or bacteria, such as the Spirochaetales, Closely
resemble protozoa. Others, such as the Actinomycetales, have some interesting
resemblances to th,e molds. Still others (Rhodobacteriineae) have some funda-
mental properties (photosynthesis) of the algae. Although lines are drawn to
make convenient diagrammatic distinctions between these groups, the dis-
tinctions in nature are far from clear. There are many puzzling overllippings
The Microscopic World 5
and morphological and physiological resemblances between apparently dis-
tant groups.
In Figure 1-1 three groups of microscopic organisms are shown outside the
animal-vegetable circle: the viruses, the rickettsiae and the pleuropneumonia-
like organisms (PPLO). Although some of these have certain properties of
some bacteria, they defy accurate classification in either animal or vegetable
kingdom.
In order to orient the beginner in this Alice-in- Wonderland universe of the
microscopic, it is desirable to describe briefly the principal characteristics of
each of the eight groups with which we shall deal (protozoa, algae, bacteria,
viruses, rickettsiae, pleuropneumonia organisms, yeast and molds), and to
show how the groups are interrelated, and their place in the living world
as a whole. These properties are summarized in Table 1.
BACI'ERIA AND TIlE ANIMAL KINGDOM
If we examine the animal kingdom as a whole, we find the vertebrates, with
man at the top of the list, representing the latest and most complex develop-
ment in organic evolution. Proceeding downward we pass through groups of
lower animals, such as the Arthropoda, Annelida, Rotifera, and Porifera,
each less and less highly developed functionally and structurally, and finally
we come to the protozoa, the least complex of all animals and the most primi-
tive in an evolutionary ~~nse. Only among the protozoa do we find any forms
in the least resembling any of the bacteria, and this resemblance is not con-
fusing except, perhaps, among certain spirochetes.
Bacteria and Protozoa. In water in which a bit of hay has been allowed to
soak for a few days, or in sewage, not only do many kinds of bacteria abound,
but also various fantastic and elegant creatures which resemble bacteria in
some respects but which can, as a rule, be readily differentiated by their rela-
tively huge size, their elliptical or ovoid form and other distinctive features.
These are protozoa (Fig. 1-2)-microscopic animals, each consisting of only
a single cell. .
Protozoa vary greatly in size, according to species and phy~iological state.
Most of them are hundreds of times as large as most bacteria. A commonly
studied type, P.qramecium, is roughly elliptical in shape and has dimensions of
about 2oo,u by 40,u. * Protozoa are the most primitive members of the animal
kingdom, just as bacteria are the lowliest group in the vegetable kingdom.
As contrasted with bacteria structurally, the protozoa are very complex and
usually contain well-defined portions performing the functions of specialized
organs in more highly organized animals. Bacteria do not contain such or-
ganelles, except flagella.
There are thousands of species of protozoa, and they may be found in
almost every habitable situation on the earth; stagnant water, pond mud,
surface waters, feces, the soil, dust, the ocean and so on. They live, in part,
upon other minute living things, including bacteria, and most of them also
take in soluble foods by diffusion through the cell membrane. A few are
pathogenic.
The protozoa seem to have evolved in the direction of complexity of physi-
• ",'is the symbol used for micron, the unit of length commonly employed in microbiology.
A micron is 0.001 millimeter or about 1/25,400 inch. The dimensions of Escherichia coli, a
cylindrically shaped bacterium common in sewage, vary around I", by 101'.
ological function without increase in number of cells. Thus, in many free-

living protozoa a portion of the cell is somewhat thickened, rigid and some-
times chitinous* in composition. This serves as a skin or outer integument.
It often contains (or forms) bristles (cilia or flagella) which have the functions
of swimming appendages of higher animals. Although there are no proper
muscles, certain portions of the protoplasm may be contractile and may act
the part of muscles in moving the cilia or flagella. The integument excretes
waste products much as do the skin and kidneys of a human being, and thus
serves the function not only of swimming oars, muscle and skeleton, but also
of kidney, intestine and sweat glands. In so far the protozoa and bacteria have
much in common. Inside the cell membrane of protozoa, however, there are
usually a well-defined nucleus, nucleolus and cytoplasm, the last often con-
taining various vacuole!: which collect waste products eventually to be ex-
Fig. 1-2. Various forms of protozoa. A, B, forms of amoeba. C, D, forms of trypano-

some. E, Giardia lamblia. F, G, types of ciliates. (After Hegner, Cort and Root, from Burdon,
"Medical Microbioiogy," by p.:rmission of The Macmillan Co., publishers.)
• Chitin is a rigid skeletal substance common in the animal kingdom and is represented
bv the shells or exoskeletons of insects, lobsters, etc.
8 The Relationships of MicTooTganisms to Each Other and the Living World
creted through the cell wall, thus serving the purpose of bladder and colon of
higher animals. Other vacuoles contain food particles in the process of diges-
tion and thus are analogous to stomachs. There is often an opening which
serves as a mouth and gullet. Bacteria contain none of these. In protozoa
there are also granules of stored-up reserve material equivalent to fatty de-
posits or liver-glycogen reserves in mammals. Bacteria often store up such
granules of food reserve.
Although no such complex structures as nose, eyes or nerves are found in
protozoa, many protozoa are nevertheless quite sensitive to heat, chemicals,
gravity, electricity and light; and in one species at least (Euglena), a portion
of the cell is differentiated into a specially sensitive area called a "light spot."
This, although not a separate organ, serves the function of a primitive eye.
There are also some bacteria which appear to possess a light-sensitive spot
or zone (see Rhodobacteriineae). Furthermore, protozoa in general accept
or reject food particles or waste, and have quite a delicate sense of touch so
that they recoil on contact with hard objects. turning aside quite as though
they were highly sensate and responsive creatures; all of which shows the
marvellous possibilities contained within a single, microscopic droplet of
protoplasm. Reproductive processes are, in some protozoa, similar to those
observed in the higher arumals. inasm uch as there are sexual phenomena, with
conjugation, nuclear interminglings, etc., in all essential respects analogous
to the sexual phenomena of fertilization in higher plants and animals. How-
ever, there are usually no special sexual organs. There are no well-defined
sexual phenomena in bacteria, though this is much debated.
The cells of all living creatures possess the power of simple, sexless, or
asexual multiplication. It is spoken of as cell fission. In the unicellular forms,
as in bacteria and protozoa, each cell resulting from fission remains a separate
organism. In complex forms, different cells group themselves together as into
"guilds" and tissues (the organs of the body, for example), such as the liver,
the brain, the heart. In the single-celled organism each cell fission leads to the
formation of two entire, complete, new individuals; in many-celled organisms
cell fission generally adds only to the size of the individual of which the cell
is a part.
Classification of Protozoa. A complete classification of, the protozoa
would be beyond the_scope of this bo'ok. A brief outline of the main groups;
will, however, be useful in later discussion. All of the protozoa which we
shall discuss are motile during at least one stage of their existence, the means
of motility being different in eachgroup. These differences are used as a basis
of classification. There are four great groups of protozoa and one or more
species in each group is pathogeniC. The groups may be listed, with one or
two common examples, as follows:
CLASS I. Sarcodina (move with pseudopods·).
A. Endamoeba hislolytic~auses amoebiasis.
B. Endamoeba coli-not pathogenic.
CLASS II. Mastigophora (move with long, whip-like lashes called flagella).
A. Intestinal tract:
(a) Giardia lamblia-possibly causes enteritis.
(b) Trichomonas hominis-not pathogenic.
• Irregular, lobular or finger-like projections constantly being thrust out from, or re-
tracted into, the cell at any part of the cell surface.
B. Urogenital tract:
(a) Trichomonas !oetus-a cause of abortion in cattle.
(b) Trichomonas vaginalis-may cause vaginitis in women.
C. Blood stream:
(a) Trypanosoma rhodesiense-causes African sleeping sickness.
(b) Trypanosoma gambiense-causes African sleeping sickness.
(c) Trypanosoma equiperdum-causes dourine ("equine syphilis") of
horses.
(d) Trypanosoma cruzi-causes Chagas' disease.
D. Blood stream and tissues:
(a) Leishmania donovani-causes kala-azar.
(b) Leishmania tropica-causes oriental sore.
(c) Leishmania braziliensis-causes espundia or South American leish-
maniasis.
CLASS III. Sporozoa (move with pseudopods only in immature stages; male gamete is flagel-
late).
A. Plasmadium vivax (also P. maiariae, P. faiciparum and P. ovale)-cause
malaria.
CLASS IV. Ciliata (move with cilia).
A. Balantidium coli-causes enteritis and ulcerations.
B. Paramecium caudatum-not pathogenic.
Some authors include a fifth class, Suctoria, in which young stages are
ciliated while adult stages are provided with tentacles.
Reproduction and Life Cycles of Protozoa. Some protozoa multiply both
sexually and asexually, others only by binary fission. Cell fission is said to be
binary when the cell divides into two approximately equal parts, each part
having all of the physiological and genetic equipment of the parent cell.'"
Unlike the bacteria, the flagellate protozoa divide lengthwise (longitudinal
fission) rather than crosswise (transverse fission). Ciliate protozoa divide trans-
versely. The nucleus undergoes changes very much like mitosis. Bacteria
exhibit only transverse binary fission.
Many protozoa, during their lifetime, pass through a definite and readily
demonstrable series of developmental stages and are'thus quite different from
the bacteria. These series of developmental stages are called life cycles and
often are quite complicated. For example, in the case of the malarial parasite
and some other protozoa, especially those which live in the blood, it is neces-
sary for the parasite in its various stages to live in an insect hostt before it is
mature and ready to infect man or animal again. The cycle involving sexual
reproduction usually takes place in one host (the definitive host) while the
asexual development occurs in another (intermediate host). This phenomenon
of the developmental cycles is spoken of as alternation of generations. In the
case of the malarial parasite the definitive host is a certain kind of mosquito,
while the intermediate host is man. In the trypanosomes the invertebrate hosts
may be tsetse flies' and other insects, the mammalian host depending on the
species of parasite, Bacteria, with a few possible exceptions, exhibit no such
complex life cycles, if any at all.
Mode of Nutrition. One property which distinguishes most plants (in-
cluding bacteria) from most animals (including protozoa) is the manner of
• Sometimes in bin,ary fission the two daughter cells are not exactly alike.
t In speaking of parasites and disease-producing (pathogenic) microorganisms, the crea-
ture on which the paiasite or pathogen lives is called the host, though usually an unwitting
or unwilling one.
taking nourishment. There are some exceptions, like parasitic protozoa and
worms, but typically, animals are able to take solid masses of food into the
body (be it a many-celled or a single-celled body), there digesting it and turning
it into soluble substances which nourish the cell or cells of the animal. This
holozoic mode of nutrition is obvious enough in large animals, but may readily
be observed in the smallest forms such as ameba or paramecium with the aid
of a microscope. * With a few exceptions, like "Venus's fly trap," the food of
all plants, from the apple tree to the fern, seaweed, fungus or bacterium, is
absorbed through the cell walls of root or thallus, and this absorption can
occur only when the food is in the form of relatively simple compounds soluble
in· water or cell contents; plants cannot ingest solid food. Their mode of
nutrition is holophytic.
BACfERIA AND THE VEGETABLE KINGDOM
Let us now examine the vegetable kingdom as a whole and consider the
different divisions and their relations to each other and to the animal king-
dom, especially the protozoa. Opinions differ as to the most logical method
of classifying the organisms of the vegetable kingdom and no system can be
said to be the best for all purposes. For the present, the schema given below
is a convenient arrangement.
THE VEGETABLE KINGDOM
Phylum I. Tracheophyta (vascular plants). Contain chlorophyll.t
Subphylum 1. Pteridopsida. Ferns and seed plants.
Class 1. Angiospermae or Spermatophyta (flowering plants).
Subclass 1. Monocotyledoneae (plants with 1 seed leaD-lilies, grasses, etc.
Subclass 2. Dicotyledoneae (plants with 2 seed leaves)-roses, zinnias, maples, etc.
Class 2. Glymnospermae (naked seeds)-pines, etc.
Class 3. Filicineae-ferns.
Subphylum 2. Sphenopsida-horsetails.
Subphylum 3. Lycopsida-dub mosses.
Phylum n. Bryophyta-Liverworts and true mosses. Contain chlorophyll.
Class 1. Musci-mosses-Sphagnum, Polytrichum, etc.
Class 2. Liverworts-Marchantia, etc.
Phylum m. Thallophyta-no roots, stems, leaves or flowers.
Subphylum 1. Algae-contain chlorophyll.
Class 1. Chlorophyceae-Green algae.
Class 2. Rhodophyceae-Red.a1gae.
Class 3. Phaeophyceae-~rown algae (and Diatoms1).
Class 4. Myxophyceae-Blue-green algae.
Subphylum 2. Fungi-Do not contain chlorophyll.
Qass 1. Basidiomycetes-mushrooms, puffballs, tree fungi, etc.
Class 2. Ascomycetes-yeasts, blue molds, mildews, etc.
Oass 3. Phycomycetes-"bread molds," ,etc.
Class 4. Fungi imperfecti-heterogeneous asexual fungi of classes 2 and 3.
Class 5. Schizomycetes-fission fungi, bacteria.
Phyla I and II may be disposed of as including only macroscopic green
plants of relatively complex structure and sho,¥ing well-developed sexuaI dif-
ferentiation. None of these morphologically resembles bacteria or protozoa.
• If we consider the tissue cells of large animals, even this difference largely disappears, be-
cause the tissue cells, like bacteria and most plants, depend on food in solution.
t ChioroplIyll is the green pigment of familiar plants like grass and trees. By means of
chlorophyll the plants absorb the energy of sunlight and use that energy to synthesize the
plant structures. The process is called photosynthesis.
The Microscopic World II
Phylum 111, subphylum I, contains many plants of relatively simple struc-
ture. Flowers and seeds are unknown. In spite of the fact that some of them
(as the kelp-algae or seaweeds of the Pacific) may attain very large size
(Macrocyslis-600 feet in length), none is possessed of any well-differentiated
root, stem or leaf structure, although certain seaweeds approach this com-
plexity.
Bacteria and Algae. As shown in Figure 1-1 the nearest relatives of the
bacteria in the vegetable kingdom are the algae and the fungi. A brief descrip-
tion of algae and of fungi will show the principal similarities and differences.
Algae are mainly aquatic and marine plants, found in sunlit waters over a
wide range of latitude. Like all thallophytes, they have no definite roots,
vascular stem, flowers or seeds. Each plant consists of only one kind of tissue,
which is distinctive for that species. Leafy, lacy and illamentous forms found
on beaches, on shallow lake bottoms, the beds of streams, etc., are often called
"sea lettuce," "seaweed," "pond or brook slime," and the like. Algae fre-
quently also grow at the surface of stagnant, sunny ponds, forming a bright
green scum. Scenedesmus is a good example. Rocks and trees in damp sunlit
places in the woods frequently carry a green film of algae, and some species
of algae grow on the surface of moist, fertile farm soils.
Algae are like other green plants in having cellulose (or cellulose-like) cell
walls, and in using solar energy in photosynthesis. With minor exceptions,
bacteria differ from algae in not having cellulose cell walls and in having no
chlorophyll. Except for one small group, bacteria do not use solar energy
but obtain energy from chemical reactions. They are independent of, and often
killed by, sunlight.
Algae are important as sources of food for many fish and other forms of marine life
dependent on fish; as sources of plant food in the soil; as a source of the jellylike agar used
to solidify bacteriological culture media (seaweed Gelideum); and as nuisances when they
grow in impounding reservoirs for city water supplies or in the private cistern. In the latter
they may be controlled by exclusion of sunlight. In reservoirs" they are often controlled by
dissolving minute amounts of copper sulfate in the water. Algae have been cultivated arti-
ficially on a commercial scale and may become important sources of food for livestock and
man in the future.
THE CYANOPHYCEAE. The algae most closely related to bacteria are the
blue-green algae or Cyanophyceae (cyano =blue; phyceae=algae) or Myxophy-
ceae. The name Myxophyceae is derived from a slimy capsule or outer enve-
lope (myxo = slime). With a few exceptions, these algae contain green chI oro-
• • • <:-
Fig. 1-3.' Synechococcus, a blue-

green alga th~ cells of which are solitary
except during fission. Several stages of
fission are shown as well as a single cell
about ready for division. (Reprinted by
;t:i;n~/1~~7
permission, from Holman and Robbins,
"Textbook of General Botany," pub-
lished by John Wiley & Sons, Inc.)
12 TM Relationships of Microorganisms to EAch DtMr and tM wing World
Fig. 1-4. Various forms of bacteria,

magnified about )000 times. These have
been stained to make them more readily
visible. Shown here are spherical cells
(coc:ci) in chains, irregular clumps. pairs
and groups of four; straight, rod-like
cells (bacilli) variously shaped: some
very thin, some thick, some with dor-
mant enclosures (endospores); a few
curved, rod-like cells (vibrios); some
spiral and wavy cells (spirochetes); some
branched cells (actinomycetes); and
some odd-shaped, variant forms.
phyll and a blue pigment called phycocyanin (hence the name Cyanophyceae).
These are the simplest, smallest and most bacteria-like of the sunlight-de-
pendent plants. Good examples are Synechococcus (Fig. 1-3), Gloeocapsa, and
Dscillatoria.
The cyanophyceae, like bacteria, multiply by binary fission and have no
sexual cycle. They are coated by a slimy capsule or sheath.· Their nucleus is
indistinct and presumably consists of granules of nuclear matter diffused
throughout the cell (a diffuse nucleus). Their chlorophyll is also diffuse, not
confined to chloroplasts as it is in higher plants. The bacteriochlorophyll-
containing bacteria (Rhodobacteriineae) are shown in Figure 1-1 as nearest
to the algae.
Bacteria ancl Fungi. Turning our attention to the fungi, we are struck by
the absence of chlorophyll from all. t Among the fungi are microscopic forms
which do not resemble ordinary plants in size or appearance and which are
indifferent to, or even killed by, sunlight. Among these are the yeasts, the
molds, and the smallest and simplest of all of the fungi: the bacteria.
All fungi, including bacteria, lack the power of utilizing sunlight. t They
make use of energy derived from chemical reactions to live and multiply.
They synthesize their cell substances from soluble substances, using such
chemical energy. Such synthetic processes are called chemosynthesis.
Among the large, fleshy fungi such as mushrooms, bracket fungi and puff-
balls, or the woolly molds seen on decaying organic matter in warm weather,
and even among the microscopic cells of yeas~ there are rudiments of sexual
differentiation.
Recapitulation. When we descend to the lowly bacteria we encounter fungi
(a) generally devoid of chlorophyll and therefore chemosynthetic; (b) only
1 or 2 microns in diameter; (c) existing as relatively simple, single cells or as
small, easily-broken-up agglomerations of independent cells; (d) devoid of
readily demonstrable sex; (e) without definite nucleus; (f) multiplying mainly
by transverse, binary fission and hence called fission fungi or Schizomycetes
(schizo = fission; mycetes = fungi) (Fig. 1-4).
• In many bacteria this is readily demonstrable, in otherI 0D]y with difficulty.
t With tbe =:eption of the RbodobacteriiDeac, wbich contain bacteriochlorophyll.
'I'he Microscopic WorM 13
DEFINITION OF TERMS
Before proceeding further to compare bacteria with other forms of life we
may describe briefly certain common properties of bacteria to which it is
frequently necessary to refer in such comparisons.
S;:::! R Variation. First is the smooth;:::! rough (S;:::! R) variation; two
alternate forms in which most bacteria often appear (and which probably
have their analogues in most other microorganisms).
The terms rough and smooth describe the appearance of colonies· of
bacteria. The surface of S colonies is smooth, often glistening or satiny. The
margins of S colonies are even and regular. The cells in S colonies usuall}
occur singly and are often covered with a coating of protective substance
called a capsule or slime layer. They exhibit other distinctive properties to be
described later. R colonies are rough, "crumbly," granular, rugose, irregular.
The margins of the colonies are irregular, serrated, notched, scalloped. The
cells usually occur in chains or clumps and are usually devoid of capsule and
have other distinctive properties to be described (Figs. 1-5 and 1-6).
Rand S forms are two of many kinds of variants which occur in many
species of bacteria and possibly other organisms. Any bacterial species may
produce either R or S form, or both at once.
Aerobic and Anaerobic Growth. A noteworthy property of many micro-
organisms (including bacteria) is their ability to thrive on lifeless media in the
IOtal absence of air or free oxygen. This astonishing fact was discovered by
Pasteur in 1861. He referred to the phenomenon as anaerobiosis, or "life-
without-air." Some are capable of normal life in either the presence or absence
of air. Such are said to be "facultative" with respect to oxygen. Others are
restricted to anaerobic conditions and are, indeed, killed by exposure to air or
Fig. 1- 5. Fig. 1-6.

Fig. 1-5. Smooth colonies of gram-positive rod: same species as Fig. 1-6.
Fig. 1-6. Rough colonies of a gram-positive rod : same species as Fig. 1-5.
These colonies are growing on a jelly-like (agar) surface containing nutrient substances
lor the bacteria. Each colony contains billions of living bacterial cells. Compare the smooth
lIIJ'face and regular margins of the "S" colonies with the rough surfaces and irregular mar-
.... of the "R" colonies. These pictures are about five times actual size (indicated by the
IJll'lbol X 5).
• Colonies are small, visible masses of bacteria growing on a solid, nutrient surface (Fig .
...S).
free oxygen. Such organisms are said to be strict or obligate anaerobes. Still
other microorganisms can live only in the presence of air or free oxygen. These
are called obligate aerobes. Some microorganisms thrive best in an environ-
ment of partial anaerobiosis or reduced oxygen tension. These are spoken as
of microaerophilic. The biological significance and biochemical mechanisms
of anaerobiosis and aerobiosis will be discussed later on.
These relationships may be summarized as follows:
RELATIONS OF MICROORGANISMS TO OXYGEN
I. Anaerobiosis
A. Strict-canilOt grow in presence of free oxygen.
B. Facultative-can grow in presence or absence of free oxygen.
II. Aerobiosis
A. Strict-cannot grow in absence of free oxygen.
B. Facultative-see under anaerobiosis.
III. Microaeropbilism
Grow best in reduced oxygen tension.
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Christensen, C. M.: The Molds and Man. University of Minnesota Press, Minneapolis,
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Coulter, M. C.: The Story of the Plant Kingdom. University of Chicago Press, Chicago,
1935.
Faure-Fremiet, E.: Morphology of protozoa. Ann. Rev. Microb., 1953, 7:1.
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Fogg, G. E.: The Metabolism of Algae. John Wiley and Sons, New York, 1953.
Hegner, R. W.: Big Fleas Have Little Fleas. Williams & Wilkins Co., Baltimore, 1938.
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Kluyver, A. J., and van Niel, C. B.: The Microbe's Contribution to Biology. Harvard Uni- )
versity Pr~s, Cambridge, Mass., 1955.
Lwoff, A.: Biochemistry and Physiology of Protozoa. Academic Press, Inc., New York, 1,
1951; II, 1955. ,
McBee, R. H., Lamanna, C., and Weeks, o. B.: Definitions of bacterial oxygen relation-
ships. Bact. Rev., 1955, 19:45.
Milner, H. W.: Algae as food. Sci. Amer., 1953,189:31.
Pringsheim, E. G.: The relationship between bacteria and myxophyceae. Bact. Rev., '1949,
11:47.
Rogers, J. S., Hubbell, T. H., and Byers, f2. F.: Man and the Biological World. Rev. ed.
University of Chicago Press, Chicago, 1952.
Schwimmer, M.: The Role of Algae and Plankton -in Medicine. Grune and Stratton, New
York, 1-955.
Smith, G. M.: Cryptogamic Botany. I. Algae and Fungi, 2nd ed. McGraw-Hili Book Co., I
New York, 1955.
Villee, C. A.: Biology, 3rd ed. W. B. Saunders Co., Philadelphia, 1957.
Weatherwax, P.: Botany. 3rd ed. W. B. Saunders Co., Philadelphia, 1956.
Weiss, F. J.: The useful algae. Sci. Amer., 1952, 187:15.
2
2. GROWTH OF BACTERIOLOGY
THE TERM bacteria as used today comprises about 1600 species of micro-
scopic, unicellular fungi having a variety of biochemical and physiological
properties and a relatively wide range in microscopic size and shape. Since
about 1667, when bacteria were first observed, we have learned several things
about them. For example, some are motile by means of flagella; others not.
The individual cells of some species are spherical. Others are cylindrical, like
a cigarette or sausage; some are spirally curved.
We now know that, although bacteria are extremely minute, measuring
often about 1/50,000 of an inch (0.5 p,) in diameter and weigh\ng as little as
4/10,000,000,000,000 (four ten-trillionths) of a gram, they are not unimportant
and they do not experience undue difficulty in the'struggle for existence.
Persistence of a species does not depend on great size but upon the power to
live and multiply under a variety of conditions.
Let us imagine creatures, for example, which can function in a range of
temperatures from freezing to almost boiling, and regardless of whether free
oxygen and food, as we know it, be present or not; creatures which, in addi-
tion to actively carryIng on the business of life under these circumstances,
can double their numbers every twenty minutes or, if required, cease growth
and go into a state of what seems to be completely suspended animation, in
this state surviving cold so intense as to liquefy hydrogen, extreme drought
prolonged for manY, years, heat so intense as to coagulate blood or "hard
boil" an egg, high pressures and high vacua. Let us imagine, furthermore,
that the creatures are so minute as to be invisible to any enemies possessed
of eyes with which to hunt them. Such creatures, it is clear, would be far
more likely to surviye the rigors of environmental change, and competition
with larger forms of life, than unwieldy and relatively vulnerable structures
like men, animals and trees. Such gifted organisms are to be found among
the bacteria and related microorganisms. Some of them are admirably adapted
to survive under conditions such as those described, some of which probably
existed in the early geological history of the earth; and to survive all the
known succeeding vicissitudes of climatic change since those periods to the
15
16 The Relationships of Microorganisms to Each Other and the living World
Fig. 2-1. The world before life. (Field Museum of Natural History.)
exclusion, if need be, of other, more advanced, forms of life. It seems not
unlikely that, unless some grand cataclysm destroy all living things suddenly,
such bacteria may be the last survivors of the pageant of life on this globe.
The Primitive World. Bacteria are primitive and it may be supposed that
they represent the type of living beings which appeared very early in the
earth's history of life. Indeed, bacteria have been regarded as the first cellular
inhabitants of this planet. Probably, however, creatures much smaller, and
sir. ':ar to genes or viruses, preceded bacteria in the evolutionary procession.
The bacteria of primeval times probably lived in the sea or mud, and from
them probably evolved many of the microorganisms familiar to us today.
Familiarity with microorganisms, however, is of rather recent achievement
since they could be studied accurately only after the development of lenses of
sufficient magnifying power to make them visible. The role of the microscope
in microbiology is, therefore, important .
._. The First Microscopes. Leeuwenhoek. By the end of the seventeenth cen-
tury lenses had already been exploited in various ways for many years, Roger
Bacon having used them experimentally before 1294. Such lenses, however,
did not magnify very highly. The man mainly responsible for revealing the
whole, hitherto unknown and unseen world of microorganisms was the
Dutch investigator (Fig. 2-2), Antonj van Leeuwenhoek (1632-1723), a linen
merchant by trade and influential in civic affairs as well. Leeuwenhoek was
an active, intelligent man of public and commercial affairs in the city of
Delft. He was not a trained scientist but was self-educated and of rare intel-
lectual powers. He amused himself by means of his skill and craftsmanship
in glass blowing, fine metal work and other occupations. He came of a well-to-
do family and lived in relatively easy circumstances with plenty of leisure for
his avocation of making minute but powerful lenl>~s. With these he delighted
in examining a great variety of objects. He examined saliva, pepper decoctions,
cork, the leaves of plants, circulating blood in the tail of a salamander, sem-
inal ftuid, urine, cow dung, scrapings from the teeth and so on. In many of
these he saw living creatures, some of which we now know were protozoa and
bacteria but all of which he called "animalcules."
In spite of the fact that his microscopes were not compound he obtained
7'M Microscopic World 17
remarkable results with them. " he showed rare ingenuity and expert
craftsmanship in the grinding and mounting of his simple lenses, a skill
which he zealously kept to himself; and in spite of the requests of his learned
friends, he refused to disclose the secret of his success." " ... Leeuwenhoek's
instruments are not true microscopes at all in the sense in which we think of
microscopes, but rather simple magnifying glasses generally consisting of a
small, single, biconvex lens. The object, and not the lens, was moved into
focus by means of screws" (Fig. 2-3). "To adjust the lens to the object was so
long and tedious a task that it is not surprising that Leeuwenhoek used an
individual lens for each object .. .." "The magnification varied and at best
did not exceed two hundred to three hundred diameters." "The size of objects
which Leeuwenhoek examined was determined by comparison. For this
purpose he used at various times a grain of sand, the seed of millet or mustard,
the eye of a louse, a vinegar eel, and still later hair or blood corpuscles. In
this way he secured fairly accurate measurements of a great variety of objects."
" ... he was forced to admit that the sand grain was more than one million
times the size of one of the animalcules."
Leeuwenhoek was so interested in the things he observed that he wrote
minutely detailed reports about them to the Royal Society in London. His
first letter was dated in 1674. He was later elected a Fellow of the Royal
Society. Some of his observations are at once quaint and epoch-making. For
example, after examining material which he scraped from between his teeth,
he said, "Though my teeth are kept usually very clean, nevertheless when I
view them in a Magnifying Glass, I find growing between them a little white
FJa. 2-2. AntOl\i van Leeuwenhoek. A fanciful delineation based on a famous portrait.
The picture shows accurately the size and shape of the first microscopes, the manner in
which they were used, and the simple laboratory apparatus of the "Father of Bacteriology."
(Courtesy of lambert Pharmacal Co.)
18 The Relatioll&hips of Microorgallisrru to Each Other and the Uving Worla
Fig. 2-3. One of Leeuwenhoek's microscopes: side, back and front views.
The tiny spherical or bemisphericallens is held in the slightly raised structure in tbe upper
part of the metal plate. The object to be examined was mounted at the tip of the sharp-
pointed mounting pin. Focussing was accomplished by means of the three thumb screws to
which the mounting pin is attached. Tbese are approximately actual size.
matter as thick as wetted flour ; in this substance, though I could not perceive
any motion, I judged there might probably be living Creatures.
"I therefore took some of this flour and mut it either with pure rain water
wherein were no Animals; or else with some of my Spittle (having no Air
bubbles to cause a motion in it) and then to my great surprise perceived that
the aforesaid matter contained very small living animals, which moved them-
selves very extravagantly. The biggest sort had the shape of A (Fig. 2-4).
Their motion was strong and nimble, and they darted themselves through the
water or spittle, as a Jack or Pike does through the water. These were generally
not many in number. The second sort had the shape of B. These spun about
like a top, or took a course sometimes on one side, as is shown at C and D.
They were more in number than the first. In the third sort I could not well
distinguish the Figure, for sometimes it seem'd to be an Oval, and other times
a Circle. These were so small they seem'd no bigger than E and therewithal
so swift, that I can compare them to nothing better than a swarm of Flies
or Gnats, fiying and turning among one another in a small space." (E. B.
Fred.)
When microorganisms were thus first brought within the range of human
vision, they were viewed by students of natural philosophy from various
standpoints. Some considered them the original and lowest forms of life;
others hailed them as the cause of disease. Many debates arose, some of
which still go on. Those concerning the origin of bacteria and their relation
to the problem of the s>rigin of life were especially vigorous and make inter-
esting reading. It is desirable to review some of these, since a number of
discoveries were made, during the experimental studies on the subject, which
are important in present-day microbiology. Without a knowledge of these
earlier discoveries, the student of microbiology works at a disadvantage.
file Microlcopic World 19
Andeot 1beories Concerning the Origin of Life. The ancients knew nothing
of microorganisms, or of evolution or of the fact that only living things could
beget living things. They believed in spontaneous generation, i.e., that crea-
tures like frogs, mice, bees and other animals sprang fully-formed from fertile
mud, decaying carcasses, warm rain or fog, and the like. Van Helmont (1577-
1644) devised a method for manufacturing mice. He recommended putting
some wheat grains with soiled linen and cheese into an appropriate receptacle
and leaving it undisturbed for a time in an attic or stable. Mice would then
appear. This observation may still be experimentally confirmed but the con-
clusions drawn from it differ today. But belief in spontaneous generation lived
on for years.
For example, an elderly lady of the writer's early acquaintance complained
bitterly that she had been cheated by a merchant who sold her a woolen coat
which was of such a quality that it turned entirely into moths when left undis-
turbed in a closet for some months!
Such empirical theories of spontaneous generation of living beings were
later discarded; but only after a most dramatic intellectual and experimental
struggle, which will be detailed presently. All such theories assumed that life
begins as fully-formed creatures of complex structure. It was later realized
&hat life must have originated in much simpler forms.
Fig. 2-4. Leeuweohoek's drawings of ~eria.

Here may be seen cocci, bacilli and (probably) a spirochete. The motion of one of the
IIIIcilli is clearly indicated. Today such observations are commonplace. But Leeuwenhoek
was seeing them for the first time in the history of the Human Race! It was as momentous
• diIIcovery as that of Columbus-a New World!
THE DISPUTE OVER SPONTANEOUS GENERATION
In the earlier years, in the absence of exact knowledge of microorganisms
or chemistry, there had already arisen much skepticism and bitter feeling
over the question of the origin of life. One "scientist" who still held to the
ancient ideas says of the views of another, who doubted, "So may we doubt
whether, in cheese and timber, worms are generated, or if beetles and wasps in
cow dung, or if butterflies, locusts, shell-fish, snails, eels, and such life be
procreated of putrefied matter which is to receive the forms of that creature
to which it is by formative power disposed. To question this is to question
reason, sense and experience. If he doubts this let him go to Egypt and there
he will find the fields swarming with mice begot of the mud of Nylus, to the
great calamity of the inhabitants." There was a great deal of such acrid dis-
cussion by wordy savants of the times who tried to settle everything by argu-
ment. Experimentation was regarded as rather undignified and even smacking
of relations with The Evil One. *
Francesco Redi (1626--1679). The experimental method was, however,
being invoked here and there. For example, it had always been supposed that
the maggots in decaying meat were derived spontaneously from transforma-
tions of the putrid meat itself. Redi, a physician of Arezzo, questioned this
and placed meat and fish in jars covered with very fine gauze. He saw flies
approach the jars and crawl on the gauze. He saw the eggs of the flies caught
on the gauze and observed that the meat then putrefied without maggot forma-
tion. Maggots developed only when the flies' eggs were deposited on the meat
itself. Obviously the meat itself did not turn into maggots. Redi's work was
not widely noted, however, and it was not until much later that another series
of experiments was made.
Louis Joblot (1645-1723). After Leeuwenhoek's discovery it was thought
·by many who believed in the spontaneous generation of life that animal or
vegetable matter contained a "vital or vegetative force," capable of converting
such matter into new and different forms oflife. Leeuwenhoek's "animalcules"
were hailed by many as proof of this. In 1710, Louis loblot observed that
hay, when infused in water and allowed to stand for some days, gave ris~ to
countless animalcules or infusoria (bacteria and protozoa). The hay was
thought to change into "animalcules." Anyone can observe the development
of the "animalcules" for himself. In ~hose times, however, most people re-,
garded this as conclusive evidence of spontaneous generation. Joblot, how-
ever, boiled hay infusion and divided it into two portions, placing one iIi a
carefully baked (sterilized) and closed vessel, the other in an open vessel. Th~
infusion in the open vessel teemed with living creatures in a few days. In the
closed vessel no life appeared as long as it remained closed, thus showing
that the infusion alone, once freed of life by heat was incapable of generating
life anew spontaneously'.
John Needham (1713-1781). Similar experiments, carried out by an Eng-
lish scientist, John Needham (1749), gave conflicting results. Life developed in
• The student will note three main phases of the question of spontaneous generation:
(a) That concerning the formation of complete and highly developed animals like bees,
mice, frogs and the like from putrid matter, mud, cheese, dirty linen, etc.; (b) that which
concerns the cause of putrefaction and fermentation; and (c) that concerning the origin of
life through che'llical evolution early in the history of life on this planet.
Needham's heated, closed vessels as well as in the open, unheated ones. He,
therefore, believed in spontaneous generation. We shall see later that this
result was due to insufficient heating, which failed to kill heat-resistant forms
of bacteria called spores. But nothing was known about spores at that time.
Lazzaro Spallanzani (1729-1799). Spallanzani, an Italian naturalist, pub-
lished the results of a whole series of the same type of experiments, which
disagreed with those of Needham. He showed that if heating was prolonged
sufficiently, and the vessels kept closed to exclude dust and air, no animalcules
developed in hay infusions, or in any other kinds ·of organic matter like urine
and beef broth. Needham, in reply, said the prolonged heating destroyed the
"vegetative force" of the organic matter which, he said, was necessary for the
spontaneous generation of life. Spallanzani answered Needham's objections
by showing that the heated infusions in the closed flasks could still develop
animalcules when exposed to air (i.e., when microorganisms were introduced
with dust).
In 1775 Lavoisier discovered oxygen and the relation between air and life.
This renewed the controversy about spontaneous generation, the objection
to Spallanzani's results beingraised that it was the exclusion of air (oxygen)
from the flasks which prevented the development of life.
Schulze and Schwann. New experiments were performed, in which air was
admitted to the previously heated infusions of meat or hay, but only after
passing through sulfuric acid or potassium hydroxide solutions (Schulze,
1836) or through very hot glass tubes (Schwann, 1836), the idea being that
the air itself introduced the germs of life into the infusions. When the infusions
exposed to air so treated failed to develop any life, it was claimed by others
that this was not 9ue to a destruction of any germs of life in the air by the
sulfuric acid or hot glass, but to the fact that the "life-giving" power of the
air had been destroyed by these methods, thus preventing spontaneous gen-
eration. Schroder and von Dusch (1854-61) overcame this objection by per-
forming similar experiments in which the air was merely filtered through
cotton wool. This method prevented the appearance of animalcules in the
heated broth or infusions until the vessels were opened. It was therefore
apparent that the method of treatment of the air had nothing to do with the
development of animalcules, and that these did not develop spontaneously but
that there were particles of living matter, floating on dust in the air, which not
only were killed by heat, acids and alkalis but which could be caught and
withheld by the cotton wool alone. The presence of the microorganisms in the
cotton wool was lflter proven by Pasteur. The experiments of Schroder and
von Dusch were the origin of our present-day use of cotton plugs for bac-
teriological culture tubes and flasks.
In spite of thes\! demonstrations long and bitter contf()versies still raged.
Schroder and von Dusch were themselves not convinced by their own ex-
periments and admitted the possibility that spontaneous generation might
occur under natural conditions.
/Louis Pasteur (1822-1895). Pasteur, the most famous, and considered by
many. the greatest, French scientist of all time, was born in Dole, December
27, 1822. Son of 'a moderately prosperous tanner who had fought for, and
been decorated ob the battlefield by, Napoleon, Pasteur had a great admira-
tion for his father's soldierly accomplishments. He later was moved to man)
22 The Relatio1lShip& of Microorganisms to Each Other and the Uving World
Fig. 2-5. Pasteur in his laboratory.

Dr. Pasteur is examining a specimen of
spinal cord from a rabid animal. The ma-
terial at the bottom of the jar is sodium
hydrollide (drying agent).
of his best scientific achievements by his patriotic zeal. In his boyhood he

was an indifferent student but later became an enthusiastic scholar, devoting
his energies to a study of chemistry. He discovered the relationship of the
stereo-isomeric forms of tartrate crystals and revealed a whole new series of
possibilities in physical chemistry.
Pasteur, however, was not one to gloat over such successes and rest on his
laurels. He sought for other fields of investigation. His choice was guided
largely by patriotic motives. An Englishman had said to him: "People are
astonished in France that the sale of French wines should not have become
more extended here [in England] since the Commercial Treaties. The reason
is simple enough. At first we eagerly welcomed French wines, but we soon had
the sad experience that there was too much loss occasioned by the diseases
[souring] to which they are subject."· Germany was. at that time, making a
much better beer than France, and Pasteur undertook to make France a
successful rival in that respect. In order to do so he made a long study of beer
manufacture and of the cause of souring and spoilage ("diseases") of beer
and wines. As a result of these stqdies he arrived at far-reaching conclusions.
"Might not the diseases of wines," he said at the Academie des Sciences in
January, 1864, "be caused by organized ferments; microscopic vegetations,
of which the germs would develop when certain circumstanCes of temperature,
of atmospheric variations, of exposure 10 air, would favour their evolution
or their introduction into wines? . . . I have indeed reached this result, that
the alterations ("diseases") of wines are coexistent with the presence and
multiplication of microscopic vegetations." Pasteur had found that acid
wines, "ropy" wines, bitter wines, sour beer and so on, were due to the
growth in them of undesirable contaminating organisms which produced
these so-called "diseases."·
The solution of the problem, as later proven by Pasteur (Fig. 2-5), lay in
• From ''The Life of Louis Pasteur," by Rene Vallery-Radol, reprinted with permission
from Doubleday, Doran " Company, Inc.
TIle Microscopic World 23
preventing the growth of foreign organisms, "wild" yeasts, bacteria, etc.,
which caused the undesirable conditions. After considerable experimentation
along these lines he discovered that the wine did not spoil in transit, if it were
held for some minutes at a temperature between SOO C. and 60° C. He said,
"I have ... ascertained that wine was never altered by that preliminary opera-
tion (heating), and as nothing prevents it afterwards from undergoing ...
improvement with age-it is evident that this process (heating) offers every
advantage." His experiments were so successful that a practical test of the
efficacy of his methods was made. He wrote to a friend, " ... experiments on
the heating of wines will be made by the Minister of the Navy. Great quan-
tities of heated and nonheated wine are to be sent to Gabon so as to test the
process; at present our colonial crews have to drink mere vinegar... •
Pasteur laid down three great principles:
1. Every alteration, either of beer or of wine, depends on the development
in it of microorganisms which are ferments of "diseases" of the beer or wine.
2. These "germs or ferments" are brought by the air. by the ingredients,
or by the apparatus used in breweries.
3. Whenever beer or wine contains no living microorganisms it remains
unchanged.
In the same way that wines could be preserved from various causes of
alteration by heating. bottled beer could escape the development of disease
ferments by being brought to a temperature of 50° C. to 55° C. "The applica-
tion of this process gave rise to the new word, 'pasteurized' beer, a neologism
which soon became current in technical language." Today. pasteurization of
milk (heating at 62° C. for thirty minutes) is commonplace. The heating kills
undesirable microorganisms.
"Pasteur foresaw the distant consequences of these studies, and wrote in
Fig 2--{j. Two of Pasteur's flasks,

A
-
showing open neck with curves to catch
dust. One flask has been opened and a
growth of mold has developed. (Duclaux,
"Pasteur, The History of a Mind.")
• From ''The Life of Louis Pasteur," by Rcn6 Vallery-Radot, reprinted with pormission
from Doubleday, Doran & Company, Inc.
Fig. 2-7. First illustration of ampu-

tation, from Von Gersdorff (1517).
Bandages constrict, at most, superficial
veins. Two aneries are spuning uncon-
trolled. Man in background is wearing
Gersdorff's pig's bladder dressing over
his forearm stump. Bacteria had a field
day under such conditions!
his book on beer-'When we see beer and wine subjected to deep alterations
because they have given refuge to microorganisms invisibly introduced and
now swarming within them, it is impossible not to be pursued by the thOUght
that similar facts may, must, take place in animals and in man."
It was obvious from Pasteur's studies that each special kind of fermentation
or disease of beer or wine was the result of the growth and activity in it of a
special, distinct form of yeast or other microorganism depending on the type
offermentation or disease under investigation. This furthered an idea, already
old, of the specificity of biological action, and supported the view that animal
and human diseases also, like different sorts of putrefaction and fermentation,
were each caused by a single, specific type of microorganism.
After Pasteur's views with regard to the nature of fermentation had been
made public, he became involved in the bitter quarrel over the apparently
mysterious appearance of tbe "germs" in fermentable or putrescible liquids
like wine, beer, urine, broth, etc., hitherto regarded by many (Needham and
others) as resulting from spontaneous generation. Without going into detail
which would occupy too great a space, we may cite a series of experiments
which Pasteur carried out to answer the various objections and fallacies of
previous workers, and to show that the "animalcules" in spoiled beer, wine,
etc., were merely descendants of microorganisms which had gained access to
the fluids from dust in the air and which, by their growth and metabolism,
caused fermentation and putrefaction. First he redemonstrated that living
creatures float in the air attached to particles of dust. Then he showed, as
Schulze and Schwann had done, that when they could be excluded from vari-
ous things like sterilized broth and urine, these substances did not ferment or
putrefy. By using flasks with long, open necks having several vertical bends in
them, he showed that, although unheated and untreated and unfiltered air
communicated freely with the interior, the dust was caught by gravity in the
bends of the neck and no life appeared in the infusions (Fig. 2-6). Not until
fa. Microscopic World 25
the flask was tilted so that the fluid came into contact with this dust and was
allowed to run back into the flask, or until the neck of the flask was broken
off close to the body, did growth occur in the fluids. Some of Pasteur's flasks
wbich were sterile in 1864 have been preserved apd are still sterile (if they have
not been destroyed by wars) after over 100 years!
IAri IJster (1827-1912). One of the many important applications of the
work of Pasteur was made in 1867 by the English surgeon Lister. He realized
that wounds become infecJed, during surgical operations, by bacteria floating
on particles of dust in the air, or clinging to instruments or to the hands of the
surgeon (Fig. 2-7). Obviously, in order to prevent such wound infections it
was only necessary to render all surgical appliances sterile and, by antiseptic
dressings and careful technique, completely to prevent the entrance of bac·
teria into surgical wounds. Lister'S original method of preventing infection
during surgical operations was to work in a field and atmosphere continuously
wet with a fine mist of carbolic acid solution emanating from a nearby ap-
paratus (Fig. 2-8). This must have made the surgeon's work difficult but the
results obtained were the foundation of our modern, aseptic surgery (Fig.
2-9). The student may judge for himself whether Lister's contribution was
of importance to human life and medicine.
THE BEGINNINGS OF PRECISE BACTERIOLOGY
One reason, perhaps, why more species of bacteria were not discovered by
Pasteur and others prior to about 1870 was that microbiological methods
were very crude. In nature, microorganisms seldom occur alone, or " pure,"
but in mixtures of many species together. It was very difficult, at that time, to
aeparate, in a pure and uncontaminated state, any given sort of microorgan.
Pia. 2-8. lister operating with carbolic spray. Representing the general arrangement
IIII'JeOIl, assistants, towels, Ipray, etc., in an operation performed with (supposed) com·
aseptic (antiseptic) precautions (t 882). Note the carbolic spray playing over t~ field
operation. {W. Watson Cheyne.}
26 TM Relationships of Microorganisms to &rch Otlrer and the Living Worla
Fig. 2- 9. Modem surgery. Note the sterile masks, caps, gowns, rubber gloves, and sheets.
The instruments have all been sterilized. (St. John's Hospital, Brooklyn. Courtesy Ewing
Galloway.)
ism from a mixture of microorganisms. The preparation of such "pure cul-

tures" is absolutely essential to the exact study of microorganisms just as pURl
~ubstances are necessary to the analytical chemist. For the development or
pure culture technique, and for many of our modern bacteriological methods
we are indebted to that precise German scientist, Robert Koch.
v Robert Koch (1843-1910). While Pasteur, and others were engaged in
their polemics and experiments on fermentation and spontaneous generation,
Koch was practicing medicine and was Provincial Health Officer in Wollstein.
He had occasion, in an official capacity, to investigate anthrax (a disease of
animals and man caused by Bacillus anthracis, a species of cylindrically-shaped
bacteria). He decided to study the disease in his laboratory during his spare
time.
At first Koch and other scientists examined all of their microorganisms in
the living state, usually in drops of fluid mounted on a bit of glass. They thus
became familiar with bacterial motility when present, and they observed re-
fractile granules inside various bacteria. But the constant motion of some of
the bacteria (either that purposeless oscillation, due to molecular impact and
known as brownian movement, or real, progressive motion due to the action
of flagella) as well as their transparency, made accurate and prolonged study
most difficult. Koch realized that it would be much better for his drawings,
and especially for his photographs (both of which, by the way, were excellent;
Fig. 2-10) if the bacteria could be made to remain still. He tried spreading out
his drops of anthrax-infected fluid in thin films and allowing them to dry,
and met with immediate success. Not only were the anthrax bacilli motion-
J'if: MICroscopic World 27
leBa, but they apparently had not shriveled or changed in any visible way.
However, the bacteria were transparent and colorless. It was very difficult
to observe the fine details of their structure, and equally difficult to photo-
graph them. He obtained ideas from other workers, a procedure commended
to all investigators.
DEVEWPMENT OF STAINING METHODS

WeIpI1 and EbrIiclL Weigert, a German scientist contemporary with
Koch, had observed the use, by Cohn and others, of various dyes to make
clear the details of cell structures in histological preparations (histology =
microscopic anatomy). This procedure had been in use for some time, the
natural dyes carmine and lrematoxylin being widely used. Ehrlich, a renowned
chemist, bad recently improved methods, discovered by William Perkin, a
brilliant British chemist, of preparing very fine dyes from coal-tar distillates.
These were the first "coal-tar" or aniline dyes. Weigert, the bacteriologist,
tried the methods of the histologists with the dyes invented by the chemist.
His fint success was in 1875, when he found that the dye methyl violet could
be used to reveal bacteria in histological preparations. This method of making
bacteria easy to find and study, where before they had been colorless and
transparent and therefore almost invisible, was adopted by Koch and soon
came into wide use. Modified, it is one of our best methods today.
Flaoresc:eoee MiCl'OllCOPY. A very interesting modem variation on this
theme is the use of fluorescent compounds to stain certain specific kinds of
F .. 2-10. Koch's photograph of an-

thrax bacilli.
The upper picture shows the bacilli
ODDtaiDing endospores (dormant, heat-
llliltant bodies) and strung together in
Joea chains sometimes called streptoba-
cilli. The lower picture shows the ba-
~ as individual cells and in short
1!baIns of 2 to 6 cells. Many present-day
IIicrophotographs are no better than
taken about three quarters of a
810·
28 The Reiatiouhips of Microorganisms to Each Other and the wing World
Fig. Z-II. One of the groups of famous scientists who studied microbiology under
Koch. Standing, left to right: Alphonse Laveran (1845-1922), discoverer of the malarial
parasite; Emile Roux (1853-1933), codiscoverer of diphtheria toxin; Edmund Etienne
Nocard (l85G--1903), French veterinarian and mycologist; George H. F. NuttaU (186Z-
1937), British microbiologist. Sitting, left to right: Robert A. Koch (1843-1910), discoverer
of-the tubercle bacillus and pioneer microbiologist; Karl Joseph Eberth (1) (1835-1926),
discoverer of the typhoid bacillus; Elie Metchnikoff (1845-1916), Russian zoologist and dis-
coverer of pbagocytes and phagocytosis. (Courtesy of Wiley A. Penn, Director of Labora-
tories, Department of Health, Savannah, Georgia.)
bacteria which are particularly difficult to see under ordinary conditions.

When the fluorescent organisms are illuminated with ultraviolet light and
viewed through a microscope they are as easily found as bright stars in the
midnight sky! This is called fluorescence microscopy.
Development of Gram's Stain. In 1884, a Danish scholar, Gram, found
that if anthrax bacilli were stained with Ehrlich's methyl-violet solution, and
then treated with iodine, the dye was "fast" and that even alcohol could not
remove it. In attempting to apply the method to certain other bacteria, how-
ever, he found that the dye was not fast and that as soon as alcohol was ap-
plied the color was removed and the bacteria were as colorless and transparent
as ever. They could even be dyed an entirely different color (counterstjlined)
with some other dye. The bacteria retaining the purple stain were called gram-
positive, while the others were called gram-negative. The method is now used,
generally with some slight modification, by every bacteriologist as one of the
first steps in the process of identifying an unknown bacterium.
Koch, using the methods of Ehrlich, Weigert, Cohn, Gram. Petri (see
Chap. 13) and others, became one of the foremost bacteriologists of his day.
His discoveries attracted scholars from all over the world (Fig. 2-11). In an-
other chapter there is outlined the epoch-making development of solid media
and the covered plate for the pure-culture cultivation of bacteria, which are
methods used daily in every bacteriological laboratory in the world. The
De Microscopic World 29
methods and principles which originated in the laboratories of Koch, Pasteur,
Lister and others have made possible the conquest of disease and the building
up of great industries. Greatest of all, they have left us a foundation of fact
and inspiration upon which to build greater achievement and this, possibly, is
the richest legacy which any man can leave to posterity.
Otber Developments. Leeuwenhoek, Pasteur, Koch and their contempo-
raries laid foundation stones of basic importance in the science of Micro-
biology and their names are writ large in the Halls of Fame. However, it must
not be supposed that they were the last great microbiologists. They were
among the first. Later workers, equally ingenious, learned and courageous,
have carried on the work along an ever-increasing number of lines of investi-
gation. Although the five years following Koch's demonstration of the value
of solid media in plates for isolating bacteria witnessed the discovery of the
organisms causing diphtheria, cholera, erysipelas, lockjaw. pneumonia and a
number of other diseases, discovery of a new species was, by that time, only a
matter of patient "plating out" of suspected materials and was only one,
rather limited, phase of the subject.
One of the greatest discoveries in medical history was made when, in 1890,
von Behring, Kitasato and Fraenkel found that guinea pigs could be im-
munized by injections against tetanus and diphtheria toxins and that their
blood then contained antitoxin which would protect other animals. The ex-
tension of this principle to other diseases was an obvious possibility and soon
bore fruit.
In the realm of soil microbiology, equally important advances were going
on simultaneously. 8eijerinck, in 1888, had already isolated the bacteria
which, in the nodules of leguminous plants, aid them in the direct utilization
of atmospheric nitrogen, a physiological property essential to the existence
of most other forms of life. Winogradsky, Beijerinck, Omeliansky and others
during the decade following 1890 worked out the processes by which ammo-
nia, formed in the soil by various biological processes, is oxidized by suc-
cessive stages, first into nitrites by organisms of the genera Nitrosomonas,
Nitrosococcus, etc., and then into nitrates by bacteria of the genera Nitro-
bacter and Nitrocystis. As nitrate, nitrogen is readily available to most plants
of agricultural value and without it the human race might have had a very
different history. The equally important transformations in the soil of sulfur,
carbon and other elements were also studied by these workers.
In 1893 to 1894 Winogradsky demonstrated that atmospheric nitrogen is
fixed in the soil by the anaerobic bacterium Clostridium pasteurianum. Aerobic
organisms having a like property, Azotobacter chroococcum and A. agile,
were discovered by Beijerinck in 1901. These organisms are of absolutely
fundamental importance to the history of the human race since, without fix-
ation of atmospheric nitrogen in the soil, there would be no human race at all.
This rather deflationary fact will be explained later.
In 1892 Iwanowski discovered the existence of a disease-producing agent
(virus of tobacco-mosaic) which was invisible and not cultivable on inanimate
media and which traversed filters capable of holding back the smallest bac-
teria. Many other viruses have since been discovered and great strides have
been made in our knowledge of that class of organisms. For example. Walter
Reed in 1900 to 1902 demonstrated not only the mode of transmission but the
30 The Relationships of Microorganisms to Each Other and $he Living World
viral nature of the cause of yellow fever, the first-known viral disease of man.
Later, about 1933, workers of the International Health Division of The Rocke-
feller Foundation developed a fully effective vaccine against yellow fever and
to date it has been given to millions of persons.
A whole series of interesting and valuable discoveries has resulted more or
less directly from the studies of Koch, Ehrlich, Weigert and.;the others with
the staining of bacteria. From continued investigation of the effects of dyes
upon bacteria there has arisen, from the work of Domagk (Nobel prize win-
ner) 1932, knowledge of such dye-like compounds as sulfanilamide and sulfa-
diazine, the use of which in treating bacterial infections is a matter of common
knowledge. In 1929 Sir Alexander Fleming (Nobel prize winner) made the
momentous discovery of penicillin, and Waksman (Nobel prize winner) et aI.,
a little later, discovered streptomycin, thus opening up the huge field of anti-
biotics, the importance of which is still growing. In 1949 Enders, Robbins and
Weller (Nobel prize winners) showed how to cultivate poliomyelitis virus in
monkey tissues in flasks. By 1955 the polio vaccine developed on this basis
by Salk (awarded a Presidential citation and Congressional Medal) was
known and used all over the world. Each began as a young student, making
notes in a classroom, listening to teachers who had trod the paths before him;
trying, failing at times, persevering, succeeding. .
So Science grows. The microbiological history of tomorrow is being written
in the laboratory notebooks of students today. And who knows which notes
will prove to be the basis of the greatest discover~?
REFERENCES
Allison, V. D.: Sir Alexander Fleming. Obituary Notice. J; of Gen. Microb., 1955, 13:i.
Aron, H. C. S.: Paul Ehrlich: His contributions to medicine. J.A.M.A., 1954, 154:969.
Barrell, Schuchert, et al.: The Evolution of the Earth. Yale University Press, New Haven,
1922.
Beutner, R.: Life's Beginnings on the Earth. Williams & Wilkins Co., Baltimore, 1938.
Bulloch, Wm.: The History of Bacteriology. Oxford University Press, London, 1938.
Calvin, M.: Chemical Evolution and the Origin of Life. Am. Sci.. 19:56, 44 :248.
Chapman, V. J.: Seaweeds and Their Uses. Pitman, New York, 19:52.
Cohen, B.: On Leeuwenhoek's method of seeing bacteria. J. Bact.,1937, 34:343.
Cohen, B.: The Leeuwenhoek Letters. Society of American Bacteriologists. Williams &
Wilkins Co., Baltimore, 1937. ,
Cohn, F.: Bacteria: The Smallest of Living.Things. (Trans. C. S. Dolley.) Johns Hopkins
Press, Baltimore, 1939.
Dobell, c.: Antony van L~uwenhoek and his Little Animals. Harcourt, Bra~e and Co.,
New York, 1932.
Dodson, E. 0.: A Textbook of Evolution. W. B. Saunders Co., Philadelphia, 1952.
Dublin, L. I.: Robert Koch, A Centenary, 1843-1943. The American Scholar. 1943-44,
13:95. I
Dubos, R. J,: Louis Pasteur, Free Lance of Science. Little, Brown & Co., Boston; Mass.,
19:50.
Ford, W. W.: Bacteriology. Paul B. Hoeber, Inc .• New York, 1939.
Fred, E. B.: Antonj van Leeuwenhoek. J. Bact., 1933,25:1.
Goodlee, R. J.: Lord Lister. The MacMitlan Co., New York, 19J8.
Haggard, H. W.: Devils, Drugs and Doctors. Garden City Publishing Co., Garden City,
New York, 1929.
Haldane, J. B. S.: The Origin of Life. In "The Inequality of Man." Harper and Bros., New
York,1928. .
Horowitz, N. H.: On the evolution of biochemical syntheses. Proc. Nat. Acad. Sci., 1945
31 :153.
33
Kudo, R. R.: Protozoology. 4th ed. Charles C Thomas, Springfield, III., 1954.
Lamanna, C., and Mallette, M. F.: Basic Bacteriology. Williams & Wilkins Co., Baltimor.
1953.
Lacy: Biology and Its Makers. 3rd ed. Henry Holt and Co., New York, 1930.
Muller, H. J.: Life. Science, 1955,121:1.
Oparin, A. J.: The Origin of Life. (frans. Morgulis). The MacMillan Co., New York, 1938.
Read, J.: Sir William Perkin. Sci. Am., 1957,196:110.
schroder, H., and van Dusch, T.: Ann. d. Chern. u. Pharm., 1854,89:232.
Vallery-Radot, R.: The Life of Pasteur. (Trans. Devonshire,) Doubleday-Doran and Com-
pany, Inc., New York, 1926.
van Niel, C. B.: The "Delft school" and the rise of general microbiology. Bact. Rev., 1949,
13 :161.
van Niel, C. B.: In memoriam Prof. Dr. Ir A. J. Kluyver. Antonie v. Leeuwenhoek J. Serol.
and Micr., 1956, 22:209.
Waksman, S. A.: Contribution of "A Simple Bacteriologist" to humanity. (Obit. Sir Alex-
ander Fleming.) Science, 1955, 121 :580.
Waksman, S. A.: Sergi Nikolaevitch Winogradsky: 1856-1953. (Obit.) Science, 1953,118:
36.
Winslow, C-E. A.: Some leaders and landmarks in the history of microbiology. Bact. Rev.,
1950, 14:99.
3
3. YEASTS AND MOLDS
GENERAL CHARACTERISTICS
ALTHOUGH NEITHER.yeasts nor molds are included in the class Schizo-
mycetes, they belong in the same major division of the vegetable kingdom,
namely, the Mycophyta or fungi. Many yeasts and the structural features of
molds are microscopic in size and the microscope is used in studying them.
They make very good study material for microbiology because of the relatively
large size and evident structural details of their cells.
The place of yeasts and molds in the organic system is fairly well defined.
They are distinguished from algae by their lack of chlorophyll and growth
in the dark. They are readily differentiated from protozoa by their characteris-
tic morphology and by the motility of most protozoa; also by their ability to
grow readily on artificial media which protozoa rarely do. Their relatively
large size and obvious nuclear structures readily distinguish them from
bacteria. There is DO chance of confusing them with PPLO, viruses or ric-
kettsia because of differences in size and structure and failure of the last two
to grow on an artificial media (see Table 1).
Many common species of yeasts and molds are of great importance m
industry. Some cause fermentations which yield valuable substances like
ethyl and isopropyl alcohols, acetone, etc. Others cause damage through
decay, "mildew," etc. Molds are exceedingly active in the decay of wood and
other organic matter. Their destructive action on wood in damp soil is a cause
of considerable economic loss to telephone and electric power interests as
well as to farmers with wooden fence posts. These, therefore, are concerned
with experiments with antifungal preservatives of wood (Fig. 3-1). Many
molds and yeasts are causes of diseases of plants, animals and man. Some
molds, such as Penicillium, have assumed enormous importance as the sources
of antibiotics. Molds and yeasts are often encountered by the bacteriologist
as contaminants in laboratory cultures· because the fungi are ubiquitous and
their spores or conidia are constantly present in dust, soil, air, etc.
• A culture of microorganisms consists of a flask, tube or other vessel containing a
nutrient material such as meat-broth, milk or other food substance (solid or liquid) in (or
on) which microorganisms are growing. The nutrient is spoken of as a medium or culture
medium (pI. = media). A pure culture is one in which only one kind of microorganism is
growing. A contamillilled culture is one to which an unwanted, extraneous microorganism
has accidentally gained entrance.
31
7"- Microscopic World 33
Yeasts and molds may be cultivated on much the same sort of materials
as are used for bacteria and they are stained and manipulated in much the
same way. Certain media are especially recommended for the cultivation of
tbese organisms, a good illustration of the general type being that of Sabour-
aud.· Most of the media of choice for molds and yeasts contain considerable
amounts of carbohydrate. The preferable reaction of this type of medium is
slightly acid (PH about 5.5). Slight acidity seems to favor the growth of many
molds and yeasts and inhibits the growth of some kinds of contaminating
bacteria. In general, the yeasts and molds have nutrient requirements similar
to those of saprophytict bacteria.
Structurally and in methods of reproduction yeasts and molds are more
complex than bacteria and may, therefore, be regarded as more highly evolved
plants.
QassificatioD of Fungi. Differentiation between yeasts and molds is some-
times difficult because the transition in form and in manner of reproduction,
from one group to another, is so subtle that it is difficult to draw sharp lines
of demarcation.
For present purposes it is sufficient to indicate the main divisions of the
p-oup of fungi, leaving finer systematization for more advanced students of
Fig. 3- 1. Blocks of wood taken from

telephone poles preserved by different
methods. The wood remains on the
damp soil under conditions simulating
actual exposure of poles to the action of
soil microorganisms ; especially fungi.
Such tests are part of the search for bet-
ter wood preservatives. (Photo courtesy
of Bell Telephone Laboratories, New
York.)
• Sabouraud's agar:
Water. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 1000 ml
Peptone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 gm
Glucose (or Maltose)....... . ... . ..... . ... . .... . . ..... .. .. .. . 40 gm
Agar. .. ................. ................. .. . ..... . ... .... . 20gm
Dissolve all ingredients in water. Sterilize by autoclaving. The pH should be about
t Causing decomposition of dead organic matter. Generally harmless.

the subject. The following outline, while incomplete, is a satisfactory working
basis for the present discussion. (See Table 2.)
Table 2. Classification of Fungi

Thallophyta (no sterns, roots, flowers, leaves)
I
I
Algae (chlorophyll) I . (no chlorophyll)
Fungi
I
Schizomycetes Eumycetes
(bacteria) yeasts, molds,
mushrooms, puff-
balls, etc.
I
I
1
Phycomycetes Ascomycetes Fungi imperfecti
Basidiomycetes
Not sac-formers. Sac-formers. Properties of Not sac-formers.
I both yeasts and
molds.
Mycelia not sep- Yeast forms Filan~entous Fungi having no Mycelia septate.
tate. Mainly unicell- forms o bserved sexual
ular or bud- Mycelia septate. cycle are found
ded. in this group.
Asexual spores Asexual spores Asexual spores Asexual spores
held in sporan- blastopores. conidia. conidia of
gia. many types.
Sexual spores Sexual spores in Sexual spores in No sexual phe- Sexual spores on
free (zygo- asci. asci. nomena basidia.
spores or known.
oospores).
Familiar exam-
ples:
Aquatic forms Saccharomyces Aspergillus Many fungi Mushrooms.
cause diseases Schizosaccharo- glallcuS causing plant Bracket or tree
on fish and myces (green mold, diseases belong fungi.
plants. (Above are typi- common on in this group. '. Rusts (e.g.,
Example: Sapro- cal y~sts). bread). Many fungi caus- wheat rusts).
Jegnia. Penicilliilm itali- ing diseases of Smuts (e.g., com
Common terros- cum (blue man and ani- smuts).
trial forms are mold, com- mals also are
Mucormllcedo mon on ie- placed here
("manure caying citrus pending more
mold," white) fruit). complete stud-
and Rhizopzis ies.
nigricans
("bread
mold," black).
THE YEASTS
Ordinarily we think of yeasts as unicellular, microscopic plants with ovoid
or elliptical cells, each cell living as a separate, complete individual. We often
differentiate yeasts from molds by the fact that molds typically form greatly
elongated structures growing in branched filaments or hyphae and forming
tangled masses of hyphae called mycelia. A difficulty in such classification
arises from the fact that some yeasts form more or less definite mycelia under
certain conditions of growth and nutrition; i.e., they are dimorphic.
Dimorphism. The ability of some yeast-like fungi to grow in either (I) the
yeast (Y) phase or (2) the filamentous (F) phase is spoken of as dimorphism.
Various factors may induce this phenomenon. For example, low temperature
and ageing generally fa vor the F phase, while certain nutrients (blood, glucose,
compounds with reduced sulfur, like -SH groups, etc.) or absenCe of air
(anaerobiosis) often favor the Y phase. There are various substances (fusel
oil, cobalt ions etc.) which induce the Y -? F variation. Camphor exhibits this
effect so markedly that its action has been called the CF reaction.
The Y -= F variation in yeast-like fungi may be related to the smooth (S)
;:= rough (R) variation in bacteria; the Y and F phases of yeasts correspond-
ing to tl\e Sand R phases of bacteria, respectively. *
Structure of Yeast Cells. Yeast cells are, on the average, much larger than
bacterial cells. Some oval yeast cells have a volume hundreds of times that of
Micrococcust cells and a long diameter up to 20~ or more. There is a well-
differentiated nucleus and a thick cell wall, composed of cellulose-like sub-
stance. Yeast cells may, by appropriate techniques, be sliced into exceedingly
thin sections. These are stained differentially and then examined in enlarged
photos of electron microscope pictures (electronographs) at magnifications up
to 100,000 diameters. It is possible in such preparations to see that the yeast
cell wall consists of at least two, and probably three or more, layers. Within
the.cytoplasm are numerous vacuoles containing food or waste substances and
there are granules of-various kinds, some evidently related to the formation
and storage of glycogen, others composed of volutin (Figs. 3-2, 3-3). Yeasts
sometimes contain large quantities of fat, of which commercial use may be
made. /
On the other hand, yeasts resemble most bacteria in being unicellular, non-
motile, devoid of chlorophyll, and plant-like in requiring food material which
is wholly soluble and which can pass through the cell wall only by diffusion.
Multiplication of Yeasts. Yeasts may multiply by one or more of four
methods.
l. BUDDING. First, they multiply by a method called budding in which
large, mature cells divide, each giving rise to one or more daughter cells which
are at first much smaller and which may cling to the parent cell (Fig. 3-3),
often even after the daughter cell has divided. Clumps and chains of cells
sometimes called rudimentary filaments or pseudomycelia, aTe thus formed.
2. FISSION. Some species of yeast, in the genus Schizosaccharomyces, di-
vide by equal (binary) fission, much as do the bacteria.
3. AsCOSPORES. Ascospores (spores within a sac or ascus) are formed with-
in a single cell when t~e nucleus undergoes 1, 2 or 3 divisions without partici-
pation of the cell wall; forming 2, 4 or 8 ascospores in the sac. There is no ap-
parent involvement of sexes in this process. Because yeasts form spores en-
• If you arc familiar with genetics it is of interest to note there is a suggestion of a par-
allelism between Y ;::= Fill molds and yeasts, R ;:::! S in bacteria and the haploid;::::! diploid
state.
t Common, spbencal bacteria about 1 !' in diameter.
36 The Relationships oj Microorganisms to Each Other and the Living World
Fig. 3-2. Yeast (Saccharomyces cere-

visiae). A, single cell highly magnified; B,
cell in process of budding; C, chain of
cells fonned as result of rapid budding
and growth; D, formation of ascospores;
E, germination of ascospore and the de-
velopment of new plants by budding.
(Redrawn from Curtis. Reprinted by
permission, from Holman and Robbins,
"Textbook of General Botany," John
Wiley & Sons, Inc., publishers.)
Fig. 3-3. Electronograph of a longitudinal section through a buddina yeast cell (Sac-
charomyces cerevisiae). A is a bud with its cytoplasm continuous with that of the mother
cell. B is a mature bud with the developing cross wall betwccn mother and daughter cell.
C is the extension of cell wall material into the cytoplasm; a phenomenon which appears to
be characteristic of the later stages of the budding process. D is a bud scar, the surface of
which is always convex. (From the collection of the Society of American Bacteriologists,
courtesy of Hilda D. Agar and H. C. Douglas in J. Bact., 1955, vol. 70.)
closed in an ascus (Fig. 3-4), they are classed in the group of Ascomycetes or
sac-forming fungi.
The ascospores are, in some respects, analogous to bacterial spores, being
resistant to climatic heat, drought, and other unfavorable environmental con-
ditions. They are not so thermoresistant as bacterial spores, being killed by
60° C in a short time. Bacterial spores resist boiling and even much higher
temperatures for hours. Since ascospores of yeasts are generally produced in
groups of two or more per cell, they represent a process of multiplication as
well as preservation, thus differing from bacterial spores, of which only one
is produced by each individual cell.
4. SEXUAL PROCESSES. The sacs or asci of yeasts of many species often
result from readily discernible sexual processes in which two cells send out
projections which meet and form a copulation canal; the two cells form a
zygospore.· The nucleus of this cell divides within the sac to form a number of
ascospores (Fig. 3-5).
CHLAMYDOSPORES. Another type of asexual spore is called chlamydospore
(Chlamydo is from a Greek root meaning a protective covering). Many yeasts
and molds (and probably bacteria) are capable of forming these bodies
(Fig. 4-3). Active growth of the cell ceases, food is stored, the cell acquires a
thick protective wall, dehydration takes place, and the resulting dormant cell
tides the plant over unfavorable conditions. No increase in numbers of nuclei
occurs as in ascospore formation and the chlamydospore is therefore not
reproductive in function. Such bodies are not as heat-resistant as bacterial
spores.
Habitat of Yeasts. Yeasts and yeast-like fungi are widely distributed in
nature. They commonly occur on grapes and other fruits, vegetables, etc.,
which they participate in decomposing. The spores pass the winter in the soil.
The kind of wine made from grapes depends to some extent on the varieties
of yeasts occurring upon them naturally_ Yeasts and torulaet may also be
found in dust, dung, soil, water and milk, and are not infrequently observed
in cultures made with swabbings from the throat. They are also found in
insects, flowers, honey, etc. Many species are found as contaminants in
brewers' and picklers' vats and many appear to live like bacteria, on the
human skin.
Activities of Yeasts. Yeasts, especially the very common bakers' and
brewers' yeasts, Saccharomyces, are characteristically fermentative organisms,
producing chiefly alcohol and carbon dioxide from sugar under anaerobic
conditions of growth. Their alcohol-forming power is used in the manufacture
of wines and beer and was formerly used in production of industrial alcohol.
Industrial alcohol is now obtained mainly from the petroleum industry.
Pia. 3--4. Saccharomyces cereVisiae showing asci with two and four ascospores, as well as
vegetative cells. (Lindegren, C. c., in Bact. Reviews, vol. 9.)
• A zygospore is a cell, dormant in character, produced by two similar gametes or repro-
cells. Zygospores are commonly produced by certain algae and molds.
t Asporogenous, yeast-like fungi.
38 The Relationships of Microorganisms to Each Other and the Liv;nz World
Fig. 3-5. Diagrammatic representation of two types of reproductive cycle in yeast. A to

E, Zygosacclwromyces and Schizosaccharomyces. A. is the vegetative yeast plant, repro-
ducing asexually by budding. The cells in this stage are haploid. * In B is seen sexual con-
jugation of gamete-like cells, with formation of diploidt fusion nuclei. In C nuclear division
occurs, resulting in the formation of haploid ascospores within asci. In D tbe ascospores
germinate and grow into the asexually reproducing plant (E) again. Growth of these genera
is most commonly in the haploid, budding stage.
In F to I are seen reproductive stages of the common baker's yeast, Saccharomyces. F
shows tbe asexual, budding plant. G shows cells in which budding has ceased and nuclear
division occurred, with formation of haploid ascospores which are later liberated from the
asCi. H-shows sexual conjugation of the ascospores, with formation of diploid fusion nuclei.
After separation the-;e germinate and continue to grow as diploid, asexually-reproducing,
budding, yeast plaDl.~. This is the usual form of growth of Saccharomyces.
Varieties especially adapted to each purpose are, used. Thus, there are "dis-
tillery yeasts," "top yeasts," for beer, and "bottom yeasts" for beer and wine.
Distillery yeasts are the better alcohol producers. The yeasts grow in the
beer-wort or fruit juice, utilizing the nutrient substances there. Sterilized
beer-wort .agart is commonly used for the cultivation of such yeasts.
The carbon dioxide-producing power of yeasts is important in baking.
Some yeasts synthesize several vitamins, especially those of the B complex.
Others require a number of vitamins in order to grow and this makes them
useful in the assay of vitamins by fermentation methods (see Chapter 44). The
cell physiology of yeasts -and molds, except as related to reproducti9nr is
basically much like that of bacteria.
CLASSIFICATION OF YEASTS
A scheme of possible relations between some groups of common yeasts,
molds and fungi is seen in Figure 3-6. The yeasts and some other yeast-like
fungi are seen as being derived. by successive. loss of filament formation and
spore formation, from molds like Endomyces. The yeasts may be regarded as
* In haploid cells the nuclei contain half of the number of chromosomes characteristic
of the fertilized or somatic cells of that species.
t In diploid cells the nuclei contain the full number of chromosomes characteristic of the
fertilized or somatic cells of that species.
:j: Agar is a vegetable gum often used to prepare solid, jelly-like culture media.
Ascomycetes* which have lost the property of filament formation. Yeasts
and yeast-like fungi are found in two major divisions of the Eumycetes:
Class Ascomycetes and Class Fungi lmperfecti (see Table 2).
The yeast-like Ascomycetes are found in the order Endomycetales, and
mainly in the family Saccharomycetaceae. There are several genera and many
species (see TabJe 3).
The yeast-like Fungi lmperfectit comprise several groups which do not form
ascospores. These fungi are included in the order Moniliales, families Nec-
A SUGGESTED SYSTEMATIC RELATIONSHIP OF SOME MOLDS AND YEAST-

LIKE FUNGl
ASCOMYCETES FUNGI IMPERFECTI
Endomyces, a typical illamentous mold. --Candida. a yeast-like. dimorphic fungus.

forms both mycelium and 'budding single common in the environment. This forms both
cells. The mycelium forms asci by fusion of mycelium and single budding cells. but fails
7
,i
contiguous cells. Losing the power to form toformascospores.Losingthepowertoform
spores, it becomes:--,---------l mycelium. it becomes:
Losing the power to form mycelium, it be-
comes:_ g-~ (P
cJ
~ @ (YP
,/
I
Saccharomyces (brewer's and baker's - ....Torulopsis and related forms common in
yeasts) and related forms; true yeasts, never soil, dust, etc. These are the false yeasts
forming mycelium. existing as single cells growing as single cells, reproducing only by
reproducing by budding and by spores budding, never forming either mycelium or
formed either by thel conjugation of neigh- spores.
boring cells or by parthenogenesis. Losing
the power to form spores, they become:
I
Fig. 3-6. (Reprinted by permission from Henrici, "Molds, Yeasts, and Actinomycetes,"
John Wiley & Sons, Inc., 2nd ed., 1947.)
• A group of funJi, largely filamentous, in which spores are formed inside of distinctive
sacs called asci (singular = ascus). hence ascospore and Ascomycetes.
t Knowledge oJ life cycle imperfect; especially sexual reproduction.
40 The Relotionships of Microorganisms to Each Other and the Living World
taromycetaceae and Torulopsidaceae. They are often spoken of coUectively as
Torulae. They are differentiated from each other on the basis of the presence
or absence of asexually produced conidia, pigments, pseudomycelium, form,
habitat, etc. (see Table 3).
Table 3. A Classification of Yeasts.

Class: Ascomycetes (ascospore-fonning fungi)
Order: Endomycetales (yeast-like Ascomycetes)
Family Saccharomycetaceae (mycelium scanty or lacking)
(1) Saccharomyceteae
(a) Saccharomyces 1
(b) Torulaspora
«~) :;,chia l I Yeasts of commercial importance: some as
abnsenu a nuisances, some as valuable aids to bak-
(e) D e aryomyces
(f) Schwanniomyces
I ers, brewers, d·1Stillers, commerCIa
. I aIcoh 0 I
(g) Schizosaccharomyces I production, wine makers, etc. Some of
these species are common in soil, dust,
(2) Nadsonieae
(a) Saccharomycodes
(b) Hanseniaspora
II etc.
(c) Nadsonia }
Class: Fungi Imperfecti (fungi of which the reproductive cycle, especially the sexual stage,
is not completely known)· "
Order: Moniliales (conidia, when formed, produced in chains more or less definite)
Family Nectaromycetaceae (form conidia; occur in nectar of flowers)
Family Torulopsidaceae (no conidia; no pigments)
Tribe:Torulopsidoideae (no pseudomycelium)
Cryptococcus (one pathogen; mostly saphrophytes in soil)
Pityrosporum (skin saprophyte)
Kloeckera (saprophyte)
Trigonopsis (saprophyte)
Torulopsis (saprophyte)
Tribe Candidoideae (form pselldomycelium)
Candida
Trichosporon
Family Rbodotorulaceae (pink and rose-colored pigments)
Rhodotorula, etc. (often cause red spoilage of foods, etc.)
• It bas often been pointed out that the imperfections of this group are more in the obser-
vations than in the fungi.
t
Family Saccharomycetaceae. The sac-formers, i.e., the ascosporogenous
yeasts, include the tribes Saccharomyceteae and Nadsonieae, in both of which
mycelium formation is reduced to a minimum.
THE TRIBE SACCHAROMYCETEAE includes the genus Saccharomyces, which is
the largest and most familiar group and in which are found most of the
common yeasts/0f commerce, such as S. cerevisiae and S. ellipsoideus.
The Saccharomyces are the most common and valuable industrial yeasts,
The cells are oval or elliptical, and oval buds are produced at any part of the
cell surfaces. As mentioned above, their .ermentative powers are very ·useful.
They are the servants of the baker and brewer. There are numerous species
having various special properties useful in industrial processes. Other genera
in the tribe are To'rulaspora, Pichia, Hansenula, Debaryomyces, Schwan-
niomyces and Schizosaccharomyces. All of these produce buds at various parts
of the parent cell, i.e., budding is not bipolar whereas the cells of the tribe
Nadsonieae are characterized by bipolar budding.
THE NADSONIEAE are mainly saprophytic and are of little industrial or agri-
cultural importance.
Genera of Nadsonieae are Saccharomycodes, Hanseniaspora and Nadsonia.
The various genera in each tribe may be differentiated from one another by
fermentation and other biochemical methods and by the shapes of their
ascospores or by their modes of germination or modes of formation of asco-
spores. Some are found in beer, others in wine vats, dung, soil, etc. (Fig. 3-7).
The Non-sporeforming Yeasts (The Fungi imperfecti). There are several
subdivisions of yeast-like plants forming no ascospores. A small group, rather
specialized as to form and habitat, the Nectaromycetaceae, occurs mainly in
honey and insects. The largest group, containing several genera, is called
Torulopsidaceae. These organisms closely resemble the yeasts in most respects
except that they have not been observed to produce ~scospores by any method.
They are, therefore, part of the heterogeneous group known as Fungi im-
perfecti which contains a heterogeneous lot of fungi mainly because their
perfect or sexual stage is unknown. The cells of Torulae are usually more
nearly spherical than are the cells of yeasts. Their classification is not complete.
The largest and commonest genus is Torulopsis. Various kinds of torula have
geoff @@(;3
c ~
~" ~
4> C1 ,
@~§
@ ~&' @ @
2 3
Fig. 3-7. Various forms of yeasts, showing vegetative and budding cells and ascospore
I. Hansenula; 2. Hansl1niaspora; 3. Torulaspora; 4. Debaryomyces; 5. Pichia; 6. Schiz(
saccharomyces; 7. Sclm;anniomyces; 8. Saccharomyces; 9. Nadsonia.
been found causing "diseases" of beer and other fermented foods. They are
common in soil, water and dust. One species of Cryptococcus is pathogenic,
infecting the brain and meninges.
Another interesting group, called Rhodotorulaceae, produces brightly
colored pigments. Various species in this group, particularly red, pink, or
salmon-colored varieties, have been described as agents of spoilage in various
organic materials and uncooked foods like canned oysters. They are found
as nuisances in places where foodstuffs are prepared, as in butcher shops,
and oyster-shucking establishments. They are not pathogenic. Most of the
torulas have little fermentative ability and consequently are of little com-
mercial value.
REFERENCES
Alexopoulous, C. J.: Introductory Mycology. John Wiley and Sons, New York, 1952.
Bartholomew, J. W., and Levin, R.: The structure of Saccharomyces carlsbergensis and S.
cervisiae as determined by ultra-thin sectioning methods and electron microscopy. J.
Gen. Mic., 1955, 12:473.
Connell, G. H., and Skinner, C. E.: The external surface of the human body as a habitat for
non-fermenting non-pigmented yeasts. J. Bact., 1953,66:627.
de Backze, G. I.: A micrObiological process report. Yeasts: I. Morphology. Ap. Mic., 1956,
4:1.
Etchells, J. L., Bell, T. A., and Jones, 1. D.: Morphology and pigmentation of certain yeasts
from brines and the cucumber plant. Farlowia, 1953,4 :265. -
Hunter, Albert C.: A pink yeast causing spoilage in oysters. U. S. Dept. Agric. Bull. No. 819,
'March 10, 1920. "-
Huxley, M. J., and Hurd, R. C.: Pink yeasts isolated from human skin surfaces. J. Bact.,
1~56, 71:492.
Lodder, J., and Kreger-Van Rij; N. J. W.: The Yeasts, a Taxonomic Study. Interscience
Publishers, New York, 1952.. _
Scherr, G. H., and Weaver, R. H.: The dimorphism phenomenon in yeasts. Bact. Rev., 1953,
17:51.
Skinner, C. E., Emmons, C. W., and Tsuchiya, H. M.: Henrici's Molds, Yeasts and Actino-
mycetes. 2nd ed. John Wiley and Sons, New York, 1947.
White, J.: Yeast Technology. John Wiley and Sons, New York, 1954.
Wickerham, L. J.: Recent advances in the taxonomy of yeasts. Ann. Rev. Micr., 1952,6:317.
Wickerham, L. J., and Burton, K. A.: Hybridization studies involving Saccharomyces sp.,
and Zygosaccharomyces sp. J. Bact., 1956, 71 :290 and 296.
4
4. THE MOLDS
THE TERM "mold" is a convc;_nient one but, strictly speaking, has no exact
definition. For present purposes it may be taken to include most of the woolly,
cobweb-like, cottony or powdery growth, black, green, yellowish, or white,
commonly seen on stale bread, or on old piles of manure, or on books or
shoes in the summertime when the humidity is high. We shall also include
certain,mycelium-producing organisms which have some yeast-like characters.
Molds are common on the tops of jams and jellies which have been imper-
fectly sealed and have stood for a long time. Their spores are as ubiquitous as
those of yeasts and bacteria. Molds are able to grow in situations where bac-
teria cannot survive because of high osmotic pressures, acidity, or low mois-
ture content. They are characteristically strict aerobes and thus cannot com-
pete with anaerobic microorganisms. In general, their metabolic activities are
much like those of yeasts and bacteria. /
SIRUCfURE OF MOLDS
The woolly growths consist of more or less compact masses of intertwining,
branching, hair-like filaments, called hyphae, which grow up into the air. In
many species, when circumstances permit, there are also developments of
surface or subsurface hyphae on or within the material on which the mold
may be growing. These serve to anchor the plant but, except for being more
restricted in extent, are not otherwise especially differentiated from the aerial
mycelium. The aerial mycelium carries the fertile hyphae which form the re-
productive organs while the remainder of the mycelium absorbs moisture and
food material.
The filaments of molds may consist of elongated cells arranged end-to-end,
and separated by walls (septa) as in the Ascomycetes. In the Phycomycetes,
the whole mycelium;consists of one continuous, branching, protoplasm-filled
tube with no apparent cross walls. These filaments are said to be non-septate
or coenocytic. In mature plants septation or non-septation is a fairly stable
characteristic and serves as an aid in differentiation among molds. As com-
pared with bacteria; 'the diameter of the filaments of most species of mold is
relatively large; sometimes 30 microns.
43
44 TM Reialioruhips oj Microorgllnisms 10 ElIch Olher fJIUllhe Li'lling World
Fig. 4-1. Oidium formation. Seg-

mentation seems to be stimulated so that
numerous sma1l cells, often called ar-
throspores are given otT. They are not par-
ticularly resistant, but serve the purpose
of propagation.
The nuclei of molds, within the filaments, are minute, but generally quite
definite. In the Ascomycetes (septate) each cell contains one nucleus except
during phases of reproduction. In the Phycomycetes (non-septate) the nuclei
are not separated by definite cell walls except as a result of the formation of
conidia.
The cytoplasm of molds is granular and contains droplets of fat, carbohy-
drate and nitrogenous material, including volutin. The cell wall of some
species probably consists of cellulose; in most, however, the cell wall is made
of a chitin-like substance.
As indicated in the table on page 34, molds corresponding to the general
description given above may be found in three large divisions of the Eumy-
cetes; namely, Ascomycetes, Phycomycetes, and Fungi imperfecti. Peculiar-
ities of structure, already noted and to be discussed, and methods of repro-
duction, enable us readily to distinguish filamentous Ascomycetes from
Phycomycetes. The Fungi imperfecti will be described later.
REPRODUcnON OF MOLDS
Molds reproduce both asexually and (except in Fungi imperfecti) sexually.
We may note at least five well-defined methods of asexual reproduction.
1. Formation of oidia· or artbrospores. Septate filaments often form a
number of divisions rather closely spaced, resulting in the separation of a
number of short, ovoid, cells. These tend to leave the parent filament by·
fragmentation. They continue vegetative growth, each starting a new plant
(Fig. 4-1).
2. BIastospores. Some of the filamentous molds form buds or blastospores
along the hyphae. These develop much as do yeasts. Indeed some of these
fungi grow readily in either yeast or filamentous form, the form of growth
depending on such factors as presence of oxygen and temperature (Fig. 4-2).
3. Cblamydospores are formed by many types of microorganisms. One or
more cells acquire thick walls, and become filled with granular reserve ma-
terial. They probably lose water. In this form they remain dormant and resist
drying and sunlight for long periods (Fig. 4-3).
• Oidium is from a Greek root meaning "egg," i.e., an ovoid body.

4. Sporangiospores* are formed only by the Phycomycetes. They are mi-
nute, thick-walled, dehydrated, dormant bodies and are formed in huge num-
bers by repeated nuclear divisions within globular envelopes called sporangia.
They are formed at the free ends of fertile hyphae. They resist drying and
sunlight for long periods. When the sporangia rupture, the sporangiospores
are released. The details of their development and structure are given in the
description of the Phycomycetes.
5. Cooidiospores, t or conidia, resemble sporangiospores in many ways.
They are formed by Ascomycetes at the free ends of fertile hyphae, but instead
of being enclosed in sporangia they are free, sometimes being produced in
chains like strings of beads. The form and arrangement of the fertile hyphae
and the chains and color of conidia are distinctive of the different genera and
species. They are described more in detail in discussing the Ascomycetes.
CULTURE MEDIA FOR MOLDS
One of the difficulties in the study of molds and yeasts is their isolation in
pure culture. In their natural habitats these microorganisms are practically
always intermingled with many other species. A specimen of soil, for example,
is inoculated onto a plate of Sabouraud's medium with a view to isolating a
particular species of mold in pure culture, i.e., entirely separate from all other
microorganisms. In such a situation other molds, yeasts, and especially hac-
Fig. 4-2. A dimorphic, yeast-like.

pathogenic fungus (Candida albicans)
showing mold-like filaments, a few yeast-
like cells, blastospores in clusters like
grapes near hyphal septa, and circular,
thick-walled chlamydospores. (Photo
courtesy of Dr. Lynferd J. Wickerham,
U. S. Dep. of Agriculture, Agricultural
Research Service.)
Hg. 4-3. Chlarnydospores. These are torme<1 trom mycelial cells by enlargement and
by thickening of the cell wall. (Photo courtesy of the U. S. Public Health Service, Communi-
cable Disease Center, Atlanta, Ga.)
• Angium is from a Greek root meaning bag or envelope; hence, Sporangium.

t Conidio is from a Greek root meaning dust.
Filo 4-4. ODe method or studyina f'uDaI in pme culture. A layer or qat' medium is
or
allowed to solidify as the flask lies fiat. Inoculation then is made in the ccoter the apr
surface with a single touch of an infected needle. After incubation the characters of tile
sinIIe "aiant colony" are readily noted. These are distinctive in various species. (Ori&inal
pr4IIIU8tiOD by Dr. Libera Ajeno. Photo courtesy of tile U. S. Health Service, C0mmuni-
cable Disease Center, Atlanta, Ga.)
tcria which grow much more rapidly than molds, often overgrow and sup-
press and kill the desired species. To prevent this arowth of undesired micro-
organisms various inhibitory agents are used, and various selective nutrients
which favor growth of the desired species may be used if they are known. For
example, a medium especially recommended for isolating fungi from sewage
is made of the following: glucose, 10 gm; peptone,· 5 gm; KH2PO", I gm;
MaSO", 0.5 gm; agar, 20 gm; water, 1000 mI; rose bengal, 10.035 gm;
streptomycin, 30.0 Pit per mi. Rose bengal is a dye which inhibits growth of
many bacteria. Streptomycin, an antibiotic, also inhibits many unwanted
organisms. The glucose and peptone furnish organic carbon and nitrogen
foods as well as organic sources of energy. The KH2PO.. maintains a suitable
degree of acidity (PH) wbile MgSO.. furnishes magnesium and sulfur; both
essential to the formation of protoplasm.
Other combinations of inhibitory .agents like oxgall, crystal violet and
streptomycin have been found very useful. One containing cycloheximide,
streptomycin, and penicillin is particularly valuable in medical mycology.
Cycloheximide is an antibiotic which, curiously enough, inhibits most com-
mon, air-borne, aaprophytic molds which contaminate medical specimens, but
permits the pathogenic species, with very few exceptions, to multiply freely.
Among the most distinctive properties of molds are morpbology of mycelia,
sexual and asexual reproductive mechanisms, pigments and manner of colony
llfowth.
GIaDt CoIoaIes An excellent method of demonstrating gross morphologi-
cal details, pigment, etc., is in the form of giant colonies. Appropriate culture
media especially deaigned to favor development of reproductive mecba-
• Partly cIiIIIted proteln.
t III is thlaymbol ror microgram: 1 millionth of a 8J'IIIII.
71N Microscopic World 47
nisms, pigments, etc., are used. For example, thiamine-enriched casein agar is
especially useful in stimulating conidia formation in certain skin-infecting
fungi (dermatophytes). Whatever medium is selected, it is poured into plates,
flasks or bottles of about 200 ml capacity, to a depth of about V3 inch, steril-
ized and allowed to solidify. This gives a broad surface and the volume of
agar serves as a reserve of moisture. A speck of inoculum (pure culture) is
transferred to a point in the center of the agar. It is well to do this with the
bottle or flask in an inverted position to avoid contamination by dust. Plugged
with cotton, the flask is held at appropriate incubating temperature for some
days. Giant colonies grow out, showing various details of color, form, etc.,
characteristic of the different organisms. A colony of this kind is seen in
Figure 4-4. Giant cultures may be examined with low power lenses in situ, or
portions may be teased out on a slide in a drop of mounting fluid· and ex-
amined under a clover slip. Drying produces shrinkage and distortion, while
manipulation, such as teasing out, causes fracturing and loss of conidia. It is
much better to examine growth in situ with the microscope.
Sllde Cultures. For the microscopic examination of molds the slide culture
is an excellent method. A good arrangement is to cement a sterile cover slip
to an ordinary (previously sterilized) slide with two other bits of glass arranged
so that the cover slip is raised about I millimeter above the slide. The desired
nutrient agar, melted, cooled to about 45° C. and inoculated with spores, is
admitted between the slide and the cover slip and the culture incubated (Fig.
4-5). This permits examination of the growth with fairly high power lenses
/
I
Fig. 4-5. Microscope slide arranged for small cultures of molds, etc. Agar of appro-
piate composition, mixed, while still fluid, with spores or parts of the desired fungus, is
allowed to run under the cover slip. Growth occurs at the margin under the cover slip.
Microscopic examinOilion of the fungus as it grows is thus possible. (Courtesy of Dr. Richard
N. Shoemaker, Science, 1950, vol. 112.)
• An excellent mounting fluid is lactophenol-colton blue solution, made as follows:

ml
Phenol crysta ls (melted) ..... .. . . ... . .. . ..' .. . . .. ... . .. . 20
Lactic acid ....... . ........... .. . . . ... . .. . . .. ..... . " 20
Glycerin ..... ..... . .. . . ......... . ....... . . .... ... . " 40
Dist. H20 ... . . .... ... ... . . . .. ... . . .. . . .. .. .. . .... . . . 20
DiIsolve by heating mixture at al.out 70' C. Add 0.05 gm of dye called Poirrier's blue or
ClOiton blue. •
A drop of this solution is placed on a slide and a small part of a fungus colony is teased
IIIlUt in it with needles. A thin cover slip is placed over it, and the preparation is examined
with the microscope.
-r l~
\,,:.
1 ··
··
A
Fig. 4-6. Zygospore formation (sexual reproduction) by Rhizopus. A, two swollen

hyphae grow toward each other and meet. B, each produces a special cell (gamete). These
then fuse or conjugate. The resultant fertilized cell develops into a dormant zygospore (C)
which later germinates, sending out a hypha which produces conidia asexually (D). (From
Swingle, D. B., Plant Life, 2nd ed. D. Van Nostrand Co., Inc.)
and in a living, undisturbed condition. This slide may be placed in a covered

Petri dish with a bit of moist cotton.
The best conditions of incubation for most molds are a moist atmosphere
and a temperature around 30" C. There should be free access of oxygen. For
general purposes the best pH is around 6.5-7.0. Other slide-culture techniques
have been described by several mycologists.
HABITA.T AND GENERAL PROPERTJES OJ' MOLDS

Mold conidia are found in all dust as every housewife knows who has had
preserves spoiled by molds. Molds occur on decomposing organic matter like
manure piles, "compost" heaps, dead plants and animals. The sea has a most
interesting, though somewhat limited, indigenous flora of molds. They par-
ticipate in the destruction of ropes, timbers, etc., exposed at water level. Some
have done enormous damage by infecting commercially valuable fish, shell-
fish, and animal and fish foods (such as "eel grass") on which many edible
marine forms live.
Molds decompose cellulose and ruin paper and wood products not pro-
tected from them. They grow in and under paint on walls and cause flaking
and deterioration of the paint. They grow on the surfaces of lenses in binocu-
lars, etc., in the tropics, diminishing clarity of vision. They also grow on the
surface of electrical insulators, causing them to transmit electricity. Some
TM Microscopic World 49
molds grow well on rubber and ruin it. Molds are merry jvkers of the micro-
scopic world!
Physiologically, molds are, as a group, active in causing very rapid oxida-
tions and decompositions of carbohydrates, fats and proteins, and other bio-
chemical changes in a great variety of substances. They are of great com-
mercial value in the production of various organic compounds which are used
as foods, flavors and drugs. From carbohydrates they produce hundreds of
substances not easily made by artificial processes, and of great usefulness.
Among these are penicillin, etc., acetone, butanol, sorbitol, takadiastase, and
so on. This is an excellent field for research.
THE PHYCOMYCETES
The group of Phycomycetes contains several genera, two of which are very
commonly observed and often very troublesome, namely Mucor and Rhizopus.
These are placed in a group called Zygomycetes because they form zygospores
(see Fig. 4-6). One aquatic genus of Phycomycetes, called Sapro/egnia, is
parasitic on fish.
Genus Mucor. This genus contains several dozen species, many of them
Fig. 4-7. One of the Mucorales. Stages in formation of a sporangium filled with spores.
I, sporangiophore and columella; 2, multiplication of spores from nuclei of the columella
and formation of the retaining membrane; 3, completion of sporangium formation; 4,
beginning of segregation of multiplied nuclei to form the individual spores or conidia; 5,
final structure of the sporangium showing the thickened retaining membrane, the free
conidia ready for dispersion on ruplure of the membrane, and the now inert, rounded
columella which will be lcft behind. (From Swingle's Plant Life, D. Van Nostrand Co., Inc.>
so TM Relationships oj Microorgtmisms to Each Other and the Living World
Fig. 4--8. RhizopllS nigrlcans. Plant spreading by stolons and rooting itself by means of
rhizoitls. (From Swingle's Plant Life. D. Van Nostrand Co., Inc.)
very similar to one another. One of the best known is Mucor mucedo, a coarse,
woolly, white mold seen on piles of rotting manure or other decaying organic
matter. Mucor mucedo reproduces asexually by means of sporangiospores.
Each sporangium is borne on a short hypha called a sporangiophore· at the tip
of which is an enlarged portion called a columella which varies in shape
according to the species. The spores are formed in a mass about the columella
within the envelope (Fig. ~7).
In sexual reproduction zygospores are formed (Fig. 4--6). The cells fusing
to form zygospores are usually on two separate plants, which produce the
spores wherever hyphae come into contact, suggesting that the plants are of
opposite sexes. Neither plant alone produces zygospores. Such plants are
said to be heterothallous, and their "sexes" are called + or -. When hyphae
of the same plant fuse, the plant is said to be homothallous.
Mucors and related genera of molds are common contaminants of bac-
teriological cultures. A few have been found as the cause of disease in man
and animals.
Genus Rbimpas. The molds of this genus, all typical Phycomycetes, are
well exemplified by R. nigricfDll. the common, black, bread mold familiar to all
who have seen bread after it has stood in a humid place for some days during
the summer. it spreads rapidly because it sends out stolons or runners (Fig.
4-8) like some Mnds of grass ("Bermuda grass") and strawberry plants. These
runners take hold of the substrate by means of "holdfasts" or root-like
byphae.
Like Mucor mucedo and its allies, R. nigricans is prominent in bringing
about decomposition and spoilage of various fruits, vegetables, and other
orpnic materials.
Molds of the genera Mucor and Rhizopus may be differentiated by at least
three characters as follows:
• A ItaIIt OIl wbicb the sporanaium is borDe.

TIle MicrolCopic World 51
Mucor Rhizopus
RUllIlCrs ........... . ... Absent Present
Sporangiophores ........ May arise at any point in the Arise only at the "holdfasts"
mycelium. where the runners touch the
substrate.
COlumella . . ... . ....•.. Never hemispherical; contin- Hemispberic:al; a single. dif·
uous with the sporangio- ferentiated cell.
phore.
THE FlLAMENTOVS ASCOMYCETES

We have already encountered sac-forming fungi (Ascomycetes) as yeasts.
We now review the filamentous Ascomycetes.
The group , of Ascomycetes as a whole contains many widely differing
plants. varying from the yeasts through filamentous wooly molds, to fleshy
mushroom-like morels and some of the beautifully formed and colored fungi
which grow on rotting logs. All have in common the formation of asci with
ascopores and all are modifications, or evolved and elaborated patterns, of
the same basic growth design. They have septate mycelia.
Among the best known Ascomycetes are the common genera Aspergillus
and Penicillium. These are distinguished from Phycomycetes Qike Mucor and
Fis. ~9. Aspergillus sp.• showing typical structures: near upper left a fertile hypha or
CINIid/op}wre arises by branching almost at a right angle from a short hypbal cell caIIcd a
(qot cell: near low center is seen a large conidiophore. from the rounded tip (YUick) of
which arises a cluster of spindle-shaped supporting cells called slerigmallJ: from the sterig.
mata arise chains of spherical conidlospores. A denuded conidiophore is seen near the top
of the picture. (Photo courtesy of Dr. Lynferd J. Wickerham, V. S. Dep. of Agriculture,
Aaricultural Research Service.)
52 The Relationships oJ Microorganisms to Each Other and the Living World
Fig. 4-10. A perithecium of Alle-

scheria boydii, a pathogenic, fi1amentous
ascomycete occurring in the soil, has
matured and ruptured, liberating many
ascospores. (Courtesy of Dr. Libero
Aje110, in Am. J. Trop. Med. and Hyg.,
1952, vol. I.)
Rhizopus) by (I) forming numerous sexual spores in sacs (ascospores) ; (2)

producing conidiospores in groups of various sorts such as chains, masses of
characteristic shape, etc., (Fig. 4-9); and (3) being ~ptate.
Genus Aspergillus. During sexual reproduction (rarely seen) hollow
structures called perithecia, which contain asci with spores, are formed. It is
for this reason that the aspergilli are included in the same order (Ascomycetes)
as the yeasts. Both form ascospores.
PmuTHECIUM FORMATION. In the formation of a perithecium two hyphal
cells twist about each other. The separating walls dissolve and the resulting
fusion-cell gives rise to a nodule of branched hyphae among which a mass of
asci is formed. The whole mass is often enclosed in a sort of hull or protective
cellular covering formed by the surrounding mycelium (Figs. 4-10, 4-11).
THE ASEXUAL FRUITING BODIES, or conidiophores, of aspergilli consist of tall
hyphae arising as a branch of a cell in the mycelium called a "foot-cell."
The fruiting hyphae have enlarged globular tips (vesicles). From the surface
of these numerous small stems, called sterigmata, radiate in all directions.
On the tips of these the conidiospores are borne in long chains (Fig. 4-9).
There is no sac or envelope as in the Zygomycetes. Classifications of aspergilli
are based to some extent on the structure, but more oarticularly on the color,
of the conidial heads.
CoMMON SPECIES. One of the commonest species of Aspergillus is A.
glaucus. It forms green or grey-green conidia and yellow perithecia and is
frequently seen on bread, preserves and clothing during the summer or in
the tropics. Another common environmental species, sometimes found as a
laboratory nuisance, A. niduians, is recognized because of its bright green
color. The perithecia are reddish in color. A. niger, also an extremely common
The Microlcopic World 53
species, is recognizable by its very large round masses of black conidia. It is
sometimes confused with Rhizopus nigricans which produces black sporangia.
There are some pathogenic species. For example, a pulmonary infection
of birds due to Aspergillus /umigatus is not uncommon.
Genus PeDidlliUDl. The penicillia are widely distributed and contribute to
the spoilage of various objects and materials composed of organic matter,
especially ripe fruits. The conidiophores are composed of hyphae which
branch at the tip into fingerlike clusters, the whole roughly suggestive of the
bony structure of the hand. The spores extend in chains from the ends of the
"fingers" (sterigmata). This arrangement gives the whole conidiophore with
its chains of conidiospores a form suggestive of a tiny paint brush, from which
the generic name is derived (Fig. 4-12, B). As in other groups of molds, the
color and form of the fruiting body furnish characters of differential value in
classification. Sacs or perithecia are formed by some penicillia in much the
same manner as by the aspergilli, but most species of Penicillium are classed
as Fungi imperfecti.
Some species of Penicillium are differentiated chiefly by their habitat. Thus,
the green molds found in Roquefort cheese (P. roque/orti), Camembert cheese
(P. camemberti) and other cheeses of the same nature are distinguished chiefly
because of their occurrence there. The molds grow in or on the cheese, pro-
ducing various enzymatic changes in the fat, carbohydrate and protein of the
cheese which result in characteristic aromas, flavors and textures. P. roque-
forti grows weD under conditions of temperature and humidity found in the
limestone caverns in the province of Roquefort, France. It is micro-aerophilic,
growing in the interior of the cheese masses provided holes are punched in
the cheese to admit small amounts of air. (These holes must not be confused
with the holes in Swiss cheese produced by gas formed by the Propionibacter.)
Penicillium camemberti, which closely resembles P. roque/orti, is strictly
aerobic and grows only on the exterior of Camembert cheese, so that the
ripening of this delicacy proceeds from without inward.
There are many other species of penicillia and they are frequently seen on:
old bread, cheese, lemons, and other fruits. They may usually be recognized
as members of the genus by their sky-blue or green color. The individual
Fig. 4-11. Structure of a perithecium

of recently discovered Westerdykella or-
nata, an East African ascomycete. Note
the thick perithecial wall and the con-
tained asci, each baving 32 ascospores
inside. (X 2(0). (Courtesy of Dr. Amelia
C. Stolk; in Trans. Brit. Mycolog. Soc.,
1955, vol. 38.)
54 TIM RelatiolU/Iip, 0/ Microorgtmimu to Each Other tmd the wing World
B ---
10)&
F ... 4-12. A, PetdcJlll_ 1]1. The rapidly growing giant colony (about }i life size) is at
first white, thm becomes bluish-p'eeo and ~ powdery due to abundant conidia or spore
production from the aeriallJl)"Qelium. B, Penicillium ap. Spore-bearing hyphae character-
istically fonn a "peoicillus" (Latin for brush). The conidia occur in unbranched chains (e)
formed at the tips oftlask-shaped sterigmata (d) which are verticillately arr:anaed (in whorls)
from the ends of metuJae (c) arising from branches (b) of the conidiophore (a). Although
species of Penicillium differ in gross appearance (size of colony, color, texture, etc.), the
ICIlIJ8 may he identified by the characteristic structure of the COnidiophore and peoicillus.
(Cooant, Martin, et al, Manual of Clinical Mycology.)
species may be differentiated by the various arrangements of the conidio-

phores.
Penicillium notatum and P. chrysogeRum, very similar species, have come
into great prominence as sources of penicillin.
FUNGI IMPERFEcrI
Fungi imperfecti comprise nonphycomycetous (septate) fungi of which the
sexual reproductive cycle is not known. The grouping is wholly artificial. The
membership of the group is, therefore, rather heterogeneous since imperfect
forms of Ascomycetes, Basidiomycetes, etc., are numerous. Most Fungi im-
perfecti, however, are really Ascomycetes of which our knowledge is in-
complete.· Many of the species of Fungi imperfecti probably have complete
sexual cycles if we only knew the proper kind of medium and growth con-
ditions which would permit their development. The imperfection lies not so
much in the fungi as in our knowledge!
Since there are about 1,330 genera and some 20,000 species, a complete
discussion of the Fungi imperfecti here is out of the question. We may, how-
ever, give attention to a few common species of filamentous fungi of this
group which any bacteriologist may from time to time encounter in his
laboratory work.
Gan111'rldloclenIIa. The conidia of this genus occur in compact masses
on tips of well-developed conidiophores which branch like trees or shrubs.
They are bright green in color. Trichoderma is common in soil and as a con-
taminant in laboratory cultures of the microbiologist.
The best-known species of this genus is T. koningi or T. viride, a common
• MOlt or the ~ and PenicUliIIm species are Fungi imperfecti. Since these geaera
haw already been deacribed they are not discussed here.
TIre Microscopic World 55
soil and environmental species which often enters laboratory cultures unin-
vited (Fig. 4-13). It forms largl: amounts of ammonia, and an ammoniacal
odor gives a clue to its identity.
Genera Candida, Trichosporon, Geotrichum. These three genera are sub-
divisions of a large group sometimes called Oidium. sometimes Monilia, * and
often Candida. In the main, these organisms are neither clearly filamentous
molds nor are they exclusively yeast-like, but partake of the characters of
both. The yeast-like characters are more often seen.
The group may be divided into three genera based upon mode of repro-
duction: (1) Geotrichum, reproducing by fragmentation or disarticulation of
the mycelium into separate cells called arthrospores; (2) Candida, reproducing
by development of yeast-like cells budding from the ends or sides of the
mycelium, called blastospores .. (3) Trichosporon, reproducing by both methods.
GEOTRICHUM. One common species is the usually harmless Geotrichum
candidum, often called Oidium lactis. It is a saprophyte of soil, ~tc. , and is
often found in the oral cavity of normal persons. It can cause serious infections
under some conditions. This species readily metabolizes lactic acid and is of
common occurrence in soured dairy products,like sour milk, cheese or butter,
and in sauerkraut and silage, where lactic acid is formed by other organisms.
It · grows as a white, felt-like, adherent membrane on the surface of solid
media like Camembert and other cheeses, clotted milk, or agar (Chapter 44).
Aerial mycelium forms as the plant grows older.
CANDIDA ALBICANS is commonly found on the skin or on the oral or vaginal
mucous membranes or in the feces of normal individuals. It also occurs in
soil, dung, etc. Growth on Sabouraud's agar is best at 37° C. The colonies are
creamy and have a yeasty odor. The growth consists mainly of small (2 to
41-'), yeast-like cells. Mycelial elements may occur, with blastospores. The
formation of clusters of budding cells and distinctive, round cblamydospores
on cornmeal agar or other suitable agar are used in identification. (Fig. 4-2.)
This species can cause a number of serious infections of man and animals,
especially of mucous membranes (thrush, vulvovaginitis, etc.), and the skin
(cutaneous candidiasis) and lungs (pulmonary candidiasis). Microscopic ex-
aminations of scrapings from lesions usually reveals the organisqlS but diag-
nosis should be made only by a skilled mycologist, especially as these and
similar species are so common in normal individuals.
Genus Alternaria. Several kinds of dark-green or brownish-green molds
from soil and dust are often met with as contaminants in laboratory media
Fig. 4-13. Trichoderma koningi. (Re-

drawn from Hennci.)
• MoniJe is from the Latin for oeck)a(:e; a striDa of beada.

56 The Relationship, of Microorganisms to Each Other and the Living World
Fig. 4-14. Alternaria, showing muri-

form multicellular conidia.
Fig. 4-1 S. Hormodendrum: dark, smoky

or black colonies.
and on Petri-dish cultures. Among the commonest forms are members of the
genus Alternaria. They are not so large and extensive as Aspergillus or Penicil-
lium. The colonies of Alternaria are wooly but are more compact, and the
underside and mycelium are very dark colored. The conidia are relatively
large, and their 8 to 14 or more large, component cells occur packed in dis-
tinctive, muriform,· conical masses so that the cells are irregularly shaped.
These groups of conidia are often arranged in long rows, or occur along the
mycelial filaments (Fig. 4-14).
Genus HOI'IIlOdeaftum or Cladosporium. These molds, like Alternaria,
form relatively small, dark-green or brownish-green colonies with a fine·
textured, velvety surface. They produce conidia by growth at the tip of the
chains (apically ) rather than at the base of the chains as in Aspergillus and
Penicillium. Thus, in contrast to the last two, the youngest conidia in Hor-
modendrum are at the tips. The conidia are rather oval or elongated, and the
chains of conidia are often branched (Fig. 4-15).
SOME PAmOOENIC FUNGI
Blastomyces dermatitidis, also sometimes called Cryptococcus gilchristi or
Oidium dermatitidis, is a dangerous pathogen, causing the disease known as
North American blastomycosis. B. dermatitidis is one of the dimorphic,
• Muriform is from tho Latin word _lIS (wall) meaning "arranged like bricks in a wan. ..
yeast-like fungi capable of invading superficially or throughout the whole
body. In tissues and in cultures at 3ro C only very thick-walled, budding,
yeast-like cells occur. In cultures at 22° C mold-like filaments occur.
A very similar organism, called Cryptococcus neojomlllllS, causes a similar
disease known as "European" blastomycosis, but world-wide in distribution.
It does not form filaments in cultures but is distinguished by its very large,
gummy capsules.
Coccidioides immitis is the cause of a disease called coccidioidomycosis, oc-
curring chiefly in the San Joaquin Valley of California and elsewhere in arid
regions. In nature the organisms live in the soil and their resistant chlamydo-
spores and arthrospores from mycelial filaments are blown about with dust
and inhaled. Many of these infections pass unnoticed ot result in a febrile
disease in association with bronchitis, "rheumatism," or pneumonia. The
disease is often confused with tuberculosis in x-ray and clinical examinations.
When C. immitis is specially cultivated away from free oxygen, or when
invading the tissues of the body, it forms cells resembling those of B. derma-
titidis, but they never form buds. On the contrary, the cell contents divide
into many smaller cells, within the cell wall. The sporangium wall ruptures,
liberating htrge numbers of small cells. These are then distributed by the
blood throughout the body and repeat the cycle. When cultivated aerobically,
or in the soil, mycelial filaments are generally formed. Thus, it is a dimorphic
fungus. No conidia are formed (Fig. 4-16).
Histoplasma capsulatum. This organism resembles B. dermatitidis and
Coccidioides immitis in several respects. It causes infections in man which
apparently may pass unnoticed in certain individuals, yet may cause fatal
generalized infections in other persons. The infection (called histoplasmosis)
appears to be widespread in the Ohio, Mississippi and Missouri river basins
and elsewhere all over the world. These infections are pulmonary in char-
acter, in some respects resembling tuberculosis. The infection has been found
naturally occurring in dogs, rats, skunks and cattle, soil (another soil sapro-
phyte as a pathogen), and dust .of the air.
Fig. 4-16. C. immilis stained with

lactophenol cotton blue. Filaments,
probably with arthrosporcs, are seen as
well as a germinating sporangium or
spherule. (Burke, R. C., P.roc. Soc. Exp.
Bioi. & Med., 1951, vol. 76.).
10
.. -~ u" Qo»
-(1)-18
20
'O~
dt?hl.~ ~ 1\1. 61' 22
Fig. 4-17. Life cycle of HistoplasmIJ capsulaturn. 1, yeast cells from blood agar incubated
at 37° C. 2, yeast cells germinating with a single tube in 48 hours in Sabouraud's glucose
agar culture at room temperature. 3, yeast cells with tube germinating from each cell on
Sabouraud's glucose agar. 4, single yeast cell with three germ tubes on Sabouraud's agar.
5, single yeast cell with two germ tubes on Sabouraud's agar. 6, budding yeast cell which
has germinated with three tubes on Sabouraud's agar. 7,20, smooth-walled chlamydospores
which have germinated in Sabouraud's broth and produced chlamydospores in eight days.
8, intercalary chlamydospores on Sabouraud's glucose agar. 9, 10, 15, round tuberculate
chlamydospores from Sabopraud's glucose agar. 11, Pyriform tuberculate chlamydospore
from Sabouraud's agar. 12, 14, small, round, smooth-walled sessile and terminal chlamydo-
spores with oil droplets. 13, largll round smooth-walled chlamydospores on Sabouraud's
glucose slide culture in fourteen days. 16, small pyriform tuberculate chlamydospores from
Sabouraud's glucose agar. 17, small round tuberculate chlamydospores on aerial hyphae.
18, 21, 22, optical view of thick-walled chlamydospores. 19, small tuberculate chlamydo-
I spore germinating in cell culture. (Conant, N. F; in J. Bact., vol. 41.)
This organism may be cultivated on 10 per cent blood infusion agar at 37°
C. Under such conditions the cells are yeast-like. In infected tissues only the
yeast-like form is seen.
When cultivated at room temperatures on such media as Sabouraud's or
glucose agar, a cottony, white, filamentous growth appears, another dimorphic
fungus. The filamentous form is septate and in older cultures there appear
large, round, or pear-shaped, thick-walled ·''Ilamydospores covered with
rounded projections (tuberculate chlamydospores) (Fig. 4-17). These are
especially characteristic of H. capsulatum.
THE DERMATOPHYTES
Mycotic (fungal) infections of the skin are common; in fact skin mycoses
are among the commonest of infectious diseases. The molds most commonly
involved are a rather well-demarkecl group called dermatophytes. In general
they belong to the Fungi imperfecti.
Attempts to classify the dermatophytes have left the taxonomy and nomen-
clature of this group in a somewhat confused state. For convenience we may
list some pathogenic fungi as follows:
I. YEAST-LIKE FORMS
Torulopsis (meningitis; European blastomycosis)
Candida (thrusb; moniliasis; paronychia; vaginitis)
Blastomyces
rr.~OUSFORMS
A. Ringworm (tinea) fungi.
1. Microsporum
M. canis (animal type; tinea capitis in children)
M. aUdouini-(human type; tinea capitis in children)
M. gypseum (animal type; tinea)
2. Trichophyton
T. schoenleini (favus)
T. mentagrophytes (kerion; tinea sycosis)
T. rubrum; T. megnini; T. violaceum (various forms of tinea, kerion, etc.)
T. tonsurims ("barber's itch" or tinea capitis)
3. Epidermophyton
E. floccosum (mycosis of hands, feet and groin. Similar conditions caused
by various Trichophyton species)
B. Other pathogenic filamentous fungi.
1. Coccidioides immitis
2. Sporotrichum
REFERENCES
/
Ajello, L.: Soil as a natural reservoir for human pathogenic fungi. Science, i956, 123:876.
Benham, R. W.: The genus Cryptococcus. Bact. Rev., 1956,20:189.
Conant, N. F., Martin, D. S., Smith, D. T., Baker, R. D., and Callaway, J. L.: Manual of
Clinical Mycology. 2nd ed. W. B. Sannders Co., Philadelphia, 1955.
Cooke, W. B.: Fungi in polluted water and sewage: I, II, III. Sewage and Ind. Wastes, 1954,
26:539,661,790.
Cutter, V. M., Jr.: The cytology of fungi. Ann. Rev. Microbiol., 1951. 5:17.
De Yay, J. E.: Mutual relationships in fungi. Ann. Rev. Micr., 1956, 10:115.
Emerson, R.: Molds and Men. Sci. Amer., 1952,186:28.
Georg, L. K.: The role of animals as vectors of human fungus diseases. Tr. New York Acad.
Sci., 1956, Ser. If, 18:639.
Georg, L. K., Ajello, L., and Papageorge, C.: Use of cycloheximide in the selective isolation
of fungi pathogenic to man. J. Lab. Clin. Med., 1954,44:422.
Gordon, M. A., and :Cupp, H. B., JI.: Detection of Histoplasma capsulaturn and other
fungus spores in the environment by means of the membrane filter. Mycologia, 1953,
45:241.
Haley, D. L.: Culture Inedia used in a diagnostic medical mycology laboratory. Am. J. Med.
Techno!., 1954,20:4.
Hotchkiss, M.: Methods for the isolation of pathogenic fungi from clinical material. Am. J.
Med. Techno!., 1953,19:154.
Lilly, V. G., and Barnett, H.: Physiology of the Fungi. McGraw-Hill Book Co., Inc., New
.york, 1953. I
Littman, M. L., and Zimmerman, L. E.: Cryptococcosis (Torulosis). Grune and Stratton,
New York, 1956.
O'Hem, E. M., and Henry, B. S.: A cytological study of Coccidioides immitis by electron
microscopy. J. Bact, 1956, 72:632.
Raper, K. B., and Thorn, C.: Manual of the Penicillia. Williams & Wilkins Co., Baltimore,
1949.
Raper, K. B., and others: Speciation and variation in asexual fungi. Ann. New York
Acad. Sci., 1954-55, 60(Art. 1):1.
Reiss, F., and others: Medical Mycology. Ann. New York Acad. Sci., 1948-1950,50:1209.
Ritchie, D.: A fungus flora of the sea. Science, 1954,120:578.
Skinner, C. E., Emmons, C. ·W., and TSllchiya, H. M.: Henrici's Molds, Yeasts and Actina-
mycetes. 2nd ed. John Wiley and Sons, New York, 1947.
Smith, C. E., (Chairman), Proc. Conf. on Histoplasmosis. Pub. Health Servo Publ'n.
No. 465,1956. Gov't. Printing Off., Washington 25, DC
Smith, G., and Raistrick, H.: An Introduction to Industrial Mycology. Edward Arnold &
Co., London, 1942.
Thatcher, F. S.: Foods and feeds from fungi. Ann. Rev. Microb., 1954,8:449.
Thorn, c., and Raper, K. R: A Manual of the Aspergilli. Williams & Wilkins Co., Balti-
more, Md., 1947.
Vishniac, H. S.: Marine mycology. Tr. New York Acad. Sci., 1955, Ser.II, 18:352.
Wolf, F. A., and Wolf, F. T.: The Fungi, Vols.I and II. John Wiley and Sons, New York,
1947.
5
S. THE VIRUSES.* A. GENERAL DISCUSSION
IN 1891, BACTERIA were the smallest, simplest and lowest forms of life
known. Physiologically and structurally they were viewed as the boundary
between the living and the, inanimate. Investigators of that time felt that they
had probed the depths of the mystery of life and discovered its extreme lower
limit with respect to size and simplicity of organization. Yet many times the
clear, colorless, and seemingly sterile fluids through which their searching
lenses swept, teemed with billions of living particles (viruses) which escaped
their vision and their knowledge. Three large groups of viruses are now known.
Plant Viruses. In 1892, Iwanowski demonstrated tliat a disease of the
tobacco plant called "tobacco mosaic" could be transmitted to healthy plants
by the sap from diseased plants after the sap had been passed through filters
of porcelain so fine as to remove all bacteria. No living thing grew from the
sap of diseased plarits on any culture media in the laboratory and nothing
could be seen in the crystal-clear fluid with a microscope. But we now know
that the sap from the diseased plant contained millions or billions of particles
of the virus of tobacco mosaic; the first-known viral disease of plants. Iwan-
owski had opened the door to the world of the ultramicroscopic, much as
Leeuwenhoek's discoveries had opened the door to the world of the micro-
scopic. Nearly 200 viral diseases of plants are now known.
Bacterial Viruses. ' The group of viruses which attack bacteria was first
described in 1915 byithe British scientist Twort and more fully studied about
1917 by the French investigator, d'Herelle. d'Herel1e named these viruses
bacteriophage. t Bacteriophage is one of the most interesting and important
living organisms, as will be seen farther on in this discussion.
I
• The word virus is derived from a Latin root meaning a slimy, poisonous liquid (e.g.,
snake venom). Today our meaning of the word virus is somewhat different but not much
more specific.
t The word bacteriophage is derived from the word bacterium and a Greek root phagein,
meaning to eat. The bacteriophage was originally thought of as eating bacteria from within
The shorter term 'phage is commonly used for bacteriophage and will so be used here.
61
Animal Viruses. In 1898, the first-known viral disease of Jowelr animals'
(foot-and-mouth disease of cattle) was discovered by Loeffler and Frosch,
while in 1900 Walter Reed, et al. discovered the .virus of yellow fever, first
known viral disease of man. Today many viral agents of disease of animals,
both vertebrate and invertebrate, are wen known.
Arthropod (Insect) Viruses. We may regard these as a subdivision of the
animal viruses. Viruses were first recognized as agents of diseases of insects
(silkworms) by B. Wahl, by von Prowazek and by Escherich (1909-1913).
Since then many viral diseases of insects have been recognized, important
among which are sacbrood of honey bees and diseases of agriculturally and
medically important insect pests. Insects are important also as vectors of
both plant and mammalian viruses.
GENERAL PROPERTIFS
With few exceptions viruses are characterized by: (1) size so minute as to
make bacteria seem enormous by comparison; (2) inability to propagate out-
side of living cells (i.e., unlike bacteria, yeasts or molds they are obligate
parasites). As a result of their minute size they cannot be seen with ordinary
microscopes. They are. however, visible by means of electron microscopes.
Their minute size also permits them to pass through clay, paper, asbesto§ or 4
porcelain filters which entirely withhold bacterial cells. Because they are obli-
gate parasites no saprophytic viruses are known but only those which produce
recognizable disease.
One of the most distinctive differential properties of all viruses is that of
host specificity. Most viruses under natural conditions. can infect only one
single species or genus of animal or plant, or a very closely similar species; for
example, polio virus infects monkeys and men but not cats. Tobacco mosaic
virus may be made to infect the related tomato or potato plants but it does not
infect any mammal, and mammalian virus does not infect any plant. Some
insect-borne viruses appear to infect both the transmitting insect and the plant
or animal host. But these relationships are highly specific and restricted.
ME11IODS OF VIROLOGY
Microscopy of Viruses. OrdiQ.aty microscopes magnify at best not much
over 1000 to 1500 diameters. This is far 'below the range of visibility of most
viruses. By the use of electron microscopes (Chapter 9) magnifications up to
25,000 diameters are possible. Electronic images (which we call eleclrono-
graphs or electron micrographs) can be enlarged ,up to 100,000 as photo-
graphs. Photographs of electronographs of several viruses are shown in this
chapter. Some jdea of the range in size and form of viruses may be gained
from a study of Figs. 5-1 to .5-10.
Some of the very largest mammalian viruses: smallpox, psittacosis ("parrot
fever" or ornithosis), lymphogranuloma venereum, are large enough to be
just visible with ordinary microscopes. They appear in tissue fluids as minute,
refractile granules, often called elementary bodies. There is controversy as to
whether these granules are actually the virus particles themselves or minute
globules of tissue protein to which the viruses are attached by absorption, or
clumps of virus particles. In dealing with mammalian viruses, even the largest
ones, this difficulty of confusion between tissue elements and actual virus
always arises. However, there is reason to think that, in several instances,
mammalian viruses have been electronographed in a fairly pure state.
Many of the plant viruses can be purified by precipitation and recrystalliza-
tion. Their purity is well established and they lend themselves well to exact
physical and chemical investigations. For example, tobacco mosaic virus, one
of the most widely studied plant viruses, occurs as needle-shaped liquid crystals
(the paracrystalline state). The crystals are doubly refractive. and possess
positive electrical birefringence. The dimensions, as determined by calcula-
tion from sedimentation rates, specific viscosity and other physical data, are
about 15 mJL by 120 to 280 mJL. Electron microscope findings give approxi-
mately the same figures. X-ray analysis yields information on molecular
arrangements, etc.
Chemistry and Physics of Viruses. Chemically, the tobacco mosaic virus
appears to consist of a central core of nucleic acid surrounded by a protective
coating of protein. The two are probably loosely combined as nucleoprotein.
Neither lipids nor carbohydrates, so common in all cellular organisms, are
present.
Nothing resembling nucleus, cytoplasm or chromosomes is known though
there certainly are definite and stable hereditary mechanisms. The virus struc-
ture appears to be non-cellular as we at present define a cell.
Egg Albumin MoleCule 9 X 3

Poliomyelitis Virus 12"
Fowl Plague Virus 90
Vaccinia Virus 225 o
Psittacosis Virus
Rickettsiae of Typhus
400
o
t=J
o
300 X 800
Micrococcus Pyogenes
900
Escherichia Coli
7000 X 1800
Human Red Blood Corpuscle

8000
Fig. 5-1. Comparati~e sizes and forms of some microorganisms. The diagram gives
only an approximate idea and is intended merely to suggest dimensional relationships. The
dimensions are stated in millimicrons (mil = 0.001 micron).
Fig. 5-2. Electronographs of representative animal viruses. A and B show the cuboidal
form and relatively large size of one of the "large" viruses (vaccinia or smallpox-vaccine
virus). The basic magnification was 24,800. C shows type I poliovirus. Note the smaller size
(in spite of magnification of 73,5(0) and different form. Each separate poliovirus particle
is about 33 mil in diameter; those in clumps about 25 mil. The particles may be spherical
or polyhedral in form. (A and B courtesy of Drs. F. P. O. Nagler and G. Rake, in J. Bact.,
vol. 55. C, from the collection of the Society of American Bacteriologists, Parke Davis and
Co., Detroit.) (The small portrait of Antonij van Leeuwenhoek is in the emblem of the
Society of American Bacteriologists.)
A good deal has been learned about animal viruses, without purifying
them, by subjecting them in vitro* to various drugs, disinfectants, heat, radia-
tions, etc., and then testing tbem in vivot to see if they have been inactivated
or have undergone mutation (heritable cbange) and tbe like. Even though
they may not be alive (or infective) they may, as Salk found concerning polio
virus, stimulate immunity in animals and serve as vaccines.
Viruses and Nucleoproteins. It is of especial importance to note the pres-
ence of nucleic acids in viruses. In all known forms of reproduction, whether
sexual or asexual, from man to bacterium, genetic material consists largely of
nucleic acids, probably loosely combined with protein as nucleoprotein.
It is therefore reasonable to assume that the nucleic acids of viruses con-
stitute their genetic principle. (A diagram of the chemical makeup of nucleo-
proteins is shown in Figure 5-5.) Thus, tobacco mosaic virus, which in
this respect exemplifies all other plant viruses, consists wholly (or almost
wholly) of genetic material. This is of especial importance when we note that
the principal, if not the only, physiological activity of viruses is self-duplica-
• In vitro is derived from the Latin word, vitrum, for glass. It is commonly used to indi-
cate experiments in the laboratory, in test tubes, not involving injection into experimental
animals.
t In vivo indicates the experimental use of live animals.
The Microscopic World 6S
lion. With few exceptions viruses appear to have no other physiological func-
tion and no metabolism. In short, most viruses behave very much as though
they are (living?) genetic material.
Animal viruses which have been studied are like sperm cells, in that they
appear to contain a little carbohydrate and perhaps some lipid (fat-like) and
other substances. * But they also, like plant viruses, consist largely, if not en-
tirely, of nucleoproteins (genetic material). t
SITE OF NUCLEIC AClOS IN VIRUSES. Observations, suggestive of the
structure of some mammalian viruses (smallpox, herpes, etc.) have been made
Fig. 5-3. A representative plant vi-

rus, the virus of tobacco mosaic disease.
Note the crystal-like structure. Compare
with bushy-stunt virus. The crystals are
probably hexagonal in cross section.
(Photo from the collection of the Society
of American Bacteriologists, courtesy of
Drs. R. C. Williams and R. W. G. Wy-
ck.off, in Science, 1945, vol. 101.)
Fig. S-4. Crystalline bushy stunt vu"uS. X 224. (Stanley, J. BioI. Chem., vol. 135.)
* As already indicated, the exact chemical composition of animal viruses cannot be too
cIrarIy stated because of the difficulty in separating them in a chemically pure state from the
blood. tissues, etc., in which they occur.
t The sperm reproductive cells of all animals contain large amounts of nucleoproteins;
some carbohydrates and lipids.
66 TIre Relationships of Microorganisms to Each Other and the Living World
Nucleoprotein
I
J
Protein 1
Nucleic acid ..... H)P04
!
Nucleoside _,
r d·ribose (RNA)
l or
/ ~-deoXyribose (DNA)
2 Purine bases: 2 Pyrimidine bases:

adenine cytosine
guanine uracil (RNA)
or
thymine (DNA)
Fig. 5-5. Diagram of the structure of nucleoproteins as shown by hydrolysis. On being
disintegrated by the action of hot acid or enzyme action, nucleoprotein first splits into a
molecule of protein and one of nucleic acid. The latter, on further acid or enzyme treatment.
yields a molecule of phosphoric acid, then a nucleoside which yields a molecule of carbo-
hydrate: d-ribose if the nucleic acid is of the so-called "plant" or cytoplasmic type (ribo-
nucleic acid or RNA), d.2-deoxyribose if the nucleic acid is of the so-called "animal" or
nucleic type (deoxyribonucleic acid or DNA). The nucleoside further splits up into two
purine bases, and two pyrimidine bases: cystosine and uracil if RNA; cystosine and thymine
if DNA.
by preparing very, very thin sections of virus-infected cells and staining by

differential methods. Briefly, it is noted in such sections that each virus particle
appears to consist of a central core of nucleic acid enclosed in a sort of memo
brane or capsule of protein. This type of structural form is also character·
istic of viruses which infect insects (polyhedral insect viruses). Bacteriophages
have a tadpole-like or sperm-like form with enlarged "head" and slender
"tail." They also appear, pretty clearly, to consist of an inner core of DNA
and an outer protective coat of protein. In each virus examined, there seems
to be an inner structure which, we have good reason to believe, is the specific,
active, self-replicating, nucleoprotein of the virus. This is protected by an
outer coating of protein which appears to be inactive, non-specific, non-self-
replicating (Figs. 5-6, 5-7, 5-8, 5-9).
It is important to note that the nucleic acid of the nucleoproteins of plant
viruses (with one main exception) is ribosenucleic acid (RNA) while animal
viruses contain either ribonucleic acid or deoxyribose nucleic acid (DNA) or
both. Bacteriophages contain DNA.
Tlssuio CULTURES ANI) V,RUSIiS
Studies of viruses which involve animals or men (as was the case when
Walter Reed used human volunteers to study the deadly yellow fever virus)
are expensive and laborious. For many purposes it is now not necessary to
nse entire animals. All that animal viruses require for growth are living animal
tissue cells.· Now, we can easily induce animal tissue cells, for example
monkey-kidney cells, to grow in test-tube cultures like bacteria. 'then we can
• Similarly, plant viruses require living plant cells, bacteriophages require Iivins bacterial
eeIls.
TM Micro6Copic World 67
infect them with virus. By examining a few of the cells with microscopes
every day we can observe the course of the infection. If the cells are damaged
we say the virus is cytopathogenic.
Fig. 5-6. IlJectronoIp'ap of a very thin cross aectioo of a kidney ceJl infected with
herpes B virus. In the cytoplasm arc seen numerous rounded virus particles (V), with central
"oore" and out« "("oat" or coats. (N m .. nuclear membrane; N = nucleus.) Similar par-
ticles are aIao found in coil nuclei as well as outside tbe tells. These particles in the tell
constitute a typical intraceDuJar inclusion. The original masniticatioo was X 36,000.
(photo courtesy of Drs. M. Reissia and 1. L. Melnick; from 1. Exp. Med., 1955, vol. 101.)
Fig. 5-7. EJec::tronoIrapbs of tobaa:o mosaic VU'US (TMVI. If IIIows the virus as
usua1Iy seen (X 78,000). These rods appear to consist of hollow, round or bclIa,onal tubes
of protein (94%) containing a thin oore of ribonucleic acid (RNA) (W"). The RNA is the
vital, ~ific, brndity-bearina part of the virus structure. It may exist as intertwininB
strands. B shows the TMV particles after treatment with a surface tension reduceut which
mooved part of the protein coat from many of the rods, revealing the RNA core (X 60,(00).
Separated, neither coat nor oore can infect. Recombined, they can infect. (Courtesy of Dr.
1L G. Hart. in Proc. ~ Acad. Sci., 1955, vol 41.)
68 The Relotionships of Microorganisms to Each Other and the UlIUrg World
Fig. 5-8. ElectronogTaphs 01" a representative insect virus (granulosis ot the variegated
cutworm). In A are seen complete virus particles, with a few bacteria to show comparative
sizes and densities. In B the outer coats of the particles have been partly destroyed by wash-
ing with Na~C03 solution, revealing the inner core in some of the particles. In C the outer
coat has been completely removed, leaving the rod-like inner cores. (From the collection
of the Society of American Bacteriologists, courtesy of Drs. E. A. Steinhaus, K. M. Hughes
and H. B. Wasser, in J. Bact., vol. 57.)
MULTIPUCATION OF VIRUSES
Nothing resembling cell fission is known among viruses. Reproduction ap-
pears to be of a very special sort. It has been most thoroughly studied in
bacteriophage because this virus is easily manipulated in vitro. We may use
this as a model of viral multiplication though the process may differ consider-
ably in some other viruses. Also, current ideas as described here vary some-
what and may alter as a result of future research. The basic ideas, though, are
probably generally applicable.
Let us consider a cell of a common, harmless. bacterial species known as
Escherichia coli. This bacterium is susceptible to infection by a certain, specific
kind of 'phage (T2) of the group of 'phages designated as the T series.·
CeU Receptors. The exterior wall of the bacterial cell has a chemical struc-
ture which we may think of at present as a sort of molecular mosaic. This is
made up of various arrangements of atomic groups or radicles (R·OH,
R-COOH, R·NH2, etc.), with various electrical charges, polar groups, etc.,
associated with them. Let us suppose that a 'phage particle, infective for that
bacterial cell, is carried close to the cell (probably by diffusion currents and
brownian movement). Certain molecular arrangements and electrical charges
at a particular site on the virust correspond exactlyt to a particular, specific,
molecular pattern on the surface of the bacterium. This specific site on the
cell is spoken of as a receptor for that 'phage. The 'phage tail attaches itself
to the specific receptor of the cell (Fig. 5-10).
Within a few seconds a digestive (pepsin-like?) mechanism in the tail of the
'phage has made an opening in the bacterial cell wall. Through this the DNA
• A series of these 'phages, which infect different varieties of E. coli, has been much used
in experimental studies. They are usually designated "a s T" T 2, T), and so on.
t In bacteriophage this site is at the tip of the tail.
t Like a mirror image or like a key to a lock.
The Micro8Coplc World 69
(genetic material) from inside the head of the 'phage is forced down through
the tail, through the opening in the cell wall and so into the bacterial cell.
The protein coating of the 'phage remains as an empty, inert shell on the
outside of the cell as a "ghost" and eventually floats away. Its mission has
been accomplished (Figs. 5-10 and 5-11).
1be Latent Period. The nucleic acid of the 'phage disappears as such,
inside the bacterial cell, for about twelve minutes. If the cells are ruptured
experimentally during this time no 'phage, as such, can be demonstrated. This
initial period, the duration of which is around twelve minutes, is quite con-
stant for any given 'phage-bacterium system. It is the first half of what is
called the latent period in viral infection of any cell. The latent period for
influenza virus is from five to nine hours. It is more or less in other virus-cell
systems.
FormatioD of New 'Phage. During the first twelve-minute period, in some
unknown manner, with incredible rapidity, tbe 'pbage enters the genetic
government of the cell. 'Phage DNA takes over the synthetic mechanisms and
causes them to synthesize, at first 'phage nucleic acid instead of bacterial
material, and then 'phage protein coatings. At the end of the latent period,
these portions begin to combine as 'phage particles. In about twelve minutes
more the essential bacterial cell contents are wholly converted into 'phages.
Fig. 5-9. Electronographs of bacteriophage (T2) of Escherichia coli. A shows the nor-
mal, tadpole-like, probably polygonal heads and tails in their usual form ( X 64,(00). B
IIbows the 'phage after being frozen and thawed once. Note one ruptured head (upper right).
The heads and tails often become separated (not shown here). The prominent caudal fibrils
ue believed to be normally twined closely around a protein core inside of the tail. Extruded,
they appear to facilitate attachment of the virus panicle to the bacterial surface and to
remove the core from inside the tail. They are nol fibers of RNA. (X 68,000.) (Counesy
of Drs. R. C. Williams and D. Frazer, in Virology, 1956, vol. 2.)
70 The ReiatiotUhips of Microorganisms *' Etu:h Other tIlId the Ulling World
Fia. '-10. Bacteriophage action on cells of Escherichia colL X 25,000. 'Phage particles
adsorbed to a bacterium by their tails. Some heads are empty because the contents have
passed through the tails into the bacterium. (Courtesy ofT.F. Anderson from Cold Spring
Harbor Symposium, vol. 18.)
Fig. '-II. Development of a virulent 'phage in a susceptiblo bacterium. At A. the 'phage

particle is seen attaching to the outer surface of the bacterium by its tail. ]n B, within a few
seconds or minutes the bacterial cell wall has been penetrated and the DNA of the 'phage
has entered the bacillus, leaving the inert protein coat on the outside of the cell. At C, about
12 minutes later, the 'phage as such is no longer demonstrable but protein coats are being
synthesized. At D, about 12 minutes later, virulent 'phage particles are demonstrable, 'phage
DNA having been synthesized by this time. At E, about 12 minutes later, the synthesis of
'phage is complete and the DOW "eviscerated" cell, an inert sac, ruptures liberating new
'phqe particles; their number is characteristic of the burst size of the particular phage
involved ("m this case 8). The whole process, from A. to E, occupies about 30 to 40 minutes.
Cell Lysis. Within about thirty minutes after attachment of the 'phage to
the outside of the bacterial cell, the cell wall ruptures and the new 'phages are
set free to begin the cycle anew. The bacterium is said to have undergone lysis.
The growth and metabolism of the 'phage have been cleverly investigated.
As shown by radioisotope methods, the virus ~ms to derive some of its
phosphorus and nitrogen from the medium in which the bacterium is growing,
but about one fifth of each of'these elements in the virus is derived from the
bacterial cell itself.
Burst Size. The number of 'phage particles formed per cell in any given
bacterium-'phage system is more or less constant. It may range around 20,
around SO, 100, or 200 or more. This number is spoken of as the hurst size
for that 'phage-cell system. In the coli-T 2 'phage system just described it is
about 200.
ResIstaDce. In preceding paragraphs we have described infection of a
bacterial cell by a virulent 'phage, resulting in lysis of the cell. However,
several other events may occur when a bacterial cell comes into contact with
a 'phage to which it is usually susceptible.
First (continuing with our E. coli-T2 system), the E. coli may, as a result of
Itome mutation, have no receptors for T 2. It is said to be a resistant strain. No
'phage T2 can enter such a cell (though some other 'phage may do so).
Second, the cell may be too old. It is important to note that only multi-
plying bacterial cells, that is, cells in a stage when their genetic material is
active and their outer coating presumably in a newly-formed condition, are
susceptible to 'phage action or any virus action. Old, dormant bacteria are
not affected.
Viruiellt aDd Temperate 'Phage. Third, the 'phage may contact the cell
and the nucleic acid may enter, yet lysis not occur because the 'phage is not
fully virulent for that cell; i.e., the virus is not able to (or for some other
reason does not) produce lysis of the particular cell. Such a 'phage would be
designated as a temperate 'phage or weak 'phage.
REDUCTION. Such a 'phage undergoes a curious change called reduction.
It quietly enters the genetic structure of the cell and remains there, apparently
as a gene.
The reduced virus often profoundly alters the heritable characteristics of
the cell so that it seems like a new species. The 'phage is duplicated just like
the bacterial DNA and is transmitted to the daughter cells when bacterial cell
fission occurs. In all respects the temperate 'phage acts as though it were a
normal part of the cell.
Prophage. It may remain there, latent for years and hundreds of millions
of generations. In this state, it is spoken of as a latent 'phage or symbiotic
'phage or prophage. Unless some peculiar influence is brought to bear, as
described under Induction, the presence of proph~ge in a species of bacteria
may never be suspected. It is essentially a permanent pact of the genetic
mechanism (genome) of the cell.
LysogeDiclty aDd Induction. A bacterial strain, in the cells of which pro-
phage exists, is said to be lysogenic. If the prophage-carrying cells of that
strain are subjected to certain influences such as ultraviolet irradiation,
H 20 2, excess vitamin C, and the like, the prophages are immediately stimu-
lated into aetivity. They multiply as new, active, virulent 'phages, causing
immediate lysis of their hosts, and liberating active, infective 'phages into the
medium. The prophages are said to have been induced. Not all prophages are
inducible. Inducibility may, like many other properties be altered by biological
mutation. Induction of prophages often occurs as a result of slight, unnoticed
influences during ordinary laboratory manipulation. Thus, cultures of bac-
teria in which lysogenicity has never been suspected may suddenly undergo
partial or complete lysis, revealing the presence of prophage in them. The
culture is discovered to be lysogenic.
Whether an viruses multiply in exactly the same manner as 'phage is not
entirely clear but available evidence indicates that they behave in a similar or
analogous manner.
Like 'phage, they all behave like transmissible mutagenic agents; all* have
only a reproductive physiology; all are wholly dependent on other living cells
for their life; all multiply only inside of those living cells, producing death
and disintegration or mutation, directing the synthetic mechanisms of the
infected cell to the making of virus substance. Animal viruses, like 'phages,
show a latent period, varying from minutes to hours in various systems.
Provirus. Phenomena among animal viruses parallelling lysogenicity are
exemplified by the virus of herpes simplex (fever blister). Nearly everyone has
suffered the indignities inflicted by this virus. Persons who have had one
attack apparently carry the virus in their tissue cells in a latent, prophage-like
or provirus state. The provirus appears to be induced readily enough by such
influences as sunburn (ultraviolet irradiation), increase in body temperature
due to other infection (fever)-hence, "fever blisters." There are other viruses
which appear to exist as inducible proviruses.
Resistance of Viruses. In general, viruses are killed in a few minutes by
temperatures like'that of pasteurizatio1). t; some even as low as 56° C. There is
at least one important exception: the virus of homologous serum jaundice
(see Chapter 45). This stands boiling for some minutes. They are highly re-
sistant to intense cold and will remain alive at _76 C for a year or longer.
0
They are readily preserved by desiccation in vacuo after rapid freezing (freeze-
drying or lyophilization). Ordinary strengths of disinfectants like phenol,
cresol, formaldehyde and halogens quickly inactivate them. Surface-active
agents like soap and some detergents appear to inactivate some viruses
rather readily in vitro, not others. Ultraviolet light is rapidly destructive to
all viruses.
Viruses and Antibiotics. In general, viral infections do not yield. to treat-
ment with antibiotics, sulfonamide drugs or other chemotherapeutic agents.
We may postulate two reasons for this. First, since viruses multiply only
inside the cell, chemotherapeutic ,drugs would not readily reach them. Second,
all chemotherapeutic drugs appear to act by interfering with certain vital,
specific, metabolic reactions in bacterial cells. Now, viruses appear to have
no such vital, specific metabolic reactions: Thus, with two or three exceptions,
there is nothing in viruses for any known chemotherapeutic agent to attack.
The two or three exceptions to this generalization are of interest. They are
the large viruses, for example, psittacosis, lymphogranuloma venereum and
others like them. These usually respond well to the so-called broad-spectrurr'
antibiotiCs (Chapter 20). These viruses, as has been noted, are large enougr.
to be just over the borderline of visibility with ordinary microscopes. Their
structure, as seen with th'e electron microscope, is more complex than that of
• With the possible exception of some of the large viruses.
t 62° C for 30 minutes.
other viruses, though not like what we ordinarily think of as a cell. Neverthe-
less, the fact that they yield to antibiotics suggests that they have some
autonomous energy-yielding metabolism. This places them as a sort of inter-
mediate form of life between the wholly dependent, degenerate, purely repro-
ductive, small viruses and the wholly self-maintaining, cellular microorgan-
isms like bacteria.
Parasitic Status of Viruses. Most bacteria can live independently of
higher plants and animals. They thrive very well in the outer world. They
are hardy and self-dependent, synthesizing their cell substance from simple
foods from the outer environment. Species of bacteria which have become
highly parasitic appear to have lost some of their rugged independence and
their resistance to the outer world, through long generations of life in a
sheltered environment such as the animal body. Here much of their food is
synthesized for them. They tend to become dependent. Such bacteria must
receive from their host in an already-synthesized state complex nutrient ma-
terials from the blood, or very complicated substances like vitamins, depend-
ing on how high a degree of parasitism (or dependence on host) they have
evolved. They do not as'a rule require, like viruses, to live only inside the host
cells. Bacteria are mainly extracellular parasites.
Advancing a step further, we may imagine that certain bacteria have under-
gone such mutations in size and metabolism that they can actually enter the
host cell and become an obligate intracellular parasite, capable of living only
there at the expense of the proteins and other essential constituents of the cell,
and losing more and more of their own synthetic and other physiological
powers. * As these powers are lost through mechanisms which induce cell
variation, size diminishes until nothing remains but a bit of substance, prob-
ably nucleoprotein in nature, ultramicroscopic in size, able to pass through
the finest filters, capable only of reproducing its kind through dominance
over the genetic mechanism by inducing the host cell to alter its own substance
to become the parasite; capable of life only if furnished with the cell-sub-
stances of some particular animal or plant to which it has become adapted.
It would, "as it were, live a borrowed life, truly the supreme summit of para-
sitism."t Such a parasite would possess, as characteristic of itself, only the
nucleoprotein transmitting the specific properties of the virus. It would re-
semble the gene (or plasmagene?) of the geneticists, independent and in-
carnate!
So viruses may be supposed to have originated through the development of
parasitism to its ultimate perfection; an evolutionary process of a highly
successful sort if We regard first-rate parasites as the goal of Nature; an in-
volutionary or degenerative trend if we regard the independent, self-support-
ing creature as the luniversal ideal. The latter, carried to its logical, non-
parasitic extreme could be only of the completely autotrophict type.
* Indeed, just such visible but extremely minute, intracellular, bacterium- and virus-like
parasites are well known and constitute an important group of organisms known as the
rickettsiae which are discussed in Chapter 7.
t Laidlaw, "Virus 'Di~eases and Viruses," 1939. By permission of The Macmillan Com-
pany. I
::: Capable of living oli exclusively inorganic matter. Many such organisms are known.
They are common in the soil.
Classification of Viruses. Classification of microorganisms is in a constant
state of change and uncertainty. Classification of viruses is still in an embry-
onic, or at most early fetal, stage.
The International Committee on Bacteriological Nomenclature appointed
a Subcommittee to deal with classification and nomenclature of viruses. There
are three subcommittees: one for animal viruses, one for plant viruses (like
tobacco mosaic) and one for bacterial viruses (bacteriophage). Work on all
three projects is incomplete so that extensive discussion of classification of
viruses, from the standpoint of proper nomenclature and taxonomy is not
warranted here. In the 1948 edition of "Bergey's Manual," the viruses are
included as the order Virales, divided into three sub-orders named Phagineae
(the bacterial virus or bacteriophage); Phytophagineae, the viruses, like to-
bacco mosaic, causing plant diseases; Zoophagineae, the viruses, like yellow
fever, causing diseases of man and animals. This classification, while having
the advantage that nomenclature is at least documented, has not been gen-
erally accepted. The International Subcommission on animal viruses tenta-
tively adopted 8 criteria for classification:
1. Morphology and method of reproduction. (These might well be sepa-
rated).
2. Chemical composition and physical properties.
3. Immunological properties.
4. Susceptibility to physical and chemical agents.
5. Natural methods of transmission.
6. Host, cell and tissue tropisms.
7. Pathology.
8. Symptomatology.
Interim names have been given some viruses, such as Poliovirus hominis
(Type I, II, III). One group, called Myxovirus, includes influenza-like viruses
(Types A and B). But so little is known for certain about the criteria used that
final classification of all viruses awaits much more research.
A convenient, provisional "classification" of the viruses of animal disease
may be based on the types of tissues or organs principally affected. Another
sort of classification could be based on mode of transmission and another on
type of disease caused. Such a list may be constructed as shown in Table 4.
Any such classifications, while convenient for purposes of'discussion, are
arbitrary and incomplete_and cannot take into consideration the fact that
viruses often become modified and cause atypical or entirely different sorts
of disease. Further, the tissue affinities of some viruses can change completely.
Finally, one is at a loss to place some viruses, such as that of mumps and of
fowl plague in the table. Further, sho,uld the large viruses, which may be
more closely rela~ed to rickettsiae, be included at all?
Mutual Interference by Viruses. It is clear that, if the specific receptors of
cells are pre-empted by one virus, another having an affinity for the same
receptors is excluded and cannot infect. This is mutual interference. The
second virus could infect were it not restricted to certain receptors for its
entry. For example, once certain plants become infected with the virus' of
tobacco mosaic they cannot be infected with a closely related variant of that
virus. If monkeys are infected with a neurotropic variant of yellow fever virus,
they are immediately resistant to the normal yellow fever virus. If rabbits are
infected with fibroma virus they are immediately wholly resistant to myxoma
virus. In simultaneous inoculations with two viruses, especially if closely
related, one pre-empts or infects; the other fails. Even inactive (irradiated)
virus of influenza will preempt the susceptible cell receptors and prevent in-
fection by live influenza virus. Viruses of canine distemper modified by passage
Table 4. A Crude Classification of Some Animal Viruses (Zoophagineae).

-
TISSUE PRINCIPALLY TYPES OF MODE OF
GROUP
AND VISIBLY AFFECTED· DISEASE CAUSED TRANSMISSION·
Dermotropic Skin; mucous mem- Various pox-like dis- Close contact; probably
branes of nose and eases (smallpox, fowl sputum, fomites
mouth pox), herpes, warts,
measles
Neurotropic Nervous tissues Poliomyelitis Feces; sputum

Rabies Bites of animals
Various encephalitides Mosquitos and other
arthropods
Pneumotropic Respiratory tract Influenza, "colds," etc. Nasal and oral dis-
, Psittacosis charges
Pneumonitis
Yiscerotropic Various internal or- Yellow fever Mosquitoes

gans Dengue Mosquitoes
Loupiiig ill Ticks
Rift Valley fever Mosquitoes
Neoplastic Various Fowl sarcoma Unknown

Fibroma of rabbits' Unknown
Myxoma of rabbits Dust, fur, contact
Fowl leukemia Unknown
Enteric Gastrointestinal tract Epidemic diarrhea, Probably by hands,

nausea and vomiting foods and objects
(?) contaminated with
Polio-like diseases feces
• Under natural conditions.
through ferrets, and of low virulence for dogs and foxes, will eliminate fully-
virulent distemper 'virus, not only if injected first, but even if given severa]
days after the virulent virus.
The va]ue of these observations in relation to the prevention and cure of
viral infections is obvious. It may be, for example, that the true effect of
Pasteur treatment for rabies lies less in development of antibodies than in
blocking susceptibie cells with a dead or attenuated virus so that the active
virus from the bite 1of a rabid animal cannot take hold.
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Rivers, T. M., Editor: Viral and Rickettsial Infections of Man. J. B. Lippincott Co., Phila-
delphia, 1952. .
Schlesinger, R. W.: Developmental stages of viruses. Ann. Rev. Microbiol., 1953, 7:83.
Smith, K. M., and Lauffer, M. A., Editors: Advances in Virus Research. Academic Press,
Inc., New York, 1953, 1954, 1955.
Society for General Microbiology Symposium No.2: The Nature of Virus Multiplication.
Fildes, Sir Paul, and van Heyningen, W. E., Editors, 1952, Cambridge University Press.
Stanley, W. M.: Virus composition and structure-25 years ago and now. Fed. Proc., 1956,
15:812.
Stent, G. S.: The multiplication of bacterial viruses. Sci. Am., 1955, 188:36.
Various Authors. Conference of the similarities and dissimilarities between viruses attacking
animals, plants and bacteria. Pasadena, Calif., California Institute of Technology, 1950.
von Magnus, H., Gear; J. H. S., and Paul, J. R.: A recent definition of poliomyelitis viruses.
Virology, 1955, 1 :185.
Weiss, E.: The nature of the psittacosis-lymphogranuloma grollp of microorganisms. Ann.
Rev. Microbiol., 1955.9:227.
6
6. THE VIRUSES. B. BACTERIOPHAGE
BECAUSE OF the ease, speed and economy with which they may be culti-
vated in living bacterial cells, their relative resistance to long storage in re-
frigerators and to general laboratory manipulation, and their distinctive
appearance and properties, the bacteriophages have been much used as
experimental models of viruses in general. Their peculiarities have also been
used for several practical purposes; for example, several 'phages are of con-
siderable industrial importance. For these reasons there are described here
some details concerning 'phages that were not given in the previous chapter
on viruses.
Bacteriophage was first described by Twort (1915) and later (1917) by
d'Herelle and its action is often called the Twort-d'Herelle phenomenon.
Bacteriophage may be found in many situations in nature where bacteria are
growing and are especially abundant in the intestine of man, animals and
insects, sewage and soil.
Isolation of 'phage. If a small amount of sewage, feces or ground-up flies
or roaches is emulsified with broth and passed through a filter fine enough to
retain bacteria, the filtrate* will often be found to contain this virus. The
bacteria may also be removed by high-speed centrifugation or by differential
heating. The 'phage may be oemonstrated in the bacteria-free fluid by adding
a drop or two to a very young, actively growing, slightly turbid, broth culture
of appropriate bacteria, such as virulent dysentery or typhoid bacilli.
The culture will be found, after a few ho~rs' incubation, to have become
nearly or entirely clear and the live bacilli in it either reduced in numbers or
entirely absent. If, now, this broth be rendered bacterium-free and a drop of
it added to another young broth culture, this in turn will clear, and filtrates
from it will induce the clearing phenomenon in other cultures. It is necessary
to use young broth cultures because 'phage does not multiply in old cells or
in any which are not actively multiplying; i.e., in any in which the genetic
mechanisms are not active. The lytict potency of the filtrates increases with
• Fluid passing through the filter.
t Dissolving or digesting.
78
each transfer, the 'phage multiplying enormously at each transfer and ac-
complishing the clearing much more rapidly and completely than at first. In
addition to the increased activity of the 'phage, it usually becomes more and
more specific for the bacterium on which it grows and less active on other
species.
If a loopful of the partly lysed (cleared) broth culture is smeared on an
agar plate, * any surviving bacteria that grow into colonies are often found
to be distinct variants from the original. This may be the effect of reduction of
'phage in some of the cells. It has been postulated that new species may origi-
nate in this way. Of course, they also may have been present all the time as
mutants in the culture, being merely revealed by the lysis of their fellows.
As pointed out in Chapter S, 'phages (and probably other viruses) can act
like living mutagens or migratory genes once they attach themselves to sus-
ceptible cells.
Transduction. In addition to entering the genetic mechanism of the bac-
terial cell, the 'phage apparently can acquire some portion of the bacterial
genetic material and transfer it to another bacterium. For example, 'phage
from a motile cell of a certain species can "infect" the cell of a closely related
but non-motile species, causing the non-motile species to become a motile
species! Biological Magic! The phenomenon of transfer of genetic material
by 'phage is called transduction. It is discussed more fully in Chapter 15.
Plaque Formation. Multiplication of 'phage may also be made evident by
smearing an agar plate with a,drop of the young, active culture before putting
in the 'phage, and then smearing another agar plate with the same culture a
few minutes after the addition of the 'phage. The first plate will show profuse
and evenly distributed normal growth in a smooth, gray sheet, unbroken
except in the most thinly seeded areas where there are separate colonies.
These will have smooth and regular edges. The second plate will show growth
dotted here and there with "pinholes" or places where/no visible growth has
occurred. These "pinholes" are called 'phage plaques. Each may be regarded
as a "colony" consisting of billions of particles of 'phage, which has destroyed
the bacteria around it (Fig. 6-1). Isolated bacterial colonies on this plate may
show crescentic irregularities in their margins, as though pieces had been
eaten out of the edges. This is due to the lytic action of the 'phage in those
,<olonies. The phenomenon of plaque formation is most easily demonstrated
with 'phage but is not restricted to the bacterial virus. It has been shown to
occur in an analogous manner with animal viruses multiplying in a layer of
animal cells. For example, cells of a rat sarcomat have been cultivated as a
layer in a flask lined with plasma clot. The growing cells settled and formed a
smootb, gray, unbroken sheet of growing cells on the bottom of the flask.
This is wholly analogous to the sheet of bacterial growth on an agar plate as
described above. Instead of 'phage, a virus capable of infecting the rat sarcoma
cells was introduced. Plaques formed in the sheet of sarcoma cells exactly as
in 'phage plaque formation. Similar plaque formation has been much used
in studies Of polio virus, using sheets of actively growing monkey-kidney cells
• A nutrient jelly surface on which bacteria can grow.

t A neoplasm related tl? cancer.
80 The ReIat~nships of Microorganisms to Each Other and the Living World
A B,
Fig. 6-1. Plaque formation by phage. The dark "holes" are plaques. A is a "small
plaque" variety of 'phage. B, a large plaque variety of 'phage. Note the irregular edges of
the plaques. Minute colonies of resistant bacteria are often found in the area of the plaque ..
'Phages often undergo mutations which can be recognized as distinctive appearances in the
size, form, margin, etc., of the plaques formed. (Courtesy of Society of American Bacteri-
ologists.)
in cultures (in place of bacteria) and polio virus (in place of 'phage). The
plaque technique has been applied to several other virus-cell systems.
Enumeration of Bacteriophage. It is obvious that the number of plaques
formed will be related to the number of 'phage particles in the 'phage-bearing
fluid. This suggests the possibility of actually counting the 'phage particles
and expressing their number (per unit volume of fluid) as 'phage titer. * This'
may be done by two methods:ln one, graded quantities of the 'phage suspen-
sion are added to constant amounts (say 5 ml) of young broth cultures of the:
susceptible bacterium. After appropri!lte,incubation, the highest dilution of
'phage suspension producing complete or perceptible clearing in the cultur~s
is noted as the titer of 'phage.
The second method depends on counting the number of plaques produced
on the surface of plates, inoculated as described above, with graded diluti<,)lls
of the 'phage suspension. A similar procedure is used to enumerate virus
particles in various other virus-cell systems; e.g., a polio-virus-monkey kidney-
cell system.
• Titer is from the French word titre, meaning a standard of value or measure. It is gen-
erally used to indicate concentration or amount per volume.
The MiCroscopic World 81
Effect of Colloidal Matter on Bacteriophage. When bacteriophage was first
discovered, it was thought that disease could be cured with it., If, for example,
persons with cholera could swallow cholera 'phage or have it injected into
them, it was thought that the 'phage would destroy the cholera vibrios in
the intestine and thus cure the disease. Similarly, it was thought that persons
suffering from boils could have anti-Micrococcus pyogenes 'phage injected, or
applied in compresses, with a resulting destruction of the micrococci and cure
of the boils. It was soon found that, unfortunately, bacteriophage will not act
well in the presence of blood, pus, fecal or any colloidal material. It is not
generally used in therapy of infections.
Varieties of Bacteriophage. Bacteriophages active against certain kinds of
bacteria have been found, but none against others.
. 'Phages active against the mold-like group of bacteria, Actinomycetales,
are rare. However, an actinophage against Streptomyces griseus, the organism
from which the antibiotic streptomycin is obtained, has been isolated. Seen by
means of electronographs some of the particles of this virus appear to have
two tails each. The 'phage_attacks the Streptomyces growing in the industrial
culture vats used for production of streptomycin and can be an expensive
nuisance.
Another industrially important 'phage is one active against the bacteria
(Streptococcus lactis and related species) which are used to cause souring of
,2.
Fig. 6-2. Plaques' made in monkey epithelial cells cultivated in bottles in fluid culture
medium. The cells were mixed with a suspension of virus particles and then held in place
in a thin layer by addin~ agar which then solidified. Each virus particle attacked the sur-
rounding tissue cells, causing the formation of a plaque analogous to 'phage plaques. I,
polio-virus (type 3) plaques; 2, ECHO virus (type 6) plaques. (photos courtesy of Drs. G. D.
Hsiung and J. L. Melnick; from Virology, 1955, vol. 1.)
Fig. 6-3. Use of highly specific bacteriophages to type typhoid bacilli. Separate drops
of a culture of Salmonella typhi of unknown type have each been mixed with a different type
of Salmonella typhi bacteriophage (indicated by letters) on the plate. Lysis (circular dark
area in the white growth) occurs only where 'phage type and bacillus type correspond; in
this case, type M. The lysis in the center of the plate is a control test. Closely parallel proc-
esses are used in the 'phage typing of staphylococci, Shigella, and other bacteria. (Specimen
prepared by Dr. Philip R. Edwards. Photo courtesy of U. S. Public Health Service, Com-
municable Disease Center, Atlanta, Ga.)
milk and cream for butter and cheese making, and to produce lactic acid in
other dairy products like yoghurt and buttermilk. When such a 'phage (and
they are widely distributed) gets into the creamery vats whole batches· of
valuable culture-soured dairy products are spoiled. The control of such
'phages is an important industrial problem. One method consists in selecting
genetically-resistant strains of milk-souring streptococci.
Bacteriophages have been found in nature active against a wide variety of
bacteria, including tubercle bacilli, diphtheria bacilli, certain very valuable
bacteria (Azotobacter) of the soil (a seriohs problem to agriculture), spore-
forming bacilli of the genus Bacillus, and numerous others.
There is said, by some students of the subject, to be only one bacteriophage
and that, by suitable adaptation (i.e., continuous propagation in contact with
the desired bacterial species), it can be made to attack selected, specific
species of bacteria. There is no doubt that 'phages are readily capable of
adaptation in very marked degree. Others explain this apparent adaptation
as resulting from selective growth of one kind of 'phage (though perhaps
initially present as only one mutant particle among billions of other particles).
When placed in contact with bacteria suitable to only that one mutant particle
all others are killed while that one multiplies. This is a matter stilI in dispute.
"Typing" of Bacteria with Bacteriophage. The process of adaptation (or
selective CUltivation) of bacteriophage may be carried to a degree where a
given strain of 'phage becomes so selective with respect to a single species of
bacterium, or even on a certain type or subdivision of that species, that it will
not act on any other. Thus, it will distinguish subdivisions or types among
apparently identical strains which are indistinguishable by any other means.
For example by propagating a given 'phage on a certain selected V* straint
of Salmonella typhi~ (typhoid bacillus), the 'phage becomes so specif.c for that
particular strain that, when appropriately diluted§ it will not act on any other
strain of the same species. The first highly specific typhoid 'phage of this sort
was designated as "typhoid 'phage A" and the corresponding susceptible
strain of typhoid bacilli as '''phage-type A of S. typhi." By a like process
several other 'phage types of S. typhi were discovered, and were designated by
letters A, B, C, D, E. etc. The use of such a system of 'phages to detect an
M type of S. typhi is seen in Figure 6-3.
Similar bacteriophage typing systems have been developed for several
other species of bacteria, notably Micrococcus and dysentery bacilli.
REFERENCES
Bennett, F. W., and Nelson, 'F. E.: Action of aerosols of certain virucidal agents on lactic
streptococcus bacteriophage. J. Dairy Sci., 1954, 37:840.
Brown, E. R., and Cherry, W. B.: Specific identification of BacilJus anthracis by means of
a variant bacteriophage. J. Inf. Dis., 1955, 96 :34.
Brown, E. R., Cherry, W, B., Moody, M. D .• and Gordon, M. A.: The induction of motility
in Bacillus anthracis by means of bacteriophage Iysates. J. Bac!., 1955, 69 :590.
Cherry, W. B., Davis, B. R., Edwards, P. R., and Hogan, R. B.: A simple procedure for the
identification of the genus Salmonella by means of a specific bacteriophage. J. Lab. and
Clin. Med., 1954, 44:51. /
Cooper, P. D.: A method for producing plaques in agar suspensions of animal cells. Virol-
ogy, 1955, 1 :397. •
d'Herelle, F.: The Bacteriophage and Its Behavior. Williams & Wilkins Co., Baltimore,
Md., 1926.
Dulbecco, R., and Vogt, M.: Biological properties of poliomyelitis viruses as studied by the
plaque technic. Ann. New York Acad. Sci., 1955,61 :790.
Evans, Alice C.: Inactivation of anti-streptococcus bacteriophage by animal fluids. Pub.
Health Rep., 1933,48:411.
Felix, A.: 'Phage typing of Salmonella typhimurium: its place in epidemiological and epi-
zootiological investigations. J. Gen. Micr., 1956,14:208.
Fogh, J., and Lund, R. 0.: Plaque fotmation of poliomyelitis viruses on human amnion cell
. cultures. Proc. Soc. Exp. BioI. & Med., 1955, 90:80.
1
• Varieties having a certain form of colony called "smooth!'· There are four forms of V
'phage: I, II, III and IV. ',Phages I and IV attack any V-strain of S. typhi. The highly specific
'phages are developed from II. Rough colony forms of S. typhi are spoken of in this system
as W forms. They are not susceptible to 'phage and cannot be typed. In Figure 6-3 the cen-
trallysis is by 'phages I and IV to show that a V form of S. typhi is being dealt with.
t Any designated specimen or culture, or progeny of same.
t This term, while not official in the 6th edition of Bergey's Manual, is proving more
acceptable and will probably be made official in the next edition of the manual.
§ If not diluted, the selective specificity is masked by an overwhelming action on all V
forms of typhoid bacilli.
Fusillo, M. H., Rierig, R. N., Metzger, J. F., and Ernst, K. F.: 'Phage typing antibiotic-re-
sistant staphylococci. Am. J. Pub. Health, 1954,44:317.
Granoff, A.: Plaque formation with influenza strains. Virology, 1955, 1 :252.
Hsiung, G. D., and Melnick, J. L.: Plaque formation with poliomyelitis, Coxsackie, and
Orphan (ECHO) viruses in bottle cultures of monkey epithelial cells. Virology, 1955,
1 :533.
McAllister, R. M., and Coriell, L. L.: Cultivation of human epithelial cells in tissue culture.
Proc. Soc. Exp. BioI. and Med., 1956, 91 :389.
Price, W. H.: Bacterial viruses. Ann. Rev. Microbiol., 1952,6:333.
Takemori, N., Nakano, M., and Hemmi, M.: Plaque formation with Rift Valley fever virus.
Virology, 1955, 1 :250.
Wasserman, M. M., and Saphra, I. : The use of bacteriophage in typing Salmonella cultures.
J. Bac!., 1955,69:97.
Whitehead, H. R.: Bacteriophage in cheese manufacture. Bact. Rev., 1953,17:109.
,
7. THE RICKETTSIAE
DISCOVERY
HOWARD TAYLOR RICKETTS, an American medical scientist, while
studying Rocky Mountain spotted fever in 1909, described as the causative
agents of that disease a group of microorganisms which differed from any
previously known. A year later he discovered similar organisms as the cause
of typhus * fever while working in Mexico. During the latter studies he con-
tracted the disease and died.
In 1916, H. da Rocha-Lima, a Brazilian scientist, made further observations
of the organisms described by Ricketts and named them Rickettsia in honor
of their discoverer. He also gave the name of prowazekii to the rickettsiae
associated with typhus fever, in honor of another scientist, Stanislaus von
Prowazek, of Hamburg, who had lost his life in the study of that disease.
The causative agent of louse-borne typhus fever is, therefore, called Rickettsia
prowazekii. It is the type species of the genus Rickettsia.
CHARACTERISTICS OF RICKETISIAE
The rickettsiae have some properties of viruses and also some properties
of bacteria. In size they are intermediate between the large viruses and the
bacteria. They are clearly not yeasts, molds, protozoa, PPLO or algae. Most
probably they are neither viruses nor bacteria, but an intermediate group.
(See Table 1.)
Morphology. Th;e organisms are very tiny, having diameters of about 0.3
II. and lengths seldom exceeding 2 J.I and often less than this. However, they
are larger than the large viruses and can readily be seen with ordinary micro-
scopes. They appear to mUltiply by fission like other cellular organisms, but
this is not certain. They are variously shaped, like minute bacilli, cocci, or
• Do not confuse typhus fever, a blood and tissue disease due to rickettsiae, with typhoid
fever, an intestinal infection due to a bacterium, Salmonella typhi. The name of typhoid
fever was given it by a' French physician contemporary with Marie Antoinette (Dr. Pierre
Louis) because the stuporous symptoms of typhoid in some ways resemble those of typhus.
The word typhus means stuporous or drowsy.
8S
86 The Relationship' of Microorganirms to Each Other and the Living Worla
..
Fig. 7-1. Epithelial cell showing

rickettsiae in the cytoplasm. Typical
rickettsiae are indicated by arrows.
(About X 3000.)
Fig. 7-2. Electronographs of various

representative rickettsiae. Note the bac-
terium-like form and structure; with cell
wall, cytoplasm with intracellular gran-
ules, and what appears to be binary fis-
sion. Note also the small size as com-
pared with bacteria. 1, R. mooseri; 2, R.
(Coxiella) burneti; 3, R. rickettsii; 4, R.
prowazekii. From yolk-sac cultures.
(From the collection of the Society of
American Bacteriologists, courtesy of
Drs. H. Plotz, J. E. Smadel, T. F. Ander-
son, and L. A. Chambers, in J. Exp.
Med., vol. 77.)
diplococci- (Figs. 7-1 and 7-2). Sometimes relatively long filaments are
formed. No sporest are produced. The organisms are not motile.
Unlike viruses, rickettsiae are not filtrable: and in this respect resemble
bacteria.
Unlike bacteria, it is difficult to stain them with ordinary basic aniline dyes.
They can, however, be colored with Giemsa's stain.§ The rod forms tend to
stain more intensely at the tips, often giving short rods the appearance of a
pair of diplococci.
• When cocci occur in pairs, they are called diplo-cocci.
t Spores are dormant, seed-like bodies produced by some species of bacteria and hiaher
*
fungi.
With the single exception of Rickettsia bumeti, the cause of Q fever.
t A special stain used for blood and protozoa.
Photographic enlargements of electronographs, and specially designed
light microscopes (phase microscopes, see Chapt. 9) indicate that rickettsiae
have internal structures suggesting the existence of nuclei or nucleus-like
mechanisms. They also appear to contain both ribonucleic acid and deoxy-
ribonucleic acid, components of cytoplasm and nuclei, respectively (Fig. 7-2).
Growth. In the matter of growth, rickettsiae resemble the viruses; i.e.,
they are not cultivable on non-living material. However, rickettsiae are easily
cultivated in live chick embryos and in living tissue-cultures like those used to
cultivate viruses (see Chapt. 13). They appear to grow best in the live cells
lining the egg-yolk sac. Yolk-grown rickettsiae are widely used in procedures
for the diagnosis of rickettsial diseases and for the preparation of rickettsial
vaccines.
Rickettsiae and Antibiotics. Some rickettsiae have been shown to have a
definite, autonomous, metabolic activity, although they have not been culti-
vated on lifeless media. This probably explains why they are markedly suscep-
tible to antibiotics, since antibiotics, as already noted, act by interfering with
certain essential metabolic processes of living cells. The large viruses, as previ-
ously indicated, are also susceptible to antibiotics, and appear to have some
independent metabolic functions and some structures like rickettsiae. Some
authorities would classify the large viruses with the rickettsiae.
Table 5. Some Typical Rickettsial Diseases.
DISEASE RICKETTSIAE
I ARlliROPOD VECTOR MAMMAUAN HOST
Rocky Mountain R. rickettsii Dog tick (Dermacentor Rabbits, dogs, sheep;

spotted fever variabilis); Rabbit tick (Man)·
(Dermacentor anderson;)
Murine (endemic) R. mooseri Rat flea (Xenopsylla che- House, barn and sewer
typhus opis) rats; (Man)·
Classical (epidemic) R. prOl"azekii Body louse (Pediculus cor- Man

typhus paris)
Tsutsugamushi (Jap- R . orientalis Harvest mite (Trombicula Field mice and other
anese, or Oriental akamushi) small rodents; (Man)·
swamp or river
fever}
---- -. ---_ - - ------- ----"
~--.
Rickettsialpox R. akari Mouse mite (Alloderman- House mouse; (Man)·

yssus sanguineus)
- _------
Brill's disease (re- R. prowazekii Body louse Man
crudescent ty-
phus)t
- _.
Q fever R. burneti Ticks, milk, dust Cattle; (Man)·
• The human mammalian host is placed in parentheses (except in classical typhus) because
he is not the usual or natural host but usually a chance or accidental victim of the vector
insect.
t Represents reactivation of an old infection, the organisms having remained in the body
in a quiescent state, sometimes for several decades.
Habitat. The rickettsiae are obligate, intracellular parasites, like viruses.
They characteristically inhabit the cells lining the intestines and other tissues
of insects, both bloodsucking and non-bloodsucking. Rickettsiae not patho-
genic for man have been found in ticks, fleas, lice, bedbugs, spiders and mos-
quitoes. Human pathogenic species of rickettsiae primarily inhabit insects
which bite man or animals or both. Many pathogenic rickettsiae are found
only in the salivary glands of the insects, whence they may be transmitted to
man. However, they sometimes occur in the intestinal contents of bloodsuck-
ing insects. They therefore appear in the feces. Transmission of rickettsiae to
animal hosts may thus be obtained by rubbing or scratching the fecal deposits
of insect vectors into the skin.
RICKETTSIAL DISEASES
Some of the principal diseases caused by rickettsiae are shown in Table 5.
As seen in this table they are primarily diseases of lower animals. Some of the
rickettsiae contain potent poisons (toxins) .
. REFERENCES
Bell, E. J., and Philip, C. B.: The human rickettsioses. Ann. Rev. Microbiol., 1952,6:91.
Bovamick, M. R.: Rickettsiae. Sci. Am., 1955.192:74.
Buddingh, G. J.: Chick embryo techniques. In Rivers', T. M., Viral and Rickettsial Infec-
York Acad. Sci., 1952,55:236.
Hopps, H. E., et al.: .Metabolic studies of rickettsiae. J. Bact., 1956, 71 :708.
Neva, F. A., and Snyder, J. C.: Studies on the toxicity of typhus rickettsiae. J. Inf. Dis.,
1955, 97:73.
Price, W. H., Johnson, J. W., Emerson, H., and Preston, C.: Rickettsial-interference phe-
nomenon: a new protective mechanism. Science, 1954,120:457.
Rivers, T. M., et al.: Viral and Rickettsial Infections of Man. 2nd ed. J. B. Lippincott Co.,
Philadelphia, 1952.
Smadel, J. E., et al.: Symposium on viral and rickettsial diseases. Bact. Rev., 1950,14:195.
Zinsser, H.: Rats, Lice and History. Little, Brown and Co., and The Atlantic Monthly, Bos-
ton, Mass., 1935.
!
8
8. THE PLEUROPNEUMONIA AND PLEUROPNEUMONIA-LIKE

ORGANISMS
PRINCIPAL GROUPS
Pleuropneumonia Organisms. In 1898 Nocard and Roux, French scientists,
discovered, in pleural * fluids from cattle with a disease called infectious
pleuropneumonia, filtrable organisms of the same range of magnitude as the
larg~r, rickettsia-like viruses, being just within the range of visibility of ordi-
nary, light microscopes. However, the pleuropneumonia organisms differ
from all viruses (as well as from rickettsia) in being cultivable on lifeless media
(Table 6). On agar ~ultures minute, round granules, not unlike the elementary
bodies of the large viruses, are seen in colonies. Long, mold-like but very thin
filaments, and stellate structures are also formed by some species. Often they
are spherical or ovoid. It is clear that an outstanding,character of these organ-
isms is pleomorphism (Figs. 8-1 and 8-2). Borrel (for whom the genus of
Borrelia is named) called the species discovered by Nocard and Roux Astero-
coccus mycoides, meaning "rounded and stellate forms with radial, mold-like
filaments." These organisms are commonly called pleuropneumonia organisms,
which we may, for convenience in this discussion, abbreviate to PPO.
Pleuropneumonia-like Organisms. After the original description of the
bovme pleuropneumonia organisms, similar species were found in various
other animals: sheep, goats, dogs, rats, mice, human beings. They are as-
sociated with various pathologic conditions, especially rheumatic or arthritic
diseases, infections of the mammary glands, respiratory tract and adjacent
tissues, and inflammations of the genito-urinary system. In addition, one
group of similar organisms was found growing as saprophytes in decaying
organic matter. All of these organisms are now referred to as pleuropneu-
monia-like organisms, commonly called PPLO.
L Forms. A grbup of organisms resembling PPO and PPLO in several
respects, and usually included with them, are often called L bodies or Lforms.
There appear to be three distinct types, Lt. L3 and L 4 •t The L bodies seem to
* Surrounding the Idngs.
t Some authors have called certain PPLO "L2," "L3." "14." causing some confusion in
the literature.
89
90 The Reilltionship! of Mic,oorgani!fIU to Each Othe, and the Living Wo,1d
A
B
Fig. 8-1. EIectronographs of pleuropneumonia organisms. A shows characteristic
branching mycelium with terminal nodes and endomycelia) condensations of protoplasm.
(Photo from collection of Soc. Am. Bacteriologists, courtesy Dr. E. A. Freundt, in Acta
Path. et Micr. Scand., 1952, 31: 508.) B shows very yOUDg growth, on solid medium, of a
saprophytic species (Laidlaw). These show the small filtrable elements (MRU). (photo
courtesy of Drs. E. Klineberger-Nobel and F. Cuckow, Lister lnst. Prevo Med., London.)
be derived from, and to be closely related to, certain well-recognized species

of bacteria.
The term PPLO is often used (as a matter of convenience) to designate all
of these organisms: PPO, PPLO, and L forms.
General Properties. The PPO, PPLO and L forms are clearly neither
I yeasts nor molds; neither protozoa nor algae. They are obviously neither
viruses nor rickettsiae. With the possible exception of the L forms, which
appear to represent stages in the developmental cycle of certain bacteria, they
cannot be classified as bacteria. They possess some of the properties of all of
these groups yet not all of the properties of any. Their exact position in the
organic system is anomalous but seems to be near the bacteria. Because long,
filamentous structures have been described in the developmental cycle of the .
PPO and PPLO they have often been considered as belonging to the group of
mold-like bacteria, the Actinomycetales. Their classification is still under dis-
cussion. It bas been suggested that they be placed in a separate order, the
7JIe Mkroscopk ",orld 91
Mycoplasmatales, family Mycoplasmataceae, genus Mycoplasma. This is
probably not the end of the argument!
The distinctive characters of these three groups of organisms may be
tabulated, and compared with those of some other microorganisms, as shown
in Table 6.
Cultivation. A very favorable medium for all of these organisms is beef-
heart infusion broth, pH about 8.0, containing about 2S per cent animal
serum or certain lipoprotein derivatives of serum. Growth occurs best on solid
media. Medium containing less than I S per cent serum will rarely support
growth of PPO or PPLO but may support bacterial L forms.
Important contributions in this field are media for PPLO of completely
known composition, such as one containing no serum, made up of a solution
of glucose, several minerals like KH 2P04 and FeS04, with I S amino acids
and a vitamin. · Another medium consists of a lipoprotein factor from serum,
9 amino acids, several vitamins, inorganic salts and several other pure sub-
stances of known composition. Agar is generally used for solidification. Tin
ions appear to be requisite. Incubation is at 37° C for 2 to 10 days or more.
The atmosphere in the plates must be saturated with moisture. PPO and
PPLO are facultative with regard to oxygen. Most L forms also appear to be
so.
Quantitative methods for determining rate and amount of growth are based
on (a) measurements of turbidity: (b) colony d iameters; and (c) enumeration
of colonies.
SELECflVE CULTIVATION. Most PPO and PPLO are markedly resistant to a
number of substances which are completely inhibitory to 'all bacteria or cer-
tain large groups of them. For example, thallium acetate, sulfonamide drugs,
penicillin and other antibiotics, and certain antibacterial aniline dyes are
often used to suppress growth of extraneous bacteria when isolation of PPLO
Fig. 8-Z. One form of PPW. This strain was isolated from a case of uret hritis in a
human being by Dr. L. Dienes. Cultivated on agar medium as described in text. (X 20,000.)
Note the relatively large, thicken«! granule in the larger organism. These are frequently
seen. Then: is no evidence of a definite cen wall. (Counesy of Drs. H. E. Morton, J. G .
Leece, J. J. Oskay and N. H. Coy, in J. Bact., 1954, vol. 68.)
• Media of completely known and reproducible composition are callcd synthetic media.
92 The Relationships of Microorganisms to Eaclr Otlrer and tire Living World
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TIle Microscopic World 93
is attempted from materials heavily contaminated with ordinary bacteria. In
this respect the PPO and PPLO resemble the small viruses. Yet it has been
demonstrated that they have a definite, autonomous metabolism, in this
resembling the larger viruses. L forms are also highly resistant to penicillin
and some other antibacterial agents but not to all antibiotics.
PPO and PPLO are also readily cultivable in living chick embryos. like
bacteria, viruses and rickettsiae, and in media containing aqueous extracts of
finely divided, whole chick embryo instead of serum. They sometimes con-
taminate biological materials like cultures of human tissue cells.
CoLONIES (ALL FORMS)

Minute colonies, so small as usually to be invisible to the naked eye and
visible only with a hand lens or low power of the microscope. appear along
the lines of inoculation on solid media (Fig. 8-3). These true colonies must
not be confused with similar-looking structures ("pseudocolonies") com-
posed of spherocrystals and other aggregations of lifeless material which
sometimes appear on the surface even of sterile serum-agar plates (Fig. ~).
These occur as a result of various physicochemical factors acting locally,
including scratching the surface of the agar with a needle. They have no
relation to the inoculum. The genuine colonies are definite in outline and
slightly raised and have a dark, vacuolated or granular central portion. The
central density of colonies on solid medium is due to growth of the organisms
below, as well as on, the surface of the agar. The central portion above the
surface contains well-defined elementary bodies which may be stained in situ.
A B
Fig. 8-3. Colonies of pleuropneumonia-like organisms (L bodies) on 20 per cent horse-
serum dextrose-starch agar. (48 hours at 37° C) (X 70). Note the minute size and granular
structure. A and B are from different cases of rat-bite fever, a disease caused by Streptobo-
eilllU monili/ormls. (Brown and Nunemaker, BuUetin of the Johns Hopkins Hosp., vol. 70.)
Fig. 8-4. A, Pseudo-colonies in 30 per cent rabbit-serum agar incubated 2 weeks at

37° C (X lIS). B, similar medium incubated 6 weeks. Both media were completely sterile.
(Brown, Swift and Watson, J. Bact., vol. 40.)
Certain very tiny colony types, which appear to grow only beneath the surface,
are called T-form colonies.
PROPAGATION. For propagation, a small agar block containing the de-
sired colony is carefully cut out of a agar plate. It may be used to inoculate an
appropriate fluid medium such as one of the synthetic media mentioned
above. It is usually more satisfactory to inoculate agar on the same or another
plate by sliding the piece of colony-containing agar, inverted, over the surface
of the fresh medium.
Staining and Morphology. Like most microorganisms, the minute, color-
-less and transparent PPO, PPLO and L forms can be seen much more readily
if they are stained with an aniline dye. A good method of staining these or-
ganisms is to cut out a block of agar on which the colonies are growing and
place it upon a slide. Then invert upon it a coverslip on which is dried methyl-
ene blue-azure (deposited from alcoholic solution). The microscope is focused
on the coverslip.
The elementary bodies in the central part of the colonies are seen to be
minute, round or elongated forms ranging in diameter from around O.()(») J.I
to 7J.1. The smallest are about 1/1000 the diameter of bacteria. But this is much
larger than many viruses. They may also be ring-shaped or sometimes irregular
or bacillary, but always exceedingly minute.
Life Cycle. The fundamental units of growth in the colonies are the tiny
elementary bodies. They are sometimes spoken of as minimal reproductive
units (MRU). Electron micrography has yielded much of interest in studies of
/ these minute organisms.
(a) PPO AND PPLO. In these organisms the MRU enlarge to form what
are called "large bodies." Many small, new, elementary bodies develop inside.
These are readily liberated by rupture of the large body since there appears
to be no definite, rigid cell wall as in bacteria. The new organisms repeat the
reproduction cycle. There are some variations from this simple theme but
mostly they concern size and shape of the elements in the cycle. Sometimes
the large bodies develop bud-like protrusions or filamentous extensions
which later break up into, or liberate, small, new MRU.
(b) L BoDIES. In bacteria with L forms, a basically similar cycle occurs.
7716 Microscopic World 95
The bacterial cell swells up and forms a "large body." Inside, there develop
many L forms, much like the MRU mentioned above. They are liberated,
and grow as MRU in typical, PPLO-type colonies. They may also grow
directly into bacterial cells.
In developing L forms bacterial cells develop irregular protusions and
undergo obscure internal developments. The cell eventually breaks apart into
minute bacillary forms if the cell was a rod; spheres if it was a coccus. Some
of these developmental cycles have been followed in single, individual cells
by means of time-lapse, phase microphotography and appear to be fairly
definite. (See Figs. 8-5 and 8-6.)
L bodies are not motile and flagella have not been seen even though the
L bodies may have been derived from a motile bacterium. Internal structure
of L bodies is obscure.
Occurrence of L Bodies. L bodies have been demonstrated in numerous
species of bacteria, bacillary as well as coccal in form. They occur notably in
Proteus species, Bacteroides, Escherichia, hemolytic streptococci, Pasteurella,
Neisseria. Streptobacillus moniliformis (also called 'Murimyces streptobacilli-
Pia. S-S. Formation and development of L forms from a common species of bacterium
mirabiJis). Similar to Figure 8-6 but showing the reverse process. Note especially
in organisms marked by arrows, which produce L forms by budding. Most others
internal granules (possibly elements like MRU?) and finally develop into
Growth is on agar containing 1000 units of penicillin per ml. Intervals
inoc:ula:tion of agar arc (Min.): A, 30; B, 165; C, 420; 0, 540; E, 1775. (X 1188.)
of Miss Lilybett Valentin and Dr. M. A. Mcdill, Dept. of Botany, Univ. of Penn-
Unpublished.)
96 TM Relationships of Microorganisms to Each Other and the Linng World
Fig. 8-6. Reversion of L forms of a common bacterium (Proteus mirabilis) into rod
forms. The series of microphotographs of the same L forms (taken by time-lapse motion
picture on Cine Kodak XX film using phase microscopy, Chapt. 9) shows successive stases
in the process. Intervals after inoculation of agar are (Min.): upper left, 30; upper right,
109; lower left, 160; lower right, 186; center, 225. (About X2400.) (Courtesy of Drs. M. A.
Medill and W. O. Hutchinson, in J. Bact., 1954, vol. 68.)
moniliformis, Haverhillia multiformis and other names) and others. These are
all common bacterial species, discussed farther on. In many of these bacteria
the L bodies appear to develop as a response to certain unfavorable agents,
especially penicillin and increased salt content of the culture medium. They
grow on the same media and under the same cultural conditions as the parent
bacteria, and are antigenically (chemically) similar to the parent bacteria.
Relatioos of PPO, PPLO and L Forms. Because of these curious similar-
ities it seems permissible to regard PPO, PPLO and L forms as closely re-
lated. L forms may provisionally be regarded as stages in the developmental
cycle of recognized species of bacteria. PPO and PPLO may be thought of as
representing similar developmental cycles in which, due possibly to a higher
I degree of parasitic evolution, the clearly bacterial stage has disappeared or
become rudimentary.
Streptobacillus MooDiformis. One of the most interesting of the L organ-
isms is generally referred to as "L .." following the nomenclature of Kliene-
berger who first described it in 1935. It is present in nearly all cultures of the
very pleomorphic, filamentous bacterium usually called Streptobacillus
moniliformis.
S. moniliformis and the L, organisms grow well under the same conditions
as other L forms. S. moniliformis may be obtained from pus of the ears of
rats where it frequently causes otitis media, and from the blood of human
7TIe Microscopic World 97
beings suffering from rat-bite fever, one type of which it also causes. It grows
on meat-infusion media, especially with blood 'or serum added. The colonies,
superficially examined, are much like those of ordinary bacteria. Smears
show bacillus-like rods and also filaments in which branching is common.
Swellings. globules and granulations of the cells are a prominent feature as
the colonies age.
After several days of incubation of the colonies of S. moniliformis, dense
areas appear under them. By washing away the earlier growth of S. monili-
formis with broth, the denser portions are found to remain adherent to the
agar. These are the growths of the Ll elements. The agar containing these
elements may be cut out, macerated in broth and used to inoculate new agar
or broth, as described earlier in this chapter. On agar media the organisms
retain their Ll form; in broth the filamentous growth of S. moniliformis
reappears.
The elementary granules can pass bacteria-retaining filters, and S. monili-
formis colonies will develop from such filtrates.
REFERENCES
Abrams, R. Y.: A method for the cultivation of L forms in liquid media. J. Bact., 1955,
70:251.
Allbritten, F. F., Sheely, R. F., and Jeffers, W. A.: HaverhiJIia multiformis septicemia.
J.A.M.A., 1940, 114:2360.
Carter, G. R.: Pleuropneumonia-like organisms isolated from bronchopneumonia of cattle.
Science, 1954, 120:113.
Cuckow, F. W., and KJieneberger-Nobel, E.: Further studies of organisms of the pleuro-
pneumonia group by electron microscopy. J. Gen. Micr., 1955,13:149.
Dienes, L.: Isolation of L type cultures from Bacteroides with the aid of penicillin and their
reversion into the usual bacilli. J. Bact., 1948,56:445.
Dienes, L., and Sharp, J. T.: The role of high electrolyte concentration in the production
and growth of L forms of bacteria. J. Bact., 1956, 71 :20&.
Dienes, L., and Weinberger, H. J.: The L forms of bacteria. Bact. Rev., 1951,15:245.
Edward, D. G., and Freundt, E. A.: The classification and nomenclature of organisms of
the pleuropneumonia group. J. Gen. Micr., 1956,14:197.
Heilman, F. R.: A study of Asterococcus muris (Streptobacillus moniliformis). I and II.
J. Infect. Dis., 1941, 69 :32 and 45.
Klieneberger-Nobel, E.: Filtrable forms of bacteria. Bact. Rev., 1951,15:77.
Medill, M. A., and Hutchinson, W. G.: The reversion of the L form of Proteus mirabilis
into the rod form. J. Bact., 1954,68:89.
Medill, M. A., and O'Kane, D. J.: A synthetic medium for the L type colonies of Proteus.
J. Bact., 1954,68:530.
Morton, H. E., Leece, J. G., Oskay, J. J., arid Coy, N. H. : Electron microscope studies of
pleuropneumonia-like organisms isolated from man and chickens. J. Bact., 1954,68:
697.
Morton, H. E., Smith, P. F., and Keller, R.: Prevalence of pleuropneumonia-like organisms
and the evaluation of media and methods for their isolation from clinical material.
Am. J. Pub. Health, 1952,42:913.
R.obinson, L. B., Wichelhausen, R. H., and Roizman, B.; Contamination of human cell
cultures by pleuropneumonia-like organisms. Science, 1956, 124:1147.
Sabin, A. B.: The filtrable microorganisms of the pleuropneumonia group. Bact. Rev., 1941,
$:1,331.
Shepard, M. C.; T -form colonies of pleuropneumonia-like organisms. J. Bact., 1956, 71 :362.
Smith, P. P.: Amino acid metabolism by pleuropneumonia-like organisms. J. Bact., 19S5,
7O:S52.
Smith, P. F.; Synthetic media for pleuropneumonia-like organisms. Proc. Soc. Exp. BioI. '"
Med., 1955,88:628.
Smith, P. F.: Quantitative measurement of the growth of pleuropneumonia-like organisms.
Appl. Micr., 1956,4:254.
Smith, W. E., Mudd, S., and Hillier, J.: L-type variation and bacterial reproduction by large
bodies as seen in electron micrographic studies of BacteroIdes funduliformis. J. Bact.,
1948,56:603.
Idem: Electron micrograph studies of two strains of pleuropneumonia-like (L) organisms
of human derivation. Ibid., p. 589.
Stempen, H.: Demonstration of a cell wall in the large bodies of Proteus vulgaris. J. Bact.,
1955,70:177.
Turner, A. W.: Study of morphology and life cycles of the organism of pleuropneumonia
contagiosa bovis. J. Path, and Bact., 1935. 41 :25.
Warren, J.: Observations on some biological characteristics of organisms of the pleuropneu-
monia group. J. Bact., 1942,43:211.
Wittler, R. G., Cary, S. G., and Lindberg, R. B.: Reversion of a pleuropneumonia-like
organism to a Corynebactenum during tissue culture passage. J. Gen. Micr., 1956,
14:763.
Yamamoto, R., Adler, H. E., and Cordy, D. R.: The propagation of a virulent goat pleuro-
pneumonia-like organism in the chick embryo. J. Bact., 1955,69:472.
SECTION 2
Methods and Phenomena
of Microbiology
SECTION 1 of this book gave a synoptic view of microorganisms in general.

The student learned about the size, structure, activities, and particularly the
major properties which distinguish microorganisms of each of eight great
groups from one another. In this section more details are given. The student
acquires a working knowledge of microbiology. He learns about microscopic
methods, more of the structural peculiarities, how one handles microorgan-
isms in the laboratory, how they grow, their biochemical chanlcters, physi-
ology, etc. Bacteria are used as the basis for most of this discussion because
they have been very thoroughly studied and because they are easily manipu-
lated and controlled in the laboratory. What is true of bacteria is, in general,
true of most other living cells, with modifications r{ecessitated by environ-
ments of various kinds. Where contrasts occur as, for example, between
bacteria and algae or protozoa or viruses, these are pointed out. All of this
infotmati.on i'i> of \)a'i»c m).'i'>ot\"'nc~ to \h~ 'i>\'Ildent o~ micto\)io\og'_j, t~gatd\e'i>'i>
of his anticipated field of specialization. The student thoroughly familiar with
the contents of sections 1 and 2 will have little trouble with the remainder of
the book and should find the subject of growing interest. The more proficient
we become in any game or intellectual activity, the more pleasurable we find
it; even chess. bridge, or ice hockey!
99
9
Optical Methods In Microbiology
LIGHT MICROSCOPY
FOR MAKING "enlarged" images, simple lenses like "magnifiers" or
"reading glasses" have been in use for centuries, especially in ancient China.
These are forms of simple microscopes. Such simple magnifiers had definite
limitations due to their crude construction. About 1590 a Dutch spectacle
maker, Zacharias Janssen, used a second lens to magnify the image produced
by a primary le,ns. This is the basic principle of the compound microscope
used by every microbiologist today. Galileo invented an improved compound
microscope in 1610. In attempts to see still more, better microscopes have
been built, until today Wee possess complicated, compound instruments which
give magnifications up' to 3000 diameters; ten times that of the ancient,
simple instruments. (Fig. 9-1.)
THE COMPOUND MICROSCOPE
General Description. A complete description of the optics of compound
microscopes will be found in any good, modern, college textbook of physics.
As shown in Figure 9-2, light is reflected upward by the adjustable mirror at
the foot of the instrument. The light is refracted in the condenser, just above,
so as to bring the rays to a central point with as little diversion from the
vertical (and consequent loss) as possible at the plane of the object. The con-
. denser is equipped with an iris diaphragm which is used to eliminate excessive
light and reduce the aperture of the condenser for various lenses. Usually the
light rays emerge from the top side of the condenser as a cone of light with
the apex downward. The rays which are not too divergent pass through the
object. If they do riot diverge at too wide an angle they enter the first and
most "powerful" le~s of the microscope: the objective. The wider the scope
(or numerical aperture or N.A.) of this lens (or the wider the angle it can
survey) the more o~ the divergent rays (those at the periphery of the cone of
rays) it can bring ini*
I
* A good camera leris, for example, is a wide-angle lens, and brings in much light (it is
"fast"). Such lenses have N.A. of around 2.5 and a relatively short focal length: It to 2 or
3 inches. An oil-immetsion objective lens has a focal length of only a few millimeters and
a N.A. of around 0.9. it is a very "short-sighted" lens with limited N.A. and works only a
millimeter or two fron'! the object in order to get as many of the divergent rays from the
object within its narrow scope as possible.
101
102 Method8 and Pheno_1UI of Microbiology
f
Fig. 9--1. Method of using a high-power microscope. 1bis is a binocular instrument
(B and L TBV-8) but. except for two eyepieces, is constructed fundamentally like a monocu-
lar instrument. Note the natural position of the head, lack of facial strain, position of the
hand on the coarse adjustment and the location of the slide under the low-power lens. (Cour-
tesy Bausch and Lomb Optical Co., Rochester, New York.)
Oil Immersion. As the rays of light emerge from the upper surface of the
condenser, some (especially peripheral rays) are refracted beyond the scope of
the objective and lost. Others are reflected away from the underside of the
glass slide on which the object is mounted, and lost. Others are refracted from
its upper surface. Others are lost by refraction and reflection in the object,
/ and at the surface of the objective lens. A considerable part of these various
losses and distortion of the light rays can be prevented by eliminating the
optical effect of most of these surfaces. This is done by placing a clear, color-
less fluid (immersion oil), having the same refractive index as glass, between
condenser and slide and between slide and objective lens; hence the use of
oil immersion lenses as objectives for high-power microscopy. Immersion oil,
in effect, can increase the N.A. of a lens because it brings in more light rays.
Several lenses, close above the objective, are used to correct difficulties in-
herent in such objectives. namely, spherical and chromatic aberration (dis-
tortions of image due to lens curvature, etc.).
Real ud Virtual Image. ThereaJ image produced by the objective is brought
Optical Mf!thods in Microbiology 103
-------------------------------
•
J>
.: L
Fig. 9-2. Chart showing path of light through laboratory microscope. (Courtesy Bausch
& Lomb Optical Co., Rochester, N. Y.)
The entering pencils of light (1, 2, 3) are reflected upward by the mirror through the
diaphragm (CD) opening. In passing through the condenser lenses they are focussed on
the object (01) on a glass slide. Passing through the object they are refracted by the lenses
in the objectil'C. They are brought to a focus at, and then diverge from, the upper focal
plane of the objective (Ft). Refracted by the lower lens of the eyepiece they form a series
of real and virtual images (03, 02) in the eyepiece focal plane (F2) and eyepiece aperture.
These are magnified by the upper lens of the eyepiece and focussed at the eyepoint (EP).
The object on the glass slide is then seen by the eye as an enlarged, virtual image (04) which
seems to be at a level a little below the condenser.
104 Methods and Phenomena of Microbiology
Fig. 9-3. Hanging drop preparation. The size of the oil droplets is exaggerated.
to a focus within the eyepiece. This is a system of 2 or 3 lenses near the top of
the microscope. This real image is magnified by the topmost lens (ocular) and
appears as a greatly enlarged virtual image, seeming to be projected to a posi-
tion just above the reflecting mirror. .
Resolving Power. The real measure of a microscope is its resolving power.
This is based on the fact that the sensory endings (receptors) of the optic nerve
in the retina of the eye have dImensions of several JL. If light rays from two
tiny objects, very close together (say 0.2 JL) strike one of these retinal receptors,
the image conveyed by the optic nerve will be that of only one object. The
two objects blur together. The unaided eye does not perceive that the objects
are separate; i.e., it does not resolve them. ,
By magnification, resolution is increased but, as we shall see later, there are
definite limitations to resolution by ordinary microscopes (around X 1200)
due to the nature of visible light rays. An oil immersion objective lens magni-
fying the object about 90 to 100 diameters is generally used. The ocular or
eyepiece should magnify the real image, formed by the objective, 10 to 12.5
diameters. The total magnification of between 900 and 1125 diameters thus
obtained is most useful, higher magnifications tending to give "fuzzy" out-
lines due in part to poor resolution.
"Hanging Drop" Preparations. Bacteria in a natural, living state are best
viewed when suspended in a clear fluid of some sort; usually water, saline
solution or broth. The cells are transparent, colorless and refractile and so
tiny that they are often difficult.to find and even to identify as bacteria in the'
drop of fluid. This is especially true of the spherical types (cocci). .
To observe bacteria .in this state it is necessary to put a loopful * of the fluid
/ in which they are suspended in the center of a 'thin cover slip. On each of the
four corners place a ,tiny droplet of mineral oil. Hold a "hollow-ground"
slide, depression down, over the drop and bring the two into contact. Invert
the slide quickly so that the drop cannot run otrto one side. A tiny additional
drop of clear mineraLoil may now be run under the edges of the cover slip at
each corner if needed. This spreads under the cover slip and prevents drying
of the drop (Fig. 9-3),..r·· -~~.:::...
• By a "loop" is meant the space included by a tiny ring or loop made at the end of a
thin wire. The wire is fixed into some sort of handle so that it may be sterilized in a flame.
Loops are usually about 2 mm. in diameter and a loopful is a smaJl drop.
Optical Methods in Microbiology 105
In the hanging drop we may see the size, shape and arrangement of bacteria
and their motion if they are motile. Sometimes bright, refractile granules and
spores may be seen within the living cells. Due to the fact that bacterial cells
contain .no chlorophyll and are colorless and transparent it is extremely diffi-
cult to study them since this lack of color prevents details from showing
clearly. Further, when suspended in fluid, they move about, either because
of their own motility, currents in the fluid, or brownian movement. The
observation of bacteria in hanging drop preparations, therefore, yields limited
though valuable information.
Smears for Staining. To overcome this difficulty, Koch's device of im-
mobilizing bacterial cells and coloring them with aniline dyes is generally used.
In order to accomplish this, the material containing the bacteria (soil, urine,
pus, milk, feces, saliva) is diluted with water, saline solution or broth. A small
drop of the suspension is spread evenly in a thin film, with a loop, over an
area about I cm. in diameter. This film is commonly called a "smear."
Nature of Dyes. Most of the dyes used in microbiology may be thought
of as salts of two kinds: (I) acidic,' those in which the color-bearing ion (called
the chromophore) is the anion, e.g., sodium+ eosinate- (the dye, eosin); (2)
basic,' those in which the chromophore is the cation, e.g., methylene blue+
chloride-. The first type of dyes is acidic in the sense that, as an acid, the
chromophore combines with a base (NaOH) to form the dye salt; the second
is basic because the chromophore acts like a base, combining with an acid
(Hel) to form the dye sait.
In general, acidic dyes combine more strongly with cytoplasmic (basic)
elements of cells; the basic dyes with nucleic (acidic) elements of cells.
Some dyes do not depend on forming salts or chemical combinations with
the stained material. They merely coat the surface by the process of adsorp-
tion. or they may merely dissolve or precipitate in the surrounding substrate.
Probably both physical and chemical interactions occur during staining and
probably different processes are involved in staining different species. None
is fully explained or understood.
STAINING BACTERIA
Simple Stains. For general bacteriological work basic stains are most
used, because bacteria react toward stains as though composed principally
of nucleic acid, which takes basic dyes most readily. Methylene blue, safra-
nine. crystal violet, basic fuchsi.n, thionin, etc., are very widely used. All
belong to the group of aniline (coal tar) dyes. Any simple aniline dye solution
may be applied by flooding the smear with it. Loff!er's methylene-blue solu-
tion * is very widely j.lsed and reveals many details of form and structure.
Other solutions and colors are a!so available for special purposes. The dye
is allowed to remain ~n contact with the smear for about one minute and is
• LOffler's stain (Soc. Amer. Bact. "Manual of Methods").
I
Solution A
Methylene blue ... ; ......................................•.. 0.3gm
Ethyl alcohol (95 per cent) .................................. . 30.0 mJ
{, Solution B
KOH (0.01 per cerit) ....................................... . 100.0 mJ
Mix solutions A and B. The mixture keeps well.
lO6 Methods and Phenomena of Microbiology
then washed off with a gentle stream of cool water. The slide is then blotted
(not rubbed) between two pieces of filter or blotting paper and when dry is
ready for examination with the oil immersion lens as previously described.
Now, a simple stain such as Loffier's is of great value for many purposes.
But another staining procedure, devised by the Danish scholar Gram, is more
valuable because it enables us to differentiate between kinds of bacteria which
may be of different species yet of the same general form and size. It is therefore
called a differential stain.
*-
Gram's Stain. To the smear which, for purposes of discussion, we shall
assume to contain a variety of bacteria (e.g., saliva), crystal violet solutiont
is applied for thirty seconds. This is gently rinsed off and an iodine solutiont
is applied for thirty seconds. This, in turn, is rinsed off. Ninety-five per cent
ethyl alcohol is applied and renewed until all but the thickest parts of the
smear have ceased to give off dye. (This usually takes from twenty seconds to
one minute.)
The differential feature of the method is now apparent. Examination with
the microscope will reveal the fact that, as Gram found, while many bacteria
retain the violet-iodine combination, others will have yielded it largely to
the alcohol and are almost as nearly invisible as before. Those species of
bacteria which retain the stain are called gram-positive. Those which yield it to
the alcohol are called gram-negative.
But the staining process is not yet complete. There is stilI the important
final step of applying the counterstain§--a dye of some contrasting color,
usually eosin (red), safranine (red), brilliant green or Bismarck brown. Any
one of these dyes colors the gram-negative species and they become as visible
as the gram-positive ones, but are readily differentiated by their color.
Thus, by applying Gram's stain, which takes but five or six minutes, we
can learn a great deal about any bacterium. We make visible not only the
form and size and certain other structural details, but we can also at once
assign the organisms which are present in the material being examined, to
one of two great artificial groups of bacteria: the gram-negative or gram-
positive. As will be seen later, this is very helpful in identification procedures.
• Gram's stain (Hucker's modification, Soc. Am. Bact. "Manual of Methods,,:>.

t , Solution A t
Crystal violet (85 per cenf dye content). . . . . . . . . . . . . . . . . . . . . . . . 2.0 gm
Ethyl alcohol (95 per cent).............................. ..... 20.0 ml
Solution B
Ammonium oxalate......................................... 0.8 gm
DistiIIed water. ~ ................... ,"".. ..................... 80.0 ml
Dilute solution A about I in 5 with distilled water and mix with an equal volume o.
solution B. I
~ Lugo)'s iodine
Iodine ......................................... ,........ . . 1 gm
Potassium iodide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 2 to 5 gm
DistiIIed water. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300 ml
Allow to stand 24 hours for the iodine to dissolve. It may be necessary to
add a few more crystals of potassium iodide.
§ Counterstain: '
Safranine (2.5 per cent solution in 95 per cent alcohol) . . . . . . . . . . 25 ml
DistiIIed water. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7S m.I
It should be noted, however, that some organisms are "borderline" cases in
respect to Gram's stain, sometimes being positive, other times negative; and
sometimes both positive and negative cells are seen in the same culture. As a
rule, repeated tests will reveal the true nature of the bacterium. Often slight
variations in cultural conditions or staining technique will affect theres ult. F.or
example, many bacteria are not definitely gram-positive unless cultivated in
the presence of at least 5 per cent blood or serum. The acidity or alkalinity
(pH) of the fluid in which the bacteria are suspended will also markedly affect
their reaction to the Gram stain. It has been suggested that bacterial species
can be arranged in a gradient based on their reaction to the Gram stain. In
this gradient the strongly and constantly gram-positive species take up and
hold measurably more crystal violet than the permanently and definitely
gram-negative species. Gram-variable species hold variable, intermediate
amounts.
MECHANISM OF THE GRAM STAIN. If we could learn just why some organ-
isms are gram-positive and others gram-negative, we could perhaps clarify
questions surrounding the nature and distribution of the nucleus of bacteria,
the alterations in Gram staining properties of an organism under different
conditions of growth, the structure of the cell wall or cell membrane, and
vital questions on bacterial physiology. The actual mechanism, though in-
vestigated for years, is still not clear. The property of gram-positiveness
appears to reside in the cell wall. In most gram-positive bacteria the cell wall
appears to have some special affinity for the crystal-violet-iodine complex.
The cell walls of gram-negative bacteria do not have this affinity. In general,
it seems that the crystat violet and iodine penetrate both gram-positive and
gram-negative cells . In most gram-positive cells the dye and iodine form a
compound in the cell wall which is relatively insoluble in the decolorizing
agent (usually alcohol) and which passes out from the cell wall with difficulty.
Removal of various components of the cell wall (magnesium ribonucleate,
lipids, carbohydrates, etc.) by various agents (ribonticlease, hot water, ether,
etc.) make the ceIl gram-negative. That the cell wall plays a decisive role in the
Gram stain is shown by the fact that mechanical disruption of gram-positive
cells makes the entire cell gram-negative. No part of the disrupted cell retains
gram-positiveness.
CORRELATION OF GRAM REACTION WITH OTHER PROPERTIES. Whatever the
explanation of the Gram reaction it is important to note that there are certain
characteristic differences between most gram-positive and gram-negative
bacteria. Several of these are shown in Table 7. There are exceptions to some
of these generalizations but it is evident that the property of gram-positiveness
is related to very fut'ldamental physiological properties. The reasons are not
entirely clear.
Ziehl-Neelsen Stain. Another differential stain is that of Ziehl-Neelsen. It
is used especially for staining tuberculosis bacilli (Mycobacterium tubercu-
losis) and related organisms (genus Mycobacterium) having the chemical com-
position peculiar to these bacilli, i.e., an abundance of a particular waxy
material in the cell. Such organisms are gram-positive but this method does
not give as useful information about them as the Ziehl-Neelsen or "acid-fast"
stain. .
In using this stain a smear of the material to be examined is made as uS).lal,
108 Metlwds and Phenomena of Microbiology
Table 7. Some Differences BelM'een Gram-positive and Gram-negative
Bacteria.
PR.OPER.TY OR.AN-POSTI1VI! GR.AN-NEOATIVE
Susceptibility to sulfonamide drugs and penicillin Marked Much less
Inhibition by basic dyes like crystal violet Marked Much less
Susceptibility to low surface tension Marked Much less
Susceptibility to anionic detergents Marked Much less
Digestion by trypsin or pepsin (dead cells) Marked Much less
1% NaOH
Dissolved by Marked
. Not resistant
Ratio of RNA to DNA in the cell (approx.) 8 :1 Almost equal
dried, and fixed by heat. The smear is then flooded with a special solution of
carbol fuchsin and heated to 90.00 C, over a steam bath for four minutes.
This softens the wax and the dye supposedly penetrates. After washing off the
excess dye, the smear is treated for five minutes with cold 95 per cent alcohol
containing 5 to lO per cent hydrochloric acid. Presumably, the wax retains
the red dye in spite of the acid-alcohol, which removes the color from every-
thing else. Organisms retaining the red stain are said to be acid-fast. If, now,
methylene blue or brilliant green be applied as a counterstain, the acid-fast
bacilli stand out as bright red objects in a blue or green field. The Ziehl-
Neelsen stain· is a differential stain because it differentiates acid-fast organ-
isms from nonacid-fast ones (Fig. 9-4).
MECHANISM OF THE ACID-FAST STAIN. Acid-fastness, like gram-positive-
ness, disappears with physical disruption of the cell. It has been suggested that
acid-fastness is a matter of relative solubilities. For example, the red dye,
fu:::hsin, is more soluble in phenol than in water or acid-alcohol. Phenol, in
turn, is more soluble in lipids or waxes, such as are present in tubercle bacilli,
than in water. In the acid-fast staining procedure, the phenol, with red fuchsin
in it, leaves the water-alcohol of the carbol-fuchsin dye mixture and enters the
cell lipids in which it is more soluble. Here it remains because it is here more
soluble than in the decolorizing agent (acid-alcohol) and because the intact
cell membrane prevents lipids from leaving the cell to dissolve in the de-
• Ziebl-NeeJsen acid-fast stain (c:arboI rucbsln)

Solution A
Basic fuchsin. . ... . ... .... . . ..... ... . ..... . . .. . . ... .. . .... .. . 0.3 gm
Ethyl alcohol (95 per cent) . .. . . . ... . .. . . .... . . .. .. . . ... ... .. . . 10.0 ml
Solution B
Phenol (melted crystals) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.0 ml
Distilled water. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 95.0 ml
Mix solutions A and B. The mixture keeps weD. A counterstain of LOffler's methylene
blue is aenerally used, although some workers use brilliant green or saturated aqueous solu-
tion of picric acid for better contrast.
colorizer. If the cell membrane is broken, the lipids leave the cell and the
acid-fast property disappears.
Negative Staining. As generally used, this is not really a method of stain-
ing bacteria, but of staining the background a solid black, usually with
nigrosin. * This dye fails to penetrate the bacteria at all, and leaves them un-
stained to appear as light areas in the darkened field (Fig. 9- 5). Only the out-
lines of the organism are made apparent by this method, and consequently it
has a limited application, being used mainly for such forms as cannot be
stained by any of the ordinary methods, especially spirochetes.
A source of error in this method is shrinkage and distortion of bacterial
cells during the drying process. This often makes them look larger than they
are.
In addition to the various stains described above, there are ethers designed
to bring out special d: t:J ils such as srores, capsules, flagella and so on. De-
scription of each of these methods wil , however, be reserved to the discussion
,
:- .~
v'
,
I ... ~.
I
,JD
" ..,, , ,
I
7 .., '
1" ,
,,
'/
"'~
~ '.
~,
,,
(
-,I ;'1;.
'" ..... ...
... , ,.~r
.-
,.--'.
~
J
Fig. 9-4. Tubercle bacilli in sputum. Ziehl-Neelsen ( X 900). The bacilli have been
stained red. The mucus and pus cells in the sputum are stained blue for contrast. Note the
granular appearance of some of the bacilli and their arrangement in clusters. The many
bacilli pictured here are in a very tiny fraction of a droplet. (Comet a nd Meyer.)
• Domer's method for negative staining

(Grease-free slides must be used)
Nigrosin. .. . . . ...... . .. . .............. . . . .. . . ... . . . . ... . . ... 10 gm
Water .. . . ... . . . . . .. ........... . .. . . . . . .. .. .. . .... . ... . . .... 100 ml
Boil 30 minutes and add 0.5 ml formalin . Filter through paper and store in
2 ml amounts in sterile tubes. Place a loopfu\ of the suspension to be examined
on a slide. lmmediately add an equal amount of the nigrosin solution. mix.
and spread out in a thin film. (Thick films will crack and peel.)
Dry in air. Do not wet the slide. Do not heat it I
Fig. 9-5. Appearance of various bac-

teria as seen when prepared by negative
staining method (X 900).
of the structural features of the bacteria to which it applies and to which we

shall give some attention in a later chapter.
Darkfield Method. A method analogous in principle to the "negative stain-
ing" procedure is that of darkfield illumination. The ordinary compound
microscope may easily be equipped for this work by substituting a darkfield
condenser (purchasable from microscope manufacturers) in place of the Abbe
condenser.
In a darkfield condenser an opaque stop is arranged to prevent the entrance
of any direct rays of light from the mirror straight upward into the tube of the
microscope. All peripheral rays are, however, reflected obliquely to the center
of the upper surface of the microscope slide, emerging from the upper surface
of the slide at such an angle that they do not enter the objective lens unless
some object be present to reflect them upward (Fig. 9-6). The empty field,
therefore, appears dark. When a fluid containing any particles such as dust,
bacteria or spirochetes is placed on the slide at the focal point of these oblique
rays, each particle becomes visible as a brightly illuminated speck due to the
light reflected upward from its surface into the barrel of the microscope
(Fig. 9-7). The remainder of the field appears dark, hence the term darkfield.
Unlike the process of negative staining, the darkfield shows not only outward
form but motility, since the organisms, covered with a cover slip, are ex-
amined in a moist, living state. As outward form and motility are the chief
means by which certain important microorganisms, especially spirochetes,
are distinguished, the method is very valuable in types of work where such
/ species are involved: for example, diagnosis of syphilis.
ELECTRON MICROSCOPY
After the discovery of tobacco mosaic virus by Iwanowski in 1892, it
became apparent that there are creatures so small as to make bacteria appear
enormous by comparison. These tiny creatures cannot be seen even with the
most highly developed lens system depending on visible light.
The difficulty lay not with the lensm akers but in the fact that the wave
length of visible light is around 6000 A (A = angstrom unit = 0.000,000, I
mm.). Particles with a diameter of less than one half of this wave length, or
Optlc41 MetluHb ill MicrobiolDrY
Abb6 Condealcr with DarJdleld Srop
Paraboloid CondeDser CardioidCondenscr

Fig. 9--6. Various forms of condensen for oblique illumination of the darldield. In tbe
upper picture some object on the slide is mlectina liaht up through the objective lens. Note
tbat only pcriphcrallight rays pus the condcDser. (Bausch and Lomb 0ptiI:al Co.)
Fig. 9-7. Darldield preparation (X

9(0). This picture shows a group of spirO-
chetes and other bacteria from a lung
abscess. Note tbat tile thicker bacteria
are seeD only as luminous outlines. This
is because they are visible only by means
or liaht reftected from their outer sur-
races.
112 MetluHb and PMIfOIMIIII of Microbiology
Fig. 9-8. This electron microscope, developed by the Radio Corporation of America,
permits viewing of particles smaller tban one la-millionth of an inch in any diameter. It
provides magnification SO per cent greater than any heretofore possible. This magnification
is so enormous that a human hair would take on the dimensions of the Lincoln Tunnel.
(Courtesy of Radio Corporation of America, Camden, N. J.)
lines separated by a distance less than about 2000 A, cannot be " resolved,"
that is, they are not perceptible as separate particles because light waves
around 6000 A length entirely "skip over" the individual particles, just as a
/ man might not be aware of pebbles in his path because of the length of his
stride.
While it has long been known that electromagnetic radiations such as
x-rays have very short wave lengths, the preparation of glass lenses or re-
flectors for electron beams is impossible because glass is opaque to electrons.
However, it was observed that electrons are deflected from their line of propa-
gation by magnetic fields and, second, it was found that electrons moving with
the speed imparted by 60 KV have a wave length of only about 0.05 A, or
1/100,000 that of visible light. The latter discovery made plain the possible
usefulness of electronic waves in microscopy, while from the former it was
clear that circular magnetic fields could be used for "refracting" electron
Optical Metllod& in Microbiology 113
beams, much as a lens refracts light rays, forming electron images in the same
manner that visible light images are formed by lenses.
From these two basic discoveries the modem electron microscope (Fig.
9-8) has evolved. The units in this instrument are nominally analogous
with units in an ordinary compound microscope but deal with electron beams
rather than light rays. The diagram and legend in Figure 9-9 show the general
• Lamp
A B
Fig. 9-9. Diagrammatic outline of the path of electron beams in the RCA electron
microscope (A) as compared with the path of light rays in an ordinary light microscope (B).
The similarity of the two is clearly evident. In A a beam of electrons traveling from the
filament at high velocity correspond to the light rays from the lamp in B. The electron rays
(A) or light rays (B) are focussed on the specimen (S in B) by condensing systems. In A
this is a circular electromagnetic condenser; in B it is the lens system' L •. After passing
through the specimen the magnetic objective lens coil (corresponding to the objective lens
system, L7., of the light microscope) forms a first image (corresponding to I in B), enlarged
about 100 times. The magnetic intermediate image projector (corresponding to the ocular
lens system, L3, of the light microscope) then magnifies this image about 250 times (cor-
responding to the virtual image (/z in B), produciJlg an overall magnification of 25,000.
The final enlarged electronic image can be viewed directly by causing it to strike a ftuores-
cent screen which makes it visible (corresponding to the eye of the observer using a light
microscope), or it can be made 10 record the image on a photographic plate for permanent
record and for enlargetl\Cnt up to 100,000 or more. (A, courtesy of the Radio Corporation
of America; B, courtesy of the Society of American Bacteriologis~ collection, print 31.)
arrangement of the focusing systems and other parts of an electron microscope
compared with those of a light microscope. The electron source is a tungsten
filament at 30-100 KV potential. The electrons enter the magnetic condenser
(analogous to an ordinary microscope condenser) and are converged on the
specimen. After passing through the specimen, the magnetic objective-lens
coil focuses them into a first (real) image, enlarged about 100 times. The
magnetic intermediate-image projector then magnifies the real image about
250 times, making an over-all magnification of 25,000. The final, enlarged
image can be viewed directly by causing it to strike a fluorescent screen which
makes it visible. The image can also be thrown upon a photographic plate
for permanent record. Portions of the photographs may be enlarged 4 times
without undue loss of detail, thus giving a picture 100,000 times as large as
the object. This degree of magnification is almost inconceivably great. A
vague notion of it may be gained by imagining the page on which this is
printed to be enlarged to the same degree. It would become a slab of spongy
material as thick as a 2-story building is high, over 8 miles wide and about
13'n miles long. A human hair magnified to this degree would take on di-
mensions approximating those of the Holland Tunnel. A good comparison
of electron microscopy with ordinary microscopy is seen in Figure 9-10.
Several details of the operation of the instrument arc of interest. Because the motion of
electrons is impeded by air, the interior of the microscope must be maintained at a vacuum
by means of suitable pumps. This necessitates air locks for the insertion and mnoval of
objects and photographic plates. The operator can look into the main tube, by means of
portholes, and sec the images on the fluorescent screens, manipulate the object, make suit-
able adjustments of field strength of the focusing magnets, alignment, etc. The object is
mounted on an extremely thin (0.000001 em. thick) cellulose film, since glass slides would
be opaque to electrons.
Under conditions of electron microscopy living organisms cannot survive

and physiological processes in live cells cannot be studied by this means at
present.
A word of caution may be spoken in closing our discussion. The organisms
to be viewed in an electron microscope must be dried and this produces
shrinkage and distortion. In addition, many important biological structures
may contain little or no matter which obstructs the electrons and so cast no
shadows and remain invisible when electrons are passed through them.
In order to visualize detail, it is necessary to produce contrasts in the object.
This may be done in one way by selective staining (as phosphotungstic acid).
Then some parts of the object are made more "opaque" to the electrons than
others.
/ Artifacts having the most deceptive appearances, and very suggestive of
living things, are found in lifeless materials (see Figure 9-11). The objects
seen in photographs of electronic images are merely shadows, much like
shadows on an Jr-ray plate, and depend for visibility only on degree of opacity,
to electrons, of parts of the object. They may represent actual structures, or
mere thickenings of the object, or artifacts. In any case, our interpretations
of them should be extremely guarded until much more is known of electronic
microscopy and its pitfalls.
ShadowiDg TedmJque. One of the most va1uable techniques for the micro-
biologist is that called electron shadow-micrography. This is best described
Opdcal M~thotb in Microbiology 115
by the inventors as "depositing an exceedingly thin (ca. 7 ml') layer of metal
(chromium or gold) obliquely (in vacuo) on the preparation ... High points
on the surface intercept more than an average number of the condensing
atoms (of metal) and at the same time shield areas immediately behind them.
These shielded areas, devoid of deposited metal, appear as 'shadows' of the
elevated regions responsible for them. Just as an observer ... in a plane sees
many details of a landscape at sunrise, or sunset, which would be invisible to
him at noon, so metal shadow-casting brin~ out much detail that is not to be
seen in an unshadowed (or in a vertically coated) preparation." The method
applied to viruses is wonderfully revealing (Fig. 9-·12). Many other interesting
,. , \
......... ".
,. / •
t I
~ - ,'"
I / "'" \ I
• ...
B
Filo 9-10. Two pictures of the lockjaw orpnism (Clostridium tetQ/ll) as seen magnified
X 2000 by ordinary microscope (A) and magnified X 14,000 by electron microscopy in B.
Jbe terminal spores are seen as Iarp dark bodies. (A, Ford, Textbook of BacteriololD';
•• S. Mudd and T. F.Anderson, J.A.M.A., 1944.)
Fig. 9-11. A. unwashed preparation (rom sterile tissue fiuid. Filaments do not arise from
a cell-like imaae here. but from a long, thick grouping of closely-packed. adjoininl: NaCl
crystals. Near the side of the picture some of the crystals simulate virus-like particles
(X 14.(00). B. isolated filaments and fibers from sterile tissue fluid. They have the same
appearance as those observed during disintegration of erythrocytes. Many small particles
as well as a large ODe resembling elementary bodies of some viruses are seen in the back-
ground (X 14.(00). (photo courtesy of Dr. J. H. L. Watson. Edsel B. Ford Institute for
Medical Research. Detroit, Mich., 1950, voL 60.)
rig. 9-12. Electronograpbs of a virus (bacteriophage). A. without metal shadowing; B.

with metal shadowing. (Courtesy of Dr. D. Gordon Sharp. Duke University.)
116
Optical Metlwd& in Microbiology 117
"shadowed" pictures have been made, showing, for example, the details of
structure and size of poliomyelitis virus, tobacco mosaic virus and several
species of bacteria.
FLUORESCENCE MICROSCOPY
8y fluorescence is meant the property of reflecting rays having a wave
length different from that of the incident rays. Thus, substances having a cer-
tain color by ordinary light appear of a totally different color by ultraviolet
light. Objects invisible when "illuminated" only by ultraviolet light may be-
come brilliantly luminous if painted with a fluorescent substance such as
quinine sulfate (fluoresces violet in ultraviolet light) or the dye auramine
(fluoresces yellow in ultraviolet rays). 8y staining bacteria with a fluorescent
dye and observing them in a field illuminated only with ultraviolet light, they
can be made visible as luminous objects readily seen in a black field and
differentiated from nonftuorescent objects.
One adaptation has been in the study of tuberculosis. The fluorescent dye,
auramine, has a strong affinity for wax-like substances in tubercle bacilli.
When the bacilli are stained with auramine, and examined in the dark by
ultraviolet light, they fluoresce with a luminous yellow light.
Fig. 9-13. Relation of incident light waves (1), transmitted waves (A) and diffracted
waves (B) in phase microscopy. Retardation in phase (broken lines) and dimming (reduc-
tion of amplitude) of A after passing through the object (0) are shown in comparison with
I. These differences croate contrast between object and surround. However, these differences
are not readily perceptible to the unaided eye. The effects of adjusting the difference in
phase between A aad I by means of the phase-altering plate in the microscope are seen in
C as additive (rc&oDaDCe) (bright contrast), and in D as subtractive (interference) (dark
CODtrast). This optical manipulation of phase differences produces contrast between object
aud surround which is RIldily perceptible to the eye when the various waves are brought
to a focus as an imap. B shows waves not passing through the object but diffracted in pass-
iDa near it. For pUI"p08ClS of this discussion B and I are considered as one because dilfrac-
doll (B) plays a smaII:roIe ia phase contrast as compared with change of phase (A).
118 Methotb and Phenomellll of Microbiology
I,
I,
I
I
I
I
~TA~o
L~
•
LIGHT SCIIICE
Fig. 9-14. Image formation by phase contrast. An QllllUfar aperture in the diaphragm,
placed in the focal plane of the substage condenser, controls the illumination on the object.
The aperture is imaged by the condenser and objective at the reor focal plane of the objective.
In this plane the phase-shifting disk (diffraction or phase plate), is placed.
With the particular plate shown, light waves A (solid lines) are transmitted through the
object and pass through the phase-altering ring on the phase plate. At this point they acquire
a one-quarter-wave-Iength advance over light waves B (broken lines) which do not pass
through the object but are partly diffracted around it. Waves B do not pass through the
wave-altering ring on the phase plate. The resultant interference or resonom:e effects of the
two portions of light form the final image. Altered phase relations in the illuminating rays,
induced by otherwise invisible elements in the specimen, are translated into brightness
/ differences (Corrtrast) by the phase-altering plate; hence phase contrast. (Courtesy of Dr.
J. R. Benford, Bausch and Lomb Scientific Bureau.)
PHAS):; MICROSCOPY
If one examines the smaller microorganisms, such as bacteria, in their
living state suspended in a hanging drop of tluid, not only is it difficult to see
the organisms but it is next to impossible to discern clearly any of the internal
structures. This is due to the fact that there is almost no difference in refractive
index between protoplasmic structures and their surrounding tluids. The
situation is almost like trying to see a tiny fragment of ice in a bowl of water.
However, in biological materials, 'the slight differences that do exist can, by
Optical Methods ill Microbiology 119
means or special optical devices, be greatly enhanced so that readily per-
ceptible contrast (phase contrast) is produced between these objects and their
surroundings. This may be described briefly as follows.
A special, ring-shaped diaphragm is used in place of the usual iris dia-
phragm, and a light-wave-altering filter is placed back of the objective lens.
The light waves passing through the object are dimmer than, and out 0/ phase
with, incident waves (I), and diffracted waves (B) (Fig. 9-13). The difference
in phase and intensity between transmitted wave and non-transmitted waves
are intensified by the filter in the objective lens and are seen by the eye in
distinct outlines of light and dark phase contrast. The relations of the phases
and amplitude of two light waves are shown in Figure 9-13. The optical
arrangements are shown in Figure 9-14.
REFERENCES
Bartholomew, J. W., and Mittwer, T.: The Gram stain. Bact. Rev., 1952, 16: 1.
Benford, J. R.: The Theory of the Microscope. Bausch & Lomb Optical Co., Rochester,
N . Y., 1952.
Bennett, A. H ., Osterberg, H., et aJ.: Phase Microscopy. John Wiley and Sons, New York,
1951.
Conn, H. J.: The History of Staining. Biotech Publications, Geneva, N. Y., 1933.
Conn, H. J.: Biological Stains. 6th ed. Geneva, N. Y., 1953.
Conn, H. J., Bartholomew, J. W., and Jennison, M. W.: Staining Methods. Manual of
Methods, Pure Culture Study of Bacteria, 11 th ed. Leaflet IV. Biotech Publications,
Geneva, N. Y., 1954.
Cosslet, V. E.: Practical Electron Microscopy. Academic Press, New York, 1951.
Fischer, R. B.: Applied Electron Microscopy. Ind. Univ. Press, Bloomington, Ind., 1953.
Hall, C. E.: Introduction to Electron Microscopy. McGraw-Hili Book Co., New York, 1953.
Lamanna, C., and Mallette, M. F.: Basic Bacteriology. Williams & Wilkins Co., Baltimore,
Md., 1953.
Libenson, L., and McIlroy, A. P.: On the mechanism of the Gram stain. J. lnf. Dis., 1955,
95:22.
Newton, B. A.: The properties and mode of action of the polymyxins. Bact. Rev., 1956,
20 :14.
Oster, G., and Pollister, A. W. : Physical Techniques in Biological Research: I. Optical
Techniques. Academic Press, Inc., New York, 1955.
Reynolds, F. W., and Hesbacher, E. U.: Darkfield microscopy. J. Ven. Dis. Inform., 1950,
31 :17.
Richards, O. W. : Phase microscopy 1954-1956. Science, 1956,124:810.
Richards, O. W., and Miller, D. K.: An efficient method for the identification of M. tuber-
culosis with a simple fluorescence microscope. Am. J. Clin. Path., 1941, 11:1.
Richards, T.: The use of the microscope; I, 11, Ill. Laboratory Practice, London, 1955,
4:488; 1956,5:56.
Various Authors: Manual of Methods for Pure Culture Study of Bacteria. Biotech Publi-
cations, Geneva, N. Y.
Vickers, A. E. J.: Modem Methods of Microscopy. Butterworth, London, 1956.
Werkman, C. H ., and Wilson, P. W. : Bacterial Physiology. Academic Press, New York,
1951.
Zemike, F.: How I discovered phase contrast. Science, 1955,121 :345.
10
Morphology and Structure of Bacteria
MORPHOLOGICAL TYPES OF BACfERIA

AS SOON as microscopes and microscopy were sufficiently well developed
to permit detailed studies of microorganisms, it was found that three main
forms of bacteria may be differentiated: the spherical, the cylindrical or rod-
like, and the spiral or helicoidal (Fig. 1-4).
Spherical bacteria are called cocci (singular:;: coccus; derived from a Greek
word for berry). Cocci ar.e classified in several groups on the basis of the man-
ner in which they cling together after fission. First, there are diplococci, which
remain in pairs. Obviously these divide in one plane. Then there are the strep-
tococci which, like the diplococci, divide in one plane but which cling together
in chains, looking much like strings of beads. Other cocci divide irregularly in
three planes, and cling together in masses shaped like bunches of grapes.
These are called micrococci. Still others divide regularly in three planes at
right angles to each other, forming cubical groups. These are named sarcinae
(Latin for packet). Some cocci divide at right angles but in only one plane.
They form groups of four, called tetrads. Since they were first described by
Gaffky, they are called Gaffkya.
The spherical form of cocci is often distorted by various influences, so that
oval, elliptical, conical and other modifications are frequently ~een.
Cylindrical or rod-like bacteria are called b~cilli (singular = bacillUs; a Latin
word meaning "little stick': or rod). Bacilli arc straight, cigaretl,e-shaped or
sausage-shaped organisms. The cell wall appears to be a fairly rigid tube or
inelastic sac since growth occurs predominantly lengthwise and variations in
size affect mainly the long axis. Some are curved. Sometimes bacilli are so
short that the length is no greater than their diameter. These are often referred
to as cocco-bacilli. It is difficult to distinguish them from cocci. However, in
pure cQltures this is not such a great problem, since a few of the cells are
usually long enough to reveai the bacillary nature of the culture.
One other form, which may be thought of as derived from the cylindrical,
is the fusiform bacillus, shaped like a long, thin cigar pointed at both ends.
Helicoidal or spiral bacteria are shaped like a coiled wire spring, rangirig
from a portion of a coil to 15 or more complete helices. Two main groups
may be recognized: the flexible spiral bacteria (classified as the order Spiro-
chaetales and commonly called spirochetes), and the rigid spiral bacteria
120
Morphology and Structure of Bacteria 121
(classified in the family Spirillaceae of the order Eubacteriales). A subgroup
of the Spirillaceae comprises species which are so short as to consist of only
part of a spiral turn; in fact they are shaped much like a slightly twisted com-
ma. These are grouped in the genus Vibrio of which Vibrio comma (cause of
Asiatic cholera) is an important member.
The relations of the morphological types are shown in the following outline:
1. Coccus forms (spherical or derived from spheres)
Diplococcus· (pairs)
Streptococcus (chains)
Micrococcus (irregular clusters)
Gaflkya (tetrads)
Sarcina (cubical packets)
2. Bacillus or rod forms (straight, rod-like or cylindrical).
(Fusiform bacillus)
3. Helicoidal forms
Spirochetes (flexible; several complete spiral turns).
Spirillum (rigid; 1 or more spiral turns)
Vibrio (rigid; less than 1 spiral tum)
As bacteria grow they are subject to many influences, such as surface

tension, osmotic pressure, nutrition and rate of growth, which affect their
size and shape, much as various factors affect the size and shape of potatoes
or peppers. Age also plays its role.
Referring to the straight, rod-shaped, cylindrical forms, it should be
pointed out that the term "bacillus" was originally employed for all. How-
ever, when endosporest were discovered by Cohn in 1875 in the strictly
aerobic organism now "known as Bacillus subtilis, he differentiated sporulating
aerobic organisms from other cylindrical bacteria and adopted the term
Bacillus as a genus name. For this reason, the term Bacillus, capitalized and
printed in italics, is now employed only for aerobic, rod-shaped bacteria
which produce endospores. In general, the term "baCillus" and "bacterium"
are often used interchangeably for all rod-shaped cylindrical forms. The
term "bacterium" is also used in a general sense to mean any organism be-
longing to the class Schizomycetes.
SIZE OF BACfERIA
The drawings and reports by Leeuwenhoek (Fig. 2-4) gave men an idea of
the size and form of certaIn common types of these organisms.
Bacteria are incpnceivably small. Their minute size may be emphasized
by various comparisons. For example, it is estimated that a cubic inch would
hold nine trillion (9,000,000,000,000) cells of a medium sized bacillus (Sal-
monella typhi). It is common practice to magnify bacteria 900 to 1000 diam-
eters with microscopes. They then look no bigger than a period on this page,
or an exclamation' point (!). A man magnified to the same degree would be
over a mile high arid 500 yards wide.
Specifically, the Isize of bacteria ranges around a diameter of about 1.0,u
• Terms in italic~ are titles of genera named for the morphology of the organisms.
t Endospores are oval, refractile, thick-walled, protoplasmic bodies which develop inside
certain species of bacterial cells. Only one is formed in each cell. It represents a dormant
stage which,like arthrospores of molds, is resistant to drying, heat, disinfectants, etc. Sporc;s
will be discussed more fully later.
and a length, in the cylindrical forms, around 8}.t. Lengths of 200 to 500}.t
are not unknown, however, in certain species. Diameters usually do not
exceed about 2}.t.
Size and Fission. The size of the individual bacterial cell bears an im-
portant relationship to the nutrition of the cell and possibly to cell fission.
This is because the volume of the cell increases many times more rapidly than
the surface area. The volume of a cylindrical cell increases as the square of its
radius (1rr 2 Xalt.); the volume of a spherical cell increases as the cube of its
diameter (diam. 3 XO.5236). Yet foods for the inner parts of either cell must
pass through the cell surface since there is no mouth. As the spherical cell
grows there arrives a critical point in the ratio of volume (which must be fed)
to surface (which supplies food) when the favorable initial relationship be-
tween surface and content must be renewed. This is accomplished by fission,
which increases the ratio of surface to volume and results in the formation of
two small daughter cells. These grow and divide and so the process continues.
When fission (as sometimes occurs) proceeds much more rapidly than cell
growth, then the size of an individual cell diminishes as the numbers increase.
The amount of foodstuffs which can diffuse in, and the amounts of waste
products which can diffuse out, through the cell coverings, have definite limi-
tations determined by the nature of the cell wall, cell membrane, etc.
Whether wastes, nutritive requirements, reproductive cycle or other factors
initiate the stimulus for cell fission is not known. Possibly all of these factors
playa combined role.
The evolution of cylindrical cells, straight or spiral, with diameters more
or less fixed by radially rigid (or at least unexpandable) cell walls may be re-
garded as a step toward efficiency and more complex organisms. In a bac-
terial or mold cell or filament the axial center of the protoplast* i~ never more
than a micron or so from the source of food supply and the means of waste
disposal. The length and branching can extend (with or without division into
ceH,) indefinitely with no loss to the protoplast.
BACTERIAL STRUCTURE
We may consider five anatomical divisions, proceeding ;rom exterior in-
ward: (I) flagella, (2) capsule and/or slime layer, (3) cell wall, (4) protoplast,
with (a) protoplast membrane (equivalent to cytoplasmic membrane); (b)
cytoplasm (including various cytoplasmic inclusions, granules, etc.); (c)
nuclear structures (see Fig. 10-12).
I. Flagella. Flagella are long, very fine threads of complex protein at-
I tached to the cell in various locations, the number and position being char-
acteristic, according to the species (see Figs. 10-1 and 2). The point of origin
of flagella is difficult to determine. Many electronographs indicate a cytoplas-
mic origin of flagella. It probably differs in different species.
FLAGELLA AND MOTION. Motion offlagella appears to be associated with
a tiny granule at their point of insertion into the cell. This is seen clearly in
electronographs. Some workers regard flagella merely as twirls of gummy,
capsular polysaccharide which swirl and exhibit wavy movements passively
as the bacteria move by some other mechanism, possibly twisting on their
• All of the material within the cell wall.

Morphology IUId Stnu:"".. 0/ Btlcu"" 123
long axis. This view is not widely held. The mechanism by which flagella are
- activated is not clear.
Flagella propel the bacteria by spiral waves along the length of the flagella.
Not all bacteria possess flagella. Most non-flagellate species are non-motile.
The power to move by means of flagella, like the power of spore formation,
appears to have evolved mainly with elongation of form. Very few (if any)
spherical bacteria have flagella or form spores.
In addition to the swimming motion produced by tlagella there are some
non-tlagellate species which move slowly by creeping, as do some algae. Some
of the spirochetes appear to move without tlagella but in others flagella
appear to have been demonstrated.
BACTERIAL MoTIUTY. Whatever their mechanism of motility, motile
bacteria can move only in fluids. They cannot leave a fluid surface. They may
pass through the air, but not of their own volition. It is only as passengers
or riders on particles of oust in the air, or on objects like pencils, eating
utensils, books, etc., or in droplets such as those given off by a person when
sneezing or coughing, or in streams of water, airplanes, ships or trains that
they can travel great distances.
Their speed is sometimes very great when measured by their size. Some of
them cover a distance equal to scores of times their length in a second. An
automobile would have to speed at a hundred miles an hour to accomplish
the same thing. Some bacteria move very slowly and sedately, sometimes al-
most imperceptibly. It has been estimated that the energy required to move a
bacterium through an aqueous medium is of the order of 56 electron volts per
second; not great horsepower!
DEMONSTRATION OF MOTILITY. Motility is easily visible by direct observa-
Fig. 10-1. Types offtaaeIJation: a, monotrichate; b, amphitrichate; c and d, lophotrichate;

e, peritricbate.
124 Metlrod8 IIItd PMrIOIM1III of Microbiology
Fig. 10-2. Electronograph of a common bacterium (Proteus vulgaris) showing peritrich-

ous flageUation. Note the fineness of the flagella, their apparent insertion through the cell
wall of the bacterium and the apparent origin of several flageUa in a small granule, sug-
gestive of a blephatoplast. Note also the diffuse, granular chatacter of the cell contents.
(From the collection of the Society of American Bacteriologists, courtesy of Drs. A. L.
Houwink and C. F. Robinow and Miss W. van lterson.)
tion of motile bacteria in a droplet (hanging drop) of the fluid in which they
are living. In any culture of bacteria, especially old cultures, motile cells
may be difficult to find among thousands of dead or senescent cells. Young
cultures should always be used. In cultures acidified by fermentation, bacteria
lose their motility. Strict anaerobes lose motility on contact with air.
It is necessary to distinguish carefully between true motility and brownian
movement. Truly motile bacteria progress definitely and continuously in a
given direction. Brownian movement is a purposeless oscillation within a
very limited area. It is due to molecular forces entirely external to the bac-
teria. Unless the bacteria are fairly active, brownian movement is sometimes
rather difficult to distinguish from true motility.
DEMONSTRATION OF FLAGELLA. Bacterial flagella easily become detached
and are so fine as to be visible only when stained, or delineated with the elec-
tron microscope (Fig. 10-2).
A useful means of staining flagella is that recommended by Leifson.· It
must be pointed out that students will ordinarily not find it easy to stain
flagella on the first try. The factors involved are not all understood. However,
!
• Lelfson'. method fOl' stIIinIDg ftageDa
Stain: Solution: I. NaC.......... . .. . . . . . ...... 1.5% in distilled H20
II. Tannic acid . .... ..... ... . .. . . 3% in distilled H20
III. Pararosaniline acetate. . . . . . . . .. 0.9 gm
Pararosaniline hydrochloride .... 0.3 gm
(or 1.2 gm certified basic fuchsin)
Ethyl alcohol. ........... . . . . . 100 ml
Mix I, n and III in equal proportions and hold in tightly stoppered bottle at about 4° C.
On smear prepared as noted above quickly place 1 mI of stain. Leave at room tem-
perature for 7-15 minutes. (Try several smears; various intervals). Rinse generously but
gently with tap water-do not pour off stain first. Counterstain with diluted methylene blue
will often improve the results.
MorpltOlDgy IIItd StnlCture of Bacterill 125
a little perseverance is usually rewarded with success. Essentially the method
consists of precipitating tannic acid on the flagella. The tannic acid acts as
a mordant; i.e., an agent which causes the stain to stick to the stained object.
FORMS OF FLAGELLA. There may be several forms of flagella: coiled,
curly, normal, or wavy. Several forms may occur on one cell, and even on one
flagellum. The type of curvature may be partly due to distortion during drying
and processing incident to staining, and to other extraneous factors. However,
the wave lengths and amplitudes of these coils (or curves or waves) appear to
be fairly constant and to bear some significant relationship to each other and
possibly to species. For example, the wave iength of a curly flagellum may be
one half the wave length of a wavy or normal flagellum on the same cell.
This may be distinctive of the species (see Fig. 10-3).
2. Capsule (Slime Layer, or Sheath). Outside of the cell wall proper there
appears to be, on most bacteria, a layer of slimy, viscous or gummy substance
varying in thickness and consistency according to species, growth conditions,
phase of variation, or genetic characters which will be discussed later on. The
chemical composition of the capsule varies from species to species (or variety)
but appears to be a remarkably constant and distinctive characteristic of that
species or variety, governed by genetic mechanisms. The electron microscope
has aided greatly in the study of the slime layer or capsule, especially in those
species in which it is a very thin sheath. Some organisms on which a capsule
is not discernible may nevertheless possess the same chemical substance, in a
very thin layer on the cell surface, as determined by immunological (serum)
tests. It appears to be closely linked with the cell and may be inapparent
merely because it is not formed in great quantity. These surface complexes
Fig. 10-3. Bacteria with ftagella stained with Leifson's flagella stain. Various arrange-
ments of flagella are seen. Note that in the bacillus with two flagella the wavelength in one
flagellum is twice that in the other. Knowledge of the wavelengths of flagella is assuming
importance as a means of diagnostic identification. Magnification about X 3000. (Courtesy
of Dr. Einar Leifson, Stritch School of Medicine, Loyola University. Chicago, Ill.)
126 Metlwtb and Plle1lO1M1fIl of Microbiology
Fig. 10-4. Streptococci, showing well

developed capsules. Background is India
ink (X 15(0). The use of India ink is a
form of ne,ative staining. (Courtesy Dr.
Sidney Rothbard. J. Exp. Med., 1948,
vol. 88.)
are of great importance in many aspects of nUcrobio]ogy as we shall see later

on, especially in medical microbiology.
THE RELATION OF CAPSULE TO CELL is not always clear. In some species
capsules seem to be a thickened layer of the cell wall; in others, a secreted,
mucoid coating (Fig. 10-4). Some bacteria secrete much slimy substance into
their environment. This may not be a true capsule and may bear no more true
structural relation to the cell proper than any excreted waste product. Some
bacteria produce slime in addition to capsules.
In many species large, easily seen capsules seem to be called forth only by
certain particular circumstances of environment such as growth in the animal
body, or on certain media. Some, for example Leuconostoc, form their cap-
sular material best in sugar vats or in milk. There is good reason to believe
also that capsule formation may be stimulated, in some species, by unfavor~
able environments such as the resistance of an infected host. For example,
capsules are often found well developed on, and demonstrably correlated with,
virulence of pathogenic forms such as pneumococci and the organisms of
Whooping cough, gonorrhea, meningitis, anthrax and other diseases. The
capsules seem to enable these bacteria to evade the defensive mechanisms of
the infected body. The bacteria often cease forming capsules when removed
from the body to laboratory test~tube media.
All capsules, whether occurring on pathogens or saprophytes, may well be
regarded as a form of protective coating produced by the organisms in re-
sponse to unfavorable external conditions or other stimulus.
CoMPOSmON OF CAPSULES. The chemical composition of capsules varies
I widely with species. Most capsules are composed mainly of carbohydrates or
gums; others also contain nitrogen and phosphorus and may be of the nature
of mucus or the mucins. The capsule of some species appears to consist of
fibrous polypeptide micellae. In others the capsule may be composed of
curiously cross-striated fibers similar to the collagen of human connective
tissue.
Capsular substance often has specific chemical properties which enable us to
distinguish between various closely similar types of organisms which could not
otherwise be differentiated. This is an important point to remember. The
methods used will be made clear later.
DEMONSTRATION OF CAPSULES. There are several methods of demonstrat-
127
ing the presence of capsules on bacteria. As capsules of various species differ
in composition, however, they are not all amenable to the same process.
A method useful for staining many capsules is given below. •
Capsules, like tlagella, do not have a marked affinity for basic dyes and
some capsules are easily removed by mechanical means or by washing with
water.
3. Cell WaD. As with most bacterial structures it is not safe to generalize
about the structure and function of the cell wall. What is true of one species
may be quite different in another. By cell wall is meant (for bacteria) the thin,
sharply-defined, relatively tough (though tlexible), somewhat rigid structure
just inside the slime layer or capsule. The cell wall is generally physically and
chemically distinct from the capsule and from the inner cytoplasmic mem-
brane. It is estimated to range in thickness around 0.02". Rough analogies of
a cell wall would be the casing of an automobile tire (not the tubeless type) or
the leather covering of a basketball.
The cell wall in some microorganisms may be beautifully shown by special
6taining procedures and by the electron microscope. Methods of diJferenti-
ating the cell wall and cytoplasmic membrane have been described. Both stain
with difficulty and generally require a mordant such as tannic acid or phos-
phomolybdic acid.
The eoll wall varies in thickness, rigidity, strongth and chemical com-
position with species and growth conditions and other unknown factors. A
good idea of the character of some bacterial cell walls is obtained with the
electron microscope, examining ruptured bacteria and also very thin slices
of bacteria embedded in material like wax or soft plastic (Fig. 10-5).
TIm Cm!MJsTR.y Of THE CaL WALL is not well established. In general bac-
terial cell walls appear to be rather stable and resistant to the action of most
substances except strong acids and alkalies. In some fungi the cell wall ap-
pears to be mainly chitin. Most studies of bacteria indicate the presence of
carbohydrates, or carbohydrate complexes, chitin or chitin-like compounds,
mucinoids, hemicellulose and similar substances, though species differ
greatly. Cellulose is not common. In gram-positive bacteria the cell wall is
thought to contain the complex (ribonucleic acid?) responsible for the gram-
positive reaction.
• Capgle stain [Welch methocl (modified)] :
A. CcyMal violet
Saturated alcoholic crystal violet. . . . . . . . . . . . . . . . . . . . . . . . . . 5 ml
Distilled water .. . . . . . . . .... . . .. . .. .. . . . .. . . . . . . . .. . . . . . . 9S ml
B. 10 per cent CuS04 . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 liter
I. Make thin smear. '
2. Dry in air.
3. Fix by ptle heat.
4. Cover with crystal violet solution.
S. Heat to steaming for 1 second. (Do not boil!)
6. Use no water.
7. Rinse immediately and copiously with copper sulfate solution.
8. Use DO water.
9. Blot dly immediately.
10. Examine.
The orpnisms lIe stained purple and the capsule appears as a faintly blue
halo. See also other methods referred to.
128 Metllod& and Plleno,.,." of Mkrobiology
FiB- 10-5. IJaclIIus cereus, a common, barmIess bacterium foUDd in dust ~hen'
has been cut 1eDsthwise in a very thin section (see scale of 1 ,. at left). This is an e1ectrono-
graph at a magnification, originally, of X 60,000. The section shows two ad,jacent cells and
beginning formation of a third. A, cell wall showing evidence of the shrinking of the cyto-
plasm. C, indicating four peripheral bodies cut at different levels. D, beginning of a cen-
tripetally growing transverse cell wall. E, completed transverse cell wall before thickening.
F, low-density fibrous component of supposed nuclear apparatus. G, dense body in nuclear
apparatus which may be inclusion of cytoplasmic material. H, small dense particles which
appear to be main constituent of cytoplasm. I, unidentified cytopJasmic inclusions. (From
the collection of the Society of American Bacteriologists, courtesy of Dr. G . B. Chapman,
RCA Laboratories Division, Princeton, N . J.)
THE STRUCTUlU! OF THE CELL WALLis sometimes thought of as open, like

the ribs of a ship. This would indicate that the cell wall is chiefly an inert
supporting structure. Others regard it as more continuous; a mosaic of
various molecular complexes. Probably both structures occur.
Whatever the makeup of the cell wall it is, in all plant cells (including
bacteria), one of the structures through which must pass inward not only
the nutrient material for the cell, but outward all waste matter, digestive
enzymes and other products given off by the cell during its life. In order to
help retain within the cell the proper substances in the proper proportions,
and to prevent the entrance and allow the escape of improper or waste sub-
stances, it must exert a very fine discrimination or seJective permeability at
all times. In this it is doubtlessly aided by the cytoplasmic membrane.
Any slight defect or alteration in the discriminatory properties of the cell
wall must, therefore, have a profound effect on the cell contents. When, as is
the case with bacteria, the entire individual consists of the cell contents and
depends for its existence as a living entity on the chemical and physical equi-
librium of its protoplasm, then the maintenance of the integrity and uniform-
ity of the cell wall is a matter of the greatest moment. Substances or forces
which tend to alter the wall or overcome its selective powers must influence
the well-being of the cell. These will be discussed later on.
4. Protoplast. The protoplast comprises all of the material inside the cell
wall. The protoplast appears to have, at its outer surface, a thin, enclosing
membrane: the protoplast membrane. This is equivalent to what is often
called the cytopJasmic membrane. The protoplast content is equivalent to
cytoplasm plus nuclear structures plus other inner, essentially cellular
materials. The protoplast is the "living stuff" of the cell; i.e., it is protoplasm
(see Fig. 10-12).
The intact protoplast is liberated when the cell walJ is dissoJved. This may
be accomplished, in one way, by means of the enzyme lysozyme. • The proto-
plasts of bacilli assume a spherical shape after the cell wall is dissolved, show-
ing that the cell wall of such organisms is a rigid, supporting structure without
which the protoplast is a limp, protoplasmic mass. Flagella of motile bacilli
are not removed with the cell wall but normally protrude through it, being
rooted in the cytoplasm.
The protoplast membrane appears to have closely associated with it certain
enzyme systems which mediate respiratory functions involving oxygen at the
cell surface. These are discussed further in Chapter 13.
Freed from the cell wall the protoplast appears to be able to carryon all of
the essential functions of the complete cell, including growth and fission, with
only slight modifications. For example, it cannot effectively move its flagella.
Its osmotic properties also appear to be altered since free protoplasts tend to
lose water. The most obvious functions of the cell wall are thus revealed as:
(a) accessory to flagellar motion, possibly in the role of "oar-locks" for
8.agella; (b) protection of the protoplast; (c) maintenance of the elongated
form of bacilli; for reasons given elsewhere this seems to be a physiologically
more efficient form than the spherical; (d) supplementation of the osmotic
properties of the protoplast membrane.
(a) PROTOPLAST MEMBRANE. When, in plasmolysis, the bacterial cytoplasm
shrink!. from the cell wall, it appears to be enclosed in an exceedingly thin,
but distinct, membrane (the protoplast membrane). This is normally il! inti-
mate contact with the inside of the cell wall like the inner tube of a tire. The
chemical nature of this cytoplasmic membrane is not clear. It may consist of
a condensation or slight thickening of certain surface-active elements of
cytoplasm where it is in contact with the cell wall. The remarks on the im-
portance of selective permeability and alterations in structure made in con-
nection with the cell wall also apply to the cytoplasmic membrane. The two
are undoubtedly of fundamental import in acting as mediators between the
living cytoplasm within, and the world outside.
(b) CYTOPLASM. This is one part of the protoplast of all typical cells.
The cytoplasm is usually clear, watery or slightly viscous, and homogeneous
in appearance although granules of varioys substances, such as stored food,
oil, etc., may be suspended in it. It consists of a complex mixture including
~ "'U""JW:i, lipids, carbohydrates, minerals and water. The organic matter is
in the colloidal state, and so intimately intermingled as to defy syn-
and any analysis but the grossest sort. In larger cells than bacteria the
part of protoplasm is in constant circulatory motion called protoplasmic
.# rf!tl1.ninf!. This has not been observed in bacteria though it may occur.
NUCLEAR. STRUCTURES. It is generally thought that something in bac-
cells must act like a nucleus in order to carry on and control such
I\ui~ctllons as various chemical activities, reproduction, heredity, and life itself.
H(]lwever, neither the nature nor the existence of the nucleus of bacteria is
A chief difficulty is the minute size of bacteria. A nucleus, if it exists, would

barely within the limits of visibility of the best of light microscopes.
the combined use of electron and light microscopy. and certain
Lysozyme was discovered in 1922 by Fleming who later discovered peniciI1in. Lysozyme
in egg-white, various bodily secretions and may be produced by certain bacteria.
130
A B
Fig. l(Hi. A, a-h are interpreted as successive stages in the development of a coccoid cell
with a broad (double?), central, chromatinic structure into a typical rod-shaped two-cell
bacteriwn with two pairs of chromosomes. Note the V-shaped division stage at d. The
chromosome forming the left limb of the V is so much broader than the right one that it
seems plausible to assume that it is preparing to divide. At e a thin strand of cliromatinic
matter is seen connecting recently divided chromosomes. A difficulty lies in not knowing
the order of events; for example, whether a normally precedes b or vice versa. Since each
is a dilferent cell and all were photographed simultaneously one has to /Usume the order of
development. (Robinow, C. F.) B, these difficulties are eliminated to some extent by con-
tinuous observation of single cells. In B are seen nucleus-like bodies in living, multiplying
bacteria (&cheriehla coil) photographed microscopically at intervals, a difficult procedure.
The nwnbers in the pictures indicate a~ in minutes of the cells from the moment of first
observation. No evidence of mitotic figures was observed and DO pattern of nuclear division
could be formulated from such series of observations. (B, courtesy of Drs. 0.1. Mason and
D. M. Powelson, in 1. Bact., 1956, vol. 71.)
chemical staining methods, a number of clever scientists have revealed

/ granules and remarkable internal changes in bacteria which suggest nuclear
structures and activities (Figs. 10-6 and 7). Doubt persists because of the
difficulty in proving their nuclear nature unequivocally.
The principal views on bacterial nuclei (except for those denying the
presence of any nucleus) may be summarized as follows:
1. There is a true nucleus and cytoplasm. This has been discussed above.
2. There is a true nucleus but no cytoplasm. When ordinary stains are used,
the bacterial cell as a whole stains like nuclear material and no differentiation
between nucleus and cytoplasm can be made with fue microscope. When
special stains are used, differentiations of the cell contents may be seen but
their exact nature is not known with certainty.
Morphology and Structure of Btlcteritl 131
3. TIre nucleus consists of minute particles of nuclear material, or granules
of chromatinic material, diffusely scattered in the cytoplasm, perhaps attached
to a mitochondrial reticular network of threads; or perhaps strung on a long
mitochondrial thread, the whole resembling a tangled string of beads enclosed
within the cytoplasmic membrane. This is a widely held view at present. In
order to explain the appearance, in electronographs, of fairly large, discrete
bodies said to be nuclei or analogous structures, it has sometimes been sug-
gested that the scattered nuclear granules may coalesce into functional
nuclei just before cell fission. This is a not unreasonable view and is consistent
with many observations.
While it must be admitted that we do not k.now very much about the
morphology of bacterial nuclei, we cannot deny that something carries on the
runctions of a nucleus in a fairly efficient manner.
MitocboDdrla-Uke Particles. In some bacteria, protein particles of definite
size, distinctive specific gravity, and demonstrable enzymatic activity have
been differentiated in the cytoplasm. The exact nature of these is not clear
but they appear to be, or to act as though they contain, enzymes. Some have
called them mitochondria, since they appear to resemble these cytoplasmic
granules which occur in the cells of animal tissue.
Spores. Only three genera of true bacteria (Bacillus, Clostridium and
Sporovibrio) form spores.
When a rod of the genus Bacillus sporulates, an area of altered cytoplasm
appears in the cell. This undergoes modifications and develops an outer wall.
The exact nature of the process is not clear. Some data indicate that spores
develop from a single primordium of nuclear material; others that it includes
nuclear granules. It has been suggested that spore formation is accompanied
by sexual functions, such as automixis, or nuclear fission or fusion. Whether
this is so or not, part or all of the vital cell content is finally condensed and
FiI. 10-7. Structures interpreted as bacterial nuclei in a common species of Bacillus

CNeUS). In A note division of nuclei in association with division of the ceUs. These are
as paired, compound, probably sister nuclei. In B and b note the internal structures
COInpc)Ulltd nuclei which ~p from the nuclei shown in A. (Photos courtesy of Dr.
Knaysi, in J. Bact., 1955, vol. 69.) These are from electronographs; each cell is
5" in Jcngth.
surrounded by a relatively thick shell. The material in the spore appears to be
packed inside the spore membrane in a very orderly manner,like the germ in a
seed. It probably consists largely of nuclear material or DNA (see Fig. 33-2).
In the genera of bacteria named above one spore is formed inside each cell,
and each spore is properly called an endospore. Some spore-forming species
are also said to occur in the gram-negative genera Vibrio and Spirillum and
the gram-positive Sarcina.
PAIlASPORAL BoDY. During spore formation by some (possibly all) spore-
forming, aerobic bacilli a small granule, called a parasporal body, appears.
It is not inside the spore proper, but is closely associated with it and seems to ..
be attached to the outer surface. It is spheroid and, in diameter, about lIs of
the length of the spore. Its significance is unknown.
The state of the water in the spore is not certain. There is evidence that the
spore is dehydrated. Contrary data indicate that the water content is un-
changed but that the water in the spore is not in a free state, being bound·
or imbibed by the lyophilic colloids in the spore. Dehydration seems more
probable. Either, as well as an increased calcium content of spores, would
seem to account for the heat resistance of endospores.
The cell with fully formed spore is called a sporangium. The empty cell
wall of the bacillus may separate off, like the hull of a seed, leaving the spore
as a free, round, or oval body. Disinfectants cannot readily penetrate into
spores, nor can aniline dyes.
STAINING SPORES. A rod with a spore inside, when stained by ordinary
methods may thus appear to have a hole in it (Fig. 10-8). However, the outer
shell of the spore may readily be colored so that, in stained smears, free spores
may appear as tiny, blue or red rings. There are special stains for spores. t
SPORE FORMATION. Endospores are formed most readily under good
growth conditions. Their formation appears to require some energy food such
as glucose. In some species manganese ions are requisite. In others iron and
manganese greatly increase spore formation. Apparently at least part of the
spore contents are newly synthesized and are not merely old material of the
vegetative cell. Certain specific nitrogenous food substances (as certain
amino acids) are required. The fact that by far the largest numbers of spores
are usually found in well-matured cultures several days old indicates that
spores generally represent a mature stage of cell development.
There is a curious difference between aerobic (Bacillus) and anaerobic
(Clostridium) sporulation. The aerobic bacilli do not sporulate under strictly
anaerobic conditions; the anaerobic bacilli do not sporulate under aerobic
conditions.
/ GERMINATION OF SPORES. When spores are placed in nutrient fluids at
favorable temperatures the spore sac splits and a vegetative, active, bacterial
cell emerges (Fig. 10-9). Germination is said to have occurred. The method
of splitting is thought to be distinctive of species. Probably water is taken up.
• The apparent dryness of a hard-boiled egg is a good illustration of bound water.

t Spore 1ItahI: Apply saturated malachite green (about 7.6%) for 10 minutes.
F1ame until warm.
Rinse with tap water for about 10 seconds.
Apply 0.25% aqueous safranine for IS seconds.
Rinse with tap water, blot, and dry.
Certain food substances favor germination: manganese ions, I-alanine, and
other amino acids, glucose, etc.
Spores of anaerobes will germinate readily under aerobic conditions and
aerobe spores will germinate under anaerobic conditions.
Dormant spores show little metabolic activity, but they are alive and do
show a low rate of respiration. .
FUNCflON OF SpORFS. Sporulation of bacteria obviously is not a necessary
function since most species reproduce perfectly without demonstrable sporu-
lation. Further, in bacteria, it is generally thought, only one spore is produced
by each cell and this spore produces only one cell; i.e., spore formation is
not a reproductive mechanism. In this respect bacterial spores differ from the
spores of true fungi and higher plants. Bacterial spores appear to represent
merely a dormant and resistant state which enables the bacteria producing
them to survive during periods unfavorable to active vegetation. However,
some workers maintain that more than one spore is formed per cell. The
question is in debate.
REsISTANCE OF SpORES. When protein or protoplasm is moist or well
hydrated, as is its condition in an actively growing. vegetative cell it coagu-
lates readily and is therefore vulnerable with respect to heat, chemicals, etc.
The "white" of fresh egg is a good illustration of a hydrated protein. It coagu-
lates ("hard boils") readily enough.
When protein is ,iehydrated, or when the protein and water are bound, it
Fia. l~. Various types of bacterial spores. Some of the spofCI. have escaped from the
1pOrangia. Stained )Vith methylene blue, which does not penetrate inside the spore, only
the outer surface of the spore is stained.
134 Mdhods and Phenomena of Microbiology
Fig. 10--9. Electronograph of B. my-

coides showing rupture and germination
of a spore. The "cap" of the spore wall
is seen at the left, while the bacillus arises
out of the remainder of the spore shell
below. (Courtesy of Knaysi, Baker and
Hillier, J. Bact., vol. 53.)
is no longer readily coagulable. It is then attacked only with difficulty b}

various chemicals, and withstands high temperatures and other conditions
which would soon destroy it were it allowed to become hydrated. Egg powder
is a good example of dehydrated protein.
Partly because of their apparently dehydrated state spores can survive
where the active, vegetative, or hydrated stage would soon perish. So re-
sistant are some bacterial spores that they will live for decades, and probably
for centuries, on dry splinters of wood, grow after treatment in strong dis-
infectant solutions, survive an hour or more of boiling, or even of pressure-
ccoking (autoclaving) at 1200 C, and remain unharmed by an hour or more in
a hot oven. Not all spores are so resistant, however, some being killed at
temperatures little above those lethal to vegetative cells (60° C-70° C). How-
ever, most bacterial spores are quite resistant.
It is sometimes difficult or impossible to demonstrate spores by means of
the microscope, even when staining methods are employed. Resort must then
be had to tests for resistance of the culture to heat. A culture which resists
85° C for 20 minutes almost certainly contains spores although they may
not be visible.
/ There is some evidence that high growth temperatures of the spore-forming
bacilli enhance the resistance of their spores to heat. For example, thermo-
philic species (those growing only at high temperatures (60° C-75° C) usually
produce the more heat-resistant spores.
Granules. In many bacteria stained by ordinary methods, granules are
readily seen. They are distinguished by the fact that they stain more intensely
than the rest of the cell, or are of a different color. In this case they are often
spoken of as metachromatic granules. We have already mentioned a number
of kinds of granules, some resembling mitochondria. The composition and
function of some types of granules is a matter of debate. As shown by their
reaction to reagents such as iodine, some of theJ;ll are of starch (blue), gly.
Morpltology and Struclll1'e of Bacteria 135
cogen (brown) or closely related compounds and doubtless often represent
reserve food substances since they tend to disappear during periods of food
scarcity. In some species globules of pure elemental sulfur are stored as food
in the cell.
VOLUTIN. In many \>acteria, granules of volutin are commonly observed
(Fig. 10-10). These have often been confused with nuclei. Volutin is a food
material, probably consisting of inorganic polyphosphates and nucleic acid-
like compounds. These are not identical with the protein of the nucleus, as
they are dissolved in hot water (800 C) instead of being coagulated by it, and
are not digested by trypsin.
FAT. Some bacteria synthesize and store much/at, especially if fed with
sugar or other assimilable food; in this showing a striking resemblance to
many human beings. Some species of the genus Bacillus are outstanding in
this respect, but other bacteria, yeasts and fungi also store up much fat (see
Fig. 10-11). A 24-hour culture on glycerol agar, if properly stained, will show
large amounts of fat.· The method offers a good field for investigation of
problems in metabolism morphology, taxonomy, etc.
Fig. 10--10. A common, hannless bacterium (Aerobacter aerogenes) stained by the

Albert-Laybourn method for demonstrating volutin. It is interesting to note the striking
similarity of these granules to the bodies interpreted as nuclei by some workers. Magnifica-
tion originally at X 3000. (Courtesy of Drs. J. P. Duguid,l. W. Smith and J. F. Wilkinson,
Dep't of Bacteriology, University of Edinburgh, Scotland. In J. Path. and Bact., 1954, vol.
67; also J. Bact., 1954, vol. 68.)
• Fat staID (Burdon)

1. Prepare the film, dry and fix by gentle heating as for the Gram stain.
2. Ho..'ld the entire slide with Sudan Black (0.3 per cent dry dye in 70 per cent CzHs(>H)
and leave the slide for 5 to 15 minutes. Drying of the dye will not hann the prep-
aration. Use of a Coplin jar is less satisfactory.
3. Drain off excess dye and blot dry.
4. Clear in xylol for about I minute, dipping in and out.
5. Counterstain with 0.1 per cent aqueous safranin for 10 seconds. Overstaining with
counterstain will mask the fat stain. It may even he omitted.
The fat appears as dark, blue-black masses; the rest of the cell is pink.
The stock of solution of Sudan Black keeps well in a clean, stoppered bottle. It should
he occasionally shaken and aUowed to settle overnight before use. Sudan III and
Scharlach R are much less satisfactory.
Methods and Phenometrll of Microbiology
- -
Fig. 10-11. Various species of bacteria stained for fat with Sudan black. Note the sim-
ilarity of many of these masses to PK.-tures representing what is said to be nuclear material.
(Burdon, K. L., in J. of Bact., vol. 52.)
Staining Granules. There are many methods of bringing granules into

prominence, the method depending largely on the chemical nature of the
granules themselves. One commonly used to demonstrate volutin granules
is that of Albert. •
Chemkal Composition of Bacteria. Bacteria are not markedly different in
chemical composition from other living cells. However, the exact chemical
analysis of living matter is difficult, if not impossible, since to analyze it one
must kill it and disturb the interrelations of various components of the cell.
Bacterial protoplasm is like all other protoplasm in that it is composed
chiefly of the elements C, 0, H, S, Nand P, with smaller amounts of many
other elements as Na, K, Ca, Fe, Mg, Cl and so on. Species differ in the actual
amounts of each element present. The actual proportion of each of these
elements usually varies greatly with the composition of the medium on which
the bacteria are growing at the time they are analyzed.
In vegetative bacterial cells, water is an important component, constituting,
roughly, from 75 to 98 per cent of the whole weight. The proportion is quite
variable, depending on environment.
I
or
• Albert's medIocI staInIag volutin I!I'8JIII)es
A. Toluidin blue ...... . .. .. . . ....... . . .. ........... .. ...... . 0.15 gm
Methyl green .. . ....................... . ................. . O.20gm
Acetic acid (glacial). • • . . . . . . . . . • . • • . • . . . . . . . . • . . • . . . • •• .. l.OOml
Alcohol (95 per cent) ...... . .. ... ................... . . . ... . 2.00ml
Water . .... .. . . ... . .... . . .. .............• . .....•.. .. ..... 100.00 m1
Allow to stand 24 hours. Filter through paper.
B. Iodine as in Gram's stain.
l. Prepare smear as usual.
2. Flood with solution A for I minute. Wash with water and blot dry.
3. Stain with solution B for 1 minute, wash and blot dry.
Granules black; other parts dark or liBht green.
Morphology lind Structure 0/ Bacteria
137
Growth in saline media increases ash content. The ash content (inorganic
matter) may vary in amount from 2 to 30 percent of the dry weight of bacteria;
organic matter from 40 to 90 per cent.
The organic matter consists of:
I. Proteins (including nuc1eoproteins) . ....... . ..... . .. 40-80

%
2. Carbohydrates· .......... .. ...................... 10-30
3. Lipids ............... . ......... . ... . ............ 1-30
4. Nucleic acids (RNA and DNA t) ............. . ..... 5-30
5. Miscellaneous organic compounds occur in lesser and varying
amounts.
The cell walls contain chitin or chitin-like compounds in some species,

carbohydrate complexes in others. Chitin was formerly regarded as dis-
tinctively an animal substance.
Many complex, Don-protein, nitrogenous substances are also found, among
them various purine bodies, polypeptides, and amino acids. All but two of the
known amino acids have been found.
PROTOI'IAST
Fig. 11}-12. Structures commonly seen in cells of representative: species of rod-shaped

bacteria. For discussion see text. N == nuclear material; form and distribution indeter-
minate. Cy = cytoplasm. G == dense, intracellular granules, probably containing RNA;
often closely associated with enzyme activity. V == vacuole-like structures, possibly emptied
of lipid, volutin or other material during preparation of the specimen for microscopy. Fo ==
granules of volutin, fat, polysaccharide or other stored food substances. CW = cell wall.
A = flagellum. B = basal granule or blepharoplast-like granule associated with attachment
and/or function of flagella. Cp == capsule. PM == protoplast membrane. The space (Sp)
between protoplast membrane and cell wall is ordinarily non-existent. It has been exag-
gerated here for diagrammatic purposes. An appearance such as that in the diagram might
occur as a result of plasmolysis. Note also that, while flagella and well developed capsule
are here shown together for diagrammatic purposes, they do not commonly occur together
on the same cell.
• Cultivation in carbohydrate-rich media increases carbohydrate and lipid content. Curi-

ously. glycogen is found among the list of carbohydrates. Glycogen was formerly regarded
as a distinctively animal substance.
t DNA was for some time thOUght of as a distinctively animal substance. Nucleopro-
teins often constitute up to 80"1. of the total organic matter in the cell. DNA is more to be
associated with the nucleus, RNA with cytoplasm.
: This is hypothetical because of the wide differences between the various orders of bac-
teria. However, it is reasonably typical and serves as a useful basis for elementary discus-
sion.
The general structure of nucleoproteins and nucleic acids may be seen in
Figure 5-6.
Pigments of Microorganisms. Colonies of most bacteria growing on dye-
free media are greyish, whitish and translucent or opaque. However, there are
some notable exceptions. Pigments are a prominent feature in some species.
In most of these their exact function is not known nor is their chemistry,
except for photosynthetic pigments of the few alga-like species. In color they
range the spectrum.
Some pigments may be excreted as colorless compounds called leukobases,
becoming colored only on oxidation after excretion. A good example is the
blue pyocyanin, a phentzine derivative produced by a common saprophyte
(sometimes pathogen) Pseudomonas aeruginosa.
Many of the pigments are of the group of red, yellow or orange lipochrome
compounds, common in flowers, butter, egg yolks, corn, etc. <:;ytochrome, the
respiratory pigment of aerobic bacteria, contributes no visible coloration to
them. Bacteriochlorophyll gives a purple or greenish color to the alga-like
photosynthetic species (Rhodobacteriineae). These are discussed in Chapter
29.
Pigments in general vary greatly in amount and intensity of color, even in
the same organism. Growth at various temperatures and on various media
affects either the mechanism responsible for pigment formation or the color
of the pigment itself. Pigments usually develop best on solid media in close
association with air. Anaerobic growth usually results in lack of, or discolora-
tion of, bacterial pigment. Pigmented species may give off non-pigmented
variants. Pigments may be intracellular, or they may pass through the mem-
brane of the cell and so be extracellular.
REFERENCES
Alexander, M.: Localization of enzymes in the bacterial cell. Bact. Rev., 1956, 20:67.
Bartholomew, J. W., and Clare, P.: Parasporal bodies of bacillus spores. J. Bact., 1956,
71:158.
Bartholomew, J. W., and Miuwer, T.: Simplified bacterial spore stain. Stain Techno!., 1950,
25:153.
Bissett, K. A.: The Cytology and Life History of Bacteria. The Williams & Wilkins Co.,
Baltimore, Md., 1955.
Bradfield, J. R. G.: Electron microscope observations on bacterial nuclei. Nature, 1954,
173:184. ,
Burdon, K. L.: Fatty material in bacteria and fungi revealed by staining dried, fixed slide
preparations. J. Bact., 1946,-52:665.
Chance, H. L.: A study of bacterial substances stainable by the hydrogen chloride method.
J. Bact., 1956, 71 :285.
/ Chapman, G. B.: Electron microscopy of ultra-thin sections of bacteria. II. J. Bact., 1956,
71 :348.
Church, B. D., Halvorson, H., and Halvorson, H. 0.: Studies on spore germination: its
independence from alanine racemase activity. J. Bact., 1954, 68 :393.
Clark. J. B., and Webb, R. B.: Ploidy studies on the large cells of Micrococcus aureus.
J. Bact., 1955, 70:454.
DeLamater, E. D.: A conceptual formulation of the structure'and divisional mechanism of
the bacterial nucleus. Tr. New York Acad. Sci., 1954, Ser.II, 16:366.
DeLamater, E. D.: The bacterial nucleus. Ann. Rev. Micr., 1954,8:23.
Dondero, N. C., Adler, H. I., and Zelle, M. R.: Quantification of the Feulgen reaction in
bacteria. J. Bact., 1954,68:483. )
, Duguid, J. P.: The demonstration of bacterial capsules and slime. J. Path. and Bact., 1951,
63:673.
Morphology and Strtlcture oj Bacteria 139
EI-Bisi, H. M., and Ordlll, Z. J.: The effect of certain sporulation conditions on the thermal
death rate of Bacillus coagulans var. thermoacidurans. J. Bact., 1956, 71:1; see also
p. 11.
Foster, J. W., and Perry, J. J.: Intracellular events occurring during endotrophic sporula-
tion in Bacillus mycOides. J. Bact., 1954, 67:295.
Hachisuka, Y., et al.: Studies on spore germination. J. Bact., ,1955,69:399.
Henrici, A. T.: Morphologic Variation and the Rate of Growth of Bacteria. Charles C
Thomas, Springfield, III., 1928.
Hewitt, L. F.: Effect of cultural conditions on bacterial cytology. J. Gen. Micr., 1951,5:293.
Hoffman, H.: The cytochemistry oS bacterial nuclear stroctUTes. l. Bact., 1~51, 62 :56).
Knaysi, G.: Cytology of bacteria. Ann. Rev. Microbiol., 1956, 10:253.
Knaysi, G.: The structvre, composition, and behavior of the nucleus in Bacillus cereus.
J. Bact., 1955,69:117.
Md., 1953.
Leifson, E., Carhart, S. R., and Fulton, M.: Morphological characteristics of flagella of
Proteus and related bacteria. J. Bact., 1955, 69 :73.
Leifson, E., and Plaen, M. I.: Variations and spontaneous mutations in the genus Listeria in
respect to flagellation and motility. J. Bact., 1955, 70:233.
Lessler, M. A.: The natlJre and specificity of the Feulgen nucleal reaction. Internat. Rev. of
Cytol., 1953, 2.
Levinson, H. S., and Hyatt, M. T.: The stimulation of germination and respiration of B.
megaterium spores by manganese, I-alanine and heat. J. Bact., 1955,70:368.
Mason, D. J., and Powelson, D. M.: Nuclear division as observed in live bacteria by a new
'. technique. J. Bact., 1956, 71 :474.
Mitchell, P., and Moyle, J.: Liberation and osmotic properties of the protoplasts of Micro-
coccus /ysodeikticus and Sarcina /utea. J. Gen. Micr., 1956,15:512.
Morowitz, H. J.: The energy requirements for bacterial motility. Science, 1954, 119:286.
Mudd., S.: The bacterial cell. Ann. Rev. Micr., 1954,8:1.
Oginsky, E. L., and Urnbreit, W. W.: An Introduction to Bacterial Physiology. W. H.
Freeman and Co., San Francisco, 1954.
Peterson, R. G., and Hartsell, S. E.: The lysozyme spectrum of the gram-negative bacteria.
J. Inf. Dis., 1955, 96:75.
Pijper, 0., and Neser, M. L.: The wavelengths of helical bacterial flagella. J. Gen. Micr.,
1956,14:371.
Roth, N. G., and Lively, D. H.: Germination of certain aerobic bacilli under anaerobic con·
ditions. J. Bact., 1956,71 :162.
Schmidt, C. F.: The resistance of bacterial spores with reference to spore germination and
its inhibition. Ann. Rev. Micr., 1955, 9:387.
Sneed, 1'., and Halvorson, H. 0.: 1'he Sats oS Aerobacter cloacae. Appl. Micr., l~54, 2 :1'05.
Spooner, E. T. C., and Stocker, B. A. D., Editors: Bacterial Anatomy. 6th Sympos., Soc.
Gen. Micr., 1956. Cambridge Univ. Press, New York.
Stedman, E9" and Stedman, EI.: The cytological interpretation of the Feulgen reaction.
Biochem. J., 1950,47:508.
Waldham, D. G., and Halvorson, H. 0.: Studies on the relationship between equilibrium
vapor pressure and moisture content of bacterial endospores. Appl. Micr., 1954, 2 :333.
Weinberg, E. D.: The effect of Mn++ and antimicrobial drugs on sporulation of Bacillus
subtilis in nutrient broth. J. Bact., 1955, 70:289.
Werkrnan, C. H., and Wilson, P. W.: Bacterial Physiology. Academic Press, New York,
N. Y., 1951.
Widmer, C., King, T. E., and Cheldelin, V. H.: Particulate oxidase systems in Acetobacter
suboxidans. J. Bact., 1956, 71:737.
Widra, A.: Studies on the cytochemistry of bacteria. J. Bact., 1956, 71 :689.
11
Classification and Taxonomy of Bacteria
WHILE ALL agree that classification and taxonomy are desirable and
necessary phases of biological work there is much disagreement as to the best
method of classifying and naming living things. The zoologists a"nd the bota-
nists have arranged matters in their fields fairly satisfactorily, largely on the
basis of morphological characters. These are a good guide to natural or
evolutionary relationships because they are based on a long, connected and
demonstrable history of evolutionary development and phylogenetic re-
lationships. The microbiologist can only speculate as to the evolutionary and
phylogenetic history of microorganisims; virtually nothing is known of them.
Hence he has not arrived at systems of classification in which the natural
relations of the organisms are used as a basis.
Development of Bacterial Classification. Nevertheless, he has systems of
classification. They have been built up largely as arbitrary descriptive keys for
identification of organisms according to whatever information about them
may be available. These descriptive keys cannot properly be called natural
classification. They are indispensable, but their artificial and entirely arbitrary
nature must not be forgotten. They have no bearing on natural or evolution-
ary relationships of microorganisms. ,
Organisms may be arranged in systematic groups, called taxa, according to'
shape, motility, or other criteria. The science and art of naming such groups
is called taxonomy. Form, motility, spore formation, growth under certain
absolute conditions such as complete lack of oxygen (anaerobiosis), and
similar properties are qualitative, unit characters. They are most useful in
classification because they are not relative and variable like size or rate of
growth. They are present or not present.
NATURE OF DIFFICULTIES. In dealing with recently discovered organisms,
having clearly described properties which can be readily determined by
generally available methods; and of which sample cultures are available for
other workers to study, classification may not be too difficult a task. If, how-
ever, the person originally naming a supposedly newly discovered organism
failed to keep cultures available for study, used undescribed or misleading
methods to determine color, motility, biochemical properties, etc., and if, in
addition, he was unacquainted with the rules of nomenclature (rules govern-
ing the naming of microorganisms) and used incorrect names or names al-
ready given to other·organisms, it can be seen that this worker has introduced
confusion and disagreement into the field of the taxonoPlist and has thrown
140
Morphology and Structure oj Bacteria 141
EI-Bisi, H. M., and Ordal, Z. J.: The effect of certain sporulation conditions on the therm..
death rate of Bacillus coagulans var. thermoacidurans. J. Bact., 1956, 71:1; see also
p. 11.
Foster, J. W., and Perry, J. J.: Intracellular events occurring during endotrophic sporula-
tion in Bacillus mycoides. J. Bact., 1954,67:295.
Hachisuka, Y., et aJ.: Studies on spore germination. J. Bact., 1955,69:399.
Henrici, A. T.: Morphologic Variation and the Rate of Growth of Bacteria. Charles C
Thomas, Springfie1d, Ill., 1928.
Hewitt, L. F.: Effect of cultural conditions on bacterial cytology. J. Gen. Micr., 1951,5 :293.
Hoffman, H.: The cytochemistry of bacterial nuclear structures. J. Bact., 1951, 62 :561.
Knaysi, G.: Cytology of bacteria. Ann. Rev. Microbiol., 1956, 10 :253.
Knaysi, G.: The structure, composition, and behavior of the nucleus in Bacillus cereus.
J. Bact., 1955,69:117.
Lamanna, c., and Mallette, M. F.: Basic Bacteriology. Williams & Wilkins Co., Baltimore,
Md., 1953.
Leifson, E., Carhart, S. R., and Fulton, M.: Morphological characteristics of flagella of
Proteus and related bacteria. J. Bact., 1955, 69 :73.
Leifson, E., and Plaen, M. I.: Variations and spontaneous mutations in the genus Listeria in
respect to flagellation and motility. J. Bact., 1955, 70:233.
Lessler, M. A.: The nature and specificity of the Feulgen nucleal reaction. Internat. Rev. of
CytoJ., 1953, 2. •
Levinson, H. S., and Hyatt, M. T.: The stimulation of germination and respiration of B.
megaterium spores by manganese,l-alanine and heat. J. Bact., 1955, 70:368.
Mason, D. J., and Powelson, D. M.: Nuclear division as observed in live bacteria by a new
technique. l. Bact., 1956, 71 :414.
Mitchell, P., and Moyle, J.: Liberation and osmotic properties of the protoplasts of Micro-
coccus lysodeikticus and,Sarcina lutea. J. Gen. Micr., 1956,15:512.
Morowitz, H. J.: The energy requirements for bacterial motility. Science, 1954, 119:286.
Mudd., S.: The bacterial cell. Ann. Rev. Micr., 1954,8:1.
Oginsky, E. L., and Umb~eit, W. W.: An Introduction to Bacterial Physiology. W. H.
Freeman and Co., San Francisco, 1954.
Peterson, R. G., and Hartsell, S. E.: The lysozyme spectrum of the gram-negative bacteria.
J. Inf. Dis., 1955, 96:75.
Pijper, 0., and Neser, M. L.: The wavelengths of helical bacterial flagella. J. Gen. Micr.,
1956,14:371.
Roth, N. G., and Lively, D. H.: Germination of certain aerobic bacilli under anaerobic con-
ditions. J. Bact., 1956,71:162.
Schmidt, C. F.: The resistance of bacterial spores with reference to spore germination and
its inhibition. Ann. Rev. Micr., 1955, 9:387.
Sneed, T., and Halvorson, H. 0.: The fats of Aerobacter cloacae. AppJ. Micr., 1954, 2 :285.
Spooner, E. T. C., and Stocker, B. A. D., Editors: Bacterial Anatomy. 6th Sympos., Soc.
Gen. Micr., 1956. Cambridge Dniv. Press, New York.
Stedman, Ed., and Stedman, El.: The cytological interpretation of the Feulgen reaction.
Biochem. J., 1950, 47:508.
Waldham, D. G., and Halvorson, H. 0.: Studies on the relationship between equilibrium
vapor pressure and moisture content of bacterial endospores. Appl. Micr., 1954, 2 :333.
subtilis in nutrient broth. J. Bact., 1955, 70:289.
Werkman, C. H., and' Wilson, P. W.: Bacterial Physiology. Academic Press, New York,
N. Y., 1951. '
Widmer, c., King, T. E., and Cheldelin, V. H.: Particulate oxidase systems in Acetobacter
suboxidans. J. Bact., 1956, 71:737.
Widra, A.: Studies on the cytochemistry of bacteria. J. Bact., 1956, 71 :689.
I
11
Classification and Taxonomy of Bacteria
WHILE ALL agree that classification and taxonomy are desirable and
necessary phases of biological work there is much disagreement as to the best
method of classifying and naming living things. The zoologists and the bota-
nists have arranged matters in their fields fairly satisfactorily, largely on the
basis of morphological characters. These are a good guide to natural or
evolutionary relationships because they are based on a long, connected and
demonstrable history of evolutionary development and phylogenetic re-
lationships. The microbiologist can only speculate as to the evolutionary and
phylogenetic history of microorganisims; virtually nothing is known of them.
Hence he has not arrived at systems of classification in which the natural
relations of the organisms are used as a basis.
Development of Bacterial Oassification. Nevertheless, he has ·systems of
classification. They have been built up largely as arbitrary descriptive keys for
identification of organisms according to whatever information about them
may be available. These descriptive keys cannot properly be called natural
classification. They are indispensable, but their artificial' and entirely arbitrary
nature must not be forgotten. They have no bearing on natural or evolution-
ary relationships of microorganisms.
Organisms may be arranged in systematic groups, called taxa, according \0
shape, motility, or other criteria. The science and art of naming such groups
is called taxonomy. ,Form, motility, spore formation, growth under certain
absolute conditions such as complete lack of oxygen (anaerobiosis), and
similar properties are qualitative, unit characters. They are most useful in
classification because they are not relative and variable like size or rate of
growth. They are present or not present. .
! NATURE OF DIFFI<;:ULTIES. In dealing With recently discovered organisms,
having clearly described properties which' can be readily determined by
generally available methods; and of which sample cultures are available for
other workers to study, classification may not be too difficult a task. If, how-
ever, the person originally naming a supposedly newly discovered organism
failed to keep cultures available for study, used undescribed or misleading
methods to determine color, motility, biochemical properties, etc., and if, in
addition, he was unacquainted with the rules of nomenclature (rules 'govern-
ing the nam,ing of micro()rganisms) and used incorrect names or names al-
I ready given to other organisms, it can be seen that this worker has introduced
confusion and disagreement into the field of the taxonomist and has thrown
140
Classification and Taxonomy of Bacteria 141
difficulty into the path of the microbiologist. Unfortunately, the field and the
path are filled with such debris. In many instances original cultures have
been lost and no one knows exactly what was being dealt with. Many older
descriptions are wholly inadequate and so ambiguously expressed that they
might fit a dozen different species. On trying to duplicate them no exact
descriptions of the methods used are to be found. Many "definitions" of
genera (groups of similar species) are very indefinite and are merely vague
general descriptions. For example, in describing a genus, it helps little to say
that some species in a genus are motile, many are gram-negative, most are
encapsulated, several are pigmented, a few forms tend to be microaerophilic,
and so on.
Even if a complete and generally acceptable system were finally agreed
upon, a new difficulty would arise because microorganisms are variable and
an organism having one set of characters today may have others tomorrow.
This would be a serious obstacle by itself, but it is still further complicated
by the fact that some sorts of microorganisms may have some of the char-
acters of others. The classifists and taxonomists (those who bring order out of
such seeming chaos) finally set up arbitrary divisions among the creatures
they are classifying and rule that the possession or absence of certain char-
acters shall differentiate between such and such organisms. This is convenien~
until it is found that certain of the creatures have lost some of those charac-
ters, but have acquired others. Finally, a man-made difficulty arises when it is
found that by means of irradiation with ultraviolet light and by means of
transfers of genetic material (transformations, trltnsductions, genetic re-
combinations, etc.) and other means it is possible to transmute one "species"
of microorganism' into another! However, such variation has definite limi-
tations under ordinary circumstances. Certain characters of most species are
reasonably constant. The fact that certain organisms may be classed as
species is due to this fact. As pointed out by Skerman, "recognition of them
as species has come through the gradual recognition of the constancy of cer-
tain characteristics Which they display ... " "Variants have been recognized
only because certain characters were selected as representative of a species."
Classification is obviously possible but it depends on cooperation among
microbiologists, a knowledge of variable properties, and of the principles and
problems of classification, taxonomy and nomenclature.
Classification Schemes. Many systems of classification have been brought
forward but none has remained long without revision and enlargement. One
of the first schemes for bacteria was devised by Cohn in 1872 based on mor-
phologyonly. In 1897 Migula devised a scheme based not only on form but
on color and some physiological characters like nitrogen fixation. OrIa-
Jensen in 190~ made ,up a system based largely on physiological properties
and this has served aSIa model for all later schemes. The suggested scheme of
Stanier and Van Niel, including the establishment of a new kingdom, the
Monera, is most stimr!lating. A very excellent critique of the whole problem
has been drawn up by Van Niel. Modern schemes use all known properties for
classification: form, arrangement, Gram stain, motility, physiology, bio-
chemistry, etc. The re~ative values of various kinds of characters and the dif-
ficulties with them and the whole philosophy of classification are very read-
ably presented by Cowan and others.
A system widely used by American bacteriologists and which still has an
international standing was formerly sponsored by the Society of American
Bacteriologists. It is now published in a volume generally known as "Bergey's
Manual." This classification is based on the botanical code of the International
Congresses of Botanists. The Bacteriological Code of Nomenclature was
approved by the International Committee on Bacteriological Nomenclature
in 1947.
This system, in its 6th edition (1948), divided the entire group of bacteria
(Class Schizomycetes) into 5 major subdivisions or orders, which are outlined
below. There will be numerous changes from this in a new edition which is
in preparatio.1. Each order is divided into families, and these into tribes,
genera and species. Groups of similar species constitute genera; groups of
similar genera constitute tribes, and so on. The names of orders end with the
suffix ales; names of families with aceae; names of tribes end with eae.
Some names of species and genera are changed with successive revisions of
the systems of classification, and different names are therefore sometimes
used by different authors for the same organism, or two different organisms
may be called by the same name. Thus there is some confusion in bacteriolog-
icalliterature. This is one of the signs of progress, like the confusion attendant
on the repair of a downtown thoroughfare.
OUTLINE CLASSIFICATION OF THE SCHIZOMYCETES AND RELATED
MICROORGANISMS*
Gass SCmzOMYCETES Niigeli
ORDER 1. EUBACTERIALES Buchanan
SUBORDER I. EUBACTERIINEAE Breed, Murray and Hitchens
Family I. Nitrobacteriaceae Buchanan
Tribe I. Nitrobacterieae Winslow et al.
Genus I. Nitrosomonas Winogradsky
Genus II. Nitrosococcus Winogradsky
Genus III. Nitrosospira Winogradsky
Genus IV. Nitrosocystis Winogradsky
Genus V. Nitrosog[oea Winogradsky
Genus VI. Nitrobacter Winogradsky
Genus VII. Nitrocystis Winogradsky
Tribe II. Hydrogenomonadeae Pribram
Genus I. Hydrogenomonas Orla-Jensen
Tdbe III. Thiobacilleae Bergey, Breed and Murray
Genus' I. Thiobacillus Beijerinck
Family Il. Pseudomo_nadaceae Winslow et al.
Tribe I. Pseudomonodeae Kluyver and Van Niel
Genus I. Pseudomonas Miguia
/ Genus II. Xanthomonas Dowson
Genus III. Methaizomonas OrIa-Jensen
Genus IV. Acetobacter Beijerinck
Genus V. Protaminobacter den Dooren de Jong
Genus VI. Mycoplana Gray and Thornton
Tribe II. Spirilleae Kluyver and Van N;iel
Genus I. Vibrio Muller
Genus II. Desulfovibrio Kluyver and Van Niel
Genus III. Cellvibrio Winogradsky
,
• From Bergey's Manual of Determinative Bacteriology. 6th Ed., 1948. The Williams
and Wilkins Co., Baltimore, Md.
Genus IV. Cellfalcicula Winogradsky
Genus V. Thiospira Vislouch
Genus VI. Spirillum Ehrenberg
Family III. Azotobacteriaceae Bergey, Breed and Murray
Genus I. Azotobacter Beijerinck
Appendix: Genus A. Azotomonas Stapp
Family IV. IVzizobiaceae Conn
Genus I. IVzizobium Frank
Genus II. Agrobacterium Conn
Genus III. Chromobacterium Bergonzini
Family V. Micrococcaceae Pribram
Genus I. Micrococcus Cohn
Appendix: Genus A. Methanococcus Kluyver and Van Niel
Genus B. Pediococcus Balcke
Genus II. Gaffkya Trevisan
Genus III. Sarcina Goodsir
Subgenera:
Zymosarcina Smit
Metharwsarcina Kluyver dnc. Van Niel
Sarcinococcus Breed
~ Sporosarcina Orla-Jcnsen
Family VI. Neisseriaceae ~evot
Genus I. Neisseria Trevisan
Genus II. Veillonelfa Prevot
Family VII. Lactobacteriaceae Orla-Jensen
Tribe.!. Streptococceae Trevisan
Genus . I. Diplococcus Weichselbaum
Genus II. St~eptococcus Rosenbach
Genus III. Leuconostoc Van Tieghem
Tribe II. Lactobacilleae Winslow et a!.
Genus I. Lactobacillus Beijerinck
Subgenera:
Thermobacterium Orla-Jensen
Streptobacterium Orla-Jensen
Betabacterium Orla-Jensen
Appendix: Genus A. Leptotrichia Trevisiim
Genus II. Microbacterium Orla-Jensen
Genus III. Propionibacterium Orla-Jensen
Genus IV. Butyribacterium Barker
Family VIII. Corynebacteriaceae Lehmann and Neumann
Genus I. Corynebacterium Lehmann and Neumann
Genus II. Usteria Pirie
Genus III. Erysipelothrix Rosenbach
Family IX. Achromobacteriaceae
Genus 1. Alkaligenes Castellani and Chalmers
Genus II. Achromobacter Bergey et a!.
Genus lli. Flavobacterium Bergey et a!.
Family X. Enterobacteriaceae Rahn
Tribe I~ Eschericheae Bergey, Breed and Murray
Genus I. Escherichia Castellani and-Chalmers
Genus II. Aerobacter Beijerinck
Genus III. Klebsiella Trevisan
Appendix: Genus A. Paracolobactrum Borman, Stuart and Wheeler
Tribe II. Erwineae Winslow et al.
Genus I. Erwinia Winslow et a1.
Tribe lli. Serrateae Bergey, Breed and Murray
Genus I. Serratia Bizio emend. Breed and Breed
Tribe IY. Proteae Castellani and Chalmers
Genus 1. Proteus Hausel"
,-~ ~
Tribe V. Sa/monel/eae Bergey, Breed· and Murray

Genus 1. Salmonella Lignieres "
Genus II. Shigella Castellani and Chalmers
Family XI. Parvobacteriaceae Rahn
Tribe 1. Pasteurelleae Caste\lani and Chalmers
Genus I. Pasteurella Trevisan
Genus II. Malleomyces Ha\lier
Genus III. Actinobacillus Brumpt
Appendix: Genus A. Donovania Anderson et al.
Tribe II. Brucelleae Bergey, Breed and Murray
Genus I. Brucella Meyer and Shaw
Tribe Ill. Bacteroideae Breed, Murray and Hitchens
Genus 1. Bacteroides Caste\lani and Chalmers
Genus II. Fusobacterium Knorr
Appendix: Genus A. Fusiformis Hoe\ling
Tribe IV. Hemopkileae Winslow et al.
Genus I. Hemophilus Winslow et al.
Genus II. Moraxella Lwoff
Genus Ill. Noguchia Olitsky, Syverton and Tyler
Genus IV. Dialister Bergey et a1.
Appendix: Tribe Mimeae DeBord
Family XII. Bacteriaceae Cohn
Genus I. Bacterium Ehrenberg
Subgenera:
Kurthia Trevisan
Cellulomonas Bergey et at.
Saccharobacterium Sickles and Shaw
Agarbacterium Angst
Photobacterium Beijerinck
Methanobacterium Kluyver and Van Niel
Appendix: Suborder Eubacteriineae: Overlooked Species and
Synonyms ..
Family XIII. Bacillaceae Fischer
Genus I. Bacillus Cohn
Genus II. Clostridium Prazmowski ,
SUBORDER II. CAULOBACTERllNEAE Breed, Murray and Hitchens
Family I. Nevskiaceae Henrici and Johnson
Genus I. Nevskia Famintzin
Family II. Gallionellaeeae Henrici and Johnson
Genus I. Gallionelfa Ehrenberg
Family III. Caulobacteriaceae Henrici and Johnson
Genus 1. Caulobaeter Henrici and lohnson
Family IV. Sideroeapsaceae Pribram
Genus 'I. Sideroeapsa Molisch
Genus II. Sideromonas Cholodny
Appendix: Family Pasteuriaceae Laurent
Genus 1. Pastellria Metchnikoff
, Genus II. Blastocaulis Henri~i and.1ohnson
SUBORDER III. RHODOBACTERIINEAE Breed, Murray and Hitchens
Family I. Thiorhodaceae Molisch
, Genus 1. Thiosarcina Winogradsky
Genus II. Thiopedia Winogradsky
Genus III. Thioeapsa Winogradsky
Genus IV. Tkiodictyon Winogradsky
Genus V. Thiothece Winogradsky
Genus VI. Thiocystis Winogradsky
Genus VII. Lamprocystis Schroeder
denus VIII. Amoebobacter Winogradsky
Genus IX. Thiopolycoccus Winogradsky
Genus X. Thiospirillum Winogradsky
Genus XI. Rhabdomonas Cohn
Genus XII. Rhodothece Molisch
Genus XIII. Chromatium Perty
Family II. Athiorhodoceae Molisch
Genus I. Rhodopseudomonas Kluyver and Van Niel emend. Van
Niel
Genus II. RhodospirilJum Molisch
Family Ill. Chlorobacteriaceae Geitler and Pascher
Genus I. Chlorobium Madson
Genus II. Pelodictyon Lauterbom
Genus III. Clathrochloris Geitler
Genus IV. Chlorobacterium Lauterbom
Genus V. Chlorochromatium Lauterborn
Genus VI; Cylintirogloea Perfiliew
ORDER II. AcnNOMYCETALES Buchanan
Family I. Mycobacteriaceae Chester
Genus I. Mycobacterium Lehmann and Neumann
Family II. Actinomycetaceae Buchanan
Genus 1. Nocardia Trevisan
Genus II. Actinomyces Harz
Family III. Strep.tomycetaceae Waksman and Henrici
Genus I. Streptomyces Waksman and Henrici
Genus II. Micromonospora Orskov
ORDER III. CHLAMYDOBACfllRIALES Buchanan
Family I. Chlamydobacteriaceae Migula
Genus I. Sphaerotilus Kutzing
Genus II. Clonothrix Roze
Genus III. Leptothrix Kiltzing
Family II. Crenothricaceae Hansgirg
Genus, I. Crenothrix Cohn
Family)II. Beggiatoaceae Migula
" Genus I. Thiothrix Winogradsky
Genus II. Beggiatoa Trevisan
Genus III. Thiospiril/opsis Uphof
Genus IV. Thioploca Lauterbom
Appendix: Family Achromatiaceae Massart /
Genus I. Achromatium Schewiakotf
Genus II. Thiovulum Hinze
Genus Ill. Macromonas Utermohl and Koppe
Appendix: Order Caryophana/es Peshkotf
Family I. Pontothricaceae Peshkoff
Genus I. Pontothrix Nadson and Krassilnikow
Family II. Arthromitaceae Peshkoff
Genus I. Arthromitus Leidy
Genus II. Co/eomitus Duboscq and Grasse
Family III. Oscil/ospiraceae Peshkoff
Genus I. Oscillospira Chatton and Perard
Family IV: Caryophanaceae Peshkoff
Genus I. Caryophanon Peshkoff
ORDER IV. MYXOB1CfllRIALllS Jahn
Family I. Cytophagaceae Stanier
Genus I. Cytophaga Stanier
Family II. I Archangiaceae Jahn
Genus I. Archangium Jahn
Genus II. Ste/angium Jahn
Family III. Sorangiaceae Jahn
Genus I. Sorangium Jahn
Family Iv'. Po/yangiaceae Jahn
Genus I. Po/yangium Jahn
Genus II. Synangium Jahn
Genus III. Melittangium Jabn
Genus IV. Podangium Jabn
Genus V. Chondromyces Berkeley and Curtis
Family V. Myxococcaceae Jabn
Genus I. Myxococcus Thaxter
Genus II. Chrondrococcus Jahn
Genus III. Angiococcus Jabn
Genus IV. Sporocytophaga Stanier
ORDER V. SPIROCHAETALES Buchanan
Family I. Spirochaetaceae Swellengrebel
Genus I. Spirochaeta Ehrenberg
Genus II. Saprospira Gross
Genus III. Cristispira Gross
Family II. Treponemataceae Schaudinn
Genus I. Borrelia Swellengrebel
Senus II. Treponema Swellengrebel
Genus III. Leptospira Noguchi
Species and Genus. In bacteriology the terms species and genus are used,
but the concept of these is somewhat vague as there is little knowledge of
genetics in relation to bacteria and no knowledge of evolutionary and phylo-
genetic relationships. Species of animals and plants are generally established
on the basis of their inability to cross-fertilize. But cross-fertilization' in bac-
teria is very doubtful indeed, except possibly in one strain of Escherichia toli, "
and no other sexual phenomena of recognized types occur in bacteria. How
then define a species? In bacteriology a species is theoretically a sIngle kind
of bacterium, all individual cells of which are identical or nearly so. 'In actual-
ity this identity of cells rarely exists . .In any culture of a given species mutant
cells may be found which, while having the outward form, staining properties
and other characters of most of the cells in the group, possess different meta-
bolic properties, different antigenic composition, and so on. Usually these
differences are not extreme and may represent only temporary fluctuations
from the principal type. It must be remembered that many ordinary test-tube
cultures of a species consist of billions of individuals and represent a "popu-
lation" many "generations" old even in 18 hours of growth. It would be
strange if there were the high degree of uniformity among microorganisms
that is seen in inorganic crystals, for example.
When two bacteria have one or more well-marked morphological, metabolic
or other differences between them which are constant, the two may be re-,
garded as distinct species. Here we run into an arbitrary difficulty. Who is to
determine what character or characters shall constitute "well-marked," and of
sufficient distinction to be the basis for differentiation between species or
genera? The same differences may be' u~ed as a basis of generic or tribal or
even familial distinction between some other kinds of bacteria. Bacterial
s:;>ecies, therefore, are rather ill defined.
The concept of genera is, in many instances, equally nebulous. A genus is
theoretically, and ideally, a group of species all of which bear sufficient re-
semblance to one another to be considered closely related and easily distin-
guishable from mempers of other groups or genera. The boundaries of some
genera are sharply defined by as few as three characteristics, as in the genus
Bacillus: (I) aerobic; (2) spore-forming; (3) rods. These are very definite,
distinct, constant and readily determined characters. The boundaries .of other
~
genera are sometimes more difficult of definition; for example, the genera
Salmonella, Escherichia, Shigella, and Aerobacter, all of which are nonspore-
forming, gram-negative, facultative, aerobic rods of identical appearance,
non-pigmented and fermenting glucose. An organism of one genus may thus
possess several of the important (?) characters of two or three or more other
genera, and its proper allocation to one of these is often difficult and must be
decided on an arbitrary basis.
Strains. A term frequently used in microbiology is "strain." A strain of
microorganisms is a particular example, specimen or "culture" of a given
species. For example, we might isolate a culture of Micrococcus pyogenes, var.
aureus, from Mr. Jones and, later, another culture of the same species from
Miss Smith. We may call the first culture the "Jones" strain; the latter the
"Smith" strain. They mayor may not show temporary or minor differences
which are referred to as "strain differences." .
Clones. A clone is a strain of microorganism derived from a single cell
and, therefore, asexually propagated. Can sex appear in a clone? How?
Bacterial Nomenclature., The selection of the proper name for an organism
requires a knowledge of the rules of nomenclature, of previously used names,
and an exhaustive knowledge of the characteristics of the organism and of
similar organisms already named. The name of an organism is (or should be)
a descriptive symbo). It should convey a definite idea of the organism named.
This saves words and time and confusion. But it requires meticulous care
not to use the same name for different organisms, and to describe the organ-
ism itself fully and accurately.
In naming a bacterium, certain definite conventions are followed. Each
species is allowed a '~first" and "last" name only. The two-name scheme is
called the binomial system and originated in 1760 under the leadership of
Linnaeus. The first name of a bacterium refers to the genus~ and is usually a
Latin or latinized word generally a noun, based on the morphology of the
organism, the name of the discoverer or some other distinguishing character,
habitat, etc. It is written with a capital initial letter. The last name is the
species name and is usually descriptive of the noun, referring to its color,
source, disease production, discoverer or some other distinguishing point. It
is not capitalized. Genus and species names are generally italicized. For ex-
,ample, the name Bacillus anthracis indicates that the organism is a spore-
bearing aerobic rod (properties of the genus Bacillus), while anthracis calls
attention to the fact that this species of the genus Bacillus produces the disease
anthrax:. The name Spirillum rubrum shows that the organism is a true, sapro-
phytic bacterium, rigid, spiral in structure, non-sporeforrning, motile, and
gram-negative (all properties of the genus Spirillum), and that the species
named is characterized by a red color (rubrum). The name Clostridium novy;
indicates a gram-posi~ve, sporeforrning, rod-shaped organism, saprophytic or
parasitic, and restricted to growth in the total absence of free oxygen. These
are properties of the genus Clostridium. This particular species bears the
name novyi in honor of Dr. F. G. Novy of the University of Michigan, who
discovered the organi.sm and its relation to a disease, gas gangrene. The prac-
tice of using personal names for newly discovered species of bacteria is
obsolescent, althougli'many generic names are derived from the discoverers
or original students of the genus; for example the genus Salmonella, from an
American bacteriologist named Salmon; Escherichia from Escherich. a famous
German bacteriologist.
In writing of bacteria, it is customary to abbreviate the generic name,
using only the initial letter if it is clear what genus is meant; for example B.
anthracis for Bacillus anthracis. Sometimes the abbreviation may be longer,
as Br. abortus for Brucella abortus. Medical bacteriologists often dispense with
these rules and designate organisms by the disease with which they are most
frequently associated, for example, meningococcus, pneumococcus, typhoid
bacillus, etc. This is a convenient but loose custom not in accord with the
rules of nomenclature.
Type Species. There are certain central types of bacteria as, for ex-
ample, Streptococcus pyogenes. Bacillus subtilis, Clostridium butyricum, and
the like. Each of these is a well-known, thoroughly-studied, easily-identifiable
species representative of a genus or a group of species. It is spoken of as the
type species of that genus or group of organisms. Usually it is the first-de-
scribed member of the species. To every experienced bacteriologist each of
these names conveys a very definite idea as to the characters of the group.
However, ill-defined, partly-studied organisms distinguishable from the type
species, or from each other, only with tlle greatest difficulty or not at all, are
often included in such groups. On the other hand, organisms differing so "
markedly from the type species that the relationship seems very vague may
also be included. Endless arguments often arise concerning such matters.
Biochemistry and Classification. With the increase in knowledge of chem-
istry in general, there 'came an increase in knowledge of the biochemistry of
bacteria. Mter Koch had showed the way to pure-culture study, it became
possible to differentiate between morphologically identical organisms by their
biochemical properties, such as ability to ferment certain sugars, and so on.
These properties soon found their way into classification as the bases'of sub-
divisions of previously large and heterogeneous morphological groups. Today
many important distinctions are based on biochemical properties of morpho-
logically indistinguishable organisms.
Antigenic Structure and Classification. Briefly, antigens are molecules,
mainly of proteins but also of some carbohydrates which, when gaining en~
trance to the deeper tissues of the body, stimulate those tissues, to produce
similar molecules called antibody. Antibodies are found in the blood serum.
Antigen and antibody molecules are structurally reciprocally related, as.
mirror to image or key to lock. Antibodies combine with antigens which,
stimulate their production. All antigen-antibody reactions are highly specific;
that is, by virtue of the curious key-lock lelationship, the antibody produced
in response to a given antigen will combine only with that antigen. This
specificity offers' a tool for differentiating microorganisms by means of anti-
bodies evoked from tissues by antigens of the microorganisms, such as
flagellar proteins, capsular polysaccharides, etc. The method, often called
antigenic analysis, has been extensively used. In attempts to introduce ,still
greater accuracy into systems of classification, immunology* has been dra,wn
~pon. Striking biochemical and antigenic differences between organisms
... formerly believed to 'be identical have been found. These are often used to
• Immunology is in part the study of antigen-antibody relationships.

formulate new and finer and more accurately-defined groupings. For ex-
ample, all pneumococci were thought to. be identical until it was found that
the polysaccharides constituting their capsules fell into some 75 or more
distinct antigenic groups which are called types: Type I, Type II, etc. Such
types, based on antigenic differences are often spoken of as serotypes because
the antibodies used to determine the different types are found in serum. An
analogous situation was found among hemolytic streptococci. We now speak
of Group A or Group B hemolytic streptococci, etc., depending on the anti-
genic nature of their carbohydrates.
REFERENcFS
Bergey, et. al.: Manual of Determinative Bacteriology. 6th ed. Williams & Wilkins Co.,
Baltimore, Md., 1948.
Buchanan, R. E.: Taxonomy. Ann. Rev. Microbiol.,1955,9:1.
Gould, S. W.: Permanent numbers to supplement the -binomial system of nomenclature.
Am. Sci., 1954,42:269. ,
International Bul. of Bact. Nomencl. and Taxon. Iowa State College Press, Ames, Iowa.
Knight, B. C. J. G., and Standfast, A. F. B., Editors: Symposium on the principles of micro-
bial classification. J. Gen. Micr., 1955, 12:314.
Skerman, V. B. D.: A mechanical key for the generic identification of bacteria. Bact. Rev.,
1949,13:175. _
Van Niel, C. B.: The classification and natural relationships of bacteria. Cold Spring Harbor
Symp. on Quant. BioI., 1946, 11 :285.
12
Effect of Chemical and Physical Agents

on Microorganisms
THE WIDE distribution of life on the globe today is due to me tendency of

living things to change or mutate so that they may, if the properties of the
mutant form are appropriate, live in new situations or under new conditions "
to which the oider forms were not adapted. The mutants may, on the other
hand, be so ill-adapted, even to their initial situation, that conditions of life
prevent their multiplication or kill them. These two phenomena: (a) mutation
followed by (b) selection of those able to multiply in the environment are the
basis of evolution. As a result of evolutionary processes microorganisms have
appeared, capable of utilizing what seem to us the most indigestible of foods
(pure sulfur, kerosene, carbolic acid, etc.), and thriving in the (to us) most
remote, dismal and uninhabitable places, such as the depths of ocean ooze,
subterranean slimes, petroleum wells, hot sulfur springs and arctic seas, as
well as inside animal intestines, and in the tissues of animals and plants. Sp
complete and highly specialized is the adaptation that many forms of micro-
organisms perish when transferred suddenly to other environments. In this
chapter we shall discuss some factors in the environment which affect micro-
organisms.
'TEMPERATURE
Tempera~e and Growth. Temperature is one of the most important fac--
tors influencing all forms of life. The relations of temperature to the growth
of bacteria are somewhat complex. Many species indigenous to the soil,_
waters, and the animal body grow well at temperatures from 20° to 40° C
(human body temperature is 37° C). Species growing well at such tempera-
tures are called mesophilic (moderation-loving). Some soil and marine bacteria
and species pathogenic for fish or plants grow best at temperatures very little
above the freezing point (4° to 10° C) and these are referred to as psychrophili'c
(cold-loving). Certain marine forms, adapted to life at around 4° C, will die!if
held at about 30° C for more than a few minutes. These could be called obli-
gate psychrophils, i.e., they are limited to low temperature as a condition of
life. There are, in addition, several species of bacteria which thrive only at
high temperatures (45° to 75° C). Such species are called thermophilic (heat-
150
Effect of Chemical and Physical Agent& on Microorgani&m& 151
loving). Some species of thermophilic bacteria are found in hot sulfur springs
while others occur in milk, soil, manure, and related situations.
A distinction should be made in all cases between ability to endure a given
temperature and ability to grow well under the same conditions. Many
bacteria, for example, can live for months or years frozen in "dry ice," yet do
not grow at all. Thermoduric (heat-enduring) organisms survive well at higher
temperatures, but only thermophilic species grow well under such conditions.
It is worth noting that the optimal temperatures for growth of nearly all
microorganisms are near the upper, maximal limits of their range. Most
microorganisms have a wide tolerance to temperatures below optimal.
GROWTH TEMPERATURES AND ENZYMES. It is noteworthy that the optimal
and limiting growing temperatures for microorganisms, and indeed for all
living cells are, in general, the optimal and limiting temperatures of their
enzymes..
Enzymes are protein complexes within the cell which are basically re-
sponsible for the chemical reactions of life. Each vital chemical reaction in the
cell, and there are many in each cell, is mediated by an enzyme which is
specific for that reaction.
Now, enzymes have minimal, optimal and maximal reaction temperatures.
Some operate best at around 10° C. Such enzymes are found in cold-blooded
animals, arctic plants, psychrophils, etc. Others function best at temperatures
of the mammalian body (around 37° C); some at summer, outdoor tempera-
tures (20° C-40° C) or at temperatures of natural hot springs or the interior
of piles of actively "rotting" manure (50° C-75° C). The latter are found in
thermophils.
Some curious temperature effects on synthetic and other enzymes have
been noted with respect to nutrition. For example, cultivated at about 41 ° C,
a strain of Esch. coli was found to require, absolutelYl the vitamin pantothenic
acid. At 37° C it can synthesize this vitamin for itself. Some organisms were
found to utilize certain vitamins more efficiently at lower~than-optimal
temperatures. It is thus clear that, when we note a temperature as "optimal"
for a given organism, we must remember that the optimal temperature may
vary under different nutritive conditions. At optimal temperatures most of
the cell enzymes act favorably for the cell. As the temperature departs from
optimal, one after another enzyme ceases to function. Unless the metabolites
whose formation each enzyme mediates are supplied ready-made, the cell
ceases growth and may die.
At temperatures well below the optimum, enzymes function more slowly
or not at all. This is, in part, because low temperatures generally cause in-
creases in viscosity bf fluids, hardening of lipids, and slower chemical re-
actions. Growth is: retarded or inhibited, but low temperatures are not \.
destructive as are higb temperatures.
Thermal Resistan~e. With respect to mere survival of heat, most micro-
organisms in an actively-growing (vegetative) state are readily killed by ex·
posures to temperatures of around 70° C for I to 5 minutes. A good'many are.
killed by 10 minute~ in water at as low as 54° C; the diphtheria bacillus
(Corynebacterium diphtheriae) for example. The conditions of commercial
pasteurization (62° C for 30 minutes) kill most vegetative pathogens in milk,
including tubercle bacilli (Mycobacterium tuberculosis). Numerous sapro-
phytes in milk survive pasteurization. Thermophils are quite resistant, some
vegetative cells surviving 80° C to 90° C for as long as 10 minutes. Boiling
kills all non-sporeforrning bacteria in a few moments.
Bacterial spores are much more resistant, as has already been pointed out.
Spores may survive boiling or higher temperatures for many hours. Killing
of spores is more fully discussed in the chapter on disinfection.
THERMAL DEATH POINT. The temperature at which all the bacteria of a
given species are killed after ten minutes' exposure is sometimes spoken of as
the "thermal death point" of that species. This is an inexact expression be-
cause, at a given temperature, say 70° C, the bacteria in a given culture or
. situation do not all die simUitaneously and suddenly just as the clock registers
the expiration of ten minutes. Unless the temperature is catastrophic, as
when dropping a culture into a furnace, "thermal death point" merely tells
us when the last survivor of all has expired, the number of live cells having
been decreasing continuously during the exposure period. Further, in meas-
uring thermal death point much depends on the species and n,umbers of
bacteria originally present, the age of the "bacteria, the acidity of the sus-
pending fluid, its osmotic pressure, its composition, etc., so that the term
"thermal death point" of a certain species can be correctly used only if the
exact conditions of a given experiment are known. The time required to kill
in a moderately acid fluid, such as tomato or orange juice for example, would
be very much shorter than in a neutral or slightly alkaline fluid such as :milk [
or serum. However, for certain purposes, where the exact conditions are care-
fully controlled, information on thermal death points is of practical"use, as in' I
the commercial preservation of certain foods, milk, etc. f
THERMAL DEATH TIME. This is the length of time required to killi all of the
bacteria of a certain species in a given substance at a stated temperature. The
thermal death time of various species of microorganisms is influenced' by the
"sal,le factors that affect thermal death point, but uhder known con,ditions
knowledge of thermal death time is of practical use. Thus, in pasteurizing,
milk is heated to 62° C and held there for thirty minutes. This is considerably
in excess of the thermal death time, at 62° C, in market milk, of all the patho"
genic microorganisms known to be commonly transmitted by milk. Many
non-pathogenic bacteria survive pasteurization so that pasteurized milk is
safe but not necessarily sterile. 1 -
Rate of Death. As noted 'above, death of all the bacteria of any given
species in a given material do,5!s not occur simultaneously unless the lethal
influence be overwhelming. On the contrary, death occurs in a definite rela-
! tionship to time, the rate being determined by various factors such as numbers
and kind of cells 'initially present, temperature, moisture, acidity, etc.
Mature (not senescent) cells are more resistant than young ones. Moisture,
and acidity greatly increase the vulnerability of cells to heat. Cells inside of
solid material such as mucus or canned meats may escape heat longer than
the same cells in distilled water or in broth, becauSe the heat does not pene-
trate immediately into the center of solid masses.
Most killing processes, whether by heat or chemical disinfectant, behave
like a monomolecular" chemical reaction, progressing in accordance with
.. A monomolecular reaction is one in which only one kind of molecule is broken down
or changed into one or more other molecules.
Effect of Chemical and Physical Agents on Microorganisms 153
the laws governing mass action. If the numbers of organisms surviving in a
disinfection test are determined at various intervals, and plotted against the
intervals, regular curves are formed. If the logarithms of the numbers are
similarly plotted, a straight line is formed (Fig. 12-1). Whether killing proc-
esses really are monomolecular reactions is not certain. Probably they are
complex reactions whose net result simulates a monomolecular reaction.
Under certain influences these theoretical straight lines may be much dis-
torted; rapid at first, slow toward the end, and vice versa.
Extreme Cold. Many species of bacteria are highly resistant to extremes
of cold, even when in the vegetative state. In this respect they are almost
unique among living things and it may be due to this that certain species of
bacteria may have survived glacial epochs in the earth's history. Many
species, even parasites like the typhoid organism, will survive (not grow) for
weeks frozen in ice. The fragile syphilis spirochetes and numerous viruses
have been maintained frozen in "dry ice" at -76 0 C (- 169 0 F) for years
without loss of infectivity. Some species of bacteria will grow, apparently un-
0
affected, even after subjection to the temperatures of liquid hydrogen ( - 252
Cor -486 F). Larger cells than bacteria are often entirely disrupted by the
0
crystallization (freezing) of their fluids but some protozoa and metazoa and
0
sperm cells have been held at - 70 C for long periods.
HYDROGEN ION CONCENTRATION·
Another factor profoundly affecting microorganisms is the acidity or alka-
linity (reaction) of the fluid in which they are suspended. In biology this is
usually expressed in terms of hydrogen ion concentration (PH) since it is the
, z 4 6 7 8 9 '0
MIN,UTES
Fig. 12-1. Relation I between numbers of organisms and time in a 1 per cent solution 01
phenol containing'typhoid bacilli. The logarithms are of numbers of bacilli per mt The
straight-line curve is characteristic of the relation between time and survivors under any
definitely adverse conditions when no growth occurs.
• It is assumed the student has a working knowledge of pH and its determination. It may
be reviewed in any good textbook of elementary physics.
154 Methods and Phenomena oj Microbiology
concentration of dissociated or ionized hydrogen (or hydroxyl) that determines
the biologically-effective acidity (or alkalinity) of a given solution.
pH IN MICROBIOLOGY
It is important for the microbiologist to remember that, as a rule; increases
in temperature cause increases in dissociation of acid hydrogen so that a
solution which is neutral or slightly alkaline at room temperature (about
22 0 C) may become definitely acid at incubator temperature (37 0 C). If a
solution is prepared at a definite pH while near the boiling point, it will be
more alkaline when cool. It is also important to note that a change in pH
equivalent to 1 on the pH scale, represents a 10-fold change in hydrogen ion
concentration, while a change from pH 7.0 to 7.3 represents a 2-fold change
(Table 8).
Table 8. Relationships of Hydrogen Iort Concentrations Expressed in Various
Ways.
REACI10N I FRACI10N OF
NORMALITY·
HYDROGEN IONS PER
LITER
(grams)
LOGARITHMS
OF HION
CONCENIRA-
EXPRESSED
AS pH
nONS
Acid .........
Acid ........ ,
Nil
Nlto
1.0
0.1
-- °1 0.0
1.0
Acid ......... Nll00 O.ot - 2 2.0
Acid ......... Nll,OOO 0.001 - 3 3.0
Acid ......... N/to,OOO 0.000,1 -,4 4.0
Acid ......... N/lOO,OOO 0.000,01 - 5 5.0
Acid ......... N/l,OOO,OOO 0.000,001 - 6 6.0
Neutral. ..... Pure water 0.000,000,1 ,- 7 7.0
Alkaline ...... NIl ,000,000 0.000,000,01 - 8 8.0
Alkaline ...... Nll00,OOO 0.000,000,001 - 9 9.0
Alkaline ...... NI 10,000 0.000,000,000,1 -10 10.0
Alkaline ...... NI 1,000 0.000,000,000,01 -II 11.0
Alkaline ...... N/lOO 0.000,000,000,001 -12 12.0
Alkaline ...... NII0< 0.000,000,000,000,1 -13 13.0
Alkaline ...... NIl 0.000,000,000,000,01 -14 , 14.0
I
* With respect to hydrogen or hydroxyl ions.

Unfavorable influences of many sorts may be much enhanced in fluids
having an a.cjd pH. For example, coagulation by heat occurs more readily
in acid solutions. Thus, milk which is only very slightly sour may curdle on
being warmed. I
Enzymes are as sensitive to alterations in pH as they are to temperature,

or even more so. They have definite minimal, optimal and maximal zones and
limits in respect to pH, just as they do to temperature. What is true of enzym~s
is largely true of living cells. The reaction of bacteriological culture media
must, therefore, be very carefully adiusted with respect to pH, the degree <of
acidity depending on the bacteria to be cultivated.
In addition to controlling enzyme action the pH undoubtedly affects the
state of colloidal suspension of protoplasm, the permeability of cell wall and
membrane, and indeed, every important physiological function. The vast
maiority of living forms, plant, animal, and microorganismal, have an
optimal pH near the neutral point of pH 7. As with temperature, some
species have become adapted to grow at a pH considerably at variance with
this. Some of these will be mentioned later on. A range from about pH 6.0
(slightly acid) to pH 8.0 (slightly alkaline) includes the optimal ranges of the
vast majority of species.
BUFFERS AND BUFFER ACTION
In the ordinary titr!ltion of, let us say, 500 ml of N/l0 acid with N/l0
alkali in distilled water, using phenol red as indicator, there comes a point in
the titratiQn when a single drop of NJI0 alkali from the buret changes the
whole volume of fluid from distinctly yellow or acid (PH around 3.0) to defi-
nitely alkaline as evidenced by the appearan~ of a deep magenta color
(PH around 10). Thus, a single drop ofN/lO alkali (or acid) produces a very
large alteration in pH.
Now, it is often necessary in microbiology to change the pH of culture
fluids ("adjust the pH"). In attempting an adjustment of the pH of bacterio-
logical media such as meat-infusion broth it is found that, unlike the titration
of the aqueous solutions of acid or alkali, no sharp end point is reached.
The change from an acid to an alkaline reaction or vice versa is very gradual,
requiring the continuous addition. of relatively large amounts of acid or
alkali; commonly thousands of times as much N/l0 acid or alkali as an aque-
ous solution of inorganic acid or base. In other words, even at, or near, the
neutral point, the solution being titrated shows a marked tendency to resist
any change in its reaction. This is due to what is known as "buffer action"
on the part of certain constituents of the broth.
These buffer substances, neither strongly acid nor alkaline in themselves,
combine with the acid or alkali as fast as it is added,and so prevent change
of reaction of the medium until they are all used up. The same sort of buffer
actions maintain the pH inside every living cell within very narrow limits
and is absolutely essential to life.
There are several sorts of substances which have the property of acting as
buffers. Important among these, from a biological standpoint, are amino
acids and their compounds, the polypeptides, proteoses, proteins, etc. These
are neither strongly acid nor alkaline but will combine with either acid or
alkali.
To cite a simple example of buffer action by an amino acid, we may note
that glycine combineS with Hel or NaOH as follows:
H H H2.H
"'-/ ~/
N N".
H-LH + HCl-+ H_J_~I

Ho...-Lo Ho-L
Glycine Glycine hydrochloride
or, I,
CH2(NH2)COOH + NaOH - + CHz(NHz)COONa + H 20.
Glycine Sodium glycinale
One of the most commonly used buffers in biological laboratories is a
mixture of the monobasic and dibasic phosphates, KH2P04 and K2HP04, in
aqueous solution. These dissociate relatively little in aqueous solution. When
acid is added the dibasic salt absorbs H+, changing into the monobasic and
forming a potassium salt with the acid. When alkali is added, the monobasic
salt gives up H+ to form H20 with the OH-, and the incomplete salt combines
with the cation of the alkali.
Other important buffer constituents of organic media are carbonates and
bicarbonates:
gas
/'
Na2C03 + 2HO' ----+ 2NaO + H20 + IC021
NaHC03 + NaOH ---> Na2C03 + H 20.
Lumps of marble (CaC0 3) are sometimes added to cultures containing
fermentable substances like glucose, sucrQse or lactose, to maintain the pH
unchanged, that is, to neutralize the acid produced by fermentation. Other-
wise the organisms in the culture would quickly cease growth or die. Colloidal
substances such as charcoal, precipitates of phosphates, kaolin, etc., which
adsorb hydrogen or hydroxyl ions directly, are also active buffers and are
used in biological systems.
OTHER IONS
The hydrogen and hydroxyl ions are not by any means the only ones having
a marked influence on microorganisms. The well-being and life of all cells is
dependent on the absence or presence, and, suitable concentration, of ions
of many different sorts.
Cations. Ions of heavy metals, such as gold, silver, lead, nickel, tellurium,
copper, mercury, and iron are usually more toxic to bacteria than are ions of
light metals, such as sodium, potassium, and calcium. However, generaliza-
tions are difficult. Toxicity of cations depends greatly on the species of micro-
organism being dealt with; the presence of chelating* agents and other sub-
stances (such as H 2S) with which the metal ions can combine; pH; etc. As a
rule, chlorides of light metals like sodium, potassium, lithium, strontium,
magnesium, calcium and of ammonia are, harmless and even desirable and
stimulating to most bacteria in concentrations up to about ].5 per cent. On
the other hand, chlorides of many heavy metals (Hg, Cu, Fe, Pb, Zn) usually
exert definitely harmful effects, even in very low concentrations (HgC12 ,
0.0007%).
The less toxic salts act favorably in 4 great variety of ways, sometimes by
effectively suppressing the ionization of unfavorable substances, or by reacting
with these substances so as to prevent them from affecting bacteria un-
favorably. For example, the presence of calcium chloride is effective in re-
ducing the toxicity of sodium oxalate, by forming an insoluble precipitate of
calcium oxalate, thus removing the toxic oxalate radicle from' the solution.
• From the Greek word, chela, for claw. Chelating agents are generally thought of as
orianic compounds which form a complex compound with a heavy metal, enclosing it in a
molecular, ring-like structure as though held in a claw. The position of iron in the porphyrin
group of hemin is a typical example (Fig. 12-2).
The magnesium ion (Mg++) appears to be of special importance. It has
been shown to lower the toxicity of Ni, Co, Zn and Mn for several species of
bacteria. It appears to be an essential factor for some species, having a con-
trolling effect on cell fission. It has already been mentioned as having a critical
relationship to the Gram stain. Many ions, including Mg++, probably act
also by affecting the permeability of the cell membrane and its electrical
charge. Mg++ is known also to be neCessary to the functioning of several im-
portant enzymes in mimy b~cterial species.
It appears that certain divalent cations (Ca++, Mg++, Sr++) are essential to
the penetration of bacteriophage into staphylococci (Micrococcus), possibly
by activating an enzyme in the tail of the !phage particle. Cobalt (Co++), a~so,
is apparently essential to the action of certain enzymes in bacteria. Iron
(Fe++) is involved in the activation of other enzymes. Some enzymes, on the
other hand, are inhibited by heavy metal ions (Hg++, Cu++, Fe++ and Zn++)~
ION ANTAGONISMS. Some cations are biologically antagonistic to each
other. For example, Li+ and Zn++ are definitely toxic to certain bacteria of
importance in the dairy industry (Lactobacillus and Leuconostoc). These ions
appear to drive H+ from its normal position, and to replace it in certain
enzymes. They are said to antagonize the H+. By increasing the concentration
of H+, this toxicity of Li+ and Zn++ is reduced.
As will be seen later, knowledge of this sort of antagonism and competition
CH,
.. ./N'
./
"~'Fe:: ",
.N
elI, CR. CH.

I I
CH. CH,
I I
CooH CooR
Fig. 12-2. The hemin molecule.
Cheiating agents, by combining with harmful metallic ions, perform a function analogous
to that served by buffers ih maintaining pH. Indeed, hydrogen ions are analogous to metallic
ions in such situations. Some pH buffers also act as chelating agents, for example, amino
acids, certain vitamins, etc.
158 Method& and Phenomena of Microbiology
of certain substances for key physiologic positions in living cells is of funda-
mental importance in disinfection, the action of chemotherapeutic drugs like
sulfanilamide and antibiotics, as well as for industry and other activities. It
will be well to keep it in mind.
Anions. The desirability or toxicity of any compound, from the stand-
point of microorganisms, is determined in part by the anion as well as by the
cation. In order of favorable action with regard to bacteria we may list several
anions (with sodium as the cation) as follows: sulfate, tartrate, chloride,
nitrate, acetate, citrate, oxalate, iodide, benzoate, salicylate, tellurite; sulfates
the least toxic, tellurites the most unfavorable.
The toxicity of alkalies varies, but depends mainly on the concentration of
hydroxyl ions (pOH). The cation of alkaline substances also plays its role, as
it d,oes in the action of acids and salts. This 'is illustrated by the fact that
barium hydroxide, a much weaker (less dissociated) alkali than sodium hy-
droxide, is nevertheless much more toxic at the same concentration of hy-
. droxyl ions. This is partly because the barium ion is more toxic than the
sodium ion.
It is very important to remember that species vary greatly in respect to
sensitivity toward various ions.
Complete Molecules. The biological activity of most compounds, in-
organic as well as organic, depends not only on anion and cation but also on ..
the undissociated molecule. For example, the toxic effects of benzoic and
acetic acid are much greater than would be expected from equivalent concen-
trations of H+. Thus, hydrochloric acid and sulfuric acid, although "strong"
acids, are much less poisonous to most bacteria at a given pH than benzoic
acid or acetic acid at the same pH. About 7.5 to 7.7 parts per million of the
first two are required to produce the same toxic effect as 0.1 and 1.2 parts per
million, respectively, of the last two. The reasons are not clear. The important
point here is that one can never, with certainty, predict the effect of a sub-
stance on a given species of microorganism without some previous knowledge
of biological action of that substance, supplemented by direct experimental
test.
l.\-fAGNETISM
The subjection of microorganisms to magnetism has not been demonstrated
to exert any important influence on them.1
- ELECIRICITY
The passage of an electrical current through a suspension of microorgan-
isms probaoiy has little effect by itselLIf a current of great intensity is passed
for a long time" however, electrolysis of some of the constituents of the
medium will result, their nature and concentration depending on the voltage,
and the composition of the medium and of the electrodes. Some of the~e
products of electrolysis have deleterious effects. Heat, also, will be generated
and, if sufficient, may kill the organisms.
Electrophoresis. The student familiar with colloids knows that any very
minute particles, including microorganisms, when suspended in 'aqueous solu-
tions, acquire an electrical charge on their surfaces. Therefore, when an
electrical current is passed through a suspension of such particles, those with
a negative charge travel toward the positive electrode (anode), while those
with a positive charge travel toward the negative electrode (cathode). Those
with a strong charge travel faster than those with a negative charge. This
migration is termed electrophoresis.
The electrophoretic migration of microorganisms may be observed directly
with a microscope focussed on a thin, hollow cell with electrodes at each
end (Fig. 12-3) or by testing for their presence in the legs of a U-tube with
electrodes at each leg, the particles being introduced at the middle of the U.
Other methods (paper electrophoresis, the Tiselius moving boundary method)
are also used.
This phenomenon has been used in the study of the electrical charge of
various microorganisms. For example, it has been shown that a modified
form of yellow fever virus with special affinity for nervous tissues has electro-
phoretic properties which differ markedly from those of the mosquito-borne
form of the virus. Pathogenic micrococci migrate differently from micrococci
of little pathogenicity. Different mutants in a culture may be differentiated by
their rate and direction of electrophoretic travel.
MOISTURE AND DESICCATION
It is obvious that abundant moisture is necessary for active, vegetative
existence of all things. Most large plants and animals require abundant water
at all times. Some plants in desert areas and many species of microorganisms,
however, can survive complete drying or desiccation for long periods, though
they do not grow under such conditions. Many substances such as hay, fruits,
fish, meat, etc., "preserved" by drying, contain large numbers of living bac-
teria which are dormant but which soon grow and cause spoilage if the dried
product becomes moist. On the other hand, some microorganisms, especially
delicate pathogenic species, are quickly killed by dying. Spores, conidia,
/
)"'-_ _..J ~ '--_--'~
Fig. 12-3. The essential parts of an electrophoresis, cell. A, reservoir of the suspension
of bacteria to be observed. B, outlet tube, for passing off water used in flushing out the appa-
ratus, etc. C, D, reservoirs for saline solution bathing the electrodes and canying the electric
current from them to the suspension of bacteria which extends from A through J, G and K.
E, F, electrodes. G, the ;electrophoresis chamber proper within which actual microscopic
observation of electrophoretic motion is made. H, I, glass-insulated platinum electrodes for
measurement of the potential gradient across the electrophoresis cell. J, K, three-way stop
cocks controlling flow of electrode fluid, bacterial suspension, or wash water.
160 M.thod& IUId PIwIfoIMIfIl of Microbiology
artbrospores, etc., of course, are cells specially adapted to withstand drying

for long periods without any special assistance.
DesIeeatlon .... Vacuum. Not only do many kinds of bacteria in a vegeta-
tive state withstand desiccation, but they may be frozen at sub-arctic tempera-
tures and subjected to the highest possibJe vacuums without harm. These facts
are of the greatest interest from both a philosophical and practical stand-
point.
In a state of virtually complete desiccation and in a high vacuum, there must be an unim-
qiDably small vital activity. The metabolic processes must stop almost completely since
theIO depend largely on osmosis, diffusion, ionization and the colloidal state, all of which
arc depeodent, in turn, on hydration. How, then, can bacteria exist IrozieD and desiccated in
a vacuum? How can living thinp surviw. as it seems, in the entire abseoce ofvitaJ activity?
There arc no known answers to these questions. The.condition of bacteria froan and desic-
cated in a vacuum, must be as near an approach to suspended animation as can be imagined.
If time (for human beings) be measured by succ:eaaive events such as heart beats, thought
traiD, action of enzymes, wars, etc., then, when these ~vities or their dfects cease to be
obIerved, time ceases for tire individual involved. It begins again only when the individual
COIIICiousness again begins to pace exterior events or when vital activity produces the
efJects of age. A penon deeply preoccupied by study,· pain, play or worry, or who has
been wholly unCOIIICious, or in an abnormal mental state (delirium), in which COIIICious-
ness of the course of events is temporarily lost, has a different idea of time from that of
normal persons, or at least a very distorted one. An analogous situation exists for bacteria
in a ~ vial; since they have no COIIICiousness of the pasa8ae of time and appear to
underao no physiological aging, when the air and moisture are withdrawn from their environ-
ment time may be imagined as ceasing to exist for them. Time (for them) begins again only
when air and ftuid arc added. In the vacuum jar their chronological aae may become great,
as ~ by human standards, but they apPear to remain physiologically young and
unchanaed even for many years during their sojourn in vacuity.
PRFSERVATION OF BACI'ERIA
1. DesiecatloD In Vacuo. From a practical standpoint, the survival of
bacteria when desiccated in a vacuum is of great importance. In the labora-
tory, in the past, in order to preserve pure cultures of non-sporeforming
bacteria. it was necessary to transfer the organisms frequently to fresh culture
medium, incubating and then maintaining them in a vegetative state in the
refrigerator. This procedure involves danger of contamination of the cultures.
and alterations in character due to changing environment and mutation.
These difficulties as well as loss of time, labor and cost of such methods can
be eliminated where it is known that the species will withstand desiccation in
vacuo. Simple methods of drying and preserving bacteria in vacuo have been
described. Once preserved by desiccation in vacuo a large number of cultures
may be stored in a pint fruit jar and many bacteria will survive for years.
Species of pathogenic streptococci, for example, have survived unharmed for
twenty-five years, while diphtheria bacilli survive for fifteen years; tubercle
bacilli for 17 years. Many can survive much longer under these conditions.
However, while many species of bacteria resist vacuum and drying this is not
true of alJ.
2. FreezIng. Many bacteria will survive in the frozen state at -760 C that
do not withstand desiccation. "Quick freezing" is done by placing the organ-
isms in a suitable suspending fluid (blood? serum, milk. glycerol, etc.•) in a
small tube and immersing in a bath of "dry ice" (solid CO2) in alcohol. Freez-
• For example, serious students of microbiolo&Y!
E,If.ct of CMmical and Phpical Agent' Oil MicrooTgtuW1fU 161
ing must be almost instantaneous to avoid the mechanical disruption which
would occur if time were allowed for the formation of large ice crystals. The
tube is then promptly removed to a storage chest of dry ice ( - 76° C).
This method is in routine use for the preservation of bull sperm for cattle
breeders, human sperm for artificial insemination, cultures of cancer cells for
cancer and virus research, animal parasites of various sorts, viruses, bacteria
and so on.
3. Freeze..clrying. A method of pn:serving very unstable substances such
as living microorganisms, enzymes extracted from the inside of living cells,
bacterial toxins, etc., all of which are inactivated on storage in a moist state
or by exposure to the air, depends on (a) extreme cold; (b) rapid evaporation;
and (c) vacuum. The materials to be preserved are suspended in a suitable
fluid and distributed in small glass ampules connected to a high-vacuum
pump. The contents of the ampules are frozen almost instantaneously by im-
mersion in a bath of dry ice in methylcellosolve (- 76° C). The frozen con-
tents of the ampules are then dried from the frozen state by the application of
a high-vacuum. When all vapor has been removed, the necks of the ampules
are sealed with a needle of flame before destroying the vacuum. The product
is a highly lyophilic (water-loving) powder (Fig. 12-4).
meets of Autolysis. In the preservation of microorganisms by freezing,
desiccation or other such means, the preserving agent must act quickly in order
Pig. 12-4. One form of f~ apparatus. The desired microorganisms from

appropriate cultures are emulsified in blood serum. Each suspension is then placed inside
• sterile, narrow, labeled, glass tube (mounted in wooden holder at left). Tho cotton plugs
are re-insened and the tubes, lubricated with a tiny drop of oil, are inserted in the rubber
.~ of the apparatus as shown. They are then immersed in the rectanauJar bath (below
the tubes) containing alcohol or MethyJcelJosolve and "dry ice" (- 76° C) where the
ImIIIl freezes almost instantly. The vacuum pump is started immediately. The water is sub-
limated from the serum and, as the ice bath is warmed to -So C, drying is compJeted in a
few minutes. Moisture is condensed and held frozen in a dry-ice trap before it reaches the
V8CUUD1 pump. The lower portion of each tube is then sealed off by means of the two-jet,
CI"OII8-fire, oxy-acetyJene torch while evacuated. Tubes at the right of the apparatus have
aIRady been seaJed off and the lower portions, with the dried suspensions, are shown laid
in a row on the table. (Photo courtesy of Drs. W. C. Haynes, L. J. Wickerham and C. W.
IIeaeItine, Northern Utilization Research Branch, U. S. Department of Apicuhure.
..."., ill., from AppL Micr., 19S5, voL 3.)
to prevent deleterious effects due to autolysis. Autolysis is a process of dis-
solution, due to inherent enzyme-like mechanisms, which takes place in dead
(or inert, live) cells. Autolysis may occur if cells undergoing preservation are
held at the threshold between active and inactive existence too long.
OSMOTIC PRESSURE
If living cells are immersed in fluids having extremely high or low osmotic
pressures, water will be drawn out till the cell collapses, or forced in till the
cell ruptures.
These effects of unfavorable osmotic pressures are plasmolysis and plas-
moptysis. Largely due to their minute volume and thin cell wall, most bacteria
are not highly sensitive to variations in salt concentrations between about 0.5
and 3 per cent. Concentrations much above this may adversely· affect some of
the more sensitive strains. Much depends on the previous environment of the
organisms. Some marine bacteria, adapted to the salinity of ocean water
(about 3.5 per cent) are quite sensitive to lower or higher salinities and will
not grow if the salinity is less than about 2 per cent or over about 15 per cent.
On the other hand, no colonies will be formed by certain fresh-water species
in media with salinities in excess of 1 per cent. Bacteria from either source,
however, may be adapted to the environment of the other. Spores do not
appear to be much affected by changes in osmotic pressure. 4
Halophilic Organisms. There are bacteria which have become adapted to

the high salinity (around 29 per cent) of various salt waters such as the Dead
Sea and the Great Salt Lake of Utah. These organisms will not grow in lake
water diluted to a salinity ofless than 13 per cent. Such organisms are spoken
of as halophilic (salt-loving). On the other hand, the growth of soil, sewage or
mouth bacteria is inhibited by Salt Lake water and ordinary marine, bacteria
are killed by a few minutes' exposure to it. There are halophilic bacteria which
grow in commercial pickling brines. Some are a cau'se of spoilage of various
commodities preserved with salt, such as fish, meat, and hides. Concentrations
of salt of around 25 and 30 per cent are used in these brines. Just how much
of the effects of such brines are due to ions per se and how much to osmotic
pressure it is difficult to be sure. UndOUbtedly osmotic effects play the major
role in brines of NaC!.
Sugar syrups !hust be much more concentrated (around 60 per cent by
weight of sugar) to achieye the same preserving effects as salt brines (around
20 per cent). This,is probably due to the larger molecular size of sugar and the
combined ionic effects of salt:
Effects of Evaporation. When a solution containing salt or non-volatile
compounds is exposed to the air so tha,t evaporation occurs slowly, the water!,
departing, leaves behind a more and more concentrated solution, the osmoti9
pressure becoming greater and greater. Microorganisms suspended in such ,iI.
fluid are unfavorably influenced by the prolonged effect of increased osmotic
tension. In the cultivation of any microorganisms, therefore, excessive evapo-
ration of moisture from the medium is to be avoided. This may be accoII;l-
plished by maintaining a very humid atmosphere in the incubator..
Alterations in Perme,ability. Even though the osmotic pressure of a given
culture medium may be appropriate, there often appear in it, as a result of
aging, substances that alter the permeability of the cell wall in some way so
that the cells become swollen and distorted. Such alterative substances may be
the waste products (acids, alcohol, etc.) of bacterial cells which have grown in
the culture. The cell wall may also become altered through aging so that it
fails to function properly, very likely permitting the passage outward of im-
portant cell constituents and the passage inward of water or of deleterious
compounds and probably, through localized weakenings, causing the swell-
ings, knoblike protrusions and other irregularities often observed in bacteria
in old cultures.
Invo]ution Forms. Some distorted forms of bacteria are generally spoken
of as "involution forms." In certain species (e.g., V. comma) such distortions
may be brought about to a very striking extent by cultivation of the organisms
on media containing high concentrations of sodium chloride.
Some modern students, however, attach importance to some of the irregular
forms of bacteria as representing stages in life cycles and complicated repro-
ductive processes. In some species they probably represent stages ("large
bodies") in the life cycle of pleuropneumonia-like organisms (L forms).
RADIANT ENERGY
The Electromagnetic Spectrum. As seen in Figure 12-5, electromagnetic
radiations may be arranged, according to wave length, in a spectrum which
includes the visible solar spectrum (red to violet), the latter being a small part
of the whole system of electromagnetic waves. The physiological effects of
these various radiations differ greatly, those in the shorter ranges being of
most known importance. Very long waves are used in radio.
The longest waves having known physiological effect are (l) the infra-red or
"heating waves." (2) The next shorter are the visible red rays, which affect
the retina of the eye and also bacteriochlorophyll. (3) Then come, successively,
yellow, blue and violet, which affect the retina and have photodynamic and
possibly other biophysical actions; then (4) the invisibkultraviolet rays, which
are shorter than the visible violet and have marked physiological and bio-
chemical activities to be discussed. Still shorter waves of known properties
are (5) x-rays ("soft" and "hard"); (6) alpha, beta and gamma rays, and
finally (7) the little understood cosmic waves.
BIOLOGICAL EFFECTS OF IRRADIATIONS
Effects of Irradiations. Radiant energy, especially soft x-rays and ultra-
violet light which are absorbed by protoplasm, produce two principal effects
on living cells: (a) lethal; (b) mutagenic. These two are not the same and often
vary independently. :
In irradiating a cell we may divide the events into three periods: (1) the
actual instant or period of irradiation during which the ionizations and/or
excitations occur; (2) the middle or latent period, after cessation of the irradia-
tion, before the results;of that irradiation become apparent. It is characteristic
of biological radiation: effects in general that there is a delay between irradia-
tion and appearance of the effect: death or mutation. (3) the final period,
during which the effects of the irradiation become evident.
Actively multiplyin~ cells are most susceptible to irradiation. They are
usually the most susceptible cells to other deleterious agents also: 'phage,
antibiotics, various bacteriostatic agents, heat, etc. Bacteria in a dormant
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Effect of Chemical and Phy~ical Agents on Microorganisms 165
condition, or in a mature, slow-growing stage are markedly resistant compared
with fast-growing cells.
Mechanisms of Irradiation Effects. When radiations like soft x-rays are
absorbed by living cells the energy is dissipated by removal of electrons from
atoms, thus ionizing those atoms. These ions appear to be the agents which
actually mediate the more important effects of ionizing radiations, which are:
(a) genetic; and (b) lethal. Ionization may occur in a molecule of actual
protoplasm, in which cases the effect (genetic or lethal) of irradiation is said
to be direct. The direct effects appear to be due mainly to impingelllent of
energy on nucleoproteins. These absorb radiant energy directly. If the ioniza-
tion occurs in the materials such as enzymes, etc., dissolved in the fluids sur-
rounding the actual protoplasm, the resultant effects are said to be indirect
(genetic or-lethal). Genetic effects (mutations) appear to be mainly direct
effects. Lethal effects could be direct or indirect.
The effects of irradiation are complex but appear to be related largely to
biological oxidations. Conversely, chemicals and physical agents which inter-
fere with biological oxidations markedly interfere with the effects of radiation.
ULTRAVIOLET RADIATION AND EXCITATION. Though we speak of irradi-
ation in a general sense it should be noted that there are differences in action
and effect of the various types of radiation. These need not be detailed at
present. For example, x-rays contain higher energy than ultraviolet and their
effects are due to atomic disruption and consequent ionization. Ultraviolet
rays cause excitation but not ionization and their .effects are due more to a
generalized distribution of excited atoms than to ions.
Recovery from Irradiation. Studies on prevention and recovery from ir-
radiation effects are encouraging with respect to atomic warfare, accidental
irradiation, sunburn, etc. Death of cells due to irradiation is often (as Mark
Twain remarked about premature reports of his own death) "grossly exag-
gerated." Cells may ,be revived or, more correctly, reactivated after apparent
death due to irradiation.
One method is exposure to visible violet light in the wave length zone
around 4500 A. Similar reactivation has been demonstrated for some viruses.
This is often called photoreactivation. Another method is incubation of the
irradiated cells in a favorable growth medium at temperatures 5° to 30° C
lower than optimal. A third is by treatment of the cells with various chemical
agents: indoacetate, pyruvate, and a long list of others. Many theoretical ex-
planations of these curious phenomena exist but none is proven and we may
at present only contemplate the facts and refrain from theorizing unless, like
Sherlock Holmes, w,e expect to put the theories to experimental proof.
There are so many variables which affect the reactivation: species of micro-
organisms; the culture medium; age and physiological state of the cells;
chemical composition of the test medium after irradiation; pH and tempera-
ture during and after irradiation, and so on.
Special Uses of Ultraviolet Light. Ultraviolet light, like visible light, con-
sists of a spectrum of different wave lengths from about 4000 Angstrom units
(A) down to about 200 A (see Fig. 12-5). Those between 2800 A and 2300 A
are most effective b,lologically, the most effective length depending to some
extent on the species of organism tested and the conditions of test. Ultraviolet
is an effective component of sunlight and produces "sun tan" and "sunburn."
r

, r
It also acts on chlorophyll. Ultraviolet light has very slight powers of penetra-
tion. It passes through ordinary glass very little. It is therefore effective
mainly at surfaces.
From the microbiologist's viewpoint the most obvious, but not the most
interesting, biological effect is death. Microorganisms of most sorts, if irradi-
ated with sufficiently intense ultraviolet (in the range of 2650-2750 ") (or
sunlight) for longer than a certain time (the limits depending on the organism
and the environment) die. This is valuable information and has been put to
practical use in medical, public health, agricultural, commercial and research
applications. Radiating tubes giving off light of wave length 2537 A are used
in preventing surface spoilage of meat, tobacco, bakers' goods and the like,
due to molds and bacteria. Other uses of the radiations are the disinfection of
glassware in restaurants, of "conditioned" air, the air in schools, hospitals and
operating rooms, and in the control of insects (see section on disinfection).
Use is sometimes made of ultraviolet light in the treatment of water in swim-
ming pools and for drinking but is not generally approved for public water
supplies. A method of sterilizing serum and vaccines for use in prevention and .
cure of disease exposes the serum or vaccine in very thin films, to the ultra-
violet light.
It is of interest to note that the effects of ultraviolet radiation are different
from those of heat, since very thermostable spores are easily killed with ultra-
violet light. Ultraviolet light is as effective in the absence of oxygen as in its
presence.
Ionizing Radiations and Food Preservation. One of the most important
developments in the study of lethal effects of ionizing radiations is their value
in food preservation. Foods like meat pies, complete dinners, hamburgers,
etc., now sold frozen (either cooked or raw) or cooked and packed in cans,
may be completely sterilized and held indefinitely at r90m temperature,with
no refrigeration, in a "fresh" state if properly irradiated after packaging. The
prospect should give the refrigerator and "deep freeze" industries the "cold
shakes" (see Chapter 44).
Genetic Effects of Irradiations. Among the most interesting and important
effects of exposure to radiant energy, especially to ultraviolet light (aside from
death) are those involving changes in genetic structures so that mutations
(new species) are formed. The nature of the genetic changes induced, and the
mechanisms involved, are discussed more fully in Chapter 15.
Photodynamic Sensitization. Yisible light rays (4,000 to 8,000 A) do nbt
appear to exert rapidly lethal or other striking influences per se, but under
certain circumstances they produce death. For example, bacteria will often
grow in contact with low concentrations of bacteriostatic dyes such as eosin,
fuchsin, etc., provid,ed no light reaches them. The combined effect oflight and
dye for only a few minutes usually kills the organisms. Methylene blue, in the
light of an ordinary electric lamp, rapidly kills certain gram-positive bacteria
which withstand much higher concentrations of the' dye in the dark. Gram~
negative bacteria are not so affected. Other organisms, as viruses, and enzyme-
like substances, proteins, etc., are also affected. The exact mechanism is not
clear. It has been shown that oxygen is essential to photodynamic action,
I since the effects are not observed if the microorganisms are placed in a vacuum
or in atmospheres devoid of oxygen.
Sunlight. The disinfectant ("purifying") action of sunlight has been known
for centuries and has been thought to be due mainly to the ultraviolet rays
in solar light. The phenomenon of photodynamic sensitization suggests that
the visible light waves may play some role in the action of sunlight. It will be
seen in other sections of this book that even in the visible range different
colored lights have different effects. Red light, for example, is most effective
in activating bacterio-chlorophyll.
The heating and drying effects of sunlight are also important in its bac-
tericidal effect.
HYDROSTATIC PRESSURES
If living microorganisms suspended in aqueous fluid are placed in a strong
steel cylinder and a piston is pressed down upon the suspension with great
force, the organisms mayor may not be affected, depending on the species of
organism. While some will withstand pressures of 5000 pounds per square
inch without obvious injury such pressures are, in general, injurious. In the
range above 9,000 pounds, death of many species occurs within one hour.
Pressures around 100,000 pounds denature proteins and inactivate en-
zymes. Other changes include increases in rate of some chemical reactions,
diminution in volume of organic colloids, enzymes, and molecules, increases
in viscosity of many fluids, and increased electrolytic dissociation. Probably
all of these changes are involved in the biological effects of high pressures,
but exact details are obscure.
Pressure and Temperature. There is a compensatory relation between
pressure and temperature. Unduly high incubation temperatures may be
thought of as causing deleterious expansion of enzymes (colloids). Increased
pressures tend to prevent the expansion and so lessen this unfavorable effect
of high temperatures. Cooling may be thought of as ~ausing undue contrac-
tion, and hence loss of activity, of enzyme colloids. High pressures enhance
the retarding effect of low temperatures while relief from pressure tends to
overcome the contraction. For example, organisms normally growing best at
20-30 C were completely inhibited at 20° C by 4500 lbs. pressure. They grew
Q
well at 40° C under the same pressure.

Marine bacteria are resistant to pressures at the bottom of the sea, which
may reach 15,000 Ibs. per square inch. Some deep-sea bacteria and others from
deep oil wells appear to be favorably influenced by such pressures; they may
be said to be barophilic (baro = pressure; philic = loving).
CRUSffiNG. Most I microorganisms of cellular organization are easily
crushed by the impingement upon them of solid particles like steel or glass
balls. Shaking in a v~ssel with steel balls (baIlmilI) is_ a method used for the
mechanical disruption of bacteria. They shatter especially readily if made
brittle by freezing and this method is used for certain chemical investigations
of bacteria. Shaking f<;>r several hours mixed in a bottle with fine beads of glass
accomplishes. a complete disruption of the cells.
SURFACE FORCES
I
As we have seen, the protoplasm of the living cell is permanently enclosed
within a cell wall which mediates many or all
of the relationships and com-
munications between the protoplast and the outer world. The exterior surface
Fig. 12-6. DiagraIIl showing how

surface tension acts. The molecules a
and a' are attracted equally from all di-
rections and are in a state of equilibrium.
Molecules at the surface, like b, are under
a greater tension from below than above
and the entire surface therefore tends to
pulUnward much as though the surface
film were a rubber membrane.
of the cell wall must, therefore, be especially adapted to carry on various

physical and chemical reactions which enable the protoplast to 'live. In
biology ~any of the most significant chemical and physical changes, indeed,
the entire chemistry of life, take place at, and because of, surfaces. The same
forces which act at the outer surfaces of cells also act at the surfaces of the
intracellular, protoplasmic, colloidal particles (enzymes). It is evident, there-
fore, that surface forces are of the most profound importance in all cell life
and that we should know something about them.
Surface Tension. One of the most important is called surface tension, which
results from the attraction or cohesive force between moleCUles. Under its
influence the fluid surface acts, in some respects, like ali elastic membrane.
Many substances, such as soap, bile, certain substances classed as "deter-
gents," can weaken this intermolecular pull and so lower surface tension.
Such substances are called surface tension reducents ..
Adsorption. Of the many phenomena which result from the action of
surface tension, probably the most important and best understood is adsorp-
tion.
Suppose a substance (e.g., soap) which reduces surface tension is added to
the water in the beaker shown in Figure 12-6. It must, according 10 physical
laws, tend to carry the reduction of surface tension as far as possible. In this
case, the surface tension redudmt can lower the surface tension to the greatest
extent by accumulating at the_ surfaces of the fluid. This accumulation is
known as adsorption. Deposition of the surface tension reducent occurs on
I the air-fluid snrface, at the surface in contact with the glass walls of the con-
taining vessel and on the surface of any particles, such as bacteria, suspended
in the fluid.
ADSORPTION AND RATE OF REACTION. It has been shown that substances·
adsorbed upon a surface are often greatly concentrated and that, in many
cases, their solubilities increase. It is probable that some gases are actually
liquefied when adsorbed. It is clear that such condensations and increases
in solubility must, from the law of mass action, greatly facilitate chemical
reactions, since the law of mass action states that the greater the concentration
of the reacting substances the more rapid the reaction between them. Catalytic
agents (including the enzymes in all living cells), which greatly speed up
chemical reactions, depend upon this law.
Surface Tension and Wetness. An important result of the action of surface
tension reducents is that many important fluids: cell fluids, disinfectants, cul-
ture media, paints, cosmetics, and drugs, are really wetter than solutions with
high surface tension. This wetness brings fluids of low surface tension into
more intimate and effective contact with surfaces. Mercury, for example, at
room temperatures has a very high surface tension (466 dynes per cm). As a
result, as everyone who has held mercury in. the palm of the hand knows from
experience, mercury possesses the property of wetness in a negligible degree.
The inward pull between the molecules at the surface causes liquid mercury
to draw inward into globules of itself in a very unsociable manner. It does not
spread out and come into intimate contact. Water has a moderately high sur-
face tension (77 dynes per cm) and a moderate degree of wetness, while alcohol
has a much lower surface tension (about 28 dynes per cm). Alcohol, therefore,
wets or spreads when in contact with surfaces much better than does water
and thus comes into more intimate contact with objects which it touches. It is
wetter than water: physiologically, politically and socially!
The importance of surface tension and adsorption to the living cell* can
hardly be exaggerated. For example, fission of some species of bacteria
growing in media of abnormally low·surface tension is delayed so that large
spheres and very long filaments and bacilli are formed. Spore formation may
be delayed or abolished. Some bacteria will not grow in media having an
excessively low surface tension.
A serious difficulty in studies of the effect of low surface tension as a bio-
logical factor lies in the difficulty of differentiation between the physical
effects of a surface tension reducent (low S.T.) and its effects as a chemical
agent. For example, at the same surface tension, say about 40 dynes per cm,
peptone will nourish, phenol will kill.
The actual roles of surface tension and adsorbtion in microbiology will be
referred to more in detail at various places farther on. Many of the substances
which nourish living cells are potent surface tension reducents; so are some
very important disinfectants. The reactions in the blood and tissues which
protect us from disease are all surface reactions involving soluble proteins.
All soluble proteins are surface tension reducents. Protoplasm itself has a
low surface tension.
RAPID VIBRATIONS
Bacteria, because of their small mass and their relatively tough cell walls,
are not readily disrupted by slow vibration. The "high" limit of audible
tone has a vibration rate of around 10,000 per second, the lowest audible
tone has a vibratiqn rate of around 100 per second. This is the range of sonic
vibrations. t ;
Production and Effects of Vibrations. Vibrations for microbiological pur,
poses are generally produced in fluids by vibrating disks of nickel or quartz
crystals, under th~ influence of alternating electric currents. Sonic or super-
L
* and to the student of microbiology!
t Much depends on the intensity and quality of the sound and the individual ear.
sonic waves, to be effective, must be of such pitch that the cells to be disrupted
vibrate in harmony with them. The vibrations must also be of considerable
intensity.
Immersed in fluid sustaining vibrations of appropriate rate and intensity,
bacteria are torn, the protoplasm disrupted, and the cells killed. Much of the
damage is believed due to cavitation: the formation of a foam of minute
bubbles of the gas which is ordinarily in solution in the protoplasm or in the
fluids at the bacterial cell surface.
Bacteria differ greatly in their susceptibility to vibration. Some, like the
diphtheria bacillus (Corynebacterium diphtheriae) appear to be very tough
indeed, while the gonococcus (Neisseria gonorrhoeae) is fragile and easily
ruptured.
Mechanical methods of shattering bacteria (crushing, vibrating, etc.) are
of value in experiments in which it is desired to kill and disrupt the organisms
without producing such profound chemical and physical changes as result
from heating, chemical destruction, exposure to sunlight, or 'age. Delicate
poisons, proteins, carbohydrates or enzymes inside the cells are liberated in
an unaltered. state by sonic and ultrasonic vibrations and crushing and may
thus be studied in an unchanged condition.
NATURAL ENVIRONMENTS
Many of the factors influencing microorganisms, which have been dis-
cussed in this chapter, occur in Nature in the environment of microorganisms,
and many of these factors are, in turn, influenced and altered by neighboring
microorganisms. For example, shortages of food, moisture or oxygen; un-
favorable pH; concentration of various metallic and other ions, antibiotic
substances, etc., may result from the activities of one ,microorgani~m near
another. On the other hand, one microorganism may create ideal conditions
of growth for another, perhaps by synthesizing a vitamin or producing a food
substance (e.g., NaN0 3) by oxidation of its own food substance (e.g., NaN02)
(see Nitrobacter). If we eliminate these naturally occurring factors, as is
generally done in pure culture studies in the laboratory, we obtain a curiously
unbalanced knowledge of microorganisms.
Consider, for example, some minute recess a few inches below the surface
of a soil rich in organic matter and moistu~e. Here we find strict aerobes and
anaerobes growing side by.Side decomposing cellulose and proteins to simple
substances available to many other soil microorganisms. Fermentation of the
cellulose derivatives results in -acid production. which may inhibit many
bacteria in the neighborhood and may injure crops. Other microorganisms
may metabolize the acids, relieving the-acid condition of the soil. These and
other organisms produce compounds of an endless variety, all available to
some part of the soil flora alld fauna. The growth of the anaerobes may be
favored by the utilization of excess oxygen, or catalase production, by the.
aerobic bacteria. The fertility of the soil may be increased because autotrophs
grow, forming nitrites from ammonia produced from proteins by the prq-
teolytic heterotrophs; and nitrobacter can then grow, oxidizing these same
nitrites to nitrates (a plantJood). At the same time, all of these bacteria may be
held in check and perhaps killed by the poisonous substances poured forth, by
certain of the Actinomyces, molds and Pseudomonas group, spore-forming
E.Dect of Chemical and Physical Agent& on Microorganisms 171
organisms alone outliving all others. There is constant necessity for adjustc
ment to changing temperatures, osmotic pressures, surface tensions, pH, as
well as changing composition of suspending fluid due to fluctuation of water.
Such conditions are held fair9' constant in most artificial media. Species that
cannot adapt themselves to these competitive situations, or form spores,
perish.
It is plain that environmental vicissitudes and interactions between various
forms of life are the usual experiences of free-living microorganisms, as con-
trasted with growth in isolated tubes and completely segregated artificial
- communities (pure cultures) under the most abso]11tely uniform and favorable
conditions possible in the laboratory.
Even those strict parasites which never survive in nature outside the animal
or plant body seldom find the site of their entrance into, or grOWth upon or
in, the body devoid of other bacteria. One need only think of the mixtures of
organisms in the oral, nasal and intestinal cavities; The science of immunology
has taught us something about reactions between pathogenic bacteria and
their animal hosts, and we know that each produces alterations in the other.
Our knowledge of influence exerted upon one another by microbial species
in soil, intestine, milk, etc., is still limited, and so is our knowledge of the
effects of those influences. To what extent, for example, can transfer of genetic
material by 'phage (transduction) occur among bactena pullulating in rotting
organic material? There are high mysteries in a dung heap!
The point is, that laboratory studies generally fall far short of being exact
investigations of microorganisms because they do not reproduce the-natural
environments of the organisms.
GERM-FREE LIFE-
As we know, microorganisms constantly occur almost everywhere in our
environment as well as> on all part, of the body surface, the entire alimentary
canal, the upper respiratory system, the eyes, the ears, and the genital open-
ings. The newborn animal is contaminated at birth and remains an involuntary
and often an unknowing host to billions of various species of microscopic
parasites throughout his life. This relationship has existed since archeozoic
periods, and animal life is well adapted to existence in contact with most
microbial life. Only a few species of microorganisms cause much disturbance
in modern man; among them are pathogens like typhoid bacilli, the pneumo-
coccus, ,the gonococcus, and the tubercle bacillus. It would be of great interest,
and extremely useful to know how this age-long contact between animal and
microorganism has affected the hereditary, physiological, biochemical, ana-
tomical and other p,,"operties of each of the symbionts.
What effect does: the enormous number of supposedly harmless bacteria
in the intestine have on our nutrition? Do they synthesize vitamins for us?
Do they produce lj-ntibiotics which are taken into our systems? Do they
produce poisons which damage us subtly and shorten our lives, or do they
exert favorable or even absolutely necessary influences?" Do they immunizf.
us to infection? What is the role of each of the species?
We begin to rea,lize their importance when it is found that suppressing
certain groups of bacteria which normally inhabit the gastrointestinal tract (as
sometimes occurs during antibiotic therapy) permits other groups, generally
172 Method& and Phenomena 0/ Microbiology
Fig. 12-7. Operatina unit (right) connected for aseptic transfer of caesarean-delivered
young to germ..free rearing unit (left). Presnant animal enters at (5). a compartment kept
separate from inner operating compartment (2) by a cellophane sheet (4). Animal (I) is
brought into close contact with cellophane and young are lifted through it aseptically. (3)
Cautery; (6) sterile passage; (7) pan with youna; (8, 9, 10) sterile food, supplies, etc.
held in abeyance, to grow, often with evil and sometimes fatal results. Could
we answer some of these questions by producing an animal entirely free from
microorganisms and then contaminating it with a single species of bacterium
at a time?
To do such a thing it would be necessary, by use of fantastically rigorous
aseptic technique, to separate an animal from all demonstrable microorgan-
isms at the beginning of life, to maintain it for months or years free from any
demonstrable live microorganisms, and then to observe how it fares in life
free from its usual living mates, the microorganisms. Such an animal might
be said to represent "germ-free" life, or axenic life (a = free from; xenic =
foreign substance), or gnotobiotic life (gnote = known or exactly defined;
bios == life.)
Pasteur realized the value of such investigations as early as 1885. Due to
the great difficulties involved, only four workers prior to 1928 had attempted
to rear germ-free animals. In 1928 Reyniers started work on chickens and,
with his co-workers, has succeeded in raising numerous germ-free animals.
Completely germ-free insects, fish, chickens, rodents, dogs, pigs and monkeys
have been born and held germ-free for many months. Second generations of
white rats and chickens have been reared completely devoid of any living
microorganisms.
The VfIrJ expensive apparatus for such work consists of large steel cylinders which can
he steam sterilized; with closed, J)ass observation ports; hand-hotes fitted with air-tight,
arm-Jeoath, seamless, rubber gloves; and air-tight systems of outer chambers or locks
through which sterilized food, water and equipment are passed into the chamber while
waste materials are passed out. Air is passed through sterilizing filters and conditioning
apparatus (Fig. 12-1). Such equipment and its operation have necessitated solution of some
diftIcuIt enaineerina problems. One of the newest chambers is bia eoouah for an attendant
to enter, dreaed in a divina suit, throuah a deep tank of disinfectant (Fia. 12-8). Careful
FJlect of Cltemiclll tutti PltY8icaJ Agent8 Oil MicroorgiUWnu 173
control is necessary at an times, and an animals and their feces, and bodies, and the dust,
feed, water, etc., in the genn-free compartments are examined bacteriologicaUy at sbort
intervals to detect any contamination.
Some of the technical difficulties are very great, as for example, feedina yauna rats
delivered aseptically at Caesarean section. They require mille about every hour, 24 hours
a day, for weeks on end. It took much research to synthesize a satisfactory substitute for
mother rat's milkl These studies on genn-free life have been carried on at the Laboratories
of Bacteriology at the University of Not 'e Dame (Lobood).
The germ-free animals in general live longer and seem healthier than
ordinary animals. However, they are often.very susceptible to fatal infections
with many, usually-harmless bacteria. The living cells of germ-free rats and
chickens seem to remain "younger" than those of "normal," contaminated
animals. Is this because they are germ-free '? If antibiotics are fed to farm stock,
this possibly reduces their bacterial burden. They certainly grow much faster.
A very important observation was made when it was found that germ-free
guinea pigs did not develop dysentery when infected with the ameba, Entamoeba
histolytica, which usually causes a particularly bad sort of dysentery in guinea
pigs. Now, it is known that E. histolyt;ca feeds on bacteria. In germ-free
guinea pigs there were no bacteria. The infected germ-free guinea pig,free from
disease, quicldy developed dysentery when fed ordinary "harmless" intestinal
bacteria and died I It is suggested that certain antibiotics are effective in
curing amebic dysentery not because they effect amebae but because they
deprive the amebae of their food, the bacteria.
Fig. 12-8. Arrangement of germ-free animal quarters. At right is the standard rearing
caae. with rubber arm-aloves, and sterile lock opening into main compartment. The traps
beneath are for removal of waste without back-flow of contaminated material; overhead,
a sterile air supply. The operator in air-tight dress enlen the main chamber through a spray
and tank pf strong disinfectant, dragging an air hose like a diver. Air conditionina is elec-
tricaUy automatic. (Figures 12-7 and 12-8 courtesy of Dr. James A. Reyniers, University
of Notre Dame, Notre Dame, Ind., 1950.)
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Bates; R. G.: Electrometric pH Determinations. John Wiley and Sons, Inc., New York,
1954.
Bawden, F. C., and Kleczkowski, A.: Studies on the ability of light to counteract the inac-
tivating action of ultraviolet radiation on plant viruses. J. Gen. Micr., 1955, 13:370.
Bellamy, W. D., and Germain, M. T.: An attempt to photoreactivate ultraviolet inactivated
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Bellamy, W. D., Goldblith, S. A., Colovos, G. C., Niven, C. F., Jr.: Symposium on applica-
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Ellison, S. A., Erlanger, B. F., and Allen, P.: The chemical reversal of ultraviolet effects on
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Goucher, C. R., Sarachek, A., Kocholaty, W.: A time-course respiratory inactivation asso-
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Hollaender, A., Editor: Radiation Biology, I, II, III. McGraw-Hill Book Co., New York,
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Hugo, W. B.: The preparation of cell-free enzymes from microorganisms. Bact. Rev., 1956,
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Kasai, G. J.: Growth response of microorganisms to vitamins at different temperatures.
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Kelner, A., Bellamy, W.oD., Stapleton, G. E., and Zelle, M. R.: Symposium on radiation
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Kempe, L. L., Graikoski, J. T., and Gillies, R. A.: Gamma ray sterilization of canned meat,
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Koh, W. Y., M_orehouse, C: T., and Chandler, V. L.: Relative ~esistance of microorganisms
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Kushner, L. M., and Hoffman, J.l.: Synthetic detergents. Sci. Am.; 1951, 185:26.
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Loebeck, M. E., and Klein, H. P.: Substrates for Myxococcus virescens with special refer-
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Phillips, B. P., Wolfe, P. A., Rees, C. W., Gordon, H. A., Wright, W. H., and Reyniers,
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13
Nutrition, Metabolism, and Cultivation

of Microorganisms
NUTRITION
Food. Napoleon is reputed to have said, "An army marches on its stom-
ach." In establishillg this military aphorism he missed the opportunity to
become a biological immortal. He could have stated the obvious truth that
all life depends on food. To Bonaparte, food meant army rations. However,
the word means various things as indicated by the cliche, "one man's food
is another man's poison." Here we shall mean, by food, any substance which,
taken into a living organism, yields energy to that organism in a physio-
logically useful and beneficial manner and/or furnishes material by means of
which the organism may synthesize itself, either directly or after modifying
the food by digestive and/or other processes to fit the physiological require-
ments of the organism. The reader may at first be inclined to consult a lawyer
in attempting to interpret this definition. However, it is hoped that a perusal
of the following paragraphs will obviate this necessity.
Holozoic Nutrition. Most biologists will agree that not only soldiers but
all animals, from man to protozoa, with few exceptions have the ability to
take solid food materials into the organism. After being ingested the food is
first digested by hydrolytic enzymes. This takes place in some form qf gastro-
intestinal tract (of larger animals) or intracellular food-digestion vacuole (of
animal cells like protozoa). I
If the food is a complex, organic material such as protein, fat or carbo-

hydrate digestion results in its being enzymatically decomposed into its con-
stituent molecules of various soluble substances such as amino acids, fatty
acids and glucose. Unlike the original solid food mass, these molecules are
small enough to pass from the gastrointestinal tract (or digestive vacuole)
through ,the cell wall into the living protoplast. This mode of nutrition is
characteristic of the animal kingdom and is said to be holozoic.
Virtually all complex foodstuffs are reduced by digestion to the common
denominator of relatively small, simple and soluble molecules before they can
be used as nutrients * in any cell. Once inside the protoplast proper they are
:lsed either in cell synthesis or as sources of energy.
* The term nutrient is often used as though it were synonymous with food, and indeed it
may be. Food is probably a more inclusive term. We shall use nutrient here to refer to
wluble substances capable of furnishing energy or cell substance which pass through the cell
176
Nutrition, Metabolism, and Cultivation oj" Microorganisms 177
Holophytic Nutrition. Unlike animals, plants, including bacteria, yeasts,
molds (and, probably, rickettsiae, PPLO and viruses), have no such "built-in"
digestive mechanisms as those described above. The digestive mechanisms of
plant cells are obviously less well organized than are animal digestive mecha-
nisms. Plants * cannot take solid food particles into the organism. All of their
food must be in aqueous solution. This sort of nutrition, characteristic of
plants, is said to be holophytic. t
Many species of bacteria, yeasts and molds can, nevertheless, use the same,
solid foods that soldiers, horses and protozoa enjoy. This they do by what
has been called "extracorporeal digestion."t
Bacterial digestive enzymes, for example, are not concentrated in a gastro-
intestinal tract or vacuole but are, to a large extent, excreted aimlessly into the
surrounding fluid where they mayor may not come into contact with food.
They may be wholly dissipated by dilution, convection currents, or other
factors. Assuming that the digestive enzymes come into contact with digestible
food, the food is decomposed (very much as occurs in the animal digestive
tract) into the same sorts of relatively small, simple, soluble molecules as
result from animal digestive processes. As in animal nutrition, these molecules
pass through the bacterial cell wall into the protoplast.
From this point on the processes' of food utilization (metabolism) in plant
and animal cells (including the cells of our own bodies) are astonishingly
alike though by no means identical. They are all obviously modifications of
the same fundamental plan. Yet each species of plant and animal in this
respect differs to some degree from every other species of plant and animal.
A thousand different tunes may be made using only eight notes of the diatonic
scale.
In spite of their hit-or-miss digestive systems, an interesting advantage
possessed by many species of yeasts, molds, bacteria and related micro-
organisms over animals is their indifference in matters of taste. For example,
a species of bacterium or mold, given time and numbers (and if it possesses
the proper enzymic equipment) may use as food with equal avidity a railroad
tie, crab shells, a dead horse's hoof, a defunct cow's horn, feces, paper,
leather, crude transmission grease, sawdust, old rubber tires, and so on.
Digestion of the solid organic food stuff is accomplished outside of the cell.
All that the cell takes in are the soluble products of the digestive process:
glucose, amino acids, fatty acids, vitamins, minerals, and sci on. The cell is
indifferent to their source. It never has indigestion or ulcers!
Few if any bacteria or molds are actually so very versatile in their ability
* Except·a few curious carnivorous plants like Venus's fly trap and the pitcher plan!.
t If we consider the tissue cells of large animals, even this difference largely disappears,
because the tissue cells depend on food in solution in the blood stream.
:j: Digestion which takes place outside the body proper.
wall or which occur temporarily in the cell during metabolism. This would include not only
simple carbohydrates and related compounds (glucose, alcohols, and the like) simple nitro-
genous compounds (amino acids) fatty acids, minerals, vitamins and related substances
derived from food but various temporary, intermediate products of metabolic processes
which take place in the cell. Glucose, for example, could be regarded as a nutrient or as a
food. But starch, which is not metabolized per se but from which glucose is derived by
digestion, is a food.
to use all of the varied foods indicated above but some approach this degree
of versatility, and all of the substances mentioned, plus hundreds of others
equally distasteful or poisonous to us, are food for one or other species of
microorganisms or combinations of them acting together. In contrast, other
microorganisms are highly restricted and fastidious in the matter of food and
can thrive only upon certain particular compounds. Examples of the various
kinds of microorganisms will be described farther on.
NUTRITIONAL TYPES
General Requirements. While plant and animal species differ somewhat in
the details of their various food requirements, we may note one universal
necessity: water. The presence of water in adequate amounts will be assumed
in all discussions of nutrition and metabolism which follow.
Elemental Requirements. The evolution of microorganisms (represented
here b3"bacteria)from the most primitive forms oflife, and their kinship with
all other forms of life from man to virus, are revealed by the universal, basic,
protoplasmic requirement for certain elements: C, H, 0', N, P, K, Na, S, Fe,
Mg, Ca, and various others. Protoplasm is made up of compounds containing
these elements. To serve as nutrients for most microorganisms they must be
in forms soluble in water and capable of passing through the cell wall and "
membrane'!. (assimilable forms).
The exact list of elemental requirements for living cells is not fully known
since some elements are required in such minute amounts (trace elements) that
it is very difficult to detect them by analytical methods. The requirements may
vary slightly from species to species but are pretty much the same for alL
4.utotrophs. Some bacteria require as food nothing but a few, simple,
inorganic substances such as NaCl, K 2HP04, FeCI 3, MgS04 and (NH4)S04
to live and mUltiply. They obtain their carbon from atmospheric CO2 or from
carbonates. Such organisms are said to be autotrophic. * From such simple
inorganic materials autotrophic microorganisms synthesize their complex
chemical structures: proteins, fats, carbohydrates, vitamins, enzymes, cell
walls, genes, cytoplasm and so on.
CHEMOSYNTHETIC AUTOTROPHS. The primeval earth is believed to have
been completely dark due to heavy clouds of vapor in the skies. It may (or
may not!t) be due to this fact that some primitive organisms, suchas the auto-
trophic bacteria, do not depend on sunlight as their source of energy as do
familiar green plants. They oJ:>tain the energy for their life processes from
chemical reactions involving oxidations of inorganic materials such as H 2S,
NaNOz, arrd NH40H: FOf"example:
2HN02 + Oz ~ 2HN0 3 + 35~OOO calories
2NH3 '+ 30 z ~ 2HNO z + 2H 20 + 66,500 calories
S + H 20 + 3NaN0 3 ~ H 2S04 + 3NaN02 + Energy
Because they obtain the energy to synthesize themselves from chemical re-
actions they are said to be chemosynthetic.
,* Auto is from a Greek word meaning self; trophic is from a Greek word meaning,nour-
ishing.
t You may argue on either side with equal profit!
Nutrition, Metabolism, and Cultivation of Microorgamsms
PHOTOSYNTHETIC AUTOTROPHS. A few species of autotrophic bacteria,
characteristically occurring in sewage-polluted sea and brackish 'waters,
possess the ability to derive energy from the sun much as do green plants.
They contain chlorophyll-like, photosynthetic pigments. These alga-like
species are discussed more fully in a later chapter (Chapt. 29). They may have
evolved from chemosynthetic autotrophs after sunlight appeared on earth.
Heterotrophs. There is good reason to believe that some of the earliest
forms .of life on earth developed in primeval oceans, lakes and muds in close
association with simple, spontaneously-occurring* organic compounds such
as aldehydes, glucose, ammonium carbonate, urea, amino acids, etc. From
such compounds very complex organic structures may be built up by poly-
merizations, simple conjugations, etc. Indeed, the earliest forms of life (much
simpler than bacteria) probably evolved from such self-initiating processes.
Be that as it may, microorganisms eventually developed which utilized such
compounds as foods. Their descendants today are characterized by absolutely
requiring at least one organic compound as a source of energy. Such micro-
organisms are said to be heterotrophic. t
Glucose is probably most commonly used as an energy source by hetero-
trophs, but is not necessarily the only one.
Many heterotrophs have the ability (some absolutely require) to use CO2
as cell building material, at least for part of their needs. This CO2 require-
ment of heterotr6phs may represent a vestigial inheritance from an auto-
trophic ancestry of millions of years ago.
Heterotrophs are by far the' commonest, most widely distributed, and
numerous types among microorganisms.
CHEMOSYNTHETIC 'HETEROTROPHS. It seems likely that the early hetero-
trophic microorganisms developed under conditions of darkness similar to
those surrounding the development of chemosynthetic autotrophs. Thus, the
commonest forms of heterotrophic microorganisms are chemosynthetic.
They derive their energy only from oxidations of organic compounds.
PHOTOSYNTHETIC HETEROTROPHS. We have noted that among the auto-
trophic bacteria there are a few species which have developed the ability to
utilize sunlight as a source of energy by means of photosynthetic pigments
resembling the chlorophyll of green plants. Similarly, some species of hetero-
trophs have developed the same form of metabolism. These curious photo-
synthetic bacteria, both autotrophic and heterotrophic, are discussed in
Chapter 29.
Saprophytes. Most of the earliest heterotrophic bacteria, yeasts and molds
lived entirely upon Inert organic compounds; either those spontaneously
occurring around them or available from the wastes and dead remains of
other organisms. Such heterotrophic forms are today commonplace and are
to be found very usefully engaged in sewage-disposal plants, the soil, and in
every situation wher~ decomposition and decay of inert, waste and dead
I
• Several kinds of organic compounds such as aldehydes, glucose, amino acids and the
lIke have been shown to form in appropriate solutions under presumably primeval, geologi-
cal conditions without tl1e intermediation of any living thing, enzyme or other organic
formative mechanism; Le., spontaneously.
t From two Greek words meaning nourished by others. This refers to the fact that organic
foods are generally deriv~d from the wastes or substance of other living things.
organic matter are going forward. They are collectively spoken of as sap-
rophytes (from the Greek roots sapro, meaning decay, and phyte, meaning
plant). Most of the bacteria, yeasts and molds known today are of this type.
Parasites. Probably still later there appeared, either through progressive
or regressive evolutionary stages or both, bacteria, etc., which could live not
only upon dead and waste organic matter, but which could also live in living
plant and animal tissues. They caused disturbances of the delicate chemical
and physical equilibria of the creatures in which they lived. This was disease
and often resulted in the death of the invaded creatures. Such organisms are
well known today and are said to be parasitic* or pathogenic. *
RECAPITULATION
We may simplify the relationships of these various groups of microorgan-
isms by tabulating them as follows:
I. Autotropbs. Require inorganic food only.
A. Chemosynthetic: obtain energy only from chemical reactions (oxida-
tions); no photosynthetic pigments.
B. Plwtosynthetic: obtain energy from sunlight; ·contain chlorophyll-
like pigments (alga-like).
II. Heterotropbs. Require organic source of carbon and energy.
A. Chemosynthetic
1. Saprophytes: as a rule live entirely on inert organic matter; not
ordinarily involved in production of disease.
2, Parasites or pathogens: mayor may not be able to live as
saprophytes but can and do live in, or upon, other living
organisms, causing disease.
B. Photosynthetic: obtain energy from sunlight; cOntain chlorophyll-
like pigments (alga-like}.
NUTRITIONAL REQUIREMENTS AND CULTURE MEDIA

In order to cultivate microorganisms, i.e., to induce them to grow and
multiply under laboratory or other specified conditions it is necessary to
know not only about the distinguishing requirements of the major nutritional
groups but the actual, specific, food requirements of each family, genus or
species which it is desired to cultivate. Any culture medium, i.e., any nutrient
fluid or material in (or on) which we expect to cultivate living organisms.: man,
fish or bacteria, must contain: (1) sources of energy; (2) sources of cell-
building material; and (3) for many forms other than autotrophs, certain
accessory substances which do not yield energy or any significant amount of!
cell substance but which are absolutely essential to normal growth. These
/ are exemplified by vitamins, various amino acids, certain minerals or the
ions derived-therefrom, and the like. The result of dissimilation is a yield of
energy to the cell.
The utilization of food by living organisms for such purposes is called
metabolism.
METABOLISM
1. NUTRIENTS AS SOURCES OF ElIIERGY
The intracellular reactions involved in the utilization of nutrients as sources
of energy are all of the exothermic or energy-yielding type. Such processes are
* (Parasitic is from Greek roots signifying to eat the food of another; pathogenic is from
Greek roots meaning to produce disease).
Nutrition, Metabolism, and Cultivation of Microorganisms
often designated by the terms biological oxidation or respiration. These )l"e-
actions result in the dissimilation* of the nutrient. i
Basically, all respiratory processes are modifications of only two types Of
chemical change: (a) oxidation by dehydrogenation; (b) oxidation by oxygen
uptake. It is doubted by some that the latter is a source of energy in micro-
organisms. Both result in the same thing: a yield, to the cell enzyme systems,
of energy as a result of the transfer of electrons. Electron transfer in this
manner is the basis of all life. The two kinds of respiratory process may be
described briefly as follows.
Dehydrogenation. This is accomplished by the enzymic removal of hydro-
gen from a nutrient molecule (or substrate) containing it. The particular
enzymes involved are called dehydrogenases. The substrate being robbed of its
hydrogen (oxidized) is called a hyrJrogen donor because it 'yields up its hydrogen
to the dehydrogenase, which is thus reduced. The reduced dehydrogenase
passes the hydrogen on to another molecule in the cell called a hydrogen
acceptor and is thus ready to take up more substrate hydrogen. This process
goes on continuously during life.
It is worth noting at this point that the actual transfer of the hydrogen is
mediated by a part of the dehydrogenase called its coenzyme. The part of the
coenzyme actually combining with, and passing on, the substrate hydrogen is
the nicotinic acid C'niacin") molecule contained in the coenzyme. (See Fig.
13-1.) Thus the role of at least one vitamin in metabolism is clear. It is repre-
sentative of most others.
In the cell the hydrogen from the substrate is transferred from one hydrogen
acceptor molecule to another in a systematic sequence until the hydrogen is at
last transferred to some final hydrogen acceptor outside of the cell. As we
shall see later, the nature of the final hydrogen acceptor is determined by the
enzymic equipment of the cell and is a very constant and distinctive property
of various types of cell.
When hydrogen is thus removed from a substrate molecule, an electron is
transferred at the same time. Energy is thus released from the substrate and
becomes available to the cell. This energy is immediately taken up into the
cell by means of a complex system of enzymes. It is absorbed into a particular
sort of organic phosphate bond (high-energy phosphate) and is later used as
life energy. Thus, the net result of dehydrogenation is decomposition of the
\
mbstrate and transfer of its energy to the living cell.
Aerobic Respiration. If air is present (aerobic conditions) the final hydro-
gen acceptor may be oxygen. H 20 or H 20 2 may be formed from the hydrogen,
depending on the species of organism and its enzyme system. Aerobic bacteria
are generally equipped with the enzyme cytochrome oxidase which enables
them to utilize atmospheric oxygen as a final hydrogen acceptor.
COMPLETE AND Il'1COMPLETE OXIDATION. The respiratory oxidation of the
substrate may be complete, as illustrated by the aerobic utilization of glucose
by baker's yeast (Saccharomyces cerevisiae) and by many heterotrophic
bacteria: '
C6H1206' + 602 ~ 6C0 2 + 6H20 + 689,800 calories
* In dissimilation th,e nutrient is decomposed, oxidized, and the products excreted as
wastes. Dissimilation is the opposite of assimilation, in which the nutrient is absorbed,
stored, and eventually built into the cell substance. Parts of some nutrient molecules, for
example glucose, are dissimilated; other parts of the same molecule are assimilated.
H
b
O
H-c0_d Nicotinic
""NH2 acid
N-C NH2
) - )-
1
amide H-C C=N> Adenine
H-hl bH group "" C-H group
~/ N-C-N
H~
N I
H-C~
Ho-t-H \ HO-t-H \
Pentose \
group HO-C\ H
°I H\ H\ HO-C\ H
\ °I
H-C 0 0 H-C
\ \ \ \
H-C-----O-p-O--p--O-----;C-H
k ~ ~ k
Fig. 13-1. Diagram of the structure of the molecule of coenzyme 1. At upper left is seen
the nicotinic acid amide ("niacin" vitamin). This is connected with a carbohydrate (pentose)
group and this, in turn, with two phosphate groups. At the right these are connected to
another pentose group and this, at upper right, to an adenine group. The intricate synthesis
necessary to the construction of such a molecule is suggested. It can readily be understood
why, if an organism cannot synthesize nicotinic acid amide (as is the case with us poor
humans) it must have the vitamin fed to it.
or by the oxidation of sulfur by certain autotrophs:

2S + 2H 20 + 302 -'> 2HzS04 + 237,000 calories
Such oxidations release all of the energy available from the substrate.
However, the oxidation may be incomplete. Much depends on the species
and the enzymic equipment pf the cell involved. As an examp1e, the produc-
tion of acetic acid by the~oxidation of alcohol by Acetobacter may be noted:
C 2H sOH + O2 __; CH 3COOH + H 20 + 118,000 calories
! Much of the available energy of the alcohol is left in the acetic acid. An ex"
ample of the incomplete oxidatiol) of an inorganic substrate (NH 3 to HNO z)
has previously been given.
STRICT AEROBES. A number of common species of microorganisms possess
respiratory mechanisms capable of utilizing only free oxygen as final hydrogen
acceptor. These must have free access to air for growth. They are called strict
or obligate aerobes.
I Anaerobic Respiration. Many species of microorganism are anaerobic.
That is, they do not possess the cytochrome oxidase enzyme which ~ould
enable them to utilize atmospheric oxygen as a final hydrogen acceptor.
Nutrition, Metabolism, and Cultivation of Microorganisms 183
STRICT ANAEROBES. As we shall see later, some anaerobes not only cannot
utilize, but are actually poisoned by, atmospheric oxygen. These are called
strict or obligate anaerobes and they must be cultivated in the complete
absence of air.
When air is not present anaerobic conditions are said to exist. Under these
conditions microorganisms must obviously use some final hydrogen acceptor
other than atmospheric oxygen. This is usually some compound which
readily yields oxygen or combines with hydrogen. For example, NaN0 3
serves as an effective final hydrogen acceptor under anaerobic conditions
because it readily yields up oxygen to be combined with the substrate hydro-
gen. It is reduced to NaN02 in the process. Many bacteria can reduce nitrates
to nitrites and the property is commonly tested for in identifying- bacterial
species. Some other final hydrogen acceptors used by anaerobes are H2S04
(reduced to S or H 2S) and C02 (reduced to CH 4). Others are used, depending
on the species of cell involved and its enzymic equipment.
INTERMOLECULAR RESPIRATION. Respiration in which the final hydrogen
acceptor is some extraneous substance like NaN0 3, or possibly some organic
compound, is spoken of as intermolecular respiration because the hydrogen is
transferred from one molecule (substrate) to another.
INTRAMOLECULAR REsPIRATION. This is often called fermentation. It is
characteristic of anaerobic respiration. It differs from intermolecular respira-
tion mainly in that the final hydrogen acceptor is derived from the nutrient
substrate itself. ·Part of the nutrient molecule is oxidized, part is reduced. For
example, in the fermentation of glucose (diagrammatically shown here) the
6-carbon-atom chain (hexose) is first combined with phosph'}te (phosphory-
lation*) and is then split into two interconvertible, 3-carbon, phosphorylated,
sugar-like compounds called trioses. From one of these (3-phospho-glycer-
aldehyde) pyruvic acid is produced after a series of chemical transformations.
All of these changes are catalyzed by enzymes. /
OH
\
H-C- H-C=O C=oH 3
, I I
H-C-OH H-C-OH + Hz -> H-C-OH
1 I
HO-C-H
I _ /
/' H2=C-OP04Hz Ho-t=a
H-C-OH 0 + 2 H3P04 3-Phosphoglyceraldehyde Pyruvic
I Lactic
acid
H_t_ _ 1
I
Hz=C-OH
"'", H2=LoH
I
C=O
acid
1
I
H 2=C-OP04H2
Glucose Dihydroxyacetone Alcohol
phosphate
It is evident in the :diagram that, in the process of dissimilation of glucose,
hydrogen is removed from certain parts of the molecule (resulting in oxida-
tion) and shifted to other parts of the same molecule (resulting in reduction).
In a later stage of ~he process, and depending on the species of organism
involved and on its ~nzymes, the pyruvic acid may act as hydrogen acceptor
• As previously noted, the phosphate is involved in the energy transfer.
I. Glucose + ATP glucose-6-phosphate + ADP
(hexokinase)
2. Glucose-6-phosphate fructose-6-phosphate
(phosph ohexoisomerase)
3. Fructose-6-phosphate + ATP ---> fructose-l,6-diphosphate + ADP
. aldolase)
.
j I
4. Fructose-l,6-diphosphate.===>3-phosphoglyceraldehyde+dihydroxyacetonephosphate
(isomerase)
I. T
J
s. 3-Phosphoglyceraldehyde + H3P04 ~ (1,3-diphosphoglyceraldehyde)
6. (l,3-Diphosphoglyceraldehyde) + DPN <====! l,3-diphosphoglyceric acid + DPNHz
7. l,3-Diphosphoglyceric acid + ADP ~ 3-phosphoglyceric acid + ATP
8. 3-Phosphoglyceric acid <====! 2-ph!,sphoglyceric acid
9. 2-Phosphoglyceric acid ~ phosphoenol pyruvic acid + HzO
10. Phosphoenol pyruvic acid + ADP;::::==:! pyruvic acid + ATP
Fig. 13-2a. The Meyerhof-Embden Scheme for dissimilation of glucose.
C.HI20.
glucose ATP } (PhOSph oryIa'
(+2H,PO.): lion)
~
2C3HSO, + 2PO.H3(-Hzj: Coenzyme I (Dehydrogenation)
Triose Pjho[sphote c::::;:H3 + ~ -+ Ico,1 + [!!iJ

ADI>: (-H,PO.) ~H(o ~iIJ ~!;}
?, '-(Trans- c:::::H3 c::::;:H3

H-C-NH,' _ + NH3 _ ~ ~ + H ....H-l.-oH
o-l.-oH aniination) a=LoH od:.-op
Alanine PYRUVIC Lactic acid
,/ ACID '"
(Decarboxylation) + (Decarboxylation)
,! C·O, '"
CH 3
l+co,1 O=LH (The Od.-OH c::::;:H3
1' I +CO,I
Wood- ....L O==C-H
~€ ~ (Retu~J::h~Yde
Aldehyde
,/
(Condensation)
H c::::;:H,
I
,/
C:::::H 3 O=C-OH ! 1
Oxalacetic C:::H] O::::C-OH
H-l.-oH acid I C-H 3 Acetic acid
l'
~
+2H 2 H 2==C-OH 1=
(Reduction) Ethyl alcohol O==C-H + O·
i
c:::::H3
Acetyl
O=~.-OH
1 '
j (Di~mu-
tation)
C=H
1- 3
J, '" e::::=M3
Methyl \ H-C-H O==C-H I'
Carbinol 1 I +H 20 I 2 Aldehyde Hz=C-OH
,/ • H-C-H Ethyl alcohol
+H,
(Reduction) Od-OH
,/ Succinic
C<=H 3 acid
H-L.oH
H-b--<JH
6 3
2-3 Butylene
Glycol
Fig. 13-2b. Common metabolic pathways illustrated by the dissimilation of glucose by
Klebsiella aerogenes. This is a "chemical map," indicating successions of reactions; it is
in no sense a balanced equation. Pyruvic acid is seen as a sort of biochemical tum-table,
reached .through a set of transformations of glucose involving dehydrogenation, phosphory-
lation, etc., as described briefly in Fig. 13-2a and in the text. Pyruvic acid may undergo one
or more of several changes, the end products of which are shown in italics. Types of chemical
change are in parentheses. Note that amino acids, represented by alanine, can enter this
system as well as glucose. Hydrogen, carbon dioxide and water, as wastes, are seen in boxes.
Pathways are not fully agreed upon, but end-products are all well esiablished.
184
and be reduced to lactic acid (as in the souring of milk") or, while accepting
hydrogen, it may be further decomposed to ethyl alcohol and C02 (as in the
making of beer and wine) (see diagram). The net result of these oxidation
and reduction processes is a release of energy from the glucose to the cell.
REsULTS OF FERMENTATION. Fermentation generally results in only partial
oxidation of the substrate. Many of the products (ethyl and butyl alcohols,
lactic and acetic acids, acetone), unlike the CO 2 and H 20 resulting from oxi-
dative or aerobic respiration, retain much of the original energy of the sub-
strate. For example, compare the yield of energy from fermentation by yeast:
C6H1206 ~ 2 C2H sOH + 2 CO2 + 31,200 calories
with that from aerobic dissimilation of glucose (689,800 calories).
Stages and Products of Energy Metabolism. For those readers who have
had organic chemistry there are shown in outline the Meyerhof-Embden
scheme (Fig. 13-2a) showing the chemical stages in the dissimilation of glu-
cose by yeasts and (often in modified form) by many other microorganisms.
These readers may also be interested in the diagrammatic illustration of some
of the terminal dissimilative changes following the formation of pyruvic acid
(Fig. 13-2b).
DISTINCTIVE METABOLIC PRODUCTS. As seen in the latter diagram, once
pyruvic acid is formed, it may undergo 1 or more of several transformations,
assimilative or dissimilative, depending on the species of cell and the physical
and chemical conditions of the environment. While it is not important, at
this point, to understand the chemistry indicated in the diagram or to mem-
orize the scheme, it is necessary for the microbiologist to know something of
the different end-products of energy metabolism in different species of cells.
A good illustration is the common, saprophytic, gram-negative rod, Klebsiella
aerogenes.
In the case of this organism the following final products of glucose dissimi-
lation under ordinary cultural conditions have been' demonstrated to occur:
hydrogen, carbon dioxide, ethyl alcohol, acetic acid, acetyl-methyl-carbinol,
2-3-butylene glycol, trimethylene glycol, lactic acid, glycerol and succinic acid.
Whether all of the reactions that occur in their formation are exactly as indi-
cated in the diagram is not certain, but many are well established. Some ofthe
end-products of metabolism, for example acetyl-methyl-carbinol, H2 and
CO 2 , are easily tested for in a culture tube and are often very distinctive of
certain species and, therefore, are of use in identifying certain microorganisms.
We have already indicated that some of these products are of great industrial
value and will be referred to later in the chapter on Industrial Microbiology.
ALTERNATIVE METABOLIC PATHWAYS. An important concept in this con-
nection is that bf aiternative metabolic pathways. Although a series of physio-
logical chemical reactions, such as those diagrammed to occur in the utili-
'zation of glucose, may usually follow a definite sequence, this is not necessarily
fixed. Under aIter~d conditions of nutrition or environmental factors, (e.g.,
pH, temperature, presence of essential metabolites or substitutes, "blocks"
such as antibiotics, sulfonamides, etc.), a different set of reactions may occur
by different chemi~al pathways, and with different growth rates, waste prod-
ucts, etc. The alternative pathways may be more or less effective in the release
of energy and synthesis of cell substance than the normal pathways.
Facultative Respiration. Most of the common types of bacteria and yeasts
* In souring, each molecule of milk sugar (lactose) is first decomposed to yield one mole.
cule of glucose and one of galactose.
are capable of utilizing both atmospheric oxygen and reducible compounds as
final hydrogen acceptors. They possess both aerobic and anaerobic respiratory
mechanisms. They are said to be facultative with respect to respiration since
they have the faculty of growth under either condition.
Direct Oxidation. Some species of microorganisms apparently can bring
about a direct combination between atmospheric oxygen and the substrate
molecule. An example sometimes given is the oxidation of the alcohol in hard
cider, wine, beer, etc., to acetic acid as in vinegar manufacture. The bacteria
used in this industrial process are Acetobacter species (see Chapter 44). The
over-all reaction is:
CH 3 ·CH20H + O2 ~ CH 3·COOH + H20 + 118,000 calories
In effect, this is in part a dehydrogenation followed by oxidation of the hy-
drogen. But oxygen is also added to the substrate.
There are several species of microorganisms which obtain energy by the
oxidation of such substrates as molecular hydrogen, carbon monoxide, sulfur,
methane, ammonia, etc. For example,
H2 + !02~H20
CO + i02~C02
CH~ + 202 ~ CO2 + 2H20
In this sort of respiration free, atmospheric oxygen appears to oxidize the

substrate directly (via enzyme action). Dehydrogenation does not appear to
be involved.
All of the biological oxidations described above proceed relatively slowly,
in a stepwise fashion, molecule-by-molecule, so that.no explosively rapid and .
damaging release of energy occurs as might be expected, for example, when
hydrogen is made to combine with oxygen to form water (see reaction given
above). All proceeds quietly and gently but efficiently because the reactions
are regulated by enzymes.
Oxi~ation-Reduction. When we discuss anaerobes later on (Chapter 34)
it will be worth remembering that when any substrate molecule is oxidized,
some other substance is reduced. Every biological oxidation is accompanied
by a simultaneous reduction, just as every action is accompanied by a reaction.
2. NUTRIENTS AS SOURCES OF CELL BUILDING MATERIALS
It is evident that the various types of organisms differ greatly in their nu,
trient requirements. Some autotrophic organisms, like Nitrobacter in. the
soil, have very great synthetic ability, forming amino acids, proteins, fats,
carbohydrates, enzymes, vitamins, and all of the many other complex organic
compounds which make up a living cell, from CO2 of the atmosphere and
a solution of inorganic compounds similar in composition to sea .water.
Some common heterotrophic organisms possess almost as great a synthetic
ability. They differ from the complete autotroph described above only in
requiring, in addition to the simple, mineral solution, one organic f~od sub-
stance such as glucose or sodium citrate, as a source of carbon and energy.
In contrast to these "almost-autotrophs," many heterotrophs are highly
restricted and demanding ill the matter of foods. Such are represented by
human beings or, among microorganisms, by various species ·of parasitic
bacteria like gonococci or influenza bacilli. These must have blood or tissues,
or complex organic extractives thereof, which they cannot synthesize for
themselves because of their enzymic limitations.
Assimilation. In the synthesis of cell substance certain atoms and mole-
cules from the food substances are selected, rearranged, and put together
again in new structures constituting the protoplasm, cell wall, and other por-
tions of the new organism. This is assimilation. Unused residues of the nutrient
molecules are given off as wastes.
The synthetic reactions involved are endothermic since energy is stored in
the complex molecules resulting from the building processes. The energy is
derived from the exothermic dissimilative or respiratory reactions previously
described.
Some of the endothermic reactions, viewed in an over-all sense, appear to
be the reverse of hydrolysis, i.e., complex molecules seem to be built up from
pairs of simpler ones by the abstraction of hydrogen from one and oxygen
from the other of the pair. A molecule of water is formed from each pair of
complex molecules brought together. An example is given below.
(enzymes)
CH 3 ·COOIH + HOICH2·CH 3 ~ H 20 + CH 3·COO.CH2 ·CH 3
I i
Acetic Ethyl Ethyl acetate
acid alcohol
However, in general, biosynthetic processes appear to be much more complex
and to involve a number of chemical steps other than the mere reverse of
hydrolysis. The entire synthetic process is not yet completely known.
With respect to the energy involved, an analogy is seen between assimilation
and the building up of a column of bricks. Each brick piled up is analogous to
a chemical synthetip step in the building up of some energy-bearing substance
in the cell, such as starch or fat. Potential energy is accumulated in the
column of bricks and in the molecules of starch or fat. When the column of
bricks is knocked down energy is liberated in the form of heat, sound,
motion, perhaps light, etc. When the fat or starch is broken down chemically
(oxidized or dissimilated) by the cell, the energy released is manifested as life.
DUAL-PURPOSE UTILIZATION OF NUTRIENTS. It has been found that or-
ganic nutrients like glucose and amino acids are generally not used exclusively
for either carbon or energy. Parts of the nutrient molecules may be incor-
porated into the cell substance "as is," while other parts are oxidized as
sources of energy and given off as waste. The total caloric energy in a molecule
of nutrient such as, glucose is, therefore, not necessarily an exact indication
of how much energy it will yield to a given type of cell.
3: NUTRIENTS AS ACCESSORY SUBSTANCES*
k
As indicated in foregoing paragraph these are substances which do not
yield energy to the cell or contribute materially to its bulk, but which are
effective in very minute quantities and absolutely essential to growth. They
range in structur~ from simple elements to complex organic compounds.
• These are also often spoken of as essential metabOlites, nutrilites or growth factors.
Vitamins are good examples of growth factors, as well as certain metallic
elements:
Certain amino acids also have the status of growth factors; that is, they are
among the substances absolutely essential to the growth of a number of bac-
teria. A good example is tryptophan, without which no growth of the typhoid
bacillus (Salmonella typhi), the lockjaw organism (Clostridium letani), the
diphtheria bacillus (Corynebacterium diphtheriae) and several others can
occur, even though their culture medium be complete in all other respects.
On the other hand, there are many bacteria which can synthesize their own
tryptophan and so do not need to have it fed to them. Among these are auto-
trophic bacteria and numerous others the cells of which, though grown in a
tryptophan-free medium, nevertheless contain this amino acid. '
A number of other growth factors are known or are under investigation.
Among them are inositol, hemin, glutamine and vitamins such as nicotinic
acid, riboflavin, thiamine, and pantothenic acid.
One probable role of such compounds in bacterial nutrition was described
by Fildes as early as 1939. Assume that an organism requires pantothenic
acid, a vitamin. It is able to carry out all of the preliminary steps necessary
to the formation of pantothenic acid, beginning with simple ammonia (see
steps 1-3 in the diagram below). However, it is not able to synthesize beta
alanine (one of the amino acid radic1es in the complex pantothenic molecule).
This is indicated in step 2 in the diagram. Given a minimum quantity of
B-alanine, completion of the pantothenic acid is provided for (step 4) and
growth occurs. Without B-alanine, no growth is possible.
(2) B-alanine
(1) /' '\.,.
NH3 - (R-NH2) (4) Pantothe!1ic acid
'\.,. /'
(3) Di-hydroxy-
valeric 'acid
A strain or species able to synthesize B-alanine as well as the other compo-
nents of pantothenic acid encounters no such difficulty and we say that "it
does not require B-alanine." Both may require B-alanine, but one can manu-
facture it internally, the other cannot. -
CULTURE MEDIA
Natural and Empirical* Culture Media. In the early days of microbiology
natural culture media were widely used: milk, urine, diluted blood, vegetable
juices, etc: Some media were artificially prepared: beef or vegetable broths,
meat "infusions," and the like. Such media are still wi<;lely used. They contain
a rich assortment of soluble organic and inorganic compounds comprising all
of the elements and accessory substances for many (but not all) microorgan-
isms. Such media are convenient, inexpensive, and serve for many routine
purposes.
Synthetic Culture Media. Some culture media, developed as a result of
increased knowledge of microorganismal nutrient requirements, consist
• Used on the basis of experience and not on the basis of exact knowledge of their com-
position and action.
wholly of dilute, accurately known and reproducible solutions of chemically
pure, inorganic and/or organic compounds. They have special uses in re-
search, industry and the like. Artificial media of exactly known, reproducible
composition are called synthetic media. In order to use them it is necessary to
know the exact nutritional requirements of the organism for which they are
designed. Such knowledge is, unfortunately, available for relatively few
organisms.
Living Culture Media. Living culture media consist of live, animal or
vegetable cells cultivated in special nutrient fluids. The live cells serve as foods
especially for viruses and rickettsiae which, as previously noted, cannot
multiply in inanimate, artificial culture media.
Examples and uses of each of the foregoing types of culture media are given
in the following discussion of cultural methods.
CULTURAL METHODS
It is evident from the foregoing that no single cultural medium or metbod
is suitable for all forms of microorganisms. Space would not permit a presen-
tation of the thousands of kinds of culture medium which have been devised
for the many various objectives of microbiology. For illustrative' purposes we
will describe some of the media most commonly used for microorganisms
described in this book. Theses are representative of many others, which are
mere modifications of the same general types.
1. Synthetic Inorganic Media. These are among the simplest of media. A
medium used for a common, sulfur-oxidizing autotrophic species of bacteria
found in the soil (Thiobacillus thiooxidans) is as follows:
(NH4hS04 ...................................... 0.2 gm
MgS04 . 7H 20.. .. . . .. .. . . .. . .. . . . . . . . . . . . .. . . . .. 0.5 gm
KH 2P04 ••.••..•.•.....•...••....••.••.......•.. 3.0 gm
CaCI2 ••••.•...••....•••..•..••.•.••....• ,••..••• 0.25 gm
Powdered sulfur ................................. 10.0 gm
Distilled H 20 ................................... 1000 gm
The phosphate in the medium is a buffer and maintains a suitable pH.
It also serves as a source of phosphorus and potassium for use in the cell.
Additional trace elements (Cu, Fe, Mn, Na, and others) are usually present
as impurities in the water or ingredients. The powdered sulfur is the source of
energy. Incubation is at about 25° C, common in the soil in summer. Note
that there is no source of carbon and that nitrogen is provided in an inorganic
form. Carbon is ob~ained as CO2 in the atmosphere. This is a typical, syn-
thetic, inorganic medium. That is, each of the ingredients is a chemically
pure, inorganic substance, the exact chemical composition of which is known
and under the control of the worker. Such media (as well as any other fluid
media) may be soli~ified with agar* or silica gel, the choice depending on the
organisms. Some autotrophs are inhibited by organic substances such as agar.
* A carbohydrate having some of the properties of gelatin in that, dissolved in water in
about 2 per cent concentration, it fluidifies on boiling and sets to a firm jelly at about 40° C.
It is attacked by few cOplmon bacteria and is therefore useful for solidifying bacteriological
media. Silica gel is often used in its place to solidify media for strict autotrophs since it i~
inorganic. Strict autotrophs cannot grow in the presence of organic matter.
2. Synthetic, Organic Media. By way of contrast, a synthetic medium
devised for a rather fastidious, pathogenic heterotroph, Corynebacterium
diphtheriae (cause of diphtheria), is given as follows:
SYNTHETIC MEDIUM FOR C. DIPHTHERIAE (Pappenheimer, Mueller and
Cohen).
Solution A
glycine .......................................... ; . -....... . .5 gm
valine ................................................... . 1.0 gm
leucine ................................•.................. .5 gm
glutamic acid ............................................. . S.Ogm
methionine ............................................... . .2gm
tyrosine .................................................. . .1 gm
NaCl. .................................................. ,. S.Ogm
KzHP04 .... ·.· .......................................... . 2.0gm
Water ............................................ : ...... . 500.0ml
Solution B (add to A)
cystine (an amino acid) .. 0.2 gm (in 30 per cent HCI, minimal quantity to dissolve).
Addition 1 (add to A after adding B)
( pimelic acid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. I mg
Vitamins I b~ta ~I~in~. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. I mg
l mcotimc aCId .................................. ; . . . .. 2 mg
Adjust to pH 7.S. (This is done by adding dilute NaOH to the desired pH.)
Solution C (add 0.3 ml of this to A, after B and I and pH adjustment)
Source of Ca++ {CaCh....................................... 33 gm
HzO ........................................ l00ml
Boil gently 10 minutes. Filter through paper.
Addition 2 (add to the above)
S fM ++ C ++ S {MgS04.7HzO...................... .3 gm
ources 0 g , u , . CuS04.5HzO ............... : ....... 5.0 mg
tryptophan (an amino acid) .......... 100 mg
water to make total volume of 1000 m!.
Dispense in flasks and autoclave. •
Just before inoculation, to each 100 ml add aseptically 2 ml of
Solution D
Organic sources
d b
Of{ sodium lactate (Merck U.S.P.} ........... .
glucose C.P............................ .
37.0 ml
7.5 gm
energy an car on maltose (purified) ...................... . 15.0gm
CaCh ................................ . 03gm
H20 to make ....... l .................. . 100.Oml
Autoclave.
The advantages of synthetic-media are that they are exactly reproducible

and, in some instances, much less expensive and troublesome to prepare
I than media made with meat and meat products such as peptone. Also, they-
do not contain proteins and therefore have no antig~nic or allergenic proper-
ties when injected'into man or animals in vaccines or for experimental pur-.
poses. By virtue of these properties they lend themselves well to exact experi-
mental research and to medical and commercial uses. (See sections on
vitamin assay, Chapter 44.) The great difficulty is that a solution suitable f\)r
one species is usually not suitable for another, and it is often difficult to
det!!rmine the exact reqvirements for a given species.
• Sterilize with compressed steam.
Empirical, Complex, Organic Media. While synthetic media have much to
recommend them for special work they are not so commonly used for routine
purposes as less exact media because (I) they are often expensive and time
consuming to prepare and (2) we know the exact requirements of relatively
few microorganisms. It is, therefore, customary in most laboratories to use
less exact organic media which, as we know from experience, support good
growth of a wide varie_ty of organisms (except strict autotrophs inhibited by
organic matter). Such organic mixtures are very useful, not too expensive and
quickly prepared. Many are commercially available in dehydrated ("add
water, heat and serve") form from laboratory supply houses.
PEPTONE. Such media nearly always contain meat- or casein-digest de-
rivatives such as peptone. These have in them all the basic mineral content
of living material as well as organic carbon and organic nitrogen in numerous
complex and soluble forms. Such media furnish a wide variety of substances
derived from living material and satisfy a wide range of nutritional require-
ments. Examples of two commonly used media of this type are given here.
MEAT EXTRACfS AND INFUSIONS. "Beef tea" (beef extract) or an aqueous
extract made by soaking (infusing) ground meat in water are common sources
of culture media for a wide variety of microorganisms. They are rich in
minerals, vitamins, proteins, carbohydrates, etc. They are often mixed with
peptone, a buffer and sterilized after adjusting the pH to near neutral. Media
made with "beef tea" are called extract media; those made from fresh meat
are called infusion media. Both, or any or all, media are sometimes loosely
included in the general term "nutrient broth" or "nutrient solution."
Meat is not the only useful source of organic matter. Extracts of vegetables
of various kinds are, often used. Media made with vegetable extracts are
available ready-prepared in convenient tablet form. Some workers use the
flesh or juices of shell fish, while those interested in the bacteria of milk
sometimes use whey, skim milk, etc. Eggs are often lJsed also, especially for
tubercle and diphtheria bacilli. An infusion of partly digested soybeans has
been shown to be as good as meat infusion for many purposes. Some media
are made by adding bits of kidney, spleen, or other tissues freshly removed
from dead animals under aseptic precautions to tubes of broth.
ADSORPTION OF NUTRIENTS AT SURFACES. The incorporation, in bacterio-
logical culture media, of small amounts of some solid substance such as
ground meat, or even sand. is often advantageous as many microorganisms
seem to grow best in the crevices of, or in contact with, the surfaces of such
matter, forming little nests or niduses there. It is probable that oxygen and
food substances concentrate at such points by adsorption. Furthermore, di-
gestive enzymes secr¢ted by the microorganisms into their environment to
digest foodstuffs do not di£ruse away so quickly (Fig. 13-3) in such protected
niduses. Indeed, in very dilute media, growth may occur only at such surfaces.
Special Media. To any of the organic media various test and experimental
or nutrient substancds may be added. Certain carbohydrates may be included
to test the fermentative powers of various organisms. Organic esters, blood,
glucosides, and many other compounds are put into the medium for a great
variety of experimental purposes. The medium is then referred to by the name
of the special substance; for instance, "blood-infusion-broth"; "serum-
dextrose-extract-agar"; "starch-carrot-whey-agar" and so on.
.. ..
'I. •• 8
,
..
.. 8'\";-:.
I
ELL ... • •
•
•
•
• ,.
.-
"
., , •
•
••
...
II • 'p • ft' .
« •• JI
c.
•
.. '
• •. 1
0. • . I
Fig. 13-3. A, a free-floating bacterial cell surrounded by a few suspended particles of

food (dark circles) which must be hydrolyzed by the exoenzyme (helicoidal lines) before
the resulting hydrolysates (dots) can be assimilated. B, particles of food concentrated in a
monomolecular layer on a solid surface. C, food particles are more available to the cell on
the solid surface where the interstices at the tangent of the bacterial cell and the solid surface
retard the diffusion of exoenzymes and hydrolysates away from the cell. D, multiple cells
form additional interstitial spaces. (ZoBell, Claude E., in J. Bact.,vol. 46.)
PURE CULTURE METHODS

Difficulties with Mixed Cultures. Early microbiologists had to contend
with the difficulty that as soon as anyone attempted to cultivate organisms
from a particular source such as blood, soil, a lesion on a plant, or to handle
them with instruments, the microorganisms under study became mixed with
a variety of extraneous microorganisms from the instruments, from dust, or
from some other source. It Wl\.s often impossible to distinguish one kind of
microorganism from anotheJ: in such contaminated cultures, since many en-
tirely different kinds look-exactly alike when viewed with the microscope, even
when stained by Gram's method. Pasteur, Koch and many others had the
same difficulty. No one could be sure that the chemical or physiological prop-
erties he assigned to a given culture were due to one kind of microorganism
alone and would remain constant or whether the observed reactions were due
to a combination of organisms growing together in a culture vessel. It was lik~
trying to determine the properties of a salt or element in solutions which con-
tained other salts and elements. Such mixed cultures would change their
properties as soon as one or the other of the different kinds of microorganisms
died off or gained the ascendancy. But no good method of separating or isola-
t(on of organisms was then known.
Origin of Pure CultUre Technique. Koch had observed the growth of molds
and of different sorts of bacteria in isolated masses (called colonies) of various
colors on slices of decaying potato in his wife's kitchen. One day, being in an
investigative mood, he thrust a sterile platinum wire into one of the gray
bacterial masses and put a bit of it in a little water under his microscope. He
saw that all of the bacteria in this particular colony looked exactly alike.
Koch examined the bacteria from a yellow colony on the potato, and then
those of a red one and of a violet one. The organisms from one colony were
all round; from another they all had the appearance of tiny, immobile cyl-
inders; from a third they looked like minute, living, highly motile, spiral
springs; but all the microorganisms in anyone colony were always exactly
the same. It was obvious that, by cultivating microorganisms on solid food,
he could obtain isolated colonies of any single kind; a pure culture!
The Use of Gelatin. Extending this principle it was but a step to the use of
gelatin to prepare a transparent, solid, sticky, nutrient surface on flat pieces
of glass. In addition, various nutrient infusions and test substances could be
added to the gelatin before it was allowed to "set." Here was a very important
advance, a revolutionary advance, one that has been the basis of all our
present-day bacteriology. Thenceforth the study and discovery of bacteria in
pure culture and by relatively exact methods became a matter largely of pa-
tience and hard work.
In summer, however, and when held in body-temperature incubators, the
gelatin melted and this spoiled everything. Being a protein it was often di-
gested and f1uidified by the metabolic processes of the bacteria. Besides that,
particles of dust settled on it with various microorganisms from the air or
soil which contaminated it, obscuring and confusing the results as badly as
ever.
First Use of Agar-Agar. Many students flocked to Koch's laboratory from
all over the world to l)!arn his methods. One of these was W. Hesse. To the
wife of this man the science of microbiology is indebted for suggesting, in
1883, as a substitute for gelatin, the jelly-like substance agar-agar, derived
from seaweed and used at that time for making jellies. Agar is transparent,
colorless, is not digested or liquefied by most bacteria, melts only at boiling
temperature and once melted, does not set again till about body temperature.
Agar has not been improved upon as a solidifying agent for culture media and
is in general use for this purpose today.
Origin of the Petri Plate. In order to prevent contamination of the pure
cultures by dust, another student in Koch's laboratory, R. J. Petri, suggested
the simple expedient of pouring the melted, nutrient agar into circular,
shallow dishes and immediately covering them with a glass cover. This per-
mitted prolonged ex<\mination of the cultures but excluded dust. Such dishes
are widely used today and are called Petri plates.
The preparation and study of pure cultures then proceeded at a great rate
all over the world. :
For many years the use of Petri dishes imposed a burden of cost and dish-
washing on bacteriological laboratories. Plastic, disposable dishes of this type
are now available apd, for many purposes, solve these knotty and long-
standing problems.
Preparation of Solid Media. The preparation of solid media is compar-
atively simple. To an~ of the fluid organic media prepared as indicated above,
gelatin may be added in 10 per cent concentration while the fluid is still hot.
Since, as Koch found, this liquefies so readily, agar is usually substituted
194 Metltotb and PMnomelUl oj Microbiology
ASS£M8tEO IN TH( FiElD

FOR STERILIZATlON BY
FORMALDEHYDE FUMES
RESULTING FROM PARTIAL
OlQOATION OF METHANOL
ASSEMBlED
VIEW
Fie. J3-4. One form of filtering device for mounting a Millipore filter disk. The porous
carbon base is placed beneath the fragile filter disk for support. It plays no role in the filtra-
tion process. In the field assembly, sterilization by formaldehyde fumes is suggested s~
the membranes are sensitive to heat except under very carefully controlled conditions in
the laboratory. (CourtesY of the Millipore Filter Corporation, Watertown, Mass.)
following Frau Hesse's suggestion. Agar is used in 1.3 to 2 per cent concen-
tration.
Silica Gels. Silica can be made into jelly-like material with surfaces suffi-
ciently solid to be inoculated like agar or gelatin. The advantages of silica as a
solidifying agent are that, supplemented with an appropriate mixture of inor-
ganic nutrient minerals, it will support the growth of colonies of a number of
species of autotrophic soil microoganisms which are injured by organic
substances, such as the agar, eggs, sennn or gelatin ordinarily used as solidi-
fying agents. Silica gels are much used by microbiologists studying bacteria
of the sea or soil.
Cultivation OD Fine Pore Filters- A means of cultivating bacteria on a solid
surface without the use of a special solidifying agent has been developed
through the use of very thin, porous membranes of cellulose acetate, collodion
/ or similar materials. Such membranes· can be prepared with pores of the
magnitude of 0.5 I/o or smaller. Mounted on a perforated plate to support it in
a funnel-shaped apparatus such a membrane acts as a sieve to filter bacteria,
or even viruses, from any fluid as desired: drinking water, dilute feces, blood,
digested, centrifuged sputum, dilute broth cultures, etc. (Fig. 13-4). After
the fluid has passed through, depositing the microorganisms on the surface of
the membranes (Fig. 13-5), the membrane is carefully removed with sterile
forceps from the supportfng device and laid on a disk or pad of sterile blotting
paper saturated with any desired nutrient solution. The nutrients diffuse
• Avai1able commercially.
195
through the membrane and support growth of colonies on its surface just as
though these were on an agar surface. Various selective or special media may
be used to cultivate special sorts of bacteria. The disks of cellulose acetate or
collodion are sometimes sterilized by exposure to ethylene oxide or carboxide,
but may also be sterilized by heat with suitable precautions recommended by
the manufacturers.
Interesting deviations from the standard procedures of cultivating bacteria
are possible. One may incubate organisms on a membrane laid on a pad
moistened with one solution for a time, and then transfer the membrane, with
its colonies, to a different medium, or to a stain or to a pad saturated with a
reagent to test for some particular growth product or property of the organ-
isms growing on the membrane. One may easily filter a liter of river water
through a membrane, place the membrane on a pad containing a special se-
lective medium, or a general-purpose nutrient broth, and isolate typhoid
bacilli from the water or enumerate the cultivable organisms in the total
sample. Many profitable applications and intriguing possibilities will suggest
themselves to the industrious and ingenious student, once he starts working
with the filters. (See also Chapt. 37.)
SeIecme Cultivation. This was discussed in Chapters 3 and 4 in connection
with yeasts and molds. The basic principle is simple: preparation of a medium
which will support good growth of the desired organism but containing sub-
stances to suppress the growth of undesired organisms. For example, in isolat-
ing gram-negati've Salmonella (typhoid) and Shigi!lla (dysentery) bacilli from
feces of patients (Chapt. 39) a selective "indicator" dye, 2, 3, 5 -triphenyl
tetrazolium chloride, is added to nutrient medium. This permits growth of
many gram-negative rods but inhibits many gram-positive species common in
feces. In isolating the gram-negative Brucella (undulant fever) organisms from
heavily contaminated soil or manure suspensions a good nutrient agar is used
Fig. 13--5. Disposable plastic Petri dish containing a used Mil1ipore filter disk resting
OD a pad saturated with culture medium The whole has been incubated and the bacteria
caught on the disk have grown into visible colonies. Actualme.(Courtesy of The Millipore
Filter Corporation, Watertown, Mass.)
to which are added: Polymyxin B, Actidione, Bacitracin, Circulin and crystal
violet, with good effect. The first four are antibiotics, the last a dye, none of
which inhibits Brucella. Scores of similar selective media are to be found in
the literature.
Enrichment. In this process inhibiting agents are not ordinarily used.
Conditions are made particularly favorable for one particular species in a
mixture. Continued incubation results in predominance ("enrichment") of
the desired species.
USE OF LIVING CELLS FOR CULTIVATION OF MICROORGANISMS
Virtually all yeasts and molds, most PPLO, some protozoa and, with the
exception of some Spirochaetales, Chlamydobacteriales and sulfur bacteria,
all bacteria are known to be cultivable upon lifeless artificial media; i.e., upon
material devoid of living cells. * Some of these microorganisms, mainly patho-
gens, require dead tissue or inert body fluids such as blood serum or ascitic
fluid for optimum growth. While many will also grow in contact with living
tissue and in so doing sometimes cause disease, none is restricted to live tissue.
This is one of the properties which distinguish viruses and rickettsiae from all
of the above-mentioned groups of microorganisms. Viruses and rickettsiae
are obligate parasites; i.e., they cannot mUltiply outside of. or in the absence
of intimate contact with, living cells.
PROPAGATION AND CULTIVATION OF VIRUSES AND RICKETISIAEt
Viruses and rickettsiae are often propagated in their natural hosts; that is,
by Nature's method of infection. In the laboratory we imitate Nature by
transmitting infectious material (blood, serum, pus, tissue, sap of plants, etc.)
from one animal or plant to another by injections, scratches, punctures
insect bites, etc. This is frequently necessary for research but it is laborious,
expensive and sometimes very dangerous to laboratory personnel unless they
can be vaccinated against the organism they are working with, as, for example,
Rocky Mountain spotted fever rickettsiae or yellow fever virus. This sort of
propagation, in live creatures, is usually called propagation in vivo.
Tissue Cultures. In vitro propagation (in culture flasks, tubes, etc.)
eliminates the use of living animals (or plants).lt does not eliminate the neces-
sity for living animal or plant cells. In vitr,o propagation of viruses and rick-
ettsiae thus requires knowledge of how to cultivate living cells in vitro. A
culture of tissue cells in vitro is called a tissue culture. Methods of'cultivating
animal tissues will illustrate the basic ideas. For plant tissues appropriate
modifications are made in details such as nutrients supplied, temperature, etc.
Bacterial viruses (bacteriophage) require special, modified procedures which
have been discuss,ed in Chapter 6. All tissue culture work requires the most,
strict precautions and considerable technical skill to keep out contaminating
bacteria, molds, yeasts, etc. These can grow vigorously in the nutrient solu-
tions furnished for the tissue cells. This, of course, is detrimental. Antibioti~s
are generally used to suppress them.
* Erythrocytes may be regarded as lifeless since they carry on no known metabolic
processes in bacteriological media.
t Methods for cultivating rickettsiae are included at this point since they are the same,
basically, as those for cultivating viruses.
The viruses grow in the multiplying tissue cells. As the infected cells dis-
integrate the viruses and rickettsiae are liberated into the suspending fluid.
The tissue cells show visible changes and, seen daily under the microscope,
the progress of their infection and disintegration can readily be followed.
Viruses which thus damage tissue cells are said to be cytopathogenic (Fig. 13-6).
PLASMA CULTURES. Tissue cells may readily be cultivated in plasma'" clots.
Briefly, a drop of sterile, fluid plasma is mixed with a nutrient fluid contain-
ing serumt (20%); extract of embryonic tissue (5%); Earle's solution (70%).
A tiny fragment of the freshly cut tissue to be cultivated is then placed in the
center of the plasma drop on a glass slide. Clotting occurs almost immediately.
The drop is then placed in a tightly-sealed, glass container and held at 37° C.
Transfers of the growing tissue to new plasma drops (explants) must be made
every two or three days. Growth of the tissue is observed on the slide with the
microscope.
Such cultures have limitations due to their minute size. Excellent slow-
motion pictures have been made of polio virus cultures in plasma clots,
showing effects of the virus on the cells.
ROLLER TuBES. In a modification of the plasma culture called roller-tube
cultures, tubes or bottles of any desired shape and size are wetted inside with
plasma. A score or more of fragments of live tissue are then placed in the
plasma. When clotting has occurred nutrient fluid is placed in the vessel
which is then sealed. It is placed in a nearly horizontal position and rotated
in a "roller" at the rate of about 7 r.p.m. in an incubator at 37° C (Fig. 13-7).
The tissues' are thus bathed in the nutrient fluid seven times per minute as
the bottles rotate. These cultures can be maintained for long periods by re-
placing the nutrient fluid every two or three days. Relatively large amounts
of tissue and of virus may thus be obtained' and prolonged experiments are
possible.
In 1950 Robbins, Enders and Weller cultivated thf virus of poliomyelitis
in monkey-kidney tissue by such methods and thus made the basic obser-
vation from which the Salk polio vaccine was derived. Viruses of herpes
simplex (fever blister), feline pneumonitis (a large virus), influenza, and
numerous others have been cultivated in vitro by similar methods.
It is of interest to note that cancer (neoplasm!) cells can be cultivated in
tissue cultures and that several viruses are highly fatal to such cells. Many
persons have thought of using viruses especially adapted to neoplasm cells
(but harmless to normal cells) to cure neoplasms in human beings. Veryex-
citing results have been obtained. These hold promise, but the method requires
much more study. !
CULTURES ON GLASS. Clot-culture methods were long used because mam-
malian cells grow b~st in contact with some solid support. It was later found
that they grow well directly on glass, spreading in a sheet over the inner sur-
face of flasks or tubes. This method, in numerous variations, is now widely
used. In a typical procedure, susceptible tissues are minced and suspended in a
• The fluid portion of the blood after removal of all white and red cells. Plasma can clot,
just as does whole blood.
t The fluid portion of. the plasma after clotting (fibrin formation) has occurred.
: Neo is from a Greek: root meaning new; plasm is from a Greek word meaning formations.
A neoplasm is a new gi"owth or new tissue.
198 Methotl4 and PMII01MIUI of Microbiology
Fig. 13--6. Cytopathogenic effect of poliovirus (type 0 on monkey-kidney tissue cells

in a roller-tube culture. In A are seen normal, young cells forming a continuous, cellular
structure. The cells are stellate in form and the nuclei visible in most, but not conspicuous.
In B the same preparation is seen 48 hours after infection with poliovirus. Note open spaces
where cells have separated and/or been destroyed, "rounding" of the cells and conspicuous,
denser nuclei. In C the same preparation is seen 4 days after the infection. Almost total
destruction of the tissue cells has occurred. The virus has multiplied enormously. (photos
courtesy of Dr. Mtlrris Schaeffer, Virus Laboratories, Communicable Disease Center, U. S.
Public Health Service, Montgomery, Alabama.)
growth medium commonly consisting of Earle's or Hanks' solution· at pH

about 7.2 (75 per cent); about 20 per cent suitable mammalian serum; some
type of extract of selected embryonic tissue (5 per cent); antibiotics. Partly
/ synthetic media are being used more as knowledge of tissue-cell requirements
accumulates. These must contain at least: 7 specified vitamins; 6 salts includ-
ing buffers; 13 specified amino acids; serum; glucose. (See references.)
Trypsin is added to the suspension of minced tissue to digest non-living
material and separate the cells. Action of the trypsin is s,.topped in fifteen
• Earle's solution (Compare with media used for autotrophic bacteria and mineral
content ofbuman blood!):
NaO ..... . . . . . ... . . 6.8 gm NaH2P04. .. . . . . 0.125 gm
KO .... .. ... . . . ... . O.4gm NaHCOJ. • • . . . . 2.2 gm
eaoz .. .. .... ...... 0.2 gm Glucose. . . . . . . . 1.0 gm
MaSO•. .. . . ... .. ... 0.2 gm H2O to • • •..•..• 1000 mI
Nutrition. Metabolism. and Cultipotion of Microo'1lanUltU 199
minutes by soy-bean trypsin inhibitor. The tissue cells are then removed from
the fluid by centrifugation. The "clean" cells are now resuspended in fresh
culture medium and placed in small tubes held motionless in a slanting posi-
tion at 37° C overnight. In twenty-four hours the cells attach themselves to
the glass and start to grow. The culture may then be handled as are roller
tubes previously described.
CULTIVATION IN ClUCK EMBRYOS
Fertile eggs must be used. They are incubated as for hatching until the em-
bryo is well developed (from five to fourteen days). The shell is cut by means
of a dentist's fine carborundum disk on a flexible shaft, rotated by a dental
motor (Fig. 13-8). The square of cut shell is then gently lifted off with a
sterile forceps. exposing the shell membrane immediately underneath. The
shell membrane is pierced. the chorioallantois drops away from it. The shell
membrane may now be tom off like paper against the cut edge of the shell,
leaving the vascular embryonic membranes exposed for inoculation.
This is done simply by applying the inoculum with a loop. a dropper, or
other gentle means. The hole in the shell is surrounded by sterile melted
petrolatum or paraffine and immediately covered with a sterile coverslip of
glass, cellophane or gummed transparent plastic strip. The egg may then be
incubated as for hatching.
Modifications of this method of inoculation include injection through the
membrane directly into the amniO.tic and chorionic cavities, yolk sac, em-
Fig. 13-7. Roller-tube technique of tissue culture. Poliomyelitis virus used in the prepa-
ration of vaccine is being tested in tissue cultures, whicl;! the virologist is placing in the roller-
tube apparatus prior to incubation. (parke, Davis & Co.)
Fig. 13-8. Cutting window in shell with rotating carborundum disk. (Goodpasture and
Buddingh, Am. J. of Hyg., vol. 21.)
a
sm
5
al
e c
c-am
y
ca
/
Fig. 13-9. Methods of inoculating chick embryos. A, injection into the yolk sac for
cultivation of rickettsiae. The normal air space is seell at a, the shell membrane at sm, and
the shell at s. The living chick embryo is shown at e, while the yolk sac and yolk are seen
at ys and y, respectively. B, many microorganisms can infect the chorioallantoic membrane,
seen at c-am_ An artificial air space (aa) is made by allowing air from the normal air space
to escape through the small opening seen at the right end of the egg. The collapsed air sac
(ca) is then sealed up. Other important parts of the embryonic membranes and sacs are the
allantoic sac (al), and the chorion (e) _ (Courtesy of E. R. Squibb and Sons. From Kelley
and Hite.)
Nutrition, :Metabolism, and Cultivation of Microorganisms 201
bryonic tissue, etc. For some of these purposes a single tiny hole in the shell
is all that is necessary (Fig. 13-9). Many saprophytic microorganisms, espe-
cially yeasts, molds and bacteria, which may enter as contaminants are able
to mUltiply as well in the fluids of the chick embryo as in the culture tube.
The embryo is usually killed and destroyed by such contaminants. Extraneous
contamination with such organisms from the air, shell dust, implements, or
from contaminated inocula such as feces or saliva, is therefore an ever present
source of error in the use of chick embryos for the study of viruses and rickett-
siae. As in handling tissue cultures, rigid precautions against such contami-
nations and considerable technical skill are required. It is customary in both
tissue cultures and egg cultures to mix small amounts of penicillin and strep-
tomycin, or other antimicrobial drugs with such materials before injection.
These agents control bacterial contaminants but permit the growth of viruses
and rickettsiae since these are not affected by the drugs.
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Takemoto, K. K., et al.: Primary isolation of influenza A, B, and C viruses in monkey kid-
ney tissue cultures. Proc. Soc. Exp. Biol. and Med., 1955, 89 :308.
The Staff of the Tissue Culture Course, 1949-1953, Cooperstown, N. Y. An Introduction to
Cell and Tissue Culture. Burgess Publishing Co., Minneapolis 15, Minn.
Thomas, H. A., and Woodward, R. L.: Estimation of coliform density by the membrane
filter and the fermentation tube methods. Am. J. Pub,Health, 1955,45:1431.
Thorne, C. B.: Metabolism ofnitrogenous"compounds. Ann. Rev. Microbiol., 1956,10:329.
Tidwell, W. L., and Gee, L. L.: Use of membrane filter in blood cultures. Proc. Soc. Exp.
Biol. and Med., 1955,88:561.
Tomlinson, A. J. H., and Mitchell, E. R.: Isolation of poliomyelitis virus in a routine bac-
terioiogicallaboratory. Monthly Bull., Min. of Health and Pub. Health Lab. Serv.,
London, 1956,15:53.
Vallee, B. L.: The metabolic role of zinc. J.A.M.A., 1956,162:1053.
Waksman, S. A. Ed.: Perspectives 'lind Horizons in Microbiology. Rutgers Univ. Press, New
Brunswick, N. J., 1955. .
Wayne, L. G.: Cultivation and visualization of mycobacteria on molecular filter membranes.
J. Bact., 1955, 69 :92.
Weinberg, E.: Selective inhibition of microbial growth by incorporation of triphenyltetra-
zolium-CI in culture media. 1. Bact., 1953,66:241.
1951.
White, P. R.: The Cultivation of Animal and Plant Cells. The Ronald Press Co., New York,
1954.
Wright, L. D.: Nutrition of bacteria and fungi. Ann. Rev. Microbiol., 1956,10:141.
Zitcer, E. M., Cailleau, R., and Kirk, P. K.: Serial cultivation of normal human embryonic
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ZoBell, C. E.: The effect of solid surfaces upon bacterial activity. J. Bact., 1943,46:39.
14
The Growth of Bacteria
REPRODUCTION
Binary Fission. The most obvious means of reproduction by organisms of
the class Schizomycetes is transverse division of each cell into two approxi-
mately equal cells. This is called simple or binary fission (Fig. 14-1).
Bacterial Multiplication by Means Other than Binary Fission. Other forms
of mUltiplication of bacteria, especially by means of intracellular bodies,
variously called gonidia, microcysts and regenerative granules, have been
described. In these types of reproduction, multiplication may be regarded
as resulting from intramural divisions of the cell material so that all of the
new organisms are contained within the original cell wall and are necessarily
very tiny. Rupture of the cell wall liberates the tiny granules. There is no con-
vincing evidence that such minute bodies occur in relation to the reproduction
of bacteria unless the formation ofL forms ofPPLO in some species (Chapter
8) be so interpreted. Some spirilla are said to multiply by formation of many
"baby" spirilla inside a parent cell. "
In attempting to demonstrate nuclei, gonidia, etc., one is very apt to be
misled by the presence of volutin granules, fat droplets and, in dead, dying or
artificially mounted cells, of globules of coagulated protein. Diffraction and
refractive effects of minute granules are much enhanced in bacteria due to the
acute curvature of their minute surfaces. These optical effects are undoubtedly
a source of some error in such' studies. The electron microscope has yielded
exceedingly valuable informatiol! in this field. Yet the existence and nature of
reproduction other than binary fission is still not clear. .
Sexual Multiplication of Bacteria. The question of the existence of sexes
among bacteria is .;_ much debated one. Evidence based on what appears to
be mitosis, meiosis"crossing over and recombination of genes and segregation
of characters is very persuasive but, again, is open to question and other
possible interpretation.
Protrusions and zygospore-like and nucleus-like bodies undoubtedly ap-
pear, but their role in sexual reproduction is not firmly established. They, or
appearances very much like them, can be induced by various artificial agents:
irradiation with ultraviolet.light, colchicine, excess salt, and others. Although
I many persuasive arguments have been advanced one could wish for more
conclusive proof. From an evolutionary point of view, sex in bacteria would
204
The Growth of Bacteria 205
Fig.I4-1. Diagrammatic representa-
tion of binary fission as it occurs in rod-
shaped and in spherical bacteria. A, ma-
ture cells ready to divide. B, early stage
in formation of a wall dividing the two
new cells. Note that the intracellular
matter (nuclear or hereditary material,
gene-like structures, enzymic granules,
food vacuoles, and the like) are approxi-
mately evenly divided between the two
daughter-cells-to-be. C, further develop-
ment of new cell walls and division of
all essential materials; the two cyto-
rl\.,~ ~ .--
__ '. • .;
l! A
fG:\
~
plasms are still connected. D, almost
complete separation. The connection be-
tween the cytoplasms is now very tenu-
to """
~
"""'. GIl;,;,_._
~-. ".'J~
II
B E:I0
ous, constituting what is sometimes
called a plasmodesma. By means of this ~ ~
structure cells often cling together in ~"@.~.JJ C ~.• ~o
long chains after fission; forming strep- ~- - •
tobacillj in the case of rod forms, strepto-
coccj in the case of spherical bacteria. In 0 ~
E, fission and separation are complete; ~
new intracellular developments are oc-
currin g· In sOhme specie~ the edntfiire prrn:-
ess 0 r growt , maturatIOn an sSlOn IS
~
~
~.'.
'.'';''''.'~,
~--
... '' E (;:).
\W ®
~
completed every 20 to 30 minutes.
be an anachronism. According to Coulter, " ... sex is not an essential feature

of reproduction [in the plant kingdom]. Historically it was the last method
of reproduction attained among plants, and when it appeared it did not re-
place older methods [fission] but was added to them ... In the evolutionary
sequence of plant groups the sexual cells appear for the first time far above the
most primitive known plants ... [bacteria] ... The beginnings of sexual cells
are seen among both the green algae [as Ulothrix) and the brown algae ...."
BACI"ERIAL "POPULATIONS"
Regardless of the problem of the sexual life of bacteria, the little creatures
find no difficulty in multiplying under optimal conditions. If left to their own
devices they can,_ within 24 hours or so, produce inside of a thimble-full of
broth a population of astronomical proportions, running into thousands of
millions and billions. You may think it queer, like oxidation without oxygen
0
(Chapt. 13), life in a Ivacuum at 400 below zero F (Chapt. 12), and reproduc-
tion without sex, but we can count these billions with reasonable accuracy, in
duplicate or quadruplicate, quite easily within a few hours or even minutes!
If you want to kno~ more, read on.
The numbers (or:"populations") of bacteria present in various natural
materials such as soil or river water vary constantly due to changing con-
ditions. In an "ideal" situation, however, such as a pure laboratory culture
under uniform, optimal physical and chemical conditions, the numbers of
organisms present vary in a perfectly regular and predictable way in accord
with general biologicallaVJs.
As an illustration'we may select a culture flask containing 50 ml of sterile
0
infusion broth at 35 C.
The Enumeration of Bacteria. Let us introduce, by means of a sterile
pipette, a drop of fluid containing about 10 cells of the common, harmless
bacterium of the intestinal tract: Escherichia coli. Let us assume that these 10
cells are from an inactive or "dormant" stock culture h:eld for weeks in the
refrigerator. The new culture is held at 35° C. The problem before us is to
measure the population at regular intervals by counting the numbers of living
cells present. The numbers present at the different periods are then plotted in
relation to time, and a "growth curve" is obtained. Enumeration may be done
by several methods, none of which is exact.
One, the hemacytometer or counting chamber method, is to place a minute drop of the
fluid in a tiny, shallow, rectangular, glass vessel (hemacytometer) partitioned off by ridges
into regular cubical chambers of exactly known volume. By counting the individual cells in
each chamber under a microscope, the numbers of organisms per m!. may be computed.
This is a total count of live and dead organisms.
Another procedure is to smear an exact volume of the culture over an exact area on a
slide, stain with methylene blue and count the organisms in a known portion of the total
area. Knowing from previous measurements (by means of a stage micrometer) the diameter
of the microscopic field, it is easy to calculate the numbers of organisms per m!. of culture.
This, also is a total count since no distinction is readily made between living and dead
organisms.
Still another method is to place a fixed volume, say 10 ml, of the culture in a kind of test
tube having a narrow, hollow, cylindrical column projecting from the bottom and graduated
in mm. The organisms are packed into the column by centrifugation at a standard speed and
for exactly measured time and their total volume read on the graduated scale. From a
knowledge of the average volume of the individual cells an estimation of numbers is pos-
sible. This also is a total estimate.
If 1 ml of blood and 1 ml of culture are well mixed, and a stained smear of the mixture
prepared, an estimate of the numbers of bacteria may be obtained by counting both blood
and bacterial cells in a certain number of fields and noting their relative proportions. Know-
ing that male human blood contains about 5 million erythrocytes per cubic millimeter an es-
timate of the numbers of bacteria is merely a matter of arithmetic. This is a total estimate.
A widely used technique is turbidometric, i.e., measurement of the turbidity

in the fluid due to the accumulation of cells in it. A measured volume of the
culture is placed in a special, clear, glass tube of known diameter. This is
interposed between a unit source of light and a photoelectric unit which is
attached to a galvanometer. The reading on the galvanometer depe_nds on the
interference with the passage of light from the unit source, due to the cells
suspended in the tube. The-method is subject to errors due to variation in size
and shape and clumping of cells) as well as to different degrees of translucency
of various species and other materials in cultures. However, the method is one
. of the quickest and simplest and is reasonably accurate. It is a total estimate.
Turbidity readings may be standardized in terms of numbers of cells by
hemacytometer cO,unts.
The method of serial dilutions is widely used to estimate numbers of bacteria in water"
milk, various foods, soil, blood, etc, Into tubes of broth are placed I-ml quantities of the
sample (of, let us say, milk), diluted in decimal, 4-fold, 2-fold or other convenient series.
After incubation of the tubes of broth numbers of organisms are recorded by noting pres-
ence or absence of growth. For example, in a IO-fold dilution series suppose there is growth
in the tube which received'the 1 :1000 dilution but no growth in the tube receiving, the
I :10,000 dilution. Then there were between 1,000 and 10,000 organisms per ml of the sample
of milk tested. This is spoken of as the indicated number (the reciprocal of the highest
dilution showing growth). But it is not a very exact estimate. It measures ol1l), lire cells
viable (capable of growth) under the conditions provided.
MOST PROBABLE NUMBER. In the above example there must be 1,000 organisms but there
may be, theoretically, any number up to 9,999 per ml in the sample. What is the true number?
This cannot be staled. However, mathematIcians have shown th.at. the number most probably
present may be calculated if the results from duplicate or triplicate simultaneous determina-
tions are known. They have prepared tables showing the most probable /llImber indicated by
all possible combinations of results in such series. These are much used in sanitary examina-
tion of water. Tables are found in "Standard Methods for the Examination of Water,
Sewage and Industrial Wastes"· with directions for use. It is to be borne in mind that these
are most probable numbers; not necessarily exact numbers.
The Colony Count. The colony count is a widely applicable procedure

used daily for determining approximate numbers of bacteria in milk, water,
soil, foods, etc.
Continuing the examination of our flask of culture inoculated with Esche-
richia coli we may, at any desired moment, withdraw exactly I ml of the
culture from the flask with a sterile measuring pipette and transfer it to a
sterile Petri dish. Immediately afterward, about 15 ml of nutrient agar of
appropriate composition, previously melted (and cooled to about 40° C so
as not to kill the bacteria), are poured into the dish. The culture is thoroughly
mixed with the still-fluid agar by a gentle horizontal rotation of the dish.
In a few minutes the agar will have solidified. This plate culture is beJd in
an incubator at about 35° C for twenty-four hours and is then examined for
the presence of colonies, t distributed throughout the agar. Each of these
represents, theoretically, the progeny of a single cell which was in the original
inoculum and which was imprisoned in (or on) the agar at that point. Actually
several organisms, if stuck together in a clump, will give rise to only a single
colony. The colony count, therefore, does not give a wholly accurate enumer-
ation of the live, individual cells present in the material under investigation.
However, the errors in the plating method are known, in a general way, and
within its limitations the plate count is one of our most useful means of
enumerating bacteria. It is very widely used and should be fully understood
at this point. It measures only organisms viable under the conditions of
growth (i.e., kina of medium, temperature, etc.) provided.
The counting of colonies in agar after only a few hours incubation is greatly
facilitated by the use of a stereoscopic microscope with both direct and in-
direct illumination.
As the number of bacteria in the sample increases toward several hundreds or thousands
per ml, the J ml of material removed for the plate count is diluted so that plates are obtained
which show only about 50 to 300 colonies. This is easy after a little experience. It avoids
lethal crowding of the colonies and separates the colonies so that counting is easier. The
number of colonies, multiplied by the dilution, gives the "number per mJ."
,I
Determination of Growth Curve. Let us suppose that we make plate
counts on our culture !Jf Escherichia coli every two hours at first, and plot the
logarithms of the numbers of colonies (roughly, live organisms per ml) against
time. If we were to plot actual numbers instead of logarithms, we should
,. American Public Health Association, New York, 10th ed., 1955.
t It is often desirable to examine the plate by means of a special illumination and a 2 X
or 3 X magnifying glass so as to make even the smallest colonies visible.
TIM!!
Fig. 14-2. Growth curve of hypothetical culture of any microorganisms under optimal
conditions of growth. For explanation see text.
need a very large sheet of paper because the numbers often run into billions
per mI. We may continue making plate counts until no further significant
changes in numbers occur. At the end of this time a curve will have been
obtained which will look somewhat like that seen in Figure 14-2.
A totally different type of curve would be obtained if we were to count
the bacteria in the fluid by means of one of the total count methods described
above. This is because many of the bacteria die in the culture during the 24
hours of incubation and, while appearing in the total count, cannot produce
growth or colonies in the dilution-tube series or the colony count in Petri
plates since these are viable counts. A curve showing total counts as compared
with viable counts is seen in Figure 14-3. '
Phases of the Growth Curve. The curve shown in Figure 14-2 has several
portions which deserve discussion. These are shown by brackets and labels
in the figure.
INITIAL PHASES. Portion A, usually called the latent or initial stationary
phase, represents a period during which the dormant organisms are probably
imbibing water and/or becoming adjusted to the new environment, much
as might occur when a dormant tree is set out in the spring. The exact details
of this "awakening" or "reallimation" process are not known. There is no
immediate growth (increase in numbers). The dotted line indicates that some
few of the cells may actually die off during this period, only the more vigorous
going on to multiplication.
PHASE OF ACCELERATED GROWTH. Once growth begins it is soon mani-
fested in the rising inflection of portion B which is properly called the phase
of accelerated growth but is often referred to as the lag phase. The first two
phases together also are often called the lag phase. During this early period,
when fission is slow, the size of the cells is large: hear the maximum for the
species. This probably represents inhibition of water with consequent swelling,
and the beginning of metabolic activity before fission has occurred.
CELL WALL AND FISSION. It is not unlikely, though not proven, that the
cell walls of the old or dormant cells of the inoculum are thicker and less
elastic and permeable than those of very young, actively-multiplying cells;
the older, mature cells possibly becoming somewhat like the arthrospores of
higher fungi. Certainly, mature and dormant bacterial cells are well known
to be much more resistant to most deleterious influences: heat, disinfectants,
radiations, antibiotics, 'phage, etc., than young, reproducing cells, which
are notoriously vulnerable to such agents.
This thicker wall, if it actually occurs, might interfere with active fission.
After inhibition of sufficient water the osmotic pressure within the cell may
help initiate fission. Thus, growth and synthesis of protoplasm probably
begin very quickly but fission, and consequent increase in numbers, do not
become evident so soon.
INTERFERENCE WITH FISSION. Fission of growing bacteria is readily inter-
fered with by numerous factors, some known, others obscure. The effect is
especially obvious in bacilli. In rod-shaped organisms growth without fission
(or at least without division into separate cells) forms long, tubular filaments.
This suggests a radially rigid tube with open, or less rigid, ends where growth
occurs.
Fission is retarded by the presence of surface-tension reducents, by Mg++
deficiency, by colchicine, by ultraviolet irradiation, by certain variations in-
ducing rough (or R) variants and other factors. The development of fila-
mentous cells is accompanied by profound physiological alterations, some
known (e.g., loss of capsule formation and changed antigen content), others
not known.
During the phase of accelerated growth the time required for each cell to
divide gradually decreases, and fission occurs more and more rapidly as·the
organisms become adapted to the new culture medium.
LOGARITHMIC PHASE. As growth continues the cells reach their maximum
rate of fission, which may become so rapid that the number of organisms
doubles every twenty to thirty minutes. The average size of the cells is at its
minimum for the species during this time. It is conceivable that cell walls are
thinnest during this period. Fission rate varies greatly with different species
and under different conditions of growth. Tubercle bacilli, for example,
probably divide only about once a day at the highest rate of growth.
During this period of most active multiplication (C) the logarithms of the
------- _,-------------------------------------------------------
.,
i'
~
:
~
.
,~ lfoU~s 15
,0 '0
Fig. 14-3. The relation of total to viable counts of bacteria in a pure culture under optimal
conditions of growth.
numbers of live organisms counted at short intervals, plotted against time,
produce a straight line as shown in Figure 14-2. This period is spoken of as the
phase of logarithmic increase. Were this to continue uninterrupted, the culture
would become a solid mass of bacteria in a few hours. A single cell of Esch-
erichia coli allowed to continue such growth for a year or so, would produce
a mass weighing more than the sun!
During this phase most of the cells are physiologically young and biologically active. If a
subculture is made from the flask to a new flask of warm, sterile broth, growth continues at
the logarithmic rate; there is no lag or dormant phase. The lag and dormant phases are
reproduced in more or less degree in subcultures made during any other phases of the growth
cycle than the logarithmic. The young logarithmic cells are apt to be more easily killed
or adversely affected by various inimical agents than older or dormant cells. Biochemical
peculiarities used for identification of organisms are also usually most manifest during this
period. It is important to remember this, as it has a significant bearing on principles of
disinfection, chemotherapy and other phenomena to be discussed.
PHASE OF NEGATIVE GROWTH ACCELERATION. Within a few hours after

the commencement of the logarithmic phase, the organisms begin to encounter
difficulties. Food begins to run out, poisonous waste products accumulate, pH
changes, hydrogen acceptors are used up, respiration becomes difficult, and
the cells interfere with each other. The rate of fission begins to decline and
the organisms die in increasing numbers, so that the increase in number
of live cells slows, as shown in the portion of the curve labeled D of Figure
14-2. This is spoken of as the phase ofnegative growth acceleration.
A number of workers have tried to prevent the development of this phase
by arranging the culture apparatus so that waste products and exhausted
medium may be drawn off at regular intervals without removing the bacteria.
New, fresh medium is added at the same times. Theoretically, a solid mass
of cells should finally result. Actually, growth eventually ceases far short of
this, and the population curve develops as usual, with only temporary changes
(Fig. 14-4). Evidently, like other creatures, microorganisms must have a
certain amount of space for the individual to thrive.
IMMORTALITY (?) OF MICROORGANISMS. It is interesting to speculate upon the relationships
between the age of cells, their multiplication and their continued existence without fission.
Let us compare the fate of two cells just produced by the fission of their "parent." Under
favorable conditions one undergoes certain unknown physical and chemical changes which
are the equivalent of "maturation" and, after a few minutes or hours, undergoes fission like
its predecessor. What is the age of the new daughter cells? How can a cell undergo the mys-
terious changes of maturation and aging and yet by the process of fission produce two young
cens~ Theoretically, such creatures never grow "01"" or undergo senescence. Are they, by
virtue of cOlitinual renewal of youth, immortal~
/ The other of our two original cells fails to reach a state in which fission occurs, and remains
intact. What is its physiological age status? If transferred to a new culture it may begin
multiplication at once and become "young" or it may die. It may die even if left in the orig-
inal culture. Does it die of "old age"? The explanation of its fate is quite obscure. It is
evident that chronological age and physiological age may be very different matters among
microorganisms.
FINAL PHASES. Eventually (the time depending on the temperature, the

size .of the flask and volume of fluid, the composition of the medium and
numerous other factors), the nwnber of cells dying balances the rate of in-
crease, and the total viable population remains unchanged for a time. The
The Growth of Bacteria 2If
total count continues. to' increase~ but not as rapidly as at first. This phase, the
maximwn. stationary phase, is- shown at E.
As conditions become more and more inimical to the bacteri~ the cells
reproduce more slowly, and death overtakes them in ever-increasing numbers,
as shown at F. This is the phase of accelerated decrease or accelerated death
phase.
This progresses,into the logarithmic death phase (G), during which decrease
in number occurs at a regular, unchanging rate.
Finally, conditions begin, to reach an' equilibrium such that both rate of
death and rate of increase tend, to balance each other again at a very low
population'level, and the phase of readjustment (H) and: a final dormant phase
are attained. Complete sterility of the culture may not ensue for weeks or
months, depending on the kind of organism, whether or not the culture is
very acid, etc.
Factors Affecting Growth Phases; The form of the growth curve may be
affected by many factors. For example, if the culture is suddenly plunged
into icewater, the curve at once ceases its upward trend, remains flat for a
time and then begins to decline. If held at 22° C instead of 35° C (for E.
coli) the rise in the positive phases is much less abrupt and much more ex-
tended. Other factors such as pH, concentration of food, and so on, have
their effects also. If the crowding and toxic growth products in the culture are
slightly abated by addition of new. food solution during the maximum sta-
tionary phase, growth immediately resumes at a logarithmic rate for a while.
If a continuous supply of food solution (and dilution of toxic products) is
continued, the maximum stationary phase is longer. If food is then discon-
tinued, the phase of logarithmic death sets in very soon.
20
18
"
...o0.14
-12
"
~IO
G
~8
~
~6
~
~4
2
100 200 300 400 500 600 700

HOURS
Fig. 14-4. Mean grO:wth curves (or Escherichia coli on constant food supply of 15.2 mg
Difco broth per hr at 35° C, showing effect of renewal of food on growth curve. (Jordan,
R. C., and Jacobs, S. E. in J. Bact., vol. 48.)
212 Methods and Phenomena" of Microbiology
Colony Growth. In the foregoing paragraphs we have considered the de-
velopment of a microbial population in a flask ofjluid medium. The surface of
a solid medium, like agar, presents a somewhat different situation. We must
think of each bacterium in the inoculum as starting a new growth in the very
thin film of fluid on the fresh agar surface. Since the new cells* cannot float
and swim very far away from the site of initial growth in the rapidly evap-
orating film, they start to pile up on one another in a restricted area, ~me
times in a pile several thousands of cells high (up to 2 mm). Each heap of
cells is a colony.
The obvious limitations to colony expansions are the facts that: (a) nutrient
solution can diffuse from the agar to the uppermost cells in the colony to only
a limited extent; (b) the available supply of nutrient in the agar in the im-
mediate area is soon exhausted; (c) wastes do not readily diffuse away and
therefore accumulate in the colony and in the agar beneath. The cells at the
top of the heap are obviously at a disadvantage, and soon the upper and
central portions of the colony undergo the effects of aging and senescence and
other effects of unfavorable nutrition and removal of wastes. Apparent muta-
tions or adaptations to the changed conditions may occur here. The growth
of these may appear as new, excrescent colonies on the surface of the old.
They are called secondary colonies. (See Fig. 16-5.) Active growth may con-
tinue at the margin of the colony for some time afterward.
Colonies assume a variety of forms and colors, depending on the species
of bacteria, the kind of nutrients in the agar and the conditions of growth.
Little is known of the forces which cause one species to form smooth, round,
flat colonies, while another produces rough, irregularly shaped colonies with
very high conical or domed or "crumbly" form. This is an excellent field for
investigation by an ingenious graduate student!
REFERENCES
Buchanan, R. E. : Growth Curves of Bacteria. In The Newer Knowledge of Bacteriology and
Immunology. University of Chicago Press, Chicago, 19,28.
Clifton, C. E. : A stereoscopic method for counting bacterial colonies. J. Bact., 1955, 69: 107.
Coulter, J. M.: The Evolution of Sex in Plants. University of Chicago Press, Chicago, 1914.
Deibel, R. H., Dowing, M., Niven, C. F., Jr., and Schweigert, B. S.: Filament formation by
Lactobacillus leishmannii when desoxyribosides replace vitamin B12 in the growth
medium. J. Bact., 1956, 71 :255. \ -
Dickenson, P. B., and MacDonald, K. D.: An electron microscope examination of the
initial cell stage in Streptomyces spp. J. Gen. Micr., 1955, 13 :84.
Henrici, A. T.: Morphology, Variation and the Rate of Growth of Bacteria. Charles C
Thomas, Springfield, Ill., 1928.
Jennison, Marshall W., and Wadsworth, George P.: Evaluation of the errors involved in
estimating bacterial numbers by the plating method. J. Bact., 1940,39:389.
Jordan, R. C., and Jacobs, S. E.: The effect ofterllperature on the growth ofB. coli at pH 7.0
with a constant food supply. J. Gen. Micr., 1947,1 :121.
Lewis, I. M.: Secondary colonies of bacteria with special reference to Bacillus mycoides.
J. Bact., 1933, 25 :359.
Novick, A.: Growth of bacteria. Ann. Rev. Microbiol., 1955, 9:99.
Pease, P.: The gonidial stages in Spirillum spp. and Vibrio spp. J. Gen. Micr., 1956, 14:672.
Powell, E. 0.: A rapid method for determining the proportion of viable bacteria in a cul-
ture. J. Gen. Micr., 1956, 14:153.
Webb, M.: Effects of magnesium on cellular division in bacteria. Science, 1953, 118:607.
• With the exception of only a few species capable of swarming.

Wenrich, D. H., Lewis, I. F., and Raper, J. R., Editors: Sex in Microorganisms. American
ASSoclation for the Advancement of Science, Washington, D. C., 1954.
1951.
Winslow, C-E. A.: The Rise and Fall of Bacterial Populations. In The Newer Knowledge of
Bacteriology and Immunology. University of Chicago Press, Chicago, 1928.
Winslow, C-E. A., and Walker, H. H.: The earlier phases of the bacterial culture cycle.
Bact. Rev., 1939,3:147.
Wollman, E. L., and Jacob, F.: Sexuality in bacteria. Sci. Am., 1956, 195:109.
15
Variation of Microorganisms
HEREDITARY MECHANISMS
ALL MICROORGANISMS undergo variations. In bacteria variations are
frequently quite obvious: color, form, motility and so on. Other variations
are demonstrable only under certain conditions or by special tests: resistance
to antimicrobic drugs, virulence, ability to digest certain proteins, and the
like. Many of these variations are genetically stable (heritable) and appear to
depend on alterations in the genetic mechanisms of the microorganisms. In
the cells of higher organisms the genetic mechanisms include chromosomes*
and genes organized in demonstrable nuclei.
Some bacteria may have nuclei, with chromosomes. Probably most do
not, though the matter is in debate. Viruses, rickettsiae and PPLO do not
appear to have definite nuclei. However, all of these microorganisms certainly
contain nucleoproteins, and chromatinic bodies more or less suggestive of
nuclei and possibly of chromosomes (see Chapt. 10). It is obvious that the
functions of heredity are carried on by these microorganisms regardless of the
exact form of the mechanism involved.
Genes. It seems probable that microorganisms contain genes, or some-
thing very like them. Two distinguishing characters of genes are: (1) self-
duplication, and (2) susceptibility to change (mutability). Whether or not
grouped in a chromosome or nucleus of the conventional form, genest de-
termine the distinctive characters of the microorganism, be it-the smallest
virus or the largest animal or plant: all synthetic (growth) reactions, all respir-
atory (energy yielding or exothermic) reactions, all reproduction, chemical
structure, form, motility, color, and, indeed, every conceivable physiological
process or attribute of the organism. By duplicating themselves, genes trans"
mit those ~haracters to the daughter mi<;roorganism in the process of repro-
duction, whether by sexual or other means.
MUTATION. As long as the genes remain unchanged they continue, like
type in a printing press, to make exact replicas of themselves, and so the
.. The term is derived from the Greek words chroma, for color, and soma, for body. J'his
refers to the fact that when cells are stained with certain dyes for the purpose of studying
their structure and emphasizing structural details, the genetic nucleoproteins and related
structures take the colors very intensely. They are often spoken of as chromatin (color
sj.tbstance). Chromosomes are elongated, rod-like structures in the nucleus.
t The term gene here is extended to include macromolecules of nucleoprotein or nucleic
acids having genetic properties.
214
Variation of Microorganisms 215
progeny "breed true" through many generations. If some disturbing in-
fluence acts upon them, then the genes are altered and make disturbed (i.e.,
different) replicas of themselves. One or more of the genes may be destroyed.
as often happens under ultraviolet radiation. Then certain properties of the
microorganism may be abolished in its progeny as a result. The heritable
characters of the progeny will be permanently different from those of the
parents. In either case a genetic mutation is said to have occurred. The mutant
progeny continues to replicate the mutant form, until a new disturbance of
genes occurs.
The mutant forms may remain stable and permanent, they may immediately
mutate into some new form, or they may revert to the parent form. What
occurs is dependent on: (a) the nature of the disturbing agent (which is often
called a mutagenic agem); (b) the microorganism; and (c) the environment.
Sometimes one gene may control a whole series of successive biochemical
reactions produced by a set of individual enzymes. Much depends on whether
the enzyme controlled by the gene is in an important "key position" in tbe
biochemistry of the microorganism. Some genes act only to modify, suppress,
or enhance the action of other genes. Sometimes a mutation produces effects
which are highly beneficial to the mutant organism, sometimes of only. in-
different significance under existing conditions, sometimes detrimental and
someLmes immediately lethal; i.e., the daughter microorganism is not
viable. * This process of survival of the more fit mutants has been called
natural selection. Mutation and natural selection are the basis for the origin
of new species and are the main spring of organic evolution (Darwin).
The same basic principles and mechanisms apply equally among elephants,
whales, mice, men and the most minute microorganisms. Indeed, the more we
study and compare, the more likeness we find in fundamental life processes
among all creatures, from viruses to man. The chemistry of all is strikingly
similar: the digestive, synthetic and respiratory chemical processes; the en-
zymes; the cell structures; genetic and cell growth mechanisms; responses to
environmental influences; and so on and on. Of course, the patterns are
modified to fit various types of environment, life history, size, etc., but the
basic rules and mechanisms are all obviously derived from the same primitive
pattern.
INDUCED MUTATIONS. When we produce mutations by some known means
under our own control, ultraviolet irradiation for example, we call them
induced mutations. .
SPONTANEOUS Ml!TATIONS. In the process of reproduction genes often be-
come translocated :with respect to each other and this definitely affects their
action and the characters of the microorganism involved. Further, genes
sometimes seem to! undergo minor chemical changes within the microorgan-
ism, possibly due to molecular collisions, adhesions, etc. Thus, while theo-
retically progeny inherit parental genic equipment exactly, the genes may be
somewhat change~ in the very process of multiplication and so the progeny
may differ genetically from their progenitors. Thus, genetic mutations may
result from intrinsic causes which we cannot control or define. Such mutations
are often said to occur
I,
"spontaneously."
• Capable of living;
Spontaneous mutations may be caused also by extrinsic causes: some
chemical effect of the surrounding medium, cosmic rays or what you will.
Plasmagenes. In addition to genes, certain other character-determining
agents almost certainly exist in the cytoplasm, apart from the genes localized
in the nucleus. They are given various names, a convenient one being plasma-
genes. Many of these appear to have at least two of the most distinctive prop-
erties of genes: self-duplication and mutability. Some plasmagenes may be
produced by genes. However, a number are known which gain entrance to
the cell from outside, and of which the chemical composition is relatively
simple, for example, certain polymerized deoxyribonucleic acids. Some may
be extracted and transferred from one cell to another by artificial or natural
means. They act as mutagenic agents, which are discussed below.
MUTAGENIC AGENTS
These are the agents (physical, chemical or biological) that induce muta-
tion. We may group them for convenience as follows:
A. Extrinsic mutagens
I. Microbiological agents
a. Macromolecules like certain deoxyribonucleic acids;
b. viruses, and virus-like factors;
c. bacteria.
II. Radiations, especially ultraviolet and x-rays.
III. Injuries or prolonged irritations of various kinds:
These appear to act more obviously on tissue cells in larger animals and
to result in cancer, but certain irritants, e.g., nitrogen mustard, are widely
studied as mutagenic agents in bacteria.
IV. Chemicals, especially methylcholanthrene, arsenic, chromium, urethane, mineral
oils, creosote, tar, nitrogen mustard, organic peroxides and H202, MnCh,
etc.
V. Sex hormones P)
B. Intrinsic Mutagenic Agents
I. Recombination of nonidentical chromosomes or portions of chromosomes {in
cells possessing chromosomes}.
II. Translocations of genes within a .chromosome or crossing over of genes between
chromosomes.
III. "Spontaneous" changes in the gene itself; possibly due to molecular impact,
adhesions, chemical modifications, etc.
A. E?CTRINSIC MUTAGENS
1. Microbiological AgentS. Among the extrinsic mutagens those included
in Groups a and b are of especial interest at this point.
a. MACRO.MOLECULES AND MICROBIAL TRANSFORMATION. This brings us
immediately into a fascinating "never-never land"; the twilight world where·
heredity and disease, living and non-living, virus, macromolecule and gene
are distinguishable' with difficulty, like shadowy objects -seen under sunlit,
shimmering water.
ENTRAINMENT AND TRANSFORMATION. As early as .1925, data were published
indicating that if one species of bacterium were cultivated in the presence
of another (of a rather closely related species) the first would acquire one or
mor,e properties of the slfcond. Apparently something passed from one to the
other. This was originally spoken of as "entrainment." For example, in one
study, a harmless streptococcus from cheese was shown to acquire the
property of forming erythrogenic (scarlet fever) toxin when grown in contact
with scarlet-fever streptococci. Similarly, in another study the virus of rabbit
fibroma* was changed into myxomat virus by bringing the fibroma virus
into contact with dead myxoma virus or virus-free extracts thereof. In another
study it was found that extracts of dead, encapsulatedt (smooth), type-III
pneumococci§ would induce live, non-encapsulated (rough), non-type-
specific cells (derived from type-II pneumococci) to change into smooth,
type-III pneumococci (see Fig. 15-1). Similar changes have been induced in
encapsulated strains of Escherichia coli and other bacteria. All of these changes
were, in all appearances, biological (genetic) mutations. Genetic changes in-
duced in this way are now spoken of as transformations.
THE TRANSFORMING PRINCIPLE. In 1944 the substance in the extracts of
smooth, type-III pneumococcus responsible for the.'. change of the rough,
Fig. 15-1. (I) Colonies of R variant of Pneumococcus Type II plated on blood agar from
a culture grown in serum broth in the absence of the transforming substance (X 3.5).
(2) Colonies of the same cells after transformation during growth in the presence of active
transforming principle isolated from Type III pneumococci. The smooth, glistening, mucoid
colonies shown are characteristic of Pneumococcus Type III. (Avery, O. T., MacLeod,
Colin M., and McCarty, Maclyn in J. Exper. Med., vol. 79.) The photograph was made by
Mr. Joseph B. Haulenbeck.
!
* A slowly-progressive, not highly-lethal, disease of rabbits resulting in the formation of

fibrous tumors of the skin.
t A very malignant, 'rapidly-fatal, disease of rabbits causing slimy disintegration of rabbit
tissues (from Greek myxa, for slime).
t The capsule of many bacteria has a very distinctive chemical composition in various
species and in subdivisions of species called types. Without its capsule the bacterium be-
comes "rough" and loses type characteristics. Encapsulated bacteria are usually found to be
in the "smooth" phase.
§ The cause of lobalj pneumonia and numerous other infections of the respiratory tract:
sinusitis, middle ear, etc. (Diplococcus pneumoniae), commonly found in the normal throat.
218
.
Methods and Phenomena of Microbiology
type-II pneumococci to smooth, type-III pneumococci was found to be a
macromolecule of highly polymerized* deoxyribonucleic acid (DNA).
Thus,. the transference of specific, hereditable characters from one micro-
organism to another is not necessarily dependent on sexual functions, or
even on what we ordinarily think of as genes. As exemplified by the trans-
formation of pneumococcal types, here is a substance (DNA) of definite and
relatively simple composition, taken from a dead cell, applied to the exterior
of a living cell of a different type, which enters the living cell and genetically
changes its type to that of the first cell, permanently. Is this a mutation?
Is it Lamarckian? The chemistry of the altered cell is such that it goes on
manufacturing more of the mutagenic substance (DNA) which, in turn,
maintains the new type-specificity and can transmit it. The DNA becomes a
self-replicating (or ceil-replicating?) part of a new cell; an artificially trans-
missible gene? .
Does this suggest prophage? Can DNA, transferred from one cell to an-
other, "come alive"? Is such a relationship similar to infection by a virus?
Both virus and DNA appear to be self-replicating (or cell-replicated?) genetic
material, dependent on a live cell for continued reproduction. Does the DNA
enter the nucleus, or a gene, or remain in the cytoplasm? Can any cytoplasmic
agent exert a genetic effect? It appears so, though the matter is not settled.
Such a cytoplasr;nic genetic agent is caIIed a plasmagene.
b. VIRUSES AND VIRUS-LIKE FACTORS: 1. PROPHAGE (PROVIRUS). We have
seen, in our discussion of bacteriophage, how DNA enters a 'phage-infected
bacterial cell and, as prophage, takes up a position and function as a normal
gene or heritable genetic constituent (or a symbiont?) of the cell and confers
new, heritable characters upon it. An obvious difference' between this phe-
.nomenon and the transformation of pneumococci, etc., is that, whereas the
pneumococcus-type-transforming DNA is extracted by artificial means from
dead pneumococcus cells and is artificially transferred by applying it to the
exterior of the transformed cells, the prophage DNA is transferred by a
microorganism ('phage) and is placed within the transformed cell by the 'phage
as part of a natural process of infection.
2. KAPPA PARTICLES. These are visible particles, about I J.I in diameter,
which contain DNA. They may be introduced, either artificially or during
sexual conjugation, into the cytoplasm of la ciliate protozoan, Paramecium
aurelia. When so introduces! these particles apparently reproduce themselves
(or are they replicated by the Paramecium ?).
Like a virus, Kappa can develop only in living cells of Paramecium. Note
that Kappa exist and are transmitted in the cytoplasm regularly at each cell
division-a type of plasmagene. They. lnduce the cell containing them to
undergo an apparent mutation which produces a -poisonous agent (a toxin)
which is called pararnecin. This kills other paramecia, not infected with
Kappa (but not those containing Kappa). Thus, Kappa acts as a mutagenic
agent.
Is Kappa a virus? An errant gene? Is it even alive? At any rate, it is some-
what more than a mere macromolecule of DNA like the pneumococcus
.. A polymelized substance is one in which the unit molecules have combined with each
other into large, complex molecules having the Same proportionate composition of elements
but a multiple of the molecular weight of the unit molecules.
Variation oj Microorganisms 219
transforming principle. It is a truly cytoplasmic genetic agent, but whether
it is a living virus is not yet clear.
There are several known (and probably many still-to-be-discovered) similar
cytoplasmic genetic agents, such as the Sigma of Drosophila, the mammary
gland tumor agent of mice, and chondriosomes of plants (see references).
3. BACTERIOPHAGE AND TRANSDUCTION. In 1951 a discovery of funda-
mental importance to microbial genetics, and to studies of heredity in general,
was made. It was observed that bacteriophage, propagated in toxigenic*
strains of Corynebacterium diphtheriae* and then completely separated from
them and later propagated in atoxigenic* strains of C. diphtheriae induced
toxigenicity as a mutation in the formerly atoxigenic strains. It was very
puzzling, however, to find later that toxigenicity could be induced by 'phage
derived from ataxigenic strains. We would like to know exactly how. Perhaps
there are "incomplete" genes which can be completed by 'phage.
The phenomenon of transfer of any genetic character by bacteriophage
from one bacterium to another is called transduction. The mechanism of this
transfer is not clear. The idea arose that the 'phage in some way picked up or
adsorbed DNA from one bacterial strain and introduced it into another
strain. This may be so, but treatment of the 'phage with a powerful enzymet
which destroys DNA did not interfere with the 'phage or the transmission
of toxigenicity. However, the DNA might be inside the 'phage particles,
protected from the enzyme.
'Phage transduction can transmit numerous other genetic characters in
other organisms. Apparently any gene-controlled characteristic may be thus
transmitted. In the genus Sa/mone//a,t for example, various nutrient require-
ments, enzymic functions, resistance or susceptibility to antibiotics, motility,
the chemical makeup of the flagella, and so on, have been transduced. In the
genus Bacillus§ flagella and motility were transduced by 'phage, from motile
B. cereus (a harmless saprophyte) to non-motile E., anthracis (cause of the
• disease anthrax). Curiously, in many instances the 'phage which transduced
was derived from a non-motile, lysogenic, strain of B. anthracis. In this last
series of tests, DNA-ase applied to 'phage during the transduction completely
inhibited the transduction. Presumably the DNA in this example of trans-
duction was on the exterior of the 'phage particles, unlike the DNA in the
C. diphtheriae toxigenicity transduction or the Salmonella transduction, which
were not affected by DNA.
C. BACTERIA. One of the most interesting examples of Jiving mutagen is
the bacterium (Agrabacterium tumefaciens) which causes "crown gall" dis-
ease, a malignant growth on the Paris daisy and related plants. Once the
growth is initiated it can continue to grow, like a cancer. The initiating bac-
I
• C. diphtheriae is the bacillus which causes diphtheria (see Chapt. 35). The principal
effects of diphtheria on the patient are due to the diphtheria toxin, a poisonous waste
product which the toxigenic bacteria secrete whenever they grow in a suitable medium, in a
laboratory or a patient's throat. Strains of C. diphtheriae are often found which are atoxi·
genic: that is, they appear never to have had (or to have lost) the power to produce toxin.
t Deoxyribonuclease-(DNAase)
~ The group containing the bacillus of typhoid fever (gram-negative rods).
§ Gram-positive, spore-forming, aerobic rods.
terium need no longer be present. Apparently a plasmagene-like agent is
transferred from bacterium to plant cell.
Under ordinary circumstances the factors continue to "grow" in the cells
but can be eliminated by certain experimental procedures so that the cells
return to normal. Is such a cure possible in human cancer?
Curiously enough, the plant-tumor-inducing property can be transferred
from Agrobacterium tumefaciens to several species of closely related, but
harmless bacteria by: (I) extracts of infected crown-gaIl-tumor tissue; (2)
killed A. tumefaciens; (3) nucleic acid and deoxyribonucleic acid from A.
tumefaciens. These are all good examples of bacterial transformation.
Fertilization as a Mutagenic Agent. The essential feature of sexual fertiliza-
tion in all forms of life exhibiting sex is at the microscopic, single-cell level. It
is the complete fusion of the nucleus of a haploid, male gamete with a hap-
loid, female gamete. A diploid cell results. This, in essence, is as true for the
simplest sexually reproducing protozoan or fungus as it is for violets, human
beings or whales. * The essential event is the transference of genetic DNA
from male to female gamete. The male DNA enters the genetic mechanism
of the female cell, becomes self-replicating and contributes to the genetic
character of the resulting diploid cells. Does this suggest infection with a
virus? Transduction? Transformation with DNA? Kappa in Paramecium,
etc. ?
We may if we wish, for purposes of argument, regard sex in higher animals
as an evolved, improved, very selective, very certain means of transmitting
certain, particular, genetic material from one particular cell to another par-
ticular cell. On the unicellular level of protozoa, yeasts, etc., simpler and less
perfected means exists: simple cell fusion or conjugations" without differentia-
tion of male and female. At the still lower level of bacteria and viruses, the
mere carrying of a bit of genetic DNA by any hit-or-miss, catch-as-catch-can
means to any "competent" cell in the vicinity appears to suffice: by 'phage,
by mere bathing of the cell in a solution of sterile DNA, by agents like Sigma, •
etc.
Disease and Mutation. It may be that some DNA is excessively active, or
is associated with protein or cytoplasmic structures that make it incompatible
with normal cell development and then it causes disease and we call it apatho-
genic virus. The line between a pathogenic yirus and a plasmagene seems very
vague. The true relation of sexual fertilization to living mutagens like 'phage
and other viruses, to Kappa, Sigma, DNA, and to (probably) many other
DNA-transmitting agents is, of course, not yet clear. That they are all pieces
of a grand biological picture-puzzle, which is being gradually put together by
researchers, into a' beautiful picture Of .N~ture seems an almost inescapable·
conclusion.
Genetic Recombination. One of the principal pieces of evidence favoring
the existence of nuclei, chromosomes, genes and sex in bacteria is based on
observations interpreted as crossing over and recombination of genes in a
particular strain of Escherichia coli, as follows. A certain strain' of Escherichia
coli, known as K-12, was irradiated with ultraviolet light to produce muta-
tio~s. Among the mutapt strains were two which had lost certain synthetic
• 1n parthenogenesis, an exception common in some invertebrates, an egg cell can de-
velop and grow without fertilization by a sperm cell.
abilities and, to live, absolutely required certain nutrient substances which
they were able to synthesize before mutation.
As a result of the irradiation, one strain (A) was unable to synthesize
biotin and methionine; the other strain (B) threonine and proline. The two
nutritionally "deficient" (auxotrophic*) strains were then cultivated together
in a medium supplying the required pairs of amino acids: biotin and methio-
nine for A; threonine and proline for B. Thus, both strains were able to grow
side by side, providing opportunity for sexual contact. Test for sexual fusion
was then made by looking among the progeny of these cells for recombination
of hereditary characters. From the mixture there were isolated strains capable
of synthesizing all four amino acids. This suggested that sexual fusion be-
tween A and B had occurred, resulting in offspring exhibiting synthetic
properties of both A and B. The new, fully-synthesizing (prototrophict)
types CAB) normally occurred too rarely (if at all) as mutants in either of the
auxotrophic strains to be detected before growth together.
While such data are highly suggestive and stimulating, they may well be
susceptible to other interpretations than sexuality and fusion of cells; for
example, transduction or transformation by DNA. The matter is one for
students of genetics.
Summary. In summary we may say that: (1) nucleoprotein (with deoxy-
ribonucleic acid: DNA) appears to be the material basis of heredity. (2) We
may postulate that there are several means by which this hereditary material
can be transmitted from one receptive cell to another: (a) by sperm during
sexual fertilization among higher plants and animals; (b) by various means of
nuclear fusion, with or without perceptible sexual differentiation, as exempli-
fied by conjugation. in certain protozoa, yeasts and molds and other fungi
(possibly in some bacteria like E. coli K 12); (c) by mechanical transfer of
DNA as in pneumococcus type transformation; (d) by biological transmission
of genetic material as seen in 'phage transduction of toxigenicity in C. diph-
theriae; (e) by latent infection with viruses as illustrated by lysogeny in bac-
teria, herpes simplex infection, Sigma, Kappa, chondriosomes, etc.
These processes may be thought of (temporarily at least, for purposes of
argument) as representing stages in the evolution of the sexual process; each
representing improvements in means of transmitting heredity material by
developing from the hit-or-miss, catch-as-catch-can processes toward the
more and more specific, exclusive and certain methods represented by sperm-
ovum mechanisms .. There appears to be a gradation in the complexity both of
material transmitted and of transmitting agency.
I
REFERENCES
I
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t Proto is from a Greek root meaning original or primitive.
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16
Some Methods of Studying Variations;

Common Variational Types
Rates of Mutation. "Spontaneous" mutation occurs among bacteria at

widely varying rates. Take, for example, mutation to 'phage resistance. In an
actively growing culture of a 'phage-sensitive bacterium a mutant cell, re-
sistant to 'phage, may occur as rarely as I cell in 105 • This is a fairly high
mutation rate. In another culture only one mutant cell may be found among
10 10 cells or more. On the other hand, one culture was reported in which 1.3
to 15 per cent of aU live cells were mutants. Mutation rates range commonly.
around 10 7 •
It is evident that unles~ large numbers of cells are examined mutants are
not likely to be found. In a small culture (5 ml), the billions of cells (5x109)
would contain only around 500 mutant cells. The detection and isolation of
these 500 mutant cells among the 5 billion is not easy without special tech-
niques.
Detection and Isolation of Mutants. Detection of "obvious" mutants such
as sporeforming or non-"sporeforming is simple. Anyone who knows about
culture methods and the resistance of spores to heat can devise a simple
laboratory procedure to detect the presence or absence of spore-forming
mutlmts in a culture. Detection of mutants resistant to various unfavorable
factors is obviously done by SUbmitting a culture, containing a billion or so of
actively growing cells, to graded concentrations of the unfavorable factors
such as an antibiotic or disinfectant. Survivors at each level of concentration
obviously must be resis(ant to that concentration. An adaptation of this
principle especially for de~ection and isolation of mutants of graded resistance
to antibiotics consists of the gradient plate.
1. THE GRADIENT PLATE. In this method one pours a shallow layer of
nutrient agar (20 ml) containing an appropriate concentration of the desired
antibiotic into a Petri piate and allows it to solidifj in a sloping position
(Fig. 16-1). When solid, a second 20 ml of agar is poured over the first layer
and the plate is held horizontally till it is solid. Owing to the reciprocating
gradations in the thickn~ss of the layers of agar, the concentrations of anti-
biotic on the top surface will be graded from high at one side of the plate to
low at the other.
223
UlliA@\\\\1\\®\ID)J»)WJlWJ2:2?~_IJ~
Fig. 16-1. Use of the gradient plate to detect drug-resistant mutants and to measure
their degree of resistance. In the Petri plate above, the bottom layer (N) consists of ordinary
nutrient agar allowed to solidify in a slanting position. The upper layer (A), poured and
allowed to solidify with the plate in a level position, consists of the same sort of agar but
with a drug (say, Aureomycin or chlortetracycline) added in measured concentration. The
antibiotic diffuses from the upper into the lower layer, leaving a gradient of concentrations
of the drug at the surface; greatest at the left of the plate shown above, least at the right.
Broth cultures of four different species of bacteria are streaked across the plate, with a
sterile brush, parallel with the slope of the agar (in this case, left to right). After incubation
it is seen that all organisms grow well at the very lowest concentrations of the drug but react
differently in the increasingly greater concentrations. Organism 0 shows moderate resistance
of most colonies, a few more than others, but none thriving much more than the majority.
Species M shows some resistance by most cells but contains some that are wholly resistant
and even appear to be stimulated by the drug. Organism C shows complete indifference to
the drug. Species T contains many slightly resistant cells, but all are completely inhibited
by exactly the same concentration of the drug. (Adapted from W. Szybalski, in Science,
1952, vol. 116.)
A culture is introduced at the area of low concentration and spread thinly

toward the area of high concentration. Of the cells which are deposited in the
areas of higher concentration only those of higher and higher resistance will
grow and form colonies. They are th!ls revealed as' resistant mutants. Their
degree of resistance is indicated by their position on the gradient plate.
2. REPLICA PLATING. This is really a very clever adaptation of the printers
art to bacteriological purposes. One prepares a wooden disk with a fiat
surface; in diameter about 9 cm (1 cm less than that of a Petri plate). A piece
of velvet about 12 cm square is placed smoothly over the surface of the disk,
drawn firmly down over the sides and fastened in place with a band. 'The
whole is sterilized. .
, Let us now select a Petri plate containing nutrient agar, on the surface of
which are several hundred colonies. How determine which colony is one of
Some Methods of Studying Variations; Common Variational Types 225
(a) streptomycin-resistant mutants? (b) "threonineless" auxotrophs? Their
appearance in no wise distinguishes them. We could pick a portion of each
of the several hundred colonies with a needle and transfer it to (a) a medium
containing streptomycin and (b) a medium containing threonine and (c)
also one without threonine. If inoculum from any colony grows on the strep-
tomycin-containing medium it is obviously streptomycin-resistant. If it
grows on the medium with threonine and not on the same medium minus
threonine, it is a threonine-deficient auxotroph. But this entails at least 300
(usually thousands) of inoculations (requiring 28 hours of work per dayl)
in order to find-the one or two mutant colonies among the hundreds of col-
onies on the plate.
Much time and labor is saved if we press the original plate gently down
on the sterile, velvet disk. Each colony leaves a small spot of cells where
it touches the velvet. The plate is removed. If, now, we press the surfaces
of three sterile plates successively down on the velvet we have, in three
motions, "printed" all of the colonies on the original plate in three replica
plates; (a) one containing medium with streptomycin, (b) a plate with agar
containing threonine and (c) a plate containing agar devoid of threonine.
The three replica plates are then incubated and colonies develop. Each
colony is in its own, readily-determined location on the surfaces of each of
the "selective" replica plates (a, b, and c). Only the colony of streptomycin-
resistant mutants will grow on the streptomycin-containing plate (a); the
threonine-deficient mutant will grow on the threonine plate (b); but not on
the plate without threonine (c). The mutant colonies are thus easily identified
by their growth and locations on the replica plates (Fig. 16-2).
3. ISOLATION BY PENICILLIN. This method depends on the fact, previously
emphasized, that organisms in the logarithmic phase of growth are most sus-
ceptible to various deleterious influences, including the action of penicillin.
Keeping this in mind let us irradiate a culture of bacteria with ultra-
violet light. Suppose purineless auxotrophs are to be looked for. (As shown
in Chapter 12, they are not immediately apparent. Several generations of
growth must be allowed before the mutational lag is overcome.) After the
proper, post-irradiation incubation, all of the cells are removed from the
culture me(iium by centrifugal force. They are resuspended in an inert saline
solution in which they have no food and so do not grow at all; they are thus
"starved" for some hours. Now, to separate the auxotrophs from the pro-
totrophs, 'penicillin is added to the suspension, along with just enough purine-
free medium to permit the prototrophs to grow but not the purineless, auxo-
trophic mutants. :
As soon as the prototrophs start to grow the penicillin kills them. The still
"dormant" auxotrop~s are now removed from the -penicillin to a purine-
containing medium where they grow vigorously and are proudly exhibited to
the class.
It might be mentiorled that several precautions are necessary in each stage
of the procedure, especially against a "joker" called syntropism. *
SYNTROPISM. This little scientific joke is played by the prototrophs in the
final stage of the above procedure. At this point the unsuspecting prototrophs
are being done to death by feeding them in the presence of penicillin. As they
.. From Greek roots signifying "nourishing each other." \
o
l
Fig. 16-2. Use of the replica plate for detection of colonies of mutants. Plate A contains
agar complete in all essential nutrients and free of penicillin. It was inoculated with a culture
of a certain species of bacillus. It was then incubated. It is desired to determine which of the
colonies (if any) consists (I) of mutants able to grow in the absence of the amino acid, thre-
onine, and (2) of mutanls resistant to penicillin. Plate A is inverted over, and lightly pressed
down upon, the sterile velvet disk Vand then removed, leaving spots of live organisms in
positions corresponding to the positions of the colonies on plate A. Immediately thereafter
plate T, containing sterile agar like that in plate A but devoid of threonine, is pressed upon
the velvet disk, removed and incubated. Plate P, containing agar in all respects like that in
plate A but with penicillin added in measured concentration, is likewise inoculated by pressing
it upon the velvet disk. After incubation, plate Thas only one colony (t) obviously consisting
of mutants able to synthesize their own threonine (or able to grow without it). Plate P has
a colony (P) resistant to penicillin in the concentration, at least, in which it exists in plate P;
perhaps wholly resistant.
start to grow they synthesize just enough purine to start the auxotrophs to
growing and so the auxotrophs, too, are killed by the penicillin; a dastardly
trick!
It is worth interrupting at this point to emphasize the fact that syntropism
is of very great importance in' natural ecological relationships. For example,
in the soil, one organism NitrosQmonas will produce a substance (e.g., NaN0 2)
without which another (Nitrobacter) cannot grow. Nitrobacter is an autotroph
requiring Na-N02 as a source of energy. The NaN0 21is oxidized to NaN0 3,
the most valuable and expensive nitrogenous food for agricultural crops.
Thus, Nitrosomonas nourishes Nitrobacter nourishes crops nourishes cattle
nourishes man; a quadruple play at home plate! '
Syntropism is an important ecological factor operative among bacteria in
the soil, the sea, and in any natural, mixed population.
Adaptation. Various forms of life living in curious habitats such as ocean
ooze, oil wells, hot springs or Great Salt Lake have "adapted" themselves to
such situations as though the organisms, aware of the peculiar qualities neces-
sary to inhabit such an environment, actively developed those properties for
the specific purpose; an idea based on the doctrine of entelechy or teleology,
Some Methods oj Studying Variations; Common Variational Types 227
now obsolete in biology. The inhabitants of such situations are, however,
descendants of mutants which, purely by chance, possessed particular pbysi-
ological properties and, being carried by some purely chance mechanism
(wind currents, underground seepage, birds, etc.), were able, because of those
peculiar properties, to survive in the environment in which they found them-
selves. Other celis, not so endowed, perished.
Thus "adaptation" in microorganisms is usually a concise expression of the
fact that mutation and natural selection have operated.
ADAPTIVE ENZYMES. Very interesting changes, almost suggestive of·en-
telechy, may be induced by cultivating organisms in the presence of certain
food substances that they ordinarily do not use. For example, if you con-
tinuously cultivate a strain of bacteria not known to ferment lactose, in
medium containing lactose, the strain eventually exhibits the property of
fermenting the lactose promptly. The cells are found to contain an enzyme
lactase, which they did not possess in demonstrable amounts before. Removed
from contact with the lactose, the power to ferment it may persist, like a mu-
tation, or it may soon disappear. Any enzyme thus appearing in response to
an environmental factor is called an adaptive enzyme.
The production of adaptive enzyme has been shown to occur in some in-
stances in "resting" vegetative cells; Le., cells that are alive but not multi-
plying. This is obviously not selection of a mutant by the environment since
there is no growth.
Sometimes it is possible to detect minute amounts of an adaptive enzyme
in cells before adaptation; in such instances adaptation seems to be merely
an enhancement of an existing though imperceptible function. In other in-
stances, no enzyme is detectable before adaptation. In these cases adaptive
enzyme formation seems to represent the induction of a new function. (But
failure to find an enzyme does not prove its absence!)
The actual mechanism of adaptive enzyme formation and the nature of the
stimulus which starts that mechanism working are not known. That actual
synthesis of new enzyme protein often occurs is indicated by the fact that
amino nitrogen and an cnergy source are usually required for adaptive
enzymes to develop.
CONSTITIJTIVE ENZYMES. Any given species of bacterium possesses certain
tairly definite and stable properties by means of which we can identify it.
Among these properties are form, motility, Gram reaction, and certain in-
herent enzymic properties such as ability to ferment certain carbohydrates,
digest certain proteins,ietc. These enzymes are present under all ordinary con-
ditions of growth, whether the substrate on which they act is present or not.
The enzymes are part pf the permanent constitution of the cell and as such
are called constitutive enzymes. Perhaps the only difference between a con-
stitutive enzyme and an adaptive enzyme is one of degree of functional
activity.
Mutations Meeting ~ole Populations. Changes which appear to involve
whole populations of organisms are in many instances demonstrably due to
mutation. For example, in liquid-medium cultures of smooth (S) Brucella*
species the amino acid<, alanine, not initially present in the medium, accumu-
.. Small, gram-negative, non-motile, non-sporeforming rods, the cause of "Bang's dis-
ease" (brucellosis or undulant fever) in domestic animals and man.
228 Methods and PMnomena oj Microbiology
lates as a waste product of the growing cells. d-Alanine (not I-alanine) has the
effect of suppressing the growth of the smooth (S) cells used for the initial
inoculation. But it markedly favors the growth of rough (R) cells. Thus, the
whole population appears to change from S to R with age, whereas, actually,
the R cells were initially present as mutants in the culture to the extent of
about I: 107 cells. Thus, vast population changes can result from apparently
trifling causes initiated by the populations themselves. This is only one illus-
tration of many such alterations.
It is interesting to imagine what might happen if human beings should
be now present on the earth, as mutants, who are totally insensitive to the
radiation effects of A and H bombs. Radiation-resistant mutants of bacteria
occur. Who knows but that you, fair reader, may be radiation-resistant your-
self? It is not recommended that anyone stand near an exploding H bomb to
ascertain this point about himself. If total war were to occur (perish the
thought!) such mutants could be the only survivors. This would be mutation
and natural selection with a bang!
Sectors in Colonies. On a solid agar surface a bacterial colony grows
radially. The oldest (and senescent) growth is at the center. The newest (and
most active) growth is around the periphery. In the development of a colony
the single cell initiating the colony may produce billions of progeny. If a
visible type of mutation (for example, production of a bright red pigment)
occurs in a single cell during the radial expansion of the colony, a roughly
triangular, and visible, sector of red cells appears. The apex of the triangle
is toward the center ofthe colony and is the point at which mutation occurred.
The base of the sector is at the periphery of the colony. The cells in the sector
are progeny of the mutant which initiated the sector (Fig. 16-3). Another
visible type of sector in a colony is produced by loss (or gain) of the property
of spore formation. If, in a colony of cells producing spores as they grow,
sporeless mutation ~urs, the progeny are sporeless and they appear as a
translucent sector in the colony (Fig. 16-4).
V ARIATlONS IN COLONY FORM
Rough and Smooth Colonies. These have been described briefly in an
earlier chapter. Here we may give a little more detail. Let us consider as an
example the colonies produced on infusion agar by a certain strain of gram-
positive bacillus. The colonies ordinarily produced by the organism on meat-
extract agar are about 2 mm in diameter, gray and translucent. They appear
in two different forms: Sand R.
IN THE S FORM the colonies are perfect!y smooth, moist and homogenous,
convex, circular and glistening and have regular margins (Fig. 1-6). They are
butyrous (butter-like) in consistency.
THIl R TYPE OF CoLONY is not glistening but dull, and has rough or wrinkled
surfaces, and irregular edges. It is dry and crumbly in consistency (Fig. 1-7).
It is commonly observed that the cells in rough colonies form themselves
into long tangled filaments on the surface of solid media whereas, in smooth
colonies, the cells tend to occur singly.
In broth the rough growth is usually granular or tlaky, often growing in a
thick scwn or pellicle on the surface or settling to the bottom. Long filaments
are seen. fluggesting a defect in fission, or separation of cells, although growth
Some Methods of Studying Varilltion.ri Common Variational Types 229
obviously occurs. The defect, if it is a defect, may affect only some factor in
the cell walls. The smooth growth produces an even turbidity in broth.
The changes in colony form are also intimately related to profound chemical
changes in the cells, especially at their surfaces. These in turn are related to
defense against outside influences, reactions to electrolytes, virulence, capsule
formation and many other characteristics: some visible; some detectable only
by selective methods like replica plating; others unknown or not clearly
understood.
Practically all species of bacteria vary in this way. There are often inter-
mediate degrees of "roughness" or "smoothness." As suggested in Chapters
3 and 4, this may be related to a haploid-diploid phenomenon, or to a di-
morphic phenomenon in some molds.
In many species the change from S to R or from R to S is readily induced
by any of several stimuli and often occurs "spontaneously." It is thought by
some that the alternation in form is not an oscillation between the two
(R ~ S) but a series of progressive mutations R -+ S -+ R -+ S. The exact
facts remain to be revealed.
Frequently S forms seem to represent a reaction to unfavorable environmental factors,

though this is not necessarily always so. Contact with disinfectants, certain salts, etc., may
stimulate the appearance of S forms. Among bacteria capable of infecting animal tissues,
U\e appearance of the S form is favored by contact with the infected host which resists the
infection. Virulence is thus commonly associated with the S form, and this, in turn, with the
formation of a visible capsule or with analogous defensive modifications in the surface of the
organisms.
These surface alterations, in turn, affect the colloidal properties of the cells and hence
Fig. 16-3. Colonies of one species of

bacterium grOwing on the same agar
plate, originating from the same inocu-
Jum. Variation between white pigmenta-
tion (light) and non-pigmentation or
ttansparent (dark) is clearly evident.
Clearly shown also is the appearance of
mutants during the development of each
type of colony, with resulting pigmented
(or non-pigmented), triangular sector in
the colony. (photo courtesy of Dr. Mary
Barber, in J. Gen. Micr., 1955, vol. 13.)
16-4. Colonies of a species of spore-forming aerobic bacillus showing sectors (light

of nonsporulating variants of the same species. (Photographs courtesy of Dr. J.
Brown.) (X about IS.)
230 M~tlrotb anti PMnoIlfllfll of Microbiology
Fig. 16-5. Different morphological types assumed by a single kind of organism (X Il00).
their immunologic· and antigenict properties (since these are aU largely surface phenomena).
These alterations in the antigenic properties of organisms are of the utmost importance.
Wbole systems of results may depend on whether S or R forms are being used.
When S forms of bacteria are cultivated on artificial media in pure culture, removed
from competition witb other bacteria tbat occur in natural environments like tbe soil or
throat, under wholly uniform, benign and favorable conditions, they frequently tend to
lose their virulence and their protective surface structures and revert to the saprophytic,
or possibly less highly differentiated, R form. There are exceptions to this, but in artificial
cultures it is common to observe this transition.
Other differences between R and S variants are frequently noted. For example, in the
spore-forming organism referred to above, colonies are frequently observed having sectors
of non-sporulating cells (Fig. 16-4). By means of a very fine needle and an instrument called
a micro-manipulator, these two types of cells (sporulating and non-sporulating) can be
separated in pure culture. It was found that a single cell of eitbertype can produce the other,
in either the R or S form. Thus, sex is not involYed. No reason has been found to account
for the variations. They seemed to occur spontaneously. Loss of spore {ormation is a com-
mon observation. In addition to this, ceUs of the organism in different colonies, ofttn of the
same type, varied so widely in size and form as to be unrecognizable as the same organism
(Fig. 16-5).
/ Changes in the cell form of the same general nature are of common occurrence in many
species of bacteria, but are not always exactly the same as these, often being much less
extreme. Such morphological variants are often very transitory, and can be made to appear
and disappear by manipulation of the medium (osmotic pressure, pH, temperature, etc.).
However, they sometimes appear in an uncontrollable manner. Some may represent stable
mutations, others rapidly progressive reverse mutations, some transitory changes due to
environment,like adaptive enzyme formation .
• /1If1IIJJ1IO/OKY, in this sense, is tbe study of the reaction of infected animals toward infect-
ing microorganisms. It involves study of the effect of substances called antibodies, which
oocur in blood due to infection, upon the infecting microorganisms.
t AntigeM, as used here, are portions of infecting microorganisms which stimulate the
production of antibodies (see footnote above).
50_ Metlwds olStudying Variations; Common VariCitioMI Types 231
Mucoid Colonies. The spore-forming organisms just discussed, when
cultivated on infusion agar containing a little phenol, produced colonies which
were large, viscous and slimy. This kind of colony is common in many species
of bacteria. and is called "mucoid" or M. The mucoid material is like an
exaggerated capsule or slime layer. It is secreted by the cells in response to
various irritating external stimuli (in this case phenol) and,like the slime coat-
ing on a garden slug or the mucous secretions of the nasal tract, has protective
properties. Mucoid colonies are often called "M" colonies. Like R and S
colonies, they not infrequently appear "spontaneously."
Minute or Small Colonies. These are very minute, often just at the limit
of the unaided vision. Cultures derived from minute colonies often have
properties (such as virulence) different from cultures from large colonies. A
commonly used term for them is dwarf or minute colonies. These colony
variants often tend to revert to the larger colony forms. Minute colonies may
also be R, S or mucoid.
H aDd 0 Forms were first described in connection with Proteus vulgaris. a
non-sporeforming, gram-negative, motile rod commonly found in feces,
stagnant water and decaying organic matter. When cultivated on agar plates
or any moist, solid medium, P. vulgaris usually spreads over the surface quite
widely in a thin, smooth, gray, translucent film. The individual cells in such
growth are motile, i.e., they have flagella and swim out from the edge of the
colony in the film of moisture on the surface of the agar. Such extension of the
margin of the colony by motile cells is sometimes called "swarming." This
form of growth (it can hardly be called a colony) of the organism has been
designated as the Hauch or "H" form (the German word Hauch meaning a
film or veil). It is analogous to the S form of most other organisms.
A second form of growth is observed in which the colonies are small and
discrete. In these the Proteus bacilli are found to be non-motile, i.e., they
possess no flagella. These are called the ohne Hauch or "0" form (Ohne =
German for "without").
Motile and non-motile variants of other organisms are common. Although
all motile variants may not have the .spreading character (Hauch form) of
Proteus, they are often referred to as H forms, meaning flagellate or motile,
while the ohne Hauch forms are called 0 forms because they have no flagella.
As will be seen later, the term H is often used to refer to the flagella proteins
(H proteins) and the letter 0 to designate the body or somatic· substances
(0 substances) of various bacteria. The meaning of these terms should be
clearly understood here. The importance of Hand 0 substances will appear
farther on.
Secondary Colonies. The formation of small excrescences, papillae or out-
growths from ordinary colonies of many species of bacteria, after the first
growth is mature and begins to age, is a common phenomenon. The out-
growths are called secondary colonies or "daughter" colonies (Fig. 16-6).
They may appear on the surface, develop from within, or grow out from the
edges. They vary in size, form, numbers, and appearance. The cells in sec-
ondary colonies vary from the original in many properties, both morpho-
logical and physiological. They represent mutations or adaptations occurring
• Somatic is from the Greek root soma, a body.

Fig. 16-6. Mutant growths appearing as papillae or secondary colonies of entirely differ-
ent character from the original colonial growth. (photos courtesy of Dr. F. J. Ryan, Colum-
bia University, in J. Bact., 1955, vol. 69.)
within the colony during its growth, usually very late, analogous to the non-
smooth mutants found in matured broth cultures of Brucella due to d-alanine
formation. They are also analogous to the sector-forming mutants in col-
/ onies, described previously.
REFERENCES
Adelberg, E. A., and Myers, J. W.: Modification of the penicillin technique for the seIec-
tion of auxotrophic bacteria. J. Bact., 1953,65:348.
Braun, W.: Bacterial Genetics. W. B. Saunders Co., Philadelphia, 1953.
Bryson, V., and Szybalski, W.: Microbial selection. Science, 1952,116:45.
Gale, E. F., and Davies, R.: Adaptation in Microorganisms. 3rd Symposium, Soc. General
Micr., 1953. Cambridge Univ. Press, New York.
Lederberg, J., and Lederberg, E. M.: Replica plating and indirect selection of bacterial
mutants. J. Bact., 1952,63:399.
Ryan. F. J., Schwartz, M., and Fried, P.: The direct enumeration of spontaneous and in-
duced mutations in bacteria. J. Bact., 1955,69:552.
17
The Systematic Study of Bacteria
IN VIEW of the mutability of bacteria mentioned in preceding chapters, it

might be thought that the exact identification and description of a species of
bacterium would be an impossible task. This is not so because variation usu-
ally occurs within certain, well-known· limitations. Practically all forms of
life vary to some visible extent, some more than others. Creatures like cats
or zinnias, with which we are thoroughly familiar, may vary considerably
yet we recognize them without difficulty because (a) we are familiar with the
genotypes and phenotypes and (b) we recognize certain basic similarities
among them.
So with microorganisms; although they may at times vary greatly in one
or more respects, they seldom do so in all details at once and, normally, under
the uniform conditions of laboratory study, they remain quite recognizable
and retain fundamental distinguishing characteristics. It is necessary to know
what these are, and how they are usually determined in the laboratory.
PROCEDURES IN IDENTIFICATION
For purposes of discussion let us proceed as though we have been given an
"unknown" organism for complete identification and systematic study.
The first important step in the systematic study of an unknown micro-
organism is to determine to which of the 8 large groups of microorganisms
it belongs and whether other species are present. Let us assume that the
organism we are investigating is a bacterium. It is readily visible with ito
ordinary compound microscope and an oil immersion lens. Its size and shape
are within the range of bacterial dimensions and form. It does not require sun-
light for growth. It grows readily on simple, organic, non-living culture media
without serum. It shows no evidence of yeast-like budding, sex, or animal
structure and none of the structures distinctive of molds. It does not produce
large bodies or filtrable L forms of the PPLO. From descriptions given
earlier in this book there should be no difficulty in identifying an unknown
organism as a bacterium and determining that it is not a protozoan, a yeast,
a mold, a virus, a rickettsia, an alga or a pleuropneumonia organism (see
Tables 1, 2 and 6).
• Supposedly!
233
Purification of Culture. The next step in an exact study of any bacterium is
to separate it from other forms with which it might be mixed. These would ~
introduce error into various biochemical experiments or tests of pathogen-
icity, pigmentation, etc., performed in the course of the identification. This
process is spoken of as isolation in pure culture. Many a student* has fallen
into difficulties by assuming the purity of a culture and neglecting this very
important step. Microscopic examination of a smear stained by Gram's
method may sometimes reveal the presence of contaminants and very often
gives a valuable clue as to the genus or family of the organism but cannot be
depended upon entirely since many different bacteria look and stain exactly
alike. The culture must be purified.
This is done by spreading a drop of the material containing the desired
organism on some solid nutrient substance which is known to support growth
of a wide variety of organisms. A medium like meat-infusion agar is very use-
ful in such situations. If the unknown will not grow on it, then it may be
necessary to prepare a medium especially to resemble as closely as possible the
material on which the organism originally grew, if this is known. If not known,
autotrophic and heterotrophic media must be furnished with blood, or yeast
extract which contains vitamins and growth factors needed by some species
of bacteria. If contaminating bacteria are present, they will probably be evi-
dent as colonies of distinctive appearance, after incubation of the plates.
However, many different bacteria produce colonies very similar in appearance
and it may be necessary to examine smears made from selected colonies in
order to obtain the desired organism. Even this may yield no useful informa-
tion, as different species may not only produce colonies closely resembling
each other, but possess identical morphological and staining properties as
well. In this case we must either decide which of the two kinds of bacteria is
the "contaminant" and which the "unknown," or iden~fy both and 'decide
afterward.
For present purposes let us select one of the colonies as the "unknown,"
and inoculate it on to three plates of blood-meat infusion agar at pH 7.4.
Such a medium supports the growth of a wide variety of heterotrophic, sapro-
phytic and parasitic bacteria. It cannot be depended on to support autotrophs.
Let us also inoculate three plates of meat- or yeast-extract agar, without
blood, in the same manner. If thought necessary, three silica-gel plates may
be prepared from some of the solutions noted as serving for the cultivation
of strictly autotrophic bacteria such as Nitrosomonas. Similar media with
organic energy source and agar will serve for such autotroph-like species as
Azotobacter.
/ OBSERVATIONS OF INITIAL GROWTH. Qne plate of each kind of medium
may now be incubated at 20° C, one at 37° C and one at SSo C, After 24 hours
there may be no growth, in which case we may continue incubation for several
days. But let us suppose that after 24 hours there is no growth on the plates
prepared for autotrophs and near-autotrophs, very sparse or no growth on
the extract and infusion plates incubated at 20° C and Sso C, while good
growth occurs on plates of both the extract and infusion media held at 37° C
This" then, tells us the approximate optimum temperature for growth and
,. As well as veteran researchers!

The Systematic Study oj Bacteria 235
also give us an idea as to the kind of media and pH likely to be of use in
dealing with our organism. It is clearly aerobic, mesophilic as to temperature
and pH, heterotrophic, and probably a saprophyte (though possibly a
pathogen) not too fastidious in the matter of organic nutrients,
An inspection of the growth on the agar gives an idea as to the size, shape,
color and consistency of colonies. Let us say that the colonies are about 1
to 2 mm in diameter, glistening, convex, circular, opaque, butyrous (butter-
like) in consistency and lemon yellow in color. * A convenient way of recording
these facts is to use one of the charts published by the Society of American
Bacteriologists.
By carefully transferring a portion of one of these colonies, with a sterile
needle into a tube of extract broth, we provide ourselves with a pure culture,
which may be studied further as described below.
If no growth occurred on any of the plates inoculated with the original
material, we may assume that:
(a) No living bacteria were present in the inoculum; or
(b) The 'temperatures used were not suitable; or
(c) Some otller medium, possibly with a different pH, is necessary; or
(d) The bacteria may have been strict anaerobes.
Suitable adjustments of conditions must then be made until growth is ob-
tained.
Let us assume that good growth was obtained on the plates and that a pure
culture was obtained. The next step in identification is a study of motility,
arrangement, morphology, and staining reaction. The first three characters
may be determined by observation in a hanging-drop preparation of the
growth in broth. Let us assume that our organism is a coccus, although this
must be confirmed by microscopical examination of a stained preparation.
Arrangement and Motility. A hanging drop prepared with a young broth
culture is a useful means of determining how bacteria are arranged and their
motility. Chains of cocci or bacilli are readily observed, while cubical packets
of Sarcina can be seen turning over and over like little bundles floating in the
medium. Micrococcus groups appear to be very irregular, like bunches of
grapes but may often appear in pairs. Motility, if present, is easily seen in
hanging drops. Let us assume that our unknown is non-motile and a Micro-
coccus.
Staining Reaction and Morphology. The next step is to confirm the mor-
phology and observe the staining reaction. Gram's stain is of great value, but
one must bear in mind that the method requires experience and that the dif-
ferentiations it gives are not absolute. Some organisms are gram-positive
only when young or when cultivated on blood or serum media. Others are
variable under nearly all conditions. Many lose gram-positiveness with aging,
or in acid (fermented) cultures. Let us assume that the organism under dis-
cussion is gram-positive.
Other morphological features may be looked for at this point. If the organ-
ism is rod-shaped it may produce spores; especially if it is gram-positive.
No truly gram-negative species of bacteria forms spores. This is best deter-
mined by staining ~n old (at least one week) agar-slant culture with methylene
• Potato Medium. Pigment is often beautifully shown on cubes of potato sterilized with
a few drops of water iD the bottom of a tube.
blue or by a method of spore staining. A surer test is to emulsify some of
the growth in 2 ml of sterile water and heat it at 85° C for ten minutes, after-
ward inoculating some of the heated material into broth and incubating for
several days. If growth occurs, it is practically certain that spores were present
since no known vegetative cells withstand 85° C for 10 minutes.
Capsules mayor may not be visible, "depending largely on the culture
medium and whether we are dealing with an R or S form. Sometimes capsules
are seen only on organisms in pathological material. They may be determined
by capsule stains or darkfield methods. Let us say our organism shows no
demonstrable capsules.
The morphology of anyorganism always varies to some extent. A given
variety of bacteria may, inany one culture, produce cells of varying size just
as, in a group of men or horses, some may be large and some small. If the
bacteria are cocci, some of the individual cells may not be perfectly round
and some may be oval. If bacilli, some may be long and thin, others short,
oval and thick; some may occur singly, others in pairs or chains or long
filaments. But the predominating form, size and arrangement of the cells in a
pure culture are usually quite apparent especially when seen growing in
different kinds of media, both fluid and solid. Form, size and arrangement of
the individual cells are, important and reliable differential characters.
Summarizing our knowledge at this point, we may state that we are dealing
with a non-motile, gram-positive, unencapsulated coccus, growing well
aerobically in irregular clusters on plain, extract medium as well as on blood-
infusion medium, at a pH of about 7.4, producing opaque, glistening, lemon-
yellow colonies and preferring a temperature of about 37° C. There is still,
however, a good deal to learn about our unknown before indentification is
complete.
Biochemical Tests. The experienced bacteriologist would know at once
exactly what peculiarities to look for in dealing with a culture of gram-positive
cocci which have the characters we have enumerated. For the present, how-
ever, we shall put ourselves in the place of a person to whom a gram-positive
coccus is a hitherto unknown bacterium.
Since certain enzymes are highly characteristic of certain genera and
species of bacteria, the power of our unknown organism to ferment or hydro-
lyze some of the carbohydrates such as glucose (dextrose), sucrose' (saccharose
or cane sugar), and lactose (milk sugar), ~hould be tested; also its power to
utilize, as hydrogen acceptors, such substances as sodium nitrate and soqium
nitrite. Its power to hydrolyze gelatin, coagulated serum, the casein in milk,
starch, fat, etc., should also be investigated. All of these are simple procedures.
In any of these tests it is important that the medium and conditions of
incubation support good vigorous growth of the organism. For example, for
certain bacteria to produce gas by the decomposition of lactose, it has been
shown that from 40 to ¢O million cells of this species must be present in each
ml of the test medium. A negative result in the absence of normal, good
growth is obviously of no value. In order that uniform, standard, and autho.r-
itative results may be obtained, it is recommended that recognized, docu-
mented procedures sU(;;h as those outlined in the "Manual of Bacteriological
Methods," published under the auspices of the Society of American Bac-
teriologists be used throughout. It should be emphasized that results obtained
The Systematic Study of Bacteria 237
in empirical media of the complex organic type, like extract or infusion broth,
can vary with changes in the quantity and quality of such ingredients as pep-
tones, yeast extracts and meat extracts; presence or absence of certain kinds
of hydrogen donors and acceptors; pH; temperature; and other factors.
These variables are always standardized as far as possible and are carefully
recorded for comparative purposes by competent workers.
FERMENTATION TESTS. When dealing with an unknown, each tube of
broth should contain a small inverted vial (placed there before sterilization)
(Fig. 17-1) to catch any gas that may be formed. Gas might otherwise pass
off into the atmosphere and not be detected. Two organisms, both of which
ferment the same carbohydrate, may be sharply differentiated if one forms gas
while the other does not.
When gas is produced from carbohydrates by bacteria, it is prima facie
evidence of fermentation and is always accompanied by souring or acid
formation since the gas is derived from the formic acid resulting from fer-
mentation. However, fermentation often occurs without gas production, and
then acid formation is our only evidence that the organism has metabolized
the carbohydrate. Sometimes only alkaline substances are produced from
carbohydrates.
Acid or alkali may be detected by adding to the medium an indicator or dye,
such as brom-cresol purple which changes from purple to yellow in the
presence of acid. Phenol red turns from yellow to red in the presence of alkali.
The change in color of the indicator is our proof of fermentation or metabolic
use of the carbohydrate. However, some species can metabolize the acids
produced by fermentation. Others may produce ammonia from amino acids
in sufficient quantity to mask acid production. In other instances, if too much
buffer is used in the medium it, too, will mask acid or alkali production. These
possibilities can be allowed for, but must be kept in mind.
Fig. 17-1. Types of abparatus used for collecting gas produced by bacteria. A, Durham
tube (gas has collected at X). B, Smith tube (gas has collected at X').
Observations of growth should be made every twenty-four hours in order
that the culture may not revert to an alkaline reaction before acid formation
has been noted. Variant strains of the same species may differ considerably
in the rate at which these reactions are brought about and ample time for
observation must therefore be allowed.
GASES PRODUCED BY BACfERIA. The nature of the gas formed by bacteria
may be of differential value. Two gases commonly given off by bacteria
during fermentation of carbohydrates, etc., are carbon dioxide and hydrogen
in various proportions. It is often of importance to determine the ratio of
carbon dioxide to hydrogen. The differentiation is easily made by adding
strong NaOH solution to the fermentation tube. This absorbs the CO 2,
leaving H. The difference in level of fluid in the fermentation vial is easily
estimated if the level was marked before adding NaOH.
Many organisms, in their metabolism of proteins or protein-digestion
products, e.g., cystine, taurine and other sulfur compounds, set fr\!e hydrogen
sulfide, often in large amounts. This gas is one of the most noticeable in con-
nection with putrefactive processes. Some organisms may be identified or
differentiated from others by their power to produce H 2S. Thus, Salmonella
schottmulleri, * a cause of gastroenteritis, produces hydrogen sulfide in its de-
composition of amino acids, while Salmonella paratyphi, t a similar species,
does not. The test therefore has differential or diagnostic value.
Methane is another gaseous product of bacterial metabolism. It results
usually from the metabolism of carbohydrates and other organic compounds
under anaerobic conditions. Carbon is a good hydrogen acceptor and is
readily reduced. In swampy places anaerobic bacteria attack the carbohy-
drates derived from dead vegetation and sometimes large amounts of the
gas are given off. The bubbles seen arising during the summer time in wood-
land swamps are nearly always largely methane. Microorganisms in sewage
digestion tanks at sewage disposal plants produce such large quantities of
methane that it is economically feasible to collect it in tanks and use it as fuel
in furnaces, etc.
Some organisms may produce poisonous gases under certain circumstances.
For example, Pseudomonas aeruginosa is said to produce hydrogen cyanide
(HCN), while a number of species of Corynebacterium, especially C. diph-
theriae, produce hydrogen telluride when cultivated, as is frequently done,
upon medium containing potassium tellurite. Many bacteria, especially
saprophytic species, also produce large amounts of ammonia and nitrogen.
These result from decomposition of proteins and other nitrogenous com-
pounds.
I PROTEOLYSIS. In addition to the above. tests, tubes containing common
proteins may be inoculated to determine the ability of our unknown to attack
different proteins. These may be incubated with the carbohydrate tubes.
GELATIN. A tube of solidified nutrient gelatint is inoculated by "stabbing"
a wire, having the desired bacteria upon it, down Into the depths. Some
workers prefer to incubate the gelatin at 20° C rather than at body tempera-
ture. At the lower temperature the gelatin remains solid except where di-
* Now S. paratyphi B. (See footnote on page 244.)
t Now S. paratyphi A. (See footnote on page 244.)
t Ordinary extract or infusion broth containing 10 per cent gelatin.
gested by the organism, and the shape of the portion liquefied may be ob-
served. This was formerly regarded as of great significance, but is really of
much less value than other tests. The time lost in waiting for growth to occur
at 20° C (if indeed it occurs at all) is much more valuable than the information
as to the form of the liquefied area. Gelatin cultures held at 37° C liquefy
completely due to the temperature but may be placed daily in the refrigerator
for a sufficient time to allow them to solidify if undigested. This should be
continued for two weeks unless evidence of digestion is obtained sooner.
An uninoculated tube of gelatin is incubated with the others to serve as a
guide in refrigeration time. Those tubes in which the gelatin fails to solidify
may be marked "+" or "digested." The tubes should not be shaken while
warm, as growth, and a small amount of gelatin digestion, frequently occurs
only in the surface layer and this would be masked were it mixed with the
bulk of the warm, fluid culture.
SERUM DIGESTION. Coagulated serum may be prepared by mixing three
parts of horse or beef serum with one part of nutrient broth. Glucose is
sometimes incorporated in a concentration of 0.25 per cent. The serum is
coagulated and sterilized at the same time. The slants are inoculated when
cool, by smearing a loopful of broth culture or growth from agar over their
surfaces.
When attacked by bacteria the serum usually becomes brownish and
translucent and the growth appears to sink inward. Total liquefaction some-
times occurs in forty-eight hours at 37° C, especially in cultures of aerobic
sporeforming bacilli, but is often delayed for as long as two weeks.
ACTION ON MILK. Milk has a pH of around 6.8 when fresh. It IS an ideal
culture medium for many bacteria.
For use as a bacteriological culture medium, skimmed milk is tubed in
5-ml amounts and sterilized by autoclaving (steam pressure cooker: 120° C
for 10 minutes). If an indicator, such as litmus or bromcresol purple, be
added, fermentation of the lactose may be detected. Rennet production may
be inferred if the milk is curdled (provided this is not due to souring; a point
difficult to determine if fermentation of the lactose also occurs). Hydrolysis of
the casein often follows coagulation. The milk then becomes brownish and
translucent and the clot disappears.
Another method of testing the ability of many organisms to hydrolyze test
substances like serum fat, or starch is to mix the test substance with nutrient
agar, pour into Petri plates and, when solid, heavily inoculate the surface in
.streaks or spots. After good growth has occurred a reagent, reacting with the
test substance to prodube some distinctive appearance, is flooded over the
surface of the agar. If hydrolytic enzymes have been produced the colonies
will be surrounded by Zones where no reaction (or a distinctive reaction)
occurs. For example, geliatin or serum may be added to the agar. By flooding
the plate (after incubation) with several ml of a solution consisting of H 20,
100 ml; HgCI2, 15 gm; IHCl, 20 ml, unhydrolyzed gelatin or serum will be
coagulated to a white 9paque appearance. The colonies of hydrolyzing organ-
isms will be surrounded by a clear zone. Starch plates may be made similarly,
and treated with Lugol'sl iodine solution. The starch-hydrolyzing colonies will
be surrounded by colorless zones. The remainder of the plate will turn dark
blue.
240 M~/hods and PhelWm~1IQ of Microbiology
DETERMINATION OF LIPOLYSIS. Many organisms produce enzymes capable
of hydrolyzing one or more fats or oils (lipids). Methods for detecting lipolysis
have not been widely developed or used. One of the inconveniences in studying
lipolysis is difficulty in bringing the lipid substrate into intimate contact with
the organism. This contact is desirable since lipolytic enzymes often do not
diffuse well into culture media. Another difficulty is in making the hydrolytic
effect evident.
In a method eliminating some of the difficulties a plate of nutrient agar is
streaked with the organism to be tested. The oil (or melted fat) is then applied
to the surface of the plate in a fine spray. After incubation, examination of the
lipid droplets is made with a low power microscope. Those that have been
hydrolyzed are readily detected by their opaque appearance. This and an-
other method are seen in Figure 17-2.
BACTERIAL REDUCTIONS. A physiological property characteristic of many
bacteria is the power to reduce various compounds.
NITRATE REDUCTION. Incubate the bacteria being investigated in broth
containing about 0.1 per cent of sodium nitrate (NaN0 3) . After forty-eight
hours and at other intervals, a test is made for the presence of nitrites (NaN0 2)
by withdrawing a little of the culture from the bottom of the tube and im-
mediately adding to it a drop of sulfanilic acid solution· and a drop of di-
methyl-alpha-naphthylamine solution . t Or allow the drops to settle to the bot-
tom of the whole culture. It is better to test the bottom layers of the culture
rather than mixing the whole culture, because slight reduction sometimes
occurs at the bottom of the tube and nowhere else, due to lower oxygen
tension in the depths of the tube. The presence of oxygen interferes with
nitrate reduction. Oxygen is present in greater concentrations in the upper
layers of fluid.
The development of a red or brown color denotes the presence of nitrites,
but the failure of this color to develop raises a question. Either (a) the nitrate
has not been attacked, so that no nitrite is present or (b) nitrite has been
formed but also attacked and reduced to free nitrogen or ammonia. A test
to see whether any nitrate remains may be made by adding a little pulverized
zinc. This reduces any remaining nitrate to nitrite (which may be tested for
as above) and tells us whether or not the organism has reduced all of the
nitrate. If the test with Zn is positive (i.e., nitrate is still present), the original
test for nitrites having been negative, then it is clear that the organism did
not attack the nitrate at all. If both the nitrate and nitrite tests are negative
then it is obvious that the organism reduced all the nitrate as well as nitrite to
I'
nitrogen or ammonia.
NITRITE REDUCTION. The latter point, i.e., ability of the organism to attack
nitrite, may be determined separately by testing the ability of the organism to
• Sulfanilic acid solution

Glacial acetic acid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100.0 ml
Water ....... . .. . . ... . .... . . . • ...... •. .. . •..... ..•. .. ... . . . 250.0 ml
Sulfanilic acid. . .. . . . . . . . .. . . . . . . . . . . . • . . . . . . . . . . . . . . . . . . . . . 2.8 gm
t Dlmetbyl-alpba-naphthylamine solutioo
Glacial acetic acid ............... . ....... . .. ...... ... . . ... . . 100.Oml
Water .. . . • .. .. ... ... . .. .. . .. . . .....•.. . . . . . ......... . . . .. . 250.0 ml
Dimethyl-alpha-naphthylamine ..... . . . ........ . .. . ..... . . . . . . 2.1 ml
Fig. 17-2. Lipase production and its detection. At A is seen the margin of a colony of
Micrococcus sp., on a plate of agar sprayed with a fine mist of olive oil. No lipase is evident
since all of the droplets of oil have remained unchanged, even in contact with the colony.
At B is seen the irregular margin of a colony of Serratia- on agar sprayed with oil. Lipase
activity has caused the oil droplets to become deformed and optically dense and refractive
near. as well as at some distance from. the colony. At C is shown a plate containing agar in
which fat is emulsified and to which has been added a small amount of Nile blue sulfate (a
dye which turns blue in the presence of lipolysis). On this plate are seen a strain of Micro-
coccus producing lipolysis (zone of color change and emulsion destruction, 1), and a strain
not producing lipolysis (no zone of color change or emulsion destruction, n). (A, courtesy
of Dr. C. J. E. A. Bulder, in A. van Leeuwenhoek J. Micr. and Serol., 1955, vol. 21 ; B, same
author, unpublished; C, courtesy Dr. M. E. Davies, in J. Gen. Micr., 1954. vol. 11.)
- A genus of red-pigmented Enterobacteriaceae.
destroy the nitrite in cultures known to contain it. This is done by incubating
cultures containing quantities of nitrite so small (about 0.002 per cent) as just
barely to give a positive test for nitrites. If the organism is capable of reducing
nitrite, the culture will soon lose its power of reacting positively to the nitrite
reagent, because the organism will quickly reduce all of such a small amount
of nitrite. A sterile control tube should be tested at the same time, since
illuminating-gas fumes, as from Bunsen burners, often contain nitrous acid
which may be absorbed by the medium and give a slight reaction.
REDUCTION OF LITMUS. The ability of an organism to reduce other sub-
stances than nitrates and nitrites is often investigated. Litmus, for example, is
often used as an acid-indicator in milk cultures, but it also serves to show
whether or not the organism has strong reducing powers by becoming entirely
decolorized when reduced. Just enough is added to the medium before sterili-
zation to give a definite color.
THE "REDUCTASE" TEST. Methylene blue is similarly decolorized by many
organisms. The dye is used as a hydrogen acceptor in respiration (see section
on bacterial respiration). Many other compounds are similarly utilized.
Standardized solutions of methylene blue are often added to market milk
samples to estimate, roughly, whether a few, a moderate number, or enor-
mous numbers of bacteria are present. When great numbers are present, the
blue color may disappear almost immediately. Several other oxidation-
reduction dyes are now used besides methylene blue: tetrazolium salts,
neutral red, resazurin, etc.
INDOLE. Indole is derived from the amino acid tryptophan as a result of
hydrolysis by certain species of bacteria. Most peptones contain tryptophan
but if a medium is used in which it is not known to be presept it must be added
if one is to test for indol~ production. Cultures are incubated for forty-eight
to seventy-two hours. Indole reacts with acidified solution of Ehrlich's
reagent (para-dimethyl-amido-benzaldehyde)* to produce a pink compound.
The culturdo be tested is first shaken with 1 ml of xylol. Indole is soluble
in xylol and is concentrated in it and carried to the surface by the solvent
after a minute of standing. Six drops of Ehrlich's reagent are then gently added
to the culture and are made to remain in a layer between the xylol and the
medium. If indole is present a pink color forms in a few minutes as a "ring"
at the junction of the xylol and the reagent, Pink compounds not soluble in
xylol sometimes develop in the presence of Ehrlich's reagent if _the xylol is
not added first. These are not due to indole.
There are many other tests which are used in microbiology; some only for
very specific purposes in diagnostic work; others in specjal phases of research.
I A few are described in other parts of this book, and others in the literature.
Rapid Microtechniqves. By certain modifications of technique, many of
the tests described above may be made more economically and also greatly
speeded up. Three general types of rapid microtechnique are available. First,
since bacteria rapidly multiplying in the logarithmic phase are most active in
all enzyme functions, one may greatly speed up their effects by adding heavy
• Ehrlich's reagent
Ethyl alcohol (95%) ..' ........................................ . 380ml
Hel (cone.) ................................................ . BOml
Para-dimethyl-amido-benzaldehYde ............................ . 4gm
suspensions of young, actively-growing organisms from an agar-slant culture
to small amounts of the test medium (previously warmed to the temperature
desired and held in a water-bath during the period of incubation). In this way,
instead of incubating cool, sparsely inoculated test cultures for one or more
days slowly to develop populations sufficiently large to induce the desired
change, the ready-made population consisting of billions of young, active
organisms in the heavy suspension from the agar slant set to work im-
mediately and bring about the desired characteristic changes within a few
minutes or hours. By using 1.0 ml amounts of test fluids in small tubes and
adding a few drops of very heavy bacterial suspensions, a great many tests
may be done economically in a small space and short time.
Second, one may centt:ifuge 10 ml of a young broth culture, remove the
supernatant broth with its various unknown ingredients, and resuspend the
bacteria in a few drops of saline solution or distilled water. For a fermentation
test, a drop of this heavy suspension of young, active cells is placed in a small
(5 x 50 mm) tube with 0.5 ml of acid-alkali indicator and a drop of the desired
sugar solution. The results may be read in 6 to 12 hours. Modifications of this
procedure permit tests to be made quickly for urease production, nitrate re-
duction, gelatine hydrolysis, etc.
A third type of procedure involves inoculation of 15 ml of melted, nutrient
agar at 40° C, in a tube, with a heavy suspension of young, active, cells. The
agar is then poured into a Petri dish and allowed to solidify. One then places
on the surface small, paper disks, previously dried after saturation with solu-
tions of test carbohydrates and suitaDle indicators. The test substance diffuses
into the agar around the paper disk and is acted upon by the bacteria 'in the
agar.
Identification of the Unknown Organism. After completion of the tests as
described above, a tabulation is made of the results. For the organism under
investigation, let us assume that they are as follows: /
Pigment (as observed in colonies on agar)-lemon yellow.

Lactose-fermented; no gas.
GI ucose-fermented; no gas.
Sucrose-fermented; no gas.
Salicin-not fermented.
Gelatin-slowly liquefied.
Agar slant and cubes of potato-good growth, soft, moist and glistening,
lemon yellow in color.
Broth-turbid, sediment is yellow; faint pellicle (scum).
Milk-coagulated,1 acidified.
Nitrates-reduced :to nitrites.
Indole-not produced.
How are we now t~ determine the genus and species of the cocci which we
have been studying?
Need for Keys. Although every experienced bacteriologist has at his finger
tips, so to speak, all/of the distinguishing cultural reactions and other identi-
fying characters of the organisms with which he is working, it is unusual, to
say the least, to nnli one who knows all the characters of all the species.
244 Methods tmd PMnome1lO of Microbiology
When an unknown organism is encountered which must be identified, the
main morphological and tinctorial features are determined in some such
manner as just described and then recourse is had to keys or other reference
works. Probably the most useful key for general bacteriological use in the
United States is "Bergey's Manual of Determinative Bacteriology."·
USE OF KEYs. In order to use "Bergey's Manual" we should first determine
to which of the five orders of the class Schizomycetes our culture belongs.
On pages 65 and 66 of the 1948 edition of the manual is to be found a brief
synopsis of the characters used to differentiate the five orders and suborders.
Obviously the species in question is neither spiral, elongate or flexuous,
filamentous, sheathed nor characterized by iron or sulfur deposits (Orders
III, IV, and V). Neither is it branching (Order II). It does not possess photo-
synthetic pigment nor form long stalks, hence does not belong in suborders
II or III of Order l. We are thus left to consider Suborder I, the Eubacteriineae.
On pages 67 and 68, we find a synoptic description of the families of the
Suborder Eubacteriineae. Our organism is spherical and may therefore fall
into either family I (Nitrobacteriaceae), family V (Micrococcaceae), family
VI (Neisseriaceae) or family VII (Lactobacteriaceae). It is obviously not a
member of the other families, which include only rod-shaped or filamentous
bacteria. To settle the choice between families I, V, VI, and VII, more exact
information is necessary and we turn to page 69. On page 69 et seq., we find
that in only one genus of family I, the Nitrosococcus, are the organisms spher-
ical. But the organisms of this genus do not grow on ordinary heterotrophic
culture media, whereas our "unknown" does, and will not grow autotrophi-
cally. This, therefore, leaves us to consider families V, VI, and VII and to
them we turn our attention.
Reference to page 295 makes it clear that we are not dealing with Neisseri-
aceae (family VI) since they are gram-negative; and we eliminate at present
also the Lactobacteriaceae (page 305, tribe Streptococceae) since our organ-
isms grow well on ordinary extract agar without blood and do not occur
predominantly in chains but in irregular clumps and masses as well as in
pairs.
Family V (Micrococcaceae), therefore, would seem to be our objective,
and we are referred to page 235. Here we find that the descriptions of genera
II and III (GafJkya and Sarcina) do not correspond with the organism in
question, and that we must search in genus I. The organisms of this genus
resemble each other so closely that it is necessary to consider carefully the
characters of the individual species.
A short study of the data we have already obtained by our cultural tests
shows that our organism corresponds closely with the description of Micro-
coccus citreus (page 242), since it produces a lemon-yellow pigment, liquefies
gelatin, ferments lactose, grows on potato with lemon-yellow pigment, reduces
nitrates to nitrites and fails to produce indole.
A further check upon the identity of the culture may be made by testing
other fermentable substances and comparing various characters of organisms
closely resembling it, such as Micrococcus flavus. A few repetitions of the
• AU IIlUDe8 of bacteria given in this book are based on the 1948 edition of "Bergey's
Manual" unless otherwise noted except in a few instances where official groups have more
_tly adopted superseding names.
'J"M Systematic Study of Bacteria 245
Fig. 17-3. Injection of a chick to test the toxigenicity of diphtheria bacilli. The injection
is intraperitoneal (abdominal cavity). The material is 4 ml of 48-hour broth culture of the
organism to be tested. Guinea pigs, rabbits, and other animals are often used. (Photo cour-
tesy of the U. S. Public Health Service, Communicable Disease Center, Atlanta, Georgia.)
tests usually serve to confirm the diagnosis, or prove it to be in error, neces-

sitating further study.
Pathogenicity may be determined by injecting 0.5 ml of a twenty-four-
hour broth culture intravenously and subcutaneously into a rabbit, guinea
pig or other animal (Fig. 17-3). If our unknown is truly pathogenic, abscesses
will probably form or the animal may die. Some strains are more pathogenic
than others. Further details concerning micrococci will be given when these
genera are taken up specifically later.
REFERENCES
Adelberg, E. A. : The use of metabolically blocked organisms for the analysis of biosynthetic
pathways. Bact. Rev., 1953, 17:253.
Bergey, et al.: Manual of Determinative Bacteriology. 6th ed. Williams & Wilkins Co.,
Baltimore, 1948.
Bulder, C. J. E. A.: Some observations on the lipolytic activity of microorganisms and a
new method for its detection. Antonie van Leeuwenhoek J. of Micr. and Serol., 195~,
21 :28.
Conn. H. J., Editor: Manual of Bacteriological Methods. Biotech Publications, Geneva,
N. Y., and/or Soc. of Amer. Bacteriologists, c/o Williams & Wilkins Co., Baltimore,
Md.
Cowan, S. T.: Rapid micromethods for bacteriology. Lab. Prac., London, 1953,2:241.
Greene, R. A., and Larks, G. G. : A Quick method for the detection of gelatin IiQuifying
bacteria. J. Bact., 1955, 69 :224.
Ordal, E. J., and Earp, B. J.: Cultivation and transmission of etiological agent of kidney
disease of salmonid fishes. Proc. Soc. Exp. BioI. and Med., 1956,92 :85.
Schaub, T. G., and Foley, M. K.: Methods for Diagnostic Bacteriology. 4thed. C. V. Mosby
Co., St. Louis, 1952.
Smith, N. R., Gordon, R., and Clark, F. C.: Aerobic Sporeforming Bacteria. Agriculture
Monogr. No. 16, U. S. Dept. of Agric., Washington, D. C., 1952.
Underwood, W. B., and Perkins, J. J.: Textbook of Sterilization. 3rd ed. Charles C Thomas,
Springfield, II!., 1955.
Verhoeven, W.: Studies on true dissimilatory nitrate reduction. V. A. Van Leeuwenhoek
J. Micr. and Sero!., 1956, 22 :385.
Weaver, R. H., et a!.: Rapid micro-technics for identification of cultures. A series of
papers. Am. J. C1in. Path., 1951,21 :195; J. Bact., 1947,54:28; J. Lab. and C1in. Med.,
1948.33:1338.
249
. inhibiting,
i.,ich
Destruction, Removal and Inhibition

of Microorganisms
1. BASIC PRINCIPLES
AN IMPORTANT phase of microbiology is knowledge of methods for kill-

ing, inhibiting (preventing growth of), and removing microorganisms. As
species of microorganisms vary in the ease with which they may be destroyed,
inhibited or removed, and as the situations in which they may occur differ
greatly (e.g., blood', foods, water, sewage, soil, milk, bedding, etc.), no One or
two methods are generally applicable. Each situation is a problem in itself,
and the methods employed must depend on the knowledge, ingenuity and
skill of the operator. There are basic facts, however, ,,:hich guide the procedure
in any given situation.
There are four main reasons for killing, inhibiting or removing micro-
organisms. They are: (1) to prevent infection of men, animals and plants;
(2) to prevent spoilage of food and other commodities; (3) to prevent inter-
ference by microorganisms in various industrial processes depending on pure
cultures; (4) to prevent contamination of materials used in pure-culture work
in laboratories (diagnosis, research, industry, etc.), so that studies of the
growth of one kind of organism in a particular medium or infected animal
may not be confused by the presence and growth of others, at the same time.
i DEFINITION OF TERMS
Several new, spe~ial terms are used in this chapter. These may be explained
as follows: i
Sterilization. Sterilization means the freeing of any object or substance
from all life of anyl kind. This is accomplished usually by heat but sometimes,
in special cases, by the use of chemicals, ultraviolet irradiation or filtration.
Bactericide. Any substance or agent killing bacteria is a bactericide or
bactericidal agentJ The suffix, -cide, indicates "killer" and is used with germ,
virus, etc.
Disinfection. "Fhis means the killing or removal of organisms capable of
247
causing infection and does not necessitate sterilization. However, some proc-
esses of disinfection accomplish sterilization. Disinfection is usually accom-
plished by chemicals like carbolic acid (phenol), formaldehyde, chlorine,
iodine or bichloride of mercury. In the case of milk, disinfection, but not
sterilization, is brought about by pasteurization, a beating process to be de-
scribed.
A DISINFECfANT is an agent accomplishing disinfection. The term is often
used synonymously with antiseptic. One ordinarily thinks of disinfection and
disinfectants as applicable mainly to situations and objects not part of the
body: floors, dishes, laundry, bedding, etc. However, one often hears of
"disinfecting a wound."
Sepsis is the growth of harmful microorgan!sms in living tissue.
Asepsis. In a strict sense, asepsis is the· absence of infectious microorgan-
isms from living tissue, but the term is usually applied to any technique de-
signed to keep all unwanted microorganisms out of any field of work or ob-
servation. The work of a microbiologist and of a surgeon involves aseptic
technique. The surgeon and his' assistants have sterile instruments, handle
them with sterilized gloves, cover the patient with sterilized sheets except for
such area as is necessarily uncovered, and wear sterilized caps, gowns and
masks to prevent infected dust, droplets of saliva, perspiration or sputum
from entering the sterile field and possibly infecting the patient. The patient's
skin cannot be absolutely sterilized without injury, but the site of the opera-
tion is disinfected as thoroughly as possible by applications of some suitable
disinfectant.
In microbiology the worker uses sterilized culture media and sterilized
glassware kept sterile, until the moment of use, by coverings of paper and
cotton plugs and by aseptic technique; i.e., avoidance of touching sterile ma-
terials with hands or unsterile objects, exclusion of dust, etc.
Antiseptic is an ill-defined term, closely allied to "disinfectant." A disin-
fectant is often used as an antiseptic and vice versa. Antiseptics are substances
which kill or inhibit microorganisms, especially in contact with the body.
Bacteriostasis; Bacteriostatic Agents. These are substances or conditions
which do not immediately kill the bacterial cell but which inhibit multiplica-
tion so that the microorganisms die only after hours, days or years without
significant increase in number. Important bacteriostatic agents are desicca-
tion, very low temperatures, <tntibiotics (Terramycin, penicillin, Aureomycin,
etc.), sulfonamide drugs, and certain dyes like crystal violet. Others will be
discussed later. -
/ The distinctIon between a disinfectant and a bacteriostatic agent is interesting but entirely
arbitrary and depends on what one may call killing "quickly," and what one may mean
by inhibiting or, what amounts to the same thing, killing "slowly." One could say that
death within a time less than the maximal for one generation (under optimal growth con-
ditions) might be regarded as more than mere prevention of multiplication and, therefore,
"true killing" or "disinfectant" action; while, if death were delayed beyond such time, one
might regard death as due only to factors other than the agent applied, acting on cells which
have merely been prevented from multiplying. This could represent "true bacteriostasis."
There are many obvious difficulties in making such a distinction. The term bacteriostasis is,
however, widely used and generally means delayed death, i.e., killing only after some hours,
,days, or longer, rather than in 10 to 30 minutes.
Destruction. Removal and Inhibition of Microorganisms 249
PRINCIPAL METHODS
We may consider four general classes of means of killing and/or inhibiting,
as follows:
1. Destruction of Physical Structure. There are many substances which
act upon colloidal suspensions (of which protoplasm largely consists) to cause
the suspended particles (discontinuous phase) to change their relationship to
each other and to the suspending fluid (continuous phase). The mass becomes
solid like hard-boiled egg or like "clotted" (soured) milk. Coagulation is said
to have occurred. In the case of the egg, heat coagulation occurred; in the
milk, acid or chemical coagulation.
Colloidal particles may be thrown out of suspension if their negative
charges are neutralized; for example, by certain metallic cations: hydrogen
ions (H+), copper ions (Cu++), zinc ions (Zn++), iron ions (Fe+++), etc.
Several heavy metals are in common use as disinfectants or bactericides,
e.g., CUS04, AgN0 3, HgCI2, ZnO. Of metallic ions it may be said that, in
general, coagulating power is exponentially related to valency and atomic
weight; the trivalent, heavy metals having much greater than one third more
coagulative power than bivalent metals.
Certain organic substances are also important as coagulants; among
them, alcohol, phenol, formaldehyde, and related and derived substances.
All of these reactions, organic and inorganic, are included in the term
chemical coagulation.
The general rule may be stated that any agent inducing coagulation, or
any change like it, is lethal to living cells.
2. Non-specific Chemical Combinations. Various chemically active sub-
stances will combine indiscriminately with any and all proteins and proto-
plasm. Chlorine is such a substance; iodine another. Creosotes, carbolic acid
(phenol), and formaldehyde are others. Lye (strong alkali) and strong acids
are destructive of nearly all organic matter. We will discuss details of the
practical uses of such substances later. Such substances are entirely non-
specific in their action. That is, they will combine as readily with body tissues,
or with casein, feces, mucus, blood, wood or leather, as with bacterial pro-
toplasm.
3. Specific Chemical Combinations and Bacteriostatic Agents. There is a
group of substances which, in relatively low concentrations, can enter certain
cells and interfere with, or completely stop, the action of one or more specific
molecular groups in a particular enzyme or enzymes in such cells. Substances
of this nature are represented by sulfonamide drugs and antibiotics which
are discussed more fully farther on.
4. Non-specific Bacteriostatic Methods. There are several methods of
stopping (or. very greatly slowing) cell function which are entirely physical
and affect the cell as a whole. All are fundamentally methods of depriving the
cell of liquid water;: (1) drying; (2) immobilizing the water by changing it
into solid ice; (3) dr~wing most of the water out of the cell by immersing it in
a fluid of high osmotic pressure such as pickling brines or preserving syrups.
These methods were discussed in Chapter 12.
The action of bacteriostatic agents is characteristically reversible; that is,.
the organisms can ~e reactivated if the bacteriostasis has not been too pro-
longed. This differs for different agents. For example, organisms treated with
bichloride of mercury can be reactivated by treating them with H 2S, up to
an hour or so following treatment with HgCI 2• The H 2S precipitates the Hg
as HgS. Many species of bacteria held dormant by desiccation in vacuo can be
reactivated after 25 years or longer.
Combined and Variable Effects. While we have cited several distinct kinds
of deleterious actions on living matter: (a) coagulation, (b) non-specific chem-
ical combinations; (c) specific chemical combinations; and (d) dehydration,
actually many widely used and effective disinfectant substances and chemo-
therapeutic drugs act by more than one of these mechanisms. In fact, many
microbicidal and/or microbistatic actions are, at best, only partly understood.
1HE ROLE OF HYDRATION IN DISINFECTION

The role of hydration in coagulation is clearlv shown by experiments on the
effect of heat on egg albumen under various degrees of hydration. The results
of some of these experiments are shown in Table 9.
Table .9. Effect of Hydration and Heat on Egg Albumen. *
WATER CONTENT APPROX. COAGULATION

(Per Cent) TEMPERATURE (" C.)
50 56
25 76
15 96t
5 149
o 165t
• Albumen is one form of protein, closely resembling protoplasm.

t Boiling water = 1000 C. .
t 165 0 C. = Oven temperature.
Obviously coagulation proceeds best where protein is well hydrated. The

less the degree of hydration, the more resistant is the protein to coagulation.
The same principle holds true in coagulation by chemicals.
The resistance of bacterial endospores tol heat and chemical disinfectants
may probably be explained-as being due either to the dehydrated condition
of their protoplasm or to the fact.that the free water of the protoplasm became
closely united or bound* when the spore was formed. In either case the water
no longer functions· as' a separate aqueous phase. Examples of the resistance
of dehydrated protein to the action of coagulative agents are seen in the
difficulty of making dried-egg powder coagulate. Heated in a test tube it will
turn brown or char, but it will not coagulate unless a considerable quantity
of water has been added. Of course, mixing dry egg with dry chemical dis-
infectants such as powdered bichloride accomplishes nothing at all till mois-
ture is added. The same is true of dried-milk powder. Chemical reactions
necessary to the action of a variety of disinfectants, are facilitated by the
pre~ence of water, since ionization is essential, as well as hydration of protein.
• The apparent dryness of a hard-boiled egg is a good illustration of "bound water."

Destruction, Removal and Inhibition of Microorganisms 251
FACTORS AFFECTING CHEMICAL DISINFECTION
Of basic importance in disinfection with chemicals is contact between the
'crobicidal agent and the microorganism. In dealing with aqueous solutions
of isinfectants, "wetness" (low surface tension) is a determining factor. There
are 0 aspects of this factor: first, the accumulation (adsorption) of surface-
tensio -reducing disinfectants on the surfaces of cells; second, the effect of
surface ension reducents on the wetting and spreading properties of the
solution. oth affect contact between disinfectant and microorganisms.
Contact. Upon adding phenol to a broth culture of bacteria, contact be-
tween disinfe ant and bacteria is immediate. The bacteria float naked, as it
were, and are ached by the disinfectant in effective concentration without
delay, partly bec use phenol lowers surface tension and is therefore adsorbed
upon their surface Once the surface-active substance is in contact with the
organism, further action depends on specific toxicity of the anion, cation, pH,
temperature, species bY organism, whether or not the disinfectant penetrates
readily inside the cell, and other factors.
WETNFSS. Surface te'nsion reducents enhance the effectiveness of disin-
fectants by increasing their wetness. The reason for this has been given in
Chapter 12.
Soap is a good surface tension reducent. Soapy, or saponated, solution&
therefore wet surfaces thoroughly. Phenol and related compounds, like cre-
sols, also lower surface tension: .and are, in and of themselves, powerful germi-
cides besides. A combination of soap, carbolic acid and cresols would, there-
fore, seem to have exceptional possibilities as a disinfectant. Indeed, mixtures
containing these substances in effective proportions are widely used in hos-
pitals, laboratories, etc. Products of this type are available on the market,
or can be made up as Liquor Cresolis Compositus from the U. S. Pharma-
copeia. Solutions of iodine with low ~urface tension are now available com-
mercially; e.g., Wescodyne. \
COMPETITIVE ADSORPTION. An exces\ of soap, however, will interfere
with the adsorption of disinfectants. Th(\ addition of more than minimal
amounts of a bactericidally ineffective but ~ ry surface-active substance like
soap to disinfectant solutions is likely to be isadvantageous. The relatively
inert soap is adsorbed on the bacterial surfac to the exclusion of the dis-
infectant. Such displacement of one surfactant by another is often called
competitive adsorption.
If organisms are coated with an oily or waxy fil ,as is the case naturally
with tuberculosis bacilli, even when a disinfectant eaches them it affects
them with difficulty; unless it possesses the special phy 'cal property of lower-
ing surface tension, !thus allowing it to wet the wax. \
I ,
There is another aspi:ct of the property of wetness, relating to nu~ition. If certain harm-
less surface-tension reducents are added to nutrient solutions, they facilitate contact of the
nutrient fluid with thei organisms and thus stimulate growth. The use of this principle in
cultivating waxy tubercle bacilli and diphtheria bacilli and other organisms is discussed in
the sections dealing with those organisms.
• Term commonly llsed for any substa!]ce active at surfaces; surface-tension reducents
in general. '
Synthetic Detergent * -Disinfectants. There is a whole class of disinfectants
which are very active surface-tension reducents. The first was discovered by
Domagk who, in 1936, described the properties of dodecyl-dimethyl-benzyl-
ammonium chloride. This is a quaternary ammonium salt, and is representa-
tive of a large number of similar compounds which have since been developed
commercially as disinfectants and/or detergents. t Many combine microbi:'
cidal and soap-like properties.
Essentially, they are ammonium halides in which the hydrogen atoms have
been replaced by organic radicles. A commercially familiar example is
Zephiran. (See Fig. 18-1.)
Chemical Structure and Activity. In general, the more effective of these
compounds are those in which the long-chain alkyl group is a chain of 12 to
16 carbon atoms, while the less effective are those oflower or higher molecular
weight. The germicidal efficacy of many organic compounds is similarly
greatly affected by chemical structure. For example, in compounds such as
alcohols, resorcinols, etc., it increases with molecular weight up to a certain
point beyond which it decreases with increased molecular weight.
There are many detergent-disinfectants now available, and more are being
marketed constantly. It is not necessary to discuss all of them here. Each
has its own advantages and disadvantages. As a class, the quaternary am-
monium halides have certain important properties in common. They are:
(1) microbicidal (or bacteriostatic?) in fairly high dilutions;
(2) not highly irritating or toxic;
(3) not corrosive and do not stain or discolor;
(4) not ill-tasting or malodorous;
(5) readily soluble;
(6) stable per se but are partly inactivated in the presence of organic matter
or colloids;
(7) relatively inexpensive;
(8) powerful surface tension reducents; therefore good wetting agents.
Their microbicidal action may be more apparent than real as they are
probably very effective bacteriostatic agents. Furthermore, caution is neces-
sary in interpreting the test data, because at least some of them have the
property of causing microorganisms to become sticky and to stick to solid
objects, and together in clumps. This prod]lces an apparent reduction in
numbers, partly through agglutination and not entirely through actual
killing. However, tincturest of quaternaries appear to be very effective indeed.
The mechanisms of the antimicrobial actions of quaternaries are not
completely uAderstood but are believed to be analogous to the action of sul-
/ fonamides, antibiotiCs, etc., i.e., they may act as specific enzymic poisons;
They must also have marked occluding or smothering effects because of
their tendency to acCumulate on cell surfaces. One other very striking effect
is lysis of many species of bacteria. This will be discussed in more detail later.
CATIONICS, ANIONICS AND NON-lONIes. There are three types of these
comp~unds: (1) those in which the organic radicle is a cation (quaternary
• Detergent is from the Latin detergere, to wash away.

t They are known in the industry as "quaternaries" or, more simply, as "quats."
t Solutions in alcohol.
ammonium halides); (2) those in which the organic group is the anion, e.g.,
sodium-lauryl-sulfate, and (3) those which do not ionize. These compounds
are, therefore, classified as cationic, anionic, and non-ionic, respectively
(Fig. 18-1). The cationic compounds tend to react with anions, and vice
versa. The cationic compounds appear to be more generally effective as dis-
infectants. The non-ionic compounds do not ionize in water. They are often
used as adjuncts in commercial detergents.
Concentration, Time, Temperature and pH or pOH. Disinfectant solu-
tions are not magical. Their values differ in different situations, depending on
the kind of bacteria to be destroyed and their location (open wounds, sewage,
skin, floors, dishes, clothing). No matter what disinfectant is selected for use
under any given circumstances, at least four important factors besides surface
tension playa part in the results. These are:
I. CONCENTRATION. In general, the more concentrated a disinfectant, the
more rapid and certain its action.
The effectiveness is, however, not linearly related to concentration but
exponentially. For example, doubling the concentration of phenol in a solu-
tion does not merely double the killing rate for bacteria but may increase it by
as much as 98 per cent. Thus, concentration of disinfectant beyond a certain
point accomplishes increasingly less and is wasteful.
The same effect as concentration may often be obtained by adding to a
disinfectant solution some reagent (e.g., NaCl) which tends to push the dis-
infectant out of solution, Le., the saturation point is reached so far as the
disinfectant substance is concerned. It then tends to accumulate elsewhere,
such as in the cell or on its surface.
When a disinfectant is in a colloidal state (emulsion) the material in each
minute globule is highly concentrated. The bacterial cell in contact with these
colloidal globules is, therefore, in contact with a high concentration of dis-
infectant.
OLIGODYNAMIC ACTION. It is well known that many substances which
are toxic in relatively large amounts actually stimulate growth at certain low
concentrations. To illustrate, if a bright piece of a heavy metal, as copper,
silver or gold, is placed on a plate of extract agar previously heavily inoculated
with an organism such as Micrococcus pyogenes, and the plate incubated,'
small quantities of the metal diffuse into the agar and inhibit growth of the
organisms in a zone around the metal piece. This phenomenon is ascribed to'
what is called oligodynamic* action of the metals (Fig. 18-2). \
At the periphery of this barren zone one might suppose that, as the con-
centration of metal ions diminishes with distance from the metal piece-
-
I
Rl
R-N-R2 ·Cl-
-+ I -
"
0
I
O-S-O-N-R2
Rl
. Na+
j-
I
R3 o" I
R3
-Cationic - Anionic Nonionic
A B C
Fig. 18-1. Diagrams of the structure of three types of quaternary compound. For explana-
tion see text.
• Action in minute concentrations.

Fig. 18-2. Biological effect of minute amounts of certain metals (oligodynamic action).
The agar medium was heavily inoculated with micrococci while warm, well mixed, and
poured into the plate. When solid, the metal disks (coins) were placed on the agar surface
and the plate was incubated. The bacteria grew where they could, producing a greyish,
granular appearance. Note that the silver disk (quarter) is surrounded by a clear zone where
'10 growth occurred, while the nickel disk shows little or no zone of inhibition. Note also
the increased density of the growth at the outer margin of the clear zone around the quarter.
This may be due to: (a) less competition for food at the edge of the sterile area; (b) stimula-
tion by a critically small concentration of the metallic ions; or Cc) both. The difference in
action between nickel and silver is not necessarily a general one. With another species of
test organism the situation could be reversed. Further, the coins are not pure silver or nickel.
(photo courtesy of Dr. Walter C. Burkhart, Department of Bacteriology, University of
Georgia, Athens, Ga.)
growth would increase: slightly, close to the zone; more and more with in-
creasing distance; and finally reach a density equal to normal in all areas
beyond the zone where the metal ions had not yet migrated. On the contrary,
in many cases growth is heaviest at the periphery of the sterile zone, where
con,centration of the toxic metal ions is minimal. Here a narrow but distinct
opaque ring of extra dense growth is often clearly evident. The same phe-
nomenon is seen in similar experiments with many other antimicrobial sub-
stances.
" II. TIME. No disinfectant, as ordinarily used, acts instantly. Sufficient
ti~e for contact, and for whatever chemical and physical reactions occur,
,~ must be allowed. The time required will depend on concentration, tempera-
ture; nature of the organisms; existence in the ba"terial population of cells
having varying susceptibilities to the disinfectant due to different ages and
other physiological 'factors, mutation, etc, I
RATE OF DISINFECTANT ACTION. As in sterilization by heat, chemical dis-
infectant action is 'an orderly, continuous process. U'nder ideal and uniform
conditions of temperature, concentration of disinfectant, pH, etc., the rate
at which death of the organisms in a pure culture occurs is often constant and
the 'number alive at any moment is theoretically a function of time only. In
such cases the rate of death suggests that the disinfectant is reacting wi~p
molecules of a single substance in organisms of identical susceptibility to the
disinfectant, under the laws of mass action-a monomolecular reaction.
Theoretically in such cases, if the logs of the number of surviving organisms
are plotted against units of time, a straight line should be obtained. If this
line is nearly perpendicular obviously the rate of killing is high; if it slopes
gently, the rate is low. The rate (high or low) under ideal, theoretical condi-
tions, tends toward uniformity throughout and in such cases the slope of the
line is affected only by such extraneous factors as temperature, pH, and con-
centration of disinfectant (Fig. 18-3).
Unfortunately, disinfectant action is not so simple. The form of the curve,
and also its slope, are dependent to a large extent on the species of organism
and on the presence of some cells which are more resistant or more sensitive
than the majority, to the disinfectant. Usually both sorts of cells are present.
Usually, also, several kinds of reaction occur simultaneously; coagulation,
non-specific chemical combinations, various enzyme blockages, etc. At times
the reaction between disinfectant and organism may proceed as a bi, tri-, or
quadrimolecular process or one of an even higher order, or mixed orders,
suggesting that (a) the reactions between protoplasms of different species
and different disinfectants are very complex, and (b) what appears to be a
reaction of a monomolecular type probably is the algebraic sum of a number
of simultaneous reactions, the net result of which superficially resembles a
monomolecular reaction. Curves representing numbers of organisms sur-
viving at any moment may not be straight lines but deflected in various ways
due to factors already mentioned and others not fully understood.
Of course, if very high concentrations of disinfectants, or very high tem-
per,!ltures, are applied, all of these gradual depopulations and measurable
fluctuations are masked and lost in one instantaneous, catastrophic stroke.
III. 'TEMPERATURE. As a rule, the warmer a disinfectant, the more effective
it is. This is based partly on the principle that chemical reactions in general
are speeded up by raising the temperature. As a rule, within ranges near the
100 ----------------
<I> 70
0:
o
>
; 60 80%
0:
:>
!II 50
.....
z
u 40
0:
'"
Q.
30
20
10
5 10 15 20
/. MINUTES
Fig. 18-3. Relation between time and death of organisms in a situation in which the I~al
agent acts at a constant rate under uniform conditions. .
lethal point for microorganisms and below, a rise in temperature of \00 C
increases reaction rates two to eight times. This is particularly true of coagula-
tion by hydrogen ions.
However, since many disinfectant actions are partly physical in character,
the' laws governing chemical reactions do not apply exclusively. Higher
temperatures generally lower surface tension, increase pH, decrease viscosity,
diminish adsorption and produce combinations of effects which are little
understood.
IV. pH AND pOH. As a general rule, the lethal or toxic action of harmful
agents, both physical and chemical, is affected by (H+) or (OH-). The syner-
gistic relationship between heat and (H+) has already been discussed. Heat
tends to cause greater dissociation of acids. The activity of phenol is markedly
increased by increases in hydrogen ion concentration.
The increase in effectiveness of benzoates and salicylates in acid solution
is an excellent example of the effect of (H+) on antiseptic action (see Chapter
43). In the case of these and other weak organic acids, the undissociated
molecule, and not the anion per se, is the active agent. The effect of in-
creased (H+) is to suppress ionization of the weaker acid, thus increasing the
concentration of the undissociated toxic molecules. Probably H+, OH-,
and heavy-metal cations are the most important ions in relation to disinfection
by chemical agents.
Inactivation of Antimicrobial Agents. NON-SPECIFIC ORGANIC MATTER.
Most common disinfectants are quite general in their affinity for protoplasm,
protein, etc. Substances like phenol, bichloride, strong acids, chlorine, etc.,
tend to combine with and destroy or coagulate all proteins or colloids. The
presence of considerable quantities of proteins or colloids such as blood
serum, mucus, feces, etc., in any material being disinfected will, therefore,
protect the organisms to a great extent by combining with the disinfectant
before it reaches the organisms. This is an important ,point to remember. In
addition to combining chemically with such organic substances the disinfec-
tant is to a great extent removed by them. These substances (proteins, mucus,
feces, blood, etc.) are largely colloidal. The colloidal structure presents a very
large surface at which surface-active disinfectants are adsorbed and removed
from the bacteria, ~ven after coagulation has occurred.
CHEMICAL ANTAGONISMS. Many antimicrobial agents may be inactivated
by the extraneous presence of certain specific substances with which they react
readily. Simple examples are the inactivation of acid by the presence of a
carbonate or hydroxide; the precipitation of toxic sodium oxalate by calcium
chloride. These are specific inactivators. Bichloride of mercury, while exerting
irreversible coagulative effects in high concentrations (1: 1000 or stronger),
exerts a specific and reversible chemical effect in dilutions of I : 100,000 or more.
The toxic action of dilute mercuric chloride is due to specific combinations
with the sulfhydryl (-SH) group in vital portions of the cells. The -SH
group is a very important, functioning part of many enzymes. When one
enzyme is stopped or blocked a whole series of dependent enzyme reactions
may also stop, both above and below the blocked enzyme. Mercury may thus'
be '(iewed as a specific prptoplasmic poison. The toxic action may be completely
neutralized by putting -SH compounds such as glutathione and cysteine into
the solution. These combine with the HgCh. Micrococcus pyogenes in contact
.
c . .
1 ! '"
.i '! 2 .2~
.
! 1
. 'I
~. ~ ..
7.
.2 ;;;
x
1 Q
I
3
.. 1
~ ~ ~l
:
:!
a
0. '!. '!.
1 ;i;
X
258 Methods and Phenomena of Microhiolof(Y
/N~ /N~ H H
H2N-C C C-H H ~ ~ H H H2 H2
I
N
II
C
I
c-c--,,-c
i
C-Q-N-C-C-
/ - ~ I I II L cf'#0
~c/ ~N/!\ ~C=C/ I ~OH
I H2 I I C
OH
Pterin.
H
Para-amino-
H
°/~ OH
Glutamic acid.
benzoic acid.
Folic (Pleroyl glutamic) acid.
-C/o °\I
C--S-NH2
/C~~H /~g
H-C C-H H-C C-H
H-C
I 11
C-H H-l ~-H
'c/ 'c/
I
NH2 ~H2
Pyridine-3 sulfonic acid. Para-aminobenzoic acid. Sulfanilamide.
Nicotinic acid amide.

Fig. 18-5. Diagram showing the molecular structure of several important vitamins,
and of some drugs used in chemotherapy. Note the similarity between sulfonamide, the
drug, on the one hand, and th_e vitamins: nicotinic acid amide and para-aminobenzoic acid,
on the other hand. ~ote also the position of para-aminobenzoic acid in the molecule of the'
folic acid molecule. For explanation see text.
for 72 hours with 0.001 per cent HgCl2, and apparently dead, can be "re-
vived" by treatment with H2S, which 'precipitates the Hg as HgS. The lethal
action of dilute phenol on micrococci and on Sa/mo,nella typhi may be stopped
and the apparently dead ol:ganisms "revived" by removal of the phenol with
activated charcoal or ferric chloride. In Chapter 12, we have seen how micro-
organisms apparently dead due to ultraviolet irradiation can be reactivate4 by
visible light and other agents.
THE SULFONAMIDE DRUGS. The sulfonamide drugs are synthetic com-
pounds all based on the same central molecule, sulfanilamide:
NH20S01NH2. The amino group (NH2) in the ortho position (shown
at left in the diagram above) is most important. (See also Fig. 18-4.)
Strictly speaking, the sulfonamides include only those derivatives of sulfa-
nilamide which structurally so closely resemble para-amino benzoic acid
(PABA) (Fig. 18-5) that they can antagonize that substance; that is, they can
act as a sort of counterfeit PABA. It appears that they can take the place of
PABA in a vital, enzymic function in the bacterial cell but, being physio-
logically inert in that situation, the enzymic function is blocked.
Let us suppose a series of synthetic reactions in a cell is carried on by a
series of enzymes A, B, C, etc. (Fig. 18-6, series I). Let us suppose, also, that
enzyme C is one which contains an absolutely essential, functional group
called folic acid.* Now, looking more closely at a graphic formula of folic
acid (Fig. 18-5) we see the molecule PABA occupying a prominent place in it.
Comparing the formula of PABA and sulfanilamide it takes no Sherlock
Holmes to see the similarity between the two and the possibility that a sul-
fonamide drug can play the imposter and take the place of PABA in the folic
acid molecule.
Enzyme C, then, is one which depends on folic acid and this, in turn, on
PABA to function. The result of the blockage of folic acid in enzyme C is a
pathologic accumulationt or "backing up" of partly completed metabolic
products at enzymes A and B. A complete stoppage of D, E, and F results
through lack of material from C on which they can work. The entire enzymic
production line ceases to operate.
CHEMOTHERAPY. Referring again to our production line analogy, let us
suppose that we replace the "dummy" (sulfonamide) at C with the functioning
agent by (1) adding enough PABA (the essential metabolite) to displace and
exclude the sulfonamide. This is chemotherapy. It also illustrates the principle
of metabolite antagonism.
We may also offset the blockage at C by (2) supplying complete folic acid,
or (3) supplying, in the culture medium, the product that would have been
formed by C (or any subsequent enzyme in the chain) or, (4) supplying the
finished product of the enzyme series.
The cell itself may be quite capable of doing this very thing. For example,
let us suppose that in a parallel series of enzyme reactions (series II, Fig. 18-6)
some of the substance usually made by C is produced as a side reaction or
intermediate stage in the function of H. This product becomes available to
D-and voilA! Such a cell is more or less sulfonamide-resistant, depending on
how much of the product needed by D is available from H.
ENZYME SERIES I: A .... B 70-> E .... F-'
ENZYME SERIES II : G -> H ---+ I ---+ J ---+ K --> L .....

i Fig. 18-6. For explanation see text.
• This is an important/vitamin, necessary for synthesis of the amino acids methionine,.

serine etc., which go into the synthesis of protein and this, in turn, into the making of
protoplasm. Such absolutely essential specific substances, whether vitamins, amino acids or
other compounds necessary to the completion of a particular functional part of the cell, are
called essential metabolites.
t Examples of such accumulations are dietary (vitamin) deficiency diseases such as
scurvy (vitamin C or ascorbic acid) and beri-beri (vitamin B or thiamine).
Numerous other such by-passings are known to occur.
DRUG-FASTNESS. If certain microorganisms are kept in contact with
small, sub-lethal concentrations of toxic substances (various salts, phenol,
antibiotics, sulfa drugs, etc.), the organisms often become highly resistant to
the effects of toxic substances. Drug-resistant organisms are often designated
as drug-fast. As indicated in the preceding paragraph, drug-fast strains of
microorganisms are generally thought, on very sound experimental grounds
(Newcomb's experiment; Luria-Delbriick experiment, etc.), to represent sur-
vival and growth of resistant mutants pre-existing in the exposed population
in imperceptible numbers. These dominate the microbial population after the
susceptible majority die off. The drugs themselves appear rarely to induce the
mutation to resistance.
DRUG DEPENDENCE. The appearance of strains wholly dependent on drugs
seems as strange as the emergence of resistant strains. The two are probably
not related except that they are two manifestations of the phenomenon of
variation. Dependent mutants obviousl) could not survive and develop
significantly except in an environment containing the factor on which they are
dependent. It is only because we have tried to cultivate microorganisms in
contact with antibiotics that these curious mutant forms have been revealed.
It is conceivable that strains dependent on all sorts of other unsuspected substances (such
as unicorn's hom or powdered emeralds ?) are also constantly occurring, but we never
learn about such strains because we never think to test for the presence of such curious
organisms by growing our cultures in media containing unicorn's hom or pulverized jewels.
However, we might profitably try various amino acids, traces of certain metals, lipids, etc.
Some valuable new organisms might be found. This sort of investigation is like treasure
hunting.
The mechanism for dependence is not entirely clear. Dependence may result
from the absence, in some mutants, of certain molecular gaps in key enzymes,
which can be filled only by certain molecular groupings in the drug on which
the microorganism is dependent. Such organisms are veritable microbial drug
addicts!
CHEMOTHERAPY AND CANCER. A very interesting aspect of chemotherapy
concerns the possibility of selectively poisoning the cells of neoplasms. The
cells of neoplasms are thought to have metabolic mechanisms different from
those of normal cells. The obvious possibility presents itself of finding some
metabolite antagonist which will poison an enzymic mechanism peculiar
to the neoplasm cell and not present in the normal cell. Thus, it is conceivable
that we could cure cancer overnight by means of a few tablets or injections
of some antibiotic-like drug.
This rosy dream may not be too far from reality. Stranger things have
happened. Already 'some drugs have been found which, if not the whole
answer, at least point definitely to the possibilities in research on cancer
metabolism. Fame, Fortune and Honor are to be won in this field!
THE EVALUATION OF DISINFEcrANTS
One often hears a disin(ectant spoken of as "strong" or "weak" or "mild."
These terms are inexact and convey different meanings to different people.
To one they mean a "disinfectant odor"; to another, pain on application to
a scratch; to still another, corrosive action; to another a definite color.
Rarely does the untrained person think of disinfectants in terms of microbi-
cidal activity, or toxicity for human beings or animals. He has to judge them
mainly by subjective criteria. Actually, the value of any substance as a disin-
fectant depends on a number of factors, important among which are: the
microorganisms concerned; the effect of the disinfectant on various materials
such as discoloration, corrosion, irritation of tissues, and toxicity in contact
with the body. Surface tension, cost, effectiveness or bactericidal action under
the conditions surrounding its use, stability and odor are also important fac-
tors. Not all disinfectants are equally effective, and some of them are more
effective against some bacteria than against others. Some are effective in pure
cultures in the test tube but not in contact with organic matter like blood,
feces or dead tissues; some are effective in the vapor phase but not as liquids
(~ee Chapter on Microbiology of the Atmosphere).
The effectiveness of many disinfectants may be estimated by mixing them,
in certain concentrations, with cultures of certain bacteria and measuring the
time required for the substance to kill the organisms. If this is done under
carefully standardized conditions, i.e., using a constant quantity of culture
medium of a stated composition, a fixed temperature, and a suspension of
measured numbers of certain bacteria of known and constant resistance, the
results will be quite accurate and reproducible.
The Phenol Coefficient. This is based on a comparison of the effectiveness
of the tested disinfectant with that of pure phenol under conditions carefully
standardized as noted above. The result obtained is called the phenol coeffi-
cient of the tested disinfectant. In this procedure 5 ml amounts of a series of
dilutions of the disinfectant to be tested (here called X) are placed in a row of
tubes of standard size. A similar series of dilutions of pure phenol (here
called P) of I :80, I :90, and I :100, is prepared. The temperature of all is
brought to 20 0 C in a water bath. To each tube is added 0.5 ml of a standard
suspension of Salmonella typhi having a resistance to phenol such that it is
killed in ten minutes by the 1:90 dilution. At intervals of 5, 10 and 15 minutes,
a standard loopful (4 mm loop of no. 23 Band S wire) is transferred from
each tube, in succession, to tubes containing 10 ml of sterile broth of standard
composition.
After 48 hours of incubation at 370 C growth in the broth tubes is recorded.
The phenol coefficient is the ratio of the highest dilution of X not killing the
organisms in five minutes (evidenced by growth in the corresponding broth
tube), but killing in ten minutes (evidenced by no growth in the broth), to the
corresponding dilutions of P. The values obtained are shown in Table 10.
In this experiment, the 1 :90 dilution of P failed to kill in five minutes but
killed all the S. typhi cells in ten minutes. This is compared with X which did
the same in a dilution of 1:450. The ratio of X to P is ~~ or 5, the FDA *
,
phenol coefficient of X. The determination of phenol coefficients requires a
high degree of skill.
The significance of 'a phenol coefficient has very definite limitations. For
example, a disinfectaJt dissolved in distilled water may have a phenol co-
• U. S. Food and Drug Administration.
262 Methods and Phenomena of Mzcrobiology
efficient as high as 50. However, it may be wholly ineffective if applied in the
blood, or used in contact with matter such as pus, saliva, feces, milk, etc., as
these may combine with the disinfectant and remove it from the bacteria.
Further, it may have a coefficient of only 2 or 3 when tested against some other
organisms, such as Micrococcus pyogenes. When a substance is said to have a
certain phenol coefficient, the limitations of the method must be kept in mind.
Other factors affecting the phenol coefficient are the composition and pH
of the broth and variations in the organism. Most important, since the com-
parison is made with pure phenol, the method is properly applicable only to
Table 10. Typical Data from a Phenol Coefficient Determination.
DISINFECTANTS
5 MINUTE 10 MINUTE 15 MINUTE
DILUTIONS
SUBCULTURES SUBCULTURES SUBCULTURES
80 _* _ _
Phenol 90 +*
_ _
100 + + +
350
_ _ _
400 +
_ _
450 +
_ _
'~Unknown"
. 500 + +
_
550 + +
_
600 + + +
650 + + +
* + = growth; _ = no growth.
water soluble substances -chemically related to phenol or acting on micro-

organisms in the same way. However, in practice, many sorts of disinfectants
are (often improperly) evaluated in terms of phenol coefficient.
In order to overcome some of these difficulties the 'Food and Drug Admin-"
istration Laboratories make tests of cb~mercial disinfectants with cultures
of various organisms and under circumstances d~signed to imitate actual
situations. Thus, tests are often conducted in the presence of blood serum.
For testing insoluble or oily products, special methods are used which are
intended to approach practical conditions of use. Among these are the filter
paper methods (wet and dry) and the agar plate (plain and cup) methods.
Inactivators. It is jmportant to note that many substances do not kill in
ten minutes but merely prevent growth of the test organisms in the broth sub-
cultures. The apparent killing and seemingly very high phenol coefficient by
some mercurials, for example, can be prevented if the transfers from the dis-
infectant tubes are made to broth containing thioglycollate. Thioglycollate
inactivates the mercury. Growth may then occur in all tubes and the phenol
coefficient is nil. This is a good example of (1) bacteriostasis and (2) reactiva-
tion by removal of the poisonous substance. With substances like HgCI2 , the
quaternaries, and the su1fonarnides. the active agent is closely attached to, or
inside of, the cells and cannot be removed or stopped by dilution. A definite
antagonist or inactivator is used. For sulfonamides para-aminobenzoic acid
has been mentioned, as have sulfur compounds for HgC1 2 • For quaternaries
no entirely satisfactory inactivator is available although "Luramin sodium,"
combined with "Tween-80" and Azo1ectin and other substances have been
found useful.
The Use-dilution Test.. No single method of testing gives a complete pic-
ture of the advantages and disadvantages, or actual value, of any given disin-
fectants under all conditions of use. In practice it has been the custom to use a
disinfectant in a dilution (use-dilution), represented by 20 times the phenol
coefficient. If the phenol coefficient is 5, the substance is arbitrarily used in
dilution of 1 :100. This was formerly thought to provide a wide margin of
safety.
THE USE-DILUTION CONFIRMATION TEST. In an attempt to give some fur-
ther idea of the activity of disinfectants under actual conditions the use-dilu-
tion confirmation test was devised. In this test small, polished, steel cylinders
(about 7 x 2 mm) are dipped into a young broth culture of Salmonella cho-
leraesuis (a typhoid-like bacillus) and then dried for about 60 minutes at
37° C. Of these contaminated rings, one is placed in each of 10 tubes contain-
ing the disinfectant being tested diluted to 100 times its phenol coefficient.
The temperature is held at exactly 20° C. After exactly 10 minutes the rings
are removed and transferred to broth culture to see if any of the organisms
originally dried upon them remained alive. An inactivator of the disinfectant
is included in this culture, to prevent any continued lethal or bacteriostatic
action of the disinfectant which might still adhere to'the steel cylinder. *
Some interesting data were collected by this method. Some disinfectants
with phenol coefficients of 5, ·ordinarily used in a dilution of 1 :100, were
found to have a steel-ring use-dilution of 1 :100 vs. S. choleraesuis and 0
against Micrococcus pyogenes. Others, with a phenol coefficient of 2.5 and
ordinarily used in a dilution of 1 :500, were found to have steel-ring use-
dilutions of 0 against both S. choleraesuis and M. pyogenes.
Modifications of this use-dilution method have been devised for testing
disinfectants under conditions of actual use, such as on steel surfaces, lin-
oleum, asphalt tile~ etc. Such data are invaluable in the hands of hospital and
institution administrators, sanitary engineers, etc., who must provide satis-
factory disinfection procedures on a large scale.
Many other methods of evaluation have been described. (See references.)
Toxicity of Disinfectants. One of the great problems in the selection and
use of disinfectants for use in contact with living tissues (i.e., for superficial
applications) is the avoidance of excessive toxic effects. Several methods are
* If no inactivator is used. the cylinders are dipped into a preliminary tube of broth to
remove as much disinfectant as possible before putting them into the final culture tube.
Unless all ten cylinders are sterilized by the disinfectant, certification of that dilution of the
disinfectant as a proper use-dilution is withheld.
available to test the toxicity of disinfectants. The results are sometimes ex-
pressed a~ toxicity index, i.e., ratio of minimal toxic dose to minimal effective
germicidal dose. One may determine the toxic dose of a disinfectant in various
ways; for example, by observing the smallest quantity necessary to stop com-
pletely the action of phagocytes (white blood corpuscles) in a test tube in a
given time. Other methods measure the inhibitory or lethal effect of the tested
substance on various tissues, while still others measure the respiration quotient
of tissue cells in contact with the germicides. One method is based on observ-
ing the survival time of chick embryos into which the tested substances are
injected. A time-honored, simple and effective method also, for many sub-
stances, is the direct trial on living animals.
Probably no single test gives a true result, and species of bacteria, as well
as of tissue cells, vary greatly in their susceptibility to different substances.
For any disinfectant, the toxicity index will therefore be different for different
tissues and bacteria.
FACfORS AFFECTING STERILIZATION BY HEAT
Heat may be applied for sterilization in three ways: (a) by steam or hot
water (moist heat); (b) by prolonged baking in the oven (dry heat); and (c)
by complete incineration. The last needs no comment beyond pointing out
that (1) common sense will direct what may'be burned up and what should
not; and (2) care must be taken to see that the material is completely burned.
At the temperatures commonly used for sterilization (100° C to 170° C)
several factors affect the rate at which microorganisms are killed. Important
among these are the time, the temperature, numbers of organisms, pH and
hydration.
Time and Temperature. Obviously these are inversely related. The.lower
the temperature, as a rule, the longer the time required to kill the organisms.
At 120° C in an autoclave (compressed steam), for example, it may require
only 15 minutes to kill everything, even resistant spores; whereas at 100° C
in the same autoclave the same spores may survive six hours or more. Even
increasing the temperature from 100° C to 105° C may diminish the killing
time to two hours. The difference is due to hydration and pressure. These are
discussed a little farther on.
Numbers of Organisms. As has been detailed in the section on disinfection
with chemical agents, sterilization is an orderly, continuous, process in which
there is a definite relationship between initial numbers of organisms, the effi-
cacy of the lethal process, and the elapsed time. In other words, under ideal
/ conditions, for any uniformly acting lethal agent the number of organisms
remaining alive after a given interval is 'a ftinction of elapsed time. This is
shown on page 265. ,
If these relations be drawn on a chart we have something like Figure 18-3,
which is a representative form of curve for many lethal agents. This shows
that the majority of cells are killed quickly but that some may survive for 11
considerable time, depending on lethality of the agent, hydration, pH, os
motic pressure, age of cells, etc.
pH, Hydration, etc. We have already indicated the relation of hydration
I to coagulation by both chemical disinfectants and heat. In addition, heat in"
SURVIVORS OF 100° C AFTER VARIOUS INTERVALS
Elapsed time Surviving organisms
(minutes) (per ml)
o 90 million
5 12
7 8
9 5
It 3
15 1
20 200 thousand
25 20
30 o
creases dissociation of acids (pH) and the effectiveness of (H+) in disinfection
processes.
The close relation of pH and temperature in lethal processes is illustrated
by the following:
Table 11. Relation of pH and Temperature to Survival of Tetanus Spores.

--
SURVIVAL (MINUTES) AT
pH
105° C 100° C 95° C 90° C
1.2 4 5 6 6
4.1 6 11 14 23
6.1 9 14 38 54
7.2 11 29 53 65
10.2 5 11 21 24
Here it is seen that (OH-) is also an adjuvant of heat, but to a lesser degree
than (H+). Acidity and alkalinity both increase the lethal effect of heat.
Fluids of high osmotic pressure tend to dehydrate the cell contents and so
increase the heat resistance. The age of the organisms is also important, older
cells being more heat resistant than young ones. The species or organism is a
determining factor, the form of survival curve varying markedly with different
kinds of organism exposed to the same lethal influence.
REFERENCES
Brochure. Reading ori Cancer. (A Bibliography.) Pub. Health Servo Publication No. 457,
1955. U. S. Government Printing office, Washington 25, D. C.
Cobb, J. P. : Tissue culture observations on the effects of chemotherapeutic agents on human
tumors. Tr. New York Acad. Sci., 1955, Ser. II, 17:237. .
Glassman, H. N.: Suiface active agents and their application in bacteriology. Bact. Rev.,
1948,12:105. '
Jawetz, E.: Antimicrobial chemotherapy. Ann. Rev. Microbiol., 1956, 10 :85.
Julius, H. W.: The mode of action of chemotherapeutic agents. Ann. Rev. Microbiol., 1952,
6:411. I,
Keller, R., and Mortqn, H. E.: The effect of a hand soap and a hexachlorophene soap on
the cultivable treponemata. Am. J. Syphilis, Gon. and Ven. Dis., 1952,36:524.
Klarman, E. G.: Recent U. S. Developments in the "Official" Testing of Disinfectants.
Mfg. Chemist, London, Feb., 1955.
Lester, W., Jr., and Dunklin, E. W.: Residual surface disinfection. J. Inf. Dis., 1955,96:40.
Litchfield, J. H., and Ordal, Z. J.: A study of the Stuart method for the evaluation of germ-
icides. Appl. Micr., 1955,3:67.
Martin, G. J.: Biological antagonism. The Blakiston Co., Inc., Garden City, N. Y., 1953.
Moore, A. M., and Boylen, J. B.: Patterns of Growth Inhibition. J. Bact., 1952,64:315.
Perkins, J. J.: Principles and Methods of Sterilization. Charles C Thomas, Springfield, 111.,
1956.
Reddish, C. F., Editor: Antiseptics, Disinfectants, Fungicides and Sterilization. Lea &
Febiger, Philadelphia, 1954.
Rhoads, C. P., Editor: Antimetabolites and Cancer. American Association for the Advance-
ment of Science, Washington 5, D. C., 1955.
Ritter, H. W.: The germicidal effect of a quaternary ammonium compound (cetylpyridinium
chloride) on Mycobacterium tuberculosis. Appl. Micr., 1956, 4:114.
Salle, A. J., and Amesur. B. R.: Evaluation of germicidal efficiencies of a group of anti-
biotics tested by tissue culture technique. Proc. Soc. Exp. Bioi. and Med., 1956, 93 :67.
Sevag, M. G., Reid, R. D., and Reynolds, O. E., Editors: Origins of Resistance to Toxic
Agents. Academic Press, Inc., New York, 1955.
Shive, W.: Biological activities of metabolite analogues. Ann. Rev. MicrobioL, 1952, 6:437.
Spencer, S. M.: Can chemicals conquer cancer? Sat. Eve. Post, 1956,228:32.
Stedman, R. L., Kravitz, E., and Bell, H.: Studies on the efficiencies of disinfectants for use
on inanimate objects: I, II, Ill, IV. Appl. Micr., 1954,2:119,322; 1955,3:71,273.
Various Authors: Conference on Mechanisms and Evaluation of Antiseptics. Ann. New
York Acad. ScL, 1950,53:1.
Various Authors: Symposium on gaseous ethylene oxide. Am. J. Hyg., 1949,50:270.
Walter, W. G.: Symposium on methods for determining bacterial contamination on the
surfaces. Bact. Rev., 1955, 19:284.
Wright, J. C.: Chemotherapy of human cancer. Tr. New York Acad. Sci., 1955, Ser. II,
17:210. .
269
'. The
llle,
1~

of Microorganisms
2. PRACTICAL APPLICATIONS
USES OF HEAT
A. Moist Heat: 1. BOILING IN WATER. The use of boiling for preserving
foods and disinfection is very simple. It is only necessary to remember that
spores may remain alive even after an hour of boiling. For ordinary household
purposes of disinfection, but not sterilization, five minutes' boiling of dishes,
clothes, etc., is usually sufficient, provided the hot water actually comes into
contact with the microorganisms, and not merely with the outside of lumps
of food or packets of instruments or other objects containing bacteria, etc.
Boiling can never be depended on for sterilization" especially at high eleva-
tions above sea level as shown below.
Table 12. Boiling Point at Various Altitudes.
LOCATION ALTITUDE· BOILING POINTt
New York City ............ . () 100

Chicago, Ill......... :....... . 589 98.9
Denver, Col. ........ ,....... . 5,280 94.3
Fort Laramie, Wyo .. ~ ...... . 7,380 92.2
Tahoe, Nev......... ~ ...... . 10,000 89.1
• Feet above sea level.

t Degree C .
I
2. FREE-FLOWING ("LIVE") STEAM. "Live" or free steam is usually applied
in a covered container which will hold steam, but without pressure. Boiling
water and free steam never reach a temperature above 100" C (212" F). In
mountainous regions it is lower than 100° C (see table). Free steam is some-
times used to accomplish fractional sterilization or tyndallization.
267
Fig. 19-1. Diagrammatic illustration of steam jacketed autoclave. (Surgical Supervisor,

Dec., 1946. American Sterilizer Co.) Steam enters the jacket, a double-walled Shell, at two
places just beneath the cylinder. It passes out the top through a pipe to which are.attached:
a wheel valve admitting the steam to the inner chamber; a safety valve; a gauge showing
pressure in the jacket. The steam enters the inner chamber at the right of the diagr'lm, filling
the upper portion. Its pressure registers on the chamber gauge. It may be allowed \0 escape
rapidly by the exhaust valve. If this is closed, steam pushes the cooler air in the lower portion
out at the bottom (left), where the thermometer registers proper temperature only when the
air is gone and is followed by the hot steam. The escaping steam may be allowed to flow out
/ without building up any pressure if the by-pass valve is,fully opened. If the by-pass valve is
closed and the shut-off valve is opened, steam passes through the thermostatic trap where
the heat shuts off all but a,pinhoie opening. This causes pressure to build up in the chamber,
yet prevents stagnation by permitting a constant minute flow of steam through the apparatus.
TYNDALLIZATION. Tyndall, a famous British scientist, noticed that a period

of about twenty-four hours at ordinary room temperatures is usually sufficient
to enable dormant and heat-resistant spores to germinate and to grow into the
vegetative state jn whjch they are sensiHve to heat. He devjsed a process of
'sterilization based on these observations. It consists in steaming for a few
minutes at 100° C on three or four successive occasions, separated by twenty-
four-hour intervals at a temperature favorable to spore germination. The
intervals permit the dormant, resistant spores to become active, vulnerable,
0
vegetative cells, readily killed by 100 C. This process renders an infusion
sterile whereas one single, continuous boiling for one hour may.not, since many
spores remain in their dormant and resistant state during this time. This·_
intermittent process is put to practical use in the home canning of foods. A
disadvantage of the method is the time required to achieve complete sterili-
zation. An advantage is that it requires no special apparatus. In some fluids,
as water, spores may not germinate promptly. Also, if the material is freely
exposed to air, anaerobic spores may not germinate and may survive the
process. If not freely exposed to air, aerobic spores will not germinate freely.
3. COMPRESSED STEAM. AUTOCLAVING. Anyone familiar with the opera-
tion of a home pressure cooker is familiar with an autoclave because the cooker
is a simple form of autoclave. In the autoclave (Fig. 19-1), be it a small and
simple home pressure-cooker or an apparatus for a hospital, large enough to
fill a room and fitted with various gauges, pipes, valves, clocks and wheels,
the object of both is alike: to heat the articles to be sterilized by means of
steam under considerable pressure.
Steam under pressure is hotter than boiling water or tree-flowing steam
such as is used in tyndallization. The higher the steam pres'Sure, the higher
the temperature.
The relation of steam pressure to temperature is shown in the following
table:
Table 13. Relation of Temperature and Pressure of Pure Steam.

Temperature
°C Pressure
(at sea level) (Pure steam)
tOO................................... 0
109................................... 5
ll5 ................................... 10
118 ................................... 12
121 .................•................. 15
124 ................................... 18
126 ...................•............... 20
It must be remembered that it is the compressed steam (moisture, hydration)

that sterilizes and not compressed air (dry and usually not as hot as steam).
For example, pure steam at 15 pounds pressure has a temperature of
121 0 C. If the steam is mixed with an equal amount of air, at the same pressure
the temperature is only 110° C while if the mixture is 2/3 air, the temperature
is only 109° C. I
Steam hydrates and thus promotes coagulation. Air does neither. Steam,
being water vapor, also produces hydrolysis at autoclave temperatures. Dry
air cannot do this at lany pressure or temperature. In autoclaving, therefore,
as in using a pressure cooker, a valve is left open for the escape of all air
before the steam pressure is allowed to rise.
The actual amount of water present as steam in an autoclave is small and
articles soon dry off;after removal, especially if removed from the autoclave
while hot.
The thermometer on the autoclave is the important guide to the process of
autoclaving, not the pressure gauge. However, the latter, as well as a steam-
escape or safety valve, is essential to safety ..
The common practice in autoclaving fluids or freely exposed surfaces such
as those of dishes and instruments is to apply 1IS° to 125° C (10 to 20 pounds'
pressure) for twenty minutes. The pressure must be allowed to subside
slowly after the heating is over or the superheated fluids in the vessels will
boil over. Any large, solid masses must be.heated a longer time, to allow for
heat penetration. Packages must be spaced so as to allow free circulation
of steam. Substances like mineral oil or petrolatum, sand, etc., or any dry
objects in tight jars, or which are impervious to moisture, cannot be satis-
factorily sterilized in the autoclave. The temperature may rise as high as
125 0 C, but, in the absence of moisture, this is ineffective. Such materials are
more effectively sterilized by dry heat in an oven.
Acid materials, canned tomatoes, acid fruits, pickles, sauerkraut, etc.
(pH more acid than 4.5) require much shorter periods and Jor lower tempera-
tures for sterilization than low acid foods (pH 6.5 to 7) like milk, corn, meats,
etc. This is because of the synergistic action between pH and temperature.
B. Dry Heat. Dry heat is used in oven sterilization. It is necessary to bear
in mind that significant coagulation does not occur when moisture is not
present.
Articles in ovens are very dry and therefore, in order to be freed of live
0 0 0
spores, must reach a very high temperature (165 to 170 C; 329 to 338 F). 0
It is customary to apply 165° C for a period of two to three hours. This ac-
r.omplishes coagulation but, what is more effective, slight charring.
A home oven can easily be used for sterilization. A mod.erate temperature
(330° F) is satisfactory, and the heating should be allowed to proceed for
three hours after reaching that temperature. Paper wrappings should be
slightly browned but not brittle; muslin or string should be yellO\y, due to
the heat.
Only dry articles not injured by baking (glassware, bandages, instruments,
mineral oils, petrolatum, and the like) may be thus sterilized. Solutions con-
taining water, alcohol or other volatile substances will, of course, boil away
and be ruined.
Thorough Heating Necessary. In any process of disinfection or steriliza-
tion by heat it is absolutely ·essential that the object be heated through and
through and that the center of thl< object be held at a killing temperature long
enough to destroy the bacteria.
Thus, in canning, a quart jar of spinach may be held in free steam (100 0 C)
for five to ten minutes and when grasped with the hand will feel very hot.
However, large masses of non-fluid materials like quart jars of canned vege-
tables and roast meat, in which the contents cannot circulate, require a long
time (1~ to 2 hours), even in the autoclave, to be heated so thoroughly that
the center reaches a sporicidal temperature. Pieces of meat or vegetables to
be sterilized in jars by heating should be loosely packed, allowing space for
circulation of the fluid in the jar. Air pockets in the jars should be carefully
removed. Penetration of the heat is facilitated if the cans or jars or pieces of
I roasting meat, etc., be small and not packed too closely together, promoting
free circulation of steam or hot air around and between them.
STERILIZATION BY FILTRATION
Clay and Paper Filters. An exceedingly useful method of freeing fluids
from bacteria is by means of filtration. Specially graded and· purified clays,
and fine unglazed porcelain are used commonly. For certain filters (Seitz, etc.),
compressed paper or asbestos disks take the place of clay. In some filters the
clays or porcelain are shaped into convenient, hollow cylindrical forms, closed
at one end, and mounted on funnel-shaped stems. They are sterilized by heat
before use, so that bacteria in the filter or glassware will not contaminate the
filtrate (filtered product).
Various forms oflaboratory filter are used, one being shown in Figure 19-2.
A convenient filter, using compressed paper disks as the filtering agent, is the
Seitz type (Fig. 19-3).
Membrane or Ultra-Filters. A particularly useful and versatile type of
filter is the so-called membrane filter made of collodion, cellulose acetate, or
similar substance. A common form is a disk about 50 mm in diameter and 0.1
mm in thickness. It has very fine, tubular openings from upper to lower sur-
face. These may be varied in diameter by the manufacturer from over 1 micron
to less than 0.005 p.. These filters permit aqueous fluids to pass freely but
withhold microorganisms in the fluid.
A particular advantage of the membrane filter is that all of the microorgan-
isms in a relatively large volume of fluid (water, milk, urine, diluted blood,
etc.) may be collected on one small disk where they may be observed directly
or cultivated in situ. There is no need to handle hundreds of tubes or flasks
with large-volume cultures. Uses of these filters have been mentioned in
Chapter 13.
Fig. 19-2. Typical filtration apparatus. The hollow "candle" of filtering clay, surrounded
by the fluid to be filtered, ~nclosed in a glass cup or mantle, is seen at A. The fluid passes
through the clay and is dniwn into the flask B by suction developed by the pump D. The
flask C is to trap any fluid. that might be sucked back from D or over from B. The cotton
plug in the side arm of B is to prevent ingress of dust when B is disconnected from the tubing.
(Belding and Marston, A frextbook of Medical Bacteriology, D. Appleton-Century Co.,
Publishers.)
272 Methods and Phenomena of MicTo~i%gy
Fig. 19-3. Seitz type filter. The fluid

to be filtered is poured into the cylindri-
cal cup (1). The fluid passes through the
bacteria-retaining disk of compressed
paper or asbestos (2) which is supported
on a wire screen (3). The stem of the fun-
nel-shaped base (4) is inserted in a suc-
tion flask through a rubber stopper and
the whole assembly sterilized before use.
SOME USEFUL DISINFECfANTS

A few substances commonly used as disinfectants may be briefly discussed
here. For purposes of discussion we may arrange them in 9 groups, as shown
in Table 14.
1. KMnO. and H101• These are mild disinfectants, sometimes used ex-
ternally on tissue surfaces. They are non-irritating and of low toxicity to tis-
sues. They are very unstable and their effect is quite temporary. They are of
some value for washing deep, anaerobic wounds. Certain compounds of
H 20 2 , such as the glycerite, are said to be of similar value in local applica-
tions.
2. Halogens. Iodine and .chlorine are the most widely used of this group.
Chlorine gas is used to disinfect filtered water at all municipal water-purifica-
tion and many sewage-disposal plants. It is usually handled in tanks or tubes
like oxygen, etc. It is applied to drinking water in a final concentration of
about 1.0 Part per million.
A more convenient form of chlorine for the individual and household user
is calcium or sodium hypochlorite. Solutions (5%) of these are purchasable
in all grocery stores under various names like Clorox, etc. They have a
multitude of household and sanitary uses. They depend for their effectiveness
probably on their liberation of free chlorine.
Various theories ascribe the antimicrobial action of hypochlorites to HOO, HCl, "rias-
cent" oxygen and free chlorine. Thus: 2CaOOz + 2HCI-> 2CaCh + 2HOCl; HOCI +
HCI -> HzO + Ch; or 2HOCI _, 2HO + +
Oz; or, 2CI HzO _, 2HCl + 102; or Clz +
HzO _, HCl + HOCI.
Directions for use of these solutions are on each bottle. Four points need
to be remembered in their use: (1) The chlorine tends to evaporate from the
solution; (2) they give a bad odor to the hands; (3) chlorine is poisonous;
(4) it is readily inact~vated by any and all organic matter.
CHiORIDE OF LIME (0.5 to 5 percent aqueous solution) is excellent for
similar purposes and is inexpensive-. The ordinary chloride of lime of com-
merce is unstable and soon loses most of its free chlorine. Many organic
chlorine compounds which liberate their chlorine more slowly are very
effective. Azochloramid is one of these; dichloramine toluol is another. The
odor of chlorine may be objectionable.
In addition to the above substances, there are many new proprietary
chlorine compounds on the market based on principles given abov:e.
IODINE is the most actively antimicrobial of'the halogens. However, it is
not commonly used in the same situations as chlorine because of its physical
properties and its cost. Formerly used widely as a 7% tincture (alcoholic
solution), this strength is far too caustic and poisonous for local application
and does more harm than good. The 2% tincture is much better and is the
most generally useful for small cuts and abrasions. If the alcohol causes too
much pain, the tincture may be diluted with twq to four volumes of water.
An aqueous solution containing approximately 1.85% of iodine and 2.2% of
potassium iodide is excellent but has a high surface tension. Gershenfeld
and Witlin recommend iodine for a variety of purposes, from clinical uses
to sanitization of dishes with aqueous solutions containing 200 parts per
million. Alcoholic iodine, 1 per cent, is sometimes recommended for disin-
fecting well-wiped clinical thermometers.
Aqueous solution of iodine with added surface-tension reducent has
many uses comparable to saponated cresol; for example, Wescodyne. Its
effect, however, is transitory due to volatility of iodine.
Iodine, like chlorine, combines rather indiscriminately with organic matter;
its solutions are unstable, and like chlorine, it is poisonous.
3. Inorganic Acids and Alkalies. These are not commonly used except in
special situations where rather violent and destructive action is desired.
4. Heavy Metal Salts. Nearly all of the heavy metal salts have strong
coagulative activity and consequently are possible microbicidal agents. How-
ever, only HgCh, CUS04 and AgN03 are in common use.
Silver nitrate (1.0%) is used principally for instillation into infants' eyes at
birth to prevent infection. Silver is also used as a local disinfectant in the form
of organic colloidal preparations like "Argyrol" or "Protargol," in which
form it is non-irritating and may be used in adult eyes at 20 per cent concen-
tration. It is not fori use in infants' eyes in place of AgN0 3•
Copper sulfate is: used chiefly to control growth of algae in open water
reservoirs. I
Solution bichloride of mercury (1: 1,000 aqueous) is useful for general pur-
poses, but its strong coagulative action must be borne in mind. Like phenol,
it will cause irritation and corrosion of skin, mucous membranes and wounds
if left too long in contact (hence the old name "corrosive sublimate"). All
mercury compounds are poisonous and should never be used internally
except under very ~pecial conditions. The action of mercury on bacteria may
Table 14. Some Common Disinfectants.
GROUP SUBSTANCES PRINCrPAL ACTION APPROPRIATE SITUATIONS

FOR USE
1. Oxidation. Superficial treatment of infected

tissue surfaces; lavage of infected
wounds.
Halogens Oxidation; formation of External: laundry, sewage, water,

Hypochlorite. protein halides. floors, utensils.
(Various laundry Municipal drinking water supplies.
bleaches, as Iodine tincture (mitis) much used
2. "Clorox.") for minor abrasions, etc.
Pure chlorine gas. Wescodyne used for hands, skin,
Iodine. general household (purposes.
(Iodine solutions
with surface ten-
sion reducent,
such as
Wescodyne.)
3. ACids} . Hydrolysis; coagulation. Not much used per se. Too de-
Alkalies Inorgamc structive except in special situa-
tions.
Heavy metal salts Coagulation; specific Instruments, inanimate surfaces,

HgClz toxic effects. tissue surfaces (very dilute).
AgNOJ (HgCIz)
CUS04 (1.0~;';) in eyes to prevent ophthal-
4. Hg(organic) mia of newborn. (AgNOJ)
In water reservoirs to suppress
algae. (CUS04)
Organic mercurials useful in ther-
apy an'd as preservatives of
serum, vaccines, etc.
Phenol and Coagulation; specific Among the most generally useful

derivatives toxic effects. disinfectants. Generally have
"Lysol" quite low surface tensions. In
Saponated cresol saponated solutions may be used
5. "Hexachloro- in 0.5% to 2% for hand wash,
phene" superficial wounds, objects, in-
Cresols, etc. struments, tables, floors, etc.
Hexylresorcinol
("ST 37")
Orthpphenylphe-
nol ("Q-syl")
Radiations Oxidation; coagulation; Limited use of UV to control air

Ultraviolet unknown changes; es- borne microorganisms in food
6. Cathode rays pecially absorbed by establishments, packing houses,
nuc1eoproteins. refrigerators, schools (7), hospi
tals (7), etc. Very microbicida
but difficult to apply in an effec
tive manner.
Cathode rays being develo1)ed for
industrial use--sterili7ation of
packaged foods, etc.
Destruction,. Removal and Inhibition of Microorganisms 275
Table 14. Some Common Disinfectants (continued).
APPROPRIATE SITUATIONS
GROUP SUBSTANCES PRINCIPAL ACTION
FOR USE
Alcohols Coagulation; toxic Usep mainly for local, mild dis-

7~ Ethyl-70% effects(?) infection of skin, thermometers,
Isopropyl-70% instruments, etc.
---
Quaternary Probably specific proto- Same as phenol and derivatives.
8. compounds.. plasmic poisons. May
act largely as bacterio-
static agents.
--
Carboxide. Oxidation (?). In:special autoclaves, for materials
9. injured by heat and/or moisture.
Used. mainly for special, medical
or research purposes.
be reversed by iodine and some sulfur compounds provided coagulation has

not occurred.
ORGANIC MERCURIALS. Attempts have been made. to decrease the toxic,
corrosive and irritating qualities of mercuric disinfectants by incorporating
mercury in complex organic molecules. This has yielded a number of products
which are less toxic and irritating than HgCl2 and which may even be used
internally within limits. Some of these compounds are highly effective in
vitro," in the presence of organic matter they are less effective but are better
than HgCI 2 • The value of all of this class of compounds is still in question.
Other useful mercurials are phenylmercuric nitrate, very effective and of
sufficiently low toxicity for limited internal use; and ammoniated mercury,
long used as a 10 per cent ointment for external woun~s, fungal infections of
the skin, etc.
5. Phenolic Compounds. There are hundreds of derivatives of phenol
which have some value as disinfectants. Their value is in great part related to
their chemical structure. Some of these have uses only in special situations.
The most generally useful compounds are the cresois, and some proprietary
qerivatives of phenol.
Weak aqueous phenol solution (0.5 to 1.0%) has a lower surface tension than
water and may be use~ for washing and dressing in emergencies, but carbolic
dressings should never be left in contact with skin, mucous membrane or
wounds for more than thirty minutes to an hour unless a very dilute solution
is used, since, like bichloride, they will cause coagulation of the live tissues
(gangrene) which may be followed by serious infection. Aqueous 1 to 2 per'
cent crude carbolic solutions (also cresol and similar compounds) make ex-
cellent disinfectants for household use, as well as in the barn.
Crude cresols form 60110idal (milky) suspensions in water and are therefore
specially effective because each colloidal droplet consists of concentrated
cresol. This is true of many colloidal disinfectants. Their uses are similar to
those of phenol and ;they are more effective and less expensive. There are
many excellent cresol ,preparations on the market. They have a clinging odor.
Most of them contain a surface tension reducent. Good examples are Lysol,
276 MethDtb IlIId PMnDmt!1IIJ oj Microbiology
l'iIo 19-4. Efficacy of electron Irradiation in food preservation. The control slice of
bread was not irradiated. Aft« three months of incubation it is abnost completely decom-
posed and blackened by growth of molds, bacteria and related microorganisms. The irradi-
ated slice remains "fresh." Cereals. grains. and other foods can be freed from weevils.
trichina worms and other pathogenic and/or damaging parasites by proper irradiation.
(Photo courtesy of General Electric, X-ray DepartmeIlt.)
and saponated cresol solution, U .S.P. The preparation of such solutions is

an important industry.
6. Radiations. Ultraviolet light is actively microbicidal when applied
properly. It has very slight powers of penetration. Bacteria hidden behind a
particle of dust may escape it. It cannot penetrate glass. Exposure must be
direct and suffiCiently prolonged. Eyes must be protected from it, as it can
severely damage them.
Cathode rays (electron beams) are expensive to apply but can be developed
on a commercial scale. They can be made to penetrate thin metal, paper or
plastic sheets and have been used successfully to sterilize packaged meats,
vegetables and other foods (Fig. 19-4). (See also Chapt. 43.)
7. Alcohols. Several alcohols have been found to have some value as
disinfectants, but only two are in general use; these ate ethyl and isopropyl
alcohols. like the phenol derivatives and quaternary compounds, disinfectant
value is closely related to chemical structure. Isopropyl alcohol is used as
"rubbing alcohol," purchasable in any drug store. As disinfectants, these
alcohols are of about equal value and usefulness. They have the disadvantage
of other coagulative substances, in that they are inactivated by mucus, pus,
etc. Tests in pure cultures of bacteria show these alcohols (70% volumetric)
to be quite effective against a wide variety of non-sporeforming bacteria. In
the presence of excessive extraneous coagulable material, they cannot be relied
on for strong disinfectant action. They are effective for disinfecting properly
IHstrw:tion, Removal and Inhibition of Mic,oorganisnu 277
wiped clinical thermomenters. As a hand wash they are commonly recom-
mended in 70% solution.
As an adjuvant, ethyl alcohol is useful because it increases the wetting
properties of aqueous solutions of other disinfectants. In some instances, as
in certain of the quaternaries, it greatly enhances their apparent effectiveness.
It is frequently used as a hand wash, with small amounts of green ~oap, per-
fumes, etc., or other disinfectants, mixed in.
8. Quaternaries. These compounds have been discussed in detail in the
section on surface tension and wetness. The value of these substances as
microbicidal agents is still in question. They are readily inactivated by com-
petitive adsorption with soap, by hard water, by acids and alkalies, and by
organic matter. They are more effective in warm than in cool water. Anionic
substances precipitate with cationic substances, and each with H+ or OH-,
respectively. There seems no doubt that many quaternaries exert bacteriostatic
action in dilutions as high as I: t 20,000. Probably they are bactericidal in
greater concentrations. As commonty used, they do not kill spores. In this
they resemble nearly all other chemical disinfectants except concentrated acids
and alkalies and ethylene oxide. In aqueous solutions they appear to be of
limited value in dealing with higher fungi or with tubercle bacilli. Tinctures
(solution in alcohol) appear to be highly effective, especially against tubercle
bacilli.
Ethylene Oxide. The use of gases for disinfection and control of disease
by fumigation has its origin in very early history. The use of glycol vapors to
disinfect ai, has been the subject of much recent investigation. In 1949 Phillips
and Kaye proposed the use of gaseous ethylene oxide for disinfection of sur-
gical instrurrrents, bedding, and other objects. Ethylene oxide has the formula
CH 2-CH 2 and is liquid at temperatures below 10.80 C. (51.40 F.), its boiling
"-./
°
point. It is inexpensive and may be obtained in small cylinders. It is some-
what poisonous and highly inflammable, but this property may be diminished
by diluting it with 90% CO 2, A commercial mixture of this sort. called Car-
boxide, is available. However, even this form of ethylene oxide must be
handled carefully. Special autoclaves are available in which carboxide may
be used instead of steam. Pressures range around 20 pounds. This is especially
valuable for plastic, rubber and leather goods, and delicate instruments which
would be ruined by heat and steam.
It is possible to sterilize fluid culture media by mixing fluid ethylene oxide
with them. The oxide disappears on warming, leaving no apparent trace except
an increase in acidity. It apparently kills spores and viruses.
REFERENCES
Adelberg, E. A.: The use of metabolically blocked organisms for the analysis of biosynthetic
pathways. Bact. Rev., 1953, 17:253.
BoUamy, W. D ., and Lawton, E. J.: Problems in using high-voltage electroDS for steriliza-
tion. Nucleonics, 1954, 12:54.
Brochure. Chilean Iodine Educational Bureau, Inc. Iodine Abstracts and Reviews, 1955,
3:No.2. .
Brochure. Wescodyne: The All-purpose Iodine Disinfectant for Hospital Use. West Disin-
fecting Co., Long Island City, N. Y.
Butterfield, C. T., Wattie, E., and Chambers, C. W.: Bacterial efficiency of quaternary
ammonium compounds. Pub. Health Rep'ts, 1950,65:1039.
Frobisher, M., Jr., Sommermeyer, L., and Blackwell, M. J.: Studies on disinfection of
clinical thermometers. App!. Micr., 1953,1 :187.
Gershenfeld, L., and Witlin, B.·: Iodine as a sanitizing agent for food and eating utensils.
Am. J. Pharrn., 1951,123:87.
Joint Committee on Atomic Energy, 84th U. S. Congress. Radiation Sterilization of Foods,
May 9; 1955. U. S. Government Printing Office, Washington, D. C.
Judge, L. F., Jr., and Pelczar, M. J., Jr.: The sterilization of carbohydrates with liquid ethy-
lene oxide for microbiological fermentation tests. Appl. Micr., 1955,3:292.
Klarman, E. G.: The role of antagonisms in the evaluation of antiseptics. Ann. New 'r ork
Acad. Med., 1950,53:123.
Klarman, E. G., and Wright, E. S.: Are "quats" fungicidal? Am. J. Pharrn., 1954, 126:267.
Klarman, E. G., and Wright, E. S.: Are quaternary ammonium compounds sporicidal?
Am. J. Pharm., 1950, 122 :330.
Migaki, H., and McCulloch, E. C.: Survivor curves of bacteria exposed to surface active
agents. J. Bact., 1949,58 :161.
Miller, O. T., Schmitt, R. E, and Phillips, G. B.: Applicatioru. of germicidal ultraviolet in
infectious disease laboratories. Am. J. Pub. Health, 1955,45:1420.
Oetlingen, W. F.: Phenol and its derivatives.N. 1. H. Bu!. No. 190, 1949.
Sommermeyer, L., and Frobisher, M., Jr.: Laboratory studies on disinfection of rectal ther-
mometers. Nursing Research, 1953, 2 :85.
Stewart, J. A., and Clark, B. S. (Pilcher, R. W., Ed.): The Canned Food Reference Manual.
Am. Can Co., New York, 1947.
Van Ese1tine, W. P., and Hucker, G. J.: The effect of organic matter on germicidal action of
the quaternary ammonium compounds. New York State Agr. Exp. Station Tech. Bu!.,
282, 1948.
Vera, H. D.: Sterility testing: The control of efficiency of sterilization techniques. App!.
Micr., 1953, 1 :117.
20

of Microorganisms
3. ANTIBIOTICS
FOR DECADES microbiologists have known that certain air-borne, antago-

nistic, saprophytic microorganisms got into their cultures as contaminants
and suppressed the growth of the species which they desired to cultivate.
The suppression of desired organisms was, of course, due to antimicrobial
substances ,(antibiotics) given off by the saprophytes (Fig. 20-1). But the
phenomenon was so commonplace and attention was so fixed on other prob-
lems, that it was pushed aside as merely an inevitable nuisance. The true
significance of the observation was overlooked until Fleming (afterwards
Sir Alexander Fleming, Nobel Prize Winner) in 1929 appreciated its possi-
bilities and acted upon the basis of his idea (Fig. 20-2).
PENICILLIN
The air-borne, antagonistic, saprophytic contaminant which first attracted
Fleming's attention was the common mold, Penicillium notatum. It occurred
to Fleming to experiment with its antimicrobial action. By passing broth
cultures of the mold through filters he removed the mold filaments. Thus he
was able to study the activity of the growth products alone as they occurred
in the broth. He found that the clarfiied, sterile broth contained a highly
potent, antimicrobial principle, the activity of which was readily demonstrated
in contact with se~sitive microorganisms. He called this principle penicillin.
In a much purified' and refined form it is the penicillin used therapeutically
today. :
Fleming realized, the practical possibility of his discovery but was not in a
position to develop it more fully and for some years it remained relatively
unknown. Attention was called to it again in 1935 but still it awakened little
interest.
Realizing that many pathogenic organisms are rapidly destroyed in the
soil, Waksman (Nbbel prize winner) suggested that the search for an effective
antagonistic microorganism be carried to the soil. In 1939 Dubos found, in
279
Pig 20-1. Tbreo 1arge colonies of PetdcIIllum notatum grown 00 a plato of apr heavily
inoculated with a penicillio-scnsitive orpuism. Tbe dark zone around the three colonies,
covering about one third ot the total area of the plate, represents bacterial inhibition. This
is the phenomenon noted by FJeming in 1929 which led to his discowry of penicillin. (Merck
& Co., The Story of Penicillin. 1944.)
Fia- 20-2. Prof. Sit Alexander Flemin& cliscoverer of peaicillbl; Nobel Prize winner.
Dr. Fkmi1I& is pointina with his inocu1ntilll needle at a aimt colony of PenJdlUum notatum
-
on an qarp1ate. (From Pfizer Spectrum. appearina in the Journal of the American Medical
AlIOdation.)
Destruction, RemoNl and lnIrlbitimt of Microorgcurimu 281
bogs, organisms (Bacillus bre,is) which produced valuable antibacterial sub-
stances (gramicidin, tyrothricin, etc.). But these, while of enormous value
therapeuticany and of great interest scientifically, were too toxic for internal
use. Dubos's discoveries reawakened interest in Fleming's observations on
Penicillium notatum and work 0., penicillin was begun in England on a large
scale by Florey, Chain, Abraha.m. and others (the Oxford group) in 1940.
SiDcc that time peoicillin bas played a role in the most titanic war in human history, in
the most prodigious piece of coopetative reeean:h ever orpnized (prior to atomic reeean:h);
in a areat industrial de¥eIopment; in the most coaip1ete control (WeI' a variety of diaeases
ever achieYed by man in a short time; and, bec:auae of its impact on ~ diseaaes, in a
trcmeodous new soc:ial and moral trend. the possibilities of which are sti1J only partly real-
ized. 1'bemiJitary importaDceofpeDicil1in in World War n. thaljust betinnina. can hardly
be estimated; but the importance of American facilities for IDIIlIS production of penicillin,
then not available in Europe, was soon of great benefit to all c:oncemcd.
Prodactloa. Production of penicillin involves cultivation of Penicillium

notatum under conditions most favorable to growth and penicillin production,
i.e., at around 24° C. Penicillin production is best at a pH between 7 and 8.
As the mold is strictly aerobic, exposure to air is essential.
Commercially, submerged growth in vigorously aerated tanks holding
thousands of gallons of medium (Fig. 20-3) is most widely used today.
In all methods suitable medium is inoculated with spore suspensions.
During the 7 to 14 days of in~ubation in the large vat of aerated, fluid
medium the mold excretes at least three waste substances which are of im-
portance: (a) the yenow pigment chrysogenin. which must be removed by
adsorption with charcoal ; (b) penicillin .. (c) notatin or penicillin B (this occurs
especially if the acidity of the medium is too great). This is removed during
the final purification process.
MEDIUM. Different formulae are doubtless in use by various manufac-
turers but basically they are similar and are mainly modifications of the
following:
Com-steep liquor- . .. . . . . .. . . .. . .. .. .. . .. . . .. . .. . .. . . .. . 40 ml
Lactose monohydrate...... . .. . . . . .. . . .... . ... . .. . ...... . 21.S am
NaN03............ .. .. .. .... .................... ... ... 3.0 am
MsS04. H:z().. .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . .. . .. . . . . 0.25 am
KHzP04 .... .... . . . . . . . . . . .. . . . ... , . . . ... . • .. ... . . . . . . . 0.50 am
ZDSO... H:z() . . . ... . ........ . ...... . . . . . .. . ..... . . .. . .. . 0.044 am
MnSO... H:z().............. .. ... . . .. .. ... . . ...... ... .. . . O.02Ogm
Glucose monohydrate. .... . .... . ....... . . . . .. . ..... . .. . . 3.0 gm
Water ad . . .. . .... .. . . . . ... .... ... . .... .. ... . . . ........ 1000 ml
After about 7 days, growth is complete and pH rises to 8.0 or above and
penicillin production ceases.
When no more penicillin is being formed, the masses of mold growth are
separated from the culture fluid by centrifugalization and filtration and the
process of extracting the penicillin from the clear fluid begins. This is quite
- Corn-steep liquor is a by-product of the distilliDJ industry, beinc the water !lied to
soak (steep) the com prior to fermentation. It contains various srowth factors (vitamins),
proteins, carbohydrates, etc., and is one of the beat soun:ea of nutriment for peoicilliD
production by many strains of the mold.
282 MetluHb and PIrno1lflNl of Microbiology
Fig. 2G-3. Like a steel igloo, tho top of a 9,OOO-gallon aerated-growth tank rises above
the floor level of tho penicillin plant at Lederle Laboratories Division, American Cyanamid
Company, Pearl River, N. Y. Like an iceberg, nine tenths of tho huge fermentation tank is
out of sight below tho floor. Small tank at the right contains chemicals to prevent excessive
foaming of tho liquid containing tho mold. (Peter Winkler, Lederle Laboratories, Inc.)
complex and involves various extractions with organic solvents, crystalliza-

tions, etc.
Properties. The "pure" substance (as will be shown later, in reality a mix-
ture of related penicillins called X, G, F, dihydro F, ftavicidin and K) occurs
as an acid. Penicillin is readily soluble in water and alcohol but the latter inac-
I tivates it. It is quickly destroyed by acid. In the crystalline form it is quite
stable, even to several days at 100° C and for years or more refrigerated in the
dark.
Among the most valuable properties of penicillin is its relatively low toxicity
for man and animals even in large doses, whereas its potency is such that a
concentration of 0.000001 gm per ml will exert marked bacteriostatic effects.
It may be administered subcutaneously, intravenously or locally. Although
the acidity of gastric juice destroys it to some extent, as well as certain bacteria
in tile intestines, and although it is not so well absorbed, much penicillin is
given orally because of the convenience. About five times the intramuscular
dose must be given. Buffers are often added to offset gastric acidity.
283
M_ of Aetloa. PenicilliD probably acts as a specific cell poison (for
certain species only), probably by the inactivation of one or more components
of their respiratory enzyme system in a manner analogous to sulfonamide
drugs. It appears to act primarily as a bactericidal agent; to a lees extent bac-
teriostatically. Much depends on the species of organism involved and the
concentration of penicillin.
Any cbcmotherapeutic ascot which acts bacteriostatically in the body gives
the normal defense mechanisms of the body (1eukocytes, etc.) the upper hand
in the struggle against invading microorganisms. This will be more fully
clarified farther on.
ChemiIItry ... Vuieties of PenidIHn The penicillins are strong, mono-
basic acids, pH about 2.8. The general chemical structure provisionally as-
signed to them is:
s
/'-..
NH-CH-CH C(CHV2
~<~-l_jCONa
(-) ~ (+) H
The R varies in the different peoicilliPs as foUows:
REl..A11ONSHlPS OF NATURAL Pl!NJCJWNS
PENICIUJN SIDE CHAIN <R>

ForI
Gorll
Xorm
KorIV
Dihydro F
Plavicidin
Laboratory synthesis of penicillin was announced shortly after the formula

was known. The substance actually produced was penicillin G. A number of
other substances closely related, which also had antibiotic properties, were
produced experimentally during the research, thus revealing the possibility
dtat better tll'tiftcial penir.illins and other antibiotics may be made than are
available from natural sources.
lIoiyathelill& of PeaIdJIhw. In addition to artificially synthesizing penicillin,
it has been found possible to induce Penicillium cnltures to synthesize new
forms of penicillin. The method used involves two main steps: <a) to "spHt"
the penicillin molecule chemically into its constituent radicles; (b) to "feed"
to the cultures of Penicillium notatum these various parts of the penicillin
molecule, or chemically related substitutes. By extra feedings of the parts of
tile normal peni~ molecule (called preCUl'sora), the production of normal
penici11ins has been greatly increased. By feeding substances chemically re-
lated to normal precursors, but of sHghtly different molecular structure, new
284 MetluHb and PhenolMltIJ 0/ Microbiolov
and more valuable penicillins have been synthesized by the mold, using these
abnormal precursors.
This same sort of thinking has been applied to other processes in which a
wanted product is synthesized by a living organism. The field of biosynthesis
is clearly wide open for the ingenious, the well-trained and the energetic
student.
Various StndDs of Peaidllium Notatum. Fleming's strain of P. notatum for
a time was thought to be unique. It was later found that some "wild" strains
of this organism were better producers of penicillin, and that other, closely
related organisms, especially P. chrysogenum, also produce penicillin. These
findings led to a very extensive search for better strains of P. notatum and for
penicillin-producing strains of other organisms. Many thousands of cultures
were tested and it was found that the penicillin-producing property is widely
distributed in the P. notatum-chrysogenum group and that some strains are
better than others, especially for the various methods of production. i.e .•
some were good in tanks. not so good on surfaces. and vice versa.
Later. naturally-occurring variants of the best strains were tested and it
was found that strains might be of two types: a conidial or C type, producing
relatively little mycelium, many conidia and much penicillin; and an M or
mycelial type. producing much mycelial growth. but relatively fe~ conidia
and little penicillin. Much care is, therefore. exercised to maintain cultures
in the C.condition and to save stocks of spores of these strains for "seed."
The search for a "super" strain continues, Irradiation with ultraviolet light
has furnished many thousands of mutant strains for testing. The method has
yielded some very potent penicillin-producing mutants. It is especially interest-
ing to note the usefulness of this method in inducing the development of
variants which produce large amounts of one or another of the various peni-
cillin isomers, F, G, X or K.
Factors IDbibiting PeniclOin. PENICILLINASE. Many microorganisms pro-
duce an enzyme, penicillinase, which rapidly destroys penicillin. In the pres-
ence of such species the action of penicillin is suppressed or eliminated.
Among the species producing penicillinase are Escherichia coli, members of
the genus Bacillus and some species of Shigella. Many species which do
not produce penicillinase are, nevertheless, resistant to penicillin for other,
mostly obscure, reasons.
Development of Resistance or Drug-Fastness; Dependence. An undesirable
aspect of the continued clinical use of antibiotics (as well as of sulfa drugs)
is the emergence of variant strains: (a) having increased resistance to the drugs
I (drug-fast strains); or (b) wholly dependent on the drugs. Drug-fastness and
drug dependence have been described in Chapter 18.
Laboratory Uses of PeoicllliD. The value of penicillin as a selective bac-
teriostatic agent in laboratory bacteriology was recognized by Fleming and
others. It is a convenient aid to the isolation of penicillin-resistant organisms
(such as the whooping cough and influenza bacilli) from throat cultures since
it suppresses the growth of unwanted, penicillin-sensitive organisms. It is also
used for suppressing bacterial growth in studies of viruses and riclcettsiae and
pleuropneumonia organisms, the latter three groups of microorganisms being
almost wholly resistant to most antibiotics (see Chapters 5 and 13).
1be dhaieal uses of peaicllIln are beyond the scope of this discussion. but
Dutl1lCtion, Remoral and InIdbitio" fJf Microorgflllisnu 285
some of the common infectious organisms against which it is more or less
effective are listed here.
Highly susceptible
B. anthracis
Various clostridia including CI. tetani and CI. welchii
D. pneumoniae
Neisseria spp., including gonococci and meningococci
Micrococci
Streptococl..us pyogenes, etc. (Fig. 20-4)
C. diphtheriae (in vitro)
Moderately susceptible
Borrelia of relapsing fever
Various alpha ("viridans") type hemolytic streptococci
Leptospira spp.
Treponema pallidum
Note that all of these except Neisseria are gram-positive species.

It is generally ineffective in salmonellosis and shigellosis (typhoid fever and
bacilli!.r7 dysentery), influenza, pertussis, tularemia, brucellosis, and other
infec60ns caused by gram-negative bacilli, as well as viral, rickettsial, and
PPLO iafectioDS and infections willi protozoa, yeasts, and molds.
StudmUzatioD. The Oxford or Florey Unit of penicillin is the least
amount necessary to inhibit completely the growth of a certain strain 0 f
Micrococcus pyogenes, var. aureus in 50 mI of a standardized, meat-extract
;broth.
In 1944 an international unit was defined as 0.6 microgram of an arbitrary
International Standard Sample, this quantity having been found to equal I
Oxford unit.
Methods of Assay and SeDsitil'ity TestIDg. Five types of procedure are in
Fi&. 20-4. Child with seveR infection of tissues of tbI: lo-r face and chin and around
eyes, due probably to hemolytic streptococci. At left, at be8iJmina of treatment with penicil-
lin; center, 96 hours later; right, 9 days after beainniDa treatment. (Herren, Proc. Staff Meet.,
Mayo Clinic. vol. 18.)
286
rig. 2O-s. Testina sensitivity of a bacterium to antibiotics or other cb!lmotherapeutic

agents by the "tube dilution" method. The top row of tubes of broth culture contain "Agent
A" in increasina amounts as shown by the figures on the tubes. The second row contains
I "Agent B" in the same amounts while the third row contains "Agent C' in the same dilu-
tions. All tubes were inoculated with ''Organism X" from the same culture at the same time
and incubated togetheF. Luxuriant growth, as'!ihown by white turbidity, has occumd even
in the hiIbest c:onccotration of Apt A. showini that Orpnism X (perhaps from a patient
very ill with this infection) is not in the least affected by this drug. The drua of choice will
be Agent C which prevents growth of Organism X even in the smallest concentration. Agent
B is sliahtly effective. The tube at the left of each row is a control tube containing no inhib-
itory &.galt. (Courtesy of The Abbott Laboratories, North Chicago, m.)
use for assaying the potency of, or sensitivity ofmircoorganisms to, penicillin
and other antibiotic preparations: (I) the serial dilution method in broth; and
(2) the serial dilution method in agar (agar-streak method); (3) the cylinder-
plate method; (4) the disk or tablet method; (5) the blood-plate method.
These methods, modified, are also used for measuring sensitivity of "un-
Destruction, Removal and Inhibition of Microo,.,animu 287
known" organisms to standardized, graded amounts of antibiotics and sul-
fonamides. This is commonly done in hospital laboratories.
There are sources of error in all of these methods unless used and inter-
preted by experts.
1. THE SERIAL DILUTION ASSAY METIIOD IN BROTH. Serial dilutions of the
"unknown" antibiotic sample are made in uniform amounts of standard
broth in culture tubes. These are then inoculated with a uniform amount of
the test organism, which is selected on the basis of its known degree of sensi-
tivity. Turbidity (or its abstnce) is noted and may be measured by means of
a photoelectric turbidimeter. The turbidities (amounts of growth) are com-
pared with a dilution-series made in exactly the same way but with a "known"
antibiotic reference standard.
The method is relatively laborious and requires sterilization of the sample,
which destroys some of its potency. However, it is one of the most accurate
procedures and results can be stated in exact terms of units of antibiotic.
This same procedure, reversed, is often used to measure the sensitivity of
an "unknown" organism, isolated from an infected patient, to various anti-
biotics. Graded, known, strengths of antibiotics are placed in series of tubes.
F.,. 20-6. Antibacterial activity of streptomycin. A, Esclrerfchia coli. B, Salmonella

typ/Il. C. ProlelU sp. D, Klebsiella pneumoniae. E, Pseudo1lll1fllU aeruginosa. F, Mycobac-
lerillm lubucu/osir. The !etters refer to streaks of growth on the plates. The numerals indi-
cate micrograms of streptomycin per mi. of agar. Note the great sensitivity of Mycobac-
terillm ,ubucu/osls (F) to the drug. Streptomycin is particularly valuable in tbe treatment of
tubemJlosis. (Courtesy of Merck & Co., Inc.)
288
Each tube is inoculated with a drop of the organism whose sensitivity is being
tested. IT growth occurs in all tubes, the organism is resistant; if no growth
occurs, the organism is sensitive. Growth in some of the tubes permits a
measure of the sensitivity of the organism (Fig. 20-5).
2. SERIAL Dn.unON (STlU!AK. AssAy) MElllODS IN AGAR. These are rapid,
do not require a sterile antibiotic sample, permit the use of several test organ-
isms at once, and can be used to test substances not dissolved in water.
The method is much used for preliminary assays of crude material.
Graded dilutions of the substance to be tested are placed in a series of
Petri dishes. Into these are poured about 10 ml of melted and cooled agar
which is thoroughly mixed with the antibiotic dilution by tilting the plates
back and forth. After the agar has hardened, the plates are marked into several
sectors, on each of which is streaked a different test organism. After incu-
bation, the highest dilution of the antibiotic inhibitory to each organism is
recorded (see Fig. 20-6).
This method, reversed, also is often used for sensitivity testing by using
known strengths of antibiotics and unknown organisms.
3. THE CYLINDER. PLATB MElllOD. This is one of the earliest procedures
used for antibiotics assay. A series of steel, glass or porcelain cylinders about
10 by 6 mm in dimensions, with perfectly square-cut ends~ like a drainage
tile, are sealed, by touching them, while slightly heated, to the upper surface
of already inoculated, solid agar in a Petri plate. The solutions of antibiotic
to be tested are placed in the cylinders (Fig. 20-7). The plate is then incubated.
Diffusing into the agar, the antibiotic solution reveals its potency by the width
of the zone of inhibition of growth in the agar around the cylinder. The test
organism, agar, density, time, temperature, pH, etc., must be very exactly
standardized. This is true in all of the methods described here.
4. THE DISK OR. TABLET MElllOD. This method is less exact than the
procedures just described but is quick, convenient and economical. It is
therefore a preferred and widely used method for many purposes not re-
quiring high degrees of accuracy. The principle is the same as the cylinder
plate method. The cylinders are replaced by disks or tablets. This method has
been adapted to: (I) assaying the potency of new preparations of antibiotics;
(2) determining the amount of antibiotic in body fluids of patients; (3) testing
the resistance or susceptibility of strains of bacteria to antibiotics.
In disk and tablet procedures nutrient agar, of a composition appropriate
to the organism being tested, is heavily inoculated with the desired organism.
The inoculum may be streaked all over the surface of the solidified agar or
mixed with the agar while still fluid before pouring the plate. IT an antibiotic
/ solution of unknown potency is being assayed (e.g. manufacturer's sample,
blood, urine, etc.) the organism used is a stock strain of kno ....n sensitivity to
standard doses of that antibiotic.
The antibiotic solution to be tested is applied to the inoculated agar in
measured quantities in disks of sterile filter paper or in small "tablets,"
which are placed on the surface of the agar plate before incubating. The
antibiotic diffuses outward from the disk or tablet and inhibits growth in the
agar around it. The width of the zone of inhibition is a resultant of: (a) the
concentration of antibiotic in the solution; (b) the sensitivity of the organism
Dutructio", Remoral and lnIribitioll of Microorgllllimu 289
used; (c) ability of the drug to diffuse into the apr; (d)time and temperature
of incubation; pH and composition of the medium, etc.
The converse of this procedure is seen in the common practice of testing
the antibiotic resistance of "unknown" strains of bacteria causing infections
in patients. In such tests the "unknown" organism, instead of a stock strain,
is used to inoculate the agar. Various disks with several antibiotics in known,
graded dosages are placed on the agar. After incubation some of the disks
will have growth in contact with their edges, showing that they have exercised
no inhibition (the organisms are completely resistant). Other disks will be
surrounded by clear zones varying in width (Fig. 20-8). The presence or
absence of a zone of inhibition, rather than its width, will guide selection of
the antibiotic to be used in the patient. Appropriate disks, with measured
amounts of all commonly available antibiotics and sulfonamide drugs, are
available commercially.
5. BLOOD AoAR. PLATE MBTHOD FOR RAPID SBNSmVITY TEmNo. Sterile
blood agar is poured into a Petri dish to a depth of about 2 mm (base layer).
Fia. '1D-7. CyUnder plate IItaIIdardIzatl or pemcIIHn. The c:uJture plate contains a Vf:C'J
carefully standardized medium, uniformly and heavily seeded with staphylococ:x:i or spores
of B. subti/is. In each cylinder was placed a measured quantity of penicilIin-containing
utract. Following iDcubation under standard conditions, growth of bacterial colODies pve
the medium the pebbled, gray appearance seen everywhere except· around the cy1inden.
Here, growth bas been inhibited by penicillin. MeasuIanent of these zones of inhibition
permits standardization of penicillin in"Oxford units by comparison with zones produced
UDder identical conditions of test by standard solutions of known unit streoath. Tho 1IeIIIl·
tivity of an unImown orpnism to any antibiotic is tested in exactly the same way; inocula·
doD of tho plate is made with the "unImown" in place of the staphylococ:x:i or B. 8IIbtIIU
routinely used. Known amounts of various antibiotics may be placed in the cylinders.
(Therapeutic Notes. March, 1944, Parke, Davis &; Company.)
290
Fia. 20-8. A simplifted procedure for the disk method of determiniDa sensitivity of an
orpnism to antlDiotics or other drugs. The plato was prepared by inoculating the surface
of the agar with the orpnism to be tested. The stanbapeci piece or filter paper was then
placed on the surface and the plate was incubated. The disk at the tip of each arm or the
star contains a diIferart drug. Obvioualy drug E is most effective in inhibiting the organism,
drug A much leas so, while the organism is whoDy resistant to drugs P, T, and TE. Many
other combinations of drugs in similar ~ts are available. (photo courtesy or The
Abbott Laboratories, North Chicago, ID.)
Similar fluid agar is heavily inoculated with the organism being tested. After
tkoroughly mixing, this is poured over the base layer to an equal depth
(seed layer). When solid, antibiotic disks are placed on the surface and the
plate is placed in the incubator. The plate may be examined as early as two
hours and every hour thereafter. Diffuse growth of the organism, whether
actual colonies are visible or not, produces a marked discoloration and/or
hemolysis of the blood except where inhibited under and around disks con-
taining effective antibiotic concentrations. Here the blood remains unchanged.
ANTIBIODCS FROM STREPTOMYCES

Streptomyces (see Chapter 30) are bacteria which have some of the struc-
tural and biochemical properties of molds, like Penicillium notatum. Strepto-
/ myces are, however, minute in size as compared with true molds and have
other characters relating them to the Schizomycetes.
A number of species of Streptomyces have been found to produce antibiotics
of medical and commercial value. Important among these are streptomycin;
chlortetracycline (Aureomycin); oxytetracycline (Terramycin); tetracycline
(AcIrromycin); erythromycin (llotycin, ErYI/vocin); carbomycin (Magnamy-
em); and chloramphenicol (Chloromycetin). (See Table 15.) There are others,
and more are constantly being introduced. Some of these are said to be active
against certain of the large viruses, others against certain viral neoplasms,
some against certain protozoa and so on. A description of streptomycin will
Destruction. Remo'llal and Inhibition of MicroorganisllU 291
exemplify all of the group, although each has its own special properties and
values.
Streptomycin. The discovery of streptomycin dates only from 1944 and
followed naturally in a long series of researches by Waksman and his col-
laborators (Fig. 2~9) into the numbers and kinds of antagonistic microorgan-
isms in the soil. The source organism produces conidia and aerial mycelia
and is one of the order Actinomycetales: Streptomyces griseus, whence the
name streptomycin. •
The principles underlying production, purification, assay and standardi-
Table 15. Partial List of Antibiotics. •
ANTIlIlonCS
CHARACTERlmcst SOURCE
COMMON NAME TRADE NAME
Penicillin! Gram-positive bacteria; Penicillium nototum

Treponema; Neisseria
Fumagillin Entamoeba histolytlca Aspergillus fumigatus
Streptomycin Myco. tuberculosis; Streptomyces griseus

Gram-negative bacteria
Dihydrostreptomycin Like streptomycin Streptomycin
Tetracycline Achromycin Broad-spectrum Chlortetracycline

Oxytetracycline Terramycin Broad-spectrum Streptomyces rimosus
Chlortetracycline Aureomycin Broad-spectrum Streptomyces aureofadens
Chloramphenicol Chloromycetin Broad-spectrum Streptomyces venezuelae
Erythromycin llotycin, Eryth- Broad-spectrum (not Streptomyces erythreus
rocin Enterobacteriaceae)
Carbomycin Magnamycin Like erythromycin Streptomyces halstedii
NeomycinB: Aavomycin Mycobacteria Streptomyces fradille

Viomycin Viocin Like penicillin Str.floridae .. Str.funiceus
Chrysomycin Bacteriophage Streptomyces sp.
Oligomycin Fungi Str. diostatochromogenes
Netropsin Some viruses Streptomyces netropsis
ADisomycin Protozoa Streptomyces sp.
Nystatin Fungi Streptomyces noursei
Cycloheximide Actidione Saprophytic fungi Streptomyces griseus
- - --- -
Bacitracin Like penicillin Bacillus licheniformis (1)
Polymyxin B: Gram-positive bacteria Bacillus polymyxa
Xerosin Some viruses Achromobacter xerosis
Pyocyanin'l Miscellaneous Pseudomonar aeruginosa
• Several not listed here are valuable commercially, agriculturally, horticulturally, and
80011.
t Not necessarily the only activity.
: Several of these antibiotics are in reality mixtures consisting of related compounds like
the penicillins, polymyxin A, B, C, D, etc., carbomycin A and B and so on.
II Not used medicinally. One of the first known antibiotic substances.
292
Pia. 20-9. Dr. Selman A. WaksmaD. (Courtesy Society of American Bacterioloaists.)
zation of streptomycin are analogous to those pertaining to penicillin, differ-

ing mainly in respect to cultural details and technical procedures appropriate
to the organism and substance involved.
Aeration of the culture is essential for rapid streptomycin production,
which is at its maximum in two to three days. Various strains of Streptomyces
griseus are more or less active producers of the drug.
The unit of streptomycin is analogous to the unit of penicillin. The S unit,
originalJy proposed, is the least amount which, when present in 1 ml of
nutrient medium, just inhibits a standard strain of Escherichia coli. A new
/ llIlit is based on crystalJine material, of which 1 gamma (1 microgram:
0.000001 gm) is equal to 1 unit (1 Oxford unit of penicillin = 0.6 gamma).
For detection of streptomycin. the streak-plate method serves as a pre-
liminary determination. More accurate definitions of potency depend on the
cylinder-plate method, using a certain strain of Micrococcus pyogenes, VQT.
t1III'eus, or the disk method. A broth dilution method has also been utilized.
The drug may be inactivated with cysteine or by semicarbazide.
The fonnuJo of streptomycin is C21H3g()lZN,. The drug is much more
stable than penicillin, both in dry form and in solution. In the refrigerator
the solutions retain potency for months, and at 37° C for about two weeks.
Destruction. RemoN' tmd Inhibition of Mkl'OOrgtmimu 293
Boiling for 10 minutes produces about SO per cent loss of potency. Strepto-
mycin in vitro is bactericidal in high concentrations; bacteriostatic in low
concentrations.
ScoPE OF ArnON. Its scope of action is in some respects broader than that
of penicillin, being active against a variety of gram-negative and gram-
positive organisms, some of which are listed below.
Gram-positive Gram-negative
Mycobacterium tuberculosis Aerogenes species
Mkrococcus pyogenes Salmonella typhi
Diplococcus pneumoniae PasteurellD tularensis
Streptococcus, species Klebsiella species
Bacillus subtilis (and related species) Brucella abortus
Erysipelothrix Proteus vulgaris
Not: Salmonella paratyphi B
Clostridia Hemophilus pertussis
Hemophilus injluenzae
Pseudomonas aeruginosa
Escherichia species
It has been used in tuberculosis with striking yet disappointing results. The
disappointment resulted only because too much was expected. It is a valuable
drug in this disease when properly used.
Streptomycin has been used effectively in many infections with gram-
negative rods not susceptible to penicillin or sulfonamides. It is not so rapidly
destroyed in, or excreted from, the body as penicillin. Some toxic effects
(vertigo, deafness, etc.), have been described. It must be used only under
medical supervision.
1'HB MODE OF ArnON OF STREPTOMYCIN is not entirely clear. Like penicillin,
its effect appears to depend on blocking certain phases of aerobic respiration
or synthesis. Both streptomycin and dihydrostreptomycin are much less active
anaerobically than aerobically. Both are mainly bactericidal in their action,
rather than bacteriostatic.
No enzyme such as penicillinase has been reported, but resistance (drug-
fastness) of organisms may be induced and, unfortunately, often occurs in
tubercle bacilli in patients during treatment. Strains of tubercle bacilli wholly
dependent on streptomycin are also found in treated patients.
0I0ramphea1c:0l. The organism producing chloramphenicol is much like
other species of Sireptomyces. It was found in 1947 by Burkholder in soil col-
lected near Caracas, Venezuela, and is called S. venezuelae. The drug is a by-
product of growth quite analogous to streptomycin, but it is inhibitory to a
much wider range of organisms (except tubercle bacilli). It may be given either
by mouth or intravenously.
It affects many species of gram-positive and gram-negative bacteria in
high dilutions. Unlike either penicillin or streptomycin, it is effective against
the largest viruses, such as those of lymphogranuloma, "parrot fever" (psitta-
cosis) and trachoma; and also against rickettsiae, such as those causing Q
hr, the typhus fevers, and Rocky Mountain spotted fever. It is the first
antibiotic discovered to have definite viricidal and rickettsicidal properties. It
©
CH,'\../OIAI
CH,-N-CH,
I
I
H_/7''''/o~,!', /'~-OH
H_I: II' I I/OH :tC-NH,
':/~:/'y'~l/ II
OH II
o
I
OR
"
0
0
Fig. 20-10. Tetracycline group formula. For explanation see text.
has proven of inestimable value in the treatment of many infectious diseases.

Because of its wide range (spectrum) of activity, including bacteria, viruses
and rickettsiae, it is classed as a broad-spectrum antibiotic. It is bacteriostatic
in action.
The therapeutic excellence of chloramphenicol led to intensive chemical
studies, as in the case of penicillin. The formula was soon learned, and the
drug synthesized (Chloromycetin) in 1949 in the laboratories of Parke, Davis
and Company.
Chloramphenicol must be used only under medical supervision.
The Tetracyclines. This group includes three antibiotics: tetracyclinq,
(Achromycin) , chlortetracycline (Aureomycin) and oxytetracycline (Terra-
mycin). These have similar molecular structures based on tetracycline which
is shown in Figure 20- IO.
Chlortetracycline differs in having a chlorine atom attached to carbon
atom No.7 (encircled). Oxytetracycline has an hydroxyl group attached to
carbon atom No.5 (encircled). All have similar antibiotic properties. They are
bacteriostatic in effect. They are effective against many gram-negative and
gram-positive species of bacteria, some viruses and some rickettsiae. For this
reason, along with chloramphenicol, erythromycin and carbomycin, they are
called "broad spectrum" antibiotics and have similar ranges of therapeutic
activity. However, in any given patient, anyone of these drugs may at times
show surprising irregularities and give unexpectedly brilliant results or fail.
They are not identical.
1
ANTIBIOTIC~ FROM THE GENUS BACILLUS ule
Several useful antibiotics have been derived from gram-positivCl &robic,
spore-forming bacteria closely reiated to, or identical with, Bacillus subtiUs.
/ One of these antibiotics is bacitracin; discovered by F. L. Meleney.
Bacitracin Was first described in 1945. The'source organism is a particular
strain of Bacillus sub~ilis, a common and harmless organism widely distributed
in dust. It was found contaminating a wound in a patient na,med Tracy; hence
baci-tracin. Bacitracin is a yellowish powder readily 'soluble in water. Like
penicillin it is active clinically mainly against infections due to gram-positive
bacteria. However, it also affects the gram-negative meningococcus (cause of
meningitis), gonococcus (cause of gonorrhea) and Hemophilus injiuenzae, as
well as Treponema pallMum (cause of syphilis). Unlike penicillin, streptomycin,
I chloramphenicol and the tetracyclines, it is best used only for topical applica-
tions, although it has been used internally in pneumonia.
Destruction, Removal atld Inhibition of Microorganisms 295
Bacitracin is representative of a number of polypeptide antibiotics with
similar properties, derived from other species of the genus Bacillus. Among
these are polymyxin B (B. polymyxa); bacillomycin (B. subtilis); biocerin (B
cereus); and circulin (B. circulans). They are mainly bactericidal in action.
NON-MEDICAL USES OF ANTIBIOTICS
Industrial Uses. In Chapter 44 some industrial uses of antibiotics are dis-
cussed. Here we need only point out that some of the antibiotics not suitable
for medical use (as well as useful drugs) are finding uses as preventives of
microbial spoilage of industrial commodities: paper, leather, etc. The use of
chlortetracycline as a preservative of dressed poultry for retail was approved
by Federal authorities on November 30, 1955. Doubtless other foods will be
preserved with antibiotics. Some antibiotics are useful in research and special
work of various sorts such as isolation of viruses and rickettsiae (see Chapter
5). Several are very useful in controlling diseases of commercially valuable
plants: fruit trees, crops, and so on. Medicinal uses are only one aspect of
the field of antibiotics.
Antibiotics as Growth Stimulants. Une of the most interesting and unex-
pected developments in connection with antibiotics has been the discovery
that a number of them greatly enhance the rate of growth and well-being of
chickens, pigs, turkeys, and rats. They are of no benefit to ruminants because
of the difference in anatomy and physiology. The tetracyclines, penicillin,
streptomycin and chloramphenicol, all exhibit this property. They are being
used in commercial stock feeds.
The exact mechanism of growth stimulation is not clear. It might be due to
suppression of growth of bacterial parasites in the intestinal t,ract, but this is
not finally proven. The usual dosage is below such inhibitory levels.
An undesirable result of feeding antibiotics to stock is the development of
drug-fast strains of bacteria capable of producing drug-resistant infections of
man and animals, thus offsetting the beneficial effects of feeding the drugs to
farm animals. However, difficulties arising from this source appear to be rare.
In 1952 it was observed that certain antibiotics have a marked stimulating
effect on the growth of plants and plant tissues. The importance of this to
agriculture, horticulture, etc., can scarcely be estimated. Such studies should
stimulate the graduate student in the fields of botany and zoology as well as
in microbiology.
Antibiotics for Plant Diseases. The stock and poultry raiser is not the only
beneficiary of the ,research on antibiotics. Bacterial and fungal diseases of
plants which, as recently as 1956, bid fair to wipe out pear growing, chrysan-
themum propagatipn and other valuable plant industries, now yield to anti-
biotics. StreptomyCin, the tetracyclines, chloramphenicol and others, but espe-
cially the first because of its low cost, .are being marketed to control the
microbial pests. Fireblight, a devasting bacterial scourge of pomaceous fruits
(pears, apples, etc.), is controlled with sprays of streptomycin and oxytetra-
cycline. Bacterial blight of beans can be controlled very well by spraying with
streptomycin solution. Other antibiotics (helixin, griseofulvin, etc.) of no
medicinal value, llre very effective in such diseases as gray mold of lettuce
seedlings, early blight of tomatoes, and so on. The antibiotics are in some
cases applied to the surface of plants as sprays; in others, cuttings are dipped
into antibiotic solutions; in others the roots are immersed.
Results differ, depending on rate of penetration of the antibiotic into the
plant (which in turn depends on the kind of antibiotic and species of plant),
the microorganism involved, and other factors. It is puzzling to find that some
antibiotics having no action against certain microorganisms when tested in
vitro, are very effective against those same microorganisms in certain plants.
REFERENCES
Alcorn, S. W., and Ark, P. A.: Movement of certain antibiotics in cuttings of Pyracantha
and carnation. App!. Micr., 1956,4:126.
Anonymous: A rapid method for the determination of microbial susceptibility to antibi-
otics. Dept. of Microbiology. U.S.A.F. School of Aviation Medicine, Randolph Field,
Texas, 1955.
Arnstein, H. R. V., and Grant, T. P.: The metabolism of the penicillia in relation to peni-
cil1in biosynthesis. Bact. Rev., 1956, 20 :133.
Boyd, J. W., Bluhm, H. M., Muirhead, C. R., and Tarr, H. L. A.: Use of antibiotics for the
preservation of fish and sea foods. Am. J. Pub. Health, 1956,46:1531.
Collier, H. O. J.: Chemotherapy of Infections. John Wiley and Sons, New York, 1954.
Collins, A. M., Craig, G., Zaiman, E., and Roy, T. E.: A comparison between disk-plate
and tube-dilution methods for antibiotic sensitivity testing of bacteria. Can. J. Pub.
Health, 1954,45:430.
Durbin, C. G.: Antibiotics in food preservation. Am. J. Pub. Health, 1956,46:1306.
Eagle, H., and Saz, A. K.: Antibiotics. Ann. Rev. Micr., 1955,9:173.
Eagle, H., Levy, M., and Fleischman, R.: The effect of the pH of the medium on the anti-
bacterial action of Penicillin, Streptomycin, Chloramphenicol, Terramycin, and Baci-
tracin. Antibi. and Chemoth., 1952, 2 :563.
Editorial: Antibiotics "against neoplasms. Brit. Med. J., 1954, Feb. 6.
Editorial: Antibiotics and growth. Brit. Med. J., 1954, Sept. 11,635.
Editorial: Chlortetracycline, a food preservative. J.A.M.A., 1956,160:779.
Editorial: Modem antibacterial agents. J.A.M.A., 1955,159:1458.
Elek, S. D., and Hilson, G. R. F.: Combined agar diffusion and replica plating techniques
in the study of antibacterial substances. J. Clin. Path., 1954, 7:37.
Gottlieb, D., Carter, H. E., Legator, M., and Gallicchio, V.: The biosynthesis of chloram-
phenicol. J. Bact., 1954,68:243.
Grove, D. C., and Randall, W. A.: Assay Methods of Antibiotics. Medical Encyclopedia,
Inc., New York, 1956.
Hamilton, J. W., Szkolnik, W., and Sondheimer, E.: Systemic control of cherry leaf spot
fungus by foliar sprays of actidione derivatives. Science, 1956, 123:1175.
Hartman, F. W., Horsfall, F. L., and Kidd, J. G.: Dynamics of Virus and Rickettsial Infec-
tions. Blakiston Division, McGraw-HilI Book Co., New York, 1954.
Heilman, F. R.: Antibiotics. Ann. Rev. Microbio!., 1953, 7:219.
Karel, L., and Roach, E. S.: A Dictionary of Antibiosis. Columbia University Press, New
York, 1951.
Kirby, W. M. M.: Antibiotics. Ann. Rev. Microbio!., 1952, 6:387.
Ley, H. L., Jr., and Smadel, J. E.: Antibiotic therapy of rickettsial diseases. Antibiotics and
/ Chemoth., 1954,4:792.
Martin, G. J.: Biological Antagonism. The Blakiston Co., Philadelphia, 1953.
Newton, B. A.: The properties and mode of action of the polymyxins. Bact. Rev., 1956,
20:14.
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Pramer, D.: Antibiotics against plant disease. Sci. Am., 1955,192:82.
Schatten, W. E., and Parker, R. F.: Evaluation of agar dilution method for determination
of sensitivity of bacteria to antibiotics. Proc. Soc. Exp. Bio!. and Med., 1953, 83 :574.
Spaulding, S. H., and Anderson, T. G.: Selection of antimicrobial agents by laboratory
means. J.A.M.A., 1951, 147-:1336.
pmbreit, W. W.: Mechanisms of antibacterial action. Ann. Rev. Micr., 1954, 8:167.
Destruction, Removal and Inhibition 0/ Microorganisms 297
Waksman, S. A.: Associative and antagonistic effects of microorganisms. Soil Sci., 1937,
43:51.
Waksman, S. A., Editor: Perspectives and Horizons in Microbiology. Rutgers Univ. Press.
New Brunswick, N. J., 1955.
Waksman, S. A., and Lechevalier, H. A.: Guide to the Classification and Identification of
the Actinomycetes and Their Antibiotics. Williams & Wilkins Co., Baltimore, Md.,
1953.
Welch, H., and Ibanez, F. M., Editors: Antibiotics Annual, 1955-1956. Medical Encyclo-
pedia, Inc., New York, N. Y.
Welch, H., et aI.: Principles and Practice of Antibiotic Therapy. Medical Encyclopedia, Inc.,
New York, 1954.
Wiseman, R. F., Sarles, W. B., Benton, D. A., Harper, A. E., and Elvehjem, C. A.: Effects
of dietary antibiotics upon numbers and kinds of intestinal bacteria in chicks. J. Bact.,
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Wyss, 0., et al.: Symposium on the mode of action of antibiotics. Bact. Rev., 1953, 17:17.
SECTION 3
Immunology: Reaction of the
Organism to Substances of
Extraneous Origin
THE TERM Immunology, used here largely because of tradition, and for
convenience, was derived from studies on resistance to disease made more than
a century ago. The modern concept of Immunology is that of a science having
fundamental, biological significance which extends well beyond me~ical
horizons. One might substitute for Immunology a phrase such as: Study of the
reactions between tissue cells of living macroorganisms (especially verte-
brates) and other organisms, especially (but not exclusively) microorganisms,
living or dead, cellular or non-cellular, including parts and metabolic products
thereof. This phrase is not only awkward but inadequate. Indeed, it would be
difficult to define Immunology in a single sentence. One may more easily gain
a true meaning of the term by reading this section.
Discussions of infection and disease are introduced in this section.not only
because they were originally the basis for the development of our knowledge
of immunology, but also because they convey valuable practical information
which will be useful to anyone as soon as he acquires it.
299
21
Immunology and Microbiology
1. PRINCIPLES AND METHODS
IMMUNOLOGY DERIVES its name from an early concept that the reac~
tions of animal tissue to substances extraneous to them were all mechanisms
of defense against infectious disease, with little other use or significance. It is
important for the student to realize that modern concepts of immunology
embrace a much wider range of ideas, including many fundamental physical,
chemical and physiological phenomena. Disease, and defense against it, are
only a segment of modern immunology, though certainly a very considerable
one. Further, modern methods of immunology are so widely used as labora-
tory tools in sO,many fields of work, and are based on such fundamental
biological principles, that one must have a clear grasp of them before a
detailed study of microbiology will prove profitable.
BLOOD
Before discussing the nature of immunity, and methods for its study, it will
be advisable to devote a little space to a description of blood and its con-
stituents, since blood is a very important' factor in immunological reactions
and is much used in immunological investigations.
IMPORTANT BLOOD CONSTITUENTS
1. Plasma. For the purposes of this discussion, blood may be considered
to have seven important constituent parts. First, there is the plasma; the
yellowish, transparent, fluid part of the blood. It consists of a solution of
proteins, salts, buffets, and other soluble substances including food for, and
wastes from, the body cells. The plasma has in solu!ion, also, the ~ubstances
necessary to the formation of a clot.
'2. Fibrin Components. The material responsible for clotting of blood is a
complex, elastic prqtein called fibrin. Fibrin forms a microscopically fine,
fibrous network in the blood after the blood leaves the body. This is seen as
clotting. The fibrin meshwork soon shrinks to about half the original volume
of the blood, squeezing out of its meshes the fluid portion of the blood, much
as a wet, contractile~sponge would exude water. Most of the blood cells (and
bacteria if any are present) are caught and held in the fibrin clot (Fig. 21-1).
301
302 lmmunology
Fig.21-1. Tube with clot (A) which

has shrunken, enmeshing virtually all of
the blood corpuscles and exuding clear
serum (B).
3. Platelets. Associated with fibrin production are small bodies called

p/clfeiets, or thrombocytes (thrombus is from a Greek root meaning clot).
These are amorphous particles, variable in size, smaller than red corpuscles,
deeply staining, which tend to form stellate groups. They may be cells or
possibly fragments of marrow cells. Their role in coagulation of blood is
important though not entirely clear. Platelets appear to facilitate clotting.
The ability of blood to clot appears to be directly related to the number of
platelets in the blood.
4. Serum. The fluid portion of blood, after it is separated from the fibrin,
is called serum. Serum is essentially plasma minus the components of fibrin.
Since fibrin usually enmeshes the blood corpuscles, the remaining serum is
yellowish and transparent, like plasma. Serum contains most of the soluble
substances, especially gamma globulins, in which the immunologist is inter-
ested. After blood has clotted the serum may be withdrawn in pipettes, centri-
fuged to remove stray red blood corpuscles, and stored in the refrigerator. It
must be handled with every precaution to keep it sterile, for microorganisms
gaining access to it would soon ca1_.lse it to decompose. Immunologists often
add minute quantities of preservatives.
5. Lymph. Lymph is very much like blood which has been deprived of
red corpuscles by passingJhroug4 the thin walls of the smlilller blood vessels
/ as through a fine filter.,One sort of white blood corpuscles (lymphocytes) are
found in it. The fluid thus seeping out of the vessels travels slowly in Hie fine
spaces surrounding the'blood vessels, and between the tissue cells and various
organs. It eventually collects from all parts of the body in large drainage
vessels (the lymphatic ducts) and is returned to the blood stream.
6. Erythrocytes. In the plasma, before clotting, are suspended the red
corpuscles or erythrocytes (erythro is from the Greek word for red; cyte from
the Greek word for cell). These are non-nucleated * cells which give to blood
! '
• • Mammalian erythrocytes are non-nucleated. In birds, amphibians and reptiles they are
nucleated.
Immunology and Microbiology 303
its opacity and red color and which carry oxygen from the lungs to the tissues.
The color of erythrocytes is due to the red, oxygen-carrying substance, hemo-
globin, which they contain.
Erythrocytes are about 8 J.I in diameter and number about 4 to 5 millions
per cubic rom of blood (Fig. 21-2). When they are ruptured by plasmoptysis or
other means, the hemoglobin is released and the blood becomes transparent,
like red ink. The cells are said to have been "laked" or hemolyzed. Certain
bacteria are very active in producing hemolysis by means of toxins. Some
snake venoms are very hemolytic.
7. Leukocytes. In addition to erythrocytes, blood contains colorless,
ameba-like cells called leukocytes (leuko is from the Greek word for white)
which, in some cases, act as phagocytes (phago is from the Greek word for
eat). Leukocytes are relatively large cells, 10 to 20 J.I in diameter, which have
a definite nucleus and a means of locomotion like amebae. They normally
number about 7 to 8 thousand per cubic rom of blood (Fig. 21-2). In many
infectious processes, such as appendicitis, they greatly increase in numbers
and the patient is said to have a leukocytosis. They act as scavengers and
"policemen" in the blood.
Like amebae, leukocytes can ingest solid particles and, in the body, they
serve by ingesting and destroying any small foreign particles such as bacteria,
dead body cells, tissue detritus and the like, that may gain entrance to the
tissues or blood stream (Fig. 21-3). The leukocytes, by their ameboid mo·
bility, can leave the blood vessels and congregate in the tissues wherever any
irritation exists.
IMMUNOLOGY IN RELATION TO DISEASE
Defensive Mechanisms. The most important defensive mechanisms of the
body against microorganisms and infectious disease may be divided into
several categories as follows: .
I. Non-specific Resistance (not directed against any specific disease but against infec-
tion in general).
A. Genetic
B. Physiological
I. State of general health, age, etc.
2. Mechanical and chemical
3. Phagocytosis
Fig. 21-2. Drawing of a smear of

blood stained with Jenner's stain, show-
ing common forms of blood cells. A, B,
C, polymorphonuclear'leukocytes with
2, 3, and 4-lobed nuclei, respectively. D,
eosinophil, showing lo~ular nucleus and
prominent, eosinophilic (red-staining)
granules. E, lymphocyte. F, G, H, vari-
ous forms of monocytes ("large lympho-
cytes"). I, lymphocyte with horseshoe-
shaped nucleus ("transitional cell"). J,
erythrocytes (red blood/corpuscles); note
biconcave-ljisk shape, thin at center. K,
platelets. (r.1agnification about X 1000.)
304
Fig. 21-3. Blectronopaph ot an ultra-thin (around 3soA) MICtion of a part of a human

polymorphonuclear leukocyte showing phagocytosis of virulent Micrococcus pyogenes VIU.
aureus. N is part of one lobe of the nucleus of the leukocyte; note the well-defined nuclear
membrane. B are cells of M. pyogenes; note that one bacterium is undergoing fission. The
bacteria at the upper right are just being surrounded or grasped by the finger-like pseudo-
podia of the leukocyte. The bacteria will be "ingested" or drawn completely inside the
leukocyte. They will be surrounded by a vacuole wall such as is seen to be fonning around
the bacterium at the left of center. Note the granular structure of the cytoplasm of the
leukocyte and its differentiation from the nuclear plasm. P is a platelet; R is part of a red
cell. Note the line indicating 1 micron. (X 21,500.) (Courtesy of Drs. J. R . Goodman and
R. E. Moore, V. A. Hospital, Long Beach, Calif.)
4. Tissue immunity
S. Properdin system
/ D. Specifk ResistaDc:e (developed by, and directed against, specific, infectious diseases).
A. Actively acquired
1. Natural (due to recovery from infectious diseas,e)
2. Artificial (due to vaccination, injection of bacterins, etc., Chapter 22)
B. PasSively acquired
1. Natural (due to passage of maternal antibodies into fetus).
2. Artificial (due to injection of antibody-containing serum).
C. Allergic (Chapter 23).
I. NON-SPECIFIC REs1srANCE
A. Genetic. By this is meant racial or species resistance to disease, illus-
trated by the facts that horses do not have measles, man does not contract
fowl pox, etc. The reasons for this type of resistance are not always clear.
They are probably quite complex, and differ in different species. For example,
"cold-blooded" (poikilothermic) animals, and birds (normal temperatures
around 104° F), are not susceptible to microorganisms which grow only at
mammalian temperatures (normal temperatures around 98° F). However, as
Pasteur proved in a very dramatic demonstration, chickens will die of an-
thrax (to which they are ordinarily resistant) if they are infected and then
cooled by partial immersion in ice water.
B. Physiological. 1. STATE OF HEALTH AND AGE. Persons weakened by
long overwork, starvation, exposure, age, disease, etc., are well known to
become more susceptible than they are normaUy, to various infections such
as tuberculosis, pneumonia, etc. Age is also an important factor. Children
often suITer little from diseases fatal to older persons, and vice versa.
2. MECHANICAL AND CHEMICAL. These mechanisms are of a general
nl\ture, and otTer protection against a variety of injurious agents.
rrhe outer skin, especially of adults, is an obvious mechanical barrier
to the entrance of foreign agents. It is chemically aided (against microorgan-
isms) by its oily secretion and the acidity of perspiration. Acetic acid in
perspiration is quite toxic to many bacteria. The skin may be by-passed by
hypodermic needles, wounds, and by entrance of certain microorganisms via
hair follicles and sweat glands.
The hairs in the ears and nose and the mucus on mucus-secreting mem-
branes like those in eyes, nose and throat, mechanically entangle or enmesh
particles of dust, insects, bacteria, etc. Secretions of mucus cause all of the
respiratory and other surfaces, in contact with the exterior, to be stjcky.
Foreign bodies accumulate in the mucus. Removal to the exterior is then ac-
complished by sneezing, coughing, salivation, tears, etc. The deeper air
ptmages are lined with ciliated epithelial (surface) cells. The cilia of these cells
maintain a constant upward-waving movement which pushes mucus (with
entrapped bacteria, dust, etc.) up to the larynx and throat where the mucus is
either coughed up or swallowed. Further, these mucous surfaces are always
··policed" by small numbers of leukocytes.
[II the gastroinOtestinal tract, acidity of the stomach kills many swallowed
microorganisms. The upper intestine is freed of microorganisms to a great
CIIltent by the bile and other digestive juices. The lower smaU intestine and
IafIe bowel contain great numbers of bacteria, many of which are highly
pathogenic if they gain entrance to the blood or body tissues. They are
aormally held in check by the thick mucous membranes lining the intestines
by and other mechanisms. A ruptured intestine, stomach or
,8II'PC·ndllx is a source of serious infection which, untreated, is usually fatal.
adult genitourinary tract is protected against most bacteria mainly by
mucous membranes, leukocytes, and the flow of urine, which is normally
PHAGOCYTOSrs. Centers of inflammation such as the bacteria in a

of wood or other foreign particle, exert a strong attraction
Ilmlm~'5I.~ especially UQlymorphonuclear leukocytes. These cells rapidly
lIlII:eoltra1te in such areas. The nature ofThis a ractiOniSnot fully known. It
chemotaxis (chemo is from the Greek w~rd for chemical; taxis is
the Greek word for orientation toward). The white cells try to dispose
306
of the foreign particles by ingesting them and also by giving off enzymes
which will kill and destroy or digest them if possible. Often many leukocytes
are in turn killed by poisons of the bacteria they ingest. and also by crowding
and lack of oxygen in the region of concentration and activity.
The white, creamy material in a boil or other infected lesion, or around a
"festering" splinter, is made up largely of dead and living white corpuscles,
dead and living bacteria, tissue debris, lymph, fibrin and serum. It is called
pus and the dead leukocytes in it are called pus cells.
I TYPES OF PHAGOCYTES. Phagocytic cells are of two main types: ed and
wandering. The fixed phagocytes are found "built into" the linings 0 the
I blOM vessels, especially in the liver, spleen and bone marrow. They are often
called histiocytes (histio is from the Greek word for tissue). They take foreign
particles from the blood as it streams by. They are part of what is called the
reticula-endothelial system of phagocytes.
The wandering pbagocytes are the true white corpuscles or leukocytes,
escribed above, which floatfree in the blood stream.
PHAGOCYTOSIS A FIRST LINE OF DEFENSE. The leukocytes and other phago-
cytic cells constitute an extremely important mechanism of defense against
microorganisms and other foreign particles which may gain entrance to the
blood and/or tissues.
CELLULAR AND HUMOIlAL IMMUNITY. The relation of phagocytosis to im-
munity was pointed out by Elie Metchnikoff about 1882 and was the origin
of the doctrine of "cellular immum . Itt about the same period specific,
soluble proteins (globulins, notably gamma globulins), called (!!!tibodies, were
discovered by Buchner, von Behring and others, in the serum ons and
animals who ba 'vaccinated" or who had recovered from specific dis-
eases. These antibodies were found to exert deleterious action against micro-
organisms. Antibodies were, therefore, hailed as the real basis of immunity to
disease and gave rise to the doctrine of "humoral (fluid) immunity."
PHAGOCYTES AND ANTIBODIES. Long and intensive studies have shown that
both antibodies and pbagocytes are important in immunity. The antibodies,
with one or two possible exceptions (antitoxins and cytolytic antibodies),
appear to act mainly, if not exclusively, as adjuvants to phagocytosis.
Phagocnes are like a standin& arllU'_; ready at all tiJnIUor immediate duty.
They are a first line of defense. Antibodies, on the contrary, are a physio-
logical response to a stimuTus and appear oBly hours, days or weelcs after an
infection has bej_un. Obviously they can be of only seconda!1 though usually
critical) istance.
ANTIBODIES AND SURFACE PHAGOCYTOSIS. The work of phagocytes is
I greatly facilitated by several factors, of which two important ones are:
(a) presence of antibodies; (b) presence of surfaces against which phagocytes
can hold or entrap bacteria in order to phagocytize them. .
a. Presence of Antibodies. Many microorganisms, especially pathogenic
species, have slimy surfaces which make it difficult for the leukocytes to
grasp them with their pseudopodia preparatory to engulfing them. This is
true especially if the microorganisms are floating freely in a fluid like blood.
One may visualize the situation by trying to grasp wet watermelon seeds,
especially when they are floating in water. Antibodies prepare the surfaces
Immunology IIIIIl Microbiology 307 •
of bacteria, especially those which are coated with slippery, slimy capsules,
so that the bacteria are sticky and do not readily slip out of the grasp of the
phagocytes. This preparation of the bacteria to be eaten more readily by the
hagocytes is often referred to as opsonization (opsonin is from the Greek word
meanmg""to prepare food for;.
b. Presence of Surfaces. If the phagocytes can get the bacteria "on the
ropes," to use a prize-fighter's term, that is, against a surface where the
organisms cannot back away, the phagocytes can grasp the bacteria much
more effectively. The surfaces of other tissue cells or of any uneven or rough
surface, or strands of fibrin, serve the purpose for the phagocytes very well.
Thus, by means of surface phagocytosis, even in the absence of antibodies,
the phagocytes can take up fully encapsulated bacteria very effectively.
Phagocytosis is, therefore, not dependent on antibodies. Doubtless anti-
bodies, if present, enhance the effectiveness of surface phagocytosis (Figs.
21-4 and 21-5).
4. TIssUE IMMuNITY. Infection under natural conditions brings the in-
fecting microorganism immediately into contact with the living tissue cells
of the host. Ordinarily these tissue cells, aided by phagocytes, localize and
destroy the invader. The reaction is manifested as a local inflammation
(defensive struggle), visible or imperceptible. This disappears with successful
repulse of the invader.
But suppose the local tissues and phagocytes, while holding some, are
unable to cope with all of the invaders. Some escape into the blood stream
or lymph channels. "Blood poisoning" or bacteriemia (bacteria in the blood
stream) is present. Let us suppose that the phagocytes in the blood and
tissues cannot immediately control these invaders either. The inflammatory
reaction at the site of the initial infection, and wherever else the invading
organisms may have localized, becomes more intense.
Fia. 2l-4. Phagocytes surrounded by thousands of virulent pneumococci (Diplococcus

".lI1ItOIIiae), most frequent cause of lobar pneumonia. Note that nolll! of the bacteria is
..... of the phagocytes. The phagocytes have been unable to engulf the microorpnisms
t.:au.e all are lying on a smooth, wet, slippery glass surface on which the phagocytes can
. . DO "purchase" or foothold which will enable them to brace than8elws to hold the
pIIIUIIIO(lOCCi to iDaeat them. Compare with Fiaure 21-5. (Courtesy of Dr. W. B. Wood,
Jr.. IIIId M. R. Smith, Washington University School of Medicine, St. Louis, Mo.)
• 308 Immunology
Fig. 21-5. A mixture ot JJ. pneumoniae and pbagocytes similar to that seen in Figure
21-4 except that in this situation there are tissue surfaces, angles, comers, fibrin shreds,
irregularities and roughnesses where the phagocytes can trap and hold the slippery bacteria,
enabling the phagocytes to ingest them. This phenomenon is caJJed surface phagocytosis.
Note that virtually aU of the bacteria are inside of the phagocytes. (Courtesy of Dr. W. B.
Wood, Jr., and M. R. Smith, Washington University School of Medicine, St. Louis, Mo.)
LOCAUZlNG ACfION OF INFLAMMATORY TISSUE. Now, the inflammatory

tissue (or other reacting tissues) begins to form antibodies probably within a
few minutes or hours after infection. It is probably due to this (in part at
least) that inflammatory tissue has very marked power to localize and hold
the invaders. Specific combinations oa:ur between tissue cells and organisms.
Increased phagocytosis of antibody-covered (opsonized) microorganisms, also
occurs. If the local tissues, antibodies and phagocytes can then hold and
destroy the invader, the infection is suppressed. If not, then the victim may
succumb.
5. THE PRoPERDIN SYSTEM. This is a group of serum components which, to-
gether, apparently play an important role in primary, non-specific resistance
to infection. There are three components in the system: (a) properdin, a serum
protein; (b) magnesium ions; (c) complement, a complex of serum proteins
(C'l, C'2, C'3, C'4) which are very important in complementing the action of
specific antibodies. Complement is discussed more fully later on. .
Properdin itself is an enzyme-Iik~.nt. It appears to be closely related to,
if not an actual part of, the complement complex. The amount of properdin
in the blood seems to be directly related to the degree of non-specific resistance
to numerous types of infection: bacterial, protozoal and viral. Injection of
properdin-rich serum from animals having naturally high levels of properdin
in their serum increases resistance to infection. Injection of certain carbo-
/ hydrate complexes (zymosan) found in the cell walls of certain bacteria and
yeasts greatly reduces properdin levels and, with them, resistance to infection.
Electromagnetic irradiations and infection with certain bacteria also lower
properdin levels.
In order for properdin to affect microorgani&ms it is necessary for Mg++ to
be present. This is also true of ~emen Complement, also, is necessary
to the action of properdin. The exact mechanism of the action of the properdin
system is not yet fully understood. Indeed, it seems to be so sadly true that
the more we learn the less we understand. Shall we eventually know every-
thing and understand nothing?
II. SPECIFIC REsIsTANCE
A. Active Immunity
I. Natural Active Immimity. ANTIGENS. After recovery from many dis-
eases such as Rocky Mountain spotted fever (rickettsial), measles (viral),
diphtheria, whooping cough, typhoid fever (bacterial), the patient is so
changed that he may never again, under ordinary circumstances, acquire the
specific disease. He is said to possess natural, active immunity to the specified
infection. This is because agents of infectious disease contain certain irritating
foreign- substances which stimulate the production of antibodies. Such anti-
body-generating subs~ are cane<T antIgens.
The irritation (or disease) caused by tlle antigens may be so mild (chronic
or subacute or subclinical) as to pass unnoticed by the individual. On the
other hand, the reaction may be rapid and violent (acute), with high fever.
In any case, upon making proper tests, the patient's blood is found to contain
protein substances called antibodies, specific for the antigens of the infecting
microorganism. These antibodies neutralize the toxins or unfavorably affect
the microorganisms, or both.
orten antibodies continue to be produced by the body cells long after the
stimulus which excited their production disappears. It is in part to this that
prolonged immunity, such as follows mumps, measles, and diphtheria, is due.
Nature of ADtigeas. In general, the most important antigenic substances
are soluble polysaccharides and soluble proteins from any outside source,
harmless or not: microorganisms, animal blood, egg, milk, serum or tissues,
plant juices, etc.
All of these polysaccharide and protein substances act as ll]ltig_ens .. i.e.,
all stimulate production of antibodies. They appear to enter into and/or
irritate sue cells whicTi prodUce the gamma globulinS of the blood plasma.
The globulins produced in response to any given antigen almost exactly
resemble that particular antigen structurally and chemically. These globulins
are called antibodies because they tend to neutralize, inactivate, or destroy
antigens.
SPECIFICITY. The structural reseDlUlance Detween an antigen and the
specific antibody which is produced to combat it is analogous to that between
a lock and its specific key or between type and print.
Thus, the s~ially-designed antibody &lobulins can combine and react -
only with their own antigen and no other. A similar relationship is seen in
that .between an enzyme and its substrate. The relationship in both cases is
spoken of as specificity.
The action of the antigen in impressing its physicochemical pattern on the
• For the purposes of this discussion, the interior of the gastrointestinal tract is considered
external to the body proper, as it is open to tho outer world at &ath ends and has no direct
CODDOCtion with the principal body cavities or tissues. We shall, therefore, consider as
fordgrJ substances only those which get into the body parenterally (i.e., otherwise than
tbrouah the gastrointestinal tract). This includes, generally, infecting micio- or macroorgan-
isms, substances or bodies injected into the tissues with hypodermic needles, or introduced
by means of splinters or wounds, or forced into the peritoneal (abdominal) or pleural (chest)
c:avity, or released or absorbed into the blood or lymph from mucus and othor surfaces, or
in any situation where they escape the diaestive enzymes of the mouth, stomach or intestines.
310 Immunology
globulin-forming cells is not fully understood. Since the globulin-forming
cells die and are replaced, the successors presumably inherit the effects of the
antigen. Or can it be that the antigen enters their genetic mechanism in
somewhat the same relationship as prophage? Can infection, prophage, sexual
fertilization, antigenicity, transduction, etc., all be different "variations on a
theme?" These are merely hypothetical questions to stir up argument!
NATURE OF SPECIFICITY. Specificity (either in enzyme-substrate relation-
( ship or antigen-antibody relationship), is a matter of chemical and physical
structure. For example, a "synthetic protein" antigen (of which several have
been prepared experimentally by combining protein with certain inorganic
radicles) having a given chemical structure (e.g., NH2C6H4 . As03H2 + pro-
tein) will, upon injection into the body, engender antibodies in the serum
which react only with that compound. Let us alter the antigen, say by substi-
tuting a -S03H group in place of the -As03H2 group. Antibodies to the
first antigen will not react with this altered antigen.
Almost any sort of chemical alteration in a protein will alter its specificity.
A slight reaction may occur if, instead of substituting a -S03H group, we
introduce, say, a -CI atom in place of the -NH2 group. The antibodies
produced in response to the original antigen are said to cross-react with the
chlorinated antigen. Many cross-reactions occur between closely related
antigens. In a natural example, serum of an animal injected with horse blood
will react strongly with horse blood and, to some extent, with mule blood;
but not at all with chicken blood. The biological relationship is close between
horse and mule, but not between horse and bird.
Antibodies and Adaptive Enzymes. While not absolutely alike in every
detail, antibody formation and adaptive enzyme formation are remarkably
similar phenomena. In each type of response the cells react to a chemical
stimulus by forming a complex, protein substance, the molecules of which
are so constructed as to combine and interact specifically· with the chemical
agent which stimulated their production. When the stimulus is not poisonous,
and/or is nutrient in character, we say that an adaptive enzyme has been
produced. Antibodies are not all enzymes, but they are very enzyme-like,
especially if we regard complement as a sort of kinase or enzyme-activator.
ANTIGEN-ANTIBODY REACfIONS
When mixed in a test tube, the antigen and antibody colloids (if they are
specific for each other), coming into contact, orient themselves with respect
to their positive and negative charges so that an absolute "fit" is obtained.
/ Thus. we may imagine an antigen molecule or particle (Ag) to be represented
hy t he figure :
:~::I
• There are some minor deviations from strict specificity in adaptive enzyme formation;
hut there are al~o some in antigen-antibody relationships.
its corresponding antibody (Ab) by the figure:
+~
.[i_
and the antigen-antibody combination by the figure:
'm'
.
A. ...
.
The compound colloidal particle, formed as a result of the interaction be-
tween these two would be electrically neutral and very large. As a result, it
would become unstable in solution and would therefore go out of solution.
In a test tube reaction, it would become visible as a cloudy precipitate (Fig.
21-6). This sort of reaction is commonly seen when antibody reacts with a
-
soluble protein or carbohydrate antigen. It is called a "precipitin reaction." A
weak cross-precipitation might occur between antigen Ag and some antibody
(x) closely similar to Ab, but not exactly like it:
ro
. '" .
Stability of Antigen-Antibody Combinations. A few additional points con-
cerning antigen-antibody reactions are worthy of mention at this point.
1. TYPE OF UNION. The union of the twu is firm but not necessarily irre-
versible since they may often be separated by certain procedures. The com-
bination is principally a physical one, depending mainly on adsorptiorr.
2. ANTIGEN-ANTIBODY REAcnONS OCCUR AT SURFACES. Surface st~uc
tures are in great part the determining factors in antigen-antibody reactions.
It is the arrangement of the surfaces of molecules, and of cells, and surface
Fig. 21-6. Precipitin test in narrow-

bore glass tubes. The antigen solution is
the cloudy zone above; the antiserum is
the clear zone below. At the interface be-
tween them, in the two right-hand tubes,
a definite white floc or precipitate has
formed, representing a reaction between
specifically related antigen and antibody.
The same antigen fails to react with a
different (non-specific) serum in the two
left-hand tubes (X S). (Preparation by
Dr. Elaine L. Updyke. Photo courtesy of
the Communicable Disease Center. U. s.
Public Health Service, Atlanta. Georgia.)
312 Immunology
structure and charges of colloids, which dominate reactivity and specificity.
This is of especial importance in relation to cells such as bacteria. Only the
surfaces of cells are of significance in reacting with antibodies since antibodies,
being colloids, cannot gain access to the interior of the intact cell. Change in
chemical or physical structure of the surface of a bacterial cell (for example,
presence or absence of capsule) therefore determines whether or not it will
react with a particular antibody for that cell.
3. ANTIGENS MAY BE INTRACELLULAR. Although only the surfaces of
antigens react with antibodies, the antigens in the interior of complex struc-
tures like bacteria stimulate production of antibodies because the bacteria
are disintegrated in the body and their intracellular proteins, etc., are released.
Thus, a serum may contain a number of antibodies stimulated by a bacterium
but only that which is specific for the bacterial surface antigen can act.
4. ANTIGEN-ANTIBODY REAcnONS OCCUR IN STAGES. The first is com-
bination, presumably due to adsorption (or accumulation) of antibodies on
antigen surfaces. This stage may proceed rapidly at temp!!ratur~s around
37° C. The second stage is visible reaction (precipitation, cell lysis or other
effect). This stage often develops slowly and may be demonstrated best after
twelve to eighteen hours at 4 to 6° C. The presence of electrolytes (MgCI2 ,
NaCl, etc.) and of certain other, enzyme-like components of serum (comple-
ment) (to be described farther on in this chapter), is often necessary for the
second stage.
Factors Influencing Development of Active Immunity. MALNUTRITION.
Faulty nutrition has long been known to lower general health, strength, and
resistance to disease. This is a non-specific factor. Nutrition is thought. by
some to have some effect also on specific immunity although the evidence for
this is generally rather confusing and not conclusive. Some studies indicate
that deficiency of certain vitamins interferes with specific antibody fon:itation.
IRRADIATION. Irradiation with x-rays is well known to lower the number
and/or effectiveness of phagocytic cells in the blood and in the reticuloen-
dotheiial system. It also appears to suppress formation of antibodies. It has
already been mentioned that irradiation lowers properdin levels. Infection
occurring post-irradiation is ther~fore likely to progress.
Bacteria of intestinal origin an/not held in check in irradiated animals so
that fatal bacteriemia by the intestinal hordes is important as a cause of death
following excessive irradiation ..
CORTISONE. This hormone, derived from certain cells forming the
cortex of the adrenal gland, is sometimes used for treatment of certain dis-
I eases of the supporting and connective tissues of the body. These diseases are
sometimes manifested in joints (arthritis). This hormone has a number of
physiological effect~, many of which are little understood, some unknown
and some dangerous. One of these effects is diminution of circulating anti-
bodies. Cortisone appears to suppress antibody formation, both during the
antigenic stimulus and afterward. This may be related to the well-known.
power of cortisone to inhibit or suppress inflammatory reactions, but the
mechanisms involved are obscure.
~tigens in Nature Are Usually Mixed. In Nature, antigens seldom occur
as pure compounds. This greatly increases the difficulties of the study of
antigen-antibody reactions involving such antigen mixtures as bacterial cells.
A bacterial cell may consist of many antigens (cytoplasm, flagella, capsules,
etc.). The serum of a person or animal, following injection with such cells,
contains a mixture of antibodies specific for each separate antigen.
. To complicate matters further, the cells of two different species, whether
closely or distantly related, may contain certain antigens in common so that
antiserum * prepared by injecting cells of one species will react with both
kinds of cell.
For example, three related species of dysentery bacilli may each contain
four antigens as follows:
Soecies I AlBlC/D
Species II C/D/E/F
SPecies III E/F/G/H
Obviously, upon injecting species I into a rabbit, antibodies a, b, c, d, will

be engendered, corresponding to antigens A, B, C, D. Upon injecting species
II into another rabbit, antibodies c, d, e, f, will be called forth. Likewise,
species TIl will stimulate production of antibodies e, f, g, h. Now, the serum
of rabbit I will react best of all with bacterial species I when these bacteria
and serum of rabbit I are brought into contact. Serum II will similarly react
best with species II, and serum III with species III. However, since serum I
contains antibodies c and d, it will cross-react to some extent with cells of
species II, since the latter have these antigenic compounds in common with
species I. There will be no cross-reaction between serum I and antigen_ III,
but serum II will cross-react with species III. Such cross-reactions are com-
monplace.
Antibody Adsorption. If a given volume of serum I (say 5 ml) be mixed
with a heavy suspension of cells of bacterial species II, then antibodies c and
d, and antigens C and D will combine, leaving antibodies a and b still free
in the serum. By means of centrifuging, the bacteria with their attached
antibodies c and d can be removed, leaving serum I free from antibodies
c and d and specific with regard to species I; i.e., the serum will no longer
cross-react with species II and will react only with bacteria containing antigens
A and B. If we further adsorb antibody b by treatment of the serum with some
species having only antigen B in common with species I, then we obtain a pure
A serum. t Many such pure and specific sera are thus prepared and it has been
possible to make very extensive analyses of the antigenic structure not only
of bacteria, but of higher plants and animals, and to detect antigenic relation-
ships hitherto unsuspected.
Even such unr,::lated organisms as type XVI pneumococci and the ery-
throcytes of certain human beings contain antigens common to both. These
are detectable by just such methods as those indicated in the preceding para-
graph. Students of evolution may not be humiliated to learn that their blood
contains antibodies related to proteins in certain fish. The relation of cow to
whale by immunological methods is perhaps an unexpected revelation. While
this may not be 1'0 evident, who can doubt the relation of horse to mule?
I
• Serum containing antibodies.
t Such a serum is said to be monovalent.
314 Immunology
Methods like these are of immense value in unraveling complex evolutionary
lines and species relationships.
Labeled ADtlbodies. ISOTOPFS. One of the most interesting technical
advaqces in the field of immunology is the "labeling" of antibodies so that
their place and activity can be observed. Antibody proteins may be combined
. with radioactive isotopes; for example 1131. Material treated with the radio-
active, specific antibody solution (infected tissues, suspensions of microorgan-
isms, soluble antigens, etc.) takes up the labeled antibody, if there is a specific
antigen present to react with it. By means of a device like a Geiger counter the
occurrence of a specific antibody-antigen combination can be detected and
the location and amount of specific antibody can be determined with a useful
degree of accuracy.
FLUORESCENT· ANTIBODIES. Another exceedingly valuable and important
means of labeling antibodies is to conjugate the antibody protein with a
fluorescent dye (usually fluorescein). Let us suppose a single bacillus of a
certain species is suspected to be present in a large section of tissue from an
infected patient. It is impossible to find the one tiny organism, even though
gram-stained, because the large mass of surrounding tissue also takes the
stain and the bacillus is lost like the needle in the proverbial haystack.
Let us flood the tissue section with specific, fluorescein-labeled antibody.
Fluorescent antibody attaches itself to the single, hidden bacillus. Then we
wash out all of the unattached antibody. When the tissue section is illuminated
with ultraviolet light. the hidden bacillus reveals itself by its brilliant, yellow,
fluorescent light like the,full moon at midnight (Figs. 21-7, 21-8). This method
will eventually acquire an international importance.
ANTIGENIC STRUcruRE OF BACI"ERIAL CELLS

Somatic (0) Antigens. Soma is the Greek word for body. We may think
of the cell wall and its contents as the body or soma of a microorganism.
Somatic anti ens therefore, are those associated with the cell wall or interior
of the cell. They are sometimes very poisonous proteins and are, therefore,
often called ewiotoxins. meaning toxins inside the cell. They are character-
istically unaffected by boiling. .
There are often several kinds of somatic antigens per cell. Somatic antigens
may be quite distinctive ofa given species of cell. As often happens, however.
the same protein may be found in several, closely related species or even in
very remotely related orders or even kingdoms. For this reason. these shared
antigens are often spoken of as group antigens.
/ Somatic antigens are often called 0 antigens. (See also Chapter 16.)
Flagella (H) antigens are localized in the flagella of motile species. In con-
trast to somatic antigen they are destroyed by boiling. They often exist in one
of two different degrees of specificity called phases. In the specific phase, phase
I, they are specific for the species in which they occur. In the less specific or
group phase, phase II, they rese.nble antigens in one or more closely related
species or types.
They appear to vary, often unpredictably, from one phase to the other for
• By fluorescence is meant the property possessed by certain substances of reftectina

rays baviDa a wave length different from tbat of the incident rays.
/l1UIJUIIOlogy and Microbiology 315
Fia- 21-7. UIC of f1uo~ anti-

bodies to locate specific antiIen in mea-
sle8-infecteci, moakey-ltidDey cella. Anti-
bodicI from a measles CCIIlvalellcent have
bcco ~ with a f1~ dye
and applied to the tissue ccIls. Free, ex-
cess dye has bcco washed away. On view-
iDg the tissue cells in the JDicro8cope witb
ultraviolet liabt the antibodies arc clearly
seen by their brilliant, yellow-green f1uo-
rcsceucc (light areas in the pbofOarapb).
These fluorescent areas reprcaent specific
antip-antibody combinatioos. They
arc seen espcciaJly in masses in (or on)
the ccn nuclei and, to a lesser extent, in
the cytoplasm. (X 700.) (Microphoto-
grapb courtesy of Drs. S. M. Cohen, L
Gordon, F. Rapp, J. C. Macaulay and S.
M. Buckley, in Proc. Soc. Exp. BioI. and
Med., 1955, vol. 90.)
Fig. 21-8. Usc of fluorescent antibody to detect 'SpcctbC antigeo-antibody combination

in mixed materials. A shows a sample of soil infcCted with Malleomyces pseudomaJlel, a
dangerous, pathogenic bacterium. A smear of the infected soil. on a slide, has been treated
with fluorescent antibody specific for M. pseudomaJJel. muminatcd by ordinary liabndarlc-
field illumination) (A) ot is impossible (without glancing at B) to distinguish the sinjlc cell
of M. pseudomallel from the many saprophytic soil bacteria and soil particles which arc
present in the sample. In the same specimen, illuminated by ultraviolet light (B), combina-
tion between the fluorescent M. pseudomaJlel antibodies and the single cell of M. pseudo-
mallei present in the mixture is strikingly evident. C shows typhoid bacilli, treated with
fluorescent antibodies specific only for the ftasclla of tbis species. lliuminated by ordinary
light the ftagcIla are not very prominent and there is no distinction in visibility between the
ftaaclla and the bacilli to which they arc attached. In D, illuminated by ultraviolet liabt,
the specificity of the combination of fluorescent H antibody with /ftlgeUa only is clearly
demonstrated. The rapidity and specificity of this method of dctccting pathogenic micro-
orpnisms. or of particular structures in organisms, arc obvious advantages over prolonged,
laborious and uncertain cultural and ordinary staining procedures. (Courtesy of Drs. B. A.
Thomason. M. D. Moody and M. Goldman, in J. Bact., 1956, vol. 72.)
316 Immunology
various reasons, some known, some not. This is spoken of as phase variation.
This is of great importance in identifying certain bacteria, especially certain
intestinal pathogens (notably the genus Salmonella). There may be several
different flagellar antigens in a given organism.
Flagellar antigens are, for reasons previously explained (Chapt. 16), often
called H antigens.
Capsular (K) Antigens. These are usually polysaccharide in composition
and are situated in (or wholly compose) the capsule. In those species which
possess capsules, the capsular substance dominates the surface and often
determines antigenic specificity of a very high order. The localization of an-
tigenic specificity in the capsular substance of such organisms as pneumo-
cocci, influenza bacilli, streptococci and Friedlander's bacillus is a striking
phenomenon. Stripped of this specific surface antigen, the exposed antigens
of the naked cells cross-react very widely with antisera of related and often
unrelated species. For example, the somatic or cellular antigens of all pneu-
mococci are immunologically alike. Antibodies for one react equally well with
all. But the antibodies of the capsules are not all alike and it has been found
that there-are about 75 different very distinct types of pneumococcus capsular
antigens. Each antigen determines a'different type of pneumococcus. Similar
series of capsular types are found in influenza bacilli (Hemophilus injluenzae,
types A, B, C, D, E); of meningococci (Neisseria meningitidis, types I, II,
II alpha) and a number of other species of microorganisms having important
agricultural, industrial and geological relationships.)
Extracellular Antigens. Some of the waste products produced by living
cells are antigeruc. For example, diphtheria and tetanus toxins are waste
proteins excreted extracellularly. Toxic, extracellular antigens like diphtheria
toxin are call~exotoxins. - - _ ~~-. -
TYPES OF ANTIBODY RESPONSE AND REACTION

There are different manifestations of antigen-antibody reaction, some of
them demonstrable in test tubes as visible reactions, some of them not visible
except indirectly by secondary tests. The different manifestations are spoken
of as though different antibodies were involved, but there are probably fewer
antibodies than types of reaction. The kind of reaction seems to depend in
great part on the kind and size of antigen molecule or cell, the physical con-
ditions of the suspending fluids, the presence of electrolytes, and other factors.
For convenience, we shall speak of_ "types of antibody," including in that
term "types of antigen-antibody reactions." Among the best understood types
of antibody or antigen-antibody manifestations are antitoxins, cytolysins,
agglutinins and precipitins. Other antibodies (or' antibody manifestations) to
be considered are so-caUed immobilizing antibodies and the' immune adherence
phenomenon.
Antitoxins. When bacteria gain a foothold in the body and secrete toxin
into the blood, the toxin, being a protein antigen, stimulates the production
of antibody. This antibody neutralizes the toxin and is therefore called anti-
toxin. The reaction inay be thought of as a precipitin reaction in which -the
particles 'of precipitate, as a rule, are too small to be seen. Under certain con-
ditions iIi which the 'quantitative relations are carefully adjusted, visible flakes
or floccules are produced. The reaction is quantitative but seems to obey
primarily the laws which govern physical adsorption rather than chemical
combination. The toxin-antitoxin reaction is obviously a kind of precipitin
reaction.
QUANTITATIVE RELATIONS (THE FLOCCULATION REACTION). As mentioned
above, when exactly the right proportions of toxin and antitoxin are brought
together in a test tube a visible precipitate or flocculation occurs. This fact is
made use of in determining the concentrations or "strengths" of toxin or
antitoxin. For example, we may set up a row of ten tubes. In the first we may
put serum cqntaining a quantity of diphtheria antitoxin arbitrarily spoken
of as 2 units. * In the next we place 4 units, in the next 6 units, and so on. We
then add to each tube a fixed amount, say I ml of filtered broth culture which
contains diphtheria toxin in unknown amount. After a short time flocculation
appears in one of the tubes, let us say the sixth tube. Since this contained 12
units of antitoxin, we have a measure of the potency of the toxin broth. We
say that it contains 12 flocculation units (12Lr) of toxin per ml. This is an
arbitrary unit of potency and is used for convenience to define diphtheria,
tetanus and other toxins. It shows combining and flocculating potency but
not necessarily toxicity. Partly deteriorated toxins (toxoids) will still give
the same, undiminished Lr value.
Conversely, by using a series of tubes containing known, graded amounts
of toxin, we may determine the number of Lr units of antitoxin in a serum of
unknown potency.
ZONE REACTION. The above r-eaction illustrates a curious but common
manifestation called a zone phenomenon (sometimes called pre-zone and post-
zone, or zones of inhibition.
Observe in the above experiment that flocculation failed to occur in a zone
of 5 tubes before the 6th tube and in a "zone" of 4 tubes after the 6th (pre-
zone and post-zone). This phenomenon depends in part on the fact that for
maximal amounts of visible precipitate (or flocculation or other antigen-
antibody reaction) to occur most rapidly optimal relative proportions of
antigen and antibody must be present. If there is too great an excess of either,
less precipitate, or none at all, will appear. When we use a series of graded
quantities of antibody serum with a fixed dose of antigen, we might expect the
highest concentrations of antibody to give the strongest reactions, the smaller
quantities correspondingly less. This is a common experience. However,
often the zone phenomenon is manifest and no reaction occurs with the
greater, or with the weaker, concentrations but only with the middle con-
centrations. Many factors are involved, among which are type of animal serum
used, nature of antigen and antibody, pH, electrolytes, and so on.
"BLOCKING ANTIBODIES." Pre-zones may also occur due to the presence 01
partial, imperfect or· partly deteriorated antibodies. These are sometimes
called "toxoids," "agglutinoids" or "blocking antibodies." They have the
ability to combine w~th the antigen, to the exclusion of perfect, complete
antibodies. They are,[themselves, incompetent to cause whatever distinctive
manifestation is involved. When they are present they prevent the true anti-
bodies from acting.
• A unit (approximately) of diphtheria antitoxin is the least amount necessary to protect
standardized (250 to 300 grn) guinea pigs against 100 minimal lethal doses (M.L.D.) of
diphtheria toxin. An M.L.D. kills about 75 per cent of such pigs in from four to five days.
318 . Immunology
They do not manifest themselves in the higher dilutions of antibody-
serum because the defective antibodies are made too dilute to interfere;
i.e., they are "diluted out."
Haptenes. This term, or partial antigens, is applied to substances which
are related to antigens, but are not actually capable of stimulating production
of antibodies. They can, however, combine with antibodies, thus blocking or
excluding the complete antigen. A visible manifestation of the combination
may occur but generally does not. The situation is the reverse of that seen in
blocking antibodies.
Thus, zone phenomena may be produced by partial or imperfect antibodies
or partial or imperfect antigens (haptenes).
The practical importance of zone phenomena is that, in determining the
activity of any antibody-containing serum or antigen by testing it in a series
of dilutions, the series should be made sufficiently long, and the intervals
between dilution steps not too great, or the zone of optimal proportions
may either not be reached or passed over. Dilutions must be sufficient to
eliminate partial, or blocking, antibodies as well as haptenes. The same con-
siderations hold for most other serological tests involving antigen-antibody
reaction.
, ~ Cytolysins and Complement. The somatic antigens of any given bacterial
cell may call forth a_gtibodies which assist in the lysis of that c~ll. These anti-
bodies are termed cytolysins. They are sometimes calletlsensitizers (see
below).
The action of cytolysin may be described briefly as follows: the cytolytic
ant~~y first combines with th_e_for:ejgILc~~ called it into being, probably
by a(isorption on the surface. The cell may be a bacterium, an erythrocyte or
a ceu: of any oUier nature. *"This simple combination is, however not sufficient
to destroy the cell. There is no visible reaction.
Complement. A second substance, called complement, which is normally
present in all mammalian blood and which is entirely non-specific, is necessary
to complete (hence complement) the lytic action. It, too, combines with the
cell which, in order for it to act, must already have been sensitized by the
cytolytic antibody. Lysis then results.
Complement behaves as though it might be a partial enzyme. As in the
properdin system certain ions must be present: Mg++ and possibly Ca++.
Complement, like properdin, is inactivated by zymosan. It is also inactivated
by a few minutes' ~xposure to 60° C and a few hours at 37° C, by shaking,
by certain chemicals and by otlier factors which inactivate enzymes.
~ Complement cannot; by itself, destroy foreign cells, but must act through
I the intermediation of the sensitizer. The sensitizer is a specific antibody, but
complement is non-specific; it helps any sensitizer to complete its work.
COMPLEMENT FIXATION. After complement has combined with the sensi-
tized cell the complement is no longer active. It is said to be fixed. It is ad-
sorbed on the sensitized cells. Complement fixation is the basis of several
valuable tests used in the study of microorganisms, disease, immunology, etc.
THE COMPLEMENT-FIXATION REACTION. The fixation of complement was
first demonstrated in 1~01 by Bordet, a famous Belgian scientist. Bordet
• Not all types of cells appear to be equally subject to cytolysis in this manner.
discovered that if the serum of a person who has recovered from bubonic
plague (and therefore containing cytolysins specific for Pasteurella pestis,
the plague bacillus), were mixed with plague bacilli, the free complement in
the serum was all used up (fixed) in destroying the bacilli. When the com-
plement was tested for by an appropriate method, none was to be found free
in the serum.
Complement fixation may occur in various other antigen-antibody re-
actions. Many of these do not necessarily result in cell lysis. This is because
complement is readily adsorbed on the surfaces of any finely divided particles,
visible or invisible, whether they be immunological precipitates, agglutina-
tions, proteins or inert colloidal particles such as clay or powdered animal
charcoal. For example, complement is adsorbed (fixed) by the fioccules re-
sulting from the interaction of toxin and antitoxin described above. Com-
plement may thus be fixed in any antigen-antibody reaction but is not a
necessary component of such reactions as it is in cytolysis.
The phenomenon of complement-fixation enables an investigator to identify
the antigens (microorganisms) causing certain diseases, or to detect and
identify the corresponding antibodies which appear in the blood as a result of
disease. For example, if antibody, antigen and complement are mixed in a
tube, we can determine whether antigen and antibody have combined by
testing to see whether the complement has been fixed or not. If complement
has been fixed, then we know that an immunologically specific, antigen-
antibody reaction has occurred. Knowing the identity of antigen or anti-
body, we can identify the other.
The Wassermann Test. A well-known use of the complement-fixation
test in this manner is in the diagnosis of syphilis. It was first described by
Wassermann. It is subject to serious errors due to non-specificity of "arti-
ficial" antigens used in it. (See Chapt. 31.)
Immobilizing Antibodies. The etiological (causative) agent of syphilis is a
spirochete, Treponema pallidum. The organism is actively motile, rotating on
its long axis and bending and flexing vigorously. It may also have flagella.
In the serum of patients with syphilis T. pallidum-specijic antibodies appear
some days or weeks after initial infection. These antibodies promptly im-
mobilize and kill the spirochetes within a few hours when mixed with them in
test tubes. This effect is readily seen by examining the mixture with a dark-
field microscope. It is commonly spoken of as the TPI (T. pal/idum immobiliza-
tion) test. The immobilizing action does not take place unless complement
is present. Curiously, little or no complement is fixed in this reaction. The
role of the antibody is evidently that of a sensitizer, but the action of the
complement is not so obvious, as no lysis or other visible effect (except death
and loss of motility) 'occurs.
The TPI test is ode of the most important immunological developments
in syphilology. It is 'the first specijic serological test for the disease. When
technical difficulties (which at present interfere with its general, routine use)
are overcome, it will help to replace non-specific serological methods com-
monly used for the diagnosis of syphilis. These difficulties are being rapidly
eliminated. I
Immobilization by specific antibodies also occurs in other microorganisms.
This is readily seen in Entamoeba histolytica (the cause of amebic dysentery)
320 InrllllllWlogy
when the active trophozoites of this protozoan are treated with the serum of
dysentery-immune animals. Similarly, there are specific immobilizing anti-
bodies for the ciliated larval state (miracidium) of a pathogenic worm (the
fiuke, Schistosoma mansoni) and for motile bacteria other than T. pollidum.
In the immobilization phenomena other than the TPI, complement is not
always necessary and death of the organisms does not necessarily follow im-
mobilization. They may, on the contrary, recover completely.
The Immune-Adherence Pbeoomeoon. This, also, is an immunologically-
specific reaction in which various species of bacteria, sensitized with specific
antibody, adhere strongly to erythrocytes. Complement is essential to the
immune-adherence.
A very important feature of this reaction is the fact that the microorganisms
adhering to erythrocytes are much more readily and actively phagocytized.
This is another illustration of the important relationship between phagocyte
and antibody.
THE PARTICULATE ADHESION PHENOMENON . The immune adherence
phenomenon just described is probably a particular manifestation of the more
general particulate adhesion phenomenon. Certain antibodies affect the sur-
faces of organisms for which the antibodies are specific by making them
sticky. As a result, not only homologous bacteria stick together (specific ag-
lutination) but non-specific particulate matter of various sorts sticks to them:
leukocytes, erythrocytes, heterologous bacteria, gamboge, collodion, yeast
cells, etc. Complement appears to be involved in the particulate adhesion
phenomenon whereas it is not essential to specific agglutination.
Agglutinins. In addition to antitoxins, cytolysins, immobilizing antibod1es,
immune adherence, and complement, there are antibodies which cause bac-
teria to stick together in flocks or clumps as though they were coated with
some glutinous substance. Such antibodies are called qgglutinins. Like all
other antibodies, they are ~i.Dc. They 00 not necessarily kill bacteria but
aid the leukocytes by gathenng the latter's prey into groups. A leukocyte, or
other phagocytic cell, can engulf 50 agglutinated bacteria fifty times as easily
I as 50 separate ones, and in much less time. Furthermore, the agglutinins

appear to opsonize the surfaces of the bacteria so that the phagocytes can
grasp and engulf them more readily.
DIAGNOSTIC USE OF AGGLUTININS. Agglutinins are very widely used in
the identification of bacteria and the diagnosis of disease. Let us assume that a
patient has a febrile disease which has remained undiagnosed for a week or
I more, during which time antibodies have formed. We draw a little blood from
a vein and allow it to clot. We then remove the clear serum and mix it, suitably
diluted (1 :20, I :40; 1:80; ... 1:2560). in a series of test tubes with, for ex-
ample. a suspension of typhoid bacilli (Salmonella typhi). These bacteria are
commonly maintained in diagnostic laboratories for this purpose. If the
patient has typhoid fever his serum will contain typhoid agglutinins and the
bacilli will be found in flocks or clumps (Fig. 21-9). Ifno agglutination occurs.
either the patient does not have typhoid fever or has not yet had time to
develop antibodies. However, antibodies can usually be detected after a week
of fever. Another test will be made later.
This means of diagnosing typhoid fever is called the Widal reaction after
Widal, who first published upon the subject. The term Widal test is sometimes
(improperly) applied to any agglutination test.
IDENTIFICATION OF BACTERIA BY THE AGGLUTINATION REACTION. Con-
versely to the Widal test, in the identification of an unknown organism,
sera containing various known antibodies are mixed with suspensions of the
unknown bacterium, and agglutination is looked for. Suppose, for example,
that we have a gram-negative rod which, by its cultural reactions, we know to
belong to the typhoid-dysentery group. We may, as a preliminary test, set up
two series of tubes: A, containing serial dilutions of serum of a typhoid-
immune animal; B, containing dilutions of serum of a dysentery-immune
animal. A drop of our "unknown" bacterial suspension is added to each
tube. If, after several hours, no change has occurred in the first series of tubes
while the serum in the tubes of series B has caused the bacilli to fall to the
bottom of the tubes in clumps, we know that, since the serum in B contained
only dysentery agglutinins, our unknown organism must be some species of
Shigella (dysentery bacilli). Many other bacteria, saprophytic and parasitic, of
agricultural, industrial and other special interests, may be identified in this
way.
THE HEMAGGLUTINATION REACTION. This procedure, widely used in im-
munology, illustrates very nicely the fact that antigens at the surface of a
cell determine its immunological specificity. Erythrocytes (sheep, cow, etc.)
are washed free from their serum and suspended in a saline solution con-
taining any desired soluble antigen; for example, an antigen extracted from'
tuberculosis bacilli. This antigen is adsorbed by the surfaces of the erythro-
cytes and covers them as a coating. The cells are removed from this suspension
and excess antigen is removed.
A series of dilutions is now made with serum containing antibodies specific
for the tuberculosis antigens with which the erythrocytes are coated. Into
each serum dilution is introduced a drop of the suspension of antigen-coated
erythrocytes. These behave as though they were tubercle bacilli! Within a
short time the coated sheep erythrocytes are agglutinated. Tuberculosis anti-
bodies have no visible effect whatever on the normal, untreated erythrocytes
of a sheep. It is interesting that totally inert particles of plastic, gum arabic,
etc., can be similarly coated with antigen and agglutinated by antibodies.
F... 21-9. Macroscopic agglutination test. Control tube C contains bacterial suspension
oaI.y. Numbered tubes contain bacterial suspension plus the following dilutions of serum:
1:100, 1 :200, 1 :500, 1:1,000, 1 :2,000, 1 :S,OOO, 1 :10,000, and 1 :20,000. Agglutination is
evident in dilutions 1 :100 to 1 :10,000 (tubes 1 through 7) but not in 1 :20,000 (tube 8). The
titer of the serum is therefore 1:10,000. (From Burrows, Textbook of Microbiology.)
322 Immunology
VIRAL HEMAGGLUTINATION. Many viruses have the property of causing
hemagglutination. This appears to be due to the existence of multiple, specific
receptors on erythrocytes and/or viruses. One virus particle can thus combine
with more than one erythrocyte simultaneously and vice versa, thus causing
visible clumping of the erythrocytes. This is often referred to as Hirst's
phenomenon after its discoverer.
The phenomenon is commonly used in diagnosis to titrate the virus content
of virus-infected fluids such as serum, nasal washings, and fluids from experi-
mentally infected chick embryos. The hemagglutinating powers of serial
dilutions of such fluids are tested. The amount of hemagglutination produced
by each dilution is recorded and the result may be expressed in terms of
"units" of virus present in the fluid.
HEMAGGLUTINATION INHIBITION (HI). If the specific receptors of the virus
particles are preempted by specific antibodies, obviously they cannot combine
with erythrocytes. Viral hemagglutination is thus specifically inhibited by
antibodies.
It is commonplace diagnostic procedure to measure the HI power of anti-
body-containing fluids (for example, serum of patients). Serial dilutions of
the patient's serum are mixed with standardized doses of the virus being
studied. After allowing time for combination, equal amounts of a standardized
erythrocyte suspension are added to each tube. The extent to which hemag-
glutination is inhibited gives a measure of the concentration of specific anti-
bodies in the serum .
•Precipitins. We have previously described the precipitin reaction, using it
as a model of a typical antigen-antibody reaction.C!_>recipitins seem to be very
closely related to agglutinins and are probably merely a different manifesta-
tion of the same antibodies. They cause the clumping and precipitation of
invisible molecules of protein (instead of whole cells) so that a visible tur-
bidity of flakiness ;is formed.
USES OF PRECIPlTINS. Because of the very high degree of specificity of
precipitin reactions, serological differentiation between soluble proteins of
closely similar composition is possible, a feat impossible of attainment by
chemical means.
An interesting application is seen in the use of precipitin tests to determine
the animal (host) from which a mosquito had its most recent blood -meal.
In determining mosquito-host blood, one first catches his mosquito (full
of the blood to be tested). The mosquito is crushed in 1 or 2 ml of saline solu-
tion and the blood is thus extracted. This constitutes the antigen to be tested.
The sera with which to test it are previously prepared in rabbits by injecting
the rabbits with blood from various animaispecies. One rabbit receives bovine
blood, another equiJ;le blood, and so on. The serum of each rabbit contains
precipitins against a certain species of animal. By bringing into contact a
little of the mosquito extract (antigen) with each of the rabbit sera (specific
antibody) in turn, one serum will be found which causes a definite precipita-
tion. If that serum is from a rabbit immunized with bovine serum, then we
may say the mosquito probably got its blood meal from one of the nearby
cattle. This information is of use in the control of mosquito-borne diseases.
It guides efforts toward eradication of the mosquitoes which bite mall.
THE PRECIPITIN TEST APPLIED TO SYPHILIS. -It seems to be well establisqed
that, in complement fixation tests, the complement is fixed because it is
adsorbed onto finely divided particles of antigen-antibody precipitate. In
the Wassermann test" for syphilis, the precipitate formed is not visible. It
would be much better if we could avoid having to test for the presence of this
invisible precipitate by adding complement and then being forced, in turn, to
test for the presence of complement by adding sensitized red corpuscles. It
would be a great advantage if we could see the precipitate directly as in other
precipitin reactions. This has been accomplished.
A specially prepared, and very concentrated, alcoholic antigen is used in
which the reactive substances are present, but in the form of large, unstable,
colloidal complexes. These are brought (by proper dilution with saline solu-
tion), to a state where, in contact with syphilitic serum, they precipitate in a
visible form. Generally, no precipitation occurs in the presence of normal
serum under proper circumstances. Tests based on this principle are the Kahn
test, Eagle test, Hinton test, Mazzini, V.D.R.L., etc.
THE UNIVERSAL SEROLOGIC REACTION. The antibodies concerned in these
tests are not related to syphilis at all but are produced by deteriorated tissues
resulting from normal "wear and tear" of the body. Certain materials inside
of such normal but "worn-out" tissue cells appear to act as antigens which
call forth antibodies (precipitins). Similar antigens may be extracted from the
tissues of normal animals. The tissue antigens are found especially in heart
muscle and are lipid in nature. Commercial antigens commonly used in
testing for syphilis are, therefore, derived from beef hearts and are called
cardiolipins. They can give false, positive reactions, with always pistressing,
sometimes tragic, results. The advantages of tests using specific antibodies,
previously mentioned, are therefore obvious.
Now, there are 3 controlling factors in the precipitin test for syphilis using
these antigens: (a) temperature, (b) concentration of NaCl in the saline dilut-
ing fluid and (c) dilution of the serum to be tested. By suitable arrangements
of these factors the serum of syphilitic persons usually can be made to give
a positive precipitin test with the cardiolipin (and similar) antigens. By
making other adjustments, the reaction can be made to occur in normal
serum. By making still other adjustments, the reaction is found to occur in
tuberculosis, malaria and leprosy and also in different species of normal
animals. Because of its wide occurrence under proper adjustments of the test
procedure, the phenomenon has been called the Universal serologic reaction.
Antigen-Antibody (Precipitin) Reactions·in Gels. A very important method
of demonstrating precipitin reactions makes use of agar gels. Reactions not
demonstrable by other methods can readily be made visible by this method.
To show the reacti,on, one mixes a specific antiserum with warm, fluid agar,
half fills a, narrow :tube with it and allows it to solidify. The corresponding
antigen, which must be in solution (not cellular), is placed in contact with the
agar. As the antig~n diffuses downward into the agar, a white band of pre-
cipitate appears in ~he agar at the zone of optimal proportions. If the antigen
is a pure substance, only one band appears. If the antigen is a mixture, and
the serum contains antibodies for each antigen in the mixture, then several
bands may appear, corresponding in number to the number of antigen-
antibody systems present. The possible value of this technique in detecting
impurities in anti~ens, in protein analysis, etc., are evident. Here is a research
324 Immunology
Fig. 21-10. The in vitro test for virulence. Serum agar was poured into the dish at about
45· C. Before it hardened, the strip of filter paper, saturated with diphtheria antitoxin, was
pressed to the bottom of the agar in the dish. After the agar hardened it was inoculated on
the surface in long streaks at right angles to the paper. As the growth developed, at 37· C,
toxin diffused from the culture into the agar. Simultaneously, antitoxin diffused from the
paper strip. Where the toxin and antitoxin me:, in proper concentration {or reaction, pre-
cipitation occurred. This is seen as thin white lines between the growths of the cultures.
This reaction with diphtheria antitoxin is produced only by toxigenic C. diphtheriae. The
growths nearest the ends of the strip of paper are not C. diphtheriae. (Specimens prepared
by Miss Elizabeth o. King Photo courtesy of the u. S. Public Health Service, Communi-
cable Disease Center, Atlan~, Ga.)
tool, simple and inexpensive to use, which should prove attractive to any
scientificaUy curious student.
THE IN VITRO TEsT FOR TOXIGENICITY. A very useful application of the
principle bs been made to studies of the toxigenicity of diphtheria bacilli
without th2 use of animals. A strip of sterile filter paper saturated with diph-
theria antitoxin is embedded in the center of a plate of nutrient agar, while
I the agar is st1ll warm and fluid. After the agar has solidified, the culture of
diphtheria bacilli to be tested is inoculated in a single straight streak across the
surface of the agar at an angle of 90° to the strip of paper. The plate is then
incubated.
The diphtheria antitoxin diffuses through the agar from the paper strip in
diminishing concentrations, an infinite series of dilutions, from the paper 'Out-
ward. Diphtheria toxin, given off by the growth on the agar surface, likewise
diffuses into the agar, but at right angles to the direction of diffusion of the
antitoxin; also, in an infinite series of dilutions. Wherever the two meet in
optimal proportions for precipitation to occur, a white point appears in the
agar. Theoretically, all of these points should occur on a line bisecting the
0
90 angle, more intense near the intersection of paper and line of growth,
growing fainter with distance from the angle of intersection. This actually
occurs, and all of the points of precipitation coalesce into a single straight line.
This is a very pretty "living graph," the paper strip being the origin of the
ordinates (concentrations of antitoxin); the line of growth, the origin of the
abscissae (concentrations of toxin) (Fig. 21-10).
The method determines toxigenicity (virulence) of diphtheria bacilli, a
property hitherto demonstrable only by expensive and time-consuming
inoculation of animals. In many instances the in vitro test is more dependable
than the in vivo test. This agar-plate diffusion method, in several ingenious
modifications, has been used in many very clever and fruitful investigations
of proteins, natural antigens, antibodies, antigen-antibody physical and
chemical relationships, etc.
Protective Antibodies. All of the immune reactions so far mentioned are
demonstrable by in vitro methods. It was mentioned that immunity does not
necessarily result from the presence of such antibodies. Indeed, it seems that
(as previously indicated) most of them aid in the process of phagocytosis.
Some are clearly lytic, some antitoxic, some immobilize. The action of others
is not demonstrable in vitro. The only reliable method of detecting and meas-
uring such antibodies is to infect experimental animals (e.g., mice) and give
them doses of the serum to be tested to see whether they are thereby pro-
tected. This measures protective power directly, regardless of whether this
power depends on agglutinins, cytolysins, or some still undiscovered anti-
body. Such a test is known as a protection test and is widely used to measure
the antigenic virtues of antigens and the protective power of sera.
REFERENCES
Anonymous: Nonspecific defense mechanisms of the body (the properdin system). J.A.M.A.,
1956,162: 1271.
Boone, I. Y., Woodward, K. T., and Harris, P. S.: Relation between bacteriemia and death
in mice following x-ray and thermal column exposures. J. Bact., 1956, 71 :189.
Boyd, W. C.: Fundamentals of Immunology. 3rd ed. Interscience Publishers, Inc., New
York,1956.
Burnet, Sir M.: How antibodies are made. Sci. Am., 1954, 191 :74.
Carpenter, P. L.: Immunology and Serology. W. B. Saunders Co., Philadelphia, 1956.
Coons, A. H.: Labeled antigens and antibodies. Ann. Rev. Microbiol., 1954,8:333.
Cox, C. D.: Hemolysis of sheep erythrocytes sensitized with leptospiral extracts. Proc. Soc.
Exp. BioI. and Med., 1955,90:610.
Cushing. J. E.: The Biological Aspects of ImmunololY. McGraw-Hili Book Co., Inc.,
New York, 1956.
Editorial: Microbes and Metchnikoff. Am. J. Med., 1952,12 :261.
Elberg, S. S.: Factors affecting resistance to infection. Ann. Rev. Microbiol., 1956, 10:1.
Gordon, A. S., and others: Leukocytic functions. Ann. N. Y. Acad. Sci., 1954, 59 (Art. 5):
665.
Gordon, L. E., Cooper, D. B., and Miller, C. P.: Clearance of bacteria from the blood of
irradiated rabbits. Proc. Soc. Exp. BioI. and Med., 1955,89:5n.
Hatch, M. H., et al.: Response of x-irradiated mice to intraWllOllS inoculation of intestinal
bacteria. Proc. Soc. Exp. BioI. and Med., 1952,80:632.
Haurowitz, F.: The immunological response. Ann. Rev. Microbiol., 1953, 7:389.
Hollander, A. G., Frobisher, M., Jr., and Kalisch, K.: Clinical evaluation of the hemagglu-
tination reaction. Am. Rev. Tub., 1953,67:497.
I .amanna, C., and Hollander, D. H.: Demonstration ·of particulate adhesion of the Riecken-
berg type with the spirochete of syphilis. Science. 1956, 123:989.
326 ImmuIWlogy
Ledbetter, R. K.: The Treponema pal/idum immobilization test. A diagnostic aid to the
clinician. I.A.M.A., 1956, 160:1392.
Lennette, E. H., et al.: Symposium on newer knowledge of viral and rickettsial diseases.
Am. J. Trop. Moo. and Hyg., 1956,5:419.
Liu, C.: Rapid diagnosis of human influenza infection from nasal smears by means of
fluorescein-labeled antibody. Pr~c. Soc. Exp. Bio!. & Med., 1956,92:883.
Magnuson, H. J., and Portnoy, J.: The Treponemapallidum complement-fixation test. Am.
J. Pub. Health, 1956,46:190.
Marrack, J.: The structure of antigen-antibody aggregates and complement fixation. Ann.
Rev. Microbio!., 1955, 9:369.
McDermott, W., and others: Natural resistance to infection. Ann. N. Y. Acad. Sci., 1956.
66(Art. 2) :233 et seq.
Miller/l. N., and Carpenter, C. M.: Treponema pallidum immune adherence (TPIA) test
in diagnOSis of syphilis. l.A.M.A., 1957, 163 :112. '
Neter, E.: Bacterial hemagglutination and hemolysis. Bact. Rev., 1956, 20: 166.
Nungester, W. J.: Nonspecific factors in immunity. Ann. Rev. Microbio!., 1954,8:363.
Raffel, S.: Immunity. Appleton-Century-Crofts, New York, 1953.
Sawyer, W. D., Smith, M. R., and Wood, W. B., Jr.: The mechanisms by which rnacro-
phages phagocyte encapsulated bacteria in the absence of antibody. J. Exp. Med., 1954,
m~~ ,
Schwartzman, G., Editor: Symposium No.6, New York Acad. Med., 1. The Effect of
ACTH and Cortisone upon Infection and Resistance. Columbia University Press,
New York, 1955.
Smith, E. L., and lager, B. V.: The characterization of antibodies. Ann. Rev. Microbio!.,
1952,6:207.
Suter, E.: Interaction between phagocytes and pathogenic microorganisms. Bact. Rev.,
1956, 20:94.
Talmage, D. W.: Effect of ionizing radiation on resistance and infection. Ann. Rev. Micro-
bio!., 1955,9:335.
Tomcsik, J.: Antibodies as indicators for bacterial surface structures. Ann. Rev. Microbiol.,
1956, 10 :213.
Topley, W. W. C., Wilson, G. S., and Miles, A. A.: Principles of Bacteriology and Immun-
ity. 4th ed. Williams & Wilkins Co., Baltimore, Md., 1955.
Vennes, J. W., and Gerhardt, P.: Immunologic comparison of isolated surfp.ce membraneS'
of Bacillus megatherium. Science, 1956, 124:535.
Wilson, M. W., and Pringle, B. H.: Experimental studies of the agar-plate precipitin test of
Ouchterlony. J. Immuno!., 1954,73:232.
Wright, W. A. and others: Nutrition in infections. Ann. N. Y. Acad. Sci .. 1955-1956.
63(Art.2):145.
I
22
2. ARTIFICIAL IMMUNITY
IN THE PREVIOUS chapter it was pointed out that persons often become
immune to certain infectious diseases by surviving natural attacks of those
maladies. The body actively acquires specific resistance against such infections.
This sort of immunity is not present in the normal person. Since it is acquired
in the course of natural events it is often spoken of as natural active immunity.
In this chapter we shall speak of purposefully induced immunity, or artificial
active immunity.
Even though subclinical (or inapparent*) infections often occur nat)Jrally,
with convenient, painless immunity as a result, natural infections causing
immunity are all too frequently very severe, may be disabling and disfiguring,
or even fatal. It would be much better if we could bJ!come immune by some
means which we can control. Thus we could avoid the dangers and dis-
comforts inherent in the natural process. Furthermore, we should like to
become safely immune to disease early in life and not have to wait for acci-
dental natural infection, occurring perhaps at a very inconvenient time in
adult life: In addition, it is often desirable to be able to produce immunity to
certain diseases at certain definite times. For example, a person desiring to do
laboratory research with yellow fever virus would like to be able to immunize
himself before starting the work, since infection with the virus might other-
wise result fatally. So also, physicians and nurses or others working with
polio or diphtheria patients should be immunized safely and comfortably
against these diseases in time to begin their work. All this, however, is too
much to expect of Nature.
In view of these meeds, man has devised means of developing specific im-
munities "artificially" and safely. The methods involve natural processes,
but are used under modified and carefully controlled conditions and are
therefore called "artificial immunization." Two types of artificial immunity
are used: active artificial immunity and passive artificial immunity.
j
* Inapparent infections are those in which symptoms are so very mild that no special
attention is paid to them. The vast majority of infections, fortunately, are of this type.
327
328 Immunology
ACfIVE ARTIF1CIAL IMMUNITY
In active artificial immunity the patient's body is stimulated to develop
resistance by being injected with certain kinds of antigens. These are of three
general types, as follows: (A) sterile bacterial exotoxins .. (B) sterile microbial
cellular* antigens (proteins and carbohydrates) in the form of, or derived
from, dead microorganisms; (C) living, infectious microorganisms, the viru-
lence of which has been reduced or attenuated by various procedures so that
no serious infection results.
PRIMARY AND SECONDARY STIMULUS
Before describing details of artificial active immunization. the matter ot
primary and secondary antigenic stimuli must be gotten in mind. In the ordi-
nary course of life one is constantly being exposed to repeated doses of in-
fectious organisms such as pneumococci, streptococci, and diphtheria bacilli
from carriers and from ambulatory, subclinical, mild and inapparent infec-
tions. These repeated antigenic stimuli serve to keep one's immunity in a good
state.
The phenomenon is used in artificial processes also. For example, suppose
that a child be given a single dose (1 ml) of diphtheria antigen such as alum
toxoid or a "shot" of Salk polio vaccine. In about two weeks his blood, tested
by appropriate methods, shows very few antibodies. After four to six weeks,
however, his blood is found to· contain a satisfactory amount of diphtheria
antitoxin, or polio antibodies. The development of immunity, however, has
been relatively slow! A year later the amounts of antibodies in his blood are
found to have declined to a very low level or to have disappeared entirely.
This diminution of antibody concentration in the serum is very common.
However, immunity has not necessarily disappeared.
"Booster Doses." After time has thus reduced the .effect of the first
antigenic stimulus, let us give the child a second dose of diphtheria toxoid
(or polio vaccine) and test his blood for antitoxin (Qr polio antibodies) at
short intervals. A surprisingly rapid and extensive response is now noted.
After the first or primary .f.limulus given a year before, response was slow.
Response to this dose given as a secondary stimulus, occurs at once and in a
few hours the child may be found to have one or more units of diphtheria
antitoxin (or ample polio antibodies) per ml of,blood (Fig. 22-1). In p.ractice,
the secondary stimulus is oftel! referred to as a "booster dose."
The body cells react as though, having once had an "antigenic experience,"
they are more alert and expert to form antibodies of this particular sort and
do so with great facility whenever called upon.
I Ability to withstand disease largely depends upon this very rapid reactivity.
This explains, in part, why resistance to disease, on the ·one hand, and con-
centration of demonsfrable antibodies in the serum, on the other hand, are
not necessarily related.
The principle of the primary and secondary stimulus is a generally ap-
plicable one and should be borne in mind. It is not restricted to exotoxins but
works equally well with living or dead bacteria, egg white, viruses and vir-
tually :,111 other antigenic substances.
• Somatic or 0, capsular, flagellar or H, etc.

(A) Immunization by Means of Exotoxins. Immunization with exotoxins
will be readily understood by those who have read the preceding chapter.
Cultures of toxin-producing bacteria, like C. diphtheriae, are made in broth
and, after sufficient growth of the bacteria, are passed through porcelain filters
which remove the organisms. The filtrate (broth passing through the filter)
contains the exotoxin. This may be injected hypodermically (under the skin)
in from 1 to 5 very minute doses at weekly intervals into the persons to be
immunized. Eventually (usually after two to six weeks) their blood will be
found to contain antitoxin which pntects them from the toxin in question.
This procedure or a modification of it was formerly used in immunizing chil-
dren against diphtheria. There is considerable danger, however, and many
fatal accidents occurred due to overdoses of toxin even when antitoxin was
mixed with it.
TOXOIDS. In 1890, von Behring, Frankel and Kitasato discovered that
diphtheria and tetanus (lockjaw) toxins, which had been heated for 1 hour
at 700 C, were no longer poisonous but could stimulate antibody production.
The possibilities for the prevention of tetanus and diphtheria were immedi-
ately recognized. In 1924, Ramon found that formaldehyde affects the toxins
of diphtheria and tetanus much as does heat. Such detoxified but antigenic
exotoxin is today spoken of as toxoid. In order to avoid the dangers attendant
upon the use of toxin, formaldehyde-treated toxoid came into general use for
active immunization against diphtheria. The same considerations apply to
other exotoxins, such as that of the tetanus (lockjaw) bacillus, etc.
In 1933 Havens and Wheeler found it possible to improve such antigens
still further. The addition of alum to broth containing the toxoid precipitates
the latter and it may then be collected, concentrated and purified to some
extent. Alum-precipitated toxoid is highly effective and only two initial in-
jections are needed as compared with three injections of fluid toxoid. Similar
discoveries have been made concerning other toxins used in active artificial
immunization, notably the toxins of scarlet fever and tetanus. The principle
0.03
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0U 0.02 I
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~ I
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Fig. 22-1. Curves showing rate of antitoxin production following a primary injection
and following a secondary injection. Note that after the secondary injection antibody pro-
duction is much more qlpid and extensive than after the first or primary stimulus. The rate
and extent of the secondary reaction vary with different individuals, antigens and species.
330 Immunology
has also been adapted to bacterial vaccine against whooping cough. It is
now common practice to mix several toxoids, as diphtheria and tetanus
toxoids as well as bacterial antigens, especially whooping-cough bacilli.
The combination of three antigens is even more effective, with respect to
each, than anyone of the three alone. In addition, the number of separate
injections is reduced.
ACTION OF ADSORBED TOXOlDS. It is important to understand why alum-
precipitated toxoid is more effective than plain, fluid toxoid. When the latter
is injected under the skin, the soluble material and fluid are quickly taken
into the body fluids and destroyed and eliminated inside a few hours. The
antigenic stimulus is very transitory, and repeated injections are necessary.
If the toxoid could be held in situ for several days, being released continu-
ously, little by little, into the body, the antigenic stimulus would be prolonged
and continuous and there would be less need for further injections.
This is accomplished by means of the alum-toxoid. The toxoid is adsorbed
and held on the surfaces of the alum precipitate, which is insoluble. The alum
precipitate remains where injected, undissolved, releasing its adsorbed toxoid
slowly, little by little, giving the patient a prolonged and continuous antigenic
action which is highly effective.
This principle is not confined to diphtheria immunization but is of broad
significance. For example, in a modified form it is used with antibiotics to
maintain a high concentration of antibiotics in the blood over a long period
without repeated injections. The persistence in the body of any antigen,
living or dead, maintains immunity by its continuous antigenic action.
(B) Immunization with Dead Microorganisms. A second procedure of
artificial active immunization is very similar to the foregoing except that,
instead:of using excreted growth products (e.g., exotoxin) of bacteria, the
microorganisms themselves are used. For convenience, greater yield, and
avoidance of foreign matter the microorganisms are usually cultivated on
agar (if bacteria), or in suitable fluid medium. In the case of viruses (e.g.,
polio virus) the microorganisms are cultivated in tissue cultures. For rick-
ettsiae, live chick-embryo yolk sacs are much used.
In dealing with bacteria, the growth on agar is removed to pl1ysiological
saline solution. Whether bacteria, virus or rickettsiae, the fluid containing
them is then heated to about 60° C (or irradiated with ultraviolef1ight, or
treated with formaldehyde or other substances) to kill the microorganisms.
A very minute amount (0.25%) of phenol, tricresol, or some other antimi-
crobic agent is added to insure sterility.
Such suspensions of killed microorganisms are frequently referred to as
<:acc~. If prepared from bacteria, they.fire correctly termed bacterins. The
term "vaccine" is properly restricted solely to the immunizing agent against
smallpox. However, it is widely used for any immunizing agent and is so used
here.
Bacterins are so effective in preventing typhoid and paratyphoid fever that
their use against these diseases in the United States military forces has long
been a matter of regulation. The principle of the secondary stimulus is widely
made use of in connection with these bacterins. After a person has received
an o'rdinary course of three weekly inoculations as a primary stimulus, his
resistance is maintained by single, annual, intradermal injections of 0.1 mlof
Immunology and Microbiolog! 331
bacterin. These cause little or no reaction, are quick, inexpensive and easy
and, above all, effective in reinforcing waning immunity.
The hypodermic injection of carefully controlled doses of suspensions of
killed micrococci (staphylococci) is sometimes used to increase resistance of
persons susceptible to boils, styes, etc., which are usually due to micrococci.
The use of Hemophilus pertussis bacterins in preventing and modifying whoop··
ing cough is of great value also. As pointed out previously, these are often
combined with toxoids.
(C) Immunization with Attenuated, Living, Infectious Agents. The third
method of artificial active immunization consists in actually infecting the-
person or animal to be immunized with the desired organism which has been
so treated that the virulence is greatly attenuated or diminished.
There are at least three means of lowering the virulence of pathogenic
organisms so that they can be safely used to induce active, artificial immunity.
These are (1) animal passage; (2) treatment with unfavorable agents, such as
desiccation; and (3) cultivation on special media or under special conditions
such as abnormally high temperature. An example of each will be described.
ANIMAL PASSAGE. This is well illustrated by the development of vaccine
against fox distemper, a scourge to fox-fur farmers. The virus from a sick fox
was injected into a ferret. Infectious fluid from the sick ferret was injected into
another ferret. This ferret-to-ferret transfer (animal passage) was continued
through a long series. The virus became adapted to, and enormously virulent
for, ferrets When fluid from the last ferret in a long series was tested in a fox,
it had little or no virulence for the fox. However, the fox was afterward found
to be completely immune to natural fox-distemper virus. The virus -had
become highly adapted to ferrets but modified or attenuated with respect to
foxes. The ferret-passaged vaccine has saved fox farmers from enormous
losses.
Another illustration of the effect of animal passage on virulence is seen
in preparation of smallpox vaccine. It was originally thought that cows
became infected with smallpox but developed only the relatively mild cow-
pox. Contact with the cow (animal passage) was supposed to have modi-
fied the virulence of the original smallpox virus. It was then called vaccinia *
virus. Vaccinia virus causes a mild infection in man. t
Jn 1798, Jenner, then a country doctor but later a famous British scientist,
observed that many dairy workers, associated with cows having cowpox, did
not succumb during epidemics of smallpox. Experimenting, he found that if
some of the serum or lymph from the pustules on the udder of a cow with
cowpox were scratched into the arm of a human being, a very mild disease
(vaccinia) resulted, with the formation of a single, localized, poxlike lesion.
This soon healed, leaving a distinctive scar. Smallpox never developed in
persons after infection with cowpox. The person thus safely became immune
to smallpox througp. infection with cowpox. The resistance to smallpox
generally lasts 3 to 1i years.
Vaccinia virus, as used today, is prepared by scratching the virus into the
shaved and disinfected skin of a calf. When the pustules are "ripe" the lymph
* Vacca is the Latin *",ord for cow.
t It may also be that the two viruses are distinct "species," but sufficiently closely related
so that the one immunizes against the other.
332 Immunology
Fig. 22-2. Children of one family who were brought to the Municipal Hospital of Phila-
delphia with the mother and father. who had smallpox. The child in the center had been
considered too young to be vaccinated. The other children had been vaccinated a year
before; they remained free from the disease, although for several weeks they lived in the
wards of patients with smallpox. (From Welch and Schamberg. "Contagious Diseases,"
Lea and Febiger, publishers.)
is collected from them and put up in glass tubes ready for use. Its potency
and cleanliness are carefully controlled by the National Institutes of Health
at Bethesda, Md. Its value is made evident by experience and by a glance at
Figure 22-2.
"Modern instances" of the attenuation of virulence by animal passage are
seen in the use of what is termed "avianized" rabies virus, and the preparation
of yellow fever vaccine. As pointed out in Chapter 13, many microorganisms
will multiply vigorously in living chick embryos. When rabies, yellow fever,
smallpox and other viruses are thus "passaged" from egg to egg, they lose
much of their virulence for human beings and, while still capable of infecting
mildly and thus immunizing, have lost the power of producing severe disease.
The avianized rabies virus is widely used for the protection of dogs from
rabies. The modified yellow fever virus (17D) has immunized millions of
persons safely and effectively against this dread disease of certain tropical
countries.
Modification of the virus of viral diarrhea of cattle by passage through
I rabbits likewise appears to furnish a very effective vaccine. Similar animal
passage of hog-cholera virus, to produce a vaccine, is under investigation.
Avianized canine-distemper vaccine is available; avianized smallpox vaccine
will probably come into use. Studies on modified, live polio vaccine are being
made by a number of investigators.
DPSICCATION. Pasteur's name is immortalized in the term "Pasteur treat-
ment" for rabies ("mad-dog bite"). Pasteur's process was not really a curative
"treatment" but a course of immunizing injections with the living, attenuated
virus of rabies. *
• In modem "Pasteur treatment" of human beings a killed virus is used.
If started soon enough after the bite of the infected animal, resistance (or
interference) develops from the injections, before the virus from the infectious
bite can cause disease.
For years Pasteur experimented with rabbits, dogs and guinea pigs (one of
the many brilliant illustrations of the value of animal experimentation) until
he felt certain that his method of attenuating rabies virus was safe and
effective. It consisted in passing the virus from an infected dog to the brain
of a rabbit and then from rabbit to rabbit (animal passage) to establish
attenuated virulence for man. Like the fox-distemper virus which became very
fatal for ferrets, the rabbit rabies virus acquired a maximum and fixed virul-
ence for rabbits. Pasteur called this virus "virus fixe." The potency of the virus
for man was further reduced by desiccation. Desiccated virus fixe was injected
in increasing doses.
Pasteur's first human immunization against rabies, in 1885, was an ex-
tremely dramatic event and marked an epoch in the progress of the war on
disease. Indeed, Pasteur is referred to as "the father of immunology." His
experiments on rabies have been movingly described by Vallery-Radot,
Pasteur's grandson.
CULTIVATION IN SPECIAL MEDIA. IMMUNIZATION AGAINST TuBERCULOSIS.
A method for immunization against tuberculosis with living, attenuated
tubercle bacilli has been known for some years. The procedure is known as the
"Calmette-Guerin process" and the attenuated cultures as "BCG" (Bacillus
Cal mette-Guerin *). The process consists of the injection of live tubercle
bacilli, the virulence of which has been reduced by cultivation of the organ-
isms on certain media containing bile. This method of cultivation induces the
development of a stable variant of the tubercle bacillus having low virulence.
This is the only important use, at present, of immunization of human beings
with living bacteria. Its safety and effectiveness have frequently been ques-
tioned.
The W.H.O. t regards BeG vaccination as the most important means for the eradication
of tuberculosis in countries where other approaches to the problem are not practical. The
American Trudeau Society, in 1949, adopted appropriate recommendations regarding the
use of BeG. It is felt by some that there is a tendency to rely too much on BeG, and as a
result, to neglect other time-tested and sure means of control such as proper nutrition,
x-ray surveys, hospitalization, sanitation, etc.
ATTENUATION BY CULTIVATION AT UNFAVORABLE TEMPERATURES. Al-

though this method is not much used at present, its discovery and demon-
stration by Pasteur constitute an episode of great dramatic interest in the
history of bacteriology. The principle was first used with Pasteurella avicida,
the bacterial cause of fowl cholera. It was later developed in connection with
studies of anthrax which, in the nineteenth century, decimated the sheep
flocks of France and still does so in some parts of Europe. Following numer-
ous preliminary experiments by the tireless Pasteur, on variants of Bacillus
anthracis, cultures of the organisms were prepared by incubation at un-
favorably high temperatures of 39° to 43° C (optimum around 35° C). This
* Calmette and Gl1erin are the names of the two French scientists who developed the
method.
t W.H.O. = World Health Organization, United Nations.
334 Immunology
caused them to lose some of their virulence (and also, often, their power of
spore formation). * The immunizing agent finally adopted by Pasteur con-
sisted of living, broth cultures of these attenuated strains. The first great
public demonstration of the value of anthrax immunization gave the in-
domitable Pasteur some 'uncomfortable hours, because his ideas had been
unfavorably regarded by many and his experiments were closely and critically
watched. He injected about 100 sheep with his attenuated organisms. After a
suitable period had been allowed for immunity to develop, these animals, as
well as an equal number of normal, unprotected animals, were publicly
inoculated with large doses of fully virulent anthrax bacilli. This experiment
has been dramatized in a Hollywood motion picture. The test injections were
made on May 31, 1881, at Pouilly Ie. Fort. There were many skeptics in the
audience when the injecting began.
A day or two later, when Pasteur arrived at Pouilly Ie Fort, "the carcasses
of twenty-two unvaccinated sheep were lying side by side; two others were
breathing their last; the last survivors of the sacrificed lot showed all the
characteristic symptoms of splenic fever (anthrax). All the vaccinated sheep
were in perfect health."t .
PASSIVE IMMUNITY
ARTIFICIAL PASSIVE IMMUNITY
In some cases it is necessary that a large supply of antibodies appear in
the blood immediately in order to combat an overwhelming infection. This
is especially well illustrated in such diseases as diphtheria and tetanus. The
chief symptoms in these diseases are caused by the bacterial toxins in the body
of the patient. The poisonous action is very rapid. When the patient is already
ill there is no time to lose waiting for him to develop active immunity, natural
or artificial. He must passively receive ready-made antibodies. Immunity re-
sulting from injections of these ready-made antibodies is called passive
immunity.
It is now possible to purchase, at all well-stocked pharmacies and health
departments, syringes or ampules already filled with antitoxic serum, prepared
for just such emergencies. Such antibody-containing serum is obtained from
animals, usually horses, which weeks or months previously have received re-
peated injections of the speci~l antigen against which antibodies are desired.
Passive Immunity in the Prevention of Disease. Passive immunity is used
in the prevention (prophylaxis) as well as in the cure of disease. For example,
if it is knoWn or suspected that a person is likely soon to become exposed or
! has very recently been exposed to certain diseases it is, under some circum-
stances (to be determined by the physician) an excellent plan to inject a small
quantity of serum, or some derivative of serlim (e.g., gamma globulin), con-
taining the appropriate antibodies as a preventive or prophylactic measure.
Diseases against which this form of prophylaxis is most commonly used are
• The method has since been shown to vary in result so that neither loss of "spores nor
cultivation at 4r C should be assumed to have deprived these organisms of their dallgerous
properties. •
t From "The Life of Louis Pasteur," by Rene Vallery-Radot, reprinted with permission
from Doubleday, Doran & Company, Inc.
measles, diphtheria and tetanus. Effective sera are available for some otber
infections (infectious hepatitis, rabies, pertussis) but are not so widely used.
Some are still under investigation, like the serum (gamma globulin) for
poliomyelitis.
Passive Immunity and Sermn Jaundice. Attention has already been called
to the fact that a considerable number of apparently normal and healthy
persons carry, in their tissues and blood, the virus of serum hepatitis or homo-
fogus serum jaundice. It is a good example of a latent viral infection. Unless
suitable precautions are taken to prevent, the virus is readily transmitted in
the blood of donors, in gamma globulin, and in other blood derivatives. It
is also easily transmitted by improperly sterilized syringes, needles, razors,
and other objects carrying blood or serum or lymph from one person to
another.
NATURAL PASSIVE IMMUNITY
Most antibodies can pass the placenta and consequently they are com-
monly found in the blood of the infant at birth. They serve to protect the child
for some months after birth. After that the child becomes susceptible to
many infectious diseases. It is therefore advisable to begin active immunization
of the child early (second month to sixth month) against pertussis and diph-
theria. Later, or simultaneously, tetanus toxoid may be given and still later
immunization against polio and less common diseases if desired.
It is important to note that antibodies in the newborn infant imply im-
munity in the mother. The expectant mother who does not have good anti-
body titers to diphtheria, tetanus, pertussis and possibly salmonellosis and
polio would do well to receive immunizing injections to confer passive
immunity on her child.
Transitory Nature of Passive Immunity. The antibodies in all artificial
passive immunity disappear from the body in two to three weeks, but while
they are present they may entirely prevent an infection or greatly lessen its
severity. In infants the maternal antibodies seem to persist for one to several
months longer, probably because they are of human origin.
REFERENCES
Brown, G. c.: Effect of booster inoculations on the serologic status of children vaccinated
with poliomyelitis vaccine. Am. J. Pub. Health, 1955,45:1401.
Carpenter, P. I.: Immunology and Serology. W. B. Saunders Co., Philadelphia, 1956.
Diphtheria and Pertussis Vaccination. World Health Organization Technical Report Series,
No. 61, May, 1953. ,
Editorial: Typhoid in vaccinated persons. J.A.M.A., 1954, 154:1265.
Edsall, G.: Immunization. Ann. Rev. Microbiol., 1955,9:347.
Koprowski, H., and others: Immunization of infants with living, attenuated polio virus.
J.A.M.A., 1956, 162r1281.
Paul, J. R.: Indications f9r vaccination against poliomyelitis. J.A.M.A., 1956,162:1585.
Peck, F. B. Jr., Powell,\ H. M., and Culbertson, C. G.: Duck-embryo rabies vaccine.
J.A.M.A., 1956,162;1373.
Qu-::ries and Minor Notes: Tetanus immunization. J.A.M.A., 1955, 15/:1663.
Raffel, S.: Immunity. Appleton-Century-Crofts, New York, 1953.
Sabin, A. B.: Present status of attenuated live-virus poliomyelitis vaccine. J.A.M.A., 1956,
162:1589. I,
Sauer, L. W., and Tucker, W. H.: Immune response to diphtheria, tetanus, and pertussis
antigens, aluminum'phosphate adsorbed. Am. J. Pub. Health, 1954,44:784.
336 Immunology
Strode, G. K., Editor: Yellow Fever. McGraw-Hili Book Co., New York, 1951.
Vaccination Against Tuberculosis. World Health Organization Technical Report Series,
No. 88, 1954. Columbia University Press, New York.
Vallery-Radot, R.: The Life of Louis Pasteur. Doubleday-Doran & Co., New York, 1926.
Various Authors on rabies control. Am. J. Pub. Health, 1955,45:998,1005.
Various Contributors: Discussion of prospects for control of poliomyelitis. J.A.M.A., 1955,
158:1249,1258,1266,1271,1274.
Volk, V. K., Top, F. H., and Bunney, W. E.: Observations on the effectiveness of various
scarlet fever antigens in multipieantigen preparations. Am. J. Pub. Health, 1953, 43:833.
Volk, V. K., Top, F. H., and Bunney, W. E.: Reinoculation with multiple antigen prepara-
tions of free-living children previously inoculated with multiple antigen preparations.
Am. J. Pub. Health, 1953,43:821.
W. H. 0.: Yellow Fever Vaccination, Monograph Ser. No. 30, 1956. Columbia Univ. Press,
New York 27, N. Y.
Yi-Yung Hsia, D., Lonsway, M., Jr., and Gillis, S. S.: Gamma globulin in the prevention of
infectious hepatitis. New England J. Med., 1954,250:417.
339
~ox but a
3. THE TISSUES IN RELATION TO IMMUNITY
THE ALLERGIC STATE

WE HAVE pointed out that tissue cells which have responded to an antigenic
stimulus are immunologically different from tissue cells which have not so
responded. The antigenically stimulated cells often retain antibodies in or on
them for long periods of time. Under proper circumstances, when the specific
antigen is again brought into contact with antibody-containing cells, the cells
react in a very distinctive manner. They are said to be in a hypersensitive or
allergic* state. The particular tissues or organs involved in any allergic,
antigen-antibody reaction are often called the shock tissues or shock organs.
The Induction Period. A period of several weeks must elapse between the
first contact with antigen (the sensitizing stimulus) alid that (the toxic dose)
which causes the allergic reaction. This induction period allows time for anti-
body formation.
Passive Allergy. In some types of allergy normal tissue cells which have
not had any active antigenic stimulus may passively acquire the antibodies
necessary to an allergic reaction by merely adsorbing them from the blood
stream. The antibodies which sensitize may be experimentally introduced into
the blood of a normal animal by injecting serum from an immune animal.
Since, in such cases, the antibodies are passively received this mode of
becoming allergic is, often spoken of as passive allergy. It is analogous to
passive immunity. In passive allergy the involved tissues are ready to react
within an hour or s6 after they have adsorbed the antibodies.
Allergens and Reagins. In some common forms of allergy (represented by
"hives" and the like) the antibodies involved appear to be precipitins or other
familiar types of antibody. They are: (a) demonstrable in the serum and (b)
readily transferred from one animal to another.
In other forms of allergy the antibodies are quite unlike any that we have
yet described. They remain closely associated with the cells that produce them
and are not readily'demonstrable in the blood stream. Not much that is
• From the Greek words alios, for changed, and ergon for activity.
337
338 immunology
definite is known of these antibodies. They are often spoken of as allergic

reagins. The antigens that engender them also differ from s;,tbstances com-
monly thought of as antigens, and are commonly called allergens." Examples
of some of these allergens are sulfonamides and other drugs, certain cosmetics,
certain dyes on fabrics, some plastics, antibiotics, etc. Hypersensitivity to these
substances is commonplace.
While these allergens are not antigens as previously described, it is
thought that they may become conjugated with proteins and other body com-
ponents so that they act like complete antigens in sllch relationships.
TYPES OF ALLERGIC REACTION

There are hundreds of different manifestations of allergy. The nature of an
allergic reaction depends OJ' ,nany factors: the shock tissue; the nature of the
allergen; the nature of the reagin; the anatomy of the animal involved (i.e.,
the location of the shock tissue); the dose of antigen; the degree of hyper-
sensitiveness; and so on.
We may classify allergic reactions into two general types: (1) the immediate
wheal or anaphylactic type, and (2) the delayed or tuberculin type. In general,
the immediate type of allergy is produced by soluble-protein antigens, like
egg-white or serum, while delayed allergy is evoked by cellular antigens such
as bacterial cells. The types of allergy are not always easily differentiated.
Sometimes both appear to be present together, and the same antigen may at
times produce one and at other times another. General characteristics of each
kind of reaction may be tabulated as follows: •
I. Immediate Allergy
(a) Visible reaction of sensitive tissues Occurs within secol1ds or minutes after contact
with antigen.
(b) Is due to antibodies which circulate in the blood stream (notably precipitins)
and which, therefore, are readily transferable, in serum, from one animal to
another.
(c) Tissues affected are primarily smooth muscle, blood vessels and supporting tissues
like cartilage, fibrous tissue, etc.
(d) Desensitization is relatively easy.
II. Delayed AUergy'
(a) Visible reaction of sensitive tissue occurs only hours following introduction 0,
antigen and develops fully during ohe or more days.
(b) Is due to ,antibodies (reagins) closely attached to, or inside of, cells, not readily
demonstrable or transferable in serum. Transferable only in cells.
(c) Any tissue may be affected.
I (d) Desensitization is difficult or impossible.
MANIFESTATIONS OF IMMEDIATE ALLERGY

Anaphylaxis.t This is one of the best known, though still incompletely
understood, manifestations of immediate allergy_ The antibodies mainly in-
volved appear to be precipitins and/or antibodies like precipitins. The anaphy-
lactic reaction received its name because it was first thought to be a para!'
~ Some workers use the terms reagin and allergen for all antibodies and antigcm referred
to in allergy.
t ana is from a Greek word meaning reverse; phylaxis is from a Greek word meaning
protection.
doxical immune reaction against immunity. It is, however, no paradox but a
sort of perversion or overreactivity of a truly protective mechanism.
t Anaphylaxis results usually from antigen-antibody combination throughout
the sensitized body following rapid, wide distribution of the specific antigen
in blood and tissues. This rarely occurs in nature but is generally brought
about by intravenous injection of considerable doses of antigen. It can also
result from absorption of antigen from a subcutaneous hypodermic deposit
or even from antigen inhaled (hay fever), ingested (food allergy) or absorbed
through the skin (allergic dermatitis).
THE ANAPHYLACTIC REACTION. Within about five minutes after intro-
duction of the toxic or shock dose of specific antigen, the animal (if a guinea
pig) becomes uneasy and scratches at its nose, coughs, and is evidently em-
barrassed for air. Gagging movements occur, and the animal gasps for breath.
Urination and defecation take place, the animal falls on its side and ceases to
breathe. Death may supervene within a few minutes. If the attack is not fatal,
recovery is often abrupt and seemingly complete within an hour or two and
the animal,will not exhibit hypersensitiveness for some days or weeks after-
ward. The animal is said to be desensitized.
Most of the symptoms are due to histamine-induced contraction of smooth
muscle fibers, i.e., smooth muscle is the most obvious shock organ in anaphy-
laxi~. Large amounts of smooth muscle are present in the lungs, thus con-
stricting the air passages. The fibers are also present in the intestines and
bladder. Reductions in temperature, swelling, inflammation, and other signs
are probably due to the release of histamine or H-substance, by the tissues
involved. Hence, antihistaminic drugs are of particular value in the immediate
types of allergic reaction, represented by anaphylaxis. In rabbits, dogs, and
other animals the picture varies, partly due to differences in anatomical
location of smooth muscle. In pregnant animals abortion often occurs because
the uterus consists largely of smooth muscle. In dogs there is much damage
to the liver and this alters the clinical picture markedly in them. In immediate
allergy there is also damage to blood vessels, resulting in escape of fluid into
the tissues, causing swelling (edema). There is also decreased ability of the
blood to coagulate (hemophilia).
OTHER REACTIONS OF IMMEDIATE ALLERGY
In human beings, typical anaphylaxis rarely occurs, but many other
manifestations of immediate allergy are commonly seen. All are closely
relateQ in basic mechanism.
Dermal Reactions. WHEAL-AND-FLARE. A common manifestation of the
immediate dermal type of allergic reaction is seen when a small amount of a
specific antigen (e.g., horse dander) is introduced into the superficial layers
of the skin of a person or animal whose tissues contain antibodies or reagins
for that antigen. Within a few seconds or minutes there appears a hard, white
swelling or wheal 'Yith irregular margins. This is quickly surrounded by an
irregularly shaped, and often quite extensive, zone of redness called a ''flare.''
These two signs (wheal-and-flare) are due to (a) edema; (b) dilation of the
locally affected bldod vessels. Both responses are characteristic of all types
of immediate allergy, except the Arthus 'phenomenon, in which the vessels
340 Immunology
contract. The whole allergic reaction itches intensely and usually disappears
within a few hours.
"HIVES" (allergic urticaria) are closely related to this form of allergic
manifestation. They often seem to result from the introduction of antigen
into the skin by escape of some particular food substance, to which the
patient is allergic, from the gastrointestinal tract into the blood stream.
Hives may also occur in persons who have received doses of serum such as
tetanus antitoxin for therapeutic purposes, and in other circumstances.
"HAY FEVER," ASTHMA, and the early stage of the COMMON COLD are other •
common manifestations of the immediate type of allergic reaction, occurring
in ectodermal tissues in the respiratory tract.
All of the phenomena of the immediate allergic reactions appear to be
due largely to histamine formed by the reacti,ng cells. The response involves
damage to, and dilatation of, blood vessels, edema, smooth-muscle con-
tractjons, and damage to supporting and connecHve tissues.
THE ARTHUS PHENOMENON. This reaction, first described by Arthus in
1903, depends on precipitins developed by repeated subcutaneous or intra-
dermal injections of an appropriate antigen (foreign serum, egg white, etc.).
As more precipitins develop, the antigen-antibody reactions in the tissues
at the sites of the repeated injections become progressively more and more
violent. In well-marked Arthus reactions prolonged smooth-muscle con-
tractions occur in the walls of the local blood vessels, and damage to the
vessel walls develops.
Local edema and erythema occur, as in most allergic reactions. The
contracted blood vessels become occluded with clots and the area, deprived
of blood supply, becomes necrotic (dead and disintegrating). This reaction
will not occllr in tissues without blood vessels. '
MANIFESTATIONS OF DELAYED ALLE~GY
The response in delayed allergy appears many hours, even several days,
after contact between sensitive cell and allergen. The reaction involves a
local inflammation and sometimes, with large doses of allergen, local necrosis.
Histamine is not involved. One of the best known examples of delayed allergy
is the tuberculin reaction (Chapt. 30). a form of bacterial allergy.
Bacterial Allergy. Bacteria, as well as viruses and other microorganisms
entering the blood stream or tissues, sensitize certain cells of the body as well
as stimulate the production of familiar types of antibody. In many instances.
the tissues of the skin seem to become especially sensitive, a condition readily
demonstrated by intradermal injection of the antigen (allergen), which results
I in a local redness and swelling. This is due to the presence of the specific anti-'
bodies (reagins) in the cells of the skin. The allergin-reagin reaction occurs at
once but the result is' not manifest for several hours or'days.
Allergy and Disease. Whenever an infectious disease becomes subacute
or chronic, body cells may become sensitized to one or, more of the microbial
antigens. The sensitivity may be purely of the delayed type or both immediate
and delayed sensitivity may be present in varying degrees. These situations
give rise to varying symptoms and little-understood reactions. A person who
has b~en infected with tubercle bacilli remains for months or years in an
'allergic condition to the organism, an important consideration in regard to
I
Immunology and Microbiol~gy 345
his resistance to the disease. As previously pointed out, BCG vac,-o ~ not
against tuberculosis is done with living tubercle bacilli. The resulting al7\""'me
against the organisms, as manifested by development of a positive tuberculiI.
reaction, is believed to be the basis of resistance to the disease. Skin tests for
allergy to tubercle bacilli (called tuberculin tests) are of great value in the study
of tuberculosis (see Chapter 30).
HARMFUL EFFECIS· OF ALLERGY
Like many normal and beneficent physiological functions, allergic reactions
may at times be so violent in some persons as to be harmful. These few violent
reactions, like violent acts, create more comment and attract more attention
than the enormous number of normal and helpful reactions which go on
constantly unnoticed. For example, certain persons appear to become exces-
sively allergic to hemolytic streptococci. The heart and joints appear to be
"shock tissue" in such allergy. There is believed to be a close relation between
this allergy to hemolytic streptococci and rheumatic heart disease, one of the
most important causes of disability and death in the United States. Many
chronic, disabling conditions, especially forms of asthma and joint disease,
are thought to be related to allergic reactions of certain tissues to obscure
microbial infections such as chronic sinusitis. Allergy, therefore, plays an
important part in chronic infectious diseases. OtheOr diseases of this chronic
nature are syphilis, undulant fever, swine erysipelas and leprosy. Many of the
rashes and eruptions seen in bacterial and viral diseases are allergic reactions
of the delayed type.
ALLERGY AS A DEFENSIVE MECHANISM

Once the body has been subjected to an antigenic stimulus, the more super-
ficial tissues * acquire a greatly enhanced power to bind and localize the
specific antigen when later brought into contact with if. The effectiveness of
tissue immunity is thus greatly enhanced by a!lergy, as are antibody and
phagocytic defense also. The Arthus phenomenon, and dermal reactions gen-
erally, are illustr'ltions of this heightened reactivity. The antigen thus bound,
although perhaps doing considerable damage locally where bound, cannot
spread throughout the body causing injury to deeper and more vital tissues.
Non-antitoxic Immunity to Toxin. An example of this is seen when rabbits
and guinea pigs are made allergic by injections of proteins extracted from
diphtherIa bacilli. The animals are able to survive doses of live, toxigenic
diphtheria bacilli which always kill normal animals. The toxin is held and
bound in the skin. While it does severe damage locally, it cannot spread to the
deep, vital tissues and so the animal survives easily in the complete absence of
antitoxin.
The Koch phenomenon is another classical example of the binding power of
tissues. If a normal guinea pig is inoculated, in the right groin, with virulent
tubercle bacilli, the bacteria gain a foothold, form a local abscess, and then
proceed, almost unopposed, from the abscess to the lymph nodes of the ab-
dominal cavity, to the spleen, the liver, the lymph nodes of the thorax, the
I,
* Especially cutaneous tissues, and those lymph systems which drain tissues in contact
with the exterior, such as respiratory and gastrointestinal tracts.
346 Microorganisms and Disease
the leukocytes and other tissue cells, they may have an opportunity to grow
in the body.
As the microorganisms grow, they become more adapted to the host, and
better able to multiply within it. Host-resistant mutants undoubtedly occur
and are selectively favored by conditions in the host. For example, they often
develop capsules which protect them from phagocytosis and antibodies. They
can then grow into the lymph spaces, spreading widely through the tissues.
Some however, as the diphtheria organism, remain entirely localized on sur-
faces, killing tissues only locally and, unfortunately for both host and parasite,
excreting toxin which is adsorbed by the blood. Others may grow in the blood
stream and then we have the condition called viremia or bacteriemia. or rick-
ettsiemia; septicemia or "blood poisoning."
Having gained entrance by a favorable portal (i.e., a tissue favorable to
their growth or maintenance*), they may be carried by the blood (or even by
leukocytes) and localize at various other points in the body, e.g., liver, spleen,
bone marrow or lymph nodules of the intestine. Secondary abscesses (sec-
ondary foci of infection or metastatic infections) result.
3. Toxicity. Toxins of microorganisms are poisonous substances, mainly
proteins or protein-like. Some microbial toxins appear to be polysaccharides.
Others are hydrolytic enzymes.
The toxins of bacteria are of two sorts: exotoxins and endotoxins.
Exotoxins are poisonous waste products excreted by the bacteria into their
environment. Exotoxins characteristically stimulate the production of anti-
toxins when injected into animals. Diphtheria toxin is a classical example of
an exotoxin. Some exotqxins, such as those of Clostridium botulinum and cer-
tain micrococci, are harmful only when swallowed. t Others, like that of diph-
theria bacilli, can be taken by mouth with impunity, but if injected or ad-
sorbed into the blood even in very tiny doses, may cause death. Most exo-
toxins appear to have an affinity for nervous tissue and often for heart inuscle,
kidney, etc. They cause damage principally to those tissues. Most of these
toxins are much more potent than cobra venom.
Some organisms produce exotoxins which kill leukocytes. The leukocidins
of micrococci and streptococci are good examples. Others produce toxins
which kill and destroy the cells of defensive tissues which tend to develop
around foci of infection, forming retaining sacs. This seems to occur i, pro-
gressive diseases like tuberculosis, actinomycosis and sy,!'hilis .•
ENZYMES AS TOXINS. Some toxins .are enzymes 0, are' related to enzymes.
For example, virulent streptococci, such as those cauf,ing scarlet fever, ery-
! sipelas and "blood~poisoning" (septicemia) produce an enzyme-activator
called streptokinase or fibrinolysin. This helps· to digest the 1.1brin in blood
clots and this presumably enables the organism producing the enzyme to
enter the blood stream and invade the local tissues (as in erysipelas). Many
organisms also produce potent lipolytic (fat or lipid-digesting) enzymes and
trypsinlike proteolytic enzymes. Catalase, an enzyme found in many species
of microorganism and common in animal tissues~ catalyzes the decomposition
of hydrogen peroxide into water and oxygen. In several bacterial species,
.. This bas sometimes been called the "tissue of pre.dilection."

t Except under abnormal, experimental administration.
Microbial Pathogenesis 347
notably Mycobacterium tuberculosis and Pasteurella pestis (cause of bubonic
plague), catalase production seems to be definitely associated with patho-
genicity. Lecithinase is an enzyme which helps destroy erythrocytes. Hy-gLll-
ronidase de_str()ys hyaluronic acid, a clear, gummy, intercellular substance
whkh normally opposes progress of microorganisms through the tissues.
This enzyme is often spoken of as a "spreading factor."
There are many factors besides microbial exotoxins which determine
whether infection will occur. Many are still very obscure. For example,
organisms generally regarded as perfectly harmless saprophytes are some-
times found as the causative agent in a serious or fatal infection. Is this
because of a defect in host defenses, or a sudden mutation of the organism to
host-resistance and aggressiveness, or both?
ENDOTOXINS remain within the bacterial cell or are closely attached to it.
Many endotoxins are the cell protoplasm itself. Like exotoxins, most bac-
terial endotoxins are proteins or substances associated with proteins. How-
ever, some important endotoxins are carbohydrates of the capsule, as in
pneumococci and type B influenza bacilli. Just how they act in the body is not
clear.
4. Dosage. This factor in establishing infection is a simple quantitative
one, yet it also involves other factors. As a generality we may say that, under
ordinary circumstances, the larger the dose of infective microorganisms the
greater the chance that an infection will result. However, certain qualifications
are necessary. For example, very large numbers of some organisms may be
present in certain situation'S without causing any difficulty at all. The in-
testine contains thousands of billions of deadly bacteria at all times. Yet if
only a dozen or so of some of these are introduced directly into the blood
stream or injected into the brain, they can quickly set up a fatal infection.
Similarly, one might swallow 3 or 4 typhoid bacilli with impunity yet several
hundred might overcome local resistance and cause typyoid feve~With sorr:e
organisms a single cell or particle is sufficient invariably to infect. Obviously
much depends on the virulence of the particular organism involved and on
the resistance of the tissues which it contacts, as well as on dosage alone.
Infectiousness, PathogenicitY and Virulence. Organisms which can (I) be
readily transmitted from one host to another and (2) maintain themselves in
a. high percentage of the hosts which they contact may be said to be highly
infectious. They may cause much or little damage and thus be of high or low
pathogeniCity. If they have little vegetative vigor and little toxigenicity they
may have little virulence. They may have high virulence and great infectious-
ness yet in certain hosts cause little disease (low pathogenicity). For example,
yellow fever and poliomyelitis have been so widespread in some communities
that virtually all of the children under ten became infected and developed
demonstrable antibod~es yet few showed any perceptible evidence of disease
at all. Most adults are immune to poliomyelitis yet never had a recognized
case. Obviously there were many infections yet little disease. Thus, distinctions
should be made when speaking of infectiousness, pathogenicity and virulence
of microorganisms. They are commonly confused with each other. Everything
is relative and dependent on something else.
Koch's Postulates. It is not always possible to be certain that the micro-
organisms isolated from a given disease lesion or from pus, blood or feces,
are the cause of the observed disease condition. Many harmless microorgan-
isms are found growing in feces, sputum and ulcerating wounds. Some would
not grow there unless the diseased condition existed first. Such adventitious
organisms are called secondary invaders. To prove that a certain microorgan-
ism is the cause of a given disease often requires the most painstaking and
careful study.
The question as to the etiological relationship of various bacteria to specific
diseases was a very live one long before the time of Koch and there was much
loose discussion and profitless argument regarding many bacteria and their
relation to disease. When Koch established the pure-culture technique it be-
came possible to apply exact methods to the study of the etiology of disease.
He was very conservative in stating the relationships of any given organism to
any particular disease.
His ideas on the subject were crystallized largely by his studies of the
relationship of tubercle bacilli (Mycobacterium tuberculoSis) to tuberculosis.
Koch, like others before him, observed the bacilli in the lesions of animals
dead of the disease. But he was not too ready to believe that he had discovered
the cause of tuberculosis because he found certain organisms present in the
lesions of tuberculosis. Might not this bacillus appear in the tissues merely
accidentally because the animal, being so ill, is too weak to resist its invasion?
Might it not be merely a relatively harmless opportunist? Might it not repre-
sent contamination with a common saprophyte capable of living in the ne-
crotic tissue? Koch, involved in a discussiqn of the problem, finally stated
what he believed to be the evidence necessary to prove an organism to be the
cause of a disease. The evidence consists of four postulates, generally called
Koch's postulates today, and they are, essentially, as follows:
1. The organism must be associated with all cases of a given disease and
in logical pathological relationship to the disease and its symptoms and
lesions.
2. It must be isolated from victims of the disease in pure culture.
3. When the pure culture is inoculated into susceptible animals or man,
it must reproduce the disease (or specific antibodies thereto). Many such
inoculations into man have been made on courageous volunteers. In others,
accidental i'nfections have ('ccurred which have provided long-wanted-.evi-
dence. The value of animal experim!!ntation is here very evident.
4. It must be isolated in pure culture from such experimental infections.
Even today the etiological relationship of some bacteria to diseases which
they are thought to cause has not been established, firmly on the basis of
I Koch's postulates. A notable example is the relation of so-called Myco-
bacterium leprae to leprosy. I .
Rivers' Postulates in Viral Diseases. Viruses were unknown at the time of

Koch's major works, so that he failed to take these invisible, noncultivable
agents of disease into consideration in stating the criteria by which the causal
relationship of a pathogen to a disease might be determined. Rivers, in 1937,
outlined criteria similar to Koch's postulates, which might apply in the cases
of viruses Essentially these are as follows:
1. The'virus must be present in the host cells showing the specific lesions,
at the time of the disease, or in the blood or other body fluids.
2. Filtrates of the infectious material (blood, etc., or tissue triturates)
Microbial Pathogenesis 349
shown not to contain bacteria or other visible or cultivable organisms,
must produce the disease or its counterpart in appropriate animals or
plants.
3. Similar filtrates from such animals or plants must transmit the disease
REFERENCES
Bauer, T. J.: The communicable disease problem in the United States. J.A.M.A., 1955
158:1407.
Burnet, Sir Macfarlane: The Natural History of Infectious Disease. Cambridge Univ
Press, New York, 1953.
Dubos,.R. J.: Biochemical Determinants of Microbial Disease. Harvard Univ. Press, Cam-
bridge, Mass., 1954.
Dubos, R. J.: Second thoughts on the germ theory. Sci. Am., 1955,192:31.
Howie, J. W., and O'Hea, A. J., Editors: Mechanisms of Microbial Pathogenicity, 5th Sym-
posium, Soc. for Gen. Microbiol., Cambridge Univ. Press, New York, 1955.
MacLeod, C. M., and Pappenheimer, A. M., Jr.: Properties of Bacteria Which Enable Them
to Cause Disease. In Bacterial and Mycotic Infections of Man, 2nd ed. J. B. Lippincott,
Philadelphia, 1952.
Nungester, W. J., Moore, Y., Mika, L., and Summers, P. W.; Comparative study of host
resistance of guinea pigs and rats. J. Inf. Dis., 1955,96:227.
Oakley, C. L.: Bacterial toxins. Ann. Rev. Microbio!., 1954,8:411.
Wedberg, S. E.: Microbes and You. The Macmillan Co., New York, 1954.
Wilson, G. S., and Miles, A. A.: Principles of Bacteriology and Immunity. 4th ed. Williams
& Wilkins Co., Baltimore, Md., 1955.
25
Transmission of Disease
VECTORS
MANY MICROORGANISMS have some degree of self-mobility in fluids.
It is generally limited, however, to distances of the order of a few inches or
feet. They cannot travel or swim long distances, or fly or climb, of their own
volition. However, they may be carried many miles in rivers and lakes, or by
aerial or marine currents. Indeed, most microorganisms depend on various
extraneous moving agents for long-distance transportation: dust; water;
food; droplets of saliva from sneezing and coughing; saliva-contaminated
hands; articles contaminated with oral, nasal and/or intestinal matter; insects;
animals. All of these are important vectors (transmitting agents) of disease.
While pathogens of plants and insects are usually fairly durable, a great
difficulty in travel for many pathogens of mammals is that conditions in the
world outside the body are too harsh. This is a penalty of extreme adaptation.
Drying is fatal to some such as meningococci, gonococci and syphilis spiro-
chetes. Exposure to sunlight quickly kills many mammalian pathogens such
as tubercle bacilli. Others cannot live in natural bodies of water or in soil or
in feces.
Most of the infectious diseases of man are produced by organisms which
do not form heat-resistani spores. For this reason, with few exceptions, ,non-
sporeforming pathogenic microorganisms are easily killed by boiling and by
disinfectants like cresol and'the halogens.
Furthermore, not only are_infectious agents much restricted in the modes
of travel available to them, but they must find a suitable portal of entry into
a host at the end oftheir journey. The host must, in addition, be a susceptible
individual. Nevertheless, in spite of'their difficulties, like the lowly bedbug*
qlicroorganisms' get' there just the same.
TYPES OF DISEASE VEcrOR
There are two general types of disease transmission: mechanical ,and
biological. Each type of transmission may be accomplished in one or more of
several ways. For convenience we may list these as follows:
I
• The moth has wings of velvet;

The butterfiy, wings of flame.
The bedbug has no wings at all.
But he gets there just the same! (Old rhyme.)
350
Disease Vectors 351
1. Mechanical by:
£a) Fomites·
(b) Foods, including milk and water
(c) Hands
(d) Droplets of saliva or nasal secretion
(e) Dust, infected from any source
(f) Direct contact, as in the venereal diseases, or in nursing patients with dis-
charging wounds, ulcers, etc.
(g) Carrierst
(h) Bodies, feet, etc., of arthropods
2. Biological by:
(a) Human blood and blood derivatives
(b) Bites of animals
(c) Bites of arthropods~
(d) Feces of biting or coprophagous§ arthropods
It will be seen that the different modes of transmission are not necessarily
mutually exclusive. Many diseases may be transmitted by several of these
methods; others are restricted, under natural conditions, to only one or two
methods.
1. MECHANICAL TRANSMISSION
(a) Fomites. It is obvious that any object having live, infectious micro-
organisms upon it may serve to transfer the bacteria from one person or place
to another. Thus, soiled bed linen or clothing, eating utensils, toys, pencils
and similar objects are dangerous after having been used by persons harbor-
ing microorganisms causing diseases of the intestinal tract (e.g., poliomyelitis,
typhoid fever or dysentery), of the skin (e.g., smallpox, measles, boils, fungus
infections), or the respiratory tract (e.g., diphtheria, scarlet fever, measles,
influenza, pneumonia). Venereal disea'ses may be transmitted by some of
these objects, although thi~ is rare. Public toilet seats, drinking cups, hair
brushes, etc., are always to be avoided for esthetic as well as sanitary reasons.
SANITIZATION OF EATING UTENSILS. The whole problem of sanitization of
public eating utensils and eating places is receiving public discussion and
thorough scientific study by progressive health departments and agencies.
Problems involved are: methods of dishwashing; detergents, their properties
and performance; effectiveness of disinfectants; tests for concentration of
disinfectants in wash water; education of persons responsible for sanitation,
and of the public; methods of examining utensils bacteriologically and so on.
Restaurant proprietors who have the well-being of their patrons in mind
either carefully scald all dishes after washing them (Fig. 25-1) or, after thor-
oughly washing them in hot water with a good detergent, rinse them in clean,
cool water containi,ng at least 100 parts per million of available chlorine,
• Fomites are objects contaminated by persons harboring infectious organisms; for

example: hand hangers'in buses, doorknobs, money, dishes, pencils, handkerchiefs, cloth-
ing, toys, bed linen, etc'.
t Carriers are persons, often more or less recent convalescents from infectious diseases,
who, though in apparently good health, nevertheless still harbor the infectious organisms
and distribute them in their environment.
t Arthropods are invertebrates with jointed legs and feet. The Phylum Arthropoda
includes crustacea (crabs, shrimps, etc.) and the groups, commonly called "insects," com-
prising flies, mosquitoes, ticks, lice, etc.
§ Feces-eating.
352 MicToorganums IlIId DuellSe
Fig. 25-1. One form of modem, sanitary, dishwasbing equipment. The working bencb
is of stainless steel. Soiled disbes are piled on the bencb in the foreground. They are soned
and scraped, the larger scraps of food dropping into a barrel beneath the counter. Glasses
are rinsed over rotating brushes, dipped in disinfectant and placed in trays in a rack (left).
Silverware soaks in a pan of special detergent solution (right foreground). The dishes,
arranged in baskets, are soaked and then given a preliminary rinse with IuJrd streams of
hot water (right). They then pass through a macbine dishwasher (center, background). The
silver, after soaking, passes through the same process as the d;shes and is self-dried after a
dip into a drying agent. Afterward all utensils are stacked and stored in dust-proof cabinets.
Eating utensils handled in this way are virtually sterile. (Courtesy, John L. Wilson and
Wm. M. Podas, Economics Lab'y, Inc., in Modem Sanitation, May, 1950.)
and dry by drainage. The odor of chlorine around a lunch counter is a favor-
able sign. Other disinfectants, without taste or odor, are also widely used.
EXAMINATION OF UTENSILS. Methods for measuring and controlling the
amount of bacterial contamination of dishes, etc., are not yet exact. Many
technical problems are involved and are under investigation. Most of the
methods at present favored for bacteriological examination of eating utensils
center around some modification of the swab-rinse technique which involves
/ the use of a sterile cotton swab made on a wooden applicator. In a simple
procedure the swab is moistened in a bland collecting fluid (water, broth
0(" buffered saline solution) and used to wipe a certain prescribed area of the
utensils within a few hours after they have been washed and dried. The swab
is then shaken thoroughly in a known volume (10 ml) of sterile saline solution,
or better, broth, in a vial.
After shaking the swab in the collecting fluid, dilution-plate counts are
made of the bacteria in the fluid. From the numbers of colonies obtained an
estimate is made of the degree of contamination on the dishes, etc. Com-
monly, a minimum standard of 100 organisms per utensil is recommended.
SOLUBLE SWABBING MATERIALS. Many of the bacteria collected from eat-
Disease Vectors 353
ing utensils or other objects with cotton swabs are not released by the cotton
fibers to the collecting fluid in which the swab is shaken, even with violent
agitation. A special, soluble swab material, such as calcium alginate (fibers
composed of a buffer-soluble, vegetable-gum salt), releases all of the attached
bacteria by dissolving in the collecting fluid.
STICKY AGAR SURFACES have also been used to remove bacteria from eating
utenstls, and may be preferable to swabs.
STANDARD PROCEDURES. · Methods of washing and handling utensils, time
and temperature of storage, humidity, dust, insects and so on, are factors
affecting the numbers of organisms surviving on the utensils al'ld dishes.
A standardized method, taking these and other factors into consideration,
has been outlined by a Committee of the American Public Health Association.
This specifie,; fo .. mulae for media, solutions, area swabbed, method of swab-
bing, cultural de tails, etc. There are also other, direct methods (Fig. 25-2).
Unfortunately, there is no exact method of measuring the amount of dis-
ease spread by d irty dishes and eating utensils in unhygienic restaurants. We
must all have a good deal of immunity to the microorganisms so transmitted.
There is, however, much indirect evidence that unsanitary eating utensils are
responsible for a great deal of disease transmission. From an esthetic view-
point, one does not likl! to feel that a little saliva from previous patrons is
being included, gratis, \\ ith his meal.
PAPER DISHES. The use of paper cups, dishes and eating utensils is the best
step toward eliminating the sanitary evils of public glass and chinaware and
Fig. 25- 2. Warm, sterile, fluid agar was poured into the plate. The fork was placed in
the agar and moved about to dislodge contaminating material. The dish was then covered,
the agar allowed to solidify and the dish incubated. The bacteria from the fork developed
ioto colonies which are clearly visible. Among these hundreds of colonies there are undoubt-
edly pathogellic bacteria. (Photo courtesy of Dr. Richard R. Barton; from Appr. Micr.,
1954, vol. 2.)
metal spoons and forks. Not only is expensive dishwashing equipment, with
its noise, sloppiness and heat eliminated, but labor and fuel costs are reduced,
breakage costs are trifling, and esthetic and sanitary standards enormously
improved. Bacteriological studies of paper used for containers and tableware
show that the processes used in their manufacture result in a product with
negligible content of microorganisms. Of these none is pathogenic. However,
paper dishes have not yet been perfected to the point where the public willingly
accepts them in place of china and glass.
(b) Transmission by Foods. Foods that are moist and not very acid are
likely to be excellent culture media for many pathogenic microorganisms: the
typhoid bacillus (Salmonella typhi) and dysentery bacilli (Shigella species);
toxin-producing Micrococcus (staphylococcus) species; Clostridium botulinum
(one cause of food poisoning); the hemolytic streptococci (Streptococcus pyo-
genes) that cause scarlet fever and septic sore throat; the diphtheria bacillus
(Corynebacterium diphtheriae); and others.
FOODS AND CARRIERS. The infection of foods with respiratory or enteric
pathogens, represented by the few species. mentioned above, is mainly by food
handlers who are carriers of the organisms and who sneeze and cough over
food and/or handle it with unwashed hands.
Foods mayor may not be sterilized by cooking. The center of large masses
of food is not always raised to a bactericidal temperature by baking or boiling.
Further, if the cooked food is infected by handling after it has cooled, and is
left standing for hours in a warm kitchen, the persons who eat it might (in
some instances) just as well drink a culture in the laboratory. In case of
doubt, discard the food or, second best, recook it. Always keep perish':lble
foods (a) covered (avoiding contamination) and (b) refrigerated (avoiding
incubation).
EXTRANEOUS INFECTION. Some foods are infecte.d from outside sources.
A good example is seen in oysters taken from beds polluted by sewage. Al-
ways look for the certification number on packages of uncooked shell fish.
Bakers' cream pies, cream puffs, etc., after incubation in a warm show-case,
sometimes contain enterotoxin * (toxin of enteritis) formed by micrococci.
The organisms are usually from a food handler's nose, boil or ulcer.
Home-canned foods sometimes contain the deadly toxin of botulism. This
toxin is formed during storage in a warm cellar, by Clostridium botulinum.
The spores of this anaerobic organism are introduced with soil into the jars
or cans. The spores germinate and the bacilli grow if the containers were not
properly sterilized after packing (see Chapters 34 and 43). Rats, flies, roaches
/ and other vermin· can also infect foods.
INFECTION OF MILK. Sterilized milk is often used in the laboratory as a
culture medium/and it is a good one. It is thus clear why milk, infected by
careless dairymen and incubated for hours in the sun on a loading platform,
has been the vector of scores of epidemics of diphtheria, typhoid fever, dys-
entery, scarlet fever and sore throat ("strep throat").
Mi:JC may be infected not only by dirty hands of dairymen and by insanitary
implements and polluted water supplies but, like other foods, also by careless
handling in the home; by flies, etc. In addition, it may come from cows in-
* Do not confuse with endotoxin.

Disease Vectors 355
feeted with brucellosis (undulant fever), the streptococci of scarlet fever, the
ricksettsiae of Q fever, or tuberculosis. Many cases of these last four diseases
have originated in infected milk.
Pasteurization of milk, and modern methods of sanitary handling of dairy
products, control by health departments, and constant vigilance through
bacteriological examinations have eliminated nearly all of the dangers from
milk. Never drink unpasteurized milk or uncertified raw milY.
(c) Transmission by Hands. Milk supplies and the food in any kitchen
may become infected from the hands of careless milkers, dairymen or cooks
who are carriers of infectious microorganisms.
Washing the hands after defecation or urination or blowing the nose is a
partial safeguard against transmission of intestinal and respiratory diseases
but careless and ignorant persons are often very lax in this respect. Pasteuriza-
tion of milk and the thorough cooking of foods (followed by prompt eating
or prompt refrigeration) are other safeguards. Persons who handle foods for
restaurants or institutions, as well as dairy workers, should be required by
law to pass bacteriological examinations and are so regulated in many com-
munities. However, enforcement is difficult and expensive. There is danger
from enteric and respiratory infections in food handlers, but little from
venereal diseases unless in an acute or active stage.
The practice of shaking hands doubtless transmits many pathogenic intes-
tinal and respiratory organisms. If hands are to be held they should be clean!
(Fig. 25-3.)
. (d) Transmission by Droplets of Saliva, Mucus, etc. The mucous secre-
tions of the nose, throat, mouth and lungs, all combined to some extent with
saliva, constitute one of the most formidable vectors of disease. Pneumococci,
streptococci, meningococci, diphtheria bacilli and tubercle bacilli, as well as
influenza virus, poliomyelitis virus, measles virus, and other organisms of
respiratory disease, are thus transmitted. Saliva, and with it oral, nasal and
IIIDlaier AIRs,allE
VIA~.1011 ' ' '(71

Fig. 25-3. Transmission of infectious \"
microorganisms of oral and respiratory
tracts from one person to another. Ab· \
sent from the picture but active, never- \
theless, are contaminated foods, water DUST BORIIE'-
and milk. (After F. ScIiwentker, from \ ••••
"Surgical Supervisor," July 1947. Cour- . _..._~~~~,~/_.~~_...,j""
tesy of the American Sterilizer Co.) SETTUS lit DUST
___
Cook DaIryman
I
--
I \
I \
Hand.
FOOd \
,#,,,,'' / \ '''~''''''..... \
ftj t't'i
£pldtlmic of food poilon/II(I
~
Wound Pu.,p.ml hmplligus

inf.cfiOll f.v., MonafO,um. £pid.mie of up'ie SOIY 'hmo'
Fig. 25-4. Spread of streptococcal infection. (I) Develops middle ear infection; (2) de-
velops rheumatic fever; (3) develops subacute bacterial endocarditi•. (From Witten: Micro-
biology with Application to Nursing, 2nd ed., McGraw-Hili, Blakiston Division.)
/
pulmonary mucus, are of the greatest importance in disease transmission
(Fig. 25-4).
We are aU very careless in our habits in regard to saliva, far more so than
we like to realize. The case has been stated vividly by a famous physician:
"1f infection by contact is of such very great importance in the fecal-borne
diseases, how much more important must it be in diseases in which the in-
fective agent is found in the secretions of the nose and mouth, as is the case
with diphtheria, scarlet fever, smallpox, mumps, measles, whooping cough,
tuberculosis, influenza, and cerebrospinal meningitis. Everyone avoids feces
and urine. but it is only the very few who have any objection to saliva.
"Not only is the saliva made use of for a great variety of purposes, and
Disease Vectors 357
numberless articles are for one reason or another placed in the mouth, but
for no reason whatever, and all unconsciously, the fingers are with great
frequency raised to the lips or to the nose. Who can doubt that if the salivary
glands secreted indigo the fingers would continually be stained a deep blue."
Droplets of saliva are presumably responsible for much disease trans-
mission. Sneezing or coughing in public without a handkerchief is repre-
hensible but commonplace (Fig. 25-5). Every cough or sneeze results in a
microbe-laden spray. The spray droplets remain suspended for some time in
the air and may be carried many feet by draughts. They land on food, lips,
hands, furniture. The droplets usually become dry.
DROPLET NUCLEI. The mucus-coated bacteria which they contain then
constitute what are called droplet nuclei. These fioat about through the air
like dust particles.
AIR DISINFECTION. The possibilities of disinfecting air in public meeting
places, operating rooms, etc., have been the subject of intensive and large-
scale investigations. The two methods giving most promise are irradiation
with ultraviolet light and the use of bactericidal vapors, sometimes called
aerosols. Both are strongly bactericidal but neither is of significant value
for practical purposes except in special situations. These are discussed in
more detail in the Chapter on Microorganisms in the Atmosphere.
(e) Transmission by Dust. Little imagination is needed to understand how
disease may be transmitted by dust. Particles of saliva or sputum containing
microorganisms fall to the floor, bedding or clothing, and dry quickly. If not
exposed to excessive heat or sunlight or other unfavorable influences, the
organisms in the droplet. nuclei may survive for considerable periods. When
the dust is stirred up, persons inhaling it or getting it into operative or acci-
dental wounds may suffer au attack of disease. Probably the respiratory dis-
eases like tuberculosis, pneumonia, diphtheria and scarlet fever are often
transmitted by such means, since the organisms involved resist drying ar.d
Fig. 25-5. Unstifled sneeze explodes a cloud of highly atomized, bacteria-laden drop-
lets. Some droplets travel at such high speed that they are streaks even at 1/30,000 of a
second. (Counesy of M. W. JennilOOn, Depan. of Plant Sciences, Syracuse University.)
exposure to diffuse daylight. Good examples of dust-borne fungal diseases are
coccidioidomycosis and histoplasmosis (see Chapter 4).
The dust in places where psittacine birds (parrots, etc.) are raised and sold
is a source of much serious and often fatal infection with the virus of "parrot
fever" (psittacosis), since the virus occurs in feces and nasal secretions of in-
fected birds. These dry and are scattered as dust about the building.
. In barracks and hospital wards dust and lint from clothing and bedding
are important means of disease transmission, especially of respiratory infec-
tions. One method of controlling this is to impregnate bedding with imper-
ceptible oils which tend to keep the dust from flying about. Floors and sweep-
ings are also oiled. The oil merely controls dust. It does not kill microorgan-
isms (see Chapter 41). Bactericidal sweeping compounds (oiled, disinfected
sand or sawdust) are now commonly used.
([) Transmission by Direct Contact. This means of disease transmission
needs little comment. Obviously, if one rubs against infectious material he
runs a risk of infection. Usually it is easy to guard against such an eventuality,
especially if one avoids transferring the contagium to its special portal of
entry. One does not voluntarily come into physical contact with feces, sputum
or the visible sores or pustules of infected persons. If, in the course of pro-
fessional or home-nursing duties, this cannot be avoided one should wear
rubber gloves or wash and disinfect the hands immediately afterward, without
touching anything first. However, an innocent kiss may transmit tuberculosis,
pneumonia, scarlet fever, diphtheria and other respiratory-borne diseases,
and doubtless does, with tragic results especially to infants and young children
and very old persons. Common examples of contact-transmitted diseases of
adults are syphilis and gonorrhea, both spread by coitus, the former by kissing
also. •
Domestic Environments and Diseases. Infectious diseases are usually much
more frequent and widespread in crowded, unsanitary, living quarters than in
clean, spacious dwellings. This is well illustrated by insect-borne.diseases like
typhus (body lice) and plague (rat fleas) which are notoriously associated with
low-grade living conditions; often as a result of wars. It is equally true of
respiratory diseases and of enteric infections.. Microorganisms spread by oral
and nasal secretions as in sneezing and coughing and by soiled hands, can
much more readily be transmitted from person to person in a close, crowded,
cold and damp room,' than in a spacious, well-ventilated, warm and dry
apartment.
Infection by enteric viruses (polio, hepatitis) and other microorganisms of
the intestinal tract (dysentery, typhoid apd related bacilli; intestinal worms
and protozoa) are obviously transmitted by feces-soiled hands, clothing, soil,
water or food. Ii is very significant that a direct correlation has been sho~n
to exist between many of these diseases and the availability of ample clean
water for domestic purposes," especially for washing of hands and installation
of sanitary plumbing (Fig. 25-6).
2. BIOLOGICAL TRANSMISSION OF DISEASE
Biological transmi;sion of infectious agents differs from the foregoing
mainly in that the vectors are living animals or arthropods. The outstanding
exceptions to this are human blood and blood derivatives.
Disease Vectors 359
(a) Human Blood and Blood Derivatives. Blood not infrequently contains
pathogenic microorganisms. In certain infectious diseases the etiologic agents
circulate in the blood stream for varying periods. Typhoid bacilli are readily
found in the blood during the first week of the disease. Meningococci not in-
frequently occur in the blood, even in the absence of meningitis. Rickettsiae
are present in the blood during typhus and Rocky Mountain spotted fever
and can readily be transmitted by carelessly handled instruments, needles,
syringes, etc., also by blood-sucking insects. Many viruses (polio, yellow fever,
dengue, encephalitis, and so on) and protozoa (malaria parasites, trypano-
somes of sleeping sickness and others) also circulate in the blood.
HOMOLOGOUS SERUM JAUNDICE. A very important virus occurring over
long periods in the blood of apparently healthy persons is that of infectious
hepatitis or homologous serum jaundice. The virus can be transmitted in
blood of donors; in gamma globulin used to prevent diseases like measles;
in blood-bank blood; and by syringes, needles, instruments not properly
sterilized, and the like. Very rigid precautions must be taken in handling any
human blood, tissues or derivatives thereof, or blood-contaminated instru-
ments to avoid transmission of this virus.
BLOOD-BANK BLOOD. Human blood may temporarily have many organ-
isms in it immediately after any severe injury or after some tooth extractions
or surgery, or even in the absence of any injury at all. Blood drawn at such
times for blood-banI: purposes may, if not properly refrigerated, contain
large numbers of bacteria, because the few bacteria which may have been
initially present soon mUltiply to thousands. Even refrigerated blood may
support growth of some psychrophilic organisms. Sometimes blood is con-
taminated by bacteria introduced by the needle from the surface of the skin.
Being recognized, these dangers are pretty successfully avoided.
(b) Bites of Vertebrates. Any animal (or human) bite will introduce a
.
mixture of the microorganisms present in the saliva and on the teeth. Such
/
HOUSE
OUTHOUSE
KITCHEN
WINDOW
UNSCREENED
... ~ ....
~# OPEN WELL
WITH LOOSE
CURBING
Fig. 25-6. How 1m unsanitary outhouse may be a source 01 pollution of a city water
supply (over-the-surface washings and underground seepage), a household well (surface
washings and underground seepage), and a kitchen (flies).
bites are always infectious and should immediately be opened, cleaned, dis-
infected and covered with sterile gauze.
The most notorious pathogen transmitted by animal bites is the virus of
rabies or hydrophobia. Most mammals are susceptible to rabies and can trans-
mit it. Cats, dogs, foxes and wolves are particularly dangerous in this respect.
Within the last decade bats: vampire, insectivorous and fruit-eating, have
been shown to harbor and transmit rabies among themselves and to cattle,
man and other animals.
(c) Bites of Arthropods. Many microorganisms are associated with arthro-
pods. There are several diseases, the sole natural means of transmission of
which is the bite of arthropods. In 1878 a domestic mosquito (Culex quingue-
fasciatus) was shown to transmit the worm Filaria bancrofti, agent of one
form ofjilariasis (a notorious symptom of which is elephantiasis). The classical
observations of Smith and Kilborne in 1893 on transmission of Texas fever of
cattle by the cattle tick (Boophilus annulatus) were the first on tick transmission
of protozoan disease. Certain mosquitoes (Anopheles) and other insects were
later found to transmit malaria (Fig. 25-7). Usually, but not always, each
disease has its own specific insect vector.
Several arthropod-borne pathogenic agents are listed in Table 16.
(d) Insect Feces. Cockroaches were shown as early as 1914 to transfer
cholera vibrios in their intestines for at least forty-eight hours after feeding on
human cholera feces. Ants transmit cholera, and probably other enteric dis-
eases, in the same manner. Flies have long been under indictment for the
same crimes.
The feces of lice infected with typhus rickettsiae will infect if scratched into
the skin. Feces of infected fleas from rats, "prairie dogs" and similar rodents
contain plague bacilli and may contaminate small wounds or scratches. In-
deed, many bloodsucking insects may pass infective agents in the feces and
may also cause infection by being crushed on the skin near, or in, an abrasion
or wound. Engorged ticks on dogs are especially dangerous in this respect
because they contain a relatively large volume of blood which can transmit
the rickettsiae of Rocky Mountain spotted fever. House flies may transmit
poliomyelitis by fecal contamination, as the virus has been demonstrated to
occur in flies. However, the vector seems a very unimportant one compared
to human feces, which is evidently the major vector of polio.
(e) Bodies of Insects. Insects which fly or crawl from unsanitary, un-
screened and undisinfected privies to hospitals or to dwellings may mechanic-
ally transmit intestinal and other disease organisms on their feet and bodies.
/ In areas where flies abound, especially rural or city slum areas, if there is
access to infectious sewage or feces, enteric fevers are usually more prevalent
during the summer months when flies are numerous. In places where city
sewerage systems are not available, fly-borne disease can be avoided to a large
extent by the construction of screened and deep-pit or other sanitary types of
privies or, better still, by the installation of sanitary plumbing and septic
tanks. Plans and specifications for such structures can be obtained from
State Health Departments.
Vertebrate Animals as Vectors of Disease. Animals constitute an enormous
and ever-present reservoir of agents infectious for human beings. Classical ex-
amples are rabies (found in many common animal species), bubonic plague
Disease Vecto,s 361
~~- - UI:£
T_ _
-'\IIgIIIIoncIi_
_
"SQFT" T1QIS
__"
RlIapIinQ F_
, Tock......,.. ~ -lITES
.._, Donnahtio
1IOIII-1ITJN8 FLIES
fIrInItIIiI
~
COnjunctivitis
-1-)""" Types of arthropods transmitting human diseases (sel:cted examples).
Fig. 25-7. Types of insects that transmit disease. (From Stitt, Clough, and Branham,
"Practical Bacteriology, Hematology, and Parasitology," McGraw-Hili, Blakiston Divi-
sion.)
Table 16. Some Arthropod-Borne Diseases; Their Vectors and Animal Reser-
voirs.
,
ETIOLOGICAL AGENTS COMMON ANIMAL
PRINCIPAL KNOWN ARTHROPOD VECTORS
AND DISEASES RESERVOlllS
Viruses
Yellow fever virus Mosquitoes (Aedes aegypti, urban; Man
Hemagogus spegazzini and others. jungle) Jungle monkeys
Dengue virus Mosquitoes (Aedes aegypti) Possibly monkeys
Various encephalitis viruses Mosquitoes (Cu/ex species); ticks; mites; Horses, birds
others
Rickettsiae
R. rickettsii (Rocky Ticks (Dermacentor andersoni, D. Sheep, rabbits,
Mountain spotted fever) variabilis.) dogs
R. prowazekii (classical Lice (Pediculus corporis) Man

typhus)
R. mooseri (endemic typhus) Fleas (Xenopsylla cheopis) Rats
R. orientalis (tsutsugamushi) Mites (Trombicula akamushi) Field mice, etc.
Bacteria
Pasteurella tularensis
(tularemia)
Ticks (Dermacentor species),
Deer-fly (Chrysops discalis)
IRabbits, various
other wild
animals
Pasteurella pestis Fleas (Xenopsylla cheopis) Rats and

(bubonic plague) prairie dogs
Borrelia recurrenlis Tkks (Ornithodoros moubata and others) Possibly rodents,

(relapsing fever) other animals
Protozoa
Plasmodium species Mosquitoes (Anopheles species) Man
(malaria)
Typanosoma species Tse-tse. flies (Glossina 'species) Wild animals

(African sleeping sickness) -
-
/ (rats, ground squirrels, etc.), tularemia or "rabbit fever," tsutsugamushi or
mite typhUS (field mice and other animals) and ornithosis or psittacosis or
parrot fever (parrots, "love birds," etc.). Over 80 diseases found in animals
are communicable to man. These include viral, rickettsial, bacterial, fungal,
and protozoan diseases.
Even the fragile and inscrutable egg is not free from the stigma. Numerous
large outbreaks of food infection (diarrhea, etc.) due to Salmonella sDecies
have been traced to foods made with raw eggs (mayonnaise, etc.). Eggs often
contain infectious organisms when laid by an already-infected hen.
Some of the animals closest to man are constant sources of infection. For
Disease Vectors 363
example, dogs often harbor hookworms which cause creeping eruption
(transmitted by contact with ground soiled with canine feces); Salmonella
species which cause gastroenteritis (transmitted by feces of various farm
animals and poultry); Leptospira canicola and L. icterohemorrhagiae which
cause hemorrhagic jaundice (transmitted by canine and rodent urine);
Rickettsia rickettsii which cause Rocky Mountain spotted fever (transmitted
by ticks: sheep, rabbit and dog); rabies virus (transmitted by bites of numerous
species of mammals); Pasteurella tularensis which cause tularemia (trans-
mitted by flies and ticks which bite rabbits, deer and other wild animals);
the fungi of ringworm (transmitted by contact with skin of cattle, horses,
dogs and cats); and numerous other pathogenic microorganisms. Two of the
commonest serious diseases of animals which affect man are brucellosis and
(outside of the United States) bovine tuberculosis. These are transmitted
frequently by drinking unpasteurized milk of infected cows. Since brucellosis
is also acquired by contact with infected tissues of animals, it is common
among veterinarians, and cattle and swine breeders, as well as among em-
ployees of slaughter houses.
Rats are well known as vectors of plague, murine typhus, leptospirosis,
trichinosis, salmonellosis and other diseases. Rats should be eliminated by
poisoning, trapping, and deprivation of food and breeding places through
cleanliness and rat-proof construction. The fleas of rats, which transmit
plague and murine typhus particularly, may be temporarily exterminated by
dusting runways and places of refuge with DDT.
REFERENCES
Anonymous: Wood Ticks. U. S. Dep't. of Agriculture Leaflet No. 387, 1955. U. S. Gov't,
Printing Office, Washington 25, D. C.
Brandly, C. A., and Jungherr, E. L.: Advances in Veterinary Science. Academic Press, New
York, 1953.
Bureau of Entomology and Plant Quarantine: Insects. The Yearbook of Agriculture, 1952.
Government Printing Office, Washington 25, D. C.
Burns, K. F., Farinacci, C. P., Murnane, T. G., and Shelton, D. P.: Insectivorous bats
naturally infected with rabies in Southwestern United States. Am. J. Pub. Health,
1956,46:1089.
Courter, R. D.: Bat rabies. Public Health Reports, 1954,69:9.
Dack, G. M.: Significance of enteric bacilli in foods. Am. J. Pub. Health, 1955, 45:1151.
Editorial: Control of bacteria in chicken salad. J.A.M.A., 1955, 157:923.
Eklund, C. M: Ecology of mosquito-borne diseases. Ann. Rev. Microbiol., 1953, 7:339.
Hagan" W. A., and Bruner, D. A.: The Infectious Diseases of Domestic Animals. 2nd ed.
Comstock Publishing Co., Ithaca, 1951.
Horsfall, W. R.: Mosquitoes-Their Bionomics and Relation to Disease. The Ronald Press
Co., New York, 1955.
Logan, J. A.: Status <if jnsect and rodent control in Public Health. Am. Soc. of Civil Engi-
neers. Centennial Transactions, 1953, 634.
Maxcy, K. F., et al.: jpreventive Medicine and Hygiene. 7th ed. Appleton-Century-Crofts,
Inc., New York, 1951.
Ordinance and Code Relative to Eating and Drinking Establishments. U. S. Public Health
Service, Publication No. 37, 1950. U. S. Gov't Printing Office, Washington 25, D. C.
Rosebury, T.: Bacteria Indigenous to Man. In Dubos, R. J., Bacterial and Mycotic Infec-
Smart, J.: A Handbook for the'Identification of Insects of Medical Importance. Ed. 3.
British Museuml(Natural History), London, 1956.
Steinhaus, E. A.: Insect Microbiology. Comstock Pub!. Co., Ithaca, N. Y., 1946.
Tanner, F. W., and Tanner, L. P.: Food-borne Infections and Intoxications. Garrard Press,
Champa:gn, Ill., 1954.
Various Authors: Studies on mites as vectors of encephalitis viruses. Am. J. Trop. Med. and
Hyg., 1955,4:90, 106.
Watt, J.: Symposium on diarrheal diseases other than amebiasis. Am. J. Trop. Med. and
Hyg., 1954,4:716.
World Health Organization Technical Report, Series No. 41. International Sanitary Regu-
lations. Geneva, 1952.
World Health Organization, Technical Report Series No. 47. EKpert Committee on En-
vironmental Sanitation. Second Report. Geneva, 1952.
SECTION 4
The Bacteria
(Class Schizomycetes)
THIS SECTION describes severa] groups of important and interesting

bacteria. These conveniently illustrate many important biological facts. They
have some relation to almost every aspect of human affairs and, in general,
constitute a group of organisms knowledge of which is of wide general
interest and utility as well as a part of any well rounded education. Here
we see exemplified such important biological phenomena as evolution,
autotrophy, heterotrophy, photosynthesis, chemosynthesis, anaerobiosis,
primitive communal life, adaptations to the widest range of environmental
conditions, ecology of microorganisms in relation to each other and higher
forms, parasitism, pathogenesis, etc. Many of these phenomena are also
seen in other forms of life: microorganisms as well as macroorganisms. It is
clear that microorganisms illustrate the basic plan oflife.
365
26
The "Slime Bacteria"

{Order Myxobacteriales}
THE MYXOBACTERIALES (myxo is from the Greek meaning slime) are

characterized by (1) the formation of a slimy secretion in which large num-
bers of the bacterial cells live as a sort of community; a so-called "swarm
stage"; and (2) alternation between the swarm stage and a "cyst" stage in
which the cells aggregate into variously formed groups around which the
slime dries, forming a cyst filled with dormant cells. Most myxobacteria are
entirely saprophytic but there are some important exceptions. Most species
of myxobacteria normally inhabit the sea waters, the soil and decaying or-
ganic matter such as old wood, dead leaves, and dung.
1. The Swarm Stage. In this stage they multiply by binary fission and
secrete a slimy matrix in which they all live together. The cells are usually
tapered rods (fusiform) reaching a length of from 10 to 15JL and a diameter of
about O.5,u. They appear to have no distinct cell wall, in this respect resembling
many animal cells. The rods differ from ordinary bacilli in being highly
flexible, often bending themselves into loops and in this respect resembling
the Spirochaetales. The cells move forward also, by means of a slow, gliding
motion. Their motion may be due, in part at least, to the secretion of more
slime at one end than at the other. There are no flagella.
All the cells in a colony move forward together, secreting slime as they go.
"A distinct, firm, hyaline, gelatinous base is secreted by the colony as it
extends itself, over which the individuals may move or in which they may
become imbedded, and is so coherent a structure that whole colonies may be
stripped intact by means of it, from the surface of nutrient agar, for example"
(Thaxter). The swa~ stage lasts for periods varying -from a day or so to a
week. Under favorable growth conditions the entire swarm stage growth may
cover an area of several square centimeters.
2. The Encystmentl Stage. The rods begin to gather together at different
points in the slimy matrix and to heap themselves up. The heaps may become
raised above the substratum. In some species this never develops beyond a
low, rounded hump.IIn others it grows into elaborate, branched projections
(Fig. 26-1). The rods push to the uppermost portions. The rods then become
shortened and roun~ed and are spoken of as microcysts. The gelatinous slime
367
368 The Bacteria
Fig.26-1. Various mature forms of Myxobacteriales. (Redrawn from Thaxter.)
dries as an envelope about them, enclosing them in a cyst or "fruiting body"

within which they resist qrying and other unfavorable conditions. These
fruiting bodies may be as large as 0.1 mm (100 1-1) in diameter. After a resting
stage, the cysts soften and the vegetative rods emerge as new swarm stages,
leaving the empty shell.
'Enzymic Activities of Myxobacteriales. The Myxobacteriales generally
produce enzymes capable of hydrolyzing complex molecules. For example,
some readily dissolve cellulose, others chitin, still others proteins, etc.
Genus Cytophaga. Of the various genera of Myxobacteriales, the Cy-
tophaga (Family Cytophagaceae) are fairly well known. These organisms
inhabit the soil and sea and are of interest because among them are some of
the most active aerobic, cellulose-decomposing bacteria and because they
exhibit several c>ther curious phenomena.
I Most Cytophaga are strict aerobes, growing well at temperatures between
10 and 30° C; prefer slightly alkaline media (pH 7.5); and are sensitive to
acid. An important species pathogenic for commercial fish (c. columnaris)
The "Slime Bacteria" 369
has been cultivated readily on simple media containing peptone (tryptone),
yeast and meat extracts and sodium acetate. Such media may be fluid or
solidified with agar. The colonies of these organisms are beautifully stellate or
arborescent, especially when floating in a fluid medium (Fig. 26-2).
Their motility is of the creeping and flexing types and is ex,llibited only in
contact with some fixed or solid surface. Sometimes they swing by one end
from a fixed surface and oscillate like a pendulum. It is of interest that a
bacteriophage (myxoohage) active in the lysis of C. coiUf11l'1aris has been
demonstrated; the first to be observed in any s~ecies of Myxobacteriales.
Cultivation of Saprophytic Myxobacteriales. The initial isolation of most
of these organisms is not difficult. One method is to place pellets of sterilized
rabbit feces close together on the surface of sifted, fresh soil in covered dishes
and to keep the whole quite moist for a week or two at about 35° C. Many
species grow wen at 10° to 20° C. Pure cultures of some species may be ob-
tained on rabbit-dung agar or on infusion agar. The field is an interesting one
for the student of cryptogamic botany.
For initial isolation of cellulose-utilizing, soil Cy/ophaga an initial enrich-
ment in a solution containing inorganic sources of all necp.ssary rlements
except carbon will suffice. Surh a solution may consist of:
Ingredient Grams
KN03 ............ ·.·.················ 0.1
K2HP0 4 ••.•••••• .'••• •··•· •••••••••••• 0.1
MgS04 .............................. . 0.02
CaCh ................... ·············· 0.01
FeCI) ............................... , 0.002
Water. ............................... . I'JOO
Adjust pH to 7.5.
To this are added some bits of filter paper (cellulose) as a source of carbon.
Fig. 26-2. Cytophaga co/umnaris.

Clusters (or stars) formed in 0.5 per cent
tryptone solution. Living material. Semi-
diagrammatiC (X 125). (Garnjobst, L.,
in J. Bact., vol. 49.)
370
Fig. 26-3. A motile colony of Bacil-

lus (sp.). The colony originated at X and,
during the course of about 3 days at
room temperature, traveled to Y. A por-
tion of the colony then made a small cir-
cular journey before the agar was too
dry. (About life size.) (Preparation by
Dr. Elizabeth I. Paroons. Photo courtesy
of Communicable Disease Center, V. S.
Public Health Service, Atlanta, Georgia. )
At least one species of Cytophaga (c. Jermentans) bas been described as

facultatively anaerobic. It cannot utilize cell)llose, agar or chitin but does use
simple sugars and starch. It requires complex organic sources of nitrogen, and
accessory substances such as the vitamin thiamine.
Relationship to Other Forms of Life. We have already indicated certain
points of similarity between the Myxobacteriales on the one hand and the
Spirochaetales and certain protozoa on the other. These resemblances are
illustrated in the Hexibility and active bending motions of the rods of myxo-
bacteria in the swarm stage and the apparent absence of differentiated cell
wall. The translatory motion of the individual rods of slime bacteria is sug-
gestive of the same type of motion found in sulfur bacteria (Begiatoaceae), in
diatoms (Navicula), and algae (Oscillatoria). The secretion of slime, a striking
character of the Myxobacteriales, is also found in the Cyanophyceae, in true
bacteria, in the iron bacteria and in the sulfur bacteria. The remarkable com-
munal existence of tbe Myxobacteriales, in which cells may aggregate for the
common aim of transportation, reproduction, and dissemination, is remirus-
cent of higher rIant or animal life, as exemplified in Volvox and Pandorina,
although there is no differentiation of functions among the cells of slime
/ bacteria.
In this connection we may mention the curious communal motility seen in
certain species of spore-forming, aerobic rods (genus Bacillus). Examples are
B. alvei and B. rotans. These organisms exhibit motility of their colonies
similar to that of colonies of slime bacteria. This mobility may express itself
as either a rotary motion, or the entire colony may exhibit a migratory mo-
bility, moving over the agar at the rate of 0.01 mm per second (Fig. 26-3).
Motility is first noticeable as unorganized, slow motion of masses of cells
which finally move forward together. It is of interest that the motion is almost
always counterclockwise. How explain this, and how explain the exceptions?
One cannot help wondering if this motile colony might not represent a prim-
1'1re "S~ Btu:teria" 371
itive attempt at communal motility which, however, finds greater expression
in the higher order of Myxobacteriales.
In the development of a common, slimy colony and tall fruiting stalks with
a resistant encysted stage, we find a surprising degree of similarity between
Myxobacteriales and those beautiful creatures, the Mycetozoa (or Myxo-
mycetes) ("slime animals").
The Mycetozoa. In certain groups of these animals a swarm stage is found,
which consists of a mass of living protoplasm (not inert slime) in which are
many nuclei. These masses are capable of ameboid motion and, like true
amebae, can ingest solid particles of food. These are distinctly animal char-
acters. The creatures live on rotten logs, etc., in much the same situations as
myxobacteria and move about in the moisture and shade like amebae. After
several days of such existence they cease to move and reproduction begins.
The protoplasm sends up stalks on the tips of which sporangia (spore-bearing
cysts) are formed, in a great variety of the most graceful and delicate
forms and of the most brilliant colors. Each sporangium contains many
spores and each spore contains a single nucleus and is surrounded by a
cellulose wall. The spores are dispersed by the wind. In water, each spore
germinates, forming a naked, ameba-like creature. These forms mUltiply
rapidly by cell division. They later coalesce or fuse, a presumably sexual func-
tion, eventually to form the multinucleate swarm stage.
The resemblance between these two forms of life is so close, yet so super-
ficial, as to suggest the idea that a comparative study in morphology and
function might have been in progress.
REFERENCES
Bachman, B. J.: Studies on Cylophaga /ermenlans, n.sp., a facultatively anaerobic lower
Myxobacterium. J. Gen. Microbiol., 1955, 13:541.
Buchanan, R. E., and Beebe, J. M.: The Myxo"acteriales. In Bergey's Manual of Determina-
tive Bacteriology, 6th ed. The Williams & Wilkins Co., Baltimore, Md., 1948. p. 1005.
Carr, L. G.: Mycetozoa, animal-plant organisms. Sci. Monthly, 1941.53:175.
Ocdal, E. J., and Rucker, R. R.: PathOgenic myxobacteria. Proc. Soc. Exp. BioI. and Med.,
1944,56:\5.
Sussman, M.: The biology of the cellular slime molds. Ann. Rev. Microbio!., 1956. 10:21.
Thaxter, R.: On the Myxobacteriaceae, a new order of Schizomycetes. Bot. Gaz., 1892,
17:389; 1904,37:405.
27
The "Sheathed" Bacteria

(Order Chlamydobacteriales) and
"Stalk-Forming" Bacteria
(Suborder Caulobacteriineae)
THE NAME Chlamydobacteriales· refers to the order of sheath-forming

bacteria. It was given because of the presence of a cylindrical sheath or mem-
brane inside of which the organisms live. Chlamydobacteriales are aquatic,
saprophytic, filamentous forms. They are important as (I) scavengers in
sewage; (2) causes of obstructions in water pipes.
Structure. The sheath of Sphaerotilus, one of the commonest forms of
Chlamydobacteriales, is an excretion product, in some respects like a cap-
sule, but a more distinctly differentiated structure than a capsule. The sheath
is soft and flexible, looking and behaving much like a clear, cellophane or
paper cylinder such as a drinking tube. Its composition is not fully known.
It may be mucilaginous; it is not chitinous.
Growth. When the growth of Sphaerotilus natans (a species common in
sewage) is young, the filaments resemble hyphae of coenocytic molds, though
"acterial in dimensions (about 1.5 to 2 . 0~ in diameter). As the growth ma-
tures, the protoplast becomes divided up into bacilli from 2.5 to 16~ long
with lophotrichous flagella. These multiply by binary fission. The resulting
motile bacilli slip out at the ends of the sheaths or are liberated at the sides as
the sheaths disintegrate (Fig. 27-1). Sometimes the young cells cling to the
I outside of the sheath of origin and grow off at an angle. This is called false
branching. In one variety, called S. dichotomus (possibly identical with S.
natans), the false branching appears to be dichotomous.
Sphaerotilus natans is aerobic (though requiring very little oxygen), is non-
sporeforrning and is heterotrophic though it can use inorganic or organic
sources of nitrogen. It does not require vitamins.
Spbaerotilus (or Leptothrix) and the Iron Bacteria. For many years certain
filamentous bacteria occurring in iron-bearing, natural waters have been
thought to oxidize the iron as a source of energy and to deposit the oxidized
• Chlamydo IS from the Greek word meaning a cloak .
372
The "Sheathed" Bacteria and "Stalk-Forming" Bacteria 373
iron in their sheath. They have been called "iron bacteria." A widely-known
species is called Leptothrix (or Sphaerotilus) ochracea.
The yellow or reddish colored slime found in the beds of iron-bearing
waters is nearly always due to ferric hydroxide in the sheaths of these and
related iron-accumulating bacteria growing on the stream bottom. Living,
iron-accumulating bacteria are seldom found in such iron deposits since the
incrustation of the membrane with iron interferes with their reproduction.
The role of iron in the physiology of these organisms is interesting but not
wholly clear. According to a logical view, only those organisms which oxidize
inorganic,jerrous iron compounds as a source of energy should be classed as
true, iron bacteria. A reaction often given to explain this process is:
4FeC0 3 + O2 + 6H20 -+ 4Fe (OHh + 4C02 + 40 cal.
A number of similar bacteria utilize organic compounds containing iron,
but do not oxidize the iron itself. Such organisms, utilizing only the organic
radical of iron-containing compounds, should not, according to Winogradsky,
be classified as true iron bacteria since their energy is obtained by oxidation
of the organic matter, not the Fe++.
Those species oxidizing FeC0 3 will necessarily deposit much waste Fe(OHh
in the sheath since the energy yietd is onty to calories per gm. moL of FeCO ~
and only 8 to 10 per cent of this is used by the organisms. Organisms oxidizing
only the organic radicals of iron compounds obtain so much energy from the
organic matter that relatively small amounts of the ferrous food are needed
and little or no iron is deposited in the sheath. This organic iron is probably
oxidized by free oxygen, extraneously to the cell, and yields no energy to the
elCll. Such bacteria are not true, "iron bacteria."
Genus Crenotbrix. Crenothrix poiyspora, a very common and representa-
dve species of Chlamydobacteriales, starts life as a single, nearly spherical,
eon-motile cell about 2p. in diameter which divides by fission and forms a
chain, much as do streptococci. They differ from streptococci in that the cell~
' _'na"tp as they divide, so that the row shortly consists of bacillary forms.
They exude a mucilaginous, tubular sheath about O.2p. in thickness. The
Fig. 27-I. Sphaeroti{us notans, show-

chain of bacillary cells inside their
flexible, tube-like sheath.
of Dr. J. L. Stokes,
Utiliz. Br., Agr. Res. Serv.,
Dept. of Agriculture, in J. Bact.,
vol. 67.)
374 TM BIlcterill
sheath soon hardens and becomes impregnated with ferric hydroxide. This
suggests a true iron bacterium. The topmost cells are continuously being
pushed out of the mouth of the sheath by the growing cells below, tl:e fila-
ment having attached itself by one end to some solid object.
Very often, in older plants, fission of the uppermost cells takes place in
three planes before their liberation from the sheaths. The increase in volume
within the sheath forces the latter to expand at the upper end so that a trum-
pet·like appearance is produced. This is a striking characteristic of Crenothrix
polyspora.
Crenothrix can live inside piped water systems, if the water does not contain
too much dissolved oxygen and does contain iron. like Sp/ aerotilus, Crena-
thrix mayor may not be a true iron bacterium but is truly an iron-accumulating
bacterium. It forms large gelatinous masses that clog the pipes. When the
mature plants die under such conditions, they may decompose, with a re-
sultant bad odor and taste that cause discouraging days in the "complaints"
office of the head engineers of water purilication plants.
Systematic ReiatioMbips of the "Sheath-Formers." The relations of these
organisms to other groups of organisms are not entirely clear. The order as a
whole seems to be more bacterial than mold-like or alga-like. It is not photo-
synthetic. It is entirely aquatic while most molds are not. In the matter of
thread formation the Chlamydobacteriales resemble molds but the threads
are much tinier than any mold threads and are oftbe same order of magnitude
as bacteria. The absence of readily demonstrable nuclei is also a distinctly
bacterial character. The cells, removed from their sheaths, are distinctly
bacterial.
Where branching is present, it is seen to be false branching due to adhesion
of filaments to one another and not to an actual branching of individual cells
as is seen in molds or Actinomycetales. The resemblance to molds is again
seen to be merely a superficial one.
The Chlamydobacteriaceae have often been classed as "higher bacteria."
Actually, if we consider the types represented by Sphaerotilus and Crenothrix.
we find them not so very much higher but, as Ellis says, they may be regarded
as bacteria which "have taken the first step toward what may be regarded as
the communal life."
THE CAULOBACTERIINEAE·
The systematic position of these bacteria is somewhat difficult to determine.
As will be seen, iron accumulation is a feature of some, but sheath-formation
/ is absent. The organisms are characterized by forming stalks.
The majority of the Caulobacteriineae have been given little attention by
most bacteriologists. They are of importance in studies of marine and aquatic
problems such as ecology of fish and aquatic vegetation. rotting of ships and
piling, etc. Here we will describe only three species: Gallionella ferrug;nea,
Siderocapsa treuhii, and CauJobacter vibrioides.
GallioaeUa Ferruginea. This species forms a stalk which, as the plant
matures, becomes encrusted with Fe(OHh It does not form a sheath. It is
said by some to be a true, iron-oxidizing bacterium but this is doubtful. Its
exact taxonomic position is at present obscure.
• Caulo is from tbe Latin word caulis for stalk.
The cells of this organism are bean-sbaped and about 0.51' by 21' in size.
From the concave side of each cell there is excreted a fiat, mucilaginous rib-
bon or stalk. As each cell divides, branching of the stalk occurs so that com-
plex tangles, or rosettes of long stalks, are formed. The stalks are sometimes
0.2 mm to 0.3 mm in length (200-300 p; Fig. 27-2).
The stalks of Gallionella have the remarkable habit of twisting so that they
resemble a loosely coiled rubber band. Large amounts of iron are later de-
posited in these stalks, giving them the appearance of a series of (oops or
string of beads. The twisting habit renders identification of Gallionella easy,
since no other organism of similar character is known to twist in just this
way. As stated by Trumann "-the gallionellas are more notable for their
excreta than for themselves."
This organism is found in nature as widely distributed as Sphaerotilus. Like
other iron-accumulating bacteria, it can multiply in water pipes, and often
causes extensive deposits and incrustations of iron which may eventually
occlude the pipes.
Siderocapsa treubii is a cocco-bacillus about 0.5 by 2 p. This organism
envelops itself in a thick, slimy or gelatinous, extracellular substance. This
does not appear as a tangle of stalks, but in the form of a compact, flat mass
attached to some object such as a stone, the underside of a leaf fioating in a
stagnant pond, or a glass slide immersed to collect growths of such organisms
in streams. The gelatinous material may be regarded as a modified stalk or
sheath. Sometimes these growths are quite extensive and iron is deposited in
A B
PIa. 27-2. GaIlionella /em'Iinea. A shows the curved cells at the ends of twisted, appar-
ribbon-like excretion bands. These bands have not yet become heavily encrusted with
. " I(UI:I)3. Microphoto at a magnitication of X 1120. (From Cholodny, in Starkey, 1. Am.
Assoc.) B shows the organisms and the bands (here encrusted with Fe(OHh.
with the electron microscope at a magnification of X 12.000. The excretion bands
to be fibrillar in structure and to exhibit a segmented pattern. The fibers appear
.fraaiie and brittle. (photo courtesy of Drs. A. E. Vatter and R. S. Wolfe. in 1. Bact••
vol. 72.)
376
A B
Fig. 27-3. Electronographs of Caulobacter. A, C. vibrioUks, showing stalks attached
Ul a central point, forming a characteristic "rosette." (X 12,500.) (Courtesy of Dr. E. A.
. Grula, in J. Bact., 1954.68:498.) B, Caulobacter sp., showing stalks attached to, and appar-
ently inserted into, ceUs of Bacillus megatherium (a common, saprophytic sporeformer of
dust, water, etc.) Note the smaU size of Caulobacter in comparison with B. megatherium.
tX 6000.) (Courtesy of Prof. Dr. A. L. Houwink, Delft: in A. V. Leeuwenhoek J. Micr. and
Serol., 1955, vol. 21.)
large amounts around them. They are probably important in the fouling of
pipes, but have often been overlooked because their growth is not so extensive
as that of filamentous forms. Some question whether Siderocapsa is truly a
bacterium.
Caulobacter Vibrioides, The structure of the stalk of this organism is so
distinctly different from both of the foregoing that it should probably be re-
moved to an entirely different group. The body of the bacterium is spindle- or
banana-shaped, curved, and pointed at both ends. It often contains granules
of volutin, fat and "cell sap." The organisms occur in the general aquatic
habitats of Chlamydobacteriales, Gallione/la, etc., and are of the same order
of size and structure as bacteria in general. They multiply by transverse,
binary fission, are non-sporeforming and, when young, motile with polar
flagella. They have a distinct capsule or slime layer. They may be cultivated
in pure cultures using much the same procedures and organic media as are
used for other Caulobacteriineae and Chlamydobacteriales.
The peculiarity about the stal~ is that it is not an excretion but a distinct
/ part of the cell itself; a narrow, flexible, tubular outgrowth closely associated
with, or possibly (-as the cell matures) including, the polar flagellum. This
stalk-like outgrowth apparently contains live proto~lasm. At the tip is an
enlarged, button-like, hold-fast, by which the organism attaches itself to
solid objects. In electronographs, fine fibrils are often seen extending from the
hold-fast.
These protoplasmic stalks may be very short but are frequently many times
as long as the bacterial cells. Often these stalks are attached to, or even (in
a possibly parasitic manner) inserted into living (later, dead I) cells of certain
species of Bacillus. This seems to be a unique method of parasitism (Fig.
27-3).
REFERENCES
Houwink, A. L.: Caulobacter: Its morphogenesis, taxonomy and parasitism. Antony van
Leeuwenhoek J. Micr. and Serol., 1955, 21 :49.
Meier, F. E.: Plankton in the Water Supply. Ann. Rep't., Smithsonian lnst., 1939, p. 393.
Stokes, J. L.: Studies on the filamentous sheathed iron bacterium Sphaerotilus natans.
J. Bact., 1954,67:278.
Strandskov, F. B.: Slime forming organisms. J. Am. Water Wk. Assoc., 1948,40:1299.
Thimann, K. V.: The Life of Bacteria, Chapter 21. The Macmillan Co., New York, 1955.
28
The Sulfur Bacteria
THE TERM "sulfur bacteria" has had various meanings: bacteria which
store up globules of elemental sulfur, intracellularly, as reserve food material;
bacteria which reduce or oxidize sulfur compounds. As used here it signifies
bacteria (a) which oxidize sulfur or its inorganic compounds (except sulfates)
as a source of energy; and (b) which reduce sulfates, utilizing them as an
acceptor for elemental hydrogen or hydrogen removed from organic or
inorganic substrates. Many of these also store eiemental sulfur intracellularly
(see Table 17).
Sulfur available to microorganisms exists in various stages of oxidation
and reduction in inorganic compounds; ranging from the most reduced, H 2 S,
through elemental S, thiosulfates, tetrathionates, etc., to the most oxidized
form, sulfates. Any of these, except sulfates, may be oxidized as energy sources
by various bacteria; and any, except H 2 S, may be reduced by still other
bacteria.
Groups of Sulfur Bacteria. The sulfur bacteria may, for convenience of
discussion (not as a recognized arrangement), be divided into five groups,
four of which comprise oxidizers of sulfur and its compounds (except sul-
fates). The fifth group contains species which reduce sulfates. For complete-
ness Table 17 includes, as a sixth group, a variety of bacteria which decom-
pose organic sulfur compounds, producing H 2S (putrefaction).-These are
not regarded as true sulfu~ bacteria and ate not discussed in this chapter.
A. SULFUR OxIDIZERS
1. PHOTOSYNTHETIC. Two groups. of sulfur oxidizers comprise Athio-
rhodaceae, * Chlotobacteriaceae* and. Thiorodaceae. * All of these contain
bacteriochlorophyll or chlorophyll-like' photosynthetic pigments. They may
be rod, vibrio or 'spiral-shaped, much like true bacteria. In the presence of
H2 S the Thiorhodaceae store elemental sulfur in the form of conspicuous
granules inside the cells. It is later oxidized to H 2S0 4 , (See reactions below.)
The Chlorobacteriaceae do not store sulfur granules intracellularly but excrete
the sulfur in free form outside the cells. The Athiorhodaceae, as their name
implies, neither store itttracellular sulfur granules nor release free sulfur into
the surrounding medium. Some oxidize thiosulfates, some oxidize elemental
• In the suborder Rhodobacteriineae, order Eubacteriales.

378
The Sulfur Bacteria 379
hydrogen, but most of them are heterotrophs and use organic sources of
energy. These photosynthetic bacteria are more fully discussed in Chapter 29.
2. NONPHOTOSYNTHETIC. (a) SULFUR STORERS. This group includes two
other families of organisms which are sulfur-storers but not photosynthetic.
One family (Beggiatoaceae) consists of filamentous forms. These filaments
float free and unattached and have no sheath. One species (Thiothrix nivea)
resembles Crenothrix, the iron bacterium (see Chapt. 27), in forming a sheath
and in attaching itself to solid objects.
The other family (Acbromatiaceae) in this group consists of large, ovoid
or rod-shaped cells, not forming filaments.
(b) NONSULFUR STORERS. This group of sulfur oxidizers, being simple,
non-sporeforming, non-branching, non-sheathforming, gram-negative rods,
are classed in the genus Thiobacillus. * In this genus sulfur, while used in the
Table 17. Relations of the Sulfur Bacteria.
OXIDIZE SULFUR AND ITS INORGANIC COMPOUNDS
INTRACELLULAR NO INTRACELLULAR PRODUCE HzS FROM

REDUCE
SULFUR GRANULES SULFUR GRANULES ORGANIC SULFUR
SULFATES
COMPOUNDS
PHOTO- NOT PHOTO- PHOTO- NOT PHOTO-

SYNTHETIC SYNTHETIC SYNTHETIC SYNTHETIC
Thiorhoda- Beggiatoaceae Chlorobac- Thiobacillus Desulfo- Various pathogenic

ceae (filamentous) teriaceae vibrio and saprophytic
Acbromatia- Athiorho- Sporovibrio (putrefactive) spe-
ceae (noo- daceae , cies: Proteus, Serra-
filamentous) tia, Clostridium, etc.
form of HzS as well as in an elemental form as a source of energy, is not

held inside the cells. On being released during the oxidation of H2S, it ac-
cumulates as a free scum or precipitate in the medium. It is afterwards further
oxidized to H 2S04 ,
B. SULFATE REDUCERS
A fifth group of highly specialized sulfur bacteria (sulfate reducers) is
characterized by the power to utilize sulfates as hydrogen acceptors in
respiration. The sulfates are reduced to HzS. This group is comprised chiefly
by the genera Sporovibrio and Desulfovibrio. t '
The group of sulfur bacteria, as defined above, is seen to be quite hetero-
geneous and to recruit membership from widely scattered sources among the
Schizomycetales.
Habitat of Sulfur Bacteria. Sulfur-utilizing bacteria, photosynthetic and
non-photosynthetic, sulfur-storing and non-sulfur-storing, oxidizing and re-
I,
• In the suborder Eubacteriineae, order Eubacteriales.
t Suborder Eubacteriineae, order Eubacteriales.
380 TM Bacteria
ducing, are found in sewage and other polluted waters, in decomposing or-
ganic matter, and in swampy soils all over the world where putrefactive or-
ganisms are releasing H 2S from dead plants and animal wastes, or where
sulfur-reducing species (Desu/fovibrio, etc.) are reducing sulfates to H2S.
Some are found around free sulfur deposits. Some occur in acid coal-mine
waters; others in garden soil.
The photosynthetic varieties develop best (or only) when exposed to light.
In certain sunlit, polluted bays they multiply to such an extent as to cause the
entire body of water to look red or purple. Certain autotrophic, photosyn-
thetic species grow in hot sulfur-springs, and others around sulfur mines.
SULFUR OXIDATION
Beggiatoaceae and Acbromatiaceae. These non-photosynthetic, sulfur-
storing bacteria thrive in sewage and brackish muds. They utilize hydrogen
sulfide and sulfur as sources of energy, probably according to the equations
2H 2S + O2 -+ 2H2 0 + 2S
2S + 302 + 2H20 -+ 2H 2S04
Beggialoa alba, a representative species of these organisms, common in
sewage, is autotrophic. B. alba requires free oxygen as indicated in the equa-
tions above. The acid combines with chlorides, metals, etc., to form sulfates.
Sulfates are valuable as the principal sulfur compound available to higher
plants.
Beggiatoa show creeping and waving movements suggestive of the alga
Oscil/aloria. Typical filaments of B. alba range in diameter from 3 to 50~ and
up to several millimeters in lenglh. Reproduction of B. alba is by fragmen-
tation of the filaments. Fission also occurs, as in true bacteria. When plenty
of H2S is present the organism stores colloidal globules of sulfur in the cells,
giving the organisms a distinctive, milky appearance (Fig. 28-1). When H 2S
is scanty the stored S is utilized. When neither H 2S nor stored S is available
the organism dies.
The Genus Thiobacillus. Thiobacilli are classed with the true bacteria and
are typical, autotrophic, chemosynthetic Eubacteriineae. Thiobacilli are small,
gram-negative, non-sporeforming, rod-shaped bacteria, some of which are
motile, others non-motile. They thrive in mud, sea water, boggy places, coal-
mine drainage, and so on, where sulfur and its compounds occur as a result
of chemical action or due to protein decomposition or the sulfate-reducing
/ activities of Desulfovibrio and related species.
Thiobacillus oxidizes sulfur or its inorganic compounds in various ways.
1. 5Na2S203 + H 20 + 40 2 -+ 5Na2S04 + H 2S04 + 4S (Thiobaci/lus
thioparus). Probably this organism forms intermediate compounds like poly-
thionates in the course of this oxidation:
2. 2Na2S203 + ~02 + H 20 -+ Na2S406 + 2NaOH.
3. 2S + 302 + 2H 20 -+ 2H 2S04 (Th. thiooxidans).
Among the most interesting and completely studied species of this genus
are Thiobacillus Ihioparus, Th. thiooxidans and Th. denitrificans. These are
strict autotrophs. Solutions like the following meet all of their nutritive
requirements :
H20 .. . ...... . ..................... . . . .. . ... . 100.000 mI
S ....... .. . . ... . ... .. ... .... . . ... .. . .... . . .. . 1.000gm or
Na2S203.· · ·· . . . ··· . . · ·· ... ··· . . · · · .. · · ····· . O.SOOgm
(NH.)2S0 4 • ••• ••• • •• ••• • •• •••••••••••• • •• • • • • 0.03Ogm
KH2PO•. . ......... . . . . . ... .... .. .. . ... ...... 0.025 gm
CaCh ... ... · ..... .......... .. ... · ....... . . . ·· O.OSOgm
FeSO•.. .. .............. . ... . . . .. ... . .... .. ,. 0'()01 gm
Thiobacillus thioparus oxidizes sodium thiosulfate, hydrogen sulfide or

IPllfur aerobically. The last is transformed quantitatively into sulfuric acid.
Fig.28-1. upper, portions of actively motile threads of Ikggialoa alba from a picture
originally made at a magnification of 2,770. Numerous sulfur vacuoles are distributed
throughout the length of the continuous thread. Lower, a tangled mass of living Ikggiatoa,
as seen under a 10 X apochromatic objective and 15 X ocular. (Johnson and Baker, J. CcII.
and Compo Physiol., vol. 30.)
382 The Bacteria
Thiobacillus dentrijicans oxidizes hydrogen sulfide, etc., under strictly
anaerobic conditions, using nitrates as hydrogen acceptor and reducing them
to nitrites. The metabolism of Th. denitrijicans is of especial interest since
this represents one of the factors responsible for losses of fertility in certain
soils: dentrijication, or reduction of nitrates.
5S + 6HN0 3 + 2H20 _, 5H2S04 + 3N2(+ E)
Thiobacillus thiooxidans oxidizes sulfur to sulfuric acid aerobically. As
sulfuric acid is formed in considerable amounts, it might be thought that the
organisms would quickly inhibit their own further growth. This species,
however, is of interest in having a great resistance to acid. It is "distinctive in
that it is able not only to tolerate but to produce higher concentrations of
acid than any other living organism yet known" (Starkey). Some growth is
said to occur at a pH of I, and it grows readily at pH3.
Free sulfur is deposited by most species of Thiobacillus, especially Th.
thioparus, in granules outside the cell proper and forms a scum' or precipitate
in flasks of medium. Most of the species oxidize it further to sulfuric acid.
An interesting physiological question arises as to how sulfur particles,
water-insoluble, pass through the bacterial cell wall. No extracellular enzymes
are known which fluidify sulfur. A plausible idea is that the sulfur, which
must be present in very smaIl (coIloidal) particles, is soluble in certain lipid
(fat-like) components of the cell wall. Sulfur first dissolves in these and so
enters the cell. It is conceivable also that other water-insoluble (or slightly
soluble) substances may enter the cell by analogous methods.
Ferrobacillus ferrooxidans. This important, strictly autotrophic micro-
organism has most of the properties of the genus Thiobacillus but does not
readily oxidize sulfur compounds. It occurs in the acid (pH 2.3) seepage
water of coal mines. Such water contains much ferrous iron. Unlike Thio-
bacillus, this organism gains its energy by the Qxidation of ferrous sulfate; a
true iron bacterium but not a sheath former. The reactions involved may be
similar to the following:
4FeS04 + 2H2S04 + O2 _, 2Fe2(S04h + 2H 20
Fe2(S04h + 6H 20 _, 2Fe(OHh + 3H 2S04
F.ferrooxidans can grow in a solution containing: (NH 4hS04, KCI, MgS04·
7H 20, K 2HP04, Ca(N0 3)2. and FeS04·7H20; pH about 3.5. Note the ab-
sence of carbon source. _
A closely similar organism, called Thiobacillus (errooxidans, can utilize
thiosulfates as well as FeS04 as a source of energy. One of these two organ-
isms may be a variant of the other. '. '
FUNCTIONS OF SULFUR OXIDIZERS
These sulfuric-acid-forming bacteria are of great importance as agents in
the acid-disintegration of various rocks and minerals, with the liberation of
such valuable elements as phosphorus, magnesium, and sodium.
Ca3(P04h + ,6H 20 + 3H 2S04 _, 2H 3P04 + 3CaS04·2H20
Soluble phosphates are among the most important and expensive fertilizers.
The transformation of barren alkali soils in desert places to fertile ones may
be brought about through the activities of such organisms under conditions
of irrigation.
The sulfuric-acid producers playa very important role in oxidizing offen-
sive sulfur compounds in sewage and other decomposing organic matter.
They are of importance in making sulfur available for other living creatures
and thus furthering the sulfur cycle (Fig. 28-2).
On the other hand, such organisms may prove to be important nuisances
because acid production in some situations, as in coal mines and iron water
pipes, causes costly damage.
SULFATE REDUCI10N
Reduction of sulfates is the distinctive physiological character of our fourth
group of sulfur bacteria. The power of reducing sulfates is not common in
bacteria, and with respect to soil fertility, is analogous to nitrate reduction.
Distribution and Structure. Sulfate-reducing bacteria are widely dis-
tributed, especially in polluted water, the sea and marine muds. They have
not been very widely studied but are extremely important. Two of the best
known types are curved-rod (vibrio-like) organisms which are classified under
the generic names Sporovibrio and Desulfovibrio (Eubacteriineae). These are
simple, unbranched, non-filamentous forms (Fig. 28-3). Sporovibrio produces
spores. It may be a spore-forming variant of Desulfovibrio.
DESULFOVIBRIO DESULFURICANS, a well known species which occurs in
fresh water, ar!,! strict anaerobes and heterotrophic. Organic materials are
dehydrogenated and the hydrogen is transferred to sulfates which are reduced
to sulfides. An equation illustrating this type of reaction is:
2CH 3·CHOH·COONa + H 2S04 --t 2CH3·COONa + H 2S + 2C02 + 2H20
Sodium lactate Sodium acetate
(hydrogen donor)
DESULFOVIBRIO AESTUARII, found in sea water, Cilll reduce sulfites, thio-
sulfate and elemental sulfur to H 2S.
~ COMBUSTION ~
I'Sj
SULfUR OXI 0 I z- . S02.
ING B~C~ERIA \
HS 2.
SULfATE REDUC-
ING B~CTE:RIA
HSO~
2
"\ I J
MISCELLANEOUS PlANTS AND
BACTERIA ANIMALS
." DEATH AND /
i "ORGANIC VAST[S OF ~
, SULfUR - PLANTS AND
I COMPOUNDS ANIMALS
Fig. 28-2. The sulfur cycle. Sulfur in its most reduced form, H2S, is found in the soil,
waters and atmosphere as a result of decomposition of animal and vegetable remains, and
volcanic action. Free sulfur occurs as such in some soils, and in mines. Both Sand H 2S are
oxidized by combustion and by microorganisms to sulfates which are used by plants, and
these, in turn, by animals. The reduction of sulfates by Desulfovibrio, etc., is indicated by
the right-to-Ieft arrow across the center of the diagram.
384 The Bacteria
Fig. 28-3. Spiral ceUs of the sulfate-

reducing Sporovibrio desulfuricans (X
1660). (From Starkey, J. Amer. Water
Works Association.)
Some sulfate reducers can grow autotrophically. They obtain energy from
oxidation of hydrogen gas and use sulfate as the hydrogen acceptor. The sul-
fate is reduced to H 2S by means of the enzyme hydrogenase:
4H2 + H 2S04 -+ H 2S + 4H20
TheSt organisms can also oxidize petroleum hydrocarbons like kerosene, etc.,
anaerobically, utilizing sulfate as H acceptor. They are of great importance
in the petroleum industry because they cause serious losses in the petroleum
products.
The low sulfate content of oil-well brines, coupled with the fact that sulfate-
reducers may transform certain organic compounds into hydrocarbons, sug-
gests a possible association between these organisms and the formation of
petroleum. Further details may be found in the literature cited.
Rr:FERENCES
Fry, B. A., and Peel, J. L., Editors: Autotrophic Microorganisms. Fourth Sympos., Soc. for
Gen. Micr., Cambridge Univ. Press, New York, 1954.
Gleen, H., and Quastcl, J. H. : Sulphur metabolism in soil. Appl. Micr., 1953, J :70.
Griffiths, M., and Stanier, R. Y.: Some mutational changes in the photosynthetic pigment
system of Rhodopseudornonas spheroides. J. Gen. Microbiol., 1956,14:698.
Harold, R., and Stanier, R. Y.: The genera Leucothrix and Thiofhrix. Bact. Rev., 1955,
19:49.
Johnson, F. H., and Baker, R. F.: The electron and light microscopy of Beggiotoo. J. CcII.
and Compo Physiol., 1947,30: 131.
Leathen, W. W., Kinsel, N. A., and Braley, S. A., Sr.: Ferrobacillus ferrooxidans: a
chemosynthetic autotrophic bacterium. J. Bact., 1956,72:700.
Sisler, F. D., and ZOBell, C. E.: Hydrogen-utilizing, sulfate-reducing bacteria in marine
sediments. l. Bact., 1950,60:747.
Starkey, R. L.: Characteristics and cultivation of sulfate-reducing bacteria. J. Am. Water
Wks. Assoc., 1948,40:1291.
Vishniac, W.: The metabolism of ThiobociJlus fl,ioportls. J. Bact., 1952,64;363.
ZoBell, C. E. : Marine Microbiology. The Chronica Botanica Co., Waltham, Mass., 1946.
29
The Photosynthetic Bacteria
THE PHOTOSYNTHETIC bacteria are of two main types: green (Chloro-

bacteriaceae) and red or purple (Thiorhodaceae* and Athiorhodaceae). Both
types contain photosynthetic pigments. The photosynthetic pigments of these
bacteria consist of one or more forms of the bacteriochlorophyll molecule.
In some species several carotinoid pigments are also present which give vari-
ous colors (i.e., red, purple, brown) to the organisms containing them. The
carotinoid pigments aid in capturing the energy of visible light in addition to
ultraviolet.
ChIorophyUs. The bacteriochlorophylls belong to the group of chromo-
proteins. They are remarkably similar to the chlorophylls of green plants. All
contain Mg. They are chemically related to the ~espiratory, red, iron-contain-
ing hemoglobin molecule ; the blue, copper-containing hemocyanin of lobsters
and related animals; and the iron-bearing cytochrome respiratory pigments
which are found in all aerobic bacteria.
Green-plant chlorophylls absorb light in wave-length ranges around 6700
A (blue-green) and also 4570 A (violet). The green bacteriochlorophyll of
Chlorobacteriaceae absorbs waves around 7400 A (orange-yellow). The bac-
teriochlorophylls of red and purple species absorb light energy of much longer
wave lengths; in the infra-red range, around 8000 to 9000 A. The brown, red
and purple carotinoids, absorbing similar long waves, aid in this absorption
of light energy.
Habitat and General Properties. The photosynthetic bacteria are found
chiefly in marine and aquatit; habitats, stagnant lakes, polluted bays, sea
water, brackish ditch water, etc. Morphologically they are like typical cocci,
bacilli, vibrios and spirilla. They are not filamentous like Beggiatoa or Creno-
thrix. They are entirely saprophytic and have no special industrial, agricultural
or medical relationships. They are, however, of much interest to the biologist
and the biochemist shrewd enough to see their possibilities.
Use of Photosynthesis. For example, in these days of scientific search for
energy sources mankind investigates solar turnaces (vast, lens-like mirrors to
act as enormous "burning glasses"), atomic piles, nuclear reactors and still
other mechanisms. Not the least intensive is the effort to determine how
chlorophyll collects solar energy and synthesizes, quietly and efficiently,
• Thio is from a Greek word for sulfur; rhod is from a Greek word meaning roS(Xolored.
38S
386 The Bacteria
enormous quantities of valuable organic materials in plants (wood, sugar,
starch, drugs, vitamins, etc.) mainly from CO2 and H 2 0.
The structure of the chlorophyll molecule is largely known; it has been
isolated and inspected at 60,000 X magnifications; its location and arrange-
ment in the cell have been determined; its raw materials are obvious; its first
end product has been isolated; what it does is clear. But just how it (presum-
ably) splits hydrogen from wtter and transfers it to CO2 (though it does so
all day long under our very noses) is like the elusive pea in the "3-shell Monte"
game: look as closely as we like, "we can't quite figger it out!" The photo---
synthetic bacteria furnish excellent study material and are much used for such
investigations.
An interesting development in our knowledge of photosynthesis is the dis-
covery of some of the first compounds formed by the photosynthetic process.
Supplying CO 2 made with radioactive carbon (0 4 0 2) to photosynthesizing
algae (Scenedesmus), the compounds formed during the first few seconds of
photosynthesis were: phosphoglyceric acid (87%), phosphopyruvic acid (10%)
and malic acid (3%). A few minutes later in the process, the 0 4 was found in
many other compounds: amino acids like alanine; sugars like sucrose, fruc-
tose and glucose; organic phosphorus compounds; organic acids like suc-
cinic acid. The actual synthetic reactions involved are not clear, but the whole
synthetic mill, the entire "production line," goes forward smoothly, with
lightning-like speed. The first compounds mentioned were formed in five
seconds, and many more were formed in workable quantities within fifteen
seconds to a minute or more; all in silence, with no visible motion, confusion,
odors, slag piles, strikes, wars or social upheavals I
PHOTOSYNTHESIS AS A TYPE OF REACIlON

1. Green Plants. In photosynthesis by green plants hydrogen is removed
from water under the influence of sunlight. A simplified form of this reaction
is as follows:
light
CO 2 + 2H 20 ~ (CH 20)*+ H 20 + O2
Chlorophyll
Actually, what is being dealt with is electron transfer by which energy is

liberated. Any substance which, in the chlorophyll system, will yield hydrogen
(an electron), will serve the purpose. In green plants apparently only water
will serve.
2. Chlorobacteriaceae and Tbiorhodaceae. The Chlorobacteriaceae and
Thiorhodaceae utilize H2S in photosyntnesis. Hydrogen is transferred from
H 2S to CO 2 :
light
CO2 + 2H2 S ~ (CH2 0)*+ H 20 + 2S
Bacteriochlorophyll
This reaction is exactly analogous to that for photosynthesis by green

plants, H2S substituting for H 20; free S being released instead of free O2 ,
• This is a hypothetical organic compound, the true nature of which is uncertain.

The Photosynthetic Bacteria 387
Athiorhodaceae. The non-sulfur oxidizing Athiorhodaceae* use organic
sources of H, instead of H 2 S. Thus:
light
CO2 + C 3H 7COOH + H 20 ~ 5(CH 20)
Bacteriopurpurin
Such organisms do not release sulfur for obvious reasons. Hence the name
of the group. * Some of these bacteria can use molecular hydrogen directly.
General Type Reaction. In all cases the hydrogen is incorporated with
carbon dioxide in some sort of organic compound generally represented in
the reaction formulas as CHzO. The process probably varies in the various
organisms.
Bacterial photosynthesis is thus seen to be a special case of the type of
reaction developed by van Niel for photosynthesis in general:
light
CO2 + 2H2R --> (CH 2 0) + H 20 + 2R
Photosynthetic pigment
where R indicates the remainder of the molecule from which the hydrogen is
abstracted.
Some growth occurs in the dark, but photosynthesis does not occur. In
the dark, hydrogen is not transferred to carbon dioxide but to oxygen. This
necessitates aerobic conditions. Bacterial photosynthesis thus occurs only
under anaerobic conditions and in the presence of light. The metabolism, in
other words, becomes like that of non-photosynthetic aerobic bacteria.
Thus, the photosynthetic feature of the photosynthetic microorganisms
seems merely to be added to an ordinary aerobic, oxidative system of me-
tabolism. This suggests that, in the course of evolution, these photosynthetic
species were derived from more primitive forms of 1?acteria with chemosyn-
thetic systems.
As van Niel states: "If, under certain conditions, one of the non-sulfur-
storing photosynthetic bacteria t should fail to produce its prominent pig-
ment system, it would thereby become indistinguishable from a typical
Pseudomonas, Vibrio or Spirillum species." It is interesting to note that non-
photosynthetic mutants of photosynthetic bacteria have been discovered.
Sulfur Utilization by Bacteria. The sulfur liberated from H 2S is stored in
the cells of Thiorhodaceae, later to be oxidized to H 2S0 4 , The free sulfur
stored in the cell as sulfur granules is eventually oxidized to sulfuric acid and
used in photosynthesis.
2S +3C02 + 5H 0 -> 2H S0 + 3(CH 0)
2 2 4 2
The Chlorobacteriaceae release free sulfur into the surrounding medium.

This sulfur is likewise used as an electron donor in the assimilation of CO 2 ,
Some species can utilize polythionates (H2 S20, H2S406).
Other Relations of Light to Photosynthetic Bacteria. Some very interesting
observations have been made on the dynamic effects oflight on certain photo-
I,
.. Athio is from Greek and Latin words. meaning without sulfur.
t Athiorhodaceae and Chlorobacteriaceae.
388 Ihe Bacteria
synthetic bacteria. If a beam of light be thrown across a fluid culture of certain
species of these organisms, they will quickly leave the darker parts and crowd
into the lighter portion. This attraction toward light is called phototaxis.
If a spectrum be thrown on a culture of certain motile Rhodobacteriineae
the organisms will congregate. most thicitly in the infra-red portions. The
infra-red is the region in which these bllcteria absorb energy for photosyn-
thesis to the greatest extent. It is of interest that the infra-red and red rays
were probably the first to pierce the murky atmosphere of the primitive earth.
These bacteria may represent descendants of the first organisms able to make
use of such light in a photosynthetic process.
Thiospirillum jenense (see family Thiorhodaceae) has a single, polar flagel-
lum which acts like the propeller of a ship in moving the cell forward. By
arranging a patch of shadow so as to move over a swimnllng celJ of T.jenense
it has been shown that there is a definite point where sensitivity to light is
localized. If the shadow moves over the organism from "ahead" a "shock
movement" or reversal and change of direction occurs as soon as the point
of insertion (center of motivation) of the flagellum is covered. The tendency
is to stay in the light where energy is available, and to stay in that color of
light in which, for that organism, energy is most absorbed. No "shock move-
ment" occurs until the sensitive spot near the base of the flagellum is shaded.
This localization of sensitivity to light recalls vividly the red "eye spot" of
Euglena viridis and stimulates speCUlations concerning the evolutionary origin
of eyes in animals.
REFERENCES
Calvin, M., Benson, A. A., and Basham, J. A.: Artificial photosynthesis. Sci. News Letter,
1954. 66:387.
Clayton, R. K., and Delbruck. M.: Purple bacteria. Sci. Am., 1951,185:68.
Fry. B. A., and Peel, J. L., Editors: Autotrophic Microorganisms. Fourth Symposium, Soc.
for Gen. Micr., Cambridge Univ. Press, New York, 1954.
Gest, H.: Metabolic patterns in photosynthetic bacteria. Bact. Rev., 1951,15:183.
Hill. R., and Whittingham, C. P.: Photosynthesis. Monograph on Biochemical Subjects.
John Wiley and Sons, New York, 1955.
Rabinowitz, E. 1.: Progress in photosynthesis. Sci. Am., 1953, 189:80.
van Niel, C. B.: The culture, general physiology, morphology and classification of the non-
sulfur purple and brown bacteria. Bact. Rev., 1944,8:1.
van Niel, C. B.: The comparative biochemistry of photosynthesis. Am. Sci., "1949. 37:371.
van NieJ, C. B.: The chemoautotrophic and photosynthetic bacteria. Ann. Rev. MicrobioL,
1954,8:105.
Various Authors: Carbon Dioxide Fixation and Photosynthesis. Symposia, Soc. for Exp.
Riol.. Academic Press, New York. 1951.
30
The Mold-like Bacteria

{Order Actinomycetales}
OF THE VARIOUS orders into which the class Schizomycetes is divided,

the order Actinomycetales is one of the most distinctive. It differs from all
others in that true, coenocytic branching is normally found in all of its species
(Fig. 30-1). None is motile. None forms true endospores though some species
form mold-like conidia. The whole order is often referred to as actinomycetes.
THREE FAMILIES OF ACTINOMYCETALES
For convenience of discussion these branching bacteria may be grouped
in three families, as shown in the following paragraphs.
The Family Mycobacteriaceae is the smallest, containing only the bacterium-
like genus Mycobacterium. This includes the tubercle bacilli (Mycobacterium
tuberculosis) and related species. Here, branching is at'a minimum. Any short
filaments which are formed tend to break up at once into bacillus-like frag-
ments.
These organisms are distinguished by a peculiar staining property called
acid-fastness. This has been described in Chapter 9. Occasionally acid-fast
cells, or fragments, are found in other actinomycetes, especially Nocardia,
but it is not like the definite, strong and constant acid-fastness of Myco-
bacterium.
The Family Actinomycetaceae includes true mycelium-formers. The anaero-
bic genus ActinomYt;es of this family includes the pathogenic species, Ac-
tinomyces bovis, causing "lumpy jaw" or actinomycosis of cattle, and a few
other species which mayor may not be pathogenic. In the genus Actinomyces
the filaments tend to:fragment into bacillus-like segments but to a lesser extent
than in Mycobacterium. "
A second genus, 'i/0cardia, comprises aerobic filament-formers which ordi-
narily remain unfnigmented unless mechanically disturbed. They tend to
grow on (and in) the surface of solid media such as agar, rarely producing
much aerial mycelium. None of the Mycobacteriaceae or Actinomycetaceae
produces spores or conidia.
The Family Streptomycetaceae consists of three genera: (1) Streptomyces,
which form long, aerial mycelia. These are mold-like, coenocytic, non-frag-
389
390 TM Bacteri4
Fig. 30--1. Electronograph of a species of actinomycete showing branching filaments.

The irregular shadows in the filaments are of unknown significance. (Courtesy of Dr.
Katharine Polevitsky and Dr. R. F. Baker; from unpublished work.)
menting filaments. Conidia are produced in long chains at the ends of the
filaments. (2) Micromonospora, which are like Streptomyces, but produce
only a single conidium at the tip of each short, fertile hypha. They produce
much less aerial mycelium, growing more on the surface and in the substance
of solid media. (3) Thermoactinomyces is a small group of species much like
Micromnnospora but, as the name implies, they grow only at temperatures
between 50° C and 65° C. The relationships of these groups are shown below.
THE ORDER AcnNOMYCETALES (TRUE BRANCffiNG)
Family Mycobacteriaceae (acid-fast)
Genus Mycobacterium (fragment; bacterium-like)
Family Actinomycetaceae No
Conidia
Genus Actinomyces (fragment; anaerobic)
Genus Nocardia (fragment; aerobic)
Family Streptomycetaceae (filament formers; do not fragment)
Genus Streptomyces (well developed mycelia; curled chains of
conidia) Conidia
Genus Micromonospora (single conidia)
Genus Thermoactinomyces (50°-65° C)
/
CoNIDIA. The conidia of the family Streptomycetaceae (often improperly
called spores) are not as heat-resistant as bacterial spores, being killed by 10
to 3D-minute exposures to 65° C, a temperature only slightly higher than that
required to kill the vegetative mycelium. In many species of Streptomyces the
formation of conidia is accompanied by a corkscrew twisting of the filament
so that a very curious, curled appearance is given to the mycelium (Fig. 3~2).
The direction and form of the coils are constant for any given species.
Molds and Actinomycetes. Striking and constant differences between the
mold-like Actinomycetales and the true molds (Eumycetes) are: (1) the
minuteness of the filaments of Actinomycetales (1 to 2 " in diameter and sel-
The Mold-like Bocteria 391
dom more than a few millimeters in length; true molds range around 50 II-
diameter and inches in extent); and (2) the absence of readily demonstrable
nuclei in Actinomycetales; both distinctly bacterial characters. Granules of
volutin and other substances, as well as vacuoles, are often seen within the
older mycelia of Actinomycetales, and have probably been mistaken by some
investigators for nuclei.
The colonies of the filamentous Actinomycetales are usually tough, dense-
Fig. 30-2. Details of growth and structure of various Actinomycetales. I, cross section
orcolony; 2~, successive stages in growth from a single conidium; 7-11, various types of
mature mycelium, showing different kinds of branching, conidia'formation, twisting, frag-
mentation, etc. (1-6 from Lieske; 7-11 from Drechsler and Waksman.)
The Bacteria
textured, and often very adherent to the medium. They have a wooly or velvety
appearance, due to the mycelial structure. The growth of many species (except
Actinomyces) is often brilliantly colored: red, orange. yellow. Colonies range
in diameter from Jess than 1 mm to several mm ; definitely smaller than the
huge colonies of true molds.
There is no apparent differentiation of sexes, and no evidence of sexual
phenomena such as are seen in molds.
FAMILY ACfINOMYCETACEAE
Most of the members of this family are saprophytes, living in the soil as
scavengers, attacking and decomposing complex organic substances of a
great variety, such as cellulose, protein, starches, fats, and even carbolic acid,
(phenol), naphthalene, rubber and cresol as sources of energy and carbon.
Except for these curious sources of carbon and energy their food requirements
are simple minerals.
Genus Nocardia. Of this genus, several species are parasitic in man or
animals, causing tuberculosis-like diseases or ulcerative lesions (nocardiosis).
They are generally aerobic. Most species are soil saprophytes and are fre-
quently found as contaminants in laboratory cultures. The mycelial fragments
are often bacillary-looking. There are usually numerous misshapen, clubbed
and knobbed forms containing volutin granules.
The colonies of these organisms on agar are usually like those of true
bacteria, ranging in diameter from I to 10 JI. and being pasty in consistency as
a rule. Filaments are inconspicuous or absent, and the organisms in smears
often much resemble true bacilli except that branching is sometimes well
marked.
Genus Actinomyces. These differ markedly from Nocardia in: (I) being
anaerobic or microaerophilic (the only anaerobic organisms in the order
Actinomycetales); (2) requiring complex organic foods, such as chopped meat
in broth. They are associated with the disease actinomycosis in man and
animals. They are much like Nocardia in morphology and colony form.
Actinomyces bovis occurs as a normal inhabitant in the oral cavity of cattle
and other animals and man. Thence it may be introduced into the flesh of the
jaws, tongue, etc. by thorns, splinters and the like. Swellings are produced by
the growing Actinomyces and the surrounding tissues become bard and in-
durated; hence the colloquial term "wooden tongue" for actinomycosis of
the tongue in cattle. Eventually the infected tissue becomes riddled with
I abscess-like cavities which are filled with pus.
Actinomyces bovis tend to form, in the pus, large (0.5 to 5 mm diameter),
yellow masses of growth ("sulfur granules") with a central core of matted
mycelia, the tips of which project at the exterior much like the spines of a sea
urchin or a chestnut burr. These tips characteristically enlarge and become
club-shaped. Crushed between two slides, the mycelial and radial structure
of the granules is evident. (See Fig. 30-3.)
"Ray-fungi." The name of this order is derived from the term Actino-
mvces, first used by Harz in 1878 to describe the organism causing "lumpy
jaw" of cattle. Actinomyces is derived from Latin words meaning "ray fun-
gus." The name is descriptive of the radial or sunray-like arrangement of the
threads of Actinomyces which make up the yellow granules ("sulfur granules")
TIre Mold-like Bacteria 393
or colonies of the organism found in pus from lesions of actinomycosis (see
Fig. 30-3). This radial arrangement is not a common character of Actino-
mycetales, branching cells being the prime differential character of the order.
However, A. bovis was the first species to be described and is now the type
species of the genus Actinomyces.
FAMlLY STREPTOMYCETACEAE
This family includes the most mold-like of all of the Schizomycetes. These
organisms are mainly saprophytes and are active in decomposition of all
types of organic material. A few are pathogens in animals or plants. Strepto-
myces scabies, for example, produces a troublesome disease (scab) of potatoes.
Some produce valuable antibiotics; e.g., S. griseus, streptomycin. Cultivation
from soil is not difficult, but slow development may permit overgrowth by
other, more rapidly multiplying, bacteria if the latter are present in large
numbers in the culture.
The flat, tough, strongly adherent colonies of Streptomyces on infusion or
extract agar are often papillate, and frequently the surface is thrown into radial
folds or ridges. They give off a distinctive, musty odor characteristic of damp
cellars and newly turned soil.
Growth is usually best at temperatures around 25° C, although some
thermophilic soil species (genus Thermoactinomyces) grow well at tempera-
tures as high as 65° C. Unlike most true molds, optimal growth occurs at pH
8 or 9, and is greatly depressed by reactions of around pH 5.
It is obvious that liming of acid soils will encourage growth of the sapro-
phytic actinomyces, resulting in increased fertility due to the activities of these
organisms in decomposing complex organic materials so that other bacteria
and farm crops can make use of them. The various species causing "scab"
of potatoes and other root crops are also encouraged by this procedure so
Fia. ~3 . "Sulfur granule" from a

of actinomycosis (X SO). (Wright.)
DtlOt()Rn,nh by L. S. Brown.)
394 The Bacteria
that one must consider the nature of the crop to be raised and soil pH before
indiscriminate liming. As the Streptomycetaceae and other actinomycetes
are aerobic, it is apparent why draining swamp lands increases their fertility.
Streptomyces and Antibiotics. The genus Streptomyces constitutes one of
the most extensively investigated groups of bacteria because various species
produce antibiotics of value in human or veterinary medicine, in the control
of crop diseases and industrial spoilage by bacteria, and in scientific research.
For antibiotic production Streptomyces are generally cultivated in deep
tanks of aerated fluid media, much as is Penicillium notatum for penicillin
production. Growth occurs in a flocculent form and antibiotic production is
usually good (Chapter 44).
ACTINOPHAGE. The growth may undergo lysis. Normally this is autolysis,
which occurs usually at about the time antibiotic production is best. Destruc-
tive, troublesome lysis is sometimes caused by actinophage (like bacteriophage)
which destroys the growing organisms. .
SELECflON OF PRODUCflVE STRAINS. In antibiotics production great care
is used to select and maintain strains of Streptomyces which produce maximum
amounts of antibiotic; to detect and eliminate 'phage, and to find new anti-
biotic-producing strains or species. Here is a field offering opportunities to all,
from the tyro to the advanced·expert.
FAMILY MYCOBACfERIACEAE
The family consists of only the genus Mycobacterium. These are gram-
positive, noncmotile, aerobic, non-sporeforming rods. The Ziehl-Neelsen
stain is of special value in the identification of mycobacteria, since they are
unique in being strongly acid-fast. Morphologically, they are much like ordi-
nary bacteria except for being somewhat curved and spindle-shaped, and
showing beaded or granular cells. The granules are often called Much granules
after the first writer to describe them. They have been regarded as forms in a
life cycle of tubercle bacillus or nuclear apparatus. The most acceptable view
at present is that they are artifacts resulting from staining processes. The
mycobacteria branch only slightly, so that X, Y, L and similar forms are
sometimes seen. Filament formation is minimal and transitory. Most of
them are harmless saprophytes living in the soil and decaying organic matter.
None of them forms conidia or aerial hyphae.
Non-pathogenic Species Or Mycobacterium. Saprophytic mycobacteria
may be found in properly stained smears of material from preputial secretions
(Mycobacterium smegmatis) or from folds of the skin as in the buttocks
or axillae. Saprophytic acid-fast bacilli are also found in butter (Myco .
butyricum), in manure (Myco. stercorisJ, or on hay (Myco. phlei). (While
many species are named, few are fully described and most are synonymol,ls
with M. phlei). Many species are also found in the soil where they act as
scavengers and bring about decomposition of a wide variety of complex
organic compounds: proteins, fats, carbohydrates, petroleum, paraffin, ~tc.
Except for the carbon source, their nutrient requirements are relatively simple.
These saprophytic mycobacteria are readily cultivated at 25° to 30° G on
infusion agar containing glycerin, or on coagulated egg or serum. Like
Streptomyces, they will also grow well in simple mineral solutions containing
a little asparagine and glucose; Le., they are close to the autotrophs. Yellowish
The Mold-like Bacteria 395
or reddish pigments are common. Their growth is fairly rapid as a rule (48 to
96 hours). The colonies of the saprophytic species are usually soft and moist,
(pasty or butyrous-butter-like).
Some species of mycobacteria, very much like these saprophytes, are found
associated with lesions in the lung resembling tuberculosis. Their etiological
relationship to such lesions is not clear. They are often accompanied by
tubercle bacilli.
Mycobacterium Tuberculosis. This organism, causing tuberculosis, is
typical of the genus Mycobacterium. It is the most strongly acid-fast species
of the genus.
Tubercle bacilli do not form spores, but may remain alive for long periods
outside the human or animal body. In dried sputum in dark corners they may
live six to eight months. In particles of dried and powdered sputum which can
float through the air as dust, they, can remain alive for days and may be
inhaled. Exposure to sunlight for a few hours kills them and so does pasteur-
ization. This is of importance since they can infect cows' udders and hence
the milk, thus transmitting the disease from cattle to persons who drink un-
pasteurized or improperly supervised (un-Certified) raw milk.
TYPES OF TUBERCLE BACILLI. There are several kinds of tubercle bacilli,
varying according to the animal infected. For example, there is a human type
(Myco. tuberculosis, var. hominis) and a bovine type (Myco. tuberculosis. var.
bovis) which grow well but slowly (2-6 weeks) on solid media such as coagu-
lated egg, serum or blood, or on glycer\n agar at 37° C. A third mammalian
type, called the vole* bacillus, was discovered in 1937. This organism is
highly virulent for voles but in man produces a mild immunizing infection.
There is also a bird or avian type (Myco. avium) which grows well at about
40° C, and turtle (Myco. chelonei), fish (Myco. marinum) and other cold-
blooded animal types which grow well at lower temperatures (18° to 30° C).
All look alike microscopically and may be cultivateq on simila~ media. The
cold-blooded animal types do not as a rule infect the warm-blooded animals
or birds and vice versa. The avian type occasionally infects mammalian
species. Human and bovine types seem to infect man and guinea pigs with
equal ease but differ in their effect on rabbits, the bovine killing the rabbit
within four to six weeks, the human usually not killing at all, or much more
slowly. This is of diagnostic value.
TUBERCULOSIS
Tubercles and Tuberculosis. Tuberculosis is much more common than is
generally supposed. It kills a great many more people in these United States
each year, even in this enlightened age, than diphtheria, scarlet fever, typhoid
fever, mumps, measies, Whooping cough and poliomyelitis altogether.
When tubercle bacilli gain a foothold in a susceptible animal or person, the
tissues where the bacilli localize immediately begin to react against the organ-
isms in a very chara~teristic way. Numbers of tissue cells begin to grow around
the bacilli in an attempt to incarcerate them or wall them in. A tiny, pearly,
gray mass of cells is thus formed, with tubercle bacilli at the center. It is called
a tubercle. If the res,istance of the host is low, the tissues are unable to arrest
• A vole is a kind of;field mouse.

396 The Bacteria
the bacilli, which continue to grow, killing the surrounding cells and destroy-
ing the fibrous walls. Numbers of adjacent tubercles may thus coalesce. The
dead tissue at the center of such masses of tubercles becomes cheesy and
yellowish and is said to be caseated. It may rapidly involve the major part of
the lung or other infected organ. If the tubercle is in a lung, the necrosis
(death of tissue) may extend till it invades and breaks through the wall of a
bronchus. The caseous material, which may contain millions ofliving tubercle
bacilli, is discharged with the sputum by coughing (Fig. 9-4). A large cavity
is left behind (cavitation).
Sometimes the caseous process breaks through the wall of an artery and
then hemorrhage of the lung OCCUJ:S, which may be fatal. In the vast majority
of human beings these tuberculous processes tend to heal and to form life-
long scars, whether the patient recovers or not.
Tuberculin Reaction. Allergic sensitivity to the protein of the tubercle
bacilli may be demonstrated in a large proportion of adults by introducing
into (not under) the skin the tuberculo-protein in the form of dead tubercle
bacilli, or the sterile filtrate of broth in which they have grown, or purified
protein derivatives (PPD) extracted from the bacilli. These tuberculo-proteins
are called tuberculin. At the site of their introduction into the skin a red, Hell-
ing spot appears in twenty-four to forty-eight hours. The reaction is called a
tuberculin reaction, and is representative of the delayed type of allergy de-
scribed in Chapter 23.
TUBERCULIN REACTION AND REsISTANCE. When a tuberculous infection
progresses to the time when hypersensitiveness appears, a certain degree of
resistance to later infection develops. Tuberculin tests become positive at the
same time. Evidently the hypersensitive tissues are able to react locally to
bind and arrest the further progress of new infection. Similar tests for allergy
(using appropriate, specific antigens) are used in diagnosing certain other
slowly-progressive infections: brucellosis or undulant fever, tularemia or
"rabbit fever," trichinosis, histoplasmosis and coccidioidomycosis (Chapt. 4).
Immunization Against Tuberculosis. Many attempts have been made to
produce an effective vaccine to prevent tuberculosis. The most widely used
and favorably reported immunizing agent is BCG, a variant of a bovine strain
of tubercle bacilli of low virulence (Chapt. 22). .
Laboratory Diagnosis of Human Tuberculosis. Tubercle bacilli often ocb'ur
in large numbers in the sputum of persons with active pulmonary tuberculosIs.
For diagnostic purposes the .organisms may be demonstrated there or in
other material such as urine, pleurisy fluid or feces, depending on the organs
affected. The methpds available for' demonstrating the tubercle bacilli in
pathological material are (a) microscopic exami.qation of smears of the
material, stained,by the acid-fast method; (b) cultivation on media specially
adapted to the growth of the tubercle bacillus and (c) by the inoculation of
guinea pigs followed by observation of the development of tuberculosis in
these animals. These procedures are relatively simple and are described in any
diagnostic manual or textbook of medical bacteriology. The essential steps
are:
'J. Preparation and 'examination of a smear stained by the Ziehl-Neelsen
method.
2. Treatment of the specimen (usually sputum or gastric washings) with an
equal quantity of 5 per cent NaOH to destroy ("digest") the mucin and most
of the contaminating bacteria.
3. Neutralization of the "digested" material.
4. Inoculation of the digested material onto media containing egg, milk,
blood or serum, with some agent to inhibit contaminants: dyes, antibiotics.
5. Test of virulence by injection of the digested, neutralized material into a
guinea pig.
Mycobacteria and Wetting Agents. Mycobacteria have simple nutritive re-
quirements and will grow well in mineral solutions containing only I or 2
amino acids. A difficulty with such aqueous media is that mycobacteria con-
tain much wax. Therefore they are not easily wetted. Consequently aqueous
nutrient solutions do not come into intimate contact with the cell surface. In
such media the bacilli grow reluctantly in clumps and float like granules of
wax on the surface. As a result, nutrition is retarded and growth of tubercle
bacilli slow, requiring 2 to 3 weeks and the use of copious inoculum.
The addition of a wetting agent (surface-tension reducent) (for example,
one commercially named "Tween-80"*) permits the preparation of rapidly
f2--4 days) growing subsurface cultures in simple aqueous media. These cul-
tures remain in a diffuse state manifested by an even turbidity with no granule
or pellicle formation. Growth is easily initiated by minimal inocula.
The nutrient solution comes into intimate contact with the outside of each
cell and permits rapid diffusion of foods and wastes, with consequent rapid
growth.
This principle, of adding wetting agents to cultures of microorganisms, is
an important one and is worthy of further investigation in connection with
other organisms to facilitate fast, smooth, evenly distributed growth. This is of
importance in many bacteriological procedures: agglutination tests, enumera-
tion, etc. Sometimes wetting agents like "Tween 80" contain toxic impurities
which are offset by adding a little serum or serum der)vative, such as Bovine
albumin fraction V (Armour & Co.).
LEPROSY
This disease is of great interest from several standpoints: (a) historically;
(b) because of the mystery surrounding its mode of transmission; and (c)
doubt concerning its true etiology.
Etiology. In 1874 Hansen described an acid-fast bacillus, morphologically
indistinguishable from M. tuberculosis, in lesions of lepers. It is called Myco-
bacterium teprae. [t is always present in leprous tissue, and never occurs in
normal tissue. For many years this has been the only one of Koch's postulates
to be demonstrated in connection with the etiology of leprosy. Demonstration
of this organism in liistological sections or biopsy material (material cut out
during life) is the most useful and conclusive diagnostic procedure.
Several reports of! successful inoculation of human beings with leprous
material have appeared. It still is not certain that the only infectious agent
transferred in these occurrences was the bacillus of Hansen. Moreover, all
,. Tween-80 (Atlas Powder Co., Wilmington, Del.) is a polyoxyethylene derivative of

sorbitol mono-oleate. (Prepare 10 per cent aqueous stock solution in distilled water. Do
not use after 1 month. Keep in refrigerator.)
398 The Bacteria
of these people were in constant association with lepers. Inoculation of leprous
tissue into chimpanzees, etc., has never produced an authentic case ofIeprosy.
An organism which will cause leprosy has never been cultivated, although
many claim to have done so. There are several strains of so-called M. ieprae
which may be obtained on ordinary culture media. They have all the proper-
ties of saprophytic species of Mycobacterium.
The method of transmission of leprosy is obscure. Lesions and discharges
contain the bacilli in large numbers. Prolonged, close contact appears to be
necessary. Persons under 25 are more likely to contract the disease. The
incubation period appears to range from a few months to as long as twenty
years. In certain regions of the world leprosy is endemic, that is, always pres-
ent. Among these areas are tropical and subtropical Asia and Africa, Poly-
nesia and South America. It is also present in certain areas of Europe (U .S.-
S.R., Baltic States, Spain).
There are about Soo known cases of leprosy in the United States, mostly
in the National Leprosarium at Carville, Louisiana, and possibly 1000 un-
known cases. In this country leprosy appears to spread only in particular areas
where conditions are favorable. The reason for this is not clear. Clinically,
leprosy progresses very slowly, lasting for years in a patient.
History. Leprosy is well known to students of the Bible and medieval
history. The fear and horror of lepers has been a human tradition since'
antiquity. In ancient times lepers were excluded from all public contacts and
left to die of exposure and starvation by many peoples. They still are excluded
and shunned, but provisions for their comfort and well-being are usually
adequate. In former days many disfiguring diseases were confused with
leprosy: various fungal infections, yaws, protozoal infections, and so 00.
One of Conan Doyle'S most intriguing Sherlock Holmes episodes depicts the
tragic segregation of a noble young man because of a suspicion of leprosy.
Holmes, with the aid of Watson, dispels the ugly cloud by his usual astute,
deductive reasoning, and the victim is found to have nothing but a harmless
skin condition called i9hthyosis. The horror of leprosy arises from' the dis-
figurement of the body and destruction of tissue, with scar formation, which
accompanies it.
The disease is most distinctive in its so-called lepromatous phase. Nodules
and gross deformities and ulcerations occur in the skin, with thickening, dis-
colorations and wrinkling. Nerves are often affected so that wounds go un-,
noticed, and revolting and _terrifying (to the ignorant and superstitious)
disfigurations occur.
REFEREl'1CES
Bloch, H.: Acid-fast bacteria. Ann. Rev. Microbiol., 1953, ,7:19.
Cummings, M. C.: Chapter on Tuberculosis. Diagnostic Procedures and Reagents. Am.
Pub. Health Association, 1790 Broadway, New York, 3rd ed., 1950.
Dubos, R. J.: Second thoughts on the germ theory. Sci. Am., 1955,192:31.
Editorial: Whereat tuberculosis? Am. J. Pub. Health, 1956,46:895.
Gross, M.: Mycobacterium Tuberculosis: Bacteriology, Biochemistry, Laboratory Diag-
nosis and Chemotherapy. Published by the Author, Jersey City, N. J., 1955.
Long, E. R.: The germ of tuberculosis. Sci. Am., 1955,192:102.
Rosenthal, S. R.: StandaIdization and efficienl.,'y of "BCG vaccination against tuberculosis.
J.A.M.A., 1955, 157:801.
Sams, C. P.: Experiences in immunization against tuberculosis with BCG vaccine in Japan.
Am. J. Pub. Health, 1954,44:903.
Thomson, D.: Tuberculosis-The'changing emphasis. Monthly Bul., Ministry of Health
and the Pub. Health Lab'y Serv., (London), 1956,15:99
Various Authors. Symposium on B. C. G. Bull. W. H. 0.,1950,2:347.
Waksman, S. A.: The Actinomycetes. Ann. Crypt. et Phyt. The Chronica Botanica Co.,
Waltham. Mass .• 1950.
Waksman, S. A., and Lechevalier, M. A.: Guide to the Classification and Identification of
the Actinomycetes and Their Antibiotics. Williams & Wilkins Co., Baltimore, 1953.
31
The Spiral, Flexible Bacteria

{Order Spirochaetales}
THE TERM spirochete is often used in a general sense to include all members
of the order Spirochaetales. It will be so used here. This inclusive and non-
specific word must be differentiated from the name of one genus of spirochete
(Spirochaeta) and one genus of true bacteria (Spirillum) .
Genera) Characters and Structure of the Spirochaetales. The order Spiro-
chaetales is divided into two families: (a) Spirochaetaceae, which includes
three genera of relatively large, saprophytic spirochetes: Spirochaeta, Sapro-
spira and Cristispira; (b) Treponemataceae, comprising three genera of
relatively small, very slender organisms, some of which are highly pathogenic :
Treponema, Borrelia and Leptospira. The nutrient requirements of spirochetes
are complex. Only a few species, notably Leptospira and Spirochaeta, have
been cultivated to a significant extent on artificial media.
The spirochetes resemble most true bacteria (sub-order Eubacteriineae) in
being: unicellular; non-filamentous; non-branching; and without sheaths,
sulfur granules, slimy masses like the slime bacteria, or incrustations of any
metal. They do not form stalks; are without photosynthetic pigments: are
microscopic in size; without readily demonstrable nuclei; and are without
definite sex phenomena. They multiply by transverse, binary fission. Some
species may multiply by other methods involving filterable granules. None
forms pigments, spores, conidia or capsules. They differ from all other bac-
teria in being both: (a) cylindrical and spiral in form (Fig. 31-1), and (b)
flexible and contractile. •
MOTILITY in most spirochetes is in part due to their helicoidal form and
their rotation around the long axis, as well as to slow, snake-like, bending
movements. The electron microscope appears to have demonstrated structures
that look like flagella on certain species.
FIBRILLAR STRUCTURE. Studies with the electron microscope indicate that
the structure of spirochetes is more complex than that of true bacteria. The
cells of several species (perhaps all) appear to have large numbers of flagellum-
like fibrils which form bundles wound spirally from end to end of the cell,
• Note the differentiation from genus Spirillum, order Eubacteriales which, while spiral,
are rigid, and from the Order Myxobacteriales which, while flexible, are not spiral.
400
The Spiral, Flexible Bacteria 401
in a manner suggestive of the fibers of a twisted Manila hempen rope. These
bundles of fibrils appear, in some species, to be inside the cell wall. In some
species their regular convolutions, from end to end, produce what looks like a
septate or chambered structure, a character formerly assigned to some spiro-
chetes. In one species (Cristispira) the fibrils appear to be on the outside of the
cell. There they appear to be aggregated and modified into a flat, keel-like
membrane called a crista.
In some species (Leptospira, Treponema) the cell appears to be wound
spirally around a central, rod-like structure, pbssibly a bundle of fused fibrils.
The arrangement is suggestive of a thin-walled, rubber tube wound spirally
around a flexible, springy, plastic wire (Fig. 31-2).
It appears that the fibrils in all species are contractile. This could explain
the flexing and wriggling motility of spirochetes. The fibrils (and perhaps
other forms of bacterial fibrils) may be thought of, provisionally, as modified,
rudimentary or evolved forms of flagella.
SIZE. The spirochetes, while in general not much thicker thau true bac-
teria, are usually much longer. In some species of the genus Spirochaeta
individuals as long as 500 p. have been described.
402
Fig. 31-2. Electronograpbs of leptospira sh~ wing, at A, the cytoplasmic cylinder wound
helically around a thinner, rigid axistyle; at B, the released axistyle which is attached at the
ends of the cytoplasmic cylinder. It is suggested that the uxislyle is not only the "backbone"
of the leptospira but also its means of locomotion, whic'} is probably due to contractility
of the axistyle. (Photos courtesy of Dr. J. W. Czekalowski, Univ. of Leeds; from J. Path.
and Bact., 1955, vol. 69.)
MICROSCOPIC STUDY. Spirochetes as a rule are not readily stained by

ordinary dyes like methylene blue, although certain species are exceptions.
Some are gram-negative. Giemsa's stain, which colors protoloa, also stains
the spirochetes. Improved stains have also been developed. The larger species
of spirochete are conveniently observed in unstained preparations, and the
method of negative staining sometimes gives satisfactory preparations. The
darkfield apparatus is most convenient for observation of the pathogenic
species when fluid suspensions are available.
By all microscopic methods granules which may well be associated with
reproduction have been seen. The nature of these spirochetal granules is not
entirely clear. Some species, especially saprophytic ones, also contain many
volutin and fat granules.
FILTRABILITY AND REPRODUcnVE GRANULES OF SPIROCHETES. Certain of
the spirochetes (e.g., Leptospira and Borrelia) are readily filtrable through
porcelain or Seitz-type filters which hold back ordinary bacteria. This, of
course, distinguishes them from true bacteria. It is thought by some authori-
ties that the filtrability of these organisms is due to the formation, through
disintegration or otherwise, of minute granules. Possibly these are the sup-
posed reproductive granules referred to in the preceding paragraph. Material
(e.g., lake water) which has been filtered and found, on microscopic examina-
tion, to contain no visible spirochetes, has later been shown to contain spiro-
chetes by cultural methods. However, microscopic examination of a drop or
two of filtered fluid might easily fail to detect the presence of a few spiro-
chetes in the greater, unexamined portion.
Small buds or granules appear to form at the end, or on the side, of some
species of Treponema, apparently as part of a reproductive cycle. Each bud
or granule appears to give rise to a young spirochete. These granules, or
analogous structures, may also form in Leptospira and Borrelia and account
for their filtrability.
GENUS SPIROCHAETA
The name of this genus is derived from the name "Spirochaete," by which
Ehrenberg, in 1833, designated a very large, spiral and flexible organism
found free-living in stagnant water. This organism is now the type species of
the genus Spirochaeta and is called Spirochaeta plicatilis. It is a slender spiral
(0.5 JI. in diameter) but often attains the relatively enormous length of 500 Ii
(0.5 mm). The ends are blunt (compare Treponema, etc.). Large granules of
volutin and fat are present (Fig. 31-1). Motility is by creeping and twisting
movements. The organisms may be found in sewage and stagnant water.
Pure cultures have been made on medium containing extract of leaves, and
on 1.5 per cent agar with red blood cells. Spirochaeta are aerobic and grow
in a pH range of 6 to 9 at around 26° C. They grow as a thin film on the agar,
rarely forming ordinary colonies. They are entirely harmless to man as far
as is known.
GENUS SAPROSPIRA
This genus resembles the foregoing in that the organisms are found free-
living in polluted water, have blunt ends, and are curved and wavy. They are
thicker than Spirochaeta plicatilis, having a diameter of about 1 Ii, but are
shorter, seldom exceeding 85 Ii in length (Fig. 31-1). Saprospiras appear to
have septate or chambered protoplasm. This could represent separate cells
as in Spirochaeta, or convolutions of fibril bundles as previously stated.
Saprospira and Spirochaeta may not represent separate genera. Only two or
three species are known. Saprospiras have never been cultivated.
GENUS CRlSTISPIRA
In most respects the cristispiras resemble the saprospiras. They have a wavy
arrangement, blunt ends and an apparently septate protoplasm (Fig. 31-[).
Motility is active. Their length ranges from 26 to 120 Ii and their diameter
from 0.5 to 3 IA. The distinct cell membrane stains like chitin; a protozoa-like
character. The habitat of Cristispira is restricted to the intestinal tract of
OJBters or to a hyaline structure, called the crystalline style, in the esophagus
• these and related mollusks. Cristispiras are distinguished by a crista; a
IOrt of keel or membrane, which winds spirally about the organism; one edge
free, the other attached to the cell. This structure is suggestive of the undul-
ating membrane seen in one genus of pathogenic protozoa, the trypanosomes.
1ho apparently fibrillar structure of this crista has been mentioned. The
erpnisms are entirely harmless. None of the Cristispira has ever been culti-
GENUS TREPONEMA
lnipolre",w are slender (0.25 p. to 0.4 IA in diameter), and seldom exceed a
about 15 ".. Their size is, therefore, comparable with that of true
Their cell structure is much less perfectly demonstrable than is that
tile larger spirochetes. The organisms have neither crista nor septa. The
404 The Bacteria
eight to fourteen ~pirals found in Treponema are close and regular unless the
protoplasmic contractions change them. The ends of the organisms are drawn
out to extremely fine fibrils. These terminal fibrils have no function in the
motility of the organism. Motion may depend on flagella, or flagellum-like
fibrils, but the propeller-like action of the spirals when the treponemas
rotate doubtless plays an important part.
Treponema are not easily stained. Indeed, the first Treponema to be c:le-
scribed, that causing syphilis (Schaudinn, 1905), was named Treponema
pallidum because of its pale appearance. Other methods are, therefore, used
to demonstrate them microscopically. One is the method of negative staining;
another, widely used to examine exudate from lesions of syphilis for diag-
nostic purposes, is the darkfield apparatus. A third, used mainly by the pathol-
ogist to demonstrate spirochetes in infected tissues, is termed "silver im-
pregnation. "
Resistance and Cultivation. T. pallidum is a relatively fragile, highly para-
sitic organism. It has never been cultivated in artificial media, although it
may be maintained alive and virulent for several days in certain artificial
media, an important factor in the diagnosis of syphilis. Some similar species
(notably the Reiter, Nichols, Noguchi and Kazan strains) have been culti-
vated, and grow vigorously, but none of these is able to cause infection.
T. pallidum, under ordinary circumstances, can survive for only very short
periods outside of the tissues of man or experimentally-infected animals.
Hence non-venereal infection of man is rare. However, when quickly frozen
0
and maintained at _76 C, by means of solid CO 2, syphilis spirochetes, as
well as many other organisms, remain viable and fully infectious for years.
The organisms do not long survive ordinary drying. Surface-tension reducents,
such as ordinary soap and bile salts, quickly cause lysis of T. pallidum. The
organism is quickly killed by ordinary disinfectants. In citra ted blood stored
in "blood banks" the spirochetes qui~kly die out.
Syphilis. Syphilis, caused by Treponema pallidum, is primarily a ve-
nereal disease, i.e., is transmitted chiefly by sexual intercourse. When so trans-
mitted and when it develops typically it begins, within 2 to 6 weeks after
exposure, as a small ulceration on the mucosal surface of the genitalia. The
spirochete~ rapidly migrate from this to the deeper tissues of the body.
The ulcer increases in size, becoming rather hard and flat. Upon removal of
the crust serous fluid oozes from the surface. This, upon examination with a
darkfield apparatus, may be found swarming with Treponema pallidum. When
syphilis is acquired through kissing, the ulcer may appear on the lip. This
ulcer, oral or genital, is spoken of as a "primary lesion" or hard chancre
(pronounced shank/er). It tends to heal spontaneously due to the development
of antibodies after two to six weeks. The victim may believe himself cured.
Attempts are made by the local lymph nodes to arrest the migrating spiro-
chetes. The nodes become much swollen and are sometimes called "bubqes."
Their efforts, U1ifortunately, are futile.
What really happens is that by this time a certain degree of immunity
develops. The Treponema have long since migrated from the primary lesion,
'probably within less'than an hour after exposure, and have been carried all
over the body. They localize in various organs, particularly the liver, spleen,
walls of arteries, heart, brain, skin and mucosal surfaces, setting up "secon-
dary lesions." These begin to manifest themselves after two to four months.
When situated on the skin these often appear as red blotches or an extensive
rash and may be very infectious. White patches may also appear in the mouth
and genitalia. In such conditions of the mouth kissing of other persons results
in infection of the lips, tongue or gums. The teeth may loosen and come out,
as well as the hair. After a time (weeks or months) these outwardly visible,
secondary lesions slowly disappear in great part and the patient may again
believe himself cured. Spontaneous cure actually may occur but this is ap-
parently very rare.
In untreated syphilis the Treponema usually slowly cause extensive tertiary
lesions called gummata in various internal organs and also on the skin. These
tend to heal and form scars as the process continues. The liver becomes
damaged and scarred (syphilitic cirrhosis of the liver) and bulges appear in
the aorta where lesions in the layers of the vessel have weakened it. These
bulges are called aneurysms. When they burst, death from hemorrhage
ensues. Gummata also occur in many of the bones.
The Treponema also damage the brain and spinal cord. Various nerve
centers are slowly destroyed and characteristic forms of insanity and paralysis
result. Death follows, sometimes after a period of many years.
PREVALENCE AND CONTROL. This picture of syphilis is not pleasant and,
indeed, the disease is one of the most insidious and dangerous. About twice
as many persons die annually of this disease in the United States as die of
poliomyelitis. Millions of cases exist which do not come to the attention of the
recorder. A million or more new cases develop each year. "V.D." is not
beaten. It is on the increase because people have been lulled into a sense of
false security.
As in the case of gonorrhea, prostitution and sexual promiscuity are the
chief means by which syphilis is spread. In spite of ren~wed efforts by federal,
state and local authorities to educate the public to the dangers of syphilis and
to enlist the aid oflegislatures, medical and civic authorities, and of the people
themselves who are endangered by it, many new cases appear each year.
As one great physician has said, "The greatest obstacles to Public Health
are the ignorance and indifference of the public!"
SYPHILIS SEROLOGY. The diagnosis of syphilis, after the disappearance of
the primary lesion in which the spirochetes are demonstrable microscopically,
is made by means of serological tests. These have been discussed in Chapter 21.
Many non-specific reactions occur.
Three specific antitreponemal tests are now available for the exact diag-
nostic study of syphilis. These are great steps forward in syphilology. All are
dependent on specific antibodies against T. pal/idum. One is the T.P.I. test;
another, the immune adherence phenomenon; the third is a complement fixation
test, differing fundamentally from the Wassermann test in using extracts of
T. pal/idum as a specific antigen (Chapter 21).
GENUS BORRELIA
The Borrelia rese111ble the Treponema in many respects. However, many
Borrelia can be stained readily by Gram's method or by means of a special
406 The Baclerill
Fig. 31-3. Vincent's angina. Stained

smear showing fusiform bacilli and spiro-
chaetal forms. (X 900.) (Todd and San-
ford.)
polychrome stain made by mixing eosin and methylene blue (Jenner's stain,
Wright's stain *). They often have a less definite spiral form, being more·
wavy and open, especially in stained preparations. In death they seem to
relax. and lose their regular, coiled form. They are also somewhat thicker and
coarser-looking than the Treponema. Cultivation of some has been accom-
plished although it is not very satisfactory. They grow well in living chick
embryos.
Commensal species of Borrelia occur, often in large numbers, in the normal
mouth (Borrelia macrodentium, B. microdentium) and on the external genitalia
(8 . refringens). Some of these so closely resemble Treponema pallidum in ap-
pearance as to create confusion at times in the diagnosis of syphilis by micro-
scopic methods. They appear to be harmless. They are sometimes classed as
Treponema. Leeuwenhoek probably was the first to observe these.
The majority of the pathogenic Borrelia, like B. recurrentis and B. novyi,
are blood parasites causing fever of a relapsing nature (relapsing fever).
The spirochetes occur in large numbers in the blood during the numerous
febrile relapses characteristic of the disease. They are transmitted by certain
ticks and by body lice.
Trencb Mouth. Some anaerobic species of Borrelia are found in the mouth
(notably B. vincentii) associated with ulcerative conditions ("trench mouth,"
or Vincent's angina). They may be seen readily in gram-stained smears from
such conditions, mixed with fusiform bacilli (Fig. 31-3). The name "trench
mouth" originated in the frequent occurrence of outbreaks of the disease in
soldiers in trenches during World War I. It is presumably transmitted by
unclean eating utensils and other articles which carry saliva directly from
mouth to mouth. It may be associated with dietary deficiencies or the micro-
organisms may act merely as secondary invaders of lesions due to other
causes : caries, herpes, and so on.
GENUS LEPI'OSPIRA
Leptospira are the smallest of the spirochetes. Their spirals are so fine and
so closely wound that, when observed in the darkfield. only the outer curves
of the spirals are seen and the organisms appear like strings of minute, ilIumi-
• These stains aIe c:ommonly used for staining blood films to demonstrate the protozoa
of malaria. They aIe available COIJlIIICl"ciaUy.
'I'M Spiral, Fkxibk Bacteria 407
nated beads. The leptospiras are further characterized by being bent into a
hook at one or both ends (Fig. 31-4). Their motion consists ofa writhing and
flexing movement and a rapid rotation around the long axis. Their progression
from place to place is rapid and can be readily explained on the basis of their
screwlike form and their rotation. They may be cultivated readily in relatively
simple mineral solutions containing serum. Curiously, no substitute for serum
has been found, in spite of diligent search.
Leptospiras are chiefly saprophytic, aquatic organisms which are found
in rivers and lake waters, in sewage, and in the sea. Occasionally, they are
found in the normal mouth. They may be cultivated in pure culture from some
pond or swamp waters by the simple procedure of passing the water through
a porcelain or Seitz-type filter (through which they readily pass), and then
adding (aseptically) 10% sterile serum; 0.8% salt; and melted agar to yield
0.2%. Incubation is for at least two weeks at 37° C. The organisms are micro-
aerophilic and growth appears in a well-defined, greyish zone ,about 0.5 to
1.0 em below the surface of the medium.
Leptospirosis. There are a dozen or more species (or sero-types) of Lepto-
spira which are dangerous pathogens. Infection with any of them is properly
called leptospirosis though special names, of importance historically or other-
wise, have been given to certain forms of leptospirosis ("canicola fever,"
"swineherd's fever," hemorrhagic jaundice or Weil's disease. etc.). Lepto-
spirosis is fairly common, though often wrongly diagnosed.
The various forms of leptospirosis are basically alike though the symptoms
vary. In general, the infection is common among certain animals. It is trans-
Table 18. Some Common Leptospiras.
SPECIES USUAL ANIMAL H05T DISEASE·
L. icterohaemorrhagiae Rats Weil's disease

L. canicola Dogs Canicola fever
L. pomona Cattle, swine, horses Swineherd's di'lealle
L. autumnaJis Rats
L. hebdomadis Field mice, other rodents
u u " .,
L. grippotyphosa
• Terms commonly used for various leptospiroses.
Fig. 31-4. Leptospira icterohaemor-

rhagiae. Appearance of organisms in the
darkfield. (X 1000.) (Zinsser, Bayne-
Jones, Textbook of Bacteriology, D.
App1eton-Century Co., publishers.)
408 The BacteriD
mitted from animal to man through the urine of animals and by anything
(water, food, etc.) coming into contact with the skin of, or being ingested by,
man. Doubtless the same mode of transmission occurs among animals.
A list of common species of Leptospira and their usual animal reservoirs
is given in Table 18.
All of the Leptospirae are much alike. They are differentiated, mainly by
serologicaL means, by special experts.
REFERENCES
Bradfield, J. R. G., and Cater, D. B.: Electron microscope evidence on the structure of spiro-
chaetes. Nature, 1952,169:944.
Dyar, M. T.: Isolation and cytological study of a free-living spirochete. J. Bact., 1947,
54:483.
Editorial: Diagnosis of human leptospirosis. J.A.M.A., 1957,163:188.
Editorial: Venereal disease control is not automatic. J.A.M.A., 1954, 155:911.
Editorial: Venereal disease is on the rise. Am. J. Pub. Health, 1955,45:652.
Expert Committee on Venereal Infections and Treponematoses. World Health Organiza.
tion Tech, Rep't. Ser. No. 63, 1953. Columbia Univ. Press, New York, N. Y.
Geiman, Q. M.: Metabolism of spirochetes. Ann. Rev. Microbio!., 1952,6:299.
Hyde, H. van Z.: The modem story of yaws. Am. J. Nursing, 1955, 55 :450.
Keller. R., and Morton, H. E.: The effect of hand soap and a hexachlorophene soap on the
cultivable Treponemata. Am. J. Syph. and Gon. and V. D., 1952,36:524.
Sehlossberger, H., and Brandis, H.: Leptospira. Ann. Rev. Microbio!., 1954; 8:133.
Symposium on the Leptospiroses: Medical Science Pub. No.1, Army Med. Servo Grad.
Sehool, Washington, D. C., U. S. Gov't Printing Office, 1953.
Ward, M. K., McDaniel, M. B., Tatum, H. W., Starr, L. E., and Williams, H. R.: An epi-
demic of canicola fever in man with the demonstration of Leptospira canicola infection
in dogs, swine and cattle. II. Laboratory studies. Am. J. Hyg., 1956,64:59.
White, A.: They said I had syphilis. Saturday Evening Post, 1955, 227:(Apr. 30). 27.
I
32
The Spiral, Rigid Bacteria
AT PRESENT, six genera of spirilleae are recognized: Vibrio, Spirillum,

Cel/vibrio, Cellfalcicula, Desulfovibrio and Thiospira. The name of the first is
derived from its rapid motility (from Latin, vibrare, to vibrate); the name
Spirillum from its form. The names ,Cel/vibrio and Cel!falcicula are derived
from their morphology and their ability to metabolize cellulose. The names
Desulfovibrio and Thiospira are derived from the facts that the organisms are
curved and that their metabolisms involve sulfate reduction and sulfur oxi-
dation, respectively.
All of the spirilleae are non-sporeforming, and gram-negative. They are
motile by means of polar flagella; hence their inclusion in the family Pseudo-
monadaceae. They differ from spirochetes (Chapter 31) in being rigid, i.e.,
not self-flexing. The spirilleae are mainly saprophytic soil and water organ-
isms. Only a few dangerous pathogenic species are found in the group.
GENUS VIBRIO
This genus includes short rods which are always curved in a portion of a
spiral turn (Fig. 32-1). They often remain attached end to end after fission,
forming long spirals superficially resembling organisms of the genus Spirillum
(see Fig. 32-2). The length of the individual vibrios seldom exceeds 10 p, or
their diameter 1.0 to 1.5 p,. Some are strictly aerobic, some facultative and
some strictly anaerobic.
There are numerous "species" or varieties of vibrios, many of which
greatly resemble each other; having similar habitats, many physiological
properties in common, and sharing several somatic and flagellar antigens.
Many are named for the place where they were isolated: V. danubicus, V.
gindha, V. massauah, etc.
V. comma et al. Vibrio comma (discovered in the feces of cholera patients
by Koch in 1886), the cause of Asiatic cholera, resembles other vibrios in liv-
ing for long periods in polluted river and lake waters. (There is no cholera in
the Western Hemisphere.) Various strains of V. comma are recognized: the
Inaba and Ogawa varieties, and others. These differ mainly in antigenic struc-
ture and source. Another species possibly of pathologic importance is called
V. proteus; anothe~ the EI Tor* vibrio. This last vibrio is strongly hemolytic
• El Tor is the name of a town on the western side of the Sinai peninsula.
409
410
Fig. 32-1. EIectronograph of a characteristic vibrio (V. fetus). Note the polar flagella
and the curved form which is distinctive of these organisms. These cells, while somewhat
shrunken due to necessary drying, show clear differentiation between cell wall and interior
cytoplasmic, and possibly nuclear, materials. Original magnification X 30,000. (Photo cour-
tesy of Dr. Wayne Binns; in Cornell Veter., 1953, vol. 43.)
when broth cultures are mixed with a suspension of sheep or goat erythrocytes
and incubated for 4 to 6 hours (the Greig test). V. comma is not hemolytic
(Greig negative) under these conditions.
Some vibrios, like V. metchnikovi, are pathogenic for guinea pigs and
pigeons, which is not true of V. comma.
Isolation of Intestinal Vibrios. Most intestinal vibrios (and especially V.
comma) grow at the surface of nutrient liquids in response to need for oxygen.
They also tolerate an alkaline reaction (pH 8 to 9) which retards the growth
of many of the bacteria associated with vibrios in fecal material (e.g., Strepto-
coccus faeca/is, Escherichia coli, etc.). They also metabolize peptone rapidly.
The admixture of beaten, partly digested egg also favors growth. Therefore,
very rapid growth occurs at the surface of alkaline-egg-peptone solutions
(Dieudonne medium). Transfers from the surface film of such cultures after
six to eight hours of incubation often yield almost pure cultures of V. comma.
Diagnosis is often made by inoculating such medium with stools of a patient
suspected of having· cholera. This is a good example of enrichment.
Vibrio comma and its congeners grow well on ordinary laboratory media
after initial isolation. The colonies on agar are small, colorless and trans-
lucent. Most intestinal vibrios are markedly proetolytic, liquefying gelatin
and digesting casein as well as being active in the decomposition of some
carbohydrates like dextrose and sucrose.
Asiatic Cholera. Cholera is characterized by an intense diarrhea and pros-
tration, due to endotoxins of the vibrios. Great damage also is done by the
excessive and rapid dehydration of the patient which is a consequence of the
diarrhea. In the acute stage, the mucosa of the large intestine comes away in
Oakes and the stool, being thin and watery, is described as "rice-water stool."
TM Spiral, Rigid Bacteria 411
These flakes of mucosa contain large numbers of microscopically demonstra-
ble cholera vibrios. Cholera is a classical example of what results from lack of
sanitation, especially with respect to water supplies. A century or less ago,
cholera was to be found in practically every large city in the world, particu-
larly in communities where there was much transient population or in centers
for religious, military or other concentrations of large numbers of people,
with no effective sanitary provisions with regard to sewage and pollution of
water and food. In medieval Europe and later, in America, cholera was an
ever-present and often widespread and fatal scourge. It has played a sinister
and strictly non-partisan role in many disastrous military campaigns. Because
of outbreaks of cholera in the Near East and Eastern Mediterranean areas in
1947, pilgrimages to holy cities from the epidemic areas were stopped. Data
published by the World Health Organization set the total number of cases
at around 7300, with about 3200 deaths, between September 23 and October
20th. Cholera is found today mainly in the Orient. This is due only to constant
vigilance by International Health authorities and in the United States to the
activities of federal, state, and local sanitary administrations.
v. fetus. This is an economically important species because it causes
abortion and considerable reduction in fertility in sheep, cattle and horses and
consequent serious economic losses among stock raisers in the United States.
V. fetus is a rather highly adapted parasite. It apparently thrives only in the
genital organs of male and female domestic mammals (possibly also in wild
animals). The infection appears to be transmitted only by coitus. The organ-
isms grow only on very moist organic media in small, translucent, colorless
colonies. The slender, curved, individual ceUs are morphologically much like
V. comma. The best means of diagnosis is by isolation of the organisms from
the animals suspected.
Anaerobic Vlbrios. Another important group of vibrios comprises strictly
anaerobic species found in the human vagina. (If search were made they might
also be found, like V. fetus, in both sexes.) They appear to cause puerperal
(child-birth) infections of the vagina and also may reduce fertility as does V.
(etus in cattle. Similar (possibly identical) organisms (V. sputorum, V. stomati-
Fia. 32-2. Spirillum undilla (X 9(0)

412 The Bacteria
tidis) also occur in lesions in the upper respiratory tract and oral cavity. They
grow only on moist, rich, organic media, with 15% blood, anaerobically, with
a 10% C02 content of the atmosphere. Colonies are minute and colorless.
They are gram-variable and sometimes gram-positive.
GENUS SPIRILLUM
This genus contains no species known to be of special interest to the in-
dustrialist or the agriculturalist, and only one or two of minor medical interest.
All but the last mentioned are harmless saprophytes and scavengers, living,
as do most vibrios, in stagnant or polluted water and putrefying materials.
Most saprophytic spirilla are relatively large, ranging from 10 to 50 p. in
length though only 0.5 to 3 p. in diameter. They are spirally twisted through
1 to 5 complete turns (Fig. 32-2) and are motile by means of tufts of flagella
at one or both poles. They usually grow with difficulty on initial isolation.
Some of them have been obtained in pure culture by first preparing in-
fusions of stagnant water, dung or sewage enriched with peptone, meat or
fish. After a few days at room temperatures the fluid usually swarms with
many sorts of microorganisms. The fluid from such an infusion is sterilized
and used as a medium by solidifying it with 2 per cent agar. Poured into
Petri dishes, colonies of Spirillum can be obtained by inoculation with the
unsterilized infusion. Some of the organisms are remarkable in being very
sensitive to salt concentrations of even as low as 0.3% NaCI, seemingly pre-
ferring distilled (rain?) water. After initial isolation, growth on ordinary
medium is relatively easy.
These organisms and many others have also been maintained by a simple
and inexpensive procedure for teaching purposes, in a state of "suspended
animation" desiccated in a vacuum for several years.
Spirillum volutans, one of the largest species, is of interest because of its
large metachromatic granules, from which the term volutin, in reference to
the species, is derived. The motility and form of these spiral organisms is well
demonstrated in hanging-drop preparations made from cultures in peptone
solution and incubated four days at room temperature. Negative staining is
an effective means of making the organisms' arrangement visible. Beautiful
demonstrations may also be made with the darkfield apparatus.
Spirillum minus' and Rat-Bite Fever. Spirillum minus causes-in man a
disease (rat-bite fever), having several of the fundamental clinical features of
a typical spirochetal disease (syphilis). Sp. minus has never been successfl.llly
cultivated. It is gram-negative.-
Sp. minus occurs in the blood of rats and possibly other animals and is
transmitted from them to each other and ~o man by their bites.
REFERENCES
Bhaskaran, K.: Nutritional studies in Vibrio cholerae. J. Gen. Microbio!., 1956, 14:XV.
Chambers, J. S.: The Conquest of Cholera. The Macmillan Co., New York, 1938.
Kuydas, C. D., and Morse, E. V.: A selective medium for the isolation of Vibrio fetus and
related vibrios. J. Bact., 1956, 71 :251.
Lewis, I. M.: The genus Spirillum Eh'b'g., etc. J. Bact., 1940,40:271.
Myers, J.: Studies on the spirilleae. J. Bact., 1940, 40 :705.
Sen.eca, H., and Henderson/E.: Laboratory diagnosis of cholera. Am. J. Trop. Med., 1949,
29:921.
Spink, W. W.: Human vibriosis caused by Vibrio fetus. J.A.M.A., 1957, 163 :180.
33
The Aerobic Spore-Forming Rods

(Genus Bacillus*)
THERE ARE SCORES of species of bacteria which produce thermoresistant

endospores. With only one, possibly two, exceptions, these are all straight, rod
forms. The best known non-straight rod form of spore bearer is Desulfovibrio
or Sporovibrio (Chapt. 28). The straight-rod endospore formers are grouped
in only two genera: genus Bacillus (typically strict aerobes) and genus Clos-
tridium (typically strict anaerobes).
Endospores must be distinguished carefully from conidiospores and
sporangiospores of molds, ascospores of yeasts and from conidia of the
Streptomycetaceae. True bacterial endospores have a high degree of resist-
ance to chemical disinfectants and temperatures commonly used in baking-
and sterilizing. None of the other spore types approaches this degree of re-
sistance. All of the other forms of spores are means of reproduction. It is
generally thought that bacterial endospores are not, ,because each individual
cell produces only one spore. However, some authorities believe some bac-
teria produce more than one spore per cell.
GENUS BACILLUS
All of the organisms in this genus are straight, gram-positive (or gram-
variable) rods having dimensions ranging around 1.5 f.L in diameter and 10 f.L
in length. Many species contain numerous fat globules, conspicuous if prop-
erly staIned. Most species are motile. From the standpoint of cultivation in
the laboratory, or gtowth in natural habitats, they are not fastidious, growing
well on simple vegetable or yeast extracts or peptone media. They appear
generally to have requirements for complex organic sources of carbon and
nitrogen and for various vitamins.
These spore-formers are active and versatile producers of enzymes and
consequently can utilize a wide variety of substances as food. A common
physiological property is that of hydrolyzing proteins like gelatin, coagulated
blood, and dead plant and animal tissues. They also hydrolyze many different
carbohydrates and lipids and attack glucosides, alcohols and organic acids.
j,
*In order to avoid confusion of names, the student is urged to re-read the paragraph on
page 12 concerning the.'use of the terms of "bacillus" and "bacterium."
413
414 The Bacteria
Fig. 33-1. Types of sporulation. 1,

spherical; central; cell not swollen. 2,
oval; excentric; cell not swollen. 3, Oval;
central; cell swollen. 4, oval; excentric;
cell swollen. 5, oval; terminal; cell swol-
len. 6, spherical; terminal; cell swollen.
, 7, large, oval, central.
At least one species (Bacillus closteroides) can utilize carbolic acid. The
aerobic, spore-forming bacilli are thus seen to be of great importance as
scavengers. Some of their proteolytic and amylolytic 'enzymes are used in
industrial processes (leather, paper, silk, coffee, etc:). Several species are
famous and respected in the community because they produce valuable anti-
biotics: bacitracin, polymyxin, etc. (see Chapter 20). Others are infamous
and shunned because they grow in all sorts of valuable commodities (paper,
various foods and drugs, wood, leather, etc.) producing spoilage and economic
loss to human beings (about whom they care nothing f).
There is only one dangerous pathogen for man and animals in~the genus:
Bacillus anthracis, the cause of anthrax. ,
Variability. These organisms are among the most difficult to classify
because of the instability of tl!eir physiological characters and morphology.
[t requires the greatest care concerning composition of medium, its pH,
temperature of incubation, age of culture and numerous other factors to
obtain reproducibie results in the study of these species.
Thermophilic ~pecies. An interesting aspect of the variability of these
bacteria is the development of thermophilic varieties, often designated as
separate species. These thermophilic variants (or species) appear to have
developed thro1,lgh natural selection of mutants of mesophilic species. They
may also represent adaptive production of thermostable enzyme systems or
other thermostabilizing mechanisms.
;Types of Sporulation and Classification. Importance has been attached to
the form, location and size of spores as a differential character among species
of Bacillus. These relationships are shown in Figure 33-1. An excellent modem
The Aerobic Spore-Forming Rods 415
scheme divides the genus into three main groups on the basis of spore size
and form:
1. Sporangia not definitely swollen; spores oval to cylindrical; (B. subtilis,
B. cereus, B. megatherium, etc.).
2. Sporangia definitely swollen by oval spores. Spores rarely cylindrical;
(B. polymyxa, B. macerans, B. circulans, B. brevis, etc.).
3. Sporangia swollen by round spores. (B. pantothenticus, B. sphaericus,
B. pasteurii).
Spore Germination. The manner in which the spore germinates is easily
observed and is a particularly constant and distinctive species character.
Difficulties due to variation, including complete loss of sporulation, have
been mentioned. Sporulation is not always readily sccn in cultures of Bacillus
species. Certain conditions of nutrition must be present for prompt sporula-
tions. Aerobes form spores only aerobically.
Presence or absence of spores is most conclusively demonstrated by means
of heat. Growth of a culture after exposure to 90° C for 10 minutes virtually
proves the presence of spores.
Biochemical Characters. Certain biochemical characters are also of taxo-
nomic value. For example, one system of identification in the genus Bacillus
is based primarily on the power to hydrolyze urea (urease production),
secondarily on sensitivity to acidity and additionally on nutrient require-
ments, especially of ammonia and certain vitamins.
Structure of Bacillus. The electron microscope shows very clearly not
only flagella, form and size, but cell wall, protoplast, cytoplasmic mem-
brane and other structures.
A valuable method of studying cell structure is that of embedding bacteria
in a material like paraffin (or soft plastic) and making very thin slices. These
slices are then stained and pictured in the electron microscope. Some remark-
able lengthwise cross sections (sagittal sections) of some species of bacillus
have been thus obtained showing the process of sporulation (Fig. 33-2).
Distribution. These bacteria, or their spores, are ubiquitous in soil, dust,
and water. Observations of dust storms, and collections of air samples high
bver the polar oceans have made it clear that spores of these bacteria and
other forms of life are carried thousands of miles by currents of air. Darwin,
as early as 1831, on board the "Beagle," noted that the dust blown many
hundreds of miles out to sea from Africa contained spores. It is not to be
wondered at, therefore, that sporeforming, aerobic bacilli are often a source
of embarrassment to the unwary bacteriologist who finds them multiplying
unwanted in his c41tures. They, with molds, are among the most frequent
weeds of the microscopic garden. It requires assiduous care and constant
vigilance to exclude them.
It was the heat-~esistant spores of such organisms that misled Needham
and others to support the view that life began spontaneously in the infusions
which they thought they had sterilized by heating. Even experienced bacteriol-
ogists are sometimes embarrassed by the appearance of spore-forming rods
in supposedly sterile material or in pure cultures of bacteria. This is usually
due to carelessness in the sterilizing room, "short-cuts" in heating processes,
etc. I
416 The Bacteria
Fig. 33-2. Electronographs of ultra-thin sections of a bacterium (Bacillus cereus) show-

ing stages in the formation of a spore. Made with RCA, type EMU, electron microscopes.
1. Two matured cells showing possible nuclear material (N), vacuole-like inclusions (V) and
the spore primordium (Sp.) The last materializes by differentiation of a mass of the cell
content in situ. At G are seen granules (these are also seen in the other cells) in the cytoplasm
of the parent sporangium. These are of unknown significance but are possibly centers of
enzymic activity (mitochondria?). Possibly similar granules are seen inside the spore in
3 and 4. Note that the spore does not, as formerly thought, "grow" from a minute granule
such as are seen at G. The primordium of the spore probably represents a collection of
nuclear and other essential materials. The physicochemical changes involved in the differ-
entiation of the spore primordium are not yet fully known. 2. The developing spore has
changed in its consistency and its opacity to electrons. It is becoming enclosed within a
well delined spore coat. 3. Three distinct layers of the spore coat are formed: an inner one
probably derived from the spore material; the middle and outer possibly from the cytoplasm
of the parent sporangium. At 4 is seen an almost mature spore with three distinct coats.
5 shows a fully mature spore with thick outer coat. The material inside the spore appears
to be much less granular (denser?) and of varying opacity to electrons. The broken bacterial
cell (sporangium) wall is seen at W. (X 38,000). (Courtesy Dr. G. B. Chapman, in J. Bact.,
vol. 71, 1956.) -
BACILLUS ANTHRACIS
Only one species, Bacillus anthrac;is, is very pathogenic to man. It also
infects farm animals causing the disease anthrax. It differs from most common
species of Bacillus in being nonmotile. Motility can be induced in it by
transduction.
Anthrax. This is primarily a disease of farm animals but it is transmissible
to man. The name anthrax is from a Greek word meaning boil or carbuncle.
In man, the organisms most commonly gain entrance from soil, dust or
animal tissues, to the body through a cut in the skin. They first localize at
the point of entrance; forming a very rapidly progressive, angry, inflamed
pustule (malignant pustule), which, when well developed, is covered with a
black crust. This pustule teems with anthrax bacilli. It not infrequently heals,
but in other cases the bacilli invade the blood stream, multiply enormously,
and are spread through all the organs of the body where they tend to form
local lesions which serve as further centers for dissemination unless the leuko-
cytes and other defensive mechanisms of the body overcome them. When
growing in the body they produce no spores but develop capsules.
Anthrax in farm animals (cattle, sheep) may be prevented by injections of
B. anthracis bacterins (formaldehyde-killed bacilli) or by injections of spore-
vaccines made with living spores of graded, attenuated virulence or by the
use of serum for temporary, passive immunity. Animals dead of anthrax
should be handled with care to avoid contaminating the premises.
BACILLUS SUBTILIS
B. subtilis is the type species of the genus and is one of the commonest of
aerobic spore-formers. It is found in dusty places everywhere and especially
in hay. If hay be soaked in warm water for a day or two, the water will be
found teeming with organisms of many kinds, among which B. subtilis will
be prominent. Numbers of other species of Bacillus will also be found. These
are common contaminants of laboratory cultures.
Bacillus subtilis often forms long chains of bacilli sometimes called "strepto-
bacilli." Since the bacilli are motile, such chains swim with a writhing motion.
Due to avidity for oxygen, B. subtilis and many other species of Bacillus grow
in a scum or pellicle at the surface of fluid media.
Due to its active attack on organic nitrogenous compounds, its cultures
smell of ammonia. On slants of potato it grows luxuriantly, with a yellowish
or pink color and a warty or vesiculated appearance.
B. subtilis is important as the source of the antibiotic, subtilin. Bacitracin
is produced from a strain very like B. subtilis.
BACILLUS CEREUS, B. MYCOIDES, B. VULGATUS, B" MESENTERICUS, ETC.
These organisms are very much like B. subtilis. All are "hay bacilli," i.e.,
their spores are found in soil, hay, etc. B. cereus forms a spreading, grayish
growth on agar, while B. mycoides forms very characteristic, mold-like, or
nebula-like colonies on agar plates (Fig. 33-3). On potato it produces a whit-
ish, granular growth which becomes brownish later. B. mycoides is regarded
by some experts as a variant of B. cereus.
Bacillus vulgatus forms a gummy slime and sometimes causes bread to
seem "ropy" by growing in it during damp, warm weather, especially if the
bread was made with flour containing many spores of this organism. Its
growth on potato is thick, pinkish and wrinkled and turns brown.
Bacillus mesentericus is a slime-forming species also; its colonies are sticky
and mucoid. On potato it forms a moist-looking, wrinkled scum which turns
a dirty brown color 'later.
B. vulgatus and ~. mesentericus are probably identical.
BACILLUS COAGULANS
This organism is' of importance as a cause of spoilage of canned foods.
It is notable for its! ability to grow in acid foods like tomatoes. Since it pro-
duces no gas, spoilage ("souring") is not discovered until the container is
418 The Bacteria
Fig. 33-3. Colony of Bacillus my-

coides growing on nutrient agar. The
curiously curled growth is distinctive of
this organism. It is interesting to note
that the growth is always curled in a
clockwise direction. The reason for this
is one of the many mysteries of life.
(About life size.) (Courtesy of Dr. F. E.
Qark, U. S. Agricultural Research Serv-
ice, Beltsville, Md.)
opened. It is said to cause "fiat sours," because the ends of the can do not
bulge as they would if gas were formed under pressure by the fermentation.
B. coagulans is very heat-resistant and thus often survives commercial pro-
cessing. It is either a facultative anaerobe or it can grow sufficiently in the
small residuum of air enclosed in cans at the time of processing to produce its
results. A thermophilic species, B. stearothermophilus, very heat-resistant, is
also well-known as a nuisance and a source pf fiat sours in the canning
industry.
BACILLUS POPILLIAE AND B. LENTIMORBUS
"Milky Wbite" Disease. Two other species, called B. popilliae and B.
lentimorbus, are now used to combat Japanese beetles. The organisms grow
in the "blood" of the larvae and cause the disease commonly known as
"milky white" disease. For use against the beetles, the larval juices are dried
and ground and mixed with chalk dust or other powder. This is applied to
the soil as a spray or dust. Methods' of obtaining large numbers of spores of
the organisms by using artificial culture media are promising havoc in this
branch of "germ warfare" against the nefarious insects. The. beetles have
disappeared almost entirely in areas where the spores have been applied.
BACILLUS ROTANS AND B. ALVEI

These species are of especial interest because they produce colonies which
move. These motile colonies are discussed in the chapter on Myxobacteriales.
Foulbrood. ,Bacillus alvei is of especial importance because it is one of
several organisms which cause, or are associated with, a disease (foulbrood)
of bees which results in great losses to bee-keepers annually. There are several
forms of the disease. American foulbrood is caused by a related organism,
B. larvae, while B. alvei causes European foulbrood. Certain streptococci
(S. apis) also appear to cause the disease. The larvae of bees contain the
!infecting organisms ,in large numbers. This appears to parallel the infection
of Japanese beetle larvae by other species of Bacillus as noted above.
RllFERENCES
Allen, M. B.: The thermophilic aerobic sporeforming bacteria. Bact. Rev., 1953,17:125.
Bomside, G. H., and Kallio, R. E.: Urea-hydrolyzing bacilli. I, II. J. Bact., 1956,71 :627,
655.
Brown, E. R.,Cherry, W. B., Moody, M. D., and Gordon, M. A.: The induction of motility
tn Bacillus anthracis by means of bacteriophage lysates. J. Bact., 1955,69:590.
Burdon, K. L.; Useful criteria for the identification of Bacillus antllracis and related species.
J. Bact., 1956, 71 :25.
Smith, N. R., Gordon, R. E., and Clark, F. E.: Aerobic Sporeforming Bacteria. Agr. Mono-
graph 16, U. S. Dept. Agriculture, 1952. Gov't Printing Office, Washington, D. C.
Stein, C. D.: Anthrax. Farmers' Bulletin No. 1736, U. S. Dept. Agric. Gov't Printing Office,
Washington, D. C., 1955.
Steinhaus, E. A.: Living insecticides. Sci. Am., 1956,195:96.
Steinhaus, E. A.: Principles of Insect Pathology. McGraw-Hill Book Co., New York, 1956.
Steinkraus, K. R., and Tashiro, H.: Production of milky-disease spores (Bacillus popilliae
Dutky and Bacillus lenlimorbus Dutky) on artificial media. Science, 1955, 121 :873.
34
Anaerobiosis. The Genus Clostridium
ANAEROBIOSIS
THE ISOLATION of oxygen by Priestley in 1774 and subsequent obser-
vations by Lavoisier about 1775, on the role of oxygen in combustion and
respiration, as well as all previous ph).>iological experience, led to the con-
clusion that free oxygen (air) is necessary LO at: life. In 1861, however, Pasteur
proved that certain yeasts and bacteria could I. ult:ply in the absence of air.
He devised the term "anaerobiosis" to describe "life without air." This was
one of the epoch-making discoveries in biological science. Subsequent studies
in the physiology of cells living in situations devoid of free oxygen revolu-
tionized ideas of cell physiology and metabolism.
Since Pasteur's researches many microorganisms capable of living without
air have been discovered. These include many common species of bacteria and
other familiar forms of life. In a sense, the cells of our own tissues are an-
aerobic since they have access to oxygen only in combined form (oxyhemo-
globin). Yeasts common in baking and brewing grow in the center of rising
dough or at the bottom of fermentation vats completely out of contact with
air.
Relations to Oxygen. As indicated in Chapter 1, microorganisms may be
divided into several groups with respect to their relation to free oxygen.
1. Strictly aerobic species. These cannot grow without free oxygen to act
as final hydrogen acceptor. Their enzyme systems can transfer hydrogen only
to free oxygen.
2. Facultative organisms. These can use either free oxygen or some other
easily reducible substance (e.g., S, C, NaN0 3) as hydrogen acceptor; i.e.,
they have thefaculty of growing aerobically or anaerobically. This appears to
be usually because they possess both the aerobic as well as anaerobic enzymes.
3. Strictly anaerobic species, have two peculiarities: (a) they cannot grow
appreciably in the presence of free oxygen (air); (b) their enzyme systems
cannot ordinarily transfer hydrogen to free oxygen. They must use other
hydrogen acceptors. *
• Recent studies support older evidence that some species of strictly anaerobic bacteria
can use free oxygen as hydrogen acceptor to a vel)' l.imited extent. This is probably because
such organisms contain large amounts of certain ftavine enzymes which can transport some
hydrogen to atmospheric oxygen in the absence of the strictly aerobic cytochrome system.
42D
A,1IIU,obiosiJ. 'I'M GenIU CIo&tridium 421
4. Microaerophilic. These require limited or lowered oxygen tension but
not strict anaerobiosis. The mechanism of this is not fully explained.
H202 AND ANAEROBIOSIS. Now, then, why are strict anaerobes incapable
of growth in contact with free oxygen? No fully adequate reply to this question
is available. However, there is good evidence that respiration in the presence
of tree oxygen results in the formation of H202, which is very toxic. Thus
some strict anaerobes, while possibly capable of some aerobic growth, im-
mediately commit suicide when they attempt it!
"But," you say, "H20 2 is produced by many aerobic bacteria. Why do
they not die?" Ah! But these produce catalase, an enzyme which immediately
decomposes H 20 2 ! And you (being a well-informed student) say, "True,
but many vigorous aerobes do not produce catalase. Why does their H 20 2
not kill them?" So true! We reply, "These are not sensitive to H 20 2." Which
is a "finisher" worthy of Mark Twain. You would like to ask, "Why aren't
they sensitive'!" and we would say, "Because they have enzyme systems not
affected by H 20 2." Which is no real explanation and is merely another
Mark Twain "finisher" in modem form.· (By familiarizing yourself with these
pros and cons you can astonish and mystify your friends who are not taking
microbiology.)
We may list some important or familiar groups of bacteria in relation to
oxygen as follows:t
1. STRICT AEROBES
Most species of the genus Bacillus
Several species of the genera Pseudomonas, Xanthomonas
Genus Brucella (exc. Br. bronchiseptica)
Genus Azotobacter
Tribe Nitrobacteriae
Genus Thiobacillus (exc. Th. denitrificans)
Genus Acetobacter
Genus Mycobacterium
Hemophilus pertussis
Most of family Micrococcaceae
Family Streptomycetaceae
Genus Nocardia
Order Myxobacteriales (exc. Cyto.jermentans)
2. FACULTATIVE
Family Enterobacteriaceae
Tribe Streptococceae (a few are strict anaerobes)
Genus Spirillum
Some of genus Micrococcus
Genus Neisseria
Genus Alcaligenes
• Especially when we ~ that what is demonstrated for one species is not necessarily
true for all!
t In each group there are always one or more exceptions. Between the 4 groups there are
always "border-line" cases and doubtful species. Hardly anything in biology is absolute;
not _ the distinction between living and non-living!
422 The Bacteria
3. STRICT ANAEROBES
Genus Clostridium
Genus Actinomyces
Tribe Bacteroideae
Cytophaga Jermentans
Genus Desulfovibrio
Family Thiorhodaceae
Family Chlorobacteriaceae
Thiobacillus denitrificans
4. MICROAEROPHILIC
Genus Leptospira
Genus Lactobacillus
CULTIVATION OF ANAEROBIC BACfERIA

Many types of anaerobic device exist but only one fundamental purpose is
involved. This is the removal of free oxygen from the immediate environment
of the bacteria, or the maintenance of a low oxidation-reduction potential
(presence of a reducing or oxygen-absorbing agent) in the medium itself.
Oxidation-Reduction Potentials. Oxidation and reduction can occur in
the absence of oxygen and are, as previously pointed out (Chap. 13), basically
phenomena dependent on electron transfers. A substance which readily takes
up electrons is a good oxidizing agent; i.e., it is readily reduced. Conversely,
a good reducing agent is one which readily gives up electrons; i.e., it is readily
oxidized. The electron-yielding or electron-accepting potentialities of any
given material (a bacterial culture, sewage, etc.), i.e., its oxidation-reduction
potential, can readily be measured by thrusting an electrode into it and
connecting with a reference cell and a voltmeter (potentiometer) (Fig. 34-1).
O-R Requirements of Microorganisms. Anaerobic organisms require low
O-R potentials for growth. For example, an O-R potential of -0.2 volt is
optimum for the initiation of growth by most species of anaerobes. The
effects of various substances added to media to maintain low O-R potentials
are shown in Figure 34-2. Once growth has started, the O-R potentials of
cultures of all bacteria decline. This is especially marked in cultures of an-
aerobes since these use every available hydrogen acceptor (tend~to release
electrons) in their respirato_ry processes. I
ANAEROBIC METHODS
Anaerobic conditions in culture media may be brought about by three
main classes of procedure. . I
(I) Chemical. (a) Cultures may be enclosed in an airtight vessel with
sticks oj phosphorus or with a freshly made mixture of potassium hydroxide
and pyrogallol. These substances absorb large amounts of oxygen and leave
mainly the inert gas, nitrogen, and a partial vacuum.
(b) The combustion of small amounts of alcohol or the burning of a small
candle in a closed jar will use up some of the free oxygen. Combustion cea.ses
wh~n the carbon dioxide content approximates 10 per cent. This method
results in only partially anaerobic conditions. It is widely used to increase
Anaerobiosis. The Genus Clostridium 423
the carbon dioxide content of the atmosphere, a condition favorable to many
organisms, both aerobic and faculative, which require CO2 for cell synthesis.
The reduction of oxygen tension favors microaerophilic organisms rather
than strict anaerobes.
(c) A means of absolute anaerobiosis is to allow a fine stream of hydrogen
to enter a closed vessel, impinging, as it enters, on a small mass of some
catalytic agent which causes it to combine with the free oxygen, torming
water. A catalyst commonly used is "platinized asbestos" (finely divided
platinum deposited on asbestos fibers). This acts very rapidly only when
heated. In some types of modern apparatus, the necessary heat is applied to
the catalyst by means of an electric current passing through a resistance wire
surrounding the mass of catalyst. Danger of explosion due- to sparks is
eliminated by enclosing the heating element in a gastight tube inside the
Fig. 34-1. In measuring the poten- T

tial of bacterial culture, A, (in this 1:5-
sembly the culture is called a half-cell),
it is connected, by means of a glass or
plastic tube filled with electrolyte (KCI-
saturated agar) (stippled), with an-
other half-cell, B, (the standard, hydro-
gen half-cell). The potential of the hydro-
gen half-cell is fixed and is arbitrarily
stated as zero. The potential of the cul-
ture is designated as Eh since it is to be
stated in reference to the potential of the
standard, hydrogen half-cell, Est·. After
connecting the electrodes (T and T') to
the potentiometer (P) and galvanometer
(G) the EMF (E) of the potentiometer
(which is indicated on a scale) is varied
by means of the slide-wire (S) urttil it
just balances the EMF of the culturel
standard-half-cell system. At this point
the galvanometer needle does not swing
toward either pole whim the circuit is
completed. E1 is then ';read off on the
scale of the potentiometer. Obviously,
then, E) = Eh - Est or, Eh = EI + Est.
Knowing E) and ESh Ell is readily calcu-
lated. .
* In practice a calomelihalf-cell (with Eh, at 20° C and 3.5% KG = 0.254 volt) is used in
place of the hydrogen nalf-cell for reasons of convenience and economy. Appropriate
allowance is made in the,calculations for the voltage of the calomel half-cell.
424 The Bacteria
Eh ."JI,-~ I
I I
+.20
+.10
,
~
,,
,,
o I
'. _No ."g.r.... Ihi.a1y •• lldt.-A
\
-.100 \
\
\
\
-.%00 i=~~~~\
10 20 ;)0 "'0 SO HOURS

Fig. 34-2. Graphs indicating the O-R potential of growing cultures of C. per/ringens in
peptone-agar medium with and without the reducing agent, sodium thioglycollate (A, B)
and in Brewer's medium (C) containing thioglycollate. Note that, before inoculation,
medium A had a definitely positive O-R potential. After inoculation, as the Clostridium
per/ringens grew, the O-R potential became rapidly more negative,.finally equalling the solu-
tions containing a strong reducing agent. This is characteristic of anaerobic growth in gen-
eral. It illustrates, in large part, the origin of BOD in sewage (see Chapter 40). Ordinates
represent Eh and abscissa time. (Reed, G. B., and Orr, J. H., in J. Bact., vol. 45;)
catalytic mass. A drying agent is enclosed in the vessel to absorb the water
that is formed. There is no vacuum, the remaining gas being a mixture of
hydrogen and nitrogen.
(2) Replacement Methods. Another method of removing oxygen from the
atmosphere of closed "anaerobic jars" is ~imply to flush out all the air with
a stream of some inert gas like hydrogen or nitrogen. This leaves an atmos-
phere of almost pure hydr'ogen or nitrogen and no vacuum.
The difficulty of obtaining hydrogen from Kipp generators or high-pressure
cylinders is readily overcome by placing in the anaerobe jar a tube containing
0.6 gm of sodium borohydride (NaBH4), in about 40 ml of water, with 0.2 gm
of CoCl 2 as a catalyst. The NaB~ decomposes readily, liberating just enough
hydrogen to use up all the oxygen in the jar without generating the dangerous
pressure of cylinder gas.
NaBH4 2H20 --+ NaB02 4H2 + +
CoCl 2
(catalyst)
(3) Oxygen Exclusion Methods. (a) A simple means of excluding oxygen
from single culture tubes is to cover the medium with a layer of sterile petro-
latum or mineral oil several em thick. The medium in the tube under the oil
is boiled or autoclaved and cooled rapidly in ice water (so that oxygen is not
reabsorbed) just before inoculation. The heat drives off the dissolved air.
If the organism is a gas-former, the gas may blow the petroleum jelly or
cotton plug out of the tube. This method is "messy" but effective if c<lrefully
manipulated. Withdrawal of material for study from such sealed tubes is
awkward.
(b) A simple and effective means is available for obtaining pure cultures of
strict anaerobes on the surface of agar plates (formerly possible only by the
use of cumbersome, time-consuming and laborious anaerobe jars). The
modern method depends on chemical absorption of oxygen from air trapped,
by a specially shaped cover, in a very thin layer over the surface of special
agar medium in a Petri dish (Fig. 34-3). The oxygen in this air is absorbed
by sodium thioglycollate, or some similar compound having an affinity for
oxygen, incorporated in the agar. By this means the O-R potential of the
medium is held very low and even the most sensitive anaerobes will grow
on the agar surface.
(c) Deep tubes of dextrose-infusion agar are also used to cultivate an-
aerobes. Infusion agar in tubes 8 to 10 cm in depth is melted and cooled to
about 50° C. The inoculum is put in and mixed thoroughly. The agar is then
made to solidify rapidly in cold water and is incubated. Strict anaerobes will
grow only in the depths and will not appear at all within a centimeter or more
of the surface. Less strict anaerobes will grow in the depths and will also
grow somewhat nearer to the surface, while facultative anaerobes will grow
on the surface as wen as in the depths. Organisms having a narrow zone of
tolerance to both oxygen and strict anaerobiosis (microaerophils) may grow
in a narrow zone some distance below the surface (Fig. 34-4). Such prepara-
tions are often spoken of as "shake tubes" because shaking is used to mix
the agar and the inoculum.
Cultivation of Anaerobes in Media Freely Exposed to Air. Pure cultures of
the strictest anaerobes may be obtained in ordinary media with no precau-
tions as to exclusion of air, provided some substance be added to the media
to maintain a low O-R potential.
For example, anaerobiosis in tubes of broth is satisfactory if the medium
contains bits of chopped tissue, "cooked meat medium." The tissue acts as a
reducing agent. The meat also serves as pabulum for the bacteria. Most
anaerobic bacteria grow well in cooked-meat medium.
If the columns of medium in the tubes are 10 to 15 cm deep, all that is
necessary is to heat :the medium in boiling water for 10 minutes to drive off
dissolved air, cool 'fpidly, and inoculate in the depths.
-.Anaerobic Petri Dish Cover-

A1I'Space
Fig. 34-3. Cross seCtion showing Brewer anaerobic Petri dish cover in use. The anaero-
bic agar contains the reducing agent, sodium thioglycollate. Note that, at the periphery of
the agar surface, the Pl1tri dish cover is in contact with the agar, thus sealing the air space.
The thioglycollate absorbs the oxygen from the air space. (Courtesy, The Baltimore Bio-
logical Laboratories.)
426 The Bacteria
ABC D E
Fig. 34-4. Deep tubes of agar inoculated with bacteria of various oxygen relationships.
A, fairly strict anaerobe, like ct. botulinum; B, 'less strict anaerobe, like ct. periringens;
C, facultative aerobe-anaerobe, like Esch. coli; D, ml.cro-aerophilic organism like Br. abor-
tus; E, strict aerobe, like Pseudomonas fluorescens.
The addition of sodium thioglycollate (0.1%) or sodium formaldehyde

sulfoxalate (0.1 %), or both, to dextrose broth or other similar fluids adapts
them to anaerobic requirements. The addition of 0.1 per cent agar creates a
very slight viscosity which reduces aeration of the solution by convection cur-
rents. The use of 0.2 per cent cysteine hydrochloride is as effective, under
most conditions, as the reagents mentioned above. The addition of cobalt ions
(Co++) to medium as Co(N03)2 in concentration of 4 fJg Co++ per ml also
permits aerobic growth of anaerobes by markedly lowering O-R potential. In
general, milk, infusion broth and infusion agar with blood, treated with the
reducing reagents, are good culture media for anaerobic organisms, since
these organisms require media rich in organic matter and having a pH of
about 7.2. Chopped brain, fish or other tissues are also often used. The addi-
tion of dextrose provides a readily available hydrogen acceptor and source of
carbon which promotes the growth of nearly all anaerobes. '
AN~ROBIC BACI'ERIA
As shown at the beginning of this chapter there are numerous important
species of strictly, anaerobic bacteria. Actinomyces have been discussed in
Chapter 30; Desulfovibrio and Thiorhodaceae in Chapters 28 and 29, the
Chlorobacteriaceae in Chapter 29. There are a few others oflesser significance.
Here' we may describe briefly the Tribe Bacteroideae and the genus Clos-
tridium.
TRIBE BACTEROIDEAE
These bacteria are non-sporeforming, generally gram-negative, small,
usually rod-shaped, very pleomorphic. None is motile. There are only two
genera: Bacteroides and Fusiformis.
Some species are normal inhabitants of the upper respiratory tract and
genital mucosae. Most species of Bacteroides occur in enormous numbers in
the intestinal tract. Various species of Bacteroides are frequently found ap-
parently as causative agents in lesions of the mucous membranes, in septi-
cemia, in appendicitis, in abscesses ofliver, lungs, and other parts of the body.
They are often overlooked in diagnostic microbiology because they grow only
under strictly anaerobic conditions on media containing blood or ascitic
fluid. The colonies are small and colorless. The organisms are fragile, and
difficult to maintain alive. The species present in normal feces grow on ordi-
nary laboratory media.
Morphological studies reveal a rather considerable variation in form of
Bacteroides from global to filamentous. This variation in form appears to be
associated with a reproductive cycle like PPLO, involving small forms, like
L bodies.
Because of their filamentous variations Bacteroides have often been classed
with Actinomyces and other illamentous bacteria. Some authors include in
the tribe, as Bacteroides fusiformis, a species of fusiform and illamentous
anaerobic bacteria common in the normal mouth. A well known species of
these (usually called Fusobacterium plauti-vincenti) is associated with Borrelia
in Vincent's angina or "trench mouth."
Other species of Bacteroides frequently occurring in ulcers and abscesses
are B. fragiUs, B. funduliformis, and B. serpens.
GENUS CLOSTRIDIUM
The bacteria belonging to this genus are all obligately anaerobic, gram-
positive, spore-bearing rods. Nearly all are motile. They vary somewhat
in size and shape, in the manner, say, of cigars, but average around 0.5 by 10 /.l
in dimensions. They require complex organic media like cooked-meat medium,
glucose-infusion agar or broth, and the like. The group inCludes the organisms
producing tetanus (lockjaw), gas gangrene, and botulism (food poisoning).
The majority of clostridia are harmless and helpful saprophytes. Many of
them produce enzymes, chemicals and industrial fermentations of great value.
All occur widely distributed in the soil. Some of them also live in the intestinal
tract of man and animals. They are metabolically active and versatile.
Oostridium Butyricum. This is one of the earliest species of Clostridium
to be studied and is the type species of the genus. It represents the group of
industrially important clostridia which are all much alike. In general they
are plump, actively motile rods having oval, excentric spores which swell the
sporangium. These organisms are widely distributed in the soil. They grow
well in media made of dilute molasses or grain extracts, with starch and
suitable mineral (and' sometimes vitamin) supplements. All have the power
of fermenting carb06ydrates, with the production of butyl alcohol, ethyl
alcohol, acetone, amyl, ethyl, and propyl alcohols and acetic, formic, and
lactic acids, acetone, carbon dioxide and hydrogen. The products of fermenta-
tion depend on the variety of Clostridium used and the condition of the
fermentation, i.e., nutrient, pH, temperature, substrate, etc.
Auaerobic Nitrogen Fixation. An interesting property of some of these
organisms is the power to fix atmospheric nitrogen. That is, they are not
428 The Bacterill
restricted to the use of nitrogen combined in the form of ammonia, nitrates,
amino acids, etc., but possess the power to cause free nitrogen of the air to
combine in the synthesis of their protoplasm. (See nitrogen cycle, Chapter 40.)
Pathogenic Clostridia
An important paradox is that although they are highly dangerous patho-
genic organisms, Clostridium botulinum, CI. perjringens and CI. tetani are not
parasites but strict saprophytes. They grow only on dead matter and cannot
invade live tissue. They are all commonly found in the soil, the latter two
species especially in animal feces. The spores, consequently, are widespread
in manured lands.
Clostridium Tetani and "Lockjaw." Clostridium lelani is one of the strictest
anaerobes. Morphologically, the organism is usually a slender rod (0.51' by
4 to 81'). It bears a spherical spore at the very tip end (terminal) of the rod.
The round, terminal spore gives to the organism what has been called a
"drumstick" appearance (Fig. 9-10).
C/o tetani gives off a potent exotoxin. Tetanus toxin is particularly active
in the motor nerve centers, irritating them so that the muscles connected with
them are thrown into a state of violent and continuous contraction (tetanic
convulsion or tetanus). The use of antitoxin in the treatment and prevention
of tetanus is a classical example of passive immunity and was the first to be
discovered (von Behring and Frankel, 1890).
Tetanus organisms gain entrance to the body with dirt or dirty objects when
these are forced into the tissues as in gunshot or shrapnel wounds or various
accidental means. Under such circumstances some tissue is killed locally by
the mechanical injury and, in deep wounds, the low Q-.R potentials favor
growth of anaerobic bacteria. The organism cannot invade the body, but
grows as a saprophyte on the dead tissue in the wound, liberating its deadly
toxin which is absorbed by the blood or nerves or both.
Tetanus toxin is one of the most potent poisons known. It requires only
about 0.00025 gm of tetanus toxin to kill a man while it requires twenty times
as much cobra venom and about one hundred and fifty times as much strych-
nine to do the same.
TETANUS iMMUNIZATION. Alum-precipitated fluid toxoids, in all respects
analogous to diphtheria toxoids, are useful in producing active immunity to
tetanus (Chapter 22). Protection depends particularly on the action of a pri-
mary stimulus consisting of at least one, preferably two, doses of tetanus
toxoid a month or so apart. This is now routinely given on entering the Armed
Forces. A booster dose is given about a year later. To a secondary stimulus,
resulting from entrrmce of tetanus toxin into the body as the result of a wound,
the tissue cells respond quickly with the production of antitoxin. A dose of
toxoid in previously-immunized persons is, therefore, often used as prophy-
laxis in dealing with any fresh wound. It is preferable to avoid the use of
serum unless tetanus is imminent. Passive prophylactic antitoxin may then
also be used. Even if serum has to be used, toxoid should also be given. Why?
The same principles apply in diphtheria (Chapter 35). Combined diphtheria
and tetanus immunization employing mixtures of the two toxoids has been
found entirely practicable and is routinely given to young infants.
Clostridium Perfringens. C/. per/ringens is a rather short, thick rod with
A_robio8b. TM G~nus Clostridium 429
rounded ends. It usually grows singly, never in long chains or filaments. It
forms oval, central or subterminal spores which do not swell the cell. With
four or five relatively unimportant exceptions, it is the only non-motile
species in the genus. *
Certain strains of Cl. perfringens are considered by some to be the cause of
gastroenteritis when taken in large numbers in food or water. The constant
presence of Cl. perfringens in feces has led at times to its consideration as
an indicator of human fecal pollution when found in water. Because of its
pronounced gas-forming powers it is often called the "gas-bacillus."
STORMY FERMENTATION. One of its most characteristic cultural reactions
is its power to produce a condition in milk called "stormy fermentation."
If a column of milk 10 cm deep in a culture tube and containing a little peptone
be inoculated with Clostridium perfringens, covered with a layer of petrolatum
or melted agar and incubated, the lactose is then quickly fermented. Sufficient
acid is quickly formed to coagulate the casein. Gas (mainly hydrogen) is then
Fig. 34-5. Tube of milk inoculated

with Clostridium pf!r/ringens showing
"stormy fermentation." For explanatioi
see text. (photo courtesy of CommunI-
cable Disease Center, U. S. Public Health
Service, Atlanta, Georgia.)
* In attempting to determine motility of anaerobic bacteria, care must be taken not to

expose the hanging drop to the air for more thJn a few seconds as motility is destroyed
by access to free oxygen.
430
Fig. 34-6. Clostridium botulinum cul-

tivated in cooked-meat medium. (X
1000). (photo courtesy of Communicable
Disease Center, U. S. Public Health
Service, Atlanta, Georgia.)
fonned in large amounts and the clot of casein is rent asunder, giving the
appearance of a very turbulent or "stormy" reaction (Fig. 34-5). Any organ-
ism producing sufficient acid to form a solid clot, followed by abundant gas
production, could give the same reaction. There are, however, oruy a few
such organisms.
The Gas Gugrene 0rgauIsms. Ct. perfringens, being common in the soil,
always accompanies C/o tetani in wounds and, like the latter organism grow-
ing saprophytically only on dead tissue, gives off toxins.
GAS GANGRENE. In dirty wounds, in addition to Clostridium perjringens
and C/. letani, there are nearly always present one or more of about a score of
rather similar species of clostridia of the soil such as Cl. navyi, CI. histolyti-
cum, etc. Some of these are able to digest dead tissue rapidly, others produce
toxins, hemolysins, etc. Some (Cl. navyi, C/. septicum) actually invade the
blood stream. All of these bacteria are spoken of as gas-gangrene organisms.
In general, the combined unchecked growth of gas gangrene organisms in
dirty wounds such as crushed limbs, shell wounds, nail punctures, etc., where
there is much dead tissue, produces a rapidly fatal condition known as gas
gangrene. It used to be much feared by soldiers wounded on the battlefield.
Oostrldimn BotuliDam. This is one of the two organisms (Micrococcus
pyogenes and ct. botulinum) causing food poisoning. C/. botulinum is a strict
anaerobe and forms large, oval spores in a subterminal position, often giving
the sporulating rod a shape that is said to resemble a snowshoe (Fig. 34-6).
These spores are-very heat-resistant. Like the other clostridia, CI. botulinum
is widely distributed in the soil.
It derives its name from the Latin word for sausage (botulus). It was given
its name because it was first found in sausages which were the cause of fatal
food poisoning (botulism). The interior of a sausage (or canned foods if not
sterilized) obviously presents an ideal place for the growth of anaerobes
(Chapt. 43).
'RIftRENCES
Brewer,1. H., Heer, A. A., and McLaughlin, C. B.: The use of sodium borohydride for pro-
ducina hydrogen in an atIaf.\'Obejar. App\. Micr., 1955,3:136.
ArtMrobio.m. TIu! Ge1lU.f Clostridium 431
Cockburn. W. C., and Vernon. E.: Food poioot\ing in England and Wales. \954. Monthly
Bull. Ministry of Health and the Public Health Lab. Serv., (London) 1955. 14:21)3.
Deck, G . M.: Food Poisoning. 3rd ed. University of Chicago Press. Chicago. III .• \956.
[)edic, G. A .• and Koch, O. G.: Aerobic cultivation of Clostridium tetoni in the presence of
cobalt. J. Bact., 1956, 71: 126.
Dolman. C. E.~ Additional botulism episodes in Canada. Can. Med. A. I., 1954,71 :245.
Editorial: Tetanus immunization. I.A.M.A .• 1956, 161 :883.
Hewitt. L. F.: Oxidation-reduction potentials in bacteriology and biochemistry. 6th ed.
London CoUllty Council, London, 1950.
Lev, M.: Aerobic cultivation of Clostridium tetani. I. Bact•• 1956, 72 :718.
McClung, L. S.: The anaerobic bacteria with special reference to the genus Clostridium.
Ann. Rev. Microbio!., 1956,10:173.
Pulaski, E. J.: Medical progress: war wounds. New England J. Med., 1953,249:890,932.
Various Authors: Symposium OD Anaerobes. J. AppJ. Microbial. (London), 1956.19:1.
35
Family Corynebacteriaceae;
Family Lactobacteriaceae
THE CORYNEBACTERIACEAE
THE NAME of this family is derived from Greek words meaning "club-
shaped bacterium," referring to the club-shaped cells often formed by some
species, notably Corynebacteriwn diphtheriae .
. The family contains three genera: the genus Corynebacteriwn, of which
C. diphtheriae, the cause of diphtheria, is type species; the genus Listeria, of
which L. monocytogenes, a motile "diphtheroid" and the cause (1) of "glandu-
lar fever" (more properly,listeriosis) in man and animals, is the only species;
and Erysipelothrix, of which the cause of swine erysipelas, E. rhusiopathiae,
is the type species.
All are gram-positive (or gram-variable) non-sporeforming, non-motile
(except Listeria) generally short, plump, rods ranging around 1.0 by 8.0 ". in
dimensions and exhibiting various degrees of pleomorphism, especially in
Corynebacteriwn and Erysipelothrix. Most of the species in this family grow
best aerobically, on infusion media at 30 to 400 C, pH 7.0 to 8.0, particularly
if serum (or blood) and dextrose are added. Except for a few parasitic species,
they are saprophytes, widely distributed in the environment: dust, dung.
dairy products, on the skin, in decaying organic matter, etc. These are fJe-
quently encountered as contaminants in laboratory cultures.
THE GENUS CORYNEBACTERIUM
There are several animal pathogens and numerous harmle~~ saprophytes
in this genus. Some motile species have been described, but these are not true
corynebacteria. To the farmer and veterinarian important species are: C.
pyogenes, which is common in purulent lesions of cattle, swine and sheep;
C. equi, causing pneumonia in foals; and C. renale which causes a necrotic
disease of the urinary tract in cattle. In addition, the genus includes a number
of important plant pathogens, like C. michiganense, the cause of tomato
canker, and C. insidioswn, cause of a destructive disease of alfalfa.
Differentiations between species of corynebacteria are commonly made on
tbe basis of fermentation reactions, pigment formation, gelatin liquefaction
and morphology. In general, they are not very active in attacking carbo-
hydrates, proteins or fats.
431
Family Corynebacteriaceae; Family Lactobacteriaceae 433
From the standpoint of human health the most important species is C.
diphtheriae although the microaerophilic C. aeMS, implicated as a cause of
acne (so often a temporary thorn in the flesh of all who are young and beauti-
ful), has an importance in human happiness.
C. diphtheriae is usually distinguishable by: (1) great variation in length of
the cells, from coccoid to spindles or clubs 10 to 15 JJ in length; (2) great
variation in shape, from club-shaped to sperm-like, needle-shaped or boom-
erang-shaped forms; (3) conspicuous intracellular granules, bars and masses.
These have been regarded as nucleus-like structures but are more generally
regarded as volutin (Fig. 35-1); (4) irregularity of arrangement; (5) marked
affinity of the volutin in the cells for methylene blue. The result of this is that
the granules, bars, or the entire cell (depending on the distribution of the
volutin in the cell) stain a very intense blue or metachromatic (red) color.
Diphtheroids. Species of corynebacteria having a more regular length,
form and arrangement, which differentiate them from C. diphtheriae. are
spoken of collectively as "diphtheroids" (Fig. 35-2).
A number of similar gram-positive rods, now separated in the genera
Microbacterium (lactic acid producer), Butyribacterium (butyric acid pro-
ducer), and Propionibacter (propionic acid producer), (Tribe Lactobacilleae)
are closely related and similar to corynebacteria in a number of respects.
They are discussed farther on in this chapter.
The colonies of corynebacteria on solid media, such as blood infusion
agar, are generally white or yellowish, opaque, round and range in diameter
from about I mm to 3 or 4 mm. They are usually soft and butyrous but some
Fig. 3S-t. Coryneboct,rium diphtherial'. These have beer stained with LoefHer's alkaline
methylene blue solution. Note the great variation in length. the pleomorphism and the
¥OIutin arranged as bars and granules and sometimes filling the entire cell. (2S00 X).
434 The Bacteria
, .....
V...,
-
I
..J (#
- Fig. 35-2. A typical diphtheroid-

Corynebacterium acnes. (Ford, Textbook
of Bacteriology.)
species form irregular and brittle colonies. Many species form brilliant pig-
ments.
We may well give some attention to diphtheria because it illustrates a
number of important principles of medical microbiology and immunology.
What is true of one infection is often, in whole or in part, true of other in-
fections. Formerly a widespread and fatal scourge, diphtheria now kills less
than 3000 persons in the United States annu,ally.
Diphtheria
Diphtheria is a specific disease due to Corynebacterium diphtheriae. The
organisms are transmitted in the same manner as others causing respiratory
disease. Healthy carriers are not uncommon (about I per cent of the popu-
lation) and are doubtless sources of cases. The bacteria establish themselves
on the mucous membrane of the throat and nose and excrete exotoxin.
Toxin Production by Corynebacterium Dipbtheriae. C. diphtheriae secretes
one of the most powerful biological poisons known. When the organisms
grow on the tonsils this poison is absorbed by the blood and damages heart,
kidneys, adrenals and nerves and may cause death unless antitoxin is (a) al-
ready present or (b) is developed rapidly by the cells of the patient, or (c) is
injected into him from some outside source. The toxin is also very poisonous
to rabbits, mice, guinea pigs and to chicks and other birds.
Corynebacterium diphtheriae is a classical illustration of virulence depending
almost entirely on toxigenicity. This organism, although often growing exten-
sively on the surfaces of the respiratory tract, has little ability to invade the
tissues beyond the mucous membrane.
Immunity to Dijlbtheria. Most persons more than 15 years of age possess
a natural, active immunity probably through subclinical attacks during earlier
childhood, and retain their immunity throughout life, probably as a result of
repeated reinfection. Their blood usually contains a small amount of antitoxin
and this helps combat ordinary infection. In addition, as pointed out in the
chapter on Allergy, natural immunity includes a tissue-reactivity or binding
power for the bacilli and probably toxin, which is a pre-antitoxin line of defense.
THE SCHICK TEST. It is possible to determine whether or not a person's
blood contains sufficient antitoxin to help protect him. Of the amount of
diphtheria toxin which takes four days to kill a small guinea pig (l minimal
lethal dose, as it is called), one fiftieth is injected into the skin of the person
437
Family Corynebacteru,ceae; Family Lactobacteriaceae
·
whose Immumty . IS. to be teste.
d If t he person' s bl00d contaInS
. a SUI.,
,<tIled
tatot
amount of diphtheria antitoxin to protect him (about 0.01 unit· per ~o t. .
centimeter of serum), nothing happens. If his blood contains too little an.,
toxin, a small red spot appears at the site of the injection and remains for
some days. This is called a positive Schick test, the test having been devised
by the Austrian physician, Schick. It is perfectly harmless and may do some
good by stimulating antibody production.
If the natural immunizing processes of often-repeated natural reinfection,
which depend on prevalence of healthy carriers of virulent organisms, are
diminished, t,hen many persons may fail to be naturally immunized. Their
Schick test may remain positive. To supplement natural immunization proc-
esses artificial active immunization is widely used.
AcnvE IMMUNIZATION TO DIPHTHERIA. Some children giving a positive
Schick test may contract diphtheria. Whether they do or not depends on
dosage of infective bacilli, natural resistance and other factors. For safety,
they should be artificially, actively immunized sufficiently so that they give a
Fig. 35-3. Immunization to diphtheria. This picture shows the simplicity of the pro-
cedure. It is of great historical interest, since it is a portrait of the late Dr. William H. Park,
long Bacteriologist for the City of New York, who introduced diphtheria immunization to
this country (right); and Dr. Schick, the famous Austrian physician who devised the Schick
test for immunity to diphtheria (standing, white gown).
• A unit, roughly, is sufficient antitoxin to neutralize a little over 100 minimal lethal doses
of toxin.
436 TM BacteriD
negative Schick test. Any physician or health department will do this on re-
quest (Fig. 35-3). The process requires only two injections of toxoid, alum-
precipitated, given 4 to 6 weeks apart. The toxoid is often mixed with tetanus
toxoid and pertussis (whooping cough) vaccine, with excellent results in
respect to all three diseases. Since this immunity wears away in time, it is
customary to reimmunize with repeated small doses ("booster doses"), a
year later and then every two to three years.
Adults not infrequently give positive Schick tests, but do not have diph-
theria nearly so often as children, from which it may be inferred that such
persons have: (a) binding power of their tissues as a result of natural im-
munization, and (bl an enhanced, allergy-like ability to respond to infection
very rapidly by producing antitoxin in their blood. Both types of response
result from a previous slight infection or primary stimulus.
PASSIVE IMMUNIZATION TO DIPHTHERIA. A person ill with diphtheria, or a
child exposed to the disease by living in the same house with such a patient,
may have immediate need of antibodies to combat the disease or ward off
infection. Endangered persons may, under special circumstances to be judged
by the physician, receive immediate protection through injections of serum
which contains large quantities of antitoxin.
Laboratory Metbods for tbe Study of Diphtberia. Bacteriological pro-
cedures for diagnostic study of diphtheria may be grouped under three genera)
headings as follows :
I. INITIAL THROAT CULTURE. The commonest culLural procedure con-
sists in rubbing a sterile "swab" (a wooden applicator, tipped with cotton)
over the infected or suspected tonsils and nasal mucosa, and then rubbing the
swab over the surface of sterile, coagulated serum (LOftier's medium *) or
sterile coagulated eggs (Pai's mediumt). After 2 to 24 hours' incubation at
37° C a methylene-blue-stained smear is made from the mixed growth on the
surface of the medium. Organisms having the characteristic morphology of
Corynebacterium diphtheriae are sought among the myriads of other organ-
isms present. Considerable experience is necessary for this work for it is in
such material that one often encounters diphtheroids.
2. ISOLATION. There are some organisms morphologically indistinguish-
able from C. diphtheriae which are perfectly harmless. In order to be sure that
diphtheria-like organisms found in the throat are really virulent, it is some-
times necessary to isolate them in pure culture and test their virulence.
This is commonly done by injecting rabbits (Fig. 35-4).
3. THE IN-VITRO TEsT. As a result of observations by Ouchterlony, Elek,
and others, virulence (toxigenicity) of C. diphtheriae and some other toxigenic
organisms may be demonstrated, without using animals, by an in vitro toxi-
genicity test. The test illustrates a very important principle in immunology.
It is described in detail in Chapter 21. The in vitro plates may also be inocu-
lated directly with the initial swab from the patient, or with colonies picked
from the tellurite plate. This often saves 24 to 48 hours of the time required
to isolate pure cultures.
• I...OfIIeI-'s medimn. To 3 parts of serum add 1 part of 1 per cent dextrose broth. Tube
and sterilize 1 hour in a slanting position without releasing steam or air pressure.
t PIli's medimn. For the serum in the above, substitute whole hen's eggs, and for the
broth substitute distilled water. Mix well and sterilize in the same manner.
Family Corynebacterillceae;. Family Lactobacterillceae 437
TyPES OF CoRYNEBACTERIUM DJPHTHERIAE. In 1931 attention was called to
several different types of C. diphtheriae. Because of the rather constant
presence of one type in severe (grave) cases of the disease it was thOUght to
be the cause of severe cases and was called the gravis type. Another type,
found at that time in less severe cases and in many healthy carriers, was
thought to be less dangerous and was called the mitis type. A third type found
in moderately severe cases, and having some of the properties of both mitis
and gravis types, was called the intermedius type. In Baltimore, during an
epidemic in 1944, a fourth type was found, which was named the minimus
type. The differential characters of these types are shown in Table 19. Experi-
ence in the United States indicates that none of these types has any special
clinical significance, though European observations support the view that
they may, in some instances.
Alterations in Virulence of C. Dipbtberiae. Wholly avirulent (atoxigenic)
cultures can suddenly acquire high grade toxigenicity (virulence). The change
is brought about by the action of certain bacteriophages (see Transduction,
Chapter 15).
GENUS LISTERIA *
The organisms of this genus are in most respects much like Corynebac-
terium pseudodiphtheriticum and other diphtheroids except that they are
more regular in form and motile by means of peritrichous (or polar?) fla-
Fig. 35-4. Intradennal tests for toxigenicity in a rabbit. The cultures to be tested \Wre
injected iD O.2-ml. doses into the squares marked on the skin. Five hours later, the rabbit
received 1000 u!1its of diphtheria antitoxin intravenously. The test cultures were then imme-
diately reinjected, each in a square adjacent to the corresponding initial injection. The large,
circular dark areas are zones of necrosis and inflammation produced in the skiD during the
five hours before diphtheria antitoxin was injected into the ear vein. After injection of anti·
toxin the same cultures, each injected into a square below the first injection, failed to pro-
duce any necrosis and only an insignificant amount of inflammation, showing that the necro·
sis could be prevented by diphtheria antitoxin. Since the antitoxin is specific in its action,
this proves that the bacilli being tested were toxigenic (virulent) C. diphlheriae. (Specimen
prepared by Miss Elizabeth O. King. Photo courtesy U. S. Public Health Service, ("om·
municable Disease ("-enter, Atlanta, Ga.)
• Named for Lord Lister, British scientist.

438 The Bacteria
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Family Corynebacteriaceae,· Family Lactobacteriaceae 439
gella; a notable exception among the gram-positive, non-sporeforming rods
(see Table 28). Listeria grow readily on blood and serum media, at 3r C.
They are facultative.
LISTERIOSIS. Listeria appears to be regularly pathogenic for man and lower
animals, causing a febrile disease characterized especially by swollen lymph
nodes and the appearance in the blood of large numbers of white cells called
monocytes; hence the species name: Listeria monocytogenes.
GENUS ERYSIPELOTHRIX
Every farmer who has raised many hogs for market probably knows about
swine erysipelas. Only the fortunate farmer has escaped the infection himself.
Erysipelothrix rhusiopathiae, * the causative organism, is widely distributed in
soil, dung, dust and sewage and can infect sheep, birds, rodents, fish, etc.
E. rhusiopathiae in many respects resembles the diphtheroids. However, it
is often pleomorphic and filamentous, like lactobacilli, especially in the R
phase. Because of its tendency to filament formation it has sometimes been
classified in the Actinomycetales. It is facultatively aerobic. It is quite re-
sistant to drying and outdoor conditions generally and, therefore, can persist
stubbornly in the dust and dirt of animal pens, buildings and vehicles where
infected animals have been. It is transmitted by inhalation and ingestion of
infected dirt and by way of cuts and scratches.
Swine ErysipeJas. Swine erysipelas is the commonest form of disease
produced by E. rhusiopathiae. It is usually slowly progressive, though the
infection at times is highly and rapidly fatal in swine herds (and very costly
to stock raisers). It. can be isolated on blood agar from the lesions of infected
animals. Because the reddish skin lesions are often roughly diamond-shaped
swine erysipelas is sometimes called "diamond disease."
THE FAMILY LACI'OBACfERIACEAE

These organisms are discussed here because they have many points of re-
semblance to the Corynebacteriaceae and also to some of the spherical
bacteria (Streptococcus, Leuconostoc). They are of great importance in
various industries. The family Lactobacteriaceae consists of two tribes:
Tribe I Streptococceae; and Tribe II Lactobacilleae. The tribe Streptococceae
are described in the next chapter, the Lactobacilleae in this chapter.
TRIBE LACTOBACILLEAE
The tribe Lactobacilleae comprises four genera: (1) Lactobacillus; (2)
Microbacterium; (3) Propionibacterium, and (4) Butyribacterium.
All of these organisms are gram-positive, non-sporeforming, non-motile,
microaerophilic and facultatively aerobic. All are aciduric, several are acido-
philic, and nearly all are thermoduric and/or thermophilic. Morphologically
they are rods, gene~ally resembling the diphtheroids. However, Lactobacillus
is much more pleomorphic. For example, under some conditions of growth
• Erysipelo is from Oreek words meaning red skin; the suffix IMix, from a Greek word
meaning thread. Of the species name, rhusio- is from the Greek for reddish in color;
palhiae is from the Greek for disease producer. The whole name literally translated, there-
fore, means "Erysipelas-thread, reddish-disease producer."
440 The Bacterill
Lactobacillus forms rods so short as to be coccoid. When in chains they
closely resemble streptococci. At other times thcy form long chains of slender
bacilli (streptobacilli) or long filaments resembling Erysipelothrix.
Genus Lactobacillus
These important organisms thrive in sour milk, sauerkraut, pickle vats, -
etc., after these products have, been made moderately acid by the growth of
other organisms. Lactobacilli are nutritionally fastidious organisms, re-
quiring complex organic media with vitamins. In the laboratory they grow
poorly on ordinary agar; heavy inoculations are necessary to produce even a
few small, translucent, delicate colonies. They gr~w best on tomato-juice
agar and whey agar at about 25° C. Their natural habitat is on plants, in
dairy products, soil, dung, etc. They are vigorous fermenters, producing much
lactic acid. They are generally harmless and their acid production is of use in
food-producing industries.
The genus may be divided into convenient groups as follows:
GROUPS OF LACfOBACILLUS
I. Homofermentative. (Products of fermentation are almost exclusively
lactic acid: 98-99%; never CO2),
A. Mesophilic (grow at 12° C; not at 45° C) (Sometimes grouped as
Streptobacterium).
1. L. plantarum (common in dairy products and fermented
vegetable products).
2. L. casei (one of the most common lactobacilli in dairy
products).
3. L. bifidus (common in feces of breast-fed infants; anaerobic;
shows Y, Land T forms). '
B. Thermophilic (grow at 45° C; not at 20° C) (Sometimes grouped
as Thermobacterium).
1. L. lac tis (one of the most common lactobacilli in dairy
products).
2. L. acidophilus (common in milk and in intestil!al tract of
man and animals; not sensitive to surface tension re-
ducents such as bile).
3. L. bulgaricus_ (common in vagina and sour milk; not- in
intestinal tract; is sensitive to surface tension reducents,
, such as bile). _
4. L. caucasicus (found in kefir and some cheeses; see Chap-,
I
. ter 42).
II. Heterofermentative. (Products of fermentation include lactic and acetic
acids, alcohol and CO 2) (Sometimes grouped as Betabacterium).
A. Mesophilic
1. L. brevis widely distributed in nature. Less desirable
2. L. pastorianus in fermented food products because of gas,
B. Thermophilic alcohol and "off" flavors.
I. L. fermenti
Family Corynebacteriaceae; Family Lactobacteriaceae 441
Both homofermentative and heterofermentative species are used extensively
in food manufactures (dairy products, sauerkraut, etc.). (See Chapters 43
and 44.)
ORAL LACTOBACILLI. A good many of these species, and some others
(L. salivarius and L. cellobiosus) are often found in the oral cavity, especially
around the teeth. With aciduric streptococci they are important as causes of
dental caries, due to the acidity which they produce.
Genera Microbacterium, Butyribacterium and Propionibacterium

These three genera comprise several species of gram-positive rods much like
the saprophytic diphtheroids.
Genus Microbacterium includes two species: M. lacticum and M. flavum,
common in the same habitats as many saprophytic corynebacteria and lacto-
bacilli. They have little known significance except as saprophytes. They
produce lactic acid during fermentation.
Genus Butyribacterium includes only one species, closely resembling sapro-
pbytic diphtheroids. It is heterofermentative, producing butyric acid and
CO2 ,
Genus Propionibacterium is somewhat more pleomorphic than the two
foregoing genera; more like Corynebacterium xerose or Erysipelothrix.
Otherwise it shares the general characters of saprophytic diphtheroids. Some
species produce reddish pigments.
Ofsome 10 or 12 species of Propionibacterium several are commonly found
in various hard cheeses. During the ripening of these Propionibacterium con-
tributes flavors due to propionic acid; for example, the distinctive sweet and
bitter flavor of Swiss cheese is partly due to propionates. The eyes of Swiss
cheese are due to C02 and air. The origin of the eyes in these cheeses is not
fully explained, but Propionibacterium is known to prod uce CO2, being hetero-
fermentative. Pure cultures of Propionibacterium are added to milk in the
manufacture of Swiss, Munster, and similar cheeses.
THE "LACTIC BACTERIA"

The Lactobacilleae are often found in dairy products, dung, soil, plants,
etc., in company with well-known species of Streptococceae: Streptococcus
lactis, Leuconostoc mesentericus, etc. All have similar physiology, habitat,
and ecological relationships. All except Propionibacterium and Butyribac-
terium form considerable amounts of lactic acid during fermentation, and
can thrive in considerable concentrations of this acid. For this reason they
are collectively spoken of as "lactic-acid bacteria" or the "lactics."
All of the lactic acid bacteria are of great industrial and agricultural im-
portance, since they (with other microorganisms) are concerned in the souring
of milk and cream for butter and cheese making; the preparation of yoghurt
and other fermente~ milk products; the curing of ensilage; the manufacture
of lactic acid and 'of sauerkraut; and the fermentation of pickles, olives,
citrons, etc. They are more full~ discussed later on.
I. REFERENCES
Davis, G. H. G.: The classification of Lactobacilli from the human mouth. J. Gen. Micro-
bioI., 1955, 13 :481.
442 The Bacteria
Diagnostic Procedures and Reagents. 3rd ed. Chapter on Diphtheria. Am. Pub. Health
Ass'n., New York, 1950. .
Frobisher, M., Jr., and Parsons, E.!.: Studies on type-specific immunization with somatic
antigens of Corynebacterium diphtheriae. Am. J. Hyg., 1950, 52 :239
Frobisher, M., and Parsons, E. I.: Further studies of tellurite plating media for Corynebac-
terium diphtheriae. Am. J. Pub. Health, 1953,43:1441.
Hagan, W. A., and Bruner, D. W.: The Infectious Diseases of Domestic Animals. 2nd ed.
The Comstock Pub. Co., Ithaca, N. Y., 1951.
Jensen, H. L.: The coryneform bacteria. Ann. Rev. Microbio!., 1952,6:77.
Kolb, R. W., Branham, S. E., and Riggs, D. B.: Comparison of the guinea pig and chick in
evaluation of diphtheria toxin for the Schick test. App!. Micr., 1955,3:241.
Lautrop, H.: On the existence of an antibacterial factor in diphtheria immunity. Acta Path.
et Micr., Scandinavica, 1955,36:274.
Parsons, E. I.: Induction of toxigenicity in non-toxigenic strains of C. diphtheriae with bac-
teriophages derived from non-toxigenic strains. Proc. Soc. Exp. BioI. and Med., 1955,
90:91.
Parsons, E. I., Frobisher, M., Moore, M., and Aiken, M. A.: Rapid virulence test in diag-
nosis of diphtheria. Proc. Soc. Exp. Biol. and Med., 1955,88:368.
Sharpe, E.: A serological classification of Lactobacilli. J. Gen. Microbiol., 1955,12:107.
Tittsler, R. P., Pederson, C. S., Snell, E. E., Hedlin, D., and Niven, C. F.: Symposium on
the lactic acid bacteria. Bact. Rev., 1952, 16:227.
36
Spherical Bacteria
SPHERICAL, or nearly spherical, bacteria are found in several divisions of

the class Schizomycetes, such as the genus of soil autotrophs, Nitrosococcus;
the genera Thiosarcina and Thiopolycoccus, of the suborder Rhodobacteri-
ineae; and the genus Myxococcus of the order Myxobacteriales. In this
chapter we shall confine our discussion to the larger and more important
groups: Micrococcaceae, Neisseriaceae and Streptococceae. These are all
heterotrophic, non-sporeforming, and non-motile. All are gram-positive
except the Neisseriaceae.
These three groups are readily differentiated, and subdivided into genera,
as follows:
1. Family Micrococcaceae: (divide in more than I plane) (Fig. 36-1, A, C,
D)
(a) Divide in 2 planes at 90°: Gaffkya* (square groups)
(b) Divide in 3 planes at 90°: Sarcinat (cubical packets)t
(c) Divide in 3 planes, at various angles: Micrococcus (irregular
groups like bunches of grapes)
2. Tribe Streptococceae: (divide in I plane only) (Fig. 36-1, B)
(a) Cling together in long chains after fission: Streptococcus
(b) Tend to separate into short chains or pairs: Diplococcus
(c) Chains of cells like Streptococcus but typically form very large,
polysaccharide capsules or zoogleal masses in vegetable juices
(sugar vats, sauerkraut. etc.): Leuconostoc
3. Family Neisseriaceae: (gram-negative; morphologically distinctive, flat-
tened, coffeebean-shaped cocci; divide in I plane only (Fig. 36-6)
(a) Remain as pairs with distinctive flattened appearance: Neisseria
(b) Irregular groups; strictly anaerobic: Veillonella (not discussed
here).
1. TIlE FAMll..Y MICROCOCCACEAE
General Characters. The organisms of the family Micrococcaceae are
usually nearly spherical in form. Three genera are included, differentiated
• Gaffky was a famous bacteriologist who first described these organisms.
t Sarcina is from a Latin word meaning packet. .
443
444 The Bacteria
B
c D
Fig.36--1. Vanous forms of Micrococcaceae. A. Gaffkya; B. Streptococcus; C, Sarcina;
D, Micrococcus (staphylococcus). (X 1000.) (Ford, Textbook of Bacteriology, W. B. Saun-
ders Co.)
primarily by the arrangement of the cocci. Their principal differential char-

acters have been listed above.
The family includes some pathogenic species, notably Micrococcus pyogenes
(var. aureus), Micrococcus pyogenes (var. a/bus) and GajJkya lelragena. Some
of these (especially M. pyogenes) cause serious diseases of man and animals.
However, most of the Micrococcaceae are saprophytes.
All of the Micrococcaceae grow well facultatively at temperatures of from
30° to 40° C on simple organic media such as extract agar or peptone solu-
tion. A large majority form opaque, butyrous colonies colored with white or
yellow pigments; various shades of red and orange are especially common
among the saprophytic types. Micrococci have a marked tolerance for NaCI
and can be isolated from mixed cultures on media containing 5 to 8 per cent
salt, a concentration which inhibits growth of many other organisms.
Micrococcaceae mNature. Except for Micrococcus pyogenes and Gaffkya
tetragena, which are pathogenic, the Micrococcaceae are of importance
mainly as scavengers. They are widely distributed in milk, soil, air, dust and
water, especially where putrefaction and decay are in prgress. They are among
tbe common air and dust-borne contaminants in laboratory cultures. Many
digest proteins like gelatin and casein and attack various carbohydrates and
other organic substances. Several species (M. cereus, M. perflavus, M. caseo-
[yticus) are of some commercial importance .in the ripening and flavoring of
cheese since they attack casein and lactose with the production of aromatic
Spherical Bacterlll 445
substances having pleasing flavors. Some produce various undesirable condi-
tions such as "ropy milk" (M. viscosus and M. cremoriviscosf).
PATIIOOENIC MICROCOCCI
Because the name Staphylococcus is one of long standing·and common
usage, it will be continued here in a non-specific sense to mean pathogenic
micrococci. Staphylococci have the general properties of other micrococci.
The staphylococci are usually to be found on the skin or mucous mem-
branes of the animal body, especially of the nose and mouth, where they often
occur in large numbers even under "normal" conditions. The two principal
species are: (1) Micrococcus pyogenes (var. aureus), distinguished by its golden-
yellow pigment and notorious as the cause of suppurative (pus-forming)
conditions: mastitis of cows, boils, carbuncles, and internal abscesses in man;
(2) M. pyogenes (var. aJbus).
The most pathogenic staphylococci generally: (I) ferment mannite; (2)
liquefy gelatin; (3) produce coagulase (an enzyme-like principle which causes
oxalated, heparinized or citrated blood plasma to clot); (4) produce golden
yellow pigment; and (5) produce lipase. However, these properties are neither
constant nor exclusive among staphylococci, and it is sometimes difficult to
make a distinction between staphylococci and the non-pathogenic micrococci.
True staphylococci, especially M. pyogenes VQT. aureus, often produce hemo-
lytic colonies when cultivated on the surface of blood agar. However, many
non-pathogenic species of micrococci and other bacteria, including various
rod forms, are also hemolytic. In addition, staphylococci produce leuko-
cidins and several toxins which destroy certain tissue cells.
ENTEROTOXIN. Staphylococci are also notorious as a cause of a very
common type of food poisoning. This is because many strains of staphylococci
excrete a heat-stable poison caned enterotoxin.· When ingested, staphylo-
coccal enterotoxin causes nausea, vomiting, diarrhea and prostration. It is
more fully discussed in Chapter 43.
Gallkya are generally regarded as saprophytic (with few exceptJons) so far
as man is concerned. However, this species is very pathogenic for mice and
some other forms of life. It is also pathogenic for lobsters, producing a com-
mercially costly disease with a high fatality rate. The diseased lobsters have
a pink discoloration on the ventral side of the abdomen; the blood, normally
blue, is pink and watery. The organisms are found in large numbers in the
blood and tissues and Koch's postulates have been demonstrated with them.
The tetrads of Ga.ffkya in the lesions are characteristically heavily encapsu-
lated.
TRIBE STREPTOCOCCEAE
A primary subdivision of streptococci may be made into three large groups:
lactic, enterococcus, and pyogenic. Many of these produce characteristic
appearances around their colonies in blood agar in Petri plates. Since these
appearances are constantly referred to in discussing streptococci, they are
described here. along with the procedure for making bl<>od-agar plates.
Blood-agar Plates. To determine the blood-agar type a tube containing
about 15 ml of melted. meat-infusion agar. cooled to about 45° C (still fluid.
• A toxin causing enteritis or diarrhea. Do not confuse with endotoxin.
446
,. x
u w
Fig. 36-2. Colonies of hemolytic streptococci in blood agar. U, clear zones of complete
hemolysis around colonies of beta type (Strep. pyogenes, Lancefield group A), natural size.
If, one beta-type colony enlarged to show edge of colony at lower left and absence of ery-
throcytes in clear hemolyzed zone. W, small hemolytic zones of alpha-type colony, Strep.
milis, natural size. X, one alpha-type colony enlatged to show edge of colony at lower left,
with many intact erythrocytes in hemolyzed zone. (Preparations by Dr. Elaine L. Updyke.
Photo courtesy of Communicable Disease Center, U. S. Public Health Service, Atlanta, Ga.)
yet not hot enough to injure the microorganisms), is inoculated with a loopful
of pus, milk, broth culture, or other material containing the desired strepto-
cocci. About 5 per cent sterile blood is added aseptically and well mixed with
the agar. The mixture is poured into plates and incubated for twenty-four
hours; at about 20° C for lactic streptococci and 37° C for others.
BLOOD-AGAR TYPES
Four main types of streptococci are recognized on the basis of their action
in blood-agar plates: the alpha and beta hemolytic types, the double-zone
beta hemolytic type, and the gamma type.
Alpha-type Hemolytic Streptococci. These colonies are surrounded by a
zone of hemolysis and also a zone of discolored erythrocytes close in
around the deep colonies. These erythrocytes have a green or brownish-green
color. Peripheral to this inner ring of discolored cells the outer zone of clear
Spherical Bacteria 447
hemolysis may be of great or small extent, and may sometimes be so small
as to coincide with the zone of green cells. It usually widens on refrigeration
of the plate \Fig. 36-2, W, X).
Only the use of a microscope can be relied upon to make the distinction
and only colonies which are deep in the agar are always thus characterized,
surface colonies sometimes producing deceptive appearances. Pathogenic
green-producing varieties (alpha type) of hemolytic streptococci are often
called "streptococcus viridans."
Beta-Type Hemolytic Streptococci. The hemolytic zones of streptococci
of this type in blood-agar plates are seen to be entirely clear and free from
any intact erythrocytes (Fig. 36-2, U, V). Such streptococci are loosely spoken
of under the general term of "streptococcus hemolyticus" or "hemolytic
strep."
Double-Zone Beta-Type Streptococci. Certain species, almost exclusively
of bovine origin, and not uncommon in dairy products, after producing a
zone of hemolysis like that of other beta-type streptococci, on standing at
room temperature or on refrigeration produce a second ring of hemolysis
separated from the first by a ring of red erythrocytes (Fig. 36-3).
Gamma Type. When colonies of streptococci in blood-agar plates show
no visible change in the blood cells surrounding the colony they are said to
be of the gamma or indifferent or non-hemolytic type.
I. THE LACfIC STREPTOCOCCI
This group contains Streptococcus lactis, * the common, milk-souring strep-
tococcus, useful in the manufacturing dairy industry. S. lactis are usually of
the gamma type in blood agar but may produce green colonies. S. /actis is
always present in market milk, even of the best quality. It occurs in cow dung,
dust, soil, on plants, in utensils, and so on and its entrance into the milk is
easily explained. Its survival in such environments shows that it is a relatively
Fig. 36-3. Double zone beta-type streptococcus colonies in blood agar; incubated at
37° C and then held at low temperature several hours; natural size. Note the usual clear
zone of complete hemolysis, surrounded by a zone of unhemoiyzed cells, then by an outer
zone of clear hemolysis. (Preparation by Dr. Elaine L. Updyke.) (photo courtesy of Com-
municable Disease Center, U. S. Public Health Service, Atlanta, Georgia.)
• S. lactis probably represer.ts a group of variants or closely similar species, rather than a
single, well-defined species.
448 The Bacteria
hardy organism. It is quite harmless to man. Its vital role in the dairy and
food industries is discussed in Chapters 42 and 43.
It sometimes forms long chains but occurs chiefly in short chains or pairs
and the cells tend toward an oval shape. It grows rapidly, causing souring of
milk at summer temperatures; usually overgrowing and suppressing the de-
velopment of other organisms. some of which might otherwise cause the milk
to putrefy. S. lactis can readily be cultivated in sterile milk, or on agar con-
taining milk or whey or tomato juice, at about 25° C. It grows best in the
presence of glucose or lactose.
Streptococci Related to S. Lactis. There are numerous closely related
streptococci, all of them forming lactic acid. One of these is S. cremoris. It
tends to form long chains. Pure cultures of this organism, mixed with S. lactis
and some species of Leuconostoc (which are very similar), are used in making
butter and cheese. The object is to ensure that the milk or cream is soured
by organisms (S. cremoris) which yield a pleasant, "buttery" flavor. Some
other varieties of lactic streptococci are carefully avoided since they cause
undesirable flavors, or conditions of sliminess ("ropy milk").
2. THE ENTEROCOCCI
Streptococci of this group inhabit the intestine of man and animals, dung,
sewage, etc. Many of the enterococci have a tendency to produce short chains
and pairs of plump, ovoid cocci, and are commonly found in clumps sugges-
tive of micrococci. They were first described as micrococci.
The group is characterized by wide tolerance of heat and cold and other
influences unfavorable to other streptococci: 6.5 per cent NaC!; bile (low
surface tension); the presence of O. I per cent 1l"ethylene blue; pH 9.6; and
conditions of life in feces (see Table 20). Enterococci are even more hardy
than the lactic group. The enterococci are most commonly represented by
S. faecalis. Colonies of this organism usually produce alpha-type zones of
hemolysis in blood-agar plates. Some species of enterococci produce beta-
type colonies and some produce gamma-type. The group is seen, therefore,
to be somewhat heterogeneous.
Related to S./aecalis are S. liquefaciens and S. zymogenes, the last differing
from other enterococci in producing beta-type zones in blood agar. Both
species occur commonly in dung and dairy products. They grow readily on
the organic media commonly used in fhe laboratory. Unlike S. lactis they
grow better at about 35° C than at 25° C.
S. Iiquefaciens and S. zymogenes hydrolyze proteins. As a result, they give
rise to strong bitter flavors in cheese. They have been fmind occasionally in
certain pathological conditions.
3. THE PYOGENIC STREPTOCOCCI
The pyogenic (pus-producing) streptococci may be subdivided into: (a) the
beta-hemolytic, pyogenic streptococci, represented by S. pyogenes; and (b)
the alpha-hemolytic or "viridans" pyogenic, streptococci, represented by
S. mitis.
(a) The Beta Hemolytic species are very different from the lactic and entero-
coccic groups. In gerteral they are relatively fragile organisms, adapted to a
parasitic life in and on the mammalian body. Generally they are studied in
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450 The Bacteria
medical and diagnostic laboratories. They are best cultivated at 37° C in
media containing serum or blood. Some of them are among the most deadly
pathogens. There are several serological groups of these streptococci as
follows.
LANCEFIELD GROUPS OF BETA-TYPE STREPTOCOCCI. Lancefield made ex-
tracts of massive cultures of different strains of these bacteria by means of
hot, N/20 HC!. These extracts contain specific, somatic, antigen (carbohydrate
or C substances). The extracts are used as antigens in precipitin tests.
By means of such precipitin tests several distinct groups of beta-type hemo-
lytic streptococci can be differentiated with respect to origin. Lancefield
designated these groups by letters: A, B, C, D, E, F, G, etc., according to
source or other characters. They are accordingly known as Lancefield groups
(Remember, these apply distinctly to beta-type streptococci.)*
GROUP A. Biological properties distinguishing group A streptococci from
other groups are shown in Table 20. Group A streptococci include those
causing scarlet fever, septic sore throat, empyema, puerperal sepsis, and many
other serious, epidemic, and acute pyogenic diseases in human beings. These
are represented by the type species of the genus, S. pyogenes.
VARIETIES AND TYPES OF GROUP A. Two colony forms of streptococci of
group A have been discerned: a, smooth, regular, glossy form often called
the T form, and a dull, slightly-irregular form called matte or M. Each M
variant contains a different protein, somatic antigen. These evoke type-
specific precipitins. Over 40 serological types ("M types") of group A strepto-
cocci have been found.
The importance of determining serological (Lancefield) group and M type
of beta hemolytic streptococci becomes clear when it is realized that resistance
to Group Astreptococcal infection is type-specific. For example, one may have
resistance to a type 6 streptococcus, yet succumb in'a type 19 epidemic with
the same disease. The importance of typing in the study of methods by which
streptococcal disease is spread can scarcely be overestimated. For example,
the whole problem of the control of rheumatic heart disease appears bound
up with the epidemiology of streptococcal infection.
GROUP B STREPTOCOCCI differ from all others in hydrolyzing sodium hip-
purate and in producing double zones of beta hemolysis (Fig. 36-3).
Hemolytic streptococci of group B arl1 usually of bovine origin and harm-
less for man, but are occasionally found in human infections. S. agaiactiae,
an important m~mber of group B, is of particular interest to the farmer be-
cause it causes severe mastitiS in cattle and stoppage of milk flow. -
GROUP C STREPTOCOCCI. This group is of a dual nature with respect to
origin, containing (I) strains of hUIpan origin culturally indistinguishable
from group A and which cause many similar conditions; and (2) strains froin
various animal sources. The latter rarely occur in human infections.
GROUP D STREPTOCOCCI. These are members of the group of enterococci
(S. zymogenes) which are discussed elsewhere.
GROUP G STREPTOCOCCI. These strains are related to group C strains, and
closely resemble group A strains in important biochemical properties. They
, • A few alpha-type streptococci and some other organisms have been found to contain
some of these antigens.
are generally not highly pathogenic, but vary in this respect. They are frequent
in human infections.
GROUPS E, F, H, ETC. These groups contain such small numbers of strains
that they will not be discussed in detail. They have been found occasionally
in animals and human beings, and also in dairy products.
(b) The Alpha Hemolytic ("Viridans") Streptococci. These may be thought
of as comprising three groups. One consists of delicate, fragile, highly-adapted,
parasitic, pyogenic streptococci. These are found mainly in the mammalian
oral and respiratory tracts and are capable of producing serious infections.
They are represented by S. mitis, of man, S. equinus of horses, and S. bovis
of cattle.
A second group is represented by S. salivarius, a species common in the
human mouth, hardier than S. mitis, and generally saprophytic. S. salil'arius
is distinguished by forming thick, gummy colonies if cultivated on media
containing about 5 per cent sucrose. The gumminess is due to the synthesis of
levulans from the sucrose and is a good example of the tendency of many
bacterial species to synthesize polysaccharides. Another common species of
saprophytic, viridans streptococci is S. thermophilus. It is distinctive in grow-
ing well at temperatures up to about 65° C. This organism gives much
trouble to commercial milk pasteurizers because it is not destroyed by
pasteurization, surviving 30 minutes at 65° C. It can grow to large numbers in
pasteurizing vats, and appears in the milk upon examination by the health
departments. It casts unjustifiable doubt on the efficacy of the pasteurization
process. However, it can be eliminated by good sanitization with hot water.
It is not pathogenic. It occurs in dung and barn dust.
A third group of alpha type, hemolytic, streptococci is made up of species of
enterococci. These are commonly represented by S. faecalis, which has been
discussed with the enterococci. These are often pyogenic.
PATHOGENESIS BY ALPHA-TypE STREPTOCOCCI. 1lthough constantly pres-
ent in the normal mouth, usually without deleterious effects, these organisms
can at times gain a foothold in the body. They tend to cause chronic, but none
the less dangerous, suppurative diseases. They are frequently found in ab-
scessed teeth, sinuses, and diseased tonsils and are always present in saliva.
From teeth and tonsils they may be carried to the joints and produce rheu-
matic conditions. They can also infect the heart valves causing a serious heart
disease, bacterial endocarditis. S.faecalis is frequently involved.
GENUS DIPLOCOCCUS
The principal species in this genus is Diplococcus pneumoniae, cause of
lobar pneumonia,. a frequent cause of death before the advent of chemo-
therapy.
British writers logicaily refer to Diplococcus pneumoniae as Streptococcus
pnellmoniae since tpe organisms have the major characteristics of streptococci.
They often form chains, usually made up of from two to eight pairs of cocci.
They are encapsulated. The pneumococci are rarely spherical, having the
form of short artillery projectiles placed base to base. They are gram-positive.
In blood-agar pla~es they produce alpha-type zones. They are facuitative,
heterotrophic and parasitic. Methods of cultivatio::l and study are like those
used for pyogeni~ streptococci. They are found in the saliva and sputum of
452 The Bacteria
Fig. 36-4. Electronographs of Diplococcus pneumoniae. The first picture shows the cap-
sules in their normal state. The center picture shows the capsules virtually unaffected by
serum of a heterologous type. The picture on the right shows the effect of homologous type
serum on the capsules--a well-marked quel)ung reaction. (From Mudd, Heinmets, and
Anderson, in The J. Exp. Mcd., vol. 78.)
patients with lobar pneumonia and, like streptococci, also occur frequently in
the normal mouth and throat.
Pneumococci are extremely pathogenic for white mice when freshly isolated
from the body. Advantage is often taken of this fact to isolate pneumococci
from sputum of patients for diagnosis. The sputum is injected into the mice
intraperitoneally. After six to twenty-four hours the mice die or become very
ill, and enormous numbers of pneumococci are found in the peritoneal cavity
and heart blood. The cocci found on the peritoneum of the mouse may be
identified by: (1) their morphology; (2) their capsules (Fig. 36-4); (3) their
solubility in bile or in solutions of various surface-tension reduccnts, of which
bUe is one; (4) ability to ferment inulin (see Table 20).
Serological Types of Pneumococci. Pneumococci may be divided into
more than seventy serological types which are designated by Roman numerals.
These are analogous to the Lancefield groups of streptococci. Serological
type-specificity of pneumococci is conferred by their capsules. Each type has
its own, specific, capsular, carbohydrate antigen. Immune sera are available
representing each type.
QUELLUNG REACTION. If encapsulated pneumococci are mixed with a
type-specific immune serum swelling of the capsules is seen (Fig. 36-4). This
is spoken of as a quellung (German for swelling) reaction. It was first de-
scribed by Neufeld and is sometimes called the Neufeld reaction.
The quellung II!ethod of serological typing or grouping is applicable to
the study of many other species of encapsulated organisms, not only of medi-
cal but also of agricultural importance. 1f deprived of their capsules, as when
they vary toward the rough phase, such organisms are: (1) immunologically
indistinguishable; and (2) (in the case of pathogens) avirulent.
These facts relating to the occurrence of specific carbohydrate substances
in or on cells of bacteria are of much wider than medical interest because they
illustrate a phenomenon of fundamental importance which is encountered
frequently in microbiology-the occurrence within a species, of immunologi-
cal "types" or groups which differ because of the chemical differences in their
carbohydrates.
453
TRANSFORMATION OF PNEUMOCOCCAL TyPES. An interesting agent capable
of inducing genetically stable alteration in type specificity of pneumococci is
a deoxyribonucleic acid fraction extracted chemically from certain pneumo-
cocci. This has been described in Chapters 5 and 15.
Pathogenic ActiOD of Pneumococ:ci. Pneumococci are highly invasive or-
ganisms and, like beta hemolytic streptococci, can infect many different parts
~f the body and, according to their localization cause meningitis, septicemia,
peritonitis, empyema, sinusitis, etc. The name pneumococcus is, of course,
derived from the fact that they were first seen as the cause of pneumonia.
The method of transmission of pneumonia is chiefly through droplets of
infected saliva and nasal and pulmonary mucus, and by inhalation of infected
dust. Kissing undoubtedly transmits the infection as well as other respiratory
pathogens but obviously not every such infection results in disease. Romance
has a powerful ally in natural resistance to infectious disease. Pneumococci
survive desiccation very well, aDd thus are found in dust in patients' rooms,
hospital wards, etc.
GENUS LEUCONOSTOC
The name of this streptococcus-like genus is derived from the name of a
blue-green alga, Nostoc, and the Greek leukos, meaning colorless. Nostoc,
the alga, is characterized by spherical cells occurring in tangled chains with a
thick, gelatinous, outer coating. Leuconostoc, the bacteria, resemble Nostoc
in forming spherical cells in tangled chains. At least two species (L. mesen-
teroides and L. dextranicum), when growing in carbohydrate-rich media,
Fig. 36-5. Leu~onostoc mt'snrtt'roidt's. Three-day culture on 10 per cent raw sugar agar,
IlCUbatcd at room temperature. (McCleskey, C. S., Faville, L. W., and Barnett, R. 0., in
J. Bact., vol. 54.)
454 The Bacteria
synthesize thick, masses of gummy polysaccharide (dextran) about themselves
(Fig. 36-5). (Compare with Slrep. salivarius.)
A detailed study of L. mesenleroities and L. dextranicum has shown that
there are various types, differentiated serologically and by the amount and
molecular nature of gum formed, as well as by amount of CO 2 produced, etc.
Leuconostoc are widely distributed on growing plants. They ferment
actively and produce acid in such carbohydrate-rich plant materials as sauer-
kraut, ensilage, and in plant juices such as are used in making cane sugar,
beet sugar, etc. They constitute a great nuisance in the sugar refining industry,
clogging vats, pipes and machines. Indeed, so much of the specific carbo-
hydrate of Leuconostoc is left in commercial cane, beet, and other sugars that
specific precipitin tests can be obtained by mixing a little "sugar water"
with suitable immune serum.
Leuconostoc citrovorum. This organism, when not growing in sugar-rich
solutions, produces little or none of the gum which is so characteristic of the
genus. It then so closely resembles S. lactis that some have regarded it as a
streptococcus (S. citrovorum). (It has also been classified, probably most
logically, as a species of Pediococcus.) A very closely related species is called
L. paracitrovorum. These organisms are closely allied to S. Jactis and are
commonly found in its company and have some of its principal characters.
DIACETYL. The important point about L. citrovorum and its allies is the
fact that they decompose citric acid with the formation of acetic acid, CO 2
and, most important, acetyl-methyl-carbinol and diacetyl. The latter two
compounds are responsible for the pleasant, buttery flavor of dairy products.
Consequently, pure cultures of L. citrovorum, etc., are commonly added to
milk or cream that is to be made into butter, cheese and the like. Citric acid
is also often added to increase the amount of diacetyl formed.
It is important to note that diacetyl is formed by the oxidation of acetyl-
methyl-carbinol in an acid medium:
CH.COOH.2COH.COOH·CH2COOH .... CH,·CO.CHOH.CH, + 2CH,COOH + 4CO, + H20
Citric acid Ac<tyl·IMth,yl. Ac.,lc
carbinol acid
(R.ductlon) (Oxida,lon)
CH,.2CHOH·CH, - - - 2H + CH,.CO.CHOH·CH, - - - CH,.CO.OC·CH, + 2H
1 :3 bu,ylon. Aatyl·me,hyl· DiIla'yl
glycol carbinol
Sometimes a little sulfuric, lactic or other harmless acid is added to ensure

proper pH. Also, aeration is necessary or, as shown in the foregoing reaction,
the acetyl methyl carbinol, instead of being oxidized to diacetyl, is reduced
to 2:3 butylene glycol which is tasteless (see above reactions). These facts are
of vital importan'ce to manufacturing dairymen.
An important branch of the dairy industry is the selection, propagation,
maintenance and sale of pure cultures of S. factis, L. citrovorum, various
lactobacilli, and mixtures of these for the manufacture of butter, fermented
milks, yoghurt, cheeses, etc. (see Chapter 44).
THE LAcrIC BACTERIA
RECAPITULATION
We might recapitulate the main facts concerning the lactic organisms in
the following manner:
I. Produce lactic acid in significant amounts from lactose and glucose.
A. Rod-forms: Lactobacillus
I. Mainly in dairy products (Homofermentative)
L. lactis
L. acidophilus
L. caucasicus
L. casei
L . thermophilus
2. Mainly in plant products (sauerkraut, pickles, ensilage, etc.)
a. Homofermentative
L. plantarum
b. Heterofermentative
L. brevis }
L. fermenti CO 2 formed
B. Coccus forms: Streptococceae
1. Mainly in dairy products
a. Homofermentative
S. lactis
S. cremoris
} (.Important as Iactlc
. aCI'd prod ucers)
S. thermophilus (resists pasteurization)
S. liquefaciens (proteolytic) (bitter flavors in cheese)
b. Heterofermentative
Leuconostoc citrovorum } D' t I b tt fl
L. dextranicum lace y - u ery avor
2. Mainly in plant products
Leuconostoc mesenteroides (slimy growths)
THE FAMILY NEISSERIACEAE

The family Neisseriaceae includes two genera, Neisseria and Veillonella.
The latter are small, gram-negative, anaerobic cocci which occur chiefly in the
normal mouth. Veillonella has been relatively little studied and will not be dis-
cussed in detail.
GENUS NEISSERIA
This genus derives its name from a German bacteriologist, Neisser, who,
in 1879, discovered and studied one of the two most important species. The
Neisseria are rather small, gram-negative diplococci, each cell characteristic-
ally flattened where it is in contact with its mate, each cell having somewhat
the shape of a coffee bean (Fig. 3(r6). The various species of Neisseria
(about eleven) are indistinguishable morphologically. They are aerobic and
grow best at 35° to 3r C. Unlike the ubiquitous and busy lactic organisms,
the Neisseria are found only in or on the human body.
Of the several species of Neisseria, two commonly cause disease. N. gonor-
rhoeae (the gonococcus) causes gonorrhea, while N. meningitidis (the meningo-
coccus) causes epidemic meningitis. N. gonorrhoeae and N. meningitidis are
very much alike and one is probably a minor variant of the other. They are
classical examples of microorganisms that have become highly adapted to an
existence of parasitism on man.
In moist, warm material as pus, urine, serum, or cuitltres in incubators,
456 The Bacteria
Fig. 36--6. Diagrammatic enlargement of Nei:ueria gonorrhoeae. The magnification is in

the range of the electron microscope. Note the rounded, hemispherical appearance of each
cell of the pairs.
the meningococci and gonococci autolyze and die rather rapidly. They do not
survive for long periods outside the body as do typhoid, tubercle and diph-
theria bacilli or streptococci. Drying is very deleterious to gonococci; chilling
to meningococci. They grow best at 35° to 37° C, on infusion media contain-
ing blood heated to 90° C ("ehocolate agar"), in a very humid atmosphere,
with 5 to 10 per cent CO2 • The colonies are from I to 4 mm in diameter, clear,
colorless, moist and fragile.
These cultural characteristics serve to differentiate the meningococci and
gonococci from all other species of Neisseria: N. flava, N. sicca, etc. These
others grow well on blood-free media at temperatures as low as 25° C and are
moderately resistant to drying, chilling and light. These species, while para-
sites in a broad sense, being restricted to a life on a mammalian host, are
usually quite harmless. However, under certain conditions some of th.:m may
cause meningitis, and some can cause vaginal infections in little girls which
are sometimes confused with gonorrhea with tragic results. Therefore, no
diagnosis of gonorrhea can be said to be complete and accurate without a full
bacteriological study of the organism involved. Some institutional outbreaks
of VUlvovaginitis in little girls are due entirely to infections by N. catarrhalis
or N. sicca.
Except for the differences noted above the Neisseria are all very similar.
N. meningitidis and N. gonorrhoeae are especially closely related to each other,
being distinguished with difficulty even by serological means and fermentation
tests. Indeed, N. gonorrhoeae has been found to cause meningitis while N.
meningitidis has been isolated fr0111 conditions clinically indistinguishable
from gonorrhea.
SpMrical Bilcleria 457
The meningococci are separable into four main serological groups on the
basis ofagglulination reactions. These are groups I,ll, Ila, and IV. A quellung
reaction for grouping is available, a nalogous to that used in typing pneumo-
cocci, since freshly isolated strams of meningococci possess capsules.
The Oxidase Test. All of the Neisseria produce an enzyme (oxidase) whicb
causes a I per cent solution of dimethyl-paraphenylene diamine to tum,
successively, pink, rose, magenta, and finaHy black. The oxidase test is ap-
plied by moistening a colony of the suspected organism with a drop or two
of the dye solution. The changes in color begin a in few moments. The same
test for ox.idase can be made on all sorts of microorganisms besides Neisseria
and is a valuable differential method for general use.
The Catalase Test. The test for catalase is made by putting a drop or two
of H202 on any "suspected" colony. If catalase is present, bubbles of oxygen
will appear almost instantly. The catalase test is widely used in microbiology.
All Neisseria produce catalase. So do many otber aerobic organisms.
Cultures from the genitalia rarely contain more than one type of Neisseria.
If more than one type are found, all may be of diagnostic significance.
Pathogenic Action of the Neisseria
Goaorrhea. This is one of several diseases commonly spoken of as "vene-
real diseases," deriving this appellation from the name of Venus, goddess of
love. The inappropriateness ofthls term will become obvious in the discussion
of the infections.
Gonorrhea is an inflammatory disease due to infection, by N. gonorrhoeae,
the mucous surfaces and adjacent glandular structures of the reproductive
of men and women. Much pus forms, and appears as a white dis-
(leukorrhea) from the genitalia. It is an alarmingly prevalent disease,
of thousands of cases being under medical care in the United States
There are undoubtedly many other, probably a million or more,
,."..nt\rtf~11 and untreated cases .
.IJlttctllOn of the genitalia with N. gonorrhoeae occurs virtually exclusively
sexual intercourse. Gonorrhea is seldom fatal but is sometimes diffi-
to cure. Patients often believe themselves cured only to find, later, that
disease has reappeared in a chronic form. The sulfonamide drugs promised
a time to eliminate gonorrhea. but drug-fast strains of gonococci rapidly
1W~101:>ed and indiscriminate use of the drugs by the medically ignorant has
such therapy of most of its effectiveness. Penicillin now offers the
hope for cure in all cases. It can be readily understood that careless,
or malicious people can spread gonorrhea widely. Prostitution is
of the chief means by which the disease is propagated. Adequate treat-
with penicillin very close to the time of exposure will prevent many cases
developing. Inadequate treatment is, in several respects, worse than
untreated, often results in sterility. The intense inflammation

lin,nnrrh.f'Il.
by the endotoxins of tbe organism (possibly capsular substance) de-
the lining tissues of the genito-urinary tract, the tissue being replaced
1Cal'S. Such scars obstruct the fallopian tubes of the female and the vas
of the male (tubes through which the reproductive cells pass). Such
obstructions are called strictures. Stricture of the urethra in the male
458
Fig. 3~7. Gonococcus from urethral

pus, stained with methyJene..blue ( X
1000). (Ford, Textbook of Bacteriology.)
also interferes with urination and may require surgical intervention. Gono-
cocci sometimes invade the body, localizing in the joints and the heart valves.
In the former case, a very painful and stubborn type of arthritis results while
in the latter case a very damaging disease of the heart occurs, with permanent
injury and sometimes death.
Gonorrheal Opbthalmia. An intensely painful inflammatory infection of
the eye (ophthalmia) results when gonococci are rubbed into the eye. Loss of
sight usually results in a few days, unless treatment is prompt. A gonorrheal
mother may infect her child's eyes at birth. Due to this fact most cities, states
and countries require that physicians, nurses or midwives attending births,
regardless of any circumstances, instill into the eyes of the infant a few drops
of weak (1%) silver nitrate, penicillin, or other appropriate disinfectant solu-
tion. This rapidly destroys gonococci before they can start an infection in the
eye. Approved disinfectants are obtainable at any health department or drug
store, ready for use.
LABORATORY DIAGNOSIS OF GONORRHEA. The diagnosis of acute gonor-
rheal infection in the adult maJe is usually based on nllcroscopic examination
of the pus stained by Gram's method. The gonococci appear within the leuko-
cytes (Fig. 36-7). Such organisms in adult males with acute urethritis are
rarely any but gonococci. In the female genito-urinary tract many other
organisms are present and, as noted above, respiratory Neisseria may cause
infection in little girls, being transmitted by hands, towels, etc., soiled with
oral or nasal secretions. Isolation and complete cultural and serological identi-
fication of the organism is therefore of importance.
Meoingitis. The term meningitis is drawn from pathology and means,
simply, inflammation of the membranes (meninges) covering the brain and
spinal cord. It may be due to mechanical irritations, viruses, or many kinds
of bacteria, both pathogens and saprophytes, which may localize in the men-
inges. The meningococcus is the only common cause of epidemics of menin-
gitis. The organisms are transmitted as are other respiratory microorganisms,
in oral and nasal secretions.
CARRIERS of meningococci are common, but meningitis is not. There is evi-
SpMrical Bacteria 459
dence that the meningococcus often causes conditions like rhinitis, "catarrh"
or purulent "colds" which heal and attract no particular attention because the
etiological agent is unsuspected.
REFERENCES
Branham, S. E.: Serological relationships among meningococci. Bact. Rev., 1953, 17:175.
Branham, S. E.: Milestones in the history of the meningococcus. Canadian J. Micr., 1956,
2:175.
J>ack. G. M.: Food poisoning. Ann. Rev. Microbiol., 1953, 7:327.
Editorial: Albert Neisser and the gonococcus. Am. J. Pub. Health, 1955,45:95.
HeBron, Roderick: Pneumonia. Commonwealth Fund, New York, 1939.
Larkin, E. P., Litsky, W., and Fuller, J. E.: Fecal streptococci in frozen foods, I and ll.
AWL Micr., 1955,3:98, 102.
Parrino, P. S., O'Shaughnessy, E. J .• and White, J. D.: Standardization of diagnostic
methods for gonococcal infections. Am. J. Pub. Health, 1955,45:457.
Ra-veaholt, R. T., and LaVeck, G. D.: Staphylococcal diseaso-an obstetric, pediatric and
community problem. Am. J. Pub. Health, 1956,46:1287.
Rogers, D. E., and others: Staphylococca1 infections. Ann. N. Y. Acad. Sci., 1956, 65
(Art. 3):57 et seq.
Scherp, H. W.: Neisseria and neisserial infections. Ann. Rev. Microbiol., 1955,9:319.
The Child with Rheumatic Fever: Children's Bureau Folder No. 42-1955. U. S. Gov't.
Printing Office, Washington 25, D. C.
White, B.: The Biology of Pneumococcus. Commonwealth Fund, New York, 1938.
SECTION 5
The Microbiology of
Special Environments
HA VING LEARNED in sections 1 to 4 a good deal about microorganisms in

general and about bacteria in particular we turn, in this final section, to
environments in which microorganisms are found, and to the activities of the
microorganisms in those environments. We' find, ·for the most part, familiar
names~organisms previously descriged as' laboratory subjects-now seen
"at home" as it were, in their natural habitats. We see how and where they
live, what they do there, and how the,if activities affect man. We travel from
the bottoms of the most profound marine chasms to "the desert and illimitable
air" over the arctic oceans; from the warm, fertile furrow to the icy, rushing
mountain torrent; from nauseous volcanic hot springs to the whiskey dis-
tillery or dairy bucket. Everywhere we find microorganisms at their appointed
tasks. They were working at some of their secret objectives long before human
beings appeared on this planet. It seems probable that they will continue in
their mysterious round long after the disappearance of man; and wholly
oblivious of him.
461
37
The Microbiology of Water
CONTROLLING FACTORS
THE MICROSCOPIC flora of water depends to a great extent on: (1) Nutri-
ent substances in the water e.g., ferrous iron for "iron bacteria;" HzS for
"sulfur bacteria;" decaying vegetation or sewage for heterotrophic bacteria;
and so on. (2) Substances unfavorable to certain organisms. For example, sea
water is too saline for many species, while HzS, produced by many putre-
factive species, is unfavorable to several other species. Many organisms pro-
duce antibiotic substances. Acids, as H 2S04 from Thiobaci/lus, or organic
acids, alcohols, etc., resulting from fermentation in soils or stagnant ooze,
or toxic metals like copper from industrial wastes, are unfavorable to many
organisms. (3) The bacterial flora of water depends partly on the presence and
numbers of other living organisms, especially protozoa and bacteriophages,
both of which destroy billions of bacteria. (4) Physical factors such as tem-
perature, amount of dissolved oxygen, exposure to sunlight (for photosyn-
thetic species) and, as indicated above, pH. (5) The soufces from which organ-
isms are contributed.
AQUEOUSE~ON~
For convenience, we may divide natural bodies of water into (1) fresh
waters (rivers, lakes, ground waters, etc.); (2) sea waters (oceans and landward
extensions); (3) saline and hOI waters (Dead Sea, Great Salt Lake, thermal
springs). Each type of environment has its own distinctive flora which cannot
thrive in other environments (the indigenous flora). There are also numerous
species which can gro;W equally well in more than one situation. The physio-
logical adaptations of some of the more restricted species are quite curious
and interesting. :
1. Bacteria in Fresh Waters. Much depends on the location and nature
of the body of fresh Iwater; on whether it is a rapid stream high in snow-
covered mountains, almeandering lowland river in a thickly populated, agri-
cultural area, or a stagnant pool in a woodland swamp. In any case, part of
the flora of any surface water is introduced periodically by rain wash from
adjacent land surface i some by dust from the air, some by continuous growth
of indigenous organisms.
UNPOLLUTED WATERS. In lakes and rivers free from sewage pollution the
concentration of nutrients in solution is usually much lower than in polluted
463
464 The Microbiology of Special Environments
streams like the Hudson, Danube or Ganges rivers. Consider a placid wood-
land pool, fed by surface runoff and springs. The water is clear and looks
"pure." Here food is found in concentration only near the shores, bottom,
and at solid surfaces (see Chapter 13).
The numbers of bacteria floating free in the water away from such zones
are quite limited; often only a dozen or so per ml. These may include various
species of soil saprophytes which can grow to some extent in the small amount
of organic and mineral substances in solution in the water: species of Micro-
coccus, Flavobacterium, Achromobacterium, Bacillus, Proteus, Leptospira and
others. If there is much decaying organic matter at the bottom, species of
Clostridium and other anaerobes, strict and facultative, are often found,
including sulfur bacteria. Caulobacteriales and Chlamydobacteriales may be
found growing on the surfaces of rocks and logs near the shore. If H 2S is
being produced by decomposition at the bottom and if the pool is not too
shaded, species of photosynthetic bacteria may be present.
If, during a windstorm, a large tree falls into the water and stirs up the
bottom sediment, especially if it is during a warm spell in the summer, the
whole flora changes almost momentarily. Cellulose-digesters and fermentative
types thrive. The organic matter stirred up from the bottom furnishes a rich
and varied pabulum. Numerous species of saprophytes, previously present in
small numbers, multiply enormously and some, previously numerous, are
suppressed by newly multiplying, antagonistic species. Total numbers per ml
may rise to 100,000 or more until an equilibrium is again reached.
In a high mountain stream derived from melting snow the numbers and
variety of microorganisms to be found are ordinarily small. Unless the
stream runs over polluted soil, or soil rich in decaying vegetable matter, the
water is likely to be almost sterile. It may contain a few spores of Bacillus,
molds or yeasts, but they will not be very actively germinating because of the
low temperature. They have probably been caught from the air by the falling
snow. A few other microorganisms: micrococci, corynebacteria, gram-
negative rods mostly from dust of the air or from soil, caught by snow or
rain, might be found. They would not be multiplying much, due to cold and
lack of dissolved, nutrient substances.
POLLUTED WATERS. The lower Hudson River has a flora r~presentative
of sewage pollution. One may assume that Escherichia coli* and other Entero-
bacteriaceae* as well as.enterococcit and various species of intestinal Clos-,
tridium are present in large numbers. Many soil saprophytes like Spirillum,
Vibrio, Sarcina, Micrococcus, Mycobacterium, Bacillus, yeasts, molds, Micro-
monospora, Leptospria and other spirochetes, Beggiatoa, Sphaerotilus, and
many others would also find the organic, fecal matter good pablum. .
In the mud and ooze at the bottom, the O-R potential is low and anaerooic
species (Clostridium, Desulfovibrio and various facultative bacteria, their
nature depending on the physicochemical nature of the sediment) exist.
In the more aerated, surface layers strict anaerobes do not thrive, and the
odors and tastes of putrefaction and fermentation are not found. The ,total
numbers of microorganisms may reach into the millions per ml of water.
I • Gram-negative, facultative, non-sporeforming rods, which are usually fOllnd in the
intestinal tract of man or a'limals.

t Intestinal streptococci.
The Microbiology of Water 465
There is nothing constant or necessarily predictable about the flora of
such a body of water except within wide limits. Conditions in such a tidal
river change hourly and the flora changes in response to conditions. The
temporary pollution from large ships like the "Constitution" or "Queen
Elizabeth" is a case in point.
In any body of wat.er, saprophytic organisms serve the purpose of scav-
engers. They decompose organic wastes and make them available as food for
other creatures in the water: higher plants, protozoa, worms, etc. These in
turn support fish and other commercially useful marine or aquatic life, and
so contribute to human welfare. The suppression of saprophytic microorgan-
isms and other aquatic life by excessive sewage pollution and by microbicidal
industrial waste is one of the major problems of the progress of civilization.
THE PSEUDOMONADACFAE AND ENTEROBACfERIACEAE
In populous areas organisms found in fresh surface waters are often of
intestinal origin. Most of these belong to the familY of Enterobacteriaceae
and the group of enterococci. Many of these same species also live in soil and
water as saprophytes. It will be helpful at this point to consider the Entero-
bacteriaceae in detail and to compare them with several similar and related
organisms of the family Pseudomonadaceae, many of which are also common
in soil and water, sewage, etc.
FAMILY PSEUDOMONADAl.~E·
The family p,seudomonadaceae is an extensh e one, comprising some 150

(recognized) species of the genus Pseudomonas; 47 of Xanthomonas; about a
dozen of Methanomonas, Acetobacter, Protaminobacter and Mycoplana; and
about 45 species of curved or spiral forms like Vibrio and Spirillum.
General Properties. Except for some species of Spirillum, all are gram-
negative; non-sporeforming; chemosynthetic; not sheathed, stalked, or sulfur-
storing; unbranching (with the possible exception of Mycoplana). All but a
few questionable species are motile by one or more polar flagella.
This large group need not concern us unduly. We have already devoted a
chapter to the spiral and curved forms. They are common aquatic bacteria.
We may by-pass the Methanomonas,J ProtaminobacterJ and Mycoplanat at
this time because, while important scavengers, they are small groups, of
interest mainly because of their curious, specialized metabolic characters.
Acetobacter we shall meet late~ (Chapter 44) as a wealthy vinegar manufac-
turer. Xanthomonas (while very much like Pseudomonas, and often washed
into water from soil and plants) we shall dismiss at this point because these
organisms are more conveniently studied as the cause of "rots" and other
diseases of plants. This leaves us to consider in detail only the genus Pseudo-
monas.
• The Pseudomonadateae are characterized by polar flagella. Their family name is

derived from the Greek word pseudo, meaning similar to, and the word monas, which is the
name of a group of protozoa having polar flagella.
t Methanomonas: autotrophic; oxidizes CH4 (derived from cellulose decomposition) as
a source of energy; col):tmon in swamps. Protaminobacter: can decompose alkylamines;
cornmon in soil and water. Mycop/ana: cells said to branch slightly; can metabolize phenol
and related compounds as source of energy; cornman in soil.
GENUS PSEUDOMONAS
These are among the most common and widely distributed bacteria. They
are easily cultivated on any ordinary, organic, laboratory media at tempera-
tures of from 15° to 40° C (best at 25° C) at pH from about 6.5 to 8.0 (best
around 7.2). They are enzymically active, hydrolyzing a wide variety of pro-
teins, fats, carbohydrates and other organic compounds. Thus they are ex-
cellent and ubiquitous scavengers. They are principally aerobic; some are
facultative. They are found in soil, fresh waters and ocean waters and de-
composing organic matter, including sewage. Only two representative species
need be detailed at this point.
Pseudomonas fluorescens produces a greenish-yellow, fluorescent pigment
in cultures. There are numerous similar species, widely distributed.
Pseudomonas aeruginosa is type-species of the genus. In addition to the
yellow-green pigment, characteristic of m'lny Pseudomonas, it produces a
turquoise-blue pigment, pyocyanin, which may be extracted from broth culture
with chloroform.
Pseudomonas as Pathogen. Ps. aeruginosa, a common saprophyte, is not
infrequently found in wounds or ulcers which have not healed promptly.
Some outbreaks of diarrhea in adults and especially among newborn children
are said to be caused by this organism. Ps. aeruginosa is also able to cause a
leaf-rot disease in tobacco and lettuce. Ps. aeruginosa causes a fatal disease
in poultry.
FAMILY ENTEROBACTERIACEAE
The family Enterobacteriaceae includes numerous species which are com-
monly found in the intestinal tract of man and/or animals. Hence, the name
of the family. The family comprises about ten genera of closely related
bacteria subdivided into hundreds of species and serological types. Some
workers divide the family into four main groups a§ shown in Table 21.
Table 21. The Enterobacteriaceae.

------- - - ---~-- ---
PIGMENT NOT PRODUCED PIGMENT PRODUCED
-- ---------- -
Lactose fermented in 24 Escher(chia (usually no perceptible capsules) Serratia (blood red
---
hours, with gas Aerobacter} probably identical; (usually pigment on starch
Klebsiellg heavy capsules) media at 22 C)
0
Lactose fermented only Paracolobactrum (gas; not proteolytic)

after 48 hours or Erwinia (mostly no gas; mostly proteolytic)
longer
-
Lactose not fermented Salf1Wnella (motile) } (do not decompose
Shigella (non-motile) urea; not proteolytic)
Proteus (motile; decomposes urea; proteo-
lytic)
I
I General Properties, The Enterobacteriaceae are like the non-spiral Pseu-
domonas in a number of ways (Fig. 37-1). Their principal distinguishing
characters are shown in comparison with Pseudomonadaceae in Table 22.
Table 22. Comparison of Non-Spiral Pseudomonadaceae and Enterobac-
teriaceae.
CHARACTERISTICS PSEUDOMONADACEAB ENTEROBACTERIACEAE

---
J. Morphology Simple, straight rods __ Same as Pseudomonadaceae
---~-.---
2. Staining Gram-nt'gative
-- -- --
. ..
----------
_
..
---.- --_.-- - --- .
3. Spores None . ..
----,- -
..
- -_-------- ---
4. Motility All motile Species vary
5. Flagellation Polar Peritrichous
6. Growth re- Heterotrophs (complex); pH 6 Heterotrophs (simple); require organic

quirements to 8; optimum 15 to 25° C C; pH 6 to 8; optimum 25 to 30° C
7. Habitat Outer environment: soil, water, Intestinal tract; outer environment:

organic matter soil, water, organic matter
8. Enzymatic Proteolytic; somewhat active Fermentative; a few are proteolytic

activities Ij:rmenters
9. Reduction Species vary Active

of NaNO)
10. Relation to Preferentially aerobic (a few Facultative

oxygen exceptions)
--------
II. Pigments Yellowish, fluorescent; pyocy- None (except Serr~tia, blood red).
anin (blue)
B
Fig. 37-1. A, Proteus vulgaris, typical Enterobacteriaceae, plain agar, twenty-four
hours, stained with crys~l violet. B, Pseudomonas jluorescens. The morphological differ-
ences between the typical,Enterobacteriaceae and Pseudomonadaceae shown here are neither
constant nor distinctive. rhe labels could be interchanged and still be correct. (Ford, Text-
book of Bacteriology.)
468 The Microbiology oj Special Environments
Enteric Genera. The most distinctively enteric organisms of this family
arc. included in seven genera: Proteus, Escherichia, Aerobacter, Klebsiella,
Paraco[obactrum, Salmonella and Shigella. At least four genera cause disease
and are detailed in Chapter 39.
GENUS PROTEUS
This genus consists of soil and water saprophytes, common in decaying
animal or vegetable matter and often found in the human intestine or as
opportunists in infections (especially cystitis*) of man and causing diseases
in lower animals. These are also situations in which some species of Pseudo-
monas and Enterobacteriaceae are found. Proteus species occur in polluted
-water and, while not highly active in fermentation, they readily decompose
proteins and thus are valuable scavengers. The power to hydrolyze urea is a
distinctive character of Proteus, which is used in diagnosis of enteric disease
(see Salmonella). Cultures of Proteus usually have a disagreeable, foetid odor.
Proteus Colonies. Proteus colonies are distinctive in that they usually
spread rapidly over the surface of solid media, forming a thin, grey, almost
transparent film which may escape notice entirely unless it is especially sought.
This form of growth is often designated as the H form, or "swarming." An
aflagellate, non-motile variant form of Proteus occurs (called the 0 form) of
which the colonies are smaU, discrete and circular. These peculiarities are of
great importance in medical diagnostic work.
hHE COLIFORM GROUP

Genus Escherichia. t This genus comprises several species of special in-
terest to the sanitarian since the organisms occur commonly in the intestinal
tract of man and animals. Escherichia coli is adapted to a constant life in the
intestines. While found in sewage and in recently-feees-polluted materials it
is not well adapted to continuous life in the outer world.
E. freundii and E. intermedium are very similar species but commonly live
in the outer world as well as in the intestinal tract. Consequently, their pres-
ence mayor may not indicate fecal or sewage pollution. As between the
distinctly intestinal E. coli and the largely outer-environmental _forms such as
Proteus, Aerobacter or Kle~siella (to be discussed), these two species are often
spoken of as "intermediates." All of these species are so similar that they are
often spoken of together as ,the "coliform group."
ESCHERICHIA COLI. This species has all of the general properties of the
family Enterobacteriaceae. It is distinguished from all of the other coliforms
by its inability to utilize citrate in place of glucose in an otherwise wholly
inorganic medium; a reflection, perhaps, of its adaptation to a sub-para~itic
existence. Some varieties of E. coli, indeed, are genuine parasites and cause
infections of the intestinal and urinary tract.
Distinctive properties of E. coli and other organisms discussed in this
chapter are detailed in Table 23.
Genera Aerobacter and Klebsiella. These organisms are much like the
• Infectious inflammation of the urinary bladder.

t Escherich was a famous German bacteriologist, pupil of Koch.
other coliforms but are more commonly soil and water organisms than
intestinal. However, they can live in either environment.
The only species of Aerobacter is A. cloacae. Closely similar to it is Kleb-
siella aerogenes. * They differ mainly in that A. cloacae is proteolytic and
motile, while K. aerogenes is not proteolytic and usually non-motile.
Table 23. Distinctive Properties of the Coliform' Organisms. *
DEX- II ,
' ('[LATIN
MOTIL- GAS
---- - - - __
SPECIES LACTOSE TROSE SUCROSE I M V C lIT RATIO
-
--- --_ - - --
Esch. coli Ee Ee Ee + + - - - + H:C0 2
>1
Esch. [reundi.' Ee Ee Ee - + - + - + H:COz
>1
Klebs. aerogenes Ee Ee Ee - - + + - - H:COz
< I
Aero. cloacae Ee Ee Ee - - + + + + H:COz
<1
I
•+ = positive test.
- = negative test.
Ee = acid and gas formed.
SANITARY RELATIONSHIPS OF THE COLIFORM GROUP

Every well-informed person knows that Salmonella typhi (the typhoid
bacillus) is transmitted by food, milk, or water supplies which have become
polluted with the urine or feces of a person harboring this organism. In
thickly populated districts practically every body of surface water receives
some fecal pollution due to discharge of sewage or due to the careless habits
of ignorant people with respect to urination and / defecation. Sometimes
people may be perfectly healthy yet harbor typhoid bacilli in their intestinal
or urinary tract and discharge them in large numbers, thus infecting water
supplies, milk, food and other substances which they handle. Such persons
are called "carriers" of typhoid. Other pathogenic Enterobacteriaceae
(Salmonella, Shigella, etc., Chapter 39) as well as viruses of poliomyelitis,
hepatitis, and other pathogenic microorganisms, may accompany S. typhi and
be transmitted in identical fasbion.
Pollution of water; with fecal material, whether infected or not, is obviously
undesirable, both from the standpoint of danger of infection and for purely
esthetic reasons. The detection of fecal bacteria in food or domestic water is,
therefore, of importance in determining its suitability for human consump-
tion. For every typlioid bacillus or other pathogen in polluted water supplies
there are, usually millions of coliform organisms.
Indices of Fecal ljoUution. Because of their common occurrence in feces,
the coliforms serve, by their presence in water, food, etc., as an index offecal
pollution. Methods for their detection and enumeration in water and foods
I -
• The organism formerly called Aerobacter aerogene,I' is now discussed as Klebsiella
aerogel1es.
470 The Microbiology of Special Environme1lts
are carefully prescribed by the American Public Health Association, and are
used daily in every health-department laboratory. A series of steps, called
Presumptive Test, Confirmed Test and Completed Test, are carried out system-
atically. These are shown in Chart l.
Two other groups of bacteria invariably present in human (and animal)
feces are enterococci (especially S. faecalis) and Clostridium (especially C/.
perfringens). These two species are easily isolated from water by the use of
relatively simple methods of selective cultivation and are readily identified.
They, also, are used frequently, especially in British practice, as indices of
fecal pollution, both in waters and foods.
SIGNIFICANCE OF INDEX ORGANISMS. The survival time of these three indi-
cator organisms in water is of significance. The enterococci seem unable to
multiply significantly in open water and do not survive long. Their presence
in considerable numbers, therefore, suggests relatively recent pollution; a
few hours or days. The coliforms, especially E. freundii, E. intermedium,
Aerobacter and Klebsiella, generally outnumber the streptococci and, being
often able to multiply to some extent in open, polluted waters, may survive
for weeks or months, depending on conditions in the water. Cl. perfringens,
because of its resistant spores, can survive indefinitely. The presence of this
organism to the exclusion of the others, therefore, suggests pollution. a con-
siderable time ago, if it occurred at all. Clostridia grow naturally in many
soils and waters.
None of these organisms is a perfect index of human pollution because all
occur in the soil or in animal dung, or both, as well as in human excrement.
Examination of any water or food for these index species must be supple-
mented by a sanitary survey-examination of the terrain or situation to evaluate
the probability of human fecal pollution before final conclusions or legal
condemnation.
The "Membrane" or "Millipore" Filter (M.F.) Method. Some of the diffi-
culties in determining the presence of coliform organisms by the methods just
outlined lie in the facts that: (a) only relatively small quantities of water
can be examined at one·time; (b) several days are required for the incubation
of the successive cultures; (c) the test is largely qualitative, only relatively
rough estimates of numbers of coliform organisms being feasible; (d) it
requires considerable amounts of expensive medium and equipment; (e) it is
not readily done in the field "on the spbt," necessitating transportation of
samples to the distant laboratory and consequently very undesirable ch~nges
in coliform content of the samples.
By means of "membrane" or "Millipore" filter~ (see Chapter 13) it is
possible to filter, rapidly, large samples of water to be tested for coliform or,
indeed, any and all organisms. The bacteria in the sample are held on the
surface of the filter membrane (Fig. 37-2). By methods already indicated it is
possible, within 20 hours at 35° C not only to enumerate the coliform colonies
by their distinctive color but to finish the procedures involved in the Coin-
pleted test, in the Standard A.P.H.A. procedure. (See Chart 1.) Compact
field kits for use on the spot are available (Fig. 37-3).
I A difficulty is that ~he filter membrane cannot be used with waters con-
taining any considerable amount of silt or sediment likely to clog the filter.
The Standard Test and the M.F. method do not measure exactly the same
The Microbiology of Water
471
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472 The Microbiology 0/ Spea.I Emironment&
Fig. 37-2. A, membrane or ultrafilter disk with colonies of Escherichia coH. The disk is
on a circular pad saturated with Endo medium, which is selective for coliform organisms.
£Sch. coli colonies are seen here as dark and glistening (about actual size). They are actually
deep magenta in color and have a green-violet metallic iridescence. B, section of membrane
filter disk with colonies of coliform organisms. Escherichia coH colonies appear here as
whitish, opaque, and slightly mounded. Klebsiella aerogenes are larger, more mucoid, with
dark centers (magnified X 4). (photos courtesy of Environmental Health Center, U. S.
Public Health Service, Cincinnati, Ohio.)
flora and neither gives results which, on the basis of present knowledge, may
be expressed accurately in terms of the other.
DifFERENTIATION OF FECAL AND NON-FECAL TyPES

For many purposes of water and food sanitation it is sufficient to know
whether or not any coliform organisms are present. However, it is sometimes
considered desirable to distinguish between the more definitely intestinal
Esch. coli and the other coliforms. Depending on the situation, these may, or
may not, represent fecal pollution.
The procedures are technically not difficult. Having obtained satisfactory
pure cultures from either the Standard or M.F. test, they are differentiated
on the basis of indol production, the methyl red test, the Voges-Proskauer
reaction and ability to utilize sodium citrate as a sole source of carbon
(Table 23). The technical details may be found in "Standard Methods for the
Examination of Water, Sewage and Industrial Wastes" 1955, American
Public Health Association.
Imvie Formula. A mnemonic· "1M ViC" is used, with plus and minus
signs, to express the differences between the organisms in a "formula."
/ I stands for indol reaction, M for methyl red reaction, V for the acetyl-
methyl-carbinol test (originated by Voges-Proskauer), i is for euphony, and
C for growth in mineral solution containing citrate as a sole source of carbon.
Thus, an organism of the coliform group designated as "IMViC ++ - -"
would be Esch. coli since this gives positive indol and methyl red reactions
but negative Voges-Proskauer and citrate reactions. These symbols are used
in Table 23.
• A memory-aiding device.
GENUS PARACOLOBACIRUM
This group resembles Aerobacter, Klebsiella, and Escherichia in all major
respects, but differs in very slow fermentation of lactose. These slow-Iacto~e
fermenters, often called "paracolons," have long confused sanitary and medi-
cal microbiologists because they are often mistaken for the pathogenic
Salmonella (Chapter 39), which does not ferment lactose at all. Some groups
of paracolons can readily be differentiated from Salmonella by the use of
media containing 0.0075 per cent KCN. Most Salmonella do not grow in this
medium while many paracolons do. Some species of paracolons are patho-
gens, causing enteritis and diarrhea.
MARINE BACTERIA
Marine bacteria have been defined as those which will not grow on media
without sea water. For general studies of marine bacteria a recommended
medium contains: sea water (aged) 1000 ml; peptone, 5.0 gm; FeP0 4 ,O.1
gm: agar, 15 gm.
Marine Zones. The sea has been divided into biotiC' zones: the benthic
zone or sea Hoor (benthos); the pelagic. usually thought of as the upper
layers; the oceanic or open sea; the neritic or shallow (200-meter) or coastal
waters; the littoral or close-in, beach or tidal inland waters; a euphotic or
lighted zone; and an aphotic or dark zone. The largest numbers and varieties
of microorganisms are found in the shallow waters near land (neritic or
littoral). Food is more plentiful here.
MARINE ENVIRONMENTS. TEMPERATURES range from 40° C in some littoral
waters of the tropics to _2° C in polar regions (sea water freezes at about
_2° C).
Fig. 37-3. Field monitoring kit.

SALINITY. Most truly marine bacteria are sensitive to small changes in
salinity above or below that of sea water; i.e., they are stenohaline. Some bac-
teria peculiar to salt lakes (Dead Sea, etc.) require salinities of 13% or more
(halophilic).
PRESSURES at the floor of the profoundest depths become very high. At one
mile the hydrostatic pressure is around 2000 lbs. per square inch. Yet many
bacteria thrive at six miles and deeper where the temperatures range around
_2° C. They seem to be adapted to these conditions. For example, in the
0
laboratory, many grow only at 2.5 C and 15,000 Ibs. pressure per square inch.
Those requiring high pressures are said to be barophilic.
It is curious that in samples of mud from the bottom of the sea off Cali-
fornia, at depths of about 5000 feet where the temperature ranges constantly
below 10° C, obligate thermophils, growing only at temperatures of 60° C
or over, were found.
Photogenic Bacteria. There are several species of photogenic bacteria,
indigenous to the sea. They may be cultivated upon sea-water agar with
peptone. Pseudomonas phosphorescens, isolated from luminous marine fish,
is one common example.
Marine Bacteria and Petroleum. Very interesting observations have been
made concerning the possible role of marine microorganisms in the formation
of petroleum. Marine bacteria are enzymatically active, like soil and sewage
bacteria. The various organisms inhabiting deep ocean beds and marine
sediments can, as a group, attack almost any sort of organic matter. There
is some evidence, although it is not conclusive, that these marine anaerobic
microorganisms can transform certain organic substances into petroleum-like
matter.
REFERENCES
Allen, C. H., and Fabian, F. W.: Comparison of E. coli and S,faecalis as test organisms to
determine the sanitary quality of food. J. Milk and Food Techno!., 1954, 17:234,237.
Anonymous. Water pollution in the United States. Pub. Health Servo Publ. No. 64. Gov't.
Printing Off., Washington 25, D. C., 1951.
Bush, J. H.: The membrane filter applied to water supply control. Water and Sewage
Works, 1955, 102:436.
Cooper, M. L., Walters, E. W., and Keller, H. M.: Detection of a new serotype of Escher-
ichia coli 0127: B8, associated with acute diarrhea in infants. J. Bact., 1955,69:689.
Design, Equipment and Operation of Swimming Pools and Other Public Bathing Places.
American Public Health Assoc. Report, NeJ York, 1955.
Editorial: Infectious agents in-sewage. I.A.M.A., 1956,160:877.
Editorial: Pathogenicity of Escherichia coli. J.A.M.A., 1954,154:837.
Edwards, P. R., and Ewing, W. H.: Identification of Enterobacteriaceae. Burgess Publ. Co.,
Minneapolis 15, Minn., 1955.
Edwards, P. R., and'Fife, M. A.: Studies on the,Klebsiella-Aerobactcr group of bacteria.
J. Bact., 1955, 70:382.
Edwards, P. R., and, Fife, M. A.: Cyanide media in the differentiation of enteric bacteria'.
Appl. Micr., 1956,4:46.
Eliassen, R.: A realistic appraisal of membrane filters for water quality control. Wat,cr
and Sewage Works, 1955, 102:523.
Eveland, W. c., and Faber, J. E.: Antigenic studies of a group of paracolqn bacteria
(32011 group). J. Inf. Dis., 1953,93 :226.
Ewing, W. H., and Edwards, P. R.: Isolation and preliminary identification of Escherichia
I coli serotypes associated with cases of diarrhea of the newborn. Pub. Health Lab.,
1954, 12 :75.
Ewing, W. H., Tanner, K. E., and Dennard, D. A.: The providence group: an intermediate
group of enteric bacteria. J. Inf. Dis., 1954,94:134.
Greenberg, A. E., Yee, L., and Hartmann, F. W.: The present state of knowledge concern-
ing the membrane filter in water bacteriology. The Pub. Health Lab., 1956,14:73.
Heukelekian, H.: Microbiology of water and sewage. Ann. Rev. Microbio1., 1953, 7 :461.
Heyl, J. G.: On the occurrence and the significance of paracolon bacteria in The Nether-
lands. A. V. Leeuwenhoek 1. Micr. and Serol., 1954,20:406.
Kelly, S., Winsser, J., and Winkelstein, W.: Poliomyelitis and other enteric viruses in
sewage. Am. J. Pub. Health, 1957,47:72.
Lieber, M.: A critique on the membrane filter. Water and Sewage Works, 1955, 102 :400.
Litsky, W., Mallman, W. L., and Fifield, C. W.: Comparison of the most probable numbers
of Escherichia coli and enterococci in river waters. Am. J. Pub. Health, 1955,45:1049.
\ MacLeod, R. A., Onofrey, E., and Norris, M. E.: Nutrition and metabolism of marine
bacteria. J. Bact., 1954, 68 :680
McBee, R. H., and McBee, V. H.: The incidence of thermophilic bacteria in arctic soils
and waters. 1. Bact., 1956, 71: 182.
Morris, W., and Weaver, R. H.: Streptococci as indices of pollution in well-waters. App.
Micr., 1954, 2 :282.
Pederson, H. T., Jr., and Skinner, C. E.: A comparison of standard lactose broth with
lauryl sulfate broth and with the Eijkman method for demonstrating fecal coliIorm
bacteria. Appl. Micr., 1955, 3 :55.
Ringen, L. M., and Drake, C. H.: A study of the incidence of Pseudomonas aeruginosa
from various natural sources. J. Bact., 1952,64:841.
Shipe, E. L., and Fields, A.: Coliform determinations from swimming and wading pools by
membrane filtration and the most probable number method. Pub. Health Lab., 1955,
13:44.,
Slanetz, L. W., and Bartley, C. H.: Evaluation of membrane filters for the determination of
numbers of coliform bacteria in water. Appl. Micr., 1955, 3 :46.
Standard Methods for the Examination of Water, Sewage and Industrial Wastes. 10th ed.
American Public "Health Association, New York, 1955.
Ware, G. C.: Bacteriological examination of polluted water and sewage effluents. Lab.
Pract., (London), 1955,4:444.
West, M. G., and Edwards, P. R.: The Bethesda-Ballerup Group of Paracolon Bacteria.
Pub. Health Monogr. No. 22, 1954. U. S. Gov't Printing Office, Washington 25, D. C.
ZoBell, C. E.: Marine Microbiology. Chronica Botanica Co.;Waltham, Mass., 1946.
38
Sanitation of Drinking Water and

Sewage Disposal
INFECTIOUS DISEASES often transmitted by unsanitized drinking water

are typhoid/ever (salmonellosis), (Chapter 39); Asi~tic cholera (Chapter 32);
amebic (protozoan) dysentery; bacillary dysentery (shigellosis), (Chapter 39);
leptospirosis, (Chapter 31); viral hepatitis (catarrhal jaundice), (Chapter 45);
tularemia "rabbit fever," (Chapter 39); infections due to certain "tape worms"
(trematodes) may also be transmitted by water. Of these various diseases
those commonly transmitted by water in the United States are printed above
in italics. The others are only occasionally water-borne or occur only in the
tropics or the Orient.
Bodies of water are convenient places for the disposal of sewage and other
refuse and it was not uncommon, until even as late as the latter part of the
nineteenth century, for a city to pour its sewage into a body of water at one
point and build the intake for a drinking-water supply not far away. Conse-
quently, sewage got into the drinking water and epidemics of intestinal in-
fection occurred.
Springs, streams, lakes and wells also become polluted through the drainage
or seepage into them of infected surface washings. The result~is that such
waters, if used for city water supplies, must be treated so that even occasional
pollution, with its dangers of infection, may be guarded against. The water is
therefore collected in reservoirs and then is usually filtered, especially if not
perfectly clear and clean all the year round. In any case, it must always be
disinfected with chlorine, or live pathogenic microorganisms may at some time
get into the water pipes and kill not only hundreds but thousands of people.
This has happened repeatedly in the past. The small amount of chlorine*
necessary for this purpose is absolutely harmless even though it may at times
give a "chemical flavor" to the water.
FILTER PLANTS
. While the construction and operation of apparatus for filtratIon and dis-
infection of water ar~ primarily engineering problems. the processes they are
• Enough must be added to leave a residual of 0.2 to 1.0 mg/L of free CI.
'476
sanitation of Drinking Water and Sewage Disposal 477
designed to carry out are based in part on microbiological principles. There
are several types of apparatus.
The Slow Sand Filter. Large sand and gravel beds, an acre or more in
area, are built up over drain pipes, starting with coarse gravel at the bottom
and graduating in size to rather fine sand at the top (Fig. 38-1). The water
is led onto the sand and allowed to trickle slowly through. The area of the
slow sand filter is necessarily large because the water passes slowly through
it. As filtration proceeds, day after day, there accumulates, around each grain
of sand, and in the interstices, especially in the upper 3 or 4 inches of sand,
a slimy, gelatinous film composed of millions of bacteria and protozoa. This
slowly closes up the pores in the sand and makes the filter bed more and more
effective but also causes the rate of filtration to become slower and slower.
At best, slow sand filters yield about 3 million gallons of filtered water per
acre per day. The slimy layer is called the "schmutzdecke" (German for "dirt
cover"). Through the action of enzymes, biological oxidation and reduction
processes, and the ingestion of bacteria by myriads of protozoa inhabiting
the slimy film, the bacterial and chemical quality of the water is greatly
improved. When the gelatinous film finally becomes too thick, the filter is
thrown out of service and the schmutzdecke removed by cleaning machines.
A newly cleaned filter is not highly effective until the schmutzdecke begins to
form. The effectiveness of the filtration is constantly tested by bacteriologists
in the plant who determine the numbers and kinds of bacteria present in the
water during different stages of the filtration process, as well as in the finished
product. The filters can remove around 99 per cent of the bacteria present in
the "raw" water.
The Rapid Sand Filter. The rapid sand filter is similar to the slow sand
filter in principle. but its area is much less and it does not depend on the
growth of a schmutzdecke. It filters water much more rapidly per unit of
filter-bed area; about 130 million gallons per acre pe~ day.
In order to obtain rapid filtration of the water it is necessary that most of
the foreign material in the water be first removed by some other means. This
is usually accomplished by settling and coagulation. Coagulative substances,
Fig. 38-1. Vertical section of a filter

bed. The dept hi of water varies from 3 to
5 feet above the sand. The principal fil-
tering action is in the upper few inches of
sand. The filtered water is drained away
in the pipe at the bottom and stored in a
cistern. It is ~hlorinated on the way.
(Hunter and Whitman, Problems in Gen-
eral Science, American Book Co.)
Fig. 38-2. A rapid sand filter, showing, in rear comers, the different layers of sand;
under foot, the drains for filtered water and the pipes admitting wash water in a reverse
direction; at the digger's shoulders, the troughs leading away the overflow from the wash-
ing process. (From Horwood, The Sanitation of Water Supplies, courtesy of Charles C
Thomas, Springfield, lllinois, publishers.)
sometimes ammonium aluminum sulfate, or a mixture of ferrous sulfate and

lime, are added to the water. These form bulky, sticky precipitates which
adsorb or occlude silt, bacteria and coloring matter. The water is held in
large settling basins till most of the dirty precipitate has settled out. The sedi-
ment is later pumped out from the bottoms 'of the tanks. The clarified water
is run onto the filters. The particles to be removed consist chiefly of the floc
remaining after the settling process and, being large, they are readily filtered
out .by the sand. The bacterial removal is of the order of 99 per cent. The
filters remain in service for several hours or days, and then are washed by a
flow of water arid air forced upward through them. Figure 38-2 shows a
rapid sand filter. \
In neither process can the filtered water be guaranteed to be free from
pathogenic microorganisms. For this reason, in all public water systems in
enlightened civilizations, after filtration the water is treated with chlorine and
stored in underground cisterns. Highly P91luted waters may be made potable,
crystal clear and almost sterile by these devices, and it is due largely to these,
as well as to improved methods of sewage disposal, that typhoid fever,
dysentery and cholera remain at a low level or absent in cities with water
filtration plants.
Combination Process. In newer processes the extensive basins for coagu-
lation or floc formation and the long time and large tanks required for settling,
plus the necessity of loading filters with the remains of unsettled floc, and
elaborate washing apparatus, are to a great extent overcome. Tank machines
(often called "package plants") are arranged in which large, heavy particles
of floc are built up by chemical action between the coagulating chemical and
Sanitation of Drinking Water and Sewage Disposal 479
the raw water in a mixing and reaction zone. Paddles provide vigorous circu-
lation of the coagulating chemicals and the raw water in a "slurry pool."
The heavy floc particles actively adsorb color, microorganisms, and finely
divided suspended matter. Being large and heavy they rapidly settle out as
sediment in the lower parts of the slurry pool. Excess sediment is removed
in a "concentrator" or drain. The supernatant water is quite clear and is led
off to be finally filtered, chlorinated and stored in a covered reservoir. There
are various engineering devices to accomplish these purposes.
SEWAGE PURIFICATION
The "purification" of sewage in cesspools and municipal sewage-disposal
plants is almost wholly dependent on processes in which microorganisms play
the chief parts. The processes are, fundamentally, the same as those occurring
in natural. decay and putrefaction, but are controlled and exploited to the
best advantage by the sanitary engineer. Many of the species of water, soil
and intestinal bacteria already mentioned are involved in both processes.
Aerobic action and oxidative processes are promoted especially in the fluid
parts of sewage, while anaerobic processes occur in digestion of sludges,
except activated sludge, which is aerated.
Most cities have separate sewerage systems to drain off storm waters. Indus-
trial wastes (from dairies, tanneries, paper mills, etc.) are usually disposed of
in plants specially designed for the particular material involved. Such wastes
often require rather complex preliminary chemical treatment, but biological
oxidative action is usually the final "purifying" agent. Such materials are
often led into broad, shallow, open, artificial "oxidation ponds" or lagoons,
there to undergo (partly) aerobic, oxidative processes. As an interesting side-
light on conservation of food and utilization of solar energy (a pet subject
for engineering research), experimental attempts are being made to cultivate
algae (Chlorella, Scenedesmus) in such ponds, fully exposed to sunlight, on a
large and intensive scale. Protein, a very expensive stock food, may thus be
produced cheaply from sewage and sunlight, resulting eventually in spare
ribs and omelets on your table.
If "raw" domestic or "sanitary" sewage is discharged directly into natural
bodies of water, the available oxygen in the water is soon used by the micro-
scopic organisms living in the sewage, and foul-smelling, anaerobic processes
develop and create a nuisance. Further, aquatic life is impossible, and con-
siderable economic loss results. Still further, the water is ruined for drinking,
swimming or esthetic purposes.
Composition of Sewage. The sewage of a city is about 99 per cent water,
and is handled in pumps and tanks much as is drinking water.
Fresh city sewage :contains dilute excrement, along with other city wastes:
comminuted garbage, laundry water, and the like. Consequently it contains
the flora and fauna: of the intestinal tract, as well as many soil and water
species, and much organic matter. The pH usually ranges around the neutral
point; temperatu:e varies seasonally (5-25° C). Of the solid material (after
removal of silt, gravel, bottles, etc.) nearly half is cellulose.
Microorganisms in Sewage. These include many aerobes, strict anaerobes
and facultative anaerobes, mostly saprophytic heterotrophs. Common types
of bacteria from soil and intestine are: Enterobacteriaceae, enterococci,
Clostridium, Bacteroides, Cytophaga, Micrococcus, Pseudomonadaceae, spiro-
chetes, Lactobacteriaceae, Achromobacteriaceae, sulfur bacteria, iron bac-
teria, yeasts, molds and actinomycetes.
Sphaerotilus, Crenothrix, Beggiatoa, and probably filamentous Rhodobac-
teriineae, (if sunlight is present) characteristically form slimy growths on the
sides and bottoms of sewage-containing ditches, pipes, tanks, etc. They are
often called "sewage fungi." Certain real fungi (Phycomycetes), Saprolegnia
and Levtomitus, are often found among them. All of the organisms aid in
decomposition of organic matter in the sewage.
Changes in Sewage. Not all of these organisms continue to thrive. The
pH changes toward acidity due to fermentation, and acid-sensitive species
disappear. Unless air is vigorously bubbled through the sewage (a common
procedure today) the oxygen dissolved in the water is soon used up and the
0-R potential becomes strongly negative. The biological oxygen demand (com-
monly called BOD) increases. The strict aerobes cease functioning: Only
facultative and strict anaerobes can thrive. Being unable t,o use oxygen to
cause complete oxidations of organic matters they cause partial decomposition
of organic compounds, with formation of odoriferous volatile compounds:
various sulfur amines, butyric acid, hydrogen sulfide and the like. A great
nuisance is created. The sewage is said to be in a "septic" condition.
Biologically, a kaleidoscopically changing picture is presented; one physio-
logical type succeeds another in rapid succession as conditions in the sewage
develop, to which each successive form is peculiarly adapted and to which
the preceding form was not. There is also a complex interplay and competition
between various bacteria, and between them and protozoa and other small
animal forms which thrive mightily in sewage.
Biological Actions. As we have seen, many of the common saprophytes
of soil, water and the intestinal tract possess marked powers of hydrolysis.
Proteolytic forms bring about digestions of proteins; lipolytic forms hydro-
lyze fats and related compounds; nearly all forms can hydrolyze one or more
carbohydrates. The woody vegetable materials in sewage are decomposed by
numerous species capable of hydrolyzing cellulose. A piece of linen or fabric
or a thick sheet of cellulose filter paper will be digested and disappear com-
pletely in active sewage in 5 to 7 days; faster in warm weather or heated
sewage. Still other forms hydrolyze all manner of organic matter: phenol
rubber, paraffin, and so on. Some of these organisms have been mentioned.
Others, especially certain gram-negative, anaerobic, cellulose-digesters of
sludge, are still imperfectly known.
SYNTROPISM. The products of hydrolysis by one organism * are excellent
pabulum for still another species, and ~o on. This is a form of syntropism.
The original molecules of wood, faf, meat, etc" are finally changed, usually
through the combined actions of several species, into soluble, relatively simple
substances.
AEROBIC PROCESSES. Under aerobic conditions all are eventually changed
into sulfates, phosphates, ammonium salts, nitrates, CO 2 , H 20 ("mineral-
, • Say, for example, starch is hydrolyzed to erythrodextrin, or cellulose to cellobiose, or

fat to glycerol and fatty acid, or flesh protein to albumoses.
Sanitation oj Drinking Water and Sewage Disposal 481
ized"). Many of these compounds are rebuilt eventually into crop plants;
these into animals and these in turn, dear reader, into you and me!
ANAEROBIC PROCE.<;SES. If the digestive process is anaerobic (as in tank-
digested sludge) reduced compounds are formed, some of which were listed
earlier: H 2S, N, NH 3, CH 4 , etc. Others are products of partial decomposition.
These, when tran:;ferred to soil or bodies of water, are utilized as sources of
energy by the saprophytic and autotrophic microorganisms capable of oxi-
dizing them (Thiobaci//us, Nitrobacterieae, Methanomonas, Rhodobacteriineae
and the like). In the oxidized form (sulfates, nitrates, carbonates, etc.) they
are available to higher plants as noted above, and so the cycle runs.
Importance of Oxygen. It is evident that, throughout the whole process,
oxygen content of the water is the principal controlling factor. If thorough
oxidation could be maintained throughout, the offensive, anaerobic, septic
condition would not occur and complete, oxidative decomposition would
proceed much more rapidly, with little or no nuisance. Such is the object of
mortern sewage disposal plants.
SEW AGE DISPOSAL PLANTS
Sewage disposal plants (Fig. 38-3) are operated to accomplish several ends
as follows:
1. Screening,' to remove bulky foreign matter such as grit, bottles, paper,
wooden boxes and other extraneous refuse as well as allow grit and gravel to
settle out.
2. Separation by gravity of all organic matter in the sewage which will
settle to the bottom of tanks (settleable solids) or float on the surface (scum).
3. Aeration and biological treatment of the supernatant fluid and usually
final chlorination of the supernatant fluid.
4. Digestion, and drying of the sludge or sediment.
5. Collection of gas from sludge digestion.
6. Disposal of the sludge. /
Numerous methods have been devised for accomplishing these various ends.
The first is largely a matter of mechanics and does not concern us. The
second is of interest since it is here that microbial action first comes into play.
Various kinds of preliminary settling or sedimentation tanks are employed,
all designed to allow the solid matter to settle out as much as possible, and in
whi('h the sewage is held for some time (usually from 2 to 10 hours). It is a
modification of the natural process of sedimentation that goes on constantly
in rivers, lakes and ~he ocean. Flotation may also occur (fat, wood, etc.) and
surface-skimming is often an important part of preliminary settling. From 40
to 60 per cent of the solid matter of sewage settles out of suspension as sludge
in 3 hours in these t~nks. Sedimentation is sometimes hastened by the addition
of flocculating agents similar to those used in water purification.
The sludge, of which about 95 per cent or more is water, is drawn off from
the depths of the tanks through pipes and is transferred to sludge-digestion
tanks where slow, I mostly anaerobic, decomposition continues. Here the
sludge may be held. for weeks or months. It is finally transformed to a dark,
viscous fluid in which little or no further biological action can occur. It is
removed from the hlllk and disposed of on sludge-drying beds or it may be
482 The Microbiology of Special &"ironments
Fig. 38-3. A modem municipal process of sewage purification combines the best fea-
tures of activated (aerated) sludge, settling tanks and sludge digestion. It gives maximum
biologic decomposition of the organic matter in the sewage in minimum space and time.
The large round structure is a gravel bed filter (see Fig. 38-5) over which the fluid is sprayed
by the rotating arms. This permits greater aeration and speeds up the microbial action on
the sewage. (Courtesy of The American WeU Works, Aurora, Ill.)
The sewage enters at the influent (left), is screened and then passes through preliminary
settling tanks where solids settle to the bottom as sludge and grease floats on the surface.
The sludge is removed for anaerobic digestion. The partly clarified sewage is then passed
over the aerated, high-capacity filter. Passing then through an intermediate tank any remain·
ing solids settle out and are taken off for anaerobic digestion. The supernatant fluid then is
thoroughly aerated with activated sludge and flows into the final tank where the activated
sludge particles settle out and are returned to the aeration tank. The fluid is then chlorinated
and passed out through the effluent into a river or other adequate body of water. When too
much activated sludge accumulates part is put into the regular sewage for routine disposal
(waste activated sludge). Pipelines are available for bypass of any part of the system or,
indeed, the entire disposal plant in case of breakdown or emergency.
de-watered in large heated drums, and pulverized, incinerated or sold as

fertilizer. "Milorganite" is a good example.
"Tw~tory" Tanks. Some tanks are made in two compartments, an upper
and a lower. The upper portion is like a long, double, V-shaped trough and
serves to introduce fresh sewage (Fig. 38-4). The solid matter settles out
through slots at the bottom of the V's into a deep sludge-slump as the fluid
flows along the trough. The solid matter, after settling to the lower compart-
ment, is held and stored, digestion and decomposition going on there. The
sludge may be removed to other digestion tanks. Gases from the sludge
compartment are collected and led to the surface by a funnel-shaped structure
inverted over the sump. This gas contains about 75 per cent methane produced
by reduction of carbon by anaerobic bacteria. It is collected in gas tanks
and used as fuel. It often serves to furnish steam and electric power for most
of the light, heat and power requirements of the disposal plant. This energy
was once the energy of life! The digested sludge, largely mineral residue, is
pumped out from the bottom at intervals. The best known tank of this type
is the Imhoff tank.
Aention and Disposal of Fluid. The fluid part of the sewage, after passage
through the sedimentation tanks or above the sludge compartment of the
Imhoff tank, still contains much putrescible organic matter. It is often passed
through a secondary clarifier or settling tank. The clarified fluid may then be
disposed of, where a porous and dry soil is available, by surface ditches or by
a subsurface irrigation system of tile pipes, furnishing excellent fertilizer for
farm crops raised on the land. There is probably little danger of infection as
typhoid, dysentery, and cholera organisms are largely killed, either by the
antagonistic action of sapropbytic bacteria in the settling tanks or soil, or
are filtered out by the soil and die. However, tubercle bacilli, Salmonella and
polio virus have been isolated from Imhoff-tank effiuent, as well as from sec-
ondary effiuents, so that promiscuous use of undisinfected sewage effiuents is
not entirely safe.
AERATING FILTERS. The fluid from any form of settling tank may also be
allowed to drain through artificial beds of sand, or broken stone, the filtrate
being collected by under-drains much as in slow sand filtration of water. This
process of purification is similar to those used in filtering drinking water, the
sand, grains or pieces of stone becoming coated with a living film of aerobic,
strongly-oxidative microorganisms much like schmutzdecke. These feed upon,
and oxidize, the organic matter of the sewage; the result being a much less
offensive liquid. Since the fluid is initially very poor in oxygen, due to the
BOD of the organisms already in it, it must be well aerated to enhance aerobic
oxidation of organic matter and prevent foul-smelling anaerobic action. For
this reason sucb filters require draining and aeration daily to allow oxidation
processes to occur. They are, therefore, intermittent in operation.
TR.ICKllNG FILTERS. A more effective type of aerating device, operating on
similar principles, is the trickling filter in which the fluid is intermittently
sprayed over the stones by stationary nozzles or rotating spargers instead of
Fia. 38-4. Structure and operation of an Imhoff-type sewage-clarification tank. The

may be 10 by 20 feet in dimensions and perhaps 10 feet deep. The diagram shows a
lICCtion. Sewage flows slowly (vertically to the page in this diagram) through the
If and B. Solids settle through slots C and D to the sludge sump at E. Sludge is
off periodically through the drain pipe to a sludge-digestion tank. Gas bubbles risq
auund the baftIes at S and T. Scum is removed at the surface as it forms.
484 TIw Microbiology 0/ S~ciQI Environments
Fig. 38--5. Types of trickling filter. At A is seen a COllCrete tank filled with coke o r
broken stone over the aerated surfaces of which the sewage is sprayed through nozzles. In
an older manner of use the tank is alternately tilled with, and emptied of, sewage; a process
which intermittently aerates the actively oxidizing film of growth on the surfaces of the
coke or stones. At B is seen a more modem use of the same principle. The rotating sparger
applies the fluid intermittently. In this picture the sparger has been stopped to clean a
plugged nozzle. Note the dark-colored growth on the surfaces of the stones. (A, courtesy
of Dr. Gordon M. Fair in Rosenau, Preventive Medicine and Hygiene. D. Appleton-Cen-
tury Co.; B, courtesy of Communicable Disease Center, U. S. Public Health Service, At-
lanta, Ga.)
alternately flooding and draining them (Fig. 38-5). The fluid adsorbs oxygen
as it is sprayed through the air, and the interstices between the stones arc
filled with air. Oxidation is rapid and complete.
Activated Sludge. If air is bubbled actively through sewage in a tank,
aerobic conditions are maintained throughout the liquid. Particles of sus-
pended matter flocculate. after a time, into smalJ masses swarming with
aerobic microscopic life and capable of oxidizing organic matter readily.
These masses are called "activated sludge." As the aeration process continues.
the volume of the floc, or activated sludge. increases as more and more
sewage is passed through the tank.
AcnVATED SLUDGE ORGANISMS. The particles of f10('; in activated sludge
consist of mixed species of hacteria which embed themselves in a mass of
polysaccharide gum called "zooglca" (more properly. phytogloea). One of
the principal zooglea-forming species is a Pseudomonas-like species called
Zoogloea ramigera. Numerous other familiar microorganisms can also form
(or help to form) zoogloeal masses under the conditions of activated sludge:
Escherichia, various Pseudomonas, Alcaligenes, Bacillus, etc. Floc is formed by
the adherence together, in clumps, of several bacterial cells at their capsular
surfaces.
The sticky zoogloeal material gathers up, by adhesion and by adsorption,
much of the colloidal material, bacteria, color and odors of the sewage fluid.
After a good, active floc of activated (aerated) sludge is formed in a tank,
more sewage can be passed through the tank and rapid clarification, organic
decomposition, oxidation, and decrease in bacterial content is obtained, pro-
vided aeration continues and enough activated sludge is intimately mixed with
the traveling sewage. The basic principle of the process is the same as that of
aeration and filtration, the sand grains or stone of sewage filters being re-
placed by the living particles and air in the activated sludge.
The fluid part of the mixture is afterward passed into a final settling tank
or clarifier, or trickling filter, part of the active, solid sludge being retained
in, or returned to, the aeration tank as "seed."
Rapid Methods of Sewage Purification. It is clear that rapid and complete
oxidation and decomposition of the organic matters in sewage depend pri-
marily on vigorous growth of aerobic microorganisms and this, in tum, on
the presence of ample food and oxygen. Machines have been devised in
which air may be mixed with the sewage in the sewers before it even reaches
the disposal plant. This provides a well-oxidized sewage to begin with. In
other systems (after screening and removal of grit), raw sewage is immediately
mixed with activated sludge and vigorously and continuously aerated by pad-
dles in the depths of a specially designed tank. The machines are built and
operated on the same principle as the "package" water filtration plants
described in a foregoing section.
PREVENTION OF WATER-BORNE DISEASE IN ABSENCE OF FILTRATION

Disease transmission by water in the absence of elaborate filtration and
chlorination systems, as on camping trips, is easily prevented. All of the com-
mon intestinal pathogens are readily killed by boiling for five minutes as well
by contact for at least 2 hours with chlorine, iodine, or other suitable dis-
Infectants.
Ice made from polluted water is nearly as dangerous as the water itself.
Water for camp use, provided it is clear and clean, may be treated with
dIlorine by using one of the methods of field chlorination involving tablets of
CaOCl2 available on the market. A teaspoonful of fresh chloride of lime in
2S to SO gallons of water will also prove an adequate safeguard unless the
water is very heavily polluted or dirty. At least ari hour must be allowed for
chlorine to act. Ordinary laundry bleaches (Na20CI) will also serve.
,DirectiOIIlS are found on the labels of the bottlest If you are contemplating a
"Dpling trip, or travel in countries where water and sanitation are poor,
Public Health Service Publication No. 387, "Drinking Water Dis-
ItfcICti,on," 1954, Superintendent of Documents, Government Printing Office,
rtubin~lton 25, D.C. (S cents).
REFERENCES
Babbitt, H. E.: Sewerage and Sewage Treatment. 7th ed. John Wiley and Sons, Inc., New
York, 1953.
Drinking Water Disinfection. Pub. Health Servo Publication No. 74, 1954. Sup't Doc.,
Washington 25, D. C.
Editorial: Infectious agents in sewage. J.A.M.A., 1956, 160:877.
Ehlers, V. M., and Steel, E. W.: Municipal and Rural Sanitation. 4th ed. The McGraw-Hill
Book Co., New York, 1950.
Fair, G. M., Geyer, J. C., and Morris, J. C.: Water Supply and Waste Water Disposal.
John Wiley and Sons, Inc., New York, 1954.
Gainey, P. L., and Lord, T. H.: Microbiology of Water and Sewage. Prentice-Hall, Inc.,
New York, 1952.
Gottas, H. B.: Composting, sanitary disposal and reclamation of organic wastes. World
Health Org. Monogr. Ser. No. 31. Columbia University Press, New York, 1956.
Hardenbergh, W. A.: Sewerage and Sewage Treatment. 3rd ed. International Textbook Co.,
Scranton, Pa., 1950.
Heukelekian, H.: Microbiology of water and sewage. Ann. Rev. Microbio!., 1953, 7 :461.
Heukelekian, H.: Stabilization of polluted waters. Water and Sewage Works J., 1955,
102:82.
Individual Water Supply Systems, 1950. Public Health Serv., Publication No. 24. Sup't Doc.
Washington 25, D. C.
Maki, L. R.: Experiments on the microbiology of cellulose decomposition in a municipal
sewage plant. A. V. Leeuwenhoek J. Micr. and Serol., 1954,20:13.
McKinney, R. E., and Weichlein, R. G.: Isolation of floc-producing bacteria from activated
sludge. Appl. Micr., 1953, 1 :259.
Renn, C. E.: Algae research on oxidation ponds. Am. J. Pub. Health, 1954,44:631.
Van Kleeck, L.: Fertilizer value in waste disposal methods. Am. J. Pub. Health, 1954,
44:349.
Wang, W. L. L., Dunlop, S. G., and De Boer, G.: The survival of Shigella in sewage.
Appl. Micr., 1956,4:34.
Warrick, L. F., Chairman, Engineering Section. Package Sewage Treatment Plants, and
Special Disposal Problems. Am. J. Pub. Health Year Bqok, Pt. 2, 1952.
Weibel, S. R., Bendixen, T. W., and Coulter, J. B.: Studies on household sewage disposal
systems. I, II, III. Pub. Health Service Publ. No. 397. Gov't. Printing Off., Washing-
ton 25, D. C., 1955.
39
Some Pathogenic Gram-Negative Rods:

Enterobacteriaceae; Parvobacteriaceae
THE PATHOGENIC ENTEROBACTERIACEAE

SOME OF the lesser, occasionally pathogenic Enterobacteriaceae were men-
tioned in the previous chapter: some varieties of Escherichia coli; some groups
of Paracolobactrum; and occasionally Proteus. The most important pathogens
of the family are comprised in the genera Salmonella* and Shigella. *
The Salmonella and Shigella are typical Enterobacteriaceae in all morpho-
logical and physiological respects, differing from those discussed in the last
chapter mainly in:. (a) being more pathogenic; (b) failing to ferment lactose
or sucrose (there are one or two important exceptions). Salmonella are motile;
Shigella are non-motile. The genera most likely to be confused with Salmonella
because of slight, slow or absent fermentation of lactose are Proteus and
Paracolobactrum. These genera are readily differentiated by the physiological
characters shown in Table 24. t All of the Enterobacteriaceae are closely
allied, as shown by the fact that antigenic relationships are commonly found
between species in all genera in the family. Further, some "species" have
identical biochemical properties. Consequently, in identifying species, it is
frequently necessary to go_ through a complex serological process called
antigenic analysis, using adsorbed sera (Chapter 21), before the true status of
an organism is known; even then the position of many is left in doubt.
GENUS SALMONELLA
The salmonellas .cause infections (commonly called salmonellosis) in man
and many species bf domestic and wild animals. The resulting diseases are
characteristically gilstrointestinal, but may be completely generalized in the
body, and may ra!lge in severity from almost imperceptible intestinal dis-
I
• The apparently rr{eaningless generic names Salmonella and Shigella are derived from
names of famous bacteriologists. Salmon was an American scientist, noted for his wo~k
on hog cholera (1885). Shiga was a Japanese bacteriologist who first determined the cause
of highly fatal epide~ics of dysentery in Japan in 1896.
t It must be underStood that, in describing any group of bacteria, allowance must be
made for variation and mutation. Forms of any species not infrequently occur which are
aberrant with respect 'to any physiological characteristic.
487
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Some Pathogenic Gram-Negative Rods 489
comfort to fatal disease. The habitat of the organisms is mainly the intestinal
tract and tissues of infected animals, but the organisms can grow in feces-
polluted foods and may survive in polluted or infected foods, waters and on
fomites for periods of from a few hours to days.
Of the salmonellas, S. typhi (the cause of typhoid fever), and the so-called
"food-poisoning" or paratyphoid group: S. paratyphi A; S. paratyphi B;
S. paratyphi C; S. typhi murium; S. enteritidis; and S. cholerae-suis are among
the most important. There are over 300 named types (serotypes), including the
above.
Isolation of Salmonella (also Shigella and Paracolobactrum) from feces,
foods, tissues, etc., is greatly facilitated by selective cultivation. Media used
for this purpose contain: (a) a nutrient agar or broth base; (b) agents to
inhibit unwanted microorganisms; (c) lactose and some acid-indicator dye
(like litmus or phenol red) to reveal and differentiate the lactose-fermenting,
acid-producing, non-pathogenic organisms (colonies are colored by acid
indicator). The non-Lactose-fermenting Salmonella, Shigella or Paraeolo-
bactrum colonies are colorless. Numerous selective inhibitory agents are
used: sodium desoxycholate; fuchsin-sulfite mixtures combined with eosin
and methylene blue; sodium selenite; sodium tetrathionate; brilliant green,
etc. There are scores of such media. All are based on the same principles.
The steps are outlined in Chart 2.
Chart 2. Outline of Procedure for Preliminary Isolatioll and Identification at
Pathogenic Enterobacteriaceae.
1. Sample is inoculated onto plates of selective agar media:
(a) Eosin-methylene-blue agar
(b) Desoxycholate agar
(c) Bismuth sulfite agar (especially good for S. typhi)
ll. Sample is inoculated into tube of broth containing selective agents:
(a) sodium selenite
(b) sodium tetrathionate
II'. After incubation of selective broths about 18 hours, plate out as in step l,
above.
III. After overnight incubation of all plates inoculated as indicated above, pick suspected
colonies to:
(a) TSI agar slants (detect gas, acid and H2S)*
(b) Urea medium to detect urease formation.
1V. If, after 24 hours of incubation, TSr agar shows reactions of Shigella or Salmonella
(Fig. 39-1), try agglutination with polyvalent Salmonella or Shigella serum.
V. Carry out additional tests for motility and various enzymic activities (Tables 23,
24, 25 and 26).
Antigenic Analysis. With a collection of adsorbed, monovalent agglu-

tinating sera (Chapter 21), each representing a single, different. antigenic
component of the yarious species of Salmonella, one may test any given
organism for the presence of different antigenic components and assign to
it a "formula" expressing the antigenic complex of which it is composed.
This sort of antigenic analysis has been developed in the genus Salmonella to
* TSI medium contaiJ?s lactose, glucose and sucrose, with an acid indicator, and an iron
salt (FeCh) to turn black (Fe2S3) if H2S is formed. The medium is used in the form of slants
in culture tubes and gives characteristic reactions depending on the species inoculated into
it (Fig. 39-1). .
c spp TO
Fig. 39-1. Cultures of Enterobacteriaceae in an agar medium designed to differentiate
major groups. The medium is triple-sugar-iron (T-S-I) agar. It contains lactose, glucose
and sucrose with acid-alkali indicator. FeCI) is added to detect the formation of HzS, The
slants are inoculated on the surface and by a stab of the needle into the depths of the butl
of the agar. C shows the reaction of coliform organisms: gas bubbles and acid (crosses)
throughout the agar. Note that the volume of gas formed has rent the agar slant at several
places and pushed the butt of the agar slant away from the bottom of the tube, where a few
drops of bacterial suspension have collected. SPP shows the reaction typical of Salmonella
organisms and "also of some strains of Protells and Paracolobactrum: acid and gas, with
formation of HzS (shading) in the butt; alkaline slant (upper portion). TD shows the reac-
tion of Salmonella typhi and of the genus Shigella (typhoid-dysentery): acid butt, alkaline
slant; no gas, no HzS. Further differentiations are made on the basis of reactions shown in
fables 24, 25 and 26 and serological tests.
a high degree and we may now state the antigenic formula for each of some
300 or more "species" or serotypes.
KAUFFMANN-WHITE SCHEMA. By means of such antigenic analyses the
salmonellas have been arranged in a series often referred to as the "Kauff-
mann-White sch.ema." In this schema the 0 (somatic) antigens are given
Arabic numbers; The flagellar (H) antigens have 2 series of numbers, de-
pending on phase variations:* small Roman letters if in phase I; Arabic num-
bers if in phase II. The antigenic structure of any given species may therefore
be expressed in terms of these numbers and letters. For example, S. typhi
murium has the antigenic formula, I, 4, 5, 12: i; 1, ,2 (see Table 27). Species
having one or more somatic antige~s iq common are placed in convenient
groups: A, B, C, etc. '
Similar antigenic schemes are found in other groups of bacteria: Clostri-
dium, Shigella, Klebsiella, Corynebacterium, Escherichia, etc.
• In many cultures of Salmonella there appear two types of cells. Some of the cells are in
what is called the "specific phase" (phase I). In this phase they agglutinate best with H
serum specific for only that species. Other cells in such diphasic cultures are in the "group
ppase" (phase In. In this phase they agglutinate to a considerable extent in H sera for other,
closely related, species. Sometimes the culture may be almost entirely in phase I; at other
times predominantly in phase II. S0'W cultures do not alternate phases; i.e., they are
monophasic.
Table 25. Differential Properties of Some Salmonellas. *
SPECIES XYLOSE TREHALOSE I ARABINOSE H2S CITRATE AOAR GAS IN DEXTROSE

------
S. typhi V + - + - -
S. paratyphi A - EB EB - - +
S. paratyphi B EB EB EB + + +
S. paratyphi C EB EB EB + -+ +
S. enteritidis EB ED EB + I + +
S. typhi murium EB EB EB + + +
S. cholerae-suis EB - - - + +
\
* EB = acid and gas; += positive test or gas only; V = variable
Table 26. Some Cultural Reactions of the Shigellas. *
SEROLOGICAL
GROUP
PRINCIPAL SPECIES i MANNITOL ILACTOSE I SUCROSE INDOLE PRODUCTION
- _ _ _ 1_ _ _
A Shigella dysenleriae - -
-
- -
-----
\ Shigella ambiglla
-,-~---- -~
-
----- - - -
- +
------
B
I
Shigella jlexneri
---_
+ ---
_.,-----
- - ==
C I Shigella boydi;
+ - - ==
~~-I I Slowly
D Shigella sonne; + + + -
- - - - - - - _ ..
* There are =eptions to most of the reactions, in aberrant strains.
Table 27. Antigenic Formulae of Some Salmonella Species.
GROUP SPECI[S o ANTlUlNS

I
1_
H ANTIGENS
Phase I Phase II
-----
A S. paralyphi A 1,2, 12 a
B S. paralyphi B' 1,4,5, 12 b 1,2

S. (I'phi /III/rill/II I 1,2
c S. paralyphi C : 6,7. Vi' c 1,5

S. ch("rrae·,mis 6 c 1,5
1 ,7
D S. {,lIjerilidis , 1.9, 12 g, m
S.lytihi 9,12, Vi d
E
-+
S. se'l/iellberg'
-_.I, 3, 19
1 g, s, t
• Vi antigen was formerly thought to confer virulence on S. Iyphi (acting somewhat like a
capsule). It is now knoWn, to occur regularly in several other virulent Enterobacteriaceae.
It also occurs in some rionvirulent forms (so confusing!). Vi stands for virulence.
. Names of Salmonella. Many antigenic types of Salmonella closely re-
sembling the major species named above, differing perhaps in only one
antigenic component, or one biochemical character, have been given species
names. Some of these nam~s are derived from the place where the organism
was found, as, for example, "senftenberg," "newport," "kentucky" and "miss-
issippi." There is a tendency now to omit such names and use simply antigenic
formulas of the various serotypes.
Variation of Species. A confusing feature of this system is the fact that,
by various procedures, including transduction, one type may readily be
changed into another! Undoubtedly such changes also occur in Nature. New
types are constantly being found.
Salmonellosis. Salmonella, like all of the Enterobacteriaceae, are trans-
mitted by feces or urine, or both, of patients or carriers. The most common
vector for any or all of them is soiled hands. Another is food which has become
infected and allowed to stand in a warm place, after little or no cooking, so
that the organisms can grow. These organisms grow well at warm room
0 0
temperatures (65 to 100 F). Infection of the food is sometimes by unwashed
hands of a very mild case, or human carrier.
In,fection of food may be by introduction of excrement of dogs, mice or
rats which harbor, particularly, S. typhi murium as well as other salmonellas.
So-called "meat-poisoning" often results from eating or handling raw or
improperly cooked flesh of cattle, swine, poultry, fish, or other animals
suffering from infection with these organisms, especially S. cholerae-suis and
S. enteritidis. These species are among the most commonly-occurring in this
country. Animals of many sorts harbor a variety of Salmonella species. Even
hens' eggs are often infected, before being laid, by maturation in an infected
hen. The eating of raw eggs or egg products is therefore not wise, and has
frequently resulted in large outbreaks due to mayonnaise in sandwiches, etc.
Obviously, avoidance of these diseases means cleanliness in the kitchen;
sanitary habits on the part of food handlers; care to see that food is properly
cooked to kill all organisms, even those in the center of large masses; proper
refrigeration of stored food; and avoidance of uncooked foods at club suppers
or on picnics, often prepared during the morning or previous evening and
then unwittingly incubated in the kitchen or in transit. Salmonella food in-
fections are very common. Characteristically the onset is at least· 12 (usually
18 to 24) hours after eating,the infected 'food. The bacteria multiply during
this "incubation period."
TYPHOID VACCINATION. Probably many of my readers will have received
typhoid "shots" at some time before studying microbiology, possibly on
entering the armed services. These injections are a good example of a method
of active artificial immunization. The m~terial u_sually injected is saline solu-
tion containing about one billion Salmonella typhi per ml killed by heating
at about 65 0 C for 30 minutes, or with formaldehyde. Often included, also,
are killed S. paratyphi A and S. paratyphi B.
For initial immunization, 3 doses (0.5 ml, and 1.0 ml) at weekly intervals
are required. Revaccination with small doses (0.1 ml intracutaneously or
O.S ml subcutaneously) annually is recommended in order to maintain im-
munity at an effective level. This is a good example of the use of the secondary
antigenic stimulus or "booster dose" (see Chapter 21).
GENUS SHIGELLA AND BACILLARY DYSENTERY
The organism discovered by Shiga in 1896 during a frightful epidemic of
dysentery in Japan, with over 22,000 fatalities, is now called Shigella dysen-
teriae. It is type species of the genus. The principal distinguishing charac-
teristics of the genus are shown in Table 24. After Shiga's discovery many
other kinds ("species") of dysentery bacilli were discovered by Flexner,
Boyd, Sonne and others. The classification and differentiation of these
species present problems analogous to those related to classification of the
salmonellas (Tables 26 and 27).
Bacillary Dysentery. The shigellas cause intestinal disturbances ranging
from very mild diarrhea to severe and sometimes fatal dysentery with intense
inflammation and ulceration of the large bowel, often with scar formation
and stricture of the bowel after recovery. Unlike Salmonella typhi,- which
always causes bacteriemia, Shigella do not commonly invade the blood
stream. In some epidemics of hacillary dysentery, especially those due to
S. dysenteriae, the fatality rate is high. .
The transmission and prevention of bacillary dysentery are similar to those
aspects of salmonellosis, except that animals do not commonly transmit
dysentery.
DYSENTERY VACCINATION. Vaccination against bacillary dysentery ap-
pears to be much less satisfactory than typhoid vaccination.
PATHOGENIC ESCHERICHIA
If given a large enough dosage and sufficient opportunity, such as a very
dirty wound or an old, slowly healing ulcer, Escherichia coli may at times
cause disease, especially if the patient's general health and non-specific resis-
tance are low. Escherichia coli may ca'Ise more serious trouble by invading
the bladder and pelvis of the kidney, where it produces a chronic and often
very stubborn inflammation. In the bladder this is called cystitis; in the
pelvis of the kidney, pyelitis.
Extensive studies of bacteria in the feces of infants with diarrhea indicate
that many cases of infantile diarrhea are due to certain particular kinds of
Escherichia coli. These pathogenic E. coli can be distinguished only by im-
munologic studies of their antigenic structure. Some of these strains of E.
coli are designated as 026:B6, 0111 :B4, 055:B5, 01l9:B14, 0127:B8, 086:B7,
and so on. The numbers and letters refer to antigens in the organisms. There
are ten or more such strains known.
These organisms are particular nuisances in children's institutions and
nurseries. They are spread about by hands and fomites, as are other enteric
pathogens, and at timesI
are very difficult to eradicate.
PARACOLONS AS PATHOGENS
Some especially de~ignated species or groups of the Paracolobactrum are
regarded by some authorities as possible causes' of diarrheal conditions in
human beings, much as are certain specific types of Escherichia coli. Groups
of Paracolobactrum possibly so implicated are the "Arizona group," the
"Bethesda-Ballerup group," the "Providence group," the "32011 group," and
so on. The Providerlce and Bethesda-Ballerup groups comprise mainly P.
intermedium-like species, the 32011 group P. aerogenoides species.
Some of the Paracolobactrum contain antigens found in Salmonella,
Escherichia and Shigella. In fact, all of the genera in the whole grand family
of Enterobacteriaceae are related more or less to one another by the posses-
sion of antigens common to one or another species.
THE PARVOBACfERIACEAE
The family Parvobacteriaceae ("little bacteria") was established to provide
a convenient grouping for a number of species of true bacteria which are
relatively small (O.3~ by 2~), gram-negative, non-sporeforrning, non-motile
parasitic, mostly aerobic rods. As a group, they represent the highly evolved
type of bacteria, requiring complex organic media for their optimum growth.
In general they lack the synthetic versatility and the ruggedness necessary to
growth and survival in the outer world, which characterize such organisms
as the Aerobacter and Pseudomonas. No saprophytic species of any genus
in this family is ~nown. All appear to cause disease.
GENUS PASTEURELLA
Pasteurella are named for Pasteur who founded the science of immunology
on his studies of vaccination against Pasteurella avicida, the cause of fowl
cholera. P. avicida is now regarded as a variant of the type species of the
genus, Past. multocida. Other variants of P. multocida have previously been
named for animals: P. bovicida (cattle); P. suilla (swine); P. muricida (rats);
etc. They differ only slightly in biochemical properties. They are highly patho-
genic for most birds and animals; much less so for man. They cause tre-
mendous losses to stock and poultry industries (hemorrhagic septicemia,
"shipping fever"). The species causing bubonic plague in man is P. pestis.
THE MORPHOLOGY OF PASTEURELLA is rather distinctive. In pathological
material the organisms are short, oval rods, about 0.5 J.L by 3 J.L ,which tend to
stain most heavily at the tips (bipolar staining). In cultures the bipolar ap-
pearance is often less definite.
SURVIVAL OF PASTEURELLA. Pasteurella succumb readily to heat and
disinfectants, but resist drying in dust to some extent. For example, Pasteu-
rella multocida can survive in, and is transmitted by, infectious dust, fomites
and animal secretions from stables, railroad cars, stockyards, etc. The organ-
isms invade the lymphatic system and blood stream and may· easily be
cultivated on infusion media from all of the organs and body fluids of infected
animals. There are many small hemorrhages on various internal mucous sur-
faces, in the skin, and in the internal organs, hence the name "hemorrhagic
septicemia." In the animal disease, there is much exudation of fluid from nose,
mouth, eyes, etc. This fluid is highly infectious and transmits the disease in
flock, barnyard and stable. The disease'is rapidly.fatal as a rule.
CONTROL. These epizootics can be controlled only by rigid isolation and
slaughter of sick animals followed by prompt incineration or burial, and dis-
infection of the premises. 'Fhere are bacterins which are of some immunizing
value. Shipping fever may be reduced by passive immunization with serum,
given just before shipping.
fasteurella Pestis. from the standpoint of human disease the most im-
portant member of the genus is Past. pestis, the cause of bubonic and pneumo-
nic plague in man. Morphologically and culturally P. pestis resembles
P. multocida but is slower and less vigorous in its growth and somewhat less
active biochemically. In general, these organisms attack no proteins and few
carbohydrates beyond glucose.
BUBONIC PLAGUE. This malady is a classical example of a bacterial disease
transmitted by the bite of an insect. It is primarily a disease of rodents. It is
conveyed to human beings by the bite of infected fleas, most important of
which, in this respect, are Xenopsylla cheopis and Ceratophyllus jasciatus, the
rat fleas. The fleas usually derive the plague bacilli from the blood of infected
rats: Mils norvegicus (sewer rat) and Mus rattus (house rat). When the rats are
infected they show lesions similar to those found in man. Rats and rat fieas
maintain plague as an epizootic disease much like hemorrhagic septicemia
among themselves for long periods and act, therefore, as an animal reservoir
of plague bacilli. When rats become excessively prevalent in any community,
human plague is apt to occur because the opportunity for rat fleas to bite
human beings greatly increases. Crowded populations living near dumps or
in dirty, unsanitary conditions suffer most. Conditions following the devas-
tation of war, with breakdown of disease-control systems, are ideal for the
development ofrats and, therefore, ofrat-borne diseases. The pages of history
are filled with disasters to armies and civil populations attacked by plague.
Daniel Defoe's "Journal of the Plague Year" and Winsor's "Forever Amber"
give dramatic descriptions of the plague in London, 1665. The rats often die in
great numbers from the disease and the fleas tend to leave the cooling bodies,
jumping on to the first warm animal which passes. Dead or dying rats are,
therefore, potentially dangerous.
In 1900 plague was first found in human beings in this country. It has since
been found in rats and in rodents other than rats, especially ground squirrels
or "prairie dogs." Many human cases have been traced to contact with wild
rodents. The disease in-woodland or wild-living creatures is often spoken of as
sylvatic (forest) or campestral (prairies) plague. The control of plague in wild
rodents, etc., is a field problem of great importance.
Pasteurella Tularensis. This organism much resembles P. pestis and P.
multocida but is somewhat more exacting in its nutritional requirements.
Infusion media with blood or serum are used, but little or no growth occurs
unless the amino acid, cystine, or (what amounts to the same thing) some
compound containing the sulfhydryl (-SH) group is added.
P. tularensis is found in much the same ecological relationship to rodents
(rabbits, gophers, mice), biting insects (wood, dog and rabbit ticks; rabbit
lice; deer flies; hors~ flies) and man as are P. pestis, fleas, and rodents. P.
tularensis causes a disease called tularemia. (The name is derived from the
Tulare swamps in California where early observations were made on this
disease.)
TULAREMIA ("RABBIT FEVER"). This is a plague-like disease in many
American .rodents and other wild animals. As in plague and hemorrhagic
septicemia, there are! enlargements of lymph nodes, swelling of the spleen,
and the appearance of tubercle-like nodules in spleen, liver and elsewhere.
The bacilli invade the blood stream from these foci just as P. pestis does in
plague.
One common meahs of transmission of tularemia is through the handling
of infected wild rabbits, as in the marketing of these animals for food and
pelts, hence "rabbit fever." In some sections of the country the disease is
known as "Deer fly fever," being transmitted largely by the deer fly, Chrysops
discalis. In Arkansas and adjacent regions the disease is largely tick-borne.
It causes enormous losses among sheep if they graze in areas where there is
tick-infested undergrowth. P. tularensis has been isolated from forest streams.
The water is apparently infected by the carcases of infected wild animals
dying in the stream or on its nearby banks.
The term pasteurellosis is properly applicable to infections with Pasteurella.
GENUS BRUCELLA
Brucella melitensis and two closely related species, Br. abortus and Br.
suis, cause Malta/ever or undulant/ever in man, a disease common all over the
world. The generic name of the causative organisms is derived from the dis-
c')verer, Bruce, a British scientist who first (1887) found the organism now
called Br. melitensis on the island of Malta * in the spleens of persons infected
by the organisms in goats' milk.
Br. abortus was first known as the cause of abortion in farm animals,
especially cattle. It was discovered by a Danish worker, Bang, in 1895 and is
still often called Bang's bacillus and the disease in cattle, Bang's disease.
Br. suis, commonly found in swine, was first observed by Traum in the
United States in 1914. Because of the frequent presence of these organisms
in the blood and tissues of farm animals, persons in contact with them are
likely to become infected. Thus meat packers, cattlemen, hog raisers, persons
who drink un-Certified or unpasteurized milk, bacteriologists and veteri-O
narians most frequently contact the disease.
The fact that the organisms discovered in 1887 in Malta, in 1895 in Den-
mark and in 1914 in America are all closely similar species of one genus was
revealed in 1918 by Evans in the United States.
Isolation. Brucellas are very small rods without distinctive morphological
features. They grow rather slowly on first isolation from the blood, milk or
tissues of infected animals, or from the blood of man. They are best cultivated
on slightly acid (pH 6.8) liver-infusion agar, or on tryptose or trypticase-soy
agar or broth. Excellent examples of selective-type media based on antibiotics
have been devised to facilitate cultivation of these organisms from contami-
nated materials such as feces and milk. Polymyxin B, penicillin, Actidione
and crystal violet are the inhibitory agents, permitting Brucella to grow.
Brucella can also be isolated from blood by injecting it into living chick
embryos. Brucella abortus will grow at first only in an atmosphere containing
about 10 per cent CO2 , The three species are closely similar but may be
/ distinguished by special tests. , '
Survival and Distribution. . They can' survive for considerable periods in
dairy products, water, soil, dung, dust and meats, and are transmitted by
these vectors. The tissues and fluids associated with an aborted animal are
highly infectious.
The three species, though originally associated with certain animals, are
not restricted to those animals but each may occur in any of the three species
mentioned, as well as in man, dogs, horses, and possibly poultry.
i •
• Malta was called Melita by the ancients because of the fine honey (Latin = mel) found
there; hence, Melit-ensis.
GENERA HEMOPIDLUS, BORDETELLA, MORAXELLA
Originally all of these organisms were included in the genus Hemophilus.
This is named for the fact that the organisms were all thought to require
hlood * for growth. It is now clear that some do not. These have been placed in
the genera Bordetella and Moraxella. All are morphologically similar small
rods though they are pleomorphic and often vary from coccoid to long,
filamentous or distorted forms.
The genus Hemophilus includes H. inJluenzaet and several other pathogenic
species, notably H. ducreyi, cause of the venereal disease, chancroid ("soft
chancre").
The hemophils are excellent examples of the highly adapted and dependent
bacterial parasite. These cannot live without certain blood components. One
of these, long known as the "X factor," is the iron complex called heme,
which is part of the red coloring matter of erythrocytes. Besides heme,
Hemophilus require for growth a "V factor," now identified as coenzyme I,~
found in erythrocytes.
Bordetella. This genus includes B. pertussis, the cause of whooping cough,
discovered by a Belgian scientist, Bordet. On initial isolation from patients
these organisms are truly and rigidly hemophilic, but on subculture soon
become adapted to growth without either X or V factor. They do, however,
require nicotinic acid (Niacin), a part of the coenzyme molecule.
Moraxella are very similar organisms which cause infectious conjunctivitis
("pink eye").
S~MMARY OF ROD-SHAPED BACTERIA
Since all rods in this book except nitrogen bacteria (next chapter) have now
been discussed, an inclusive list of rods is given at this point for the conve-
nience of the student. This is not a classification. It is merely a systematized
collection of data.
Table 28. Rod-shaped Bacteria of the Sub-order Eubacteriineae Grouped on

the Basis of 5 Principal Characteristics: Form, Sporulation, Gram-
reaction, Motility, Flagellation.§
T. STRAIGHT RODS
A. Non-sporeforming
I. gram-positive
a. Motile
(I) Flagella, polar. ................. none known
(2) Flagella, peritrichous ............ Listeria
b. Non-motile
(aj ................................ Lactobacilleae
(b} ............................... Corynebacteriaceae
, (exc. motile Listeria)
• Herrw is from the Greh for blood; phi/us from the Greek for liking or requiring.
t Formerly thought to cause influenza. Influenza is now known to be due to viruses.
t Phosphopyridine nucleotide. This is part of an important respiratory enzyme.
§ Exceptions occur in most groups. Some of the exceptions are listed. Others are unknown
or are due to "spontaneohs" variations and therefore unpredictable. The ill-defined and
heterogeneous group listed in "Bergey's Manual" under B8cteriaceae is not included.

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~ Mabu.

MARTIN FROBISHER, Se.D.

W. B. SAUNDERS COMPANY, Philadelphia and London

CHARLES E. TUTTLE COMPANY, Tokyo

© 1957, by W. B. Saunders Company

All rights reserved

No part of this book may be reproduced in any

First printing (Modern Asia Edition), 1961

MARTIN FROBISHER, JR.

SECTION 1. THE RELATIONSHIPS OF MICROORGANISMS TO

SECTION 2. METHODS AND PHENOMENA OF MICROBIOLOGY

OPTICAL METHODS IN MICROBIOLOGY.......................... 101

MORPHOLOGY AND STRUCTURE OF BACTERIA. . . . . . . . . . . . . . . .. 120

CLASSIFICATION AND TAXONOMY OF BACTERIA ............... 140

NUTRITION, METABOLlSM, AND CULTIVATION OF MICROOR-

DESTRUCTION, REMOVAL AND INHIBITION OF MICROORGAN-

SECTION 3. IMMUNOLOGY: REACTION OF THE ORGANISM TO

MICROORGANISMS AND DISEASE. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 343

SEcrION 4. THE BACTERIA. (CLASS SCHIZOMYCETES)

AnaerobiOSIS ..................................................... 420

Bloo:~~~!:i~~~e~: ~~: ............ : ....................... ..\ 446

MICROBIOLOGY;AND INDUSTRY ................................. 556

SOME VIRAL DISEASES .................................. , ......... 575

INDEX ............................................................. 589

The Microscopic World

1. PROTOZOA, ALGAE, FUNGI AND BACTERIA

THE WORLD of microorganisms must not be thought of as sharply differ-

ological function without increase in number of cells. Thus, in many free-

Fig. 1-2. Various forms of protozoa. A, B, forms of amoeba. C, D, forms of trypano-

Fig. 1-3.' Synechococcus, a blue-

Fig. 1-4. Various forms of bacteria,

Fig. 1- 5. Fig. 1-6.

The Microscopic World

Fig. 2-4. Leeuweohoek's drawings of ~eria.

Fig. 2-5. Pasteur in his laboratory.

of his best scientific achievements by his patriotic zeal. In his boyhood he

Fig 2--{j. Two of Pasteur's flasks,

Fig. 2-7. First illustration of ampu-

ism from a mixture of microorganisms. The preparation of such "pure cul-

DEVEWPMENT OF STAINING METHODS

F .. 2-10. Koch's photograph of an-

bacteria which are particularly difficult to see under ordinary conditions.

The Microscopic World

3. YEASTS AND MOLDS

Fig. 3- 1. Blocks of wood taken from

t Causing decomposition of dead organic matter. Generally harmless.

Table 2. Classification of Fungi

Fig. 3-2. Yeast (Saccharomyces cere-

Fig. 3-5. Diagrammatic representation of two types of reproductive cycle in yeast. A to

A SUGGESTED SYSTEMATIC RELATIONSHIP OF SOME MOLDS AND YEAST-

Endomyces, a typical illamentous mold. --Candida. a yeast-like. dimorphic fungus.

Table 3. A Classification of Yeasts.

The Microscopic World

Fig. 4-1. Oidium formation. Seg-

• Oidium is from a Greek root meaning "egg," i.e., an ovoid body.

Fig. 4-2. A dimorphic, yeast-like.

• Angium is from a Greek root meaning bag or envelope; hence, Sporangium.

• An excellent mounting fluid is lactophenol-colton blue solution, made as follows:

Fig. 4-6. Zygospore formation (sexual reproduction) by Rhizopus. A, two swollen

and in a living, undisturbed condition. This slide may be placed in a covered

Bloo:~!:i~e~: ~~: ............ : ....................... ..\ 446