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Full text of "Pathogenic micro-organisms; a text-book of microbiology for physicians and students of medicine"

MEDICAL 




Gift of 



Panama-Pacific Intern 
Exposition Company 



PATHOGENIC MICRO-ORGANISMS 



M AC N E A L 



PATHOGENIC 
MICRO-ORGANISMS 

A TEXT-BOOK OF 

MICROBIOLOGY FOR PHYSICIANS 
AND STUDENTS OF MEDICINE 



BY 

WARD J. IV^ACNEAL, PH. D., M. D., 

PROFESSOR OF PATHOLOGY AND BACTERIOLOGY IN THE NEW YORK 
POST-GRADUATE MEDICAL SCHOOL AND HOSPITAL, NEW YORK 



(Based Upon Williams' Bacteriology) 



WITH 213 ILLUSTRATIONS 



PHILADELPHIA 

P. BLAKISTON'S SON & CO. 

1012 WALNUT STREET 
1914 



COMPLIMENTS 
OF 



sion's Son Co. 






COPYRIGHT, 1914, BY P. BLAKISTON'S SON & Co. 



THE- MAPLE- PRESS. YORK. PA 



PUBLISHER'S ANNOUNCEMENT 

When Professor H. U. Williams was requested to undertake 
the sixth revision of this book he expressed a wish to be relieved 
of it and of all further obligations in respect thereto because oi 
the demands that were being made upon his time by largei 
duties in connection with the University. 

As this manual is a popular one we did not feel warranted in 
letting it go out of print as suggested by Dr. Williams, therefore 
we arranged with Professor Ward J. MacNeal to continue it with 
the approval of Professor Williams. The wisdom of this choice 
should be evident in the following pages. 



100 



PREFACE. 

This volume is the outgrowth of an attempt to revise the well- 
known William's Manual of Bacteriology, undertaken at the invi- 
tation of the Publishers, Messrs. P. Blakiston's Son and Co., very 
cordially seconded by Dr. Williams, who kindly placed the mate- 
rial of the previous editions at my disposal. The text has been 
very largely rewritten and the order of treatment considerably 
altered. Many of the illustrations of Dr. Williams have been 
retained and, as they have not been acknowledged in the legends, I 
wish to express my special obligation for them in this place. 

The book is intended as an introduction to the study of patho- 
genic micro-organisms and is designed especially for the use of 
physicians and students of medicine. During the past decade, 
the parasitic protozoa have assumed an importance which places 
them almost on a par with the bacteria as pathogenic agents, and 
the extension of bacteriological methods to the study of molds, 
yeasts, filterable viruses and protozoa has tended again to re- 
unite the various portions of this field of knowledge, much as it 
was in the days of Pasteur. The attempt has here been made to 
outline the subject and to present a few examples under each 
important heading, in the hope that the student may become 
acquainted with the broad principles of the science and appreciate 
the variety of procedures, conceptions and organisms with which 
it deals. Part I is devoted to a description of technical procedures, 
Part II to the general biology of micro-organisms and Part III 
to a consideration of individual microbes. Much has of necessity 
been omitted and many topics treated only very briefly. 

In the preparation of the manuscript considerable use has 
been made of the text-books of McFarland, Jordan, Marshall, 
and of Hiss and Zinsser, and the Handbuch der pathogenen Mikro- 
organismen of Kolle and Wassermann and Doflein's Lehrbuch der 

vii 



Vlll PREFACE 

Protozoenkunde have been most extensively employed. Numer- 
ous illustrations have been copied from the last mentioned work 
and the text itself has been closely followed in many places. 

The attempt has been made to give proper credit for borrowed 
illustrations, but the numerous cuts retained from the previous 
editions of Williams have not been specially designated. Some 
references have been included, to offer the student a ready intro- 
duction to the literature of the topic under discussion, especially 
in those instances in which the topic has been only briefly men- 
tioned here. 

My thanks are due to the Press of Gustav Fischer, Jena, for 
the loan of numerous illustrations, to P. Blakiston's Son and 
Company for their uniform courtesy in our relations, and espe- 
cially to my wife whose enthusiastic assistance has made possible 
the preparation of the manuscript and has lightened the burden 
of seeing it through the press. 

W. J. MACNEAL. 
NEW YORK 
December, 1913. 



TABLE OF CONTENTS. 

INTRODUCTION. 

Bacteriology and Microbiologys, i ; Biological relationship, 3 ; Spontaneous genera- 
tion, 3; Heterogenesis, 4; Systematic relationships, 4; Fermentation and Putre- 
faction, 5; Specific fermentations, 6; Pathology and Hygiene, 7; Contagion, 8; 
Specific infection, 10; Antisepsis, n; Proof of the germ theory, n; Immunity, 
12; Parasitic protozoa, 12; Insect transmission, 13; Pathogenic spirochetes, 13; 
Filterable viruses, 13; Agriculture, 14; Biological view-point in the study of 
micro-organisms, 14. 

PART I. BACTERIOLOGICAL TECHNIC. 
CHAPTER I. THE MICROSCOPE AND MICROSCOPIC METHODS. 

Development of the microscope, 15; Lenses, 15; Achromatic and apochromatic 
objectives, 15; Ultra- microscope and dark-field microscopy, 16; Tandem micro- 
scope, 16; Principle of the microscope, 16; Pin-point aperture, 16; Relations of 
magnification, definition and brilliancy of image, 16; Lens-armed aperture, 17; 
Two lenses in series, 18; Magnification measured by the ratio of the opening 
and closing angles of a beam, 19; Simple microscope, 19; Reading glass, 19; 
Spherical aberration, 20; Chromatic aberration, 20; Diffraction, 20; Image 
formation in compound microscope, 22; Numerical aperture, 23; Illumination 
by the Abbe condenser, 24; Central illumination, 24; Dark-field, 25; Illumina- 
tion by broad converging beam, 25; Visibility of microscopic objects, 25 ; Defini- 
tion by light and shade, 26; The color picture, 28; The Bacteriological micro- 
scope, 29 ; Eye-pieces and objectives, 30; Use of the microscope, 31; Microscopic 
measurements, 31; The platinum wire, 31 ; Pasteur pipettes, 32 ; The hanging- 
drop, 33 ; Motility of micro-organisms, 34; Brownian motion, 34; Hanging- 
block, 35 ; Slide for dark-field study, 35 ; Use of dark-field, 36; Smear prepara- 
tions, 36 ; Cover-glasses, 36; Technic, 37; Slide smears, 39; Staining solutions, 39 ; 
Aniline stains, 40; Method of simple staining, 44; Gram's stain, 44; Acid-proof 
staining, 46; Sputum staining, 47; Spore staining, 50; Capsule stain, 51 ; Stain- 
ing of flagella, 52 ; Wet fixation, 54 ; Iron hematoxylin, 54 ; Blood films, 54 ; 
Staining of tissue sections, 55; Celloidin, 55; Paraffin, 55; Sectioning, 56; 
Simple staining, 58; Gram-Weigert method, 59; Tubercle bacilli, 60; Nuclear 
stains, 61. 

ix 



X TABLE OF CONTENTS 

CHAPTER II. STERILIZATION, DISINFECTION, ANTISEPSIS, FOOD 
PRESERVATION. 

Definitions, 62 ; Physical sterilization, 62 ; Mechanical removal, 62; Desiccation, 63; 
Light, 63; Cold, 64; Heat, 64; Electricity, 71; Chemical sterilization, 71 ; Soaps, 
71; Acids, 71; Alkalies, 73; Oxidizing agents, 73; Inorganic salts, 74; Organic 
poisons, 76; Antiseptics and preservatives, 79; Physical, 79; Chemical, 79; 
Testing of antiseptics and disinfectants, 80. 

CHAPTER III. CULTURE MEDIA. 

Definition, 83 ; Glass-ware, 83 ; The common media, 84 ; Nutrient broth, 84; Titra- 
tion of media, 85; Gelatin, 88; Agar, 89; Modifications, 90; Sterilizable special 
media, 91 ; Potato, 91; Milk, 92; Peptone solution, 92; Nitrate broth, 92; Blood- 
serum, 92; Loeffler's blood-serum, 94; Eggs, 94; Dorset's egg, 94; Bread paste, 
94; Media containing uncooked protein, 95; Sterile blood, 95; Ascitic fluid, 97; 
Sterilization, 97; Sterile tissue, 98; Blood-streaked agar, 98; Blood-agar, 98; 
Broth containing tissues, 99; Ascitic-fluid agar, 99; Ascitic fluid with tissue, 99. 

CHAPTER IV. COLLECTION OF MATERIAL FOR BACTERIOLOGICAL 

STUDY. 

General considerations, 100; Sampling water and foods, 100; Material from the 
body, 100 ; Sputum, 101; Urine, 101; Blood and transudates, 101; Cerebro-spinal 
fluid. 101; Feces and intestinal juice, 102; Pus and exudates, 102; Material from 
autopsies, 103. 

CHAPTER V. THE CULTIVATION OF MICRO-ORGANISMS. 

Avoidance of contamination, 104; Isolation of bacteria, 105; Plate cultures, 106; 
Roll tubes, no; Streak method, 112; Tall-tube method, 112; Colonies, 113; 
Pure cultures, 113; Stock cultures, 114; Regulation of temperature, 115; High 
temperature incubator, 115; Gas-regulator, 116; Automatic safety-burner, 120; 
Incubator room, 120; Prevention of drying, 120; Low-temperature incubator, 
121; Cultivation of anaerobic bacteria, 124; Deep stab, 124; Veillon tall-tube 
method, 124; Fermentation tube, 125; Removal of oxygen, 125; Hydrogen at- 
mosphere, 126; Further methods, 130. 

CHAPTER VI. METHODS OF ANIMAL EXPERIMENTATION. 

Value of Animal experimentation, 131 ; Care of animals, 131 ; Holding for operation, 
132; Inoculation, 133; Subcutaneous and intraperitoneal, 133; Intracranial, 
133; Into circulating blood, 133; Other sites, 134; Subcutaneous application, 
134; Alimentary and respiratory infection, 134; Collodion capsules, 134; Obser- 
vation of infected animals, 136. 



TABLE OF CONTENTS XI 



PART II. GENERAL BIOLOGY OF MICRO-ORGANISMS. 

CHAPTER VII. MORPHOLOGY AND CLASSIFICATION. 

Molds, 137 ; Yeasts, 140 ; Bacteria, 141 ; Trichobacteria, 141; Spherical bacteria, 142; 
Cylindrical bacteria, 144; Spiral bacteria, 146; Structure of the lower bacteria, 
147; Endospores, 149; Filterable viruses, 150; Protozoa, 150; Flagellates, 151; 
Rhizopods, 154; Sporozoa, 155; Ciliates, 159; Outline classification of micro- 
organisms, 160 ; Specific nomenclature, 160. 

CHAPTER VIII. PHYSIOLOGY or MICRO-ORGANISMS. 

Relations of morphology and physiology, 162 ; Conditions of physiological study, 163 ; 
Environmental factors, 164; Moisture, 164; Organic food, 164; Inorganic salts 
and chemical reaction, 165; Oxygen, 166; Temperature, 166; Microbic variation, 
167; Products of microbic growth, 168; Physical effects, 168; Chemical effects, 
168; Enzymes, 169; Toxins, 171; Relation of microbe and its environment, 171 ; 
Morphological characters, 171; Physiological tests, 173; Descriptive chart of 
the Society of American Bacteriologists, 173. 

CHAPTER IX. THE DISTRIBUTION or MICRO-ORGANISMS AND THEIR 
RELATION TO SPECIAL HABITATS. 

General distribution, 174; Micro-organisms of the Soil, 175; Pathogenic soil 
bacteria, 176; Micro-organisms of the air, 176; Micro-organisms of Water and 
Ice, 178; Self-purification of water, 179; Storage of water, 180; Filtration, 180; 
Disinfection of water, 182; Bacteriological examination of water, 182; Detec- 
tion of intestinal bacteria, 186; Bacteriological examination of ice, 188; Micro- 
organisms of food, 189; Milk, 189; Milk flora, 190; Pathogenic microbes in 
milk, 192; Milk for infant feeding, 192; Other foods, 193. 

CHAPTER X. PARASITISM AND PATHOGENESIS. 

The parasitic relation, 194; Pathogenesis, 195; Rules of Koch, 195; Infectious 
disease, 196; Possibility of infection, 196; Susceptibility and resistance, 196; 
Number of invaders, 197; Modes of introduction, 197; Local susceptibility, 199; 
Local and general infections, 199; Transmission of infection, 200; Healthy 
carriers of infection, 201. 

CHAPTER XI. THE PATHOGENIC PROPERTY or MICRO-ORGANISMS. 

Adaptation to parasitism, 202 ; Virulence, 202 ; Microbic poisons, 203 ; Defensive 
mechanisms, 204. 



Xll TABLE OF CONTENTS 

CHAPTER XII. REACTION or THE HOST TO INFECTION. 

Facts and theories, 206 ; Physiological hyperplasia, 206 ; Phagocytosis and encapsu- 
lation, 207; Chemical constitution of the cell, 207; Antitoxins, 208; Cell 
receptor of first order, 209; Precipitins, 209; Receptor of second order, 210; 
Agglutinins, 211; Phenomenon of agglutination, 211; Bactericidal substances, 
212; Cytolysins, 213; Receptor of third order, 214; Amboceptor and comple- 
ment, 214; Deviation of complement, 215; Fixation of complement, 216; 
Opsonins, 217; Anti-aggressins, 218; Source and distribution of antibodies, 
218; Allergy, 219. 

CHAPTER XIII. IMMUNITY AND HYPERSUSCEPTIBILITY. THEORIES 

or IMMUNITY. 

Immunity, 220; Natural immunity, 220; Immunity of species, 220. Racial im- 
munity, 221; Individual variations, 221; Acquired immunity, 222; Active 
immunity, 222. Passive immunity, 224. Combined active and passive 
immunity, 225 ; Mechanisms of immunity, 225 ; Hypersusceptibility or Ana- 
phylaxis, 226 ; Theories of immunity, 227. 

PART III. SPECIFIC MICRO-ORGANISMS. 

CHAPTER XIV. THE MOLDS AND YEASTS AND DISEASES CAUSED BY 

THEM. 

Mucors, 231; Aspergilli, 233; Penicillium crustaceum, 234; Claviceps purpurea, 
234; Ergotism, 235; Botrytis bassiana, 235; Muscardine, 236; Oidium lactis, 
236; Oidium albicans, 238; Thrush, 238; Achorion schoenleinii, 239; Favus, 
239; Microsporon audouini, 241; Alopecia areata, 241; Microsporon furfur, 
242 ; Tricophyton acuminatum, 242 ; Sporotrichum schenki, 242 ; Sporotri- 
chum beurmanni, 244; Saccharomyces cerevisiae, 244; Blastomycetic derma- 
titis, 244 ; Coccidioidal granuloma, 245. 

CHAPTER XV. TRICHOMYCETES. 

Actinomyces bovis, 246; Streptothrax madurae, 248; Mycetoma, 248; Cladothrix, 
249 ; Leptothrix buccalis, 249. 

CHAPTER XVI. THE COCCACE^E AND THEIR PARASITIC 
RELATIONSHIPS. 

Diplococcus gonorrheae, 250; Occurrence, 250., Culture, 250; Toxins, 253; Gonor- 
rhea, 252; Specific diagnosis, 253; Prophylaxis, 253; Diplococcus meningi- 
tidis, 253; Anti-meningococcus serum, 255; Quincke's puncture, 255; Exami- 



TABLE OF CONTENTS Xlll 

nation of spinal fluid, 256; Diagnosis, 257; Diplococcus catarrhalis, 257; 
Diplococcus pneumonias, 257; Occurrence, 257; Morphology, 258; Cultures, 
258; Pneumonia, 259; Toxins, 259; Immunity, 260; Streptococcus viridans, 
260 ; Streptococcus mucosus, 260 ; Streptococcus pyogenes, 260 ; Occurrence, 
261; Cultures, 261; Animal inoculation, 262; Surgical infections, 263; Erysip- 
elas, 263; Puerperal fever, 263; Immunity, 264; Streptococcus lacticus, 264 ; 
Staphylococcus aureus, 264; Occurrence, 264. Morphology, 264; Cultures, 
265; Toxins, 265; Animal inoculation, 266; Infection of man, 266; Immunity, 
266; Vaccine therapy, 266; Staphylococcus alb us, 267; Micrococcus tetra- 
genus, 267; Sarcina ventriculi, 267; Sarcina aurantiaca, 267; Micrococcus 
agilis, 267. 

CHAPTER XVII. B ACTERIACE^E : THE SPOROGENIC AEROBES. 

Bacillus mycoides, 268 ; Bacillus vulgatus, 268 ; Bacillus subtilis, 269 ; Parasitism 
269; Bacillus anthracis, 270; Occurrence, 270; Morphology, 271; Resistance 
272; Anthrax, 272; Human anthrax, 273; Immunity, 273; Seium, 274. 

CHAPTER XVIII. BACTERIACE.E: THE SPOROGENIC ANAEROBES. 

Group characters and habitat, 275 ; Bacillus endematis, 275 ; Putrefactive prop- 
erties, 276; Malignant edema, 276; Bacillus feseri, 276; Bacillus welchii, 
276; Occurrence, 276; Characters, 277; Emphysematous gangrene, 277; Bac- 
illus tetani, 278; Occurrence, 278; Morphology, 278; Cultures, 279; Toxin, 
279; Tetanus, 280; Immunity, 281; Antitoxin, 281; Standard unit, 282; Pro- 
phylaxis and treatment, 283; Bacillus botulinus, 283 ; Botulin, 284; Immune 
serum, 284; Botulism, 284. 

CHAPTER XIX. BACTERIACE^E: THE BACILLUS or DIPHTHERIA AND 

OTHER SPECIFIC BACILLI PARASITIC ON SUPERFICIAL Mucous 

MEMBRANES. 

Bacillus diphtherias, 285; Occurrence, 285; Culture, 285; Toxin, 288; Diphtheria, 
289; Bacteriological diagnosis, 290; Transmission of the disease, 292; Immu- 
nity, 292; Antitoxin, 293; Standard unit of antitoxin, 294; Prophylactic and 
therapeutic use of antitoxic serum, 295; Untoward effects, 295; Bacillus 
xerosis, 295; Bacillus hoffmanni, 296; Morax-Axenfeld bacillus, 296; Koch- 
Weeks bacillus, 296 ; Bacillus pertussis, 296 ; Bacillus influenzas, 297 ; Bac- 
illus chancri, 298. 

CHAPTER XX. BACTERIACE.E: THE TUBERCLE BACILLUS AND OTHER 
ACID-PROOF BACTERIA. 

Bacillus tuberculosis, 299; Human type, 300; Occurrence, 300; Morphology, 300; 
Cultures, 301; Chemical composition, 302; Toxins, 303; Resistance, 304; Tuber- 



XIV TABLE OF CONTENTS 

culin, 304; Animal inoculation, 305; Tuberculosis, 305; The Tubercle, 306; 
Mode of transmission, 307; Bacteriological diagnosis, 307; Allergic reactions, 
308; Bovine type, 310; Avian type, 311; Fish or amphibian type, 312; Bac- 
illus leprae, 312; Morphology and occurrence, 312; Leprosy, 313; Bacillus 
smegmatis, 313; Bacillus moelleri, 314; Other acid-proof organisms, 314; 
Pseudo-bacilli, 315. 

CHAPTER XXI. B ACTERIACE^E : THE BACTERIA OF THE HEMOR- 
RHAGIC SEPTICAEMIAS, OF PLAGUE AND OF MALTA FEVER. 

Bacillus avisepticus, 316; Bacillus plurisepticus, 316; Bacillus pestis, 317; Occur- 
rence and morphology, 317; Cultures, 318; Toxins, 318; Animal inoculation, 
319; Bubonic plague, 319; Epizootic plague, 320; Human plague, 320; Immu- 
nity, 320; Immune serum, 321; Prophylaxis, 321; Eradication of endemic 
centers, 321; Bacillus melitensis, 321; Malta fever, 322. 

CHAPTER XXII. BACTERIACE^E: THE COLON, TYPHOID AND 
DYSENTERY BACILLI. 

Bacillus coli, 324; Occurrence and morphology, 324; Cultures, 325; Pathogenic 
relations, 325; Bacillus aerogenes, 326; Bacillus pneumoniae, 327; Bacillus 
rhinoscleromatis, 327; Bacillus enteritidis, 328; Bacillus suipestifer, 329; 
Bacillus psittacosis, 329 ; Bacillus typhi murium, 329 ; Bacillus alkaligenes, 
329; Bacillus typhosus, 330; Occurrence and morphology, 330; Cultures, 331; 
Resistance, 332; Toxins, 332; Animal inoculation, 332; Typhoid fever, 333; 
Bacteriological diagnosis, 333; Transmission of the disease, 334; Prevention, 
335; Bacillus dysenteriae, 336; Epidemic dysentery, 336; Paradysentery 
bacilli, 337. 

CHAPTER XXIII. B ACTERIACE^E : BACILLUS MALLEI AND MISCEL- 
LANEOUS BACILLI. 

Bacillus mallei, 339; Occurrence and morphology, 339; Cultures, 339; Mallein, 
340; Glanders, 340; Bacteriological diagnosis, 340; Bacillus abortus, 341 ; 
Bacillus acne, 342; Bacillus bifidus, 342; Bacillus bulgaricus, 342; Bacillus 
vulgaris, 343; Bacillus pyocyaneus, 343; Bacillus fluorescens, 343; Bacillus 
violaceus, 343; Bacillus cyanogenus, 343; Bacillus prodigiosus, 344. 

CHAPTER XXIV. SPIRILLACE.E AND THE DISEASES CAUSED BY 

THEM. 

Spirillum rubrum, 345 ; Spirillum choleras, 345 ; Occurrence and morphology, 345 ; 
Cultures, 345; Animal inoculation, 347; Toxins, 348; Pfeiffer's phenomenon, 
348; Asiatic cholera, 348; Mode of infection, 349; Bacteriological diagnosis, 
350; Prophylaxis, 351; Spirillum metchnikovi, 352; Spirillum Finkler -Prior, 
352 ; Spirillum tyrogenum, 352. 



TABLE OF CONTENTS XV 



CHAPTER XV. 

Spirochseta plicatilis, 353; Other saprophytic spirochetes, 353; Spirochaeta recur- 
rentis, 353; Varieties, 354; Cultures, 354; Diagnosis of relapsing fever, 355; 
Spirochaeta anserina, 356; Spirochaeta gallinarum, 356; Spirochaeta muris, 
356; Spirochaeta pallida, 357; Occurrence and morphology, 357; Cultures, 
358; Luetin, 359; Syphilis, 360; Bacteriological diagnosis, 360; Microscopic 
detection of spirochetes, 360; Animal inoculation, 361; Wassermann reaction, 
361; Luetin test, 366; Spirochaeta refringens, 366; Spirochaeta microdentium, 
366 ; Spirochaeta (Bacillus) fusiformis, 367. 

CHAPTER XXVI. THE FILTERABLE MICROBES. 

The virus of foot-and-mouth disease, 368 ; The virus of bovine pleuro-pneumonia, 
368; The virus of yellow fever, 368; Occurrence and nitration, 368; Yellow 
fever, 369, Transmission, 369; Prophylaxis; 369 ; The vims of cattle plague, 
370; The virus of rabies, 370; Occurrence and nitration, 370; Negri bodies, 
370; Rabies, 372; Transmission, 372; Diagnosis, 372; Pasteur treatment, 373; 
The virus of hog cholera, 373 ; Spirochaeta suis, 374; Immunity, 374; The virus 
of dengue fever, 374; The virus of phlebotomus fever, 374; The virus of 
poliomyelitis, 374; Occurrence and nitration, 374; Resistance, 374; Cultures, 
375; Globose bodies of Flexner and Noguchi, 375; Transmission, 375; The 
virus of measles, 375 ; The virus of typhus fever, 375 ; The virus of small-pox, 
376; Filtration, 376; Small-pox, 376; Vaccinia, 376; Immunity, 376; The 
virus of chicken sarcoma, 377. 

CHAPTER XXVII. MASTIGOPHORA. 

Herpetomonas muscae, 378; Leptomonas culicis, 378; Cultures, 378; Trypano- 
soma rotatorium, 379; Trypanosoma lewisi, 381 ; Tansmission, 382; Cultures, 
383; Pathogenesis, 383; Immunity, 384; Trypanosoma brucei, 384; Occurrence 
and morphology, 384; Transmission, 386; Cultures, 386; Nagana, 386; Diag- 
nosis, 387; Trapanosoma evansi, 387; Trypanosoma equiperdum, 387; Try- 
panosoma equinum, 388; Trypanosoma gambiense, 388; Occurrence and 
morphology, 388; Transmission, 388; Animal inoculation, 389; Human try- 
panosomasis, 389; Trypanosoma rhodesiense, 390; Trypanosoma avium, 
391; Occurrence, 391; Cultures, 392; Schizotrypanum cruzi, 392; Occurrence 
and morphology, 392; Animal inoculation 394; Cultures, 394; Leishmania 
donovani, 394; Occurrence and morphology, 394; Cultures, 394; Trans- 
mission, 396; Kala-azar, 396; Leishmania tropica, 396; Cultures, 397; 
Leishmania infantum, 397 ; Trypanoplasma borreli, 398 ; Bodo lacertae, 398 ; 
Trichomonas hominis, 400 ; Lamblia intestinalis, 400 ; Mastigamceba aspera, 
400; Trimastigamceba philippinensis, 400. 



XVI TABLE OF CONTENTS 

CHAPTER XXVIII. RHIZOPODA. 

Amoeba proteus, 401 ; Occurrence and morphology, 401; Cultures, 402; Entamoeba 
coli, 402 ; Occurrence and morphology, 402; Parasitic relation, 403 ; Entamoeba 
tetragena, 404; Occurrence and morphology, 404; Parasitic relation, 405; 
Entamoeba histolytica, 405; Relation of amebae to dysentery, 406; Cultures 
of dysenteric amebae, 406; Other rhizopoda, 407. 

CHAPTER XXIX. SPOROZOA. 

Cyclospora caryolytica, 408; Occurrence and morphology, 408; Pathogenesis, 410; 
Eimeria steidae, 410; Occurrence and morphology, 410; Sexual and asexual 
cycles, 410; Coccidiosis, 411; Eimeria schubergi, 412 ; Haemoproteus columbae, 
412; Occurrence and morphology, 412; Developmental cycle, 412; Haemopro- 
teus danilewskyi, 414; Fertilization in the sexual cycle, 414; Haemoproteus 
ziemanni, 415; Developmental stages, 416; Proteosoma praecox, 417; Occur- 
rence 418; Cycle in the blood, 418; Sexual cycle, 419; Plasmodium falciparum, 
419; Morphology, 420; Sexual cycle, 422; Cultures, 423; Plasmodium, vivax, 
424; Cycle in the blood, 424; Sexual cycle, 425; Plasmodium malariae, 425; 
Developmental cycle, 425; Malaria, 425; Types of fever, 426; Diagnosis, 427; 
Mosquito carrier, 427; Prevention, 427; Plasmodium kochi, 429; Babesia 
bigemina, 429 ; Morphology, 429; Transmission, 429; Texas fever, 430; Babesia 
canis, 430; Gregarina blattarum, 430 ; Nosema bombycis, 431 ; Developmen- 
tal cycle, 431; Pebrine, 432. 

CHAPTER XXX. CILIOPHORA. 

Paramaecium caudatum, 433; Morphology, 433; Conjugation, 433; Opalina ran- 
arum, 434; Balantidium coli, 435; Parasitic relationships, 436; Balantidium 
minutum, 437 ; Sphaerophyra pusilla, 437. 

INDEX OF NAMES 439 

INDEX OF SUBJECTS 445 



LIST OF ILLUSTRATIONS. 

FIG. PAGE 

1. Image formation by means of a pin-point aperture 16 

2. Image formation by a single lens *7 

3. Image formation by two lenses in series, without magnification 17 

4. Image formation by two lenses in series, with magnification of two 

diameters l8 

5. Image formation by two lenses in series, with magnification of three 

diameters l8 

6. Microscope objectives 20 

7. Sectional view of compound microscope 21 

8. Image formation in the compound microscope 22 

9. Image formation in the compound microscope with an eye-piece of higher 

power 22 

10. Central illumination by the Abbe condenser 24 

11. Illumination by a hollow cone of light 24 

12. Illumination by a broad convergent beam 24 

13. Dark-field condenser 25 

14. Optical parts of dark-field condenser 25 

15. Production of the "dark outline picture" 26 

16. Production of the "bright outline picture" 27 

17. Obliteration of outline by homogeneous illumination 27 

18. Microscope 29 

19. Abbe condenser 3 

20. Platinum needles 32 

21. Pasteur pipettes 33 

22. Hanging-drop preparation 34 

23. Cornet cover-glass forceps 3 8 

24. Stewart cover-glass forceps 38 

25. Novy's cover-glass forceps 38 

26. Kirkbride forceps for slides 39 

27. Schanze microtome 57 

28. Hot-air sterilizer 65 

29. Koch's steam sterilizer 66 

30. Diagram of the Arnold steam sterilizer 67 

31. Steam sterilizer, Massachusetts Board of Health 68 

32. Autoclave 69 

33. Apparatus for filling test tubes 89 

34. Potato tube 91 

35. Kock's serum sterilizer 93 

xvii 



XV111 LIST OF ILLUSTRATIONS 

FIG. PAGE 

36. Pipette for collection of sterile blood 95 

37. Pipette for collection of sterile blood from an animal 96 

38. Instrument for collection of feces from infants 102 

39. Method of inoculating culture media 107 

40. Petri dish 108 

41. Colonies in gelatin plate 109 

42. Manner of making Esmarch roll-tube no 

43. Dilution cultures in Esmarch roll-tubes in 

44. Stab-culture closed with rubber stopper 114 

45. Smear culture closed with rubber cap 114 

46. Incubator 116 

47. Reichert gas-regulator 117 

48 MacNeal gas-regulator 118 

49. Pvoux bimetallic gas-regulator 119 

50. Koch automatic gas-burner 119 

51. Diagram of electric regulator for low- temperature incubator 122 

52. Anaerobic culture by Buchner's method 126 

53. Novy anaerobe jar for tube cultures 127 

54. Novy anaerobe jar for Petri dishes or tubes 127 

55. Novy anaerobe jar, improved pattern 127 

56. Tripod and siphon flask for anaerobic culture by combined hydrogen and 

pyrogallate method 128 

57. Anaerobic organism that will not grow under a cover-glass 129 

58. Method of making collodion capsules 135 

59. Common molds 138 

60. Yeast cells stained with fuchsin 139 

61. Wine and beer yeasts 140 

62. Various groupings of micrococci 142 

63. Bacilli of various forms 145 

64. Sporulation 145 

65. Various positions of spores 146 

66. Types of spirilla 147 

67. Bacteria with capsules 148 

68. Bacteria showing flagella 148 

69. Formation of spores 149 

70. Bacteria with spores 149 

71. Germination of spores 149 

72. The most important trypanosomes 152 

73. Lcishmania donovani 152 

74. Leishmania donovani in culture 153 

75. Trichomonas hominis 153 

76. Lamblia intestinalis 1 53 

77. Entamosba coli 154 

78. Developmental cycle of Eimeria sckubergi ! . . . . 156 

79. Asexual cycle of Plasmodium falciparum 15? 



LIST OF ILLUSTRATIONS XIX 

FIG. PACK 

80. Forms of Babesia muris 158 

8 1. Developmental cycle of Nosema bombycis I 5 

82. Sedgwick-Tucker aerobioscope 177 

83. Jeffer's plate for counting colonies 184 

84. Surface divided in square centimeters for counting colonies 185 

85. Receptor of the first order uniting with toxin 209 

86. Receptor of the second order 210 

87. Receptor of the third order 214 

88. Deviation of complement 216 

89. Mucor mucedo 232 

90. Mucor corymbifer 232 

91. Aspergillus glaucus 233 

92. Penicillium crustaceum 234 

93. Oidium lactis 236 

94. Oidium albicans, colony 237 

95. Oidium albicans, mycelial thread 238 

96. Scutulum of favus on the arm of a man 239 

97. Scutulum of favus in a mouse 240 

98. Achorion schoenleinii, colony 241 

99. Sporotrichum schenki, cultures on agar 242 

100. Sporotrichum schenki, forms of mycelium 243 

101. Organisms found in oidiomycosis 245 

102. Actinomyces bovis 247 

103. Gonococci and pus cells 251 

104. Meningococcus in spinal fluid 256 

105. Pneumococcus showing capsule 258 

106. Staphylococcus aureus, gelatin culture 265 

107. Bacillus subtilis 269 

108. Anthrax bacilli in capillaries of the liver 270 

109. Bacterium anthracis showing spores 271 

no. Bacterium anthracis, colony upon a gelatin plate 271 

in. Bacterium anthracis, thread formation of colony 272 

112. Bacillus welchii, in agar showing gas formation 278 

113. Tetanus bacilli, showing terminal spores 280 

1 14. Bacillus tetani, stab culture 281 

115. Bacillus botulinus 282 

116. Bacillus of diphtheria 286 

117. Bacillus diphtheria stained by Neisser's method 286 

118. Forms of Bacillus diphtheria in cultures on Loeffler's serum 287 

119. Forms of Bacillus diphtheria on agar 287 

1 20. Colonies of Bacillus diphtheria on glycerin agar 288 

121. B. diphtherias, culture on glycerin agar 289 

122. Swab and culture tube for diagnosis of diphtheria 290 

123. The Morax-Axenfeld bacillus 296 

1 24. The Koch-Weeks bacillus 297 



XX LIST OF ILLUSTRATIONS 

FIG. PAGE 

125. Bacillus tuberculosis in sputum 300 

126. Bacillus tuberculosis from a pure culture 301 

127. Tubercle bacillus showing branching and involution forms 302 

128. Bacillus tuberculosis, culture on glycerin agar 303 

129. Bacillus of bubonic plague 317 

130. Bacillus coli, showing flagella 324 

131. Bacillus coli, superficial colony on gelatin plate 325 

132. Friedlander's pneumobacillus, gelatin stab-culture 327 

133. Bacillus of typhoid fever 330 

134. Bacillus typhosus, showing flagella 331 

135. Colonies of Bacillus typhosus and Bacillus coli 331 

136. Bacillus mallei 339 

137. Cholera vibrios, short form 346 

138. Cholera vibrios, showing flagella 347 

139. Involution forms of the spirillum of cholera 347 

140. Spirochaetae of relapsing fever 354 

141. SpirochcBta recurrentis in blood of a rat 355 

142. Preparation showing Spirochata pallida and Spirochceta refringens 357 

143. Spirochata pallida stained by Levaditi method 359 

144. Aedes (Stegomyia) calopus 369 

145. Negri bodies in brain of a rabid dog 371 

146. Herpetomonas musc<z 378 

147. Leptomonas culicis 379 

148. Trypanosoma rotatorium in blood of a frog 380 

149. Trypanosoma rotatorium in culture 380 

150. Trypanosoma lewisi 381 

151. Trypanosoma lewisi, various multiplication forms . . . 382 

152. Trypanosoma lewisi, eight-cell rosette 383 

153. The most important trypanosomes parasitic in vertebrates 384 

154. Glossina morsitans, dorsal view 385 

155. Glossina morsitans, lateral view 385 

156. Trypanosoma equiperdum 387 

157. Glossina palpalis : 389 

158. Trypanosoma avium in blood of birds 39 

159. Trypanosoma avium in culture 391 

1 60. Schizotrypanum cruzi in tissues of the guinea pig 393 

161. Schizotrypanum cruzi in human blood 394 

162. Conorhinus megistus 395 

163. Leishmania donovani in spleen juice 395 

164. Leishmania donoiani in culture 396 

165. Leishmania tropica in pus 396 

166. Leishmania tropica in culture 397 

167. Trypanoplasma cyprini 397 

168. Bodo lacerta 398 

169. Trichomonas hominis 398 



LIST OF ILLUSTRATIONS XXI 

FIG. PAGE 

170. Lamblia intestinalis 399 

171. Trimastigamceba philippinensis 399- 

172. Amoeba proteus 4i 

173. Entamceba coll 4 2 

1 74. Entamceba tetragena, unstained 44 

175. Entamceba tetragena, stained preparation 44 

176. Entamceba tetragena, mature cyst 4S 

177. Cydospora caryolytica, male cells 408 

178. Cydospora caryolytica, female cells 49 

179. Cydospora caryolytica, fertilization and production of sporozoits 409 

180. Eimeria steidce, oocyst 4 10 

181. Eimeria steidce, various forms 411 

182. Hcemoproteus columbce, developmental cycle 4 T 3 

183. Hcemoproteus danilewskyi 414 

184. Hcemoproteus ziemanni, gametocytes 4 1 5 

185. Hcemoproteus ziemanni, formation of microgametes and fertilization 415 

186. Hcemoproteus ziemanni, various forms observed in blood 4 J 6 

187. Developmental cycle of Protoesoma 417 

188. Proteosoma prcecox in blood of a lark 418 

189. Midgut of a mosquito showing oocysts of Proteosoma 418 

190. Oocyst of Proteosoma 4*9 

191. Plasmodium falciparum, various forms observed in the blood 420 

192. Capillary of brain filled with Plasmodium falciparum 420 

193. Plasmodium falciparum, development of the gametocytes 421 

194. Stomach wall of Anopheles infected with Plasmodium falciparum 421 

195. Digestive tract of Anopheles, infected with Plasmodium falciparum 422 

196. Plasmodium falciparum, ripe sporozoits in oocyst 422 

197. Salivaiy gland of Anopheles, containing sporozoits of Plasmodium falci- 

parum 423 

198. Plasmodium vivax, stages in asexual cycle 424 

199. Plasmodium vivax, sporulation 424 

200. Plasmodium vivax, double infection 424 

201. Plasmodium vivax, stages in development of the gametocytes 425 

202. Plasmodium malarice, asexual cycle 426 

203. Plasmodium malaria, gametocytes 426 

204. Comparison of Culex and Anopheles 428 

205. Babesia bigemina 429 

206. Gregarina blattarum 431 

207. Nosema bombycis 432 

208. Paramcecium caudatum 433 

209. Paramcecium caudatum and Paramcecium pittrinum 434 

210. Opalina ranarum 435 

211. Balantidium coll 436 

212. Intestinal wall infected with Balantidium coll 436 

213. Sphcerophrya pusilla within a paramaecium 437 



INTRODUCTION. 



Bacteriology and Microbiology. The science of Bacteriology 
occupies a somewhat peculiar position among the natural 
sciences, partly because of its recent development and partly 
because of the overshadowing importance of its practical appli- 
cations. As bacteria are microscopic plants, some have con- 
sidered bacteriology as a minor division of botany; but the 
methods of work and the practical applications of bacteriology 
have little in common with those of the more ancient science. 
Indeed were it not for the importance of these little organisms 
to the chemist, the pathologist, the physician and the agricul- 
turist, we should hear little about them. 

The foundations of the science were laid by Pasteur (1858) 
by the introduction of media and methods for artificial culture 
of bacteria and the separation of mixtures into pure culture by 
the laborious and uncertain but nevertheless successful method 
of dilution in fluid media, thus making possible the accurate ex- 
perimental study of microbes. Robert Koch (1872-1882) con- 
tributed much to the establishment of the new science by intro- 
ducing the use of solid media and the method of plating for the 
isolation of pure cultures and especially by his wonderful 
achievements in investigation of the pathogenic bacteria by 
his new methods. Koch used potatoes, and aqueous humor 
and blood serum rendered solid by the addition of gelatin. He 
first employed the anilin dyes in staining bacteria (1877), 
microphotography of bacteria (1877), homogeneous immersion 
objectives and the Abbe illuminating apparatus (1878). Much 
of our modern technic has been devised by his pupils and 



2 INTRODUCTION 

colleagues. The commonly used meat-water-pepton-gelatin was 
introduced by Loffler; agar by Frau Hesse. 

The development of bacteriology has been promoted by the 
work of biologists, botanists, chemists, pathologists and agronom- 
ists, many of whom have been willing to include bacteriology 
as a subdivision of their own field. The practical importance of 
Bacteriology to these various fields is becoming progressively 
more evident. The relation to pathology and medicine is per- 
haps most clearly recognized, although the importance of bac- 
teria in chemical technology and in agriculture is no longer 
questioned. The relationships to general biology have not been 
so completely developed as yet, partly because these have seemed 
to offer less promise of immediate practical application, and partly 
because few well-trained zoologists or botanists have devoted 
serious attention to bacteriology. 

As a matter of fact, bacteriology must be ranked as a distinct 
science, especially because of its peculiar special technic and be- 
cause of the peculiarly critical thought necessary in the inter- 
pretation of bacteriological observations and experiments. The 
importance of these can be fully appreciated only after actual 
experience in handling microbes. Here is a science in which 
skepticism is a necessary safeguard, a skepticism which will be- 
come convinced only when overwhelming evidence compels con- 
viction; and, while regarding other conclusions with interest or 
even with enthusiasm, still carefully reserves final judgment as 
long as the observed phenomena are open to more than one 
interpretation. 

These methods of thinking and of working have been applied 
to organisms other than the bacteria, on the one hand to the 
unicellular animals, the protozoa, on the other to more complex 
plant-forms such as the yeasts and molds, and more especially to 
the study of the still undefined types of living things known as 
filterable viruses or more vulgarly as the ultramicroscopic mi- 
crobes. Inasmuch as many of these live as parasites and some 
are important in the causation of disease, they are commonly 



INTRODUCTION 3 

considered along with the pathogenic bacteria. The terms mi- 
crobe and micro-organism properly include these as well as the 
bacteria. There is thus an evident tendency to extend the field 
of bacteriology so that it becomes microbiology or the science of 
micro-organisms. There are many reasons why this is desir- 
able. It is certainly essential that the microbes included among 
the protozoa and the filterable viruses should receive more atten- 
tion in the future, both from beginning students and from trained 
investigators. Until separate instruction in these subjects is pro- 
vided for medical students, they may perhaps best be studied 
along with bacteriology. 

Biological Relationships. Since the earliest times, the essen- 
tial difference between living things and lifeless things, that is, 
the nature of life, has been an interesting subject of speculation. 
It was at first assumed as a matter of course that the transition 
from lifeless to living matter readily took place without the 
agency of preexisting living matter. This speculative assump- 
tion is still not without its able supporters. The history of actual 
observations, however, is one long record of refutation of this 
assumption wherever the facts have been subjected to accurate 
observation. The ancient Greeks held that living beings arose 
spontaneously and even Aristotle (384 B.C.) asserted that ani- 
mals were sometimes formed in this way. These ideas were dis- 
proved by more careful observation. A notable experiment was 
that of Francesco Redi (about 1650) who allowed meat to putrefy 
in a jar covered with fine wire gauze. The flies attracted by the 
odor deposited their eggs on the gauze and the maggots were 
hatched there. The assumption that the maggots arose de novo 
in putrefying meat was thus disproven. Harvey in 1650 made the 
famous statement, "Omne animal ex ovo" which was later ex- 
tended to "Omne vivum ex vivo." 

When Anthony van Leeuwenhoek, the "Father of micro- 
scopy," discovered, described and figured bacteria in 1683, the 
assumption of spontaneous generation was at once applied to this 
group of organisms and, although rendered exceedingly doubtful 



4 INTRODUCTION 

by the experiments of Spallanzani (1777) and of Schulze (1836), 
it still continued to be accepted by many scientific men until it 
was combated by Pasteur, 1860 to 1872. After the accurate 
observations of Pasteur upon fermentation and putrefaction and 
his successful defense of them through a long period of contro- 
versy, the assumption of spontaneous generation as applied to 
bacteria was discredited and has been very generally given up. 
Only a very few observers 1 still claim the existence of evidence 
in support of its application here. The more prominent advo- 
cates 2 of the assumption of spontaneous generation or abio- 
genesis seem inclined now to apply it to some group of living 
beings still beyond the limits of actual observation. 

Closely related to the assumption of abiogenesis has been the 
assumption of heterogenesis among the bacteria, the notion that 
various kinds of microbes could readily be produced from 
one species. Although very successfully combated by Pasteur, 
this idea still persisted for many years in the early bacteriological 
literature, the observed new species of microbes actually resulting 
from faulty technic by which new germs had gained entrance to a 
former pure culture. These observations are often repeated 
unwittingly by beginners in bacteriology. The validity of bac- 
terial species is now unquestioned. On the other hand, the vari- 
ability in the descendants of a single cell through a greater or 
less range, and the possibility of producing morphologically and 
physiologically different strains of the same species by appro- 
priate environmental conditions are now well known, resulting 
again very largely from the fundamental work of Pasteur in the 
production of attenuated cultures of the germs of chicken cholera, 
and of anthrax. 

The systematic relationships and the classification of bacteria 
were first studied by O. F. Mueller (1786). Ehrenberg (1838) 
made the first serious attempt at a comprehensive classification 

1 Bastian, The Evolution of Life, London, 1907. The Origin of Life, London, 

1913- 

2 Schafer, Nature, Origin and Maintenance of Life, Science, 1912, Vol. XXXVI, 
pp. 289-312. 



INTRODUCTION 5 

and many modern systematists are inclined to return to his work 
to establish authoritative terminology for present use. He re- 
garded the bacteria as animals. Ferdinand Cohn (1872) recog- 
nized the nature of bacterial spores, showed the close relation- 
ship of bacteria to the algae and established their classification 
in the plant kingdom. He distinguished six genera micro- 
coccus, bacterium, bacillus, vibrio, spirillum and spirochaeta. 
Migula (1897) undertook an extensive revision of bacteriological 
nomenclature and classification, basing it upon morphological 
characters, and his system is doubtless the most satisfactory yet 
offered. The subject is still in a very unsettled state, neverthe- 
less, and there is no system of classification generally accepted 
by bacteriologists. The problem presents so many difficulties 
and our knowledge of the bacteria is still so incomplete that many 
authorities seem prone to consign systematic classification to the 
future, and to employ names of sufficient historical prominence 
to insure their correct interpretation. 

Fermentation and Putrefaction. The relation of micro- 
organisms to the decomposition of organic matter, fermentation 
and putrefaction, was one of the first fields of applied bacteri- 
ology to be studied. Following the observation of bacteria in 
saliva by van Leeuwenhoek in 1683, micro-organisms were dis- 
covered in all sorts of decomposing material. At first, these or- 
ganisms were regarded as unimportant for the chemical process 
and interest attached chiefly to the question of their origin, 
whether by spontaneous generation or from previously living 
cells. Needham (1745) directing his attention more particularly 
to this first question, boiled an infusion of meat, and keeping it free 
from contact with the air, nevertheless observed after some days 
the presence of "infusoria." Spallanzani (1765) repeated Need- 
ham's experiments, subjecting hermetically sealed flasks of meat 
infusion to the temperature of boiling water for one hour, 
and he found no subsequent development of life and no decom- 
position of the infusion as long as it remained sealed. While 
discussion continued concerning the discrepancy between the 



6 INTRODUCTION 

results of Needham and Spallanzani and concerning the relation 
which the subsequent exclusion of the air might bear to the ab- 
sence of life in the flasks, .the method of heating was applied to 
the preservation of vinegar by Scheele (1782) and to the preser- 
vation of foods in general by Appert (1811). The method was 
quickly introduced into other countries, and developed by va- 
rious tradesmen, who attempted with more or less success to 
keep their processes secret. Success in preservation by canning 
remained somewhat uncertain, as a precise understanding of the 
underlying scientific principles was still lacking. Schulze (1836) 
showed that air might be admitted to flasks prepared by Spallan- 
zani's method, without the development of life and without 
putrefaction, provided the air were first passed through a series 
of bulbs containing concentrated sulphuric acid. The subse- 
quent work of Schroder and van Dusch (1853), who obtained 
similar success by filtering the air through cotton, of Pasteur and 
Tyndall (1860-62) who were able to preserve putrescible fluids 
directly in contact with air, provided the air were rendered per- 
fectly free from dust, has established the fact that the decom- 
position ordinarily taking place after exposure to the air is due 
to the introduction of living germs into the previously sterile 
material. 

The idea that specific kinds of fermentation are caused by 
specific kinds of microbes was first clearly put forward by Schwann 
and Cagniard-Latour (1837), who showed that yeast-cells were 
living organisms and claimed that the alcoholic fermentation of 
sugar solutions was due to their growth. The importance of this 
relationship received little recognition until Pasteur (1860-72), 
during his extensive and careful researches into the nature of 
fermentation and the causation of undesirable fermentation (dis- 
eases of wines and beers), demonstrated conclusively that the 
kind of decomposition of a fermentable substance depended upon 
the nature of the substance, the kind of microbes present and the 
environmental conditions, such as temperature and presence or 
exclusion of air. The mere introduction of a small number of 



INTRODUCTION 7 

unfavorable microbes was sufficient to change the whole nature 
and course of the fermentation. Furthermore, Van der Brock 
(1857) and Pasteur (1863) were able to collect such fermentable^ 
materials as grape juice, wine, blood, tissues of plants and ani- 
mals and preserve them free from decomposition and from all 
microbic life, merely by effectively avoiding contact with germs 
during collection and storage. 

The agency of microbes in fermentation was ridiculed by 
Liebig, the most prominent chemist of the time, who steadfastly 
continued to regard decomposition of organic material as a 
purely chemical process uninfluenced by biological activity. His 
ideas prevailed for a time because of his prominent position. 
The correctness of Pasteur's contention is now universally ac- 
cepted. Nevertheless it should not be forgotten that many 
organic substances are in themselves so unstable that even in the 
absence of microbic life they disintegrate, or become oxidized in 
the presence of the air. These changes are different from those 
ordinarily known as fermentation and putrefaction. 

Pathology and Hygiene. The history of the development of 
our ideas concerning the relation between microbes and disease 
is one of the most interesting and perhaps the most important 
chapter in the history of bacteriology. The customs and rec- 
ords of the ancients give evidence that they recognized the pres- 
ence of an unseen agency in the body of the diseased individual 
capable of causing sickness in others. This was recognized by 
the ancient Persians as recorded by Herodotus. The isolation 
of lepers by the ancient Hebrews shows that the infectious char- 
acter of the disease has long been recognized, though other affec- 
tions than leprosy were probably confused with this disease. "He 
is unclean; he shall dwell alone; without the camp shall his 
habitation be." (Lev. XIII, 46). There is, in fact, much in the 
laws of Moses that points to some knowledge of the nature of 
infection. "This is the law, when a man dieth in a tent all that 
come into the tent and all that is in the tent shall be unclean for 
seven days. And every open vessel that has no covering on it 



8 INTRODUCTION 

shall be unclean." (Numb. XIX, 14, 15). " Every thing that may 
abide the fire, ye shall make it go through the fire, and it shall be 
clean." (Numb. XXXI, 23). In Homer we read of Ulysses, that, 
having slain his wife's troublesome suitors: 

"With fire and sulphur, cure of noxious fumes, 
He purged the walls and blood-polluted rooms." (Pope's Odyssey). 

These records certainly suggest a rather advanced state of knowl- 
edge concerning the nature of contagion. It may be that they 
record customs derived from a superior knowledge of some other 
ancient people, perhaps the ancient Egyptians. During the 
middle ages, as doubtless also before the dawn of history, epi- 
demic disease was regarded as a visitation of Providence or at- 
tributed to the influence of gods, demons or other supernatural 
agencies. Epidemics were associated with the appearance of 
comets in the sky or with other evidences of divine wrath. These 
conceptions of disease have not altogether disappeared even at 
the present time. 

Hippocrates (400 B. C.) denied the supernatural causation 
of disease and held that such doctrines were mere cloaks for help- 
less ignorance. He ascribed epidemic disease to a morbid secre- 
tion of the atmosphere, and later writers have expressed this 
idea of a morbid secretion by the word miasm, its exact nature 
remaining for centuries intangible and mysterious. There is 
here a conception different from that upon which the hygienic 
measures of the Persians and Hebrews were founded and the 
distinction was clearly expressed by Pettenkofer in the nineteenth 
century, who defined contagious diseases as those which are trans- 
mitted directly from man to man or through the agency of solid 
objects, while in miasmatic diseases the causative agent enters 
from the outside world where it may live naturally or where it 
must have undergone a ripening process since its escape from the 
body of the sick person. As will be seen later these ideas apply 
very well to certain diseases, for example, small-pox and syphilis 
as contagious diseases and yellow-fever and malaria as a mias- 



INTRODUCTION 9 

matic. The ancient Greeks recognized the contagiousness of 
several diseases and Galen classed plague, itch, ophthalmia, con- 
sumption and rabies as contagious. Fracas torius (1546) during 
the period of the great epidemic of syphilis in Europe, published 
a book containing the first comprehensive discussion of the theory 
of contagion. He recognized contagion by contact, by fomites 
and at a distance. Soiled material of all kinds was included un- 
der fomites, as also those healthy individuals capable of trans- 
mitting disease, a phenomenon already recognized. Transmis- 
sion by insects and animals was also included under this head. 
The transmission "per distans" was considered due to emana- 
tions from the patient diffusing to a distance through the 
atmosphere. 

Kircher in 1658 claimed to have seen the living contagium in 
the body in the form of minute worms, and his observations were 
widely recognized. The objects he saw were not accurately 
described but it seems very certain that they were not bacteria. 
Probably they were the normal cells of the tissues. 

The discovery of bacteria by van Leeuwenhoek (1683) was 
not immediately recognized as of importance for the germ 
theory. Leeuwenhoek himself considered it impossible for his 
"animalcula" to penetrate into the blood because of the com- 
pactness of the epithelial tissues. 

Almost a century later, Plenciz (1762) maintained that each 
infectious disease must have its own specific cause. Reimarus 
(1794) also expressed the same opinion and considered these liv- 
ing organisms to be of the order of infusoria or perhaps still 
smaller beings not yet visible with the microscope. These ideas 
were not supported by objective evidence and received only 
passing attention. They were soon thrust aside by other inter- 
esting if less valuable speculations. 

The development of general knowledge of the animalcules 
in the early part of the nineteenth century, already referred to 
in the discussion of the biological relationships and of fermentation, 
was preparing the way for progress in the problem of disease. 



10 INTRODUCTION 

In 1834 the contaguim vivum of itch, the itch mite (Sar copies 
scabei), a fairly large mite to be sure, was rediscovered and its 
relation to the disease made evident. In 1837, the same year 
in which Cagniard-Latour and Schwann established the relation 
of living yeast to alcoholic fermentation, Donne described 
vibriones (bacteria) in syphilitic ulcers, and Audouin amplified 
the discovery of Bassis that muscardine, a disease of the silk- 
worm, was caused by a mold (Botrytis bassiana) which was trans- 
mitted from the sick to the healthy worms by contact or by air 
currents. These discoveries furnished a great impulse to further 
investigation. 

Henle (1840) reviewed the evidence then at hand and con- 
cluded in a very logical way that the causes of contagious dis- 
eases were to be sought for among the minute living micro-organ- 
isms. He recognized that no human disease had yet been shown 
to be caused by a micro-organism and he formulated the re- 
quirements to be fulfilled in order to prove such a relation, 
namely, that the microbe must be constantly present in the 
disease, must be isolated from the infectious material, and must 
then alone be capable of producing the disease. 

During the next twenty years, the attempts to discover the 
cause of an infectious disease and to satisfy the postulates of Henle 
were successful in several diseases due to molds, Favus (Achorion 
Schoenleinii) 1839, similar skin diseases known as trichophytosis 
and pityriasis and especially thrush, shown to be caused by 
Oidium albicans by Robin in 1847; but in all the more important 
diseases only failure resulted. The reawakened interest in con- 
tagium vivum therefore again gradually faded away. 

During this time Pollender and Davaine and Rayer (1850) 
had discovered the minute rods in the blood of animals sick with 
anthrax, and in 1863 Davaine had proved the almost constant 
presence of these rods in the disease and the possibility of trans- 
mission by inoculation from one animal to another. 

Pasteur from 1865 to 1868 investigated the fatal disease of 
silk-worms known as pebrine, discovered the microsporidium 



INTRODUCTION 1 1 

(Nosema bombycis) which occurs in the sick worms and in the 
eggs, and devised a successful method of eradicating the disease.^ 

In 1870-71 the presence of bacteria in wounds and in the 
internal purulent collections in pyemia and septicemia was first 
definitely recognized by Rindfleisch (1870), but more especially 
by Klebs in a large number of cases at the military hospital at 
Karlsruhe. The latter observed spherical bacteria arranged in 
groups or as a rosary to which he gave the name Microsporon 
septicum. His observations were quickly confirmed by other 
competent pathologists. Similar organisms were quickly found 
in a great many wounds and other inflammatory processes. 
Specific causal relationship was still unproven. 

In 1873 Obermeier described the slender but actively motile 
spirochetes seen by him in the blood in relapsing fever as early 
as 1868. 

In 1874 Billroth concluded that there was still no disease in 
which the causal relationship of micro-organisms had been con- 
clusively proven. The skin diseases due to molds were relatively 
unimportant and had not been recently studied. The microbes 
found in other diseases might just as reasonably be regarded as 
a product of the disease or as only incidental to it. Even in 
anthrax, where the evidence seemed strongest, there were cases 
of the disease without the presence of the peculiar rod-like bodies 
in the blood, and indeed these rods might be crystals and not 
living organisms at all. 

Since 1867 Lister, stimulated by the investigations of Pasteur 
on fermentation and putrefaction, had been developing and 
applying an antiseptic method to the treatment of wounds, 
which consisted of the use of carbolic acid. The results of this 
method published in 1875 were so remarkably favorable that it 
was quickly adopted throughout the world, and its success did 
much to prepare the way for the recognition of the role of microbes 
in suppuration, if it did not in itself convince. 

Robert Koch, 1876-1881, first satisfied the postulates laid 
down by Henle, and again formulated by himself, in the bacterial 



12 INTRODUCTION 

disease, Anthrax. The presence of the bacilli in the blood of 
animals suffering from anthrax had been established by a large 
number of previous workers, and the transmissibility of the dis- 
ease by inoculation with blood of diseased animals was already 
known. Koch was able to grow the bacillus in pure culture in a 
test tube, using the aqueous humor of the ox's eye as a medium. 
He was able to observe growth and division and the formation 
and germination of spores under the microscope. Finally with 
these cultures which had been propagated a long time in the 
culture medium, he was able again to cause anthrax by injecting 
them into susceptible animals. The demonstration of the causa- 
tion of disease by bacteria had been achieved. 

The introduction by Koch in 1881 of the plate method of sepa- 
rating bacteria paved the way for rapid advances in bacteriology, 
and during the next ten years the bacterial causes of several 
diseases were discovered and proven by thorough test, and since 
then the number of diseases known to be due to bacteria has 
gradually increased. 

The history of immunity extends far back into ancient times. 
For many diseases it was recognized that those who recovered 
could associate with the sick without danger to themselves. 
Recognizing this, people sometimes exposed themselves purposely 
in order to have the disease at a convenient time. Artificial 
inoculation to cause small-pox was introduced into Europe from 
the Orient in 1721. The use -of cowpox, vaccination, was discov- 
ered by Jenner in 1797. Artificial immunization by inoculation 
with altered bacterial cultures was first successfully demonstrated 
by Pasteur in chicken cholera and in anthrax in 1881. Analogous 
methods have since been devised for many other diseases. The 
discovery of the antitoxic property of the blood serum of animals 
immunized to tetanus and to diphtheria was made by von Behring 
and Kitasato (1891). 

With the discovery of amebae in the stools in tropical dysentery 
by Loesch (1875) and of the malarial plasmodium in the blood 
by Laveran (1880) the relationship of protozoa to important 



INTRODUCTION 13 

diseases was suggested. An enormous number of protozoal para- 
sites are now known, many of them associated with important 
diseases. The strict proof of causal relationship to the disease 
has presented greater difficulties here, especially the step of artifi- 
cial culture. However, the causal relationship of bacteria hav- 
ing been demonstrated, the probable causal relationship of 
the protozoa has found more ready acceptance. Cultures of 
ameba have been obtained by many workers but the successful 
cultivation of a pathogenic ameba is still questionable. Pure 
cultures of trypanosomes were obtained by Novy and his pupils 
(1903-04) and the infections again produced by inoculation with 
these cultures. 

The transmission of protozoal diseases by insects, first demon- 
strated by Salmon and Smith in Texas fever, has developed into 
a subject of prime importance. Malaria and the insect, Anophe- 
les, sleeping sickness and tsetse fly, Glossina, are important ex- 
amples of this relationship. 

Obermeier (1873) described a motile spiral organism in the 
blood of relapsing fever, the first known parasitic member of a 
group of very great importance. Very many pathogenic spiral 
organisms of this general type are now known. Their syste- 
matic relationships have not been fully worked out and further 
knowledge is necessary before they can be finally classed with 
either the bacteria or the protozoa. The discovery of practical 
methods of artificial culture for these .organisms has been very 
recent and the most successful methods seem to have been de- 
vised by Noguchi (1910-12).' Many of these parasites are trans- 
mitted by insects and they pass through a somewhat obscure de- 
velopment in the insect carriers, the forms developed being ex- 
tremely minute (Nuttall, 1912). These facts suggest a possible 
relationship of this group of organisms to the filterable viruses. 

Nocard (1899) discovered that the virus of pleuro-pneumonia 
of cattle would pass through filters impervious to bacteria. The 
number of recognized filterable viruses has grown appreciably 
since then and among them are the causes of several very im- 



14 INTRODUCTION 

portant diseases, such as yellow-fever, dengue fever, poliomy- 
elitis, measles, typhus fever, small-pox, rabies and hog cholera. 
Knowledge of this group of organisms is accumulating rapidly 
and, although microscopic methods of denning their form and 
structure are still undeveloped, they cannot with justice be re- 
garded as wholly in the realm of the unknown. 

Agriculture. The importance of microbes in soil fertility 
and agriculture has a relatively short history. Duclaux, 1885, 
showed that plants could not well utilize complex organic matter 
as food in the absence of microbic life. In addition to ordinary 
decomposition of organic matter, bacteria also bear an important 
relation to the nitrogen metabolism of plants. Hellriegel and 
Wilfarth (1886-88) showed the infectious nature of the nitro- 
gen-fixing root tubercles of legumes, and the organism B. radici- 
cola was isolated by Beyerinck in 1888. The importance for agri- 
culture of other living elements in the soil, such as amebae and 
nematodes, has been more recently recognized. 

Although it is well to recognize the many important appli- 
cations of bacteriology, a word of caution may not be amiss, lest 
we follow too eagerly the alluring applications and neglect the 
secure foundation of scientific knowledge of the biology and bio- 
logical relationships of micro-organisms, the proper training in 
logical thinking concerning these beings and in the technic of 
dealing with them. 



PART I. 
BACTERIOLOGICAL TECHNIC 



CHAPTER I. 
THE MICROSCOPE AND MICROSCOPIC METHODS. 

The development of bacteriology has depended especially 
upon the development of new methods of scientific study, and 
in a very important way upon the improvements in construction 
of the microscope and in methods of preparing objects for study 
under the microscope. Knowledge of the construction of a mi- 
croscope is not an essential part of bacteriology but the demands 
of modern microscopical methods require a skill in manipulation 
of the instrument which is best acquired after the principal struc- 
tural features of the microscope are understood. 

The Development of the Microscope. Roger Bacon, in 
1276, seems to have been the first to recognize the peculiar prop- 
erties of a lens. Spectacles began to be used about the same 
time and are said to have been invented by d'Armato. 1 Gali- 
leo (1610) probably made the first record of the use of the com- 
pound microscope. It was a lens maker, Anton van Leeuwen- 
hoek, who first saw bacteria in 1683. A method of correcting 
chromatic aberration was discovered by Marzoli in 1811, but 
became generally known through the work of Chevalier in 1825. 
The correction of the color defects was accomplished by the com- 
bination of two kinds of glass, crown glass and flint glass, in the 
1 Jour. A. M. A., Nov. 9, 1912, Vol. LIX, p. 1721. 



i6 



BACTERIOLOGY 



objective lens system, and made possible the construction of 
achromatic objectives, perhaps the most important advance ever 
made in the construction of the microscope. Abbe (about 1880) 
introduced his substage condenser which made possible the in- 
tense illumination of the microscopic field. In collaboration with 
Zeiss, Abbe (1886) devised an objective lens system with more 
perfect chromatic correction than had been previously attained. 
These objectives are constructed of several different kinds of 
glass and have in addition one lens composed of rmorite. Sieden- 
topf and Zsigmondi (1903) devised a method of illuminating the 
microscopic preparation by horizontal beams and so brought to 







FIG. i. The formation of an image by means of a simple pin-point aperture. 

(After A, E. Wright.} 

view exceedingly minute refractive particles as luminous points 
on a dark field. The various dark-field condensers introduced 
in recent years (1906) utilize similar principles, the object being 
illuminated by oblique light. Recently, Gordon has devised 
the tandem microscope, an instrument which has demonstrated 
the possibility of achieving greater microscopic resolution than 
has previously been attained and even suggests that there is no 
necessarily final limit to the degree of magnification at which 
satisfactory definition and resolution may be achieved. 

Principle of the Microscope. The formation of an image by 
means of a simple pin-point aperture is illustrated in Fig. i. It 
will be noted that the magnification achieved is the quotient of 



THE MICROSCOPE AND MICROSCOPIC METHODS 17 

aperture-image distance divided by object-aperture distance; also 
that the sharpness of outline of the image increases and the 
brilliancy diminishes as the size of the aperture is decreased. 

If the simple aperture be replaced by a convex lens and the 
object and the screen be set at the conjugate foci of the lens, it 




FIG. 2. Image formation by a single lens. Note that the image, at the right, 
is | the size of the object, in proportion to their respective distances from the lens; 
the opening angle being f the size of the closing angle. 

will be seen that magnification is again the quotient of the aper- 
ture-image distance divided by the object-aperture distance. 
The sharpness of outline, however, depends now upon the quality 
of the lens and the accurate adjustment of the distance, and 
brilliancy is not seriously impaired in attaining definition. 




FIG. 3. Image formation by two lenses in series without magnification. Note 
that the opening angle of the beam preceding from the object, at the left, is equal to 
the closing angle of the beam forming the image at the right. 

Image formation in the human eye is an example of the work- 
ing of the lens-armed aperture. The rays of light are brought 
to a focus on the retina and the image produced here is in- 
verted and actually much smaller than the object, the reduction 
(minification) being again measured by the quotient of the lens- 



i8 



BACTERIOLOGY 



retina distance divided by the object-lens distance. The longer 
the antero-posterior diameter of the eye, the larger will be the 
retinal image. Our subjective interpretation of the stimulation 
of the retina (i.e., what we see) is influenced by other psycholog- 




FIG. 4. Image formation by two lenses in series, with magnification of two 
diameters. Note that the opening angle of the beam is twice as large as the closing 
angle. 

ical elements and especially by the memory of things seen before. 
When two lenses are disposed in series so that the rays of 
light coming from a point in the object pass through both lenses 
before coming to a focus, we find the possibilities shown in Figs. 3, 
4 and 5. In the figures it will be seen that the image produced 




FIG. 5. Image formation by two lenses in series, with magnification of three 
diameters. Note that the opening angle of the beam is three times as large as the 
closing angle. 

when the first lens is in position so as to render the rays parallel 
(Fig. 4), is just five times as large as that produced when it is 
left out (Fig. 2), assuming that the second lens is capable of 
change so as to focus upon the same screen slightly divergent 



THE MICROSCOPE AND MICROSCOPIC METHODS IQ 

rays proceeding from the object. It will further be perceived 
that the sine of the angle of divergence of the beam proceeding, 
from the object varies directly with the magnification achieved, 
and further that the magnification in any such system is equal 
to the quotient of the sine 1 of the angle of divergence of the beam 
proceeding from the object, divided by the sine of the angle of 
convergence of the beam to form the image. This is capable 
of mathematical proof and is illustrated in the four figures. 
From these it is evident that magnification is a function of the 
relation of these two angles of the opening and closing limbs of 
the beam, and that the intermediate course of the rays, whether 
parallel, convergent or divergent, is negligible in this computation. 
If the second lens be that of the eye and an image is to be formed 
on the retina, then the rays proceeding from a point must be ren- 
dered parallel, or approximately so, by the first lens. This is the 
arrangement which exists in the simple microscope or in the ordi- 
nary reading glass. The magnification achieved by such a simple 
microscope is measured by the relation between the magnitude 
of the image on the retina when the lens is employed, and the 
size of such an image when the lens is left out of the path of the 
light. The value of the reading glass, entirely aside from con- 
siderations of magnification, in conditions of hyperopia and pres- 
byopia is also evident from these figures, as it of course renders 
the rays coming from a near point more nearly parallel, and thus 
enables the refracting media of the presbyopic eye to bring them 
to a focus. 

So far we have been employing in our discussion the ideal 
lens, one which refracts all light equally and brings to a focus in 
one plane all rays proceeding from one plane in the object. As 
a matter of fact the ideal lens in this sense does not exist. The 
simple convex lens has many serious optical defects. 

1 In the figures, as drawn, this statement actually applies to the tangents of the 
angles designated, rather than the sines. However, for very small angles the sine 
and tangent are approximately equal. The use of the term sine finds its complete 
justification in the fact that the plane at which the rays are bent is not flat but is 
the segment of a sphere or its optical equivalent. 



2O 



BACTERIOLOGY 



Points in the same plane in the object are imaged by the simple 
lens on a curved surface, the segment of a spherical surface. 
This defect is known as spherical aberration. It is diminished 
to some extent by combining convex and concave lenses and the 
correction may be changed by altering the distance between these 
component lenses, as, for example, in an objective equipped with 
a correction collar. Objectives corrected in respect to spherical 
aberration are designated as aplanatic. Restriction of the size 
of the field is also an important factor in making it appear flat. 

Light of different wave lengths (different colors) is refracted 
to a different degree by the simple lens, so that, for example, the 
violet rays are brought to a focus earlier than the red rays, with 






FIG. 6. Microscope objectives showing the component parts of the objective 
lens system. (After Leitz.} 

the remainder of the spectrum spread out between. This defect 
is known as chromatic aberration. It is corrected to a very con- 
siderable extent by combining biconvex lenses of crown glass 
with plano-concave lenses of flint glass (achromatic objectives), 
to a still nicer degree by combinations of lenses of several different 
kinds of glass together with a lens of fluorite (apochromatic ob- 
jectives); and finally, when desired, chromatic aberration may 
be wholly avoided by employing mono-chromatic light. 

A third defect of lenses is known as diffraction, which is a 
phenomenon giving rise to a whole group of less luminous second- 
ary images around the principal image. The influence of dif- 



THE MICROSCOPE AND MICROSCOPIC METHODS 



21 




FIG. 7. Sectional view of a compound microscope illustrating the course of 

) and indic 
After Leitz.} 



IG. 7. econa v 

two beams preceding from two points in the object (P and Q) and indicating the 
subjective interpretation of the image formed on the retina. ( 



22 BACTERIOLOGY 

fraction is most evident when the surfaces of the lens are rough- 
ened by scratches or by presence of dust, but even the most per- 
fect lens systems are not wholly free from diffraction phenom- 
ena. Some of these defects will require brief consideration in 
our discussion of the compound microscope. 

In the modern compound microscope the beam of light pro- 
ceeding from a point in the object is refracted by the lens system 




FIG. 8. Image formation in the compound microscope. Compare with Fig. 9 

of the objective (Fig. 6) so as to render the rays slightly conver- 
gent. Near the upper end of the tube of the microscope these 
rays are further refracted by the lower lens of the eye-piece and are 
converged and brought to a focus in the interior of the eye-piece. 
A screen placed at this level would show a real image, and any 
pattern (for example an eye-piece micrometer) inserted in the 




FIG. 9. Image formation in the compound microscope with an eye-piece of 
higher power. Observe that the increased magnification is accomplished by narrow- 
ing the beam of light which enters the eye and so diminishing the size of the closing 
angle. Compare with Fig. .8. 

eye-piece at this level is readily fused with the microscopic field. 
Continuing in a straight line the rays diverge from this focus to 
reach the upper lens of the eye-piece. In traversing this lens 
they are again refracted and made parallel so that they will 
enter the eye and be brought to a focus on the retina. The paths 
of two beams of light, one proceeding from the center of the mi- 
croscopic field and one from its periphery, are illustrated in Fig. 



THE MICROSCOPE AND MICROSCOPIC METHODS 23 

8. Fig. 9 shows the change which is introduced by the use of 
an eye-piece of higher magnifying power. 

It will be noted that the objective and lower lens of the eye- 
piece bring the beam to a focus forming a real image, and that 
the rays diverging again from this image are again brought to a 
focus on the retina by the upper lens of the eye-piece and the op- 
tical structures of the eye. The magnification represented in the 
first image is the quotient of the sine of the angle of the opening 
limb of the beam divided by the sine of the closing angle. The 
subsequent magnification between this and the eye is the quotient 
of the sine of the opening angle of the rays proceeding from this 
image divided by the sine of the closing angle of the rays approach- 
ing the retina. The closing angle at the formation of the first 
image and the opening angle of the beam proceeding from it are 
obviously equal, so that the total magnification equals the sine 
of the first opening angle divided by the sine of the last closing 
angle in the system. It will be noted that the eye-piece of higher 
power narrows the beam and decreases the closing angle. 

In the above discussion, the refractive index of the vitreous 
humor has been disregarded. This is not the same as that of 
air (in reality it is about 1.3) and the peripheral beam is there- 
fore bent toward the axis of the eye instead of proceeding in its 
former direction, the magnification being thereby reduced by 
precisely the fraction 



refractive index of air 



or 



refractive index of vitreous 1.3 

This brings us to a definition of numerical aperture. The 
numerical aperture of the closing limb (n.a.) is the sine of half 
the angle of the converging beam multiplied by the refractive 
index of the medium (in this instance the vitreous humor). 
This is commonly designated as n.a. The numerical aperture 
of the opening limb of the beam (N.A.), proceeding from a point 
in the object to the objective, is the sine of half the angle of this 
beam multiplied by the refractive index of the medium through 



BACTERIOLOGY 



which it passes. This is commonly designated as N.A. Many 
desirable properties of objectives, other than magnification, such 
as brilliancy of illumination, definition, and resolution in depth, 
also depend upon the numerical aperture, which is therefore 
perhaps the most important single feature of objectives of high 
power. 




FIG. io. Central il- 
lumination by a narrow 
beam. Three beams of 
parallel rays, such as 
might come from a large 
white cloud, are repre- 
sented. Note that these 
rays reach the object as 
almost vertical rays, 
varying from the vertical 
by only a narrow angle. 
Compare with Fig. 15. 



FIG. 1 1 . Illumination 
by a hollow cone of light 
converging upon the object 
at a wide angle, by use of 
the central spot stop. 
Compare with Fig. 14, and 
with Fig. 16. 



FIG. 1 2 . Illumina- 
tion by a broad beam 
converging upon the ob- 
ject at a wide angle. 
Only a few beams of par- 
allel rays from a distant 
point source of light are 
represented in the figure. 
Compare with Fig. 17. 



Another important optical part of the bacteriological micro- 
scope is the substage illuminating apparatus, consisting of the 
mirror, the iris diaphragm and the condenser. These are neces- 
sary to illuminate minute objects so that they may be satis- 
factorily studied at high magnifications. By the use of the iris 
diaphragm and of the central spot stop, the ordinary condenser 
may be made to furnish three different kinds of illumination, (i) 



THE MICROSCOPE AND MICROSCOPIC METHODS 25 

central illumination by a narrow beam, (2) illumination by a hollow 
cone of light converging on the object at a wide angle, an ex- 
ample of dark-field illumination, and (3) intense illumination by a 
broad beam converging at a wide angle upon the object. These 
possibilities are illustrated in Figs. 10, n and 12. Dark-field 
illumination is obtained in a more satisfactory manner by em- 
ploying a special condenser made for the purpose, illustrated in 
Figs. 13 and 14. The way in which these different methods of 




FIG. 13. Dark-field condenser showing optical FIG. 14. Optical parts of 

parts and centering mechanism. (After Leitz.} the dark-field condenser with 

object slide .and microscope 
objective with funnel stop in 
position. The path of light 
rays is indicated by the dotted 
lines. (After Leitz.) 

illumination affect the visibility of a colorless refractive object 
is illustrated in Figs. 15, 1 6 and 17. 

Visibility of Microscopic Objects. In the use of the mi- 
croscope it is necessary to pay some attention to the factors upon 
which visibility depends. An object may be distinguished and 
perceived by the eye only when the light coming from the object 
differs from that coming from its surroundings either in quantity 
or in quality, and the greater the extent of this difference the 



26 BACTERIOLOGY 

more distinctly visible will the object be. Uncolored trans- 
parent objects are visible by virtue of their ability to refract light 
and so to present darker and lighter zones. If the surrounding 
medium possess the same refractive power as the colorless trans- 
parent object, the latter is invisible. 1 Microscopic objects may 
conceivably be invisible or so nearly invisible as to have escaped 




FIG. 15. Showing the manner in which the "dark outline picture" is produced. 

(After A. E. Wright.) 

detection for this very reason. If, however, the object be sus- 
pended in a medium of lower refractive index, then it may be de- 
nned by light and shade, and it is most clearly defined when illu- 
minated in one of two ways, either by a rather narrow direct beam 
of light passing from behind it directly toward the eye, in which 
case the object is denned by dark outlines upon a white field; or by 

1 This may be illustrated fairly well by immersing clean, perfectly clear glass 
beads in oil of cedar wood. 



THE MICROSCOPE AND MICROSCOPIC METHODS 27 




FIG. 16. Showing the manner in which the "bright outline picture" is produced 

(After A. E. Wright.} 




FIG. 17. Showing the manner in which the outlines are obliterated when an object 
is illuminated by a homogeneous illuminating field. (After A. E. Wright.} 



28 BACTERIOLOGY 

oblique beams directed at an angle from the sides, when the object 
is defined by bright outlines on a dark background. If, how- 
ever, the object be illuminated from all sides or from behind and 
from both sides by light of similar intensity, its outlines become 
less distinct and may even be completely obliterated so that the 
object becomes invisible. These facts may be crudely illustrated 
by holding a test-tube full of water, (i) between the eye and a 
window, (2) between the eye and a dark wall between two win- 
dows, and (3) against the center of the window pane. Their 
importance in microscopy may be readily illustrated by examining 
a simple preparation of living bacteria, (i) with the iris diaphragm 
nearly closed, (2) with the dark-field condenser, and (3) with the 
ordinary condenser with the iris wide open. It will be evident 
that the third arrangement is fatal to the definition of colorless 
transparent microscopic objects. It will also be observed that 
the dark field offers an advantage in the ease with which the ob- 
jects can be seen, the small luminous outline on the dark back- 
ground being more distinct then the dark outline on the luminous 
background. The former might be compared in this respect 
to a star at night, and the latter to a sun spot in the day- 
time, which though many times larger may not be readily 
perceived. 

The method of making objects visible by a difference in 
quality of light (color) usually involves the necessity of staining. 
Colored preparations have certain very important advantages 
for microscopic study. If an object can be differentially colored, 
that is, stained a different color or a different shade of the same 
color from the material by which it is surrounded, it becomes 
clearly visible even in the absence of different refractive power. 
Refraction may be largely eliminated by replacing the fluids of 
the preparation by other fluids of high refractive index, such as 
cedar oil or balsam, and this elimination of refraction eliminates 
the opacity of the preparation, " clears" it, and makes possible the 
distinct definition of minute objects situated in the deeper optical 
planes of the preparation. A proper appreciation of this mi- 



THE MICROSCOPE AND MICROSCOPIC METHODS 2Q 

croscopical principle will at once suggest the importance of differ- 
ential staining methods in microscopy. 

The Bacteriological Microscope. The bacteriological micro^ 
scope consists of a tubular body which carries the optical parts, and 




FIG. 1 8. Microscope. 

which can be raised or lowered for focusing. The objectives should 
be three in number, and should be attached to the body by means 
of a triple nose-piece, which permits any objective to be turned into 
the optical axis at will. The eye-piece slips into the upper and 
opposite end of the body or tube. The arrangements for focus- 




30 BACTERIOLOGY 

ing consist of a rack and pinion which accomplish the coarse ad- 
justment, and a more delicate fine adjustment. The stage, upon 
which the objects to be examined are placed, has an opening in 
the middle. In this opening an iris diaphragm and Abbe con- 
denser are inserted. The iris diaphragm enables one to alter 
the size of the opening as desired. Beneath the stage is a mov- 
able mirror, of which one side is plane and the other concave. 
All of these parts are supported on a short, heavy pillar which 
is fixed in the horseshoe-shaped base. 

The essential parts of the microscope are, of course, the eye- 
piece (German, Ocular), and the 
objective. Objectives are given 
various names by different makers, 
for instance, A, B, C, etc., or i, 2, 
3, etc.; or they are named accord- 
ing to their focal distances, as f 

FIG. 19. inch, J inch, f inch, etc. In bac- 

Abbe Condenser. On the right side the, . , . , 

figure gives a sectional view. tenological work a rather low 

power" | or f inch objective, an 

ordinary "high power" J to f inch dry objective, and a high power 
-jV inch oil-immersion objective are needed. The magnification 
with the f or f inch objective is about 75 to 100 diameters; with 
the J to | inch 400 to 700 diameters; with the yV immersion 
750 to 1,000 diameters. The magnification varies according to 
the eye-piece used, as well as with the objective. A i inch and 
if inch eye-piece (Leitz No. 2 and No. 4) serve well for most 
purposes. The eye-pieces are Usually named arbitrarily, like the 
objectives. The oil-immersion objective is used in the exami- 
nation of bacteria where a very high power is desired. A layer 
of thickened oil of cedar-wood is placed between the lower sur- 
face of the objective and the upper surface of the glass covering 
the object under examination. The oil must be wiped away 
from the surface of the objective when the examination is finished. 
For this purpose the soft paper sold by dealers in microscopical 
apparatus serves admirably. Care must be taken not to scratch 



THE MICROSCOPE AND MICROSCOPIC METHODS 31 

the lower surface of this objective. Oil of cedar- wood furnishes 
a medium having nearly the same refractive index as the glass of 
the lens and the glass on which the object is mounted, and it ob^ 
viates the dispersion of light which takes place when a layer of 
air is interposed between the objective and the object, as happens 
with the ordinary dry lens. 

The microscope should be placed in front of the observer on 
a firm table. The observer should be able to bring the eye easily 
over the eye-piece when the tube of the microscope is in vertical 
position. Daylight should be employed if possible. When arti- 
ficial illumination is necessary, an ordinary lamp, a Welsbach 
burner or an incandescent electric light may be used. It is best 
to modify the artificial light by inserting a sheet of blue glass be- 
tween the light and the mirror. 

In order to focus upon any object, having first secured a satis- 
factory illumination with the mirror, it is best, beginning with 
the low power and using the coarse adjustment for focusing, to 
bring the objective quite close to the object, and then, with the 
eye in position, to raise the tube until the object comes into 
focus. The exact focusing is done with the fine adjustment. 
The observer should keep both eyes open when using the micro- 
scope, and should be able to use either eye at will. 

All measurements of microscopic objects are expressed in 
terms of a micromillimeter. This is one-thousandth of a milli- 
meter (o.ooi mm.), which is about -^-^^-Q-Q of an inch. This unit 
is designated as a micron, and is denoted by the Greek letter /*. 
For example, 5 /* = 0.005 mrn - = 5~oV<r inch. 

The Platinum Wire. The substance under examination 
is usually placed upon thin slips of glass called cover-glasses. The 
material is spread over the cover-glass by means of a platinum 
wire which has been fixed in a glass rod about six inches long. 
Such a platinum wire is used constantly in doing bacteriolog- 
ical work. The platinum wire must be stiff enough not to 
bend too easily, and yet it should not be so large that it will 
not cool rapidly after heating. A good size for most pur- 



BACTERIOLOGY 



O 



poses is No. 28, English standard gauge, diameter .014 inch. 
The wire may be straight throughout its length, or the tip may 
be bent to form a loop (German, Oese). It is well to follow, from 
the beginning, certain rules which make the use of the platinum 
wire safe and accurate. Every time it is taken into the hand 
and before using it for any manipulation, heat it 
in the flame of a Bunsen burner or an alcohol lamp 
to a red heat; and always, after using and before 
putting it down, heat it again to a red heat. After 
the needle has become wet by dipping it in a fluid 
and is to be sterilized in the flame, it is necessary 
to avoid " sputtering" of the fluid by bringing the 
wet needle gradually to the flame, so as to dry the 
material adhering to it before burning it. This 
procedure must be done with great care when the 
wire has been dipped in milk or other substances 
containing oil. When the needle " sputters," as 
it is called, from too rapid heating, particles that 
have not yet been sterilized may be thrown some 
distance. On no account should the needle touch 
any object other than that which it is intended it 
should touch. With such a platinum wire, which 
has been properly sterilized, one can easily remove 
portions from a culture of bacteria, or from a 
fluid in which bacteria are supposed to be present. 
The glass rod in which the platinum wire is fixed 
should be held between the thumb and forefinger of the right 
hand like a pen. 

- Glass Pipettes. Sterile glass tubes drawn out to form slender 
capillaries, Pasteur pipettes, are very convenient instruments 
for handling bacteriological materials, and, for many kinds 
of work, really indispensible. They serve nearly all the pur- 
poses of the platinum wire and are capable further of use to 
transfer large quantities of fluid without contamination. They 
are also especially useful in collecting material from patients 



FIG. 20. Need- 
les used for inocu- 
lating media. 



THE MICROSCOPE AND MICROSCOPIC METHODS 33 

and at autopsy. Each pipette is sterilized and discarded after 

use. 

These pipettes are made by cutting glass tubing of a suitable 
size, diameter 3 mm. to 9 mm., into pieces from 20 to 40 cm. in 
length. The cut ends are smoothed in the flame. In the tubes 
of larger caliber it is well to make a constriction about 5 cm. from 
each end. Each end is plugged with cotton. The tubes are then 
sterilized by dry heat. By heating the middle of the tube in 
a blast lamp or over a large Bunsen flame, the glass may be 
softened and then drawn out into a capillary of any desired 




FIG. 21. Drawn-out tube pipettes of Pasteur, a, Plugged, sterile tube as 
kept in stock; b, the same heated at x in blast-lamp and drawn out; then sealed at 
x; c and d, completed pipettes; e, the same with bulb. (After Novy.) 

length and caliber. This is melted in the middle and severed 
by the flame, giving two pipettes. When a large capacity is 
desired a bulb may be blown in the tube between the capillary 
and the cotton plug. This requires a little practice. The tip 
of the pipette is finally broken off with aid of a file, sterilized by 
the flame and the pipette is ready for use. The various steps in 
the preparation of pipettes are illustrated in the figures (Fig. 21). 

The Hanging-drop. Living bacteria may be studied with 
the microscope while suspended in some fluid substance. The 
platinum loop having been heated to a red heat in the flame and 
having been allowed to cool, a small portion of the culture or 

3 



34 BACTERIOLOGY 

other material may be removed with it and deposited in the center 
of an ordinary cover-glass. The needle should again be sterilized 
in the flame. When cultures on solid media are to be examined, 
a small particle may be mixed with a drop of sterilized water or 
bouillon. The cover-glass should have been carefully cleaned and 
sterilized over the flame. The cover-glass with the small drop 
of fluid material held in sterilized forceps is now to be inverted 
over a sterilized glass slide, which has a concavity ground in the 
middle of it. Around the concavity, the slide should be smeared 
with vaseline. In this manner a small air-tight chamber is made. 
This slide and cover-glass is next put upon the stage of the micro- 
scope. A good dry lens, if of sufficiently high power, is more 
convenient for examining the hanging-drop than an oil-immer- 
sion. If the latter be used, having placed a drop of cedar-oil on 
the center of the cover-glass, and a good light having been 



FIG. 22. 

secured, the oil-immersion objective should be brought down 
upon this drop of oil. The beginner often experiences difficulty 
in focusing upon a hanging-drop. It is necessary to shut off 
most of the light by means of the iris diaphragm, for as has 
already been pointed out (page 28), colorless objects may be clearly 
seen only when illuminated either by a narrow central beam or 
by oblique illumination (dark-field). Often it is well to secure 
the focus roughly upon the extreme outer edge of the chamber, 
or to find the edge of the drop of fluid with the low power and 
then focus upon this edge with the oil-immersion objective. 
Above all things guard against breaking the cover-glass by forcing 
the objective down upon it. The motility of certain bacteria is one 
of the most striking phenomena to be observed in the hanging- 
drop. It is not to be confused with the so-called "Brownian 
movement' ' which is exhibited by fine particles suspended in a 



THE MICROSCOPE AND MICROSCOPIC METHODS 35 

watery fluid. It is well for the beginner to observe the character 
of the Brownian movement by rubbing up some carmine in^a- 
little water, and with the microscope to study the trembling 
motion exhibited by these particles of carmine. It will be noticed 
that, although the particles oscillate, no progress in any direction 
is accomplished unless there are currents in the fluid. Such cur- 
rents might give rise to the impression that certain bacteria 
possessed motility when they were, in fact, powerless to move 
of themselves. In the hanging-drop the multiplication of bacteria 
can be studied, the formation of spores and the development of 
spores into fully formed bacteria. The hanging-drop is also 
used extensively for the demonstration of the agglutination 
reaction with the bacillus of typhoid fever. Sometimes bacteria 
must be watched in the hanging-drop for hours, or even days, 
and it may be necessary to keep it at the temperature of the 
human body for this length of time. Various complicated kinds 
of apparatus have been devised for this purpose, but they are 
needful only for special kinds of work. When the hanging- 
drop preparation is no longer required, the slide and cover-glass 
should be dropped into a 5 per cent carbolic acid solution and 
afterward sterilized by steam. 

The Hanging-block. Hanging-block preparations, which 
were introduced by Hill, 1 make use of a cube of nutrient agar 
instead of a drop of fluid. Bacteria are distributed on the sur- 
face of the agar, which is then applied to a cover-glass, and 
mounted like a hanging-drop. The bacteria are thus kept in a 
layer close to the glass, where growth may be studied. 

The Microscopic Preparation for Study by Dark-field Illumi- 
nation. The central portion of a clean glass slide is encircled 
with a ring of vaseline, and a drop of the fluid to be examined 
is deposited on the clean surface in the center of the ring by means 
of a capillary tube. It is then covered with a clean large cover- 
glass so that the fluid spreads out in a moderately thin layer 
beneath the cover-glass and is confined on all sides by the 

1 Journal of Medical Research, Vol. VII., March, 1902. 



36 BACTEEIOLOGY 

vaseline, thus preventing evaporation and resulting currents in 
the preparation. 

Best results with the dark-field microscope are obtained 
only in a dark or dimly lighted room. An electric arc or a power- 
ful gas-light may be employed as the source of light, and it is well 
to put a flask of water between the light and the microscope to 
eliminate the heat-rays. The substage condenser of the micro- 
scope is replaced with the special dark-field condenser and this 
is carefully centered. A large drop of immersion oil is placed on 
the upper surface of the condenser. The slide is carefully placed 
upon the stage so that the oil fills in completely the space between 
the condenser and slide and remains free from air bubbles. 
The preparation is then ready for examination. Objectives of 
numerical aperture wider than i.o cannot be successfully used 
with the ordinary dark-ground condensers and therefore it is 
necessary to stop down the aperture of the oil-immersion objec- 
tive before using it. A special funnel stop is furnished for this 
purpose. When this has been "attached the preparation may be 
studied with the oil-immersion objective in the usual way. Skill 
in this method of studying unstained microbes is quickly acquired, 
offering, as a rule, less difficulty than the method of central 
illumination which is employed for the hanging- drop and hanging- 
block. 

Smear Preparations for Staining. The examination of 
bacteria with the microscope is carried out to a very large extent 
by means of smears made upon thin slips of glass. Such slips 
of glass are generally called cover-glasses. It is best to obtain the 
kind sold by dealers as No. i, f inch squares. 

The cover-glass may be cleaned best by immersion in a mix- 
ture of sulphuric acid and bichromate of potassium solution, and 
afterward washed thoroughly in distilled water, and finally in 
alcohol. A stock of clean cover-glasses may be kept in a bottle 
of alcohol, or perhaps preferably in alcohol containing 3 per cent 
of hydrochloric acid. 



THE MICROSCOPE AND MICROSCOPIC METHODS 37 

CLEANING FLUID. 

Potassium bichromate 40 grams. 

Water 150 c.c. 

Dissolve the bichromate of potassium in the 
water, with heat; allow it to cool; then add 
slowly and with care sulphuric acid, com- 
mercial 230 c.c. 

When they are needed for use they should be wiped clean 
with a piece of linen cloth. As a rule, cover-glasses cleaned in 
this way still retain a small amount of oily matter on their surfaces, 
sufficient to prevent the proper spreading of a drop of water. 
This difficulty may be overcome by passing each glass several 
times through the flame. It is better, when time permits, to fill 
an Esmarch dish with clean cover-glasses and then heat them in 
the oven at 200 C. for half an hour. Cover-glasses treated in this 
way will allow the droplet of bacterial suspension or other material 
to spread perfectly. They must be carefully preserved in a 
covered dish from which they are to be removed only by clean 
(flamed) forceps. Carelessness in this matter may necessitate 
recleaning of the whole lot of cover- glasses. 

An ordinary pair of fine forceps may be used to pick up the 
cover-glass and insert it between the blades of such special forceps 
as those of Cornet or of Stewart. Perhaps the most convenient 
style of forceps is that devised by Novy, provided with a clasp. 
Bacteria may be placed upon the cover-glass by allowing the 
glass to fall upon one of the colonies of bacteria, on a gelatin or 
agar plate (see page no), which will adhere to it in part, produc- 
ing an " impression preparation" (German, Klatschpreparat). 
Such a preparation, after drying in the air, is to be fixed by pass- 
ing it through the flame three times. (See below.) The forceps 
with which it is handled should be sterilized in the flame. 

Generally bacteria contained in fluids, like sputum, or taken 
from the surface of a culture, are smeared over the cover-glass 
by means of the platinum wire or loop, which must be heated to 
a red heat before and after the operation. Such preparations 



BACTERIOLOGY 



are called smear, cover-glass, cover-slip, or film preparations. 
When the material to be spread is thick or very viscid, a small 
drop of distilled water must first be placed in the center of the 
cover-glass so as to dilute it. Beginners generally take too much 




FIG. 23. Cornet forceps for cover-glasses. 

material on the wire. As thin a smear as possible is made. It 
is allowed to dry in the air; this should occupy a few seconds. 
The drying may be hastened 4 by holding the forceps with the 
cover-glass a long distance above the flame, at a point where 
the heat would cause no discomfort to the hand. Having dried 




FIG. 24. Stewart forceps for cover-glass. 

the preparation, it is to be passed through the flames of a Bunsen 
burner or alcohol lamp three times, taking about one second for 
each transit. The heat of the flame serves to dry the bacteria 
upon the cover-glass and fix them permanently in position; it is 
not sufficient, however, when applied in this manner, to kill all 



FIG. 25. Novy's cover-glass forceps withe clasp. (After Novy.) 

kinds of bacteria, especially those containing spores. After it 
has been passed through the flame three times the preparation 
may be stained with one of the aniline dyes, and after washing 
in water and drying, may be mounted, face down, in Canada 



THE MICROSCOPE AND MICROSCOPIC METHODS 



39 



balsam upon a glass slide. It makes a suitable object to be 
examined with the oil-immersion objective. The slide is a thin 
slip of glass, 3 inches by i inch, with ground edges. 

The smear preparation may equally well be made directly 
upon the glass slide provided this be cleaned and heated to insure 
a clean surface free from oily matter. The fixation in the flame 
must then occupy a longer time than with the small and thin 
cover-glass. Such preparations have the advantage that several 
may be made upon one slide, and that after staining them they 
may be examined in cedar-oil, with the oil-immersion lens, 
without the use of the cover-glass and Canada balsam. They 
are also less readily broken in handling. The forceps of Kirk- 
bride will be found convenient when staining on the slide. The 




FIG. 26. Kirkbride forceps for holding slides. 

aluminium dish devised by Krauss, 1 or some similar dish, will 
be found useful when the stain has to be heated. Experiments 
have shown that the ordinary method of fixation in the flame, 
when applied to bacteria spread upon slides, has little effect on 
the vitality of many species. The beginner is, therefore, advised 
to make his preparations on cover-glasses. 

When very resistant or dangerous pathogenic bacteria are 
being handled, after fixation by heat upon the slide or cover- 
glass, the preparation may, if desired, be immersed in i-iooo 
solution of bichloride of mercury long enough to kill the bacteria, 
without injuring the preparation or its staining properties. 

Staining Solutions. The staining of bacteria is done for the 
most part with the aniline dyes. The object of staining bacteria 
is to give them artificially some color which makes them distinct 

1 Krauss, Jour. A.M. A., Apr. 6, 1912, Vol. LVIII, p. 1013. 



4O BACTERIOLOGY 

and easily visible without imparting this color to the substance 
or medium in which they are imbedded. The substances known 
as aniline dyes are derivatives of coal-tar, but not always of ani- 
line. These dyes are of great importance in bacteriological 
work. Their number is very large, but only a few are in common 
use. It is important to have the purest, and those obtainable 
from Griibler are reliable. 

It is simplest to classify the aniline dyes as acid or basic. 
Eosin, picric acid and acid fuchsin are acid dyes; they tend to stain 
tissues diffusely. Fuchsin, gentian-violet and methylene blue are 
basic dyes; they have an affinity for the nuclei of tissues and for 
bacteria; they therefore are the dyes used chiefly in bacteriolog- 
ical work. The other varieties may be employed as contrast- 
stains; another contrast-stain frequently used is Bismarck brown. 
It is best to keep on hand saturated solutions of the aniline dyes 
in alcohol, which are permanent, but cannot be employed directly 
for staining. In order to prepare the simple staining solutions, 
the alcoholic solution is diluted about ten times, or so as to make 
a liquid which is just transparent in a layer about 12 mm. in 
thickness, after filtering. These watery solutions deteriorate 
after a few weeks. 

Fuchsin and gentian-violet stain rapidly and intensely. 
Methylene blue works more slowly and feebly; it is to be pre- 
ferred where the bacteria occur in thick or viscid substances, 
like pus, mucus, and milk. 

Aniline-water Staining Solutions. The intensity with which 
aniline dyes operate may be increased by adding aniline oil to 
the solution: 

Aniline oil 5 c.c. 

Water 100 c.c. 

Mix, shake vigorously, filter through wet filter paper. The fluid 
after filtration should be perfectly clear. Add 

Alcohol 10 c.c. 

Alcoholic solution of fuchsin (or gentian violet, or 

methylene blue) i c.c. 



THE MICROSCOPE AND MICROSCOPIC METHODS 41 

Aniline-water staining solutions do not keep well, and need to 
be freshly prepared about every two weeks. The applications 
of the aniline-water stains will be given under separate headings7 
In general, however, they are employed where a stain of unusual 
power is required. 

Carbol-fuchsin. The intensity of staining may also be in- 
creased by the presence of carbolic acid. The most common 
example of this is carbol-fuchsin. 

Saturated alcoholic solution of fuchsin 10 c.c. 

5 per cent aqueous solution carbolic acid 100 c.c. 

This solution keeps for some months. It is employed especi- 
ally where very intense action is required, as in staining spores, 
flagella, and acid-proof bacteria. 

Loffler's Methylene Blue. A very useful solution, which 
keeps well, isLoffler's alkaline methylene blue: 

Saturated alcoholic solution of methylene blue. . 30 c.c. 
1-10,000 aqueous solution of potassium hydroxide 100 c.c. 

This solution stains more intensely than simple methylene blue, 
and also gives rise to useful differential staining in smears and 
even in sections of tissue. 

Nocht-Romanowsky Stain. This requires two solutions, one 
of ripened alkaline methylene blue, the other of eosin. 

Solution i. 

Methylene blue i . o gram. 

Sodium carbonate 0.5 gram. 

Distilled water 100.0 grams. 

Heat at 60 C. for two days until solution shows a slight purplish 
color. 

Solution 2. 

Eosin, yellowish, water soluble i .o gram. 

Distilled water 100.0 c.c. 

In staining, a few drops of each of these solutions are mixed with 
about 10 c.c. of distilled water in an Esmarch dish, and the smear, 



42 BACTERIOLOGY 

which has previously been fixed in absolute methyl alcohol, is 
floated on this mixture for about ten minutes. Considerable 
practice is necessary before the best results are obtainable. 
The method is especially useful in staining blood films, and 
protozoa in blood, in feces or in culture. 

Leishman's Stain. Leishman has utilized the principle of 
Jenner's stain 1 and has added to it the important additional 
constituents found in polychrome methylene blue by substituting 
this for the ordinary methylene blue used by Jenner. 

Solution A. To a i per cent solution of medicinally pure 
methylene blue in distilled water add 0.5 per cent sodium car- 
bonate and heat at 65 C. for 12 hours, then allow it to stand 10 
days at room temperature. 

Solution B. Eosin extra B. A. (Griibler) o.i per cent solution 
in distilled water. 

Mix Solutions A and B in equal amounts and allow to stand 
six to twelve hours, stirring at intervals. Filter and wash the 
precipitate thoroughly. Collect, dry and powder it. 0.15 gram 
is dissolved in 100 c.c. of pure methyl alcohol to form the staining 
solution. It keeps perfectly for at least five months. To stain, 
cover the dried but unfixed film of blood with the staining solu- 
tion. After 30 to 60 seconds add about an equal amount of 
distilled water. Allow this mixture to act for five minutes. 
Wash in distilled water for about one minute, examining the 
specimen mounted in water under the microscope. Blot, dry 
thoroughly, mount in balsam, or preserve the specimen as an 
unmounted film. 

Numerous imitations or modifications of Leishman's stain 
have been described. 

Giemsa's Stain. This stain contains certain of the essential 
constituents of polychrome methylene blue and eosin, the whole 
being dissolved in a mixture of glycerin and methyl alcohol. 
Giemsa's Azur I is the substance methylene azure and his Azur 

1 Jenner (Lancet, 1899, I, p. 370) first employed the solution of eosin and methy- 
lene blue in methyl alcohol as a stain for blood films. 



THE MICROSCOPE AND MICROSCOPIC METHODS 43 

II is this substance mixed with an equal amount of methylene 
blue. His Azur II-eosin is the compound precipitated when 
aqueous solutions of Azur II and eosin are mixed. The Giemsa 
solution is made according to the following formula : 

Azur II-eosin 3.0 grams. 

Azur II 0.8 gram. 

Glycerin 250 . o grams. 

Methyl alcohol 250.0 grams. 

Dissolve the powdered dyes in the glycerin at 60 C.; then add 
the methyl alcohol previously heated to the same temperature. 
After mixing, let it stand 24 hours at room temperature, and 
filter. To stain, mix one drop of this solution with i c.c. of water 
and immerse the film, previously fixed, for 15 minutes to 24 hours. 
Direct preparation of Romanowsky Stains. In a study of the 
essential constituents of the Romanowsky stain, MacNeal 1 
found both methylene azure and methylene violet to be present 
and participating in the nuclear staining. The preparation of 
solutions directly from the pure dyes, methylene azure, methy- 
lene violet, methylene blue and eosin, has been recommended 
as the best manner of preparing these staining solutions, as the 
proportion of the various constituents may be varied at will to 
obtain various kinds of differentiation. As a routine blood 
stain for study of leukocytes and staining of hematozoa, the fol- 
lowing is recommended : 

Solution A . 

Methylene azure 0.3 

Methylene violet (Bernthsen's, insoluble in water) . o. i 

Methylene blue 2.4 

Methyl alcohol, pure 500. o 

Solution B. 

Eosin, yellowish, water soluble 2.5 

Methyl alcohol, pure 500 . o 

These solutions keep for at least a year. They are mixed in equal 
parts and diluted by the addition of 25 c.c. of methyl alcohol to 

1 Journ. Infectious Diseases, Vol. Ill, 1906, pp. 412-433. 



44 BACTERIOLOGY 

each ioo c.c. of the mixture. This final mixture is employed 
in the same manner as Leishman's stain. It keeps for a few 
months. 

Method of Staining Cover-glass Preparations. (a) A smear 
preparation of bacteria having been made and fixed in the manner 
above described, and a watery solution of either fuchsin, gentian 
violet or methylene blue having been prepared, the cover-glass 
is to be dropped into a dish containing the dye, or the dye may 
be dropped upon the cover glass held in the forceps. 

(b) Allow the stain to act for about thirty seconds. 

(c) Wash in water. 

(d) Examine with the microscope in water directly or after 
drying and mounting in Canada balsam. 

The rapidity and intensity of staining may be increased by 
warming the solution slightly. The bacteria will usually appear 
more distinct if, directly after pouring off the stain, the prepara- 
tion is rinsed for a few seconds in i per cent solution of acetic 
acid, and then thoroughly washed in water. The acetic acid 
solution serves to remove in a measure any color which has 
been imparted to the background, and which is undesirable. 

Preparations that are mounted at first in water may be made 
permanent by moistening the edge of the cover-glass so that it 
may be easily removed from the slide, then drying and mounting 
in Canada balsam. Cover-glass preparations which have been 
stained are examined with the oil-immersion objective, employ- 
ing the plane mirror, having the iris diaphragm open and the 
condenser close to the lower surface of the glass slide. The 
purpose is to obtain the most intense illumination possible over 
a small field. 

Gram's Method. Cover-glass preparations, having been 
prepared and fixed in the usual manner (see page 38), are stained 
as follows: 

(a) Stain in aniline-water gentian violet solution, from two 
to five minutes. The intensity of the stain may be increased 
by warming slightly. 



THE MICROSCOPE AND MICROSCOPIC METHODS 45 

(b) Gram's solution, one and one-half minutes: 

Iodine , i gram. 

Potassium iodide 2 grams. 

Water 300 c.c. 

In this solution the preparation becomes nearly black. 

(c) Wash in alcohol repeatedly; the alcohol becomes stained 
with clouds of violet coloring matter; the alcohol is used as long 
as the violet color continues to come away, and until the prepara- 
tion is decolorized or has only a faint steel-blue color. 

(d) When desired, the specimens may be stained, by way of 
contrast, with a watery solution of Bismarck brown, dilute 
fuchsin or eosin. 

(e) Wash in water, and examine either in water directly or 
after drying and mounting in Canada balsam. Gram's method 
and its modifications should not be regarded as absolute means 
of distinguishing between Gram-positive and Gram-negative bac- 
teria in every case, as much depends upon the condition of the 
bacteria, and very much upon the technic of staining. When the 
Gram stain is used for diagnosis, it is well to put a smear of a 
known Gram-negative and a smear of a known Gram-positive 
organism on the same slide or cover-glass along with the un- 
known, and subject them all to the same technic. 

Some bacteria that are stained by Gram's method : 
Staphylococcus aureus, 
Streptococcus pyogenes, 
Micrococcus lanceolatus (of pneumonia), 
Micrococcus tetragenus, 
Bacillus of diphtheria, 
Bacillus of tuberculosis, 
Bacillus of leprosy, 
Bacillus of anthrax, 
Bacillus of tetanus, 

Bacillus welchii ( aerogenes capsulatus), 
Ray fungus of actinomycosis. 



46 BACTERIOLOGY 

Of these the tubercle bacillus and the bacillus of leprosy 
require a much longer exposure to the stain than other bacteria 
in the list. 

Some bacteria that are not stained by Gram's method : 
Gonococcus, 

Diplococcus intracellularis (meningitidis) , 
Micrococcus melitensis, 
Bacillus of chancroids (Ducrey), 
Bacillus of dysentery (Shiga), 
Bacillus of typhoid fever, 
Bacillus coli, 
Bacillus pyocyaneus, 
Bacillus of influenza, 
Bacillus of bubonic plague, 
Bacillus of glanders (Bacillus mallei), 
Bacillus proteus, 
Spirillum of Asiatic cholera, 
Spirillum of relapsing fever. 

Staining of Acid-proof Bacteria. A very large number of 
methods have been proposed for staining the tubercle bacillus, 
all of which depend upon the principle that, after adding to 
solutions of aniline dyes certain substances, like aniline water, 
carbolic acid, or solutions of ammonia or soda, the tubercle bacillus 
is stained with great intensity, and gives up its stain with difficulty. 
Solutions of acids will remove the stain from all parts of the prepa- 
ration excepting from the tubercle bacilli, which retain the dye, 
having once acquired it. The rest of the preparation may now 
be given a different color contrast-stain. 

Bacilli that resist decolorization by acids are called acid-proof 
or acid-fast. 

Some acid-proof bacteria: 
Bact. tuberculosis, 
Bact. leprae, 
Bact. smegmatis, 
Grass bacillus of Moeller, 



THE MICROSCOPE AND MICROSCOPIC METHODS 47 

Butter bacillus of Rabinowitsch, 

Certain strep to thrices, 

Certain bacilli common in the feces of cattle, 

Certain bacteria found in distilled water, 

Spores of many bacteria. 

Occasionally other bacteria, micrococci and horny epithelial 
cells are imperfectly decolorized, but their forms distinguish 
them from tubercle bacilli. Minute crystalline needles which 
have a shape like that of bacilli, are often encountered in sputum, 
but their nature will be recognized after a little practice. 

The stain for acid-proof bacteria is most frequently used for 
specimens of sputum from cases of suspected pulmonary tubercu- 
losis; it may be applied to other fluids and secretions equally 
well. It is not reliable, however, when applied to milk, as the 
oil present in milk interferes with its operation, and milk 
and its products quite often contain other acid-proof bacilli. 
The smegma of the external genitals also frequently contains 
acid-proof bacilli that are not tubercle bacilli. On this account 
all fluids and discharges from the genito-urinary tract need to 
be examined with particular care not to confuse tubercle bacilli 
with smegma bacilli. Too much reliance should not be placed 
on the possibility of distinguishing between tubercle and smegma 
bacilli by decolorizing in alcohol. In doubtful cases an animal 
should be inoculated. 

Patients should be given minute instructions concerning the 
collection of sputum. The bottle used should be new, wide- 
mouthed, clean, and kept tightly stoppered with a clean cork. 
The patient should be cautioned against allowing the expectora- 
tion to get on the outside of the bottle. Probably whatever 
risk is incurred by those who examine sputum comes chiefly 
from the outside of the bottle having been soiled with sputum 
containing tubercle bacilli. It is well to disinfect the exterior 
of the bottle when it is received at the laboratory. Often little 
white particles may be seen floating in the mucous portions of 
the sputum. These particles should be selected for the investiga- 



48 BACTERIOLOGY 

tion, and may be spread in a thin film on the cover-glass with the 
platinum wire, which is sterilized in the flame before and after 
using. The selection of the little white particles will be faciliated 
if the sputum be poured into a clean glass dish, which may be 
placed on a black surface. A form of porcelain dish is furnished 
by dealers, the bottom of which is black, and which is convenient 
for these manipulations. The smears may be made moderately 
thick as a larger amount of sputum may thus be examined in 
a short time. Uniform thickness is difficult to obtain and is not 
absolutely essential. It is hardly necessary to observe that the 
operator must be scrupulously careful not to contaminate the ma- 
terial under examination with any kind of extraneous matter. 
The cover-glasses and slides which are used should be new, and 
should have been cleaned with bichromate of potassium and 
sulphuric acid (see page 36). When the work is completed, the 
bottle containing the sputum should be sterilized by steam or 
boiling. 

Method for staining the tubercle bacillus: 

(a) The cover-glass or slide preparation is made, dried, and 
fixed by passing through the flame three times. 

(b) The cover-glass, held in forceps or in a watch-crystal is 
covered with steaming carbol-fuchsin for five minutes. If a 
slide is employed it may be conveniently stained in the Krauss 
staining dish, being turned face downward. 

(c) Wash in water. 

(d) Wash in alcohol containing 3 per cent of hydrochloric 
acid one minute, or longer if necessary to remove the red color. 

(e) Wash in water. 

(f) Stain with methylene-blue solution (see page 40) thirty 
seconds. 

(g) Wash in water. 

(h) Examine in water directly, and after drying and mounting 
in Canada balsam. If the preparation has been made on a slide 
it may be dried and examined directly in cedar oil with the iV in. 
objective. When the preparation is mounted in water, tubercle 



THE MICROSCOPE AND MICROSCOPIC METHODS 49 

bacilli may be obscured by refraction in the thicker portions of 
the smear. Tubercle bacilli take a brilliant red color; other_ 
bacteria and the nuclei of cells are stained blue. 

Of the numerous methods of staining tubercle bacilli only 
a few others can be mentioned. Aniline- water fuchsin, aniline- 
water gentian violet, or carbol-fuchsin may be used. The in- 
tensity of the stain must then be increased by warming the prepa- 
ration till it steams or boils, then allowing the warm stain to 
act on the specimens for from three to five minutes; the prepara- 
tion may also be left in the cold stain over night. Decoloriza- 
tion may be effected with a 25 per cent solution of sulphuric 
acid used till the red color disappears, or a 30 per cent solution 
of nitric acid, which operates very rapidly. If the red color 
persists after washing in water, dip in the acid again. After 
either acid the preparation is to be washed in alcohol until the 
last trace of the stain has been removed. An excellent de- 
colorizing agent is a 3 per cent solution of hydrochloric acid in 
alcohol, used for about a minute. The contrast stain may be 
omitted entirely if it is .desired. A suitable contrast stain after 
fuchsin staining is a solution of methylene blue; after gentian- 
violet staining, Bismarck brown. 

Those who have had experience in staining tubercle bacilli 
soon discover that the bacilli exhibit some differences in their 
resisting power to strong acids. One encounters occasionally 
bacilli that are perfectly stained side by side with others that are 
more or less completely decolorized. These facts show the 
necessity of practice with any method, and of exercising caution 
and judgment in making a diagnosis where the number of bacilli 
happens to be scanty. If tubercle bacilli are not found in the 
first preparation, other preparations should be made. Some- 
times a large number of cover-glasses must be examined. 

Various expedients have been devised to concentrate tubercle 
bacilli when only a small number may be present in a sample of 
sputum. Recently, antiformin (a preparation of chlorinated 
sodium hydroxide) has been employed for this purpose. The fol- 



50 BACTERIOLOGY 

lowing method is that of Williamson. 1 The sputum is measured 
and transferred to a clean flask of Jena glass. An equal volume of 
50 per cent antiformin is added, mixed with the sputum, and the 
mixture brought to a boil over the flame. This dissolves the 
sputum promptly. The material is then cooled and to each 10 c.c. 
of material in the flask 1.5 c.c. of a mixture of chloroform, one part, 
and alcohol, nine parts, is added. The mixture is thoroughly 
shaken. As a result the tubercle bacilli imbibe some of the chloro- 
form and become heavier. The material is next centrifugalized 
at high speed for 15 minutes, which separates it into three layers, 
antiformin above and chloroform below with the layer of sediment 
between the two. This layer is removed and mixed with egg 
albumen (egg albumen +0.5 per cent carbolic acid) on a slide 
and then spread into a smear between two slides. The smears 
are then dried and stained in the usual way. Instead of using 
albumen to fix the sediment to the slide, it is convenient to save 
some of the original sputum and mix it with the sediment for 
this purpose. 

Staining of Spores. The method is applicable to cover- 
glass preparations which may be prepared in the usual way from 
material supposed to contain spores. 

(a) After drying the smear on the cover-glass, fix it with heat 
by passing through the flame three times. 

(b) Float the cover-glass face downward on the surface of 
steaming hot carbol- fuchsin or aniline- water fuchsin for three to 
five minutes. 

(c) Wash in 3 per cent hydrochloric acid alcohol one minute, 
or less. 

(d) Wash in water. 

(e) Stain with watery solution of methylene blue half a minute. 
(/) Wash. 

(g) Dry. 
(h) Balsam. 

The spores are intensely stained by the fuchsin. The stain 
1 Williamson, Journ. A.M. A., Apr. 6, 1912, Vol. LVIII, p. 1005-07. 



THE MICROSCOPE AND MICROSCOPIC METHODS 51 

is removed from everything except the spores by the acid alcohol. 
The methylene-blue solution stains the bodies of the bacteria, 
the spores remaining brilliant red. There are various other 
methods for staining spores, but this procedure usually gives 
good results. The principle is the same as in staining the tubercle 
bacillus, except that more pains are needed to impregnate spores 
with the dye. 

When it fails, the cover-glass preparation may be treated by 
Moeller's method previous to staining. After fixation, the prep- 
aration is immersed in chloroform for 2 minutes, drained and 
dried in the air. It is then immersed in 5 per cent chromic 
acid for 2 minutes, washed thoroughly in water, and stained 
as above described. 

Staining of Capsules. The capsules which many bacteria 
possess, appear to be made of some gelatinous substance, which 
is difficult to stain. 

Method of Welch. (a) Cover-glass preparations are made 
in the usual manner. Pour glacial acetic acid over the film. 

(&) After a few seconds, replace with aniline-water gentian 
violet, without washing in water. Change the stain several 
times to remove all the acetic acid. Allow it to act three or 
four minutes. 

(c) Wash and examine in sa,lt solution 0.8 to 2.0 per cent. 
Bacteria are deeply stained, while their capsules are pale violet. 
This method has been recommended for staining the capsule 
of the pneumococcus. 

Methods of Hiss. i. (a) Cover-glass preparations are made 
in the usual manner, and fixed in the flame. 

(b) Stain for a few seconds in a half- saturated watery solu- 
tion of gentian violet. 

(c) Wash in 25 per cent solution of potassium carbonate in 
water. 

(d) Mount and study in the same. 

2. (a) Cover-glass preparations are made and fixed in the 
ordinary way. 



5 2 BACTERIOLOGY 

(b) Use the following stain, heated till it steams : 

Saturated alcoholic solution of gentian violet or fuchsin 5 c.c. 

Distilled water 95 c.c. 

(c) Wash in 20 per cent solution of cupric sulphate crystals. 

(d) Dry and mount in Canada balsam. 

The methods of Hiss are recommended to be used for bac- 
teria that have been cultivated on serum-agar with i per cent of 
dextrose. They have shown that many streptococci have cap- 
sules. The writer has had good success from the latter method, 
with preparations of the pneumococcus from animal tissues. 

Staining of Flagella. Flagella are among the most difficult 
of all objects to stain. The best-known method is that of 
Loffler. It is important to use young cultures (4 to 10 hours 
old), preferably on agar. 

(a) A small amount of the growth is gently mixed with a 
large drop of distilled water on a clean slide, so that the water is 
made very faintly cloudy. From the top of this drop one or 
two transfers are made to a second drop with a small platinum 
loop. From this second drop a loopful is transferred to a per- 
fectly clean (flamed) cover-glass, spread with minimum manipu- 
lation and dried quickly, high over the flame. 

(b) After drying, fixation is effected by passing through the 
flame three times, holding the cover-slip between the thumb and 
fore finger to avoid overheating. 

(c) The essential point in this method is the use of a mordant 
as follows : 

Tannic acid, 10 per cent solution 20 c.c. 

Saturated solution of ferrous sulphate 4 c.c. 

Saturated alcoholic solution of fuchsin i c.c. 

This solution should be freshly prepared from pure substances, 
and should be filtered at once after mixing. It may deteriorate 
in a few hours but sometimes keeps for a few days or weeks. 
A few drops are placed on the cover-glass, or the cover-glass is 



THE MICROSCOPE AND MICROSCOPIC METHODS 53 

placed, face down, in a dish containing the stain; it is then left 
for one to five minutes, warming slightly. 

(d) Wash in water. 

(e) Stain with aniline-water fuchsin, or carbol-fuchsin. 
(/) Wash in water. 

(g) Dry. 

(ti) Mount in Canada balsam. , 

(According to Loffler, certain bacteria require the addition of 
an acid solution, and certain others an alkaline solution, but many 
observers consider this unnecessary.) 

Another and very valuable method is that of Van Ermen- 



(a) Make and fix cover-glass preparations as in the preceding 
method. 

(b) Use the following mordant for one-half hour at room 
temperature or for five minutes at 50 to 60 C. 

Osmic acid 2 per cent solution i 

Tannic acid 10 to 25 per cent solution 2 

(c) Wash carefully in distilled water and then in alcohol. 

(d) Place for a few seconds in a 0.25 to 0.50 per cent solution of 
nitrate of silver "the sensitizing bath." 

(e) Without washing transfer to the "reducing and reinforcing 
bath": 

Gallic acid 5 grams. 

Tannic acid 3 grams. 

Fused potassium acetate 10 grams. 

Distilled water 350 c.c. 

(/) After a few seconds, replace the preparation in the nitrate 
of silver solution, in which it is kept constantly moving, till the 
solution begins to acquire a brown or black color. Some recom- 
mend leaving the preparation in the nitrate of silver solution for 
two minutes in the first place, and in the reducing bath for two 
minutes, without using the nitrate of silver solution a second time. 



54 BACTERIOLOGY 

(g) Finally wash in distilled water, dry, mount in Canada 
balsam. It is difficult to avoid the formation of precipitates; 
otherwise the results of this method are usually good. 

Wet Fixation of Protozoa. The fluid containing the protozoa 
is spread on a cover-glass or slide and immediately dropped upon 
a solution of the fixing agent, commonly sublimate alcohol heated 
to 60 C. This is prepared by mixing saturated aqueous solu- 
tion of mercuric chloride, 100 c.c., with absolute alcohol, 
50 c.c., and acetic acid, 5 drops. After a few minutes the prepa- 
ration is carefully washed in water, and passed through graded 
alcohols to harden. It may then be stained, dehydrated in 
graded alcohols, cleared in xylol and mounted in balsam. The 
preparation should not be allowed to dry at any stage of the 
process. 

Haidenhain's Iron Hematoxylin. The preparation to be 
stained by this method should be fixed in mercuric chloride or 
alcohol. The stain is prepared by dissolving hematoxylin crys- 
tals, i gram, in hot absolute alcohol 10 c.c., and then adding dis- 
tilled water 90 c.c. This solution is allowed to stand in an open, 
cotton-plugged bottle for about four weeks, and it is then diluted 
with an equal volume of water before using. The iron solution 
is made by dissolving 2.5 grams of ferric ammonium sulphate 
(lavender-colored crystals) in 100 c.c. of distilled water. The 
preparation to be stained is first soaked in the iron solution 
for four to eight hours, then rinsed and immersed in the hema- 
toxylin for twelve to twenty-four hours. It is again rinsed and 
now differentiated by immersion in the iron solution until black 
clouds cease to be given off. When the desired differentia- 
tion has been obtained the preparation is washed, dehydrated 
by passing through graded alcohols, and absolute alcohol, cleared 
in xylol and mounted in balsam. 

Preparation and Staining of Blood Films. Blood films are 
best made on clean, flamed slides. A small drop of fresh blood is 
received on the surface of one slide near one end. The end of 
another slide is applied to the first at an acute angle so that the 



THE MICROSCOPE AND MICROSCOPIC METHODS 55 

blood spreads laterally in the angle between the two slides. 
The second slide is then pushed along the surface of the first with 
the blood following it in the angle. The thickness of film may 
be regulated by varying the size of angle between the two slides. 

For staining blood films, either Leishman's or Giemsa's stain 
or some modification of them should be used as a general rule. 
After fixation in absolute alcohol, blood films may be stained 
with Loffler's methylene-blue or by Gram's method. 

Staining Bacteria in Tissues. Pieces of organs about i cm. 
in thickness may be taken. Alcohol is the best agent for pre- 
serving them. The hardening will be completed in a few days. 
It is best to change the alcohol. The amount of the alcohol must 
be twenty times the bulk of the tissue to be preserved. 

Ten parts of the standard 40 per cent solution of formalde- 
hyde, with 90 parts water make a good mixture for fixation; after 
twenty-four hours change to alcohol. 

Imbedding in Collodion or Celloidin. From alcohol the 
pieces of tissue are placed in equal parts of alcohol and ether 
twenty-four hours; thin collodion (ij per cent), twenty-four 
hours; thick collodion of a syrupy consistency (6 per cent) twenty- 
four hours. The specimen is laid upon a block of wood and sur- 
rounded by thick collodion, and then inverted in 70 per cent al- 
cohol. The collodion makes a firm mass, surrounding and perme- 
ating the tissue, and permits very thin sections to be cut. The 
soluble cotton sold by dealers in photographer's supplies serves 
as well as the expensive preparation known as celloidin. To 
make collodion, dissolve it in equal parts of alcohol and ether. 
Soluble cotton is also called pyroxylin, and is a kind of gun-cotton. 

Imbedding in Paraffin. (a) Pieces of tissue 2 to 3 mm. 
thick which have already been fixed in alcohol or formaldehyde 
are to be placed in absolute alcohol for twenty-four hours. 

(b) In pure xylol one to three hours. 

(c) In a saturated solution of paraffin in xylol one to three 
hours. 

(d) In melted paraffin having a melting-point of 50 C., 



56 BACTERIOLOGY 

which requires the use of a water-bath or oven, one to three 
hours. The xylol must be entirely driven off, and the tissue 
thoroughly infiltrated. 

(e) Change to fresh paraffin for one hour. 

(/) Finally, place the tissue in a small dish or paper box and 
pour the melted paraffin about it. Harden as quickly as possible 
with running water. It is important to fix the piece of tissue 
in a suitable position, if the position is of importance, before 
pouring in the melted paraffin. Sections of exquisite thinness 
may now be cut. The knife need not be wet. Paraffin im- 
bedding is especially desirable when serial sections are to be made. 

In order to mount the sections, proceed as follows: 

(a) Place the sections on water in a porcelain capsule. 
Warm slightly, when the sections will flatten nicely. Smear the 
surface of a slide with a very thin layer of Mayer's glycerin- 
albumen mixture. Dip the slide under the sections; lift them; 
and then drain off the water, leaving the sections in their proper 
positions. Let them dry for some hours in the incubator, and 
they will be firmly fastened to the slide. 

GLYCERIN- ALBUMEN MIXTURE (MAYER). 

Equal parts of white of egg and glycerin are thoroughly mixed, and then 
filtered. Add a little gum-camphor to preserve. 

(b) Dissolve out the paraffin in one of the numerous solvents 
(xylol, a few minutes) . 

(c) At this point the xylol should be washed off with absolute 
alcohol, and then 70 per cent alcohol and finally distilled water. 

(d) The section is stained. 

(e) Dehydrate in absolute alcohol. 
(/) Clear in xylol. 

(g) Mount in balsam. 

Section Cutting. Cutting is best done with an instrument 
called a microtome. The tissues may be imbedded in collodion 
or paraffin; or when they have been hardened with formaldehyde 



THE MICROSCOPE AND MICROSCOPIC METHODS 57 

they may be cut after freezing. Bacteria stain admirably in 
frozen sections. For routine work collodion imbedding will be 
found as convenient a process as any. Paraffin imbedding gives 
the thinnest sections. 

A microtome consists of a heavy, sliding knife-carrier, which 
moves with great precision on a level, and of a device for elevating 
the object which is to be cut, any desired distance after each ex- 
cursion of the knife. The thickness of the section will be the 




FIG. 27. Schanze microtome. 

distance which the object is elevated. The knife is kept wet 
with alcohol during the cutting of collodion sections, otherwise 
it is left dry. The microtome is usually provided with a special 
form of knife. A razor will serve nearly as well, after having 
had the lower side ground flat. If a razor is used, a special form 
of razor-holder must be attached to the microtome to receive the 
razor. Above all, it is necessary that the knives should be kept 
in good condition. Only occasionally will they need honing, using 
a fine water-stone or Belgian hone. The movement in honing 



58 BACTERIOLOGY 

should be from heel to toe, always placing the back of the knife 
next the hone when turning. The knife should be stropped fre- 
quently. The leather of the strop should be glued to a strip of 
wood to make a flat surface. The movement in stropping should 
be from toe to heel. Sections should be cut to a thickness of not 
more than 25 /*. Thinner sections (5 to 10 //) are to be desired. 

Staining of Sections. A watery solution of one of the aniline 
dyes is used fuchsin, gentian violet or methylene blue made 
by adding a few drops of the alcoholic solution to a dish filled 
with water. Loffler's solution of methylene blue serves very 
well. 

By this process most bacteria are stained; also the nuclei of 
cells; frequently, also, certain granules contained within some 
cells (German, Mastzellen), which may easily be mistaken for 
bacteria by the inexperienced (basophilic granules). 

(a) Place the section in the staining solution from two to five 
minutes. 

(b) Wash in water. 

(c) Place in a watery solution of acetic acid, i per cent for 
one minute. 

(d) Alcohol, one to two minutes; change to absolute alcohol. 
Touch the sections to blotting-paper to remove the superfluous 
alcohol. 

(e) Xylol until clear; xylol is to be preferred to other clearing 
agents, like oil of cloves, most of which slowly remove aniline 
colors. It has the disadvantage of not clearing when more than a 
trace of water is present; dehydration in alcohol must, therefore, 
be complete. The section should be removed from the xylol as 
soon as it is cleared; otherwise wrinkling occurs. 

(/) The section is placed upon a glass slide; a drop Canada 
balsam is placed upon it and then a cover-glass. The Canada 
balsam should be dissolved in xylol. 

The section is to be manipulated with straight or bent needles. 
The removal from xylol to the glass slide is managed best with a 
spatula or section-lifter. 



THE MICROSCOPE AND MICROSCOPIC METHODS 59 

The above statements apply to frozen sections or to sections 
imbedded in celloidin. Paraffin sections are preferably attached 
to the slide with glycerin-albumen. The different steps in the 
process follow in the same order. The stain may be poured on 
the slide, or the slide may be placed in a large dish full of staining 
fluid. (See page 44.) Celloidin sections may also be stained on 
the slide. If the section be well spread and flattened thoroughly 
with blotting paper, it will usually adhere to the slide, and is less 
likely to wrinkle. It must not be allowed to dry. 

Gram's Method may be applied to the staining of sections 
of tissues as well as to smears upon cover-glasses. 

(a) Place the section in aniline-water gentian violet, one to 
five minutes. 

(b) Rinse briefly in water.- 

(c) Iodine solution (see page 45), one and one-half minutes. 

(d) Alcohol, until decolorized to a faint blue-gray. 

(e) Xylol. 

(/) Mount on a slide in balsam. 

Weigert's Modification of Gram's Method, or Weigerfs 
Stain for Fibrin. (a) Place the section in aniline-water gentian 
violet solution, five minutes or more. 

(b) Wash briefly in water. 

(c) Place the section upon a slide by means of a section lifter; 
having straightened it carefully, absorb the water with blotting- 
paper. 

(d) Gram's solution (see page 45) one to two minutes. 

(e) Absorb the iodine solution with blotting-paper. 

(/) Add aniline oil, removing it from time to time with blotting- 
paper, and adding fresh aniline oil until the color ceases to come 
away. (Aniline oil serves in this connection both to decolorize 
and to dehydrate. It absorbs the water rapidly and efficiently. 
However, on account of its decolorizing tendency, it must be re- 
moved before the specimens can be mounted permanently.) 

(g) Add xylol; remove it with blotting-paper; and add fresh 
xylol several times, in order to extract the last trace of aniline oil. 



60 BACTERIOLOGY 

(ti) Mount in Canada balsam. 

This method is more convenient for the staining of sections 
than the Gram method. The results, however, are essentially 
the same as far as the bacteria are concerned; fibrin and hyaline 
material are stained blue, bacteria violet. It is often impossible 
to decolorize the nuclei completely without decolorizing the 
bacteria also. The parts of the nuclei which remain stained 
often present pictures that resemble bacteria, and which may 
lead to error if not recognized. Basophilic granules also retain 
the stain, as do the horny cells of the epidermis. These remarks 
apply also to Gram's method, except as regards fibrin. Very 
beautiful preparations can be obtained according to this or the 
Gram method when the sections have previously been stained 
in carmine; the nuclei will then be colored red, bacteria violet. 

Tubercle bacilli may be stained in sections as follows: 

(a) Use carbol-fuchsin, or aniline- water gentian violet for 
one-half to two hours with very gentle warming, or over night 
without warming. 

(b) Wash in water. 

(c) Decolorize with some one of the decolorizing agents men- 
tioned in connection with the staining of tubercle bacilli in cover- 
glass preparations, preferably 3 per cent hydrochloric-acid al- 
cohol. Decolorization must be continued until the red color 
has disappeared, which requires one-half to several minutes. 

(d) Wash in alcohol. 

(e) Wash in water. 

(/) Use hematoxylin as a contrast-stain for fuchsin prepara- 
tions, and carmine for gentian violet preparations, (it is bet- 
ter to stain with carmine first of all and before staining the 
bacilli. The carmine is not affected by the subsequent 
treatment.) 

(g) Wash in water. 

(h) Alcohol. 

(*) XyloL 

(/) Balsam, 



THE MICROSCOPE AND MICROSCOPIC METHODS 6 1 

Nuclear stains, which may be used as contrast-stains for 
sections: 

DELAFIELD'S HEMATOXYLIN. 

Hematoxylin crystals 4 grams. 

Alcohol 25 c.c. 

Ammonia alum 50 grams. 

Water 400 c.c. 

Glycerin 100 c.c. 

Methyl alcohol , 100 c.c. 

Dissolve the hematoxylin in the alcohol, and the ammonia 
alum in the water. Mix the two solutions. Let the mixture 
stand four or five days uncovered; it should have become a deep 
purple. Filter and add the glycerin and the methyl alcohol. 
After it has become dark enough, filter again. Keep it a month 
or longer before using; the solution improves with age. At the 
time of using, filter and dilute with water as desired. 

LITHIUM-CARMINE (ORTH). 

Carmine 2.5 grams. 

Saturated watery solution of lithium carbonate. 100.0 c.c. 

Add a few crystals of thymol. The carmine dissolves readily 
in the lithium carbonate solution. Filter the stain at the time of 
using. Sections are to be left in the stain five to twenty minutes. 

Sections stained in carmine are placed directly in acid alco- 
hol (i part hydrochloric acid, 100 parts 70 per cent alcohol) for 
five to ten minutes. They acquire a brilliant scarlet color. 
When used as a contrast-stain for tissues containing bacteria, 
it is best to use it before staining the bacteria, which might be 
decolorized by the acid alcohol. 



CHAPTER II. 

STERILIZATION DISINFECTION ANTISEPSIS- 
FOOD PRESERVATION. 

Definitions. By sterilization is meant the killing or the re- 
moval of all micro-organisms in or on a body or substance. 
Disinfection has a somewhat analogous signification, but denotes 
the destruction or removal of infectious microbes, and this may 
or may not be accomplished without complete sterilization, 
according to the nature of the particular case in hand. Anti- 
sepsis means the inhibition of growth of micro-organisms with- 
out ordinarily killing or removing them, and is especially applied 
to the checking of microbic activity in wounds and the effects 
produced thereby (sepsis). Food preservation involves similar 
principles, depending upon the prevention of microbic activity 
in dead organic matter either by sterilization or by the presence 
of inhibitive substances, similar to antiseptics, but in this instance 
called preservatives. 

In connection with sterilization we shall consider those agents 
which remove or destroy a part of the microbic flora without 
producing complete sterility, as well as the methods which insure 
complete sterilization. A few examples of each general class 
will be considered. 

Physical Sterilization. Among the physical means by which 
sterilization may be accomplished, those which are merely me- 
chanical may be mentioned first. The removal of microbes from 
an infected surface by washing them away is a method of wide 
application. Complete sterility may sometimes be attained in 
this way. In ordinary disinfection of woodwork, walls and 
floors, or of the hands, mechanical cleaning is of primary impor- 
tance, even though it does not insure complete sterilization. 

62 



STERILIZATION ANTISEPSIS FOOD PRESERVATION 63 

The process removes not only many of the bacteria, but also 
much other material which serves to protect them and even to_ 
furnish food for their development. Another mechanical method 
is that of comminution, actual crushing of the bacterial cells. 
It is of very narrow application and not to be relied upon. 
High pressures have been employed to destroy bacteria, but 
hydrostatic pressure of even 1000 atmospheres does not produce 
complete sterilization. Sedimentation is a method of primary 
importance, especially in the removal of suspended bacteria from 
the atmosphere. It also operates to remove a large proportion 
of the bacteria from drinking water when stored in suitable reser- 
voirs. Filtration of fluids is an important means of sterilizing 
them. Air may be sterilized by drawing -it slowly through a 
sufficient layer of cotton. Water becomes bacteria-free as it 
niters through the soil, so that waters from the depths of the earth 
are sterile. Liquids are commonly sterilized in the laboratory 
by forcing them through a layer of unglazed porcelain (Pasteur- 
Chamberland filter) or through a compact wall of diatomaceous 
earth (Berkefeld filter). Liquids rich in bacteria, such for ex- 
ample as cultures in broth, may be rendered bacteria-free in this 
way. These filters have also been employed for the sterilization 
of drinking water, but their use for this purpose requires intelli- 
gence and care, and when carelessly employed they are worse 
than useless. 

Dessication is destructive to many microbes, especially those 
which do not form spores. The germs of Asiatic cholera are dead 
in a few hours after complete drying. The spores of the anthrax 
bacillus on the other hand remain alive for at least ten years after 
drying. Most bacteria resist drying long enough so that they 
may be transferred by air currents as dust and still be capable of 
growth. 

Light is injurious to bacteria and direct sunlight is rapidly 
fatal to them, even in spore form. Light seems to act by pro- 
ducing powerful chemical germicides, probably organic peroxides, 
in the medium surrounding the bacteria. Such substances are 



64 BACTERIOLOGY 

known to be produced under these circumstances. They rapidly 
decompose. 

Cold appears to be fatal to some pathogenic forms, and a con- 
siderable percentage of the bacteria in a culture are usually killed 
by freezing. Cultures cannot be completely sterilized even by 
exposure to the temperature of liquid air. Cold is therefore not 
to be regarded as an efficient germicide, although it may com- 
pletely check the growth of bacteria. 

Heat is the most important of the physical means and 
doubtless the most important of all means of destroying 
bacteria. Its value as a purifying agent was recognized among 
the ancients. Heat is applied under conditions insuring 
the presence of liquid water, so-called moist heat, and in 
the absence of water, so-called dry heat or hot- air sterilization. 
The most reliable methods of sterilization by dry heat are those 
which accomplish the combustion or destructive distillation of 
organic matter in general. Actual combustion of clothing and 
bedding, and even of houses has been resorted to in the past 
as a method of disinfection. Heating to redness in the naked 
flame is the routine method of sterilizing our platinum wire, and 
glass articles, such as capillary pipettes, cover-glasses and slides 
are commonly sterilized in the flame. Flaming may even be 
employed for sterilization of surgical instruments in an emergency, 
although such treatment quickly destroys steel instruments. 
Sterilization of large objects and of combustible material by dry 
heat is generally accomplished in an oven or hot-air sterilizer. 
The common laboratory sterilizers are boxes of sheet iron with 
double walls, with air space between to allow the hot gases from 
the flame completely to surround the inner compartment. The 
door, which occupies one full side, is usually double. A tubula- 
tion through the top allows a thermometer to be inserted into the 
interior so that the temperature may be read off at any time. 
Even the best hot-air sterilizers fail to give an even temperature 
all over the interior, so that the thermometer bulb at one corner 
cannot be implicitly relied upon to record the temperature of 



STERILIZATION ANTISEPSIS FOOD PRESERVATION 65 

other parts. Ordinarily a temperature of 150 C. for one hour, 
170 C. for 30 minutes, or 200 C. for one minute will kill all 
bacteria. Such exposure browns cotton of good grade only 
slightly. One fallacy in hot-air sterilization needs to be guarded 
against. Glassware and other apparatus must be dry before it 
is put into the oven to sterilize. A tube containing water may 
be left in the oven until the thermometer records a temperature 




FIG. 28. Hot-air sterilizer. 

of 200 C. in the upper corner of the sterilizer, and subsequently 
the tube may be removed from the oven with the most of the 
water still in it. Hot-air sterilization is employed for glassware, 
tubes with cotton plugs, granite-ware, stone-ware, and for metals 
not injured by heat. 

Moist heat or heat in the presence of liquid water must be used 
whenever drying is to be avoided, especially in the sterilization of 
culture media and various solutions. It is employed as continu- 
ous sterilization at a single exposure and as discontinuous ster- 

5 



66 



BACTERIOLOGY 



ilization, heating for a short time on several consecutive days. 
The temperature employed varies according to the effect desired. 
A temperature of 60 C., maintained throughout a watery liquid 
for twenty minutes will kill most vegetative bacteria, and practi- 
cally all pathogenic bacteria which do not form spores. Such 
partial sterilization is called Pasteurization. Boiling water, 100 
C., kills vegetative bacteria in a very short time, less than two min- 
utes for most bacteria, and the 
spores of many species are de- 
stroyed by boiling for 5 to 30 min- 
utes. Some spores, however, for 
example those of some varieties of 
B. vulgatus, may survive a boiling 
temperature for several hours. 
Boiling is one of the most useful 
practical methods of disinfection. 
Nearly all pathogenic bacteria are 
quickly killed in boiling water. 
Surgical instruments are com- 
monly boiled in water to which 
sodium carbonate, i to 2 per cent, 
has been added. Rusting and 
corrosion may also be prevented 
by adding 10 per cent of borax 
to the water in which metal in- 
struments are boiled. Steriliza- 
tion of bacteriological media is 

usually done by means of streaming steam, rather than by immer- 
sion in boiling water. The Koch steam sterilizer is a compara- 
tively simple device for this kind of sterilization. It is a tall, 
cylindrical, tin vessel covered with asbestos or felt. The lower 
portion is filled with water; on the side is a water-gauge indicat- 
ing the height of the water, in order .that one may observe when 
there is danger of the sterilizer boiling dry. Over the top there 
is a tight-fitting cover. The steam is generated by a Bunsen 




FIG. 29. Koch's steam sterilizer. 



STERILIZATION ANTISEPSIS FOOD PRESERVATION 



6 7 



burner standing underneath. A perforated shelf placed some dis- 
tance above the surface of the water is for the reception of the- 
tubes and flask that are to be sterilized. The Arnold steam sterili- 
zation is somewhat more complicated but is very convenient and 
efficient. It consists of a cylinder of tin or copper with a cover, 
which is enclosed in a movable cylindrical outer cover or hood. 
The inner cylinder has an opening in the bottom through which 
steam may enter, the steam com- 
ing from a small chamber under- 
neath with a copper bottom to 
which the flame is applied. The 
peculiarity of this form of steril- 
izer consists in the fact that the 
steam which escapes from the 
sterilizing chamber condenses be- 
neath the outer cover or hood and 
falls back upon the pan over the 
chamber in which the steam is 
generated. The bottom of this 
pan is perforated with three small 
holes, which allow the water of con- 
densation to return into the cham- 
ber where the steam is generated. 
The sterilizer, therefore, to a cer- 
extent, supplies itself with 



tain 




FIG. 



30. Diagram of the Arnold 
steam sterilizer. 



water, although not by any means 
perfectly. It is, however, less 
likely to boil dry than other forms of sterilizers, and it has the 
advantage of being reasonably cheap and quite effective. The 
space inclosed by the hood also serves as a steam-jacket and helps 
to prevent fluctuations in temperature. A great improvement 
upon the ordinary Arnold sterilizer is the modification of it devised 
by the Massachusetts Board of Health. 

In the use of this, or any form of steam sterilizer, the time is 
noted from the period when boiling is brisk and it is evident that 



68 



BACTERIOLOGY 



the sterilizing chamber is filled with hot steam; or, what is better, 
when the thermometer registers 100 C., if the sterilizer be pro- 
vided with a thermometer. With a large Arnold sterilizer a 
temperature of 100 C. may not be reached intil it has been 
heated with a rose-burner for twenty to thirty-five minutes. 
When bulky articles or large amounts of material are to be ster- 
ilized, allowance must be made for the time necessary to bring the 
temperature in the middle of the mass to 100 C. 




FIG. 31. Steam sterilizer, Massachusetts Board of Health. 

Autoclave Sterilization. Sterilization in the presence of 
moisture and at temperature above 100 C., requires a 
pressure greater than that of the atmosphere and the apparatus 
used for this purpose is known as the autoclave. All bacteria 
and their spores are killed by heating at 110 C., in the presence 
of water, for fifteen minutes, and in about five minutes at 120 C. 
The steam pressures corresponding to these temperatures are 
approximately 7 J pounds and 1 5 pounds per square inch or J kilo- 



STERILIZATION ANTISEPSIS FOOD PRESERVATION 



6 9 



gram and i kilogram per square centimeter, respectively. The 
autoclave consists of a metal cylinder with a movable top, which is 
fastened down tightly during sterilization. It is furnished with 
a pressure gauge, a stop-cock, and a safety-valve which is set 
to allow the steam to escape when the desired pressure is attained 
and thus prevents it from running too high. Heat is furnished 
by a gas-burner underneath . The lower 
part of the cylinder contains water. 
The objects to be sterilized are sup- 
ported above this water on a perforated 
bottom or shelf. 

% It is necessary to follow certain pre- 
cautions in the use of the autoclave. 
Allusion has already been made to the 
necessity for having the steam saturated 
with moisture. This is effected by 
allowing the air to escape after the heat 
is applied, and in order to be sure that 
all the air has really been expelled, the 
stop-cock, with which all autoclaves are 
provided, is left open until the steam 
escapes freely. The stop-cock is then 
closed, and the pressure begins to rise. 
After leaving the articles to be steril- 
ized in the autoclave for the length of 
time desired, the apparatus must not be 

opened while the steam contained within it is still under pressure, 
as there may be a sudden evolution of steam upon the removal 
of the pressure which may blow the media out of their tubes and 
flasks. After the pressure has fallen to zero it is well to open the 
stop-cock only a little way so that air may not be drawn in 
too rapidly to replace the condensing steam. The autoclave 
may be opened as soon as the internal and external pressure 
become equal. 

The length of exposure necessary to accomplish sterilization 




FIG. 32. Autoclave. 



70 BACTERIOLOGY 

in the autoclave depends upon the protection which the article 
to be sterilized affords the bacteria. In sterilizing agar, a con- 
siderable interval elapses before the agar becomes liquified, es- 
pecially if it be in large flasks, and it is well to allow 30 to 35 
minutes at 110 C., for its sterilizaton. Closely packed surgical 
dressings serve to protect the interior, and considerable time may 
be required for penetration of a sterilizing temperature into such 
packages. In such instances it is unwise to rely upon the gauge 
as an indicator of the temperature throughout the materials 
being sterilized. It is well to test the efficiency of the steriliza- 
tion from time to time by enclosing test objects in the center of 
several packages. A convenient test object for surgical auto- 
claves may be made by spreading spores of B. subtilis or B. vulga- 
tus on a sterile cover-glass and placing it in a sterile test-tube 
plugged with cotton, and then drying the preparation thor- 
oughly in the incubator for 24 hours. A number of these may be 
prepared and subsequently kept in the refrigerator until used. 
After the test object has been exposed in the autoclave, sterile 
broth is added to the tube by means of a capillary pipette. The 
development of a culture from the spores indicates lack of effi- 
ciency in the process of sterilization. 

Discontinuous or fractional sterilization by moist heat is em- 
ployed to sterilize certain kinds of culture media, more especially 
blood serum and gelatin, which are likely to be injured by heat- 
ing above 100 C., or by prolonged heating. In this method the 
medium is exposed to a temperature deemed sufficient to kill the 
vegetative forms of bacteria but not the spores. An interval is 
then allowed for the generation of these spores, whereupon the 
heat is again applied. This sequence is repeated until, according 
to past experience, sterilization may be regarded as almost cer- 
tainly accomplished. In the case of gelatin steaming (100 C.) 
for 15 to 20 minutes on three consecutive days is the usual 
practice; with inspissated serum, exposure for i hour at 60 to 
70 C. on six successive days is usually sufficient. These methods 
are applicable only to media in which spores may germinate and 



STERILIZATION ANTISEPSIS FOOD PRESERVATION 71 

they may fail to sterilize even in case of such materials, es- 
pecially in the presence of rapidly growing spore-producing bac- 
teria and when there are spores of anaerobic bacteria in the 
material to be sterilized. On this account, materials sterilized 
in this way should not be injected into patients. 

Electricity has little or no direct demonstrable germicidal ac- 
tion. An electric current may generate sufficient heat to kill 
bacteria, or it may produce powerful germicides by electrolysis, 
such for example as acids and alkalies. 

Chemical Agents, Sterilization by means of chemicals is 
not employed in the preparation of culture media because of the 
difficulty of removing the added substance after the desired effect 
has been obtained. It is necessary in every case to consider 
the other effects which the use of chemical germicides entails, and 
their usefulness is therefore somewhat more limited than that of 
the physical agents for sterilization. Their efficiency is also 
subject to great variation according to the nature of the materials 
with which they come in contact. Nevertheless they have a 
very important place in practical sterilization and disinfection. 

The common soaps, and more particularly green soap, have 
a slight germicidal value, and this in conjunction with their sol- 
vent action upon fats and protein, and the mechanical cleans- 
ing which accompanies their use, justifies assigning them an 
important place among the chemical disinfectants. 

Acids, especially those which are strongly dissociated, are 
powerful germicides. Hydrochloric acid apparently owes its 
power entirely to its acidity, and in fairly weak solution, 0.2 to 
i .o per cent, it kills vegetative bacteria in a short time. Strong 
sulphuric acid actually carbonizes organic matter, while nitric 
acid oxidizes and also forms special combinations with protein, 
the reactions resulting in death of living protoplasm. Sulphurous 
acid (sulphur dioxide) also possesses marked germicidal proper- 
ties, probably due to oxidation effects. 

Sulphur dioxide gas has been employed extensively in the 
fumigation of rooms, and is usually prepared by burning sulphur. 



72 BACTERIOLOGY 

Much difference of opinion exists regarding the value of it as a 
disinfectant. The spores of anthrax are not killed by several 
days' exposure to the liquefied gas. Anthrax and other bacilli 
are destroyed in thirty minutes when exposed on moist threads 
in an atmosphere containing one volume per centum of the gas. 
An exposure of twenty-four hours in an atmosphere containing 
four volumes per centum of the gas will destroy the organisms 
of typhoid fever, diphtheria, cholera and tuberculosis. The 
presence of moisture greatly enhances the activity of the disin- 
fectant, owing to the formation of the more energetic sulphurous 
acid. 

For the destruction of insects, such as mosquitoes, this agent 
is superior to formaldehyde. Its application for this purpose is 
important in preventing the spread of yellow fever and malaria. 

In practice, at least 3 pounds of sulphur per 1000 cubic feet 
should be used, and moisture must be present. This latter re- 
quirement can be fulfilled by evaporating several quarts of water 
within the tightly closed room just prior to generating the gas. 
In using powdered or flowers of sulphur, the necessary amount 
is placed on a bed of sand or ashes in an iron pot, which should 
rest on a couple of bricks in a pan or other vessel containing an 
inch or two of water. The sulphur is ignited by means of some 
glowing coals, or by moistening with alcohol and applying a 
match. Difficulty is often experienced in keeping the sulphur 
burning, and for this reason it is surer and more convenient to 
use the so-called sulphur candles now on the market. In operating 
with these, a sufficient number are placed on bricks in a pan of 
water and the wicks lighted. Liquefied sulphur dioxide may be 
used, and can now be obtained in convenient tin receptacles con- 
taining a sufficient quantity for the disinfection of an ordinary 
room. The can is opened by cutting through a soft metal tube 
projecting from the top. The fluid vaporizes at the room tem- 
perature, and it is simply necessary to place the can in a con- 
venient porcelain dish and allow the fluid to evaporate. 

Sulphur dioxide is objectionable on account of its lack of 



STERILIZATION ANTISEPSIS FOOD PRESERVATION 73 

power when dry, and on account of its corrosive action on metal 
and its bleaching effect on hangings and draperies in the presence^ 
of moisture; it is, therefore, preferable to use formaldehyde for 
room disinfection when possible. 

Alkalies, especially the caustics, sodium hydroxide and potas- 
sium hydroxide, are powerful germicides. Commercial lye is also 
valuable as a disinfectant. Perhaps the most important of the 
alkalies is calcium hydroxide, Ca(OH) 2 which, because of its 
low cost, is extensively used for the disinfection of excreta. 

Lime. The addition of o.i per cent of unslaked lime to fluid 
cultures of the typhoid bacillus and cholera spirillum will render 
them sterile in four or five hours. Typhoid dejecta are sterilized 
in six hours when thoroughly mixed with 3 per cent of slaked lime; 
the addition of 6 per cent will accomplish the same result in two 
hours. A convenient form for practical use is an aqueous mix- 
ture containing 20 per cent of lime so-called milk of lime. 
Typhoid and cholera dejecta are sterilized in one hour after mix- 
ing with 20 per cent of this mixture. In practice it is safer to use 
a considerable excess of lime. From the foregoing facts it would 
seem probable that lime or whitewash as ordinarily applied would 
possess disinfectant properties. Experimental work has demon- 
strated this to be a fact. The organisms of anthrax, glanders and 
the pus cocci were destroyed within twenty-four hours by one 
application. For spore-forming organisms and the bacillus of 
tuberculosis the power is not so great, the latter organism not 
being destroyed by three applications of the whitewash. Practi- 
cally, whitewashing is an effective means of disinfecting wood- 
work, perhaps because those microbes which are not killed at once 
are caught in the whitewash and their further distribution 
prevented. 

Oxidizing agents are usually germicidal. Chlorine, bromine 
and iodine, ozone, nitric acid, potassium permanganate, chlorinated 
lime, organic peroxides and peracids, and hydrogen peroxide, 
belong to this class. Chlorine, employed as chlorinated lime, 
is a valuable disinfectant for excreta. In the form of bleaching 



74 BACTERIOLOGY 

powder it has been extensively used in the disinfection of drinking 
water and of swimming pools. Bromine and iodine have long 
been employed in surgery, and solutions of iodine are often applied 
to the skin before surgical incision. Iodine probably acts to 
some extent as a germicide in this instance, but also as an anti- 
septic, remaining in the skin for some time after its application. 
Hydrogen peroxide is a germicide, as it quickly decomposes to 
form water and oxygen. It is placed on the market in solutions 
varying in strength from 10 to 30 volumes, the mode of expression 
indicating that corresponding solutions will liberate ten to thirty 
times their volume of oxygen when appropriately treated. It 
decomposes rapidly when in contact with purulent secretions, 
setting free abundant oxygen, and on this account is much used 
for cleansing infected wounds. It deteriorates in strength so 
rapidly that only fresh solutions of known strength should be 
used. 

Potassium Permanganate. Koch asserts that a 3 per cent 
solution will destroy anthrax spores in twenty-four hours, but 
that a i per cent solution cannot be depended upon to kill patho- 
genic organisms. Its disinfectant value in practice is very low 
on account of its ready decomposition by inert material. In the 
dilute solutions usually used for medicinal injections and irriga- 
tions no disinfectant action occurs. 

lodoform. This substance possesses little if any disinfectant 
power. It is mildly antiseptic in moist wounds, due to the gradual 
liberation of small quantities of iodine. 

Inorganic Salts. Mercuric chloride, HgC^, is probably more 
commonly used than any other one germicide. But Geppert, 
whose work in this direction has been abundantly corroborated 
by others, found that the potency of corrosive sublimate as a 
germicide had been greatly overrated. The inhibitory action of 
corrosive sublimate, on the other hand, is very great, and the 
veriest trace of it left adhering to the bacteria is sufficient to 
prevent them from growing. Corrosive sublimate is difficult 
to remove by ordinary washing and traces of it remain even after 



STERILIZATION ANTISEPSIS FOOD PRESERVATION 75 

very thorough washing. But if the last traces are removed by 
treatment with ammonium sulphide or other reagents which pre^ 
cipitate the mercury salt without themselves injuring the bac- 
teria, growth takes place even where the corrosive sublimate 
solutions have been used which are apparently efficacious. Thus 
anthrax spores will not grow in culture media when they are 
exposed for even a few minutes on silk threads to the action of 
corrosive sublimate solution of the strength of yV per cent and 
then washed thoroughly in water and rinsed in alcohol; but 
Geppert showed that the spores so treated were only apparently 
killed, for it took twenty hours' exposure to corrosive sublimate 
solution of this strength where the spores were not dried on silk 
threads, but suspended in water, and where the last trace of 
corrosive sublimate was removed by treatment with ammonium 
sulphide. It is claimed that its affinity for albuminous bodies 
and the readiness with which it combines with such substances 
detract from its value for some purposes. On the other hand, 
many observers claim that the albuminous combinations formed 
under such circumstances are soluble in an excess of albuminous 
fluid, and that its value as a germicide is not affected thereby. 
To obviate this possible difficulty it is customary in practice to 
combine the bichloride of mercury with some substance that will 
prevent the precipitation of the mercury salt by albumin. For 
this purpose 5 parts of any one of the following substances to i 
part of bichloride of mercury may be used hydrochloric acid, 
tartaric acid, sodium chloride, potassium chloride, or ammonium 
chloride. A very practical stock solution for laboratory purposes 
has the following composition: 

Hydrochloric acid 100 c.c. 

Bichloride of mercury 20 grams. 

Five c.c. in a liter of water makes a solution of about i-iooo strength. 

Mercuric Iodide. An extremely high antiseptic value has 
been placed on this substance by Miquel, who claims that the 



76 BACTERIOLOGY 

most resistant spores are prevented from developing in a cul- 
ture medium containing 1-40,000. In combination, as potas- 
sio-mercuric iodide, it has been used in soaps (McClintock) 
with very favorable results. The substance is not extensively 
employed, and further investigation is necessary to determine 
its true value. 

Silver Nitrate. This salt probably occupies the next posi- 
tion to the bichloride of mercury in germicidal power. Behr- 
ing claims it to be superior to bichloride of mercury in albumin- 
ous fluids. The anthrax bacillus is killed by a solution of 
1-20,000 after two hours' exposure. At least forty-eight hours' 
exposure to a 1-10,000 solution is required to kill the spores of 
anthrax. It is very irritating, and possesses strong affinities 
for chlorides, forming with them insoluble chloride of silver, a 
salt without germicidal value. For these reasons the use of 
silver nitrate is limited. In the solutions usually employed for 
douching the cavities of the body the available silver nitrate is 
immediately converted into the insoluble chloride, and little if 
any germicidal action takes place. To this fact may be ascribed 
the varying clinical results reported. 

Many proprietary silver compounds are on the market, in- 
troduced to replace the nitrate and its objectionable features. 
The most important are protargol and argyrol, organic silver 
combinations. They do not combine with chlorides, are less 
irritating than the nitrate and, not coagulating albumin, they 
possess greater penetrating power. 

Organic Poisons. Carbolic acid is one of the most important 
and most widely used disinfectants. It is usually employed in 
strengths of from i to 5 per cent. A 3 per cent solution will 
sometimes kill the spores of anthrax after two days' exposure. 
In the absence of spores, the anthrax bacillus is destroyed by a i per 
cent solution in one hour. The less resistant pus cocci are de- 
stroyed rapidly by a 2 per cent solution. Combination with an 
equal proportion of hydrochloric acid enhances the efficacy of 
carbolic acid to a marked extent. This_is due to the prevention 



STERILIZATION ANTISEPSIS FOOD PRESERVATION 77 

of albuminous combinations, thus allowing greater penetration 
of the disinfectant. 

Many other substances closely related to carbolic acid are 
used and possess marked germicidal properties. Among them 
may be mentioned creolin, cresol and lysol. They are all slightly 
superior to carbolic acid in actual germicidal value. 

Formalin is a 40 per cent aqueous solution of formaldehyde, 
H 2 CO. Remarkable claims have been made for this substance, 
and numerous investigations have shown it to possess, both in 
the liquid and gaseous forms, wonderful disinfecting power under 
certain conditions. Later investigations indicate that its germi- 
cidal power had been somewhat overestimated. In solutions 
of i-iooo an exposure of twenty-four hours is necessary to destroy 
the staphylococcus pyogenes aureus, while 1-5000 is sufficient 
to restrain its growth (Slater and Rideal). Its use in a gaseous 
form as a house disinfectant is by far the most important applica- 
tion at the present time. 

From 250 to 500 c.c. of formalin together with 500 to 1000 c.c. 
of water should be vaporized for each 1000 cubic feet of air space 
in the room, and the room should remain tightly closed for at 
least four hours, preferably over night. Many methods of 
vaporizing formaldehyde have been devised. Some form of 
tank, provided with heating apparatus and with an outlet tube 
which passes through the keyhole into the room, is perhaps the 
most convenient where much disinfection has to be done. If 
apparatus of this sort is not at hand, good results may be obtained 
by putting the formalin and the water previously heated to boil- 
ing, in a large pail in the center of the room, and then adding 
rapidly crystalline potassium permanganate, about 200 grams to 
each 500 c.c. of formalin used. The permanganate oxidizes 
some of the formaldehyde and produces heat to evaporate the 
rest of it. From 25 to 50 per cent more formalin should therefore 
be used for a given air space. It is well also to add about 10 per 
cent of glycerin to the water so as to raise the boiling-point some- 
what and insure more complete vaporization of the formaldehyde. 



78 BACTERIOLOGY 

Formaldehyde penetrates very slightly beneath exposed 
surfaces so that everything to be disinfected should be completely 
exposed. Openings about windows and doors should be carefully 
plugged up and sealed with strips of paper. Mechanical cleansing 
supplemented by application of i-iooo solution of mercuric 
chloride to floors and walls should follow the fumigation. The 
persistent odor of formalin may be removed by fumes of 
ammonia. 

Aniline Dyes. Many of the aniline dyes, notably pyoktanin 
(methyl- violet) , possess germicidal properties. Malachite green 
is said to possess even greater germicidal value than pyoktanin. 
Methylene blue also possesses considerable germicidal power. 

Alcohol is a germicide of moderate power. It has little 
effect upon spores but in concentrations of from 50 to 95 per cent 
it destroys vegetative bacteria in a few minutes. 

Germicides destroy bacteria, as a general rule, because they 
are general protoplasmic poisons, destructive to all living matter. 
There is, nevertheless, some selective action. Thus, formal- 
dehyde kills bacteria but has little poisonous effect upon insects, 
such as mosquitoes, bedbugs, roaches or fleas. Mercuric chloride 
is rapidly fatal to bacteria when it comes into contact with them, 
but it has no very immediate destructive effect upon fly larvae 
(maggots). Some of the oxidizing agents, such as hydrogen 
peroxide and acetozone are not poisonous to man because they 
are decomposed into relatively harmless substances before they 
can be absorbed. Attempts to discover or to produce chemicals 
which would exhibit a selective destructive effect upon microbes 
in the interior of the body have not met with much success. 
Quinine is perhaps the best known example, as it may circulate 
in the blood in sufficient concentration to poison the malarial 
parasites without at the same time killing the host. The effects 
produced by mercury and by salvarsan in syphilis are perhaps 
analogous, but they evidently depend to a large extent upon a 
special susceptibility of the microbe, a susceptibility not yet 
apparent in most parasites. The specific immune substances 



STERILIZATION ANTISEPSIS FOOD PRESERVATION 79 

may perhaps be classed in the same category. These will be 
considered in more detail in a later chapter. 

Antiseptics. Antiseptic and preservative agents prevent or 
delay the development of bacteria, without killing them. Very 
much the same agents are applied to prevent the growth of mi- 
crobes in living tissues and consequent poisoning of the body 
(antisepsis) as in preventing microbic development in dead 
organic matter (food preservation). 

Of the physical antiseptics, dessication and cold are perhaps 
of greatest importance. These agencies find application to the 
living body as well as in preservation of dead material. Sub- 
stances which increase osmotic pressure, sodium chloride and 
sugar, are also employed to prevent microbic growth in foods. 

The chemical antiseptics are very numerous. In general 
a germicide in higher dilution exhibits antiseptic effect. * Small 
quantities of the inorganic acids, hydrochloric, nitric, sulphuric 
or sulphurous acid, prevent bacterial growth. Even boric acid 
which has little or no germicidal effect will delay or inhibit mi- 
crobic development. Many organic acids possess inhibitive prop- 
erties toward bacterial action. Acetic and lactic acids prob- 
ably act merely by virtue of their acidity. Benzoic and salicylic 
acids seem to be more antiseptic, probably by virtue of other 
structural features in their molecules. Other organic substances, 
such as phenol (carbolic acid) and formaldehyde in high dilu- 
tions prevent or delay bacterial growth, and weaker germicides 
such as alcohol, chloroform or ether, are fairly effective preserva- 
tives. Oxidizing agents often decompose too rapidly to be of 
much value as antiseptics. Iodine, however, is one member of 
this group having considerable antiseptic value. 

Of the inorganic salts, mercuric chloride is most important. 
Small quantities of this agent inhibit the multiplication of bac- 
teria. It is extensively employed in antiseptic treatment of 
wounds. The borates, nitrates and salicylates, the latter com- 
pounds of an organic acid, also inhibit bacterial action to some 
extent. 



8o BACTERIOLOGY 

In using these substances as antiseptic applications to wounds, 
the possible poisonous effects upon the body as a whole from 
absorption of the antiseptic must be kept in mind. Moreover, 
such substances ought not to be used as food preservatives with- 
out due regard to the changes they may induce in the food and 
the possible effects they may exert upon the consumer. 

TESTING ANTISEPTICS AND DISINFECTANTS. 

The determination of the antiseptic value of a material is a 
comparatively simple matter. A virulent culture of the organ- 
ism used as a test is inoculated into sterile bouillon containing a 
known quantity of the antiseptic. The process is repeated with 
varying strengths of the material until the smallest quantity of 
it capable of preventing growth is determined. This dilution 
may be considered the antiseptic value of the material in question 
for the organism used, under the conditions of the test. 

Determination of the disinfectant power of a substance is a 
problem of much greater magnitude, and the method used must 
be altered more or less to suit the properties of the substance 
tested. It is obvious that some of the substance tested remains 
in contact with the organisms in the method given for determin- 
ing the antiseptic value, and that we do not know whether the 
bacteria are alive and merely inhibited in growth, or actually 
killed. 

The chemical composition of the medium in which the bac- 
teria are tested may have a marked influence upon the action of 
germicides. If components of the medium enter into chemical 
union with the germicide there may be an inert compound 
formed. There may also be formed dense, flocculent precipi- 
tates which envelop the bacteria and protect them from the action 
of the germicide. It is therefore apparent that the potency of a 
germicide may appear very different when acting upon the bac- 
teria in water or in physiological salt solution or on bacteria 
dried on glass rods or on silk threads, on the one hand, and upon 



STERILIZATION ANTISEPSIS FOOD PRESERVATION 8 1 

the same bacteria in beef broth or in feces or in urine, on the other. 
For these reasons it is not always possible to draw conclusions 
from the results of laboratory experiments as to the value of a 
germicidal agent for practical disinfecting purposes. 

Method. To 15 c.c. of sterile water in a 60 c.c. Erlenmeyer 
flask add 2 c.c. of a virulent culture of the test-organism. Then 
add a solution of the substance under investigation in the pro- 
portion necessary to give the dilution wished. Mix thoroughly, 
and allow this " action-flask " to stand as long as it is desired to 
have the germicide in contact with the test-organism (action- 
period). Transfer 0.5 c.c. from the action-flask to a flask con- 
taining 200 c.c. of a solution of some chemical capable of decom- 
posing the substance being tested with the formation of inert or 
insoluble compounds. In this " inhibition-flask " the strength 
of the solution should be such that molecular proportions of the 
chemical are present in sufficient quantity to combine with all 
the germicide carried over. The inhibition-flask is shaken for 
30 seconds, and i c.c. transferred from it to 100 c.c. of sterile 
water in another, the " dilution-flask." After two minutes, 
three agar tubes are inoculated with i c.c. each from the dilution- 
flask, plated, and growth watched for. 

Control-experiments should be performed to determine that 
the dilution of the test-culture is not too great when carried through 
the three flasks. It likewise should be determined that the in- 
hibiting chemical has no effect on the bacteria. 

What the inhibiting chemical shall be must be determined 
for each individual case. For salts of the heavy metals ammo- 
nium sulphide answers well; for mercury salts, stannous chloride 
may be used; for formaldehyde, ammonium hydrate; for carbolic 
acid, sodium sulphate. 

The testing of gaseous disinfectants, such as sulphur dioxide 
and formaldehyde, must be conducted under conditions as nearly 
parallel to actual practice as possible. The test-organisms may 
be exposed on threads or cover-glasses, and acted upon by a known 
volume strength of disinfectant 'for a known length of time. 
6 



82 BACTERIOLOGY 

Subsequent treatment of the organisms with a suitable inhibitor 
is necessary when possible, and should growth occur in the cul- 
tures following, the test-organism should be recognized in order 
that possible contamination by extraneous organisms may be 
excluded. 

In determining the value of germicides for sterilizing ligatures, 
the students can apply methods based on the foregoing principles. 
Great care and ingenuity are necessary to arrive at correct con- 
clusions, particularly in the case of animal tendons. In many 
instances quite stable compounds are formed between tendon 
and germicide, and living organisms may be so imbedded in such 
a substance that subsequent growth in a test-culture is impossible. 
The use of a suitable inhibitor, and, prior to final culture-tests, 
a prolonged soaking in sterile water, will promote the accuracy 
of the results. 



CHAPTER III. 
CULTURE MEDIA. 

Culture media are substances in which microbes are artificially 
cultivated. The variety of such substances is very large, different 
materials being suited to different purposes. Particular kinds 
of media have been devised in order to bring to development or 
especially to favor the development of certain kinds of microbes. 
Various media are also used to demonstrate the physiological 
properties of bacteria, especially the physical arrangement of the 
bacterial cells as they grow under various conditions, and the 
chemical changes induced in the various constituents of the 
media by the microbic growth. 

Glassware. Micro-organisms are usually grown in glass 
test-tubes or sometimes in glass flasks. The tubes and flasks 
should be of more durable glass than those ordinarily used in 
chemical work, but heavy tubes of glass of poor quality are not 
to be recommended. For ordinary purposes, test-tubes I25X 
15 mm. are convenient. Larger tubes, 150X20 mm., are used 
to store media to be used in making plate cultures and for roll- 
tube cultures. New glassware should be thoroughly washed 
before using, and for critical work it should be boiled in dilute 
sodium carbonate, rinsed, washed in dilute hydrochloric acid, 
rinsed repeatedly in running water, finally in distilled water, 
and then inverted to drain in a warm place, such as the incubator, 
until perfectly dry. Used glassware should be sterilized in the 
autoclave at 120 C. for half an hour, emptied, cleaned with a 
swab and hot water, rinsed in distilled water and drained. In 
case of special difficulty the glassware may, after emptying and 
washing in water, be cleaned by soaking in a special cleaning fluid, 
and all organic matter may be readily removed by using this 

83 



84 BACTERIOLOGY 

fluid hot. It should not come into contact with the hands or 
with any large quantity of organic matter. 

CLEANING FLUID. 

Potassium or sodium bichromate 40 grams. 

Water 150 c.c. 

Dissolve the bichromate in water, with heat; 
allow it to cool; then add, carefully, con- 
centrated commercial sulphuric acid 230 c.c. 

Exact proportions are not necessary in making this fluid. Glass- 
ware cleaned in it must be repeatedly rinsed subsequently. 

Plugs. The clean dry tubes or flasks are plugged with raw 
cotton of a good grade which does not char too readily upon 
heating. The cotton plugs may be carefully made by rolling 
an oblong rectangular strip, of even thickness, into a firm cylinder 
of proper size, rolled plugs, or more hastily made by stuffing the 
cotton into the open end of the flask or tube, stuffed plugs. The 
latter kind of plug serves very well for tubes in which media are 
to be stored temporarily but is not so satisfactory for other 
purposes. 

Sterilization. After plugging, the tubes are placed in a wire 
basket and sterilized in the hot-air sterilizer or, sometimes, to 
avoid charring, in the autoclave. This not only renders the 
glassware free from bacteria but also gives more permanent 
form to the plugs. 

THE COMMON CULTURE MEDIA. 

Broth. Broth, bouillon or beef- tea, is best made from fresh 
meat, either beef, veal or chicken. Finely chopped lean meat, 450 
to 500 grams, is mixed with 1000 c.c. of distilled water and either 
allowed to stand over night in the refrigerator or else digested 
for half an hour at temperature of 50 to 55 C. It is then strained 
through muslin, yielding a filtrate of deep red color. Any ex- 
cessive amount of fat should be skimmed off. To the filtrate, 
which should measure 1000 c.c., are added: 



CULTURE MEDIA 85 

Peptone, Witte's 1 10 grams. 

Sodium chloride (common salt) 5 grams. 

These should be dissolved by stirring at a temperature below 
60 C. The mixture is then boiled for half an hour over the 
direct flame, cooled slightly, and filtered through paper pre- 
viously wet with warm water. The filtrate should be clear and 
light yellow in color, and should be diluted to 1000 c.c. with 
distilled water. Its reaction is acid, a reaction unfavorable to 
the growth of many bacteria, especially to many pathogenic, 
forms. 

The amount of alkali to be added is ascertained by titration. 
For this purpose exactly 5 c.c. of the broth is placed in each of 
three test-tubes. Five-tenths cubic centimeters of a 5 per cent 
solution of purified litmus (Merck's highest purity) is added to 

N 

each tube. An accurately prepared solution of sodium hy- 
droxide 2 is then run in drop by drop from a graduated burette, 
the reading of which has been recorded, into one of the tubes 
until the red color just changes to blue. The burette reading is 
taken and recorded. The alkali is then run into the second tube 
rather rapidly until the endpoint ascertained by the first test is 
nearly reached. By comparing the color of this tube with that of 
the first one and with the third to which no alkali has yet been 
added, the exact point at which the color is changing from red 
to blue may be accurately judged. When this point is reached, 
the burette reading is again recorded and the amount of alkali 
necessary to neutralize the 5 c.c. of broth ascertained. The 
third tube should then be titrated to confirm the previous result. 
The titration of the broth should now be repeated, using phe- 
nolphthalein as an indicator. For this purpose, 5 c.c. of the medium 
is transferred to a small porcelain dish, diluted by the addition 

1 Commercial peptones are mixtures of albumoses and a small amount of peptone. 

2 A normal solution of sodium hydroxide contains one gram-molecule of anhy- 
drous NaOH, or 40 grams, in a liter. A solution contains -%$ of this amount 
or 2 grams in a liter. 



86 BACTERIOLOGY 

of approximately 45 c,c. of distilled water, and boiled for a minute, 
i c.c. of a 0.5 per cent solution of phenolphthalein in 50 per cent 

N 

alcohol is now added and solution of sodium hydroxide run in 

20 

from the burette until the color changes to a faint but distinct 
and permanent pink color. The burette reading is recorded 
and the amount of alkali necessary to neutralize the 5 c.c. of 
medium in respect to phenolphthalein thus ascertained. This 
titration may well be repeated, especially by beginners. As a 
result of these titrations we shall have ascertained the amount of 
alkali necessary to neutralize the remaining broth to either indi- 
cator. For example suppose that 5 c.c. of the broth titrated as 
follows : 

0.5 c.c. of alkali with litmus as indicator. 

20 

2. o c.c. of alkali with phenolphthalein as indicator 

In order to neutralize the remaining 980 c.c. of broth to 

litmus would require or 08 c.c. of alkali. A solu- 

5 20 

tion of alkali twenty times as strong as this, namely normal 
sodium hydroxide, is employed for this purpose, and only f or 
4.9 c.c. of this are necessary to neutralize the 980 c.c. of broth 
to litmus. The reaction generally required for pathogenic 
bacteria is slightly alkaline to litmus and for this reason an excess 
of 10 c.c. of normal alkali per liter is added to the broth, 9.8 c.c. 
for the 980 c.c., making altogether 14.7 c.c. to be added. Cal- 
culation from the result obtained with phenolphthalein in the 
same way shows that 19.6 c.c. of normal alkali would be required 
to neutralize the medium to this indicator. The desired final 
reaction of the medium in respect to phenolphthalein is acid, 
usually that of 5 to 15 c.c. of normal acid per liter, or 0.5 to 1.5 
per 100 c.c., or 0.5 to 1.5 per cent, as it is commonly expressed 
after Fuller. 1 In this instance, therefore, 5 to 15 c.c. per liter, 
or 4.9 to 14.7 c.c. less than the 19.6 for the 980 c.c., would be 

1 Fuller. Journal of Amer. Public Health Assoc., 1905. 



CULTURE MEDIA 87 

added, namely 14.7 to 4.9 c.c., according to the purpose for which 
the broth is to be used. 

The amount of normal alkali finally decided upon is added to 
the broth, which is then weighed in its pan. It is then cooked 
by boiling over the direct flame for half an hour or by heating 
in the autoclave at 110 C. for 15 to 20 minutes. It is now 
cooled to about 50 C., filtered through paper, filled into tubes 
and sterilized, either in the autoclave at 110 C. for 15 minutes 
or by fractional sterilization in streaming steam at 100 C. for 
15 minutes on three consecutive days. 

Broth may be prepared from meat extract instead of meat. 
Meat extract 3 grams, peptone 10 grams and salt 5 grams are 
dissolved in 1000 c.c. of water, boiled, filtered and titrated against 

N 

sodium hydroxide. The subsequent steps are the same as in 

preparation of broth from fresh meat. 

Remarks upon Titration. The titration of bacteriological 
media made from meat or meat extract is an important step in 
their preparation. There is some confusion on this point because 
of the use of different indicators in ascertaining the reaction. 
The neutral point indicated 1 by litmus is very nearly the actual 
neutral point in respect to acidity and alkalinity, and this point 
is not appreciably displaced in either direction by the addition 
of a neutral mixture of a feebly dissociated acid and its salts to 
the solution. The end reaction indicated by phenolphthalein 
when it turns pink is actually a point at which there is a slight 
excess of alkali. This is so nearly the actual neutral point in 
inorganic solutions, when electrolytic dissociation is marked, 
that the error is not appreciable. In solutions of organic sub- 
stances, especially when considerable amounts of feebly dissoci- 
ated substances, such as are contained in peptone or gelatin, are 
present, this error becomes very appreciable. The discrepancy 
between the end point for litmus and for phenolphthalein will 

1 Washburn, E. W., The significance of the term alkalinity in water analysis and 
the determination of alkalinity by means of indicators. Report Illinois Waterworks 
Association, 191 1. 



88 BACTERIOLOGY 

vary for different lots of media. Another source of error and 
misunderstanding arises from the fact that the reaction of a 
medium changes somewhat after its neutralization, especially 
during sterilization, but also upon standing afterward at ordinary 
temperature. This change is toward decreased alkalinity and 
increased acidity and its extent is not the same for different 
media, being most marked, perhaps, in those rich in glucose. 
Where particular importance is attached to the titre of a medium, 
it is well, therefore, to determine this upon a sample of the medium 
taken from the lot at the time it is used, rather than to quote 
figures obtained before sterilization. The optimum reaction for 
most microbes is very close to the neutral point for litmus and 
preferably slightly alkaline to this indicator. 

Gelatin. Finely chopped meat, 450 to 500 grams, is mixed 
with a liter of distilled water and digested on the water bath for 
half an hour at 50-55, with stirring. It is then strained through 
muslin, yielding a filtrate of deep red color, which should be made 
to equal 1000 c.c. This filtrate is placed in the inner compart- 
ment of a double boiler (rice cooker) and to it are added 10 grams 
peptone, 5 grams sodium chloride and 100 to 150 grams of sheet 
gelatin of the best quality ("gold label" gelatin). The larger 
amount of gelatin should be used during warm weather if no low- 
temperature incubator is at hand. These constituents are dissolved 
by stirring at a temperature below 55 C, After complete solution, 
the reaction is titrated as has been described for the titration of 
broth. From 30 to 50 c.c. of normal alkali are usually required 
to give the proper reaction to a liter of the medium. After this 
has been ascertained, and the amount added, the medium is 
thoroughly mixed and then left covered and undisturbed while 
the water in the outer compartment of the cooker is boiled for 
an hour. It is well to have boiling water at hand in another 
receptacle so that the supply in the cooker may be replenished 
if it gets low, without chilling the medium. The gelatin is now 
filtered through paper wet with hot water, and should be kept 
warm during filtration by means of a funnel-heater, or by a steam 



CULTURE MEDIA 



8 9 




bath, although these are not essential. If it gets cold it may be 
poured out of the funnel and warmed again in the pan. A portion 
of the nitrate should be boiled in a test tube over the flame for a 
minute or two. It should then remain (i) perfectly clear, (2) 
alkaline to litmus paper, and (3) should solidify on cooling in tap 
water. After nitration the medium is filled into tubes and steril- 
ized in streaming steam by the fractional method, 20 minutes at 
1 00 C. for 3 consecutive days. Gelatin 
may be sterilized in the autoclave at 110 C. 
for 10 minutes, but it should be chilled in 
cold water at once after removal, and even 
then its gelatinizing property may be seri- 
ously impaired. 

In filling gelatin into tubes it is important 
that the medium should not be spilled on 
the mouth of the tube or on the cotton plug, 
as this accident causes the latter to be glued 
in position. The filling apparatus indicated 
in Fig. 33 will be found convenient for filling 
any sort of liquid medium into tubes, and 
with proper care one may fill tubes rapidly 
without soiling the mouths of tubes and their 

cotton plugs. Fio.33.-Apparatusfor 

Gelatin may be made from beef extract, filling media into tubes. 

mi I.L j i j.' It is held in a ring-stand 

The extract, peptone, salt and gelatin are sup port. 
dissolved at a temperature below 60 C. or 
the medium is cooled to this temperature after solution has been 
accomplished. It is titrated and the proper amount of alkali 
added. An egg is beaten up in water and then stirred into the 
medium. It is then boiled on the water bath for an hour, 
filtered, tested, tubed and sterilized. 

Nutrient Agar. To a liter of nutrient broth, prepared as 
above described (page 84) add 15 grams of finely cut agar 
shreds. Weigh the pan with its contents. Boil the material 
over the direct flame for one to two hours, with constant stirring 



QO BACTERIOLOGY 

to avoid burning, adding hot distilled water from time to time 
to compensate for the loss by evaporation. Instead of boiling 
it is convenient to cook the medium in the autoclave at 110 C. 
for 45 minutes to an hour. In either case, the agar should be 
very completely dissolved. The medium is then cooled to 60 C. 
and an egg previously beaten up in water is added and thor- 
oughly mixed with the agar. It is then boiled again for 10 minutes 
over the free flame, with constant stirring at the bottom, or for 
45 minutes on the water bath, or for 15 minutes in the auto- 
clave at 110 C. Distilled water is added to restore the original 
weight, and the medium is then filtered, usually through a layer 
of cotton wet with hot water, although filter paper may be used. 
Filtration is favored by keeping the funnel hot, either with the 
hot-water funnel heater or in a steam bath, and it may be hastened 
by the use of suction. The filtrate need not be perfectly clear, 
and it usually clouds on cooling unless it is acid in reaction. The 
reaction should be alkaline to litmus. After filling into tubes or 
flasks, agar should be sterilized in the autoclave at 110 C. for 30 
to 35 minutes. 

Modifications of the Common Media. Broth is made nearly 
free from sugar by fermenting the meat infusion over night at 
37 C. after inoculating it with B. coli, and then proceding with 
the filtrate in the usual way. This medium is designated as 
sugar-free broth. Various sugars or other substances are added 
to such broth in order to test the ability of bacteria to ferment 
them. Acetic acid, 0.5 per cent, is added to broth to make a 
selective medium for acid-resisting bacteria. Glycerin, 5 to 7 
per cent, is added to broth for the cultivation of the tubercle 
bacillus. Naturally sterile ascitic fluid or blood is added to broth 
to promote the growth of certain types of microbes, and to en- 
courage anaerobes. Bits of naturally sterile tissue are added 
to broth for similar purposes. 

Gelatin is modified by the addition of various sugars, especially ' 
dextrose and lactose, often with the further addition of litmus. 
The production of acid by fermentation of the sugar is at once 



CULTURE MEDIA 9 1 

evidenced by the reddening of the litmus. Glucose litmus gela- 
tin is also a useful medium for anaerobes. It is best to sterilize, 
the litmus separately and add it from a sterile pipette at the time 
the medium is used. 

Agar is modified by the addition of 5 to 7 per cent of glycerin, 
and such glycerin-agar is used extensively for cultivation of the 
tubercle bacillus and several other pathogenic bacteria. Various 
sugars, supplemented by the addition of litmus, are 
dissolved in agar to test the fermentation properties 
of bacteria. Glucose agar is extensively employed as 
such for the cultivation of anaerobes. Agar also forms 
the gelatinizing base for a number of more or less com- 
plex special media. 

STERILIZABLE SPECIAL MEDIA. 

Potato. Potatoes were perhaps the first solid med- 
ium employed in the cultivation of micro-organisms. 
Boiled or steamed potatoes kept in a moist place, such 
as a large covered glass dish, may well be employed as 
an illustration of primitive technic, and excellent cul- 
tures of the common chromogenic bacteria may be ob- 
tained in this way. For most purposes it is better to 
put pieces of potato in test-tubes where they are more 
perfectly protected from contamination, as suggested 
by Bolton. 1 The potato is carefully washed, a slice Potato 4 in 
removed from each end, and a cylinder is cut out with culture 
a cork-borer or with a test tube cut off near its bottom. 
This cylinder is divided diagonally into two pieces. The pieces 
are washed in running water for twelve to eighteen hours. 
They are placed in test-tubes containing a little water to keep 
the potato moist, and are supported from the bottom on a piece 
of glass tubing about i to 2 cm. in length (or on cotton, or in a 
specially devised form of tube with a constriction at the bottom) . 

1 Bolton, The Medical News, Vol. I, 1887, p. 318. 



g 2 BACTERIOLOGY 

The tubes are plugged, and sterilized in the autoclave at 110 C. 
for 30 minutes. Potato is best when freshly prepared; it is likely 
to become dry and discolored with keeping. 

Milk. Milk fresh as possible is placed in a covered jar, 
steamed for fifteen minutes, and then kept on ice for twenty- 
four hours. At the end of that time the middle portion is re- 
moved by means of a siphon. The upper and lower layers must 
not be taken; the upper part contains cream, and the lower part 
particles of dirt, both of which are to be avoided. About 7 to 10 
c.c. are to be run into each test tube. The tube is plugged with 
cotton, and sterilized in the autoclave at 110 C. for 30 minutes. 

The coagulation of milk, which is accomplished by certain 
bacteria, is a very valuable differential point. A little litmus 
tincture may be added to the tubes of milk before sterilizing, 
until they acquire a blue color, to indicate whether or not acids 
are formed by the bacteria which are afterward cultivated in 
the milk. 

Dunham's Peptone Solution. 

Peptone 10 grams. 

Sodium chloride 5 grams. 

Water i liter. 

Boil, filter, sterilize in the usual manner. 

Dunham's solution is valuable to- test the development of 
indol by bacteria (see Part II., Chapter VIII.). The develop- 
ment of acids may be detected after the addition of 2 per cent of 
rosolic acid solution (0.5 per cent solution in alcohol); alkaline 
solutions give a clear rose-color which disappears in the presence 
of acids. 

Nitrate Broth. Dissolve i gram of peptone in 1000 c.c. of 
distilled water, and add 2 grams of nitrite-free potassium nitrate. 
Fill into test-tubes, 10 c.c. in each, and sterilize in the autoclave 
at 110 C. for 15 minutes. 

Blood -serum. The blood of the ox or cow may be obtained 
easily at the abattoir. It should be collected in a clean jar. 
When it has coagulated, the clot should be separated from the 



CULTURE MEDIA 93 

sides of the jar with a glass rod. It may be left on the ice for 
from twenty-four to forty-eight hours. At the end of that time 
the serum will have separated from the clot and may be drawn 
off with a siphon into tubes. These tubes are sterilized for the 
first time in a slanting position, as the first sterilization coagulates 
the serum. The coagulation may be done advantageously, as 
advised by Councilman and Mallory, in the hot-air sterilizer at a 
temperature below the boiling-point. After coagulation, sterilize 
in the autoclave at 110 C. for 20 minutes. This serum makes an 
opaque medium of a cream color. Blood-serum may be more 







FIG. 35. Koch's serum sterilizer. 

conveniently sterilized in the Koch serum inspissator (Fig. 35). 
A clear blood-serum is to be obtained by sterilization at a tempera- 
ture of 58 C. for one hour, on each of six days, if a fluid medium 
is desired, or of 75 C. on each of four days if the serum is to be 
solidified. In the latter case the tubes are to be placed in an in- 
clined position. Opaque, coagulated blood-serum has most of 
the advantages of the clear medium. Blood-serum may be se- 
cured from small animals by collecting blood directly from the 
vessels, and with proper technic may be obtained in a sterile 
condition; and the serum may be separated and stored in a fluid 
state. Human blood-serum is sometimes obtained from the 



94 BACTERIOLOGY 

placental blood. The preservation of blood-serum is sometimes 
accomplished with chloroform, of which i per cent is to be added 
to the medium; in this manner the serum may be preserved for a 
long time. It may be filled into tubes, solidified and sterilized 
as required; the chloroform will be driven off by the heat, owing 
to its volatility. Blood-serum media which are sterilized at 
low temperatures should be tested for twenty-four hours in the 
incubator to prove that sterilization has been effective; if it has 
not, development of the contaminating bacteria will take place 
and be visible to the eye. 

Loffler's blood-serum consists of one part of bouillon con- 
taining i per cent of glucose, mixed with three parts of blood- 
serum. It is sterilized like ordinary blood-serum. It is used 
largely for the cultivation of the bacillus of diphtheria. 

Fresh eggs in their shells may be used without other preparation 
than washing the surface thoroughly with bichloride of mercury 
solution; or after sterilization by steam, which of course coagu- 
lates the albumen. The egg is easily inoculated through a small 
opening made with a heated needle, which may be closed after- 
ward with collodion. Hueppe recommended eggs closed in this 
manner for the cultivation of anaerobic bacteria. 

Dorset's Egg Medium. 1 Perfectly fresh eggs are washed and 
the shells sterilized with bichloride solution. The eggs are then 
carefully broken and the yolks and whites mixed in a sterile 
dish. The mixed material is poured into sterile tubes and solidi- 
fied in the slanting position by heating at 70 75 C. for two 
hours. Contamination with bacteria should be carefully avoided 
throughout the preparation of the medium. The tubes should 
be sealed with rubber caps or with wax and incubated for a 
week before use. It is well to moisten the surface with a few 
drops of sterile water from a pipette before inoculating the me- 
dium. This medium is used for growing the tubercle bacillus. 

Bread-paste. Dry or toasted bread is broken into small 
crumbs, filled into tubes or flasks, moistened with water and 

1 Dorset: American Medicine, April 5, 1902. 



CULTURE MEDIA 



95 



sterilized in the autoclave. This medium is used for cultivation 
of molds. 

MEDIA CONTAINING UNCOOKED PROTEIN. 

Culture media containing naturally sterile uncooked protein 
have made possible the cultivation 
of microbic forms not cultivable on 
other media. Many microbes 
which may also grow on cooked 
media do much better on those con- 
taining uncooked protein. It would 
seem that media of this kind are to 
play an important part in the fur- 
ther development of our knowledge 
of pathogenic micro-organisms. 

Collection of Sterile Blood. A 
few drops of blood may be obtained 
from the ear lobe. The skin is 
cleaned with soap and alcohol and 
then dried perfectly with sterile 
cotton. It is punctured with a ster- 
ilized lancet and the blood quickly 
transferred to the surface of an agar 
slant by means of a platinum loop 
or a sterile capillary pipette. It 
should be incubated before use to 
insure sterility. 

Larger quantities of sterile 
human blood may be obtained with 
far less danger of contamination 
from the median basilic vein 
other large vein at the 
i The skin is washed, disinfected 

with alcohol and bichloride and dried. An elastic bandage is 
applied about the arm to distend the veins. A sterile needle 




FIG.. 36. Pipette with needle at- 
tached for drawing human blood 
or from a vein for use in culture media. 
The glass rod inside is used to defi- 
elbow. brinate the blood. 



96 BACTERIOLOGY 

attached to a special sterilized blood pipette is thrust into 
the vein and the desired amount of blood collected (see 
Fig. 36). It may be allowed to clot if sterile serum is desired, 
or it may be defibrinated by stirring with the glass rod if a mix- 
ture of corpuscles and serum is desired, or it 
may be kept in the fluid state by the addi- 
tion of sterile 10 per cent solution of sodium 
citrate so that the final mixture may con- 
tain i per cent of citrate. The bandage is 
removed from the arm before the needle is 
withdrawn. Pressure over the wound with 
cotton wet in alcohol for five minutes pre- 
vents subcutaneous hemorrhage. No dress- 
ing is required. The inlet to the blood pip- 
ette is closed by kinking the rubber tube. 
The blood or the serum is subsequently 
handled by means of sterilized pipettes, and 
most conveniently by means of the Pasteur 
bulb pipettes. (See page 33.) 

Blood from small laboratory animals 
serves as well as human blood for most pur- 
poses. It may be drawn from the carotid 
artery by aseptic technic into a special blood 
pipette the lower end of which is drawn out 

FIG. 37 Pipette into a capillary which is inserted directly 
with capillary tip for . . , . 

drawing blood fromcaro- into the artery (see Fig. 37). This blood 




WtoNw:) an anima1 ' ma y be defibrinated, citrated or allowed to 

clot. 

Small amounts of sterile blood may be obtained from labora- 
tory animals without killing them by means of heart puncture. 
The needle of a Luer glass syringe is inserted through the chest 
wall, after anesthetizing the animal and shaving and disinfecting 
the skin, so as to enter the cavity of the right ventricle. A 
quantity of blood not greater than yV the weight' of the animal 
may be removed. The needle is withdrawn and the blood quickly 



CULTURE MEDIA 97 

forced out into a sterile tube where it may be defibrinated 
or mixed with citrate solution, or allowed to clot, as may be- 
desired. 

Very large amounts of sterile blood are best obtained from 
the jugular vein of the horse or the superficial abdominal veins 
of the cow. The skin is shaved, washed and cauterized with 95 
per cent carbolic acid. When this has dried the vein is punctured 
with the needle, which is attached to a suitable glass receptacle 
by means of rubber tubing. 

Collection of Sterile Ascitic Fluid. For this purpose a large 
trochar and canula provided with a lateral outlet, and made so 
that the trochar can be drawn back beyond this outlet without 
being completely removed, is most convenient. The instrument 
is oiled with liquid paraffin. A rubber tube about 40 cm. in 
length is attached to the outlet and the whole is wrapped in a 
cloth and sterilized in the autoclave. The site selected for punc- 
ture should be cleansed and painted with tincture of iodine and 
the skin may be frozen with ethyl chloride if desired. One man 
inserts the trochar and canula, taking care not to contaminate 
it after it is removed from the cover. Another manipulates the 
attached rubber tube, carefully guarding it from contamination 
and allowing the fluid to flow into sterilized flasks of 1000 c.c. 
capacity which are handled by an assistant. The mouth of each 
flask should^be flamed after removing the cotton plug and again 
before it is inserted after filling the flask. With proper technic 
the ascitic fluid will as a rale be found bacteria-free. It should 
be stored in a cool place, and is most conveniently handled by 
means of large Pasteur bulb pipettes. 

In collecting hydrocele fluid or other fluids to be used for 
culture media, similar aseptic technic should be employed. 

Sterilization of Contaminated Fluids. Any of the clear fluids 
may be sterilized, when this is necessary, by filtration through the 
Berkefeld filter. The filtrate will usually prove less valuable 
as a medium than the corresponding unfiltered naturally sterile 
material. 



98 BACTERIOLOGY 

Collection of Sterile Tissue. For this purpose, a healthy 
animal is first bled to death as described above (page 96) for the 
collection of sterile blood. The skin is then thoroughly wet with 
water or with bichloride solution. With sterile instruments, an 
incision is made in the median line and the skin carefully stripped 
back. It is then well to sear the abdominal wall with a hot iron 
along the median line and also crosswise and cut along these 
lines with sterile scissors, opening the abdominal cavity. The 
organs desired are quickly removed with sterile instruments 
and placed in covered sterile glass dishes. The liver, kid- 
neys and testes are the organs most frequently employed in 
culture media. They are divided into pieces of suitable size 
with sterile scissors. Brain tissue may be readily obtained from 
the rabbit. The top of the head is skinned and an opening 
made by cutting away the skull between the orbits with the bone 
forceps. An area of the anterior portion of the brain is exposed. 
This is thoroughly seared with a hot iron, as well as the adjacent 
structures. A Pasteur bulb with a large capillary (internal 
diameter at least 5 mm.) is convenient for drawing out the 
brain tissue. This large capillary is inserted through the seared 
area and the brain is broken up by moving it about in the 
cranial cavity, while the tissue is drawn into the bulb by suction. 

Pfeiffer's Blood-streaked Agar. A large loopful of naturally 
sterile human blood, freshly taken from the ear, is spread over 
the surface of an agar slant, and incubated to insure sterility. 
This medium is employed for cultivation of the influenza 
bacillus. 

Novy's Blood-agar. The agar is melted and cooled to 50 
C. The naturally sterile defibrinated blood, usually rabbit's 
blood, is warmed to about 40 C. The blood is mixed with the 
agar in various proportions, and the mixture is allowed to solidify 
in the inclined position. The medium should be fairly firm in 
consistency and some fluid should collect at the bottom of the 
slant. The medium is useful for cultivation of the gonococcus, 
the influenza bacillus, streptococcus, pneumococcus and meningo- 



CULTURE MEDIA 99 

coccus, but more especially for cultivation of the flagellated 
hematozoa such as trypanosomes and related organisms, including 
the Leishman-Donovan bodies. 

Smith's Broth Containing Sterile Tissue. Pieces of naturally 
sterile organs, usually liver or kidney, are placed in broth, more 
particularly in fermentation tubes of broth. The bits of tissue 
are conveniently handled by touching with a hot platinum wire 
or glass capillary, to which they will adhere. The medium is 
especially useful for the culture of anaerobic bacteria. Naturally 
sterile blood added to the broth also serves for this purpose. 

Ascitic-fluid-agar. This is made in the same way as the 
Novy's blood-agar except that naturally sterile human ascitic 
fluid is employed instead of blood. The medium is beautifully 
transparent, and may be employed for plating as well as for tube 
cultures. It is especially valuable for cultivation of the gono- 
coccus and also for the streptococcus, pneumococcus and 
meningococcus. 

Noguchi's 1 Ascitic Fluid with Sterile Tissue. Naturally 
sterile tissue is placed in a tall tube. A deep layer of ascitic fluid 
is added, and for some purposes this is covered with a layer of 
sterile paraffin oil. The medium is used more especially for the 
cultivation of the blood spirochetes which cause relapsing fever. 

1 Noguchi: Journ. Exp. Med., Jan. i, 1912, Vol. XV, pp. 90-100. 



CHAPTER IV. 

COLLECTION OF MATERIAL FOR BACTERIOLOGICAL 

STUDY. 

Bacteria under natural conditions are usually associated as 
mixtures of several species living together. Only under rather 
exceptional circumstances will a single kind of bacteria be found 
growing alone. This does occur in disease, however, where the 
living host may be able to keep out all but the one kind of mi- 
crobe. But even diseased tissues or exudates originally harbor- 
ing only one kind of bacteria may quickly acquire others in abun- 
dance after removal from the living body. It is well therefore 
to regard any material presented for bacteriological examination 
as potentially, and in all probability actually, harboring several 
kinds or species of bacteria. The direct planting of such material 
on a culture medium .will, therefore, in most instances give rise 
to a mixed culture, in which those forms least prominent in the 
original material may easily appear as most important. If the 
material be allowed to stand, especially if it be a favorable 
medium for bacterial growth, the relationships present may be- 
come seriously confused. It should, therefore, be examined as 
fresh as possible. When immediate examination is impossible 
the material should be kept on ice. 

Samples of water, milk or other fluid should be collected in 
sterilized tubes or bottles. Samples of solid food should be 
seared or charred all over the surface and divided with a sterilized 
knife. A small piece of the interior is then removed to a sterilized 
glass dish and covered. 

Material removed from the human or from the animal body dur- 
ing life or at autopsy may.be, bacteria-free, : or it may contain 
one or more specie^, of -microbes. Ijt is irnportaiit that the picture 

100 



MATERIAL FOR BACTERIOLOGICAL STUDY IOI 

be not confused by the addition of bacteria from the surface of 
the body, from instruments or from the air during the collection^ 
and transportation to the laboratory. Unfortunately the 
laboratory study of such material is too often rendered untrust- 
worthy or worthless through lack of attention to this point. 

When merely microscopic examination is to be undertaken, 
contamination may not be serious, and an antiseptic, such as 
two per cent of carbolic acid, may be added to the material, if 
fluid, and if solid it may be immersed in ten per cent formalin. 
The bottles used should be new and clean. Such material may 
also be spread on microscopic slides or cover-glasses in a thin 
layer, dried, fixed in the flame, and transported to the labora- 
tory. This method is not always free from danger when the 
material passes through several hands. Special precautions for 
collecting material for microscopic examination will be considered 
in discussing the specific pathogenic microbes. 

Specimens of sputum should be raised from the trachea, bron- 
chi and lungs after previously cleansing the mouth. Sputum 
should be received into a sterile wide-mouthed bottle, and stop- 
pered with a sterilized cork. The exterior of the bottle should 
then be carefully washed with 5 per cent carbolic acid. 

Urine should be collected by catheter with careful aseptic 
technic, and should be received in a clean sterilized bottle. 

Blood and transudates are collected by the technic previously 
described (page 95). Blood is drawn from the vein by means 
of the Luer syringe and is quickly ejected into several flasks of 
broth (150 to 250 c.c.) and into Petri dishes where it is mixed with 
melted agar, (cooled to 50 C. ) before clotting takes place. 

Cerebro-spinal fluid is obtained by inserting a sterilized needle 
(4 cm. long for children, 8-10 cm. long for adults, and with 
lumen i mm.) a little to one side of the median line in the back, 
so that it enters the spinal canal between the second and third, 
or between the third and fourth, lumbar vertebrae. Aseptic 
technic is essential. The fluid coming from the needle is 
received in a sterile tube. 



102 



BACTERIOLOGY 



Feces from infants and young children are best collected by 
means of a heavy glass tube closed and rounded off at the end, and 
provided with a lateral opening near the closed end. This is 
enclosed in a larger tube and sterilized. It is inserted well into 
the rectum with aseptic technic and the entrance of fecal material 
through the lateral opening is favored by 
gently moving the tube. It is then with- 
drawn and replaced in its original container 
to be transported to the laboratory. From 
adults the feces are passed directly into a 
sterilized covered agateware basin without 
other special apparatus. 

Intestinal juice from the duodenum may 
be obtained in infants 1 by inserting a sterile 
rubber catheter, closed below with a steri- 
lized gelatin capsule, through the esophagus 
and stomach into the duodenum. The cap- 
sule is then blown off by pressure from a 
sterile syringe attached at the other end of 
the catheter and the fluid contents of the 
duodenum aspirated. In adults 2 the Einhorn 
duodenal tube is employed. The tube is 
of instrument for obtain- sterilized by boiling and the lower opening 

mg feces from infants for J 

bacteriological examina- sealed with a sterilized gelatin capsule and 
by finally coating with shellac. The tube is 
inserted through the esophagus and is carried 
through the pylorus by peristalsis. Ordinarily it is inserted in 
the evening. On the following morning the seal at the lower end 
is broken by pressure of a sterile syringe attached to the free end 
of the tube and the sample of juice aspirated. Intestinal juice 
may be obtained from various levels in the jejunum also by regu- 
lating the length of tube inserted. 

Pus and other exudates are best collected in sterile glass capil- 




FIG. 38. Two types 



tion. (After Schmidt and 
Strasburger.) 



1 Hess: Journ. Infections Diseases, July 1912, Vol. XI, pp. 71-76- 
2 MacNeal and Chace: Arch. Int. Med., Aug., 1913, Vol. XII, pp. 178-197- 



MATERIAL FOR BACTERIOLOGICAL STUDY 103 

lary pipettes (see page 33). A sterilized cotton swab, made 
by winding a pledget of absorbent cotton around the end of a stiff- 
wire, enclosing it in a test-tube and sterilizing it, is also useful, 
especially when it is impossible or undesirable to employ the 
glass tube. 

At autopsies on human subjects, the same principles for col- 
lection of material apply. Fluids are best collected in sterile 
glass pipettes and even solid organs may be seared and punctured 
with a strong glass capillary into which some of the pulp is drawn 
by suction. The tubes may be sealed in the flame and trans- 
ported considerable distances to the laboratory. This is usually 
more satisfactory than the inoculation of culture media in the 
autopsy room, especially if the facilities for bacteriological work 
there are somewhat limited. Smears on slides or cover-glasses 
should also be made for microscopic examination, and pieces of 
the various organs fixed in alcohol or formalin and preserved 
for sectioning. 



CHAPTER V. 
THE CULTIVATION OF MICRO-ORGANISMS. 

Avoidance of Contamination. Micro-organisms are so numer- 
ous on the body of man and in his environment that they are likely 
to be present on all articles about us unless special precautions 
are taken to remove or destroy them. The dust blown about 
in the air contains bacteria and spores of molds. The primary 
essential in all bacteriological culture work is the exclusion of 
these extraneous micro-organisms. The unskilled or careless 
worker may quickly add some of these chance organisms to the 
material which he is attempting to study, introducing an element 
of almost hopeless confusion unless it is recognized. Another 
essential of great importance, especially when working with patho- 
genic microbes, is the complete destruction of all living bacteria 
before they are allowed to pass beyond strict and absolute con- 
trol. The unskilled or careless worker in the laboratory, who 
allows micro-organisms to escape from him while he is attempt- 
ing to study them, is a serious menace not only to himself but to 
all others in the laboratory. These two primary essentials must 
be mastered by practice in handling harmless forms. 

Every instrument with which bacteria are handled should be 
sterilized before it is used, and again after use. In the case of 
the commonly used platinum wire, this sterilization is accom- 
plished in the flame. The wire is heated to a glow and allowed 
to cool before handling bacteria, and immediately after its use, 
before it leaves the hand, it is brought close to the flame so as to 
dry the material on it and then again heated to redness. Care- 
ful drying in this way avoids sputtering and consequent scattering 
of bacteria, which is almost certain to occur if moist material, 
especially fat or protein, is immediately thrust into the flame. 

104 



THE CULTIVATION OF MICRO-ORGANISMS 1 05 

In using the Bunsen flame for sterilization, the innermost cone 
near the base of the flame may be utilized for drying material on_ 
the end of the wire. This inner cone is not burning and is com- 
paratively cool, and after a little practice the end of the wire 
is easily brought into it and dried without sputtering. Slowly 
elevating the wire brings it gradually into hotter zones of the 
flame until it glows. 

Bacteria do not of themselves leave a moist surface. They 
are not even removed by moderate currents of air unless they 
have been previously dried. Their distribution about the labora- 
tory, therefore, results from relatively gross accidents or 
gross carelessness. When material containing bacteria is acci- 
dentally spilled, it should be covered at once with disinfectant 
solution, such as i-iooo mercuric-chloride solution. As a rou- 
tine procedure it is well to wash the work table daily with bi- 
chloride solution and, when working with pathogenic bacteria, 
to wash the hands at the end of the day's work, first with the 
bichloride solution and then with soap and water. 

Isolation of Bacteria. In order to study any kind of bacteria 
it is necessary to have the particular species separated from other 
sorts with which it may be mixed. The earlier bacteriologists 
endeavored to separate bacteria of different sorts by successive 
transplantations through a series of tubes of fluid media, one 
kind of bacteria outgrowing the rest. Isolation was also ac- 
complished by diluting the material very highly and then in- 
oculating one drop into each of a large number of tubes of 
broth. Some tubes would thus receive no bacteria, others would 
receive several, and occasionally one would receive only a single 
germ and would give rise to a pure culture. Another early 
method of separating a pathogenic species was by inoculation 
of animals. The ability of the animal to prevent the development 
of all but one species contained in the inoculated material was 
utilized to obtain the first pure cultures of anthrax bacilli and 
tubercle bacilli. These methods are successfully employed only 
for relatively few bacterial species. 



IO6 BACTERIOLOGY 

Methods of isolating bacteria, which are of more general appli- 
cation, were introduced by Koch. The essential characteristic 
of these methods is the dilution of the bacteria in a fluid medium 
which quickly becomes solid so that each germ develops in a 
definite fixed position in the medium. The great progress which 
bacteriology has made during the last twenty years is largely 
owing to these methods. 

It is impossible in most cases to distinguish between bacteria 
of different varieties by microscopical examination alone. Bac- 
teria of widely different species and quite unlike one another in 
their properties may present similar appearances under the mi- 
croscope. The differences which they exhibit are usually appar- 
ent when they are grown in culture-media. The growth, called 
a colony, which results from the multiplication of a single 
bacterium, is in many cases very characteristic for the species. 
By the plate-method, the individual bacteria in a mixture are 
separated from one another by dilution. They are fixed in place 
by the use of a solid medium. They are allowed to grow, and 
from each individual there arises a colony. It is usually possible 
to distinguish between colonies arising from different species when 
it is not possible to distinguish between the individual bacteria 
of these species. A convenient comparison has been suggested by 
Abbott. A number of seeds of different sorts may appear very 
much alike, and considerable difficulty may be found in distin- 
guishing one from another with the eye. Let them be sown, how- 
ever, and let plants develop from them, and these plants will 
easily be distinguished from one another. 1 

Method of Making Plate -cultures. Melt three tubes of gela- 
tin or agar. (There is some difficulty in keeping agar in a fluid 
state while dilutions are being made. It is necessary to have 
some form of water-bath with a thermometer for the purpose.) 
Let the liquefied agar cool to 45 C. Gelatin may be used at a 

1 It must be understood that no close comparison can be drawn between higher 

"ally present in the 
the progeny of one 



'rb l/U-70- l/C> U'/ U/UUrb U&I/W&&HP ilrvgiwi 

plants, which simply complete the development of parts potentially present in the 
seed, and colonies of bacteria, which are aggregates of individuals, 



individual of the same kind. 



THE CULTIVATION OF MICRO-ORGANISMS IOy 

temperature anywhere between 28 and 40 C. Take a small 
portion of the material to be examined pus, for example and- 
introduce it with a sterilized platinum wire or loop into one of 
the tubes. The plug of the test-tube is to be withdrawn, twisting 
it slightly, taking it between the third and fourth fingers of the 
left hand, with the part that projects into the tube pointing to- 
ward the back of the hand. It must not be allowed to touch 
any object while the inoculation is going on. Pass the neck of 
the tube through the flame. If any of the cotton adheres to the 
neck of the tube, pull the cotton away with sterilized forceps, 
while the neck of the tube touches the flame, so that the threads 
of cotton may be burned and not fly into the air of the room. 




FIG. 39. Method of inoculating culture media. 

The tube is held as nearly horizontal as possible. The tube is 
to be held in the left hand between the thumb and forefinger, 
the tube resting upon the palm, and the neck of the tube pointing 
upward and to the right. Mix the material introduced thor- 
oughly with the liquefied culture-medium, taking care not to wet 
the plug. Now remove the plug again, and, having sterilized the 
platinum wire, insert it into the liquefied medium. Carry three 
loopfuls in sucession from thistube/whichisNo. i, into tube No. 2. 
When two tubes are being used at the same time, they are 
placed side by side between the thumb and forefinger of the left 
hand. The two plugs are held between the second and third and 
the third and fourth fingers of the left hand, respectively. The 
wire may now be passed into the first tube, which we will suppose 



io8 



BACTERIOLOGY 



to hold some material containing bacteria, and a little of this 
material may be removed on the tip of the wire from the first 
tube to the second. When the needle is introduced into or re- 
moved from either tube it should not touch the side of the tube 
at any point, and should only come in contact with the region 
desired. After inoculation of the second tube has been effected, 
the wire is to be heated to a red heat in the flame, the necks of 
the tubes are to be passed through the flame, and the plugs are to 
be returned to their respective tubes. In the same manner 
transfer three loopfuls from tube No. 2 into tube No. 3. The 
original material will obviously be diluted in tube No. i, more in 
tube No. 2, and still more in tube No. 3. The most convenient 
form of plate is that known as a Petri dish, a small glass dish 




FIG. 40. Petri dish. 

about 10 cm. in diameter and 1.5 cm. in height, provided with a 
cover which is a little larger but of the same form. This dish 
should be cleaned and sterilized for an hour in a hot-air sterilizer 
at 1 50 C. or higher. When it is cool it may be used. 

Such dishes having previously been prepared, the contents of 
tube No. i are poured into one dish, and those of tube No. 2 
into another, and those of tube No. 3 into a third. They are to 
be labeled Nos. i, 2, and 3. 1 In pouring proceed as follows: 
remove the plug of tube No. i ; heat the neck of the tube in the 
flame; allow it to cool, holding it in a nearly horizontal position. 
When the tube has cooled, lift the cover of the Petri dish a little, 
holding it over the dish; pour the contents of tube No. i into the 
dish, and replace the cover of the dish. The interior of the dish 

1 The labels should be moistened with the finger, which has been dipped in water. 
They should not be licked with the tongue. While working in the bacteriological 
laboratory it is best to make it a rule that no object is to be put in the mouth. 



THE CULTIVATION OF MICRO-ORGANISMS 1 09 

should be exposed as little and as short a time as possible. Tubes 
Nos. 2 and 3 are to be treated in the same manner. Burn the 
plugs, and immerse the empty tubes in 5 per cent solution of 
carbolic acid. Where much culture work is being done, it will be 
found convenient to sterilize the mouth of each tube by thorough 
heating in the flame after pouring out its contents, and then to 
replace the plug. The tube may then be placed in a special 
receptacle which is sterilized with its contents in the autoclave 
at 120 C. for 20 minutes, at the end of the day's work. 




FIG. 41. Colonies in gelatine plate showing how they may be separated and the 

organisms isolated. 

The culture-medium in the Petri dish will soon solidify. 
Petri dishes of agar should be inverted after the medium is firmly 
set; otherwise the water, which evaporates from the surface and 
condenses on the inside of the lid, may overflow the surface of 
the agar, confusing the result. Agar plates are usually developed 
in the incubator. Gelatin plates must be developed at a tempera- 
ture below the melting-point of the medium, which is usually 
between 22 and 28 C. Colonies usually appear in from one to 
two days. In plate No. i they will be very numerous, in plate 



no 



BACTERIOLOGY 



No. 2 less numerous, and in plate No. 3 still less numerous. 
Where the number is small the colonies will be widely separated 
and can readily be studied. They may be examined with a hand- 
lens, or the entire dish may be placed on the stage of the micro- 
scope and the colonies be inspected with the low power. The 
iris diaphragm should be nearly closed and the plane mirror 
should be used. Dilution-cultures prepared as described in the 
next paragraph, where the principle is the same, are shown in 
Fig. 43. In tube No. i the colonies are so numerous as to look 
like fine white dust. In tubes 2 and 3 they become less numerous 
and larger. 

Esmarch's Roll-tubes. Use liquefied gelatin or agar. The 
dilutions in tubes i, 2 and 3 are made as above. Tubes contain- 







FIG. 42. Manner of making Esmarch roll- tube. 

ing a rather small amount of the culture-medium are more con- 
venient. A block of ice should be at hand, and, with a tube filled 
with hot water and lying horizontally, a hollow of the size of the 
test-tube should be melted on the upper surface of the ice. In 
this hollow, place the tube of liquefied gelatin or agar; roll it rapid- 
ly with the hand, taking care that the culture-medium does not 
run toward the neck as far as the cotton plug. The medium is 
spread in a uniform manner around the inside of the tube, where 



THE CULTIVATION OF MICRO-ORGANISMS 



III 



it becomes solidified. Gelatin roll-tubes must be kept in a place 
so cool that there is no danger of their melting; in handling them 




FIG. 43. Dilution-cultures in Esmarch roll-tubes. In tube i the colonies are 
very close together; in tube 2 they are somewhat separate; in tube 3 they are well 
isolated. 

they are to be held near the neck, so that the warmth of the hand 
may not melt the gelatin. Agar roll- tubes should be kept in a 



112 BACTERIOLOGY 

position a little inclined from the horizontal, with the neck up, 
for twenty-four hours, so that the agar may adhere to the wall 
of the tube. 

By the plate-method as originally devised by Koch, instead of using 
Petri dishes, the gelatin was poured upon a sterile plate of glass. This plate 
of glass was laid on another larger plate of glass, which formed a cover for a 
dish of ice-water, the whole being provided with a leveling apparatus. The 
plate was kept perfectly level until it had solidified, which took place rapidly 
on the cold surface. The glass plates were placed on little benches enclosed 
within a sterile chamber. The more convenient Petri dish has now displaced 
the original glass plate. 

Streak Method of Isolating Bacteria. The isolation of bac- 
teria may sometimes be effected by drawing a platinum wire 
containing material to be examined rapidly over the surface of a 
Petri dish containing solid gelatin or agar; or over the surface of 
the slanted culture-medium in a test-tube; or by drawing it over 
the surface of the medium in one test-tube, then, without steril- 
izing, over the surface of another, perhaps over several in succession. 
This method is ordinarily less reliable than the regular plating 
method. 

Veillon's Tall-tube Method. Three to six tubes of glucose 
agar, the agar being at least 6 cm. deep, are liquefied and cooled 
to 45 C. in a water-bath. A small amount of the material to 
be examined is placed in the first tube by means of the platinum 
loop, and carefully mixed. From this dilutions are made in series 
to tubes, 2, 3, 4, 5 and 6, each being carefully mixed without intro- 
ducing air bubbles. The tubes are quickly solidified by immersion 
in cold water, and are incubated at 37 C. These culture tubes 
offer the contained bacteria a wide range of oxygen supply. 
This is abundant at and near the top, and gradually diminishes 
lower in the tube until near the bottom almost perfect anaerobic 
conditions obtain. The method is very useful in isolating B. 
bifidus from feces of infants, and in studying the oxygen require- 
ments of other bacteria. When energetic gas-forming bacteria 
are present in considerable number, the method is of no value. 



THE CULTIVATION OF MICRO-ORGANISMS 113 

Colonies are picked out with sterile glass capillaries, and deeper 
colonies are reached by breaking the tube. The successful use 
of the method requires some practice. 

Appearance of the Colonies. The colonies obtained in the 
Petri dishes or roll-tubes (Fig. 43) may be studied with a hand- 
lens or with a low power microscope. In the latter case, use the 
plane mirror with the iris diaphragm nearly closed. The colonies 
present various appearances. Some of them are white, some 
colored; some are quite transparent and others are opaque; some 
are round, some are irregular in outline; some have a smooth 
surface, others appear granular, and others present a radial 
striation. Surface colonies often present different appearances 
from those occurring more deeply. Surface colonies are likely 
to be broad, flat and spreading. If the colony consists of bacteria 
which have the property of liquefying gelatin, a little funnel- 
shaped pit or depression forms at the site of the colony. The 
appearance of colonies may be of great assistance in determining 
the character of doubtful species. The appearance in gelatin 
plates of the colonies of the spirillum of Asiatic cholera, for in- 
stance, is one of the most characteristic manifestations of this 
organism. 

Pure Cultures. From these colonies pure cultures may be 
obtained by the process called "fishing." Select a colony from 
which cultures are to be made; touch it lightly with the tip of a 
sterilized platinum wire, taking great care not to touch the me- 
dium at any other point. Introduce the wire into a tube of gelatin 
after removing the plug and flaming the mouth of the tube. 
Sterilize the wire and plug the tube. In a similar manner, and 
from the same colony, inoculate tubes of agar, bouillon, milk, 
potato and blood-serum. Gelatin tube cultures are usually inocula- 
ted by introducing the platinum needle into the medium vertically, 
making a "stab-culture." Inclined surfaces such as those of 
agar, potato or blood-serum are inoculated by drawing the wire 
lightly over the surface of the medium, making a "smear-culture" 
or "streak-culture" (Figs. 44 and 45). Liquid media are inocula- 
8 



BACTERIOLOGY 

ted by simple introduction of a small mass of bacteria and mixing 
them with the medium. At the same time it is well to make a 
smear preparation from the colony and to stain with one of the 
aniline dyes so as to determine the morphology of the bacteria. 
The growths which take place in the tubes should contain one 
and the same kind of bacteria. As seen under the microscope 
these bacteria should have the same general form and appearance 



FIG. 44. Stab-culture. 

A rubber stopper may 
be used to prevent drying, 
see page 121. 



FIG. 45. Smear-culture. 
This tube shows the 
rubber cap used to prevent 
drying. 



as those seen in the colony from which they were derived. This 
will be the case, provided the colony has resulted from the develop- 
ment of a single bacterium. 

A pure culture is a culture which contains only the descendants 
of a single cell. 

Stock Cultures. To maintain their vitality bacteria need to 
be transplanted from one tube to another occasionally; the time 



THE CULTIVATION OF MICRO-ORGANISMS 11$ 

varies greatly with different species. Many bacteria grow on 
culture-media with difficulty at the first inoculation, but having^ 
become accustomed to their artificial surroundings, as it were, 
they may be propagated easily afterward; this is especially true 
of the tubercle bacillus. After they are developed, stock cultures 
are best kept in a refrigerator, and it is well to seal them so as to 
prevent drying. Rubber caps or rubber stoppers are useful for 
this purpose (Figs. 44 and 45). 

Some bacteria flourish better on one culture-medium than 
another. The tubercle bacillus grows best on blood-serum and 
glycerin-agar; the bacillus of diphtheria grows best on Loffler's 
blood-serum; the gonococcus on human serum-agar or ascitic- 
fluid-agar. 

The virulence of most pathogenic bacteria becomes diminished 
after prolonged cultivation upon media. In some forms the viru- 
lence is lost very quickly, for example, the Streptococcus pyo genes 
and Micrococcus lanceolatus of pneumonia. 

REGULATION or TEMPERATURE. 

High-temperature Incubator. Many bacteria flourish best 
at a temperature about that of the human body, 37 C. Some 
species will grow only at this temperature. The pathogenic bac- 
teria in particular, for the most part, thrive best at a point near 
the body temperature. 

The ordinary incubator is a box made of copper, having 
double walls, the space between the two being filled with water. 
The outer surface is covered with some non-conductor of heat, 
such as felt or asbestos. At one side is a door, which is also double. 
The inner door is of glass, the outer door is of copper covered 
with asbestos. At one side is a gauge which indicates the level 
at which the water stands in the water-jacket. The roof is per- 
forated with several holes, some of which permit the circulation 
of the air in the air-chamber inside the box; some of them enter 
the water-jacket. A thermometer passes through one of these 



n6 



BACTERIOLOGY 



holes into the interior of the air-chamber, and often another into 
the water standing in the water-jacket. A gas-regulator passes 
through another hole, and is immersed in the water standing in 
the water-jacket. There are various forms of gas-regulators 




FIG. 46. Incubator. 

more or less complicated. The simplest and least expensive 
thermo-regulators for gas are made of glass and filled with mercury 
or with mercury and some lighter liquid, the heavy mercury 
serving to close the chief source of gas supply when the desired 



THE CULTIVATION OF MICRO-ORGANISMS 



temperature has been attained, while a minute opening at another 
point remains open to furnish sufficient gas to keep the flame 
alight, but not sufficient to maintain the temperature. Upon 
cooling the mercury falls and allows gas to flow again through 
the larger opening. In this way the supply of gas is made large 
whenever the temperature is a little below the 
desired temperature and very small whenever 
the temperature rises above that point, and the 
temperature varies within a slight range. The 
Reichert regulator is designed to operate ac- 
cording to these principles, and various modi- 
fications of this regulator are on the market. 
In many of these instruments the larger supply 
is only imperfectly shut off at the desired tem- 
perature, and, where the weight of the mercury 
is relied upon to stop this opening, the gas may 
often bubble out through it unless special pre- 
cautions are taken to regulate the pressure of 
the gas supply. 

A modification of this type of regulator 
devised by Mac Neal 1 overcomes this difficulty 
(see. Fig. 48). The inlet tube A leads through 
the wall of the chamber D, to which it is fused, 
into an inner upright tube, B C . Near the upper 
end of this upright is a small opening, O, which allows the minimum 
supply of gas to pass to the burner to avoid extinction of the flame. 
The lower end of this upright tube fits quite closely the bottom of 
the chamber D, around the opening leading into the capillary 
tube, EF. This end is adjusted so close to the bottom that mer- 
cury will not pass through between inner and outer tube at less 
than twenty millimeters mercury pressure, yet not so close but 
that an abundant supply of gas may pass. The proper adjust- 
ment of this part must be thoroughly tested before the instrument 
leaves the factory. The upper end of the upright, BC, is closed 

1 The Anatomical Record, August, 1908, Vol. II, No. 5. 




FIG. 47. Reichert's 
gas-regulatcr. 



n8 



BACTERIOLOGY 



by a ground glass stopper, which also closes the top of the outer 
chamber, D. In the ground surface of this stopper a gamma- 
shaped (r) groove is cut, the vertical limb extending from the 
lower tip of the stopper to the level of the opening, O. The 
horizontal limb is deep where it joins the ver- 
tical, but gradually becomes shallow and ends 
about one-quarter the way around the stopper. 
This groove serves for passage of the gas from 
the inner tube BC, to the opening O, and thus 
to the outer chamber D, and by rotating the 
stopper, the amount of gas flowing through 
this passage may be reduced to any desired 
point. The outlet tube, H, leads from the 
chamber D to the burner connection. 

The capillary, EF, leads to a bulb of suffi- 
cient size; the larger the more sensitive the 
instrument. Either the large bulb with inside 
capillary, J, to be filled with mercury and 
alcohol, or the smaller simple bulb for mercury 
alone may be used. A side arm is attached 
to one side of the capillary EF, for conveni- 
ently controlling the height of the mercury 
column. Either the curved capillary tube 
with stopcock and a cup on the end, or the 
simple tube with metal screw cemented in, 
may be used here, according to the purpose 
which the regulator is to serve. These parts 
FIG. 48. Mac Neal are s i m ii ar to those of Novy's modification of 

gas-regulator. 

the Reichert regulator. 

To fill the instrument, the air is partly driven out by heating 
the bulb and then the desired liquid is drawn in by cooling, 
repeating the heating and cooling until the instrument is full 
of the liquid. For the small bulb, mercury is always used alone. 
The large bulb, on the other hand, is filled first with either ether, 
alcohol or toluol, and then part of this liquid is forced out by 




THE CULTIVATION OF MICRO-ORGANISMS 



IIQ 



heat and replaced with mercury so that the capillary EF, the bulb 
at its lower end, and a small part of the large bulb J, are occupied, 
by the mercury. Ether may be used when the regulator is not 
to be heated above 35 C., alcohol when it is not to be heated 
above 75 C., and toluol for temperatures between 75 and ico C. 
A more satisfactory regulator is that of Roux. It is con- 
structed entirely of metal, and its 
operation is due to the unequal 
expansion and contraction of two 
metals which are riveted together. 
Fig. 49 shows this regulator. The 
gas passes in at e and passes out 
at d. The amount of gas passing 
through is regulated by a piston 
on the end of the set screw inside 





FIG. 49. Roux bime- 
tallic gas-regulator, a, Set 
screw; b, Screw collar; c, 
Clamp; d, Outlet for gas; 
e, Inlet for gas. 



FIG. 50. Koch auto- 
matic gas-burner. 



the tube from which the outlet tube branches off. This piston 
moves in or out according to the changes of temperature of the 
water jacket of the incubator into which the stem (/") of the regu- 
lator is inserted. This stem is fenestrated and has the riveted 
metallic strips running down in it. These strips are pivoted at 
the collar, g. 



1 20 BACTERIOLOGY 

The gas coming from the gas-regulator passes to a Bunsen 
burner, which stands underneath the incubator. This burner 
should have some kind of automatic device for cutting off the flow 
of gas in case it becomes accidentally extinguished by a sudden 
draught of air or from any other cause. The automatic burner 
invented by Koch is an ingenious, simple and effective device 
(Fig. 50). The coils of metal -seen on each side at the top of 
the burner are so arranged that when they expand they turn the 
disk below so as to support the arm coming from the stop-cock; 
when they cool they turn the disk in the opposite direction, and 
allow the arm to fall and cut off the gas. Some inconvenience 
will at times arise from irregularities in the flow of gas from the 
main supply-pipe. A properly constructed regulator should, 
however, compensate perfectly for all ordinary variations in pres- 
sure of artificial gas. Natural gas is commonly furnished at 
much higher pressure and it is necessary to install apparatus 
to reduce the pressure, a gas-pressure regulator, between the gas 
main and the thermoregulator. Fluctuations of the temperature 
within the incubator depend very largely upon the external 
temperature, especially if its outer walls are not well insulated. 
The incubator should, therefore, be kept in a place free from 
draughts of air, where the temperature is fairly constant. 

In large modern laboratories, the incubators are built in as 
special insulated rooms, heated by a gas stove. A regulator of 
large size is installed to control the supply of gas to the stove. 
These incubator rooms are very satisfactory and provide quite a 
range of constant temperature according to the height of shelves 
from the floor. 

Culture-tubes which are being kept in the incubator are likely 
to become dry if their stay is prolonged. In such cases they 
should be covered with rubber caps, tin-foil, sealing-wax, paraffin, 
or some other device to prevent evaporation. If rubber caps 
are used, they should be left in i-iooo bichloride of mercury 
solution for an hour, and the cotton plugs should be singed in the 
flame, before putting them on (Fig. 45). Some bacteriologists 



THE CULTIVATION OF MICRO-ORGANISMS 121 

prefer rubber stoppers, which may be boiled and stored in bi- 
chloride of mercury solution. Cut the cotton plug even with the- 
edge of the tube; singe it in the flame; push it into the tube about 
i cm., and insert the rubber stopper (Fig. 44). 

Low-temperature Incubator. An incubator regulated for so- 
called "room temperature" is very desirable for the cultivation 
of bacteria upon gelatin and for the bacteriological analysis 
of water. In our climate the temperature of the rooms of the 
laboratory often reaches a point at which gelatin melts, and 
for this reason in a low-temperature incubator provision has to 
be made for cooling when the room temperature is too high as 
well as for heating when it is too low. 

A form of incubator devised by Rogers 1 for this purpose 
consists of a refrigerator or of a specially constructed chamber 
heated by electricity and controlled by an electric thermoregu- 
lator. Below is given a description of an incubator constructed 
according to Rogers' plans. This incubator has been in use 
for some time and has given entire satisfaction since the pre- 
cautions noted below were followed. There would appear no 
reason why this incubator should not be employed for high 
temperatures as well as for low, but so far it has been run at 22 C. 
The temperature has kept very constant. The incubator con- 
sists of a refrigerator, 30 inches high, 24 inches wide, 18 inches 
from front to back, all outside measurements. Instead of the 
ordinary drip pipe, there is a coil of i-inch galvanized iron pipe 
run down the back of the cooling chamber attached water-tight 
to the ice tank. From the bottom of the cooling chamber the 
coil runs up perpendicularly nearly to the bottom of the ice 
compartment, and then runs horizontally through the wall of the 
refrigerator. A bracket on the outside supports a drip-pan. 
A thermometer encased in a fenestrated metal jacket is inserted 
about half way up on one side. A lump of ice, about 50 pounds, 
placed in the ice compartment serves to keep the tem- 

1 L. A. Rogers. On electrically controlled low temperature incubators. Cen- 
tralblatt fur Bakteriologie, etc., Bd. XV, Abt. II, pp. 236-239, Sept. 23, 1905. 



122 



BACTERIOLOGY 



perature sufficiently cool. In summer doubtless more ice will be 
required. 

For heating, an ordinary i6-candle-power electric bulb is 
used, and the electricity is obtained from the public supply. 
The wire is run through one of the walls, and a part of the current 
is made to operate a horse-shoe magnet, and another part is 
conducted through the lamp used for heating. 

The accompanying diagram (Fig. 51), will serve to- show 
the arrangement. 

A telegraph key is used to supply the horse-shoe magnet 




Ther.morea/ulator 
FIG. 51. Diagram of electric regulator for low- temperature incubator. 

which is inserted in the heating circuit in such a way that when 
the armature is attracted toward the magnet the circuit is com- 
pleted and the lamp is consequently lighted. The part of the 
current, a, supplying the magnet first passes through a small 
lamp and through two resistance coils so as to red ace the current. 
It then passes through the magnet, and is continued on to the 
set-screw, b, which is so placed that when the thermoregulator 
comes in contact with it the circuit is complete. The, regu- 
lator consists of a strip of hard rubber and a strip of brass riveted 






THE CULTIVATION OF MICRO-ORGANISMS 123 

together. One end is fixed, while the other is free, and when it 
is heated it tends to bend toward the metal side, when it cools it 
bends toward the rubber. The brass strip is 15 inches long, J 
inch thick, and \ inch wide; the rubber strip is \ inch thick, \ 
inch wide, and a little less than 15 inches long. In the diagram 
the end is fixed at d and is free at b. When it is heated, the free 
end travels away from the set-screw at 6; when it cools, it moves 
toward the set-screw. Rogers also recommends a regulator 
made of invar and brass instead of hard rubber and brass. Where 
invar is used instead of the hard rubber the dimensions for the 
two metals are the same as those given for the brass strip in the 
hard-rubber-brass regulator just described. As is evident from 
the description, the circuit controlling the magnet is closed when- 
ever the free end of the regulator comes in contact with the set 
screw at b. When this circuit is closed the magnet attracts the 
armature, and the heating circuit is closed by the contact formed 
at c between the armature and the set-screw. In the diagram 
this point of contact is put to one side for the sake of clearness, 
but as a matter of fact in the instrument in use, the set-screw is 
above and between the ends of the horse-shoe magnet, and 
comes in contact with the armature which is extended upward 
in the shape of a tongue. From the description just given it will 
be noted that the thermoregulator does not control the heating 
directly, but indirectly through the electro-magnet. 

Certain precautions have been found necessary in practice 
in order to obtain satisfactory results with this incubator. The 
set-screw against which the armature strikes at c should be so 
set that the armature does not come in contact with the magnet. 
In the apparatus described above there is a space of about J inch 
between the armature and the magnet when contact takes place 
between the set-screw and the armature. If the set-screw does 
not project far enough to prevent the armature from coming in 
contact with the magnet, the armature may adhere to the 
magnet even after the current is broken at b, and when this is 
the case of course the lamp remains lighted, and the temperature 



1 24 BACTERIOLOGY 

may run up too high. This sticking of the armature to the mag- 
net is said to be due to the residual magnetism left in the core of 
the magnet. When the current passing through the magnet is 
broken by the excursion of the end of the thermoregulator away 
from the set- screw at b, the armature is pulled away from the 
magnet by a coiled spring. Another important precaution is 
that the points at which contact is made and broken, b and c, 
should be tipped with platinum. A small piece of platinum 
wire inserted into the ends of the set-screws and a few square 
centimeters of platinum foil riveted to the opposite point of con- 
tact, meet the requirements. If platinum is not used at these 
points oxidation takes place and prevents contact. The set- 
screw at b is set by experiment for the temperature desired. 
The further the point of the set-screw projects toward the free 
arm of the regulator, the higher the temperature maintained. 

CULTIVATION OF ANAEROBIC BACTERIA. 

Deep Stab Culture. Bacteria which cannot grow in the pres- 
ence of atmospheric oxygen may be successfully cultivated by 
methods in which the oxygen is excluded or its concentration 
diminished. The simplest procedure, first practised by Liborius, 
is to make deep stab cultures into freshly solidified alkaline 
glucose agar. The agar quickly closes over the needle track and 
any traces of oxygen introduced into the depths of the agar are 
absorbed and reduced by the glucose in the presence of the 
alkali. The bacteria thus find at various points along the punc- 
ture all variations in partial pressure of oxygen from almost 
complete absence up to the concentration existing in the atmos- 
phere at the surface of the medium. Obligate anaerobes begin 
to grow near the bottom and, as the gases produced replace the 
air above, the growth extends upward, often even entirely to the 
surface. 

Veillon Tube Cultures. Isolated colonies of anaerobic bac- 
teria may be obtained by a modification of this tube method of 



THE CULTIVATION OF MICRO-ORGANISMS 125 

Liborius, which seems to have been used first by Veillon. Several 
tubes of glucose agar are melted, cooled to 45 C. and then in- 
oculated by dilution in series just as if plate cultures were to be 
made. After careful mixing the agar is quickly congealed by 
standing the tubes in cold water. The later tubes in the series 
should contain only a few bacteria so that single colonies may 
develop. The method serves for anaerobes and also for those 
kinds of bacteria which seem to require some free oxygen but do 
not grow well when exposed to the full amount in the atmosphere 
(B. abortus, B. bifidus). 

Fermentation Tube. Anaerobic bacteria grow excellently 
in the Smith fermentation tube filled with glucose broth, especially 
if a small piece of naturally sterile liver or kidney from a small 
animal, or a few cubic centimeters of naturally sterile defibrinated 
blood be added to the medium in the tube. Glucose gelatin 
to which litmus has been added also furnishes a medium in which 
anaerobes will grow abundantly without any special precautions 
to protect them from oxygen or from the air. 

Removal of Oxygen. Anaerobic conditions may be furnished 
by pumping out the air from a container in which the cultures 
have been placed, a method employed by Pasteur. The oxygen 
may be absorbed from the air by a mixture of pyrogallic acid 
and alkali. Buchner's method is carried out as follows: Into 
a bottle or tube which can be tightly stoppered, pour 10 c.c. of a 
6 per cent solution of sodium or potassium hydroxide, for each 
100 c.c. of air contained in the jar. Add one gram of pyrogallic 
acid for each 10 c.c. of solution. The culture-tube is placed 
inside of the larger bottle or tube, supported above the bottom, 
and the stopper, smeared with paraffin, is inserted. The mix- 
ture of pyrogallic acid and potassium hydroxide possesses the 
property of absorbing oxygen. 

Wright's Modification of Buchner's method: The tube of cul- 
ture-medium is to be plugged with absorbent cotton, using a plug 
of large size. The culture-medium is inoculated in the usual 
way. The plug is cut off close to the neck of the tube, and is 



126 



BACTERIOLOGY 



then pushed into the tube about i centimeter. Now allow a 
watery solution of pyrogallic acid to run into the plug, and then a 
watery solution of sodium or potassium hydroxide. Close 
quickly and tightly with a rubber stopper. Wright recommends 
that the first solution be freshly made and consist of about equal 

volumes of pyrogallic acid and water, 
and that the second solution contain i 
part of sodium hydroxide and 2 parts 
of water. With 6 inch test-tubes, f 
inch diameter, the amounts advised are 
\ c.c. solution of pyrogallic acid and 
i c.c. solution of sodium hydroxide. 

Hydrogen Atmosphere. The most 
perfect anaerobic conditions are ob- 
tained by replacing the air with hy- 
drogen in a perfectly air-tight container. 
The method of hermetically sealing such 
containers full of hydrogen by melting 
the glass in a flame is really too dan- 
gerous to be recommended. The ap- 
paratus devised by Novy is most con- 
venient and has practically superseded 
all other devices for cultivation of 
anaerobes in hydrogen. The Novy jar 
is especially valuable for plate cultures. 
In using this jar, all ground-glass sur- 
faces should be thoroughly coated with 
a fairly stiff mixture of bees wax and 
olive oil so as to make all joints air-tight. Rubber gascots or 
packing should never be employed between the ground-glass sur- 
faces, regardless of the fact that many dealers furnish them for this 
purpose. After the plate cultures or tubes have been put into the 
lower section of the jar, the cover is put on so that the flanges fit to- 
gether perfectly. A heavy rubber band may then be passed around 
the circumference of the flanges to cover the circle of contact. Fi- 




FIG. 52. Arrangement of 
tubes for cultivation of anae- 
robes by Buchner's method. 






THE CULTIVATION OF MICRO-ORGANISMS 



I2 7 




p IG 53 Bottle for tube cultures. (After Novy.) 





FIG. 54. Apparatus for Petri dishes or tubes FIG. 55. Apparatus for plates 

gasorpyrogallate method. (After Novy.) or tubes gas, pyrogallate or vac- 
uum method. (After Novy.) 



128 



BACTERIOLOGY 



nally two or three clamps, the jaws of which are cushioned with cork 
or with rubber, are fastened on the flanges, pressing them firmly 





FIG. 56. Tripod and siphon flask for anaerobic culture by combined hydrogen and 

pyrogallate method. 

together. The jar is now attached to a source of pure hydrogen 
so that the gas enters at the top of the jar. The other open- 
ing is connected with a wash bottle containing water which 



THE CULTIVATION OF MICRO-ORGANISMS I2Q 

serves as a valve. Hydrogen is passed through the jar for two 
hours or more. It is well to keep all flames away from the appa-_ 
ratus as a precaution against explosion of the hydrogen when 
mixed with air. 

The hydrogen is generated by the action of 25 per cent 
sulphuric acid on granulated zinc. It should be purified by pass- 
ing through a wash bottle of alkaline lead acetate solution, a 
second one containing a solution of potassium permanganate 




FIG. 57. An aerobic organism (potato bacillus) that will not grow under a cover- 
glass. 

and a third of silver nitrate. In diluting sulphuric acid, the acid 
must be poured slowly into the water, and the mixture cooled 
in a bath of cold water, or under the tap. Carelessness in dilut- 
ing this acid may allow violent boiling to occur, sometimes with 
serious consequences. 

For critical work in anaerobic culture it is well to combine 
the pyrogallate and hydrogen methods. This is readily accom- 
plished by placing the Petri dishes on a low glass tripod with a 
small amount (2 grams) of pyrogallic acid beneath them on the 

9 



130 BACTERIOLOGY 

bottom of the Novy jar. 1 On top of the stack of Petrid is hesis 
placed a small flask containing strong solution of sodium hydrox- 
ide, and provided with a siphon spout ( see Fig. 56). A rub- 
ber is attached to this spout and leads down to the floor of the 
jar. After hydrogen has been passed through the jar and it 
has been finally closed, a 'slight tipping to one side starts the flow 
of the alkali through the siphon and so makes the pyrogallic acid 
available to absorb the last traces of oxygen. 

Further Anaerobic Methods. Numerous other expedients 
have been employed for the cultivation of anaerobes. Koch 
covered part of the surface of a gelatin plate with a bit of steril- 
ized mica or a cover-glass. Such a method suffices to prevent the 
growth of strictly aerobic forms but rarely suffices for the success- 
ful culture of strict anaerobes. Covering the surface of the med- 
ium with sterile liquid paraffin is a more perfect means of exclud- 
ing air. 

In all anaerobic culture methods, the presence of one or more 
reducing substances in the culture medium is of great importance. 
Those commonly employed are glucose, litmus and native protein. 

1 MacNeal, Latzer and Kerr, Journ. Infect. Diseases, 1909, Vol. VI, p. 557. 



CHAPTER VI 
METHODS OF ANIMAL EXPERIMENTATION 

Value of Animal Experimentation. The importance of ex- 
perimentation upon animals in the development of our knowledge 
concerning disease-producing micro-organisms can hardly be 
over-estimated, and animals must be used in considerable numbers 
in any adequate presentation of the subject to a laboratory class 
in pathogenic bacteriology. Only in this way has it been possible 
to discover the causal relation of bacteria to disease and the way 
in which diseases are transmitted, and it is only by the use of 
animals that this information can be presented first-hand to 
students. The inoculation of animals also provides accurately 
controlled material for studying the course and termination of 
the disease as well as the gross or microscopic lesions produced 
by it. 

Care of Animals. Laboratory animals should be housed in a 
light, well-ventilated room which should be heated in winter to 
about 60 F. If possible a run-way in the open air should be 
provided. The fixed cages may be constructed with wood or 
steel frames, but at least the front and preferably both front and 
back should be made of strong wire netting to provide ample 
ventilation. For rats and mice it is well to provide an enclosed 
perfectly dark space inside the cage into which these animals 
may retire. Smaller movable cages must also be provided for 
animals acutely sick and those infected with dangerously com- 
municable diseases. These must be sterilizable, and wood should 
not be used in their construction. Glass jars with weighted 
covers of wire netting are useful for mice and rats, and for larger 
animals such as guinea-pigs, rabbits and cats, cages of galvanized 
iron and wire netting are used. Pigeons may also be kept in such 



132 BACTERIOLOGY 

cages. Very large animals such as monkeys and dogs require 
specially constructed cages. Laboratory animals should receive 
very careful attention. They should be supplied with new food 
at least once daily and with clean water twice a day. If food 
remains at the end of the day, it should be removed and a smaller 
amount given for the next day. The cages should be completely 
emptied and cleaned at least once a week, the refuse being in- 
cinerated. The animal house should be screened, and insects of 
all kinds given careful attention. It will be found practically 
impossible to control the lice and fleas, but winged insects, 
especially biting varieties, may be kept out; and bedbugs, which 
sometimes gain entrance on new lots of guinea-pigs or rats, should 
not be allowed to remain uncontrolled. These possible carriers 
of infection require serious consideration as sources of confusion 
where experimental investigations are being carried out, not to 
mention the element of danger to the human individuals in the 
neighborhood. 

Holding for Operation. Animals to be inoculated or operated 
upon must be held in a fixed position. Many special mechanical 
holders have been devised for the various animals, but these 
are not necessary or especially useful. A pair of long-handled 
hemostatic forceps with lock, or a pair of placental forceps with 
lock, will be found most serviceable in handling mice or rats, 
the loose skin of the animal's neck being caught in the forceps. 
Guinea pigs are best held by an assistant, the thumb and fore- 
finger of one hand encircling the thorax just behind the fore legs 
and the other hand holding the hind legs stretched out. Rabbits 
are held by the ears and hind legs with the body stretched over 
the knee. Monkeys are to be handled with thick gloves and 
should be caught around the neck from behind with one hand and 
by the pelvis or hind legs with the other. A second assistant 
is required to hold the fore legs. For all work which would cause 
any considerable pain the animal must be anesthetized, either 
by putting it into a closed compartment with the anesthetic or 
by use of a cone. Anesthesia is also necessary when delicate 



METHODS OF ANIMAL EXPERIMENTATION 133 

manipulations are to be carried out. For operations requiring 
some time the animal is fastened to a board with stout cords, -01 
is held by means of a specially constructed animal holder. 

Inoculation. Infectious material may be introduced into 
the animal body in various ways. The most common methods 
are injection under the skin and injection into the peritoneal 
cavity. The hair should be removed from the site selected. 
A sterilized hypodermic syringe is used, and it is again 
sterilized by boiling after use. Subcutaneous injection is 
usually made in the thoracic region as one easily avoids pene- 
trating the chest cavity. For intraperitoneal injection the 
needle is quickly thrust through the abdominal wall. 

Inoculation into the cranial cavity is practised especially in 
studying rabies. The animal, rabbit or guinea-pig, is anesthe- 
tized and the scalp is shaved. An incision through the scalp 
about 8 to 10 mm. long is made at the left of the median line 
and parallel with it, a little in front of a line connecting the 
external auditory openings. The scalp i-s then forcibly drawn 
over to the right and a hole drilled though the skull at the right of 
the median line. A sharp-pointed scalpel may serve the purpose 
of a drill. The needle is then inserted into the cerebral substance 
nearly to the floor of the cranial cavity and the material (o.i 
to 0.5 c.c.) injected. Any blood or fluid is taken up with sterile 
absorbent cotton. The skin is replaced in its original position 
and may be dressed with cotton and collodion, although dressing 
may be omitted altogether. 

Inoculation into the circulating blood is a method of special 
importance. In rabbits intravenous injection is easily done. 
The hair is removed from the ear over the marginal vein, and 
the vein is dilated by application of a hot towel, after which the 
skin is wiped dry. An assistant constricts the base of the ear 
to congest the vein and the needle is easily inserted into it. 
Other veins on the ear may be used, but they are not so easily 
penetrated by the needle. In rats, guinea-pigs or monkeys, 
intravenous injection is not so simple and it is easier to inoculate 



134 BACTERIOLOGY 

these animals by intracardiac injection. For this purpose the 
animal is etherized and the precordial region is shaved and dis- 
infected. The material to be injected is taken up into a Luer 
glass syringe. A second syringe, empty, with needle attached, 
is used to puncture the chest wall and the heart, preferably the 
wall of the right ventricle. The needle is introduced in the inter- 
costal space directly over the heart and near the border of the 
sternum. The appearance of blood in the previously empty 
syringe gives notice that the cavity of the heart has been entered. 
The syringe is now detached from the needle and the other 
syringe which contains the material to be injected is quickly 
substituted for it. The injection is made slowly. 

Other Sites for Inoculation. Many other regions are easily 
reached with the injection needle, such as the pleural cavity, the 
chambers of the eye, the spinal canal, the interior of muscles, 
and the substance of the testis. 

Subcutaneous Application. Inoculation may be accomplished 
without using a syringe if desired. The skin and mucous mem- 
branes may be scratched with a needle or other instrument and 
the infectious material applied to the slight wound thus made. 
A small pocket may be made under the skin by making a small 
incision and introducing a blade of the forceps to separate the 
skin from the underlying muscle; and into such a pocket one may 
introduce solid material, bacteria from a culture, pieces of tissue, 
garden soil or splinters of wood, ^with accompanying bacteria. 
The opening of the pocket is closed by cauterization or sealed 
with collodion 

Alimentary and Respiratory Infection. Animals are some- 
times infected by feeding the virus, occasionally by injection 
into the rectum. Infection of the respiratory tract by spraying 
infectious material^m the air breathed by the animal is rarely 
employed. 

Collodion Capsules. Bacteria may be cultivated in the 
living body of an animal, without infecting the animal, when they 
are enclosed in collodion capsules. Their soluble products are 



METHODS OF ANIMAL EXPERIMENTATION 135 

able to diffuse through the collodion, while the animal's fluids may 
pass into the sac to nourish them. These capsules were originally 
made by dipping the round end of a glass rod into collodion 
repeatedly. McCrae's method 1 is easier and more satisfactory. 
(Fig. 58.) 

A piece of glass tubing is taken, and a narrow neck drawn on it near one 
end. This end of the tube is rounded in the flame and, while still warm, the 
body of a gelatin capsule is fitted over it, so that the gelatin may adhere to 
the glass. The capsule is now dipped into 3 per cent collodion, covering 
the gelatin and part of the glass. It is allowed to dry a few minutes, and is 
dipped again. In all, two or three coatings may be given. The capsule is 
filled with water and boiled in a test-tube with water. The melted gelatin is 
removed from the inside of the capsule by means of a fine pipette. The cap- 
sule is partly filled with water or broth and sterilized. The capsule may now 
be inoculated. The narrow part of the glass tube which constitutes the neck 
must then be sealed in the flame, taking care that the neck be dry. The 



(1 



FIG. 58. Method of making collodion capsules. (After McCra.) 

sealed capsule should be placed in bouillon for twenty-four hours. No 
growth should occur outside the capsule if it is tight. It may now be placed 
in the peritoneal cavity of an animal. 

A method for making collodion sacs recommended by Gorsline 2 consists 
in the use of a glass tube, the lower end of which is rounded and closed, 
except a small hole, which is temporarily filled with collodion. This tube is 
dipped in collodion and dried, as above. It may now be filled with water. 
By blowing at the opposite end, the pressure through the hole in the bottom 
of the glass tube will cause the capsule to loosen so that it comes away easily. 
Sacs made in this way are soaked in water for 30 minutes, dried and attached 
to the glass tube by gentle heat. The joint is wound with silk thread and 
coated with collodion. The sac is then filled with distilled water, immersed 
in a tube of water and sterilized in the autoclave. 

There are also various other methods recommended for making collodion 
sacs. 

1 Journal of Experimental Medicine. Vol. VI, p. 635. 

2 Contributions to Medical Research. Dedicated to Victor C. Vaughan, Ann 
Arbor, 1903, p. 390. 



136 BACTERIOLOGY 

Collodion capsules are ordinarily placed free in the peritoneal 
cavity of the animal, by an aseptic laparotomy. The wound is 
sutured with silk or catgut and dressed with sterile cotton and 
collodion. 

Observation of Infected Animals. In nearly every case it 
will be well to keep a record of the weight of the animal from time 
to time. The temperature may be observed by means of a 
thermometer in the rectum. It should be inserted a considerable 
distance, 4 to 8 centimeters in guinea-pigs. Other examinations 
are made in special cases, such as palpation of the lymph glands 
in tuberculosis and microscopic examination of the blood in an- 
thrax, trypanosomiasis and the relapsing spirochetoses. 

The post-mortem examination of experimental animals has 
been discussed (pages 98 and 100). 



PART II. 

GENERAL BIOLOGY OF MICRO- 
ORGANISMS. 



CHAPTER VII. 
MORPHOLOGY AND CLASSIFICATION. 

The minute living things included under the general term 
microbe, are exceedingly various in form and structure as well as 
in respect to food requirements and physiological activity. 
The number of different microbes is so great and so great are 
the difficulties involved in the accurate observation of their 
various features, that the biological relationships of many of 
the various forms to each other are not yet determined, and 
much of the generic and specific terminology in common use 
rests upon insecure foundation. Nevertheless a certain kind 
of order has developed in our conceptions of the grouping of 
micro-organisms. 

Molds. The molds or hyphomycetes are multicellular or- 
ganisms characterized by the formation of a network (mycelium) 
made up of branching threads (hypha), and by their special 
fruiting organs. These threads vary from 2 to 7;* in width. 
Within the group of molds the structure of fruiting organs is used 
as the most important character from which to determine relation- 
ships. The phycomycetes, or algo-fungi, are characterized 
by the presence of sexual reproduction in which the union of 
two cells gives rise to resting cells, zygospores and oospores, 
which are enclosed in a thick wall. The ascomycetes are char- 

137 



GENERAL BIOLOGY OF MICRO-ORGANISMS 








FIG. 59. Common Molds. 

a. Penicillium glaucum. b. Oidium lactis. c. Aspergillus glaucus. d. The 
same more highly magnified, e. Mucor mucedo. (Baumgarten.) 



MORPHOLOGY AND CLASSIFICATION 139 

acterized by the occurrence of a spore-sac called the ascus which 
usually contains eight spores. The common aspergilli belong here. 
The basidiomycetes are characterized by the occurrence of a 
spore-bearing cell, the basidium, which bears four protuberances 
called sterigmata (singular sterigma) upon each of which is a 
single spore. Mushrooms and puff-balls belong to this group. 
Besides these three well-defined families, there are many kinds 
of molds and fungi concerning which definite knowledge's still 





F-Vy 



FIG. 60. Yeast cells stained with fuchsin. (Xiooo.) 

too incomplete for them to be finally placed. The common 
oidium and penicillium and many parasitic molds are included 
here. The molds 1 are especially important as causes of disease 
in plants. Relatively few diseases of man or other animals have 
been shown to be due to them, although the first diseases proven 
to be due to micro-organisms were those caused by certain molds. 
The molds possess the general morphological features of plants 
except for the absence of chlorophyll. 

1 For fuller discussion of molds in general see Marshall, Microbiology, pp. 12-27, 
article by Thorn. 



140 



GENERAL BIOLOGY OF MICRO-ORGANISMS 



Yeasts. The yeasts (Blastomycetes) are very closely related 
to the molds. In fact some stages in the growth of molds resemble 
very closely the normal development of a yeast. The yeasts, 
however, do not grow out into long filaments but remain spherical 
or ovoid. The cells vary from 2.5 to 12 ft in diameter. During 
active growth they reproduce by budding, a smaller portion being 








FIG. 61. Wine and beer yeasts. A, S. ellipsoideus, young and vigorous; B, S. 
ellipsoideus, (i) old, (2) dead; C. S. cerevisia, bottom yeast; D, S. cerevisice, top 
yeast. (After Marshall.) 

pinched off from the parent cell. The true yeasts also form spores 
inside the cell, from four to eight typical ascospores, showing 
their very close relationship to molds. Yeasts are very important 
in the fermentation industries. Very few of them are pathogenic. 
Among themselves, the yeasts are subdivided into two groups, 
(i) those which produce ascospores (saccharomycetes or true 



MORPHOLOGY AND CLASSIFICATION 141 

yeasts) and (2) those which fail to produce such spores (torula 
or wild yeasts). They are further distinguished by differences 
in the form of the cells, but especially by differences in physi- 
ological characters, such as the fermentation of sugars and the 
production of pigments. 

In the yeasts there is no definite differentiation of cells. 
Various cell structures such as cell-wall, nucleus and cystoplasm 
with vacuoles and granules, can be demonstrated. The cell 
membrane is, as a rule, more delicate than in the molds. It 
sometimes secretes a gelatinous material which forms a thick 
capsule about the cell. The nucleus is shown by appropriate 
methods of staining as a single more or less sharply defined 
mass of chromatin. Under suitable conditions the true yeasts 
produce endospores, usually multiple, and as many as eight in 
one cell. These are spherical or ovoid masses surrounded by a 
definite wall, and usually about half the diameter of the yeast 
cell. When supplied with nutriment these spores swell and burst 
the mother cell, and then begin at once to multiply by budding. 
Dry commercial yeast cakes contain spores of yeast along with 
bacteriaand molds; moist, "compressed," yeast contains vegetat- 
ing yeast cells, also mixed with other organisms. 

Bacteria. Bacteria (schizomycetes) are minute unicellular 
organisms 0.2 to 4//. in width which multiply solely by simple 
transverse division (fission), ordinarily resulting in the produc- 
tion of two cells of equal size. In many instances the cells re- 
main attached to each other so as to* form long filaments. 

Trichobacteria. Certain of them grow into long filaments 
without dividing at once into shorter segments. These forms 
which are classed as higher bacteria or trichobacteria, suggest 
a very close relationship to the molds and may, perhaps, be re- 
garded as intermediate between the molds and the lower bacteria. 
Many of them exhibit a differentiation of the filament into base 
and apex, some of them branch in an irregular fashion, and in 
some there is a suggestion of the formation of special fruit 
organs. These higher bacteria require further study to deter- 



142 GENERAL BIOLOGY OF MICRO-ORGANISMS 

mine their relationships. A few of them are important patho- 
genic agents. 

The Lower Bacteria. The lower bacteria, or true bacteria, 
are always simple in form, the transverse division producing 
cells, relatively short, and of nearly equal length. Long filaments 
are produced only by the attachment of many individual cells 
together, end to end. There are no special fruit organs. The 
special resistant form, or spore, which occurs in some forms is 
produced only inside of the vegetative cell, one cell producing 
one spore. There are three general forms of bacteria, the sphere 
(coccus, plural cocci), the cylinder (bacillus, plural bacilli), and 
the spiral or segment of a spiral (spirillum, plural spirilla). In- 
termediate forms occur, so that there is not a sharp line between 
the groups. These three forms are generally accepted as a basis 
for division of the lower bacteria into three families, the coccaceae, 
bacteriaceae and the spirillaceae. 

Spherical Bacteria. The Coccacea or cocci are spherical 
bacteria. They vary in size from about 0.3^ to 3^ in diameter. 





Staphylococci. Streptococci. Diplococci. Tetrads. Sarcinae. 

FIG. 62. 

During the process of cell division, a coccus may become elongated 
somewhat, and after division, the daughter cells may be shortened 
so that they appear as if compressed against each other. Slightly 
elongated forms are included among the cocci in certain instances, 
and especially the lancet-shaped bacteria such as the germ of 
lobar pneumonia. The recognition of genera within the family 
is still unsettled. Morphologically five genera have been dis- 
tinguished by Migula: Streptococcus, Micrococcus, Sarcina, Piano- 
coccus and Planosarcina. The first three do not possess flagella 
and are non-motile. Streptococcus includes those forms which 
divide only in one plane so that a thread or chain is produced. 
Micrococcus includes the cocci which divide in two planes at 



MORPHOLOGY AND CLASSIFICATION 143 

right angles so as to produce plates, and it also includes those 
which divide in an irregular fashion so that no definite geometric 
figure results. Sarcina includes those cocci which divide in three 
planes at right angles to each other, in turn, so as to produce 
cubical masses of cells. Planococcus is similar to Micrococcus 
in all respects except that its members are motile and possess 
flagella, and Planosarcina includes the motile forms which are 
in other respects the same as the forms included under Sarcina. 

COCCACE^E Cells spherical, without endospores. 
Streptococcus Division in one plane, forming chains of cells; 

non-motile; without flagella. 
Micrococcus Division in two planes, forming flat plates of 

cells, or irregular, forming masses of cells irregularly 

grouped; non-motile; without flagella. 

Sarcina Division in three planes, forming cubical or package- 
shaped masses of cells; non-motile; without flagella. 
Planococcus Division in two planes, forming flat plates 

of cells, or irregular, forming mass of cells irregularly 

grouped; motile; bear flagella. 
Planosarcina Division in three planes, forming cubical 

or package-shaped masses of cells; motile; bear flagella. 
These genera have not been generally adopted by bacteriolo- 
gists. The terms Streptococcus and Sarcina are, however, 
quite generally employed as the generic names for the organisms 
of their respective groups as defined by Migula, as they had 
been used in this way before. Micrococcus, however, is commonly 
employed as a general term for all the members of the family 
Coccaceae, and Planococcus and Planosarcina have not been used, 
because bacterial forms belonging to these genera are exceedingly 
uncommon and it may even be questioned whether those which 
have been described might not better be classed with the cylin- 
drical bacteria, in which motility is of frequent occurrence. 
Other terms in common use as generic names for certain cocci 
are Diplococcus and Staphylococcus. A diplococcus is a double 
coccus, two spheres attached together. This grouping by twos 



144 GENERAL BIOLOGY OF MICRO-ORGANISMS 

is very common and the generic term Diplococcus is employed 
for those forms in which it is a prominent characteristic. The 
term Staphylococcus is applied to those micrococci which are 
grouped in an irregular mass resembling a bunch of grapes. 

Cylindrical Bacteria. The cylindrical bacteria, Bacteriacece, 
have been subdivided by Migula into three genera, Bacterium, 
Bacillus and Pseudomonas. The genus Bacterium includes 
those members of the family which are without flagella and 
are non-motile. Bacillus includes those forms possessing flagella 
distributed over the surface, and Pseudomonas is the generic 
term for those forms with flagella situated at the extremities 
only (polar flagella). 

BACTERIACE^E Cells cylindrical, straight, non- 
motile or motile by means of flagella. 

Bacterium Cells without flagella, non-motile. 

Bacillus Cells motile with flagella distributed over the 
surface. 

Pseudomonas Cells motile with polar flagella. 
These genera have not been generally adopted by bacteri- 
ologists, and there are serious reasons for dissatisfaction with 
such a classification of the rod-shaped bacteria. In the first 
place the names Bacterium and Bacillus are unfortunate. The 
former has long been employed as a general term designating 
any member of the Schizomycetes and its plural, Bacteria, 
is everywhere the common term employed in designating this 
large group of micro-organisms. Its use in the narrower sense 
by Migula has not displaced the former, signification, and its 
use in the sense of Migula must necessarily result in confusion. 
The latter term, Bacillus, has long been used very generally by 
bacteriologists to designate any member of the Bacteriaceae 
or rod-shaped bacteria, regardless of the motility or distribution 
of flagella. A further serious objection is due to the lack of 
stability in the character selected to distinguish the genera. 
The flagella may disappear from bacteria ordinarily possessing 
them as a result of changes in environment and may be again 






MORPHOLOGY AND CLASSIFICATION 145 

made to appear by reversing the conditions. 1 Furthermore 
in some groups of bacteria which seem to be closely related in 
respect to other characters, morphological and physiological, 
both motile and non-motile forms occur. On the whole the pres- 
ence or absence of flagella would seem to be too fragile a character 
to serve as a sole distinction between genera among the rod- 
shaped bacteria. 






FIG. 63. Bacilli of various forms. 

The different species of rod-shaped bacteria are very numerous, 
several thousand different kinds having been described. They 
vary in width from 4/1 to. o.i or probably less, and in length from 
from6o,to o.2//. The very large ones are non-pathogenic species. 
The form is ordinarily that of a straight cylinder of equal caliber 
throughout its length. Certain slightly curved forms are never- 
theless included in the family, although they may perhaps be 
regarded as intermediate between the bacteriaceae and the 



CMD CMD 



FIG. 64. Sporulation. a, First stage showing sporogenic granules; b, incomplete 
spore; c, fully developed spore. (After Novy.) 

spirillaceae. Some of the rod-shaped bacteria are of uneven 
caliber, especially when growing under unfavorable conditions or 
when spores are produced. The ends of the rod may be pointed, 
rounded, square-cut or concave. The bacteria may remain 
attached after cell-division, forming groups of two, diplo-bacillus , 
or many cells remain attached, to form long threads, strepto- 
bacillus. Endospore formation occurs almost exclusively in 

1 Passini: Zts.f. Hyg., 1905, XLIX, pp. 135-160. 



146 GENERAL BIOLOGY OF MICRO-ORGANISMS 

the bacteriaceae and the form of the spore-bearing cell differs 
for different species and is fairly constant for any one species. 




FIG. 65. Position of spores; resultant forms (diagrammatic), a, Median 
spores; b, intermediate spores; c, terminal spores; 20,, b, c, change in form of cells 
due to the presence of the spore; 20, clostridium; 20, drum-stick form. (After Novy.} 

The spore, which is always single, may be located at the center of 
the cell, median spore, or at the end, terminal spore, or 
at an intermediate point. The spore-bearing cell may retain 
its normal outline or it may be bulged by the spore. The cell 
containing a median spore with bulging is called a clostridium; 
one with terminal spore with enlargement of the cell is spoken 
of as a drumstick or sometimes as a plectridium. 

Spiral Bacteria. The screw-shaped bacteria, Spirillacea, have 
been subdivided into four genera by Migula. The genus Spiro- 
soma includes those spirals which are rigid and without motility. 
Motile cells possessing one, two or three polar flagella are classes 
in the genus Microspira; while those possessing more than three 
are put in the genus Spirillum. The genus Spirochaeta includes 
the slender flexuous forms of spirals. 

SPIRILLACE^E Cells circular in cross-section but 

curved to form a spiral or segment of a spiral. 
Spirosoma Cells rigid, without flagella, motionless. 
Microspira Cells rigid, motile, with i to 3 polar flagella. 
Spirillum Cells rigid, motile, with polar tufts of flagella. 
Spirochaeta Cells slenders, flexuous, motile. 
Two of these generic terms, Spirillum and Spirochaeta, have 
long been used, and almost in the sense in which they are em- 
ployed by Migula. Spirillum has frequently been applied to 




MORPHOLOGY AND CLASSIFICATION 147 

all the Spirillaceae and especially to those forms which Migula 
includes in his first three genera, Spirosoma, Microspira ancf 
Spirillum. The distinction between Microspora and Spirillum 
seems of too slight importance to serve as a basis for the formation 
of two genera, and indeed the same objection exists here as in 
the Bacteriaceae to the use of flagella as a basis for generic 
distinctions. 

Cell division occurs by simple transverse fisson in all the spiral 
bacteria. Endospores are 

said to be formed by some . / 

species. $fr ,Tu ^ \f 

The group of spiro- V 
chaetes has received much O/V^W^ 

attention during the past FlG . 66. Types of spirilla. 

decade and the propriety 

of including them in the spirillaceae may be seriously questioned. 
Many investigators are inclined to regard them as more properly 
classed with the protozoa than with the bacteria. It is claimed 
that these forms multiply by longitudinal splitting and not by 
transverse fission, and this would at once remove them from the 
Schizomycetes. The observations are still in dispute and there 
are good observers who regard transverse fisson as the mode of 
multiplication. Further study is necessary to settle this impor- 
tant question. It is possible that some of these slender spirals 
may multiply by both methods, or that one species may divide 
longitudinally and another transversely, but this does not seem 
probable. For the present it would seem wise to reserve judg- 
ment and avoid encumbering the group with new genera until a 
definite' and final agreement has been reached concerning the 
exact morphological facts. (See page 353.) 

Structure of Lower Bacteria. The bacterial cell is enclosed 
in a relatively stiff cell membrane, which generally retains its form 
after plasmolysis. Under special conditions of growth many 
forms of bacteria become enclosed in a gelatinous capsule. This 
seems to be a viscid material secreted by the cell through the cell 



148 GENERAL BIOLOGY OF MICRO-ORGANISMS 

membrane. The motile bacteria possess exceedingly slender hair- 
like processes, called flagella, which serve as organs of locomotion. 
These processes apparently take origin from the cell membrane. 
Bacteria without flagella are spoken of as atrichous, those with a 
single flagellum at one end as monotrichous, those with a flagellum 
at either end as amphitrichous. When there 
^ ft is a tuft of flagella at the end, the distribution 

($) ^ /^ K is said lobe lophotrichous, and when they are 
distributed all over the surface the arrange- 
ment is called peritrichous. The internal 



structure of the bacterial cell has received 
FIG. 67. Bactena with 

capsules. comparatively little attention. The direct 

microscopic study of the living cells shows 
them to be finely or coarsely granular, or sometimes nearly ho- 
mogeneous. No constant internal structures can be distinguished. 
Ordinary simple staining w'th the basic aniline dyes colors the bac- 
terial cell diffusely and intensely, usually without any internal 
differentiation. The cell membrane between two cells in a chain 
may remain relatively colorless and so be differentiated from the 
protoplasm on either side. At times the stainable substance is un- 
evenly distributed in the cell, perhaps grouped at the ends of a rod, 





FIG. 68. Bacteria showing flagella. 

or in granules or bands. Under special conditions some bacteria 
show internal granules of special composition, distinguishable as 
pigment granules or by their microchemical reactions. Granules 
which stain with iodine, so-called granulose or glycogen granules, 
are important features of some kinds of bacteria. 

The recognition of the cell nucleus has received special atten- 
tion. Zettnow, more especially, has shown that the chromatin or 
essential nuclear substance is present in the bacterial cell as finer 



MORPHOLOGY AND CLASSIFICATION 149 

or larger granules, sometimes distributed pretty generally and 
sometimes collected together at one or more places in the elL 
The Romanowsky stain and its modifica- 
tions have been especially useful in differ- 
entiation of chromatin from cytoplasm. 

Special movements of the internal 
granules have been described by Schau- 
dinn as being associated with beginning 

cell division. For the great majority of FIG. 69. The formation of 
,-, , spores. (After Fischer from 

bacteria these have not been observed, f rost an d McCampbdl.) 

and according to our knowledge, the 

process of cell division is extremely simple. It consists of a pro- 
gressive constriction and thinning of the cell at the middle until 







FIG. 70. Bacteria with spores. 

two cells are produced. In some forms the division is completed 
by a sudden snapping movement. 

The formation of an endospore begins with the accumulation 

o C 




n 
u 



d 



FIG. 71. Germination of spores, a, Direct conversion of a spore into a bacillus 
without the shedding of a spore- wall (B. leptosporus); b, polar germination of B. 
anthracis, c, equatorial germination of B. subtilis; d, same of B. megatherium; , 
same with "horse-shoe" presentation. (After Novy.) 

of chromatin granules in one part of the cell, where they coalesce, 
lose their contained water and seem to become embedded in an oily 



150 GENERAL BIOLOGY OF MICRO-ORGANISMS 

or fatty substance and surrounded by a membrane. Very early in 
the process the spore no longer stains readily. In some forms 
(Bact. anthracis) the cell in which a spore has formed disintegrates 
rapidly, setting free the spore, while in others (B. telani) the cell 
may continue its activities after formation of the spore. The spore 
germinates when conditions again become favorable to active 
growth. The new cell may burst the spore wall into halves, or 
at the end, or the spore wall may soften and become a part of the 
new growing cell. 

Filterable Viruses. The difficulty of accurate morphological 
study is so great as to appear insurmountable in the case of cer- 
tain microbes which are very definitely recognizable by certain 
effects which they produce. This is especially true of those 
living things capable of passing through the fine filters which 
prevent the passage of small bacteria. The causes of certain 
diseases exhibit this character, and these have come to be known 
as filterable viruses. There can be little question that non-patho- 
genic filterable microbes also exist although they seem to have 
escaped observation. Accurate knowledge of the morphology 
of these forms remains to be disclosed by future investigation. 
Meanwhile, the efforts to classify them as bacteria or as protozoa 
may well be spared. The propriety of including them as living 
things is, however, only occasionally questioned. 

Protozoa. The protozoa or unicellular animals have assumed 
very great importance as causes of disease during the past dec- 
ade. Fortunately for the systematist, the free-living protozoa 
had received considerable careful study and the larger groups of 
protozoa had been well defined before the interest in pathogenic 
properties had the opportunity to over-shadow morphological 
study. The number and variety of easily recognizable morpho- 
logical characters presented by the protozoa are greater than 
those of the bacteria; and the organisms are, on the whole, 
larger. These factors make for more accurate observations of 
morphological characters, and their more successful employment 
as a basis of classification. 



MORPHOLOGY AND CLASSIFICATION 151 

The protozoan cell is generally larger and more complex in 
structure than the bacterial cell appears to be, although the 
viding line is in places indefinite or even wholly obscure. In 
general the protozoon shows the typical structure of a single cell 
of the metazoon. A well-defined nucleus is usually present, some- 
times several of them, although in some forms the nuclear ma- 
terial is more or less scattered throughout the cell. Most proto- 
zoa exhibit differentiation of the protoplasm into cell organs or 
organellae, adapted to perform certain functions. In many pro- 
tozoa sexual reproduction has been observed, a process involving 
complex morphological changes. The cells showing these evi- 
dences of complex organization resemble in most respects cells 
of the higher animals, and in fact a colony or group of protozoa 
may be regarded as representing a transition to the many-celled 
animals, just as, on the other hand, the bacteria were seen to be 
connected with the higher plants through the forms of the higher 
bacteria, the yeasts, the molds and algae. Physiologically, pro- 
tozoa differ from bacteria and other plants in requiring more com- 
plex nitrogenous food, but this distinction is far from absolute. 
Doflein divides the protozoa into two substems, (i) Plasmodroma, 
including those forms which move by means of pseudopodia or 
flagella, and which exhibit for the most part an alternation of 
asexual and sexual generations, and (2) Ciliophora, including 
those forms which move by means of cilia and in which the sexual 
fertilization gives rise to no special reproductive form of the 
organism. 

The substem Plasmodroma includes three classes, (i) Masti- 
gophora, (2) Rhizopoda and (3) Sporozoa. 

Flagellates. In the class Mastigophora, are included a great 
many different organisms, the one common feature being the 
type of locomotive apparatus, which consists of one or more fla- 
gella. The further subdivision of the class has not yet been agreed 
upon, not because of any lack of morphological differences upon 
which to base a classification, but largely on account of difficulty 
in estimating the relative importance and meaning of the many 



152 



GENERAL BIOLOGY OF MICRO-ORGANISMS 




B 





D 







H 




FIG. 72. The most important trypanosomes parasitic in mammals. A, Try- 
panosoma lewisi (Kent). B, Tr. evansi (Steele), Indian variety. C, Tr. evansi 
(Steele), Mauritian variety. D, Tr. brucei (Plimmer and Bradford). E, Tr. equiper- 
dum (Doflein). F, Tr. equinum (Voges). G, Tr. dimorphon (Laveran and Mesnil). 
H, Tr. gambiense (Button). (From Doflein after micro photo graphs of Novy.) 



FIG. 73. Leishmania donovani. Various forms obtained by spleen puncture, some 
free and some inside red blood cells. (From Doflein after Donoian.) 



MORPHOLOGY AND CLASSIFICATION 



153 



criteria presented. The genera of particular interest from the 
pathological standpoint are Trypanosoma, Leishmania,, Tricho- 
monas and Lamblia. The members of the Trypanosoma are 






FIG. 74. Leishmania 
donovani. Various forms 
of the organism in artifi- 
cial culture. (From Doflein 
after Chatterjce.} 



FIG. 75. Trichomonas hominis. 
(From Doflein after Grassi.} 



characterized by an approximately crescent-shaped body, 10 to 
40ft in length, flexible and provided with a flagellum which origi- 




FIG. 76. Lamblia inttstinalis. A, Ventral aspect. B, Lateral aspect. C. At- 
tached to an epithelial cell. (From Doflein after Grassi and Schewiakoff.) 

nates in the endoplasm near one end and passes along the border 
of the body and finally projects as a free whip at the other end 
of the cell. As it passes along the border of the cell it is enclosed 



154 



GENERAL BIOLOGY OF MICRO-ORGANISMS 



in a sheath of ectoplasm, which is drawn out into a thin sheet 
forming an undulating membrane. Multiplication takes place 
by approximately longitudinal division. Leishmania includes a 
few parasitic forms, for the most part living inside the cells of 
the host. These organisms are oval, about 2X3^, without fla- 
gellum or undulating membrane. In artificial culture outside 
the body, the protozoon grows larger, develops a flagellum and 
resembles a trypanosome. Trichomonas includes pear-shaped 




F G H I 

FIG. 77. Entamceba coli (Losch). A to C, Various forms of the free ameba. 
D, Stage with eight nuclei. to G, Cysts with various numbers of nuclei. H, 
Opening cyst. 1, Young amebae escaped from a c>st. (From Doflein after Casa- 
grandi and Barbagallo.) 

organisms 4 to 30/4 in diameter, provided with three or four fla- 
gella. Isogamic and autogamic fertilization have been described, 
and cysts containing numerous daughter cells result from the 
multiplication following this process. Lamblia resembles tricho- 
monas, but the cell is here shaped more like a beet, is provided 
with eight flagella and is hollowed out at one side near the 
rounded anterior end to form a suction cavity. 

Rhizopods. The members of the second class, Rhizopoda, 
are characterized by their ability to send out protoplasmic proc- 



MORPHOLOGY AND CLASSIFICATION 155 

esses to serve for locomotion and also to surround and engulf 
solid food particles. The two genera, Amoeba and Entamaeba, 
are of chief est interest. The organisms are masses of protoplasm 
containing a nucleus, food granules and sometimes vacuoles, 
and surrounded by a slightly denser more hyaline layer of ecto- 
plasm. The members of the genus Amoeba are free-living 
saprophytic forms, while those of Eniamosba are parasitic. 
Multiplication occurs by fission after a more or less complex di- 
vision of the nucleus. Multiple division also occurs, more es- 
pecially in an encysted condition, and subsequent to a possible 
autogamic fertilization. 

Sporozoa. The third class, Sporozoa, is made up entirely 
of parasitic forms, which at some stage in their life history multiply 
by division into numerous daughter cells, which are enclosed in a 
protective envelope to form a spore. The spores serve to dis- 
tribute the species to other hosts. In cases where there are special 
adaptations for distribution, as for example by means of inter- 
mediate hosts, the protective envelope may be absent. An enor- 
mous number of parasitic micro-organisms are included in this 
group. The genera of greatest present interest from the patho- 
logical point of view are Eimeria (Coccidium), Plasmodium 
Babesia (Piroplasma) and Nosema. 

The Coccidia. Eimeria includes a number of intracellular 
parasitic forms, perhaps better known as coccidia. The small 
parasite resulting from asexual division is called a merozoit. It 
is somewhat spindle-shaped and 5 to IOJJL long. This merozoit 
penetrates an epithelial cell of the host, grows at the expense of 
the cell to a spherical mass 20 to 50;* in diameter, and eventually 
divides into numerous (sometimes as many as 200) merozoits, 
which become free by rupture of the host cell. Besides this asexual 
mode of multiplication, there is also a sexual cycle. Some of the 
growing parasites do not divide into merozoits but become differ- 
entiated into male and female cells (gametocy tes) . The male 
gametocyte gives rise to a large number of elongated motile micro- 
gametes, one of which approaches and penetrates the ripened 



156 



GENERAL BIOLOGY OF MICRO-ORGANISMS 



jzr 




FIG. 78. Developmental cycle of Eimeria (Coccidium) schubergi. I, Sporzoit; 
II, sporozoit penetrating a cell of the host; III and IV, stages of growth; V to 
VII, asexual multiplication; VIII, agamete or merozoit beginning again the asexual 
cycle; IX and X, agametes destined to form sexual cells (gametes); XI, a to c, devel- 
opment of the macrogamete; XII, a to d, development of microgametes; XIII, 
fertilization; XIV and XV, the fertilized cell or zygote; XVI and XVII, metagamic 
division of the zygote; XVIII, formation of the sporoblasts; XIX, formation of 
the spores and sporozoits; XX, sporozoits emerging from the spores and from the 
oocyst. (From Doflein after Schaiidinn,) 



MORPHOLOGY AND CLASSIFICATION 



157 



macrogamete. The nuclei of the two gametes fuse and the 
fertilized cell quickly forms a protective wall around itself and then 
divides into eight cells which are enclosed in pairs within secondary 
cysts known as spores. This form of the organism passes out of the 
host, and after a passive existence in the external world may gain 
entrance to a new host, whereupon the spore wall ruptures and the 
enclosed cells, sporozoits, emerge to penetrate new host cells. 

The Plasmodia. Plasmodium includes the malarial parasites, 
forms parasitic in red blood cells and closely analogous to the 




FIG. 79. Forms in the asexual cycle of Plasmodium falciparum, the parasite 
of tropical malaria. A, Multiple infection of a red blood cell; B to E, various forms 
of the growing parasite; B and C show also the Maurer granulations; F, full-grown 
parasite with many nuclei; G, Segmentation. The pigment is shown in E, F and G. 

(After Doflein,} 

coccidia in the asexual cycle. The garnet ocytes are also similar to 
those of Eimeria except that the gametes are not formed within 
the mammalian host, but only after the blood has been drawn. 
The sexual cycle of development takes place in a definite secondary 
host, the mosquito. In the stomach of this insect the gametes 
unite and the fertilized cell (ookinet) actively penetrate? the 
epithelium and beneath it develops into a large oocyst, 30 to 90^ 
in diameter, enclosed in the elastic tunic of the stomach wall of the 
mosquito. As the oocyst enlarges the nucleus divides and eventu- 



158 GENERAL BIOLOGY OF MICRO-ORGANISMS 

ally the cytoplasm also. The nucleus of each of these masses 
(sporoblasts) then divides many times. Each nucleus, together 
with a small amount of protoplasm, separates and then elongates 
into a slender thread-like sporozoit (14X1/0 As many as io,ooc 
of these may be produced in one oocyst. The cyst bursts into the 
body cavity of the mosquito and the motile sporozoits circulate 
through the body of the insect and eventually assemble in the cells 
of the salivary glands. From these they escape with the secretion 
and gain entrance to the wound made by the mosquito in biting. 
Babesia. A number of parasites of the red blood cells are 
classed in the genus Babesia (Piro plasma). These resemble the 
members of the preceding genus very closely but multiple division 
(segmentation) does not seem to occur in the asexual cycle. The 

v G^~ H 






FIG. 8c. Babesia muris. A, Young form in a red blood cell. B, Form with 
two nuclei. C and D, Binary division. E and F, Multiple infection; ameboid 
forms in F. G, An exceptionally large individual (gametocyte?). H, Form with a 
thread-like process (flagellated stage?). (From Doflein after Fantham.) 

multiplication seems to be by longitudinal division into two 
daughter cells. The characteristic form is pear-shaped, but 
irregular amoeboid 'forms are also common. Flagellate stages 
existing in the blood plasma have also been described. The sexual 
cycle takes place in a tick, and is in part analogous to that described 
for Plasmodium. The stages are not fully known, but the infec- 
tivity of the tick is transmitted to the offspring in the case of the 
Texas-fever tick (Rhipicephalus (Boophilus) annulatus). 

Nosema. The sporozoa above described all belong to the 
Telosporidia, organisms which end their individual existence at 
the stage of spore formation. A second large subdivision of the 
sporozoa is named Neosporidia. In this group the spores are 
formed without terminating the existence of the individual. The 



MORPHOLOGY AND CLASSIFICATION 

parasites of this type are comparatively small and not very well 
known. They are often spoken of as microsporidia or psorosperm^, 
The best-known form is Nosema bombycis, the cause of Pebrine in 
silkworms. 

Ciliates. The second substem of the protozoa, Ciliophora, 
is distinguished by the locomotive organs, numerous cilia which 




FIG. 8 1. Diagram of the developmental cycle of Nosema bombycis. C, Cell of 
the intestinal epithelium containing asexual multiplication forms and showing their 
transition into spores, a, b, c, Spores, the last with polar thread, d, Ameboid form 
emerging from the spore to penetrate a new host cell at h. (From Dofiein after 
Stempell.} 

cover most of the body surface, and by the possession of two dis- 
tinctly different nuclei, one apparently concerned with nutrition 
of the eel] and the other definitely associated in an important man- 
ner with the sexual reproduction. Multiplication takes place by 
transverse division into two daughter cells or by budding. In the 
parasitic forms this may take place within a protecting wall (cyst). 



i6o 



GENERAL BIOLOGY OF MICRO-ORGANISMS 



The sexual fertilization is not followed by any special kind of divi- 
sion. Balantidium is the only genus of present interest as a cause 
of human disease. See Balantidium coli p. 435. 



OUTLINE CLASSIFICATION OF MICRO-ORGANISMS. 





f Phycomycetes 




Hyphomycetes 
(Molds) 


j Basidiomycetes 
\ Ascomycetes 
| Unclassified 






[ molds. 








[ Oidia 




Fungi (Plants) i 


Blastomycetes 
(Yeasts) 


J Torulae 
1 Saccharomy- 








{ cetes. 








f Trichobacteria 






Schizomycetes 


J Coccaceae 






(Bacteria) 


j Bacteriaceae 


Protista (Micro- 






( Spirillaceae 


organisms) 


/ Spirochaetes 
Not classified \ ~,, , , . , 
I Filterable microbes 






f Mastigophora 




f Plasmodroma 


\ Rhizopoda 




Protozoa 


[ Sporozoa 




(Animals) 
j Ciliophora 


f Ciliata 
1 Suctoria. 



Specific Nomenclature. A species is properly designated only 
by a binomial Latin name, the first member being that of the 
genus, such for example as Mucor mucedo, Saccharomyces cere- 
visiae, Bacillus coli, Spirochoeta pallida, Plasmodium falciparum, 
and Balantidium coli. A third term may be employed to des- 
ignate a variety of a species, but such usage should not be per- 
sisted in. It is possible to give the variety a new specific name 



MORPHOLOGY AND CLASSIFICATION l6l 

if the distinction is of sufficient importance, or to drop the dis- 
tinctive term from the Latin name altogether if the difference 
proves to be unimportant. The system of genera is in a very 
unsatisfactory state, especially in the schizomycetes where the 
number of species in one genus is much too large. Even in the 
other great groups the generic nomenclature is far from settled. 
The specific name however should be a very definite and single 
term, and it is usually either the first published name given to 
the organism or some emended adaptation of it, in proper 
grammatical agreement with the generic term employed. Thus, 
in designating the parasite of syphilis, one may employ the term 
Spirochceta pallida classing it in the genus Spiroch&ta (Ehren- 
berg), but if he adopts the proposed genus Treponema (Schau- 
dinn) the name becomes Treponema pallidum. 



CHAPTER VIII. 
PHYSIOLOGY OF MICRO-ORGANISMS. 

Relations of Morphology and Physiology. In morphological 
study observations are restricted to the relationship of various 
elements at a given time, facts relating to form and structure. 
From the physiological viewpoint one is more interested in the 
sequence of events and the relation of cause and effect. The 
possible suggestion that these two methods of study are independ- 
ent or mutually exclusive would be most unfortunate and is really 
very fallacious. The sequence of events may often best be ascer- 
tained by a series of morphological observations of a microbe 
undergoing change of form, and certainly the form and structure 
of a living organism at a given time may be properly regarded as 
an expression and result of previous physiological activity as well 
the most essential element in its potentiality for future activity. 
All must agree that difference in behavior, that is, reaction to a 
definite environmental change, is really associated with a difference 
in structure of the living organism. The important difficulty 
lies in the fact that the morphological or structural difference 
with which this difference in reaction is correlated, may not be 
capable of direct observation by any known method and may be 
ascertainable only by means of the physiological test. On the 
other hand the method of experimental physiology involves the 
factor of environment, small and unmeasured differences in which 
may grossly influence the resulting phenomenon and lead to erro- 
eous conclusions. Furthermore, the experimental conditions and 
the method of physiological observations may be wholly lacking 
in adaptation to potentialities of the organisms under observation. 
When properly employed, however, the method of experimental 
physiology yields valuable knowledge obtainable in no other way, 

162 






PHYSIOLOGY OF MICRO-ORGANISMS 163 

and it has been the most important single method in establishing 
our modern ideas of the relation of micro-organisms to infectious 
diseases, and is the method of greatest promise for the immediate 
futrue. 

Conditions of Physiological Study. The physiology of many 
organisms is subject to only very limited experimental investi- 
gation. Those organisms of very narrow biological adaptation, 
such as many of the parasitic protozoa, can be studied only in 
very close relation to their natural environment, the various 
important elements of which are not readily subject to experi- 
mental alteration and are largely unrecognizable. Our knowl- 
edge of these forms must therefore be derived almost exclusively 
from observations of form and structure, physical and chemical, 
as they exist and change under the natural conditions of environ- 
ment, and from changes which take place in the tissues surround- 
ing the parasite, which we may ascribe with more or less justifica- 
tion to their activity. Practically all that we know about the 
physiological activity of the very numerous microbes not yet 
brought into the group of artificially cultivable forms, has been 
deduced from morphological observations. Even observations 
of this kind, however, can be more successfully pursued in those 
forms capable of artificial culture, and artificial culture is a prime 
necessity for the study of cause and effect by the methods of ex- 
perimental physiology. For this reason accurate knowledge of 
what micro-organisms do is much richer in regard to the culti- 
vable forms such as bacteria, yeasts and molds. In fact the mi- 
crobic pure culture presents the most favorable object known for 
the study of cellular physiology and bio-chemistry. Further- 
more, the physiological activities of many microbes are of the 
greatest practical importance. It is not surprising, therefore, 
that, among the bacteria, many of which grow in artificial media 
under a great variety of environmental conditions, the relative 
ease of physiological experimentation, as compared with the dif- 
ficulty of observation of the minute morphological details, and 
the great practical importance of its results has lead to an enor- 



164 GENERAL BIOLOGY OF MICRO-ORGANISMS 

mo us development of knowledge gained by the first- mentioned 
method, which quite over-shadows our knowledge of morph- 
ology and structure in this group of organisms. 

THE INFLUENCE OF ENVIRONMENTAL FACTORS. 

Moisture. Moisture is indispensable to the growth of mi- 
cro-organisms. A few species will grow and multiply in almost 
pure distilled water. Drying causes the death of the majority of 
the vegetating cells, of some more readily than others, while the 
spore forms may remain alive in a dry condition for many years. 

Heim 1 found that pathogenic bacteria resist drying much 
longer when contained in pathological tissues or exudates from 
animals which have succumbed to the disease, than when they 
are taken from artificial cultures. 

Organic food. One species of bacteria, Nitrosomonas of 
Winogradsky, lives, grows and multiplies without organic food, 
utilizing the gases of the atmosphere as its source of carbon and 
nitrogen. From the standpoint of nutrition this organism is 
among the most primitive of beings. Other bacteria are known 
which may grow in water containing only mineral salts and a 
simple sugar, utilizing large quantities of atmospheric nitrogen. 
These are known as nitrogen-fixing bacteria. Most of the bac- 
teria, yeasts and molds require a small amount of nitrogenous 
organic matter as food, such as the ammo-acids or albumoses, and 
many of them flourish better when furnished a fermentable 
carbohydrate such as dextrose. The complex organic molecules 
are utilized in part to build up the substance of the bacteria, but 
a much larger part of them is broken down into simpler and more 
stable substances, such as carbon dioxide, simple fatty acids, 
ammonia and water, with the liberation of energy. Sapro- 
phytic organisms are those which grow on dead organic matter. 
Micro-organisms of still narrower adaptibility grow well in artifi- 
cial culture only if they be furnished abundant protein or nucleo- 

1 Zeitschriftf. Hygiene, Apr. 4, 1905, Bd. L, No. i, p. 123. 



PHYSIOLOGY OF MICRO-ORGANISMS 165 

protein. Some important disease-producing bacteria belong in 
this category, as well as many parasitic spirochetes and some _of 
the protozoa. Such organisms are not adapted to any natural 
saprophytic existence, and they grow in the artificial cultures 
only because the dead medium is made to resemble somewhat 
their natural parasitic habitat. Finally there are the micro-or- 
ganisms which have not yet been grown in artificial culture and 
whose food requirements are essentially unknown. Many of 
these are parasites, and are called obligate parasites. A few bac- 
teria, many of the filterable agents, and most of the parasitic 
protozoa are included in this category. 

Inorganic Salts and Chemical Reaction. Phosphorus, sul- 
phur, chlorine, calcium, sodium and potassium, in addition to 
carbon, hydrogen, oxygen and nitrogen, are present as constituents 
of the microbic protoplasm. Minute quantities of these suffice to 
supply the food requirements of micro-organisms and it is un- 
necessary to add them to culture media to serve as food. Com- 
mon salt, sodium chloride, is ordinarily employed to give the 
artificial medium an osmotic tension approaching that of the 
body fluids, and calcium carbonate is sometimes used to neutral- 
ize the organic acids which may arise in the culture as a result of 
the bacterial growth. 

The most favorable chemical reaction for most micro-organisms 
is that of actual slight alkalinity, not sufficiently alkaline to pro- 
duce a red color with phenolphthalein and not sufficiently acid 
to produce a red color with litmus. Some bacteria and many of 
the yeasts and molds will grow well in a weakly acid medium, 
but most parasitic bacteria and protozoa, which can be cultivated 
at all, require a reaction slightly alkaline to litmus or rosolic 
acid. The anaerobic bacteria do best in a medium containing 
glucose and with a reaction quite alkaline, indeed very close to 
the point at which phenolphthalein becomes pink. Organisms 
which produce acid or alkali are usually arrested in their growth as 
soon as a certain concentration is reached, and the medium may 
then rapidly kill the micro-organisms. 



1 66 GENERAL BIOLOGY OF MICRO-ORGANISMS 

Oxygen. Oxygen, either free as atmospheric oxygen or com- 
bined as in water or organic compounds, is an essential constitu- 
ent of the food of all micro-organisms. The concentration of 
uncombined oxygen dissolved in the medium, or the partial 
pressure of atmospheric oxygen, is the factor ordinarily meant 
when oxygen requirement is mentioned. Many micro-organisms 
grow best in a medium freely exposed to the air. These are 
called aerobes. Some which will grow only when there is free 
access of oxygen are called obligate aerobes. There are a few 
bacteria and some spirochetes which grow only in the absence 
of, or in extremely weak concentration of oxygen. These are 
called obligate anaerobes. Many of the bacteria grow well in 
various concentrations of oxygen or in its absence. These are 
spoken of as facultative anaerobes, or sometimes as facultative 
aerobes if they seem to prefer the anaerobic existence. Finally 
there are a few organisms, some bacteria and spirochetes, and 
perhaps some protozoa, which seem to require a fairly definite 
partial pressure of oxygen, but are not adapted to growth in 
a medium freely exposed to the atmosphere (B. bifidus, B. abortus, 
Spirochceta rossii, Plasmodium falciparum) . 

Temperature. Among the various micro-organisms are found 
types which are adapted for growth at different temperatures 
throughout a considerable range. There are some bacteria and 
perhaps some molds capable of growth at a temperature of 0.5 
C., in food substances such as milk, which are not frozen at this 
temperature. A certain yeast is said to multiply even at 6 C., 
in salted butter. Microbes which grow at very high temperatures, 
even up to 80 C., occur in the soil, in ensilage and sometimes 
in the intestine of animals. The great majority of micro-organ- 
isms grow only between o and 40 C. It is possible to recognize 
a minimum, a maximum and an intermediate optimum tem- 
perature for growth of each species. Ordinarily the optimum 
temperature is only a few degrees below the maximum at which 
growth will take place. The^following table from Marshall's 
Microbiology illustrates the relation of these temperatures. 



PHYSIOLOGY OF MICRO-ORGANISMS 



I6 7 







Temperatures 




opecies 


Minimum 


Optimum 


Maximum 


Penicillium gJaucum . . 


I.c 


25 -27 


3i-36 


As per gill us niger 


7-io 


77-37 


4o-43 


Saccharomyces cerevisicz I 


i- 3 


28-3o 


40 


Saccharomyces pasteurianus I 
Bacterium phosphoreum 


0-5 
below o 


25-30 
i6-i8 


34 
28 


Bacillus subtilis 


6 


30 


50 


Bacterium anthracis 


10 


7 -37 


43 


Bacterium ludwigii 


50 


SS-S7 


80 











Heating above the maximum temperature for growth injures 
the microbe and exposure for a short time kills it. A temperature 
of 60 C. for 20 to 30 minutes destroys most vegetative forms of 
bacteria. Cooling, on the other hand, merely checks and inhibits 
growth. Freezing destroys some of the germs contained in a 
liquid but many of them remain alive. Still lower temperatures 
seem to be entirely without further effect. Bacteria gradually 
die in frozen material. 

Germicides. Unfavorable environmental factors, germicides 
and antiseptics have been considered in an earlier Chapter 
(Chapter II). 

Microbic Variation. A microbic species is very stable in its 
characters when maintained under fairly constant conditions in 
its normal habitat. Change in environment brings about rather 
quickly change in some of the characters of a bacterial species. 
The alterations in virulence or ability to produce disease, which 
may be produced by methods of artificial culture, are perhaps 
best known. It would seem that the descendants of a single 
cell are not all identical, but they vary among themselves within 
fairly narrow limits in respect to a great many characters, fluc- 
tuating about a mean type which is that best adapted to the 
environment. With a change in surrounding conditions, this 
mean or normal type may no longer be best adapted, but a 



1 68 GENERAL BIOLOGY OF MICRO-ORGANISMS 

variation slightly removed in respect to certain characters may 
flourish better and become the mean type about which the 
fluctuating variants group themselves. Thus the pure culture 
seems to respond to environmental change. Whether the 
fluctuating variations are due to small differences in the imme- 
diate surroundings of the individual microbes, or whether they 
arise as a result of a property of variability inherent in protoplasm, 
may be disputed, but the latter view is more commonly held by 
biologists. 

THE PRODUCTS OF MICROBIC GROWTH. 

The effects resulting from the growth of a micro-organism 
depend on the one hand upon the nature of the organism and on 
the other upon the environment, more especially the medium in 
which it grows and the conditions of temperature and oxygen 
supply. Apparently slight variations in the latter may influence 
the results to a marked degree. 

Physical Effects. Heat is evolved by many actively growing 
bacterial cultures and is especially evident in the fermentation 
of such substances as ensilage and manure. Perhaps some of 
the heat may result directly from microbic activity, but the most 
of it appears to arise from secondary chemical reactions in which 
the microbic products sometimes play a part. Microbes which 
produce heat are designated as thermogenic. Light is also emitted 
by some microbic cultures. Here it seems certain that the light 
is produced by the oxidation of a bacterial product and not emitted 
directly by the micro-organisms. These phosphorescent or 
photogenic organisms occur in salt water and on fish and they 
have rarely been found in other places. 

Chemical Effects. These are the most important results of 
microbic growth. As we have just seen, the production cf 
heat and light is probably due to a secondary reaction entered 
into by some of the chemical products of growth. Almost all 
the other important practical effects of the growth of micro- 



PHYSIOLOGY OF MICRO-ORGANISMS 169 

organisms are due to chemical changes produced by them. 
Primary products are those which are produced inside the cell 
by its living protoplasm. These include all the synthetic products 
such as the substance of the germ itself, the complex bodies 
which it forms from simpler substances, such as its enzymes 
and its toxins, and also the simpler chemical substances which 
result from internal cellular metabolism, the proper excretions 
of the cell. The secondary products are those which result from 
the action of a primary product, such as an enzyme, upon some 
material outside the cell. The distinction is clear enough in 
theory but practically it is often obscure. 

Enzymes. Fermentation in its broad sense means the 
chemical changes brought about by living cells or their products. 
In its more restricted sense, it applies to the splitting of carbohy- 
drates by the action of microbes, which is accompanied by the 
evolution of gas. Organisms which cause active fermentation 
are spoken of as zymogenic. Dextrose, C 6 Hi 2 O 6 , is a readily 
fermentable carbohydrate and is decomposed in various ways 
by different microbes. In some instances a large proportion 
of it is converted into alcohol and carbon dioxide according to 
the following equation: 

C 6 Hi 2 O 6 (fermented) = 2C 2 H 6 O+2CO 2 . 

Other kinds of micro-organisms produce little alcohol or gas 
but abundant lactic acid. The reaction may be represented 
roughly by this equation: 

C 6 Hi 2 6 (fermented) = 2C 3 H 6 O 3 . 

In other instances acetic acid may be produced: 

C 6 Hi 2 O 6 (fermented) =3C 2 H 4 O 2 . 

These equations are only an approximate indication of the 
reactions which take place, as it is very doubtful that the whole 
molecule of dextrose is ever converted into a single simpler 
compound by fermentation, but they will serve to indicate the 



170 GENERAL BIOLOGY OF MICRO-ORGANISMS 

nature of the reactions involved and to suggest the variety of 
products which may arise from the decomposition of complex 
organic substances. Some of these fermentative changes take 
place to a large extent inside the microbic cell. Such is the 
case in the alcoholic fermentation produced by saccharomyces. 
The sugar-splitting or glycolytic ferments are found in the 
cultures of many bacteria and molds. Less common are the 
diastatic ferments capable of changing starch to dextrose, the 
inverting ferments which change saccharose and lactose into 
glucose and other hexoses, and the acetic ferments capable of 
causing the oxidation of alcohol to produce vinegar. 

The fermentation or decomposition of proteins usually gives 
rise to evil-swelling gases. This decomposition is called putrefac- 
tion, and the organisms which cause it are called saprogenic or 
putrefactive organisms. The nature of the products is much 
influenced by the amount of oxygen available and the foulest 
gases are produced especially in the absence of oxygen. Proteo- 
lytic ferments of the same general nature as trypsin are produced 
by many microbes. A few form rennet-like enzymes. Proteo- 
lytic ferments which act in the presence of acid, like pepsin, are 
produced by some molds and by some bacterial species. 

The decomposition of the complex protein molecules gives rise 
to an enormous variety of intermediate products before the ulti- 
mate analysis into ammonia, carbon dioxide, water, sulphates and 
phosphates is accomplished. Many of these intermediate prod- 
ucts are very unstable and of unknown chemical composition. 
Some of them are highly poisonous. Brieger and his followers 
were able to separate a number of the complex substituted ammo- 
nia and ammonium compounds in a pure state and these par- 
ticular bodies are known as putrefactive alkaloids, or as ptomains. 
A simple ptomain is trimethylamin, N(CH 3 ) 3 ; a more complex 
one cadaverin, HgN-CHa'CHa-CHa'CHa'CHa-NHa. Some of the 
ptomains are poisonous. These various decomposition products 
are for the most part secondary products resulting from the action 
of enzymes upon the decomposing material. Many of them are 



PHYSIOLOGY OF MICRO-ORGANISMS 171 

so unstable that their presence in a decomposing substance is 
influenced by access of air, temperature and moisture, and they 
may quickly disappear or decompose. 

Micro-organisms also form fat-splitting or steatolytic enzymes, 
and enzymes capable of transforming urea into ammonium 
carbonate. 

NH 2 -CO-NH 2 +2H 2 O (fermentation) = (NH 4 ) 2 CO 3 . 

Various inorganic substances undergo chemical change under the 
influence of microbic activity and some of these changes appear to 
be due to enzymes. Specific examples will be considered in the 
section on the soil bacteria. 

The toxins of bacteria are primary products built up by the cell. 
The true bacterial toxins are of unknown chemical composition, 
are labile like enzymes and stimulate the production of antitoxins 
when they are injected into animals. They are the most poisonous 
substances at present known. Analogous substances have been 
found in some plants, ricin in the castor bean and abrin in the 
jequirity bean, and the poisonous property of some kinds of snake 
venom is due to the presence of substances similar in nature to 
the bacterial toxins. These substances will be considered more 
fully in a later chapter devoted to the relation of parasitic microbes 
to their hosts. 

MUTUAL RELATIONS OF A MICROBE AND ITS ENVIRONMENT. 

Morphological Characters. It is evident that the phenomena 
of growth taking place in a microbic pure culture depend not only 
upon the particular kind of microbe present but also in a very 
important way upon the chemical and physical structure of the 
medium, the access of air and the temperature. Variations in 
these latter may even bring about considerable alteration in the 
form and structure of the individual cells. A common effect of 
high temperature is the shortening of individual bacilli and spirilla 
because of more rapid division and complete separation of the 



172 GENERAL BIOLOGY OF MICRO-ORGANISMS 

daughter cells. The presence of unfavorable influences, such as 
antiseptics or bacterial waste products in the medium, may cause 
marked irregularities in the shape and size of the cells, so-called 
involution forms. The ability to form endospores may be lost 
through growth at high temperature. The form which a micro- 
organisms presents in a given instance may not, therefore, be 
regarded as essentially typical without regard to the conditions 
under which it has been produced. 

The morphology of cell-groups is even more obviously depend- 
ent upon the conditions of the environment and the physiological 
properties of the micro-organism. A slow scanty growth on a 
given medium does not necessarily mean that the organism 
essentially lacks vigor. It may mean that the medium is not well 
adapted to the requirements. Diffuse growth through a semi- 
solid medium may be merely an expression of the motility of an 
organism. A great variety of different culture media have been 
employed to bring out more or less characteristic features in the 
gross appearance of cultures, and these appearances often depend 
upon the grouping of the cells or upon their fermentative activity 
or both. Although the characters of a cell-group of micro-organ- 
isms are really morphological characters of the same general na- 
ture as the morphological characters of higher plants and animals, 
to which so much significance is attached, in the case of micro- 
organisms in an artificial environment, such as a culture medium, 
the gross appearance or the cell-grouping is more properly regarded 
as a feature of physiological rather than morphological significance. 
Nutrient gelatin is a medium well adapted, in the case of those mi- 
crobes which will grow in it, for showing physiological differences 
in the appearance of cell-groups or colonies, and perhaps a greater 
variety of appearances may be obtained upon this medium than 
any other. Unfortunately its use entails certain difficulties, the 
most important of which is the necessity for experience and s care 
in the interpretation of the appearances observed. Important 
features in the appearance of the colonies and other cell-groups 
are brought out by the use of various other media. 



PHYSIOLOGY OF MICRO-ORGANISMS 173 

Physiological Tests. Specific tests for a simple physiological 
character require less skill and care in their observation, and are 
widely used. Cultivation in a fermentation tube of sugar broth 
as a test of ability to form gas from the sugar, titration of sugar- 
broth cultures to ascertain the ability to produce acid from various 
sugars, chemical test for the presence of indol and of ammonia in a 
culture in peptone solution, observation of the ability to hemolyze 
or discolor blood mixed with the medium, and the ability to fer- 
ment glycerin, these are some of the valuable simpler tests. 
Cultivation in milk is a somewhat more complex test, as a variety 
of fermentable substances is offered the microbe, increasing the 
difficulties of interpretation but also increasing the variety of phe- 
nomena which may occur. 

A convenient outline to use in making morphological and 
physiological observations upon bacteria and in recording the re- 
sults, has been prepared by a committee of the Society of American 
Bacteriologists. Many features of this will be found of assistance 
in the study of new or unknown bacteria, especially saprophytic 
forms. A copy of the revised descriptive chart is inserted. 



CHAPTER IX. 

THE DISTRIBUTION OF MICRO-ORGANISMS AND 
THEIR RELATION TO SPECIAL HABITATS. 

General Distribution. Micro-organisms are very generally 
distributed over the surface of the earth and in its waters, and 
are carried about as dust in the air. They flourish abundantly 
in the digestive canals of animals and on their body surfaces. 
Wherever there is organic matter, the dead remains of animal 
and plant life, there are micro-organisms in abundance living 
upon the dead material and, if the temperature and moisture be 
suitable, transforming it into simpler chemical substances. In 
the soil, bacteria, yeasts, molds and protozoa are fairly numerous, 
especially in fertile soils near the surface. Their number rapidly 
diminishes in the deeper layers, and at a depth of six to twelve 
feet they are very scarce or entirely absent. The surface waters 
of the earth contain large numbers of bacteria and protozoa, 
especially numerous where organic matter is abundant. The air 
contains considerable numbers of molds and bacteria suspended 
as dust. The deep layers of the soil and water below impervious 
rock strata are free from micro-organisms. The surfaces of snow- 
covered mountains and of the frozen polar regions of the earth, as 
well as the atmosphere in these regions, are practically free from 
microbes. The atmosphere over large bodies of water during 
calm weather, the air in damp cellars, in sewers and in undisturbed 
rooms is germ-free, because the suspended dust particles settle 
out and do not escape again into the air unless swept up by air 
currents, which must be rather violent to remove them from 
moist surfaces. 

174 



THE DISTRIBUTION OF MICRO-ORGANISMS 175 

The environment and the surfaces of growing plants and animals 
are rich in micro-organisms, especially bacteria, but the interior 
of the living tissues is generally germ-free in health. To this 
statement there are certain exceptions, namely, the occurrence 
of a few bacteria in the liver, the thoracic duct and the blood of 
animals during active digestion, which are, however, soon de- 
stroyed by the healthy tissue; and the invasion of the root tissues 
of leguminous plants byPs.radicicola and the growth of the bac- 
terium within the plant tissues, which results not in injury to 
the host but in a definite improvement of its nutrition by enabling 
it to utilize atmospheric nitrogen. 

Micro-organisms of the Soil. The germ-content of soil depends 
chiefly upon the amount of organic matter present. They may 
be present in millions per gram of soil. Bacteria, molds and pro- 
tozoa are the most numerous. Their relation to soil fertility 
seem to be important, and they probably play a large part in 
preparing the organic matter of the soil for use as food by plants. 
A great many soil bacteria decompose protein and set free am- 
monia, and the urea bacteria are especially important in the 
transformation of urea and of animal manures into ammoniacal 
compounds. The transformation of ammoniacal compounds 
into nitrates, so-called nitrification, is accomplished by the nitri- 
fying bacteria, of which a few species have been obtained in pure 
culture, Nitrosomonas of Winogradski which produces nitrite 
from ammonia, and his genus Nitrobacter which oxidizes nitrites 
to nitrates. Very many species of soil bacteria are able to change 
nitrogen in the opposite direction, reducing nitrates to nitrites 
and further to ammonia or to free nitrogen gas. Of special interest 
are the soil bacteria which are able to fix atmospheric nitrogen, 
that is, absorb nitrogen from the air and combine it so as to make 
it available for plant food. The various species of the genus 
Azotobacter (A. chroococcum, A. beyerincki) accomplish this as 
they grow in the presence of dextrose, and the organism of the 
root tubercles, Pseudomonas radicicola, fixes nitrogen as it grows 
within the tissues of the legume roots. Numerous soil bacteria 



176 GENERAL BIOLOGY OF MICRO-ORGANISMS 

ferment sugars, starches and fats, and there are several known 
species capable of dissolving cellulose. 1 

Pathogenic Soil Bacteria. Certain pathogenic bacteria are 
of common occurrence in the soil. Whether this is their normal 
habitat or whether they gain entrance to the soil with animal 
excrement, may be questioned. At any rate the pathogenic 
anaerobes, B. edematis, B. tetani, and B. welchii are likely to occur 
in garden soil, and it seems probable that they actually multiply 
there to some extent. Bact. anthracis also occurs in the soil of 
fields where the disease has prevailed, and it is not improbable 
that this organism multiplies in the ground at times. Other 
pathogenic bacteria, such as those of typhoid and cholera, seem 
to be rather quickly eliminated in the struggle for existence under 
the conditions found in surface soils. 

Micro-organisms of the air. Micro-organisms exist in the air 
only as floating particles of dust, or as passengers on small drop- 
lets of moist spray, or as parasites on or in winged aerial creatures. 
Those floating as. dust are derived from the earth's surface, and 
most of the living germs usually found in this condition are the 
spores of molds. Living tubercle bacilli are unquestionably 
suspended in the air as dust, especially in the dry sweeping of 
floors where careless consumptives have lived. The spores of 
anthrax bacilli may also be suspended in the air where hides or 
wool of anthrax animals are handled. Other pathogenic bacteria 
may at times float as dust, but their presence in the air in this 
condition is apparently rather uncommon, and should be expected 
only in the fairly recent environment of cases of the disease. The 
moist droplets, expelled from the mouth and nose in speaking, 
in coughing and especially in sneezing, may remain suspended in 
the air for many minutes and be distributed to considerable 
distances. After drying the solid material may still float as dust. 
Pathogenic micro-organisms may readily be transmitted from 
person to person in this way. 

1 For a discussion of the microbiology of the soil, see Monograph by Lipman in 
Marshall's Microbiology, 1911. 



THE DISTRIBUTION OF MICRO-ORGANISMS 177 

In a rough way one may obtain some knowledge of the charac- 
ter of the organisms in the air of a given locality by removing the 
cover of a Petri dish containing sterilized gelatin or agar for a 
few minutes, replacing it, and allowing the organisms to develop. 
In most cases a large proportion of the growths that appear will 
be molds. Yeasts are also common, and among the bacteria 
the micrococci are abundant. Chromogenic varieties are likely 
to be present. 

A few studies of this character will show that the number 
of organisms that are present depends chiefly upon whether the 
air is quiet or has recently been disturbed by drafts, gusts of 
wind, or sweeping. These facts are of fundamental importance 
in laboratory work, if we wish to avoid contaminations. Among 
various devices that have been proposed for the accurate study 




FIG. 82. Sedgwick-Tucker aerobioscope. 

of the organisms of the air, the Sedgwick-Tucker aerobioscope 
is the simplest and most accurate. It consists of a glass tube, 
one end of which is drawn out so as to be smaller than the other. 
The small end contains a quantity of fine granulated sugar; 
both ends are plugged with cotton, and the instrument is sterilized. 
A definite quantity of air is to be aspirated through the large 
end, after removing the cotton, and this may be done by means 
of a suction-pump applied to the other end, or by siphoning 
water out of a bottle, the upper part of which is connected with 
the end of the aerobioscope by means of a rubber tube. The 
sugar acts as a filter and sifts out of the air the micro-organisms 
which are contained in it. Liquefied gelatin or agar may be 
introduced into the large end of the instrument by means of a 
bent funnel; and, after replacing the cotton, it is mixed with 
the sugar which dissolves. The culture-medium may be spread 



178 GENERAL BIOLOGY OF MICRO-ORGANISMS 

around the inside of the larger portion of the tube after the 
manner of an Esmarch roll-tube. The bacteria which are filtered 
out by the sugar will develop as so many colonies upon the 
solidified medium. 

Many important micro-organisms, and certainly some germs 
of disease, are borne through the air by the winged insects, and 
to a less extent by birds. The microbes are found not only on 
the feet and outer body surfaces of these carriers, but they also 
occur on and in the mouth parts, in the alimentary canal and 
sometimes in the interior of the animal's body tissues. Certain 
pathogenic micro-organisms (Plasmodium, Trypanosoma) are 
known to be transmitted from one person to another almost 
exclusively by biting insects, and the importance of these carriers 
in air-borne disease of both animals and plants, is being recognized 
more and more. 

Micro-organisms of Water and Ice. The water of rivers, 
lakes and the ocean always contains bacteria. The number 
of organisms varies greatly in different places and under different 
conditions. The number of different species found in water 
is also very large. Some of these, the natural water bacteria, 
including many bacilli which produce pigment and some cocci 
and spirilla, seem to live in surface water as their natural habitat. 
With the addition of putrescible material these forms are in- 
creased in number and certain of them (Proteus group, fluorescing 
bacteria) become numerous. Soil bacteria are numerous in 
waters during floods and after rain, and they may persist for 
some time. Intestinal bacteria occur in waters which receive 
sewage or are otherwise contaminated with excreta. They 
persist only a relatively short time. Certain intestinal protozoa, 
Entamaba, Aalantidtum, seem also to occur in waters at times. 
Ground-water 1 contains few or no bacteria under normal condi- 
tions, and is therefore suitable for a source of water-supply, 
when a sufficient amount is available. The possibility of contami- 

1 Ground-water is the water which originally derived from rain or snow sinks 
through superficial porous strata, like gravel, and collects on some underlying, 
mpervious bed of clay or rock. 



THE DISTRIBUTION OF MICRO-ORGANISMS 179 

nation of the ground-water from unusual or abnormal conditions 
should always be eliminated before it is taken for drinking 
water. Numerous epidemics of typhoid fever have been traced 
to contamination of wells. The location of wells with reference 
to privy-vaults and other possible sources of contamination 
should be chosen with the greatest care. 

The ordinary bacteria of water are harmless, as far as is 
known. 1 Bad odors and tastes in drinking water that is not 
polluted with putrid material are usually due to minute green 
plants (algae). 2 The diseases most commonly disseminated 
by water are typhoid fever and Asiatic cholera, and probably 
also dysentery. The spirillum of cholera will usually die in 
natural water (not sterilized water) inside of two or three weeks; the 
bacillus of typhoid fever will usually die in two or three weeks. 
Under exceptional circumstances these organisms may perhaps 
maintain their vitality for a longer period. They appear, however, 
to be less hardy than the ordinary water bacteria. As 
we now understand these diseases, the organisms causing them 
will be present only in a water-supply which has been recently 
contaminated by the excreta from a case of the disease. Notwith- 
standing the rapid death of these organisms in water, they 
may exist long enough to infect individuals habitually drinking 
the water. Many epidemics of cholera and typhoid fever have 
been traced to water polluted with the discharges from cases 
of these diseases, and in a few instances the relation of the con- 
taminated water supply to the epidemic has been established 
beyond a reasonable doubt. 

By self -purification of water is meant the removal, through 
natural processes, of contaminating organisms such as might 
occur from the discharge of sewage into it. It depends upon 
the sedimentation of the contaminating material in the form 
of mud, upon the growth of the ordinary water-plants and protozoa, 

1 See Fuller and Johnson, "The Classification of Water Bacteria," Journal of 
Experimental Medicine, Vol. IV, p. 609. 

: " Contamination of Water Supplies by Algae." G. T. Moore in Yearbook 
U. S. Department of Agriculture, 1902. 



180 GENERAL BIOLOGY OF MICRO-ORGANISMS 

upon the exhaustion of the food supply by the growth of bacteria 
themselves, upon the destructive influence of direct sunlight, 
and the dilution of the contamination by a large volume of 
water. 1 It is not usually to be relied upon as a means of freeing 
the water-supply from pathogenic bacteria. 

Storage of Water. When water is kept in large reservoirs, 
the solid particles in it, including bacteria, tend to fall to the 
bottom. The number of bacteria in a water-supply may be 
considerably reduced .in this way. The use of large storage 
reservoirs also provides for the dilution of any sudden excess of 
pollution, and if the water is held in storage the pathogenic 
germs present disappear for the most part in a few days or weeks. 

Filtration. Water may be completely sterilized by passing 
it through the Pasteur-Chamberland niters of unglazed porcelain, 
or through the more porous Berkefeld niters. Such niters are 
effective only when frequently cleaned and baked, and in practical 
purification of water for household purposes they usually fail 
because of the intelligent care they require. Other types of 
domestic filters are generally worse than useless. 

Filtration on a large scale has been more commonly in use in 
the cities of Europe than elsewhere, until lately. Filtration- 
plants now exist in several cities of the United States. By this 
method 98 per cent to 99 per cent of the bacteria in water may 
be removed. 

Slow Sand Filtration. 2 The filter consists of successive layers 
of stones, coarse and fine gravel. The uppermost layers are 
of fine sand. The whole filter is from i to 2 meters thick. The 
sand should be 60 cm. to 1.2 meters in thickness. The accumu- 
lated deposit from the water and a little of the fine sand must 
be removed from time to time, but the layer of fine sand must 
never be allowed to become less than 30 cm. in thickness. The 
first water coming from the filter is discarded. The actual fil- 
tration is done largely by the slimy sediment which collects 

1 See Jordan, Journal of Experimental Medicine. Vol. V, p. 271. 

2 For a full discussion see Journal American Medical Association. Oct. 3 to 
3i, 1903- 






THE DISTRIBUTION OF MICRO-ORGANISMS l8l 

on the surface of the layer of fine sand. The filterbeds may 
be several acres in extent, and in cold climates should be pro- 
tected by arches of brick or stone. They require renewal occa- 
sionally. This kind of filtration has come largely into use since 
the cholera epidemic of 1892-93, and it appears to be very effective. 
It is important to use storage basins in connection with sand 
filtration. 

The results obtained by filtration depend greatly upon the 
intelligence displayed in operation, and must be controlled by 
frequent examinations of the water. 

Mechanical Filtration. This method of filtration is also 
called the American system. It is more rapid than the preceding 
method and does not require a large area for filter beds. Al- 
though sand is required also, filtration is accomplished by a 
jelly-like layer of aluminium hydroxide. This product is formed 
by adding to the water a small quantity of aluminium sulphate 
or of alum. The carbonates in the water decompose the aluminium 
salt and produce aluminium hydroxide. It precipitates as a 
white, flocculent deposit, entangling solid particles, including 
bacteria, as coffee is cleared with white of egg. Only a 'trace 
of aluminium should appear in the water. This method of filtra- 
tion has not been tested so extensively as slow sand filtration, 
but seems likely to prove efficient. With water poor in carbonates, 
these may have to be added. 1 

Whipple and Longley 2 found that the efficacy of mechanical 
filters with the addition of alum depends somewhat upon the 
character of the alum. They find that the alum shall be shown 
by analysis to contain 17 per cent of alumina (AljOj) soluble 
in water, and of this amount at least 5 per cent shall be in excess 
of the amount necessary theoretically to combine with the 
sulphuric acid present. It shall not contain more than i per 
cent of i soluble substances, and shall be free from extraneous 
debris of all kinds. It must not contain more than 0.5 per 

1 See Fuller, Journal American Medical Association, Oct. 31, 1903. 

2 Jonrn. Infect. Diseases, Supplement No. 2, Feb., 1906, pp. 166171. 



182 GENERAL BIOLOGY OF MICRO-ORGANISMS 

cent of iron (Fe 2 O 3 ) and the iron shall be preferably in the 
ferrous state. 

Chemical Disinfection. Various methods for the purification 
of water by means of chemicals have been proposed. The use 
of copper sulphate to disinfect drinking water was recommended 
by Moore and Kellerman, 1 and various investigators tested the 
value of their recommendation. Clark and Gage 2 came to the 
conclusion from their investigation that the treatment of water 
with copper sulphate or the storing of water in copper vessels 
has little practical value. Others also have come to practically 
the same conclusion. While the addition of copper sulphate is 
of use in preventing the growth of the algae, which sometimes 
grow so abundantly as to choke up water pipes, and is of benefit 
in this direction, the weight of evidence appears to be against 
its efficacy for purifying water for drinking purposes. More 
effective chemical disinfection has been obtained by means of 
ozone generated by electricity. More recently, calcium hy- 
pochlorite and free chlorine have been employed for this purpose 
with considerable success. 

Physical Disinfection. The most effective and surest method 
of disinfecting drinking water is by boiling it or by distillation. 

Bacteriological Examination of Water. For bacteriological 
examination samples from the water-supply of a city may be 
drawn from the faucet, but the water should first be allowed to 
run for half an hour or longer. From other sources the supply 
should be collected in sterilized tubes or bottles, taking care to 
avoid contamination. These samples should be examined as 
promptly as possible, for the water bacteria increase rapidly 
in number after the samples have been collected. When trans- 
portation to some distance is unavoidable the samples should 
be packed in ice, but even this precaution does not preserve the 
original bacteriological condition of the water at the time of 
collection; for more or less change probably takes place at all 

1 U. S. Dep. Agriculture, Bu. Plant Ind. Bulletin 64, 1904. 
8 Journ. Inf. Diseases, Sup. No. 2, Feb., 1906, pp. 175-204. 



THE DISTRIBUTION OF MICRO-ORGANISMS 183 

temperatures. If the temperature is too low, and the water 
freezes, more or less of the bacteria may be killed; if, on the 
contrary, the temperature is not low enough there will be a 
multiplication of the bacteria in transit. Special containers 
with provision for packing in cracked ice should be employed 
for shipment, and even then any considerable delay should be 
avoided. 

The number of bacteria may be determined by making plates of 
a definite quantity of the water with gelatin or agar. 1 The amount 
examined ordinarily is i c.c. When the number of bacteria is 
very large, a smaller quantity must be taken, and it may be neces- 
sary to dilute the sample ten times or more with sterilized water. 
The amount should be measured with a sterilized, graduated 
pipette. The water is mixed with melted gelatin or agar in a 
tube which has been allowed to cool after melting. After 
thorough mixing, remove the plug, burn the edge of the tube in 
the flame, hold in a nearly horizontal position until cool and pour 
into a sterilized Petri dish; or better, measure the water into 
the Petri dish, and pour the melted medium in, and mix. The 
number of colonies may be counted on the third or fourth day; 
the later the better, as some forms develop slowly and may not 
present visible colonies for several days; but the plates are often 
spoiled after three or four days by the profuse surface growths of 
certain forms, or by the rapid liquefaction of gelatin, if that be 
used. The number of colonies that develop is supposed to repre- 
sent the number of individual bacteria contained in the quantity 
measured. That will probably not always be the case, however, 
as colonies may develop from a clump of bacteria which have 
not been separated from one another by the mixing process. 
Abbott has shown that the number of colonies is usually larger 
on gelatin plates than upon agar plates, and at the room tempera- 
ture than in the incubator. This observation illustrates the fact 

1 For standard methods of water analysis see the report of a Committee of the 
American Public Health Association, Journ. Inf. Diseases, Supplement No. i, May, 
1905; also Report of Committee, American Public Health Association, New York, 
1912. 



1 84 



GENERAL BIOLOGY OF MICRO-ORGANISMS 



that there are doubtless many kinds of bacteria that do not find 
favorable conditions for development on ordinary culture- 
media. The reaction of the medium has an important influence 
upon the 'development of these water bacteria in plate cultures. 




FIG. 83. Jeffer's plate (Bausch and Lomb}. For counting colonies of bacteria on 
circular plates. The area of each division is one square centimeter. 

When the number of colonies is small, there is no difficulty 
in counting them as they appear in the ordinary Petri dish. 
When the number is large, some kind of mechanical device may 



THE DISTRIBUTION OF MICRO-ORGANISMS 185 

be used to assist counting. The Wolffhiigel plate is a large 
square of glass resting in a wooden frame painted black. The 
glass plate is ruled in squares. It was designed particularly 
with reference to the form of plate-cultures first made by Koch. 
The Petri dish, however, may be placed upon the glass plate and 




FIG. 84. Surface divided in square centimeters for counting colonies. 

the cross lines be used to assist in counting. Lafar, Pakes and 
Jeffer recommend a surface painted black, ruled with white lines 
which represent the radii of a circle, which may be still further 
subdivided by other lines. Many find counting easier when a 
black surface divided into squares is employed. An ordinary 



1 86 GENERAL BIOLOGY OF MICRO-ORGANISMS 

card with a smooth black surface divided into squares by white 
lines may be placed under a Petri dish and will be found to serve 
very well. For the mere examination of the colonies no better 
surface can be devised than the ferrotype plate used by pho- 
tographers. The examination of the colonies will be easier if a 
small hand-lens be used. Care must be taken not to mistake 
air-bubbles or particles of dirt for colonies of bacteria. 

In any case, if possible, all the colonies in the plate should be 
counted. But if this is not possible, the number contained 
within several squares may be counted and the average taken; 
knowing the size of the squares and the area of the plate, the 
number contained in the whole plate may be calculated. 

The plating may be done by rolling the medium after the 
manner of Esmarch. When the number of colonies is not large 
this may serve very well. Counting may be assisted by drawing 
lines with ink on the outer surface of the test-tube. It is obvious 
that the character of the bacteria is of prime importance; that 
pathogenic organisms may occasionally be present, even when 
the number of bacteria to the cubic centimeter is small. But 
knowing the number usually found in a good water-supply, any 
sudden variation above that number is to be looked upon with 
suspicion, as indicating a possible contamination. 

The bacteriological examination should always be accom- 
panied by a chemical examination, and by an inspection of the 
surroundings. A large number of bacteria is to be expected 
when the water has been subjected to unusual agitation from 
winds or currents which stir up the bacteria from the bottom. 

The Detection of Intestinal Bacteria. Bacillus coli is the 
organism ordinarily sought as a proof of pollution of a water- 
supply. Various quantities of the water, o.oi c.c., o.i c.c. and 
i c.c. may be inoculated into three series of fermentation tubes 
containing glucose broth or lactose bile. These are incubated 
at 39 C. Plates are made from the tubes in which gas is pro- 
duced and pure cultures obtained from the colonies for further 
study and identification. The number of tubes in which gas is 






THE DISTRIBUTION OF MICRO-ORGANISMS 187 

produced is regarded as presumptive evidence of the approxi- 
mate number of organisms of the B. coll type in the respective 
volume of water. 

The recognition of pathogenic bacteria, such as the germs of 
typhoid fever and Asiatic cholera, in water supplies has been 
accomplished very infrequently. Search for the cholera germ 
is best undertaken by adding a large volume, i to 10 liters, of the 
suspected water to one-tenth its volume of a sterile solution con- 
taining 10 per cent of peptone and 5 per cent of sodium chloride. 
After incubation for twelve to twenty-four hours at 37 C., trans- 
fers are made from the surface of this culture to tubes or flasks 
containing Dunham's solution (peptone i per cent, salt 0.5 per 
cent). At the same time gelatin plates are inoculated from 
this surface material. The cholera organism, if present, tends 
to outgrow all other bacteria in the surface film of such cultures, 
and after one or two transfers in series it will so predominate 
that it may be recognized by specific agglutination with a cholera- 
immune serum in high dilution (i-iooo). The appearance of 
the colonies on the gelatin plates is valuable as confirmatory 
evidence, and from them perfectly pure cultures may be obtained 
for further study. The search for the typhoid bacillus in water 
is usually rather hopeless. It has occasionally been detected 
by plating the water on special media such as litmus lactose 
agar or gelatin, or by culture in broth at 39 C. and inoculation 
of guinea-pigs or white rats with the culture, and subsequent 
plating of the heart's blood from the dead animal. Supposed 
typhoid bacilli isolated in this way must satisfy the biochemical 
tests for B. typhosus and furthermore must show specific agglu- 
tination with high dilutions (i -100) of typhoid-immune serum. 

If it is not already apparent from what has been said, it must 
be here emphasized that the difficulty of detecting the presence 
of pathogenic bacteria in water is very great, and the length of 
time necessarily consumed in making the tests greatly lessens 
the value of the results when obtained. Added to this is the 
further limitation of the value, that a negative result, i.e., where 



1 88 GENERAL BIOLOGY OF MICRO-ORGANISMS 

no pathogenic bacteria are found, cannot be taken as proof that 
the water-supply under examination may not be contaminated 
at times. Flugge 1 has shown that the chemical examination 
of itself also permits of no definite conclusion as to the potability 
of water. It would seem that those best suited by training and 
experience and who are capable of forming disinterested opinion 
attach but limited importance to the result of laboratory exami- 
nations of water unaccompanied by a sanitary inspection. In 
fact, many of those who have made disinterested study of the 
subject are inclined to question the value of the ordinary chemical 
and bacteriological water analysis in toto, and in view of the 
arbitrary and mechanical manner in which the results of these 
analyses are sometimes interpreted, this attitude is justified. 
It would seem, however, that after the establishment of normal 
standards for a given locality, such analyses are useful if they 
are checked by intelligent consideration of all the conditions 
entering into the case, but no hard and fast rules can be 
applied. 

Ice. The bacteriological examination of ice differs in no 
respect from that of water. Although development may be 
arrested, the vitality of bacteria is not necessarily impaired by 
freezing. Prudden found the bacillus of typhoid fever alive 
in ice after more than one hundred days. However, Sedgwick 
and Winslow have stated that when typhoid bacilli are frozen 
in water the majority of them are destroyed. 3 Nevertheless, 
it is as necessary to have the source from which ice is taken as 
carefully scrutinized as that of the water-supply, especially in 
view of the universal habit of cooling water in the summer time 
by adding ice directly to the water. It is better to cool water 
and articles of food by surrounding with ice the vessels containing 
them. 

1 Flugge: Zeitschrift fur Hygiene, Bd. XXII, 1896, pp. 445 et seq : 

2 Bolton: Sanitary Water Supplies for Dairy Farms. Public Health and 
Marine Hospital Service, Bulletin 41, February, 1908, p. 534. 

3 Clark. Bacterial Purification of matter by Freezing. Reports American 
Public Health Association, Vol. XXVII. See also Hutchings and Wheeler: Ameri- 
can Journal Medical Sciences, Vol. CXXVI, p. 680. 



THE DISTRIBUTION OF MICRO-ORGANISMS 189 

MICRO-ORGANISMS OF FOOD. 
\ 

Milk. Milk is the natural food of young mammals, and 
naturally it is taken directly from the mammary gland into 
the digestive tract of the young mammal. For many centuries, 
however, the milk of certain animals has been extensively used 
as a commercial food for man. The principal animals furnishing 
commercial milk are the cow, goat and mare. The chemical 
composition of milk is different in different animals, in the same 
animal at different periods of lactation, and even that obtained 
at different stages of a single milking shows considerable varia- 
tion. In general cow's milk has the following composition. 

Variation. Average. 

Fat 3-6 4 per cent. 

Lactose 1-3 2 per cent. 

Protein 5-8 7 per cent. 

Water 84-88 87 per cent. 

It is an excellent medium for the growth of most bacteria and is 
commonly used in the laboratory for this purpose. 

There are about 200 species and varieties of bacteria which 
commonly occur in milk. They are derived in part from the 
udder itself. Bacteria are always present in the milk ducts of 
the udder and are fairly abundant in the first portions of milk 
drawn, so that milk very carefully drawn from healthy animals 
may contain 200 to 400 bacteria per cubic centimeter. Milk 
from diseased udders may be very rich in pathogenic micro- 
organisms. As the milk is drawn, many micro-organisms usually 
gain entrance to it from the atmosphere, the hands of the milker 
and the utensils with which it comes in contact. From the 
body of the cow, particles of dust and hairs drop into the milk, 
carrying with them the flora of the intestine and of the skin of 
the cow. From the milker, the material on the hands and possibly 
also from the nose and mouth may reach the milk. The utensils, 
unless sterilized before use, contribute the microbic flora of the 
previous milkings, of the water used for cleansing and from the 



1 90 GENERAL BIOLOGY OF MICRO-ORGANISMS 

person who handles them. From the air, the milk may receive 
further contamination (i) from flies coming to drink or perhaps 
to drown without a clean bill of health from their port of last 
departure, (2) from particles suspended as dust and containing 
micro-organisms derived from manure, from hay and straw, 
and from soil, and (3) moist droplets expelled from the mouth 
and nose of the milkers and of the cattle. The subsequent 
handling of the milk may add further kinds of bacteria from 
human sources. Modern dairy practice in vogue in the produc- 
tion of the higher grades of milk eliminates some of these sources 
of contamination and minimizes the importance of the rest, but 
nevertheless fresh milk of even the better grades contains a 
great variety of micro-organisms, and often as many as 10,000 
to 100,000 per cubic centimeter when it leaves the producer's 
dairy. 

The usual milk flora derived from these various sources may 
be classed under the following heads: 

A. Lactic acid bacteria. 

1. Bacterium (streptococcus?) acidi lactici 

2. Bacillus coli and B. lactis aerogenes. 

3. Long rods of B. bulgaricus type. 

4. Streptococcus pyo genes. 

5. Micrococcus acidi lactici. 

6. Acid formers which liquefy gelatin. 

B. Gelatin-liquefying bacilli. 

7. Rapidly liquefying types B. subtilis. 

8. Slowly liquefying types. 

C. Pigment-forming bacteria. 

D. Anaerobic bacteria B. welchii, putrefactive anaerobes. 

E. Special types causing peculiar fermentations, such as 
slimy consistency, bitter taste and peculiar odors. 

F. Pathogenic organisms typhoid, tuberculosis, scarlatina, 
diphtheria, diarrhea, septic sore throat, foot-and-mouth disease, 
dysentery. 

G. Other fungi Molds, Oidia, Yeasts, Actinomyces. 



THE DISTRIBUTION OF MICRO-ORGANISMS IQI 

The development of these various microbes in the milk 
depends very much upon the temperature at which it is kept. 
At o to 10 C. the acid-forming bacteria grow very slowly or 
not at all, and the milk may remain practically unchanged for 
many days or even weeks. Eventually some of- the liquefying 
bacilli or the slime-producing types may gain the upper hand 
and change the consistency and flavor. Between 10 and 21 
the Bad. acidi lactici is almost certain to gain the dominance 
and rapidly to suppress the other types, and it produces the 
normal souring of milk. Between 21 and 35 C. the organisms 
of the B. coli and B. lactis aero genes groups are likely to pre- 
dominate and at temperatures from 37 C. to 40 C. the B. bul- 
garicus is likely to gain the ascendency, after a few days at 
any rate. These may be regarded as the normal fermentations 
of unheated milk of very good quality. The other microbes in 
the milk are not destroyed by these fermentations but their 
development is usually held in check somewhat. 

Shortly after the coagulation of the milk, which occurs when 
the lactic acid reaches a concentration of about 0.45 per cent, 
the living bacteria begin to diminish in number, and gradually 
Oidium lactis and other molds become prominent, although acid- 
resisting forms such as B. bulgaricus still continue to grow. 
Organisms of these kinds seem to be specially concerned in 
the ripening of acid curd in cheese making. Finally the acidity 
may disappear as a result of the activity of molds, and putre- 
factive bacteria find the opportunity to develop. 

If the milk be pasteurized, the bacteria which form lactic 
acid are killed, and when fermentation occurs it is likely to be 
different from the normal souring. At a high temperature, 
the stormy butyric-acid fermentation due to B. welchii may be 
observed. At a lower temperature, a slow putrefaction due to 
spore-forming putrefactive anaerobes in conjunction with other 
bacteria may occur. These fermentations are ordinarily inhib- 
ited by the lactic acid produced in the normal souring of milk. 

Alcoholic fermentation of milk occurs as a rule only when 



IQ2 GENERAL BIOLOGY OF MICRO-ORGANISMS 

special ferments are purposely added to produce this result. 
Kumyss and Kefir are fermented milks produced in this way. 
The starter or ferment contains yeasts as well as bacteria. 

The pathogenic micro-organisms in milk are derived in part 
from unhealthy cows tuberculosis, foot-and-mouth disease, 
septic sore throat (?) but in a larger measure from the people 
who handle the milk or from utensils tuberculosis, typhoid 
fever, scarlatina, diphtheria, diarrheas, dysentery, septic sore 
throat (?). The bacteria of typhoid fever, diphtheria and dysen- 
tery are known to multiply in milk. The microbes of tuberculo- 
sis and foot-and-mouth disease may persist in butter and cheese 
for several weeks at least. 

Leaving out of consideration the question of specific patho- 
genic micro-organisms, the presence of more than 500,000 bac- 
teria per cubic centimeter in the milk regularly fed to infants 
and young children is undoubtedly harmful, and especially so 
in warm weather. Doubtless many factors contribute to the 
causation of the summer diarrheas and the summer mortality 
of children, but there can no longer be any question that a milk 
rich in living bacteria as food for these children is one of the very 
important causes of their illness and death. 

Milk for infant feeding should come from clean, healthy 
(tuberculin-tested) cows, should be handled by clean healthy 
workmen, in clean stables and rooms and with clean, sterilized 
utensils. It should be bottled at the producing dairy, promptly 
chilled to 10 C. or below, and maintained at this temperature 
until delivered at the home. At this time the living bacteria 
should not exceed 30,000 per cubic centimeter. In the home, 
the milk should be kept cold. It must be handled only with 
utensils sterilized by boiling in water. Boiled water is employed 
in making the necessary dilutions. If the milk supply is not 
above suspicion the milk should be pasteurized by heating to 
60 C. for 20 minutes. The dilution is prepared and filled into 
separate bottles sufficient in number so that one may be used 
for each feeding during the succeeding 24 hours. Each bottle 



THE DISTRIBUTION OF MICRO-ORGANISMS 193 

is chilled in cool water, then ice water, and finally stored in the 
refrigerator. Immediately before feeding it is warmed by partial 
immersion in warm water. 

Other Foods. Other foods, meats, fish, eggs, vegetables 
and fruits, undergo decompositions due to more or less definite 
types of micro-organisms, and the activities of these are delayed 
or prevented by modern methods of preserving foods, in some 
instances very successfully, and in other cases with limited success. 1 
Any food, and especially that eaten without cooking, may serve 
as a passive carrier of pathogenic micro-organisms. Salads, 
green vegetables and fresh fruits may undoubtedly act in this 
way during epidemics. Oysters taken from sewage-polluted 
beds have been found to convey typhoid fever. Meats derived 
from mammals may contain specific germs causing disease in 
both animals and man, such as tuberculosis, anthrax and foot-and- 
mouth disease. The flesh of bovine animals suffering with 
enteritis at the time of slaughter seems to be particularly liable 
to develop poisonous properties, and the ill effects observed in 
these instances may have been due to a specific infection. Para- 
typhoid fever is sometimes traced to such meat as a cause. 

Meats and fish are rich in protein and their decomposition 
by saprophytic bacteria may give rise to various poisonous sub- 
stances, as has been mentioned on page 170. The usual course 
of putrefaction, however, goes on without very strong poisons 
being produced, as we may judge from the habitual use of partly 
decomposed foods of this sort. Virulent poisons are occasion- 
ally encountered and some of these are due to the presence of 
specific microbes, B. botulinus of Van Ermengen, B. enteritidis of 
Gaertner and the paratyphoid and paracolon bacilli. 2 

1 For a discussion of the microbiology of foods and of food preservation see 
MarchalFs Microbiology for agricultural and domestic science students, 1911. 

2 Consult Bolduan, C. F.: Bacterial Food Poisoning, N. Y. 1909. Also Novy, 
F. G.: Food Poisons, Osier's Modern Medicine, Vol. I, Phila., 1907. 



CHAPTER X. 
PARASITISM AND PATHOGENESIS. 

The Parasitic Relation. The presence in a living organism 
of one or several organisms of another species, which live as para- 
sites upon the first, is a phenomenon of common occurrence in na- 
ture. Those organisms such as the bacteria, which are too small 
to harbor visible internal parasites, are subject to the parasitic 
ravages of larger beings such as amebae and other protozoa, 
which engulf them bodily and digest them. Man, who is wont 
to complain of his parasitic ailments, takes all his protein, fat 
and carbohydrate from the bodies of plants and other animals. 
Parasitism in the larger sense is a well-nigh universal character- 
istic of living beings. Parasitism in a narrower sense usually 
applies to the existence of a smaller organism, the parasite, in 
or on the body of a larger, the host, a relation in which the host 
furnishes the parasite its necessary food. In many instances the 
advantages of the relation are wholly one-sided, but in others 
the two organisms seem to be of mutual benefit. In the latter 
case, the condition is called symbiosis. The infection of the 
roots of the clover with Pseudomonas radicicola, which promotes 
the nitrogenous nutrition of the plant, is an example of this rela- 
tion. In other instances the two organisms living in close associa- 
tion seem neither to help nor injure each other. They are then 
called commensals or companions at the same table. Internal 
parasites occur in all the higher animals and plants, and have 
been found even in the bodies of protozoa. Representatives of 
all the great classes of micro-organisms are found among the 
internal parasites, and many more highly organized animals 
and plants also lead parasitic lives. Man, alone, is subject to 

194 



PARASITISM AND PATHOGENESIS 195 

infestation with parasitic insects and numerous worms, in addi- 
tion to an enormous variety of microbes. Whether a parasitk 
organism is to be regarded as a symbiont, a commensal or a 
pathogenic agent depends upon the effect which it produces 
upon its host. A pathogenic organism is one whose presence 
results in definite injury to the host. 

Pathogenesis. In human pathology the phenomena of dis- 
ease have for centuries been the object of careful study and 
speculation, and in many instances the phenomena commonly 
associated together have long been regarded as a complex result 
of a single primary cause, and the condition in which such phe- 
nomena are observed has been regarded as a single morbid en- 
tity or a definite disease. Even the most ancient records indicate 
that such recognition had long been common knowledge. A 
beginner in parasitology or pathology may be inclined to ascribe 
a causal relation to a parasite which he observes in the body of a 
sick individual; in fact this has been done repeatedly. The log- 
ical requirements for the proof of such a relationship were first 
formulated by Henle, as has been mentioned in the historical 
sketch in the introductory chapter. They were reformulated 
by Koch, who, for the first time, was able to comply with them 
in respect to a bacterial disease. They may be stated as follows: 

1. The organisms must be present in all cases of the particular 
disease. 

2. The organism must be isolated from the diseased body 
and propagated in pure culture. 

3. The pure culture of the organism when introduced into 
susceptible animals must produce the disease. 

4. In the disease thus produced, the organism must be found 
distributed as in the natural disease. 

Although we may very properly consider a micro-organism as 
the probable cause of a disease with which it is associated, with- 
out satisfying all of the above requirements, the experience of 
the last three decades has served to emphasize more and more 



196 GENERAL BIOLOGY OF MICRO-ORGANISMS 

the wisdom of reserving final judgment wherever these rules or 
similar stern logical requirements have not been satisfied. 

Infectious Disease. An infectious disease is a disease due 
to the entrance of a living micro-organism and its growth in the 
body. Although conservative bacteriologists are sometimes 
loth to accept a disease as infectious until Koch's rules have 
been satisfied, most are agreed that a disease, which can be 
reproduced indefinitely by the inoculation of healthy individuals 
in series with material taken from a preceding case, is due to a 
living cause. The proof that a disease is due to a living cause 
may therefore precede the identification of the causal organism, 
often by many years. 

Possibility of Infection. Whether a parasitic organism will 
be able to enter and multiply in a new host and cause disease 
depends upon a number of circumstances, the most important 
of which may be considered under four heads, namely, the 
quality of the microbe, the resistance of the host, the quantity 
of invading parasites, and the path of entrance. The course 
and ultimate result of an infection depend also to a marked 
degree upon these same factors. In general the qualifications 
of the micro-organism depend first upon the experience of its 
ancestry under the same or similar enviromental conditions, 
factors inherent in its species, and second, upon its very recent 
history, factors affecting the virulence and general vigor of the 
individual microbe. Thus the tubercle bacillus is qualified by 
inheritance for a parasitic existence, while the common yeast cell 
is not. Yet, the tubercle bacillus, when cultivated for a long time 
on artificial media may lose its former ability to grow in the 
animal body. The factors affecting the pathogenic properties of 
a microbe will be considered in the succeeding chapter. 

Susceptibility and Resistance. Among the important things 
in the nature and condition of the host, we need also to consider 
both racial and individual characters. Certain species of animals 
have harbored certain parasites for so long that the latter have 
become adapted to growth in the particular species of host. In 



PARASITISM AND PATHOGENESIS 1 97 

some instances the adaptation is very narrow and the parasite 
may be able to exist naturally only in the one host species, as for 
example Spirochaeta pallida. Individual resistance of different 
hosts of the same species is variable. Age is one important 
factor: there are the children's diseases, measles, chicken- 
pox; the disease of active adult life, pulmonary tuberculosis, 
typhoid fever; and the diseases of the aged, pneumonia, carci- 
noma. Hunger and thirst have been shown experimentally to 
reduce the resistance to infection: pigeons, which are normally 
immune to anthrax become susceptible when starved. The 
effect of fatigue is well known: a white rat, normally immune 
to anthrax, succumbs to it after prolonged work in the treadmill. 
Abnormal chilling of hens removes their immunity to anthrax 
and abnormal heating of frogs affects them in a similar way. 
Chemical poisoning also increases susceptibility to infection, and 
cachexia and malnutrition are well-known predisposing factors 
to such infections as tuberculosis. Traumatism is very impor- 
tant, not only for its general effect upon the resistance of the host, 
but especially in the reduction of local resistance by destruction 
or injury of tissue (wounds). There are certain locations where 
resistance to infection is naturally lower, such as the ends of 
growing bones and the interior of the parturient uterus. 

Number of Invaders. The quantity of infectious material 
introduced is of importance in determining whether infection 
will or will not occur. Very few species of microbes are capable 
of causing disease when only a single individual organism is in- 
troduced into the body. A large number of microbes entering 
at the same time seems to overburden the defensive powers of 
the body so that some of the parasites succeed in establishing 
themselves and multiplying. 

Modes of Introduction. There are various avenues by which 
micro-organisms may enter the body to produce disease. In- 
fection of the ovum in the ovary with spirochetes and protozoa 
is known to occur in some insects, and Rettger has shown that 
this phenomenon occurs in the hen infected with Bacterium pul- 



198 GENERAL BIOLOGY OF MICRO-ORGANISMS 

lorum. The human ovum also seem occasionally to be infected 
with Spirochata pallida in this way. It may also become in- 
fected with the same organism derived from the seminal fluid. 
The developing fetus is sometimes invaded by pathogenic micro- 
organisms introduced through the placental circulation. The 
organisms of tuberculosis, small-pox, typhoid fever and the 
pyogenic cocci are known to be transmitted, somewhat uncom- 
monly to be sure, in this way. As a rule the germ must be circu- 
lating in the blood of the mother in considerable numbers, or 
there must be actual infectious lesions of the placenta before 
placental transmission occurs. After birth non-pathogenic mi- 
crobes gain access to the entire surface of the body and penetrate 
the various canals opening to the exterior to certain normal 
limits. Pathogenic germs may be introduced with the food and 
drink, which is the common natural mode of infection with cholera 
and typhoid fever in man and with tuberculosis in hogs and cattle. 
The barrier presented by the activity of the gastric juice is fre- 
quently passed in safety by the ingested microbes. Inhalation 
is probably the most common way in which tuberculous infection 1 
reaches the lungs in man, although there is conclusive evidence 
that tuberculosis in this location may be derived from the alimen- 
tary tract through the blood stream. Experimentally, guinea- 
pigs are much more susceptible to infection with tubercle bacilli 
by inhalation than by ingestion. Mere application of the in- 
fectious agents to the epithelial surface of the skin or mucous 
membranes results in infection in many instances and, indeed, 
infection by ingestion and inhalation may be regarded as examples 
of this. The mucous membranes of the urethra and the eye, and 
also of the rectum in young children, are especially susceptible to 
infection with the gonococcus. The unbroken skin may be infected 
with staphylococci, which seem to penetrate through the hair fol- 
licles and sebaceous glands, giving rise to boils and carbuncles; but 
to most microbes the uninjured skin presents an effective barrier. 

1 McFadyean, Journal Royal Institute of Public Health, 1910, Vol. XVIII, pp. 
703-724. 



PARASITISM AND PATHOGENESIS 1 99 

The question whether infectious agents may penetrate epithe- 
lium and gain the lymph or blood-vessels beyond without causing 
a local lesion, has received considerable attention and it seems 
to be established as certainly possible in the intestine during 
the absorption of fat, and it may perhaps occur in other locations. 

Infection through wounds, even minute breaks in the epithe- 
lial covering, is very common. Such wounds made by insects 
are the common portals of entry for the germs of malaria, plague, 
yellow fever, relapsing fever and many more diseases. Larger 
wounds nearly always become infected with pyogenic cocci 
unless they are properly cared for. The introduction of infectious 
material into the subcutaneous tissue may occur accidentally in 
deep wounds and is a common mode of inoculation in the labora- 
tory. Infection with the anaerobic bacillus of tetanus frequently 
occurs in this type of wound. 

Infections of the peritoneal cavity, pleural cavities and cavi- 
ties of the joints result from penetrating wounds, by the entrance 
of bacteria from contiguous tissues, as through the intestinal 
wall into the peritoneal cavity, and through the blood and lymph 
channels. 

Local Susceptibility. The invading parasite is favored by 
conditions of local susceptibility such as tissue destruction, 
presence of necrotic tissue and foreign bodies, and also by the 
presence of other infectious microbes. Small-pox and staphylo- 
coccus, tetanus and the pus cocci, scarlet fever and streptococcus, 
are common examples of such mixed infections. In some in- 
stances one infection predisposes to another. For example, 
measles is likely to favor the development of tuberculosis; the 
caseous tubercle is likely to be invaded by the streptococcus. 
These subsequent invasions are spoken of as secondary infections. 

Local and General Infections. The invading microbes may 
remain localized near the point of entrance, as for example in 
tetanus and diphtheria. In such cases the general effects may be 
due to disturbance in function of the local tissue, such as laryngeal 
obstruction in diphtheria, or to the absorption into the lymph 



200 GENERAL BIOLOGY OF MICRO-ORGANISMS 

and blood of poisons produced at the infected site. Such ab- 
sorption results in toxemia with symptoms due to poisoning of 
distant tissue elements. On the other hand, the infectious 
agent may pass quickly to the blood stream without appreciable 
local reaction and multiply there, as in malaria, trypanosomiasis 
and streptococcus bacteremia. Again there may first develop 
an intense local reaction, with subsequent extension to the 
blood stream with fatal issue, as in malignant pustule (anthrax). 
In other instances repeated temporary invasions of the blood 
occur, with numerous localized abscesses in various parts of 
the body, a condition to which the name pyemia has been applied. 
Of particular interest are those general infections of the blood 
stream, which finally fade away, but leave behind localized 
infections in the joints, on the heart valves, in the central nervous 
system, or other parts of the body. Sleeping sickness, syphilis, 
acute articular rheumatism and generalized gonococcus infection 
belong in this category. 

Transmission of Infection. The manner in which an infectious 
agent passes from its host to a new victim varies considerably. 
In general it may be said to occur (i) by direct contact or close 
association, transmission by contagion, (2) through the agency of 
intermediate dead objects as passive carriers, transmission by 
fomites, or (3) through the agency of a living or dead object in 
which the parasite undergoes development or multiplication, 
transmission by miasm. These terms have been employed in 
the past to designate rather hypothetical objects not to say 
abstract ideas, and their application to the facts learned by 
modern research is, perhaps, not desirable. Nevertheless, they 
may be made to fit the observed phenomena in a way. Thus, 
syphilis and gonorrhea are transmitted by contagion; diphtheria 
and small-pox by contagion and by fomites; tetanus and anthrax 
by fomites and perhaps also miasm; plague by contagion, fomites 
and miasm (through the rat and flea); malaria, trypanosomiasis 
and yellow fever by miasm. All of these are doubtless infectious 
diseases but some of them are not naturally spread by contact at 



PARASITISM AND PATHOGENESIS 2OI 

all. In studying each disease it will be necessary to consider 
the avenues by which the parasite leaves the patient, its existence 
in the external world and the means of gaining access to its new 
victim. 

Healthy Carriers of Infection. A person or animal may 
harbor virulent infectious agents without showing symptoms of 
disease, and may serve as a source of infection to others. This 
was clearly recognized in the sixteenth century by Fracastorius 
as a factor in the spread of syphilis. Only recently has its 
importance in other diseases been emphasized. 



CHAPTER XI. 
THE PATHOGENIC PROPERTY OF MICRO-ORGANISMS. 

Adaptation to Parasitism. In order to live as a parasite, an 
organism must be adapted to grow under the conditions met 
with in the body of the host, but in order to produce disease it 
must also injure the host. The most perfect adaptation of 
parasitism is probably exhibited by those micro-organisms 
which do not injure the host, the symbionts and commensals, 
as it is obviously to the interest of the parasite to keep its host 
alive. An adaptation of this kind usually requires that the 
microbe shall either grow very slowly, or shall be so situated 
that the excessive numbers resulting from its multiplication 
may readily pass out of the host or be disposed of in someway; 
otherwise the host would be physically crowded out. This sort 
of adaptation is illustrated by the normal intestinal bacteria. 
Parasites which invade the tissues of the body rarely show such 
adaptation. It is, perhaps, approached to some extent by 
the slow-growing bacilli of leprosy and tuberculosis. In most 
instances of parasitism, however, there is more or less of a struggle 
between the invader and the host for the possession of the field, 
and the phenomena of disease are incident to this combat. 

Virulence. The ability of the parasite to injure its host, is 
designated as virulence. Virulence depends in part upon growth 
vigor, but also upon other factors largely unknown. A great 
deal is known about specific methods of changing the virulence 
of micro-organisms, and various procedures are commonly em- 
ployed with this object in view. A diminution in virulence is called 
attenuation and an increase in virulence, exaltation. Attenua- 
tion was first observed by Pasteur in a culture of Bacterium 
amsepticum (chicken cholera) grown in broth in the presence of 

202 



PATHOGENIC PROPERTY OF MICRO-ORGANISMS 203 

air. Pneumococci and streptococci also attenuate rapidly in 
artificial culture. Even those bacteria which retain then- 
virulence in ordinary cultures become attenuated when grown at 
unusually high temperatures (42 C.) or in the presence of 
antiseptics, both of which methods have been employed in 
attenuating the anthrax bacillus. Attenuation also results 
sometimes from parasitism in hosts of another species. Variola 
and vaccinia present a conspicuous example of this. Mere 
dessication of a virus seems to attenuate it in some instances 
(rabies) but this is somewhat doubtful. Many pathogenic 
agents become somewhat attenuated upon long residence in the 
same host in chronic infections. Exaltation of a virus, on the 
other hand, is accomplished by rapid passage through susceptible 
animals in series. When the organism is too attenuated to 
produce an infection alone, it may be aided by the admixture of 
other organisms (mixed infection) or by the presence of irritating 
foreign bodies (splinters, stone dust ) or by mechanical protection 
in collodion capsules. 

Microbic Poisons. The weapons which the pathogenic 
agent employs to injure its host are various. The physical 
mass of the invaders may be injurious, more particularly by 
obstructing blood-vessels, as in estivo-autumnal malaria in man 
and anthrax in the mouse. Usually, however, the offensive 
weapons are chiefly chemical poisons. The soluble toxins, or 
true toxins are substances of unknown chemical composition 
produced inside bacterial cells and passed out to their surround- 
ings. These so-called extracellular toxins include the most 
poisonous substances known. Brieger and Cohn obtained a 
toxin, still impure, from tetanus bacilli, of which five one hundred 
million ths of a grams (.00000005 gram) killed a mouse weighing 
15 grams. At this rate .00023 of a gram would kill a man weigh- 
ing 70 Kilos. 1 The soluble toxins elaborated by the diphtheria 
and tetanus bacilli have been studied most, and many of our 
ideas concerning toxins in general have been derived from these 

1 Vaughan and Novy, Cellular Toxins, Phila., 1902, p. 62. 



2O4 GENERAL BIOLOGY OF MICRO-ORGANISMS 

studies. These poisons are rapidly destroyed by heat, resembling 
enzymes in this respect. They differ from enzymes in that 
they are used up in combining with tissue. Thus tetanus toxin 
may be completely neutralized by the addition of brain tissue, 
and either diphtheria or tetanus antitoxin may be quantitatively 
neutralized by its specific antitoxin. Ehrlich in his study of the 
reactions of diphtheria toxin showed that on standing it loses 
much of its poisonous property without any diminution in its 
ability to combine with diphtheria antitoxin, and to this less 
poisonous substance he gave the name toxoid. From this observa- 
tion he concluded that the toxin molecule contains at least two 
very definite atomic groups. One of these is comparatively 
stable and serves for attachment of the toxin molecule to the 
cell attacked by it, and is called the haptophorous group or 
simply haptophore. The other recognizable chemical group 
disintegrates more readily and is that which bears the poisonous 
property. To this he gave the name of toxophorous group or 
toxophore. In their reactions toxins behave in part like feebly 
dissociated chemical compounds, as has been shown by Arrhenius 
and Madsen, but the reactions by which they combine are only 
slightly or not at all reversible and, moreover, take place in 
variable proportions. Bordet very aptly compares the reactions 
of toxin to the union of a dye with a stainable material. Bacteria 
also produce poisons which are part of their own body substance, 
and set free only upon their death and disintegration. These 
are spoken of as intracellular toxins. Injurious substances may 
also be produced from the tissue of the host by a secondary action 
outside the cell of the parasite, but these secondary products 
play a very minor role. 

Defensive Mechanisms. The defensive armor of parasites 
seems also to be in part physical and in part chemical, and perhaps 
we may regard the physiological adaptation to slow growth as a 
defensive mechanism because it tends to avoid exciting the 
opposition of the host. The physical structure seems to be 
protective in case of the waxy bacteria (tubercle and leprosy) 



PATHOGENIC PROPERTY OF MICRO-ORGANISMS 205 

and the capsules of other bacteria may serve a similar purpose 
(pneumococcus). There is some indication that micro-organisms 
may produce special chemical substances to neutralize the 
agencies which the host employs against them. These defensive 
substances have been designated by Bail as aggressins. Ehrlich 
has also found evidence of the acquirement of immunity to 
chemical substances by certain pathogenic microbes, especially 
trypanosomes and spirochetes, and he ascribes this property of 
the parasites to an alteration of their cell-chemistry. 



CHAPTER XII. 
REACTION OF THE HOST TO INFECTION. 

Facts and Theories. The host reacts to the presence of a 
pathogenic agent by a number of alterations in its physiological 
activities. Some of these alterations are gross and well known 
as the clinical manifestations of an infectious disease; others 
require special search for their detection; while some, doubtless 
a considerable number, still pass unobserved. Many of these 
changes are susceptible of very accurate observation, and in 
most instances the observed facts are well established. A clear 
understanding of the relation of the various facts to each other 
involves some imaginative reasoning, and various hypotheses 
have been advanced to explain the phenomena observed, and to 
fill in the gaps in our knowledge. The student may need to be 
on his guard not to confuse facts susceptible of observation with 
hypothetical deductions based upon such observations. Both 
have their peculiar value. An understanding of the phenomena 
of pathological physiology must be based upon correct ideas of 
normal physiology and accurate knowledge has not fully replaced 
hypothesis in this latter field. 

Physiological Hyperplasia. Under normal conditions each 
cell of the human body is in close association with other cells 
and with the body fluids, and is subject to the physical and 
chemical stimulation of cells and fluids. One of the effects is 
apparently to restrain the prolif erative activity of the cells. When 
certain of these cells are destroyed, or even certain parts of them, 
this restraint is removed, and the natural tendency to prolifera- 
tion asserts itself, resulting in the production of new cells or of 
new parts to replace the old, and usually more than compensates 
for the loss. This somewhat hypothetical conception, due to 

206 



REACTION OF THE HOST TO INFECTION 207 

Carl Weigert, serves to explain tissue hyperplasia and repair 
following exercise or local destruction of tissue. Examples of 
these phemomena will occur to the reader. 

Phagocytosis and Encapsulation. The mere physical mass of 
a parasite within the tissue acts as a foreign body and it becomes 
surrounded by tissue elements. If it is minute, certain cells of 
the body (phagocytes) flow around and ingest it, as was first 
observed by Metchnikoff. If it is larger, the connective tissue 
cells proliferate and surround it, and eventually contract into a 
firm capsule. Further, the tissues produce enzymes capable 
of dissolving many foreign substances introduced in this way 
(parenteral digestion). If the foreign body is insoluble, it will- 
remain encapsulated, or, if sufficiently minute, it may be trans- 
ported considerable distances inside wandering cells and eventu- 
ally be deposited in a lymph gland. The wholly passive para- 
site or the dead body of a micro-organism is therefore either 
digested and dissolved, ingested by cells, or encapsulated in 
fibrous tissue. Most infectious agents are not passive in this 
way, as we have seen, but tend actively to grow and multiply, 
to absorb and utilize food material, and, most important of all, 
to produce various substances which stimulate or poison the 
cells of the host. Against these the physical measures of inges- 
tion (phagocytosis) and encapsulation are often inadequate de- 
fenses and may be entirely useless. 

Chemical Constitution of the Cell. Ehrlich has compared 
the living body cell to a complex chemical molecule; in fact it 
may be said that he regards the living cell as an enormous mole- 
cule, a chemical unit of great complexity. Certain atom groups 
within this molecule are pictured as relatively very stable and 
they constitue the chemical nucleus (not to be confused with 
the anatomic nucleus). Grouped about this chemically stable 
center are very many, more labile atom groups which readily 
enter into chemical reaction with substances in the surrounding 
medium. The conception is derived directly from well-known 
facts in organic chemistry. For example when benzoic acid, 



2O8 GENERAL BIOLOGY OF MICRO-ORGANISMS 

CeHVCOOH, reacts with other chemicals the reaction takes 
place at the reactive group, or side-chain, rather than in the 
nucleus. The graphic formula may illustrate this point better. 

H 

I 
H C O 

\ /\ II 
C C C OH 

I 
H C C 

\/ \ 
C H 

H 

The six carbon atoms in the ring are stable, and a strong chem- 
ical reagent, such as phosphorus pentachloride, reacts with the 
side-chain without attacking the ring. So in the living cell, 
Ehrich assumes, as a working hypothesis, the existence of a 
wonderfully complex but comparatively stable chemical nucleus, 
with abundant and various more reactive side-chains. These 
latter serve to combine with food materials in the surrounding 
lymph, and these are then utilized in the cell by an intramo- 
lecular rearrangement of atoms which is always in progress. Use- 
less atomic groups formed in the metabolism of the cell are de- 
tached and passed off as excretions. These reactions of intra- 
molecular rearrangement and molecular disintegration also 
find their analogues in carbocyclic chemistry. 

Antitoxins. Von Behring and Kitasato (1890-91) showed 
that animals injected with small non-fatal doses of toxin of the 
tetanus bacillus, produce as a result of this treatment a some- 
thing which circulates in solution in the blood plasma, which 
is capable of neutralizing the poisonous properties of the tetanus 
toxin. Soon afterward von Behring obtained analogous results 
with the toxin of diphtheria. The protective substances in the 
blood were called antitoxins. The exact chemical composition 



REACTION OF THE HOST TO INFECTION 



209 




of these substances is unknown. They accompany the pseudo- 

globulin fraction of the plasma in its chemical analysis, 1 but 

the union here is probably a mere physical adsorption or very 

unstable chemical combination. Ehrlich explains the formation 

of antitoxin on the basis of his side-chain theory as follows. 

The molecule of toxin attacks 

the body cell at one of its 

side-chains or receptors which 

is best adapted to this reac- 

tion. In the resulting intra- 

molecular rearrangement the 

toxin reveals itself as a dis- 

turbing element, causing de- 

struction of that portion of 

the cell to which it has be- 

come attached. In recover- 

ing from this disturbance the 

cell overcompensates by 

forming an excessive number 

Of the particular kind of side- 

r 

chain destroyed, and some can Medical Association, 1905, p. 955.) a, 

of the excess side-chains are 

detached, and circulate in the 

. 

blood, ready to react with 
toxin entirely apart from the cell which has produced them. 
These constitute Ehrlich's receptors of the first order and their 
sole effect upon the toxin is that of combining with it. The free 
receptors circulating in the blood give it its antitoxic property. 

Precipitins. Other chemical products of bacterial growth 
are attacked and rendered insoluble by products of the body 
cells. Kraus 2 (1897) showed that animals injected with cultures 
of bacteria produce a substance or substances, which circulates 
in the blood and is capable of causing a precipitate when mixed 

1 Banzhaf, Johns Hopkins Hospital Bull., 1911, Vol. XXII, pp. 106-109. 

2 Wiener klin. Wochensckr., 1897, X, p. 736. 

14 



. . IG - 85.-Receptor of the first order 
uniting with toxin. (Journal of the Amen- 



the toxm molecule; e, haptophore of the cell 
receptor. 



210 



GENERAL BIOLOGY OF MICRO-ORGANISMS 



with the clear filtrate of the cultures of the same bacteria. The 
parenteral introduction of any foreign protein in solution stimu- 
lates' the production of a substance which will precipitate it. 1 
These substances, which are called precipitins, resemble enzymes 
in many respects. Thus, the precipitin produced by the injec- 

tion of a milk, causes a 
change in the milk very 
similar to that caused by 
rennet. Rennet, however, 
coagulates milk from vari- 
ous animals while the milk 
precipitin is specific, within 
certain limits, for the one 
kind of milk. Precipita- 
tion results only when the 
blood serum (precipitin) is 
combined with the proper 
amount of the culture fil- 
trate or other protein *so- 

FIG. 86. Receptors of the second order and lution (pretipitinogen) 

some substance uniting with one of them. (Jour- w u pn tnn l pro -p an PYPP^ of 

nal of the American Medical Association, 1905, p. wnen to lar g e an 

1113.) c, Cell receptor of the second order; d, one or the other is used no 
toxophore or zymophore group of the receptor; 
e, haptophore of the receptor; /, food substance 

or - r ^ du f / Bacterial disintegration uniting 

the haptophore of the cell receptor. 




-, 

wit 



. . 
precipitate occurs. 

H h explains the formation 
of precipitins on the basis 

of his side-chain theory in the same way as the production of 
antitoxins was explained. The foreign protein stimulates the 
body cells to produce specific receptors capable of combining 
with it. In this instance, however, the receptor not only com- 
bines with the foreign material, but also brings about a definite 
change in it which is evidenced by the phenomenon of precipita- 
tion. The side-chain therefore contains at least two distinct 
atomic groups, one of which serves to combine with the pre- 

1 Specific precipitin tests have been employed to some extent in determining 
the source of blood stains and of meats. See Citron, Immunity, translated by 
Garbat, Phila., 1912, p. 112. 



REACTION OF THE HOST TO INFECTION 211 

cipitinogen, and is specific in nature, and another which brings 
about the change evidenced by formation of the precipitate^ 
The former of these chemical groups is called the combining or 
haptophorous group or haptophore, and the latter is called the 
ferment-bearing or zymophorous group or zymophore. This 
type of side-chain is Ehrlich's receptor of the second order. It 
is represented in the figure as possessing one smooth branch 
which serves for simple attachment, the haptophore, and one 
branch equipped with saw-teeth to suggest its property of pro- 
ducing chemical change, the zymophore. The precipitin pres- 
ent in the blood plasma is supposed to consist of such receptors 
which have become detached from the cell producing them. 

Agglutinins. Gruber and Durham (1896) found that the 
blood of animals suffering from certain infections has the power 
of causing the bacteria involved to clump together and lose their 
motility when it is added to a broth culture or a suspension of the 
bacteria in salt solution. The phenomenon has been observed 
in connection with many bacteria, not only motile but also non- 
motile species, but the most important examples are the typhoid, 
paratyphoid, cholera and dysentery organisms. In typhoid and 
paratyphoid fever the agglutination test is used as an aid in diag- 
nosis of the disease by testing patient's serum against known 
cultures, and the test with known serum is important in the iden- 
tification of cultures of any of these bacteria. Agglutinins are 
comparatively stable substances although they decompose 
rapidly at 70 to 75 C. When dried they keep for a long time. 
In Ehrlich's theory, the agglutinins are classed as receptors of 
the second order, along with the precipitins. 

The Phenomenon of Agglutination. Clear fluid blood serum 
to be tested for specific agglutinins is diluted with broth or with 
salt solution to make mixtures containing one part of the serum 
in 5, 10, 20, 40, 80 and 160 parts of the mixture. This is con- 
veniently done by means of the Wright capillary pipette, or 
graduated pipettes may be employed. To each dilution of serum 
an equal amount of a very young (preferably two to six hours 



212 GENERAL BIOLOGY OF MICRO-ORGANISMS 

old) broth culture, or a suspension of an active young agar cul- 
ture in broth or salt solution, is added. The reaction may be 
observed by mixing small quantities (loopfuls) on a large cover- 
glass and studying the mixture microscopically as a hanging 
drop, or by mixing larger quantities in small tubes and incubating 
them at 37 C. Control specimens free from serum and contain- 
ing normal serum should be set up at the same time for compari- 
son, as many bacteria may be agglutinated somewhat by normal 
serum in a dilution of one to ten, and sometimes the organisms 
in the culture, especially if it is too old, may be already grouped 
together somewhat or may spontaneously clump during the ex- 
periment. Some practice is necessary before one can estimate 
agglutinins reliably and, on the whole, accuracy is more easily 
attained with the macroscopic test. For agglutination tests 
requiring only moderate accuracy, dried blood may be used, 
the dilutions being prepared by comparison of colors with an 
empirical standard. 

Bactericidal Substances, Alexin. Nuttall (1886) showed 
that normal blood is capable of killing bacteria and that this 
germicidal property is destroyed by heating the blood to 55 C. 
for thirty minutes. Buchner confirmed these observations and 
showed further that the germicidal property is resident in the 
serum and not exclusively in the cells of the blood as taught by 
MetchnikofL To this germicidal substance Buchner gave the 
name alexin, and he ascribed the normal resistance to infection 
exhibited by the healthy animal, as well as the heightened resist- 
ance of the immunized animal, to this substance. It will have 
been noted that, historically, these discoveries followed Metch- 
nikofPs first observations on the phagocytes, and preceded 
the discovery of antitoxins, agglutinins and precipitins, and 
thus presented the first proof of the existence of soluble anti- 
infectious agents. These bactericidal substances are now con- 
sidered to be identical with the bacteriolysins and will be 
considered with them under the more general heading of 
cytolysins. 



REACTION OF THE HOST TO INFECTION 213 

Cytolysins. Pfeiffer (1896) found that guinea-pigs, when 
injected repeatedly with non-fatal doses of cholera germs, reacted 
to this treatment by producing a something which would dissolve 
these bacteria. This new property was present in the blood and 
also in the peritoneal fluid. The substance was called bacterioly- 
sin. Subsequent investigators have shown that bacteriolysins 
can be produced for a great variety of micro-organisms, although 
in none can the reaction be better demonstrated than in the 
cholera vibrio originally employed by Pfeiffer. Lysins, or 
dissolving substances, have been produced for very many other 
kinds of cells also, of which those for red blood cells (hemolysins) 
are perhaps the most important. It seems to be possible to 
produce a lysin (cytolysin) for any kind of cells by injecting these 
cells into an appropriate animal. 

Cytolysins, including bacteriolysins, are active only when 
comparatively fresh. Upon standing for a day at room tem- 
perature, or upon heating to 56 C. for 30 minutes, the cytolytic 
power disappears. This power is, however, restored in a re- 
markable manner if the cytolysin and the cells to be dissolved are 
injected together into a normal animal, for example into the 
peritoneal cavity of a guinea-pig, or if a fresh normal blood serum 
be added to the mixture in the test-tube. The experiment results 
as follows: 

Immune serum + cholera germs = Bacteriolysis. 

Immune serum (old or heated) -f cholera germs = No bacteriolysis. 
Normal serum + cholera germs = No bacteriolysis. 

Immune serum (old or heated) -(- normal serum + cholera germs 

= Bacteriolysis. 

This experiment proves that the cytolytic property of the serum 
depends upon the presence of at least two recognizably different 
substances, one of which is present in fresh normal serum and 
in fresh immune serum but is destroyed on standing or by heating, 
and a second which is present in the immune serum and which 
is not destroyed so readily. 



214 



GENERAL BIOLOGY OF MICRO-ORGANISMS 



Ehrlich explains the formation of cytolysins by the same 
kind of reasoning as was applied to antitoxins and precipitins. 
The resulting side-chain would be considered of the same sort 
as in the latter class of substances, that is a receptor of the 
second order with a haptophorous group by which to combine 
with the foreign cell, and a zymophorous group to bring about 
its solution, were it not for the observed facts given in the experi- 
ment outlined above, which demonstrate the presence of two 
distinct substances in the cytolytic complex. A new picture is 
here necessary and it is furnished by making a joint in the arm 




FIG. 87. Receptors of the third order. (Journ. A. M. A , 1905, J. 1369.) c. 
Cell receptor of the third order an amboceptor; c, one of the haptophores of the 
amboceptor with which the foreign body, /, (antigen) may unite; g, the other 
haptophore of the amboceptor with which complement, k, may unite; /?, 
haptophore of the complement; z, zymophore of the complement. 

of the receptor of the second order in which the fermentative 
property is supposed to reside, separating off the zymophorous 
group as a separate substance and leaving a branched figure with 
two combining or haptophorous elements, one capable of com- 
bining with the foreign cell and the other capable of combining 
with the cytolytic ferment of normal serum and so bringing its 
action to bear upon that particular cell. The receptor of the 
third order is called, in accordance with this conception of its 
relationships, amboceptor, because it acts as a receptor at two 



REACTION OF THE HOST TO INFECTION 



215 



points. It is also called intermediary body, immune body and 
sensitizer. The other component of the lytic complex, which 
is thermolabile and which is present in normal serum, is called 
complement or cytase, and by some authors (Bordet) alexin. 1 
It will be noted that only a part of the cytolysin is produced 
by the body in its reaction to invasion, namely, the immune body. 
Deviation of Complement. Neisser and Wechsberg observed 
that the bactericidal power of a given immune serum (bacteriolytic 
amboceptor), when combined with a constant amount of normal 
serum (complement) and a constant amount of a bacterial sus- 
pension (antigen), increased progressively with progressive 
dilution of the immune serum to a certain point, after which it 
diminished again. The following data taken from Citron illus- 
trate the experiment: 



Typhoid culture (antigen) 


Immune serum 
(amboceptor) 


Fresh serum 
i : 12 
(complement) 


Colonies produced 
by plating after 
3 hrs. at 37 C. 


0.5 c.c. 1/5000 
0.5 c.c. 1/5000 
0.5 c.c. 1/5000 
0.5 c.c. 1/5000 
0.5 c.c. 1/5000 


I/IOO C.C. 

1/5000 c.c. 

I/2OOOO C.C. 

1/30000 c.c. 
1/50000 c.c. 


0.5 c.c. 
0.5 c.c. 
0.5 c.c. 
0.5 c.c. 
o. 5 c.c. 


Many thousand 
Many thousand 
200 
o 
60 


o. 5 c.c. 1/5000 


I/2OOOOO C.C. 


o. 5 c.c. 


Many thousand 











Neisser and Wechsberg have undertaken to explain this 
result by supposing that the excessive number of amboceptors 
present in the more concentrated solutions of immune serum 
hinders cytolysis because some of them combine with the antigen 
by means of their cytophile groups while others are combining 
with the complement by means of their complementophile 
groups, and as a result the mixture contains combinations of 
amboceptor with antigen, and of amboceptor with complement, 
but practically no combinations of the three elements together. 
There are grave reasons for questioning the accuracy of this 

1 This use of the term alexin would seem to be undesirable, for Buchner employed 
the term to designate the whole bactericidal or cytolytic complex before the possi- 
bility of recognizing two separate elements was clearly recognized. 



2l6 



GENERAL BIOLOGY OF MICRO-ORGANISMS 



assumption, as it has been shown by Bordet that amboceptor 
does not unite with complement in the absence of antigen. It 
seems more probable that some other factor, such perhaps as a 
marked agglutination of the bacteria in the stronger solutions, 
may serve to protect them from the bacteriolytic action. 

Fixation of Complement.- As has been mentioned, it is pos- 
sible to produce cytolysins for red blood cells. This is commonly 
done by injecting the washed blood corpuscles of a sheep (o.i c.c. 
+0.5 c.c. salt solution) into a rabbit intravenously three or four 
times at intervals of five days. The serum of the rabbit becomes 




FIG. 88.- 



Illustrating the conception of deviation of complement. 
b, antigen; k, complement. 



a, Amboceptor; 



strongly hemolytic for sheep's cells. The blood is drawn from 
the carotid artery, the serum separated, rendered perfectly 
clear and after heating to 56 C. for 30 minutes is stored in hermet- 
ically sealed ampoules containing i c.c. each, in a low tempera- 
ture refrigerator. When this hemolytic amboceptor is diluted to 
the proper point, which must be ascertained by trial and error, 
it will just cause the complete hemolysis of a definite quantity 
of washed sheep's corpuscles (0.2 c.c. of a 5 per cent suspension) 
when combined with o.i c.c. of a 10 per cent solution of fresh 
normal serum of a guinea-pig (complement). The mixture of 
this quantity of the immune serum, which may now be called 
one unit of hemolytic amboceptor, with 0.2 c.c. of freshly prepared 
5 per cent suspension of washed sheep's corpuscles produces a 



REACTION OF THE HOST TO INFECTION 217 

reagent which serves for the detection of complement and the 
approximate estimation of its amount in an unknown mixture. 
By the use of such a reagent it is possible to show that complement 
is destroyed or used up in various specific cytolytic, proteolytic. 
and precipitin reactions. Thus Bordet and Gengou mixed 
together typhoid bacilli (antigen), heated typhoid-immune 
serum (amboceptor) and fresh normal serum (complement) 
and incubated the mixture. After an hour the hemolytic ambo- 
ceptor and sheep's blood cells were added and incubation con- 
tinued. No hemolysis resulted, showing that the complement 
added in the first place had been used up, "fixed," as a result 
of a reaction with the typhoid bacilli and typhoid amboceptor. 
This is the phenomenon of fixation of complement. Obviously 
it lends itself to use as a test for the presence of a specific 
antigen or for the presence of specific amboceptor. Its more 
definite application will require subsequent mention. 

Opsonins. Wright and Douglas (1903) showed that blood 
serum contains a something which affects bacterial cells, soaked 
in the serum, in such a way that they are more readily ingested 
by the living leukocytes. To this substance they gave the 
name "opsonin" (opsono, I prepare victuals for). Substances 
of this sort are present in normal blood, but are increased as a 
reaction following infection. It would seem that more than 
one substance may act upon bacterial cells in this manner, for 
Neufeld has shown that the opsonic power of normal serum may 
be destroyed by heating to 56 C., while the similar property of 
immune serum remains after this treatment. It is not yet con- 
clusively proven that opsonins are separate substances entirety 
distinct from bacteriolysins and agglutinins, but it has been shown 
that opsonic power of a serum does not correspond to its con- 
centration to that of the other antibodies, and some other 
element must, therefore, be a factor. Hektoen considers the 
opsonins to be distinct bodies, different from lysins and agglutin- 
ins. The study of opsonins has done much to bring about 
harmony between the followers of Metchnikoff, with their tendency 



2l8 GENERAL BIOLOGY OF MICRO-ORGANISMS 

to emphasize the importance of phagocytosis, and the followers 
of Buchner and Ehrlich, who fixed their attention largely upon 
the substances dissolved in the body fluids. 

Anti-aggressins, Specific Proteolysins. Various substances 
produced in the body as a result of infection show particular 
ability to combat the effects of the soluble products of the para- 
site to which the name aggressins has been given (see page 205). 
Knowledge of these substances and their behavior is still some- 
what incomplete, but they seem to be particularly concerned 
with the parental digestion of foreign proteins, a process in which 
cystolysis may be regarded as a beginning stage. Whereas, 
however, cytolysis is concerned with the disintegration of formed 
material, these substances now under consideration act particularly 
upon proteins already in solution. In many instances the products 
of the first stages in this parental digestion are toxic (disintegra- 
tion of tuberculin and of egg-white), and some of the symptoms 
of infectious disease, such as fever, have been ascribed to them. 
In their general characters these lytic substances are wholly 
analogous to the cytolysins and their action is due to at least two 
components, an amboceptor and a complement. 

Source and Distribution of Antibodies. The exact source 
of the antibodies dissolved in the body fluids is unknown. All 
agree that they are derived from cells. Metchnikoff regards 
the phagocytic cells as the important source; Ehrlich does not 
specify, but it would seem, in accordance with his theory, that 
any cell capable of being affected by the foreign substance should 
be capable of throwing off cell receptors (antibodies) to combine 
with it. Many investigators consider antibody formation to be 
a common property of many kinds of cells, but more especially 
of relatively undifferentiated cells such as those of the connective 
tissue. 

Antibodies are present in greatest concentration in the blood 
and lymph. They are absent or present only in small amount 
in the serous fluids of the pleural, pericardial, peritoneal and 



REACTION OF THE HOST TO INFECTION 2IQ 

joint cavities, and in the cerebrospinal fluid. 1 Parasites in 
these locations are less readily influenced by antibodies circulating 
in the blood, so that localized infections may continue in these 
regions in spite of a considerable concentration of antibodies in 
the body generally. 

Allergy. Allergy is a term invented by Von Pirquet to 
designate the condition of altered reactivity on the part of the 
body which comes about as a result of infection. A few of the 
phenomena which may be included under this term have been 
considered above in this chapter. Many of these alterations in 
bodily function are manifestly of advantage to the host in limiting 
the activities of the parasite, neutralizing its poisonous products, 
and even in destroying and removing the parasite itself. Some of 
them, such as specific precipitation, seem to serve no important 
purpose, while others, such as cytolysis and proteolysis actually 
lead sometimes to results very harmful to the host, although 
their usual effect is favorable. Many of the recognized weapons 
which the body employs in its battle against parasites are still 
imperfectly understood, and there are doubtless many factors 
involved in this relation which are not yet capable of definite 
recognition. Of those agents mentioned above, the phagocytes 
are ready for immediate defense as soon as the body is invaded 
by the parasite. Hyperplasia and encapsulation require more 
time, probably one to four weeks. The chemical antibodies, 
antitoxins, agglutinins, cytolysins and opsonins, although possibly 
present in small amounts in the normal body fluids, become 
definitely increased in from eight to twelve days after the entrance 
of the parasite, an interval approximately equal to the incubation 
period of some infectious diseases. These various agents have 
much to do in determining the manifestations and course of the 
disease as well as the final outcome, and as we shall see, they also 
play a part in immunity. 

1 See Flexner, Harbin Lectures, Journ. of the State Medicine, March, April, May 
1912. 



CHAPTER XIII. 

IMMUNITY AND HYPERSUSCEPTIBILITY. THEORIES 
OF IMMUNITY. 

Immunity. Immunity is that condition of a living organism 
which enables it to escape without contracting a disease when 
fully exposed to conditions which normally give rise to that disease. 
Immunity may depend upon many different factors, or upon 
only one of a great variety. In general, we shall find that it 
depends very largely upon those factors which we have already 
considered in the preceding chapters, such as the possession of 
anatomical structures or habits of life which render invasion by 
the particular parasite impossible, or the possession of a body 
structure, physically or chemically not adapted for the growth 
of the particular disease virus, or the ability to harbor the particular 
parasite as a commensal without suffering injury, or the ability to 
react against the invading parasite and destroy it by phagocytosis 
or by cytolysis, neutralize its poisons by antitoxins, or limit its 
activity by encapsulation. Immunity is ordinarily considered 
under two heads, Natural Immunity, or that present as a part of 
the individual's birthright, and Acquired Immunity, that which 
follows as the result of some experience of the individual. 

Immunity of Species. Natural immunity to certain diseases 
is possessed by certain species of animals. Where the morphology 
and physiology is quite different from that of the usual victims 
of the disease, immunity might be expected. Thus cold-blooded 
vertebrates, fish, amphibians and reptiles, are immune to many 
diseases of mammals, apparently because of the different tem- 
perature of their tissues. In other instances the difference 
in resistance between two species of animals seems to be correlated 

220 



IMMUNITY AND HYPERSUSCEPTIBILITY 221 

with difference in food habits. Thus the carnivorous mammals 
are relatively insusceptible to anthrax and tuberculosis, diseases 
natural to the herbivora. Many infectious diseases of man 
are not readily transmissible to animals, for example, typhoid 
fever, syphilis, pneumonia, and in some instances it has so far 
been impossible to infect animals, as for example with scarlet 
fever and gonorrhea. 1 

Racial Immunity. Within a species there is moreover a 
racial difference in resistance to natural infection. Thus the 
pure-bred dairy cattle are more susceptible to tuberculosis than 
other cattle, and Yorkshire swine are relatively less susceptible 
to swine erysipelas. In man, the relation of race to susceptibility 
is not very clear. The examples of supposed racial immunity 
have not proved to be so definite as had been assumed at first. 
Thus the supposed immunity of African natives to syphilis has 
vanished with their increasing contact with civilization and 
with this accompanying disease. In the case of malaria the 
supposed racial immunity of negroes seems to be an acquired 
immunity due to severe attacks of the disease in childhood. 
There is, however, some evidence that prolonged contact with a 
disease through many generations may result in a relative resist- 
ance, so that the disease assumes a milder form in such a race of 
people a sort of inherited acquired immunity. Such considera- 
tions have been brought forward to explain the relatively high 
resistance to tuberculosis shown by the Hebrews as compared 
with the American Indians. 

Individual Variations. Individual variations in resistance 
to infection are commonly observed. They may depend in part 
upon age, condition of nutrition, fatigue, exposure or intoxica- 
tion, but they are ascribed also to differences in anatomical 
structure (shape of the thorax in tuberculosis). Individuals 
especially susceptible to a disea.se are said to possess an idiosyn- 
crasy for it. The physiological mechanisms upon which varia- 
tions in individual resistance depend are not clearly understood. 

1 Kolle und Wassermann, II Auflage, Bd. IV, p. 693 (1912). 



222 GENERAL BIOLOGY OF MICRO-ORGANISMS 

Doubtless, the number and activity of the white blood cells and 
the nature and amount of bactericidal substances in the blood 
play a part in some instances. 

Acquired Immunity. Acquired immunity results from some 
experience affecting the individual, either an infection which the 
individual has survived or some artificial procedure of immuniza- 
tion. There are recognized two different kinds of acquired 
immunity, first, active immunity which is due to the activity 
of the cells of the individual immunized, and second, passive 
immunity which is produced by introducing into the body material 
(blood serum) from another animal, which contains substances 
conferring at once an immunity upon the new individual. 

Active Immunity. Active immunity may be acquired by an 
attack of the disease. This immunity may endure for a lifetime 
in some instances (yellow fever, small-pox, scarlet fever) or for 
many years (typhoid fever) or it may be very evanescent 
(erysipelas, pneumonia, influenza). Some diseases were at one 
time so universal that few escaped them, and individuals used 
to be purposely exposed or inoculated in order to contract the 
disease and gain the resulting immunity. Inoculation of small- 
pox seems to have been practised in China about 1000 A. D. and 
in India as early as the twelfth century, and it was introduced into 
Europe in 1721 by Lady Montague and was employed very 
extensively in Europe and America during that century. 

Active immunity may also be produced without causing a 
definite attack of the disease. This may be accomplished in a 
variety of ways. Fully virulent micro-organisms may be intro- 
duced into a part of the body unfavorable to their development. 
The subcutaneous injection of cholera cultures according to the 
method of Ferran and Haffkine has proven to be practically 
without danger, and results in immunity. The same principle 
is ultilized in immunizing cattle against pleuro-pneumonia. 1 
Introduction of virulent organisms in very minute doses has been 
employed to immunize against rabies (Hogyes method), and against 

1 Kolle und Wassermann, II Auflage, Bd. I, S. 928 (1912). 



IMMUNITY AND HYPERSUSCEPTIBILITY 223 

tuberculosis by Webb. In most diseases these methods are re- 
garded as too dangerous for extensive use. 

Living virus, altered in its virulence, was first used by Edward 
Jenner, when he inoculated with cow-pox (vaccinia) and induced 
immunity to small-pox. Cow-pox is doubtless due to the organism 
which causes small-pox, attenuated by its life in the body of the cow. 
Viruses artificially cultivable are attenuated by a variety of pro- 
cedures, and are employed to induce immunity. Pasteur's vaccine 
for anthrax, for chicken cholera and possibly the treatment of 
rabies with dried spinal cord, are examples of the application of 
this principle. Virus of extraordinary virulence is sometimes in- 
jected after previous treatment with attenuated organisms, in 
order to confer a higher degree of immunity. Thus Pasteur 
employed the most virulent rabies virus obtainable, virus fixe, in 
the immunization against rabies. 

Living virus, of full virulence, but apparently influenced in some 
way by the body fluid containing it, is employed in immunizing 
against rinderpest and against Texas fever. The bile of an animal 
dying of rinderpest is injected subcutaneously in doses of 10 c.c. 
into cattle. Kolle has shown that the virus can be separated from 
such bile in fully virulent condition; so it appears that some con- 
stituents of the bile restrain the activity of the virus. In Texas 
fever, blood of young animals containing relatively few of the 
parisites is used to inject new animals. 

Immunization by injection of dead microbic substance is now 
extensively employed in the prophylaxis of cholera, typhoid fever 
and plague. As a result of such injections there is a marked in- 
crease in specific agglutinins and bacteriolysins in the blood. The 
principle of general immunization is also employed with some suc- 
cess in the treatment of subacute, chronic or recurrent local 
infections, the production of antibodies and their circulation in 
the blood and lymph exerting a favorable effect upon the local 
lesions. The emulsions of dead bacteria employed are called 
bacterial vaccines. 

The soluble products of bacterial growth are injected into 



224 GENERAL BIOLOGY OF MICRO-ORGANISMS 

animals to immunize them, especially in the case of diphtheria 
and tetanus, the bacteria of which produce very powerful soluble 
toxins. As a result of this treatment antitoxins are produced and 
circulate in the blood of the animal. 

Bacterial extracts, either those contained in inflammatory 
exudates, the so-called aggressins of Bail, or extracts obtained by 
soaking bacteria in blood serum or in distilled water, the so-called 
artificial aggressins of Wassermann and Citron, have proved of 
value in experimental immunization of animals against many dif- 
ferent bacteria. It is claimed that the reactions to injection are 
exceptionally mild, while the resulting immunity is more solid. 
Certain products of the disintegration of typhoid bacilli have been 
obtained by Vaughan, which possess considerable immunizing 
power, but apparently only slight toxicity. None of these bac- 
terial extracts has yet passed beyond the experimental stage in the 
immunization of man against a disease. 

A certain slight grade of immunity may be secured in some 
instances by procedures which seem to bear no relation to the 
specific micro-organisms in question. Thus the injection of cul- 
tures of B. prodigiosus and B. pyocyaneus results in an increased 
resistance to infection with anthrax. Similar increased resistance 
has been observed to follow a simple surgical procedure, such as 
section of the sciatic nerve. The explanation of these results is 
not clear, but perhaps the effect may be attributed to a general 
stimulation of the body defenses, especially the phagocytes. 

Passive Immunity. Passive immunity is produced by inject- 
ing into the body a fluid taken from another animal which contains 
antitoxins, bacteriolysins, opsonins or other substances known 
as immune bodies. The animal which furnishes the immune 
bodies must be first actively immunized, and it possesses an ac- 
tive immunity. If its blood plasma be drawn and injected into 
a child, the child acquires a borrowed immunity without the 
necessity of any active participation of its own cells in the pro- 
duction of immune bodies. The possibility of producing such 
passive immunity has been demonstrated in a number of diseases. 



IMMUNITY AND HYPE INSUSCEPTIBILITY 225 

In some instances the effect of the serum is antitoxic (diphtheria 
and tetanus), in others it is bacteriolytic (cholera), while in 
other instances the nature of the dominant antibodies is not 
definitely known. 

Combined Active and Passive Immunity. Various procedures 
have been devised to produce immunity by introducing at, or 
nearly at, the same time the infectious agent or its products and 
the serum of an immune animal containing protective substances. 
The combination of immune blood with virus of full strength is 
used in immunizing animals against rinderpest, foot-and-mouth 
disease and hog cholera, all being diseases due to filterable 
agents; and also in immunizing hogs against hog erysipelas 
(B. rhusiopathice). The combined injection of attenuated 
virus and immune serum 'is employed especially in Sobernheim's 
method of preventive inoculation against anthrax. Besredka 
has employed dead bacteria combined with their specific immune 
serum in immunizing against typhoid fever, plague and cholera. 

The Mechanisms of Immunity. Certain biological factors 
in the phenomenon of immunity are now clearly recognizable 
and readily demonstrable. The activity of the phagocytes, first 
emphasized by Metchnikoff and believed by him to be the sole 
important factor in the defense of the body, is easily observed 
in immunity to many diseases. The dependence of phagocytic 
activity upon dissolved substances in the body fluids (opsonins) 
is also demonstrated beyond doubt. Phagocytosis is, perhaps, 
the factor of most general operation in immunity to all sorts of 
disease. The antitoxins stand forth prominently as powerful 
factors in immunity to two important diseases, diphtheria and 
tetanus, and the bacteriolysins are undoubtedly of greatest im- 
portance in the case of Asiatic cholera, and probably also in ty- 
phoid and plague. In most instances the immunity seems to 
depend upon several different factors, phagocytosis, opsonins, 
bacteriolysins, antitoxins, and perhaps substances of unknown 
nature. In some instances of immunity there is no particular 
excess of these immune bodies demonstrable in the blood, and 
15 



226 GENERAL BIOLOGY OF MICRO-ORGANISMS 

nearly always an immunity remains long after such an excess 
has disappeared. It would seem that the ability of the cells of 
the body to respond promptly to invasion is often developed by 
experience with one such invasion, and that this ability may re- 
main for a long time as a factor in immunity. 

Hypersusceptibility or Anaphylaxis. If a guinea-pig be in- 
jected with a small amount of a protein, such as egg-albumen or 
blood serum of the horse, and then after an interval of ten to 
twenty days be injected with a second dose of the same protein 
of good size (0.5 to 5 grams), the animal usually develops symptoms 
of nervous intoxication and often dies within a half hour. Inas- 
much as normal guinea-pigs withstand enormous doses of such 
protein substances, it is evident that the first injection has brought 
about some change in the animal, an altered reactivity, which re- 
sults in the intoxication after the second dose. That this phe- 
nomenon of hypersusceptibility or anaphylaxis ( = against pro- 
tection) bears a definite relation to immunity may be illustrated 
by an experiment in which typhoid bacilli are substituted for 
the soluble protein. If a guinea-pig be immunized by repeated 
doses of the killed micro-organisms he is able to resist inoculation 
with an ordinarily fatal dose of the living germs, which are 
quickly killed and dissolved by the specific bacteriolysins in the body 
fluids. However, if such an immune guinea-pig be injected with 
a proper dose of dead organisms, which would not kill a normal 
animal, he may quickly succumb. The ability of the body fluids 
of the immune animal to disintegrate the bacterial cells rapidly 
would seem to be the factor upon which depends not only its 
immunity to the small dose of living germs, but also its exagger- 
ated sensitiveness to dead germ substance. The products of the 
rapid parenteral digestion of the foreign protein would seem to be 
the cause of the symptoms of intoxication. The essential unity 
of the substances upon which immunity and anaphylaxis depend 
has been emphasized by Von Pirquet 1 and his co-workers. 

1 Von Pirquet: Allergy. Archives of Internal Medicine, 1911, Vol. VII, pp. 259-288 ; 
pp. 383-436. 



IMMUNITY AND HYPERSUSCEPTIBILITY 227 

Theories of Immunity. Early theories of immunity were 
based upon meager observations. The idea that an attack of a, 
disease left behind in the body something which prevented the 
subsequent entrance of that disease was formulated by Chauveau 
in 1880 as the so-called retention hypothesis. In the same year 
Pasteur expressed the idea that an attack of a disease removed 
something from the body and so exhausted the soil as far as that 
particular disease was concerned. Neither of these ideas was 
new at that time, and neither of them pretended to any very 
definite or specific application to phenomena observed in immu- 
ity, but only to the general phenomenon of immunity itself. 
The discovery of phagocytosis by Metchnikoff in 1884 was the 
first observation of a definite phenomenon which appeared to 
explain the facts of immunity. The phagocytic theory, which 
grew out of this observation, was an attempt to ascribe immunity 
in general to this one phenomenon of phagocytosis. With the 
observation of the bactericidal substances in solution in the blood 
plasma by Nuttall and by Buchner, of the antitoxins by von 
Behring and the bacteriolysins by Pfeiffer, there developed at- 
tempts to ascribe all the observed facts of immunity to these 
factors, resulting in the alexin theory and the antitoxin theory 
of immunity. More intimate study of the dissolved immune 
bodies lead to the formulation of a hypothesis to explain their 
formation, composition and action, the side-chain theory of Ehr- 
lich, which has been of great value as a working hypothesis and 
as a central conception about which to arrange the observed facts 
relating to these dissolved substances. The elementary concepts 
of this theory have been given in the preceding chapter. 

In brief, Ehrlich pictures the living cell as a chemical unit 
possessing numerous and varied combining groups or side-chains 
capable of uniting with substances in contact with the cell. 
The toxin molecule is conceived as a substance containing at 
least two distinct chemical groups, one which serves for attach- 
ment to the side-chain of the cell and the other serving to bear 
the poisonous properties. The union of the toxin with the cell 



228 GENERAL BIOLOGY OF MICRO-ORGANISMS 

results in destruction of the side-chains attacked, and in regen- 
erating these the cell over-compensates, the excess side-chains, 
receptors of the first order (see page 209), being set free into the 
blood and constituting the antitoxin, which is capable of neutral- 
izing l toxin there or in the test-tube. The assumption of two 
chemical groups in the toxin molecule is strengthened by the 
observation that diphtheria toxin changes on standing so that 
its poisonous property is much diminished without corresponding 
loss of ability to combine with antitoxin. Such changed toxin, 
in which the haptophorous group persists while the toxophorous 
group has degenerated, is called toxoid. In order to explain the 
formation and structure of agglutinins and precipitins, Ehrlich 
assigned a more complex composition to the side-chains which 
constitute these substances, leading to the conception of a receptor 
of the second order (see page 210), with its haptophorous and 
zymophorous groups. In the case of the cytolysins, a further 
amplification of the idea was necessary to explain the observed fact 
that the cytolysis is due to two components, one of which is a 
thermolabile, normal constituent of the blood and not increased 
as a result of immunization, the other being a thermostable sub- 
stance which is produced as a result of the immunization process. 
This latter immune body, the receptor of the third order, was there- 
fore pictured as a double receptor (amboceptor) capable of attach- 
ing on the one hand the foreign body (antigen) and on the other 
the normal component necessary to complete the lytic complex, 
to which component .the name complement was given. 

With the recognition of opsonins by A. E. Wright in 1903, 
the opposing theories of the French and the German schools be- 
gan to be reconciled, and the relatively simple and largely hypo- 
thetical theories of ^jfnmunity be^ga'n to give way to a more exact 
and necessarily' more complex science df immunology. Bordet 
and his -pupils Reserve credit for leading the reaction against too 
slavish adherence to theory in the study of immunity. Our 
modern ideas are no longer confined within the scope of any one 
theory and it is necessary to recognize the existence of a great 



IMMUNITY AND HYPERSUSCEPTIBILITY 22Q 

variety of phenomena in the interaction of the host cells and their 
secretions on the one hand with the parasites and their chemical 
products on the other. The elementary conceptions of immun- 
ology and the primary language of the science are derived from 
the old theories, especially from Ehrlich's theory, and these theo- 
ries are an essential part of the introduction to immunology. 1 

1 For a concise presentation in English of facts and practical experiments re- 
lating to immunity, the student is referred to Citron, Immunity, translated by 
Garbat, Philadelphia, 1912. 



COMPLIMENTS 
OF 





:>'. . V 

K) 







PART III. 
SPECIFIC MICRO-ORGANISMS 



CHAPTER XIV. 

THE MOLDS AND YEASTS AND DISEASES CAUSED 

BY THEM. 

The general characters of molds and yeasts have been men- 
tioned in a previous chapter. The generic and specific relation- 
ships of many of those commonly met with by the pathological 
bacteriologist are in a state of confusion. No claim of systematic 
arrangement is made for the material here presented. 

Mucor Mucedo. This is the most common species of mucor, 
especially about barns and on manure. It produces a network 
of threads (mycelium) in the substratum, and zygospores are pro- 
duced here by the union of two cells. The aerial hyphae are 
vertical, about 10 cm. in length and bear a spherical spore sac 
(sporangium) at the end. The sporangium is at first yellow, 
later brown and finally black and covered with crystals. The 
contained spores are 4 to 6^ wide by 7 to ID/* long. It is 
saprophytic. 

Mucor Corymbifer. Lichtheim found this mold growing on 
a bread-infusion gelatin as an accidental contamination. The 
growth is at first white and later gray. The spore-bearing hyphae 
are long and irregularly branched, and each branch bears a pear- 
shaped sporangium 10 to yo/* in diameter. The contained spores 
are small (2X3^). Intravenous injection of the spores into rab- 
bits causes severe nephritis and death in two or three days. 

231 



232 



SPECIFIC MICRO-ORGANISMS 




FIG. 89. Mucor mucedo. i, A sporangium in optical longitudinal section: 
c, columella; m, wall of sporangium; sp, spores. 2, A ruptured sporangium with only 
the columella (c) and a small portion of the wall (m) remaining. 3, Two smaller 
sporangia with only a few spores and no columella. 4, Germinating spores. 5, 
ruptured sporangium of Mucor mucilaginus with deliquescing wall (m) and swollen 
interstitial substance (z); .>/>, spores. (From Jordan after Brefeld.) 




FIG. 90. Mucor corymbifer. (From Plant after Lichthelm.} 



MOLDS AND YEASTS AND DISEASES CAUSED BY THEM 



233 



The mold has been found growing as a parasite in the auditory 
canal. 

More than a hundred species of Mucor have been described 
and several of them cause disease and death when injected into 
animals. 

Aspergillus Glaucus. This is very widely distributed in 
nature, occurring on fruits, moist bread and other food substances 
and very frequently as a con- 
tamination in laboratory cul- 
tures. The aerial spore-bear- 
ing hypha (conidiophore) is 
erect, about i mm. long, swollen 
at the end to a diameter of 20 
to 40;*. On the surface of this 
spherical head are numerous 
closely packed spore-bearing 
sterigmae, each of which bears 
at its tip a chain of spherical 
spores (conidia) which are 
budded off from it. The coni- 
dia are gray to olive green in 
color. Ascospores are also produced, grouped together as yellow 
masses, called perithecia, on the surface of the medium. The 
mold is not pathogenic. Probably a considerable number of 
different species have been included under this name. 

Aspergillus Fumigatus. The growth of this mold is at first 
bluish and later grayish-green. It is widely distributed. The 
sterigmae are unbranched, thickly set on the swollen end of the 
spore-bearing hypha. The conidia measure 2.5 to 3^. The for- 
mation of ascospores has also been observed. Aspergillus fumi- 
gatus plays a part in the heating of hay and sprouting barley, 
and is the most common of the pathogenic aspergilli. It infects 
doves and other birds naturally, sometimes causing veritable 
epidemics, and the disease has been observed in bird fanciers, 
in whom it runs a clinical course very similar to that of pulmonary 




FIG. 91. Aspergillus fumigatus from the 
lung of a parrot. (After PlauL} 



234 



SPECIFIC MICRO-ORGANISMS 



tuberculosis. Fragments of the mycelium are found in the spu- 
tum. Doubtless the human disease is contracted from the birds 
in these cases. This mold has been found as the apparent cause 
of inflammation in the auditory canal in a large number of cases 

and in the nasal fossae in a few in- 
stances. Various other mammals are 
susceptible to inoculation and natural 
infection has been observed in horses, 
cattle, sheep and dogs. 

Many other species of pathogenic 
aspergilli have been described, of less 
frequent occurrence than A.fumigatus. 
Penicillium crustaceum (glaucum) 
is the commonest contaminating mi- 
cro-organism met with in the labora- 
tory, and is probably the most widely 
distributed mold. Ascospores, similar 
to those of Aspergillus glaucus have 
been observed, but they are rarely 
produced. The aerial fruiting hypha 
(conidiophore) is erect, septate and 
branched at the upper end like a brush. 
At the end of these branches are bot- 

FIG. 92. Pemcillmm crusta- 
ceum. Conidiophore^ with verti- tie-shaped stergmae from which the 

conidia are constricted off. The 




comdia. XS4C. (From Jordan growth IS at first white and then It 
after Strasburger.) 

becomes blue-green, the development 

of color beginning at the center. Penicillium crustaceum, or at 
any rate a certain variety of it, is an important agent in the 
ripening of Rocquefort cheese. It is not pathogenic, but the 
extracts from cultures of some varieties are poisonous when in- 
jected into animals. It is possible that several distinct species 
have been included under this one name of Penicillium crustaceum. 
Claviceps Purpurea. This is a fungus parasitic upon rye 
and a few other plants. The spores gain access to the flower of 



MOLDS AND YEASTS AND DISEASES CAUSED BY THEM 235 

rye and develop a mycelial mass which grows in the utricle, dis- 
placing the grain, the rudiment of which lies above the mass of 
the mold. Closely packed conidiophores produce oval conidra 
and at the same time secrete a sweet milky fluid which attracts 
insects and thus furthers the distribution of the parasite. Later 
the mycelial mass produces sclerotia, which are masses of thick- 
walled cells containing starch and oil together with specific poi- 
sonous substances, and the whole becomes dry and hard with black 
outer covering, forming the ergot grain, which is considerably 
larger than the normal rye grain. In autumn this falls to the 
ground and remains until spring, when numerous red stalks grow 
out of it. Upon the swollen ends of these stalks, abundant as- 
cospores are produced, and these serve to infect again the flowers 
of the new crop of rye. 

This fungus is of great importance as the source of the drug, 
ergot, and as a cause of food poisoning, ergotism, in certain coun- 
tries. It is one example of a mold parasitic upon higher plants. 
There are very many different species of such parasitic fungi, 
and they are probably the best known microbic agents causing 
diseases of plants. 1 

Botrytis Bassiana. This mold was shown to be the cause of 
muscardine, a disease of silkworms, by Bassis and Audouin in 
1837, a discovery following closely the recognition of the itch 
mite, Sar copies scabei, as the cause of scabies in 1834. The in- 
fected silkworm becomes sluggish and dies, and the aerial hyphae 
of the fungus grow out from its surface and pinch off round or 
pear-shaped conidia. These spores gain the surface of other 
silkworms or butterflies by contact or by air transmission, and 
germinate, sending threads into their bodies. Sickle-shaped 
spores are produced from these inside the body, and these grow 
out into, threads, forming a mycelial network throughout the 
body of the victim and causing its death. It is possible that 

1 For a consideration of molds in relation to plant pathology, see Massee, 
Diseases of cultivated plants and trees, New York, 1910. 



236 



SPECIFIC MICRO-ORGANISMS 



several different species of molds may be concerned in the 
causation of muscardine. 

The fungus is of interest because it was probably the first 
mold to be recognized as a cause of disease, and also because 
it is an example of a large group of fungi which attack various 
insects. The disease muscardine is, moreover, one of con- 
siderable importance to the silk industry. 




FIG. 93. Oidium lactis. a, b, Dichotomous branching of growing hyphae; c, d. g, 
simple chains of oidia breaking through substratum at dotted line x-y, dotted por- 
tions submerged; e, /, chains of oidia from a branching outgrowth of a submerged 
cell; h, branching chain of oidia; k, /, m, n, o, p, s, types of germination of oidia under 
varying conditions; /, diagram of a portion of a colony showing habit of Oidium 
lactis as seen in culture media. (From Bull. 82, Bur. Animal Industry, U. S. Dept. 
Agr.) 

Oidium Lactis. Oidium lactis is very widely distributed and 
is almost always present in milk and milk products, and in brew- 
er's and baker's yeast, and it is an especially prominent organism 
in the further fermentation of acid substances, such as sauer- 



MOLDS AND YEASTS AND DISEASES CAUSED BY THEM 237 

kraut, sour milk and cheese. The organism is especially impor- 
tant in the ripening of Camembert cheese. It grows well on ordi- 
nary nutrient gelatin. The colony consists of a loosely woven, 
white network of septate, branched and anastomosing threads, 




FIG. 94. Oidium albicans. A deep colony on a plate culture of the liquifying 
variety, showing chlamydospores. (After Plant.} 

chiefly in the substratum but also extending into the air. The 
peripheral threads are divided by septa to form chains of oval 
or spherical conidia. 

This mold may be readily obtained for study by making plate 
cultures from compressed yeast. 



238 SPECIFIC MICRO-ORGANISMS 

Oidium Albicans (Monilia Candida). The thrush fungus 
was discovered by von Langenbeck in 1839 and by Berg in 1841, 
but the popular recognition of a relation between this disease and 
a mold seems to have preceded this discovery by many years. 
Berg (1841) transferred the fungus from cases of thrush to healthy 
children with positive results. His work was confirmed by numer- 
ous other investigators (1842-43). Robin (1847) accurately 
described the parasite, with illustrations, classed it as an oidium, 
and gave it the name Oidiym albicans (1853). Grawitz (1877) 
obtained the first pure cultures and successfully inoculated rab- 
bits and puppies with them. 

In the throat lesion as well as in cultures the organism con- 
sists of mycelial threads and oval yeast-like cells. It grows read- 




FIG. 95. Oidium albicans. Mycelial thread with four ripe chlamydospores; and 
conidia in the middle of the picture. (After Plant.} 

ily on various artificial media and the appearance of the growth is 
quite variable, not only because of the proportional relation 
between the oval cells and the threads, but also in pigmen- 
tation and in density of growth. Two varieties, one liquefying 
gelatin and producing large (5^) oval conidia, and the other failing 
to liquefy gelatin and producing small (2.5^) spherical conidia 
are distinguished. 

Thrush is most common on the buccal mucous membrane of 
young infants, but it also occurs on the vaginal mucosa of preg- 
nant women, and it may attack others when weakened by dis- 
ease, especially diabetics. The disease also occurs naturally in 
birds, calves and foals. The threads of the mold penetrate the 
squamous epithelium and even enter the subepithelial tissue, 
sometimes penetrating blood-vessels and giving rise to metas- 



MOLDS AND YEASTS AND DISEASES CAUSED BY THEM 239 

tases. It results in death in about 20 per cent of the cases in 
infants. The predisposing digestive disorder or other primary 
disease is, however, usually more important than the thrush, and 
demands first consideration in treatment. The thrush lesion may 
be carefully removed with a soft swab and the eroded area treated 
with silver nitrate, o.i per cent. Generalization of the disease 
is rare, but several cases have been observed. Inoculation of 
animals (mice, guinea-pigs, puppies, rabbits) is sometimes success- 
ful, and generalized thrush has followed intravenous injection of 
young rabbits. The fungus seems to exert some poisonous action, 
in addition to the mechanical effect upon the tissues. 




FIG. 96. Scutulum of favus on the arm of a man. (After Plant.) 

Achorion Schoenleinii. The fungus of favus was discovered by 
Schoenlein in the skin lesions of this disease in 1839, two years 
after the recognition of Botrytis bassiana as the cause of mus- 
cardine. Remak in 1845 g rew the mold on slices of apple and 
successfully inoculated his skin with these cultures. He named 
the organism Achorion schoenleinii. In the lesion of favus the 
threads of the fungus are found growing in the horny layer of the 
epidermis, usually about a hair, and giving rise to a dry, circular, 



240 SPECIFIC MICRO-ORGANISMS 

yellow crust with depressed center, the "Scutulum.'" By macerat- 
ing this crust in 50 per cent antiformin the elements of the 
mold are made clearly visible under the microscope. In the 
center of the lesion are doubly contoured oval or rectangular 
conidia 3 to 8/* by 3 to 4//, single and in chains. The mycelial 
threads are indistinguishable in the center, but are seen at the 
periphery as tubes of very irregular width, refractive with granu- 
lar protoplasm, often branched or knobbed at the end. The 




FIG. 97. Typical scutulum of favus in a mouse. (After Plant.) 

scutulum in its interior is a pure culture of the mold, entirely free 
from other organisms. The mold also grows in the interior of 
the hair shaft, and by macerating the hair in alkali the fungus 
may be demonstrated microscopically. 

Cultures may be obtained upon various media. Plaut recom- 
mends a medium containing pepton i to 2 per cent, glycerin 
0.5 per cent, salt 0.5 per cent and agar 2 per cent, without meat 
extractives or any addition of alkali. The cultures are incubated 
at 30 C. Mycelial threads and numerous conidia are produced. 

Inoculation into the epidermis of mice or onto the human 



MOLDS AND YEASTS AND DISEASES CAUSED BY THEM 



241 



skin gives rise to typical lesions. Intravenous injection into 
rabbits is usually followed by a pseudo-tuberculosis in the lungs, 
sometimes fatal. Similar skin lesions occur naturally in various 
animals, and the molds there present are very similar to, if not 




FIG. 98. Achorion schoenleinii. Colony developing from a favus scale. End, endo- 
conidia on submerged hyphae. Ect, ectospores on aerial hyphae. (After Plant.} 

specifically identical with, Achorion schoenleinii. The exact 
relationships of the parasites are not very exactly settled as yet. 

Microsporon Audouini. This mold is found growing in the 
hair-shaft in alopecia areata. If the hair be pulled out it breaks 

16 



242 



SPECIFIC MICRO-ORGANISMS 



near the lower end and the oval conidia and jointed threads of 
the parasite may be demonstrated by macerating this broken 
end. The disease is very contagious, chronic and resistant to 

treatment, but proceeds without inflam- 
mation or subjective symptoms, the 
conspicuous sign being loss of the hair. 
Cultures grow slowly and are snow 
white. Animal inoculation is rarely 
successful. 

Microsporon Furfur. This mold is 
found in the superficial layer of the skin 
in pityriasis versicolor, as short thick 
hyphae 3 to ^ wide by 7 to 13," long, 
together with abundant doubly con- 
toured single conidia. Pityriasis versi- 
color occurs most frequently on the 
skin of the chest and is one of the com- 
monest affections of the skin. 

Tricophyton Acuminatum. The 
mold invades the hair shaft and causes 
it to break off close to the surface of 
the skin. In such a hair long chains 
of oval cells of the parasite may be seen. 
The parasite also attacks the skin and 
produces ringworm. Several other spe- 
FIG. gg.Sporotrichum cie s of tricophyton are distinguished. 

schencki. Cultures on the Tnese para sites are concerned in the 
glucose-pepton agar of Sabour- f 

aud. (After Gougerot.) causation of barber's itch, eczema mar- 

ginatum, tinea cruris, and other skin 
affections of this type. 

Sporotrichum Schencki. Schenck, at Baltimore in 1898, de- 
scribed this parasitic mold which he found in the lesions of a 
peculiar disease, beginning as a localized ulcer, with later involve- 
ment of the neighboring lymph glands, in which cold abscesses 
formed and opened to the exterior. A second similar case was 




AND YEASTS AND DISEASES CAUSED BY THEM 



243 



described by Hektoen and Perkins. Ruediger 1 has reported a 
large series of cases of sporotrichosis and the references to American 




FIG. ioo. Sporotrichum schenki. _ Various forms of mycelum with and without 
conidia. (After Gougerot.} 

literature will be found in his paper. The organisms are not 

1 Journ. Infect. Diseases, 1912, Vol. XI, pp. 193-206. 



244 SPECIFIC MICRO-ORGANISMS 

readily found in the pus by microscopic examination and seem 
to exist there only as conidia. In cultures a branching mycelium 
with clusters of conidia is produced. Dogs are susceptible to 
inoculation. 

Sporotrichium Beurmanii. De Beurmann and Ramond at 
Paris in 1903 found this parasite in a case of lymphangitis. It 
seems to be different from the organism described by Schenck 
but may ultimately prove to be the same species. 

Saccharomyces Cerevisiae. This organism is ,the type of 
the true yeasts. The cell is spherical or ovoid, and multiplies 
by budding. Endospores are produced, usually four to eight 
in a single cell, indicating a rather close relationship to the molds. 
The organism is found widely distributed, especially on fruits 
and sugar-containing substances. It has been used for centuries in 
the leavening of bread and in the alcoholic fermentations. Varie- 
ties of the species are distinguished by differences in physiological 
characters, and especially in respect to the amounts of alcohol 
which they produce. 

Material for study may be obtained from commercial com- 
pressed yeast, which contains vegetating cells of saccharomyces 
along with other organisms, or from commercial dried yeast in 
which the spores are present. Pure cultures may be obtained by 
plating on gelatin. True yeasts also occur in the gastric juice at 
times and seem to be able to multiply in the stomach when the 
acidity of the gastric juice is diminished. 

Blastomyces Dermatidis. Doubly contoured yeast-like cells 
in human tissues were first discovered by Busse and Buschke 1 in 
1 894, in a case presenting abscesses in the bones and internal organs 
together with lesions of the skin. They obtained cultures of the 
organism and classed it as a yeast. About the same time Gil- 
christ 2 independently observed similar organisms in cases of 
dermatitis at Baltimore. The organisms have been most thor- 
oughly studied by Ricketts. 3 Most of the cases have been ob- 

1 Deutsch. med. Wochenschr., 1895, Nr. 3. 

2 Gilchrist: Johns Hopkins Hosp. Kept., Vol. I, p. 209, 1896. 
3 Journ. Med. Research, Vol. VI, No. 3. 



MOLDS AND YEASTS AND DISEASES CAUSED BY THEM 



245 



served in the United States, at Baltimore, at Chicago and in Cali- 
fornia. One type of the parasite appears to multiply in the tissues 
by a process of budding (Blastomycetic dermatitis, Blastomycosis) 
while in other cases, particularly those from California, the 
spherical bodies found in the tissue seem to multiply by endog- 
enous spore formation, an appearance which at first suggested 
the protozoal nature of the parasite and lead to the use of the 




FIG. 101. Doubly contoured organisms found in oidiomycosis (blasto mycosis). 
(From Buschke after Hyde and Montgomery.) 

unfortunate term, Coccidioidal granuloma. On glucose agar, the 
parasites usually grow without difficulty and the growth resem- 
bles that of an oidium, often with abundant aerial hyphae. Inoc- 
ulation of guinea-pigs with pus or with cultures is usually fol- 
lowed by formation of abscesses in which the typical spherical or 
ovdid parasites may be found. The tissue changes have been 
mistaken for tuberculosis. Further investigations are required 
to determine the specific relationships of the parasites found in 
different cases. 



CHAPTER XV. 
TRICHOMYCETES. 

The trichomycetes or higher bacteria are intermediate in 
morphological characters between the molds and the lower bac- 
teria. They resemble the molds in the formation of long threads, 
sometimes branching and interlacing to produce a network, and 
in the formation of oval or spherical conidia constricted off from 
the ends of the threads. They resemble the lower bacteria in 
their small transverse diameter, the delicacy of their structure 
and their mode of life. Petruschy 1 recognizes four genera, 
Actinomyces, Streptothrix, Cladothrix andLeptothrix. 

Actinomyces Bovis. Bollinger in 1877 studied the lumpy- 
jaw disease of cattle and described this parasite which occurs 
in the lesions. Israel, in the following year, found the organism 
in granulomatous lesions in man. The infection also occurs in 
horses, sheep, swine and dogs. In the tissues and in the purulent 
discharge from the lesions, the organism occurs in small yellowish 
masses, sometimes visible to the naked eye but usually smaller 
(10 to 2oo/* in diameter). Such a mass is a single colony of the 
parasite or a conglomerate of several colonies. The colony is a 
dense network of threads in the center, with radially arranged 
threads about the periphery, most of the latter being swollen, 
club-shaped, at their free ends. Spherical bodies may also be 
present, but whether these are conidia or degeneration forms of 
the parasite is uncertain. The organism is Gram-positive. 

Inoculation of pus or bits of tissue containing the parasite 
from one animal into another usually fails to transmit the dis- 
ease, although positive results have been obtained in a few in- 
stances. Attempts at culture have failed also in many instances, 

1 Kolle and Wassermann: Handbuch, 1912, Vol. V, p. 270. 

46 



TRICHOMYCETES 247 

and the difficulty here seems to depend in part upon the oxygen 
requirements of the organism. The material for culture should 
be obtained from uncontaminated tissue containing the fungus. 
If this is impossible, the granule of actinomyces should be washed 
in several changes of sterile salt solution, then crushed between 
sterile glass slides or, better, ground up in a sterile mortar with 
a small amount of sterile sand. A series of dilution cultures 
should then be made in tall tubes of melted glucose agar cooled to 
45 C., the tubes chilled in cold water and incubated at 37 C. 




FIG. 102. Actinomyces bovis. The ray-fungus from cow. (Diagrammatic.) 



Colonies of the fungus may be expected to develop some distance 
below the surface of the agar. Wolf and Israel were able to 
reproduce the disease in animals (rabbits and guinea-pigs) by the 
inoculation of pure cultures. More recently Harbitz and Gron- 
dahl 1 isolated twenty-seven strains of actinomyces, but their 
inoculation experiments were wholly negative. It would appear 
that other factors are essential to the development of actinomy- 
ces in addition to the inoculation of the specific parasite. Many 
authors regard the presence of bits of straw or sharp grains in 
wounds of the mucous membrane of the mouth or pharynx as 
important elements in predisposing to infection with actinomyces. 

1 Amer. Journ. Med. Sciences, 1911, Vol. CXLII, pp. 386-395. 



248 SPECIFIC MICRO-ORGANISMS 

The disease shows little or no tendency to be transmitted from 
animal to animal in a herd. Several varieties of actinomyces 
have been described, and possibly more than one species will 
eventually be recognized. 

Streptothrix Madurae. Kanthack (1892) and Gemy and 
Vincent (1892) discovered the fine mycelial threads in pus from 
cases of Madura foot. Granules about the size of a pin-head occur 
in the pus, and under the microscope these are found to consist of 
a network of threads i to 1.5^ in thickness, arranged radially 
at the periphery and presenting somewhat swollen ends. These 
granules are white in some cases, yellow, red and black in others. 
The nature of the disease seems to be the same in all cases, but 
the micro-organisms are apparently not the same, that found in 
the black variety probably representing a distinct species. 
Cultures may be obtained by inoculating the pus, collected 
without contamination, into several flasks of sterilized hay in- 
fusion, and shaking daily to insure abundant oxygen supply. 
It also grows upon other media. Gelatin is not liquefied. The 
growth is made up of interwoven, slender branching threads 
about i/* in thickness. Spores (conidia) capable of resisting a 
temperature of 75 C. for five minutes are produced at the sur- 
face of the culture. Inoculation of animals usually gives nega- 
tive results, but Musgrave and Klegg 1 have succeeded in infecting 
monkeys. 

The disease, Mycetoma or Madura foot, is a localized chronic 
inflammation, almost painless, and usually involving the foot, 
the hand or some exposed portion of the body. The disease 
involves the tissues by direct extension, attacking the bones as 
well as the soft tissues. It usually remains localized to one 
extremity. 

The black variety of Madura foot is due to a different organ- 
ism, the threads of which are 3 to 8/* in thickness. 2 This organ- 

1 Philippine Journ. of Science, 1907, Vol. II, pp. 477-512; A complete bibli- 
ography by Polk is included. 

2 Wright: Journ. of Exp. Medicine, Vol. Ill, pp. 421-433. 



TRICHOMYCETES 249 

ism seems to be an aspergillus, and has been named Madurella 
mycetori. 

Streptothrices have also been found in abscesses of the brain and 
in chronic disease of the lung clinically resembling tuberculosis 
in man. Many of them are Gram-positive and some are rela- 
tively acid-proof when stained with carbol-fuchsin. Such acid- 
proof forms are common in the feces of cattle where short seg- 
ments of them may be mistaken for tubercle bacilli. Organisms 
of this type are very abundant in the soil, which is doubtless 
their natural habitat. 

Cladothrix. The cladothrix forms resemble the strepto- 
thrices very closely but the cells of the threads do not branch. 
The apparent branching of the threads is explained as due to a 
transverse division of the thread with continuing growth of the 
one free end which pushes out beyond the other, giving rise to 
the appearance of branching or so-called " false branching." 
Organisms of this type have been described as occurring in ab- 
scesses of the brain and in other parts of the body. The dis- 
tinction from streptothrix has not always been clearly made. 

Leptothrix Buccalis. This is a normal inhabitant of the 
mouth cavity. It consists of slender filaments which do not 
branch. The organism has been found in abundance in small 
white patches on the tonsils, where it sometimes causes a very 
chronic but mild inflammation. Artificial culture of the organ- 
ism ordinarily results in failure. Arustamoff 1 appears to have 
obtained it on a neutral or acid agar inoculated with leptothrix 
from urine. 

1 Kolle and Wassermann: Handbuch, 1912, Bd. V, S. 290. 



CHAPTER XVI. 

THE COCCACE^: AND THEIR PARASITIC RELATION- 
SHIPS. 

Diplococcus Gonorrheae. The gonococcus was discovered 
by Neisser 1 in 1879 in the discharge of acute urethritis and he 
recognized its probable causal relationship to the disease. Cul- 
tures were first obtained by Bumm 2 in 1885 and he proved the 
relationship by inoculating the human urethra with his cultures. 
The organism naturally lives and multiplies only in the human 
body and is the microbic cause of gonorrhea and many of its 
complicating inflammations. 

The gonococcus is found in both the serum and the poly- 
nuclear cells of the purulent discharge, usually in pairs with the 
adjacent surfaces flattened. The long diameter of the pair is 
about i. 25/4. It stains readily, best perhaps with Loffler's 
methylene-blue. It is decolorized when stained by Gram's 
method, a fact of great importance in the quick recognition of 
the organism. The staining procedure has to be carefully carried 
out and a beginner should practice upon cultures of the gonococcus 
and upon samples of gonorrheal pus and staphylococcus pus 
before placing too much reliance upon the appearance of his 
Gram-stained preparation. The reaction to the Gram stain, 
together with the remarkably characteristic appearance of the 
pus cell full of diplococci are usually sufficient for the recogni- 
tion of the organism in acute urethritis. 

Cultures of the gonococcus were obtained by Bumm on coagu- 
lated human blood serum. Wertheim 3 employed serum agar 

1 Neisser: Centralbl. f. d. med. Wissenschaft, 1879, Bd. XVII, S. 497-500. 

2 Bumm: Deutsche med. Wochenschr., 1885, Bd. II, S. 910 and 911. 

3 Deutsche med. Wochenschr., 1891, Bd. XVII, S. 958; S. 1351 and 1352. 

250 



COCCACE^E AND THEIR PARASITIC RELATIONSHIPS 



251 



made by mixing human blood serum at 40 C., one part, with 
ordinary nutrient agar melted and cooled to 40 C., two parts. 
The medium may be inclined in tubes or may be employed for 
plating. Human ascitic fluid or hydrocele fluid is just as good 
as blood serum. A large drop of pus from an acute urethritis 
should be mixed with 2 to 3 c.c. of serum or ascitic fluid in a 
test-tube and from this, dilutions made to a second and a third 
tube. The contents of a tube of agar (5 to 6 c.c.), previously 
melted and cooled to about 40 C., is then added to each tube of 




FIG. 103. Gonococci and pus-cells. Xiooo. 

serum, mixed thoroughly and poured into Petri dishes to solidify. 
At 37 C., colonies appear within 24 hours and at the end of this 
time measure about i mm. in diameter. The colony is circular, 
grayish-blue and transparent and of a mucoid consistency. 
The individual cocci disintegrate rapidly, even within the first 
24 hours at the center of the colony, and for microscopic study, 
simple staining and staining by Gram's method, cultures 5 to 10 
hours old are recommended. Even under favorable conditions 
the gonococcus ordinarily dies out in the culture tube in about a 
week, although exceptionally it may survive for three weeks. 



252 SPECIFIC MICRO-ORGANISMS 

It should be transplanted every few days and a large quantity 
of growth must be transferred. When transplanted from vigor- 
ous cultures to plain agar the gonococcus grows for a few days, 
but it cannot be successfully propagated for any length of time 
on ordinary media. 

The gonococcus is very sensitive to drying and to tempera- 
tures above 40 C. It is usually impossible to recover it from 
dried pus, but in moist material it may live for i to 24 hours. 
The organism is easily killed by chemical germicides, of which 
silver nitrate is probably the most effective. 

Inoculation of animals in the urethra or on the conjuctiva 
is without result. Intraperitoneal injection of cultures into 
white mice or guinea-pigs usually kills the animals in 24 hours 
and the gonococci can be recovered from the peritoneal fluid 
and the heart's blood. These effects seem to be due to toxins of 
the injected material rather than actual infection. The specific 
poisons seem to be intracelluar and set free upon disintegration 
of the organism. The poison withstands heating to 100 C. for 
hours. Inoculation of the human urethra with cultures of the 
gonococcus has been repeatedly done and has resulted nearly al- 
ways in the production of typical gonorrhea. 

Gonnorrhea has been recognized as a contagious disease 
since the dawn of history. The most important forms are (i) 
urethritis with tendency to extension in the female to the cervix 
uteri, oviducts and peritoneum, and iri the male to the prostate, 
seminal vesicles, and epididymis, and in both sexes to the blood 
stream, heart valves and joints; (2) conjunctivitis and keratitis 
leading to scarring of the cornea and permanent blindness; 
(3) valvo-vaginitis in girl babies, an exceedingly contagious 
disease, especially in hospital wards. The disease tends to 
become chronic and eventually latent, that is, the symptoms 
subside but the micro-organisms remain alive in certain loca- 
tions, such as the prostate in the male and the cervix uteri in 
the female. The acute inflammation may be. followed by scars' 
resulting in strictures of the urethra or occlusion of the epididy- 



COCCACE^: AND THEIR PARASITIC RELATIONSHIPS 253 

mis. In the female, pyosalpinx is a not unusual complication. 
Secondary infection with staphylococci is common in chronic 
gonorrhea. 

Specific diagnosis by finding gonococci usually presents no 
difficulties in acute inflammations of the genital tract, in which 
the characteristic groups of Gram-negative intracellular diplococci 
are practically diagnostic. In chronic cases and in extra-genital 
inflammations the diagnosis presents greater difficulty. Both 
microscopic and cultural examinations should be made and if 
negative they should be repeated many times. Even repeated 
failure to find the gonococcus by these methods does not justify 
the positive assertion that it is absent. Specific diagnosis by 
the method of complement fixation has been developed by 
Schwartz and McNeill. 1 The antigen is prepared from several cul- 
ture strains of the gonococcus and in all other respects the test is 
similar to the Wassermann test for syphilis. Irons 2 has employed 
a cutaneous test, using a glycerin extract of gonoc< -cci. The tech- 
nic is similar to that of the von Pirquet test for tuberculosis. 

The prevalence of gonorrhea throughout the civilized world 
is much greater than has been popularly supposed. Erb, in a 
study of 2000 males among private patients of the middle and 
better classes, found a history of gonorrhea in 50 per cent. Many 
other students of the disease disagree with Erb, regarding his 
figures as much too low. Among women in German obstetrical 
hospitals, largely from the poorer class, gonorrhea is present in 
10 to 30 per cent. The danger to the eyes of the new-born 
infant is now overcome by the use of silver nitrate in the eyes 
when they are first cleansed. The general prevention and re- 
striction of gonorrheal infection is engaging more and more the 
serious attention of thoughtful citizens, and it is already recog- 
nized as a sanitary problem of the first magnitude. 

Diplococcus Meningitidis. Weichselbaum in 1887 examined 
the cerebrospinal fluid in six sporadic cases of meningitis and 

1 Amer. Joitrn. med. Sciences, 1912, Vol. CXLIV, pp. 815-826. 

2 Jour n. Infec. Diseases, 1912, Vol. XI, pp. 77-93. 



254 SPECIFIC MICRO-ORGANISMS 

found in all of them a very definite Gram-negative intracellular 
diplococcus, the meningococcus. He obtained cultures but his 
animal inoculatons all gave negative results. Jaeger in 1895 
seems to have found a similar organism in fourteen cases of 
epidemic meningitis and Huebner in 1896 apparently found it 
in five cases. The cultural work of these authors seems to be 
unreliable as their cultures were Gram-positive. More conclu- 
sive confirmation of the relation of this organism to epidemic 
meningitis was furnished by Councilman, Mallory and Wright 1 
in 1898. 

The meningococcus is found in the bodies of patients suffering 
from meningitis, occasionally on the nasal mucous membrane 
of healthy persons and of cases of rhinitis, and very rarely in 
other situations. In cerebrospinal meningitis the organism is 
present in the cerebrospinal fluid, in the meninges, often on the 
nasal and pharyngeal mucous membrane, sometimes in the 
blood and on the conjunctivae, and rarely in the urethra, where 
it may be mistaken for the gonococcus. It is usually found 
without difficulty in the cerebrospinal fluid in the first few days 
of the disease, but may be very difficult to find at a later stage. 

The organism is found for the most part inside polynuclear 
leukocytes and in its form, size, arrangement and behavior to 
the Gram-stain resembles very closely the gonococcus. The 
outline of the cocci is often somewhat hazy, suggesting possible 
disintegration, and this sometimes makes their recognition 
somewhat difficult in microscopic preparations of cerebrospinal 
fluid. Cultures are best made on ascitic-fluid agar or blood 
agar, upon which small dew-drop colonies appear in 24 hours 
at 37 C. The color of blood is unaltered by the growth. Cul- 
tures may be obtained on Loffler's blood serum, although ascitic- 
fluid agar is probably the best medium for continued culti- 
vation. The meningococcus grows more luxuriantly than the 
gonococcus, as a rule, and adapts itself more readily to growth 

1 Report of the Mass. Bd. of Health on Epidemic Cerebrospinal Meningitis, 
etc., Boston, 1898. 



COCCACE.E AND THEIR PARASITIC RELATIONSHIPS 255 

on ordinary media, but its cells disintegrate rapidly in the colony, 
which is viscid. In nearly every respect it resembles very closely 
the gonococcus. 

Intraperitoneal inoculation of white mice and of guinea-pigs 
usually results in fatal peritonitis and the organism can be recov- 
ered from the heart's blood. Intraspinal inoculation of monkeys 
with large doses causes typical meningitis with symptoms similar 
to those of the disease in man. In man the disease is undoubtedly 
transmitted very largely by coccus-carriers, healthy people or 
people with slight pharyngitis or rhinitis, who carry the virus 
on their mucous membranes and distribute it. 

Antimeningococcus serum is prepared by immunizing horses 
with a mixture of many typical and atypical meningococcus 
cultures injected subcutaneously. At first the cultures are 
killed by heat before injection, and only one or two loopfuls are 
given. The dose is increased and repeated every 8 to 10 days 
until the growth on two Petri dishes is being injected. Living 
cultures are then given, and finally old cultures which have 
disintegrated are also used. The serum is used after the horse 
has been treated for 8 to 10 months. Jochmann showed that 
the subcutaneous injection of the serum is without effect upon 
meningitis in monkeys but that when introduced into the spinal 
canal is specifically curative. Flexner 1 and his co-workers 
have studied this very fully and there can no longer be any ques- 
tion of the value of the serum in the treatment of meningococcus 
meningitis. 

Cerebrospinal fluid is obtained by Quincke's puncture. For 
children a needle 4 cm. long and with a lumen of i mm. is intro- 
duced near the median line upward and forward so as to enter 
the spinal canal between the second and third or the third and 
fourth lumbar vertebrae. From 20 to 50 c.c. of fluid may be with- 
drawn if it comes away under pressure, and then the curative serum 
is injected through the same needle. The fluid withdrawn should 
be examined to establish the presence of meningitis and its 

Flexner: Harbin lectures. Journ. State Medicine, 1912, Vol. XX, pp. 257-270. 



256 



SPECIFIC MICRO-ORGANISMS 



variety. In general the examination includes a macroscopic 
examination and description of the appearance of the sample, a 
microscopical numerical count of the cells present, chemical 
examination of the cell-free fluid for excessive protein 1 content, 
microscopic and cultural examination of the sediment for bacteria 
and of the filmy clot which may form after standing an hour or 



f 





* 



: 









FIG. 104. Meningococcus in spinal fluid. (After Hiss and Zitisser.) 



so for tubercle bacilli, and sometimes it includes the Wassermann 
reaction. In meningococcus meningitis the cell count is generally 
above 100 per cu. mm., and most of the cells are polynuclear 
leukocytes. Within these cells the meningococci may or may not 
be found. In case of doubt, plate cultures on blood-agar and 

1 Noguchi's test: To 0.5 c.c. of blood-free fluid add i c.c. iq per cent butyric 
acid, boil; add 0.2 c.c. normal NaOH and boil again. Set aside to cool. A floc- 
culent precipitate indicates an increase in the globulin content. 



COCCCAE.E AND THEIR PARASITIC RELATIONSHIPS 257 

ascitic-fluid agar should be made. The recognition of a Gram- 
negative intracellular diplococcus in the fluid is sufficient for a 
tentative diagnosis, and the appearance of characteristic colonies 
on the plates may be considered conclusive. 

Diplococcus (Micrococcus) Catarrhalis. This organism is 
commonly present on the mucous membrane of the upper air 
passages, especially in catarrhal inflammations. It is usually 
seen as a Gram-negative intracellular diplococcus not to be 
distinguished microscopically from the meningococcus or gono- 
coccus. In examining material from the air passages this organ- 
ism has to be considered. It is readily distinguished by cultural 
methods. On ascitic-fluid agar the colony is dry and brittle, 
quite different from the meningococcus or gonococcus. Further- 
more, it grows readily at once on ordinary agar. 

Diplococcus Pneumoniae. Sternberg in 1880 injected the 
saliva of healthy persons into rabbits and produced a rapidly 
fatal bacteremia with abundant lance-shaped diplococci in the 
blood and internal organs of the animal. Pasteur, independently 
and at about the same time, injected the saliva of a boy suffering 
from rabies into rabbits with a similar result. The organism 
was spoken of as the diplococcus of sputum septicemia or the 
septicemic microbe of saliva. Koch in 1881 demonstrated the 
organism microscopically in sections of lung. Friedlaender 
(1882-1884) found the organism microscopically in a large 
number of cases of pneumonia and accurately described its form, 
the capsules and staining properties. His cultures, however, 
which were made on gelatin at room temperature, brought to 
development not the pneumococcus but a wholly different organ- 
ism which he believed to be identical with it, Friedlaender's 
pneumobacillus. A. Fraenkel obtained the first undoubted pure 
cultures on solidified blood serum, proved the identity of the organ- 
ism in pneumonia with that of normal saliva seen by Sternberg 
and Pasteur, and distinguished it absolutely from the pneumo- 
bacillus of Friedlaender. He also succeeded in producing typical 
pneumonia by injecting cultures of moderate virulence intrave- 
17 



258 SPECIFIC MICRO-ORGANISMS 

nously into rabbits. Recently Lamar and Meltzer 1 have induced 
typical lobar pneumonia in dogs by introducing cultures of the 
pneumococcus into the bronchi. 

The pneumococcus is somewhat variable in form. In the 
animal body it occurs in pairs of lance-shaped individuals with 
the points directed away from each other, and the pair is surrounded 
by a thick gelatinous capsule. 2 The organism is always Gram- 
positive. In cultures the capsules are less well developed and 
often cannot be demonstrated at all. The individuals are often 




FIG. 105. Pneumococcus, showing capsule, from pleuritic fluid of infected rabbit, 
stained by second method of Hiss. 

less pointed and frequently resemble short bacilli in form. They 
may remain attached together in chains of six to eight cells. 

Cultures may be obtained on ordinary media but they are 
prone to die out quickly. Blood-agar, serum agar or ascitic-fluid 
agar are the best solid media, but even with these weekly trans- 
plantation is usually necessary. Broth to which serum or ascitic 

1 Journ. Exp. Med., 1912, Vol. XV, pp. 133-148. 

2 In demonstrating the capsules, the method of Hiss gives excellent results. 
Spread some blood or tissue juice on a cover -glass and as soon as the film of moisture 
has disappeared, fix the preparation by heat. Then stain with hot aqueous gentian 
violet and wash off the dye with a 20 per cent solution of copper sulphate. Examine 
in the copper solution. Blot the preparation, dry it in air and mount in balsam. 



COCCACE.E AND THEIR PARASITIC RELATIONSHIPS 259 

fluid has been added forms an excellent medium. There is prac- 
tically no growth below 25 C. On blood agar, the colony js 
surrounded by a zone of greenish discoloration, a character of 
great value in the early recognition of the pneumococcus isolated 
from the body. The virulence of the microbe diminishes very 
rapidly in artificial culture. Virulent material is best kept in 
stock by preserving in a desiccator dried blood taken from a 
rabbit dead of pneumococcus infection. The fluid blood may also 
be kept in sealed capillaries in the refrigerator. By these methods 
the virulence may be preserved for months. Rabbits, mice and 
young rats are the most susceptible animals. 

The pneumococcus is the microbic agent in from 80 to 95 
per cent of cases of acute lobar pneumonia. It also occurs in 
otitis media, mastoiditis, meningitis, peritonitis and arthritis. 
Its presence is usually associated with a fibrino-purulent exudate. 
In severe pneumonia it is often present in the circulating blood. 

Pneumonia, or inflammation of the lungs, may be caused 
by a great variety of organisms, the tubercle bacillus, the pneu- 
mobacillus of Friedlaender, the streptococcus, the typhoid 
bacillus and many others. Typical lobar pneumonia, however, 
a disease characterized by a definite sequence of pathological 
changes in the lung and by a rather typical clinical course, is 
rarely caused by any organism other than Diplococcus pneumonia. 
This is a very frequent disease in adults and doubtless the most 
frequent cause of death in persons over 50 years of age. 

The nature of the poisons produced by the pneumococcus 
is not definitely known. When killed by heat, the dead germ 
substance is not very toxic. One very remarkable property of 
the organism is its susceptibility to the action of bile and solutions 
of bile salts. These cause a complete and prompt solution of 
suspensions of pneumococci. Cole 1 has shown that a powerful 
poison is set free by this disintegration of pneumococci, the 
toxic action of which resembles that seen in the phenomenon 
of anaphylaxis. 

1 Cole: Journ. Exp. Med., 1912, Vol. XVI, pp. 644-664. 



260 SPECIFIC MICRO-ORGANISMS 

It has been possible to induce a high degree of immunity in 
horses, and the serum of these animals is protective and to some 
extent curative in animal experiments. Practically it has as 
yet no place in the treatment of human infections with the 
pneumococcus. 

Streptococcus Viridans. Schottmueller 1 has found a strepto- 
coccus, resembling in some respects the pneumococcus, in the 
blood of cases of subacute endocarditis or endocarditis lenta. 
On the blood-agar plates the colonies appear after two to five 
days as opaque granules surrounded by a cloudy but distinctly 
greenish zone. The organism is being found very frequently 
in cases of subacute endocarditis, 2 and is apparently the specific 
cause of this particular fairly well-defined type of endocarditis. 

Streptococcus Mucosus. Schottmueller 3 has isolated a strep- 
tococcus from various purulent processes, which not only pos- 
sesses a mucoid capsule in the living body, but also shows very 
distinct capsules in artificial culture. The size of the cells is ex- 
ceedingly variable. Serum agar or ascitic-fluid agar are necessary 
for successful culture. 

Streptococcus Pyogenes. Bacteria were observed in pyemic 
abscesses by Rindfleisch in 1866 and in the following years this 
observation was confirmed by numeTOUs pathologists. Klebs 
(1870-71) recognized the u Micro sp or on septicum" as the cause of 
wound infections and the accompanying fever, as well as the 
resulting pyemia and septicemia. Ogston (1882) first clearly 
distinguished between the chain-form, streptococcus, and the 
grape-form, staphylococcus, of the pus cocci, not only on the 
basis of their grouping but also in respect to the types of inflamma- 
tion with which they are associated. Pure cultures were first 
obtained by Fehleisen (1883) from erysipelas (Streptococcus 
erysipelatos) and by Rosenbach (1884) from the pus of wounds 
(Streptococcus pyogenes). The former produced typical erysipe- 
las by inoculating the human skin with his cultures. There 

1 Muenchener med. Wochenschr., 1903, (I), No. 20, p. 849. 

2 Major, Johns Hopkins Hasp. Bull., 1912, Vol. XXIII, pp. 326-332. 
*Mnench. med. Wochenschr., 1903, Bd. L, S. 849-853; S. 909-912. 



COCCACE^E AND THEIR PARASITIC RELATIONSHIPS 261 

is no specific distinction between the streptococci found in ery- 
sipelas and those found in other lesions. The difference in the 
pathological process depends rather upon the portal of entry of 
the infection, the virulence of the microbe and the resistance of 
the host. 

Streptococcus pyogenes lives naturally upon the mucous 
membranes, especially in the pharynx, nose and mouth, the 
intestine and on the vaginal mucosa. Such streptococci found 
in normal individuals are relatively non-virulent. Virulent 
streptococci occur in erysipelatous lesions of the skin, in infected 
wounds, on the inflamed pharyngeal mucosa, and in the lochia, 
uterine wall and in the circulating blood in puerperal fever. 
Streptococci are frequently found in pyemic abscesses, bacteremia, 
meningitis and pneumonia. It seems probable that these virulent 
races originate from the ordinary relatively harmless parasitic 
forms in some instances, when an opportunity is presented for 
successful invasion of tissues by a lowered resistance of the host, 
and that by successive transfer from one susceptible individual to 
another the virulence is still further enhanced. 

The individual cells of a chain vary in size from 0.6 to 1.5^ 
and in form from flattened disks to long ovals. The chains are 
variable in length and in general the more virulent types form 
longer chains in broth cultures. In old cultures the cells are very 
irregular in size, and it was once supposed that the larger spheres 
were special resistant forms, "arthrospores." They are now 
regarded as involution or disintegrating forms. The streptococcus 
stains readily and is Gram-positive. 

Cultures on ordinary media are relatively poorly developed 
and of short life. Broth or glucose broth serves very well, and 
a few cultures in series may be obtained on glycerin agar or glu- 
cose agar. Loffler's blood serum is better than these. Serum 
agar, ascitic-fluid agar and blood agar are the best solid media 
and ascitic-fluid broth is an excellent fluid medium for cultiva- 
tion of streptococci. Blood agar is especially valuable in plating 
pus or exudates because of the rather characteristic appearance 



262 SPECIFIC MICRO-ORGANISMS 

of the small colony surrounded by a very clear zone of hemolysis 
which the streptococcus produces on this medium. In making 
cultures from the blood in bacteremia, plain agar previously 
melted and cooled to 45 C. is mixed with freshly drawn blood 
of the patient and allowed to solidfy in a Petri dish. In other 
cases naturally sterile defibrinated rabbit's blood may be used, 
the technic of plating being analogous to that described for the 
gonococcus. The streptococcus grows very slowly below 20 C. 
and poorly in ordinary gelatin, which it does not liquefy. On 
solid media, agar or serum-agar, at 37 C., small round elevated 
colonies develop, 0.5 to i.o mm. in diameter, and they tend to 
remain discreet. In broth only a slight cloud develops, but 
considerable granular deposit made up of streptococci is found 
at the bottom of the tube. Various carbohydrates are fermented 
with the production of acid and without formation of gas, but 
the behavior of streptococci toward these substances seems so 
variable that the attempts to utilize the fermentative power as 
a basis for classifying the streptococci has not led to wholly satis- 
factory results. The differences in fermentative power seem to 
depend more upon vigor of growth than upon essential qualita- 
tive differences between the streptococci tested. 1 

The streptococcus is relatively very resistant to heat, at times 
requiring one to two hours heating at 65 C. or one hour at 70 
C. in order to insure sterility, according to V. Lingelsheim. 
Most investigators have found 60 C. for twenty minutes suffi- 
cient. Its poisons seem to be chiefly intracellular and set free 
upon disintegration of the organisms. Soluble poisons have 
nevertheless been found in some cultures. 

Laboratory animals are not very susceptible to inoculation 
with streptococci. White mice and rabbits are most useful, 
and they ordinarily succumb to intraperitoneal injection of 
virulent strains. 

The enormous importance of the streptococcus as a cause 
of sickness and death before the aseptic era is difficult to realize 

1 V. Lingelsheim in Kolle und Wassermann, Handbuch, 1912, Bd. IV, S. 462. 



COCCACE^E AND THEIR PARASITIC RELATIONSHIPS 263 

at the present time. Veritable epidemics of streptococcus in- 
fection in the surgical and obstetrical wards of hospitals made 
this one of the most dreaded of diseases. Even to-day the 
virulent streptococcus is held in great respect by many surgeons, 
and cases of erysipelas and other recognizable active streptococcus 
infections are commonly excluded from surgical wards. 

Erysipelas is an acute febrile disease characterized by a local 
redness and edema of the skin which tends to spread to contigu- 
ous areas. In the lymph spaces beneath the epithelium there is a 
collection of leukocytes and serum, and the streptococci are also 
found here, especially at the periphery of the reddened area. 
In follicular tonsilitis and many cases of pseudo-membranous 
angina as well as in the pharyngitis of scarlet fever, streptococci 
occur in large numbers, and doubtless bear a causal relation to 
at least a part of the pathological process. In true diphtheria, 
streptococci seem to play rather frequently the role of important 
secondary invaders. From the pharynx the streptococcus 
may gain access to the middle ear and the rnastoid cells, to the 
meninges, to the trachea, bronchi and lungs, setting up purulent 
inflammations in any of these locations. It is an important 
secondary invader in pulmonary tuberculosis. The streptococcus 
seems also to cause enteritis, particularly in infants. In the 
puerperium, streptococci are practically always present in the lochia. 
In spite of many attempts to differentiate between virulent 
and non-virulent types in this situation, it is still impossible to 
distinguish them. Probably local conditions in the uterus as 
well as the general condition of the pateint have much to do in 
determining her resistance to infection of the uterine wall with 
these normal streptococci. Undoubtedly the frightful epidemics 
of puerperal fever in some hospitals previous to 1875 was due to 
the transference of virulent organisms from patient to patient 
by the attending physicians and nurses. This was first suggested 
by Holmes (1843) an d more definitely proven by Semmelweiss 
(1861), but their ideas received little credence until the last 
quarter of the nineteenth century. Streptococcus bacteremia 



264 SPECIFIC MICRO-ORGANISMS 

is commonly a terminal phenomenon, but it may occur without 
immediate fatal issue, and may result in endocarditis and strepto- 
coccus arthritis. 

Immunity to streptococcus infection is slight in degree and 
very temporary. Koch showed that erysipelas could be repeat- 
edly produced on the same area of the skin by inoculation at inter- 
vals of 10 to 12 days. Rabbits and horses acquire a high degree 
of immunity when treated with gradually increasing doses of 
many different strains of streptococci. The serum of such 
animals has a marked protective influence when injected into 
animals and has been employed in treating human infections, 
in some cases with success, while in others the serum has appar- 
ently exerted no influence on the course of the disease. In local- 
ized chronic streptococcus infections, treatment with autogenous 
bacterial vaccines (bacteria suspended in salt solution and killed 
by heat) seems to produce favorable effects in some cases. 

Streptococcus Lacticus (Micrococcus Ovalis). This is a 
variety of Streptococcus pyogenes growing normally in the intes- 
tine and of special importance as the cause of the normal souring 
of milk. 

Staphylococcus (Micrococcus) Aureus. By the early ob- 
servers (Rindfleisch, Klebs) this organism was not distin- 
guished from the streptococcus. Pasteur in 1880 obtained it in 
broth cultures from pus. Ogston in 1882 clearly distinguished it 
from the streptococcus. Rosenbach (1884) by his extensive inves- 
tigations established the position of the Staphylococcus as a 
cause of wound infection and of osteomyelitis. 

Staphylococci have their natural habitat on the skin, in the 
mouth, in the nasal cavities and in the intestine, without the 
presence of inflammation. More virulent forms occur in in- 
fected wounds, furuncles, carbuncles, various localized purulent 
inflammations, bacteremia (staphylococcemia), endocarditis, 
osteomyelitis, meningitis and pneumonia. 

The cell is spherical, 0.7 to 0.9," in diameter. Division 
takes place in various planes, giving rise to irregular bunches of 



COCCACE^: AND THEIR PARASITIC RELATIONSHIPS 265 

cocci. The organism stains readily and is Gram-positive. Cul- 
tures are readily obtained on all the common media and growth 
occurs between 9 and 42, best at 37 C. Broth is diffusely 
clouded with abundant sediment. In gelatin stab-culture, 
growth occurs all along the line of inoculation 
with funnel-shaped liquefaction (Figure 106). 
On agar slant the growth is confluent and 
yellowish after 24 hours. There is similar 
growth on Loffler's serum, often with lique- 
faction of the medium. 

The staphylococcus is relatively resistant 
to heat and chemical germicides. It is killed 
at 62 C. in ten minutes and at 70 C. in five 
minutes. V. Lingelsheim 1 found it more resis- 
tant, requiring ten minutes at 80 C. and an 
hour at 70 C. to kill his strains, but his fig- 
ures cannot be accepted without further con- 
firmation. 2 It is about as resistant to chemical 
poisons as any of the sporeless bacteria, and 
is commonly employed as a test object in the 
investigation of germicides. Mercuric chlo- 
ride i- 1 ooo requires three to five hours to kill 
staphylococcus cultures and much longer if 
the organisms are present in pus. Carbolic FlG I0 6. Gelatine 
acid, 3 per cent, kills them in two to ten culture staphylococcus 

aureus one week old. 

minutes. 

The pigment is a lipochrome and is produced only in the 
presence of oxygen. The tryptic ferment diffuses out of the cells 
and is capable of liquefying gelatin, albumen and fibrin. The 
staphylococcus produces a soluble poison which kills leukocytes 
(leukocidin) and others which dissolve red blood cells (staphy- 
lolysin) and cause clumping of red blood cells (agglomerin) . 
These substances are true soluble toxins and they are destroyed 

1 Neisser: Kolle und Wassermann, Handbuch, 1912, Bd. IV, S. 361. 

2 Compare with similar tests on streptococci by v. Lingelsheim, p. 262. 




266 SPECIFIC MICRO-ORGANISMS 

by heating to 80 C. Other soluble poisons seem also to be pre- 
sent. The bacterial cells killed by heat are only slightly toxic, 
yet it is very probable that in the disintegration of the cocci in 
an inflammatory process more poisonous substances may be 
derived from their cell protein. 

Rabbits are the animals of choice for inoculation with staphy- 
lococci. Intravenous injection with virulent cultures usually 
causes multiple abscesses in the internal organs with death in 
4 to 8 days. Typical endocarditis has been produced by injected 
organisms from potato cultures, and with greater certainty when 
the heart valves are injured mechanically, especially in young 
rabbits. Osteomyelitis sometimes follows intravenous injection 
in growing rabbits, especially if the bone be slightly injured 
at the time of inoculation. In man, typical furuncles and carbun- 
cles have been produced by rubbing pure cultures on the skin 
(Garre 1885) and by subcutaneous injection. 

In man this organism is a frequent cause of local purulent 
inflammations, and it sometimes gives rise to pyemic abscesses 
and general bacteremia. Recurrent furuncles and carbuncles 
are ordinarily due to staphylococci. 

Animals have been immunized to staphylococci but the serum 
obtained from them has relatively slight value in treatment. 
Specific treatment by means of dead bacterial cells, bacterial 
vaccines, has been developed by A. E. Wright and has proved 
its value in the treatment of chronic furunculosis. A suspension 
in salt solution of bacterial cells from an agar slant, sterilized 
by heating to 60-65 C. for 30 minutes and standardized by 
microscopic count of the bacterial cells, is employed. Doses 
from 50 million to 1000 million bacterial cells are injected two 
or three times a week for a long period of time, the size and fre- 
quency of dosage being governed by the clinical condition of the 
patient. Determination of the opsonic index is probably un- 
necessary and is now quite generally neglected. Autogenous 
vaccines (made with the staphylococcus isolated from the patient) 
are usually superior to stock vaccines. 



COCCACE.E AND THEIR PARASITIC RELATIONSHIPS 267 

Staphylococcus Albus. This is quite similar to Staphylococcus 
aureus in all respects except pigment production. Usually, 
but not always it is less virulent. Staph. epidermidis (Welch) is 
an avirulent variety of Staph. albus, very abundant on the normal 
skin. Many other varieties of staphylococci have been described. 

Micrococcus Tetragenus. This organism occurs in lung 
cavities in phthisis, and in the sputum, usually in groups of four 
cells, tetrads, enclosed in a transparent capsule. It is Gram- 
positive, grows on ordinary media and does not liquefy gelatin. 
White mice and guinea-pigs are susceptible and ordinarily die 
of general bacteremia in two to six days after inoculation. The 
pathogenic role of the organism in man is doubtful. 

Sarcina Ventriculi. Goodsir in 1842 observed sarcines in 
vomitus. The coccus is large, 2.5^ in diameter, and occurs in 
cubes of eight cells or as large conglomerates of these. It grows 
on ordinary media, usually producing a yellow pigment. It 
is found in the stomach in some conditions in which the acidity 
of the gastric juice is diminished. It is apparently non-pathogenic. 

Sarcina Aurantiaca. This is a common saprophytic coccus 
found in fermenting liquids and occasionally in the air. It 
grows well on ordinary media and liquefies gelatin. An orange 
pigment is produced. Typical packets are produced in liquid 
media, especially in hay infusions. 

Micrococcus (Planococcus) Agilis. This organism occurs 
in surface waters. It liquefies gelatin and produces a rose-red 
pigment on agar and potato. Its remarkable feature is the 
possession of a flagellum and active motility. It is Gram-positive. 



CHAPTER XVII. 
BACTERIACE^E: THE SPOROGENIC AEROBES. 

The aerobic spore-forming bacilli are essentially inhabitants 
of the soil and the fermenting organic material likely to occur 
there. Along with a few species of this* group we shall consider 
one pathogenic sporogenous bacterium, the anthrax bacillus, 
which resembles them very closely except in its virulence for 
animals and its lack of active motion, both of which may perhaps 
justly be regarded as variations from the group type due to its 
parasitic mode of life. 

Bacillus Mycoides. This organism is universally distributed 
in fertile soils and also occurs in surface waters and in the air. 
It is a large rod with rounded ends, usually growing in threads. 
Large median spores are formed without distorting the cell. 
It is motile but rather sluggish. Growth occurs on all ordinary 
media. In gelatin stab-culture, thread-like processes extend 
out on all sides from the line of puncture giving the appearance 
of an inverted pine tree. Later the gelatin becomes entirely 
liquefied. The organism is an important agent in the decompo- 
sition of plant residues in the soil. It is without pathogenic 
properties. 

Bacillus (Mesentericus) Vulgatus. This is another widely 
distributed soil bacterium. It is commonly called the potato 
bacillus. The cell is short and relatively thick with rounded 
ends, actively motile, often in pairs or threads. Large spherical 
median spores are produced without distortion of the cell. 
These spores are very resistant to heat and germicides, sometimes 
surviving the temperature of boiling water for several hours. 
B. vulgatus grows well on all ordinary media. Gelatin is liquefied. 
Milk is coagulated and then digested. On potato a wrinkled 

268 



BACTERIACE^E : THE SPOROGENIC AEROBES 



269 



membrane is produced, so characteristic that the name "mesen- 
tericus" was applied to this species. It is not pathogenic. 

Bacillus Subtilis. Bacillus subtilis, or the hay bacillus, is 
abundant in the soil and on the surface of plants, and common 
in surface waters and in the air. It is readily obtained by boiling 
hay in water and then setting the infusion aside for a few days. 
The cell is relatively large, about 1.2," wide by 5^ long, with 
ends somewhat rounded. Long threads are commonly formed. 
It is motile with peritrichous flagella. Large oval median spores 




FIG. 107. Bacillus subtilis. Xiooo. 

are formed without distortion of the cell and these are almost 
as resistant as the spores of the potato bacillus. B. subtilis 
grows rapidly on ordinary media in the presence of air, best at 
about 30 C. Gelatin is liquefied and milk is digested. The 
organism is typically saprophytic, but it has been found growing 
in the intestine by some investigators, and has been found in a 
few instances in infections of the human eye, cases of pan- 
ophthalmitis following injury. 1 

1 Silber schmidt: Annales de V I nstitut Pasteur, 1903, Vol. XVII, pp. 268-287; 
Also see Kneass and Sailer: Univ. Penn. Med. Bull., June, 1903, Vol. XVI, pp. 



270 SPECIFIC MICRO-ORGANISMS 

Bacillus (Bacterium) Anthracis. Pollender in 1849 and 
Davaine and Rayer in 1850 observed thread-like bodies in the 
blood of animals dying of anthrax. Robert Koch in 1876 obtained 
pure cultures of the organism, using the aqueous humor of the 
ox's eye as culture medium. He saw the small rod-shaped bodies 
found in the anthrax blood elongate into threads in this medium, 
and observed the formation of the bright refractive bodies in 
these threads, which he correctly recognized as spores. Finally 
by inoculating healthy animals with his cultures he produced 



FIG. 108. Anthrax bacilli in the capillaries of the liver of a mouse. 

typical anthrax in them, thus proving conclusively for the first 
time the causal relation of a bacterium to a disease. 

The anthrax bacillus occurs in the blood and throughout the 
tissues of animals suffering from anthrax, and in the excretions 
of such animals. Its spores occur on hides and in wool derived 
from anthrax animals. Furthermore, the soil of fields where 
anthrax animals have grazed harbors these organisms for many 
years. It seems probable that the bacilli multiply in the soil 
during the warm wet seasons and it is certain that the spores 
may lie dormant for as long as ten years in dry places. 



BACTERIAC.E: THE SPOROGENIC AEROBES 



271 



The cell is about 1.25^ wide and 3 to io, long, with rounded 
ends when single, but in the threads the contiguous ends are 



.;% . * V*** /*- 

" ' ' 







FIG. 109. Bact. anthracis. Spore production. (From Marshall after Migula.} 

square-cut. In the circulating blood the bacilli are single or in 
pairs and spores are never formed in the animal body (Fig. 108). 
In cultures long threads are produced and spores are usually 




FIG. no. Bact. anthracis. Colony upon a gelatin plate. Xioo. (After Fraenkel 

and Pfei/er.) 

formed after 24 to 48 hours (Fig. 109). The anthrax bacillus 
is aerobic and grows readily on all ordinary media, best at 37 C. 



272 SPECIFIC MICRO-ORGANISMS 

Gelatin is slowly liquefied. The colony presents a very char- 
acteristic appearance, especially as it grows on gelatin, which is 
due to the large coils of long parallel threads, of which the colony 
is composed. The vegetative bacillus is rather easily killed but 
the spores may survive boiling in water for 5 minutes and in 
some instances as long as half an hour when afforded some 
mechanical protection. Chemical germicides cannot be relied 
upon to destroy the spores. Sterilization in the autoclave is 
the safest method of disposing of anthrax material. 

Anthrax is a disease which occurs spontaneously in cattle and 




FIG. in. Bact. anthracis. Showing the thread formation of colony. (After Kolle 

and Wassermann.) 

sheep and rarely in horses, swine and in man. The disease is 
produced by inoculation in many other animals. Mice, guinea- 
pigs and rabbits are susceptible in the order named. The disease 
is common in European and Asiatic stock-raising districts and 
in Argentine Republic. Several local epizootics have occurred in 
the United States and a few cases of human anthrax. Experi- 
mental anthrax is readily produced in susceptible animals by 
subcutaneous inoculation, less certainly by feeding the spores. 
In the acute form the bacilli are found in large numbers every- 
where in the blood, and this is the common picture in cattle, 
sheep, rabbits, guinea-pigs and mice. Chronic forms occur, 



BACTERIACE^:: THE SPOROGENIC AEROBES 273 

however, either because of lowered virulence of the germ or of 
increased resistance of the host, and in these cases the bacteria 
may be very scarce and difficult to find microscopically, even 
after death of the animal. Cultures from the spleen will usually 
show the presence of the bacillus there. The mechanism by 
which the bacillus causes death is unknown. In the acute cases, 
as in the mouse, the bacilli are so abundant in the blood that 
mechanical interference with the circulation seems a plausible 
explanation, but this certainly does not suffice for other types of 
the disease in which chemical poisoning must play the chief 
role. So far it has not been possible to demonstrate any powerful 
poisons in cultures of the anthrax bacillus. It is probable that 
the essential poisons are produced by a reaction between the 
substance of the bacillus and the fluids of the host, particularly 
the enzymes of the latter, which cause disintegration of the bac- 
terial bodies. 

The infection is acquired by grazing animals through the 
alimentary tract primarily, but also to some extent by inoculation 
(contact, flies, intermediate objects). In man there are three 
recognized types (a) malignant pustule, (b) pulmonary anthrax, 
and (c) intestinal anthrax. Malignant pustule results from in- 
oculation of the skin, especially in those who handle hides or care 
for anthrax animals. It is at first a local pustular and necrotic 
lesion tending to involve contiguous tissue by extension, but soon 
invading the lymph vessels and walls of the veins. The bacteria 
thus gain the blood stream and a rapidly fatal general bacteremia 
supervenes. Recovery sometimes occurs before the disease be- 
comes generalized. Pulmonary anthrax is caused by inhalation 
of anthrax spores (woolsorter's disease). Intestinal anthrax 
is uncommon in man but has occurred. Both are very fatal 
forms of the disease. 

Immunity to anthrax was first successfully produced by Pas- 
teur through vaccination with attenuated living cultures. Broth 
cultures inoculated with bacilli taken directly from the animal 
body were grown at 42C to 43C. At this temperature spores 

18 



274 SPECIFIC MICRO-ORGANISMS 

are not produced and the bacillus gradually loses its virulence. 
When it will no longer kill guinea-pigs but will still kill mice the 
strain is again grown at 37C. and injected into cattle and sheep 
as the first vaccine. Twelve days later a second vaccine is in- 
jected, which is a somewhat more virulent culture, still capable 
of killing guinea-pigs but not powerful enough to cause fatal in- 
fection of rabbits. As a result of these two treatments, nearly 
all animals become immune to the natural disease or to inocula- 
tion with fully virulent cultures. Sobernheim 1 and Sclavo 2 have 
induced a high degree of immunity in sheep and in asses by re- 
peated injections of the bacilli, and have found the serum of such 
hyper-immune animals to be protective and curative upon in- 
jection into other animals. The injection of this serum along 
with a dose of living culture of about the strength of Pasteur's 
second vaccine has been employed in immunizing cattle and 
sheep. All the necessary treatment is thus given at one time. 
The serum has also been successfully employed in conjunction 
with the appropriate medical and surgical measures in the treat- 
ment of malignant pustule in man. 3 

Sobernheim: Zeitsch. f. Hyg., 1897, XXV, pp. 301-356; Centralbl. f. Bakt., 
1899, XXV, p. 840. 

2 Sclavo: Centralbl. f. Bakt., 1899, XXVI, p. 425. 

3 For a discussion of treatment of human anthrax consult Boidin, Vignaud and 
Fortineau, Presse Medicale, Aug. 14, 1912; also Becker, Munch, med. Wochenschr., 
Jan. 23, 1912. 



CHAPTER XVIII. 
BACTERIACE^: THE SPOROGENIC ANAEROBES. 

The bacteria of this group are hindered in their development 
by the presence of free oxygen and their artificial culture is ordi- 
narily successful only when they are protected from oxygen, at 
least in the early stages of development. Like the sporogenic 
aerobes, they live in the soil, but they are associated here more 
especially with decomposing materials of animal origin, and are 
less frequently found in soils which have not received fertilizers 
from animal sources. There is good reason to believe that their 
essential habitat is the intestinal canal of animals, especially the 
mammals, and that their life in the soil does not represent the 
most active stage of their existence, but that they reach the soil 
with animal excreta and the bodies of dead animals and continue 
to live in the soil for a considerable period. 

Bacillus Edematis. Pasteur in 1877 injected infusions of 
putrid flesh into laboratory animals and produced a fatal sub- 
cutaneous edema with penetration of the bacteria into the blood 
in some instances. The organism which he called "Vibrion 
septique" was found to be an obligate anaerobe, the first anae- 
robic organism ever recognized. Koch (1881) studied the organism 
in pure culture on solid media and named it Bacillus edematis 
maligni. The recognized type organism is that studied by 
Koch. 

The bacillus is very widely distributed in soil and dust, and 
is very common in the feces of herbivorous animals. It is es- 
pecially abundant in putrefying animal matter. The cell is about 
i/x thick by 3/i in length, although considerable variation in size 
and shape occurs. It is usually slightly motile and possesses 
peritrichous flagella, stains readily, is only relatively Gram-posi- 

275 



276 SPECIFIC MICRO-ORGANISMS 

tive, some of the cells being decolorized by prolonged treatment 
with alcohol. The spores are central, or intermediate in position 
with bulging of the cell. 

In cultures B. edematis is a strict anaerobe. It liquefies gela- 
tin. Milk is slowly coagulated and the coagulum digested, the 
reaction remaining alkaline to litmus. The cultures have a foul 
odor. The spores withstand boiling sometimes for 2 to 3 hours. 
The morphological and physiological properties of this organism 
are quite variable and the many intermediate types between it 
and B. feseri make distinction between the two species somewhat 
difficult. 

In animals and man, malignant edema occurs spontaneously 
as a wound infection, but it is not very common. It has been 
observed most frequently in horses and in new-born calves. The 
guinea-pig is susceptible. In general a mere injection of the 
bacilli fails to produce serious disease. The presence of foreign 
bodies or extensive tissue destruction favors the infection. 

Bacillus Feseri. Feser and Bollinger (1875-1878) observed 
the large narrow rods in the diseased tissues and exudates of 
symptomatic anthrax or black leg, a fatal disease of cattle and 
sheep. Man is not affected. Arloing, Cornevin and Thomas 
(1884) obtained the organism in culture. The organism is a 
strict anaerobe and resembles B. edematis very closely. Black 
leg is a local emphysematous inflammation usually beginning in 
one leg of cattle or sheep, rapidly extending and resulting in death 
as a rule. Immunity is obtained by injecting small doses of the 
virulent bacteria or by injecting attenuated organisms, and also 
by injecting the virus together with an immune serum. 1 

B. Welchii. Welch and Nuttall in 1892 discovered this organ- 
ism at autopsy in a body showing general emphysema of the 
tissues and gas bubbles in the blood-vessels. They obtained 
cultures by anaerobic methods and caused similar post-mortem 
emphysema in the bodies of rabbits. The organism lives and 
multiplies in the intestine of man and other mammals, is widely 
1 Kitt, Kolle and Wassermann, Handbuch, 1912, Bd. IV, S. 819-836. 



BACTERIACE^E : THE SPOROGENIC ANAEROBES 277 

distributed in the soil and is commonly present in milk and other 
animal food products. The cell is a large rod surrounded by_a 
capsule when grown on media rich in protein or in the animal 
body. The width of the cell (without capsule) varies 1 from i.i to 
i.fu with a mean of 1.3^ and the length from 2.6 to 7.6/1, with an 
average of 4.6/z, the measurements being made on organisms 
grown in an agar stab-culture 24 hours at 37 C. When grown 
in blood broth the germ is capsulated and the measurements, in- 
cluding the capsule are as follows: width 1.9 to 2.5/1 with average 
of 2.i;u, and length, 2.8 to 6. 6 /* with average of 4.7/11. Usually the 
organism is non-motile, but flagella can sometimes be demon- 
strated. In the intestine and in protein media the organism 
forms spores, usually median without bulging of the cell, but 
these are not commonly observed in cultures. The organism is 
a strict anaerobe. Its most striking property is the enormously 
rapid production of gas in media containing dextrose or lactose. 
Cultures are obtained most readily by heating a suspension of 
feces to 80 C. for 15 minutes and inoculating it into glucose broth 
mixed with blood in a Smith fermentation tube. After 24 to 48 
hours incubation its presence will usually be revealed by abun- 
dant production of gas. Milk is coagulated and rendered acid 
with an abundant production of gas (stormy fermentation). 
On blood-agar plates incubated in hydrogen, the colony is round 
with regular outline and surrounded by a clear zone of hemolysis. 

Emphysematous gangrene occurs in man as a rapidly extend- 
ing, very fatal disease, due to the infection of wounds with this 
organism. The presence of necrotic tissue seems to be necessary 
in order that the organism may gain a foothold, but when once 
begun the inflammation may extend with great rapidity. The 
gas found in bodies at autopsy is usually the result of an agonal 
or a post-mortem invasion by the bacilli from the intestine. 

There are several other types of sporogenic anaerobes of the 
same general nature as B. edematis, B. feseri, and B. welchii, iso- 

1 The measurements are taken from Kerr, The Bacillus welchii, Thesis, Univ. 
of Illinois, 1909. 



2 7 8 



SPECIFIC MICRO-ORGANISMS 




e soil, from the feces or from 



lated by various workers froi 

putrefying material. 

Bacillus Tetani. Tetanus has 
been recognized as a complication,.^ 
wounds since the time of Hippocrates/, 
Forscher, Carle and Rattone, in 1884,) 
first proved it to be inoculable by in- 
jecting pus from a human case into 1 2 
rabbits, of which n died of tetanus. 
Nicolaier in 1884 produced tetanus by 
injecting soil into mice, guinea-pigs and 
rabbits, and found a slender bacillus in 
the animals at the point of inoculation. 
He was able to propagate the bacillus 
in mixed culture on coagulated sheep's 
serum. Kitasato obtained the first 
pure cultures by subjecting the mixed 
culture to a temperature of 80 C. for 
an hour, inoculating agar plates and 
incubating them in an atmosphere of 
hydrogen. With his pure cultures, he 
caused typical tetanus in animals. 

The organism occurs in the soil 
which has received animal fertilizers 
and in the intestine of herbivorous 
mammals. The bacterial cell is 0.3 to 
0.5^1 wide and 2 to 4/4 long, single in 
young cultures, but ,often joined end 
to end to form long threads in older 
cultures. It is motile and possesses 
abundant peritrichous flagella. The 
spore is very characteristic. It is usu- 
ally spherical, i to 1.5 ^ in diameter, 

FIG. ii2. B. welchii in agar 

culture, showing gas formation, situated at the extremity of the cell, 
giving it the appearance of a drumstick. The bacillus stains 
readily and is Gram-positive, 



BACTERIACE.E : THE SPIROGENIC ANAEROBES 279 

Isolation of B. tetani from mixed material or from wounds 
known to contain it is not always easy. The material should 
be planted in glucose broth and incubated in hydrogen at 37 C7 
for 2 to 3 days. Microscopic examination of the sediment may 
then reveal the drumsticks. Kitasato's procedure should then be 
followed, employing agar distinctly alkaline to litmus and con- 
taining 2 per cent of glucose. If many other spore-forming bac- 
teria are present in the mixture, special procedures are necessary, 
such as preliminary culture for 8 days at 37 C. in a deep stab in 
coagulated rabbit's blood with subsequent heating to 80 C. to 
get rid of B. edematis, or culture for 8 days at 37 C.^ih milk with 
subsequent heating to get rid of B. welchii. Aerobic spore-formers 
may be eliminated by successive transfers in animals. 

The spores of B. tetanic esist the temperature of boiling water 
for 5 to 30 minutes. Biological products to be introduced into 
the human body need to be sterilized in the autoclave or else 
carefully examined by anaerobic culture methods to insure their 
freedom from tetanus spores. The danger of infection from this 
source has been emphasized by Smith. 1 

The colony in glucose gelatin or glucose agar consists of a 
compact center with slender, radiating, straight or irregularly 
curved threads about the periphery. Liquefaction of gelatin 
becomes evident in stab-culture after about two weeks at 20 C. 
Milk is sometimes but not always coagulated and the casein is 
eventually digested. 

The cultures of the tetanus bacillus are extremely poisonous, 
especially so when they are developed under very strict anaerobic 
conditions. A nerve poison, tetanospasmin, and a hemolytic 
poison, tetanolysin, are present. The former is the more impor- 
tant constituent of the tetanus toxin. Neutral or slightly alka- 
line plain nutrient broth, incubated in an atmosphere of hydrogen 
for ten days after inoculation gives the most powerful toxin. 
The bacteria-free fluid from such a culture has been found to kill 
a mouse of ic-grams weight in a dose of o.ooo 005 c.c. The toxin is 

1 Journ. A. M. A., Mar. 21, 1908, Vol. L., pp. 929-934. 



280 



SPECIFIC MICRO-ORGANISMS 



unstable in solution but very stable when dried. Dry material 
of which o.ooo ooo i gram is the fatal dose for a mouse is readily 
obtained. The watery solution loses it toxicity when heated to 
60 C. for 20 minutes, but when dry the toxin withstands 
heating at 120 C. for an hour. 

Tetanus presents essentially the same picture in inoculated 
animals as in the natural disease, which is indeed, as a general 
rule, merely an accidental inoculation. The presence of insoluble 
material and of other bacteria mixed with them in a wound favors 
the development of tetanus bacilli. The tetanus bacilli always 




FIG. 1 13. Tetanus bacilli showing terminal spores. (After Kolle and Wassermann.} 

remain localized near the point of inoculation and may be hard 
to find. The poison produced by the organisms is probably ab- 
sorbed by the nerve endings 1 and transmitted to the central nerv- 
ous system through the axis cylinders or in the perineural lymph 
spaces of the motor neurones rather than through the blood 
stream. The symptoms arise after the poison reaches the central 
nervous system in sufficient concentration to stimulate the nerve 
cells. In guinea-pigs and mice the spasm always begins near the 
point of inoculation, but in man and the large mammals it often 
begins in the muscles of the jaw and neck regardless of the location 

1 Von Lingelsheim, Kolle and Wassermann, Handbuch, 1912, Bd. IV, S. 766. 



BACTERIACE.E I THE SPOROGENIC ANAEROBES 



28l 



of the wound. Wassermann and Takaki have shown that o.i 
gram of brain substance suspended in salt solution is able to neu- 
tralize 10 fatal doses of tetanus toxin, forming a loose combina- 
tion from which the toxin may be set 
free by drying. Most mammals are 
very susceptible, although cats and 
dogs are only slightly so. Birds are 
relatively resistant and some reptiles 
are wholly refractory to the tetanus 
toxin. 

Von Behring and Kitasato in 1890 
produced immunity in rabbits, and 
later in horses, by injecting into them 
toxin to which iodine trichloride had 
been added, and subsequently unal- 
tered toxin. The immunized animal 
was able to survive an injection many 
times greater than the amount neces- 
sary to kill a normal animal. More- 
over, the cell-free blood serum of the 
immunized animal was found to neu- 
tralize the poison in a test-tube and 
to protect a normal animal against 
fatal doses of it. The new substance 
of the blood capable of rendering the 
toxin harmless was called antitoxin. 
One antitoxic unit of tetanus anti- 

, . i T -r i after Fraenkel and Pfeiffer.) 

toxin, according to Von Behring, is 

the amount which will neutralize 40 million times the amount 
of fresh tetanus toxin necessary to kill a mouse weighing 15 
grams (40 million X the 15 + Ms dose) so completely that 
only a slight local contraction, indicated by a folding of the 
skin, results from subcutaneous injection of the mixture into a 
mouse (the L effect). This amount of toxin (40 million X the 
15 + Ms dose) is generally measured in practice against a standard 




FIG. 114. Bacillus tetani. 
Stab culture in glucose gelatin, 
six days old. (From McFarland 



282 SPECIFIC MICRO-ORGANISMS 

antitoxin and is designated as a toxic unit. The toxin is pre- 
served in a dry state. To test a new antitoxin one employs 
ToVo- of a toxic unit (40, ooo X the 15 + Ms dose) and ascertains 
the amount of serum which must be added so as to neutralize it 
to the L end point. Each trial mixture is diluted to i c.c. with 
salt solution and 0.25 c.c. per 10 grams of body weight is injected 
into a mouse. When the typical L effect is produced in the 
mouse, the amount of antitoxic serum employed in the prepara- 
tion of this particular mixture is said to represent ToW anti- 
toxic unit. Ordinarily the mixtutre of toxin and antitoxin is 



A < -\ 




FIG. 115. Bacillus botulinus. Some individuals containing spores. (After van 

Ermengem.) 

allowed to stand 30 minutes before injection. Comparable re- 
sults are obtained only by following a definite procedure and it 
is especially necessary to use the conventional dose of roW 
antitoxic unit and -nrVo toxic unit in the standardization of 
sera. 

The standard unit employed in the United States is some- 
what different from the Von Behring antitoxic unit. The Ameri- 
can immunity unit of tetanus antitoxin is ten times the least 
amount of antitetanic serum necessary to preserve the life of a 
guinea-pig weighing 350 grams for 96 hours against the official 



BACTERIACE^E : THE SPOROGENIC ANAEROBES 283 

test dose of standard tetanus toxin furnished by the Hygienic 
Laboratory of the U. S. Public Health Service. 1 Tetanus anti- 
toxin deteriorates with moderate rapidity. The reaction be- 
tween tetanus toxin and antitoxin seems to take place in two 
stages, first a reversible absorption and following this a specific 
chemical union. 

Tetanus antitoxin seems to be an absolute preventive of teta- 
nus if given soon after the wound is inflicted in a dose of 20 anti- 
toxic units (German) or 1500 immunity units (U. S. Standard). 
After symptoms of tetanus have appeared, antitoxin is of less 
use. At this time the poison is present not only in the vicinity 
of the wound and in the blood but also in the peripheral nerves 
and in the central nervous system. The toxin in the last two situ- 
ations is only slightly or not at all influenced by subcutaneous in- 
jection of antitoxin. That in the peripheral nerves may be reached 
by intraneural injection, and in subacute or chronic cases recovery 
may sometimes take place. Acute cases in which symptoms 
appear in a few days after infliction of the wound offer no hope. 
Prophylactic use of tetanus antitoxin in all punctured and lacer- 
ated wounds, especially those caused by gunpowder (Fourth of 
July) is an essential feature of the effective treatment for tet- 
anus. Surgical cleansing and antiseptic open treatment of such 
wounds is to be recommended. 2 

s 

Bacillus Botulinus, Van Ermengem in 1895 discovered the 
spores of this organism in the intermuscular connective tissue 
of a ham which had given rise to 30 cases of food poisoning with 
3 deaths. Other anaerobic as well as aerobic bacteria were also 
present in the meat. Its natural habitat is unknown but it seems 
to occur in the feces of swine. The bacillus is 0.9 to i.2/* wide 
by 4 to 6/z long and occurs single or in pairs. It is slightly 
motile and has 4 to 8 peritrichous flagella. It is Gram-positive. 
The spores are oval and usually nearer one end of the cell. They 

1 Rosenau and Anderson: U. S. Hygienic Laboratory, Bulletin No. 43, 1908, p. 59. 
The official test dose of toxin is 100 times the amount of a dry tetanus toxin required 
to kill a 350 gram guinea-pig in four days. 

2 Editorial, Jour. A. M. A., 1909, Vol. LIU, p. 955. 



284 SPECIFIC MICRO-ORGANISMS 

are only feebly resistant, being killed at 85C. in 15 minutes and 
by 5 per cent carbolic acid in 24 hours. 

Strict anaerobiosis is necessary for successful culture, except 
when B. botulinus grows in symbiosis with aerobes. Growth is 
best at 25-30 C., very slight at 37-38.5 C., and best in a 
medium slightly alkaline to litmus. Gelatin is quickly liquefied 
and abundant gas is produced in glucose media. The organism 
appears to be incapable of growth in the animal body. Cultures 
are very poisonous when injected into or fed to animals. 

The poison "Botulin" resembles in some of its properties the 
tetanus toxin. It is destroyed rapidly at yo -8o C., and pre- 
serves its toxicity for years when dried. It is neutralized by 
mixing with brain substance. It differs from the other pow- 
erful toxins, however, in its ability to resist the gastric juice and 
to poison by absorption through the alimentary canal. Forssman 
has immunized guinea-pigs, rabbits and goats, and has obtained 
an antitoxic serum from these animals. 

Botulism is a form of food poisoning definitely recognized as 
such as early as 1820 It has followed the consumption of sau- 
sage, hams, fish and other cured or preserved meats. The symp- 
toms are very characteristic, appearing in 18 to 48 hours after 
ingestion of the poisonous food. There is vomiting, dryness of 
the mouth and constipation, motor paralysis, especially early in 
the external ocular muscles. The involvement of the central 
nervous system may progress to complete motor paralysis and 
death. The mind is usually clear even in the fatal cases. This 
disease is evidently due to the poisons already formed in the food 
at the time it is eaten, and it is to be regarded as an intoxication 
rather than an infection. Van Ermengem designates B. botu- 
linus as a pathogenic saprophyte. 



CHAPTER XIX. 

BACTERIACE^: THE BACILLUS OF DIPHTHERIA AND 
OTHER SPECIFIC BACILLI PARASITIC ON SUPER- 
FICIAL MUCOUS MEMBRANES. 

Bacillus (Bacterium) Diphtherias. Klebs in 1883 discovered 
this organism in the microscopic study of pseudomembranes 
from fatal cases of epidemic diphtheria. Loffler in 1884 obtained 
pure cultures of the bacillus and by inoculating the abraded 
mucous membrane of susceptible animals with his cultures, he 
produced local lesions similar to those observed in human diph- 
theria, in some instances followed by death or paralysis. 

B. diphtheria occurs in the exudate (false membrane) which 
occurs in the pharynx, larynx and adjacent mucous membranes 
in epidemic diphtheria, on the mucous membranes of those who 
have recovered from the disease and, much less commonly, on 
the mucous membranes of healthy throats. It is a rod-shaped 
organism extremely variable in size, shape and staining properties. 
The width is ordinarily between 0.3 and o.8/* and the length 
varies from i to 6ju. The cell is straight or slightly curved and 
very frequently of uneven diameter, with swelling at one end or 
in the middle portion. The cell contents stains unevenly in 
many of the cells. Many different morphological types are thus 
presented which may be designated roughly as regular cylinders, 
clubs, spindles and wedges according to form, and as uniformly 
pale, uniformly dark, regularly or irregularly banded or granular 
according to internal structure of the stained cell. These varia- 
tions in form and internal structure are best seen after staining 
the bacillus with Loffler's methylene blue and are especially 
valuable in the quick recognition of B. diphtheria as it grows in 
the diphtheritic membrane or in culture on Loffler's blood serum. 

285 



286 



SPECIFIC MICRO-ORGANISMS 



On other media, such as glycerin agar, the morphological irregulari- 
ties are less marked as a rule. The organism in young cultures 




FIG. 116. Bacillus of diphtheria. X 1000. 



/ 



FIG. 117. B. diphtheria stained by Neisser's method. 

stains readily, best perhaps with Loffler's methylene blue in 
the cold. It is Gram-positive. Old cultures stain with great 
difficulty. 



BACTERIACE.E : THE BACILLUS OF DIPHTHERIA 



287 



Loffler's blood serum is the medium of choice. The colonies 
develop at 37 C. in 8 to 12 hours as grayish, slightly elevated 
points and become 2 to 3 mm. in diameter in the course of 48 

A B 










t> x 



FIG. 118. Forms of B. diphtheria in cultures on Loffler's serum. A, Charac- 
teristic clubbed and irregular shapes with irregular staining of the cell contents. 
Xnoo. B, Irregular shapes with even staining. X 1000. (After Park and 

Williams.) 

hours. Contiguous colonies become confluent. On glycerin 
agar after 24 hours at 37 C., the colony is coarsely granular 
with a somewhat jagged outline. Many variations from this 





FIG. 119. Forms of B. diphtheria in cultures on agar. A, Bacilli^small and 
uniform. Xiooo. B, Spherical forms in culture 24 hours old. On Loffler's serum 
this same organism produced granular forms. X 1410. (After Park and Williams.) 

typical appearance occur. Growth in gelatin is slow and ceases 
below 20 C. The medium is not liquefied. The bacillus grows 
in milk without producing coagulation. In broth the growth 



288 SPECIFIC MICRO-ORGANISMS 

may occur as a granular sediment, as a diffuse cloudiness or as a 
pellicle on the surface, depending upon the reaction and pepton 
content of the medium and the vigor of growth of the culture. 
The growth on the surface produces the best yield of toxin. 
Acid is produced in dextrose broth. The organism is killed 
when moist by heating to 60 C. for 20 minutes. It is fairly 
resistant to drying and has been found alive in bits of dry diph- 
theritic membrane after four months. 

Roux and Yersin in 1888 filtered broth cultures of the diph- 
theria bacillus through porcelain filters and found the filtrate 




FIG. 120. Colonies of B. diphtheria on agar. X2oo. (After Park and Williams.) 

extremely poisonous. By injecting it into animals they were 
able to produce the signs of local and general intoxication which 
are observed in the natural disease. A favorable medium for 
toxin production is a veal broth containing 2 per cent pepton 
and having a titre of 9 c.c. 1 of normal sodium hydroxide above 
the neutral point to litmus. It should be placed in flasks in a 
thin layer to allow abundant air supply. Incubation for from 
5 to 10 days gives the maximum toxicity. The filtrate from such 
a culture may kill a 250 gram guinea-pig in a dose of 0.002 c.c. 
Less powerful toxin is frequently obtained, so that sometimes 
even 0.5 c.c. or more may be required to kill a guinea-pig, and 

1 Per 1000 c.c. of the medium. 



BACTERIACE^E : THE BACILLUS OF DIPHTHERIA 



289 



some strains of bacilli morphologically indistinguishable from 
B. diphtheria seem to produce no toxin at all. The toxin is 
quickly destroyed by boiling and loses 95 per cent of its strength 
in five minutes at 75 C. It grad- 
ually deteriorates even at low tem- 
peratures. Its chemical nature is 
unknown. Ehrlich has shown that 
old toxin which has lost much of its 
poisonous property is still able to 
combine with as much antitoxin as 
before. This deteriorated toxin is 
called toxoid. He explains the phe- 
nomenon by assuming the existence 
of two distinct chemical groups in 
the toxin molecule, one serving to 
combine with antitoxin and being 
relatively stable, the other bearing 
the poisonous properties and readily 
undergoing disintegration. The 
former he has called the haptopho- 
rous group and the latter the toxo- 
phorous group. In toxoid the toxo- 
phorous group has degenerated. 

Diphtheria was recognized as a 
distinct disease by Bretonneau in 
1821. It is characterized by a local 
inflammation, usually on the mu- 
cous membrane of the throat, the 
nose, more rarely the genital mu- 
cous membrane, or the surface of 
a wound, and by an accompanying general intoxication giving rise 
to focal necrosis in various parenchymatous organs and affecting 
more particularly the heart and the nervous system. The local 
inflammation may be only a mild reddening or it may be a wide- 
spread area of necrosis. Most frequently there is an exudate 

I 9 




FIG. 121. B. diphtheria, culture 
on glycerine agar. 



2 go 



SPECIFIC MICRO-ORGANISMS 



of plasma containing leukocytes, epithelial cells and bacteria, 
and this coagulates on the mucous surface. The epithelium 
underneath also undergoes necrosis in moderately severe cases 
and is firmly attached to the exudate by the fibrin threads. In 
severer forms there is an escape of blood into the exudate giving 
it a dark color. The local lesion is largely due 
to soluble toxin formed by the bacilli. The gen- 
eral disturbance is, as a rule, due solely to the 
absorbed toxin. The bacilli remain at the site of 
the lesion and do not appear in the blood or in- 
ternal organs in any appreciable numbers. They 
are occasionally found in the spleen or kidney 
of fatal cases, but not more frequently than the 
streptococcus is found in these organs in appar- 
ently uncomplicated fatal cases of diphtheria. 

The local lesion in the throat may be simu- 
lated very closely by inflammation due to strep- 
tococci, but the general manifestations are not 
duplicated in such conditions. Mixed infection 
Swab with diphtheria bacilli and virulent streptococci 
and culture-tube mav present a clinical picture of great severity. 

used in the diag- J - J 

nosis of diphthe- Bacteriological examination is often a great help 
oT'cotton on d fhe m diagnosis even to the expert clinician, and is 
wire shown is quite generally employed. 

much too bulky. . 7 . 7 ~ . . / r .. n i T 

Bacteriological Diagnosis of Diphtheria. In 
many large cities the bacteriological diagnosis of diphtheria is un- 
dertaken by boards of health. The methods used differ somewhat 
in detail, but are similar in the main, and are based upon the pro- 
cedure devised by Biggs and Park for the Board of Health of New 
York City. Two tubes are furnished in a box. The tubes are like 
ordinary test-tubes, about three inches in length, rather heavy and 
without a flange. Both are plugged with cotton. One contains 
slanted and sterilized Loffler's blood-serum mixture (Fig. 122); 
the other contains a steel rod, around the lower end of which a 
pledget of absorbent cotton has been wound. These tubes con- 




BACTERIACE.E : THE BACILLUS OF DIPHTHERIA 2QI 

taining the swabs are sterilized. The swab is wiped over the 
suspected region in the throat, taking care that it touches nothing 
else, and is then rubbed over the surface of the blood-serum mix- 
ture. The swab is returned to its test-tube and the cotton plugs 
are returned to their respective tubes. The plugs, of course, 
are held in the fingers during the operation, and care must be 
taken that the portion of the plug that goes into the tube touches 
neither the finger nor any other object. The principles, in fact, 
are the same as those laid down in general for the inoculation 
of culture-tabes with bacteria (see page 107). In board-of-health 
work these tubes are returned to the office. When it is desirable, 
a second tube may be inoculated from the swab. The tubes 
are placed in the incubator, where they remain for from 6 to 15 
hours and a microscopic examination is then made of smear 
preparations stained with Loffler's methylene blue. After use 
the tubes and swabs should be most carefully and thoroughly 
sterilized. 

On Loffler's blood-serum kept in the incubator the bacillus 
of diphtheria grows more rapidly than most other organisms 
which are ordinarily encountered in the throat, a property 
which to a certain extent sifts it out, as it were, from them, and 
makes its recognition with the microscope easy in most cases. 
The appearance of the bacilli under the microscope is quite 
characteristic. The diagnosis of the diphtheria bacillus in prac- 
tice is made from the character of the growth upon the blood- 
serum and the microscopical examination, taking into account 
the size and shape of the bacilli, with the frequent occurrence 
of irregular forms and the peculiar irregularities in staining, and 
this usually suffices; but in doubtful cases a second culture should 
be made from the throat, and at the same time another tube of 
Loffler's serum should be inoculated from the first culture. 
On the next day plate cultures on glycerin agar may be made 
from which typical colonies should be transplanted to broth. 
After 48 hours at 37 C. the broth is injected into two guinea- 
pigs in doses of 0.5 c.c. and one of the guinea-pigs should receive 



2Q2 SPECIFIC MICRO-ORGANISMS 

at the same time diphtheria antitoxin. In this way virulent 
diphtheria bacilli may be accurately detected. 

The very large number of examinations that have been made 
by various boards of health have shown that the diphtheria 
bacillus may persist in the throat for a long time occasionally 
several weeks after the patient has apparently recovered; also 
that diphtheria bacilli are occasionally found in the throat, 
when there is an inflammatory condition without any pseudo- 
membrane, and that they not only appear in an apparently 
healthy throat, especially in hospital nurses and in children 
who have been associated with cases of diphtheria, but also in 
those who have had no traceable contact with diphtheria cases. 1 
It has been found that bacilli sometimes occur in the throat, 
which have all the morphological and cultural properties of the 
diphtheria bacillus, but which are devoid of virulence when 
tested upon animals. Such diphtheria bacilli have frequently 
been called pseudodiphtheria bacilli. A bacillus closely resembling 
the diphtheria bacillus, but without virulence, has been found 
in xerosis of the conjunctiva. It is called the xerosis bacillus. 
If not a transformed diphtheria bacillus, it is at least closely 
related. The diphtheria bacillus is subject to wide variations 
in morphology, so that, in dealing with unknown cultures where 
the forms are not characteristic and injection into animals is 
without result, it may be difficult to decide whether or not the 
organisms are diphtheria bacilli. 

The disease is undoubtedly transmitted very largely by 
immediate contact, especially with persons harboring the bacilli 
but not seriously ill, and by fomites. Children in school or at 
play readily transfer secretions of the mouth, and a cough or 
sneeze may distribute such material over a wide area. 
. Immunity to diphtheria was produced by Von Behring in 
1890 by injecting the toxin into animals, the general method of 
procedure being quite similar to that followed in the production 
of tetanus antitoxin. The blood serum of the immunized animal 

1 Sholly: Journ. Infect. Dis., Vol. IV, 1907, pp. 337-346. 



BACTERIACE.E : THE BACILLUS OF DIPHTHERIA 293 

was found to be capable of neutralizing the poisonous property 
of diphtheria toxin. The brilliant success of Roux (1884) in treat- 
ing diphtheria with antitoxic serum caused the rapid adoption' 
of antitoxin as a therapeutic agent throughout the world. Park 
and his co-workers, Atkinson, Gibson and Banzhaf, have devel- 
oped a method of concentrating diphtheria antitoxin which is 
now generally employed. 

For the production of antitoxin 1 young healthy horses are 
selected with great care. They are specifically tested for tubercu- 
losis and glanders. A powerful diphtheria toxin is then injected 
into the horses, in an amount sufficient to kill 5000 guinea-pigs, 
together with 10,000 units of antitoxic serum. The toxin is 
subsequently injected at intervals of three days and each succeeding 
dose is increased by about 20 per cent as long as the condition 
of the horse is satisfactory. At the end of two months the dose 
is about fifty times as large as the initial dose. Antitoxin is 
given only at the start. The serum of the horse is tested from 
time to time and, when the desired antitoxic strength has devel- 
oped, the blood is drawn once a week for the preparation of anti- 
toxin. A dose of toxin is given after each weekly bleeding. 
The blood is drawn from the jugular vein into jars containing a 
10 per cent solution of sodium citrate, nine parts of blood to one 
of the citrate solution. The material is mixed and allowed to 
sediment in a refrigerator. The plasma is then siphoned off 
into large bottles and heated to 57 C. for 18 hours to change 
part of the soluble globulins 2 to euglobulins, insoluble in a satu- 
rated solution of sodium chloride. An equal volume of saturated 
aqueous solution of ammonium sulphate is then added. The 
precipitate which forms consists of the globulins and nucleopro- 
teins of the plasma. This precipitate is collected on a filter 
and extracted with a saturated solution of sodium chloride, in 
which the pseudoglobulin fraction, carrying with it the antitoxic 

1 For details of the method see Park and Williams, Pathogenic Bacteria and 
Protozoa, Phila., 1910. 

2 Banzhaf: The Preparation of Antitoxin; Johns Hopkins Hosp. Bull., 1911, 
Vol. XXII, pp. 106-109. 



2Q4 SPECIFIC MICRO-ORGANISMS 

property, is dissolved. This is precipitated by the addition of 
dilute acetic acid, filtered out and again taken up in salt solu- 
tion. It is carefully neutralized with sodium carbonate and 
dialyzed for several hours against water to remove the inorganic 
salts. The residue in the dialyzer is then passed through a 
Berkfeld filter to sterilize it, a preservative is added, and it is 
ready to be tested and put up in containers for distribution. 
The final product contains 75 to 90 per cent of the original anti- 
toxic strength and is only about one-third as bulky. The serum 
albumin, euglobulin and nucleoprotein have also been to a large 
extent eliminated in the process of concentration. 

The antitoxic strength of anti-diphtheritic serum is expressed 
in immunity units and is ascertained by animal experimentation. 
The von Behring unit is contained in ten times the amount of 
serum required to protect a 250 gram guinea-pig perfectly from 
the effects of ten times the dose of fresh diphtheria toxin which 
kills a similar guinea-pig in four days. The dose of toxin is 
first ascertained by trial on guinea-pigs and the dose necessary 
to kill in four days (minimum lethal dose) determined. Ten 
times this quantity is then injected along with varying doses of 
antitoxic serum into a series of guinea-pigs until the quantity 
of serum, which not only saves the animal but prevents loss of 
weight and local induration at the site of injection, has been 
ascertained. Ten times this amount contains one immunity unit. 

Ehrlich has carefully standardized an antitoxic serum and 
has preserved it as a dry powder, of which one gram contains 
1700 immunity units. This standard is now employed as the 
official standard for comparison in the United States. In stand- 
ardizing an antitoxin by the Ehrlich method, one unit of the 
standard antitoxin is injected along with various quantities of a 
toxin to ascertain how much of the latter is required so that the 
animal dies after four days. This dose of toxin, which when 
combined with one unit of the standard antitoxin, kills a 250 
gram guinea-pig in four days is called the L + dose. One next 
injects this L + dose along with varying quantities of the new 



BACTERIACE.E : THE BACILLUS OF DIPHTHERIA 2Q5 

antitoxin, and the amount of the latter which keeps the guinea- 
pig alive for just four days, or, in other words, produces the same 
effect as the standard unit, is known to contain one immunity 
unit. In the United States, the Hygienic Laboratory at Washing- 
ton furnishes standard antitoxin to manufacturers for this official 
test and all marketed sera are tested by this method. 

Diphtheria antitoxin not only prevents the development of 
diphtheria when injected in doses of 1000 units, but it also 
exerts a marked influence as a therapeutic agent in diphtheria, 
neutralizing the poison produced by the bacilli in the body of 
the patient. It does not kill the bacilli but it nullifies their 
chief offensive weapon, the soluble diphtheria toxin. Its value 
in treatment of diphtheria is everywhere attested by clinical 
evidence. The inflammation in the throat subsides and the 
membrane disappears. The bacilli, however, may remain for a 
considerable time. Local antiseptics may assist the natural 
agencies of the body in their destruction. In some cases they 
persist for months in spite of vigorous treatment. 

Certain untoward effects have followed the injection of anti- 
diphtheritic serum. Sudden death has occurred in very rare 
instances and skin rashes are rather common. These effects 
are probably due to toxic substances set free in the parenteral 
digestion of the foreign protein and are doubtless of the same 
general nature as the phenomenon of anaphylaxis. Since the 
introduction of the concentrated antitoxin fatalities have become 
exceedingly rare or have been entirely eliminated. The serum 
rashes and cases of nervous shock do occur, especially in asthmatic 
individuals and in those who have received a previous injection 
of horse serum. In these persons it is well to give a minute 
quantity, 0.2 c.c., of the serum as a preliminary injection, wait 
two or three hours and then give the full dose. The danger of 
serious reactions due to anaphylaxis may thus be avoided. 1 

Bacillus (Bacterium) Xerosis. This organism occurs on the 
normal mucous membranes, particularly the conjunctiva. It 

1 Vaughan: Amer. Journ. Med. Sciences, 1913, Vol. CXLV, pp. 161-177. 



296 SPECIFIC MICRO-ORGANISMS 

resembles B. diphtheria very closely, simulating the granular 
morphological type. Its cultures are not poisonous. 

Bacillus Hofmanni. This organism is also called the pseudo- 
diphtheria bacillus. It occurs frequently in cultures from the 
nose and pharynx, and resembles the short solid-staining morpho- 
logical types of B. diphtheria. It does not produce toxin, nor 
does it produce acid from dextrose. 

The Morax-Axenfeld Bacillus. This is a small non-motile 
diplo-bacillus, the individuals measuring about 1X2^, which 




FIG. 123. The Morax-Axenfeld bacillus in the exudate of conjunctivitis. (From 
McFarland after Rymowitsch and Matschinsky.} 

occurs in one form of epidemic conjunctivitis. It can be cultured 
on Loffler's serum which it liquefies, and the disease has been 
produced in man by inoculation with pure cultures. 

The Koch-Weeks Bacillus. This a non-motile rod 0.25^ 
wide and i to 2/z long, which occurs in epidemic conjunctivitis. 
It is cultivated with difficulty and abundant moisture is essential 
to success. Inoculation with pure cultures causes conjunctivitis. 

Bacillus (Bacterium) Pertussis (Bordet-Gengou Bacillus). 
Bordet and Gengou in 1906 described a minute, non-motile 
bacillus 0.3X1.5^ which occurs in the sputum and on the mucous 
membrane of the trachea and bronchi in whooping cough. They 



BACTERIACE.E : THE BACILLUS OF DIPHTHERIA 297 

obtained cultures of the organism on blood agar and, employing 
these cultures as an antigen, they demonstrated an antibody 
in the blood of patients by means of the complement-fixation 
test. Klimenko 1 has further succeeded in producing a chronic 
catarrh of the respiratory passages in monkeys and puppies by 
applying pure cultures to the tracheal mucosa. The bacillus 
is a minute rod, motionless, stained with moderate difficulty, 
and Gram-negative. It occurs in large numbers between the 
cilia of the epithelial cells lining the trachea and bronchi in cases 
of whooping cough where it mechanically 2 interferes with the 




FIG. 124. Koch-Weeks bacillus in muco-pus from conjunctivitis. X 1000. 
(From Park and Williams after Weeks.} 

action of the cilia and gives rise to irritation. It is an obligate 
aerobe and at first grows well only on media containing blood, 
ascitic fluid or other protein. Later it adapts itself to artificial 
culture on ordinary media. Gelatin is not liquefied. 

Bacillus (Bacterium) Influenzae. Pfeiffer in 1892 isolated a 
small bacillus 0.25^ wide by 0.5 to 2.0^1 long from the bronchial 
secretion in cases of epidemic influenza. The bacillus occurs in 
enormous numbers in acute uncomplicated cases of influenza 
in the nasal and bronchial mucus. It is non-motile, aerobic, 

1 Centralbl.f. Bakt. Orig., 1909, Bd. XLVIII, S. 64-76. 

2 Mallory: Pertussis: The Histological Lesion in the Respiratory Tract, Journ. 
Med. Rsch., 1912, Vol. XXVII, pp. 115-124; Mallory, Hornor and Henderson, 
Journ. Med. Rsch., 1913, Vol. XXVII, pp. 391-397. 



298 SPECIFIC MICRO-ORGANISMS 

rather difficult to stain and Gram-negative. Cultures are 
obtained on ordinary agar smeared with fresh human or rabbit's 
blood or upon a mixture of blood and agar. Hemoglobin seems 
essential to growth. The bacillus is very sensitive to drying, 
and its transmission would seem to occur largely through close 
association, and the scattering of moist droplets of material 
from the nose and mouth in sneezing, coughing and talking. 
The cultures are toxic for rabbits and monkeys. The causal 
relation of B. influenza to influenza is not as yet fully established. 
Conditions resembling influenza very closely seem to be caused 
by other organisms, such as the cocci. 

Bacillus (Bacterium) Chancri (Bacillus of Ducrey). Ducrey 
in 1889 found a short bacillus in the soft venereal sore known 
as chancroid, obtained it in pure culture and produced typical 
lesions by inoculation in man. The organism is about 0.5X1.5^, 
often growing in threads. It grows on a blood-agar mixture at 
37 C. Material for culture should be obtained from an un- 
broken pustule or from a chancroidal bubo, so as to avoid con- 
taminating organisms. The bacillus possesses very little resist- 
ance to drying or to germicides. Successful inoculation experi- 
ments have been carried out on man, on monkeys and on cats. 
Other organisms 1 appear to produce soft chancre in the absence 
of the Ducrey bacillus in some cases. 

1 Herbst and Gatewood: Journ. A. M. A. t 1912, Vol. LVIII, pp. 189-191. 



CHAPTER XX. 

BACTERIACE^E: THE TUBERCLE BACILLUS AND 
OTHER ACID-PROOF BACTERIA. 

Bacillus (Bacterium) Tuberculosis. Robert Koch in 1882 
discovered the minute rods in tuberculous tissue, planted the 
tissue on slanted inspissated blood serum and obtained pure 
cultures of the tubercle bacillus, inoculated these cultures into 
animals and produced typical tuberculosis. He succeeded in 
doing this with natural tuberculosis of man and many other 
mammals and also with the tuberculosis of birds. Silbey in 
1889 observed with the microscope morphologically similar 
bacilli in a snake. Rivolta and Mafucci in 1889 pointed out the 
differences between the tubercle bacillus of birds and that of 
mammals and their work, together with subsequent confirmatory 
investigations, has established a distinct avian type of tubercle 
bacillus, B. tuberculosis var. gallinaceus. In 1897 Bataillon, 
Dubard and Terre found acid-proof bacilli in definite histological 
tubercles in a fish (carp), obtained cultures and recognized it as 
distinct from the mammalian form, and it was subsequently 
designated as B. tuberculosis var. piscium. Theobald Smith 
in 1898 published the results of a careful and extensive com- 
parative study of tubercle bacilli from human sputum and 
tubercle bacilli from tuberculous tissue of the bovine pearl 
disease (tuberculosis), and pointed out distinct differences in 
morphology, cultural characters and virulence between the 
organisms derived from the two sources. The mammalian 
tubercle bacilli were thus divided into two types, and subsequent 
investigation has fully justified the recognition of B. tuberculosis 

299 



300 SPECIFIC MICRO-ORGANISMS 

var. humanus and B. tuberculosis var. bovinus. Some, or perhaps 
all four of these types may be eventually recognized as distinct 
species. At present the designation as types or varieties seems 
more appropriate. 

Bacillus Tuberculosis var. Humanus. This organism occurs 




FIG. 125. Bacillus tuberculosis in the sputum of a consumptive; stained by Ziehl 
method. X 2100. (After Kossel.} 

in the infiltrated lung in human phthisis and also in the great 
majority of the other tuberculous lesions in man. In the ex- 
ternal world it does not grow naturally and passes there a more 
or less temporary existence in discharges from the body, of which 
the most important is the sputum. The cell is about 0.4;* in 
width and quite variable in length, 0.5 to S.oju. The longer 



BACTERIACE^E : THE TUBERCLE BACILLUS 301 

forms are often somewhat bent, and they frequently contain 
refractile granules. When stained these forms have a beaded 
or banded appearance. Spores have not been observed. Branch- 
ing forms occur sometimes in cultures, suggesting a close relation 
to actinomyces and streptothrix. There is a considerable amount 
of a waxy substance in the body of the bacillus, which makes it 
difficult to stain and also difficult to decolorize after it has been 
stained. Hot carbol-fuchsin is generally employed, applying 
it for one to two minutes. The preparation is then washed and 






* ' J 

I x;^; v . 

\'r9< ^ I 



FIG. 126. Bacillus tuberculosis, from a pure culture. X 1000. 

decolorized in dilute mineral acid (2 to 20 per cent) and in alcohol. 
Tissue elements and most other materials may be completely 
bleached by this treatment, leaving the tubercle bacilli still 
colored. B. tuberculosis is Gram-positive. 

Cultures are most readily obtained by transferring bits of 
tuberculous tissue, free from other micro-organisms, to moist 
slants of inspissated blood serum or Dorset's egg medium. If 
the available material is already contaminated, the extraneous 
organisms may usually be eliminated by inoculating it into 
guinea-pigs and making the cultures from the tuberculous guinea- 



302 



SPECIFIC MICRO-ORGANISMS 



pig tissue, about four weeks later. 1 The tubes may be sealed 
with rubber caps or paraffin and incubated at 37 C. Better 
results are obtained by leaving the tubes unsealed and in- 
cubating at 37 C. in an atmosphere saturated with moisture, 
as the bacillus is a strict aerobe, but this requires special care 
and is not absolutely essential to success. After two or three 
weeks a dry, white growth is developed which may later become 
folded. Transplants from the primary culture to glycerin agar, 
glycerin broth or glycerin potato are usually successful. Old 




FIG. 127. Tubercle bacillus showing branching andjinvolution forms. (After 

Migula.} 

cultures on potato and agar often become yellowish or even 
pink in color. 

The chemical composition of tubercle bacilli has been ex- 
tensively studied. The moisture content varies from 83 to 89 
per cent. The ash (inorganic salts) amounts to about 2.6 per 
cent of the dry substance, and about half of this is phosphor c 

1 It is possible to cultivate tubercle bacilli directly from contaminated material, 
such as sputum, by carefully washing it in sterile water and then spreading it over 
the surfaces of a series of serum tubes. Results are somewhat uncertain. For 
details of this and other methods see Kolle and Wassermann, Handbuch, 1912, 
Bd. V, S. 420-422. 



BACTERIACE.E : THE TUBERCLE BACILLUS 



303 



acid (PC>4). The waxy constituent of 
the bacterial cells is of particular in- 
terest. This makes up from 8 to 40 per 
cent of the dry substance, less in young 
and more in old cultures. The acid-proof 
staining property depends upon this 
waxy substance, for the bacilli from 
which it has been extracted by ether-al- 
cohol are no longer acid-proof while the 
wax itself exhibits this peculiarity of 
staining. It is also known that the ba- 
cilli in young cultures are on the whole 
less acid-proof than those from old cul- 
tures in which chemical analysis shows 
a greater concentration of the waxy sub- 
stance. The protein substances, largely 
nuclein, make up about 25 per cent of 
the dry cell substance. Several other 
constituents of the cell have been iden- 
tified. As in the case of other bacteria 
the chemical composition varies within 
rather wide limits according to the nutri- 
tive medium, conditions of growth and 
especially the age of the culture. 

The poisons of the tubercle bacillus 
exist to a large extent in an inactive 
form in the culture fluid and more 
particularly as an undissolved constituent 
of the bacterial cell bodies. Culture fil- 
trates exert little or no effect upon in- 
jection into normal animals. The dead 

FIG. 128 Bacillus tuber- 

bacilli, however, give rise to local mflam- culosis. Culture on glycerin 
mation and in many instances stimulate agar several month 
the formation of typical tubercles at the 
point where they lodge. Evidently the 




(From McFarla nd after 
Curtis.) 



304 SPECIFIC MICRO-ORGANISMS 

poison is set free from some substance in the dead cells by the 
action of the tissue cells or body fluids upon them, and it is quite 
certain that the bacteria-free culture fluid (old tuberculin) becomes 
toxic as a result of such an action. 

Tubercle bacilli outside the body are moderately resistant 
to harmful influences. In dried sputum, they have been found 
alive after eight months. Direct sunlight kills the bacilli in 
sputum in a few minutes if this be exposed in a thin transparent 
layer. In thicker masses the effect of light is uncertain. In 
buried cadavers the bacilli remain alive and virulent for 2 to 6 
months. In watery suspensions the bacilli are killed by heating 
to 60 C. for 15 minutes. In milk, heating at 60 C. for 20 minutes 
or at 65 C. for 15 minutes kills the tubercle bacilli, provided all 
the fluid is heated to this temperature for the full period. The 
bottle should be tightly stoppered and completely immersed 
in the hot water. Dry heat at 100 C. for 30 minutes is effective. 
Against chemical disinfectants B. tuberculosis is rather resistant, 
doubtless because of the waxy constituent of the cells. Absolute 
alcohol and mercuric chloride i to 500 fail to disinfect sputum 
in 24 hours. Five per cent carbolic acid is effective in this time. 
Formalin, 5 per cent solution, requires about 12 hours. B. 
tuberculosis remains alive in strong antiformin solutions (a pro- 
prietary preparation of chlorinated caustic alkali) for 30 to 60 
minutes, whereas ordinary bacteria are rapidly disintegrated 
by this chemical agent. 

Tuberculin is a name applied to various chemical products 
of the tubercle bacillus. The oldest and most important tuber- 
culin was described by Koch in 1890. It is made by growing 
the bacillus on the surface of 4 per cent glycerin broth in shallow 
flasks at 37 C. for eight to ten weeks, steaming the cultures 
for one hour and filtering through porcelain, or often merely 
through paper, to remove the dead bacilli. The filtrate is then 
concentrated to one-tenth its original volume by evaporation at 
90 on the water-bath. The product keeps indefinitely in sealed 
containers and is known as Koch's old tuberculin (" alt tuber- 



BACTERIACE.E : THE TUBERCLE BACILLUS 305 

kulin"). Chemical study of tuberculin has shown that the spe- 
cific active substance is a thermostable, dialyzable substance, 
insoluble in alcohol, which gives most of the protein reactions 
but not the biuret test. It is digested by pepsin and by trypsin. 1 
Koch's new tuberculin, better known as tuberculin B . E. ("Bacillen- 
emulsion") is made from the solid bacterial growth on glycerin 
broth. The growth is pressed between filter papers, dried and 
then pulverized in a ball mill for about three months, then sus- 
pended in 50 per cent aqueous solution of glycerin, 0.002 gram 
of the powder to each cubic centimeter. Finally it should be 
sterilized by heating to 60 C. for 20 minutes. This tuberculin 
is a suspension, not a solution, and must be thoroughly mixed 
each time before use. Numerous other tuberculins have been 
prepared, of which perhaps the " Bouillon filtre" of Denys is 
the most important. It is the porcelain filtrate of the unheated 
glycerin-broth culture of the tubercle bacillus. It resembles 
Koch's old tuberculin except that it is not heated and is not 
concentrated. 

Inoculation of animals with B. tuberculosis gives rise to typical 
tuberculous lesions and death in most mammalian species. 
The guinea-pig is very susceptible to subcutaneous injection 
but not readily infected by the alimentary route. The lesions 
are usually well developed four or five weeks after subcutaneous 
inoculation and death occurs as a rule in 6 to 12 weeks. Rabbits 
are less susceptible to inoculation with the human type and they 
usually recover when injected with small doses of a culture, 
o.oo i gram intravenously. Cattle are quite immune to this 
organism. Large doses of cultures or of sputum have been 
injected into calves and older bovines without producing tubercu- 
losis, and quarts of tuberculous sputum have been fed to bovine 
animals with negative results. 

Tuberculosis is, economically, the most important human 
disease. Approximately one death in every three between the 
age of 20 and 45, the active period of life, is due to it. It was 
1 Lowenstein in Kolle und Wassermann, Handbuch, 1912, Bd. V, S. 554- 555. 

20 



306 SPECIFIC MICRO-ORGANISMS 

recognized as a contagious disease by the ancients. Laennec, 1 
in 1805, by extensive post-mortem studies recognized the essential 
pathological unity of tuberculous processes. Villemin, in 1865, 
conclusively demonstrated its transmissibility by successful 
inoculation of animals with tuberculous tissue from man and from 
cattle. 

The response of the infected tissue to the presence of the 
tubercle bacillus results in a localized mass of granulation tissue, 
the tubercle, of which the histological structure is so characteristic 
that the presence of tuberculosis may be recognized by it alone. 
From the point of introduction the bacilli may be distributed 
by the lymph or blood stream or may be carried by wandering 
cells. Eventually a bacillus comes to rest and grows slowly 
in the intercellular spaces of connective tissue. Very soon, the 
neighboring fixed tissue elements, connective-tissue cells and 
endo helial cells, begin to multiply by karyokinesis and at the 
same time the cells become swollen with nuclei large and bladder- 
like, forming the so-called epithelioid cells. The bacilli are 
found in and between these cells. As the pathological process 
continues the nucleus of an occasional epithelioid cell divides 
many times without division of the cytoplasm, giving rise to a 
multi-nucleated giant cell. Very early in its development the 
peripheral portion of the tubercle becomes infiltrated with lympho- 
cytes and later, as the giant cells are formed, numerous poly- 
nuclear leukocytes are also present. Newly formed blood vessels 
are absent. With further extension, the center of the tubercle 
undergoes a caseous necrosis and liquefaction, and eventually 
this necrotic center enlarges so as to break through an epithelial 
surface to a passage to the exterior. This gives rise to open 
tuberculosis and tubercle bacilli may usually be found in the 
discharge from the lesion at this stage. 

The tubercle is the essential histological unit of tuberculosis. 
An infiltrated tissue may contain myriads of these tubercles in 

1 For a history of tuberculosis seeLandouzy: Cent ans de phtisiologie, 1808-1908, 
Sixth Internat. Cong, on Tuberculosis, Special Volume, pp. 145-189. 



BACTERIACE.E : THE TUBERCLE BACILLUS 307 

all stages of evolution. At any stage in its evolution the develop- 
ment of the tubercle may become arrested and it may retrogress- 
and heal if the infected tissue is able to overcome the bacilli. 
If this occurs early the bacilli may be entirely destroyed and the 
abnormal tissue may disappear completely or remain only as a 
little hyaline or fibrous tissue. After caseation has occurred, 
healing results in the formation of a dense fibrous nodule, usually 
with calcareous material in the center, in which living tubercle 
bacilli can usually be demonstrated. 

The mode of infection in human tuberculosis has been a matter 
of some controversy and much of the evidence concerning it 
has been derived from animal experimentation. Unquestionably 
tubercle bacilli may pass through epithelial surfaces, especially 
of mucous membranes, without production of any demonstrable 
lesion. Ingested bacilli readily pass through the intestinal 
mucosa, especially during the digestion of fat, and they may 
first produce lesions in the mesenteric lymph glands, the liver 
or in the lungs. In the latter instance, they doubtless pass with 
the absorbed fat through the thoracic duct, superior vena cava 
and right heart to the pulmonary arteries. In man, the most 
important mode of infection is through inhaling the dust of dry 
powdered sputum, as a result of which lesions develop in the 
lungs. Tuberculosis may occur in any tissue of the body, reach- 
ing it through the blood and lymph. A massive infection of 
the blood stream often leads to generalized miliary tuberculosis 
with minute tubercles in all the organs. 

The bacteriological diagnosis of the disease depends upon 
finding the tubercle bacilli in discharges from the suspected 
lesion. In sputum an acid-proof bacillus of the proper size and 
shape is almost invariably a tubercle bacillus and a diagnosis 
based upon such a finding by an experienced microscopist is 
justly regarded as very accurate. Inoculation of guinea-pigs 
will clinch the proof. The latter procedure will also sometimes 
detect tubercle bacilli when careful microscopic search has failed. 
In discharges from the intestine or urinary organs one may 



308 SPECIFIC MICRO-ORGANISMS 

meet with other acid-proof organisms (B. smegmatis), and more 
care is necessary in arriving at a diagnosis. In tuberculous 
meningitis, the tubercle bacillus may be detected by microscopic 
examination of the cerebrospinal fluid 1 in nearly every case. 
The filmy clot which usually forms in such a fluid in a half hour 
after drawing it is the most favorable material for examination. 

When a considerable amount of purulent or mucoid material 
is available for examination and one has failed to find the tubercle 
bacilli by the usual method of microscopic examination, it is 
often advisable to try some method of concentration. One of 
the common methods of general applicability is that of Uhlenbuth, 
in which antiformin is employed to dissolve the tissue elements, 
leaving the bacilli unchanged. LofHer's modification 2 of the 
Uhlenbuth method is a convenient one. The material to be 
examined is mixed with an equal amount of 50 per cent anti- 
formin and brought to a boil. This dissolves the sputum or 
other material and serves to kill the bacilli. It is then cooled 
and, for each 10 c.c., 1.5 c.c. of chloroform-alcohol (i 19) is added. 
The mixture is next violently shaken to form a fine emulsion, 
and is then centrifugalized at high speed for 15 minutes. The 
solid matter collects as a tough mass on top of the drop of chloro- 
form and beneath the watery liquid. This mass is crushed 
between slides, mixed with a little egg albumen or with some of 
the original untreated exudate, spread, fixed, stained and exam- 
ined in the usual way. The albuminous material is necessary to 
make the preparation adhere to the slide. 

Allergic reactions are extensively employed in the diagnosis 
of tuberculosis. Tuberculin is without particular effect upon 
normal individuals but in the tuberculous individual it gives 
rise to irritation and intoxication. The phenomenon is analogous 
to that of anaphylaxis, the irritant or toxic substance being set 
free from the tuberculin by the action of specific ferments pro- 



: Amer. Journ. Dis. Children, Jan. IQII, Vol. I, pp. 26-36. Hemenway: 
ibid., 1911, Vol. I, pp. 37-41. Koplik: Johns Hopkins Hosp. Bull., 1912, Vol. XXIII, 
pp. 113-120. 

2 Williamson: Journ. A. M. A., 1912, Vol. LVIII, pp. 1005-7. 



BACTERIACE^E : THE TUBERCLE BACILLUS 309 

duced and present in the body as a result of previous contact 
with the tubercle bacillus and its products. The tuberculous- 
individual is therefore sensitized to tuberculin. The sensitization 
may be local and confined to the tissue immediately surrounding 
a solitary tubercle, or it may be general as a result of more ex- 
tensive lesions. Tuberculin is applied to the skin mixed with 
an equal amount of lanolin (Moro test), or applied to a scarified 
point undiluted (Von Pirquet test), or injected into the sub- 
stance of the skin in a dose of o.i c.c. of i to 1000 dilution (Ham- 
burger intracutaneous test), or applied to the conjunctiva in a 
dose of one drop of a freshly prepared i per cent solution of old 
tuberculin (Wolff-Eisner or Calmette test), or finally it may be 
introduced into the circulation by subcutaneous injection of 
a dilution representing o.ooooi gram of old tuberculin, with 
subsequent progressive increase of the dose up to o.oio gram if 
reaction is not obtained. The local reaction is that of irritation, 
evidenced by redness and edema, sometimes by vesiculation. 
The general reaction is evidenced by malaise, irritation at site 
of the lesion (increased cough in pulmonary tuberculosis) and 
a rise in body temperature. The reaction depends upon the 
tuberculin coming into contact with the specific ferment, and 
the location, extent and activity of the tuberculous process are 
important elements influencing the outcome of the various 
tests. Tuberculosis in the eye causes such a violent reaction 
to the conjunctival test that this method should never be employed 
without first excluding ocular tuberculosis. The subcutaneous 
test will often detect tuberculosis not revealed by the other 
methods. It is, however, a more serious procedure than the skin 
tests, which are indeed practically harmless. 

The various tuberculins are now extensively employed in 
the treatment of tuberculosis, largely because of the favorable 
results obtained by Trudeau. It is given subcutaneously every 
5 to 7 days beginning first with a blank dose of salt solu- 
tion and next with o.ooooi gram of tuberculin. The dose is 
kept at the point at which the least general reaction possibly 



310 SPECIFIC MICRO-ORGANISMS 

recognizable occurs, or just below this amount, the general pur- 
pose being to induce an immunity to tuberculin. It is often 
possible to begin with a case which reacts to o.oooi gram of tuber- 
culin and after treatment for 6 months so change the sensitive- 
ness that 0.5 gram may be injected without reaction. Some 
cases do remarkably well when treated with tuberculin together 
with the usual careful hygienic-dietetic treatment 1 given in sanito- 
ria, but the value of tuberculin for treatment of the average case, 
is, perhaps, not yet fully established. 2 

Bacillus Tuberculosis var. Bovinus. The bovine type of 
tubercle bacillus is found in the lesions of tuberculous cattle 
(perlsuchi), frequently in hogs, in a considerable percentage of 
tuberculous lesions in children, and very rarely in the tubercu- 
lous lungs of adult human beings. In artificial culture on solid 
media, the cell is about i/z long, shorter than that of the human 
type, and is easily stained. In glycerin broth the length of the 
cell and the staining is more irregular. On all media the growth 
is at first much less abundant than that of the human type. 
Smith has shown that the bovine type produces alkali in glycerin 
broth during the first two months, whereas the human type 
tends rather to .produce acid. The virulence of the bovine 
bacillus is greater than that of the human type for all mammals, 
and it also infects birds. Intravenous injection of o.ooooi 
gram of culture in thin emulsion kills rabbits with generalized 
tuberculosis in about three weeks, while a similar dose of the 
human variety is without such effect. Subcutaneous injection of 
rabbits shows a similar difference. Calves are very susceptible 
to the bovine type, not to the human. 

Tuberculosis of cattle is widely distributed and is very preva- 
lent in the older European dairy regions. The lesions are 
most common in the bronchial and retropharyngeal lymph glands, 
but they may occur anywhere in the body of the animal. The 
disease may remain localized for years in a single lymph gland or it 

1 Brown: Journ. A. M. A., 1912, Vol. LVIII, pp. 1678-81. 

2 Brown: Amer. Journ. Med. Sciences, 1912, Vol. CXLIV, pp. 469-624. 



BACTERIACE.E I THE TUBERCLE BACILLUS 31 1 

may extend rapidly causing marked emaciation and death of 
the animal. The bacilli escape from the living bovine animal 
most commonly in the feces, 1 sometimes in the mucus and spray 
from the nose and mouth, in the uterine discharge and in the 
milk, and of great importance is the fact that animals may be 
excreting the bacilli without showing any gross evidence of the 
presence of the disease. Tuberculin is extensively employed in 
the detection of tuberculosis in cattle. A dose of 0.2 to 0.5 
gram diluted with 9 volumes of 0.5 per cent carbolic acid is in- 
jected subcutaneously at the side of the neck. The typical 
positive reaction includes a rise in temperature of 2 or 3 F. 
over that of the previous day. The test is very accurate when 
positive but not so reliable when negative. Tuberculous animals 
should be segregated from healthy animals and food products 
from them used only after effective disinfection, or they should 
be slaughtered under inspection. 

Great interest has been manifested in the question of suscep- 
tibility of man to the bovine tubercle bacilli and the solution 
has been reached by isolating bacilli from human tissue and identi- 
fying them. Park and Krumwiede 2 have summarized the results 
of 1511 such examinations, and conclude that somewhat less 
than 10 per cent of the deaths from tuberculosis in young children 
are due to the bovine tubercle bacillus, while in adults infection 
with this bacillus is much less frequent. 

Bacillus Tuberculosis var. Gallinaceus (Avium). This variety 
occurs particularly in the tuberculous lesions of barnyard fowls, 
but also in many other birds. The form of the bacillus is not 
specially characteristic except that in old cultures there is a 
marked tendency to the production of branching threads. In 
glycerin broth the growth is more delicate, and development 
takes place at the bottom of the flask as well as on the surface 
of the liquid. Chickens are very susceptible to intravenous 
inoculation with this type of bacilli but quite refractory to the 

1 Briscoe and MacNeal: 111. Agr. Exp. Sta. Bull. 149, 1911; Assn. for Tubercu- 
osis, Transactions, 1912, pp. 460-465. 

*Journ. Med. RscL, 1912, Vol. XXVII, pp. 109-114. 



312 SPECIFIC MICRO-ORGANISMS 

mammalian types. Mice and rabbits are also susceptible, while 
guinea-pigs are relatively resistant. The avian tubercle bacillus has 
been found in human tuberculous lesions in a very few instances. 

Bacillus Tuberculosis var. Piscium. This variety occurs 
in natural tuberculous lesions of snakes, fish, turtles and frogs. 
The bacillus is quite different from the preceding varieties, as 
it grows rapidly on ordinary media at temperatures ranging from 
12 to 36 C., and the bacilli developed on the poorer media are 
often not at all acid-proof. When grown in bouillon with fre- 
quent shaking the culture becomes diffusely cloudy, and the 
organisms of such cultures are said to be motile. Most warm- 
blooded animals are wholly refractory to inoculation, but, in 
the guinea-pig, inoculation has sometimes been followed by the 
production of typical tubercles with epithelioid and giant cells, 
usually encapsulated and tending to heal. 

Bacillus (Bacterium) Leprse. Hansen in 1873 and Neisser 
in 1879 discovered this organism in the nodular lesions of leprosy. 
Cultures were first obtained by Clegg in 1908 by inoculating 
leprous tissue onto agar along with living amebae and the vibrio 
of Asiatic cholera. Pure cultures of B. leprcz were subsequently 
obtained by heating the mixture to kill the other organisms. 
Inoculation of cultures into mice and guinea-pigs is said to pro- 
duce leprous nodules but the evidence has not appeared to be 
very convincing. More recently Duval and Couret 1 after very 
extensive investigations, in which Clegg's work was confirmed, 
have been able to produce very typical leprosy in a monkey by 
repeated injections of a pure culture, resulting in general dissemi- 
nation and death one year after the last injection. The results 
have not been confirmed and is a subsequent paper 2 Duval is 
inclined to question the value of his previous animal experiments, 
and even suggests that the organism employed plays only a 
negligible part in leprosy. 

B. leprce is a slender rod 0.2 to 0.45/4 wide by 1.5 to 6ju long 

1 Journ. Exp. Med., Vol. XV, pp. 292-306. 

2 Duval and Wellman, Journ. Inf. Diseases, 1912, Vol. XI, pp. 116-139. 



B ACTERIACE^E : THE TUBERCLE BACILLUS 313 

as it occurs in tissues, much shorter in cultures. In its staining 
properties it closely resembles the tubercle bacillus, but is less 
constantly acid-proof in cultures. The organism occurs in 
enormous numbers in most of the nodular lesions of leprosy and 
if often abundant in the nasal mucus of these cases. When less 
numerous the antiformin method of Uhlenbuth may assist in 
finding them. Duval and his co-workers have obtained pure 
cultures by the method of Clegg and also by planting uncontami- 
nated leprous tissue on serum agar to which trypsin has been 
added. Eventually the bacilli adapt themselves to growth on 
ordinary media such as plain agar. In the first cultures the growth 
may be slow and relatively meager, but later abundant growth 
may be obtained in 2 to 3 days. The color is orange. Injection 
of these cultures into mice, guinea-pigs and monkeys is ordinarily 
followed by transient lesions which have been considered by some 
to resemble those of leprosy. The one instance of the monkey 
reported by Duval and Couret, mentioned above, seems to be 
more convincing, but further work is necessary before the status 
of these cultures can be definitely established. 

Leprosy has been known since the dawn of history and has 
been considered to be transmissible for a long time. It is widely 
distributed over the earth, especially in Norway, Russia, Iceland 
and in Turkey. In the United States there are leper colonies in 
Louisiana, Minnesota and in Hawaii. Lepers are occasionally seen 
in the clinics of all the larger cities. 

Leprosy is universally considered to be due to the leprosy 
bacillus, but as to mode of transmission, whether direct from 
man to man, or from the external world, or how, little or nothing 
is really known. It seems certain that the disease is always con- 
tracted in some way from a previous case, but it is certainly not 
very readily transmitted. Segregation without absolute isolation 
is the common method of handling lepers. The disease is not 
ordinarily inherited. 

Bacillus Smegmatis. This organism occurs in the smegma 
on the genitals of man and other mammals and also in moist folds 



314 SPECIFIC MICRO-ORGANISMS 

of the skin where there are collections of moist desquamated 
epithelium. It resembles the tubercle bacillus in form and stain- 
ing properties, but is, on the average, more readily decolorized in 
alcohol. This property cannot be relied upon to differentiate 
the two organisms in any given case. Proper care in collecting 
specimens for examination usually suffices to exclude this or- 
ganism. Urines to be examined for tubercle bacilli should be 
obtained by catheter. In doubtful cases inoculation of a guinea- 
pig is necessary. B. smegmatis has been grown in artificial culture 
and after a time adapts itself to ordinary media. 

Bacillus Moelleri. Acid-proof organisms resembling the 
tubercle bacillus in form and staining properties were found on 
timothy hay by Moeller. The bacillus is likely to be found in 
milk and other dairy products. Probably the " butter bacillus" 
of Rabinowitsch is identical with it or a near relative. When 
introduced into guinea-pigs these organisms sometimes produce 
lesions resembling tubercles, but these do not progress and kill 
the animal and a second animal inoculated from the lesions of the 
first gives a negative result. Cultures are easily, obtained on 
ordinary media, and the organisms grow rapidly at 25 to 30 C. 

Other Acid-proof Organisms. Many of the strep to thrices 
which grow in the soil and upon plants are to some extent similar 
in their staining properties to the tubercle bacillus and when 
broken up into short segments may be a source of confusion. 
These are most likely to be met with in examining agricultural 
products and especially in the feces of cattle. Mere microscopic 
examination of such materials for tubercle bacilli has, as a rule, 
little value, as both positive and negative findings are question- 
->able. Brem, 1 in the Canal Zone, has made the important obser- 
vation that acid-proof bacilli may grow in distilled water stored 
in bottles in the laboratory and that, when such water is used in 
preparing the microscopic objects for examination," these extrane- 
ous bacilli may be mistaken for tubercle bacilli. Burvill-Holmes 2 

1 Journ. A. M. A., 1909, Vol. LIU, pp. 909-911. 

2 Proc. Path. Soc. Phila., 1910, N. S. Vol. XIII, pp. 154-160. 



BACTERIACE.E : THE TUBERCLE BACILLUS 315 

has made similar observations at Philadelphia. Pseudo-bacilli, 
microscopic bodies somewhat resembling tubercle bacilli, some-- 
times occur in microscopic preparations stained with carbol- 
fuchsin. These deceptive pictures seem to be common in prepa- 
rations of laked or digested blood. 1 

1 Calmette, Sixth Internat. Cong, on Tuberculosis, 1908, Spec. Vol., p. 70; see 
also Bacmeister, Kahn and Kessler, Munch, med. Wochenschr., Feb. 18, 1913. 



CHAPTER XXI. 

BACTERIACE^E: THE BACTERIA OF THE HEMOR- 
RHAGIC SEPTICEMIAS, PLAGUE AND MALTA FEVER. 

Bacillus (Bacterium) Avisepticus. Moritz 1 in 1869 observed 
this minute rod in the blood of chickens with chicken cholera. 
Toussaint (1879) and Pasteur (1880) obtained pure cultures in 
liquid media and Pasteur (1880) made the far-reaching discovery of 
the method of immunization by means of attenuated bacterial 
cultures while working with this organism. B. avisepticus occurs in 
enormous numbers in the blood, internal organs, urine and feces of 
the acutely affected birds, in far smaller numbers in those having 
the chronic form of the disease and has also been found in the in- 
testinal contents of apparently healthy birds. It is 0.3^ wide and 
0.2 to iju in length, the shorter ones being joined together. 
It is non-motile and Gram-negative. Cultures are readily ob- 
tained on ordinary media by inoculation with heart's blood. 
Gelatin is not liquefied. Minute quantities of a virulent culture 
suffice to produce a fatal infection in chickens and many other 
birds, either by feeding or by subcutaneous injection. Rabbits 
are also extremely susceptible, guinea-pigs almost immune. 
Artificial cultures kept for three to ten months in contact with air 
are no longer capable of causing a fatal infection in chickens and 
their injection is followed by recovery and a state of immunity to 
the fully virulent organism. Acute chicken cholera is the typical 
hemorrhagic septicemia of birds, with abundant bacteria in the 
blood, and hemorrhages on the serous membranes and into the 
stomach and intestine. 

Bacillus (Bacterium) Plurisepticus. This name is applied to 
an organism occurring in the hemorrhagic septicemias of various 

1 Vallery-Radot: Life of Pasteur, 1911, Vol. II, p. 75. 

316 



THE BACTERIA OF THE HEMORRHAGIC SEPTICEMIAS 317 

mammals and birds. The virulence is variable and seems to be 
especially developed for the species of animal in which the organ- 
ism is found. It does not differ essentially from B. avisepticus. 
Other minute bacteria exhibiting the same general characteristics 
and occurring as a generalized infection in diseases of animals 
are Bacillus murisepticus in mice and Bacillus (Bacterium) rhusio- 
pathicz suis in swine. 

Bacillus (Bacterium) Pestis. This organism was discovered 
simultaneously by Kitasato and Yersin in 1894 in the bodies of 
persons dying of bubonic plague in the epidemic at Hongkong. 




FIG. 129. Bacillus of bubonic plague. (Yersin.} 

The description of Yersin has proven to be the more accurate. 
The organism is unquestionably the cause of plague, as in addi- 
tion to the evidence of animal experimentation there are several 
instances of fatal infection of men working with the organism 
in laboratories far removed from any focus of the disease, and 
finally the very unfortunate accident at Manila 1 where cholera 
vaccine mixed with a culture of B. peslis by mistake was injected 
into men and caused fatal bubonic plague. 

B. pestis in the body of the patient is a short plump rod, 0.5 
to 0.7;* wide by 1.5 to i.8/z long, and rounded at the ends. When 

1 Freer: Journ. A. M. A., 1907, Vol. XL VIII, pp. 1264-65. 



318 SPECIFIC MICRO-ORGANISMS 

stained the ends become deeply colored while the equator remains 
pale (bipolar staining) . Alongside this typical form many irregu- 
lar organisms are usually found, especially longer and shorter 
bacilli, some pale, some irregularly outlined, and some swollen 
and poorly stained. The last-mentioned types of bacilli are more 
frequently found in the bodies of plague victims which have be- 
gun to decompose. They are also observed in artificial cultures. 
These irregular forms (involution forms) are important in the 
quick recognition of plague. The bacillus stains very readily, 
best with methylene blue or with a momentary exposure to carbol- 
fuchsin. Better results are obtained by fixing the spread in alco- 
hol one minute, rather than heating it. The Romanowsky stain 
gives good results. It is distinctly Gram-negative (contrary to 
the original statement of Kitasato). Capsules may be demon- 
strated on bacilli in the peritoneal exudate of guinea-pigs and 
mice, less easily in cultures. It is non-motile and flagella have 
not been demonstrated. Spores have not been observed and 
cultures are killed at 60 C. in 10 to 40 minutes. It is also easily 
destroyed by chemical germicides, for example, by 5 per cent car- 
bolic acid in i minute. Mere drying at 35 to 37 C. kills the 
bacillus in two to three days, but at 20 C. it may withstand drying 
for 20 days. It may live for months in frozen material. 

Cultures are readily obtained on ordinary media, best at a 
temperature between 25 and 30 C. Growth is moderately 
slow. Gelatin is not liquefied. On agar containing 3 per cent 
of sodium chloride, irregular involution forms areprodu ced in 24 
to 48 hours. Long chains are produced in broth. It does not 
form gas from sugars but does produce acid from dextrose, levu- 
lose, mannite and galactose, not from lactose or dulcite. 

The toxins of the plague bacillus are in part soluble and in 
part intimately combined with the bacterial cell. Filtrates of 
young broth cultures are without toxic properties but older broth 
cultures (14 days) yield a toxic filtrate. The bacterial cells killed 
by heat produce fatal poisoning in guinea-pigs and rabbits. 
The poisons obtained so far are much less powerful than the sol- 



THE BACTERIA OF THE HEMORRHAGIC SEPTICEMIAS 319 

uble toxin of B. diphtherias or the endotoxins of the typhoid and 
cholera germs. 

Rodents, especially rats and guinea-pigs, are very susceptible 
to inoculation, even a needle prick carrying the minutest quantity 
of a virulent culture being sufficient to kill in a few days. At 
autopsy the adjacent lymph nodes are found greatly swollen 
and surrounded by hemorrhagic edema. The spleen is greatly 
swollen. Everywhere are enormous numbers of the bacilli. 
Infection by feeding gives positive results in about half the ex- 
periments. Inhalation of the bacilli produces typical pneumonic 
plague in rats. Monkeys are susceptible and present lesions 
similar to human plague. 

Bubonic plague can be recognized in descriptions of epidemics 
in very ancient records. Rufus of Ephesus who lived at the time 
of Trajan (A. D. 98) mentions specifically a very fatal acute 
bubonic plague (" pestilentes bubones"). Great epidemics oc- 
curred in Europe in the 6th century (527-565 A. D.), in the four- 
teenth century (1347-1350 A. D.). Each of these was followed 
by smaller outbreaks persisting in the latter epidemic up to about 
1850. It is estimated that 25 million persons died of the plague 
in the " Great Mortality" of the i5th century. Another pandemic 
of plague began in 1893. Its progress has been slow and un- 
doubtedly hampered by the prophylactic measures made possible 
by the discovery of Yersin and Kitasato. It exists as a persistent 
infection among rodents or human beings, or both, in central 
Asia, central China, northern India, Arabia, southern Egypt, 
and, more recently, seems to be establishing itself in California. 
Outbreaks of plague in man in new localities have usually been 
preceded or associated with mortality among rodents, especially 
rats. When an epidemic begins in a seaport town, the sewer rats 
(Mus decumanus) are first attacked. Two to three weeks later 
the house rats (Mus rattus) begin to die, and about four weeks 
later the epidemic of human plague begins. The transmission 
from animal to animal and from animal to man is accomplished 
very largely by the agency of fleas. Rat fleas are rarely found 



320 SPECIFIC MICRO-ORGANISMS 

on man or at large in human habitations as long as their normal 
hosts are at hand, but when the rats sicken and die of plague, 
then the fleas leave and becoming hungry they bite human beings 
and thus 'noculate them with plague bacilli. 

In its permanent endemic centers, plague exists as an acute 
and chronic disease of rodents. It spreads from these regions 
through the agency of the wandering rats traveling along the 
routes of commerce and especially in ships. The infected rat, 
arrived at its destination, sets up an epizootic among its own 
species, which later spreads to other animals and to man through 
the agency of fleas, producing the bubonic form of the disease. 
The infection may then be transmitted from man to man by 
fomites and directly by contact, and by infectious material sus- 
pended in the air, giving rise to the pneumonic form of the dis- 
ease. A persistent epizootic of chronic plague among rodents 
in a new region may give rise to a new permanent endemic 
center. 

In man the disease occurs in two principal forms, the bubonic 
type, in which the portal of entry is on the skin or mucous mem- 
brane and the disease is manifested by swelling of the neighboring 
lymph nodes, and the pneumonic type in which the organisms 
are inhaled or aspirated into the lung. Both of these forms re- 
sult in general bacteremia, as a rule. The bubonic form is largely 
due to inoculation of the skin by bites of insects (fleas), while the 
pneumonic form is transmitted more directly. Other clinical types 
of the disease occur. The death rate is 30 to 90 per cent in the 
bubonic and 98 to 100 per cent in the pneumonic type. In the 
bacteriological diagnosis, the morphology of the organism in the 
tissues and in cultures, its effect upon rats and guinea-pigs, and, 
finally, agglutination of the newly isolated culture with a known 
immune pest serum are important points. 

Immunity, at least a relative immunity, follows recovery from 
the plague. Artificial immunity can be induced by injection 
of attenuated living cultures and by the injection of killed bac- 
teria (Haffkine's method). Many modifications of the latter 



THE BACTERIA OF THE HEMORRHAGIC SEPTICEMIAS 321 

are recommended and they constitute the practical method of 
vaccination against plague. Haffkine employs broth cultures 
incubated at 25 to 30 C. for six weeks under a covering of sterile 
oil. The cultures are killed at 65 C., and preserved with car- 
bolic acid. The dose is o.i to 0.5 c.c. for children and 3 to 4 c.c. 
for an adult man. It may be repeated after ten days. Good 
results have followed the use of this prophylactic in India. 
Kolle suspends two-day agar cultures in broth or salt solution 
and kills at 65 C. by one to two hours exposure. Five-tenths per 
cent carbolic acid is then added. The dose injected is the prod- 
uct of one agar culture. The vaccination should be taken by any 
physician who expects to handle plague bacilli, even if only in the 
laboratory. 

Horses have been immunized by Yersin, injecting first killed 
bacilli, later highly virulent bacilli, and finally the filtrates of old 
broth cultures intravenously. The serum of these horses in a 
dose of 20 c.c. confers a transient passive immunity, and has 
seemed to be of value in the treatment of a few cases of plague. 
Its preparation is so difficult and its potency so low that it has 
not come into general use. The serum has also been injected 
along with killed bacilli to confer immunity (combined active 
and passive immunization) . 

The restriction and prevention of plague require measures 
adapted to the special conditions existing. In general they include 
precautions to exclude infected animals, wholesale destruction 
of rats and other rodents and the artificial immunization of the 
human population when confronted by the disease. The eradi- 
cation of the endemic centers presents a problem of great com- 
plexity, requiring the recognition and destruction of the infected 
species of animals. 

Bacillus (Micrococcus) melitensis. 1 Bruce in 1887 dis- 
covered this organism in the spleen of persons suffering from Malta 

1 This organism is classed as a micrococcus by most authors. It is here classed 
as a bacillus because of its general resemblance in many of its characters to B. pestis. 
None of the Gram-negative parasitic cocci resemble it in respect to physiological 
characters or in the remarkable ability to change its host. 

21 



322 SPECIFIC MICRO-ORGANISMS 

fever and obtained pure cultures. Inoculation of monkeys with 
pure cultures gives rise to a disease resembling in detail 1 Malta 
fever in man. 

The organism is spherical or oval 0.3 by 0.4^ in size, and is 
classed as a micrococcus by many bacteriologists. In gelatin 
cultures the cell is somewhat longer and resembles that of a true 
bacillus. The organisms are single, grouped in pairs or sometimes 
in short chains of four to five cells. Capsules and spores have 
not been observed. It is non-motile. Flagella have been de- 
tected by Gordon but other investigators have failed to confirm 
the observation. The organism stains readily and is Gram- 
negative. 

Cultures are obtained on ordinary media and growth is possi- 
ble between the extremes of 6 and 45 C. The colonies develop 
in one to three days at 37 C. and are very homogeneous. Gela- 
tin is not liquefied and neither gas nor acid is produced in media 
containing the various sugars. The organism is killed by moist 
heat at 57 C. in 10 minutes, by dry heat at 95 C. in 10 minutes 
and in i per cent carbolic acid in 15 minutes. It survives drying 
for several months and retains its vitality in culture without 
transplantation for several years if drying is prevented. 

Many mammals are susceptible, including guinea-pigs, rabbits, 
monkeys, rats and mice. Horses, cows, sheep and goats are not 
only susceptible to inoculation but also contract the disease natu- 
rally. In all animals the course of the infection is usually chronic 
and characterized by an irregularly remittent fever. Death is 
a common outcome in monkeys. Often the subcutaneous injec- 
tion or the feeding of a minute quantity of the culture is sufficient 
to infect, but for the smaller laboratory animals intracerebral in- 
oculation may be necessary. 

Malta fever in man is a chronic disease characterized by an 

irregularly remittent fever. The spleen is enlarged and often 

the liver as well. Positive agglutination of a known culture of 

B. melitensis by the patient's serum in dilution of i to 1000 is an 

1 Eyre in Kolle and Wassermann, Handbuch, 1912, Bd. IV, S. 432. 



THE BACTERIA OF THE HEMORRHAGIC SEPTICEMIAS 323 

important aid in diagnosis, and isolation of the organism from the 
circulating blood, or from the spleen, and its identification makes 
the diagnosis certain. Positive cultures are more often obtained 
from the spleen, but the puncture of this organ by the inexperi- 
enced is not without danger. Blood cultures should be made dur- 
ing a febrile period and preferably late in the afternoon. Death 
occurs in i to 2 per cent of the cases. 

Careful investigations have shown that infection with B. meli- 
tensis is endemic among the goats of Malta, from which animals 
is obtained the milk supply of the region. The micro-organisms 
are excreted in the milk. Monkeys fed such milk acquire the 
disease, and human epidemics of Malta fever have followed the 
use of such milk under conditions closely resembling those of 
critical experimentation. Other methods of transmission have 
been tested with negative results. 

Immunity follows recovery from the disease, but artificial 
immunization is not yet a practical success. 



CHAPTER XXII. 

BACTERIACE^E: THE COLON, TYPHOID AND DYS- 
ENTERY BACILLI. 

Bacillus Coli. This organism was probably observed by sev- 
eral investigators previous to 1886 but it was either neglected or 
its significance was misinterpreted. The first important study 
of it was made by Escherich in that year, who discovered it in the 
feces of healthy infants and obtained it alone on the aerobic gela- 
tin plate cultures inoculated with this material. 




FIG. 130. Bacillus coll showing flagella. (From McFarland after Migula.} 

B. coli lives and grows in the intestinal tract of man and mam- 
mals, and organisms closely resembling it have been found in the 
intestinal canal of other vertebrates. It is discharged in large 
numbers in the feces and some of these bacilli may continue their 
growth in the external world for a time. The organism is 0.4 to 

324 



THE COLON, TYPHOID AND DYSENTERY BACILLI 325 

o.7M wide and i to 6/1 long, with rounded ends, usually single but 
sometimes occurring in threads. It is motile but not very active^ 
and many cells, even in young cultures, may be motionless. 

There are four to eight peritrichous flagella. Spores have 
not been observed. The bacillus stains readily and is Gram- 
negative. 

Cultures develop rapidly at 37 C. on all ordinary media. 
The colony is white, opaque, often somewhat heaped up in the 
center and thinner near the edge. It may be round with smooth 
outline or the border may be lobulated. Under the low-power 




FIG. 131. Bacillus coli. Superficial colony on a gelatin plate two days old. X 21. 
(From McFarland after Heim.) 

lens the colony appears brown, finely granular near the periphery 
and more coarsely granular near the center. It is soft and moist, 
easily removed from the medium and easily suspended as a dif- 
fuse cloud in water. Gelatin is not liquefied. B. coli ferments 
dextrose and lactose with the production of gas as well as acid. 
It coagulates milk in 24 to 48 hours at 37 C. and renders it acid, 
produces considerable indol in pepton solution and grows abun- 
dantly on potato, often producing a brown color. 

Intraperitoneal injection of cultures into guinea-pigs and rats 
causes fatal peritonitis. Subcutaneous injection may also cause 
death but frequently results in a local abscess. 



326 SPECIFIC MICRO-ORGANISMS 

The cultures of B. coli on ordinary media are practically free 
from soluble poisons, but there is some evidence that soluble 
poisons may be produced by this organism under special condi- 
tions. 1 The bacterial cell substance is poisonous. 

As it grows in the intestine the colon bacillus is a harmless 
commensal but with a distinct tendency to invade the living 
tissue and become pathogenic whenever the normal resistance is 
lowered. The bacilli doubtless pass through the intestinal wall 
in very small numbers during absorption of the food and are de- 
stroyed in the normal body fluids and tissues in a few hours. In 
intestinal disturbances the invasive properties and the virulence 
are increased. In many other regions of the body the colon bacil- 
lus gives rise to inflammation, often purulent in character. It 
is a common cause of cystitis and pyelitis, and is an important 
agent in the causation of peritonitis following perforation of the 
intestine. Generalized infection with B. coli is rather uncommon. 
The bacilli frequently enter the blood stream from the intestine 
during the death agony, and are often present in the heart's blood 
at autopsy, especially if this is delayed. 

The detection of B. coli in any material is ordinarily regarded 
as evidence of fecal contamination. Examinations of drinking 
water and of shell liquor from oysters are, perhaps, the most fre- 
quent applications of this principle. Fermentation tubes of 
dextrose broth are inoculated with measured quantities of the 
liquid to be tested, o.oi c.c., o.i c.c. and i c.c. Those cultures jn 
which gas is produced are plated on litmus lactose media and the 
typical colonies transplanted to gelatin, milk, fermentation tubes 
of dextrose_broth and agar slants, and for final identification the 
agglutination test with a known colon-immune serum may be 
employed. 

Bacillus (Lactis) Afe'rogenes. Escherich described this organ- 
ism in 1886 as distinct from B. coli. It is non-motile, is usually cap- 
sulated and its colonies are thicker and less spreading. In other 
respects it does not differ materially from B. coli and many authori- 
1 See Vaughan and Novy: Cellular Toxins, Phila., 1902, p. 220. 



THE COLON, TYPHOID AND DYSENTERY BACILLI 



3 2 7 



ties regard it as a variety of this species. 
B. aero genes was found by Escherich in 
the upper part of the small intestine. It 
is commonly present in ordinary cow's 
milk and has been found in the urine in 
cystitis 1 and pyelitis. 

Bacillus (Bacterium) Pneumoniae. 
This organism was obtained by Fried- 
laender in 1883 on gelatin plates inocu- 
lated with material from cases of pneu- 
monia and was confused by him with the 
organisms which he observed microscopi- 
cally in abundance in his material. The 
latter were undoubtedly pneumococci (See 
Diplococcus pneumonia page 257). B. pneu- 
monia is rather common in the upper air 
passages and occurs in the lungs in some 
cases of pneumonia. It is non-motile, 
capsulated and Gram-negative, and in 
nearly all respects quite like B. aerogenes. 
The nail-shaped culture in gelatin stab is 
regarded as specially typical. 

Bacillus (Bacterium) Rhinosclero- 
matis. This organism was described by 
von Frisch in 1 88 2 . It is readily obtained, 
often in pure culture, by incising the lesion 
of rhinoscleroma and spreading the blood 
thus obtained on an agar surface. 2 It is 
also found in abundance by microscopic 
examination of sections of rhinoscleroma 
tissue. B. rhinosderomatis is capsulated, 
non-motile and in morphology and cultural 

1 Luetscher, Johns Hopkins Hosp. Bull., ion. Vol 
XXII, pp. 361-366. 

2 Wright and Strong: New York Med. Journ., ion 
Vol. XCIII, pp. 516-519. 



FIG. 132. Friedlan- 
der'spneumobacillus; gel- 
atin stab culture, show- 
ing the typical nail-head 
appearance and the for- 
mation of gas bubbles, not 
always present. (From 
McFarland after Curtis.) 



328 SPECIFIC MICRO-ORGANISMS 

characters indistinguishable from B. pneumonia. It is Gram- 
negative when stained by the usual technic. Its etiological rela- 
tion to rhinoscleroma is somewhat uncertain. 

Rhinoscleroma is a disease characterized by the occurrence of 
circumscribed grayish nodules in the mucous membrane of the 
nose, which tend slowly to extend without ulceration. Histo- 
logically the lesion is composed of granulation tissue and fibrous 
tissue with lymphocy tic infiltration. Many of the cells appear 
swollen and vacuolated, so-called lace-cells, and in and near these 
the bacilli are present in large numbers. The disease occurs in 
Europe and has been seen in a number of Russian immigrants to 
the United States. 

Bacillus (Mucosus) Capsulatus and Bacillus Ozenae also occur 
on the mucous membranes of the upper air passages. They do 
not appear to be specifically different from B. pneumonia of 
Friedlaender. 

Bacillus Enteritidis. Gaertner in 1888 isolated this organism 
from the spleen of a man who died in an epidemic of meat poison- 
ing in which 57 persons were made ill. The meat was derived 
from a cow, sick at the time of slaughter, and this same organism 
was found in the meat which had not been sold. The bacillus is 
of the same size and shape as B. coli, but is more actively motile 
and has more flagella. It ferments dextrose with the production 
of gas, does not ferment lactose nor coagulate milk, nor does it 
produce an amount of indol appreciable by testing with sulphuric 
acid and nitrite. Its cultures are highly toxic, even after they 
have been boiled. 1 A typhoid-immune serum agglutinates B. 
enteritidis in fairly high dilutions. The cases of food poisoning 
in which it was found were characterized by vomiting and diarrhea 
and at autopsy by severe enteritis and swelling of the lymph 
follicles of the intestine. Food poisoning of this type seems to 
be rather common. 2 



1 Vaughan and Novy: Cellular Toxins, 1902, p. 207. 

2 Anderson, Poisoning from Bacillus enteritidis. The Military Surgeon, 1912, 
Vol. XXXI, pp. 425-29. See also Marshall's Microbiology, 1911, p. 414. 



THE COLON, TYPHOID AND DYSENTERY BACILLI 329 

Bacillus Suipestifer (B. Salmonii). This organism occurs in 
the intestinal contents of hogs and in the blood in the late stages 
of hog cholera, and was for a time believed to be the cause of this 
disease. More recent studies indicate that the etiological factor 
is a filterable virus (See page 373). B. suipestifer resembles B. 
enteritidis very closely. 

Bacillus Icteroides was described by Sanarelli in 1897 as the 
cause of yellow fever, a disease now known to be caused by a filter- 
able agent (page 368). It cannot be specifically distinguished 
from B. suipestifer. 

Bacillus Psittacosis was found by Nocard in 1892 to be the 
cause of an epidemic pneumonia transmitted to man from dis- 
eased parrots. It resembles B. coli but may be distinguished by 
inoculating parrots, for which it is extremely virulent. 

Bacillus Typhi Murium. L6fHer in 1890 found this organism 
to be the cause of a fatal epizootic among laboratory mice. It 
forms gas and acid from dextrose, does not produce indol nor co- 
agulate milk. Mice are very susceptible and the organism has 
been employed as a practical means of destroying mice. It seems, 
however, not to be altogether harmless for larger animals and for 
man, and it is believed that some of the paratyphoid fever fol- 
lowing food poisoning in man has been due to this particular 
organism. 

Bacillus (Fsecalis) Alkaligenes. This organism is occasionally 
found in human feces and is of importance because of the possi- 
bility of mistaking it for the typhoid bacillus, which it resembles 
in most respects. It does not produce acid from any of the sugars 
nor is it agglutinated by typhoid serum. It is not known to 
cause disease. 

Several other organisms of this general type have been found 
in pathological conditions of man or of animals and some of them 
have received specific names. In certain irregular fevers in man 
resembling somewhat typhoid fever, organisms have been found 
in the circulating blood which are agglutinated by the patient's 
serum, and which exhibit many of the characters of the B. coli or 



330 



SPECIFIC MICRO-ORGANISMS 



B. enteritidis groups. They are ordinarily regarded as inter- 
mediate between B. coli and B. typhosus and are designated 
as paracolon and paratyphoid bacilli. The diseases in which they 
occur are sometimes traceable to meat poisoning. B. enteritidis 
and B. typhi murium doubtless occur in the circulating blood of 
man at times as paratyphoid bacilli. B. psittasosis is usually 
regarded as a paracolon bacillus. 

Bacillus Typhosus. Eberth in 1880 and Koch in 1880 ob- 
served this organism in the spleen and mesenteric lymph glands 




FIG. 133. Bacillus of typhoid fever. X 1000. 

of persons dying of typhoid fever. Gaffky in 1884 obtained the 
first pure cultures. Metchnikoff 1 and Besredka in 1911 succeeded 
in causing typical typhoid fever in anthropoid apes (chimpanzees) 
by feeding them cultures of B. typhosus, thus adding conclusive 
proof of the causal relationship of this organism to typhoid fever 
to the abundant strong evidence previously at hand. 

B. typhosus is found in the intestinal contents, mesenteric 
lymph glands, spleen, blood and urine of patients suffering from 
typhoid fever. It is 0.5 to o.8/t in width and i to 4/4 in length, 

1 Annals de VInstltut Pasteur, 1911, Vol. XXV, 193-221. 



THE COLON, TYPHOID AND DYSENTERY BACILLI 



331 



commonly occurring single or in short threads, stains readily 
with anilin dyes and is Gram-negative. It is actively motile and 




FIG. 134. Bacillus typhosus showing flagella. (After McFarland.} 

possesses 10 to 20 peritrichous flagella. Spores have not been 
observed. 







A B 

FIG. 135. Colonies on gelatin plate two days old of (^4) Bacillus typhosus, and (B) 
Bacillus coli. Y^ZT.. (From Jordan after Heim.) 



The organism grows readily on ordinary media but not so 
luxuriantly as B. coli. The colony is smaller but relatively more 



33 2 SPECIFIC M^IO-ORGANISMS 

spread out and thinner than that of B. coli, and in semi-solid media 
the growth of B. typhosus may diffuse for quite a distance because 
of its active motility. Dextrose is fermented with the production 
of acid but without gas. Lactose is not fermented. Litmus 
milk is rendered slightly acid and later becomes alkaline without 
coagulation. On potato the growth is almost invisible. In Dun- 
ham's pepton-salt solution, indol is not produced in sufficiently 
large quantities to be detected, but indol can be demonstrated 
in old cultures in 5 per cent pepton. Growth is most rapid at 
37~39 C., but occurs also at room temperature. 

B. typhosus is killed by moist heat in 10 to 15 minutes, and by 
5 per cent carbolic acid or i-iooo mercuric chloride in three to 
five minutes, when exposed in aqueous suspension. It resists dry- 
ing for several days and may be alive in dry dust. The longevity 
of B. typhosus in surface waters has been studied by several in- 
vestigators without full agreement. In general B. typhosus would 
seem to survive in such water only for three to ten days except 
it be taken up by aquatic animals, such as the shellfish, when it 
may persist for several weeks. In soil and in frozen material 
the bacillus may live a much longer time. Freezing and thawing 
destroys a large percentage of the bacilli in a given liquid but 
does not destroy them all. 

The poisons are intimately associated with the cell substance, 
and it is not often that culture filtrates are found to be toxic. 
The dead germ substance is somewhat poisonous, and when it 
is disintegrated by physical comminution or by digestion with 
dilute alkali at a high temperature, or by the action of serum 1 
upon it, there are set free quite powerful poisons or perhaps differ- 
ent quantities of the same poison. 

The various small laboratory animals are very susceptible to 
intraperitoneal inoculation with B. typhosus and usually die in 
24 to 48 hours with acute peritonitis and bacteremia. The dis- 
ease produced bears no resemblance to typhoid fever in man. 
In chimpanzees a very typical attack of typhoid fever has been 
1 Zinsser: Journ. Exp. Med., 1913, Vol. XVII, pp. 117-131. 



THE COLON, TYPHOID AND DYSENTERY BACILLI 333 

caused by feeding the organisms, with resulting lesions in the in- 
testine, bacilli in the blood and spleen, and a continued fever. 

Typhoid fever exists generally throughout the temperate 
zone, is present throughout the year but most prevalent in the 
fall. The usual mode of infection is undoubtedly through food 
and drink. The bacilli swallowed survive in part the action of 
the gastric juice and so gain the lumen of the duodenum. The 
first multiplication seems to occur here 1 in a location fairly free 
from bacteria in health. The infection extends along the wall 
of the intestine, involving especially the lymphatic structures, 
solitary glands and Peyer's patches. The bacteria pass into 
the lymph stream to be carried to the mesenteric nodes, spleen 
and into the blood. At the onset of definite symptoms of typhoid 
fever the bacilli have usually reached the general blood circu- 
lation. Subsequently the infection reaches the gall bladder, per- 
haps by extension along the common bile duct and cystic duct or 
perhaps by the blood stream through the liver; the organisms 
also pass through the kidney and multiply in the contents of the 
urinary bladder. They are present in the rose spots on the 
skin. The bacilli are often present in the feces in small numbers, 
the abundance of other organisms making their isolation and 
recognition difficult. At times localized inflammations due to 
B. typhosus develop elsewhere in the body, as in the lungs. It 
is evident therefore that the bacilli may leave the body of the 
patient through many channels, but chiefly with the urine and 
feces. Even after recovery the patient may continue to dis- 
charge virulent bacilli for months or years. It is estimated that 
one per cent of recovered cases are persistent carriers of the in- 
fectious agent. 

The bacteriological diagnosis of typhoid fever depends upon 
isolation and recognition of the germ or detection of specific sub- 
stances in the blood produced by the patient as a reaction to the 
presence of B. typhosus. B. typhosus is sought by blood culture 
(see page 101) diluting the blood with large amounts of broth 
1 Hess: Journ. Infect, Diseases, 1912, Vol. XI, pp. 71-76. 



334 SPECIFIC MICRO-ORGANISMS 

(200 c.c. of broth to 2 c.c. of blood) as well as inoculating tubes of 
bile and the usual agar plates; by cultures from the rose spots, 
and by cultures inoculated with duodenal fluid. These methods 
are likely to be successful very early in the disease. Later it 
is well to make cultural examination of the feces and urine, 
especially just before discharging a recovered patient. 

The detection of B. typhosus in feces requires special care. 
Russell recommends plating the feces on Endo's medium, 1 fishing 
of the promising colonies to a slant of his double-sugar medium, 2 
inoculating both as a streak and stab, and then making the agglu- 
tination test with known serum upon the typical cultures in the 
double-sugar medium. The examination is thus completed in 
two or three days. 

The specific antibody ordinarily sought in the blood is the 
typhoid agglutinin. A few drops of blood in a Wright's capsule 
suffice for the microscopic test (see page 211). A young active 
culture (broth three hours) of a known B. typhosus is used, and 
the serum is tested in dilutions of 1:20, 1:40 and 1:80, observed 
for an hour. Normal serum rarely shows any clumping in any 
of these dilutions at the end of an hour. This agglutination test 
is of little or no value if the patient has received typhoid vaccine 
within a year. 

Transmission of the disease takes place in a variety of ways. 
To the best of our knowledge, the typhoid bacilli come only from 
human individuals infected with them. Some of these actually 

1 For Endo's medium a stiff lactose agar is prepared containing Liebig's extract 
5 grams, salt 5 grams, pepton 10 grams, lactose 10 grams and agar 30 grams in 1000 
c.c. of water. This is sterilized in flasks containing 100 c.c. each. When needed 
the contents of a flask is liquefied, enough sodium hydroxide is added [to make the 
reaction 0.2 per cent acid to phenolphthalein and to it are then added 10 drops of 
saturated alcoholic solution of basic fuchsin, and 20 drops of a freshly prepared solu- 
tion of sodium sulphite. The material is well mixed and poured into 8 or 10 Petri 
dishes, allowed to solidify and dried in the incubator to remove water from the sur- 
face before use. Fecal material is spread by means of a bent glass rod over the sur- 
face of several plates in succession. 

2 The double-sugar medium is a 2 to 3 per cent agar, neutral to litmus, to which 
has been added i per cent lactose and o.i per cent glucose. On this medium B. 
typhosus does not change the color when it is growing on the surface, but produces 
a red (acid) color about the stab. See Russell, Journ. Med. Rsch., 1911, Vol. XX, 
pp. 217-229. 



THE COLON, TYPHOID AND DYSENTERY BACILLI 335 

suffer from typhoid fever, while others are merely healthy carriers 
of the infection. From them as centers the bacilli are distributed 
by contact and by intermediate objects. B. typhosus is able to 
live for a considerable time in the external world, probably for 
one to three weeks in ordinary surface waters and longer in soil. 
It is able to grow and multiply in some foods, especially milk. 
Water supplies contaminated with feces and urine from patients 
or from healthy carriers have unquestionably been an important 
factor in the causation of typhoid fever in the past, and the pro- 
vision of a supply of drinking water free from all suspicion of 
recent mixture with sewage is the first step in the control of this 
disease in a community. The infected oyster from a sewage- 
polluted oyster bed is another source of typhoid fever. The 
contamination of food by permanent carriers of the bacilli is 
difficult to control. All possible means need to be employed to 
prevent these persons from handling foods prepared for consump- 
tion, and especially milk. Flies (Musca domestica) are important 
aids in the transfer of bacilli from discharges containing them, 
especially from open privies, to foods exposed for sale or being 
prepared in neighboring unscreened kitchens, i i 

The prevention of typhoid fever by restricting the distribu- 
tion of the bacilli has been only partially successful in civil life 
and in armies on a war footing it has proven wholly ineffective. 
Vaccination to prevent typhoid fever was first extensively prac- 
tised by Wright in the British army. Russell 1 following the method 
developed by Wright and Leishman has prepared a vaccine with 
which practically the whole U. S. army has been inoculated. 
The vaccine is a suspension of B. typhosus in salt solution, stand- 
ardized by microscopic count of the bacterial cells, sterilized by 
heating at 53 to 56 for an hour and preserved by the addition 
of 0.25 per cent trikersol. Three injections are given subcutane- 
ously at intervals of 10 days, 500 million bacilli at the first dose 
and 1000 million at each of the following doses. The results 

1 Russell: Boston Med. and Surg. Journ., 1911, Vol. CLXIV, pp. 1-8; Harvey 
Lecture, 1913. 



336 SPECIFIC MICRO-ORGANISMS 

in the U. S. army have been remarkably good, rivaling those 
obtained with the use of vaccinia in the prevention of small-pox. 

Bacillus (Bacterium) Dysenteriae. Shiga in 1898 isolated 
this organism from the feces of patients suffering from dysentery, 
showed that it is agglutinated by the blood of dysenteric patients 
in high dilutions and not by normal human blood. 

B. dysenteries is about o.6/x in width by 2 to 4/4 in length, usually 
single and non-motile. It stains readily and is Gram-negative. 
Involution forms are common in older cultures. The organism 
grows readily on ordinary media and its cultures resemble those 
of B. typhosus very closely. Gelatin is not liquefied; no indol is 
produced in pep ton solution; no gas is formed from any of the 
sugars; milk is rendered slightly acid and then alkaline without 
coagulation. It differs from the typhoid bacillus in failing to 
ferment mannite and maltose. 

When cultures are injected intravenously into rabbits severe 
diarrhea is produced, which may be bloody. The animal usually 
dies in a few days, and if it recovers often exhibits paralysis of 
the hind legs. Similar results are obtained by the injection of 
dead bacilli, indicating that the effect is toxic rather than infec- 
tious. Kittens and puppies have been infected by introducing 
dysentery bacilli into the stomach, resulting in diarrhea with the 
intestinal lesions of dysentery. The toxins seem to be intimately 
bound up in the cells in young cultures, but readily set free into 
solution after the bacilli are killed. Culture filtrates, of which 
0.02 c.c. suffices to kill a rabbit in 24 hours, have been obtained. 

Acute epidemic dysentery is the disease in which this organism 
is found. The infectious agent is found on the membrane of the 
large intestine, which is diffusely inflamed, often covered with a 
fibrinous exudate, or by a pseudo-membrane. Later numerous 
ulcers may be found. The bacilli are only very rarely found in 
the blood or internal organs. The blood of the patient aggluti- 
nates the bacillus of Shiga in dilutions of i to 50 or i to 100. The 
mortality is about 25 per cent, but variable in different epidemics. 

Horses have been immunized with cultures of B. dysenteric and 



THE COLON, TYPHOID AND DYSENTERY BACILLI 337 

the serum of these animals has been found to be antitoxic as well as 
bactericidal. Its use in treatment has given promising results and_ 
seems to cause a reduction in the death rate of about 50 per cent. 

Paradysentery Bacilli. Flexner in 1899 isolated a bacillus 
from cases of dysentery in the Philippines which at the time was 
considered to be the same as the Shiga bacillus. Kruse, although 
he found the Shiga bacillus in epidemic dysentery, found a some- 
what different organism in " asylum dysentery" or pseudo- 
dysentery, which proved to be identical with the Flexner bacillus. 
Between 1901 and 1903 a number of strains of bacilli resembling 
somewhat B. dysenteries were isolated -by different investigators 
from epidemics of diarrheal disorder, especially in the Eastern 
United States. The paradysentery bacilli are indistinguishable 
from B. dysenteries in morphology or in cultures on ordinary 
media. They are all much less toxic to rabbits than the Shiga 
bacillus, and they all ferment mannite with the production of 
acid, while the Shiga bacillus does not. 

The bacteria considered in this chapter are all inhabitants 
of the alimentary canal (mouth, pharynx, intestine) of man or 
other mammals. They are small bacilli, Gram-negative, without 
spores and without the ability to liquefy gelatin. They vary 
from each other in motility, possession of flagella, possession of 
capsules, and in their ability to form poisonous substances and 
to ferment various carbohydrates. Media containing various 
carbohydrates along with an indicator such as litmus to show 
the production of acid, and contained in fermentation tubes so 
as to measure the production of gas, are very useful in differentiat- 
ing 1 the various types of bacteria in this group. Thus, in a 

1 Hiss has devised a very useful medium for this purpose which obviates the neces- 
sity of using the fermentation tube to detect the gas. His serum- water medium is 
made by mixing beef serum, i part, with distilled water, 2 to 3 parts, and steaming 
15 minutes to destroy enzymes. Pure litmus solution (about i part of a 5 per cent 
solution to 100 parts of the medium) is then added to produce a deep blue color. 
The medium is divided into several portions and i per cent of the desired carbo- 
hydrate is added to its respective portion. The sugar serum-water media are then 
sterilized at 100 C., on three days. Fermentation is shown not only by the redden- 
ing of the litmus but also by coagulation of the liquid medium, and gas production 
is shown by bubbles caught in the coagulum. (Hiss and Zinsser: Text-book of 
Bacteriology, 1910, p. 132.) 



SPECIFIC MICRO-ORGANISMS 

broth containing maltose, B. typhosus produces acid, B. coli 
produces acid and gas, and B. dysenteries produces neither. 
Specific agglutination with the serum of an animal immunized 
with a known culture constitutes the most important test in the 
identification of unknown forms falling within this group. This 
test may be used with the capsulated species after they have 
lost the tendency to form capsules through propagation on artifi- 
cial media. 1 

1 Fitzgerald: Proc. Soc. Biol. and Med., 1913, Vol. X, pp. 52-53 



CHAPTER XXIII. 

BACTERIACE^: BACILLUS MALLEI AND MISCELLA- 
NEOUS BACILLI. 

Bacillus (Bacterium) Mallei. Loffler and Schiitz in 1882 
obtained pure cultures of this organism from glandered horses 
and produced glanders by the injection of these pure cultures. 

The bacillus is 0.3 to o.5ju wide and 2 to 5/4 long, usually 
straight with rounded ends, but sometimes irregular in shape. 
Filamentous and branched forms have been observed in cultures. 




FIG. 136. Bacillus mallei from an agar culture. 

Williams.) 



X 1 1 oo. (After Park and 



It is not motile. Spores have not been observed. B. mallei is 
stained with moderate difficulty and often stains unevenly like 
the tubercle and diphtheria bacilli. After being stained, the 
bacterium is easily decolorized in weak acid or alcohol; it is also 
Gram-negative. Cultures develop on ordinary media, better on 
glycerinated media, at temperatures ranging from 22 

339 



to 42 C., 



340 SPECIFIC MICRO-ORGANISMS 

best at 37 C. On potato at 37 C. a viscid yellowish-brown 
growth develops surrounded by a greenish stain on the potato. 
Gelatin is not liquefied. The organism is killed by moist heat 
at 55 C. in 10 minutes, and in 2 to 5 minutes by 5 per cent 
carbolic acid or i to 1000 mercuric chloride. It survives drying 
for only a few weeks and dies out quickly in water. Many 
mammals are susceptible to inoculation, including horses, guinea- 
pigs, cats and dogs. Cattle are immune. Man is susceptible 
and human glanders frequently ends in death. 

Mallein is analogous to tuberculin. A culture in glycerin 
broth incubated for six weeks is steamed and filtered, and the 
filtrate evaporated to one-tenth the original volume is the mallein. 
This substance is toxic to animals suffering from glanders but 
not poisonous to healthy animals. 

Glanders is a disease most common in horses, mules and asses. 
It begins as an inflammation of the nasal mucosa with localized 
nodular infiltrations which later ulcerate. The infection may 
become generalized at once causing acute glanders and death in 
one to six weeks, or it may progress very slowly and persist for 
years as chronic glanders. The chronic type is common in horses. 
After apparent recovery from the disease nodules containing 
living bacilli may be found in the lungs. Histologically the gland- 
ers nodule consists of granulation tissue infiltrated with leukocytes 
and tending to become purulent at the center. The bacilli leave 
the body in the nasal secretion and in the discharge from ulcers. 
Infection of equines takes place most frequently by ingestion of 
food soiled by these discharges. In man the disease seems to 
result from inoculation of small wounds in the skin. It often 
runs an acute course terminating in death, but chronic glanders 
with recovery also occurs in man. A few sad laboratory accidents 
in which workers have become inoculated with glanders have 
emphasized the necessity for caution in handling this organism. 

The bacteriological diagnosis depends upon (i) identification 
of B. mallei, (2) reaction of the animal to mallein, (3) agglutina- 
tion reaction, and (4) complement fixation. For the recognition 



BACILLUS MALLEI AND MISCELLANEOUS BACILLI 341 

of the bacillus, some of the suspected material is suspended in 
broth and injected into the peritoneal cavity of a male guinea-pig^ 
(method of Straus). If B. mallei is present a general inflamma- 
tion of the peritoneum develops and after three or four days the 
testicles of the animal become swollen, inflamed and later suppu- 
rate. They may burst through the scrotum. Cultures should 
be made from this pus on plates of glycerin agar and the colonies 
transplanted to potato at 37 C. Very few other organisms 
give rise to a similar pathological picture in the guinea-pig. At 
the same time the mallein test is carried out by injecting 0.2 c.c. 
of the concentrated mallein diluted with 0.25 per cent solution 
of carbolic acid into the suspected horse. The presence of gland- 
ers is indicated by a rise in temperature of 2 to 5 F., signs of 
general intoxication, and especially by swelling and inflammation 
at the site of injection. For the agglutination test the serum 
is diluted to i : 500 to i : 3000. Positive results with lower dilu- 
tions may apparently be given by normal horses. The comple- 
ment-fixation test follows the principles of Wassermann test for 
syphilis, a culture of B. mallei being employed as antigen. 1 At- 
tempts at immunization have not been practically successful. 

Bacillus (Bacterium) Abortus. Bang and Stribolt isolated 
this organism from the uterus of a cow suffering from the disease 
known as contagious abortion, and reproduced the disease by in- 
oculating healthy cows with these cultures. The organism is 
of interest because of its behavior toward oxygen when first iso- 
lated. It fails to grow in the air or in hydrogen, but grows in 
a partial pressure of oxygen somewhat below that of the atmos- 
phere. The bacillus is pathogenic for a number of different 
mammals, and in guinea-pigs it causes granulomatous lesions 
resembling somewhat those of tuberculosis. 2 The organism 
occurs rather frequently in market milk. It is not known to 
infect man. 

1 Mohler and Eichorn: Twenty-seventh Annual Rep. Bur. Anim. Industry, U. S. 
Dept. Agr., 1910; reprinted as Circular 191 (1912). 

2 Smith and Fabyan: Centr. f. Bakt., I, Abt. Orig., 1912, Bd. LXI, S. 549-555. 
Fabyan, Journ. Med. Rsch., 1912, Vol. XXV, p. 441-488. 



342 SPECIFIC MICRO-ORGANISMS 

Bacillus (Bacterium) Acne. This minute non-motile organ- 
ism, first described by Gilchrist, is constantly present in the pap- 
ules and pustules of the common skin affection, acne vulgaris. 
Cultures are most readily obtained by expressing, with careful 
asepsis, some of the cheesy pus from a recent papule and mixing 
it with 2 c.c. of ascitic fluid in a test-tube. Dilutions from this 
are made to similar amounts of ascitic fluid in series (about five 
tubes in all). To each tube are then added 8 c.c. of melted 
glucose agar cooled to 50 C., the contents of each tube mixed 
without introducing air bubbles and then quickly solidified by 
immersion in cold water. The colonies of B. acne develop at 
37 C. after five to ten days, beginning about 8 mm. beneath 
the surface, and they grow best in a narrow zone about 5 mm. in 
depth. The colonies attain a large size (3 mm.) and an abundant 
supply of bacillary substance for preparation of vaccine may be 
obtained by thrusting a sterile glass capillary into such a colony. 
In its behavior to oxygen when first isolated the organism exhibits 
the same peculiarity as the bacillus mentioned in the preceding 
paragraph. 

Bacillus (Bacterium) Bifidus. Tissier in 1898 showed that 
the Gram-positive bacillus predominant in the stools of healthy 
nurslings is not a form of B. coll as had been supposed since the 
investigations of Escherich (1886) but is an entirely different 
organism. He obtained cultures by making a series of dilutions 
(five to ten tubes) in tall tubes of glucose agar by the method of 
Veillon (see page 112). The colonies develop best about i to 2 
cm. beneath the surface after three to eight days at 37 C. In 
these colonies many of the bacilli show dichotomous branching. 
Bifid forms are also sometimes seen in stools and in mixed cul- 
tures in broth. The organism produces a strong acid reaction 
and the cultures soon die out. The bifid forms are doubtless 
involutions due to presence of unfavorable amounts of acid. 

Bacillus (Bacterium) Bulgaricus. This organism is a rather 
large rod i by 6ju approximately. It occurs in milk and milk 
products and is especially abundant in milk fermented at 40 C. 



BACILLUS MALLEI AND MISCELLANEOUS BACILLI 343 

for three or four days. Colonies may be obtained on plates of 
milk agar (i 12) incubated at 37 C. in hydrogen. A high degree 
of acidity (lactic acid) is produced in the cultures of this organism, 
and it is employed to some extent in the preparation of acid-milk 
beverages. 

Bacillus (Proteus) Vulgaris. Hauser in 1885 discovered 
this organism in putrefying infusions of animal matter. It is an 
actively motile rod 0.6 n in thickness and exceedingly variable in 
length, with abundant flagella. Spores have not been observed. 
It is universally distributed in the soil and is abundant in putrefy- 
ing flesh. Gelatin is rapidly liquefied. Food poisoning in man 
has been ascribed to this organism. It is also capable of infecting 
laboratory animals when injected in large doses. 

Bacillus Pyocyaneus (Pseudomonas Pyocyanea). Gessard 
in 1882 isolated this organism from green pus. It is a slender 
rod, actively motile. A soluble blue-green pigment is produced 
in the cultures. Gelatin is liquefied. Guinea-pigs are susceptible 
to intraperitoneal inoculation. In man the organism is most 
common in the pus from wounds, where its presence is considered 
as only mildly deleterious. The bacillus has also been found in 
otitis media and a few cases of fatal generalized infection with B. 
pyocyaneits have been described. 

Bacillus Fluorescens var. Putidus. This non-pathogenic 
actively motile rod is common in putrefying material. It pro- 
duces spores when grown on quince jelly. The greenish-yellow 
pigment is soluble in water. Gelatin is not liquefied. A number 
of different fluorescing bacilli have been found in the soil and 
surface waters. Some of them liquefy gelatin. 

Bacillus Violaceus. This is a non-pathogenic water bacterium 
which produces a pigment of deep violet color. It is actively 
motile and liquefies gelatin rapidly. The pigment is not soluble 
in water. Several different bacteria are known which produce 
a violet pigment. 

Bacillus Cyanogenus (Pseudomonas Syncyanea). This non- 
pathogenic actively motile organism produces a bluish-black 



344 SPECIFIC MICRO-ORGANISMS 

pigment which is soluble in water. Gelatin is not liquefied. 
B. cyanogenus sometimes causes trouble in dairies as its growth 
in milk imparts a blue color to it. 

Bacillus Prodigiosus. This small oval organism grows rapidly 
at room temperature on ordinary media and is occasionally 
observed on foodstuffs such as moist bread and potatoes. Ordi- 
narily it is encapsulated and non-motile, but it sometimes possesses 
flagella. Gelatin is rapidly liquefied. A red pigment is produced 
at room temperature but not at 37 C. This pigment is insoluble 
in water. Large doses of B. prodigiosus injected into animals 
sometimes gives rise to signs of intoxication. 



CHAPTER XXIV. 
SPIRZLLACE^E AND THE DISEASES CAUSED BY THEM. 

Spirillum Rubrum. Esmarch discovered this organism in 
the body of a dead mouse. It is of chief interest as a harmless 
example of spiral bacterium for class study. It grows rather 
slowly at room temperature without liquefying gelatin. A dull 
red pigment, insoluble in water, is produced even in the absence 
of oxygen. Growth occurs at 37 and also in the refrigerator at 
5 to 10 C. When grown at temperatures above 20 C. the 
organism is a relatively short, slightly bent rod and its spiral 
nature is not very evident. At 10 C. beautiful long spirals are 
produced in broth cultures. It is actively motile. 

Spirillum Cholerae (Microspira Comma). Koch in 1883 
discovered this organism in the intestinal discharges of patients 
suffering from Asiatic cholera, and continuing his studies in India 
in the same year established this organism as the probable cause 
of cholera. It occurs in the intestinal contents and feces of cholera 
patients, often in great abundance, rarely in the feces of healthy 
persons, and has been found at times in surface waters, and in 
drinking water during epidemics of cholera. 

Sp. cholera is a curved cylinder about 0.4 /x in thickness and 
i.5/z in length. In older cultures in broth long spiral forms occur. 
There is considerable variation in shape in cultures older than 
48 h,ours. The organism is actively motile and possesses a single 
flagellum at one end. Those short spirals showing more than 
one flagellum are not to be regarded as true cholera germs. 
Spores have not been observed. The spirillum stains readily 
and is Gram-negative. 

It grows well and rapidly on ordinary media. The reaction 
needs to be distinctly alkaline to litmus as the organism is very 

345 



346 SPECIFIC MICRO-ORGANISMS 

sensitive to acids. Colonies appear on gelatin at 22 C. in about 
24 hours as circular disks with somewhat irregular border and 
granular interior. A few hours later the gelatin begins to liquefy. 
In pep ton-salt solution both indol and nitrite are formed, so that 
the addition of sulphuric acid gives rise to the red color due to 
nitroso-indol. This has been called the cholera-red reaction, but 
it is of course not a specific test for this organism. In milk there 
occurs abundant growth without apparent change in the medium. 
In broth, growth is extremely rapid and a pellicle forms in 24 




FIG. 137. Cholera vibrios, short forms. (From Kolle and Schurmann after Zettnow.') 

hours. The rapid growth in pepton solution (pepton i per cent, 
salt 0.5 per cent) and the tendency for the organisms to collect 
at the surface are utilized in practical enrichment for purposes 
of diagnosis. The spirillum is an obligate aerobe. It is very 
easily killed. If dried on a cover-glass at 37 C., the organisms 
are all dead in two hours. It seems impossible, therefore, for the 
infection to be distributed in dry dust. Moist heat at 56 C. 
kills the cholera spirilla in 30 minutes. They are also easily 
killed by chemical germicides. Milk of lime is recommended for 
the disinfection of excreta. The organism lives for several weeks 



SPIRILLACE.E AND THE DISEASES CAUSED BY THEM 347 

in surface waters but certain waters, as for example the Ganges 
River, destroy the cholera spirilla very quickly. This property- 
has been ascribed to a weak acidity of the water. 




FIG. 138. Cholera vibrios, longer forms at higher magnification, showing long 
flagella. (From Kolle and Schurmann after Zettnow.) 

Animals are not naturally susceptible to cholera. Koch gave 
to a guinea-pig 5 c.c. of a 5 per cent solution of sodium carbonate 




FIG. 139. Involution forms of the spirillum of cholera. (Van Ermengen.) 

through a tube, and then 5 to 10 c.c. of water containing cholera 
spirilla. The animal then received i c.c. of tincture of opium 



348 SPECIFIC MICRO-ORGANISMS 

per 200 grams of body weight, injected into the peritoneal cavity. 
In this way .a condition resembling cholera in man was induced, 
and the animal died in 24 to 36 hours. Autopsy revealed severe 
enteritis, and abundant cholera spirilla in the intestine. Similar 
results may be obtained, however, when other organisms are 
substituted for the cholera germs in this procedure. Intravenous 
injection of cultures into rabbits, and feeding of virulent cultures 
to very young rabbits gives rise to rather typical cholera in many 
of the animals. Intraperitoneal injection of cultures into guinea- 
pigs gives rise to fatal peritonitis. Pigeons are relatively immune. 

The poisons of the cholera germ are intimately connected 
with the substance of the living cell. Culture filtrates are 
slightly or not at all poisonous. The dead bacterial cells are 
poisonous, but the poison in them is a very labile substance and 
readily altered by heat. It seems to become soluble when the 
cell disintegrates, and this may explain the poisonous properties 
sometimes observed in the nitrates of older cultures. 

Immunity to this organism was obtained by Pfeiffer by inject- 
ing non-fatal doses into guinea-pigs. When a small amount of 
culture is injected into the peritoneal cavity of such an immune 
animal, the bacteria become quickly clumped together and are 
then rapidly disintegrated and dissolved in the peritoneal fluid. 
This is known as Pfeiffer's phenomenon and was the first example 
of cytolysis to be observed. The solution of the bacteria sets 
free their poison and if a very large dose has been injected the 
animal may be killed by this poison regardless of his immunity 
to the living germs. 

Asiatic cholera seems to have existed in India for many 
centuries and there are reliable records of its occurrence there 
in the sixteenth, seventeenth and eighteenth centuries. The 
first recognized great world invasion of cholera began in 
1817 and ended in 1823. Succeeding pandemics occurred in 
1826-1837, 1846-1862, and 1864-1875. The fifth invasion began 
in 1883 and ended shortly after the great outbreak at Hamburg 
in 1892. The sixth epidemic began in 1902 and has involved 



SPIRILLACE.E AND THE DISEASES CAUSED BY THEM 349 

Egypt, Russia, Turkey and Italy. The fifth and sixth invasions 
have been very much restricted, largely without doubt because 
of the modern methods founded upon knowledge of its causation. 
Cholera was epidemic in the United States in 1833-35, 1848-54, 
1871-73, and there were a few cases in 1893 and again in 1910. 
The disease occurs as a protracted epidemic in which the infection 
passes from person to person, and as an explosive epidemic in 
which many people are stricken at once as a result of con- 
tamination of the public water-supply. 

The causal relationship of Spirillum cholera to human Asiatic 
cholera is no longer questioned. Several laboratory workers 
among them R. Pfeiffer and E. Oergel, have suffered typical 
attacks of the disease as a result of accidental laboratory inocula- 
tion. Dr. Oergel received some peritoneal fluid from an inocu- 
lated guinea-pig into his mouth and he died of cholera. Petten- 
koffer and Emmerich, in order to disprove the supposed causal 
relation of this organism to cholera, took some alkaline water 
and then water containing a minute quantity of a fresh culture. 
The former investigator had a severe diarrhea and the latter a 
severe and dangerous attack of typical cholera from which he 
eventually recovered. The organism was recovered from the 
stools in all these instances. 

The cholera spirilla enter the body with the food and drink 
and if they escape the germicidal action of the gastric juice they 
may establish themselves in the intestine. In an acute case of 
cholera they multiply here enormously and induce a severe 
enteritis in which large quantities of fluid are secreted into the 
lumen of the intestine and discharged from the rectum along with 
bits of desquamated epithelium and enormous numbers of cholera 
spirilla. The germs do not pass through the intestinal wall, but 
they multiply on and in the intestinal epithelium as well as in 
the intestinal contents. The general symptoms, shock, coma 
and the ultimate death, seem to be due in part to the absorption 
of poisons from the intestine and in part to the severe local irrita- 
tion in the abdomen. 



3 so SPECIFIC MICRO-ORGANISMS 

The bacteriological diagnosis depends altogether upon the 
recognition of the cholera germ in the feces. During an epidemic 
of the disease a probable diagnosis in the individual case may be 
made by mere microscopic examination of stained preparations 
of the mucous flakes in the stools. The presence of abundant 
curved rods arranged parallel to each other is sufficient for a 
probable diagnosis. The problem presents itself in a different 
phase when it is necessary to recognize the first case of cholera in 
a given locality. Here it is necessary to follow up the microscopic 
diagnosis by cultures on gelatin plates, agar plates and in pepton 
solution, and the identification of the cultured organisms by 
agglutinating them with a known cholera-immune serum in high 
dilution (i :iooo). The serum should be powerful enough in a 
dilution of i: 10,000 to agglutinate very definitely the culture 
used in producing it. The examination of immigrants for the 
detection of cholera carriers also requires culture work. The 
stool should be passed naturally, but a dose of salts is permissible 
if there is too great delay. About i gram of feces is mixed with 
50 c.c. of sterile pepton solution 1 in a flask, and this is incubated 
a t 37 C. f r six to eight hours. A stained preparation is then 
made from the surface film of the flask. If no curved rods are 
found in it, the specimen is probably negative. A loopful of the 
surface film should nevertheless be transferred to a tube of pepton 
solution which is incubated for six hours and again examined 
microscopically. If curved rods are found microscopically on 
the surface film of either the first or second culture, the problem 
of differentiating between the cholera vibrio and other similar 
organisms is presented. Plate cultures on gelatin at 22 C. and 
on agar at 37 C. should be made and at the same time the trans- 
plantation to fresh pepton solution should be continued at six- 
hour intervals. After eighteen hours, one examines the plates 
for typical colonies and subjects these to agglutination tests with 
specific serum of high titre. The bacteria from the surface film 
of the pepton solution are also tested in the same way. A rapid 

1 Pepton 10, NaCl 10, NaNO 3 o.i, NaCO 3 0.2, distilled water 1000. 



SPIRILLACE.E AND THE DISEASES CAUSED BY THEM 3$! 

clearing of the microscopic field in the agglutination preparations 
warrants positive diagnosis. 1 

Similar principles are followed in attempting to find cholera 
germs in drinking water. A solution of pep ton 100 grams, salt 
100 grams, potassium nitrate i gram and sodium carbonate 2 
grams in distilled water 1000 c.c. is prepared, filtered, distributed 
in 10 flasks each of 1000 c.c. capacity, and sterilized. To each 
flask containing 100 c.c. of this sterile solution, one adds about 
900 c.c. of the suspected water and incubates the mixture at 37 C. 
for six to eight hours. Subcultures and microscopic preparations 
are made from the surface films and any curved bacteria observed 
are tested as described above. 

The prophylaxis of cholera no longer rests upon the enforce- 
ment of quarantine regulations, for it is now known that conval- 
escents may carry the vibrio alive in their intestines for many 
weeks. The exclusion of the disease depends upon the bacterio- 
logical examination of every person coming from infected regions 
before he is allowed to land at his destination. A water-supply 
system well protected from fecal pollution is an element of safety 
for any community. The Hamburg epidemic of 1892 illustrated 
this point. The unfiltered water taken from the Elbe near the 
harbor carried the infection and distributed it throughout the city 
of Hamburg. In the presence of an epidemic the best protection 
against contact infection is provided by immunization. 

Ferran in 1884 first induced immunity to cholera in animals 
and in man by the subcutaneous injection of living cultures. 
Haffkine improved the method so as to make it reliable. He 
employed a first vaccine of attenuated virus and a second vaccine 
of high virulence with an internal of five days between the injec- 
tions. Kolle introduced the use of killed cultures, employing a 
single injection of 2 mg. of growth from an agar culture suspended 
in i c.c. of salt solution and killed by heating an hour at 58 C. 
As a result of this treatment the agglutinins, bacteriolysins and 
opsonins for the cholera vibrio are increased. Practically such 

1 Krumwiede, Pratt and Grund, Journ. Infect. Diseases, 1912, Vol. X, pp. 134-141. 



352 SPECIFIC MICRO-ORGANISMS 

vaccination has resulted in a reduction in case incidence to about 
one-half and in mortality rate to about one- fourth that observed 
among the unvaccinated. 

Spirillum (Vibrio) Metchnikovi. This curved organism was 
found by Gamaleia in 1887 in the feces and in the blood of chickens 
suffering from enteritis. Morphologically and in cultures this 
organism resembles Sp. cholera very closely. It has a single 
flagellum. The growth and liquefaction of gelatin seems to be 
somewhat more rapid in the case of Sp. metchnikovi, and it usually 
produces a larger amount of indol. Accurate differentiation is 
possible only by animal experimentation and by testing with 
anti-sera. A minute quantity of culture of Sp. metchnikovi in- 
troduced into the skin of a dove or chicken is sufficient to cause 
general bacteremia and death, whereas even large doses (4 mg.) 
of true cholera organisms introduced into such a skin wound are 
without effect. Sp. metchnikovi is also much more virulent for 
guinea-pigs. Agglutination and bacteriolytic tests with specific 
sera also differentiate the two organisms. 

Spirillum (Vibrio) Finkler -Prior. Finkler and Prior in 1885 
isolated this organism from the feces in cholera nostras. Morpho- 
logically it resembles the cholera vibrio very closely. Indol is 
not produced. It is apparently non-pathogenic. 

Spirillum Tyrogenum (Vibrio Deneke). This organism was 
isolated from old cheese. It resembles the cholera vibrio but 
does not form indol and appears not to be pathogenic. 

A large number of other cholera-like organisms have been 
isolated in the Various examinations for the cholera germ. Some 
of these can be differentiated morphologically, as they possess 
more than one flagellum. Others fail to produce indol or show 
other cultural difference from the true cholera organism. In 
some instances differentiation depends almost altogether upon 
the agglutination test. This latter has come to be regarded as 
most important in the accurate recognition of the cholera organ- 
ism and its differentiation from other vibrios. 



CHAPTER XXV. 
SPIROCH.ETVE. 

Spirochaeta Plicatilis. Ehrenberg in 1833 observed this long 
slender spiral organism in swamp water. It occurs commonly 
in stagnant water among the algae which grow there and has also 
been found in sea water. The cell is about 0.75/4 in thickness and 
20 to 500/1 in length. It moves by rotation and also by bending 
of the thread. Multiplication takes place by transverse division, 
sometimes occurring simultaneously at many points in a filament 
so that many short forms result. This organism is regarded as 
the type species of the genus Spirochceta. 

A number of saprophytic spirochetes are known. Dobell 1 
has made a careful study of several species, not only free-living 
but also parasitic spirochetes, directing special attention to their 
systematic relationships. He concludes that the spirochetes 
belong to the bacteria and that they agree with the bacteria in 
their structure in all respects except the organs of locomotion. 
Concerning the flagella he seems to be doubtful. 

Spirochaeta Recurrentis. Obermeier in 1873 described the 
slender spiral organism first seen by him in 1868 in the blood in 
cases of relapsing fever. Ross and Milne observed a similar 
organism in man in Uganda in 1904 and Button and Todd in the 
same year demonstrated the presence of a spirochete in the blood 
in the African tick fever of the Congo. In 1905 a similar organ- 
ism was found in a case of relapsing fever in New York City. 
The disease has also been recognized in Russia and in India. 
The spirochetes have been successfully inoculated into monkeys 
and into rats, and various strains from different parts of the 
world have thus been made available for comparative study in 

1 Archiv.f. Protistenkunde, 1912, Bd. XXVI, pp. 117-240. 
2 3 353 



354 SPECIFIC MICRO-ORGANISMS 

the same laboratory. There are certain differences between 
these spirochetes of human relapsing fever, and several distinct 
varieties (or species?) are recognized. We shall consider them 
as varieties of Sp. recurrentis. 

Spirochaeta Recurrentis var. Duttoni. This is the spirochete 
of Congo tick fever discovered by Button and Todd in 1904. It 
is about 0.45 /z in thickness and 24 to 30 ju in length. The organism 
has been cultivated by Noguchi 1 in ascitic fluid containing sterile 
tissue and covered by paraffin oil. The African tick fever caused 




FIG. 140. Spirochaetae of relapsing fever in blood of a man. (After Kolle and 

Wassermann.) 

by this organism is one of the most fatal of the relapsing fevers. 
The tick remains infective for a very long time and also transmits 
the infection to its offspring through the egg. Other insects, 2 
fleas and lice, are also capable of transmitting the infection. 

Spirochaeta Recurrentis var. Rossii (Kochi). This organism 
occurs in the blood of relapsing fever of East Africa. It resembles 
Sp. duttoni very closely. Noguchi obtained cultures readily in 
ascitic fluid containing sterile tissue. 

Spirochaeta Recurrentis var. Novyi. 3 This organism is 
more slender than the two preceding varieties, measuring about 

1 Journ. Exp. Med., 1912, Vol. XVI, pp. 199-210. 

2 Nuttall, Johns Hopkins Hosp. Bull., 1913, Vol. XXIV, pp. 33-39. 

3 Novy and Knapp: Journ. Inf. Diseases, 1906, Vol. Ill, pp. 291-393. 



355 

0.31 in thickness. The relapsing fever in which it occurs has 
been observed in South America. Noguchi has obtained cul- 
tures by the same methods as he employed for Sp. rossii, but the 
cultivation is more difficult. 

Several other varieties of spirochetes, which cause relapsing 
fever in man, have been recognized. The spirochete concerned 
in any case seems to be able to infect several species of insects and 




^^P^^' 

FIG. 141. Spirochczta recurrentis (novyi). Organisms of different lengths in the 
blood of a white rat. X 1500. (After Novy and Knapp.) 

to be transmitted to a new mammalian host by them. Further- 
more one species of insect seems to be capable of transmitting 
any one of these spirochetes. 1 

The diagnosis of relapsing fever depends upon recognizing the 
characteristic spirochetes in the blood during the febrile attack. 
Their recognition offers little difficulty, as a rule, but they may be 
overlooked by a beginner. In doubtful cases it is well to search 

1 Nuttall: Johns Hopkins Bull, 1913, Vol. XXIV, pp. 33-39. 



356 SPECIFIC MICRO-ORGANISMS 

the fresh drop of blood not only by direct central illumination 
with a yellow light but also by means of dark-field illumination, 
and to examine thin films made by mixing India ink 3 parts with 
the blood i part and spreading very thin. Finally thin blood 
films should be stained and examined. The inoculation of white 
rats with i to 5 c.c. of blood conveys the infection to them and 
the parasites appear in the blood of the animal 2 to 4 days after 
inoculation. The spirochetes may vanish from the blood with 
marvelous rapidity. 

Spirochaeta Anserina. Sacharoff in 1890 discovered this 
spiral organism in the blood of geese suffering from a serious 
disease in the Caucasus. Ducks and chickens are also susceptible. 
The spirochete is about o.5/z thick by 10 to 20/1 long. It is con- 
sidered by Nuttall to be indentical with the Sp. gallinarum of 
Marchoux and Salimbeni. 

Spirochaeta Gallinarum. Marchoux and Salimbeni in 1903 
discovered this organism in the blood of diseased chickens at 
Rio Janeiro. The organism is 0.5/4 thick and 15 to 20^ long. 
The disease is transmitted by means of the fowl tick Argas minia- 
tus (persicus?}, most effectively when the tick is kept at a tempera- 
ture of 30 to 35 C. In cold climates the disease is unknown. 
Leishman and Hindle have studied very carefully the changes 
which the spirochetes pass through in the body of the insect. 
They found numerous exceedingly minute "coccoid bodies" in 
the cells of the Malpighian tubules. These minute bodies are 
considered 1 to be the products of a fragmentation of spirochetes 
and to be capable of again growing into typical spirochetes. If 
the view is correct these bodies necessarily play an important part 
in the infection of the vertebrate host and in the inheritance of 
the infection in the insect species. 

Spirochaeta Muris. This is a very short spirochete which 
occurs naturally in a non-fatal relapsing fever of rats and mice. 
It possesses one or sometimes two flagella on each end and multi- 
plies by simple transverse fission. 

1 Nuttall: Harvey lecture, 1913. 



357 



Spirochaeta Pallida (Treponema Pallidum). Schaudinn and 
Hoffmann in 1905 observed this slender spiral organism in pri- 
mary syphilitic lesions, in fluid obtained from swollen lymph 
glands in syphilis and in the liver and spleen of a still-born syphi- 
litic fetus. The occurrence of the 
organism in syphilitic lesions was 
quickly and abundantly confirmed 
by other workers. Cultures were 
first obtained in collodion sacs by 
Levaditi and Mclntosh in 1907. 
Schereschewsky, and Muhlens and 
Hoffmann obtained cultures in gela- 
tinized horse serum. Noguchi 1 has 
carried out the most successful cul- 
tural work and has succeeded for 
the first time in causing syphilitic 
lesions in animals by the inocula- 
tion of pure cultures. 

Sp. pallida occurs naturally only 
in human syphilis. It is a slender 
spiral 0.2 to 0.3 5 n in thickness and 
3.5 to 1 5. 5 /z in length. Its curves 
are narrow and very regular. It is 

! ,., ,, . FIG. 142. Film preparation 

actively motile, as are all the spiro- from a gen ital syphilitic papule; in 
chetes, and has a very slender fla- e . ce . nter ar f 7 ^ wo specimens of 

; Sptrochata pallida, the other three 

gellum at each end. The Usual mo- are specimens of Spirochala refrin- 

tion is that of rapid rotation on the Schaudinn and 



longitudinal axis with progression, 

but at times there is gross bending of the filament, especially 
when the organism is living under unfavorable conditions. 
The mode of division is a somewhat vexed question as it 
is in regard to the whole group of spirochetes. Transverse 
and longitudinal division have been described. Probably 
the weight of authority 1 now favors transverse division as 

1 Journ. Exp. Med., 1911, Vol. XIV, p. 99; 1912, Vol. XV, p. 90. 



358 SPECIFIC MICRO-ORGANISMS 

the sole mode of multiplication, although able adherents to the 
opposite view are not lacking. The refractive index of the 
filament is not very much greater than that of serum, so that the 
unstained organism is difficult to see by direct illumination. 
Dark-field illumination is more satisfactory. Sp. pallida in film 
preparations stains with difficulty by ordinary methods. Schau- 
dinn employed Giemsa's modification of the Romanowsky stain. 
Good results are obtained by staining with solutions of the Roman- 
owsky staining principles in methyl alcohol provided an excess of 
methylene-violet be present (see p. 43). Tunnincliff 2 recom- 
mends staining with a mixture of saturated alcoholic solution of 
gentian violet, i part, in 5 per cent carbolic acid, 9 parts. Thin 
films are essential but the staining process requires only a few 
seconds. In pieces of tissue the spirochete is best stained by the 
method of Leviditi. For this purpose thin (i mm.) pieces of 
syphilitic tissue are fixed in formalin (10 per cent) for 24 hours or 
longer and hardened in 95 per cent alcohol for a day. The alcohol 
is then removed by soaking in distilled water and the tissue is trans- 
ferred to a fresh i to 3 per cent solution of silver nitrate in distilled 
water. This is placed at 37 C. in the dark for three to five days. 
The tissue is next washed in distilled water and placed in a re- 
ducing fluid, consisting of pyrogallic acid 3 grams, formalin (40 
per cent formaldehyde) 5 c.c. and distilled water 100 c.c., for one 
to two days. It is then washed in distilled water, dehydrated, 
embedded in paraffin and sectioned. The spirochetes are stained 
a dense black by this method. The sections may be stained to 
show histological structure also, by applying methylene blue or 
toluidin blue to them after they have been fixed on the slide. 

Cultivation of Sp. pallida has been most successfully practised 
by Noguchi. 3 He has grown the organism in a mixture of serum 
and water, to which naturally sterile tissue was added, and in 
ascitic-fluid agar with similar bits of tissue, always under strict 

1 Hoffmann: Centrabl. f. Bakt., I Abt., Orig., 1912, Bd. LXVI, S. 520-523. 

*Journ. A.M. A., 1912, Vol. LVIII, p. 1682. 

3 Journ. Exp. Med., 1911, Vol. XIV, p. 99; 1912, Vol. XV, p. 90 



SPIROCH^ET^E 



359 



anaerobic conditions. The technic of culture is somewhat diffi- 
cult and the original papers should be consulted in detail. Inocu- 
lation of the cultures into rabbits and monkeys has caused typical 
syphilitic lesions. 




FIG. 143. Spirochceta pallida stained by Levaditi method. The section shows 
an infarcted lymph vessel at the junction of two branches. The lumen is filled with 
leukocytes. The spirochetes follow the lymph vessel for the most part, but are also 
penetrating into the surrounding tissue. (From Doflein after Ehrmann.} 



Noguchi's luetin is prepared by grinding the solid medium 
rich in spirochetes in a mortar and emulsifying it in a small 
amount of fluid. This is then heated to 60 C. for an hour and 



360 SPECIFIC MICRO-ORGANISMS 

preserved by the addition of 0.5 per cent carbolic acid. The 
final preparation contains many dead unbroken spirochetes. 

Syphilis is an inoculation disease which has been widely 
prevalent throughout the civilized world since the early part of 
the 1 6th century. Transmission takes place by direct contact 
and in the great majority of instances by venereal contact, although 
many authentic cases of transmission by means of intermediate 
objects are known. The spirochete is able to live for some hours 
outside the body if drying is prevented. The primary lesion 
develops at the point of inoculation about two weeks after that 
event, first as a papule, which becomes vesicular and ulcerates, 
remaining indolent for several weeks. The neighboring lymph 
glands become swollen. The secondary manifestations occur 
about a month later as a general macular or sometimes papular 
eruption on the skin, together with sore throat and ulcerated 
patches in mouth. The skin eruption does not itch. Subsequent 
to this stage there may be local necrotic lesions (gummata) in 
various parts of the body, or low-grade inflammatory changes in 
the meninges and central nervous system. Bacteriological 
methods of diagnosis are of assistance in some cases in all the 
various stages of syphilis. Early in the disease the spirochetes 
are relatively numerous, in certain locations at any rate, while 
later the parasites may be so few as to render their detection 
practically hopeless for diagnostic purposes. In these later 
stages, however, the presence of specific and other antibodies in 
the body fluids of the patient may often be recognized and this 
recognition employed as an aid in diagnosis. 

Microscopic examination of a primary ulcer is best done by 
means of the dark-field illumination. For this purpose the ulcer 
(which should not have been treated with mercurials) is carefully 
cleansed and a few drops of freshly exuded serum collected in a 
glass capillary, and the usual slide-cover-glass preparation is made 
with this fluid. Permanent preparations are made most easily 
by mixing such serum with India ink on a slide and spreading 
the mixture in a very thin layer. Collargol, one part in nineteen 



361 

parts of water, gives even more satisfactory preparations 1 than 
India ink. It is used in the same way. Thin films of the serum - 
on slides or cover-glasses may be stained as directed above. Micro- 
scopic examination of fluid obtained by gland puncture or from 
secondary lesions on the skin or mucous membranes is carried 
out in the same way. Serious confusion in the recognition of the 
spirochete is likely to arise in the case of lesions in the mouth or 
pharynx, inasmuch as some of the normal mouth spirochetes are 
very similar in form to Sp. pallida. The presence of typical 
spirochetes in the juice aspirated from a lymph gland is practically 
diagnostic, and the recognition of typical organisms in genital 
chancres or lesions on the skin has considerable diagnostic value. 

Inoculation of animals is of little practical use in diagnosis, 
but it has been possible by this method to demonstrate the fre- 
quent presence of Sp. pallida in the circulating blood in cases of 
untreated secondary syphilis. 

The detection of antibodies in the blood of the patient is under- 
taken in two ways, first by the complement-fixation ( Wassermann) 
test and second by the luetin test. For the complement-fixation 2 
test, as performed at the Laboratories of the New York Post- 
Graduate Medical School and Hospital by Dr. R. M. Taylor, 3 
the following are employed: 

1. The red blood cells are obtained by defibrinating fresh 
sheep's blood, filtering it through paper if necessary to remove 
fragments of clot, separating the cells in the centrifuge and wash- 
ing them four times with 0.9 per cent salt solution. Finally i c.c. 
of the corpuscles as packed by the centrifuge is suspended in 
19 c.c. of 0.9 per cent salt solution; 0.2 c.c. of this suspension is 
arbitrarily taken as the unit of red blood cells. 

2. The complement is obtained by drawing 5 to 10 c.c. of 
blood from a large guinea-pig by cardiac puncture. This blood 
is transferred to a Petri dish, allowed to clot, incubated at 37 C. 

1 Harrison: Journ. Roy. Army Med. Corps, 1912, Vol. XIX, p. 749. 

2 For a detailed discussion see Citron-Garbat, Immunity, Phila., 1912; Simon, 
Infection and Immunity, Phila., 1912. 

3 1 am indebted to Dr. Taylor for the details of this procedure. 



362 SPECIFIC MICRO-ORGANISMS 

for 30 minutes and then refrigerated. The separated serum is 
then drawn off with a pipette and 2 c.c. of it are mixed with 18 c.c. 
of cold 0.9 per cent salt solution. This 10 per cent solution of 
guinea-pig's serum is kept in a cold place, preferably immersed in 
ice water. It is prepared on the day it is to be used. The unit 
of complement is contained in 0.2 c.c. of this solution. 

3. The hemolytic amboceptor is prepared by injecting 2 c.c. 
of thoroughly washed (five times) sheep's corpuscles intravenously 
into a large rabbit at intervals of three days, until four injections 
have been given. Ten days after the last injection the animal is 
allowed to fast for 12 hours and the blood is then aseptically 
drawn from the carotid artery, allowed to clot and the serum 
separated by standing at 37 C. for two to five hours. The clear 
serum is transferred to small glass ampoules in amounts of 0.5 to 
i.o c.c. and hermetically sealed. These are then heated at 56 C. 
for 30 minutes and stored in the refrigerator. The hemolytic 
power of this serum is ascertained by titration. The unit is that 
amount which, when mixed with 0.2 c.c. (i unit) of corpuscles 
and 0.2 c.c. (i unit) of complement and sufficient salt solution 
(0.9 per cent) to make a total volume of i c.c., will cause complete 
laking of the red blood cells in exactly i hour after being placed 
in the incubator (air) at 37 C. The unit of amboceptor is ordi- 
narily contained in o.i c.c. of a dilution of i part of serum in 
1000 to 2000 parts of salt solution. After the strength is ascer- 
tained the diluted amboceptor is made up so that o.i c.c. contains 
i unit. 

The amboceptor is quite permanent under ordinary refrigera- 
tor conditions, but when diluted it may deteriorate after a few 
days. The relation of complement, red blood cells and ambo- 
ceptor is tested always immediately before undertaking a comple- 
ment-fixation test. If the mixture of one unit of each of these in 
a total volume of i c.c. produces complete hemolysis at the end 
of an hour, the hemolytic system is considered satisfactory. If 
there is only a slight discrepancy this may be corrected by em- 
ploying a little more or a little less (within limits of 20 per cent) 



SPIROCILETJE 363 

amboceptor, that is, down to o.c8 c.c. or up to 0.12 c.c. as may be 
necessary in place of the usual o.io c.c. If the discrepancy 4s 
greater than this it is well to obtain a new sample of complement 
or of sheep's cells or of both. The hemolytic system should be- 
have much the same from day to day when the technic is 
accurate. 

4. The patient's serum is obtained from 5 to 10 c.c. of blood 
drawn from the elbow vein. The serum must be free from sus- 
pended matter, centrifugalized if necessary. The serum is heated 
at 54 to 56 C. for 30 minutes just before use. 

5. The antigen is a 3 per cent solution in methyl alcohol of the 
acetone-insoluble lipoids extracted by alcohol and ether from the 
heart muscle of beef. The strength of antigen to be used must be 
ascertained by careful titration. A dilution of i c.c. of the antigen 
in 9 c.c. of salt solution is first prepared. Then various quanti- 
ties, o.i c.c., 0.2 c.c., 0.3 c.c., 0.4 c.c. and 0.5 c.c. of this suspension 
are placed in separate tubes. To each tube is added i unit of 
complement and sufficient salt solution to bring the total volume 
to i c.c. The tubes are incubated i hour at 37 C. (air). Then 
one unit of corpuscles (0.2 c.c.) and two units of hemolytic ambo- 
ceptor (0.2 c.c.) are added and the tubes are again incubated an 
hour. Of those tubes in which hemolysis is not complete, the one 
containing the least antigen marks the concentration at which 
the antigen is distinctly anti-complementary. The second test 
of the ant'gen is now undertaken. Various amounts of a i to 100 
dilution, o.oi c.c., 0.03 c.c., 0.05 c.c., o.i c.c. and 0.2 c.c., are meas- 
ured into tubes. To each tube is then added i unit of comple- 
ment, 0.02 c.c. of serum from an active untreated case of syphilis 
and sufficient salt solution to make a total volume of 0.6 c.c. 
The tubes are incubated an hour. Then i unit of corpuscles 
(0.2 c.c.) and 2 units of hemolytic amboceptor (0.2 c.c.) are added 
and the tubes are again incubated one hour. Of the tubes show- 
ing no hemolysis (complete fixation), that one which contains the 
least antigen marks the lowest effective concentration of the an- 
tigen. This amount of antigen should be very much less than the 



364 SPECIFIC MICRO-ORGANISMS 

anti-complementary amount ascertained in the first test. Ordi- 
narily it is about T -J~g- of this amount. The unit of antigen to 
be employed should be chosen so that it is several times greater 
than the least effective quantity but still not more than one-fifth 
to one-half the least anti-complementary amount. Having chosen 
the tentative antigen unit, a third test is applied. One, two and 
four units of antigen are placed in tubes and a unit of corpuscles 
is added to each, together with sufficient salt solution to make the 
total volume i c.c., and these are incubated for an hour. The 
corpuscles should not be laked. If they are laked the antigen is 
itself markedly hemolytic. A satisfactory antigen should per- 
form its specific function of fixing complement in the presence 
of a syphilitic serum in an amount which is at most -g-V of the 
amount which is in itself either anti-complementary or hemo- 
lytic. It keeps well in the refrigerator as the alcoholic solution. 
The dilution for use should be freshly prepared. 

The antigen is the element in the test which is designed to 
enter into chemical reaction with the specific substance in the 
patient's blood, which is present there as a result of active syphi- 
lis. During the course of this reaction, complement is absorbed 
or destroyed. The nature of the lipoidophilic substance 1 is un- 
known. It behaves in the test very much as a specific immune 
body would be expected to behave. Experience has shown that 
an antibody of this nature is rarely present in other conditions 
than active syphilis and that it is present in this disease. Upon 
the results of this experience we have to rely in ascribing diagnos- 
tic value to the test. 

In performing a test for diagnosis, sera from several patients 
should be tested at the same time, and one, two or three sera, pre- 
viously tested and found to fix complement in varying degrees, 
and at least one serum known to give a negative result, should 
be tested along with the new samples. Four tubes are used for 
each serum to be tested. 

1 Simon: Infection and Immunity, Phila., 1912, p. 272. 



365 



Tube No. i, back row 



Tube No. 2, back row 



Complement i unit (o. 2 c.c.) 
Patient's serum 0.08 c.c. 
Salt solution 0.32 c.c. 



Complement i unit (0.2 c.c.) 
Patient's serum o.oi c.c. 
Sheep's corpuscles, i unit (o. 2 c.c.) 
Salt solution 0.59 c.c. 



Mix thoroughly and incubate at 37 C. i hour. Then add: 



Sheep's corpuscles i unit 

(0.2C.C.). 

Hemolytic amboceptor 2 units 

(0.2 c.c.). 


0.4 c.c. 1 


Nothing. 



Mix thoroughly and incubate for i hour, recording the progress of hemolysis at. 
intervals of 15 minutes. Then refrigerate 16 hours and record the final reading. 



Tube No. 3, front row 



Tube No. 4, front row 



Complement i unit (o. 2 c.c.) 
Patient's serum 0.02 c.c. 
Antigen i unit (o. i c.c.) 
Salt solution o. 28 c.c. 



Complement i unit (0.2 c.c.) 
Patient's serum o. 04 c.c. 
Antigen i unit (o. i c.c.) 
Salt solution o. 26 c.c. 



Mix thoroughly and incubate at 37 C. i hour. Then add: 



Sheep's corpuscles i unit } 

(0.2C.C.). 'o/LCC 1 

Hemolytic amboceptor 2 units ( 
(0.2 c.c.). 



unit 



Sheep's corpuscles i 

(0.2 c.c.). I , 

Hemolytic amboceptor 2 units [ ' 

(0.2C.C.). 



Mix thoroughly and incubate for i hour, recording the progress of hemolysis at 
intervals of 15 minutes. Then refrigerate 16 hours and record the final reading. 



1 The suspension of sheep's corpuscles containing i unit in o. 2 c.c. and the solu- 
tion of hemolytic amboceptor containing 2 units in 0.2 c.c. are quickly mixed to- 
gether in equal parts, and o . 4 c.c. of this homogeneous mixture is added at this point. 
This procedure results in a saving of time as well as greater accuracy. 



366 SPECIFIC MICRO-ORGANISMS 

Tube No. i should show complete hemolysis early in the second 
incubation. Tube No. 2 should remain free from hemolysis, or 
show only a very slight amount at the end of the second incu- 
bation. If these have behaved properly and the tests on the 
known sera have resulted as they did when previously tested, 
then the behavior of Tubes 3 and 4 is a measure of the amount of 
lipoidophilic substance in the serum of the patient. One dis- 
tinguishes about eight different grades of reaction, from complete 
fixation (no trace of hemolysis) to no fixation (complete hemolysis) . 

The luetin test is performed by injecting 0.05 c.c. of luetin 
intracutaneously in two places on the left arm and at the same 
time 0.05 c.c. of a control suspension, consisting of the medium 
without any growth of spirochetes, at two points on the right 
arm. Local inflammation on the left arm, appearing in two to 
ten days and sometimes resulting in the formation of a pustule, 
is regarded as a positive test. The test is often negative in the 
earlier stages of syphilis. H 

The various diagnostic tests for syphilis are now extensively em- 
ployed. Microscopic search for the spirochete is of value in the 
untreated primary and secondary stages. The complement-fixa- 
tion test becomes positive a few weeks after the appearance of the 
primary lesion and is generally regarded as indicating an active 
syphilitic process. The luetin test may be positive in latent or 
inactive syphilis when the Wassermann is negative. Further 
experience with the luetin test is necessary in order to determine 
its real significance. 

Spirochaeta (Treponema) Refringens. This is a relatively 
gross spirochete which occurs in primary syphilitic lesions along 
with Sp. pallida. It seems to have no pathogenic properties. 
Noguchi 1 has obtained pure cultures of it and found them with- 
out pathogenic properties for rabbits and monkeys. 

Spirochaeta (Treponema) Microdentium. 2 This is one of the 
common spirals of the mouth. It may be confused with Sp. pal- 

1 Journ. Exp. Med., 1902, Vol. XV, p. 466. 

2 Noguchi: Journ. Exp. Med., Vol. XV, pp. 81-89. 



367 

lida, which it resembles in size and shape. Pure cultures have 
been obtained by Noguchi. Other spirochetes of the mouth 
have also been cultivated by this investigator and there are prob- 
ably several species of them. 

Spirochaeta (Bacillus) Fusiformis (Vincenti). In an ulcer a- 
tive disease of the tonsils known as Vincent's angina there occur 
very constantly large numbers of fusiform rods 0.3 to o.Sju in 
thickness and 3 to lo/x long, associated with spiral filaments with 
rather coarse windings. Similar organisms occur in other ul- 
cerative conditions of the mouth and pharynx and rarely else- 
where in the body. The relation of these organisms to each other, 
whether they are distinct species or different forms of the same 
species, is still unsettled. Their etiological relationship to the 
disease is also uncertain. Tunnicliff 1 has observed spiral forms 
in her pure cultures of Bacillus fusiformis. It seems probable 
that the spirals seen in the ulcer are to a large extent the ordi- 
nary mouth spirochetes, but the fusiform bacillus itself is evidently 
a close relative of the spirochetes, as it requires similar conditions 
for successful culture and is able at times to assume a distinctly 
spiral form in culture. 

1 Journ. Inf. Diseases, 1906, Vol. Ill, p. 148; Rosenow and Tunnicliff: Journ 
Inf. Dis., 1912. Vol. X, pp. 1-6. 



CHAPTER XXVI. 
THE FILTERABLE MICROBES. * 

The Virus of Foot-and-mouth Disease. This filterable or- 
ganism occurs in the vesicles present in the mouth and on the 
feet of the diseased animals, and also in the milk of cows suffering 
from foot-and-mouth disease. The virus was shown to be filter- 
able by Loffler and Frosch in 1898. It is rendered inert by heat- 
ing to 50 C. for 10 minutes. Animals are immune after recovery 
from the disease. Cattle and swine are naturally susceptible 
and a few cases of the disease have occurred in man. Nothing 
definite is known concerning morphology or cultures. The in- 
fection seems to be transmitted with the food as well as by 
inoculation. 

The Virus of Bovine Pleuro-pneumonia. This organism is 
present in the- affected lungs and in discharges from the respira- 
tory tract of cattle suffering from pleuro-pneumonia. Nocard 
filtered the virus through a Chamberland "F" filter in 1899. It 
is rendered inert by heating at 58 C., but retains its virulence in 
glycerine for weeks and resists freezing. Cultures have been 
obtained by the collodion-sac method by Nocard and Roux. The 
organisms in such cultures are extremely minute and variable in 
form. Some of them are spirals and others approximately spher- 
ical. Immunity follows recovery from the disease, and has been 
induced artificially by inoculation with cultures and also by inocu- 
lation with virulent exudate from the lung of a dead animal into 
the subcutaneous tissue of the tail of the animal to be immunized. 1 

The Virus of Yellow Fever. 2 This organism occurs in the blood 
of man at least during the first two or three days of an attack of 

1 Kolle and Wassermann, Handbuch, 1912, Bd. I, S. 928. 

2 The publications of Reed, Carroll and their associates have been issued as a 
volume entitled Yellow Fever, U. S. Senate Document No. 822, 6ist Congress, 
3rd Session, 1911. 

368 



THE FILTERABLE MICROBES 



369 



yellow fever. It was shown to be filterable by Reed, Carroll, 
Lazear and Agramonte in 1901. It passes through the Chamber- 
land "B" filter. It is rendered inert at 55 C. in 10 minutes and 
even by standing at room temperature for two days. Yellow 
fever is an acute febrile disease of man usually accompanied by 
jaundice and sometimes by the vomiting of altered blood (black 



aft 




FIG. 144. Aedes (Stegomyla) colipus; female, a, Front tarsal claw. (After Reed 

and Carroll^) 

vomit). It is frequently fatal. Permanent immunity follows 
recovery. The disease is naturally transmitted by a blood-suck- 
ing mosquito, (Stegomyia, Aedes) calopus, which becomes capable 
of inoculating the disease about twelve days after sucking blood 
containing the virus. The mosquito probably remains infective 
as long as it lives, and this insect thus becomes the essential reser- 
voir of the virus of yellow fever. Prophylactic measures based 
24 



370 SPECIFIC MICRO-ORGANISMS 

upon this deduction have been remarkably successful in the sup- 
pression of the disease. 

Seidelin 1 has described a minute structure which occurs in the 
blood cells and in the blood plasma in yellow fever, which he has 
called Paraplasma flamgenum and regards as the pathogenic 
agent. The work lacks confirmation by other observers and the 
evidence is not yet convincing. The earlier papers of Seidelin have 
been severely criticised by Agramonte. 1 

The Virus of Cattle Plague (Rinderpest). This organism 
occurs in the blood, organs and excretions of cattle suffering from 
the disease. It was shown to be filterable by Nicolle and Adil- 
Bey in 1902, and is able to pass through the Chamberland "F" 
filter. The virus resists drying for four days and remains active 
for two or three months when spread on hay in a dark place. 
It is destroyed by distilled water in five days, by glycerin in eight 
days and rendered avirulent in a few hours by admixture of bile. 
The disease is an acute febrile disorder characterized by severe 
inflammation of the mucous membranes and rapid emaciation. 
It is usually fatal. Immunity follows recovery and is induced 
artificially by injecting the bile of infected animals under the 
skin of the healthy cattle. In this way an active immunity is 
acquired without an evident attack of the disease. 

The Virus of Rabies. This organism exists in the central 
nervous system, the peripheral nerves, the salivary glands, the 
saliva and less frequently in other parts of the body of persons 
or animals suffering from lyssa or rabies. The virus was filtered 
by Remlinger in 1903. It may also be dialyzed through collodion 
sacs. 3 The virus is rendered inert by drying for two weeks, and 
by heating at 55 C. for 30 minutes, by admixture of bile in a few 
minutes, and by the gastric juice in 5 hours. It remains virulent 
in glycerine for several months. Negri in 1903 described certain 
bodies which seem to occur in the central nervous system in- 
variably and exclusively in this disease. They are especially 

1 Bull. Yellow Fever Bureau, 1912, Vol. II, pp. 123-242. 

1 Medical Record, 1912, Vol. LXXXI, pp. 604-607. 

2 Poor and Steinhardt, Journ. Infect. Dis., 1913, Vol. XII, pp. 202-205. 



THE FILTERABLE MICROBES 



371 



numerous in the ammon's horn of the brain in cases of street 
rabies. Preparations should be made from the gray matter of 
the brain. A bit of this tissue is carefully spread on a slide by 
exerting moderate pressure upon it with a second slide or a cover- 
glass and at the same time moving it along the surface of the first 




FIG. 145. Section through the cornu ammonis of brain of a rabid dog; stained by 
the method of Lentz. Five Negri bodies of different sizes are shown, enclosed within 
the ganglion cells. The smallest contains only three minute granules. (After Lentz, 
Centralbl.f. Bakt, 1907, Abt. I, Vol. XLIV, p. 378.) 



slide. The film is fixed in pure methylic alcohol and stained with 
Giemsa's solution, or it may be stained directly without fixation 
with Leishman's stain. The Negri bodies are round and some- 
what irregular in outline from i/i to 27/^1 in diameter, and usually 
inside the nerve cells. In the interior of the larger bodies, smaller 



372 SPECIFIC MICRO-ORGANISMS 

spherical structures of variable size and number may be seen. 
The exact nature of the Negri bodies is uncertain. Some stu- 
dents of rabies regard them as protozoa, while others consider 
them to be products of cell degeneration. The evidence to de- 
cide the matter is not yet at hand. They seem to occur only in 
rabies and to be constantly present in this disease. 

Lyssa or rabies 1 is primarily a disease of dogs but it occurs in 
other mammals as well, usually as a result of dog bites. In ani- 
mals inoculated directly into the brain with the most virulent 
material (fixed virus), the symptoms of rabies appear in 4 to 6 
days and death occurs on the seventh day. Inoculation with the 
saliva or nervous tissue of a mad dog (street virus) rarely causes 
symptoms before three weeks and the onset may be delayed for 
a year. In fact many persons and animals bitten by rabid dogs 
may fail to develop the disease at all. This variability depends 
upon the virulence and the amount of virus and especially upon 
the part of the body into which it is introduced. Bites upon the 
face or hands, because of the rich nerve supply and the lack of 
protection by clothing, are especially dangerous. After the dis- 
ease has developed so as to cause symptoms, death is inevitable 
in the present state of our knowledge. 

Rabies may be diagnosed in an animal by observing the course 
of the disease, by autopsy and by inoculation of test animals and 
observation of the course of the disease in them. If the sus- 
pected animal be caged, the question of rabies may be settled in a 
few days, for, if he is mad, the raging stage will be quickly followed 
by the characteristic paralysis and death. If the animal has been 
killed, a careful autopsy may reveal the absence of food from the 
digestive tract and the presence there of abnormal ingested ma- 
terial (grass, wood or stones), highly suggestive of rabies. Mi- 
croscopic examination of the central nervous system may reveal 
the Negri bodies, characteristic of the disease. For confirmation 
of the diagnosis a portion of the brain or spinal cord, removed with- 

1 For a general discussion of rabies see Gumming: Journ. A. M. A., 1912, Vol. 
LVIII, pp. 1496-1499. 



THE FILTERABLE MICROBES 373 

out contamination, should be injected into the brain of guinea- 
pigs and rabbits and the effects observed. This last test caF 
ried out by an experienced observer is the most trustworthy 
of all. 

The Pasteur treatment of rabies is designed to induce immu- 
nity after the person has been bitten and before the disease has had 
time to develop. Pasteur 1 first demonstrated the possibility of 
this by experimental work on dogs, and the subsequent use of 
the method in man has been remarkably successful and the dis- 
ease is practically always prevented if the treatment is begun 
directly after infliction of the infecting wound. The first essen- 
tial is thorough cauterization of the wound, best with concentrated 
nitric acid under anesthesia. The patient is then injected sub- 
cutaneously with emulsions of the spinal cords which have been 
removed from rabbits dying of rabies after inoculation with the 
fixed virus, and which have been dried by hanging in bottles 
over caustic soda for some time. The first injection is prepared 
from cords hung for 14 and 13 days, the second from cords hung 
1 2 and 1 1 days, and so on until the three-day cord is reached on 
the seventh or eighth day of the treatment. The series from 
five-day down to three-day cords is then repeated several times, 
the whole treatment lasting about 21 days. The course of treat- 
ment is varied somewhat according to the urgency of the case and 
the severity of the wounds inflicted. It is most effectively carried 
out at special Pasteur institutes devoted to this work, but the 
material for injection may be shipped for some distance when 
necessary. 

The Virus of Hog Cholera. Dorset, Bolton and McBryde, 
continuing the investigations of de Schweinitz, demonstrated in 
1905 the presence of a filterable agent in the blood of hogs suffering 
from hog cholera, capable of causing the disease upon injection 
into healthy animals. It passes through the Chamberland "B" 
and "F" filters. It leaves the body in the urine and probably 
also in other excretions, and seems to enter the new victim with 

1 Vallery-Radot, The Life of Pasteur, 1911, Vol. II, p. 188. 



374 SPECIFIC MICRO-ORGANISMS 

the food and drink. The virus resists drying for three days, 
remains alive in water for many weeks and in glycerine for eight 
days. It is destroyed at 60 to 70 C. in an hour. 

King, Baeslack and Hoffman 1 have found a short, rather thick, 
actively motile spirochete, Spiroch&ta suis, in the blood in forty 
cases of hog cholera, together with abundant granules which may, 
perhaps, represent a stage of this organism. The spirochete has 
not been found in healthy hogs. It seems probable that this 
organism may prove to be the causative agent of the disease, but 
further evidence is necessary to demonstrate this relationship. 

Hog cholera is an extremely contagious disease of hogs, fre- 
quently fatal, characterized by fever and by ulcerations in the 
intestine. Immunity follows recovery and is induced artificially 
by the injection of serum from a hyperimmune hog (passive 
immunity) and by the injection of such serum together with viru- 
lent blood from a hog sick with the disease (combined passive 
and active immunity) . 

The Virus of Dengue Fever. Ashburn and Craig showed in 
1907 that the virus of this disease exists in the blood of the pa- 
tients and that it is filterable. The disease is probably trans- 
mitted by the mosquito Culex fatigans. Apparently the analogy 
to yellow fever is rather close. 

The Virus of Phlebotomus Fever. Doerr in 1908 demon- 
strated a filterable virus in the blood of persons suffering from 
the benign three-day fever of Malta and Crete. The disease is 
rather widely distributed in tropical countries. It is transmitted 
by the sand-fly Phlebotomus papatasii. 2 

The Virus of Poliomyelitis. Several investigators, among 
them r Flexner and Lewis, demonstrated in 1899 the presence of a 
filterable virus in the central nervous system of patients suffering 
from infantile paralysis. The virus also occurs in the nasal mucus 
and in the blood. It survives in glycerine for a month, also re- 
sists freezing for weeks, and is rendered inert at 45 to 50 C. in 

1 Journ. Infect. Dis., 1913, Vol. XII, pp. 39-47; PP- 206-235. 

2 Birt. Journ. Roy. Army Med. Corps, 1910, Vol. XIV, pp. 236-258. 



THE FILTERABLE MICROBES 375 



30 minutes. It is quickly destroyed by hydrogen peroxide and^ 
by menthol. 

Flexner and Noguchi 1 have obtained cultures of the organism 
in ascitic fluid containing sterile tissue and covered with paraf- 
fin oil, and in this medium rendered solid by admixture of agar. 
The colonies are made up of minute globose bodies 0.15 to 0.30/4 
in diameter. Similar bodies have been identified in the nervous 
tissue from cases of the disease. It seems probable that this 
structure is a living organism and the microbic cause of poliomye- 
litis, especially as inoculation of monkeys with the cultures has 
given rise to the disease. 

Poliomyelitis or infantile paralysis occurs in epidemics and 
also sporadically, attacking children and young adults. It is 
characterized by digestive disturbance and fever, which may be 
very mild, followed by paralysis of one or more extremities as a 
rule. Death may occur, but recovery with permanent paralysis 
is the usual result. The mode of transmission is unknown. 
Rosenau is inclined to ascribe considerable importance to the 
stable fly, Stomoxys calcitrans, as the transmitting agent. Other 
modes, especially direct contact, food, and healthy carriers also 
need to be considered. 

The Virus of Measles. Goldberger and* Anderson 2 in 1911 
succeeded in inoculating monkeys with measles and demonstrated 
the presence of the virus in the blood and in the secretions of the 
nose and mouth, and in filtrates of these fluids. The organism 
passes through the Berkefeld filters. The virus is destroyed at 
55 C. in 15 minutes. 

The Virus of Typhus Fever. Nicolle, Conor and Conseil in 
1910 transmitted typhus fever to monkeys by means of serum 
which had passed through a Berkefeld filter. Ricketts and Wilder 
failed to obtain infective filtrates in their study of Mexican ty- 
phus. Typhus is an acute febrile disease, widely distributed but 
not very prevalent in any locality. Apparently it is not con- 

1 Journ. A. M. A., 1913, Vol. LX, p. 362. 
2 Journ. A. M. A. } 1911, Vol. LVII, pp. 971-972. 



SPECIFIC MICRO-ORGANISMS 

tagious 1 but is transmitted from man to man by body lice (Pedi- 
culus vestimenti). Immunity follows recovery. 

The Virus of Small-pox. The virus of this disease was shown 
to be filterable by Casagrandi in 1908. The vaccine virus, which 
is generally considered to be the same organism, had been pre- 
viously filtered. The organism passes through the coarser Cham- 
berland filters. The virus resists drying for several weeks and 
remains active in glycerine for eight months, but is quickly ren- 
dered inert by bile and by sodium oleate. It is also destroyed by 
heating at 58 C. for 15 minutes. Cell inclusions, which were 
described by Guarnieri in 1892, are considered by some to repre- 
sent forms of the pathogenic agent. 

Small-pox is an acute disease of man characterized by a general 
eruption on the skin, at first papular, then vesicular and pustu- 
lar. It is highly contagious by direct association and by fomites 
and is readily transmitted by placing bits of crust from dried 
pustules on the nasal mucous membrane or on a scratch in the 
skin. Cow-pox is a milder disease which occurs naturally in cows, 
and has also been produced by inoculating calves with small-pox 
virus. An attack of either small-pox or cow-pox is followed by 
immunity to both diseases. Cow-pox in man is a comparatively 
mild disease. Inoculation results in the formation of a single 
pustule, rarely surrounded by secondary vesicles, with slight illness 
for a few days. Edward Jenner in 1798 discovered that cow-pox 
resulting from artificial inoculation (vaccination) confers an immu- 
nity to small-pox. Vaccination is now very generally practised 
in enlightened communities and in such places small-pox is practi- 
cally unknown. The inoculation is best done by making a very 
slight superficial linear incision, about 5 mm. long, in the epi- 
dermis and rubbing into it the vaccine virus. The whole pro- 
cedure should result in only a faint tinge of blood. When the 
vesicle appears it should be carefully protected from violence. 
A normal vaccination causes little inconvenience and is usually 

1 Wilder: Journ. Infect. Dis., 1911, Vol. IX, p. 9. Ricketts and Wilder :Joiirn. 
A. M. A., 1910, Vol. LV, pp. 309-311. 



THE FILTERABLE MICROBES 377 

completely healed in about 4 weeks after inoculation. Failure 
of the inoculation is not a proof of immunity. The vaccination 
should be repeated until it does take. 

The Virus of Chicken Sarcoma. Rous in 1910 discovered a 
tumor in a chicken which is histologically a typical spindle-cell 
sarcoma and which he has been able to reproduce in other chickens, 
not only by transplantation but also by inoculation of an agent 
which can be separated from the tumor cells 1 by nitration through 
Berkefeld filters, as well as by inoculation with tumor tissue which 
has been dried and powdered and preserved in the dry condition 
for months. The filterable microbe, or filterable agent as Rous 
conservatively calls it, is rendered inert by heating at 55 C. in 
15 minutes, also by the admixture of chicken bile or saponin. 
Two other sarcomata of the fowl have been shown to be due to a 
filterable agent by the same investigator. 

Our conceptions of the nature of filterable agents is at present 
beginning to become more definite. They are no longer re- 
garded as necessarily beyond the possibility of morphological study 
and there is good reason to hope that the development of improved 
methods of study and their careful application may be able to 
establish not only the important physiological properties of these 
agents but their form and perhaps to some extent their structure 
as well. The beginning already made is full of promise for the 
future. 2 

1 Rons and Murphy: Journ. Exp. Med., 1913, Vol. XVII, pp. 219-231. Pre- 
vious papers are cited there. 

2 A number of other diseases have been shown to be caused by filterable agents. 
A brief mention of these together with references to the literature will be found in 
the article by Wolbach: Journ. Med. Rsch., 1912, Vol. XXVII, pp. 1-25. 



CHAPTER XXVII. 



MASTIGOPHORA. 1 

Herpetomonas Muscae (Domesticae). 2 This flagellate proto- 
zoon is commonly found in the intestine of the house fly (Musca 
domestica). The cell body is spindle shaped (Fig. 146) and 15 to 
to 2 5 A* in length. The flagellum is of 
about equal length and contains two 
stainable filaments which terminate near 
the deeply staining blepharoplast situated 
in the anterior part (flagellated end) of 
the cell. From this blepharoplast a deli- 
cate thread extends in the cytoplasm to- 
ward the posterior end. The nucleus (tro- 
phonucleus) is at the center of the cell. 
Multiplication takes place by longitudinal 
division. 

Leptomonas (Herpetomonas) Culicis. 3 
In the digestive tract of mosquitoes, 
flagellated organisms occur which bear a 

FIG. 146. Herpetomonas 

a, Normal indi- confusing resemblance to trypanosomes. 
Th fy >ltiply abundantly in the blood 
which the insect ingests and are most 

.. f . . . 

easily found in the mosquito near the end 
of digestion of a blood meal (48 to 96 hours after feeding). The 
body is 1 6 to 45 ju in length and 0.5 to 2/4 in width. Artificial 
cultures have been obtained in the condensation water of blood- 
agar and these have been purified by streaking on blood-agar 

1 Only a few protozoal forms can be considered and those very briefly. The 
interested student should consult Doflein: Protozoenkunde, III Auflage, Jena, 1911. 

2 Prowazek, Arb. Kais. Gesundheitsamt., 1904, Bd. XX, S. 440. 
3 Novy, MacNeal and Torrey, Journ. Inf. Dis., 1907, Vol. IV, p. 223. 

378 




blepharoplast. (From 

Doflein after Prowazek.) 



MASTIGOPHORA 379 

plates. The organism is not known to be capable of infecting 
vertebrates. 

Somewhat similar flagellates are found in the alimentary 
tract of various insects, where they may be easily mistaken for 
developmental stages of hematozoa. Trypanosoma (Herpeto- 
monas) grayi which is found in the tsetse fly Glossina palpalis 
may be mentioned as another example. 




FIG. 147. Leptomonas culicis from the digestive tract of a mosquito. X 1500. 
(After Novy, MacNeal and Torrey.) 

Trypanosoma Rotatorium. This organism is the type species 
of the genus Trypanosoma, as this name was first applied to it by 
Gruby in 1843. It * s commonly found in small numbers in the 
blood of frogs. The form of the cell varies from that of a slender 
spindle to a very broad and thick structure (Fig. 148). The 
width varies from 5 to 40/1, and the length from 40 to 8oju. These 
various forms are probably stages in the growth of the parasite but 
it is not impossible that they represent different species parasitic 
in the same animal. When the larger forms are well stained the 
typical structures of a trypanosome are distinctly evident. The 
large nucleus (trophonucleus) lies near the middle of the body 
and closer to the undulating border. Posterior to it is the smaller 
and more deeply stained blepharoplast. Close to the latter a 
small clear colorless area is commonly seen. The flagellum 



SPECIFIC MICRO-ORGANISMS 




* 



FIG. 148. Trypanosoma rotatorium in blood of a frog; drawn from a preparation 
stained by Romawowsky method after dry fixation. The smaller form is feebly 
stained. 




FIG. 149. Trypanosoma rotatorium. The various forms which occur in arti- 
ficial culture. A, Crithidia form; B, trypanosome form; C, spherical form; D and 
E, club forms; F and G, spirochete forms; H, resting stage; /, resting stage with 
vacuole and double nucleus. (After Doflein.) 



MASTIGOPHORA 



originates near the blepharoplast and extends along the convex 
border of the cell, which is drawn out into a well-developed thin 
undulating membrane, to the anterior end of the cell and beyond 
it as a free flagellum. The posterior tip of the cell is usually 
drawn out to form a slender process. The other border of the 
cell is nearly straight and the cytoplasm near it usually shows 
definite evidence of longitudinal 
striation, indicating the presence 
of elementary muscular structures, 
so-called myonemes. The slender 
form resembles very closely the 
shape of mammalian trypano- 
somes. 

Cultures of Tr. rotatorium were 
first obtained by Lewis and H. U. 
Williams in the condensation fluid 
of slanted blood-agar. Various 
forms of the organism occur in 
the cultures. Many of these are 
doubtless degenerating cells. The 
mode of transmission from frog to 
frog is unknown but it is prob- 
ably accomplished by means of leeches. 

Trypanosoma Lewisi. This organism, the common rat 
trypanosome, appears to have been seen as early as 1845, but its 
modern study dates from its rediscovery by Lewis in 1879. It 
occurs in the blood of wild rats throughout the world, from i to 
40 per cent being infected. In the rat the parasite passes through 
a short period, 8 to 14 days, of rapid multiplication, which is 
followed by a period, usually several weeks or months, in which 
the organism persists without evident increase in numbers; 
further multiplication beginning upon transfer to a new host. In 
the adult or resting stage, the trypanosomes are quite uniform, 
1.5 to 2fjL wide by 27 to 28/z in length, including the flagellum 
(Fig. 150). When blood containing these adult forms is injected 




FIG. 150. Trypanosoma lewisi. X 
2500. (From Doflein after Minchin.) 



382, 



SPECIFIC MICRO-ORGANISMS 



into a healthy young rat the multiplication forms of the parasite 
appear after about three days. These forms show a great variety 
of size and shape and they stain more deeply than the adult stage 
(Fig. 151). Numerous dividing parasites are also present, some of 
them showing multiple division with the formation of rosettes. 
The division is longitudinal and essentially unequal, as one cell 
retains the old flagellum while the new one is formed for the other 




I 




FIG. 151. Trypanosoma lewisi. Various forms in the blood of a rat six days after 
inoculation. X 1125. (After MacNeal.) 

daughter cell. The rosettes arise by successive longitudinal 
divisions, and an unbroken rosette contains one cell with the old 
flagellum larger than the others (Fig. 152). 

The infection is readily transmitted to young rats by the 
injection of blood containing the parasites. Under natural condi- 
tions transmission is due to insects, especially fleas and lice. 1 The 

1 Swellengrebel and Strickland: Parasitology, 1910, Vol. Ill, pp. 360-389. 



MASTIGOPHORA 



383 



trypanosomes multiply in the digestive tract of these insects, 
producing various forms, many of them resembling herpetomonas 
and leptomonas. Fleas remain infective for a long time. 

Cultures of Tr. lewisi were obtained by MacNeal and Novy 1 
in 1902-03, in the condensation fluid of inclined blood- agar, and 
the infection was reproduced by inoculation of these cultures. 




FIG. 152. Trypanosoma lewisi. Eight-cell rosette in division. Note the long 
original or parent whip on one of the cells. Several cells show a second flagellum 
growing out preparatory to a further division. X225o. (After MacNeal.) 

The size and shape of the organism in culture is quite variable 
The actively dividing forms are usually grouped in rosettes with 
flagella directed centrally, and the cells themselves are pear- 
shaped or oval. Herpotomonad forms are common. 

The infection with Tr. lewisi rarely results in death of the rat. 

1 Contributions to Medical Research, dedicated to Victor Clarence Vaughan, 
1903, PP. 549-577- 



384 



SPECIFIC MICRO-ORGANISMS 



Other species of animals are not readily infected. Immunity 
follows recovery. Artificial immunity has been produced by 
Novy, Perkins and Chambers 1 by the injection of a pure culture 
which had been propagated for six years on artificial media and 
had lost its virulence. 

There are many other relatively harmless trypanosomes 
parasitic in the blood of various mammals. 

Trypanosoma Brucei. Bruce in 1895 discovered this organism 



D 










FIG. 153. The most important trypanosomes parasitic in vertebrates. A, 
Tr.kwisi; B, Tr. eiansi (India); C, Tr. evansi (Mauritius); D, Tr. brucei; E, Tr. 
equiperdum; F, Tr. equinium; G, Tr. dimorphon; H, Tr. gambiense. All magnified 
(From Do flein after Novy.} 



in the blood of horses suffering from Nagana, the Tsetse-fly dis- 
ease of Zululand. Pure cultures have been obtained in the con- 
densation fluid of inclined blood-agar by Novy and MacNeal 
and the injection of pure cultures into animals produces the dis- 
ease and death. 

Tr. brucei is 1.5 to 5ju wide and 25 to 35/z long, including the 

1 Journ. Inf. Dis. y 1912, Vol. XI, pp. 411-426. 



MASTIGOPHORA 



385 



flagellum. The nucleus lies near the center of the cell. It is oval 
or somewhat irregular in outline and usually occupies the who4e 
width of the cell. Near the blunt posterior end of the cell is a 

A 




FIG. 154. Glossinamorsitaus. A, Magnified. (After Dofiein.} B, Sketch showing 
natural size. (From Dofiein after Blanchard.} 

spherical granule, the blepharoplast. Near this the flagellum 
originates and it extends forward along the convex border of the 
cell, which is drawn out into a thin undulating membrane, and 
A B 





V 

FIG. 155. Glossina morsitaus-, lateral view of the resting fly. A, Before feeding. 
B, After sucking blood. (From Doflein after Austen.} 

extends beyond the anterior end of the cell as a free flagellum. 
The cytoplasm anterior to the nucleus often contains many coarse 
granules. The general shape of the trypanosome as seen in the 

25 



386 SPECIFIC MICRO-ORGANISMS 

blood of the infected animal is fairly uniform. There is, however, 
considerable variety in size, internal structure and staining 
properties. Multiplication takes place by unequal longitudinal 
division, much the same as in Tr. lewisi, but the dividing cell has 
the same general form as the others and multiple division figures 
are less common. The larger cells are usually in process of divi- 
sion. Trypanosomes with feebly staining cytoplasm and others 
with very abundant coarse granules also occur. The former are 
probably degenerating and disintegrating cells. 

Tr. brucei is taken up by the blood-sucking tsetse fly, Glossina 
morsitans and in about 5 per cent of these it multiplies in the 
alimentary canal and penetrates into the body cavity, causing a 
generalized infection of the fly. After about three or four weeks 
the salivary glands are invaded and the fly is then able to infect 
other animals by biting them, and it remains infective for a long 
time, probably as long as it lives. Other insects may possibly 
serve to transmit the parasite. The infection is also readily 
transmitted from animal to animal by the injection of infected 
blood. 

Cultures are obtained with some difficulty, but most readily 
by inoculating inclined blood-agar, 1 2:1, and incubating at 28 C. 
The primary cultures should not be transplanted until they are 
about three weeks old, and they usually fail to infect animals if 
injected into them. The virulence is regained in the subcultures. 
Culture filtrates are not toxic. The poison of trypanosomes 
seems to be set free as a result of their disintegration in the body 
fluids. 2 

Nagana occurs naturally in a great variety of the quadrupeds 
and is usually fatal. Man is not susceptible. Mice and rats 
die in 6 to 14 days after inoculation. Guinea-pigs may show one 
or more relapses, the disease lasting for two to ten weeks. 

1 The agar employed should contain the extractives of 125 grams of meat, 10 grams 
pepton, 5 grams salt and 25 grams of agar in 1000 c.c. It is liquefied, cooled to 
50 C. and mixed with twice its volume of warm defibrinated rabbit's blood and then 
allowed to solidify in an inclined position. 

2 MacNeal: Journ. Inf. Dis., 1904, Vol. I, p. 537. 



MASTIGOPHORA 



387 



Diagnosis may be made by microscopic examination of the 
blood when the parasites are numerous. At other times it is well 
to inject 5 to 10 c.c. of blood into a white rat. The distinction 
of Tr. brucei from other species of trypanosomes causing similar 
diseases is not easy and may require prolonged study. 

Immunity of susceptible animals has not yet been achieved, 
but inoculation with attenuated cultures produces a relative 
immunity in small laboratory animals. 1 

Trypanosoma Evansi. This organism was discovered by 
Griffith Evans in 1880 in the blood of horses and various other 





FIG. 156. Trypanosoma equiperdum. Blood of an inoculated rat. A, after four 
days; B, after eight days. (After Doflein.} 

animals suffering from the disease known in India as Surra. The 
trypanosome resembles Tr. brucei in most respects but is recog- 
nized as a distinct species. Surra is apparently transmitted by 
various flies, Tabanidcz, Stomoxys, and also by fleas. 

Trypanosoma equiperdum was found by Rouget in 1896 in 
the blood of horses suffering from dourine. The infection is 
transmitted by coitus and probably also in other ways. Dourine 
occurs in southern Europe and northern Africa. A few cases 
have been observed in Canada and in the United States. Small 
laboratory animals are susceptible to inoculation. 

1 Novy, Perkins and Chambers: Journ. Inf. Dis., 1912, Vol. XI, pp. 411-426. 



388 SPECIFIC MICRO-ORGANISMS 

Trypanosoma Equinum. Elmassian in 1901 observed this 
organism in the blood of horses suffering from Mai de Caderas 
in South America. It possesses a very minute blepharoplast, a 
morphological character which distinguishes it from most other 
trypanosomes. Small laboratory animals are susceptible. 

Several other species of trypanosomes have been described, 
which cause fatal diseases in quadrupeds. Most of these have 
been found in Africa. 

Trypanosoma Gambiense. Button and Todd in 1901 ob- 
served this organism in the blood of an Englishman in Gambia. 
The parasite had been previously seen by Forde. The disease, 
which resulted in death after two years, was called trypansoma 
fever. Castellani in 1903 observed trypanosomes in the cerebro- 
spinal fluid of patients suffering from sleeping sickness in Uganda. 
This organism is now known to be the same as the Tr . Gambiense 
of Button, and sleeping sickness is recognized as the terminal 
stage of trypanosoma fever. 

Tr. gambiense is very similar in form to Tr. brucei but the 
posterior end is on the average somewhat more pointed. The 
length varies between 15 to 30^ and the width from i to 3/z. The 
significance of the different forms found in the blood is not defi- 
nitely known. Multiplication takes place in the same way as 
in Tr. brucei. In the tsetse fly, Glossina palpalis, the trypano- 
somes slowly disintegrate and disappear during the first four days 
after the infected blood is ingested, and in most of the flies this 
results in extermination of the trypanosomes. In 5 to 10 per 
cent of the flies the parasites are not completely destroyed, but 
the early diminution in their number is followed by an abundant 
multiplication of the trypanosomes in the stomach and intestine of 
the insect. After 18 to 53 days these flies become capable of 
infecting new animals by their bite and remain infectious for a 
very long time. The parasites are found in the salivary glands 1 
when the fly becomes capable of causing the disease. A great 

1 Bruce, Hamerton, Bateman and Mackie: Proc. Royal Soc., 1911, Ser. B, 
Vol. LXXXIII, pp. 338-344; PP. 345-348; pp. 513-527- 



MASTIGOPHORA 389 

diversity of form is observed in the trypanosomes within the fly but 
the significance of the different types is not yet fully understood. 

Many of the mammals are susceptible to inoculation with 
Tr. gambiense. White rats usually relapse 2 or 3 times before 
finally succumbing to the infection, whereas they usually die 
within 2 weeks when inoculated with Tr. brucei. The virulence 
of the organism is somewhat variable. 

Attempts to cultivate Tr. gambiense in artificial media have 
not been fully successful. It has been possible to obtain multipli- 





FIG. 157. Glossina palpalis in natural resting position, and with wings outstretched. 

(After Dofiein.} 

cation of the organisms and to keep them alive for several weeks 
on blood-agar but such cultures'are not virulent and cannot be 
kept up indefinitely. 1 

Human trypanosomiasis is a most important and widespread 
disease in equatorial Africa. Symptoms appear long after the 
infection has taken place. The disease manifests itself in two 
forms, the trypanosoma fever and the sleeping sickness. Trypano- 
soma fever is an irregularly remittent fever lasting for several days 
at each attack, accompanied by a macular eruption, and always 

1 Thomson and Sinton: Annals of Trop. Med. and ParasitoL, 1912, Vol. VI, 
PP- 331-356. 



390 SPECIFIC MICRO-ORGANISMS 

associated with a general enlargement of the lymph nodes. The 
trypanosomes are numerous in the blood during the febrile period 
and become very scarce during the intermissions. The fever 
leads to emaciation and death, sometimes without inducing the 
terminal coma and sometimes with the production of typical 
sleeping sickness. The sleeping sickness is characterized by 
prolonged coma and progressive emaciation. At intervals the 
patient may be aroused and given nourishment, but eventually 
this is no longer possible. At this stage the trypanosomes are 
present in the cerebrospinal fluid. Bacterial infection of the 
meninges often takes place as a terminal event. It is conserva- 




FIG. 158. Trypanosoma amum in the blood of common wild birds. X 1500. 
(After Novy and MacNeal.) 

tively estimated that 100,000 natives have died of trypanosomiasis 
in Africa from 1900 to 1910. There have been several cases in 
Europeans. Recovery seems to be rather uncommon but does 
occur. 

Trypanosoma Rhodesiense. Stephens and Fantham 1 have 
studied a case of human trypanosomiasis contracted in north- 
eastern Rhodesia, where Glossina palpalis does not occur. The 
parasite differs somewhat from Tr. gambiense and is regarded by 

1 Proc.lRoyal Soc., 1910, Ser. B, Vol. LXXXIII, pp. 28-33. 



MASTIGOPHORA 



391 



these authors as a distinct species. It seems to be transmitted 
by Glossina morsitans. 1 

Trypanosoma Avium. Trypanosomes were probably seen in 
the blood of birds by earlier investigators, but the first accurate 
description of such observations is that of Danilewsky in 1885. 




FIG. 159. Trypanosoma avium in culture on blood agar. X 1500. (After Novy 

and MacNeal.} 

Infection with trypanosomes is very common in the ordinary 
wild birds. Novy and Mac Neal 2 examined 431 American birds 
representing 40 common species and found trypanosomes in 38 
individuals, representing 16 species. The indicated prevalence 

1 Kinghorn and Yorke: Annals of Trop. Med. and ParasitoL, 1912, Vol. VI, 
pp. 269-285. Kinghorn, Yorke and Lloyd: ibid., 1912, Vol. VI, pp. 495-503. 
2 Journ. Infect. Dis., 1905, Vol. II, pp. 256-308. 



3Q2 SPECIFIC MICRO-ORGANISMS 

of the infection, 8.8 per cent, is doubtless far below the actual 
percentage, as many of the birds were not tested by the cultural 
method. There are doubtless several species of bird trypanosomes 
but the most common form is Tr. avium. The length varies 
from 25 to yo/x and the width from 4 to yju. 

Cultures are easily obtained by transferring the infected blood 
to tubes of blood-agar and incubating at 25 to 30 C. The pro- 
tozoa grow abundantly and, by weekly transfers, may be kept 
under cultivation without special difficulty for an indefinite period. 
Injection of cultures into birds is only rarely followed by appear- 
ance of trypanosomes in the blood. 

The parasites persist in the blood of the birds for many months 
and probably for years. They seem to be comparatively harmless. 
The mode of transmission from bird to bird in unknown. 

Trypanosoma avium is a form of considerable importance in 
the study of systematic protozoology because of the confusion of 
trypanosomes and hemocytozoa by Schaudinn 1 in 1904, who 
regarded Tr. avium as merely an extracellular form of Hamopro- 
teus noctucB (danilewskyi?) (see page 414). This misconception, 
together with the analogous assumption of similar relationship 
between spirochetes of birds and the leukocytozoon of Ziemann, 
Hczmoproteus ziemanni, made by Schaudinn at the same time, 
has exercised a profound influence upon the course of investiga- 
tion in the groups of spirochetes, trypanosomes and hemocytozoa 
during the last eight years, and it is only recently that this error 
of Schaudinn has been recognized as such by the German and 
English protozoologists. 

Schizotrypanum Cruzi. Chagas discovered this organism 
in 1907. It occurs in the blood in the Brazilian human trypano- 
somiasis called coreotrypanosis. Multiplication takes place 
within endothelial cells, lymphocytes and other cells in the paren- 
chymatous organs, and especially in the interior of muscle cells in 
the heart and skeletal muscles. 2 The dividing parasites are without 

1 Arb. a. d. Kais. Gesundheitsamte, 1904, Vol. XX, pp. 387-439. 
2 Vianna: Memorias do Institute Oswaldo Cruz, 1911, Vol. Ill, pp. 276-293. 
Abstract in Sleeping Sickness Bull., 1912, Vol. IV, pp. 288-293. 



MASTIGOPHORA 



393 














FIG. 1 60. Schizotrypanum cruz'i developing in the tissues of the guinea-pig, 
i. Cross-section of a striated muscle fiber containing Schizotrypanum cruzi: Note 
dividing forms. 2. Section of brain showing a Schizotrypanum cyst within a 
neuroglia cell, containing chiefly flagellated forms. 3. Section through the supra- 
renal capsule, fascicular zone. 4. Section of brain showing a neuroglia cell filled 
with round forms of Schizotrypanum. (From Low, in Sleeping Sickness Bulletin, 
after Vianna.} 




394 SPECIFIC MICRO-ORGANISMS 

flagella and resemble the intracellular forms of Leishmania. From 
these cysts the parasites escape into the blood, where they are 
found as trypanosomes in the blood plasma. Slender and thick 
forms occur here, the difference probably depending upon the 
age of the parasites. 

Monkeys, rats, mice, young guinea-pigs and many other 
mammals are susceptible to inoculation. The infection is trans- 
mitted by a bug, Conorhinus megistus, 
in which the protozoon develops 
abundantly. The bedbug, Culex 

lectularius also is capable of trans- kj? C\ *~" 

mitting the disease. f*r\ *\^' 

Cultures are readily obtained %4^ ' 
on blood-agar and Chagas was p IG> , 6 1 .-Schizotrypanum 
able to infect animals with such cul- cruzi in human blood. (From 

Doflein after Chagas.) 

tures. 

Leishmania Donovani. Laveran and Mesnil in 1903 described 
this protozoon which occurs inside cells in various parts of the 
body, but is especially abundant in the spleen and liver, in the dis- 
ease known in India as Kala-Azar or tropical splenomegaly. The 
organism is oval, 2 to 4/4 in diameter, finely granular and some- 
times vacuolated. In the interior there is a large rounded nucleus 
and a smaller oval or rod-shaped blepharoplast, near which a third 
very slender short thread may usually be recognized as the rudi- 
ment of the undeveloped flagellum. These structures are doubled 
in the division stages. Multiple division also occurs. In the cir- 
culating blood the organism is found within lymphocytes and poly- 
nuclear leukocytes. Many of them may be found in a single cell. 

Cultures are readily obtained by inoculating fluid (citrated) 
blood with blood or with spleen juice containing the parasites, or 
by inoculating the usual blood-agar. In artificial culture the 
cell elongates, the rudimentary whip extends into a true flagellum 
and the organism assumes the appearance of a typical leptomonas 
(herpetomonas). Little difficulty is experienced in keeping the 
cultures alive and flourishing. 



MASTIGOPHORA 



395 



The parasite has been supposed to be transmitted from man 
to man by bugs of the genus Cimex, but this hypothesis has be_en 




FIG. 162. Conorhinus megistus, the insect carrier of Schizotrypanum cruzi. (From 

Dofltin after C ha gas.} 



** 









FIG. 163. Lieshmania donovani in the juice obtained by puncture of the spleen in 
kala-azar. (From Doflein after Donovan.} 

rendered very uncertain by recent work of Wenyon 1 and the 

1 Journ. Lond. Sch. Trop. Med., 1912, Vol. II, pp. 13-26. 



396 



SPECIFIC MICRO-ORGANISMS 



Sergents. 1 The latter investigators were able to effect experi- 
mental transmission by means of the dog flea, Ctenocephalus canis. 

Kala-Azar is 'endemic in tropical Asia 
and northeast Africa, where it occurs among 
the poorer class of people, living in squalor. 
It is characterized by irregular fever, weak- 
ness and cachexia and especially by enormous 
enlargement of the spleen, often of the liver 
also. It is frequently fatal. Dogs and mon- 
keys are susceptible to inoculation. 

LeishmaniaTropica. This organism was 
first accurately described by J. H. Wright, 2 
who found it in great abundance in the 
lesion known as Aleppo boil, Delhi boil or tropical ulcer. 
The parasites occur within the endothelial cells within the lesion 




FIG. 164. Leishmania 
donovani, various forms 
observed in artificial cul- 
ture. (From Doflein after 
Chatter jee.) 




IM 



FIG. 165. Leishmania tropica. Smear from a Delhi boil. Xisoo. (From Doflein 

after J. H. Wright.} 

and are very numerous. Leishmania tropica resembles L. donovani 

1 Sergent (Edm. & Et.), L'Heritier and Lemaire, Bull. Soc. Path. Exot., 1912, 
Vol. V, pp. 595-597- 

2 Journ. Med. Rsch., 1903, Vol. X, pp. 472-482. 






MASTIGOPHORA 



397 



very closely except in its pathogenic properties. Cultures on 
blood-agar have been obtained by Nicolle and are easily propa^ 
gated at 22 C. Dogs and monkeys are susceptible to inoculation 
and the human disease is probably contracted from dogs through 
the agency of insects. The disease is relatively benign and recov- 
ery is followed by prolonged immunity. Inoculation has been 
practised in man in order to produce immunity. 






FIG. 166. Lciskmania Iropica, 
forms observed in cultures. (From 
Dofiein after Nicolle.) 



FIG. 167. Try pano plasma cy- 
prini. Bl, Blepharoplast; N, nu- 
cleus. X 2000. (After Doflein.} 



Leishmania Infantum. Nicolle in 1908 observed this organ- 
ism in the spleen, liver and bone marrow of children dying from 
splenomegaly in northern Africa. The disease resembles Kala- 
Azar in all respects except that the patients are all very young. 
Dogs are naturally infected with this parasite and are probably 
the source of the human disease. Cultures on blood-agar are 
readily obtained and kept up indefinitely without special difficulty. 



398 



SPECIFIC MICRO-ORGANISMS 



Trypanoplasma Borreli. Laveran and Mesnil in 1901 de- 
scribed this protozoon which occurs in the blood of various species 
of fish. It resembles a trypanosome somewhat, but the blepharo- 
plast is relatively large and from it two flagella originate, one 
extending forward immediately as a free whip while the other 
runs along the convex border, ensheathed in an undulating mem- 
brane, and extends at the posterior end 
as a free flagellum. Longitudinal divi- 
sion takes place in the circulating blood. 
Transmission seems to be accomplished 
by means of leeches. T. cyprini and 
T. guernei seem to be identical with T. 
borreli, but they may prove to be dis- 
tinct species. 




FIG. 1 68. Bodo lacertce. a, 
Sketched from life; b, drawn from 
a stained preparation. (From 
Doflein after H artmann and 
Prowazek.} 




FIG. 169. Trichomonas hominis from the 
mouth. (From Doflein after Prowazek.) 



Bodo Lacertae. In the cloaca of various lizards a flagellate 
is almost constantly found. It is 2 to 4^ wide and 6 to 12.5^ 
long, lance-shaped and twisted at the posterior (pointed) end. 
The nucleus is near the anterior end. At its side is a granule 
resembling a blepharoplast and from this a thread extends to 
the anterior end of the cell where it gives rise to two flagella. 



MASTIGOPHORA 



399 






FIG. 170. Lamblia intestinalis. A, Ventral aspect; B, lateral view; C, in posi- 
tion on epithelium; D, the same enlarged. (From Doflein after Grassi and 'Sckevria- 
kof.) 

A B C 








s 



FIG. 171. Trimastigamceba philippinensis. A, Early stage of division of the 
nucleus. The polar caps are still united by a bridge. The equatorial plate has 
formed. B, Ordinary cyst. C, Vegetative form showing the nucleus and a second 
chromatin granule (split off from it?). D, Flagellated form showing remains of the 
rhizoplast between the nucleus and the basal granules. , Flagellated form with 
pseudopodia. (After Whitmore.} 



4OO SPECIFIC MICRO-ORGANISMS 

Trichomonas Hominis. Davaine observed this parasite in 
1854. It is common in the human digestive tract, especially in 
the stomach in anacidity and in the intestine in chronic digestive 
disturbances. The organism is 3 to 4/z wide and 4 to i5/z long, 
pear-shaped and provided with three free flagella, and a fourth 
thread which passes around one side of the cell in the margin of 
the undulating membrane. The parasite seems to be a harmless 
commensal, as a rule, but it may possibly bear some causal rela- 
tion to diarrhea in some cases. Animals have not been success- 
fully inoculated with it. Tr . vaginalis is very similar. It grows 
in the acid vaginal mucus. Other trichomonad forms occur in 
the intestines of animals, particularly in mice, in frogs and in 
lizards. 

Lamblia Intestinalis.- The cell has the form of a turnip with 
a wide and deep excavation in front near the anterior rounded 
end, forming a suction cup. The body is bilaterally symmetrical. 
The length is 10 to 2i/z and the width 5 to i2ju. There are eight 
flagella, each from 9 to 14/1 long. The mode of multiplication is 
not fully known. Resistant cysts are formed, probably after 
sexual union of two individuals, and these escape with the feces 
and lead to the infection of new hosts. Lamblia lives in the duo- 
denum and jejunum of man and many other mammals. It ap- 
pears to be relatively harmless in most cases but the possibility 
that it may be a cause of digestive disturbance must be con- 
sidered. It is often present in chronic dysenteries. 

Mastigamceba Aspera. This a saprophytic form, described 
by Schulze, which possesses a single flagellum, but is also capable 
of extending finger-like projections of its cytoplasm, pseudopodia, 
just as an ameba does. Whitmore 1 has described a somewhat 
similar saprophyte, Trimastigamceba philippinensis, which is at 
times ameboid without flagella and at other times possesses three 
or possibly four whips. It divides and encysts like an ameba. 
The organism is readily cultivated on the alkaline agar of Mus- 
grave and Klegg. 

1 Archivf. Prolistenkunde, 1911, Bd. XXIII, S. 81-95. 



CHAPTER XXVIII. 
RHIZOPODA. 

Amoeba Proteus. This large saprophytic ameba may be 
considered as an example of the numerous species of free-living 
amebae, the classification and identification of which is still in 
hopeless confusion. The organism is widely distributed in stag- 
nant water and is easily cultivated in the laboratory in not too 
foul infusions containing bacteria and algae. The cell is 50 to 




FIG. 172. A, Amoeba proteus engulfing a clump of small alg<2 (No). Cv, con- 
tractile vacuole; N, nucleus. B, Newly encysted ameba showing nuclear fragments; 
cy, cyst wall; w, nucleus; R, reserve food substance. C, Cyst containing man}'' young 
amebaj beginning to escape; cy, cyst wall; k, young amebae. (After Doflein.) 



across, often possesses numerous thick, blunt pseudopodia. 
The ectoplasm and endoplasm appear distinctly different, the 
latter being filled with granules, crystals, vacuoles and food parti- 
cles, such as algae and bacterial cells, and possessing a contractile 
vacuole. The nucleus is lentil-shaped and the chromatin within 
it has a very typical arrangement in a central plate surrounded 
by a network on which the peripheral chromatin is symmetrically 
26 401 



402 SPECIFIC MICRO-ORGANISMS 

placed. Binary division with mitosis of the nucleus seems to be 
the common mode of multiplication. Multiple division also 
occurs in the vegetative state. The resistant stage (cyst) is charac- 
terized by a thick, firm wall of several layers, within which the 
nucleus divides into 200 or more daughter nuclei. Each of these 
becomes surrounded by a little cytoplasm and, when the cyst 
bursts, wanders out as a young ameba. The life history is in- 
completely known. 

Cultures of saprophytic amebae are readily obtained upon 
agar plates. The medium contains agar 0.5 gram, tap water 





FIG. 173. Eutamceba coli. a, Free ameba; b, ripe cyst with eight nuclei. (From 

Doflein after Hartmann.) 

90 c.c., ordinary nutrient broth 10 c.c. Cultures are incubated 
at 25 C. Williams 1 has succeeded in obtaining pure cultures, 
free from bacteria, at 36 C. by employing agar smeared with 
naturally sterile brain substance. 

Entamoeba Coli. Loesch 2 in 1875 observed amebae in the 
human large intestine in gastro-intestinal disturbance. The 
organism is very common in the human intestine, being found in 
10 to 60 per cent of persons without digestive disturbances, 
when the examination is thorough. 

The cell in the vegetative stage is variable in shape and size, 

1 Journ. Med. Rsch., 1911, Vol. XXV, pp. 263-283. 

2 Virchow's Archiv, 1875, Bd. LXV, S. 196-211. 






RHIZOPODA 403 

the diameter measuring 10 to 70/1. The protoplasm is slightly 
granular and shows distinctly an alveolar structure. The d!s-~ 
tinction between ectoplasm and endoplasm is apparent only in 
the pseudopodia. There is no contractile vacuole. Food sub- 
stance is present in the cytoplasm, bits of vegetable material, 
bacteria and, rarely, red blood cells. The nucleus is round, ve- 
sicular and enclosed in a nuclear membrane. In its center is a 
relatively large mass of chromatin and there are numerous smaller 
masses of chromatin at the periphery beneath the nuclear mem- 
brane. Multiplication in the vegetative stage takes place by 
binary division as a rule, but multiple division preceded by re- 
peated division of the nucleus also occurs. 

E. coli discharges all food material from its cytoplasm before 
encystment so that the cell is clear and the nucleus plainly visible. 
A large vacuole in the cytoplasm usually makes its appearance 
and is present during the first and second division of the nucleus 
in the cyst. It is large in those cysts in which much chromatin 
escapes from the nuclei into the cytoplasm as chromidia, and it 
usually disappears when the four nuclei have been formed. A 
further division of the nuclei gives rise to eight and this is the 
usual number present in the fully developed cyst of E. coli, al- 
though rarely ten or even sixteen nuclei may be observed. 1 The 
self-fertilization, autogamy, described by Schaudinn as occurring 
early in encystment has not been observed by Hartmann, and 
its actual occurrence seems questionable. The developed cyst 
with eight nuclei is about i5/z in diameter and is considered to be 
definitely characteristic of this species. 

E. coli is generally regarded as a harmless commensal in the 
human intestine. It is however impossible to exclude the possi- 
bility that it may contribute to the aggravation of pathological 
conditions present in the digestive tract. (Compare with Bacillus 
coli.) Its common occurrence in healthy men speaks against 
its possessing any very specific and powerful pathogenic property. 

1 Hartmann and Whitmore: Archiv f. Protistenkunde, 1912, Bd. XXIV, S. 
182-194. 



404 



SPECIFIC MICRO-ORGANISMS 



Entamceba Tetragena. Viereck in 1906 recognized this 
organism as a species distinct from E. coli. It occurs in the intes- 
tine and in the stools of persons suffering from amebic dysentery 




FIG. 174. Entamosba telragena. The same living individual drawn at brief intervals 
while moving. (From Doflein after Hartmann.) 

and very seldom in other individuals. The cell is 8 to 6o/i in 
diameter. The ectoplasm is distinctly differentiated from the 






FIG. 175. Eutamceba telragena. a, Vegetative cell containing a red blood cell 
(near upper end). Xisoo. b and c, Drawings of nuclei showing stages of the so- 
called cyclical changes. X26oo. (From Doflein after Hartman.} 

endoplasm even when the cell is motionless, and the lobose pseudo- 
podia are made up entirely of the stiff highly refractive ectoplasm. 
The endoplasm contains food material consisting of bacteria, cell 



RHIZOPODA 405 

fragments and red blood cells. The nucleus is very distinctly 
visible in the living ameba. It is spherical and surrounded by-a- 
thick doubly contoured nuclear membrane. The chromatin is 
usually distributed just beneath the nuclear membrane in largest 
amount and in the center there is a karyosome with definite centri- 
ole. The vegetative multiplication takes place by division into 
two daughter cells. Multiple division seems not to occur. 

Cyst formation is rarely observed. The cysts are most likely 
to be found when the stool becomes formed in 
convalescence from an attack of dysentery and 
they may then be very numerous. The mature 
cyst contains four nuclei, and frequently contains 
also one or more large masses of chromidial sub- 
stance which stain black with iron hematoxylin. 




The forms of the organism commonly observed FlG - * ib 

. . , . , , . mceba tetragena. Ma- 

in the feces of dysentery are either the active ture cyst containing 
vegetative cells 1 or degenerating forms, and the ^ STto^id 
latter may lead to confusion unless their true na- substance. (After 

j Hartmann.) 

ture is recognized. 

E. tetragena is regarded as the causal agent of amebic or tropical 
dysentery and there can be little question that it is the parasite 2 
present in most cases presenting the typical clinical picture and 
pathology of the disease. It is doubtless transmitted in food and 
drinking water in the encysted stage. 

Entamceba Histolytica. Schaudinn in 1903 distinguished 
this species from E. coll and regarded it as the causal organism 
in amebic dysentery. The subsequent study of Schaudinn's 
preparations by Hartmann 3 has shown that most of the specimens 
recognized as E. histolytica by Schaudinn are in reality vegetative 
and degenerating forms of E. tetragena. Our whole knowledge 
of the species, which was founded upon Schaudinn's studies, 
therefore becomes very uncertain and even the existence of E. 
histolytica as a disticnt species may be seriously questioned. 

1 Hartmann: Arch. f. Protistenkunde, 1912, Bd. XXIV, S. 163-181. 

2 Whitmore: Arch. f. Protistenkunde, 1911, Bd. XXIII, S. 71-80. 

3 Hartmann, in Prowazek, Handbuch der Path. Protozoen, 1912, Bd. I, S. 58-61. 



406 SPECIFIC MICRO-ORGANISMS 

The belief that amebae bear a causal relation to dysentery is 
based upon the fact that certain types of amebae, E. tetragena 
(and E. histolytica?) are found in the stools, as a rule, only in 
cases of dysentery; further, that these cases of dysentery, in 
which these amebae occur, are characterized by definite clinical 
signs and typical anatomical changes in the intestine; and that 
these amebae are found penetrating deeply into the mucosa of 
the intestine, and it is possible to produce ulcerative enteritis 
in experimental animals by injecting feces containing amebae 
into the rectum or by feeding fecal material containing cysts; and 
further, the fact that abscesses occur in the liver in amebic dysen- 
tery, in which the amebae are present and in which it has been 
impossible to demonstrate the presence of bacteria. The causal 
relation seems highly probable, but it must be recognized that 
the evidence is very inconclusive and admits of other possible 
explanations. Even the relationships of the various forms seen 
in the microscopic preparations require a certain amount of specu- 
lation for their determination, and the possibility of error, even 
by the experienced protozoologist, must be recognized and has 
been well illustrated by the divergent views of Schaudinn and of 
Hartmann in studying the same slides. Greater certainty would 
doubtless be derived from the study of artificial cultures if such 
could be made available. 

Numerous cultures of amebae have been obtained from the 
stools of cases of dysentery, and some from the pus of amebic 
abscesses of the liver, the growth taking place on agar in the pres- 
ence of a single species of bacteria. With these cultures it has 
been possible to cause enteritis in monkeys. Such cultures have 
also been grown at 37 C. by A. W. Williams 1 in pure culture on 
agar streaked with brain substance and with blood, and in these 
cultures she finds that the amebae approach in their structure 
the typical entamebae, not only in nuclear structure and cyst 
formation, but also in the utilization of red blood cells as food. 

1 Soc. Amer. Bact., New York Meeting, Jan. 2, 1913. Science 1913; Vol. 
XXXVIII, p. 451; Williams, A. W., and Calkins, G. N., Journ. Med. Rsch., 1913, 
Vol. XXIX, pp. 43-56. 



RHIZOPODA 407 

Whitmore 1 has carefully studied a number of cultures of amebae 
obtained from cases of dysentery, one of them from a liver abscess, 
and has concluded that in every instance the amebae were free- 
living saprophytic forms belonging to the genus Amoeba and not 
in any case parasitic species. 

Other Rhizopoda. The remaining orders of the Rhizopoda, 
namely Helizoa, Foraminifer, Radiolaria and Mycetozoa contain 
no parasitic forms of great importance to human pathology. 
Plasmodium brassica which causes tumors on the roots of the 
cauliflower plant is of some interest. 2 

1 Archiv f, Protistenkunde, 1911, Bd. XXIII, S. 71-80; ibid, pp. 81-95. 

2 See Doflein, Protozoenkunde, 1911, S. 672-678. 



CHAPTER XXIX. 



SPOROZOA. 

Cyclospora Caryolytica. Schaudinn in 1902 discovered this 
organism, which lives as a parasite in the nuclei of epithelial cells 
of the intestinal mucosa in the common mole. It is ingested in 
the form of spores, from which the slender young sporozoites 
escape in the intestine and penetrate the nuclei of epithelial cells. 
Here the parasite becomes rounded and enlarges, becoming 
quickly differentiated into either the male or female type. The 
former type of parasite has numerous refractive granules in its 










f 



? T 

*;> ** 







FIG. 177. Cyclospora caryolytica. A, Male cells within the nucleus of the host 
cell. B and C, Reproduction by multiple division with final rupture of the host 
nucleus in (C). (From Doflein after Schaudinn.} 

cytoplasm, while the female type has a clear cytoplasm. The 
parasites grow rapidly and segment after 4 to 8 hours, the females 
earlier than the males, and the cells resulting from this segmenta- 
tion, so-called merozoites or agametes, penetrate new nuclei and 
go through the same development. Four to five days after in- 
fection of the mole, the parasites suddenly cease their asexual 
multiplication. The male parasites, microgametocytes, after 
rapid multiplication of nuclei, give rise to numerous microgametes 

408 



SPOROZOA 



409 



provided with two flagella. The female cells, macrogametocytes, 
enlarge slowly and produce numerous yolk-like granules in their 



A 





D 



FIG. 178. Cyclospora caryolytica. A, Female cell (agamete) within the host 
nucleus. B and C, Multiple division. D, A free young female agamete. (From 
Dofldn after Schaudinn.) 

cytoplasm. The nucleus undergoes two reduction (maturation) 

divisions, and one daughter nucleus remains while the others 

A BCD 







II 



FIG. 179. Cyclospora caryolytica. A, Fertilization. B, Fertilized cell. C, Fer- 
tilized cell (oocyst) with cyst wall. D, E, F and G, Division of the cyst contents to 
form two spores, each containing two sporozoits. H, Escape of the sporozoits. 

(From Doflein after Schaudinn.} 

disintegrate. Several microgametes penetrate the matured macro- 
gamete and one of them unites with the nucleus. A cyst wall 



4io 



SPECIFIC MICRO-ORGANISMS 



forms about the fertilized cell and within this the cell divides 
into two and later into four embryo parasites, which are enclosed 
in pairs in two spores within the cyst. This escapes with the 
feces of the mole and serves to infect a new host. 

The invasion of the epithelium produces a severe diarrhea in 
the mole often resulting in death. If the animal survives for 
five days, until after the spores are formed, it then 
usually recovers. 

Eimeria Stiedae (Coccidium Cuniculi). This 
very common parasite of the rabbit was first de- 
scribed by Lindemann in 1 86 5 . It lives and grows 
within the epithelial cells of the small intestine, of 
the bile passages and of the liver of rabbits suffer- 
ing from coccidiosis, and its oocysts are found in 
the intestinal contents and in the feces of such 
animals. The oocyst is an elongated oval, vari- 

able in width fr m IX to 28 ^ and in len g th from 

24 to 4Qju. It contains, when fully developed, four 

, , , . , 

sporozoits are de- spores, each of which contains two embryo para- 
s jj- es or sporozoits. These gain entrance to the 




FIG. 1 80. Ei- 
meria steidtz. 
Oocyst containing 
four spores, in 
each of which two 



pyle is 

low. ~ (From Do- intestine of a new host along with the food and the 

flein after Metz- , -, ,. -, 

ner ^ pancreatic digestion makes an opening at one end 

where the wall is exceedingly thin, the micropyle, 
and through this opening the wedge-shaped sporozoits escape. 
They penetrate epithelial cells, in which the parasite becomes 
rounded and grows to a diameter of 20 to 5o/z, destroying 
the host cell. The nucleus divides many times and after 
it the cytoplasm, so as to form numerous spindle-shaped 
young cells, merozoits of agametes, which penetrate new epi- 
thelial cells and pass through the same cycle. This cycle of 
asexual multiplication, schizogony, is repeated many times and 
may lead to extensive destruction of intestinal mucosa, of the 
epithelium of the bile ducts and of liver substance. Some of 
the growing parasites become differentiated into sexual elements. 
The female cell, macrogametocyte, accumulates numerous large 



SPOROZOA 



411 



granules in its cytoplasm, and when full-grown the chromatin 
of the nucleus is reduced by expulsion of the karyosome. The~ 
matured cell, macrogamete, is then ready for union with the 
microgamete. The growing cell destined to give rise to the male 
sexual elements attains a large size and possesses a pale cytoplasm. 
It is called the microgametocyte. Its nucleus divides many 
times, the small nuclei accumulate near the surface of the cell 
and each escapes with a small portion of protoplasm as a slender 
motile microgamete. The penetration of one of Jthese into the 
macrogamete produces the fertilized oocyst, which forms a thick 









FIG. 181. Eimeria steida. a, Young agamete (merozoit). b, Epithelial cell 
invaded by three young agametes. c, d and e, Stages in the multiple division of the 
agamete. /, Young macrogametocyte. g, Full-grown macrogametocyte. (From 
Doflein after Hartmann.) 

wall about itself and escapes to the external world. Here, the 
fertilized cell divides to form eight cells, sporozoits, which are 
enclosed within four oval spores (two in each) within the wall of 
the oocyst. If this cyst is ingested by another rabbit the cycle 
of development starts anew. 

Coccidiosis is a very common disease in rabbits. The animal 
suffers from severe diarrhea and loss of appetite, and becomes 
emaciated. Young rabbits often die of the disease. Diagnosis 
is readily made by finding the oocysts in the feces. Children 
have been found to be infected with this organism. Cattle, 



412 SPECIFIC MICRO-ORGANISMS 

horses, sheep and swine are also susceptible and serious epizootics 
of coccidiosis due to E. stiedce have been observed in cattle. 

Eimeria (Coccidium) Schubergi. This coccidium occurs in 
the intestine of a common myriapod (thousand-legged worm). 
Lithobiusforficatus. It is the organism in which Schaudinn worked 
out the life-cycle now regarded as typical for Eimeriadse, and 
which corresponds very closely to that of E. stiedce. (See Fig. 
78, page 156). 

Haemoproteus Columbae. Celli and Sanfelice in 1891 observed 
this organism in the red blood cells of doves. It is widely dis- 
tributed as a parasite of wild doves and has been found in Europe 
and in North and South America. The life-history of the parasite 
in the vertebrate host and its mode of transmission by flies of the 
genus Lynchia has been most fully studied by Aragao. 1 In the 
circulating blood of doves the organism is most commonly seen 
as a large crescent-shaped structure occupying most of the interior 
of an erythrocyte and crowding the nucleus of the latter to one 
side or encircling it. The outline of the erythrocyte and the out- 
line of its nucleus are not distorted. The parasites are definitely 
recognizable as females and males, macrogametocytes with granu- 
lar, deeply staining cytoplasm and microgametocytes with a 
paler cytoplasm. When these are ingested by the fly along with 
its blood meal, the gametes arise, fertilization takes place and 
there is produced a creeping ookinete which apparently does not 
penetrate the intestinal wall in the fly or indeed undergo any 
further development there. It gains the blood stream of a new 
host, especially young nestlings, when the fly bites them. It is 
taken up by a leukocyte which comes to rest in the pulmonary 
capillaries of the young bird. Here the parasite produces a very 
large cyst and divides to form very numerous minute sporozoits. 
When the cyst bursts these sporozoits gain the blood stream, 
penetrate erythrocytes and grow to produce the gametocytes 
again. The asexual cycle of schizogony seems to be lacking. 

This organism is important as a typical example of Hcemo- 

1 Archi. /. Protistenkunde, 1908, Bd. XII, S. 154-167. 



SPOROZOA 



413 




FIG. 182. H&moproteus columba. la to 3<z, Development of the female para- 
site in the blood of the dove; ib to 36, development of the male parasite in the blood 
of the dove; 40, 46, 50, 56, 6 to 12, development in the digestive tube of the fly 
(Lynchia); 13 to 20, development of the parasite inside leukocytes in the lung of 
the dove. (After Aragao.) 



414 SPECIFIC MICRO-ORGANISMS 

proteus, as it is the one species of this genus in which the life cycle 
has been most completely studied. 

HaBmoproteus (Halteridium) Danilewskyi. Grassi and 
Feletti 1 first clearly recognized this organism as a definite malarial 
parasite of birds. It is widely distributed and has been found in 
very many different birds, including sparrows, doves, owls, robins, 
blackbirds and crows. The life history is imcompletely known. 
In the blood of the infected bird the organism first appears as a 
small oval or lance-shaped body within the cytoplasm of an ery- 
throcyte. This enlarges, without distorting the outline or dis- 
placing the nucleus of the blood-cell, and stretches along one side 
of the cell. It curves about the nucleus and is enlarged at either 
end when fully developed. Two types, macrogametocytes and 
A B c 








FIG. 183. Hamoproteus danilewskyi. A and B, Fresh triple infection of red 
blood cells. C, D and E, Growing parasites, the last two showing vesicular nuclei. 
jF, Full-grown halteridium with two nuclei. (After Doflein.} 

microgametocytes, are easily recognizable in stained prepara- 
tions. If blood containing these mature halteridia is diluted 
with citrated salt solution and studied under the microscope the 
further changes in the sexual cells may often be followed. Each 
gametocyte bursts the erythrocyte enclosing it and assumes a 
rounded outline. In the microgametocyte the protoplasmic 
granules exhibits violent agitation and several fine filamentous 
processes suddenly shoot out from its periphery and lash about. 
After a few moments these microgametes separate completely 
and rapidly swim away. Meanwhile, the macrogametocyte has 
escaped from its erythrocyte and come to rest in a rounded condi- 
tion. A microgamete approaches and penetrates the macro- 
gamete, and in a few minutes this fertilized sphere elongates into 

1 Cenlralbl.f. Bakt. 1891, Bd. IX, S. 403-409; 429-433; 460-467. 



SPOROZOA 



415 



a curved spindle and actively creeps over the 
slide. It is then known as the ookinete. Fur- 
ther development has not been observed, but there 
can be little doubt that the further stages of 
sporogony and also the unobserved stages of 
schizogony in the bird are somewhat analogous 
to those of H. columbcE or to those of the plas- 
modia of human malaria. Whether the halteridia 
which occur in various species of birds are all of 
one species cannot be decided without further in- 
vestigations. 

Haemoproteus (Leukocytozoon) Ziemanni. 
This organism was doubtless seen by Danilewsky 
in iSgo. 1 Ziemann in 1898 described it as a para- 
site in the blood of hawks. Its known life history 
is very incomplete, and even the nature of the 
blood cell containing it is somewhat doubtful. 
The youngest stage observed in the blood is a 




FIG. 184. 
H &mo prote u s 
(Leukocytozoon) 
ziemaunni. Macro- 
gametocy te and 
microgametocy t e 
(paler.) (From 
Doflein after 
Schaudinn.) 




A B 

FIG. 185. Hamoproteus (Leukocytozoon^ ziemanni. A, Formation of micro- 
gametes from the microgametocy te; B, Fertilization of the macrogamete by one of 
the microgametes swarming about it. (From Doflein after Schaudinn.) 

1 Cenlrabl.f. Bakt., 1891, Bd. IX, S. 401, Fig. i. 



416 



SPECIFIC MICRO-ORGANISMS 



small oval parasite 1 situated at the side of the nucleus of 
the blood cell. The latter appears to be an erythroblast, an 
immature red blood cell in which there is little or no hemoglo- 




D 



FIG. i 86. Hamoproieus (Leukocytozoon) ziemanni in the blood of an owl with 
a pure infection. A , Young parasite in an erythroblast. B, Growing parasite distort- 
ing the nucleus of the host-cell. C and D, Further stages of growth with marked 
distortion of the nucleus and of the outline of the host cell. E, Full-grown macro- 
gametocyte. F, Macrogametocyte and microgametocyte in the same field. G, Forma- 
tion of microgametes from the microgametocyte. (After micro photo graphs of Prof. 
F. G. Novy.) 

bin. As the parasite enlarges, the host cell becomes swollen and 
its nucleus much flattened and distorted. The parasite itself 

1 The description here given is derived in part from unpublished observations 
by Novy and MacNeal. See Proc. Soc. Exp. Biol. and Med., 1904-05, Vol. II, 
pp. 23-28; American Medicine, 1904, Vol. VIII, pp. 932-934. 



SPOROZOA 



417 



grows long and rather slender and is differentiated to form either 
the male or the female gametocyte, readily distinguished by 
appearance in stained preparations. Meanwhile, the host cell 
becomes very much elongated and pointed at the ends. The 
explanation of this peculiar distortion of the cell is unknown, but 
it may be due to the mechanical streaming of the blood acting 
upon the bladder-like cell which has been deprived of elasticity 




FIG 187. Diagram of the developmental cycle of Proteosoma. i, Sporozoit 
entering an erythrocyte; i. 2, 3 and 4, the cycle of schizogony; 5, macrogameto- 
cyte; 5^, microgametocyte; 6, macrogamete; 6a, formation of microgametes; 7, 
fertilization; 8, ookinete; 9, formation of sporoblasts (in mosquito); 10, forma- 
tion of sporozoits; n. sporozoit. (From Doflein after Schaudinn,} 

by the destructive action of the parasite. The further stages in 
the cycle of sporogony are unknown. An asexual multiplica- 
tion probably occurs in some internal organs of the bird. Fan- 
tham has observed schizogony in the spleen of Lagopus scoticus, 
the red-game grouse of Scotland, infected with a similar parasite 
Leukocytozoon lovati. 

Proteosoma (Plasmodium) Praecox. Grassi and Feletti de- 
scribed this malarial parasite of birds and designated it as Hcem- 
27 



4i8 



SPECIFIC MICRO-ORGANISMS 



amceba prcecox. 1 The parasite is very common in the blood of 
small birds, such as sparrows, robins and larks, in all parts of the 
world. The cycle of schizogony is completed in the peripheral 
circulation. The small merozoit or agamete enters an erythro- 
cyte and enlarges, retaining its oval or circular form. The nucleus 




FIG. 188. Proteosoma prcecox in the blood of a field lark (Glauda arvensis). 
A, Young parasite in a blood cell. B, Half-grown parasite which has pushed aside 
the nucleus of the erythrocyte. C. Parasite with clump of pigment and many nuclei 
The nucleus of the erythrocyte has been lost (uncommon). D, Divisicn into eighteen 
merozoits. (From Doflein after Wasielewski.) 

of the host cell is pushed out of position but its form is not ma- 
terially altered. The full-grown parasite segments, producing 
i o to 30 merozoits and leaving behind a small residual body con- 
taining the accumulated pigment, thus completing the asexual 
cycle, which may be repeated many times. After a time some 




FIG. 189. Midgut of a culex mosquito, covered with oocysts of Proteosoma prcecox 
V, Vasa malpighii. (From Doflein after Ross.) 

of the growing parasites become differentiated to form macro- 
gametocytes and microgametocytes, which are kidney-shaped and 
do not divide nor undergo further development in the vertebrate 
host. When the blood is drawn and diluted with citrated salt so- 
lution, or taken in by a mosquito, four to eight microgametes are 

l Centrabl.f. Bakt., 1891, Bd. IX, S. 407. 



SPOROZOA 



419 



formed just as has been described for H. columbce. They are very 
slender actively motile spindles without flagella. Fertilization 
of the macrogamete and the production of an ookinete takes 
place in the usual manner. The latter penetrates the intestinal 
epithelium of the mosquito (Culex sp.) and enlarges to produce 
a spherical cyst filled with an enormous number of thread-like 
sporozoits. These escape into the body ca- 
vity of the mosquito as the cyst bursts, and 
are generally distributed throughout the 
body of the insect. They assemble, prob- 
ably as a result of some chemical stimulus, 
in the salivary glands of the mosquito, 
whence they are injected into the wound as 
the insect bites, and at once invade erythro- 
cytes to begin the cycle of schizogony. 

The discovery of the sexual cycle of pro- 
teosoma in the mosquito and the conclusive 
proof that this form of bird malaria is trans- 
mitted by a mosquito stands to the ever- 
lasting credit of Ronald Ross. His brillant 
discovery made in India in 1898, pointed FlG - X 9- Oocyst of 

. . Proteosoma prcscox, de- 

the way to the Solution of the whole prob- veloped on; the intestine 

of A'edes (Stegomyia) ca- 
lopus, showing numerous 
sporozoits. (From Do- 




flein after Neumann.} 



lem of the transmission of the malarial dis- 
eases and their practical restriction. 

Proteosoma is a favorable parasite for 
class study, as it is readily transmitted from bird to bird (spar- 
rows or canaries) by injection of infected blood, and the para- 
sites often become very numerous in the blood. There seems 
to be no good reason for placing this organism in a separate genus 
from the human malarial parasites. 

Plasmodium Falciparum (Praecox). Laveran in 1880 dis- 
covered the first malarial parasite in the blood of man and cor- 
rectly interpreted his observations. The distinctions between the 
three species was recognized by Golgi, and the life history of the 
parasites and especially their relation to mosquitoes and insects 



420 



SPECIFIC MICRO-ORGANISMS 



in general has been most thoroughly studied by Grassi. 1 PL falcip- 
arum is the parasite of estivo-autumnal or pernicious malaria of 




FIG. 191. Plasmodium falciparium, forms in the asexual cycle (schizogony). 
A, Multiple infection of an erythrocyte, showing signet rings and parasites attached 
to the external surface. B and C, Growing parasites with Mauer's granules in the 
erythrocytes. D, Growing parasite without granulation of the hemoglobin. E, Half- 
grown parasite showing pigment. F, and G, Multiple division (sporulation), rarely 
seen in the peripheral blood. (After Doflein.} 

man. The young organism is i to 1.5/4 in diameter. It pene- 
trates a red blood cell and enlarges. A vacuole appears in the 

center, giving the parasite the 
appearance of a signet ring, the 
setting being represented by 
the nucleus or chromatin gran- 
ule which stains violet red with 
the Romanowsky stains. The 
parasite attains a diameter of 
about 6/j, when it segments to 
produce 7 to 16 merozoits or 
agametes which enter new ery- 




FIG. 192. Section through a capil- 
lary in the brain, showing numerous di- 
viding forms of the non-pigmented type 
of PL falciparum. (Stained prepara- 
tion.) From Doflein after Mannaberg.) 



throcytes and repeat the cycle. 
The larger stages of this cycle of schizogony are rarely seen 
in the peripheral circulation, and the segmentation of the 

1 Grassi: Die Malaria, lite Auflage, Jena, 1901. 



SPOROZOA 



421 



parasite occurs in the capillaries of the internal organs. The 
cycle probably requires 48 hours for its completion. The ery- 
throcyte is not enlarged by the growth of the parasite within 





FIG. 193. Plasmodium falciparum. Stages in the development of the gametocytes 
(crescents). X22oo. (After Doflein.) 

it, but tends rather to become smaller. Maurer has observed an 
irregular granulation of the erythrocytes. Why the cells con- 





i . .... 

F em e c I F em e c I 

FIG. 194. Sections through the stomach wall of Anopheles showing stages in 
the development of PI. falciparum. A, Fixed a few hours after the infective feeding, 
showing ookinetes within the lumen and two in the cuticula of the epithelium. 5, 
Fixed a few days after the infective feeding, showing the partly grown oocyst in the 
stomach wall. F, Fat surrounding the stomach; em, tunica elastico-muscularis; e, 
epithelium; c, cuticula; I, lumen of stomach. 

taining the larger forms should remain in the internal capillaries 
of the body is not definitely known. 

The gametocytes develop by the growth of ordinary merozoits, 



4 22 



SPECIFIC MICRO-ORGANISMS 



marrow and 



which become crescentic early in their development and differen- 
tiated into deeply staining macrogametocytes and pale-staining 
microgametocytes. These are produced especially in the bone 
circulate in the peripheral blood. Further 
development takes place when the blood 
is taken into the stomach of a mosquito 
of the genus Anopheles. Here the mi- 
crogametes, slender actively motile 
threads, are given off by the microgam- 
etocyte and fertilize the macrogam- 
etes, producing ookinetes which ac- 
tively penetrate the epithelium. In the 
wall of the mosquito's stomach each 
ookinete gives rise to a rapidly growing 





FIG. 195. Digestive tract 
of Anopheles, the stomach of 
which is covered with numer- 
ous oocysts of PL falciparum, 
-viewed from the left side, c, 
Cloaca;s, stomach; o, oocysts 
of Plasmodium; mt, malpigh- 
ian tubules; sb, sucking blad- 
ders; sg, salivary gland. 
(From Doflein, modified after 
Ross and Grassi.) 



FIG. 196. Plasmodium falciparum. 
Ripe sporozoits arranged about residual 
bodies within the oocyst, cut in various 
directions (7 to 8 days after infection of 
the mosquito). (From Doflein after 
Grassi.} 



SPOROZOA 



423 



cyst and within this an enormous number of very slender sporozoits 
are developed. The ripe cyst bursts into the body cavity and the- 
sporozoits become generally distributed throughout the body of 
the insect and later assemble in the secreting cells of the salivary 
glands, from which they escape into the human host when the 
mosquito bites. The cycle in Anopheles requires eight days at a 
temperature of 28 to 30 C. At temperatures below iyC. the 
microgametes are not produced. 

Development of the estivo-autumnal parasite through the 



\ 




FIG. 197. Section through salivary gland of Anopheles showing numerous 
sporozoits of Plasmodium falciparum. i, Fat bodies; 2, gland duct; 3, sporozoits of 
Plasmodium; 4, Secretion in the gland cells. (From Doflein after Grassi.} 

stages of schizogony has been obtained by Bass and Johns 1 in the 
test-tube, in a medium consisting of defibrinated blood to which 
0.5 per cent glucose has been added. They were able to keep the 
organisms alive for ten days at a temperature of 40 C., during 
which period the developmental cycle was repeated four or five 
times. Their findings have been confirmed by other investi- 
gators. More recently Joukoff 2 has reported partial development 
in the test-tube, of the cycle of sporogony in the case of PL falci- 

1 Journ. Exp. Med., 1912, Vol. XVI, pp. 567-579. 

2 Compt. Rend. Soc. BioL, 1913, Vol. LXXIV, pp.Ji36-i38. 



424 



SPECIFIC MICRO-ORGANISMS 



parum, and greater success with PL malaria. Details of this work 
have not yet been published. 

Plasmodium Vivax. The parasite of tertian malaria is dis- 
tinctly different from the estivo-autumnal parasite. The young 





FIG. 198. Plasmodium vivax. Stages of growth in the asexual cycle, commonly 
seen in the peripheral blood. Three of the cells show granules in the hemoglobin, 
the stippling of Schiiffner. X22oo. (After Do flein.} 

merozoit is i to 2/4 in diameter and practically not to be distin- 
guished, but very early in its growth it becomes actively ameboid 
and extends irregular and slender processes into the protoplasm 
of its host cell. As the parasite enlarges, the erythrocyte, often 
but not always, becomes swollen, paler, and shows a coarse granu- 
A B 






FIG. 199. Plasmodium vivax. Multinu- 
cleated stage preceding division and the stage 
of. multiple division (sporulation) ; found in the 
blood just before and during a chill. X22oo. 
(After Do flein.} 



FIG. 200. Plasmodium 
vivax. Double infection of 
a red blood cell which is 
considerably enlarged as a 
result; Schuffner's stippling 
slight. X22CO. (After 
Do flein.} 



lation, the stippling of Schueffner. The parasite often attains 
a diameter greater than that of the average blood cell before it 
segments. The segmentation gives rise to from 15 to 30 mero- 
zoits which enter new erythrocytes and begin the cycle anew. 
This complete cycle of schizogony takes place in the peripheral 
circulation and requires almost exactly 48 hours. 



SPOROZOA 425 

The young parasites destined to become gametocytes ex- 
hibit relatively less ameboid movement. Their pigment exists as 
large granules, some of them even rod-shaped. The macrogame- 
tocyte attains a diameter of 15 to 25/1 and usually destroys its 
erythrocyte and escapes from it entirely. The cytoplasm stains 
deeply with methylene blue. The microgametocyte is smaller 
with paler cytoplasm. The development of the parasite in the 
mosquito (Anopheles) is wholly analogous to that of PL falciparum, 
although there are some slight morphological differences ob- 
served. Development ceases at temperatures below 16 C. 

ABC D 




FIG. 201. Plasmodium vivax. Stages in growth of the sexual cells (gametocytes). 
A and B, Young sexual cells distinguished from the agametes by the absence of 
vacuoles and the more regular outline. C, Full-grown macrogametocyte. D, Full- 
grown microgametocyte. X22oo. (After Doflein.} 

Plasmodium Malariae. The young quartan parasite is not 
characteristic, but in its growth it soon stretches as a band across 
the erythrocyte. Later it almost fills the cell and then segments, 
producing 6 to 14, most often 8, merozoits. The infected erythro- 
cyte is not enlarged or distorted nor does it become pale or show 
granulation. The gametocytes, when stained, are not very 
different in appearance from the asexual cells. In the living 
preparation they show much more active protoplasmic move- 
ment. The sexual cycle takes place in Anopheles and agrees very 
well with that of the other two malarial parasites, as far as it has 
been studied. 

Malaria is probably the most important as well as the most 
well-known human disease due to protozoa. It is characterized 



426 



SPECIFIC MICRO-ORGANISMS 



by recurrent paroxysms of fever with afebrile intervals, progress- 
ive anemia and weakness, with the accumulation of a dark brown 
or black pigment in the spleen and liver. This pigment is pro- 
duced by the parasites and set free into the blood when they 





FIG. 202. Plasmodium malaria. Stages of the asexual cycle in the circulating 
blood. Note the absence of granulation from the hemoglobin and the uniform size 
of the red blood cells. X22oo. (After Do flein.) 

segment. The estivo-autumnal malaria caused by PL falci- 
parum shows a somewhat irregular and not very characteristic 
fever curve, but usually there is fever every day (quotidian fe- 
ver). The tertian fever due to infection with PL vivax is char- 




FIG. 203. Plasmodium malaria. Sexual cells in the circulating blood. A , Young 
gametocyte. B, Full-grown macrogametocyte. C, Full-grown microgametocyte. 
X 2 200. (After Do flein.} 

acterized by febrile attacks recurring at intervals of 48 hours and 
bearing a very definite relation to the asexual cycle of the para- 
site. The segmentation of the plasmodium is coincident with 
the chill and the rise in the patient's temperature. In quartan 






SPOROZOA 427 

malaria due to infection with PL malaria, the fever recurs at 
intervals of 72 hours, again at the stage of segmentation in die- 
asexual cycle of the parasite. Obviously an association of two 
or more crops of parasites reaching maturity at different times 
may give rise to a variety of fever curves. 

The diagnosis of malaria is most conclusively established by 
recognizing the parasites in the blood of the patient. One should 
examine a fresh drop of blood, unstained, under the microscope, 
and also thin films of blood stained with some one of the Ro- 
manowsky stains. The parasites may be very scarce in old cases 
and especially in those patients who have been treated. 

The mosquitoes which transmit human malaria were first 
recognized by Ross and have been most thoroughly studied by 
Grassi. The mosquito is capable of causing malaria only after 
it has fed upon a person harboring the parasite in his blood. 1 The 
members of the genus Culex, the most common mosquitoes, do 
not permit the development of the plasmodia within them, but 
this occurs, so far as is known, only in certain species of the genus 
Anopheles, A. maculipennis appears to be the most important 
species. It is easily recognized by the four small black spots on 
each wing due to a relative accumulation of pigmented scales in 
these situations. The members of the genus Anopheles are read- 
ily distinguishable from Culex by the form and arrangement of 
their eggs, the form and position of the larvae and by the general 
form and structure of the adult insect, as well as its posture when 
at rest. 

The restriction and prevention of malaria is founded upon the 
knowledge of its nature and its mode of spread. The measures 
include (i) the destruction of malarial parasites in man by thor- 
ough treatment of the disease with quinine, (2) destruction of 
mosquitoes and mosquito larvae and the drainage, oiling or screen- 
ing of their breeding places, and (3) exclusion of mosquitoes from 
contact with infected persons and also from contact with healthy 
persons, by the use of screens. The thorough application of 

1 Fermi and Lumbau: Centrbl. f. Bakt., 1912, Bd. LXV, pp. 105-112. 



428 



SPECIFIC MICRO-ORGANISMS 

Culex Anopheles 










6 



Q 




FIG. 204. Comparison of Culex and Anopheles. Eggs, larvae (note position), 
position of insects at rest, wings, heads showing antennae and palpi. (From Jordan 
after Kolle and Hetsch.*) 



SPOROZOA 429 

these measures has demonstrated the possibility of effectively 
controlling this disease even in the tropics. 

Plasmodium Kochi. This is a malarial parasite which causes 
a mild fever in monkeys. It is not transmissible to man. Other 
species of malarial parasites have been recognized in these 
animals. 

Babesia 1 Bigemina. Smith and Kilborne discovered this 
organism in the red blood-corpuscles of cattle suffering from 
Texas fever. The parasite is pear-shaped, 2 to 4/x long and 1.5 to 
2/x wide and usually occurs in pairs within the erythrocytes. The 




J I * / * 



,/ 




'* 



FIG. 205. Babesia bigemina. Characteristic forms in the peripheral blood of cattle. 
X2000. (After Doflein.) 

cytoplasm is quite clear without granules or pigment and contains 
one or two chromatin bodies. Minute ameboid forms are also 
found. Multiplication apparently takes place by longitudinal 
division of the pear-shaped forms as well as by multiple division 
of the ameboid forms. Macrogametocytes and microgametocytes 
have been recognized. The transmission of the parasite from 
animal to animal is effected by the cattle tick, Boophilus boms, 
(Rhipicephalus annulatus) as was conclusively demonstrated by 
Smith and Kilborne, the first instance in which such a relation 

1 The generic name Pyrosoma bestowed by Smith and Kilborne in 1893 is incor- 
rect, because this is the name of a genus of marine animals belonging to the Tuni- 
cata. Babesia proposed by Starcovici in 1893 has the next claim to priority. 



43 SPECIFIC MICRO-ORGANISMS 

was proved for any blood-sucking invertebrate. The details of 
the life cycle in the tick are unknown. It is certain however 
that the infection is conveyed to the next generation of ticks 
through the eggs and that these young ticks are capable of in- 
fecting cattle. Renewed investigation of the parasite is much to 
be desired. 

Texas fever is a very important disease of cattle in the southern 
United States and a similar disease occurs in Europe, Africa and 
South America. Young cattle usually survive the disease and 
become immune. Older cattle imported into the endemic area 
contract Texas fever and usually die of it. Immunity may be 
conferred by injecting blood which contains a small number of 
parasites, taken from an animal which has passed the acute stage 
of the disease. Restriction of the Texas-fever area in the United 
States is slowly progressing as a result of systematic eradication 
of the tick. 

Babesia Canis. This organism occurs in the blood of dogs 
suffering from the so-called malignant jaundice, and has been 
carefully studied by modern methods by Nuttall and Graham- 
Smith and later by Breinl 1 and Hindle. In morphology and life 
history it agrees with B. bigemina as far as these have been worked 
out, but B. canis is incapable of infecting cattle. The infection 
is transmitted to dogs by several different species of ticks. 

Gregarina Blattarum. This organism lives as a parasite in 
the intestine of the common cockroach Periplaneta orientalis, and 
is therefore liable to be found in human food, and at times in 
specimens from human cases submitted to microscopic study, 
probably because of accidental presence of cockroaches in the 
containers employed. The vegetative cells are elongated, often 
attached together. The spore cyst results from the union of two 
cells and the subsequent repeated division of the fertilized cell 
to produce an enormous number of spores. These spores are 
discharged from the cyst when it enters a fluid medium. When 
fully developed, each spore contains eight sporozoits. 

1 Ann. Trap. Med. and Parasitol., Vol. II., pp. 233-248. 



SPOROZOA 



431 



Nosema Bombycis. This organism was discovered by Naegeli 
in 1857. It is an example of the Neosporidia and is of peculiar 
interest as the cause of pebrine, the disease of silkworms studied 
by Pasteur in 1866-1870, and largely eradicated by application 
of the methods devised by him as a result of his investigations. 




FIG. 206. Gregarina blattarium. I, Two individuals stuck together. //, Cysts 
with conjugated cells and developing spores. Ill A, Unripe spore with undivided 
contents. IIIB, Ripe spore with eight sporozoits; ek, ectoplasm; en, endoplasm; 
cu, cuticula; pm, protomerit; dm, deuteromerit; n, nucleus; pn, spores; rk, residual 
body; sk, sporozoits. (From Doflein after R. Hertwig.) 

The spore of N. bombycis is 1.5 to 2/z wide by 3/1 long. If treated 
with nitric acid it swells and reaches a length of 6/* and extends 
a slender thread which may be lo/x long. The spore is ingested 
by the silkworm and in its intestine the ameboid parasite es- 
capes and penetrates the epithelium. It may pass to any part 
of the host to undergo its further development. Multiplication 



432 



SPECIFIC MICRO-ORGANISMS 



of the small rounded agamete results in the formation of long 
chains of oval bodies inside a cell of the host. From these the 
spores are again produced. Pebrine is a disease of the greatest 




FIG. 207. Nosema bombycis. Section of intestinal epithelium of silkworm 
showing spores of Nosema and also the peculiar multiplication resembling the 
growth of a mold. (From Doflein after Stempell.} (See also Fig. 81, p. 159.) 

importance to the silkworm industry. It is effectively restricted 
by a careful microscopic examination of all the silkworm eggs 
and the exclusion and destruction of all those in which the para- 
site exists (Pasteur's method). 



CHAPTER XXX. 



CILOPHORA. 

Paramaecium Caudatum. This is the most common infusor- 
ian met with in stagnant water. Its 
length varies from 120 to 325^1. The 
cell is spindle-shaped with a deep oral 
groove which takes a spiral course on 
one side of the body. The surface is 
thickly set with active cilia. Food par- 
ticles are swept into the oral groove, 
enter the cytoplasm at its bottom and 
circulate in the cell within food vac- 
uoles. Near the center of the cell is 
a large macronucleus and near it a 
smaller micronucleus. Multiplication 
takes place by simple longitudinal or 
oblique division. 

Conjugation is isogamic. The simi- 
lar conjugating cells adhere to each 
other, the micronuclei divide twice and 
three of the four nuclei thus produced 
disintegrate, as does also the macro- 
nucleus. The remaining micronucleus 
divides into two and one of these passes 
into the other conjugating cell in ex- 
change for a similar element. The 
newly acquired element unites with the 
element which remained behind to form 
the new nucleus. The new nucleus di- 
vides three times in succession to form 
28 433 




FIG. 208. Paramcecium 
caudatum. K, Macronucleus; 
NK, micronucleus; C, gullet; 
N, food vacuoles; CV, contrac- 
tile vacuoles. (After Doflein.} 



434 



SPECIFIC MICRO-ORGANISMS 



eight nuclei, of which four enlarge to become macronuclei, one re- 
mains as a micronucleus and three disintegrate and disappear. 
The one micronucleus then divides by mitosis and the cell divides 
to form two paramaccia, each containing one micronucleus and two 
macronuclei. The next division gives rise to cells containing 
A the normal number of nuclei, one micronucleus and 

one macron ucleus. 

The paramecia are large saprophytic organisms, 
easily kept under cultivation in the laboratory, and 
they have been very extensively studied. Many 
conceptions founded upon these 
studies are considered to have a 
broad bearing upon the physiology of 
all living cells. For example Jen- 
nings 1 has found that conjugation 
serves two purposes, (i) to provide 
chemical stimulation of cell division 
and (2) to insure variety in the de- 
scendants. The variety in the de- 
scendants is a result of the exchange 
of nuclear material. Calkins 2 has 




FIG. 209. Paramaecia drawn at .. . .. ... 

the same magnification. A. Para- discovered a specialization of func- 

m&cium caudatum. 
cium putrinum. 



B. ParamcE- 



tion in paramecium in respect to con- 
jugation and concludes that in some 
of the descendants of an ex-conjugant the ability to conju- 
gate is in abeyance, thus suggesting a resemblance to the somatic 
cells of a metazoon, while other descendants retain this function 
and are therefore analogous to the germ cells of a metazoon. 

Three other species of paramecium are recognized, namely, 
P. aurelia, P. bursaria and P. putrinum. 

Opalina Ranarum. This is a common parasite in the in- 
testine (cloaca) of the frog. It reaches a large size, 600 to Sooju 
in diameter, is flattened and somewhat irregular in outline. The 



1 Harvey Lectures, 1911-12, pp. 256276. 

2 Proc. Soc. Exp. Biol. and Med., 1913, Vol. X, pp. 65-67. 



CILOPHORA 



435 



ectoplasm is striated and there are very many nuclei in the in- 
terior of the cell. In the springtime, as the frogs enter water to 
spawn, the parasites divide rapidly and give rise to cysts 20 to 40/1 
in diameter. These escape into the slime and are ingested by 
the growing tadpoles. In the cloaca the cells escape from the 
cysts. They are differentiated into male and female gametes and 
fuse to form one cell which grows and multiplies in the developing 
frog. 





FIG. 210. Opalina ranarum, showing the numerous vesicular nuclei. A, Ordinary 
form. B, Dividing form. (From Doflein after Zeller.) 

Balantidium Coli. This parasite of the human intestine was 
described by Malmsten in 1857. Its normal habitat seems to 
be in the large intestine of swine, where it is commonly found in 
large numbers. The cell is a short oval, 50 to yo/z wide and 
70 to IOOM long, rarely larger. Its surface is covered with active 
cilia, and there is a short oral groove at the anterior end. The 
cytoplasm contains drops of fat and food vacuoles, often red 
blood cells and leukocytes of the host. The principal nucleus 
is kidney-shaped and the accessory nucleus lies in contact with 
it. Multiplication takes place by simple transverse fission. 
Conjugation and cyst formation have been observed. 



436 



SPECIFIC MICRO-ORGANISMS 



Bal. coli is sometimes found in man in cases of intestinal dis- 
order with diarrhea. Its possible causal relation to the patho- 
A B c D 




FIG. 211. Balantidium coli. A. Fully developed individual, showing the 
nucleus above at the right and a food particle below. B and C, Division stages. D, 
Conjugation. (From Doflein after Leuckart.} 

logical condition is not conclusively ascertained. In some in- 
stances the cells of Balantidium have been found deeply situ- 
ated in inflamed intestinal wall. Brooks 1 observed Bal. coli in 




FIG. 212. Section through the intestinal wall in a case of enteritis due to 
Balantidium. S, Serosa; M, Muscularis; B, Balantidia. (From Doflein after Solow- 



several cases of dys'entery in Orangoutangs in the New York 
Zoological Park and Brumpt 2 has been able to transfer balan- 

^roc. N. Y. Path. Soc., 1903, Vol. Ill, pp. 28-39. 

2 Compt. Rend. Soc. Biol., 1909, Vol. LXVII, pp_. 103-105. 



CILOPHORA 437 

tidium infection from monkey to swine and back to monkey. 
Still there is perhaps some question as to the identity of the para- 
sites found in man and in hogs. 

Balantidium Minutum. Schaudinn in 1899 observed this 
organism in the human feces. It is smaller than Bal. coli, the 
greatest measurements being 20X30^, and the oral groove ex- 





FIG. 213. Spharophrya pusilla within a paramaecium. At one place there are 
four parasites"and a fifth is escaping. Higher up, one of the parasites is just pene- 
trating the host, and a single parasite is seen near the center of the paramaecium. 
(From Doflein after Butschli.} 

tends more than half way back along the side of the cell. It 
probably occurs rarely in the human small intestine. Other 
species of balantidium have been described. 

Sphaerophrya Pusilla. This organism is of peculiar interest 
because it lives as a parasite within another protozoon, the para- 
mecium. The cell of Sph. pusilla is spherical, 20 to 40/4 in diam- 
eter, and provided with sucking tentacles and cilia when outside 
the body of the host. 



INDEX OF NAMES 



Abbe, 16 
Abbott, 106 
Adil-Bey, 370 
Agramonte, 369, 370 
Anderson, F., 328 
Anderson, J. F., 283, 375 
Appert, 6 
Aragao, 412 
Aristotle, 3 
Arloing, 276 
Armato (d'Armato), 15 
Arnold, 67 
Arrhenius, 204 
Arustamoff, 249 
Ashburn, 374 
Atkinson, 293 
Audouin, 10, 235 
Axenfeld, 296 

Bacon, 15 

Baeslack, 374 

Bail, 205 

Bang, 341 

Banzhaf, 209, 293 

Bass, 423 

Bassis, 10, 235 

Bastian, 4 

Bataillon, 299 

Bateman, 388 

Becker 274 

Behring (von Behring), 12, 208, 281, 

292 

Berg, 238 
Besredka, 225, 330 
Beurmann (de Beurmann), 244 
Beyerinck, 14 
Biggs, 290 
Billroth, ii 
Birt, 374 
Boidin, 274 
Bolduan, 193 
Bellinger, 246, 276 
Bolton, 91, 188, 373 
Bordet, 204, 215, 216, 217, 228 
Breinl, 430 
Brem, 314 
Bretonneau, 289 
Brieger, 203 
Briscoe, 311 



Brooks, 436 
Brown, L., 310 
Bruce, 321, 384, 388 
Brumpt, 436 
Buchner, 125, 212, 227 
Bumm, 250 
Burvill-Holmes, 314 
Buschke, 244 
Busse, 244 

Cagniard-Latour, 6 

Calkins, 406, 434, 

Calmette, 309, 315 

Carle, 278 

Carroll, 368, 369 

Castellani, 388 

Celli. 412 

Chace, 102 

Chagas, 392, 394 

Chambers, 384 

Chauveau, 227 

Chevalier, 15 

Citron, 210, 215, 229, 361 

Clark, H. W., 182, 188 

Clegg, 312 

Cohn, 203 

Cohn, F., 5 

Cole, 259 

Conor, 375 

Conseil, 375 

Cornet, 37 

Cornevin, 276 

Councilman, 93, 254 

Couret, 312 

Craig, 374 

Cumming, 372 

Danilewsky, 391, 415 
d'Armato, 15 
Davaine, 10, 270, 400 
DeBeurmann, 244 
De Schweinitz, 373 
Delafield, 61 
Dobell, 353 
Doerr, 374 

Doflein, 151, 378,40? 
Donn6, 10 
Dorset, 94, 373 
Douglas, 217 



439 



440 

Dubard, 299 
Duclaux, 14 
Ducrey, 298 
Durham, 211 
Dusch, 6 

Button, 353, 354, 388 
Duval, 312 

Eberth, 330 

Ehrenberg, 4, 353 

Enrlich, 204, 205, 207, 208, 211, 214, 218, 

227, 289, 294 
Eichorn, 341 
Einhorn, 102 
Elmassian, 388 
Endo, 354 
Erb, 253 
Ermengem (von Ermengem), 53, 283, 

284 

Escherich, 324, 326, 327 
Esmarch, no, 345 
Evans, 387 
Eyre, 322 

Fabyan, 341 

Fantham, 390, 417 

Fehleisen, 260 

Feletti, 414, 417 

Fermi, 427 

Ferran, 222, 351 

Feser, 276 

Finkler, 352 

Fitzgerald, 338 

Flexner, 219, 255, 337, 374 , 375 

Flugge, 1 88 

Forde, 388 

Forscher, 278 

Fortineau, 274 

Fracas torius, 9, 201 

Frankel, * 257 

Freer, 317 

Friedlander, 257, 327 

Frisch (von Frisch), 327 

Frosch, 368 

Fuller, 86, 179, 181 

Gaffky, 330 
Gage, 182 
Galen, 9 
Galileo, 15 
Gamaleia, 352 
Garbat, 210, 229, 361 
Garre, 266 
Gartner, 328 
Gatewood, 298 
Geppert, 74, 75 
Gemy, 248 



INDEX OF NAMES 



Gengou, 217 
Gessard, 343 
Gibson, 293 
Giemsa, 42 
Gilchrist, 244, 342 
Goldberger, 375 
Golgi, 419 
Goodsir, 267 
Gordon, 16, 322 
Gorsline, 135 
Graham-Smith, 430 
Gram, 44, 59 
Grassi, 414, 417, 420, 427 
Grawitz, 238 
Grondahl, 247 
Gruber, 211 
Grund, 351 

Haffkine, 222, 320, 321, 351 

Hamburger, 309 

Hamerton, 388 

Hansen, 312 

Harbitz, 247 

Harrison, 361 

Hartmann, 403, 405 

Harvey, 3 

Hauser, 343 

Hellriegel, 14 

Henderson, 297 

Henle, 10, 195 

Herbst, 298 

Herodotus, 7 
^ss, 102, 333 

Hesse, 2 
Heidenhain, 54 

Heim, 164 
Hektoen, 217, 243 
Hill, 35 

Hmdle, 356, 430 
Hippocrates, 8 
Hiss, 51, 258, 337 
Hoffman, 374 
Hoffmann, E., 357 
Hogyes, 222 
Holmes, 263 
Homer, 8 
Hornor, 297 
Hubner, 254 
Hutchings, 188 

Irons, 253 
Israel, 246, 247 

Jaeger, 254 

Jeffer, 184, 185 

Jenner, 42 

Jenner, Edward, 12, 223, 376 






INDEX OF NAMES 



441 



Jennings, 434 
Jochmann, 255 
Johns, 423 
Johnson, 179 
Jordan, 180 
Joukoff, 423 

Kanthack, 248 

Keller man, 182 

Kerr, J., 130, 277 

Kilborne, 429 

King, 374 

Kinghorn, 391 

Kircher, 9 

Kirkbride, 39 

Kitasato, 12, 208, 278, 279, 281, 317 

Kitt, 276 

Klebs, n, 260, 264, 285 

Klegg, 248, 400 

Klimenko, 297 

Knapp, 354 

Kneass, 269 

Koch, i, n, 12, 66, 93, 112, 119, 195, 

257, 270, 275, 296, 299, 304, 

305, 330, 345, 347 

Kolle, 221, 222, 223, 302, 321, 351, 368 
Kraus, 209 
Krauss, 39 
Krumwiede, 311, 351 
Kruse, 337 

Laennec, 306 

Lafar, 185 

Lamar, 258 

Landouzy, 306 

Langenbeck (Von Langenbeck), 238 

Latzer, 130 

Laveran, 12, 394, 398, 419 

Lazear, 369 

Leeuwenhoek, 3, 5, 9, 15 

Leishman, 42, 335, 356 

Levaditi, 357, 358 

Lewis, G. W., 381 

Lewis, Paul A., 374 

Lewis, T. R., 381 

Liborius, 124 

Lichtheim, 231 

Liebig, 7 

Lindermann, 410 

Lingelsheim (Von Lingelsheim), 262, 

265, 280 
Lip man, 176 
Lister, n 
Loffler, 2, 52, 285, 287, 308, 329, 339, 

368 

Longley, 181 
Losch, 12, 402 



Lb'wenstein, 305 
Luer, 96 
Luetscher, 327 
Lumbau, 427 

McBryde, 373 

McClintock, 76 

McCrae, 135 

McFadyean, 198 

Mclntosh, 357' 

MacNeal, 43, 102, 117, 193, 311, 378; 

383, 384, 386, 391, 416 
McNeill, 253 
Mackie, 388 
Madsen, 204 
Mafucci, 299 
Major, 260 
Mallory, 93, 254, 297 
Malmsten, 435 
Marchoux, 356 
Marshall, 3 28 
Marzoli, 15 
Massee, 235 
Maurer, 421 
Mayer, 56 
Meltzer, 258 
Mesnil, 394, 398 

Metchnikoff, 212, 218, 225, 227, 330 
Migula ; 5, 142, 144, 146 
Milne, 353 
Mique], 75 
Mohler, 341 
MSller, 314 
Montague, 222 
Moore, 179. 182 
Morax, 296 
Moritz, 316 
Moro, 309 
Moses, 7 
Muhlens, 357 
Miiller, 4 
Musgrave, 248, 400 

Nageli, 431 

Needham, 5, 6 

Negri, 370 

Neisser, 215, 250, 265, 312 

Neufeld, 217 

Nicolaier, 278 

Nicolle, 370, 375, 397 

Nocard, 13, 329, 368 

Nocht, 41 

Noguchi, 13, 99, 256, 354, 355, 357, 358, 

359, 366, 367, 375 
Novy, 13, 37, 98, 118, 126, 193, 203, 

326, 328, 354, 378, 383, 384, 

391, 416 



442 



INDEX OF NAMES 



Nuttall, 13, 212, 227, 276, 354, 355,356, 
43 

Obermeier, n, 13, 353 
Ogston, 260, 264 
Orth, 6 1 

Fakes, 185 
Park, 290, 293,311 
Passini, 145 

Pasteur, i, 4, 6, 7, 32, 125, 223, 227, 257, 
264, 273, 275, 316, 373, 431, 

43 2 

Perkins, C. F., 243 
Perkins, W. A., 384 
Petri, 1 08 
Petruschy, 246 
Pettenkofer, 8 

Pfeiffer, 98, 213, 227, 297, 348 
Pirquet (Von Pirquet), 219, 309 
Plaut, 240 
Plenciz, 9 
Pollender, 10, 270 
Poor, 370 
Pratt, 351 
Prior, 352 
Prowazek, 378 
Prudden, 188 

Quincke, 255 

Rabinowitsch, 314 
Ramond, 244 
Rattone, 278 
Rayer, 10, 270 
Redi, 3 

Reed, 368, 369 
Reichert, 117 
Reimarus, 9 
Remak, 239 
Remlinger, 370 
Rettger, 197 
Ricketts, 244, 375, 376 
Rideal, 77 

Rindfleisch, n, 260, 264 
Rivolta, 299 
Robin, 10, 238 
Rogers, 121 
Romano wsky, 41 
Rosenau, 283 
Rosenbach, 260, 264 
Rosenow, 367 
Ross, 353, 419, 427 
Rouget, 387 
Rous, 377 

Roux, 119, 288, 293, 368 
Ruediger, 243 



Rufus of Ephesus, 319 
RusseU, 334, 335 

Sacharoff, 356 

Sailer, 269 

Salimbini, 356 

Salmon, 13 

Sanarelli, 329 

Sanfelice, 412 

Schafer, 4 

Schaudinn, 357, 358, 392, 403, 405, 408, 

412, 437 
Scheele, 6 
Schenck, 242 
Schereschewsky, 357 
Schonlein, 239 
Schottmiiller, 260 
Schroder, 6 
Schiiffner, 424 
Schulze, F., 4, 6 
Schulze, F. E., 400 
Schiitz, 339 
Schwann, 6 
Schwartz, 253 

Schweinitz (De Schweinitz), 373 
Sclavo, 274 
Sedgwick, 177, 188 
Seidelin, 370 
Semmelweiss, 263 
Sergent, Edm., 396 
Sergent, Et., 396 
Shiga, 336 
Sholly, 292 
Siedentopf, 16 
Silberschmidt, 269 
Silbey, 299 
Simon, 364 
Sinton, 389 
Slater, 77 
Smith, Theobald, 13, 99, 279, 299, 341, 

429 

Sobernheim, 225, 274 
Spall anzani, 4, 5, 6 
Starcovici, 429 
Steinhardt, 370 
Sternberg, 257 
Stevens, 390 
Stewart, 37 
Stribolt, 341 
Strickland, 382 
Strong, 327 
Swellengrebel, 382 

Takaki, 281 
Taylor, 361 
Terre, 299 
Thorn, 139 






INDEX OF NAMES 



443 



Thomas, 276 
Thomason, 389 
Tissier, 342 
Todd, 353, 354, 3^8 
Torrey, 378 
Toussaint, 316 
Trudeau, 309 
Tucker, 177 
Tunnicliff, 358, 367 
Tyndall, 6 

Uhlenhuth, 308 

Vallery-Radot, 316, 373 

Van der Brock, 7 

Van Dusch, 6 

Van Ermengen, 53, 283, 284 

Van Leeuwenhoek, 3, 5, 9, 15 

Vaughan, 203, 224, 295, 326, 328 

Veillon, 112, 125, 342 

Vianna, 392 

Viereck, 404 

Vignaud, 274 

Villemin, 306 

Vincent, 248, 367 

Von Behring, 12, 208, 227, 281, 292 

Von Frisch, 327 

Von Langenbeck, 238 

Von Lingelsheim, 262, 265, 280 

Von Pirquet, 219, 226, 309 

Wassermann, 221, 222, 281, 302, 361, 

368 
Washburn, 87 



Webb, 223 

Wechsberg, 215 

Weeks, 296 

Weichselbaum, 253 

Weigert, 59, 207 

Welch, 51, 267, 276 

Wellman, 312 

Wenyon, 395 

Wertheim, 250 

Wheeler, 188 

Whipple, 181 

Whitmore, 400, 403, 405, 407 

Wilder, 375, 3 76 

Wilfarth, 14 

Williams, A. W., 293, 402, 406 

Williams, H. U., 1 80, 381 

Williamson, 50, 308 

Winslow, 1 88 

Wolf, 247 

Wolff-Eisner, 309 

Wolffhiigel, 185 

Wright, A. E., 217, 228, 266, 335 

Wright, J. H., 125, 248, 254, 396 

Wright, Jonathan, 327 

Yersin, 288, 317, 321 
Yorke, 391 

Zeiss, 16 
Zettnow, 148 
Ziemann, 392, 415 
Zinsser, 332 
Zsigmondi, 16 



INDEX OF SUBJECTS. 



(An asterisk (*) designates pages showing illustrations.) 



Abbe condenser, 16, 24* 

Aberration, chromatic, 15 

Abiogenesis, 3, 4 

Abortion, contagious, 341 

Abrin, 171 

Abscess, 261, 266 

Absorption of oxygen for anaerobic 

culture, 125 

Accidental infection, 104 
Acetic acid, 169 
Achorion schonleinii, 10, 239, 241* 

cultures of, 240 
Achromatic objectives, 16 
Acid, carbolic, 76 

production of, 169 
Acid-proof bacilli, 46, 314 

method of staining, 46 
Acids, germicidal action of, 71 
Acne, 342 
Acquired immunity, 220, 222 

active, 222 

passive, 224 
Actinomyces, 246 

bovis, 246 
Actino mycosis, 246 
Active immunity, 222 

duration of, 222 

methods of inducing, 222 
Adaptation to environment, 167, 171 

to parasitism, 202 
Aedes (Stegomyia) calopus, 369* 
Aerobes, 166 

sporogenic, 268 
Aerobic bacteria, 166 
Aerobioscope, 177* 
Agar, 89 

ascitic-fluid, 99 

blood-streaked, 98 

glucose, 91 

glycerin, 91 

sugar, 91 

Age factor in susceptibility, 197 
Agglomerin, 265 
Agglutination, 211, 338 

technic of, 211 
Agglutinins, 211 
Aggressins, 205 



Agriculture, 14 

relation of microbes to, 14 
Air, 174, 176 

disease-bearing insects of, 178 

micro-organisms of, 176 
Albumen fixative, 56 
Alcohol, as germicide, 78 

production of, 169 
Alcoholic fermentation, 169 
Aleppo boil, 396 
Alexin, 212, 215 
Alimentary canal, bacteria of, 202 

infection, 134 

Alkalies, germicidal action of, 73 
Allergy, 219 
Alopecia areata, 241 
Alum in water filtration, 181 
Amboceptor, 214 
Ameba (Amoeba), 155 

cultures of saprophytic, 402 

in tropical dysentery, 12 
American filtration, 181 
Ammonia, 170 
Amoeba, 155 

cultures of saprophytic, 402 

in tropical dysentery, 12 

proteus, 401* 

Amphitrichous bacteria, 148 
Anaerobes, 166 

sporogenic, 275 
Anaerobic bacteria, 166 

cultivation of, 124 

cultures, 124 

Buchner's method, 125, 126* 

combined hydrogen and pyrogallate 
method, 128*, 129 

deep stab, 124 

termentation tube, 125 

in hydrogen, 126, 127* 

Novy's method, 126, 127* 

reducing substances in, 130 

removal of oxygen, 125 

under paraffin, 130 

Veillon tube, 124 
Anaphylaxis, 226 
Aniline dyes, 40 

disinfectant action of, 78 



445 



446 



INDEX OF SUBJECTS 



Aniline-water staining solutions, 40 
Animal experimentation, 131 

value of, 131 
Animals, care of, 131 

experimentation with, 131 

holding of, 132 

inoculation, 133 

observation of infected, 136 
Anopheles mosquitoes, 427, 428* 
Anthrax, 12, 270, 272 

bacillus, 270 

colony, 271 

immunity, 273 

infection, 273 

intestinal, 273 

pulmonary, 273 

pustule, 273 

serum, 274 

vaccine, 273 
Antiaggressins, 218 
Antibacterial serum, 212, 213 
Antibodies, 206, 208, 218 

distribution of, 218 

source of, 218 

Anticomplementary reaction, 363 
Antiformin method, 50 
Antigen, 217, 228 
Antimeningococcus serum, 255 
Antipneumonococcus serum, 260 
Antisepsis, 62, 79 
Antiseptic surgery, n 
Antiseptics, 79 

testing of, 80 

Antistreptococcus serum, 264 
Antitoxic serum, 208 
Antitoxin, diphtheria, 12, 208, 292, 293 

concentration of, 293 

curative value of, 295 

preparation of, 293 

prophylactic value, 295 

standardization of, 294 
Antitoxin, tetanus, 12, 208, 281 
Antityphoid vaccination, 335 
Apochromatic objectives, 16 
Arnold steam sterilizer, 67*, 68* 
Arthritis, streptococcus, 264 
Artificial culture, 163 
Ascitic fluid, sterile, 97 

agar, 99 

with sterile tissue, 99 
Ascomycetes, 137 
Asiatic cholera, 345, 349 

cairiers of, 351 

diagnosis, 350 

epidemics of, 348 

history of, 348 

prophylaxis, 351 



Asiatic quarantine in, 351 

spirillum of, 345 

transmission of, 349 

vaccine for, 351 
Aspergillosis, 233 
Aspergillus fumigatus, 233 

glaucus, 138*, 233* 
Atmosphere, bacteria of, 176 

hydrogen, for anaerobes, 126 
Atrichous bacteria, 148 
Attenuation, 202 
Autoclave, 68, 69* 

sterilization, 68 
Autopsies, 103 
Avenues of infection, 197 
Avian tuberculosis, 299, 311 
Avoidance of contamination, 104 
Azotobacter, 175 
Azure, methylene, 42, 43 

Babesia, 155, 158*, 429* 

bigemina, 429* 
immunity to, 430 
transmission of, 430 

canis, 430 

muris, 158* 
Bacilli, 145* 

acid-proof, 46, 299 

capsulated, 51, 148, 326, 338 

chromogenic, 343 

colon- typhoid, 324 

pigment-producing, 343 
Bacillus, 5, 142, 144 

abortus, 341 

acne, 342 

aerogenes, 326 

aerogenes capsulatus (B. welchii), 
276 

alkaligenes, 329 

anthracis, 10, 12, 270* 

anthracis symptomatici (feseri), 276 

ayisepticus, 316 

bifidus, 342 

Bordet-Gengou (B. pertussis), 296 

botulinus, 283 

bulgaricus, 342 

butter, 314 

capsulatus, 328 

chancri, 298 

chauvei (B. feseri), 276 

cholerse-suis (B. suipestifer), 329 
Bacillus coli, 324* 

cultures, 325 

detection of, 326 

in water supplies, 186 

pathogenic properties, 326 

poisons of, 326 



INDEX OF SUBJECTS 



447 



Bacillus, comma (Sp. cholerae), 345 

cyanogenus, 343 
Bacillus, diphtherias, 285 

animal inoculation, 285, 288, 291 

bacilli resembling, 289, 292, 295, 
296 

cultural characters, 287 

granular types, 285 

in human body, 290 

Loffler's serum for culture, 287, 
290*, 291 

mode of infection, 292 

morphology, 285 

resistance, 288 

solid types, 285 

staining of, 286, 291 

toxin of, 288 

types, 286*, 287* 

virulence, 291 
Bacillus, Ducrey's, 298 

dysenteric, 336 

edematis, 275 

enteritidis, 328 

fecalis alkaligenes, 329 

feseri, 276 

fluorescens, 343 

fusiformis (Spirochaeta vincenti), 

367 

Gartner's (B. enteritidis), 328 
gas (B. welchii), 276 
grass (B. molleri), 314 
hay (B. subtilis), 269* 
hoffmanni, 296 
icteroides, 329 
influenzas, 297 

Klebs-LOfikr (B. diphtherias), 285 
Koch-Weeks, 296, 297* 
lactici-acidi, 190 

lactis aerogenes (B. aerogenes), 326 
leprae, 3 1 2 
mallei, 339* 
melitensis, 321 
mesentericus, 268 
molleri, 314 
Morax-Axenfeld, 296* 
mucosus, 328 
murisepticus, 317 
mycoides, 268 
ozenae, 328 
paracolon, 330 
paradysentery, 337 
paratyphoid, 330 
perfringens (B. welchii), 276 
pertussis, 296 
Bacillus pestis, 317* 
cultures, 318 
immunity, 320 



Bacillus pestis in animals, 319 
morphology, 317* 
toxins, 318 

Bacillus plurisepticus, 316 
pneumoniae, 327 
potato (B. vulgatus), 268 
prodigiosus, 344 
proteus, 343 
pseudo-diphtheria (B. hoffmanni), 

296 

psittacosis, 329 
pyocyaneus, 343 
radicicola, 14 
rhinoscleromatis, 327 
rhusiopathiae suis, 317 
salmonii, 329 

Shiga's (B. dysenteriae), 336 
subtilis, 269* 
suipestifer, 329 
tetani, 278 
Bacillus tuberculosis, 299 

amphibian, 312 

avian, 311 

bovine, 310 

branching of, 302* 

chemical composition of, 302 

cultures of, 301 

fish type, 312 

human type, 300* 

morphology, 300 

poisons of, 303 

resistance of, 304 

varieties of, 299 
Bacillus typhi murium, 329 
Bacillus typhosus, 330 
agglutination of, 334 
distribution of, 330, 332, 333 
flies as carriers of, 335 
human carriers of, 333, 335 
in blood, 333 
in feces, 333, 334 
in food, 335 
in milk, 335 
in soil, 335 
in sputum, 333 
in urine, 333 
in water, 187, 335 
isolation of, 354 

pathogenic properties of, 330, 332 
poisons of, 332 
resistance of, 332 
vaccines, 335 
Bacillus violaceus, 343 
vulgaris, 343 
vulgatus, 268 
welchii, 276 
xerosis, 295 



448 



INDEX OF SUBJECTS 



Bacteremia, 200 

streptococcus, 264 
Bacteria, 141 

acid-proof, 46, 299 

adaptability, 167 

aerobic, 166 

anaerobic, 166, 275 

classification, 137, 141, 160 

colonies, 109*, 172 

cylindrical, 144 

dimensions, 141 

discovery, 3 

distribution, 174 

fluctuation, 167 

food, 189 

in air, 174, 176 

in agriculture, 175 

in food, 189 

in ice, 178 

in milk, 189 

in soil, 175 

in water, 178 

soil, 175 

spherical, 142 

spiral, 146 

structure of, 147 

variation, 167 

with spores, 149* 
Bacteriaceae, 144, 268 
Bacterial poisons, 171, 203 

vaccines, 12, 223 
Bactericidal substances, 212, 227 
Bacteriology, i 

biological relations, 3 

history, i 

hygienic, 7 

nomenclature of, 160 

scope, 2 

Bacteriolysins, 213 
Bacteriolysis, 213 
Bacterium, 5, 144 
Balantidium coli, 435 

parasitic relations, 435, 436* 

pathogenesis, 436 
Balantidium minutum, 437 
Basic dyes, 40 
Basidiomycetes, 139 
Basophile granules, 60 
Bed-bugs (Cimex), 395 
Beef-tea (broth), 84 
Berkefeld filter, 63 
Bichloride of mercury, 74 
Biological relationships of bacteriology, 

3 

Birds, malaria of, 412, 414, 415, 417 
trypanosomes of, 390* 
tuberculosis of, 299, 311 



Black death (plague), 317 
Black-leg, 276 

Blastomyces dermatidis, 245 
Blastomycetes, 140*, 244 
Blastomycetic dermatitis, 245 
Blastomycosis, 244 
Bleaching powder, 74 
Blepharoplast, 378 
Blood-agar, Novy's, 98 

Pfeiffer's, 98 
Blood, 92 

bacteria in, 200 

citrated, 96 

culture, 101 

defibrinated, 96 

films for microscopic examination, 

54 

protozoa in, 200 

sterile, collection of, 95 
Blood serum, 92 

as culture medium, 92 
Loeffler's, 84 
Blue milk, 343 

PUS, 343 

Bodo lacertae, 398* 
Boil, Delhi, 396 
Boils, 266 

Boophilus bovis, 158, 429 
Bordet-Gengou bacillus (B. pertussis), 

296 

Boric acid, 79 
Botrytis bassiana, 10, 235 
Botulin, 284 
Botulism, 284 

antitoxin for, 284 
Bouillon (broth), 84 
Bovine pleuro-pneumonia, 368 

tuberculosis, 310 
Branching bacilli, 302* 
Bread-paste, 94 
Bromine, 74 
Broth, nutrient, 84 

containing sterile tissue, 99 

sugar, 90 

sugar-free, 90 
Brownian movement, 34 
Bubonic plague, 317, 319 

diagnosis, 318 

fleas as carriers of, 319 

history of, 319 

immunity to, 320 

prophylaxis of, 320, 321 

rodents as reservoirs of, 314, 320 

serum, 321 

vaccines, 320 

Buchner method for anaerobic culture 
125 



INDEX OF SUBJECTS 



449 



Burner, Bunsen, 105 

Koch's automatic safety, 119*, 120 
Butter bacillus, 314 
Butyric acid test, 256 

Calcium oxide (lime), 73 

Calmette's test (tuberculin), 309 

Capsules, 147, 148* 

Carbol-fuchsin, 41 

Carbolic acid, 76 

Carbuncles, 266 

Carmine, 61 

Carriers of infection, 201 

Caseation, 306 

Cattle plague, 370 

tick, 158, 429 
Cedar-wood oil, 30 
Cell, chemical constitution of, 207 
Cell-membrane of bacteria, 147 
Celloidin, 55 

Cell-receptors, 209*, 210*, 214* 
Cerebro-spinal fluid, 255 

collection of, 101, 255 

examination of, 256 

in meningitis, 255 

test for globulins in, 256 
Chancroid, 258 
Charbon (Anthrax), 270, 272 
Cheese, 191, 237 
Chemical agents as germicides, 71 

disinfection, 72, 77 

effects, 168 

products, 1 68 
Chicken cholera, 316 

sarcoma, 377 
Chlorine, 73 

Chloroform, preservative action of, 79 
Cholera, Asiatic, 345 

carriers, 351 

diagnosis of, 350 

prophylaxis, 351 

transmission of, 349 
Cholera, fowl, 316 
Cholera, hog, 373 
Chromatin, 148 
Chromogenic bacteria, 343 
Ciliates, 159, 433* 
Ciliophora, 151, 159, 433* 
Cladothrix, 246, 249 
Classification, 4, 137 

of molds, 137 

of protozoa, 150 

of yeasts, 140 

outline of micro-organisms, 160 
Claviceps purpurea, 234 
Cleaning fluid, 37 

Clearing microscopic preparations, 28 
29 



Clostridium, 146* 

Coccaceae, 142*, 250 

Cocci, 142*, 250 

Cocci dioidal granuloma, 245 

Coccidiosis, 411 

Coccidium (Eimeria), 155, 156* 

cuniculi (Eimeria steidae), 410 
Coccus, 142 

Cold, effect on bacteria, 64 
Collection of material, lo'o 

of sterile ascitic fluid, 97 

of sterile blood, 95 

of sterile tissue, 98 
Collodion capsules, 134, 135* 

embedding, 55 
Colon bacillus, 324* 

cultures of, 325 

detection of, 326 

in water-supplies, 186 

pathogenic properties of, 326 

poisons of, 326 

Colonies of bacteria, ic6, 109*, 113 
Comma bacillus, 345 
Commensal. 194 
Complement, 215, 216 

deviation of, 215 

detection of, 217 

fixation of, 216, 361 
Complement-fixation test, 361 

antigen, 363 

blood cells for, 561 

complement for, 361 

hemolytic amboceptor for, 362 

patient's serum for, 363 

signification of, 366 

technic of, 364 
Condenser, sub-stage (Abbe). 24* 

dark field, 25* 
Conjunctivitis, 296, 297 
Conorhinus megistus, 395* 

as vector of cereotrypanosis, 394 
Consumption (tuberculosis), 305 
Contagion, 200 

early ideas of, 7, 8, 9 
Contagious abortion, 341 

disease, 200 

Contamination, avoidance of, 104 
Coreotrypanosis, 392 

transmission of, 394 
Cornet forceps, 38* 
Corrosive sublimate, 74 
Cotton plugs, 84 
Cover-glass forceps, 37, 38* 

preparations, 36 
Cover-glasses, 36 

cleaning of, 36 
Cow-pox, 12, 376 



45 



INDEX OF SUBJECTS 



Creolin, 77 

Cresol, 77 

Croupous pneumonia, 259 

Culex mosquitoes, 427, 428* 

Cultivation, 104 

of anaerobes, 124 

of bacteria, 104 

of protozoa, 13 

of spirochetes, 13 
Culture media, 83 

agar, 89 

bread-paste, 94 

broth, 84 

blood-agar, 98 

blood-serum, 92 

choice of, 115 

containing uncooked protein, 95 

dextrose, 90 

dextrose-free, 90 

Dorset's egg, 94 

Dunham's solution, 92 

filling into tubes, 89* 

gelatin, 88 

lactose, 90 

litmus, 90 

Loffler's blood serum, 94 

method of inoculating, 107* 

milk, 92 

nitrate-broth, 92 

peptone solution, 92 

potato, 91 

preparation of, 84 

special, 91 
Cultures, plate, 12, 106 

pure, 113 

roll-tube, no*, in* 

sealing of, 120 

smear, 113, 114* 

stab, 114*, 124 

stock, 114 

streak, 113, 114* 

tube, 112, 114*, 124 
Cutaneous tuberculin test, 309 
Cutting sections, 56 
Cyclospora caryolytica, 408* 

life cycle of, 408 

parasitic relations, 408 

pathogenesis, 410 
Cystitis, 327 
Cytase, 215 
Cytolysins, 213 
Cytolysis, 213 

Dark-field microscopy, 16, 35 
Decomposition, 5, 6, 7, 170 
Deep stab-cultures, 124 
Defects of lenses, 20 



Defensive mechanisms of microbes, 204 

Delafield's hematoxylin, 61 

Delhi boil, 396 

Deneke's spirillum, 352 

Dengue fever, 374 

Denitrification, 175 

Descriptive chart, Soc. Am. Bact., 174 

Desiccation, 63, 164 

Development of bacteriology, i 

Deviation of complement, 215 

Dextrose, fermentation of, 169 

media, 90 

Dextrose- free media, 90 
Diffraction, 20 

Dilution cultures, 105, 106, no, 112 
Dimensions of bacteria, 141 
Diphtheria, 289 

antitoxin, 273, 293 

bacillus (B. diphtheriae), 285 

carriers, 292 

diagnosis, 290 

immunity to, 292 

mixed infection, 290 

prophylaxis of, 295 

streptococcus in, 290 

toxin, 288 

transmission of, 292 
Diplobacillus, 145 

of Morax-Axenfeld, 296* 
Diplococcus, 142* 

catarrhalis, 257 

gonorrheae, 250 

meningitidis, 253 

pneumoniae, 257 
Disease, causation of, 195 

contagious, 8 

infectious, 196 

miasmatic, 8 

phenomena of, 195 

theories of, 9, 206 
Disinfectants, 71 

testing of, 80 
Disinfection, 62 

of feces, 73 

of rooms, 72, 77 
Distribution of micro-organisms, 174 

in animal body, 175 

in atmosphere, 174, 176 

in foods, 189, 193 

in milk, 189 

in plant tissues, 175 

in soil, 175 

in water, 174, 178 
Diversion of completement, 215 
Doflein's classification of protozoa, 150 
Dorset's egg-medium, 94 
Dourine, 387 






INDEX OF SUBJECTS 



451 



Drinking water, 178 

Drying (desiccation), 63 

Ducrey's bacillus, 298 

Dum-dum fever (kala-azar), 394, 396 

Dunham's peptone solution, 92 

Dust, 176 

Dyes, 40 

acid, 40 

aniline, 40, 78 

basic, 40 
Dysentery, ameba of, 404 

amebic, 406 

entamebic, 406 

bacillary, 336 

bacillus, 336 

diagnosis, 336 

serum therapy, 337 

Eberth's bacillus, 330 

Edema, malignant, 276 

Egg, fresh, as culture medium, 94 

Dorset's egg medium, 94 
Ehrlich's conception of chemical struc- 
ture of cell, 207 

side chain theory, 208, 227 

theory of immunity, 227 
Eimeria, 155, 156* 

schubergi, 156*, 412 
Eimeria steidae, 410* 

life cycle in rabbit, 410 

pathogenesis, 411 

Electricity, germicidal action of, 71 
Electric thermostat, 122 
Emmerich's bacillus (B. coli), 324* 
Emphysematous gangrene, 277 
Encapsulation, 207 
Endocarditis, 260 

lenta, 260 

staphylococcus, 266 

subacute, 260 
Endo's medium, 354 
Endospores, 146, 149, 155 
Endotoxins, 204 
Entamceba, 154*, 155 
Entamceba coli, 154*, 402* 

morphology, 403 

occurrence, 402 

parasitic relation of, 403 
Entamceba histolytica, 405 
Entamceba tetragena, 404* 

cyst of, 405* 

morphology, 404* 

questionable cultures of, 406 

relation to dysentery, 405, 406 

transmission of, 405 
Entamcebic dysentery, 406 
Enteric fever (typhoid fever), 333 



Environment, 164 

mutual relation of microbe and, 171 
Enzymes, 169 

diastatic, 170 

glycolytic, ^170 

proteolytic, 170 

steatolytic, 171 
Eosin, 40 

Epibemic meningitis, 253 
Epithelioid cells, 306 
Erysipelas, 263 

immunity to, 264 

streptococci in, 260 
Escherich's bacillus (B. coli), 324 
Esmarch roll-tubes, no*, in* 
Estivo-autumnal malaria, 419, 426 
Extracellular toxins, 203 
Exudates, 102 
Eye-piece, 23, 29, 30 

narrowing of the beam by, 23 

Facultative serobe, 166 

anaerobe, 166 

Fatigue predisposing to infection, 197 
Fats, fermentation of, 171 
Favus, 10, 239*, 240* 
Feces, collection for examination, 102* 

disinfection of, 73 

typhoid bacilli in, 354 
Fermentation, 5, 6, 7, 169 

acetic, 169 

alcoholic, 169 

of milk, 191 

tube, 125, 186 
Film preparations, 36 

of blood, 54 
Filters, Berkefeld, 63 

Pasteur-Chamberland, 63 

sand, 1 80 

Filterable micro-organisms, 13, 150, 368 
Filterable, virus, 13, 150, 368 

of bovine pleuro-pneumonia, 368 

of cattle plague, 370 

of chicken sarcoma, 377 

of dengue fever, 374 

of foot-and-mouth disease, 368 

of hog cholera, 373 

of measles, 375 

of phlebotomus fever, 374 

of pleuro-pneumonia, 368 

of poliomyelitis, 374 

of rabies, 3 70 

of rinderpest, 370 

of small-pox, 376 

of typhus fever, 375 

of yellow fever, 368 
Filtration of bacterial cultures, 63 



452 



INDEX OF SUBJECTS 



Filtration of media, 85, 88, 90, 97 

of water, 180 

sterilization by, 63, 97 
Finkler and Prior, spirillum of, 352 
Fishing colonies, 113 
Fixation of complement, 216 

in glanders, 341 

in gonorrhea, 253 

in syphilis, 361 
Fixation of protozoa, 54 
Fixative, albumen, 56 
Flagella, bacterial, 148* 

staining of, 52 

Flagella of protozoa, 151, 153* 
Flagellates, 151 
Fleas, 319, 382, 396 
Flies, 154, 335, 387, 388 
Fluctuating characters, 4 
Focusing, 30 
Fomites, 200 
Food, bacteria in, 189, 193 

of micro-organisms, 164 

poisoning, 195, 284, 328 

preservation, 6, 62, 79, 193 
Foot-and-mouth disease, 368 

filterable virus of, 368 
Forceps, cover-glass, 37, 38* 
Formaldehyde, 77 
Formalin, 77 
Fowl-cholera, 316 

tuberculosis, 311 
Fractional sterilization, 70 
Fraenkel's diplococcus, 257 

cultures of, 258 
Freezing, 64, 166 
Friedlander's bacillus, 257, 327 
Fuchsin, 40 

carbol-, 41 
Furuncle, 266 
Fusiform bacillus, 367 

Gametes, 156* 
Gametocytes, 155, 156* 
Gangrene, emphysematous, 277 
Gartner's bacillus (B. enteritidis), 328 
Gas bacillus (B. welchii), 276 
Gas-burner, Koch's safety, 119*, 120 
Gas-formation, 169 
Gas-regulator, 116, 117*, 118*, 119* 
Gastric juice, gerroicidal action of ; 71 
Gelatin cultures, 106, 113, 114* 

nutrient, 88 

plates, 1 06 
Gentian violet, 40 
Germ carriers, 201 
Germicidal serum, 212 
Germicides, 71, 78 



Giemsa's stain, 42 
Glanders, 340 

bacillus, 339 

diagnosis, 340 
Glassware, 83 
Glossina morsitans, 385*, 386 

palpalis, 388, 389* 
Glucose media, 90 
Glycerin media, 90 
Gonococcus, 250 

cultures of, 251 

in pus, 251* 
Gonorrhea, 252 

diagnosis of, 253 

prophylaxis of, 253 
Gonorrhea! arthritis, 252 

ophthalmia, 252 

vulvo-vaginitis, 252 
Gram-negative bacteria, 45 
Gram-positive bacteria, 46 
Gram's stain, 44 

Gram-Weigert method of staining, 59 
Granuloma, coccidioidal, 245 
Grass bacillus (B. molleri), 314 
Green pus, 343 

Gregarina blattarum, 430, 431* 
Gruber-Widal reaction, 211, 338 

Hsemoproteus columbae, 412 

distribution, 412 

life cycle of, 42, 413* 

transmission of, 412 
Haemoproteus danilewskyi, 414* 

ziemanni, 415*, 416* 
Haffkine's prophylactic inoculation, 320 

for cholera, 351 

for plague, 3 20 
Halteridium, 414* 
Hanging-block, 35 
Hanging-drop, 33, 34* 
Haptophore, 204, 211 
Hardening of tissues, 55 
Hay bacillus (B. subtilis), 269* 
Healthy carriers of infection, 201 
Heat in food preservation, 6 

production of, 168 

separation of bacterial species by, 
' .278,^279 

sterilization by, 64 
Hematoxylin, Delafield's, 61 

Heidenhain's iron, 54 
Hematozoa, 379, 412 
Hemolysins, 216 
Hemolysis, 216 
Hemolytic amboceptor, 216 

serum, 216 

titration of, 216 



INDEX OF SUBJECTS 



453 



Hemorrhagic septicemia, 316 
Herpetomonas muscae, 378 

culicis, 378 
Heterogenesis, 4 

High-temperature incubator, 115, 116* 
Higher bacteria (trichobacteria), 141 
Hiss capsule stain, 51 

serum- water medium, 377 
History of bacteriology, i 
Hoffmann's bacillus (B. hoffmanni), 296 
Hog cholera, 373 

bacillus of (B. suipestif er) , 329 

immunity, 374 

serum, 374 

spirochete, 374 

virus of, 373 

Hogyes treatment of rabies, 222 
Honing of knives, 57 
Hot-air sterilization, 64 

sterilizer, 65* 
House disinfection, 72, 77 
Hunger predisposing to infection, 197 
Hydrochloric acid as germicide, 7 1 
Hydrogen atmosphere for anaerobes, 126 

peroxide, 74 
Hydrophobia (rabies), 370, 372 

diagnosis, 372 

Negri bodies in, 370, 371 

Pasteur treatment, 373 

treatment of wound, 373 
Hyperplasia, 206 
Hypersusceptibility, 226 
Hypha, 137 
Hyphomycetes, 137 
Hypochlorite, 73, 182 
Hypodermic inoculation, 133 

Ice, 1 88 

bacteriological examination of, 188 
Illumination by broad beam, 24* 

by hollow cone, 24*, 25* 

central, 24* 

dark field, 24*, 25* 
Image formation, 17*, 18* 
Imbedding, 55 
Immune body, 2I4 1 
Immunity, 12, 220 

acquired, 220, 222 

active, 222 

antiaggressive, 224 

antitoxic, 224 

bacteriolytic, 213 

combined passive and active, 225 

duration of, 222 

Ehrlich's theory of, 227 

following vaccination, 223 

individual, 221 



Immunity, mechanisms of, 225 

natural, 220 

of species, 220 

passive, 224 

racial, 221 

theories of, 227 

unit, 281, 294 
Impression preparation, 37 
Inactivated serum, 213, 363 
Incubator, 115, 116* 

low temperature, 121 

rooms, 1 20 
Infection, 196 

avenues, 197 

general, 199 

healthy carriers of, 201 

local, 199 

possibility of, 196 

secondary, 199 

transmission of, 197, 200 
Infectious disease, 196 

facts and theories of, 206 

phenomena of, 206 
Influenza, 298 
Inoculation, animal, 133 

into the circulating blood, 133 

into the cranial cavity, 133 

intracardiac, 133 

intraperitoneal, 133 

subcutaneous, 133 

Inorganic salts as microbic food, 165 
Insects, 13 

destruction of, 72 
Instruments, sterilization of, 66 
Intermediary body, 214 
Intermittent sterilization, 70 
Intestinal amebae (entamcebae), 402 

anthrax, 273 

juice, collection of, 102 
Intestine, infection through, 199 
Intrauterine infection, 198 
Intravenous inoculation, 133 
Invisible microbes, 9, 26 
Iodide of mercury, 75 
Iodine, 74 

antiseptic value, 79 
lodoform, 74 
Iris diaphragm, 24 
Iron hematoxylin, 54 
Isolation of bacteria, 105, 112 

plate method, 105 

streak method, 112 

Veillon method, 112 
Itch (scabies), 10 

Jaw, lumpy (actinomycosis), 246 
Jeffer's plate, 184 



454 



INDEX OF SUBJECTS 



Jennerian vaccination, 223, 376 
Jenner's stain, 42 

Kala-azar, 394, 396 

parasite of, 394 

transmission of, 395 
Kefir, 192 

Kirkbride forceps, 39* 
Klebs-Loffler bacillus, 285 
Koch-Eberth bacillus, 330 
Koch's safety burner, 119* 

plate cultures, 106 

postulates, 195 

steam sterilizer, 66 
Koch- Weeks bacillus, 296, 297* 
Koumiss, 192 

Lactic acid, 169 
Lamblia, 153 

intestinalis, 153*, 399*, 400 
Leishman-Donovan bodies (L. dono- 

vani)', 152*, 394 
Leishmania donovani, 152*, 153*, 394 

cultures, 394 

occurrence, 394 

transmission, 395 
Leishmania infantum, 397 
Leishmania tropica, 396* 

cultures of, 397* 

immunity to, 397 
Leishman's stain, 42 
Leprosy, 313 
Leptomonas culicis. 378 
Leptothrix, 246, 249 

buccalis. 249 
Leukoddin, 265 
Levaditi's silver stain, 358 
Light, effect on bacteria, 63 
Lime, 73 

Lithium carmine, 61 
Litmus, 85, 87 
Lockjaw (tetanus), 278, 280 
Locomotion, 34 
Loffler's bacillus (B. diphtheria?) , 285 

blood serum, 94 

flagella stain, 52 

methylene blue, 41 
Lophotrichous bacteria, 148 
Lower bacteria, 142 
Luetin, 359 

test, 366 
Lumpy jaw, 246 
Lungs, infection of, 198 

inflammation of (pneumonia), 259 
Lysins, 213 
Lysol, 77 
Lyssa (rabies), 370, 372 



Lyssa (rabies), diagnosis of, 372 
Hogyes treatment of, 222 
Pasteur treatment of, 373 

Macrogametes, 156* 
Macrogametocytes, 156* 
Madura foot, 248 
Madurella mycetori, 249 
Magnification, 16*, 17*, 18*, 19, 23 
Malachite green, 78 
Malaria, 425 

ayian, 412, 414, 415, 417 

diagnosis of, 427 

estivo-autumnal, 419, 426 

mosquitoes in, 427, 428 

prophylaxis, 427 

quartan, 425, 426*, 427 

tertian, 424*, 426 

transmission of, 427 
Malarial parasites of birds, 412, 414, 

415, 4i7 

of man, 419, 424*, 425, 426* 

of monkeys, 429 

transmission of, 472 
Mai de Caderas, 388 
Malignant edema, 276 

pustule, 273 
Mallein, 340 
Malta fever, 321, 322 

diagnosis of, 322, 323 
Mammalian tuberculosis, 299 
Marmorek's serum (antistreptococcus 

serum), 264 

Mastigamceba aspera, 400 
Mastigphora, 151, 3 78 
Mayer's glycerin-albumen, 56 
Measles, 375 
Mechanical filtration, 181 

sterilization, 62 
Media, culture, 83 
Mediterranean fever (Malta fever), 321, 

322 

Membranous croup (diphtheria), 289 
Meningitis, 253 

diagnosis, 255, 257 

serum, 255 

serum treatment, 255 
Meningococcus, 253, 256* 

cultures of, 254 
Mercuric chloride, 74 

iodide, 75 
Metchnikoff's phagocytic theory, 225, 

227 

Methyl violet, 78 
Methylene azure, 42, 43 
Methylene blue, 41 

germicidal power of, 78 



INDEX OF SUBJECTS 



455 



Methylene violet, 43 
Miasm, 200 
Microbe, 3 

relation of, to environment 171 
Microbiology, 3 
Micrococcus, 5, 142, 143 

agilis, 267 

catarrhalis, 257 

gonorrhea?, 250 

melitensis, 321 

meningitidis, 253 

tetragenus, 267 
Microgamete, 156* 
Microgametocyte, 156* 
Micro millimeter, 31 
Micron, 31 
Micronucleus, 433 
Micro-organisms, 3 

distribution of, 174 

in air, 176 

in food, 193 

in ice, 188 

in milk, 189 

in soil, 175 

in water, 178 
Microscope, 15, 21*, 29* 

development of, 15 

principle of, 16, 22* 

tandem, 16 

use of, 31 
Microscopic definition, 24 

measurements, 31 

resolution in depth, 24 
Microspira, 146 

comma, 345 
Microsporon audouini, 241 

furfur, 242 

septicum, n, 260 
Microtome, 57* 

Migula's classification of bacteria, 142 
Miliary tuberculosis, 307 
Milk, 189 

acid, beverages, 343 

as culture medium, 92 

bacteria of, 189 

blue, 344 

collection of samples of, 100 

composition of, 189 

for infant feeding, 192 

pasteurization of, 192 

micro-organisms of, 190 
Milzbrand (anthrax), 270, 272 
Mixed infection, 199 
Modes of entry of infection, 197 
Moisture requirement of bacteria, 

164 
Molds, 137, 231 



M oiler's grass bacillus (Bacillus molleri), 

3H 

spore stain, 51 
Monilia Candida, 238* 
Monotrichous bacteria, 148 
Morax-Axenfeld bacillus, 296* 
Morphology, 137 

relation of, to environment, 171 

relation of, to physiology, 162 
Mosquitoes in malaria, 427, 428* 
Motility, 34 
Movement, 34 

Brownian, 34 

real, 34 
Mucor, 231, 232*, 233 

corymbifer, 231, 232* 

mucedo, 138*, 231, 232* 
Muscardine, 10, 236 
Musgrave and Clegg's medium for 

ameba, 402 
Mycelium, 137 
Mycetoma, 248 

Nagana, 384, 386 

diagnosis of, 387 

immunity to, 387 

occurrence of, 386 

transmission of, 386 

trypanosome of, 384* 
Natural immunity, 220 

individual, 221 

mechanisms of, 225 

of species, 220 

racial, 221 

Negri bodies, 370, 371 
Neisser's gonococcus, 250 
Neisser-Wechsberg phenomenon, 215 
Neosporidia, 158, 431 
Neutralization of culture media, 85, 87 
Nitrate of silver, 76 

Nitrates, production of, by bacteria, 175 
Nitrification, 175 
Nitrifying bacteria, 175 
Nitrites, formation of, 175 
Nitrogen fixation, 175 
Nitrosomonas, 164 
Nocht-Romanowsky stain, 41 
Nodule bacteria (root tubercles), 175, 

194 

Nomenclature, 160 
Normal solution, 85 
Nosema, 155, 158 

bombycis, n, 159*, 431, 43 2 * 
Novy's anaerobic method, 126, 127* 

blood-agar, 98 

cover-glass forceps, 38* 
Nuclear stains, 61 



45 6 



INDEX OF SUBJECTS 



Nucleus of bacteria, 148 

of protozoa, 151 
Number of bacteria in milk, 189 

in water, 183 

required to infect, 197 
Numerical aperture, 23 
Nutrient agar, 89 

Obermeier's spirillum, 353 
Objectives, achromatic, 16, 20*, 29, 30 

apochromatic, 16, 20 

defects of, 20 

immersion, 30 

Ocular tuberculin reaction, 309 
Oculars, 23, 29, 30 
Oidiomycosis, 245 
Oidium albicans, 10, 238* 

lactis, 138*, 236* 
Oil, aniline, in stains, 40 
Ookinete, 157 

Opalina ranarum, 434, 435* 
Opsonins, 217 
Organic poisons as germicides, 76 

food requirements, 164 
Oriental sore (Delhi boil), 396 
Osteomyelitis, 266 
Outline classification, i6q 
Ovum, infection of, 197 
Oxidizing agents as germicides, 73 
Oxygen, 166 

requirement, 166 

removal of, 1 25 
Oysters as source of typhoid, 335 

Panophthalmitis, 269 
Paracolon bacilli, 330 
Paraffin, 55 

imbedding, 55 
Paralysis, infantile, 374 
Paramaecium aurelia, 434 

bursaria, 434 

caudatum, 433 

conjugation, 433, 434 

division, 434 

form and structure, 433 
Paramascium putrinum, 434* 
Paraplasma flavigenum, 370 
Parasite, 165 

obligate, 165 
Parasitism, 194 
Paratyphoid bacilli, 330 
Parenteral digestion, 207 
Passive immunity, 224 
Pasteur pipettes, 33 

treatment for rabies, 373 
Pasteur-Chamberland filter, 63 
Pasteurization, 66 



Pathogenesis, 194, 195 
Pathogenic bacteria, 195 

organisms, 195 

protozoa, 378 

soil bacteria, 176 

Pathology, relation of bacteriology to, 7 
Pearl disease (bovine tuberculosis), 310 
Pebrine, 10, 431, 432 

parasite of, 431, 432* 

restriction of, 432 
Penicillium crustaceum, 234* 

glaucum, 138*, 234 
Peptone solution, 92 
Peptonizing ferments, 170 
Peritrichous bacteria, 148 
Perlsucht (bovine tuberculosis), 310 
Permanganate of potassium, 74 
Peroxide of hydrogen, 74 
Pertussis, 296 
Petri dishes, 108 
Pfeiffer's phenomenon, 213 
Phagocytic theory, 225, 227 
Phagocytosis, 207, 227 
Phenol, 76 

Phenolphthalein, 86, 87 
Phenomena of disease, 195 
Phenomenon, Pfeiffer's, 213 
Phlebotomus fever, 374 
Phosphorescence, 168 
Photogenic bacteria, 168 
Phy corny cetes, 137 
Physical sterilization, 62 
Physiological method, 163 

hyperplasia, 206 

tests, 173 
Physiology of micro-organisms, 162 

relation to morphology, 162 
Pipettes, glass (Pasteur pipette), 32, 33* 

for drawing blood from animal, 96* 

for drawing blood from man, 95* 
Piroplasma (Babesia), 155, 158* 

bigeminum, 429* 

canis, 430 

muris, 158* 
Pityriasis, 242 
Placental transmission, 198 
Plague, 317 

bubonic, 319 

diagnosis, 318 

fleas as carriers of, 319 

Haffkine's prophylactic, 320 

immunity, 320 

in animals, 319, 320 

pneumonic, 320 

prophylaxis of, 320, 321 

serum, 321 

transmission, 319, 320 



INDEX OF SUBJECTS 



457 



Plague vaccines, 320 
Planococcus, 142, 143 

agilis, 267 

Planosarcina, 142, 143 
Plants, diseases of, 235 
Plasmodium, 12, 155, 157* 

brassicae, 407 
Plasmodium falciparum, 157*, 420 

asexual cycle in man, 420* 

cultures of, 423 

pathogenic relation of, 426 

sexual cycle in anopheles, 422* 

transmission of, 423, 427 
Plasmodium kochi, 429 

malariae, 425, 426* 

praecox, 420 

vivax, 424*, 425* 
Plasmodroma, 151 
Plasmolysis, 147 
Plate cultures, 12, 106 

Koch's original method, 112 
Platinum wire, 31, 32* 
Pleuro-pneumonia of cattle, 13, 368 

filterable virus of, 368 
Plugs, cotton, 84 
Pneumococcus, 257, 258* 

immunity to, 260 

poisons of, 259 
Pneumonia, 259 

micro-organisms in, 259 

serum, 260 
Poisoning, food, 193, 284, 328 

botulism, 284 

enteritidis type, 328 

proteus vulgaris as cause of, 343 
Poisons, 193, 203 
Poliomyelitis, 374 
Porcelain filter, 63 

Post-mortem examination, 98, 103, 136 
Postulates of Henle, 10, 1 2 

of Koch, 12, 195 
Potassium permanganate, 74 
Potato cultures, 91 

bacillus (B. vulgatus), 268 

medium, 91* 
Precipitation test, 209 
Precipitinogen, 210 
Precipitins, 209 
Predisposition, 197 
Preservation, 6, 62, 79 
Preservatives, 79 
Pressure, effect on bacteria, 63 

filter, 63 
Products of bacteria, 168 

chemical effects, 168 

enzymes, 169 

physical effects, 168 



Products of primary, 169 

ptomaines, 170 

secondary, 169 

toxins, 171 
Protective inoculation for anthrax, 273 

for cholera, 351 

for diphtheria, 295 

for plague, 320 

for small-pox, 376 

for typhoid, 335 
Proteolysins, 218 
Proteosoma praecox, 417 

development in blood, 418* 

development in the mosquito, 419* 
Froteus vulgaris (Bacillus proteus), 

343 

Protista, 160 
Protozoa, 12, 150 

relation to disease, 13 

wet fixation of, 54 
Pseudo-diphtheria bacillus, 296 
Pseudomonas, 144 

radicicola, 175, 194 
Ptomain, 170 
Puerperal fever, 263 
Pulmonary anthrax, 273 
Pure culture, 113 
Purification of water, 179 
Pus, collection of, 102 
Pustule, malignant, 273 
Putrefaction, 5, 170 
Putrefactive alkaloids, 170 

bacteria, 170 

products, 170 
Pyemia, n, 200 
Pyoktanin, 78 
Pyrogallic-acid anaerobic method, 125 

Quartan malaria, 425, 426*, 427 
Quarter evil (symptomatic anthrax) 

276 

Quincke's puncture, 255 
Quotidian malaria, 426 

Rabies, 370, 372 

diagnosis of, 372 
Hb'gyes treatment of, 222 
Negri bodies in, 370, 371* 
Pasteur treatment, 373 
treatment of wound, 373 

Racial immunity, 221 

Rats, relation to bubonic plague, 319, 

320, 321 
trypanosomes 01, 381 

Rauschbrand (symptomatic anthrax), 
276 

Ray fungus (actinomyces), 246, 247* 



458 



INDEX OF SUBJECTS 



Reaction, cutaneous, 309 

of culture media, 165 

of host to infection, 206 
Reading glass, 19 
Receptor of first order, 209* 

of second order, 210* 

of third order, 214* 

theory of immunity, 227 
Reducing substances, 130 
Regulation of temperature, 115 
Regulator, electric, 122* 

Roger's, 122*, 123 
Regulator, gas, 116 

Mac Neal, 117, 118* 

method of filling, 118 

Reichert, 117* 

Roux, 119* 
Relapsing fever, n, 353 

diagnosis, 355 

spirochetes, 353 
Resistance to infection, 196 
Respiratory infection, 134 
Rhinoscleroma, 328 
Rhipicephalus annul atus, 158, 429 
Rhizopoda, 151, 154*, 401 
Ricin, 171 

Rinderpest, 223, 370 
Roll-tubes, no*, in* 
Romano wsky stain, 41, 43, 149 
Rooms, disinfection of, 72, 77 

incubator, 120 

Root-tubercle bacteria, 14, 175, 194 
Rubber caps, 114*, 115, 120 

stoppers, 114*, 115, 120 
Rules of Koch, 195 

Saccharomyces, 140 

cervisiae, 140*, 244 

ellipsoideus, 140* 
Sanarelli's bacillus (Bacillus icteroides), 

329 

Sand filtration, 180 
Saprogenic bacteria, 170 
Saprophyte, 164 
Saprophytic, 164 
Sarcina, 142*, 143 

aurantiaca, 267 

ventriculi, 267 
Sarcoma, chicken, 377 
Sarcoptes scabei, 10 
Schizomycetes, 141 
Schizotrypanum cruzi, 392, 393* 

cultures of, 394 

transmission of, 394 
Sealing culture tubes, 114*, 115, 120 
Secondary infection, 199 
Section-cutting, 56 



Sections, 58 

staining of, 58, 59 

tubercle bacilli in, 60 
Sedg wick-Tucker aerobioscope, 177* 
Sedimentation, 63 
Self-purification of water, 179 
Semen, transmission of infection by, 198 
Sensitizer, 215 
Septicemia, n, 200 

hemorrhagic, 316 

sputum, 277 
Serum, anthrax, 274 

antibacterial, 212 * 

antimeningococcus, 255 

antipneumococcus, 260 

antistreptococcus, 264 

antitoxic, 208 

bactericidal, 212 

blood, 92 

cytolytic, 213 

dysentery, 337 

hemolytic, 216 

immune, 213 

Loffler's, 94 

normal, 217 

plague, 321 

Yersin's, 321 
Shiga's bacillus (Bacillus dysenteriae) , 

336 

Side-chain theory, 208, 227 
Silver nitrate, 76 
Sleeping sickness, 388, 390 

transmission of, 388 

trypanosome of, 388 

tsetse fly concerned in, 388, 389* 
Slides, forceps for, 39 

glass, 39 

method of cleaning, 39 
Small -pox, 376 

inoculation, 12 

vaccination, 223, 376 

virus of, 376 
Smear culture, cover-glass, 38 

preparations, 36 

slide, 39^ 

Smegma bacilli, 314 
Soaps, germicidal action of, 71 
Sodium hydroxide, normal solution of, 85 
Soft chancre, 298 
Soil bacteria, 175, 176 
Solutions, normal, 85 
Soor (thrush), 10, 238 
Sore, Oriental (Delhi boil), 396 
Souring of milk, 191 
Species of bacteria, 167 

stability, 167 

variation, 167 



INDEX OF SUBJECTS 



459 



Specific nomenclature, 160 
Sphaerophrya pusilla, 437* 
Spherical bacteria, 142* 
Spirilla, 142, 147*, 345 
Spirillacese, 146, 345 
Spirillum, 5, 142, 146 
Spirillum choleras, 345 

agglutination, 350 

cultures of, 345 

immunity, 348, 351 

in feces, 350 

in water, 351 

poisons of, 348 

resistance of, 346 

transmission of, 349 
Spirillum, Deneke's, 352 

metchnikovi, 352 

of Finkler and Prior, 352 

rubrum, 345 

tyrogenum, 352 
Spirochaeta, 5, 13, 146 

anserina, 356 

culture of, 13 

duttoni )> 353, 354 

fusiformis, 367 

gallinarum, 356 

kochi, 354 

microdentium, 366 

muris, 356 

novyi, 354, 355* 

obermeieri, 353 

of relapsing fever, 353 
Spirochaeta pallida, 357*, 359* 

animal inoculation of, 359 

antibodies, 361 

cultures of, 357, 358 

in blood, 361 

microscopic demonstration of, 360 

morphology, 357 

staining of, 358 
Spirochaeta plicatilis, 353 
Spirochaeta recurrentis, 353 

transmission of, 354, 355 

varieties of, 353 
Spirochasta refringens, 357*, 366 

suis, 374 

vincenti, 367 
Spirochetes, 5, 13, 146, 147, 353 

cultivation of, 13 

of mouth, 366 

of relapsing fevers, 353 

of syphilis, 357* 

saprophytic, 353 
Spirosoma, 146 

Splenic fever (anthrax), 270, 272 
Splenomegaly, tropical (kala-azar), 394, 
396 



Spontaneous generation, 3 
Sporogenic aerobes, 268 

anaerobes, 275 
Spores, 146, 149*, 155 

formation of, 145*, 149* 

germination of, 149* 

resistance of, 66 

staining of, 50 
Sporotrichum beurmanni, 244 

schencki, 242*, 243* 
Sporotrichosis, 242, 244 
Sporozoa, 151, 155 
Sporulation, 145* 
Sputum, 47 

collection of, 47, 101 

examination of, 48 

septicemia, 257 
Stab-culture, 113, 114*, 124 
Staining, 38, 44 

acid-proof bacilli, 47 

anilin-water gentian violet, 40 

blood films, 55 

capsules, 51 

dish, 39 

flagella, 52 

Gram's method, 44 

Romano wsky, 41 

solutions, 39 

spirochetes, 358 

spores, 50 

tissues, 55 

tubercle bacillus, 47, 49 
Standard antitoxin, 282, 294 
Standardization of antitoxins, 281, 294 

diphtheria, 294 

tetanus, 281, 282 
Staphylococcus, 142*, 143, 264 

albus, 267 

aureus, 264 

epidermidis, 267 

immunity, 266 

infections, 264 

toxins, 265 

vaccines, 266 
Staphylolysin, 265 
Steam sterilization, 66 

sterilizer, 66* 

Stegomyia (Aedes) calopus, 369* 
Sterilization, 62 

autoclave, 68 

by desiccation, 63 

by filtration, 63, 97 

by light, 63 

by moist heat, 65 

by sedimentation, 63 

chemical, 71 

fractional, 70 



460 



INDEX OF SUBJECTS 



Sterilization, hot air, 64 

mechanical, 62 

of blood-serum, 93 

of glassware, 84 

physical, 62 
Sterilizer, Arnold, 67*, 68* 

hot-air, 65* 

Koch's, for serum, 93* 

steam, 66* 

Stewart's forceps, 38* 
Stick-culture (stab-culture), 114*, 124 
Stock cultures, 114 
Stomach, germicidal action of, 71 

infection through, 198 
Stomoxys calci trans, 387 
Stools (feces), 102 
Straus's test for glanders, 341 
Streak cultures, 113, 114* 
Streptobacillus, 145 
Streptococcus, 142*, 143, 260 

erysipelas, 263 

erysipelatos, 260 

hemolytic, 261 

immunity, 264 

infections, 263 

lacticus, 190, 264 

mucosus, 260 

pyogenes, 260 

vaccines, 264 

viridans, 260 

virulence of, 261 
Streptothrix, 246 

madurae, 248 

Structure of bacteria, 147 
Subcutaneous application, 134 
Sugar-free media, 90 
Sugars in culture media, 90 
Sulphur dioxide, 71 
Sunlight as germicide, 63 
Surra, 387 _ 
Susceptibility, 196 

local, 199 

Swine erysipelas, 317 
Symbiosis, 194 
Symptomatic anthrax, 276 
Syphilis, 357, 360 

diagnosis, 360, 366 

fixation of complement in, 361, 366 

in animals, 359, 361 

luetin test in, 366 

spirochete, 357, 360 

transmission of, 360 

Wassermann test for, 361 
Systematic relationships, 4, 137 

Telosporidia, 158 
Temperature, influence of, 167 



Temperature, maximum, 166 

minimum, 166 

optimum, 166 

regulation of, 115 

requirements, 166 

sterilizing, 68 
Tertian malaria, 424*, 426 
Test, Calmette's, 309 

complement- fixation, 216, 361 

luetin, 366 

mallei n, 340 

precipitin, 210 

Straus's, 341 

tuberculin, 308, 311 

von Pirquet's, 309 

Wassermann's, 361 

Testing antiseptics and disinfectants, 80 
Tetanolysin, 279 
Tetanospasmin, 279 
Tetanus, 278, 280 

antitoxin, 281 

bacillus, 278, 280* 

immunity, 281 

immunity unit, 281, 282 

prophylaxis, 283 

spasm of, 280 

standard antitoxin, 283 

toxin, 203, 279 

treatment of, 283 
Tetrad, 142 
Texas fever, 13, 429 

control of, 430 

immunity to, 223 

parasite of, 429 

restriction of, 430 

tick, 429 

Theories of immunity, 227 
Thermogenic bacteria, 168 
Thermostat (thermoregulator), 116, 122 
Thrush, 10, 238 
Tick, cattle, 429 

fever, 354 
Tinea, 242 

versicolor (pityriasis), 242 
Tissues, examination of, 55 
Titration of culture media, 85, 87 
Tongue, wooden (actino mycosis), 246 
Torula, 141 
Toxemia, 200 
Toxin, 171, 203 

. chemical nature of, 171 

diphtheria, 288 

extracellular, 203 

intracellular, 204 

soluble, 203 

standardization of, 281, 294 

tetanus, 279 



INDEX OF SUBJECTS 



461 



Toxoid, 204 

Toxophore, 204 

Transmission of disease, 13, 197, 200 

Treponema pallidum (Spirochaeta pal- 

lida), 357* 

Trichobacteria, 141, 246 
Trichomonas, 153,* 400 

hominis, 398,* 400 
Trichomycetes, 246 
Tricophyton, 242 
Trimastigamoeba philippinensis, 399,* 

400 
Tropical dysentery, 406 

malaria, 419, 426 

splenomegaly (kala-azar), 394 

ulcer, 396 
Trypanoplasma borreli, 398 

cyprini, 397,* 398 

guernei, 398 

Trypanosoma, 152,* 153, 379 
Trypanosoma avium, 390,* 391* 

cultures of, 392 

occurrence of, 391 
Trypanosoma brucei, 152,* 384* 

cultures of, 384, 386 

form and structure, 384, 385 

immunity to, 387 

multiplication of, 386 

occurrence, 386 

poisons of, 386 

transmission of, 386 
Trypanosoma equinum, 152,* 388 

equiperdum, 152,* 387* 

evansi, 152,* 387 
Trypanosoma gambiense, 152,* 388 

cultures of, 389 

form and structure, 388 

in animals, 389 

in man, 389 

in the fly, 388 

transmission of, 388 
Trypanosoma lewisi, 381* 

cultures of, 383 

division of, 382 

occurrence of, 381 
Trypanosoma rhodesiense, 390 

rotatorium, 379, 380* 
Trypanosomes, 152,* 379 
Tsetse-fly (Glossina morsitans), 385,* 
386 

disease (Nagana), 384 
Tubercle, 306 
Tubercle bacillus, 299 

amphibian, 312 

avian, 311 

bovine, 310 

branching of, 302* 



Tubercle bacillus, chemical composition 
of, 302 

cultures of, 301 

fish type, 312 

human type, 300* 

in sections, 60 

poisons of, 303 

resistance of, 304 

stain for, 48 

transmission of, 307 

varieties of, 299 
Tuberculin, 304 

reaction, 308, 311 

test, 308, 309, 311 

treatment, 309 
Tuberculosis, 305 

avian, 311 

bacillus of, 299 

bovine, 310 

diagnosis of, 307 

fowl, 311 

immunity, 310 

mammalian, 299 

mode of infection in, 307 

tuberculin test in, 308 

tuberculin treatment in, 310 
Typhoid bacillus (B. typhosus), 330 

carriers, 333 

detection in water, 187 
Typhoid fever, 333 

diagnosis of, 333 

immunity to, 335 

in animals, 332 

prophylaxis of, 335 

transmission of, 333, 334 

vaccines, 335 

vaccination, 335 
Typhus fever, 375 

Udder, bacteria in, 189 

Ulcer, tropical, 396 

Ultramicroscope, 16 

Ultramicroscopic organisms, 150, 368 

Unit, immunity, 281, 294 

of diphtheria antitoxin, 294 
of tetanus antitoxin, 281, 282 

Urethritis, 252 

Urinary bladder, inflammation of, 327 

Urine, collection of, 101 

Vaccination, anthrax, 223 

Asiatic cholera, 223 

small-pox, 12, 223, 376 

typhoid, 223 

Vaccines, bacterial, 12, 223 
Vaccinia, 12 
Vagimtis, gonorrheal, 252 



462 



INDEX OF SUBJECTS 



Van Ermengem's flagella stain, 53 
Variola, 376 
Vibrio, 5 

choleras, 345 

Deneke's, 352 

metchnikovi, 352 

of Finkler and Prior, 352 

tyrogenum, 352 

Vibrion septique (B. edematis), 275 
Vincent's angina, 367 

spirillum, 367 
Vinegar, 170 
Violet, anilin-water gentian, 40 

gentian, 40 

methyl (pyoktanin), 78 

methylene. 43 
Virulence, 202 

factors influencing, 202 

loss of, in cultures, 115 
Virus, filterable, 13, 150, 368 
Visibility of microscopic objects, 25 

by light and shade, 26*, 27* 

by quality of light (color), 28 
Von Pirquet test, 309 
Vulvo-vaginitis/ 252 

Warmth, 115 
Wassermann test, 361 
Water, bacteria, 178, 183 

cholera germs in, 187 

collection of samples, 100 

disinfection of, 182 

examination, 178 

filtration, 182 

intestinal bacteria in, 186 



Water, self-purification of 179 

storage of, 180 

typhoid bacilli in, 187 
Watery solution of aniline dyes, 40 
Weigert's stain, 59 
Welch's bacillus (B. welchii), 276 

capsule stain, 51 
Whooping cough, 296 
Widal's test (agglutination), 211, 338 
Wire, platinum, 31, 32* 
Wolff hiigel's colony counter, 185 
Wooden tongue (actino mycosis), 246 
Woolsorter's -disease (pulmonary an- 
thrax), 273 
Wounds, 197 
Wright's method for anaerobes, 125 

Xerosis bacillus, 295 
Xylol, 55, 59 

Yeasts, 139,* 140* 
Yellow fever, 368 

immunity, 369 

mosquito, 369* 

prophylaxis of, 370 

transmission of, 369 

virus, 368 
Yersin's serum, 321 

Ziehl's solution (carbol-fuchsin), 46 
Zwischenkorper (intermediary body), 

214 

Zygospore, 137 
Zymogenic bacteria, 169 
Zymophore, 211 



DATE DUE SLIP 

UNIVERSITY OF CALIFORNIA MEDICAL SCHOOL LIBRARY 



THIS BOOK IS DUE ON THE LAST DATE 
STAMPED BELOW 



MAY 7 1924 



$EP 3 U 

?9 1930 

5 

2 3 1931 
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JAN 13 1932 

FEB 4 1932 
.SEP ? ' 



MAR '9- 1939 



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Library of the 
University of California Medical School and Hospitals