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A TEXT-BOOK OF

BACTERIOLOGY

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A TEXT-BOOK OF

BACTERIOLOGY

A PRACTICAL TREATISE
FOR STUDENTS AND PRACTITIONERS

OF MEDICINE

PHILIP HANSON HISS, Jr., M.D.

LATE PROFESSOR OF BACTERIOLOGY, COLLEGE OF PHYSICIANS AND SURGEONS,

COLUMBIA UNIVERSITY, NEW YORK CITY

AND

HANS ZINSSER, M.D.

PROFESSOR OF BACTERIOLOGY, COLLEGE OF PHYSICIANS AND SURGEONS, COLUMBIA
UNIVERSITY, NEW YORK CITY; FORMERLY PROFESSOR OF BACTERIOLOGY
AND IMMUNITY, STANFORD UNIVERSITY, CALIFORNIA

WITH ONE HUNDRED AND FIFTY-FIVE ILLUSTRATIONS IN THE
TEXT, SOME OF WHICH ARE COLORED

SECOND EDITION

ins'

NEW YORK AND LONDON
D. APPLETON AND COMPANY

1915

PRINTED IN

New ‘Ydrk, X?.‘& A.

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PREFACE TO THE FIRST EDITION

The volume here presented is primarily a treatise on the funda¬
mental laws and technique of Bacteriology, as illustrated by their
application to the study of pathogenic bacteria.

So ubiquitous are the bacteria and so manifold their activities
that Bacteriology, although one of the youngest of sciences, has
already been divided into special fields — Medical, Sanitary, Agricul¬
tural, and Industrial — having little in common, except problems of
general bacterial physiology and certain fundamental technical pro¬
cedures.

From no other point of approach, however, is such a breadth of
conception attainable, as through the study of bacteria in their rela¬
tion to disease processes in man and animals. Through such a
study one must become familiar not only with the growth character¬
istics and products of the bacteria apart from the animal body, thus
gaining a knowledge of methods and procedures common to the study
of pathogenic and non-pathogenic organisms, but also with those
complicated reactions taking place between the bacteria and their
products on the one hand and the cells and fluids of the animal body
on the other — reactions which often manifest themselves as symptoms
and lesions of disease or by visible changes in the test tube.

Through a study and comprehension of the processes underlying
these reactions, our knowledge of cell physiology has been broadened,
and facts of inestimable value have been discovered, which have
thrown light upon some of the most obscure problems of infection
and immunity and have led to hitherto unsuspected methods of
treatment and diagnosis. Thus, through Medical Bacteriology — that
highly specialized offshoot of General Biology and Pathology — have
been given back to the parent sciences and to Medicine in general
methods and knowledge of the widest application.

It has been our endeavor, therefore, to present this phase of our
subject in as broad and critical a manner as possible in the sections

PREFACE

dealing with infection and immunity and with methods of biological
diagnosis and treatment of disease, so that the student and practi¬
tioner of medicine, by becoming familiar with underlying laws and
principles, may not only be in a position to realize the meaning and
scope of some of these newer discoveries and methods, but may be
in better position to decide for themselves their proper application
and limitations.

We have not hesitated, whenever necessary for a proper under¬
standing of processes of bacterial nutrition or physiology, or for
breadth of view in considering problems of the relation of bacteria
to our food supply and environment, to make free use of illustrations
from the more special fields of agricultural and sanitary bacteriology,
and some special methods of the bacteriology of sanitation are given
in the last division of the book, dealing with the bacteria in relation
to our food and environment.

In conclusion it may be said that the scope and arrangement of
subjects treated of in this book are the direct outcome of many years
of experience in the instruction of students in medical and in advanced
university courses in bacteriology, and that it is our hope that this
volume may not only meet the needs of such students but may prove
of value to the practitioner of medicine for whom it has also been
written.

It is a pleasure to acknowledge the courtesy of those who furnished
us with illustrations for use in the text, and our indebtedness to Dr.
Gardner Hopkins and Professor Francis Carter Wood for a number
of the photomicrographs taken especially for this work.

P. H. H., Jr.,

H. Z.

PREFACE TO THE SECOND EDITION

Inquiry in the field of bacteriology is so active at the present day
that no general text-book can maintain its usefulness long without
frequent revision. In preparing the second edition of this book it has
been our purpose to correct omissions and to incorporate the more im¬
portant researches of the last three years, rather than to alter exten¬
sively the plan of the text. From the wealth of material which these
years have brought, we have attempted to glean those facts which
have seemed to us most important and most directly bearing upon
medical problems, since this book was planned, from the beginning, to
meet especially the needs of the student of infectious disease.

The most extensive changes and additions have been made in the
chapters on streptococci, tuberculosis, plague, leprosy, syphilis, rabies,
and poliomyelitis. Short sections on typhus fever, on the plague¬
like disease of rodents, and on rat leprosy have been added, and we have
inserted a tabulation of our knowledge of filtrable virus, adapted
largely from the summary recently published by Wolbach. The Ander¬
son and McClintic method for the standardization of disinfectants,
and Churchman’s recent work on anilin dyes and bacterial growth,
have been added. Many minor corrections and additions have been
made throughout the text. In preparing these changes, valuable as¬
sistance has been given us by Dr. J. Gardner Hopkins, Associate in
Bacteriology at Columbia University, and many helpful suggestions
have been made by Drs. Dwyer and Bliss.

It has been gratifying to note how much of the work which seemed
to us particularly valuable and enlightening has emanated, during these
three years, from American laboratories.

We have purposely omitted making any extensive changes in the
section on immunity. The function of this part of the book is to give
the beginner a basis for further reading and introduce him, as simply as
possible, to the difficult problems of the field. We have felt that the
addition of much more detail and theory would render this section un¬
suited to the needs of a general text-book.

It is a sorrowful necessity that this revision must be put forth with-

vii

Vlll

PREFACE TO THE SECOND EDITION

out the wise counsel of one of its authors. Since the first edition of this
book was published Prof. Philip Hanson Hiss, Jr., has died. By his
death we have lost a dear friend and a valued teacher, and American
bacteriology has been deprived of a worker who was entering into the
most brilliant period of his scientific maturity.

New York, 1914

H. Z.

CONTENTS

SECTION I

THE GENERAL BIOLOGY OF BACTERIA AND THE
TECHNIQUE OF BACTERIOLOGICAL STUDY

CHAPTER PAGE

I. The Development and Scope of Bacteriology . 1

II General Morphology, Reproduction, and Chemical and

Physical Properties of the Bacteria . 9

II. The Relation of Bacteria to Environment, and Their

Classification . 25

IV. The Biological Activities of Bacteria . 40

V. The Destruction of Bacteria . 62

VI. Methods Used in the Microscopic Study and Staining of

Bacteria . 93

VII. The Preparation of Culture Media . 113

VIII. Methods Used in the Cultivation of Bacteria . 141

IX Methods Determining Biological Activities of Bacteria . 164

X. The Bacteriological Examination of Material from

Patients . 174

SECTION II

INFECTION AND IMMUNITY

CHAPTER PAGE

XI. Fundamental Factors of Pathogenicity and Infection . . 181

XII. Defensive Factors of the Animal Organism . ' . 189

XIII. Toxins and Antitoxins . . 203

XIV. Production and Testing of Antitoxins . 216

XV. Lysins, Agglutinins, Precipitins, and Other Antibodies „ 224

XVI. The Technique of Serum Reactions . . . . 249

CONTENTS

CHAPTER PAGE

XVII. Phagocytosis . . . 275

XVIII. Opsonins, Leucocyte Extract, and Aggressins ..... 281
XIX. Anaphylaxis or Hypersusceptibility . . 295

XX. Facts and Problems of Immunity in their Bearing upon

the Treatment of Infectious Diseases . 305

SECTION III

PATHOGENIC MICROORGANISMS

CHAPTER PAGE

XXI. The Staphylococci ( Micrococci ) . 321

XXII. The Streptococci . . 335

XXIII. Diplococcus pneumonias . 352

XXIV. Micrococcus intrace llularis meningitidis ( Meningococcus ) . 371

XXV. Diplococcus gonorrhoea ( Gonococcus ), Micrococcus ca-

TARRHALIS, AND OTHER GrAM-NEGATIVE COCCI . 380

XXVI. Bacilli of the Colon-Typhoid-Dysentery Group — Bacillus

COLI COMMUNIS, . 388

XXVII. Bacilli of the Colon-Typhoid-Dysentery Group ( continued )

—Bacillus of Typhoid Fever . 399

XXVIII. Bacilli of the Colon-Typhoid-Dysentery Group ( continued )

— Bacilli Intermediate between the Typhoid and Colon
Organisms . 428

XXIX. Bacilli of the Colon-Typhoid-Dysentery Group ( continued )

— The Dysentery Bacilli . . . 435

XXX. Bacillus mucosus capsulatus . 447

XXXI. Bacillus tetani . 456

XXXII. Bacillus of Symptomatic Anthrax, Bacillus of Malignant
Edema, Bacillus aerogenes capsulatus, Bacillus botu-

linus . 465

XXXIII. The Tubercle Bacillus . 479

XXXIV. The Smegma Bacillus and the Bacillus of Leprosy . . . 503

XXXV. Bacillus diphtherias, Bacillus Hoffmanni, and Bacillus

xerosis . . 512

XXXVI. Bacillus mallei . . 528

XXXVII. Bacillus influenzae and Closely Related Bacteria . . . 536

CONTENTS xi

CHAPTER PAGE

XXXVIII. Bordet-Gengou Bacillus, Morax-Axenfeld Bacillus, Zur

Nedden Bacillus, Ducrey Bacillus . 543

XXXIX. The Bacilli of the Hemorrhagic Septicemia Group and

Bacillus pestis . 551

XL. Bacillus anthracis and Anthrax . 563

XLI. Bacillus pyocyaneus . 577

XLII. Asiatic Cholera and the Cholera Organism . 582

XLIII. Diseases Caused by Spirochetes . 592

XLIV. The Higher Bacteria . 618

XLV. The Yeasts . 629

XLVI. Hyphomycetes . 635

SECTION IV

DISEASES OF UNKNOWN ETIOLOGY

CHAPTER PAGE

XL VII. Rabies . 646

XL VIII. Smallpox . 657

XLIX. Acute Anterior Poliomyelitis . . 664

L. Yellow Fever . 668

LI. Measles, Scarlet Fever, and Foot-and-Mouth Disease . . 675

SECTION V

BACTERIA IN AIR, SOIL, WATER, AND MILK

CHAPTER PAGE

LII. Bacteria in the Air and Soil . 683

LIII. Bacteria in Water . 689

LIV. Bacteria in Milk and Milk Products, Bacteria in the

Industries . 699

INDEX OF AUTHORS . 719

INDEX OF SUBJECTS . 731

LIST OF ILLUSTRATIONS

FIGURE PAGE

1. Types of bacterial morphology . . . 10

2. Bacterial capsules . 12

3. Arrangement of bacterial flagella . 15

4. Various positions of spores in bacterial cell . 17

5. Germination of spores . . . . 17

6. Degeneration forms of Bacillus diphtheriae . . . 19

7. Degeneration forms of Bacillus pestis . . 20

8. Hot-air sterilizer . 69

9. Arnold sterilizer . . . 70

10. Low- temperature sterilizer . 71

11. Autoclave . 72

12. Lentz formalin apparatus . 90

13. Breslau formaldehyde generator and section of same ....... 91

14. Hanging drop preparation . 94

15. Florence flask . . . 114

16. Erlenmeyer flask . ..114

17. Petri dish . 115

18. Test tubes, showing method of stoppering . 116

19. Burette for titrating media . 117

20. Tubing media . 118

21. Media in tubes . 119

22. Berkefeld filter . 120

23. Berkefeld filter . 121

24. Reichel filter . 122

25. Kitasato filter . 123

26. Maassen filter, for small quantities of fluid . 124

27. Platinum wires . 142

28. Taking plugs from tubes before inoculation . 143

29. Inoculating . 144

30. Pouring inoculating medium into Petri dish . 145

31. Streak plate . 147

32. Deep stab cultivation of anaerobic bacteria . 148

33. Deep stab cultivation of anaerobic bacteria . 149

34. Cultivation of anaerobes in fluid under albolin . 150

35. Wright’s method of anaerobic cultivation in fluid media . 151

36. Novy jar . 152

37. Wright’s method of anaerobic cultivation by the use of pyrogallic-acid

solution . .... . 153

38. Jar for anaerobic cultivation . . 154

xiii

XIV

LIST OF ILLUSTRATIONS

FIGURE PAGE

39. Apparatus for combining the methods of exhaustion, hydrogen replace¬

ment, and oxygen absorption . 155

40. Simple apparatus for plate cultivation of anaerobic bacteria . . . . .156

41. Incubator . 157

42. Thermo-regulator . 158

43. Thermo-regulator . 158

44. Moitessier gas-pressure regulator . 160

45. Variations in the conformation of the borders of bacterial colonies . .161

46. Wolffhiigel counting-plate . : . 162

47. Types of fermentation tubes . 165

48. Types of gelatin liquefaction by bacteria . . . . . 169

49. Intraperitoneal inoculation of rabbit . .....170

50. Intravenous inoculation of rabbit . 170

51. Intraperitoneal inoculation of guinea-pig . 171

52. Guinea-pig cage . 171

53. Rabbit cage . 172

54. Blood-culture plate showing streptococcus colonies . 179

55. Toxin and body cell . 206

56. Toxin and antitoxin . 214

57. Ehrlich’s conception of cell-receptors, giving rise to lytic immune bodies 227

58. Complement, amboceptor or immune body, and antigen or immunizing

substance . 227

59. Microscopic agglutination reaction . 229

60. Macroscopic agglutination . 230

61. Ehrlich’s conception of the structure of agglutinins and precipitins . . 238

82. The structure of cell-receptors and immune bodies, according to Ehrlich’s

conception . 239

63. Neisser and Wechsberg’s conception of complement deviation .... 245

64. Schematic representation of complement fixation in the Bordet-Gengou

reaction . 247

65. Capillary pipette for removal of exudate in doing the Pfeiffer test . . 256

66. Wright’s capsule for collecting blood . . 284

67. Pipette for opsonic work . 285

68. Pipette with three substances, — corpuscles, bacteria, and serum, as first

taken up . 285

69. Staphylococcus pyogenes aureus . . . 322

70. Staphylococcus colonies . 323

71. Micrococcus tetragenus . . 334

72. Streptococcus pyogenes . . 336

73. Streptococcus colonies on serum agar . 339

74. Streptococcus colonies from blood culture on blood-agar plate .... 345

75. Pneumococci . 354

76. Pneumococci . 354

77. Meningococcus, pure culture . 372

78. Meningococcus in spinal fluid . 373

79. Meningococcus culture (streak plate) . . 375

80. Gonococcus pus from urethra . . 381

LIST OF ILLUSTRATIONS xv

FIGURE PAGE

81. Gonococcus . 382

82. Gonococcus colony . 383

83. Bacillus coli communis . 390

84. Bacillus coli communis on various media ........... 396

85. Bacillus coli communior on various media ........... 397

86. Bacillus typhosus . 400

87. Bacillus typhosus, showing flagella . 401

88. Surface colony of Bacillus typhosus on gelatin . 402

89. Bacillus coli; deep colonies on Hiss plate medium . 407

90. Bacillus typhosus; deep colonies in Hiss plate medium . 408

91. Bacillus typhosus; colony in Hiss plate medium, highly magnified . . . 409

92. Colon and typhoid colonies in Hiss plate medium . . 410

93. Scheme of fermentations of the dygentery-typhoid-colon-bacilli in carbo¬

hydrate serum-water media . 445

94. Bacillus mucosus capsulatus . 448

95. Bacilli of rhinoscleroma . 452

96. Bacillus tetani . 457

97. Young tetanus culture in glucose agar . 458

98. Older tetanus culture in glucose agar . 459

99. Bacillus of symptomatic anthrax . 466

100. Bacillus of symptomatic anthrax, culture in glucose agar . 467

101. Bacillus of malignant edema . 469

102. Bacillus of malignant edema, culture in glucose agar . 470

103. Tubercle bacilli in sputum . 480

104. Culture of Bacillus tuberculosis in flask of glycerin bouillon . 485

105. Bacillus diphtherise . . 513

106. Colonies of Bacillus diphtherise on glycerin agar . 518

107. Bacillus Hoffmanni . . 523

108. Colonies of Bacillus Hoffmanni on agar . 524

109. Bacillus xerosis . 525

110. Glanders bacillus . 529

111. Glanders bacilli in tissue . 531

112. Bacillus influenzae; smear from pure culture on blood agar . 537

113. Bacillus influenzae; smear from sputum . 538

114. Colonies of influenza bacillus on blood agar . 539

115. Koch- Weeks bacillus . 542

116. Bordet-Gengou bacillus . . 544

117. Morax-Axenfeld diplo-bacillus . 546

118. Bacillus pestis . 555

119. Bacillus pestis, involution forms .... ......... 556

120. Bacillus anthracis; pure culture on agar . 564

121. Bacillus anthracis, in kidney tissue . 565

122. Bacillus anthracis, in spleen tissue . . 566

123. Anthrax colony on gelatin . 567

124. Anthrax colony on agar . . 568

125. Bacillus subtilis . . . . 569

126. Cholera spirillum . . . . . 573

xvi LIST OF ILLUSTRATIONS

FIGURE PAGE

127. Cholera spirillum; stab cultures in gelatin, three days old . . . . . . . 586

128. Cholera spirillum; stab culture in gelatin, six days old . 586

129. Spirochaete pallida; smear from chancre . 594

130. Spirochaete pallida, in spleen of congenital syphilis . 600

131. Spirochaete pallida, in liver of congenital syphilis . 601

132. Spirochaete of relapsing fever . 605

133. Spirochaete of relapsing fever . 606

134. Spirochaete of relapsing fever . 607

135. Spirochaete of Dutton, African tick fever . 609

136. Smear from the throat of a case of Vincent’s angina . 611

137. Throat smear, Vincent’s angina . 612

138. Spirochaete gallinarum . 616

139. Cladothrix, showing false branching . 620

140. Streptothrix, showing true branching . 621

141. Actinomyces granule crushed beneath a cover-glass . 624

142. Actinomyces granule crushed beneath a cover-glass . 625

143. Branching filaments of actinomyces . 626

144. Yeast cells . 630

145. Mucor mucedo . . 636

146. Mucor mucedo . 637

147. Mucor mucedo . 638

148. Mucor ramosus . 639

149. Penicillium glaucum . . 640

150. Aspergillus glaucus . 641

151. Thrush . 642

152. Achorion Schoenleinii . 643

153. Method of drying spinal cord of rabbit for purposes of attenuation . . . 653

154. Stegomyia fasciata . 671

155. Bacillus bulgaricus . . 714

SECTION I

THE GENERAL BIOLOGY OF BACTERIA AND THE
TECHNIQUE OF BACTERIOLOGICAL STUDY

CHAPTER I

THE DEVELOPMENT AND SCOPE OF BACTERIOLOGY

As we trace back to their ultimate origins the lines of development
of living beings of the animal and plant kingdoms, we find them con¬
verging toward a common type, represented by a large group of uni¬
cellular organisms, so simple in structure, so unspecialized in function,
that their classification in either the realm of plants or that of animals
becomes little more than an academic question. However, even such
microorganisms, in which the functions of nutrition, respiration, loco¬
motion, and reproduction are concentrated within the confines of a
single cell, and in which adaptation to special conditions more readily
brings about modifications leading to the production of a multitude of
delicately graded transitional forms, fall into groups which, either in
structure or in biological attributes show evidence of a tendency
toward one or the other of the great kingdoms.

Most important of these unicellular forms, for the student of medical
science, are the bacteria and the protozoa.

The former, by reason of their undifferentiated protoplasm, their
occasional possession of cellulose membranes, their biological tendency
to synthetize, as well as to break down organic compounds, and because
of the transitional forms which seem to connect them directly with the
lower plants, are generally placed in the plant kingdom. The latter,
chiefly on the basis of metabolism, are classified with the animals.

Knowledge of the existence of microorganisms as minute as the
ones under discussion, was of necessity forced to await the perfection of
instruments of magnification. It was not until the latter half of the
seventeenth century, therefore, that the Jesuit, Kircher, in 1659, and
the Dutch linen-draper, van Leeuwenhoek, in 1675, actually saw and
2 1

BIOLOGY AND TECHNIQUE

described living beings too small to be seen with the naked eye. There
can be no doubt that the small bodies seen by these men and their many
immediate successors were, at least in part, bacteria. And indeed the
descriptions and illustrations of several of the earliest workers cor¬
respond with many of the forms which are well known to us at the
present day.

During the century following the work of these pioneers, the efforts
of investigators lay chiefly in the more exact morphological description
of some of the forms of unicellular life, already known. Conspicuous
among the work of this period is that of Otto Friedrich Muller. In the
generation following Muller’s work, however, a marked advance in the
study of these forms was made by Ehrenberg,1 who established a
classification which, in some of its cardinal divisions, is retained until
the present day.

Meanwhile the regularity with which these “animalcula” or “in¬
fusion animalcula ” were demonstrable in tartar from the teeth, in intes¬
tinal contents, in well-water, etc., had begun to arouse in the minds of
the more advanced physicians of the time a suspicion as to a possible
relationship of these minute forms with disease. The conception of
“contagion,” or transmission of a disease from one human being to
another, was, however, even at this time, centuries old. The fact had
been recognized by Aristotle, had been reiterated by medieval philos¬
ophers, and had led, in 1546, to the division of contagious diseases by
Fracastor, into those transmitted “per contactum,” and those con¬
veyed indirectly “per fomitem.” It was for these mysterious facts of
the transmissibility of disease, that clinicians of the eighteenth century,
with remarkable insight, saw an explanation in the microorganisms dis¬
covered by Leeuwenhoek and his followers.

In fact, Plenciz of Vienna, writing in 1762, not only expressed
a belief in the direct etiological connection between microorganisms
and some diseases, but was the first to advance the opinion that each
malady had its own specific causal agent, which multiplied enormously
in the diseased body. The opinions of this author, if translated into
the language of our modern knowledge of the subject, came remark¬
ably near to the truth, not only as regards etiology and transmission,
but also in their suggestion of a specific therapy for each disease.

The conception of a “contagium vivum ” was thus practically es¬
tablished with the work of Plenciz and many others who followed in

1 “ Die Infusionstierchen/’ etc., Leipzig. 1838.

DEVELOPMENT AND SCOPE OF BACTERIOLOGY

his train, but the astonishingly shallow impression which the acute
reasoning of these men left upon the medical thought of their day
furnishes an excellent example of the futility of the most penetrating
speculation when unsupported by experimental data.

The real advancement in the scientific development of the subject
was destined to be carried on along entirely different lines. In 1837,
Schwann, a botanist, showed that the yeasts, found in fermenting sub¬
stances, were living beings, which bore a causal relationship to the proc¬
ess of fermentation. At almost the same time, similar observations were
made by a French physicist, Cagniard-Latour. The opinions advanced
by these men on the nature of fermentation aroused much interest
and discussion, since, at that time and for a long period thereafter,
fermentation was ascribed universally toproteid decomposition, a process
which was entirely obscure but firmly believed to be of a purely chemical
nature.

Although belief in the discovery of Schwann did not completely
master the field until after Pasteur had completed his classical studies
upon the fermentations occurring in beer and wine, yet the conception
of a “ fermentum vivum ” aroused much speculation, and the attention
of physicians and scientists was attracted to the many analogies ex¬
isting between phenomena of fermentation and those of disease.

The conception of such an analogy, however, was not a new thought
in the philosophy of the time. Long before Schwann and Cagniard-
Latour, the philosopher Robert Boyle, working in the seventeenth
century, had prophesied that the mystery of infectious diseases would be
solved by him who should succeed in elucidating the nature of fermenta¬
tion.

Nevertheless, the diligent search for microorganisms in relation to
various diseases which followed, led to few results, and the successes
which were attained were limited to the diseases caused by some of
the larger fungi, favus (1839), thrush (1839), and pityriasis versicolor
(1846). During this time of ardent but often poorly controlled etiolog¬
ical research, it was Henle who formulated the postulates of conserva¬
tism, almost as rigid as the later postulates of Koch, requiring that
proof of the etiological relationship of a microorganism to a disease
could not be brought merely by finding it in a lesion of the disease, but
that constant presence in such lesions must be proven and isolation and
study of the microorganism away from the diseased body must be car¬
ried out.

It was during this period also that one of the most fundamental

BIOLOGY AND TECHNIQUE

questions, namely, that of the origin of these minute living beings, was
being discussed with much passion by the scientific world. It was held
by the conservative majority that the microorganisms described by
Leeuwenhoek and others after him, were produced by spontaneous
generation. The doctrine of spontaneous generation, in fact, was
solidly established and sanctified by tradition, and had been applied

in the past not alone to microorganisms.1 And it must not be forgotten

♦

that without the aid of our modern methods of study, satisfactory
proof for or against such a process was not easily brought.

Needham, who published in 1749, had spent much time in fortify¬
ing his opinions in favor of spontaneous generation by extensive ex¬
perimentation. He had placed putrefying material and vegetable in¬
fusions in sealed flasks, exposing them for a short time to heat, by
immersing them in a vessel of boiling water, and had later shown them
to be teeming with microorganisms. He was supported in his views
by no less an authority than Buffon. The work of Needham, however,
showed a number of experimental inaccuracies which were thoroughly
sifted by the Abbe Spallanazani. This investigator repeated the ex¬
periments of Needham, employing, however, greater care in sealing his
flasks, and subjecting them to a more thorough exposure to heat.
His results did not support the views of Needham, but were answered
by the latter with the argument that by excessive heating he had pro¬
duced chemical changes in his solutions which had made spontaneous
generation impossible.

The experiments of Schulze, in 1836, who failed to find living organ¬
isms in infusions which had been boiled, and to which air had been
admitted only after passage through strongly acid solutions, and similar
results obtained by Schwann, who had passed the air through highly
heated tubes, were open to criticism by their opponents, who claimed
that chemical alteration of the air subjected to such drastic influences,
had been responsible for the absence of bacteria in the infusion. Similar
experiments by Schroeder and Dusch, who had stoppered their flasks
with cotton plugs, were not open to this objection, but had also failed to
convince. The question was not definitely settled until the years im-

1 Valleri-Radot, in his life of Pasteur, stated that Van Helmont, in the six¬
teenth century, had given a celebrated prescription for the creation of mice
from dirty linen and a few grains of wheat or pieces of cheese. During the centu¬
ries following, although, of course, such remarkable and amusing beliefs no longer
held sway, nevertheless the question of spontaneous generation of minute and
structureless bodies, like the bacteria, still found learned and thoughtful partisans.

DEVELOPMENT AND SCOPE OF BACTERIOLOGY

mediately following 1860, when Pasteur conducted a series of experi¬
ments which were not only important in incontrovertibly refuting the
doctrine of spontaneous generation, but in establishing the principles
of scientific investigation which have influenced bacteriological re¬
search since his time.* 1

Pasteur attacked the problem from two points of view. In the
first place he demonstrated that when air was filtered through cotton¬
wool, innumerable microorganisms were deposited upon the filter. A
single shred of such a contaminated filter dropped into a flask of pre¬
viously sterilized nutritive fluid, sufficed to bring about a rapid and
luxuriant growth of microorganisms. In the second place, he succeeded
in showing that similar, sterilized “ putrescible ” liquids, if left in con¬
tact with air, would remain uncontaminated provided that the en¬
trance of dust particles were prohibited. This he succeeded in doing by
devising flasks, the necks of which had been drawn out into fine tubes
bent in the form of a U. The ends of these U-tubes, being left open,
permitted the sedimentation of dust from the air as far as the lowest
angle of the tube, but, in the absence of an air. current, no dust was
carried up the second arm into the liquid. In such flasks, he showed
that no contamination took place but could be immediately induced
by slanting the entire apparatus until the liquid was allowed to run
into the bent arm of the U-tube. Finally, by exposing a series of
flasks containing sterile yeast infusion, at different atmospheric levels,
in places in which the air was subject to varying degrees of dust con¬
tamination, he showed an inverse relationship between the purity of
the air ancl the contamination of his flasks with microorganisms.

The doctrine of spontaneous generation had thus received its final
refutation, except in one particular. It was not yet clear why com¬
plete sterility was not always obtained by the application of definite
degrees of heat. This final link in the chain of evidence was supplied,
some ten years later, by Cohn, who, in 1871, was the first to observe and
correctly interpret bacterial spores and to demonstrate their high powers
of resistance against heat and other deleterious influences.

1 In a letter to his foremost opponent, at this period, Pasteur writes: “In
experimental science, it is always a mistake not to doubt when facts do not compel
affirmation. ”

The critical spirit pervading the scientific thought of that time in France is
also well expressed by Oliver Wendell Holmes, who said that he had learned three
things in Paris: “Not to take authority when I can have facts, not to guess when

I can know, and not to think that a man must take physic because he is sick.”

BIOLOGY AND TECHNIQUE

Meanwhile, Pasteur, parallel with his researches upon spontaneous
generation, had been carrying on experiments upon the subject of
fermentation along the lines suggested by Cagniard-Latour. As a
consequence of these experiments, he not only confirmed the opinions
both of this author and of Schwann concerning the fermentation of beer
and wine by yeasts, but was able to show that a number of other fer¬
mentations, such as those of lactic and butyric acid, as well as the de¬
composition of organic matter by putrefaction, were directly due to the
action of microorganisms. It was the discovery of the living agents
underlying putrefaction, especially, which exerted the most active
influence upon the medical research of the day. This is illustrated by
Lister’s work. The suppurative processes occurring in infected wounds
had long been regarded as a species of putrefaction, and Lord Lister,
working directly upon the premises supplied by Pasteur, introduced
into both the active and prophylactic treatment of surgical wounds,
the antiseptic principles which alone have made modern surgery possible.

There now followed a period in which bacteriological investigation
was concentrated upon problems of etiology. Stimulated by Pasteur’s
successes, the long-cherished hope of finding some specific microorgan¬
ism as the causal agent in each infectious disease was revived.

Pollender, in 1855, had reported the presence of rod-shaped bodies
in the blood and spleen of animals dead of anthrax. Brauell, several
years later, had made similar observations and had expressed definite
opinions as to the causative relationship of these rods to the disease.
Convincing proof, however, had not been brought by either of these
observers. Finally, in 1863, Davaine, in a series of brilliant investi¬
gations, not only confirmed the observations of the two authors men¬
tioned above, but succeeded in demonstrating that the disease could
be transmitted by means of blood containing these rods and could never
be transmitted by blood from which these rods were absent. Anthrax,
thus, is the first disease in which definite proof of bacterial causation
was brought.

Speaking before the French Academy of Medicine at this time,
Davaine suggested that the manifestations of the disease might in
reality represent the results of a specific fermentation produced by the
bacilli he had found. This, in a crude way, expresses the modern
conception of infectious disease.

Within a few years after this, 1868, the adherents of the parasitic
theory of infectious diseases were further encouraged by the discovery,
by Obermeier, of a spirillum in the blood of patients suffering from

DEVELOPMENT AND SCOPE OE BACTERIOLOGY

relapsing fever. It is not surprising that the successes attained in these
diseases, fostering hope of analogous results in all other similar condi¬
tions, but without the aid of adequate experimental methods, should
have led to many unjustified claims and to much fantastic theorizing.
Thus Hallier, at about this time, advanced a theory as to the etiology
of infectious diseases, in which he attributed all such conditions to the
moulds or hyphomycetes, regarding the smaller form or bacteria as
developmental stages of these more complicated forms. Extravagant
conjectures of this kind, however, did not maintain themselves for any
length of time in the light of the critical attitude which was already
pervading bacteriological research.

Progress was made during the years immediately following, chiefly
in the elucidation of suppurative processes. Rindfleisch, von Reckling¬
hausen, and Waldeyer, almost simultaneously, described bodies which
they observed in sections of tissue containing abscesses, and which they
believed to be microorganisms. Notable support was given to their
opinion by similar observations made upon pus by Klebs, in 1870. In
view, however, of the purely morphological nature of their studies, the
opinions of these observers did not entirely prevail. Satisfactory
methods of cultivation and isolation had not yet been developed, and
Billroth and his followers, with a conservatism entirely justified under
existing conditions, while admitting the constant presence of bacteria
in purulent lesions, denied their etiological significance. The contro¬
versy that followed was rich in suggestions which greatly facilitated
the work of later investigators, but could not be definitely settled until
1880, when Koch introduced the technical methods which have made
bacteriology an exact science. By the use of solid nutritive media, the
isolation of bacteria and their biological study in pure culture were made
possible. At about the same time the use of anilin dyes, developed
by Weigert, Koch, and Ehrlich, was introduced into morphological study
and facilitated the observation of the finer structural details which had
been unnoticed while only the grosser methods employed for tissue
staining had been available.

With the publication of Koch’s work, there began an era unusually
rich in results held in leash heretofore by inadequate technical methods.
The discovery of the typhoid bacillus in 1880, of the bacillus of fowl
cholera and the pneumococcus in the same year, and of the tubercle
bacillus in 1882, initiated a series of etiological discoveries which, ex¬
tending over not more than fifteen years, elucidated the causation
of a majority of the infectious diseases.

BIOLOGY AND TECHNIQUE

Coincident with the elucidation of etiological facts began the inquiry
into that field which is now spoken of as the science of immunity. The
phenomena which accompany ’the development of insusceptibility
to bacterial infections in man and in animals, first studied by Pasteur,
have become the subject of innumerable researches and have led to
results of the utmost practical value.

The problems which were encountered were first studied from a
purely bacteriological point of view, but their solution has shed light
upon biological principles of the broadest application. Investigations
into the properties of immune sera, while making bacteriology one of
the most important branches of diagnostic and therapeutic medicine,
have, at the same time, inseparably linked it with physiology and
experimental pathology.

By the revelations of etiological research, and by the study of the
biological properties of pathogenic bacteria, contagion, an enemy hitherto
unseen and mysterious, was unmasked, and rational campaigns of public
sanitation and personal hygiene were made possible. Upon the same
elucidations has depended the development of modern surgery — a
science which without asepsis and antisepsis would have been doomed
to remain in its medieval condition.

Apart from its importance in the purely medical sciences, the study
of the bacteria has shed beneficial light, moreover, upon many other
fields of human activity. In their relationship to decomposition, the
conditions of the soil, and to diseases of plants, the bacteria have been
found to occupy a position of great importance in agriculture. Knowl¬
edge of bacterial and yeast ferments, furthermore, has become the scien¬
tific basis of many industries, chiefly those concerned in the production
of wine, beer, and dairy products.

The scope of bacteriology is thus a wide one, and none of its various
fields has, as yet, been fully explored. The future of the science is rich
in allurement of interest, in promise of result, and in possible benefit
to mankind.

CHAPTER II

GENERAL MORPHOLOGY, REPRODUCTION, AND CHEMICAL
AND PHYSICAL PROPERTIES OF THE BACTERIA

Bacteria are exceedingly minute unicellular organisms which maj
occur perfectly free and singular, or in larger or smaller aggregations,
thus forming multicellular groups or colonies, the individuals of which
are, however, physiologically independent.

The cells themselves have a number of basic or ground shapes which
may be roughly considered in three main classes: The cocci or spheres,
the bacilli or straight rods, and the spirilla or curved rod forms.

The cocci are, when fully developed and free, perfectly spherical.
When two or more are in apposition, they may be slightly flattened along
the tangential surfaces, giving an oval appearance.

The bacilli, or rod-shaped forms, consist of elongated cells whose
long diameter may be from two to ten times as great as their width,
with ends squarely cut off, as in the case of bacillus anthracis, or gently
rounded as in the case of the typhoid bacillus.

The spirilla may vary from small comma-shaped microorganisms,
containing but a single curve, to longer or more sinuous forms which
may roughly be compared to a corkscrew, being made up of five, six,
or more curves. The turns in the typical microorganisms of this class are
always in three planes and are spiral rather than simply curved.

Among the known microorganisms, the bacilli by far outnumber
other forms, and are probably the most common variety of bacteria in
existence. Many variations from these fundamental types may occur
even under normal conditions, but contrary to earlier opinions it is
now positively known that cocci regularly reproduce cocci, bacilli
bacilli, and spirilla spirilla, there being, as far as we know, no mutation
from one form into another.

The size of bacteria is subject to considerable variation. Cocci may
vary from .15 y to 2 . y in diameter. The average size of the ordinary
pus coccus varies from .8 p to 1.2 y in diameter. Fischer has given a
graphic illustration of the size of a staphylococcus by calculating that
one billion micrococci could easily be contained in a drop of water hav-

BIOLOGY AND TECHNIQUE

ing a volume of one cubic millimeter. Among the bacilli tne range of
size is subject to even greater variations. Probably the smallest of the
common bacilli is the bacillus of influenza which measures about .5 /i in
length by .2 y in thickness. The limit of the optical possibilities of the
modern microscope is almost reached by some of the known micro¬
organisms, and it is not at all out of question that some of the diseases,
for which, up to the present time, no specific microorganisms have

Fig. 1. — Types of Bacterial Morphology.

been found, may be caused by bacteria so small as to be invisible by any
of our present methods. In fact, the virus causing the peripneumonia of
cattle has been shown to pass through the pores of a Berkefeld filter,
which are impenetrable to the smallest of the known bacteria.1

MORPHOLOGY OF THE BACTERIAL CELL

When unstained, most bacteria are transparent, colorless, and ap¬
parently homogeneous bodies with a low refractive index. The cells
themselves consist of a mass of protoplasm, surrounded, in most in¬
stances, by a delicate cell membrane.

The presence of a nucleus 2 in bacterial cells, though denied by the
earlier writers, has been demonstrated beyond question by Zettnow,
Nakanishi,3 and others. The original opinion of Zettnow was that the
entire bacterial body consisted of nuclear material intimately inter¬
mingled with the cytoplasm. The opinion now held by most observers

1 Nocard and Roux, Ann. Past., 12, 1898.

2 A. Fischer, Jahrbiicher f. wissen. Botanik, xxvii.

3 Nakanishi, Munch, med. Woch., vi, 1900.

MORPHOLOGY, REPRODUCTION, ETC.

who have studied this phase of the subject favors the existence of an
ectoplasmic zone which includes cell membrane and flagella, but is
definitely a part of the cytoplasm, and an entoplasm in which is con¬
centrated the nuclear material. Biitechli 1 claims to have demonstrated
within this entoplasmic substance a reticular meshwork, between the
spaces of which lie granules of chromophilic or nuclear material.
Confirmation of this opinion has been brought by Zettnow2 and others.
Nakanishi, working with a special staining method, asserts that some
microorganisms show within the entoplasmic zone a well-defined,
minute, round or oval nucleus, which possesses a definitely charac¬
teristic staining reaction.3

In the bodies of a large number of bacteria, notably in those of the
diphtheria group, Ernst,4 Babes,5 and others have demonstrated
granular, deeply staining bodies now spoken of as rnetachromatic granules,
or Babes-Ernst granules, or, because of their frequent position at the ends
of bacilli, as polar bodies. These structures are irregular in size and
number, and have a strong affinity for dyes. They are stained dis¬
tinctly dark in contrast to the rest of the bacterial cell with methylene
blue, and may be demonstrated by the special methods of Neisser and
of Roux.6 Their interpretation has been a matter of much difficulty
and of varied opinion. Those who first observed them held that they
were a part of the nuclear material of the cell. Others have regarded
them as an earty stage in spore formation, or as arthrospores.7 Again,
they have been interpreted as structures comparable to the centrosomes
of other unicellular forms. As a matter of fact, the true nature of these
bodies is by no means certain. They are present most regularly in
microorganisms taken from young and vigorous cultures or in those
taken directly from the lesions of disease. It is unlikely that they repre-

1 Butschli, “ Bau der Bakterien,” Leipzig, 1890. 2 Zettnow, Zeit. f. Hyg., xxiv, 1897.

3 The method of Nakanishi is carried out as follows: Thoroughly cleansed
slides are covered with a saturated aqueous solution of methylene blue. This is
spread over the slide in an even film and allowed to dry. After drying, the slide
should be of a transparent, sky-blue color. The microorganisms to be examined are
then emulsified in warm water, or are taken from the fluid media, and dropped upon
a cover slip. This is placed, face downward, upon the blue ground of the slide. In
this way, bacteria are stained without fixation. Nakanishi claims that by this
method the entoplasm is stained blue, while the nuclear material appears of a reddish

or purplish hue.

4 Ernst, Zeit. f. Hyg., iv, 1888. 5 Babes, Zeit. f. Hyg., v, 1889.

6 See section on stains, p. 107. 7 See section on sporulation, p. 16.

BIOLOGY AND TECHNIQUE

sent structures in any way comparable to spores, since cultures con¬
taining individuals showing metachromatic granules are not more
resistant to deleterious influences than are others. Their abundant
presence in young vigorous cultures may indicate a relationship between
them and the growth energy of the microorganisms. There is no proof,
however, that these bodies affect the virulence of the bacteria.

Cell Membrane and Capsule. — Actual proof of the existence of a cell
membrane has been brought in the cases of some of the larger forms
only,1 but the presence of such envelopes may be inferred for most

bacteria by their behavior during
plasmolysis, where definite retrac¬
tion of the protoplasm from a
well-defined cell outline has been
repeatedly observed. The occur¬
rence, furthermore, of so-called
“shadow forms” which appear as
empty capsules, and of, occasion¬
ally, a well-outlined cell body,
after the vegetative form has en¬
tirely degenerated in the course
of sporulation, make the assump¬
tion of the presence of a cell
membrane appear extremely well
founded. Differing from the cell
membranes of plant cells, cellulose
has not, except in isolated instances, been demonstrable for bacteria,
and the membrane is possibly to be regarded rather as a peripheral
protoplasmic zone, which remains unstained by the usual manipula¬
tions. Zettnow,2 who has carefully studied the structure of some of
the larger forms, takes the latter view, and regards the “ectoplasmic”
zone as a part of the cell protoplasm devoid of nuclear material. Zett-
now’s opinion is borne out by the greatly increased size of the bacterial
cells as seen by means of special stains.

Many bacteria have been shown to possess a mucoid or gelatinous
envelope or capsule . According to Migula,3 such an envelope is present
on all bacteria, though it is in only a few species that it is sufficiently
well developed and stable to be easily demonstrable and of differential

Fig. 2. — Bacterial Capsules.

1 Biitschli, loc. cit. 2 Zettnow , loc. cit.

3 Migula, “Systeme d. Bakterien,” 1, p. 56.

MORPHOLOGY, REPRODUCTION, ETC.

value. When stained, the capsule takes the ordinary anilin dyes less
deeply than does the bacterial cell body, and varies greatly in thickness,
ranging from a thin, just visible margin to dimensions four or five
times exceeding the actual size of the bacterial body itself. This struc¬
ture is perfectly developed in a limited number of bacteria only in which
it then becomes an important aid to identification. Most prominent
among such bacteria are Diplococcus pneumoniae, Micrococcus tetra-
genus, the bacilli of the Friedlander group, and B. aerogenes capsulatus.
The development of the capsule seems to depend intimately upon the
environment from which the bacteria are taken. It is most easily de¬
monstrable in preparations of bacteria taken directly from animal tis¬
sues and fluids, or from media containing animal serum or milk. If
cultivated for a prolonged period upon artificial media, many otherwise
capsulated microorganisms no longer show this characteristic structure.

Capsules may be demonstrated on bacteria taken from artificial
media most successfully when albuminous substances, such as ascitic
fluid or blood serum, are present in the culture media, or when the
bacteria are smeared upon cover slip or slide in a drop of beef or other
serum,1 Most observers believe that the capsule represents a swelling
of the ectoplasmic zone of bacteria. By others it is regarded as an
evidence of the formation of a mucoid intercellular substance, some of
which remains adherent to the individual bacteria when removed from
cultures. It is noticeable, indeed, that some of the capsulated bacteria,
especially Streptococcus mucosus and B. mucosus capsulatus, develop
such slimy and gelatinous colonies that, when these are touched with a
platinum wire, mucoid threads and strings adhere to the loop. Exactly
what the significance of the capsules is cannot yet be decided.

There is, however, definite reason to believe that there is a direct
relation between virulence and capsulation; capsulated bacteria are
less easily taken up by phagocytes than are the non-capsulated mem¬
bers of the same species. Also, as Porges and others have shown,
capsulated organisms are not easily amenable to the agglutinating action
of immune sera. Many bacteria (plague, anthrax) which are habitu¬
ally uncapsulated on artificial media acquire capsules within the in¬
fected animal body. Also in some species (pneumococci), the loss
of capsule formation as cultivated on the simpler media is accompanied
by a diminution of virulence.

Organs of Locomotion. — When suspended in a drop of fluid many
bacteria are seen to be actively motile. It is important, however, in

1 Hiss , Jour. Exp. Med., vi, 1905.

BIOLOGY AND TECHNIQUE

all cases to distinguish between actual motility and the so-called Brown¬
ian or molecular movement which takes place whenever small particles
are held in suspension in a fluid.

Brownian or molecular movement is a phenomenon entirely ex¬
plained by the physical principles of surface tension, and has absolutely
no relation to independent motility. It may be seen when particles of
carmine or any other insoluble substance are suspended in water, and
consists in a rapid to and fro vacillation during which there is actually
no permanent change in position of the moving particle except inas¬
much as this is influenced by currents in the drop.

The true motility of bacteria, on the other hand, is active motion
due to impulses originating in the bacteria themselves, where the actual
position of the bacterium in the field is permanently changed.

The ability to move in this way is, so far as we know, limited almost
entirely to the bacilli and spirilla, there being but few instances where
members of the coccus group show active motility. In all cases, with
the exception of some of the spirochetes, where motility may occasionally
be due to an undulating membrane marginally placed along the body,
bacterial motility is due to hair-like organs known as flagella. These
flagella have rarely been seen during life, and their recognition and study
has been made possible only by special staining methods, such as those
devised by Loeffler, van Ermengem, Pitt, and others.

In such stained preparations, the bacterial cell bodies often appear
thicker than when ordinary dyes are used, and the flagella apparently
are seen to arise from the thickened ectoplasmic zone.

The flagella are long filaments, averaging in thickness from one-tenth
to one-thirtieth that of the bacterial body, which often are delicately
waved and undulating, and, judging from the positions in which they
become fixed in preparations, move by a wavy or screw-like motion.
In length they are subject to much variation, but are supposed to be
generally longer in old than in young cultures. Very short flagella have
been described only on nitrosomonas, one of the nitrifying bacteria
discovered by Winogradsky.1

As to the finer structures of flagella, little can be made out except
that they possess a higher refractive index than the cell body itself,
and that they can be stained only with those dyes which bring
clearly into view the supposedly true cytoplasm of the cell.
Whether they penetrate this cytoplasmic membrane or whether they

1 Winogradsky, Arch, des sci. biologiques, St. Petersburg, 1892, I, 1 and 2.

MORPHOLOGY, REPRODUCTION, ETC.

are a direct continuation of this peripheral zone of the bacterial
body, can not be decided.

The manner in which bacteria move is naturally subject to some var¬
iation depending upon the number and position of the flagella possessed
by them. Whether bacteria exercise or not the power of motility de¬
pends to a large extent upon their present or previous environment.
They are usually most motile in vigorous young cultures of from twenty-
four to forty-eight hours' growth in favorable media. In old cultures
motility may be diminished or even inhibited by acid formation or by
other deleterious products of the bacterial metabolism.

At the optimum growth-temperature motility is most active, and a
diminution or increase of the temperature
to any considerable degree diminishes or
inhibits it. Thus actively motile organisms^
in the fluid drop, may be seen to diminish
distinctly in activity when left for any
prolonged time in a cold room, or when
the preparation is chilled. Any influence,
in other words, chemical or physical, which
tends to injure or depress physiologically the bacteria in any way, at
the same time tends to inhibit their motility.

Messea1 has proposed a classification of bacteria which is based
upon the arrangement of their organs of motility, as follows:

I. Gymnobacteria, possessing no flagella.

II. Trichobacteria, with flagella.

1. Monotricha, having a single flagellum at one pole.

2. Lophotricha, having a tuft of flagella at one pole.

3. Amphitricha, with flagella at both poles.

4. Peritricha, with flagella completely surrounding the bac¬

terial body.

Bacterial Spores. — A large number of bacteria possesses the power of
developing into a sort of encysted or resting stage by a process commonly
spoken of as sporulation or spore formation. The formation of spores
by bacteria depends largely upon environmental conditions, and the
optimum environment for spore formation differs greatly for various
species. It is usually necessary that a temperature of over 20° C.
exist in order that spores may be formed. Unfavorable factors, like
acid formation, accumulation of bacterial products in old cultures, or

Fig. 3. — Arrangement of
Bacterial Flagella.

1 Messea, Cent. f. Bakt., I, Ref. ix, 1891.

BIOLOGY AND TECHNIQUE

lack of nutrition, frequently seem to constitute the stimuli which lead
to sporulation. In the case of some species, notably the anthrax bacillus,
spores are formed only in the presence of free oxygen and are therefore
never formed within the tissues of infected animals. It is claimed that
some of the pathogenic anaerobes, like B. tetani and the bacillus of
malignant edema, may form spores anaerobically. Nevertheless it has
been observed that when an absolute exclusion of oxygen is practiced
in the cultivation of these bacteria, vegetative forms only are seen in
the cultures.1

The process of sporulation is by no means to be regarded as
a method of multiplication, since it rarely occurs that a single bacil¬
lus produces more than one spore. In some species of bacteria the
formation of several spores in one individual has occasionally been
observed, but there can be no question about the fact that such a
condition is exceptional.

Varieties of spores are often recognized, the so-called arthrospores
and the true spores or endospores. It is seriously in doubt whether the
structures once spoken of as arthrospores should be considered as in any
way comparable to true spores. They are represented by the granular
and globular appearances occasionally observed in old cultures of some
bacteria, notably streptococcus, cholera spirillum, diphtheria bacillus,
and others. It was believed that they were due to a transformation of
certain individuals of the cultures into more resistant forms. It is
probable, however, that such structures are merely to be regarded as
evidences of involution or degeneration, since it has never been demon¬
strated that cultures containing them are more resistant either to dis¬
infectants or to heat, than cultures showing no evidences of such forms.
The true spores or endospores are most common among bacilli, and
are rarely observed among the spherical bacteria. They arise within
the body of the individual bacterium as a small granule which probably
represents a concentration of the protoplasmic substance. Nakanishi2
claims that there is a definite relation between these sporogenic globules
and the nuclear material of the bacterial cell. At the time at which
sporulation occurs there is usually a slight and gradual thickening of the
bacillary body. After the formation of this thickening, a spore mem¬
brane appears about the same thickened area. The completed spore is
usually round or oval, has an extremely high refractive index, and a

1 Zinsser, Jour. Exp. Med., viii, 1906, p. 542.

2 Nakanishi, Munch, med. Woch., 1900, p. 680.

MORPHOLOGY, REPRODUCTION, ETC.

membrane which is very resistant. Muhlschlegel 1 believes that the spore
membrane is a double structure, and, as stated before, Nakanishi believes
that the spore contains nuclear material.

The position of the spore in the mother cell is of some differential
importance in that it is usually con¬
stant for -one and the same species.

Thus, the spores of the tetanus
bacillus are regularly situated at
the _ extreme ends of the bacillary Fiq_ 4__Various Positions'of Spoees
bodies, while those of anthrax are IN Bacterial Cell.

situated at or near the middle.

Physiologically, sporulation is probably to be regarded as a method
of encystment for the purpose of resisting unfavorable environment,

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and it is indeed true that species
of bacteria the vegetative forms of
which are rather easily injured by
heat, light, drying, and chemicals
have a comparatively enormous re¬
sistance to these agents after the
formation of spores. Thus, while a
10-per-cent solution of carbolic acid
will kill the vegetative forms of
anthrax bacilli within twenty min¬
utes, anthrax spores are able to resist
the same disinfectant for a long
period in a concentration of over 50
per cent; and while the vegetative
forms of the same bacillus show little
more resistance against moist heat
vegetative

a O &0

Fig. 5.— Germination of Spores. than other vegetative forms, the
A, Bacillus subtilis, equatorial spore spores will withstand the action of
germination; B, Bacillus anthracis, live steam for as long as ten to twelve
germination by simple transition; C, minutes and more

Clostrydium butyricum, polar germi- -rTT1 £

.. Whenever the spores ol any mi-

nation. . .

croorgamsm are brought into an en¬
vironment suitable for bacterial growth as to temperature, moisture,
and nutrition, the spores develop into vegetative forms. This process
differs according to species. In general it consists of an elongation of

1 Muhlschlegel, Cent. f. Bakt., II Abt., vi, 1900, p. 65.

BIOLOGY AND TECHNIQUE

the spore body with a loss of its highly retractile character and resist¬
ance to staining fluids. The developing vegetative cell may now
rupture and slip out of the spore membrane at one of its poles, leav¬
ing the empty spore capsule still visible and attached to the bacillary
body. Again, a similar process may take place equatorially instead
of at the pole. In other species again, there may be no rqpture of
the spore membrane at all, the vegetative form arising by gradual
elongation of the spore and an absorption or solution of the mem¬
brane which is indicated by change in staining reaction. Division by
fission in the ordinary way then ensues.

REPRODUCTION OP BACTERIA

Bacteria multiply by cell division or fission. A young individual
increases in size up to the limits of the adult form, when, by simple
cleavage, at right angles to the long axis, without any discoverable
process of mitosis or nuclear changes, it divides into two individuals.
In spite of the claims of various bacteriologists, notably Nakanishi, 1
traceable analogy to the karyokinesis of other cells has not been
definitely established. In the case of the spherical bacteria a slight
change to the elliptical form takes place just before cleavage and
this cleavage may occur in one plane only, in two planes, or in three
planes. According to the limitations of cleavage direction, the cocci
assume a chained appearance (streptococci), a grape-like appearance
(staphylococci), or an arrangement in packets or cubes having three
dimensions (sarcinse). In the cases of bacilli and spirilla, cleavage
takes place in the direction of the short axis. The individuals, after
cleavage, may separate from each other, or may remain mutually
coherent. The cohesion after cleavage is pronounced in some species of
bacteria and slight in others, and, together with the plane of cleavage,
determines the morphology of the cell-groups. Thus among the cocci
diplo- or double forms, long chains and short chains may arise and fur¬
nish a characteristic of great aid in differentiation. Similarly among
the bacilli there are forms which appear characteristically as single
individuals and others which form chains of varying length.

The rate of growth varies to a certain extent with the species, and
also with the favorable or unfavorable character of the environment.
A generation, that is, the time elapsing in the interval between one

1 Nakanishi, Cent. f. Bakt., I, xxx, 1901.

MORPHOLOGY, REPRODUCTION, ETC.

cleavage and the next, has been estimated by A. Fischer1 as being
about twenty minutes for the cholera spirillum and 16-20 minutes for
bacillus coli communis, under the most favorable conditions. The same
author has calculated that under these conditions a single cholera
spirillum would yield 1600 trillions in a single day. Such a multiplica¬
tion rate, however, is probably not usual under natural or even artificial
conditions, both on account of lack of nutritive material and because of
inhibition of the growth of the' bacteria by their own products.

VARIATIONS OF BACTERIAL FORMS

Variations from the basic forms considered in the preceding sec¬
tion may occur, but are not common among bacteria under normal
conditions. Thus the formation of club shapes by a thickening of the

Fig. 6. — Degeneration Forms of Bacillus Diphtheria. (After Zettnow.)

bacillary body at one or both ends has been frequently observed among
bacteria of the diphtheria group, and in the glanders bacillus, and an
irregular beading is not infrequently observed in tubercle bacilli under
normal conditions. Such pictures can not, in these cases, be regarded
as degeneration or involution forms, since they are visible in young,
actively growing cultures under ordinary conditions. It is a well-known

1 A. Fischer, “ Vorlesungen iiber Bakt.,” Jena, 1903.

BIOLOGY AND TECHNIQUE

fact, furthermore, that the sizes and contours of bacteria may vary to
some extent according to the medium on which they are grown. This
may, to a degree, be due to osmotic relations. On fluid media, for in¬
stance, many bacteria may appear larger and of a less dense consistency
than do members of the same species cultivated upon solid media.

Degeneration Forms. — When bacteria are grown under conditions
which are not entirely favorable for their development, or when they
are grown for a prolonged period upon artificial culture media without
transplantation, there may occur variations which often depart consider¬
ably from the ground type, known as degeneration or involution forms.

Fig. 7. — Degeneration Forms of Bacillus Pestis. (After Zettnow.)

Thus, in the case of the diphtheria bacillus, old cultures may contain
long, irregularly beaded forms with broad expansions at the ends.
In the case of B. pestis the fact that large numbers of oval, vacuolated
bodies in old cultures are formed regularly has become of differential
value.1 These degeneration forms are shown most characteristically
when the bacteria are cultivated on agar containing 3 to 5 per cent NaCl.

Among the cocci, marked evidences of involution are often seen in
cultures of the meningococcus in the form of large, swollen poorly-
staining spheres, and in the case of the pneumococcus in the so-called
shadow forms which have the appearance of empty capsules. There are

1 Hankin and Leumann, Cent. f. Bakt., I, xxii, 1897.

MORPHOLOGY, REPRODUCTION, ETC.

few microorganisms indeed, in which prolonged cultivation on artificial
media or other unfavorable influences do not produce variations from
the ground type which may often make the cultures morphologically
unrecognizable. In the case of many of the spirilla (spirillum Milleri,
spirillum Metchnikovi, etc.) the degeneration forms may appear within
so short a time as two or three days after transplantation.

CHEMICAL AND PHYSICAL PROPERTIES OF THE BACTERIAL CELL

Chemical Constituents. — -The quantitative chemical composition of
bacteria is subject to wide variations, dependent upon the nutritive
materials furnished them.

Approximately 80 to 85 per cent of the bacterial body is water.
The remainder consists chiefly of proteids which constitute roughly from
50 to 80 per cent of the dry substances. Remaining, after extrac¬
tion of these, are fats, and in some cases true wax (fatty acid combina¬
tions with higher alcohols) , traces of cellulose (in some bacteria only) ,
and the ash which makes up usually about 1 to 2 per cent of the dry
substances.'

The extensive researches of Cramer1 have shown how widely at va¬
riance quantitative analyses may be when made of cultures of the same
species of bacteria grown upon different media. Thus the dry sub¬
stances of the cholera vibrio were found to be made up of 65 per cent
of proteids when the microorganisms were grown upon nutrient broth
as against 45 per cent when the same bacteria had been grown upon
the proteid-free medium of Uschinsky. Analyses made by Kappes2 of
B. prodigiosus and by Nencki3 and Scheffer of some of the putrefactive
bacteria, may serve to illustrate the approximate proportions of the
substances making up the bacterial body.

B. Putrefactive

prodigiosus Bacteria

Water . 85 . 45 per cent. 83.42 per cent.

Proteids . 10.33 “ “ 13.96 “ “

Fats . 0.7 “ “ 1. “ “

Ash . 1.75 “ “ 0.78 “ “

Residue . 1.77 “ “ 0.84 “ “

1 Cramer, Arch. f. Hyg., xii, xiii, xvi, xxii, xxviii.

2 Kappes, “ Analyse der Massenkulturen,” etc. Diss., Leipzig, 1889.

s Nencld und Scheffer, Jour. f. prakt. Chemie, new ser. xix, 1880.

BIOLOGY AND TECHNIQUE

Analyses of the tubercle bacillus by Ruppel,1 Hammerschlag,2 Weyl,3
and others, have yielded the following approximate results (calculated
from results of above-mentioned authors).

Tubercle bacillus

Water . 85 to 86 percent.

Proteids . 8 . 5 to 9

Fat and waxes . 3 . 5 to 4

Ash and carbohydrates . 1.2 to 1.4 “

The proteids which are contained in the bacterial dry substances
consist partly of nucleoproteids, globulins, and proteids differing ma¬
terially from those ordinarily met with. Ruppel, in an analysis of
the tubercle bacillus, obtained the following values, for 100 grams of
dried tubercle bacilli:

Nucleic acid .

(Tuberculinic acid)

Nucleoprotamin .

Nucleoproteid .

Albuminoids .

(Keratin, etc.)

Fat and wax .

Ash .

8.5 grams.

25.5 “

23 “

8.3 “

26.5 “

9.2 “

A true globulin has been isolated from bacteria by Hellmich,4 and
true proteids, coagulable by heat, have been demonstrated by Buchner,5
in the “ Presssaft ” or juice obtained by subjecting bacteria to mechanical
pressure. In this connection, too, we should not fail to consider the
thermolabile toxic substances contained in many bacteria, the endo¬
toxins, which though of uncertain chemical nature, are probably pro-
teid in composition.6

The fats which are demonstrable both by microchemical methods,
staining with Sudan III., Scharlach R., Osmic acid, and by alcohol-
ether extraction, consist of fatty acids, true fats, and, in the case of the
tubercle bacillus at least, of waxy substances.7

1 Ruppel, Zeit. f. physiol. Chemie, xxvi, 1898.

2 Hammer schlag, Zeit. f. klin. Med., 1891.

* Weyl, Deut. med. Woch., 1891.

* Hellmich , Arch. f. exper. Pathol., etc., xxvi.

* Buchner, Munch, med. Woch., 1897.

6 Shattock, Lancet, May, 1898.

! De Schweinitz and Dorset, Cent. f. Bakt., I, xxii, 1897.

MORPHOLOGY, REPRODUCTION, ETC.

The carbohydrates isolated from various bacteria consist chiefly of
small quantities of cellulose and allied bodies, presumably concerned in
the formation of the bacterial cell membrane. The demonstration of
these substances has been successful only in isolated cases and has not
found universal confirmation.

Cdycogen-like substances haw) been demonstrated, according to
A. Fischer,1 in B. subtilis and B. eoli. These bacteria stained a reddish
brown color when stained with iodin, and after treatment with weak
acids were shown to contain dextrose.

The bacterial ash, remaining after removal of other substances, con¬
sists largely of phosphates and chlorides of potassium, sodium, cal¬
cium, and magnesium.

Osmotic Properties of the Bacterial Cell. — Like all other animal and
vegetable cells, the bacterial cell forms in itself a small osmotic unit
which reacts delicately to differences of pressure existing between its
own protoplasm and the surrounding medium. The perfect and normal
morphology of a microorganism, therefore, can exist only when the
osmotic pressure within the protoplasm of the cell is isotonic or equal
to that of its own environment. The changes produced in the morpho¬
logical relations of a cell when transferred from one environment into
another of varying osmotic pressure, depend intimately upon the
“permeability” of the cell membrane for different substances. When
such a membrane is permeable for water and not for substances in solu¬
tion, it is technically spoken of as “semi-permeable.” Now, as a matter
of fact, the bacterial cell membrane is easily permeable for water, but
its permeability differs greatly in various species of bacteria for other
substances. Thus, for instance, the cholera vibrio shows great perme¬
ability for common salt and B. fluorescens liquefaciens shows a lower
permeability for potassium nitrate than do many other bacteria.2

When a microorganism is suddenly removed from an environment
of low osmotic pressure into one showing a high pressure, say, from a
dilute to a concentrated solution of NaCl, an abstraction of water from
the cell occurs, with a consequent shrinkage of the protoplasm away
from the cell membrane. This process is spoken of as “plasmolysis.”
Cell death does not usually occur with plasmolysis, but by slow diffusion
of the salt itself into the protoplasm, the equilibrium may eventually
be restored and the normal morphology of the cell resumed. In all cases

1 A. Fischer , “ Vorlesungen fiber die Bakt.," Jena, 1903.

2 Gottschlich , in Flugge, M ik roo rgan ismen / ’ i, p. 91.

BIOLOGY AND TECHNIQUE

the speed and completeness of the return to normal depends upon the
permeability of the cell membrane for the dissolved substances. There
is no evidence to support the view that the internal pressure of a cell
may be in any way increased by an inherent power of the protoplasm
independently of the laws of diffusion. As a general rule, old cultures
are more susceptible to plasmolysis than are young and vigorous strains.
Spores and, according to A. Fischer,1 flagella are much less susceptible
to osmotic changes than are the vegetative bodies.

When, on the other hand, bacteria are suddenly removed from a
medium possessing a high osmotic pressure to one comparatively low,
say, from a concentrated salt solution to distilled water, a bursting of the
cell may occur, a process spoken of as “plasmoptysis.” Plasmoptysis
leads to cell death, and is probably the cause of the death of micro¬
organisms so often observed in distilled-water emulsions of bacteria.

Other Physical Properties of Bacteria. — The refractive index of the
vegetative bacterial body is low, in contrast to the highly refractive
character of the spores and flagella. According to Fischer, the ectoplasm
or cell membrane shows a higher index than does the endoplasm.

The specific gravity of various microorganisms has been investi¬
gated by Bolton,2 Rubner,3 and others. Some of Rubner’s results are
the following:

Gelatin fluidifiers . Sp. gr. 1.0651

Gas formers . “ “ 1.0465

Cultures from potato . “ “ 1.038

M. prodigiosus . “ “ 1.054

1 A. Fischer, quoted from Gottschlich in Fliigge, “ Mikroorganismen/’ I, p. 91.

2 Bolton, Zeit. f. Hyg., i, 1886. 3 Rubner, Arch. f. Hyg., xi, 1890.

CHAPTER III

THE RELATION OF BACTERIA TO ENVIRONMENT, AND THEIR

CLASSIFICATION

NUTRITION OF BACTERIA

Like all protoplasmic bodies, bacteria consist of carbon, oxygen,
hydrogen, and nitrogen, to which are added inorganic salts and varying
quantities of phosphorus and sulphur. In order that bacteria may
develop and multiply, therefore, they must be supplied with these sub¬
stances in proper quantity and in forms suitable for assimilation. To
formulate definite laws based on chemical structure as to the compounds
suitable, and those unsuitable for use by the bacteria, is obviously im¬
possible owing to the great metabolic variations existing within the
bacterial kingdom, and notable attempts to do so, such as those by
Loew,1 have not successfully withstood critical inquiry.

Carbon. — The carbon necessary for bacterial nourishment or ana¬
bolism may be obtained either directly from proteids, carbohydrates,
and fats, or from the simpler derivatives of these substances. Thus, the
amido-acids, leucin and tyrosin, ketons, and organic acids, like tartaric,
citric, and acetic acids, glycerin, and even some of the alcohols, may
furnish carbon in a form suitable for bacterial assimilation. A limited
number of bacterial species, furthermore, notably the nitrobacteria of
Winogradsky, are capable of obtaining their required carbon from
atmospheric C02, and possibly from other simple carbon compounds
added to culture media.2

Oxygen. — Oxygen is obtained, by the large majority of bacteria,
directly from the atmosphere in the form of free 02. For many micro¬
organisms, moreover, the presence of free oxygen is a necessary condi¬
tion for growth. These are spoken of as the “obligatory aerobes.”
Among the pathogenic bacteria proper, many, like the gonococcus,
bacillus influenzae, and bacillus pestis, show a marked preference for a
well-oxygenated environment. Probably there is no pathogenic micro-

1 Loew, Cent. f. Bakt., I, xii, 1892.

2 Muntz, Compt. rend, de l’acad. des sciences, t. iii.

BIOLOGY AND TECHNIQUE

organism which, under certain conditions of nutrition, is entirely
unable to exist and multiply in the complete absence of this gas. The
conditions existing within the infected animal organism cause it to
seem likely that all incitants of infection may, at times, thrive in the
complete absence of free oxygen.

There is another class of organisms, on the other hand, for whose
development the presence of free oxygen is directly injurious. These
microorganisms, known as “ obligatory anaerobes/’ obtain their supply
of oxygen indirectly, by enzymatic processes of fermentative and. pro¬
teolytic cleavage, from carbohydrates and proteicls, or by reduction
from reducible bodies. Among the pathogenic microorganisms the class
of “ obligatory anaerobes ” is represented chiefly by Bacillus tetani, the
bacillus of malignant edema, the bacillus of symptomatic anthrax,
Bacillus aerogenes capsulatus, and Bacillus botulinus.

Intermediate between these two classes is a large group of bacteria
which thrive well both under aerobic and anaerobic conditions. Some
of these, which have a preference for free oxygen but nevertheless
possess the power of thriving under anaerobic conditions, are spoken
of as “facultative anaerobes.” In others the reverse of this is true;
these are spoken of as “facultative aerobes.” These varieties of
bacteria are by far the most numerous and comprise most of our
parasitic and saprophytic bacteria.

The relation of microorganisms to oxygen is extremely subtle, there¬
fore, and not to be biologically dismissed by a rigid classification into
aerobes, facultative anaerobes, and obligatory anaerobes. Both Engel-
mann,1 by a method of observing motile bacteria in the hanging drop
as to their behavior in relation to the oxygen given off by a chloroph}dl-
bearing alga, and Beijerinck,2 by a macroscopic method of observing
similar bacteria as to their motion away from or toward an oxygenated
area, were able to demonstrate delicately graded variations between
species, favoring various degrees of oxygen pressure.

The discovery by Pasteur that certain bacteria develop only in the
absence of free oxygen, produced a revolution in our conceptions of
metabolic processes, since up to that time it was believed that life could
be supported only when a free supply of 02 was obtainable. Pasteur's
original explanation for this phenomenon was that anaerobic conditions
of life were always associated with some form of carbohydrate fermenta-

1 Engelmann, Botanische Zeitung, 1881.

* Beijerinck, Gent, f. Bakt., I, xiv, 1893.

RELATION TO ENVIRONMENT— CLASSIFICATION

tion and that oxygen was obtained by these microorganisms by a split¬
ting of carbohydrates. As a matter of fact, for a large number of micro¬
organisms, this is actually true, and the presence of readily fermentable
carbohydrates not only increases the growth energy of a large number
of anaerobic bacteria, but in many cases permits otherwise purely
aerobic bacteria to thrive under anaerobic conditions.1 On the
other hand, the basis of anaerobic growth can not always be found
in the fermentation of carbohydrates or in the simple process of
reduction.

The favorable influence of certain actively reducing bodies, like
sodium formate or sodium-indigo-sulphate, upon anaerobic cultivation
is probably referable to their ability to remove free oxygen from the
media and thus perfect the anaerobiosis.2 A number of strictly anae¬
robic bacteria, however, may develop in the entire absence of carbohy¬
drates or reducing substances, obtaining their oxygen supply from other
suitable sources, some of which may be the complex proteids. Thus
the tetanus bacillus mav 3 thrive when the nutritive substances in the
media are entirely proteid in nature. (See p. 28.)

As Hesse 4 has shown, the respiratory processes of aerobic bacteria
consist in the taking in of oxygen and the excretion of C02. The C02
excretion has been shown, in these cases, to be markedly less than is
represented in the intake of oxygen.

Anaerobes, likewise, show an excretion of C02 which must, in these
cases, be a result of bacterial katabolism.

Certain bacteria, like the red sulphur bacteria, have the power of
utilizing atmospheric oxygen in the same way in which this process
takes place in the chlorophyll-bearing plants.

While a profuse supply of oxygen absolutely inhibits the growth of
most anaerobes, a number of these may, nevertheless, develop when only
small quantities of oxygen are present. Minute quantities of free oxy¬
gen in culture media have been shown by Beijerinck5 and others not to
inhibit the growth of Bacillus tetani and Theobald Smith 0 has recently
demonstrated that when suitable nutritive material in the form of fresh
liver tissue is added to bouillon, a number of anaerobic bacteria may be

1 Theobald Smith , Cent. f. Bakt., I, xviii, 1895.

2 Kitasato and Weyl, Zeit. f. Hyg., viii, 1890.

s Chudiakow , Cent. f. Bakt., Ref., II, iv, 1898.

A Hesse, Zeit. f. Hyg., xv, 1897.

6 Beijerinck, Cent. f. Bakt., II, vi, 1900.

Th. Smith, Brown , and Walker, Jour. Med. Res., ix, 1906.

BIOLOGY AND TECHNIQUE

induced to grow in indifferently anaerobic environment. Ferran,1
moreover, succeeded in gradually adapting the tetanus bacillus to an
aerobic environment. In this case, however, the virulence of the bacil¬
lus was lost.

Nitrogen. — The nitrogen required by bacteria is taken, in most cases,
from proteids. Most important in this respect, of course, are the dif¬
fusible proteids.; but many of the non-diffusible albumins may be
rendered assimilable by the proteolyzing enzymes possessed by many
microorganisms. Among the pathogenic, more strictly parasitic bac¬
teria, moreover, a delicate specialization may be observed as to the
particular varieties of animal albumin which may be utilized by them.
Thus the gonococcus grows more readily only upon uncoagulated human
blood serum; the Pfeiffer bacillus requires hemoglobin, and the diph¬
theria bacillus outgrows other bacteria upon a medium composed for
the greater part of coagulated beef serum. For bacteria that do not
absolutely require native animal proteid for their development, the
most common nitrogenous ingredient of culture media is pepton, added
in solutions of varied concentration.

A large number of bacteria (pathogenic and saprophytic), on the
other hand, may thrive on media containing absolutely no proteid, in
which case, of course, a synthetic proteid production by the micro¬
organisms must be assumed. A medium which has been extensively
used to demonstrate this phenomenon is that devised by Uschinski,2
containing ammonium lactate, glycerin, asparagin (the amide of amido-
succinic acid), and inorganic salts.

Creatin, creatinin, urea and urates, and even ammonia compounds
and nitrates, may serve as. adequate sources of nitrogen for many of the
less parasitic bacteria. A limited number of species, moreover, the bacilli
in the root tubercles of the leguminosse and the nitrogen-fixing organ¬
isms of the soil, possess the power of obtaining their supply of nitrogen
directly from the free N2 of the atmosphere.

Although the sources of carbonaceous and of nitrogenous food supply
have been separately treated in the preceding paragraphs, it should not
be forgotten that, in many instances, both elements are taken up within
the same compound, and that separate supplies are a necessity in isolated
cases only.

Hydrogen. — Hydrogen is obtained by bacteria largely in combina-

1 Ferrari, Cent. f. Bakt., I, xxiv, 1898.

2 Uschinski, Cent. f. Bakt., I, xiv, 1893.

n 7 is

RELATION TO ENVIRONMENT— CLASSIFICATION 29

tion as water and together with the carbon and nitrogen containing
substances.

Salts. — The phosphatic constituents of the bacterial body are taken
in, chiefly, as phosphates of magnesium, calcium, sodium, or potassium.
The phosphates seem to be necessary constituents of culture media,
while chlorides, on the other hand, according to Proskauer 1 and Beck
are not absolutely essential. Sodium salts, as a rule, seem to be more
advantageous for purposes of bacterial cultivation than potassium salts.

The uncombined sulphur, which is a constituent of the bacterial body
in many cases, is usually supplied by soluble sulphates. In the case of
the thiobacteria of Winogradsky, however, the presence of free H2S is
necessary for its formation.2

The iron contained in some of the higher bacteria is taken in in the
form of ferrous compounds, and is oxidized in the bacterial body into
ferric compounds. *

The relative quantities of the various nutritive substances in culture
media are of importance only in so far as too high concentrations may
have a distinctly inhibitory influence. In this respect, however, separate
species may show widely divergent tastes.

The development of bacteria in any given medium, it may be noted,
is far oftener arrested by the accumulation of waste products than by an
exhaustion of nutrient materials.

PARASITISM AND SAPROPHYTISM

When we speak of bacteria as parasites or as saprophytes, we classify
them, primarily, according to their relationship to the bodies of higher
animals. “ Parasites ” are those bacteria which are capable of living and
multiplying within the human or animal body, whereas the term “ sapro¬
phytes ” refers to the multitude of microorganisms which are unable to
hold their own under the environmental conditions found in the tis¬
sues of higher animals, but are found, almost ubiquitously, in air, soil,
manure, and water. The separation is by no means a sharp one and
carries with it other implications, which the use of these terms always
conveys. While parasites are usually very fastidious as to nutritional
and temperature requirements, most saprophytes are easily cultivated
upon the simplest media. Thus certain parasitic bacteria, such as the

1 Proskauer and Beck, Zeit. f. Hyg., xviii, 1895.

2 Voges, Cent. f. Bakt., I, xviii, 1893.

BIOLOGY AND TECHNIQUE

bacillus of influenza, the gonococcus, and others, are dependent upon
specific forms of animal proteids for their food supply, while typical
saprophytes, like Bacillus proteus, may thrive and multiply upon even
the simplest organic proteid derivatives.

Between the strict parasites and the saprophytes, however, there is a
large class of bacteria, to which the majority of our pathogenic varieties
belong, the members of which are capable of developing luxuriantly
under both conditions. These bacteria are often spoken of as facultative
parasites.

More recently the question of parasitism and saprophytism has
become closely interwoven with our conceptions of virulence. Bail
(see section on Aggressins) has classified parasites into strict parasites
and half parasites. By the first term he designates bacteria like Bacillus
anthracis, which actually invade all the tissues of their host, while,
by the term “half parasites,” he refers to microorganisms like the spiril¬
lum of cholera which gain a foothold upon some part of the body of the
host, but do not actually penetrate into the general circulation.

All pathogenic bacteria, therefore, must be grouped as parasites,
strict or facultative, while the saprophytes, as a class, perform the far
more thankful task of breaking up organic matter outside of the animal
body, by putrefaction and fermentation. Absolute separation between
the two classes, however, can not be maintained, since many ordinarily
saprophytic bacteria may display parasitic qualities if administered in
large numbers to animals or man in whom resistance to bacterial
invasion is at a low ebb.

ANTAGONISM AND SYMBIOSIS OF BACTERIA

The ubiquity of bacteria in nature naturally carries with it the simul¬
taneous presence of many species in all places where special conditions
have provided a favorable environment for growth. Thus bacteriological
investigation of water, milk, manure, soil, or organic infusions, always
reveals the presence of a large number of different varieties within the
same substance. If the food supply in such a natural culture is at all
limited in quantity, or the removal of waste products is prohibited, it
will usually be found that gradually the numbers of varieties will dimin¬
ish and a few species, or even only one, will prevail. In the case of milk,
for instance, after standing for three or four days at a suitable temper¬
ature, two or three varieties will be found to have taken the place of
the twenty or thirty, which may have been present originally.

RELATION TO ENVIRONMENT— CLASSIFICATION

This behavior is due to the influences which various microorganisms
exert upon each other and is known as antagonism. Such antagonism
probably depends upon the fact that the metabolic products of the pre¬
dominant species (the one or ones for whom the special cultural condi¬
tions are most favorable) inhibit the growth of the less vigorous varieties.
Many examples, experimentally supported, of such antagonism, can be
given. Thus, the gonococcus is distinctly inhibited by the soluble pro¬
ducts of Bacillus pyocyaneus,1 while in the presence of pyogenic cocci it
develops luxuriantly, and the bacillus of pktgue is completely inhibited
when streptococci are present in the culture.2

Mutual inhibition may also be due to the monopolizing of the nutri¬
tion in the medium by the predominating species or to the change in re¬
action produced by its growth. This last consideration is probably the
secret of the frequently noticed inhibitory effect exerted by acid-pro¬
ducers upon bacteria of putrefaction, and has received practical thera¬
peutic application in Metchnikoff s lactic-acid bacillus therapy, which
see.

When the simultaneous presence of two bacterial species within the
same environment favors the development of both species, the condi¬
tion is spoken of as symbiosis. Such dependence is not so frequent as
antagonism, but it does occur. Examples of such a condition have been
observed in cultures containing diphtheria bacilli and streptococci 3 and
have been frequently observed in cultures containing both aerobic and
anaerobic bacteria, where the former favor the development of the latter
by monopolizing the supply of free oxygen. Symbiosis may also take
place in cultures in which complex food products are split up by one
species, furnishing substances for ingestion by species with a lesser
digestive ability.

RELATIONS OF BACTERIA TO PHYSICAL ENVIRONMENT

Relation to Temperature. — Like all other living beings, bacteria
develop and multiply by virtue of a series of chemical and physical
processes, by means of which growth energy is obtained by destruction
or catabolism, and the lost tissues resupplied by absorption of nutritive
materials. It is natural, therefore, that the conditions of external

1 Schafer, Fortschr. d. Med., 5, 1896.

2 Bitter, Rep. Egypt Plague Com., Cairo, 1897.

3 Hilbert, Zeit. f. Hyg., xxix, 1895.

BIOLOGY AND TECHNIQUE

temperature should intimately affect the metabolic processes. The
range of temperature at which bacteria may grow is subject to wide
variations among different species. Each species, on the other hand,
may thrive within a more or less elastic range of temperature, each one
having an optimum, a minimum, and a definite maximum tempera¬
ture. When the optimum temperature is present in the environment,
the functions of absorption and excretion keep pace with each other, and
the chemical balance is well preserved. When the temperature is lower
than the optimum, all metabolic processes take place more slowly, and
the bacterium gradually enters into a resting or latent stage, at which ac¬
tual growth may be exceedingly slow or entirely inhibited. When the
temperature is higher than the optimum, the destructive processes are
carried on more rapidly than the substitution of waste products by ab¬
sorption, and a gradual weakening of vital energy, or even a gradual
death of the bacterium, may take place. When certain bacteria form
spores, they become very much more resistant against both high and
low temperatures, probably because a true resting stage has been
reached, during which metabolism has been reduced to a minimum,
there being practically no nutritive material taken in and corresponding¬
ly little destruction taking place within the body of the microorganism.

The optimum temperature for various bacteria depends upon the
habitual environment, in which the particular species is accustomed to
exist. Thus, for the large majority of bacteria pathogenic for human
beings, the optimum temperature is at or about 37.5° C. There are
a large number of bacteria common in water, however, which grow
hardly at all at the body temperature, but thrive most luxuriantly at
temperatures of about 20° C. F. Forster,1 moreover, described certain
phosphorescent bacteria, isolated from sea-water, which grow readily at
0° C., or a little above. On the other hand, Miquel2 has described non-
motile bacilli, which he isolated from the water of the Seine, which grew
rapidly at temperatures ranging about 70° C., and the so-called “muce-
dinees thermophiles,” described by Tsildinski,3 develop most readily at
temperatures very little above this. It is thus plain that the tempera¬
tures favored by various bacteria depend to a large extent upon an
adaptation of these bacteria through many generations to specific en¬
vironmental conditions. A good illustration of this is furnished by the
bacillus of avian tuberculosis, a microorganism differing essentially

1 F. Forster, Cent. f. Bakt., ii, 1887.

2 Miquel, Bull, de la Stat. Munic. de Paris, 1879.

3 Tsildinski, Ann. Past., 1889.

RELATION TO ENVIRONMENT— CLASSIFICATION

from the bacillus of human tuberculosis in that its optimum growth
temperature lies at 41°-42° C., a temperature which exceeds the op¬
timum temperature for the human type by as much as the normal tem¬
perature of birds exceeds that of man. The same principle is illustrated
by the facts that the bacteria which have a very low optimum tem¬
perature are usually those isolated from water, and the so-called ther-
mophile or high-temperature bacteria are obtained from hot springs and
from the upper layers of the soil, where, according to Globig,1 occasion¬
ally temperatures ranging from about 55° C. occur.

As stated before, one and the same species may develop within a
wide temperature range, and it may be possible, by persistent cultiva¬
tion at special temperatures, to adapt certain bacteria to grow luxu¬
riantly at temperatures removed by several degrees from their normal
optimum. In such cases it may often occur that special characteristics
of the given species may be lost. An example of this is the loss of viru¬
lence and of spore-formation which takes place when anthrax bacilli
are cultivated at 42° C., or the loss of the power to produce pigment
when bacillus prodigiosus is grown at temperatures above 30° C.

The vegetative forms of most of the pathogenic bacteria may grow
at temperatures ranging between 20° C. and 40° C. This can, however,
by no means be regarded as applicable to all of the pathogenic bacteria,
as some of these, like the gonococcus, the pneumococcus, the tubercle
bacillus, and others, are delicately susceptible to temperature changes
and have the power of growing only within limits varying but a few
degrees from their optimum. Others, on the other hand, like bacilli of
the colon group, Bacillus anthracis, Spirillum cholerse asiaticse, etc.,
may develop at temperatures as low as 10° C. and as high as 40° C., or
over. The range of temperature at which saprophytic bacteria may
develop is usually a far wider one. When temperatures exceed in any
considerable degree the maximum growth temperature, the vegetative
forms of bacteria perish. Thus, ten minutes’ exposure to a temperature
of between 55° and 60° C. causes death of the vegetative forms of most
microorganisms. Death in such cases is due probably to a coagulation
of the protoplasm, and since all such processes of coagulation take place
best in the presence of water, the thermal death point of most bacteria
is lower when heat is applied in the form of boiling water or steam,
than when employed as dry heat. (See section on Sterilization.)

When spores are present in cultures, the resistance to heat is enor-

4 Globig, Zeit. f. Hyg., iii.

BIOLOGY AND TECHNIQUE

mously increased. Exactly what the explanation of this is can not be at
present stated. It may be that the high concentration in which the
protoplasmic mass is found in the spores renders it less easily coagulable
than is the protoplasm of the vegetative body. A more detailed discus-
sionof these relations will be found in the section on Heat sterilization.

The thermal death points of a large number of bacteria have been
very carefully studied by Sternberg,1 by a special technique described
elsewhere.

The thermal death points ascertained by him in this way, with an
exposure of ten minutes in a fluid medium, for some of the more common
non-sporogenic bacteria are as follows:

Spirillum cholerae asiaticse . 52° C.

Diplococcus pneumoniae . 52° C.

Streptococcus pyogenes . 54° C.

Bacillus typhosus . 56° C.

Bacillus pyocyaneus . 56° C.

Bacillus mucosus capsulatus . 56° C.

Bacillus prodigiosus . 58° C.

Staphylococcus pyogenes aureus . 58° C.

Gonococcus . 60° C.

Staphylococcus pyogenes albus . 62° C.

The bacillus tuberculosis, though not a spore bearer, seems to be slightly
more resistant to heat than other purely vegetative microorganisms.
Thus, according to the researches of Smith 2 and others, ten and twenty
minutes’ exposure to a temperature of 70° C. is necessary to destroy
tubercle bacilli in a fluid medium. For the effectual destruction of spores
by moist heat, a temperature of 100° C., or boiling point, is usually
necessary.

Low temperatures are much less destructive than the high ones,
and are even in a number of cases useful in keeping bacteria alive for
long periods, inasmuch as metabolic processes are inhibited and life is
maintained without actual development in a sort of resting state.
Actual destruction by low temperatures rarely takes place. The
exposure of diphtheria, typhoid, and other bacilli to temperatures as
low as 200° C. below zero has been carried out without destruction
of the microorganisms, a fact which is of great importance in considering
the possibility of infection by the vehicle of ice. Meningococci and
gonococci, on the other hand, die out rapidly when exposed to 0° C.

1 Sternberg, “Textbook of Bacteriology/’ New York, 1901.

2 Th. Smith, Jour, of Experimental Med., No. 3, 1899.

RELATION TO ENVIRONMENT— CLASSIFICATION

Relation to Pressure. — High pressure does not exert any noticeable
effects upon bacteria. In the experiments of Certes,1 a pressure of two
atmospheres seemed to have no influence upon the growth and motility
of anthrax bacilli suspended in blood.

Relation to Moisture. — For the growth and development of all bac¬
teria, the presence of water in the culture medium is necessary. It is
self-evident that nutritive materials can not be absorbed by an osmotic
process unless in a state of solution. While complete dryness does not
permit growth, its destructive action upon various bacteria is subject
to great differences. The effect of complete drying upon bacteria will
be found more fully discussed in the section upon the destruction of
bacteria by physical agents. (See page 62.)

In the same section may be found a discussion of the effects of light,
electricity, x-ray, and radium rays upon bacteria.

THE CLASSIFICATION OF BACTERIA

Too simple in structure, too varied in biological properties to be
definitely identified with either the vegetable or animal kingdom, the
bacteria are placed at the bottom of the scale of all living beings. Closely
linked on the one hand to the plant kingdom by the yeasts and the
molds, and on the other to the animal kingdom by the protozoa, they
themselves combine, within one and the same division, attributes so
widely divergent as to structure, metabolism, and biological activity that
their grouping is more a matter of working convenience than of actual
scientific classification. Thus, for instance, all stages of metabolic ac¬
tivity fill in the gap between the synthetizing sulphur and nitrifying
bacteria and the purely katabolic activities of some of the aerobic and
anaerobic microorganisms which cause putrefaction. Growth takes
place within the limits of a wide temperature range, and the specific
modes of life and cultural conditions are subject to the widest varia¬
tions, from those of an indisputably useful saprophytism to those of the
most exquisite parasitism. Although, therefore, strictly speaking, the
bacteria can be classified as a whole neither in the animal nor in the
veget able realms, being non chlorophyll-bearing, they are for conve¬
nience classified with the fungi or colorless plants.

The relationship of the bacteria to other simple plants may be
graphically represented by the following scheme:

1 Certes, Compt. rend, de Tacad, d, sc., 99, Paris, 1884,

BIOLOGY AND TECHNIQUE

Cryptogamia.

Thallophyta.

_ l_ _

1 'I I

Algas. Lichens. Fungi.

SCHYZOMYCETES BlASTOM YCETES HYPHOMYCETES

(Bacteria). (Yeasts). (Moulds — Oidia).

Coccacea. Chlamydobacteria.

Bacteria: eae. (Higher bacteria.)

Spirillaceai. Streptothrix.

Cladothrix.

Leptothrix.

Actinomyces.

The special classification of the bacteria has offered still greater
difficulties, for the lower we proceed in the phylogenetic scale of living
beings, the less specialized the morphological and biological charac¬
teristics of any group become, and the more difficult it is to establish a
classification which can in any way be regarded as final. It is, there¬
fore, quite impossible to classify the bacterial varieties or species on any
basis which can hope to satisfy all the demands of scientific accuracy
and it is necessary to resort to the expedient of utilizing some one
characteristic which remains constant for the individual genus and to
base upon this an attempt at grouping. When bacteria were first dis¬
covered, and for many years following, numerous observers contended
that the form of the microorganism observed was not a constant one
for each genus, but that cocci could be converted into bacilli or spirilla
accord in >• to environmental conditions. It was Cohn 1 who, in 1872,
first recognized the constancy of the morphology of bacteria and es¬
tablished, upon morphological basis, a classification which, with minor
changes, has been retained until the present day. Such classifications
can not, however, be regarded as anything more than a convenient
make-shift pending the clay when the finer structure and true biological
relations of the various bacteria shall have been more accurately inves¬
tigated. The scheme most commonly accepted at present is the one
given below, proposed by Migula2:

1 Cohn, “Beitrage zur Biol. cl. .Pfianzen,” Heft 1 u. 2, 1872.

2 Migula, i: System d. Bakt.,” Jena, 1897.

RELATION TO ENVIRONMENT— CLASSIFICATION

Bacteria (Schizomycetes) . — Fission fungi (chlorophyll free) with cell
division in one, two, or three directions of space. Many varieties
possess the power of forming endospores. Whenever motility
is present, it is carried on by means of flagella, or, more rarely,
by undulating membranes.

Family I. Coccace^e. — Cells in free state perfectly spherical.
Division in one, two, or three directions of space, by which each
spherical cell divides into two, four, or eight segments, each
of which again develops into a perfect sphere. Endospore
formation rare.

Genus I. Streptococcus . — Cells divide in one direction of space only,
for which reason, if they remain connected after fission, bead¬
like chains may be formed. No organs of locomotion.

Genus II. Micrococcus (Staphylococcus). — Cells divide in two
directions of space, whereby, if the cells remain connected after
fission, tetrad and grape-like clusters may be formed. No organs
of locomotion.

Genus III. Sarcina. — Cells divide in three directions of space,
whereby, if they remain connected after fission, bale-like packets
are formed. No organs of locomotion.

Genus IV. Planococcus. — Cells divide in two directions of space,
as in micrococcus, but possess flagella.

Genus V. Planosarcina. — Cells divide in three directions of space
as in sarcina, but possess flagella.

Family II. Bacteriaceal— Cells long or short, cylindrical, straight,
never spiral. Division in one direction of space only, after pre¬
liminary elongation of the rods.

Genus I. Bacterium. — Cells without flagella, often with endospores.

Genus II. Bacillus. — Cells with peritrichal flagella, often with
endospores.

Genus III. Pseudomonas. — Cells with polar flagella. Endospores
occur in a few species, but are rare.

Family III. Spirillace^e. — Cells spirally curved or representing a
part of a spiral curve. Division in one direction of space only,
after preceding elongation of cell.

Genus I. Spirosoma. — Cells without organs of locomotion. Rigid.

Genus II. Microspira. — Cells rigid, with one or, more rarely, two
or three polar undulated flagella.

BIOLOGY AND TECHNIQUE

Genus III. Spirillum. — Cells rigid, with polar tufts of five to twenty
flagella usually curved in semicircular or flatly undulating curves.

Genus IV. Spirochcete. — Cells sinously flexible. Organs of locomo¬
tion unknown, perhaps a marginal undulating membrane.

Family IV. Chlamydobacteriace^. — Forms of very varying stages
of evolution, but all distinguished by a rigid sheath (Hiille) or
covering, which surrounds the cells. The cells are united in
branched or unbranched threads.

Genus I. Streptothrix. — Cells united in simple, unbranched threads.
Division in one direction of space only. Reproduction by non-
motile conidia.

Genus II. Cladothrix. — Cells united or pseudodichotomously branch¬
ing threads. Division in one direction of space only. Vegeta¬
tive multiplication by separation of entire branches. Repro¬
duction by swarming forms with polar flagella.

Genus III. Crenothrix. — Cells united in unbranched threads, at
first with division in one direction of space only. Later the cells
divide in all three directions of space. The daughter cells be¬
come rounded and develop into reproductive cells.

Genus IV. Phragmidiothrix. — Cells at first united in unbranched
threads, dividing in three directions of space, thus forming a
rope of cells. Later some of the cells may penetrate through the
delicate sheath, and thus give rise to branches.

Genus V. Thiothrix. — Unbranched, non-motile threads, inclosed
in fine sheaths. Division of cells in one direction only. Cells
contain sulphur granules.

Family V. Beggiatoace^e. — Cells united in sheathless threads.
Division in one direction of space only. Motility by undulating
membrane as in Oscillaria.

Genus Beggiatoa. — Cells with sulphur granules.

It will be seen in reviewing the classification just given that the sub¬
divisions are based upon questions of form, motility, and situation of
flagella. While these characteristics, so far as we know, are constant,
there are, nevertheless, many instances in which types entirely similar
in these respects must be differentiated. This can be done only by care¬
ful study of staining reactions, finer structure, cultural characteristics,
and biological activities.

RELATION TO ENVIRONMENT— CLASSIFICATION

As a matter of fact, while the botanical classification of the bacteria
offers almost insurmountable difficulties, actual identification is not so
complicated a task as this would indicate. Identification, once roughly
made on a morphological basis, is further carried on by the aid of cul¬
tural characteristics, such as the conditions favorable and unfavor¬
able for growth, appearance of growth on different media, and pigment
formation, by biochemical reactions and by pathogenic properties.
The bacteria occupy so important a place in agriculture, in medicine,
and in hygiene, that it rarely becomes necessary for a worker in any par-
icular field to survey the entire group. The habitat of a large number of
pecies is so well known that this consideration alone often gives a clew
nvaluable for actual identification.

CHAPTER IV

THE BIOLOGICAL ACTIVITIES OF BACTERIA

While the bacteria pathogenic to man and animals largely usurp
the attention of those interested in disease processes, this group of micro¬
organisms is after all but a small specialized off-shoot of the realm of
bacteria, and, broadly speaking, actually of minor importance. Sur¬
veying the existing scheme of nature, as a whole, it is not an extrava¬
gant statement to say that without the bacterial processes which are
constantly active in the reduction of complex organic substances to
their simple compounds, the chemical interchange between the animal
and vegetable kingdoms would fail, and all life on earth would of
necessity cease. To understand the full significance of this, it is neces¬
sary to consider for a moment the method of the interchange of matter
between the animal and vegetable kingdoms.

All animals require for their sustenance organic compounds. They
are unable to build up the complex protoplasmic substances which form
their body cells from chemical elements or from the simple inorganic
salts. They are dependent for the manufacture of their foocl-stuffs,
therefore, directly or indirectly, upon the synthetic or anabolic activi¬
ties of the green plants.

These plants, by virtue of the chlorophyll contained within the cells
of their leaves and stems, and under the influence of sunlight, possess
the power of utilizing the carbon of the carbonic acid gas of the atmos¬
phere, and of combining it with water and the nitrogenous salts ab¬
sorbed by their roots, building up from these simple radicles the highly
complex substances required for animal sustenance.

These products of the synthetic activity of the green plants, then,
are ingested by members of the animal kingdom, either directly, in the
form of vegetable food, or indirectly, as animal matter. They are
utilized in the complex laboratory of the animal body and are again
broken down into simpler compounds, which leave the body as excreta
and secreta.

The excreta and secreta of animals, however, are, in a small part
only, made up of substances simple enough to be directly utilized by
plants. The dead bodies, moreover, of both animals and plants would

THE BIOLOGICAL ACTIVITIES OF BACTERIA

be of little further value as stores of matter unless new factors inter¬
vened to reduce them to that simple form in which they may again
enter into the synthetic laboratory of the green plant. Agents for
further cleavage of these compounds are required, and these are supplied
by the varied activities of the bacteria.

On the other hand, bacteria are also important in the process of
synthesis. The main supply of nitrogen available for plant life is found
in the elementary state in the atmosphere — a condition in which it
can not be utilized as a raw product by the plant. This gap again is
bridged by the bacteria found in the root bulbs of the leguminous plants
— bacteria which possess the power of assimilating or aiding in the as¬
similation of atmospheric nitrogen and its preparation for further use by
the plant itself. Another bacterial activity which may be classified as an
anabolic process is the oxidation of the ammonia, released by decomposi¬
tion. into nitrites and nitrates. This is carried on by certain bacteria of
the soil. These are to be treated of in greater detail in another section.

There is a constant circulation, therefore, of nitrogen and carbon
compounds, between the plant and the animal kingdoms, by virtue of an
anabolic or constructive process in the one, and a katabolic or destruc¬
tive process in the other, rendering them mutually interdependent and
indispensable. The circuit, however, is not by any means a closed one;
there are important gaps, both in the process of cleavage and in that of
synthesis, which, if left unbridged by the bacteria, would effectually
arrest all life-activity of plants and eventually of animals.

Far from being scourges, therefore, these minute microorganisms
are paramount factors in the great cycle of living matter, supplying
necessary links in the circulation of both nitrogenous and carbon com¬
pounds.

KATABOLIC ACTIVITIES OF BACTERIA

The katabolic activities of bacteria, then, consist in the fermentation
of carbohydrates and in the cleavage of proteids and fats.

Fermentation is carried out to a large extent by the yeasts, but also
to no inconsiderable degree by bacteria. Proteid decomposition and the
cleavage of fats are carried out almost exclusively by bacteria.

For our knowledge of the fundamental laws underlying these phe¬
nomena of fermentation and proteid decomposition, we are indebted
to the genius of Pasteur,1 who was the first to prove experimentally the

1 Pasteur, “ Etude sur la bi re,” Paris, 1876.

BIOLOGY AND TECHNIQUE

exclusive and specific parts played by various microorganisms in these
processes. While the observations and deductions made by Pasteur have
not been greatly modified, a large store of information has been gained
since his time, which has thrown additional light upon the chemical de¬
tails and the more exact manner of action of the factors involved.

The actual work of cleavage in both fermentation and proteid cleav¬
age is carried out by substances known as enzymes or ferments, the nature
of which we must further discuss before their manner of action can be
fully comprehended.

Bacterial Enzymes or Ferments. — A ferment or enzyme is a substance
produced by a living cell, which brings about a chemical reaction with¬
out entering into the reaction itself. The enzyme itself is not bound to
any of the end products and is not appreciably diminished in quantity
after the reaction is over, although its activity may be finally inhibited
by one or another of the new products. The action of bacterial enzymes is
thus seen to be closely similar to that of the chemical agents technically
spoken of as “katalyzers,” represented chiefly by dilute acids. Thus,
if an aqueous solution of saccharose is brought into contact with a
dilute solution of sulphuric acid, the disaccharid is hydrolyzed and is
decomposed into levulose and dextrose.

Thus:

C12H22 011 + H20 — C6H1206 + C6H1206
In contact with Dextrose Levulose

dilute H2 S 04

During this process, which is known as “inversion,” the concentration
of the sulphuric acid remains entirely unchanged. While theoretically
the changes brought about by enzymes and katalyzers are usually
such as would occur spontaneously, the time for the spontaneous oc¬
currence would be, at ordinary temperatures, infinitely long. The defini¬
tion for enzymes and katalyzers is given by Ostwald, therefore, as
“ substances which hasten a chemical reaction without themselves taking
partin it.” Exactly the same result which is obtained by the use of dilute
sulphuric acid is caused by the ferment “invertase” produced, for
instance, by B. megatherium. Were a solution of saccharose sub¬
jected to heat, without katalyzer or ferment, a similar change would
occur, but by the mediation of these substances the inversion is pro¬
duced without other chemical or physical reinforcement.

This analogy between enzymes and katalyzing agents is very
striking. Thus, as stated, both katalyzers and enzymes bring about

THE BIOLOGICAL ACTIVITIES OF BACTERIA

changes without themselves being used up in the process, both act
without the aid of heat, and the reactions brought about by both
have occasionally been shown to be reversible. While this last phe¬
nomenon has been variously shown for katalyzers, the process of re¬
versibility has been demonstrated for bacterial enzyme action only in
isolated cases. Thus, it has been found that by the action of the yeast
enzyme maltase upon concentrated dextrose solutions, a re-formation of
maltose may occur. In both cases, moreover, the quantity of enzyme
or katalyzer is infinitely small in proportion to the amount of material
converted by their action.

There is a close similarity, furthermore, between the bacterial en¬
zymes and the ferments produced by specialized cells of the higher ani¬
mals and plants. For instance, the action of the ptyalin of the saliva or
of the diastase obtained from plants is entirely analogous to the starch¬
splitting action of the amylase produced by many bacteria.

The action of all enzymes depends most intimately upon environ¬
mental conditions. For all of them the presence of moisture is essential.
All of them" depend for the development of their activity upon the exist¬
ence of a specifically suitable reaction. Strong acids or alkalies always
inhibit, often destroy them. Temperatures of over 70° C. permanently
destroy most enzymes, whereas freezing, while temporarily inhibiting
their action, causes no permanent injury, so that upon thawing, their
activity may be found almost unimpaired. Direct sunlight may injure,
but rarely destroys, ferments. Against the weaker disinfectants in com¬
mon use, enzymes often show a higher resistance than do the bacteria
which give rise to them.

The optimum conditions for enzyme action, then, consist in the
presence of moisture, the existence of a favorable reaction, weakly acid
or alkaline, as the case may be, and a temperature ranging from 35°-
45° C.1

Proteolytic Enzymes. — In nature, the decomposition of dead animal
and vegetable matter occurs only when the conditions are favorable for
bacterial development. Thus, as is well known, freezing, sterilizing by
heat, or the addition of disinfectants will prevent the rotting of organic
material.

In the laboratory, the presence of proteolytic enzymes is determined
chiefly by the power of bacteria to liquefy gelatin, fibrin, or coagulated
blood serum. These ferments are not always secretions from the bac-

1 Oppenheimer, “ Die Fermente,” etc. Leipzig, 1900.

BIOLOGY AND TECHNIQUE

terial cell, but in some cases may be closely bound to the cell-body and
separable only by extraction after death. In such cases they are spoken
of as endoenzymes. Whenever they are true secretory products, however,
they can be obtained separate from the microorganisms which form them
by filtration through a Berkefeld candle. From such filtrates they may,
in some cases, be obtained in the dry state by precipitation with alcohol.
When obtained in this way the precipitated enzyme is usually much
more thermostable than when in solution, for while soluble enzymes in
filtrates are usually destroyed by 70° C., and even less, the dried powder
may occasionally withstand 140° C. for as long as ten minutes.1

Apart from the general conditions of temperature and moisture, the
development of these enzymes seems to depend directly upon the presence
of proteids in the culture media. The number of bacterial species
which produce proteolytic enzymes is legion. Among those more com¬
monly met with are staphylococci, B. subtilis, B. proteus, B. fsecalis
liquefaciens, Spirillum cholerse asiaticae, B. anthracis, B. tetani, B. pyo-
cyaneus,and a large number of others. The inability of any given micro¬
organism to liquefy gelatin or fibrin by no means entirely excludes the
formation by it of proteolytic enzymes, since these ferments may often
be active for one particular class of proteid only.

In order to study the qualitative and quantitative powers of any
given bacterial proteolyzing enzyme or protease, it is, of course, neces¬
sary to study these processes in pure culture in the test tube with media
of known composition. In the refuse heap, in sewage, or in rotting
excreta, the process is an extremely complicated one, for besides the
bacteria which attack the proteid molecule itself, there are many other
species supplementing these and each other, one species attacking the
more or less complex end-products left by the action of the others.

Exactly what the chemical reactions are which take place in these
cleavages is not entirely clear. It is believed, however, that most of the
cleavages are of an hydrolytic nature.

In general, the action of the proteid-splitting ferments is comparable
to that of the pancreatic ferment trypsin, and they are most often active
in an alkaline environment. They differ, among themselves, chiefly in
the form of proteid which they are competent to attack, and in the
extent to which they are able to reduce it toward its simple radicles.

A distinction is occasionally made between the terms putrefaction
and decay, the former being used to refer to the decomposition taking

1 Fuhrmann, “ Die Bakterienzyme/’ p. 45.

THE BIOLOGICAL ACTIVITIES OF BACTERIA

place under anaerobic conditions, that is, in the absence of oxygen, a
process usually resulting in incomplete cleavage of the proteid medium;
the latter being used to signify decompositions under aerobic conditions
and leading to a more complete splitting, the end-products often being
represented by such simple compounds as carbon dioxide, water, and
ammonia. In general, the products of putrefaction are largely repre¬
sented by the amino-acids, leucin and tyrosin, fatty acids, mercaptan,
indol, and skatol. The gases generated in such decomposition are largely
made up of CO 2, Hydrogen, NIT4 and H2S. The coincident presence,
furthermore, of the carbohydrate-splitting bacteria and of denitrifying
microorganisms renders the actual process of putrefaction a chaos of
many activities in which the end-products and by-products are qualita¬
tively determinable only with much inexactitude, and which com¬
pletely defies any attempt at quantitative analysis.

Ptomains. — There are certain products, however, resulting from the
proteolytic action of bacterial enzymes upon proteids which claim more
than a purely chemical interest because of their toxic action upon the
animal organism, and their consequent importance as incitants of dis¬
ease. Pre-eminent among these are the ptomains. The word ptomain
(from 7 TT&tia, a dead body) is used to designate organic chemical
compounds produced by the action of bacteria, which are basic in char¬
acter; that is, are able to combine with an acid to form a salt. They
should be definitely distinguished from the so-called leucomains, a
term employed to designate similar substances formed in the course
of proteid metabolism within the animal body, and not bacterial in
origin. Both in their basic characters and in their nitrogenous constitu¬
tion, the ptomains resemble the vegetable alkaloids, and for this reason
are sometimes spoken of as “animal alkaloids.”

The ptomains must be sharply distinguished from the bacterial
toxins, which are products of the bacterial growth irrespective of the
medium in which they are grown, except in so far as this hinders or
abets the development of the microorganisms. Thus, toxins may be
developed by diphtheria organisms, for instance, in proteid-free media.
As will be seen in a subsequent section, the true toxins are comparable
to the enzymes themselves, rather than to their cleavage products, rep¬
resented in this instance by the ptomains.

A great number of ptomains are chemically known. Many of
these possess little or no toxicity. Others, however, like putrescin
(tetramethylenediamin, C4H12N2) and cadaverin (C5H14N2) are very
highly poisonous. It is to one or another of these ptomains that most

BIOLOGY AND TECHNIQUE

cases of so-called meat poisoning (kreatoxismus) , cheese poisoning
(tyrotoxismus) , or vegetable poisoning (sitotoxismus) are due.

In each individual case the variety of ptomain resulting from a bac¬
terial decomposition varies with the individual species of microorganism
taking part in the process and with the nature of the proteid upon which
its development takes place.

In breaking down animal excreta, the task of the bacteria is rather
a simpler one than when dealing with the cadavers themselves, for here a
part of the cleavage has already been carried out either by the destruc¬
tive processes accompanying metabolism, or by partial decomposition by
bacteria begun within the digestive tract. This material outside of the
body is further reduced by bacterial enzymes into still simpler sub¬
stances, the nitrogen usually being liberated in the form of ammonia.
One example of such an ammoniacal fermentation may be found in
the case of the urea fermentation by Micrococcus urese, in which the
cleavage of the urea takes place by hydrolysis according to the follow¬
ing formula:

(NH2)2 CO + 2H2 O = C02 + 2NH3 + H2 O

Similar ammoniacal fermentations are carried out, though perhaps
according to less simple formulae, by a large number of microorganisms.
Perhaps the most common species which possesses the power is the group
represented by B. proteus vulgaris (Hauser).

From what has been said it follows naturally that, so far, the decom¬
position of the proteid molecule from its complex structure to ammonia
or simple ammonia compounds is an indispensably important function,
not only for agriculture, but for the maintenance of all life processes.
It is clear, on the other hand, that a further decomposition of ammonia
compounds into forms too simple to be utilized by the green plants Avould
be a decidedly harmful activity. And yet this is brought about by the
so-called denitrifying bacteria which will be considered in a subsequent
section.

Lab Enzymes. — There are a number of ferments produced by bacteria
which, although affecting proteids, can not properly be classified with
the proteolytic enzymes. These are the so-called coagulases or lab
enzymes, which have the power of producing coagulation in liquid pro¬
teids. Just what the chemical process underlying this coagulation is,
is not known. If Hammarsten’s 1 conclusions as to the hydrolytic

1 Hammarsten , ‘'Textbook of Physiol, Chemistry,” Translation by Mandel.

THE BIOLOGICAL ACTIVITIES OF BACTERIA

nature of the changes produced by them are true, these enzymes are
brought into close relationship to the proteolyzers, although a coagula¬
tion can hardly be regarded as a true katabolic process. In milk where
the lab-action becomes evident by precipitation of casein, a strict dif¬
ferentiation must be made between this coagulation and that brought
about by acids or alkalies. In the former case, casein is not only pre¬
cipitated and converted into paracasein, but is actually changed so that
when redissolved it is no longer precipitated by lab.1

Coagulating enzymes for milk proteids, blood, and other proteid
solutions are produced by a large variety of bacteria. They have been
observed in cultures of the cholera vibrio, B. prodigiosus, B. pyocyaneus,
and several others.2

The lab enzymes are easily destroyed by temperatures of 70° C. and
over, and are very susceptible to excessive acidity or alkalinity.

Fat-Splitting Enzymes (. Lipase ). — The fat-splitting powers of bac¬
teria have been less studied than some of the other bacterial func¬
tions and are correspondingly more obscure. It is known, nevertheless,
that the process is due to an enzyme and that it is probably hydrolytic
in nature. The following formula represents the simplest method in
which some of the molds and bacteria produce cleavage of fats into
glycerin and fatty acid.

- C3 H5 (Cn H2n_1 02)3 + 3H2 O = C3 H5 (OH3) + 3Cn H2n 02

Glycerin Fatty acid

Some of the bacteria endowed with the power of producing lipase
are the spirillum of cholera, B. fluorescens liquefaciens, B. prodigiosus,
B. pyocyaneus, Staphylococcus pyogenes aureus, and some members of
the streptothrix family. The methods of investigating this function of
bacteria, originated by Ejkmann, 3 consists in covering the bottom of a
Petri dish with tallow and pouring over this a thin layer of agar. Upon
this, the bacteria are planted. Any diffusion of lipase from the bacterial
colonies becomes evident by a formation of white, opaque spots in the
tallow. Carriere4 was able to demonstrate a fat-splitting ferment for the
tubercle bacillus. Apart from the importance of these enzymes in
nature for the destruction of fats, they are industrially important be-

1 Oppenheimer, “ Die Fermente u. ihre Wirkung,” Leipzig, 1903.

2 Torini, Atti dei laborat. d. sanita, Rome, 1890.

3 Ejkmann, Cent. f. Bakt., I, xxix, 1901.

4 Carriere, Comptes rend, de la soc. de biol., 53, 1901.

BIOLOGY AND TECHNIQUE

cause of their action in rendering butter, milk, tallow, and allied prod¬
ucts rancid, and are medically of interest for their action upon fats in
the intestinal canal.

Enzymes of Fermentation {The Cleavage of Carbohydrates by Bacteria).
— The power to assimilate carbon dioxide from the atmosphere is
possessed only by the green plants and some of the colored algae,
and the sulphur or Thiobacteria. All other living beings are thus
dependent for their supply of carbon upon the synthetic activities
carried on by these plants to the same degree in which they are de¬
pendent upon similar processes for their nitrogen supply. The return of
this carbon to the atmosphere is, of course, brought about to a large ex¬
tent by the respiratory processes of the higher animals. The carbon,
which, together with nitrogen, forms a part of proteid combinations, is
freed, as we have seen in a previous section, by the processes of proteid
cleavage. That, however, which is inclosed in the carbohydrate mole¬
cule, is set free by the action of yeasts, molds, or bacteria, by an enzy¬
matic process similar in every respect to that described above for the
process of proteid cleavage.

Fermentation. — The power of carbohydrate cleavage is possessed
by a large number of the yeasts and bacteria. The process, as has
been indicated, is of great importance in the cycle of carbon compounds
for the return of carbon to its simplest forms,, and is, furthermore, as
will be seen in a later section, of great utility in the industries. In each
case the power to split a particular carbohydrate is a more or less specific
characteristic of a given species of microorganism, and for this reason
has been extensively used as a method for the biological differen¬
tiation of bacteria. In the course of much careful work upon this
question it has been ascertained that the specific carbohydrate-splitting
powers of any given species are constant and unchanged through
many generations of artificial cultivation. Thus, differentiation of the
Gram-negative bacteria, the members of the pneumococcus-streptococ¬
cus group, and the diphtheria group, can now largely be made by a study
of their sugar fermentations.

In most of these cases, as far as we know, the cleavage is produced by
a process of hydrolysis. A convenient nomenclature which has been
adopted for the designation of these ferments is that which employs the
name of the converted carbohydrate adding the suffix “ ase ” to indicate
the enzyme. There are thus ferments known as amylase, cellulase, lac¬
tase, etc.

' A mvlase ( Diastase or Amylolytic Ferment). — Amylases or starch-

THE BIOLOGICAL ACTIVITIES OF BACTERIA

splitting enzymes are formed by many plants (malt) and by animal
organs (pancreas, saliva, liver). Among microorganisms amylase is
produced by many of the streptothrix group, by the spirilla of Asiatic
cholera and of Finkler-Prior, by B. anthracis, and many other bacteria.
A large number of the bacteria found in the soil, furthermore, have
been shown to produce amylases. By cultivating bacteria upon starch-
agar plates, amylase can be readily demonstrated by a clearing of the
medium immediately surrounding the colonies.1

Since, of course, there are several varieties of starches, it follows that
the exact chemical action of amylase differs in individual cases. The
determination of the structural disintegration of starch by these fer¬
ments is fraught with much difficulty, owing to the polymeric constitu¬
tion of the starches. Primarily, however, a cleavage takes place into
a disaccharid such as maltose (hexobiose) , and the non-reducing sugars
and -dextrin. Beyond this point, however, the further cleavages are
subject to much variation and are not entirely clear. The dextrins
upon further reduction yield eventually dextrose.

Cellulcise. — Cellulose is fermented by a limited number of bacteria,
most of them anaerobes. The chemical process by which this takes place
is but poorly understood.2

Gelase. — An agar-splitting ferment has been found by Gran.3

Invertase. — The enzymes which hydrolytically cause cleavage of
saccharose into dextrose and levulose are numerous. The chemical
process takes place according to the following formula:

C12HMOn + HaO = C6H12 06 + C6H1206
Saccharose Dextrose Levulose

Invertase is produced by many of the yeasts. It is one of the most
common of the enzymes produced by bacteria, and has been found in
cultures of B. megatherium, B. subtilis, pneumococcus, some strepto¬
cocci, B. coli, and many others. Invertase is usually very susceptible to
heat, being destroyed by temperatures of 70° C. and over. A slightly
acid reaction of media abets the inverting action of these enzymes.
Strong acids and alkalies inhibit them. Inverting enzymes may be
precipitated out of solution by alcohol. Antiseptics even in weak con¬
centrations will inhibit their action.

1 Ejkmann, Cent. f. Bakt., xxix, 1901, and xxxv, 1904.

2 Omelianski, Lafar’s “ Handb. d. techn. Mykologie,” Bd. iii, Chap. 9.

3 Gran, Bergens Museum Aarbog, 1902, Hft. I.

BIOLOGY AND TECHNIQUE

Lactase. — Lactose-splitting ferments are extremely common both
among bacteria and among the yeasts. The process is here again a
hydrolytic cleavage resulting in the formation of the monosaccharids
as dextrose and galactose.

Maltase. — A maltose-splitting ferment has also been found in the
cultures of many bacteria, leading to the formation of dextrose.

Lactic Acid Fermentation. — Lactic acid (oxyproprionic acid, C3H6 03)
is one of the most common substances to appear among the prod¬
ucts of bacterial activity, both in media containing carbohydrates
and in those consisting entirely of albuminous substances. In most of
these cases, the lactic acid is formed merely as a by-product accom¬
panying many other more complicated chemical cleavages. In some
instances, however, lactic acid is produced from carbohydrates, both
disaccharids and monosaccharids, as an almost pure product due to a
specific bio-chemical process. The reactions taking place in this phenom¬
enon may be briefly expressed according to the following formulae:

C12 H22 On + H2 O = 4C3 He 03
Lactose Lactic acid

C6 H12 06 = 2C3 H6 03
Dextrose Lactic acid

In the same way lactic acid may be produced by bacteria from levu-
lose.

Examples of lactic acid formation are furnished by the streptococcus
lacticus, and B. lactis aerogenes. In the case of the former, the fer¬
mentation may indeed proceed by the simple chemical process indi¬
cated in the formulae, since the action of the bacillus is entirely unac¬
companied by the evolution of gas.

Numerous other bacteria produce large amounts of lactic acid from
lactose, possibly by chemical processes less simply formulated. Among
these are bacilli of the colon group, B. prodigiosus, B. proteus vulgaris,
and many others. Although lactic acid is usually the chief product in
the bacterial fermentation of the simpler carbohydrates, acetic, formic,
and butyric acids may often be found as by-products in variable
amounts.1

Oxydases ( Oxydizing Enzymes). — The most common example of
oxidation by means of bacterial ferments is the production of acetic acid

1 Buchner und M eisenheimer , Ber. d Deut. chem. Gesellsch., xxxvi, 1903.

THE BIOLOGICAL ACTIVITIES OF BACTERIA

from weak solutions of ethyl alcohol. This process, which is the basis of
vinegar production, is universally carried out by bacterial ferments.
While possessed to some extent by a considerable number of microorgan¬
isms, acetic acid formation is a function pre-eminently of the bacterial
groups described by Hansen, including “ Bacterium aceti ” and “ Bac¬
terium pasteurianum.” To these two original groups a number of others
have since been added.

The organisms are short, plump bacilli, with a tendency to chain-
formation, and occasionally showing characteristically swollen centers
and many irregular involution forms. In the production of vinegar, as
generally practiced by the farmer with cider or wine, these bacteria
‘accumulate on the surface of the fluid as a pellicle or scum which is
popularly known as the “mother of vinegar.” Destruction of these
bacteria by disinfectants or by sterilization with heat promptly arrests
the process of vinegar formation. Chemically, the conversion of the
alcohol consists in a double oxidation through ethyl aldehyde into acetic
acid as shown in the following formulae:

1. C2H5 (OH) + O = CH3 (COH)

Alcohol Ethyl aldehyde

2. CH3 (COH) + O = CH3 (COOH)

Acetic acid

Alcoholic Fermentation [Zymase). — The formation of alcohol as an
end product of fermentation is of great importance in a number of the
industries, primarily in the production of wine and beer. While accom¬
plished by a number of bacteria, this form of fermentation is carried
out chiefly by the yeasts.

Expressed in formulae the simplest varieties of alcoholic fermenta¬
tion, from mono- and disaccharids, may be represented as follows:

C6H1206 = 2C2H5 (OH) + 2 CO 2
Dextrose Ethyl alcohol
or

C12H22Ou + H20 = 4C2H5(OH) + 4C02

Saccharose Ethyl alcohol

In all cases the process may not be so simple as indicated by the equa¬
tions, since by-products, such as higher alcohols, glycerin, succinic
and acetic acids, may often be found in small traces among the end-
products of such fermentations. The conditions which favor alcoholic

BIOLOGY AND TECHNIQUE

fermentation by the yeasts are extremely important, since, upon obser-
servance of these, depends much of the uniformity of result which is so
desirable in the industries mentioned above. The optimum concentra¬
tion of sugar for the production of the highest quantity of alcohol is at
or about 25 per cent. The temperature favoring the process ranges
about 30° C. Under such conditions fermentation may continue until
the alcohol forms almost a 20-per-cent solution. Most of the fermenta¬
tions important in the wine, beer, and spirit industries, take place under
anaerobic conditions, since the carbon dioxide which is formed soon
shuts out any excess of air.

In the industrial employment of yeasts for fermentative purposes, it
is necessary to work with specific strains, and in scientifically conducted *
vineyards, breweries, and distilleries the study and pure cultivation of
the yeasts form no unimportant part of the work. Certain races of yeasts
are more uniform in their fermentative powers than others, and the by¬
products formed by some races differ sufficiently from those of other
races to cause material differences in the resulting substances. In the
wine industries, the yeasts differ much from one another according to
climatic and other environmental conditions. In vineyards, natural
inoculation of the grapes occurs by transportation of the yeast from
the soil to the surface of the grapes by wasps, bees, or other insects,
through whose alimentary canals the microorganisms pass uninjured.
In the autumn the yeast is returned to the soil by falling berries and
remains alive in the upper layers of the ground throughout the winter
months. In actual practice this natural yeast inoculation is not de¬
pended upon, but pure cultures of artificially cultivated yeasts are
employed for inoculation. In some of the wine-growing countries these
are supplied by special government experiment stations.

Denitrifying Bacteria. — Nitrogen is most readily absorbed by plants
in the form of nitrates. These are furnished to the soil chiefly by the
proteid decomposition induced by the proteolytic bacterial enzymes.
It is self-evident, therefore, that any cleavage which reduces nitrog¬
enous matter beyond the stage of nitrates, to nitrites and ammonia,
detracts from the value of the nitrogen as a food stuff for plants,
and the eventual setting free of nitrogen in the elementary state ren¬
ders it entirely valueless for any but the leguminous plants.

Nevertheless, this process of nitrogen waste or denitrification is
constantly going on in nature. In the course of ordinary decomposition,
there is a constant reduction of nitrogenous matter to nitrites and salts
of ammonia, actively taken part in by a host of bacteria, as many as

THE BIOLOGICAL ACTIVITIES OF BACTERIA

85 out of 109 investigated by Maassen 1 being found to possess this power.
This, however, is not nearly so harmful a source of nitrogen waste as
the process technically spoken of as true denitrification, in which
nitrates are reduced, through nitric and nitrous oxides, to elementary
nitrogen.

This phenomenon, more widely spread among bacteria than at first
believed, depends essentially upon simple oxygen extraction from the
nitrates by the bacteria, and for this reason goes on most actively when
the supply of atmospheric oxygen is low. The first bacteria described
as possessing this power of denitrification were the so-called B. denitri-
ficans I and II, the first an obligatory anaerobe, the other a facultative
aerobe. Since then numerous other bacteria, among them B. coli and
B. pyocyaneus, have been shown to exhibit similar activities. It is
important agriculturally, therefore, to know that many species which
are able to utilize atmospheric oxygen when supplied with it, will get
their oxygen by the reduction of nitrates and nitrites when free oxygen
is withheld. It is thus clear that a loss of nitrogen is much more apt
to proceed rapidly in manure heaps which are piled high and poorly
aerated. There are other factors, however, in regard to the physi¬
ology of these microorganisms, which must be considered for practical
purposes.

In order that these bacteria may develop their denitrifying powers
to the best advantage, it is necessary to supply them with some carbon
compound which is easily absorbed by them. This, in decomposing
material, is furnished by the products of the carbohydrate cleavage
going on side by side with the proteolytic processes. It is still more
or less an open question whether the facilitation of denitrification
brought about in manure heaps by the presence of hay and straw is due
to the carbon furnished by these materials, or whether it is due to the
fact that bacilli of this group are apt to adhere to the straw which acts
in that case as a means of inoculation.

The actual danger of nitrogen depletion of the soil by denitrifying
processes is probably much less threatening than was formerly supposed ;
for, in the first place, the conditions for complete denitrification are
much more perfect in the experiment than they ever can be in nature,
and the nitrifying processes going on side by side with denitrification
make up for much of the loss sustained.

1 Maassen, Arb. a. d. kais. Gesundheitsamt, 1, xxviii, 1901.

BIOLOGY AND TECHNIQUE

ANABOLIC OR SYNTHETIC ACTIVITIES OF BACTERIA

Nitrogen Fixation by Bacteria. — The constant withdrawal of nitroge¬
nous substances from the soil by innumerable plants would soon lead to
total depletion were it not for certain forces continually at work re¬
plenishing the supply out of the large store of free nitrogen in the atmos¬
phere. This important function of returning nitrogen to the soil in
suitable form for consumption by the plants is performed largely by
bacteria.

It is well known that specimens of agricultural soil when allowed to
stand for any length of time without further interference will increase
in nitrogenous content, but that similar specimens, if sterilized, will
show no such increase.1 The obvious conclusion to be drawn from this
phenomenon is that some living factor in the unsterilized soil has aided
in increasing the nitrogen supply. Light was thrown upon this problem
when Winogradsky,2 in 1893, discovered a microorganism in soil which
possessed the power of assimilating large quantities of nitrogen from
the air. This bacterium, which he named “ Clostridium Pasteurianum,”
is an obligatory anaerobe which in nature always occurs in symbiosis
with two other facultatively anaerobic microorganisms. In s}^m-
biosis with these, it can be cultivated under aerobic conditions and thus
grows readily in the upper well-aerated layers of the soil.

Although, until now, no other bacteria with equally well-developed
nitrogen-fixing powers have been discovered, yet it is more than likely
that Clostridium Pasteurianum is not the only microorganism endowed
with this function. In fact, Penicillium glaucum and Aspergillus niger,
two molds, and two other bacteria described by Winogradsky, have been
shown to possess this power slightly, but in an incomparably less marked
degree than Clostridium Pasteurianum.3 According to the calculations
of Sachse,4 unsterilized soil may, under experimental conditions, gain
as much as 25 milligrams of nitrogen in a season, a statement which
permits the calculation of a gain of twelve kilograms of nitrogen per
acre annually.5 It is very unlikely, however, that such gains actually
occur in nature, where nitrogen-fixation and nitrogen-loss usually
occur side by side.

1 Berthelot, Compt. rend, de la soc. de biol., cxvi, 1893.

^ Winogradsky, Compt. rend, de la soc. de biol., cxvi, 1893, ibid., t. cxviii, 1894.

3 Tacke, Landwirtsch. Jahresber., xviii, 1889.

& Sachse, u Agr. Chem.,” 1883.

5 Pfeffer, Pfliigers Physiologie, p. 395.

THE BIOLOGICAL ACTIVITIES OF BACTERIA 55

Agriculturally of even greater importance than the free nitrogen¬
fixing bacteria of the soil are the bacteria found in the root tubercles of
a class of plants known as “leguminos2e.,, It has long been known that
this class of plants, including clover, peas, beans, vetch, etc. , not only does
not withdraw nitrogen from the soil, but rather tends to enrich it. Upon
this knowledge has depended the well-known method of alternation of
crops employed by farmers the world over. The actual reason for the
beneficial influence of the leguminosse, however, was not known until
1887, when Hellriegel and Wilfarth 1 succeeded in demonstrating that
the nitrogen-accumulation was directly related to the root tubercles of
the plants, and to the bacteria contained within them.

These tubercles, which are extremely numerous — as many as a
thousand sometimes occurring upon one and the same plant — are formed
by the infection of the roots with bacteria which probably enter through
the delicate root-hairs. They vary in size, are usually situated near the
main root-stem, and, in appearance, are not unlike fungus growths.
Their development is in many respects comparable to the develop¬
ment of inflammatory granulations in animals after infection, inas¬
much as the formation of the tubercle is largely due to a reactionary
hyperplasia of the plant tissues themselves. They appear upon the
seedlings within the first few weeks of their growth as small pink
nodules, and enlarge rapidly as the plant grows. At the same time,
later in the season, when the plants bear fruit, the root tubercles begin
to shrink and crack. When the crops are harvested, the tubercles with
the root remain, rot in the ground, and re-infect the soil.

Histologically the tubercles are seen to consist of large root cells
which are densely crowded with microorganisms.

The microorganism itself, “ Bacillus radicicola,” was first observed
within the tubercles by Woronin 2 in 1866. The bacilli are large, slender,
and actively motile during the early development of the tubercles, but
in the later stages assume a number of characteristic involution forms,
commonly spoken of as “ bacteroids.” They become swollen, T and Y
shaped, or branching and threadlike. Their isolation from the root
tubercles usually presents little difficulty, since they grow readily upon
gelatin and agar under strictly aerobic conditions. On the artificial
media the bacillary form is usually well retained, involution forms
appearing only upon old cultures.

1 Hellriegel und Wilfarth, Cent. f. Bakt., 18S7.

2 Woronin , Bot. Zeit., xxiv, 1866.

BIOLOGY AND TECHNIQUE

The classical experiments of Hellriegel and Wilfarth conclusively
demonstrated the important relation of these tubercle-bacteria to nitro¬
gen assimilation by the leguminosse.

These observers cultivated various members of this group of plants
upon nitrogen-free soil — sand — and prevented the formation of root
tubercles in some, by sterilization of the sand, while in others they
encouraged tubercle formation by inoculation. An example of their
results may be given as follows:1

Lupinus luteus was cultivated upon sterilized sand. Some of the
pots were inoculated with B. radicicola, others were kept sterile. Com¬
parative analyses were made of the plants grown in the different pots

Root tubercles
present .

N o root tubercles

king result:

N . added in seed,

Harvested

soil, and soil-

Gain or

dry weight

N. present

extract

loss of N.

(a) 38.919

.998

.022

+ .975

(b) 33.755

.981

.023

+ .958

.016

.020

— .004

(d) 0.828

.011

.022

— .009

The great importance of this process in agriculture is demonstrated,
furthermore, by a comparison made by the same observers between a
legume, the pea, and one of the common nitrogen-consuming crops, oats.2

Nitrogen contents Nitrogen contents

of seed and soil. of crop. Gain or loss.

Oats 0.027 grams 0.007 grams — .020

Peas 0.038 “ 0.459 “ +.421

Exactly what the process is by which the bacteria supply nitrogen to
the plant is as yet uncertain. Although the degenerating bacteroids in
old nodules are bodily absorbed by the plant, this can not be con¬
ceived as the only method of supply, since the total nitrogen gain many
times exceeds the total weight of bacteria in the nodules. It is probable
that the microorganisms during life take up atmospheric nitrogen and
secrete a nitrogenous substance which is absorbed by the plant cells.

Although formerly the relationship between plant and bacterium
was regarded as one of symbiosis and of mutual benefit, the opinions as
to this subject show wide divergence. While, according to some authors,
the entrance of the bacteria into the plants is regarded as a true in¬
fection against which the plant offers at first a determined opposition as
evidenced by tissue reactions, other observers, notably A. Fischer, regard

1 Pfeffer, “ Planzenphysiologie,” Leipzig, 1897.

2 Hellriegel und Wilfarth, Zeit. d. Ver. f. d. Riibenzucker Industrie, 1888. Quoted
from Fischer, “ Vorles. iiber die Bakt.,” Jena, 1903.

THE BIQL.OGICAL ACTIVITIES OF BACTERIA

the plant as a parasite upon the bacteria, in that it derives the sole
benefit from the relationship and eventually bodily consumes its host.

Nitrifying Bacteria. — A process diametrically opposed in its chem¬
istry to denitrification and reduction is that which brings about an
oxidation of ammonia to nitrites and nitrates. The actual increase
of nitrates in soil allowed to stand for any length of time and examined
from time to time has been a well-established fact for many years; but
it was believed until a comparatively short time ago that this increase
was due to a simple chemical oxidation of ammonia by atmospheric oxy¬
gen. The dependence of nitrification upon the presence of living organ¬
isms was finally proved by Muntz and Schlossing 1 in 1887, who demon¬
strated that nitrification was abruptly stopped when the soil was
sterilized by heat or antiseptics. It remained, however, to isolate and
identify the organisms which brought about this ammonia oxidation.
This last step in our knowledge of nitrification was taken in 1890, by
Winogradsky. Winogradsky 2 found that the failures experienced by
others who had attempted to isolate nitrifying bacteria were due to the
fact that they had used the common culture media largely made up of
organic substances. By using culture media containing no organic
matter Winogradsky succeeded in isolating free from the soil, bacteria
which have since that time been confirmed as being the causative factors
in nitrification. During his first experiments this author observed that
in some of his cultures the oxidation of ammonia went only as far as the
stage of nitrite formation, while in others complete oxidation to nitrates
took place. Following the clews indicated by this discrepancy, he
finally succeeded in demonstrating that nitrification is a double process
in which two entirely different varieties of microorganisms take part,
the one capable of oxidizing ammonia to nitrites, the other continuing
the process and converting the nitrites to nitrates. The nitrite-forming
bacteria discovered by Winogradsky, and named Nitromonas or Nitro-
somonas, are easily cultivated upon aqueous solutions containing am¬
monia, potassium sulphate, and magnesium carbonate. According to
their discoverer they develop within a week in this medium as a gelat¬
inous sediment. After further growth this sediment seems to break
up and the bacteria appear as oval bodies, which swim actively about
and develop flagella at one end. Upon the solid media in ordinary use
they can not be cultivated. Special solid media suitable for their cul-

1 Muntz und Schlossing , Compt. rend, de l’acad. des sciences, 1887.

2 Winogradsky, Ann. Past. Inst., iv and v, 1890, 1891.

BIOLOGY AND TECHNIQUE

tivation and composed of silicic acid and inorganic salts have been
described by Winogradsky and by Omeliansky.1

Other nitrite-forming bacteria have since been described by various
observers, all of them more or less limited to definite localities. Some
of these are similar to nitrosomonas in that they exhibit the flagellated,
actively motile stage. In others this stage is absent.

The nitrite-forming bacteria, apart from their great agricultural im¬
portance, claim our attention because of their unique position in rela¬
tion to the animal and vegetable kingdoms. Extremely sensitive to
the presence of organic compounds, they are able to grow and develop
only upon media containing nothing but inorganic material; and this
entirely without the aid of any substances comparable to the chlorophyll
of the green plants. The source of energy from which this particular
class of bacteria derive the power of building up organic compounds
from simple substances is to some extent a mystery. The carbon
which they unquestionably require for the building up of organic mate¬
rial may be, as Winogradsky believed, derived to a certain extent from
ammonium carbonate. But it is also quite certain that they are capable
of utilizing directly atmospheric C02. In the absence of chlorophyll
or of any highly organized chemical compound, it seems likely that
the energy necessary for the utilization of the carbon obtained in this
simple form is derived from the oxidation of ammonia during the proc¬
ess of nitrification.

The conversion of nitrites into nitrates is carried on by other species
of bacteria also discovered by Winogradsky. These bacteria are much
more generally distributed than nitrosomonas and probably include a
number of varieties. The organism described by Winogradsky is an
extremely small bacillus with pointed ends. Capsules have occasionally
been made out. It may be cultivated upon aqueous solutions con¬
taining :

Sod. nitrite .

Potass, phosphate
Magnesium sulph.
Sodium carbonate
Ferrous sulphate.

per

cent

.05

.03

.04

The development of the organism is slow and sparse, and is directly
inhibited by the presence of organic matter. It is strongly inhibited by
the presence of ammonia.

The Liberation of Energy by Bacteria. — Like all other living beings,

1 Omeliansky, Cent. f. Bakt., II, 5, 1899.

THE BIOLOGICAL ACTIVITIES OF BACTERIA

bacteria in their metabolic processes liberate energy. It has been
shown by several observers that slight quantities of heat are given
off from actively growing cultures. The functions, furthermore, of
reproduction, motility, and enzyme formation may be looked upon as
forms of energy liberation. In addition to this, certain bacteria have
been observed which may liberate energy in the form of light.

Light Production by Bacteria. — The production of light by bacteria
is a power possessed chiefly by certain species inhabiting salt water.
Thus, much of the phosphorescence observed at sea, though more fre¬
quently due to Medusa and other invertebrate animals, is caused by
these bacteria. Numerous species which produce this phenomenon
have been isolated, too many, and too unimportant, to be individually
described. All of them are aerobes and require highly complex food
stuffs. They are closely allied to the putrefactive bacteria, and in
the sea are usually found upon rotting animal matter.1 The production
of light seems directly dependent upon the free access of oxygen, since
no light appears under anaerobic conditions. Their luminous quality,
moreover, is not a true phosphorescence, in that it does not depend
upon previous illumination and develops as well in cultures kept in the
dark as in those which have been exposed to light.2

The Formation of Pigment by Bacteria (Chromobacteria) . — A large
number of bacteria, when cultivated upon suitable media, give rise to
characteristic colors which are valuable as marks of differentiation.
For each species, the color is usually constant, depending, to a certain
extent, upon the conditions of cultivation. In only a few of the
pigmented bacteria is the pigment contained within the cell body, and
in only one variety, the sulphur bacteria, does the pigment appear to
hold any distinct relationship to nutrition. In most cases, the coloring
matter is found to be deposited in small intercellular granules or globules.
The absence of any relationship of the pigment to sunlight, as is the case
with the chlorophyll of the green plants, is indicated by the fact that
most of the chromobacteria thrive and produce pigment equally well in
the dark as they do in the presence of light. Among the most common
of the pigment bacteria met with in bacteriological work are Staphy¬
lococcus pyogenes aureus, Bacillus pyocyaneus, Bacillus prodigiosus,
and some of the green fluorescent bacteria frequently found in feces.

The chemical nature of these pigments has been investigated quite
thoroughly and it has been shown that they vary in composition.

1 Pfliiger’s Arch. f. Phys., xi, 1875. 2 Fischer, Cent. f. Bakt., iii, 1888.

BIOLOGY AND TECHNIQUE

Some of the pigments, like that of Staphylococcus aureus, are probably
non-proteid and of a fatty nature.1 They are insoluble in water but
soluble in alcohol, ether, and chloroform. Because of their probable
composition, they have been spoken of as “ lipochromes.” Other
pigments, like the pyocyanin, which lends the green color to cultures
of Bacillus pyocyaneus, are water soluble and are probably of proteid
composition. Pyocyanin may be crystallized out of aqueous solu¬
tion in the form of fine needles. The crystals may be redissolved in
chloroform. Aqueous solutions retain their color. Solutions in chloro¬
form, however, are changed gradually to yellow.

The power of pigment production of various bacteria depends in
each case upon cultural conditions. In most cases, this simply signifies
that pigment is produced only when the microorganism, finding the most
favorable environmental conditions, is enabled to develop all its func¬
tions to their fullest extent. Thus, a too high acidity or alkalinity of
the culture medium may inhibit pigment formation. Oxygen is neces¬
sary for the production of color in some bacteria, since the bacteria them¬
selves often produce the pigment only as a leuko-body which is then
oxydized into the pigment proper. A notable example of this is the pig¬
ment of B. pyocyaneus. In other cases, temperature plays an impor¬
tant role in influencing color production. Thus, Bacillus prodigiosus
refuses to produce its pigment when growing in the incubator. By
persistent cultivation in an unfavorable environment, colored cultures
may lose their power of pigment production.

Sulphur Bacteria. — Wherever the decomposition of organic matter
gives rise to the formation of II2 S, in cess-pools, in ditches, at the bottom
of the sea, and in stagnant ponds, there is found a curiously interesting
group of microorganisms, the so-called sulphur or thiobacteria. Red,
purple, and colorless, these bacteria all possess the power of utilizing
sulphuretted hydrogen and by its oxidation into free sulphur obtain
the energy necessary for their metabolic processes. The colorless sul¬
phur bacteria, the Beggiatoa and Thiothrices, usually appear as threads
or chains which, in media containing sufficient H2 S, are usually well-
stocked with minute globules of sulphur. If found upon decomposing
organic matter, they often cover this as a grayish mold-like layer.
The red sulphur bacteria, of which numerous species have been described
by Winogradsky, may appear as actively motile spirilla (Thiospirillum)
or as short, thick bacillary forms.

1 Schroeter, Cent. f. Bakt., xviii, 1895.

THE BIOLOGICAL ACTIVITIES OF BACTERIA

The physiology of all the sulphur bacteria, and especially of the
colored varieties, is of the greatest interest in that these microorganisms
are among the few members of the bacterial group which behave meta-
bolically like the green plants. The higher organic substances play lit¬
tle or no part in the nutrition of these microorganisms. Strictly aerobic,
the colorless thiobacteria are independent of sunlight, while the red and
purple varieties exhibit their physiological dependence upon light by
accumulating under natural conditions in well-lighted spots. Both
varieties possess equally the power of oxidizing sulphuretted hydrogen
as a source of energy. The sulphur is then stored as elemental sulphur
within the bacterial body and when a lack of food stuffs sets in, the
store of sulphur can be further oxidized into sulphurous or sulphuric
anhydrides. With this sole source of energy, these bacteria are capable
of flourishing aerobically, while an absence of H2S, even in the presence
of organic food stuffs, leads to a rapid disappearance of their sulphur
contents and an inability to develop.

In the case of the colored thiobacteria, the red pigment appears to
fulfil, to some extent, a function comparable to that of the chlorophyll
of the green plants.

Engelmann,1 who has studied this pigment spectroscopically, has
found that besides absorbing the red spectral rays there is an absorption
of rays' on the ultra-red end of the spectrum. The absorption of the
red rays between the lines B and C of the spectrum, and of violet rays
at the line F, is the same as that of the absorption spectrum of
chlorophyll, and it is in the zone of these rays that the physiological
effects of chlorophyll are most active. In addition to these absorp¬
tion bands, the bacteriopurpurin of the red sulphur bacteria shows
absorption of the invisible ultra red rays of the spectrum.

Engelmann, with a microspectroscope, projected a spectrum into
a miscroscopic field in which green algae or, in the case under discussion,
red sulphur bacteria had been placed. Other sources of light were, of
course, excluded. By adding emulsions of strictly aerobic bacteria to
such preparations, an accumulation of microorganisms was observed
at those points in the spectrum at which most oxygen was liberated. In
the case both of chlorophyll and of the red sulphur bacteria such areas
of bacterial accumulation (in oxygen liberation) occurred in the zones
of the absorption bands mentioned above.

1 Engelmann, Bot. Zeit., 1888.

CHAPTER V

THE DESTRUCTION OF BACTERIA

GENERAL CONSIDERATIONS

No branch of bacteriology has been more fruitful in practical appli¬
cation than that which deals with the factors which bring about the
destruction of microorganisms. Upon the study of this branch has
depended the growth and the development of modern surgery.

The agents which affect bacteria injuriously are many, and are both
physical and chemical in nature.

When a procedure completely destroys bacterial life it is spoken of as
sterilization or disinfection, the term disinfection being employed more
especially to designate the use of chemical agents. When the procedure
destroys vegetative forms only, leaving the more resistant spores un¬
injured, it is spoken of as “incomplete sterilization.” When an agent,
on the other hand, does not actually kill the microorganisms, but merely
inhibits their growth and multiplication, it is spoken of as an antiseptic.
The term deodorant is indiscriminately applied to substances which
mask or destroy offensive odors, and may or may not possess disinfectant
or antiseptic value. Some deodorants act chemically on the noxious
gases, destroying them.

PHYSICAL AGENTS INJURIOUS TO BACTERIA

The principal 'physical agents which may exert deleterious action
upon bacteria are: drying, light, electricity, and heat.

Drying. — Complete desiccation eventually destroys most of the path¬
ogenic bacteria, yet great differences in resistance to this condition are
shown by various microorganisms. Ficker,1 who has made a systematic
study of the influence of complete drying upon bacteria, concludes that
the resistance of bacteria to desiccation is influenced by the age of the
culture investigated, the rapidity with which the withdrawal of moisture

1 Ficker, Zeit, f. Hyg., xxix, 1896.
62

THE DESTRUCTION OF BACTERIA

is accomplished, and the temperature at which the process takes place.
Microorganisms like the gonococcus and the Pfeiffer bacillus, are
destroyed by drying within a few hours. The cholera vibrio dried upon a
coverslip was found by Koch 1 to be killed within four hours; by Burck-
holtz,2 to survive about twenty-four hours. The spore-forms of bacteria
are infinitely more resistant to this influence than are the vegetative
forms, though they may be destroyed by rapid and complete drying in
a desiccator.

It is self-evident that many discrepancies in the experimental
results of various authors may depend upon the technique of investiga¬
tion, since the degree of drying attained depends intimately upon the
thickness and consistence of the material investigated, and upon the
methods employed for desiccation.

Light. — Direct sunlight is a powerful germicide for all bacteria except
a limited number of species like the thio- or sulphur bacteria, which
utilize sunlight for their metabolic processes as do the green plants.

Koch 3 has shown that exposure to sunlight will destroy the tubercle
bacillus within two hours or less, the time depending upon the thick¬
ness of the exposed layers and the material surrounding the bacilli.
Confirmatory researches have been published by Mignesco 4 and others.
The powerful disinfecting influence of sunlight upon bacteria suspended
in water has been shown b}^ Buchner.5 Observations in regard to the
influence of sunlight upon anthrax spores have been made by Arloing,6
and similar observations upon a number of other microorganisms have
been carried out by Dieudonne, Janowski, v. Esmarch, and many
others. All these observers, while differing somewhat as to the time
necessary for bacterial destruction, agree in finding definite and pow¬
erful bactericidal action of sunlight. Diffuse light, of course, is less
active than direct sunlight. According to Buchner, typhoid bacilli are
inhibited by direct sunlight in one and one-half hours, by diffuse light
in five hours. A remarkable statement is made by Arloing, who claims
to have found that anthrax spores are more quickly destroyed by
direct sunlight than are the vegetative cells. This fact would call for
further confirmation.

1 Koch, Arb. a. d. kais. Gesundheitsamt, iii, 1887.

2 Burkholtz, Arb. a. d. kais. Gesundheitsamt, v, 1889.

3 Koch, X Internat. Med. Congress, Berlin, 1890.

4 Mignesco, Arch. f. Hyg., xxv, 1896.

6 Buchner, Cent. f. Bakt., I, xi, 1892.

6 Arloing, Compt. rend, de l’acad. d. sci., c, 1885.

BIOLOGY AND TECHNIQUE

It has been shown by various authors that the influence of sunlight is
not to be attributed in any way to temperature, nor always to a direct
action of the light upon the bacteria, but depends largely upon photo¬
chemical changes produced by the light rays in the media. Richardson 1
and Dieudonne 2 conclude that under ordinary aerobic conditions in fluid
environment peroxide of hydrogen is formed under the influence of light.
Novy and Freer 3 believe that the bactericidal effects in fluids noticed
as a result of exposure to light are too strong to be explained by the
formation of small quantities of peroxide of hydrogen, and attribute
this action to organic peroxides formed under the described conditions,
such as the peroxides of diacetyl, benzoylacetyl, and others. These
views are somewhat strengthened by the fact that exclusion of oxygen
from media markedly diminishes the bactericidal power of light.4 That
the photochemical changes alone, however, do not explain this action
follows from the fact that dried bacteria, not surrounded by media, are
subject to a similar action.5

In analyzing sunlight in regard to its bactericidal power, it has been
found by various observers that the most powerful action is exerted by
the ultraviolet spectral rays, whereas the yellow, red, and ultra-red rays
are practically innocuous.6

It is of importance to note that sunlight has been found also to have
a strong attenuating influence 7 upon some bacterial poisons, as shown
by the experiments of Ferri and Celli upon tetanus toxin.

Electric light exerts a distinct bactericidal action when applied in
strengths of 800 to 900 candle power for seven or eight hours.8

Rontgen or x-rays are said by Zeit,9 Blaise 10 and Sambac, and others
to be without appreciable germicidal power. Rieder,11 on the other
hand, has reported definite inhibition of bacterial growth after exposures
of half an hour to x-rays.

1 Richardson, Jour. Chem. Soc., i, 1893, Ref. Deut. chem. Gesells., xxvi.

2 Dieudonne, loc. cit.

3 Novy and Freer, 3d Ann. Meeting Assn. Amer. Bacteriologists, Chicago, 1901.

4 Roux, Ann. Inst. Past., ix, 1887.

5 Dieudonne, loc. cit.

6 Ward, Proc. Royal Soc., 52, 1893.

7 Ferri and Celli, Cent. f. Bakt., I, xii, 1892.

8 Dieudonne, loc. cit.

9 Zeit, Jour. Amer. Med. Assn., xxxvii, 1901.

10 Blaise and Sambac, Compt. rend, de la soc. de biol., 1896.

11 Rieder, Munch. med. Woch., 1898.

THE DESTRUCTION OF BACTERIA

Radium rays have a distinct inhibitory and even bactericidal
power when applied at distances of a few centimeters for several
hours.1

Electricity. — If we exclude the' indirect actions of heat and electro¬
lysis, it can hardly be said that the direct bactericidal action of electric
currents has been satisfactorily demonstrated. Such action, however,
has been claimed by d’Arsonville and Charrin,2 and by Spilker and
Gottstein.3

Heat. — The most widely applicable and efficient physical agent for
sterilization is heat.

The dependence of bacteria for growth and vitality upon the main¬
tenance of a proper temperature in their environment, and the ranges
of variation within which bacteria may thrive, have been discussed in a
preceding section, in which a table of so-called “ thermal death points ”
has been given. In the method of expressing these values it was seen
that two elements entered into the destruction of bacteria by heat,
namely, that of the degree of temperature which is applied, and that of
the time of application.

The prolonged application of moderately high temperatures, in other
words, may in certain instances, accomplish the same result as the brief
use of extremely high ones. In general, the death of bacteria following
prolonged 'exposure to temperatures but slightly exceeding the optimum
is due to the inability of the anabolic processes to keep pace with the
accelerated katabolic processes, gradual attenuation resulting in death.
At somewhat higher temperatures death results from coagulation of
the bacterial protoplasm, and at still higher degrees of heat, applied in
the dry form, direct burning of the bacteria may be the cause of their
destruction.

Heat may be applied in the form of dry heat or as moist heat, these
methods being of great practical value, but differently applicable ac¬
cording to the nature of the materials to be sterilized. The two methods,
moreover, show a marked difference in efficiency, temperature for tem¬
perature. For the recognition of this fact we are largely indebted to the
early researches of Koch and Wolffhiigel,4 and of Koch, Gaffky, and
Loeffler.5

1 Personal observations.

2 D’Arsonville and Charrin, Compt. rend, de la soc. de biol.

3 Sjpilker and Gottstein, Cent. f. Bakt., I, 9, 1891.

4 Koch und Wolffhiigel, Mitt. a. d. kais. Gesundheitsamt, 1, 1882.

5 Koch, Gaffky and Loeffler, ibid.

BIOLOGY AND TECHNIQUE

These observers were able to show that the spores of anthrax were
destroyed by boiling water at 100° C. in from one to twelve minutes,
whereas dry hot air was efficient only after three hours’ exposure to
140° C. Extensive confirmation of these differences has been brought
by many workers. An explanation of the phenomena observed
is probably to be found in the changes in the coagulability of
proteicls brought about in them by the abstraction of water.
Lewith,1 working with various proteids, found that these sub¬
stances are coagulated by heat at lower temperatures when they
contain abundant quantities of water, than when water has been
abstracted from them. On the basis of actual experiment with egg
albumin he obtained the following results,2 which illustrate the point
in question:

Egg albumin in dilute aqueous solution, coagulated at 56° C.

with 25 per cent water, “ “ 74-80° C.

18 “ “ ft “ “ 80-90° C.

6 “ “ “ “ “ 145° C.

U U

(6 U

Absolutely anhydrous albumin, according to Haas,3 may be heated
to 170° C. without coagulation. It is thus clear that bacteria exposed
to hot air may be considerably dehydrated before the temperature rises
sufficiently to cause death by coagulation, complete dehydration neces¬
sitating their destruction possibly by actual burning.

Bacteria exposed to moist air or steam, on the other hand, may ab¬
sorb water and become proportionately more coagulable.

The same principle, as Lewith points out, probably explains the great
resistance to heat observed in the case of the highly concentrated pro¬
toplasm of spores.

Apart from the actually greater efficiency of moist heat when com¬
pared with dry heat of an equal temperature, an advantage of great
practical significance possessed by moist heat lies in its greater powers of
penetration. An experiment carried out by Koch and his associates
illustrates this point clearly. Small packages of garden soil were sur¬
rounded by varying thicknesses of linen with thermometers so placed
that the temperature under a definite number of layers could be deter-

1 Lewith, Arch. f. exp. Path. u. Pharm., xxvi, 1890.

2 Lewith, loc. cit., p. 351.

3 Haas, Prag. med. Woch., 34-36, 1876.

THE DESTRUCTION OF BACTERIA

mined. Exposures to hot air and to steam were then made for com¬
parison, and the results were as tabulated:1

Tempera-

Time of

Temperatures Reached within
Thicknesses of Linen.

tures.

Application.

Twenty

Thicknesses.

F orty

Thicknesses.

One Hundred
Thicknesses.

Hot air .

130-140° C.

4 hours.

86°

72°

Below 70°

Incomplete

steriliza¬

tion.

Steam .

90-105.3°

3 hours.

101°

101.5°

Complete

steriliza¬

tion.

This great penetrating power of steam is due presumably to its com¬
paratively low specific gravity which enables it to displace air from the
interior of porous materials, and also to the fact that as the steam comes
in contact with the objects to be disinfected a condensation takes place
with the consequent liberation of heat. When a vapor passes into the
liquid state it gives out a definite amount of heat, which in the case of
water vapor, at 100° C., amounts to about 537 calories. This brings
about a rapid heating of the object in question. Following this
process the further heating takes place by conduction, and it is, of
course, well known that steam is a much better heat conductor than air.2

Moist heat may be applied as boiling water, in which, of course,
the temperature varies little from 100° C., or as steam. Steam may be
used as live, flowing steam, without pressure, the temperature of which is
more or less constant at 100° C., or still higher efficiency may be attained
by the use of steam under pressure, in which, of course, temperatures
far exceeding 100° C. may be produced, according to the amount of
pressure which is used.

The spores of certain bacteria of the soil which can not be killed in
live steam in less than several hours may be destroyed in a few minutes,
or even instantaneously, in compressed steam at temperatures ranging
from 120° to 140° C.3

In all methods of steam sterilization, it is of great practical impor-

1 Koch, Gaffky und Loeffler, loc. cit., p. 339.

2 Gruber, Cent. f. Bakt., iii, 1888.

3 Christen, Ref. Cent. f. Bakt., V, xiii, 1893.

BIOLOGY AND TECHNIQUE

tance, as v. Esmarch 1 has pointed out, that the steam shall be saturated,
that is, shall contain as much vaporized water as its temperature per¬
mits. Unsaturated, or so-called “ super-heated' steam ” is formed when
heat is applied to steam, either by passage through heated piping or over
heated metal plates. In such cases the temperature of the steam is
raised, but no further water-vapor being supplied, the steam exerts
less pressure and contains less water in proportion to its volume than
saturated steam of an equal temperature. The super-heated steam,
therefore, is heated considerably over its condensation temperature and
becomes literally dried. In consequence, its action is more comparable
to hot air than to saturated steam, and up to a certain temperature its
disinfecting power is actually less than that of live steam at 100° C.
v. Esmarch, who has made a thorough study of these conditions, con¬
cludes that up to 125° C., the efficiency of superheated steam is lower
than that of live steam at 100° C. Above this temperature, of course,
it is again active as in the case of ordinary dry heat.

Practical Methods of Heat Sterilization. — Burning. — For ob¬
jects without value, actual burning in a furnace is a certain and easily
applicable method of sterilization. Flaming, by passage through a
Bunsen or an alcohol flame, is the method in use for the sterilization of
platinum needles, coverslips, or other small objects which are used in
handling bacteria in the laboratory.

Hot air sterilization is carried out in the so-called ahot air chambers,”
simple devices of varied construction. The apparatus most commonly
used (Fig. 8) consists of a sheet-iron, double-walled chamber, the
joints of which, instead of being soldered, are closed by rivets. The inner
case of this chamber is entirely closed except for an opening in the top
through which a thermometer may be introduced, while the outer has a
large opening at the bottom and two smaller ones at the top. A gas-
burner is adjusted under this so as to play directly upon the bottom of
the inner case. A thermometer is fitted in the top in such a way that it
penetrates into the inner chamber. The air in the chamber is heated
directly by the flame and by the hot air, which, rising from the flame,
courses upward within the jacket between the two cases and escapes at
the top. To insure absolute sterilization of objects in such a chamber,
the temperature should be kept between 150° and 160° C. for at least an
hour. In Sterilizing combustible articles in such a chamber, it should be
remembered that cotton is browned at a temperature of 200° C. and

1 v. Esmarch, Zeit, f, Hyg., iv. 1888,

THE DESTRUCTION OF BACTERIA

over. This method is used in laboratories for the sterilization of Petri
dishes, flasks, test tubes, and pipettes, and for articles which may be in¬
jured by moisture. Both heating and subsequent cooling should be done
gradually to avoid cracking of the glassware.

Moist Heat. — Instruments, syringes, and other suitable objects may
be sterilized by boiling in water. Boiling for about five minutes is amply
sufficient to destroy the vegetative forms of all bacteria. For the de¬
struction of spores, boiling for one or two hours is usually sufficient,
though the spores of certain saprophytes of the soil have been found

occasionally to withstand moist heat at a temperature of 100° C. for
as long as sixteen hours.1 The addition of 1 per cent of sodium car¬
bonate to boiling water hastens the destruction of spores and prevents
the rusting of metal objects sterilized in this way. The addition of car¬
bolic acid to boiling water in from 2 to 5 per cent usually insures the
destruction of anthrax spores, at least, within ten to fifteen minutes.

Exposure to live steam is probably the most practical of the methods
of heat sterilization. It may be carried out by simple makeshifts of
the kitchen, such as the use of potato-steamers or of wash-boilers. For

1 Christen, loc. cit.

BIOLOGY AND TECHNIQUE

laboratory purposes, the original steaming device introduced by Koch
has been almost completely displaced by devices constructed on the
plan of the so-called “ Arnold ” sterilizer (Fig. 9) . In such an appara¬
tus, water is poured into the reservoir A and flows from there into
the shallow receptacle B, formed by the double bottom. The flame
underneath rapidly vaporizes the thin layer of water contained in B ,
and the steam rises rapidly, coursing through the main chamber C.
Steam which escapes through the joints of the lid of this chamber is
condensed under the hood and drops back into the reservoir. Exposure

to steam in such an apparatus for fifteen
to thirty minutes insures the death of
the vegetative forms of bacteria.

In the sterilization of media by such
a device, the method of fractional sterili¬
zation at 100° C. is employed. The prin¬
ciple of this method depends upon
repeated exposure of the media for fif¬
teen minutes to one-half hour on three
succeeding days. By the first exposure
all vegetative forms are destroyed. The
media may then be left at room tem¬
perature, or at incubator temperature
(37.5° C.) until the following day, when
any spores which may be present will
have developed into the vegetative stage.
These are then killed by the second ex¬
posure. A repetition of this procedure
on a third day insures sterility. It must
always be remembered, however, that
this method is applicable only in cases
in which the substance to be sterilized is a favorable medium for
bacterial growth in which it is likely that spores will develop into vege¬
tative forms.

Exceptionally the method may fail even in favorable media when
anaerobic spore-forming bacteria are present. Thus, it has been ob¬
served that anaerobic spores, failing to develop under the aerobic con¬
ditions prevailing during the intervals of fractional sterilization, have
developed after inoculation of the media with other bacteria, when sym¬
biosis had made their growth possible. Tetanus bacilli have, in this way,
occurred in cultures of diphtheria bacilli employed for toxin production.

THE DESTRUCTION OF BACTERIA

In noting the time of an exposure in an Arnold sterilizer, it is
important to time the process from the time when the temperature
has reached 100° C. and not from the time of lighting the flame.

The principle of fractional sterilization at low temperatures is ap¬
plied also to the sterilization of substances which can not be sub¬
jected to temperatures as high as 100° C. This is especially the case
in the sterilization of media containing albuminous materials, when
coagulation is to be avoided, or when both coagulation of the medium
and sterilization are desired.

In such cases fractional sterilization may be practiced in simply con¬
structed sterilizers, such as a Koch inspissator or, in the case of fluids,
such as blood serum, by immersion in a water-bath at a temperature

varying above 55° C., according to circumstances. Exposures at such
low temperatures may be repeated on five or six consecutive days, usu¬
ally for an hour each day.

The use of steam under pressure is the most powerful method of heat-
disinfection which we possess. It is applicable to the sterilization of
fomites, clothing, or any objects of a size suitable to be contained in the
apparatus at hand, and which are not injured by moisture. In labora¬
tories this method is employed for the sterilization of infected appa¬
ratus, such as flasks, test tubes, Petri plates, etc., containing cultures.
The device most commonly used in laboratories is the so-called auto¬
clave, of which a variety of models may be obtained, both stationary
and portable. The principle governing the construction of all of these

BIOLOGY AND TECHNIQUE

is the same. The apparatus usually consists of a gun-metal cylinder
supplied with a lid, which can be tightly closed by screws or nuts,
and supplied with a thermometer, a safety-valve, and a steam pressure
gauge. In the simpler autoclaves, water may be directly filled into
the lower part of the cylinder, and the objects to be sterilized supported

upon a perforated diaphragm. In this
case the heat is directly applied by means
of a gas flame. In the more elaborate
stationary devices, steam may be let in
by piping it from the regular supply used
for heating purposes. Exposure to steam
under fifteen pounds pressure (fifteen in
addition to the usual atmospheric press¬
ure of fifteen pounds to the square inch)
for fifteen to twenty minutes, is sufficient
to kill all forms of bacterial life, including
spores.

In applying autoclave sterilization
practically, attention must be paid to
certain technical details, neglect of which
would result in failure of sterilization. It
is necessary always to permit all air to
escape from the autoclave before closing
the vent. If this is not done, a poorly
conducting air-jacket may be left about
the objects to be sterilized, and these
may not be heated to the temperature
indicated by the pressure. It is also nec¬
essary to allow the reduction of pressure,
after sterilization, to take place slowly.
Any sudden relief of pressure, such as
would be produced by opening the air-
vent while the pressure gauge is still above zero, will usually result in
a sudden ebullition of fluid and a removal of stoppers from flasks.

The temperature attained by the application of various degrees of
pressure is expressed in the following table :

. t4-

i i

■ i
• *

• i

1 1

•

r v — i
_ _ _ _

Fig. 11. — Autoclave.

Lbs. Pressure Temperature

1 . 102.3°

2 . 104.2

3 . 105.7

4 . . . 107.3

Lbs. Pressure Temperature

5 . 108.8°

6 . 110.3

7 . 111.7

8 . 113

THE DESTRUCTION OF BACTERIA

Lbs. Pressure

9 .

10 .

11 .

12 .

13 .

14 .

15 .

16 .

Temperature

Lbs. Pressure

Temperature

. . 114.3°

17 .

. 123.3°

. . 115.6

18 .

. 124.3

. . 116.8

20 .

. 126.2

. . 118

22 .

. 128.1

. 119.1

24 .

....... 129.3

. 120.2

26 .

. 131.5

. . 121.3

28 .

. 133.1

. 122.4

30 .

. 134.6

CHEMICAL AGENTS INJURIOUS TO BACTERIA

Since the time of Koch;s 1 fundamental researches upon chemical
disinfectants, the known number of these substances has been enor¬
mously increased, and now embraces chemical agents of the most varied
constitution. It is thus manifestly impossible to refer the injurious in¬
fluence which these substances exert upon bacteria to any uniform law of
action. The efficiency of a disinfecting agent, furthermore, is not alone
dependent upon the nature and concentrations of the substance itself, but
depends complexly upon the nature of the solvent in which it is employed,
the temperature prevailing during its application, the numbers and bio¬
logical characteristics of the bacteria in question, and the time of ex¬
posure. All these factors, therefore, must be considered in testing the
efficiency of any given disinfectant. While it is true, furthermore,
that all substances which in a given concentration exert bactericidal or
disinfecting action upon a microorganism, will in greater dilution act
antiseptically or inhibitively, no definite rules of proportion exist be¬
tween the two values, which in each case must be determined by experi¬
ment.

Disinfectants Used in Solution. — The actual processes which take place
in the injury of bacteria by disinfectants are to a large extent unknown.
In the case of strong acids, or strongly oxidizing substances, there may
be destruction of the bacterial body as a whole by rapid oxidation.
Other substances may act by coagulation of the bacterial protoplasm;
others again by diffusion through the cell membrane are able to enter into
chemical combination with the protoplasm and exert a toxic action.
Again, in other cases, a difference in tonicity between cell protoplasm
and disinfectant may tend to withdrawal of water from the bacterial
cell and consequent injury of the microorganism.

Among the inorganic disinfectants the most important are the metallic

1 Koch, Arb. a. d. kais. Gesundheitsamt, i, 1881.

BIOLOGY AND TECHNIQUE

salts, acids, and bases, the halogens and their derivatives, and certain
oxidizing agents like peroxide of hydrogen and permanganate of potas¬
sium.

It has been shown by Scheuerlen and Spiro,1 Kronig and Paul,2 and
others, that in the case of the salts, acids, and bases, there is a distinct
and demonstrable relationship between the disinfecting power of these
substances and their dissociation in solution.

According to the theory of electrolytic dissociation, when bodies of
this class go into solution they are broken up or dissociated into an
electro-positive and an electro-negative ion. Thus, metallic salts are
broken up into the kation, or positive metal, and into the anion, or
negative acid radicle (AgN03 = Ag, + ion and N03, — ion). In the
case of the acids, ionization takes place into the hydrogen ions and the
acid radicles, while in the case of the bases the dissociation occurs into
the metal, on the one hand, and the OH group on the other. The de¬
gree of dissociation taking place depends upon the nature of the sub¬
stance in solution, its concentration, and the nature of the solvent.
Thus, in any such solution there appear three substances, the undis¬
sociated compound as such, its electro-negative ion, and its electro¬
positive ion, their relative concentrations depending upon an interrela¬
tionship calculable by definite laws. It goes without saying, therefore,
that any chemical or physical reaction, taken part in by such a solution,
may be participated in, not only by the dissolved undissociated residue
as a whole, but by its separate ions individually as well. In the case of
many disinfectants, the writers referred to above have been able to
demonstrate a relationship between the degree of dissociation and the
bactericidal powers. According to Kronig and Paul, double metallic
salts, in which the metal is a constituent of a complex ion and in which
the concentration of the dissociated metal-ions is consequently low,
have very little disinfecting power. Thus potassium-silver-cyanide,
which is a comparatively weak disinfectant, dissociates into the kation K
and the complex anion Ag (CN) 2, this latter further dissociating to a very
slight degree only. The same writers conclude that the bactericidal
action of mercuric chloride and of halogen combinations with metals is
directly proportionate to the degree of dissociation. This considera¬
tion, moreover, explains why aqueous solutions of such substances are
more active than are solutions in the alcohols or in ether, since it is well

1 Scheuerlen und Spiro, Munch, med. Woch., 44, 1897.

2 Kronig und Paul, Zeit. f. Hyg., xxv, 1897.

THE DESTRUCTION OF BACTERIA

known that metallic salts are ionized in these substances to a much
slighter degree than they are in water.1

On the other hand, the addition of moderate quantities of ethyl
and methyl alcohol or acetones to aqueous solutions of silver nitrate or
mercuric chloride, definitely increases the disinfecting action of such
solutions. In the case of mercuric chloride, Kronig and Paul obtained
the most powerful effects in solutions to which alcohol had been added
in a concentration of 25 per cent. For this empirical fact a satisfactory
explanation has not yet been found. Kronig and Paul suggest that low
percentages of alcohol may facilitate the penetration of the disinfectant
through the cell membrane and thus increase its efficiency, while high
percentages of alcohol have the opposite effect, by decreasing the degree
of dissociation. In this connection it has been suggested, however,
that absolute and strong alcohols possibly act as desiccating agents,
thus actually rendering the bacteria dry and less susceptible to dele¬
terious chemical influences.

In the case of acids and bases the same authors have determined
that the powers of disinfection of these substances are again directly
proportionate to the degree of their dissociation: that is, to the concen¬
tration of the hydrogen or hydroxyl ions, respectively. The hydrogen
ions are more powerfully active than the hydroxyl ions in equal con¬
centration; acids, therefore, are more efficient disinfectants than bases.

A fact which appears to strengthen the opinion as to the relationship
between bactericidal powers and dissociation, is that brought forward
by Scheuerlen and Spiro, that the addition of NaCl to bichloride of
mercury solutions reduces the disinfecting power of such solutions, in¬
asmuch as it diminishes the concentration of free ions. In practice,
however, NaCl or NH4C1 is added to bichloride of mercury solutions,
since these substances aid in holding in solution mercury compounds
formed in the presence of alkaline albuminous material, blood serum,
pus, etc.

In regard to the halogens, Kronig and Paul have shown that the
germicidal power of this class of elements is inversely proportionate to
their atomic weights. Thus, chlorine with the lowest atomic weight is the
strongest disinfectant of the group. Next, and almost equal to this, is

1 Water is the strongest dissociant known. Methyl alcohol has about one-half to
two-thirds the dissociating power of water (Zelinsky, Zeit. f. physiol. Chemie, xx,
1896). Ethyl alcohol allows dissociation much less than methyl alcohol; ammonia
allows dissociation to about one-third to one-fourth the extent of water. See Jones,
“ Elements of Physical Chemistry,” p. 371. Macmillan, New York, 1902.

BIOLOGY AND TECHNIQUE

bromin. Iodine with a much heavier atomic weight than either of the
former is distinctly less bactericidal.

Chloride of Lime. — Of the halogen compounds commonly used
in practice, the most important is what is popularly known as
chloride of lime or bleaching powder. As to the composition of this
substance, there is some difference of opinion. It was formerly be¬
lieved to be a mixture of calcium hypochlorite, Ca(C102), and of
calcium chloride, CaCl2. The fact that the substance is not deliques¬
cent, however, speaks against the presence of calcium chloride as such,
and it is probable that it consists of a single compound with the for¬
mula CaOCl2. The action of acids or even of atmospheric C02 upon
this substance results in the liberation of chlorine. For instance,

Ca(Cl20) + 2HC1 = CaCl2 + 2HC10.

2HC10 + 2HC1 = 2H2 + 2C12.

Bleaching powder is readily soluble in about twenty parts of water.
According to Nissen,1 solutions of 2 in 1,000 of this substance, destroy
vegetative forms of bacteria in five to ten minutes.

Terchloride of iodine (IC13), another halogen derivative, is an
extremely strong disinfectant, being efficient for vegetative forms in
solutions of 0.1 per cent in one minute and a 1 per cent solution de¬
stroying spores within a few minutes.2

Surgeons have found that painting with tincture of iodine (10 per
cent) is a simple and reliable method of sterilizing the skin. It is now
used in many clinics as the sole disinfecting agent in sterilizing the field
of operation.

The oxidizing agents most commonly employed are peroxide of hy¬
drogen (H202) and permanganate of potassium (KMn04).

Peroxide of hydrogen is formed by the action of dilute sulphuric
acid upon peroxide of barium. It readily gives up oxygen and acts
upon bacteria probably by virtue of the liberation of nascent oxygen.
In the presence of organic matter such as blood, pus, etc., associated
with bacteria, H202 is quickly reduced and weakened. It is important
that the H202 come in immediate contact with the bacteria. In prac¬
tice, therefore, blood and pus should be removed from wounds when
applying the H202 or a large excess of H202 should be used.

Permanganate of Potassium, acting probably in the same way, is
a powerful germicide. It also is readily reduced by many organic sub¬
stances often associated with bacteria, being rendered weaker thereby.

1 Nissen, Zeit. f. Hyg., viii, 1890.

2 v. Behring, Zeit. f. Hyg., ix, 1891.

THE DESTRUCTION OF BACTERIA

Among organic disinfectants those of most practical importance are
the alcohols, formaldehydes, iodoform, members of the phenol group
and its derivatives, carbolic acid, cresol, lysol, creolin, salicylic acid, cer¬
tain ethereal oils, and, more recently introduced, organic silver salts
such as protargol, argyrol, argonin, and others.

The alcohols are but indifferent disinfectants. Koch 1 in 1SS1
found that anthrax spores remained alive for as long as four months
when immersed in absolute and in 50 per cent ethyl alcohol. On the
other hand, while absolute alcohol possesses practically no germicidal
powers, possibly because of the formation of a protecting envelope by
the coagulation of the bacterial ectoplasm, or, as suggested above, by
desiccation due to the abstraction of water, dilute alcohol in a concen¬
tration of about 50 per cent is distinctly germicidal, destroying the vege¬
tative forms of bacteria in from ten to fifteen minutes or less.2
Attention has already been called to the fact that moderate ad¬
ditions of alcohol to aqueous solutions of mercuric chloride enhance
the germicidal power of this disinfectant. Additions of ethyl and
methyl alcohol to carbolic acid or formaldehyde solutions, on the
other hand, progressively decrease the bactericidal activities of these
substances.3

The value of boiling alcohol for the destruction of spores — especially
in the sterilization of catgut — has been investigated by Saul,4 who
found that boiling in absolute ethyl, methyl, or propyl alcohol is prac¬
tically without effect, while spores are destroyed readily in boiling
dilute alcohol, the most effectual being propyl alcohol of a concentra¬
tion of from 10-40 per cent.

Iodoform (CHI3)5 is weakly antiseptic in itself, but when introduced
into wounds where active reducing processes are taking place — often
as the result of bacterial growth — iodine is liberated from it and active
bactericidal action results.

Carbolic acid (C6H5OH), at room temperature, consists of color¬
less crystals which become completely liquefied by the addition of 10
per cent of water. In contradistinction to most inorganic disinfectants,
the action of carbolic acid and other members of the phenol group is

1 Koch . Arb. a. d. kais. Gesundheitsamt, i, 1881.

2 Epstein, Zeit. f. Hyg., xxiv, 1897.

3 Kronig und Paul, loc. cit.

4 Saul, Archiv f. klin. Chir., 56, 1898.

5v. Behring , “ Bekaempfung d. Infektions-Krankh.,” Leipzig, 1894,

BIOLOGY AND TECHNIQUE

not in any way dependent upon dissociation.1 According to Beckmann 2
and others, carbolic acid acts as a molecule and not by individual ions.
The proof of this is brought out by the fact that the addition of NaCl
to carbolic acid solutions, an addition which would tend to decrease
the concentration of free ions, markedly increases the bactericidal
powers of such solutions. On the other hand, as stated above, addi¬
tions of alcohol progressively diminish the efficiency of the phenols.

Other members of this group of disinfectants are ortho-, meta-, and
paracresol (C6H4CH3OH), isomeric compounds differing only in the
position of the OH radicle. Tricresol is a mixture of these three. The
cresols are relatively more powerfully germicidal than is carbolic acid,
but are less soluble in water. Lysol is a substance obtained by the
solution of coal-tar cresol in neutral potassium-soap. Dissolved in
water it forms an opalescent easily flowing liquid. According to Gru¬
ber,3 its germicidal action is slightly greater than that of carbolic acid.
Creolin, another combination of the cresols with potassic soap, forms
with water a turbid emulsion, v. Behring 4 expressed the relative
germicidal powers of carbolic acid, cresol, and creolin for vegetative
forms by the numbers 1:4 : 10, in the order named.

Formaldehyde (H-COH), or methyl aldehyde, is a gas which is
easily produced by the incomplete combustion of methyl alcohol. The
methods of actually generating it for purposes of fumigation will be
discussed in a subsequent paragraph. In aqueous solution this substance
forms a colorless liquid with a characteristic acrid odor, and in this form
is largely used as a preservative for animal tissues and as a germicide.
It is marketed as “formalin,” which is an aqueous solution containing
from 35 to 40 per cent of the gas and which exerts distinctly bactericidal
action on vegetative forms in further dilutions of from 1 to 10 to 1 to
20 (formaldehyde gas 1 : 400 to 1 : 800) . Anthrax spores are killed
in 35 per cent formaldehyde in ten to thirty minutes.5 Unlike the
phenols, the addition of salt to formaldehyde solutions does not increase
its efficiency, but similar to them, additions of ethyl and methyl alcohol
markedly reduce its germicidal powers.

The essential oils which are most commonly used in practice —
largely as intestinal antiseptics — are those of cinnamon, thyme, eucalyp-

1 Scheuerlen und Spiro, Munch, med. Woch., 44, 1897.

2 Beckmann, Cent. f. Bakt., I, xx, 1896.

3 Gruber, Cent. f. Bakt, I., xi, 1892.

* v. Behring, loc. cit., p. 111.

6 Kronig und Paul, loc. cit.

THE DESTRUCTION OF BACTERIA

tus, and peppermint. Omeltschenko 1 believes that the employment of
these oils in emulsions is illogical, inasmuch as their bactericidal powers
depend upon their vaporization. He classifies the oils in decreasing
order of their efficiency as follows: Oil of cinnamon, prunol, oil of thyme,
oil of peppermint, oil of camphor, and eucalyptol.

Methods of Testing the Efficiency of Disinfectants. — The efficiency of
any given disinfectant depends, as we have seen, upon a number of
factors, any one of which, if variable, may lead to considerable differences
in the end result. Thus, as far as the bacteria themselves are concerned,
it is necessary to remember that not only do separate species differ in
their resistance to disinfectants, but that different strains within the
same species may show such variations as well. This fact largely ac¬
counts for the widely varying reports made by different investigators
as to the resistance of anthrax spores, and depends possibly upon tem¬
porary or permanent biological differences produced in bacteria by the
conditions of their previous environment.

The numbers of bacteria exposed to the disinfectant, furthermore,
is a factor which should be kept constant in comparative tests. The
medium, moreover, in which bacteria are brought into contact with the
disinfectant is a matter of great importance, inasmuch as either by
entering into chemical combination with the disinfectant it may detract
from its concentration or by coagulation it may form a purely mechanical
protection for the microorganism. Thus bacteria which may be de¬
stroyed in -distilled water or salt-solution emulsion with comparative
ease, may evince an apparently higher resistance if acted upon in
the presence of blood serum, mucus, or other albuminous substances.
Temperature influences bactericidal processes in that most chemical
disinfectants are more actively bactericidal at higher than at lower
temperatures, a fact due most likely to the favorable influence of tem¬
perature upon all chemical reactions.2 As far as merely inhibitory or
antiseptic values are concerned, however, the temperature least favor¬
able for the reaction of the antiseptic is that which represents the opti¬
mum growth temperature for the microorganism in question and the
inhibitory effects of any substance are less marked at this point than at
temperatures above or below it.

The important influence exerted by the solvent in which the

1 Omeltschenko, Cent. f. Bakt., I, ix, 1891.

2 v. Behring, “ Bekaempf. der Infektions-Krankh., Infektion u. Desinfection,”
Leipzig, 1894.

BIOLOGY AND TECHNIQUE

disinfectant is employed has already been discussed. For ordinary
work it is customary to express absolute and comparative antiseptic
and bactericidal values in terms of percentages based upon weight, and
this, beyond question, is both simple and practical. For strictly scien¬
tific comparisons, however, as Kronig and Paul 1 have pointed out, it
is by far more accurate to work with equimolecular solutions.

Rideal and Walker 2 have devised a method of testing disinfectants,
in which an attempt is made to establish a standard for comparisons.
They choose, as the standard, carbolic acid, and establish what they call
the “carbolic-acid coefficient.” This coefficient they obtain in the fol¬
lowing way: the particular dilution of the disinfectant under investiga¬
tion which will kill in a given time, is divided by the strength of carbolic
acid which, under the same conditions, will kill the same bacteria in
the same time. We quote an example of such a test, given by Simpson
and Hewlett,3 comparing formalin and carbolic acid.

BACILLUS PESTIS.

Sample.

Dilution.

Time in Minutes.

2.5

7.5

12.5

Formalin . j

Carbolic acid . j

lin 30
lin 40

1 in 100

1 in 110

growth

In the above table, formalin 1 in 30 killed in the same time as
carbolic acid 1 in 110. Thus the carbolic-acid coefficient of formalin
in this test = 3%io = .27.

The Rideal-Walker method has been much used and is recommended
by many workers.4

The most precise method of standardizing disinfectants is that now
in use in the U. S. Public Health Service. It is a modification of the
Rideal-Walker procedure devised by Anderson and McClintic.5

Stock 5 per cent solutions of the disinfectant in question and of the

1 Kronig und Paul , loc. cit

2 Rideal and Walker, Jour, of the Sanitary Ins. London, xxiv.

3 Simpson and Hewlett, Lancet, ii, 1904.

4 Sommerville, Brit. Med. Jour., 1904.

6 Anderson and McClintic, Jour, of Inf. Dis., 1911, viii, 1.

THE DESTRUCTION OF BACTERIA

standard (phenol) are first prepared and a series of accurate dilutions
made with distilled water using graduated pipettes. (To make 1:70 take
4 c.c. of stock and 10 c.c. distilled water; 1:80 = 4 c.c. of stock + 12
c.c. distilled water; 1:90 = 4 c.c. stock + 14 c.c. distilled water; 1:500
= 2 c.c. of stock + 48 c.c. of distilled water. Complete dilution tables
are given in their original article.) The series should include dilutions
strong enough to kill B. typhosus in two and a half minutes and weak
enough to fail to do so in fifteen minutes. If dilutions greater than 1-
500 are required, a second 1 per cent stock solution is prepared. They
adopted the following scale for their tests: Dilutions up to 1:70 should
vary from the next in the series by a difference of 5 (i.e., 5 parts of water).

From 1:70
From 1: 160
From 1:200
From 1:400
From 1:900
From 1 : 1800

to 1 : 160 by a
to 1 : 200 by a
to 1 : 400 by a
to 1 : 900 by a
to 1 : 1800 by a
to 1 : 3200 by a

difference of 10
difference of 20
difference of 25
difference of 50
difference of 100
difference of 200

and so on if higher dilutions are necessary.

Short wide test tubes 1 inch by 3 inches are used in making the test.
These are placed in a rack in a water bath at 20° C. Five c.c. of each
dilution are measured into a series of these tubes beginning with the
strongest specimen and rinsing the pipette once with each dilution
before the 5 c.c. are measured out. For inoculation, a 24-hour broth
culture of B. typhosus is prepared which has been transferred daily for
at least 3 days. Before use it is shaken and filtered through sterile
filter paper. The wide test tubes containing diluted disinfectant are
inoculated with fo c.c. of this culture with a graduated pipette. The
tip of the pipette is held against the side of the tube to insure accurate
measurement and the tube immediately shaken to mix the bacteria
thoroughly with the disinfectant. Test inoculations are made from
this mixture at proper intervals into tubes containing 10 c.c. of standard
extract broth of + 1*5 acidity, using loops 4 mm. in diameter. At least
four such loops should be at hand, supported on a rack or wooden block
so that a fan-tail Bunsen burner may be placed under each wire in turn.
Each one is sterilized after a plant is made and allowed to cool while the
other three are being used in order.

The test is conducted as follows: A row of ten wide tubes containing
dilutions of the antiseptic is placed in the water bath at 20° C. and time
allowed for them to reach the temperature of the bath. They are then

inoculated in order at intervals of exactly 15 seconds. Fifteen seconds
7

BIOLOGY AND TECHNIQUE

after the last tube has been inoculated a subculture is made from the
first tube of the series (be., 2j/^ minutes after this first tube was inocu¬
lated) and from the other tubes in order at 15-second intervals. Fifteen
seconds after this first series of subcultures is completed a second series
of subcultures is begun which will givethe result of a 5-minute exposure to
the antiseptic and the subinoculations continued at 15-second intervals
until all dilutions have been tested for fifteen minutes. If the strength
of the antiseptic is known approximately subcultures of the lower dilu¬
tions for the longer periods may be omitted. It is convenient to have
an assistant at hand to call time and to label the subcultures as soon as
made. The tubes may, however, be placed in order in suitable racks

DETERMINATION OF THE CARBOLIC-ACID COEFFICIENT

OF A DISINFECTANT.

(Anderson and McClintic)

Name . . “A”

Temperature of Medication . 20° C.

Culture Used B. Typhosus . 24-hr., Extract Broth, Filtered

Proportion of Culture and Disinfectant . 0.1 c.c. + 5 c.c.

Organic Matter, None; Kind, None; Amount, None.

Subculture Media . Standard Extract Broth

Reaction . : . . + 1.5

Quantity in Each Tube . . . 10 c.c.

Sample.

Dilu-

Time Culture Exposed to Action
of Disinfectant for Minutes

Phenol Coefficient.

tion.

23^

7 H

123^

Phenol .

1:80

1:90

—

80)375

1:100

—

4.69

1:110

—

110)650

5.91

Disinfectant “A”. .

•1:350

1:375

—

2)10.60

1:400

—

5.30 =

1:425

—

coefficient

1:450

—

1:500

—

1:550

—

1:600

—

1:650

—

1:700

1:750

THE DESTRUCTION OF BACTERIA

without labelling. The subculture tubes are incubated for 48 hours at
37° C. and those in which growth is observed are recorded positive.

To obtain the coefficient the weakest dilution of the unknown
antiseptic which kills in 2^ minutes is divided by the weakest dilution
of phenol which kills in the same time. The same is done for the weak¬
est strength that kills in 15 minutes and an average is taken. The
results of such a test are shown in the table on page 82.

As only the 2j^-minute and 15-minute intervals are used in deter¬
mining this result it seems unnecessary to make plants at the intervening
periods except in special cases where more detailed information is desired.

The procedure may be modified by adding some organic substance
such as killed bacteria to the diluted antiseptic. For many substances,
e.g., bichloride of mercury, the antispetic value in presence of organic
matter is much lower than in watery solution. Anderson and McClintic
insist that great care in making the dilutions and rigid adherence to a
uniform technique are necessary to obtain consistent results in such tests.

Determination of Antiseptic Values. — The antiseptic or in-
hibitive strength of a chemical substance, sometimes spoken of
as the “coefficient of inhibition/7 is determined by adding to
definite quantities of a given culture medium, graded percent¬
ages of the chemical substance which is being investigated and plant¬
ing in these mixtures equal quantities of the bacteria in question.
The medium used for the tests may be nutrient broth or melted gelatin
or agar. If broth is used, growth is estimated by turbidity of the
medium and by morphological examination; if the agar or gelatin is
employed, plates may be poured and actual growth observed.

Thus, in the case of carbolic acid, a 5 or 10 per cent solution is
prepared and added to tubes of the medium, as follows:

Tube

contains

carbolic

((

2 c.c. + broth 8 c.c. = 1: 1,000 carbolic acid.
1 c.c. + broth 9 c.c. = 1:200 “ “

.5 c.c. + broth 9.5 c.c. = 1:400 “ “

7 c.c. + broth 9.8 c.c. — 1:1,000

.1 c.c. + broth 9.9 c.c. = 1:5,000 “

To each of these tubes a definite quantity of the bacteria is added
either by means of a standard loopful of a fresh agar culture, or better by
a measured volume of an even emulsion in sterile salt solution. The
inoculated tubes are then incubated at a temperature corresponding to
the optimum growth temperature for the microorganism in question.
The tubes are examined for growth from day to day. From tubes
containing higher dilutions, in which no growth is visible, transplants

BIOLOGY AND TECHNIQUE

INHIBITION STRENGTHS OF VARIOUS ANTISEPTICS.
Adapted from Flugge, Leipzig, 1902.

Acids

Sulphuric. . . .
Hydrochloric

Anthrax Bacilli.

1 : 3,000
1 : 3,000

Other Bacteria.

Choi. spir. 1 : 6,000
B. diph. 1 : 3,000
B. mallei 1 : 700

Sulphurous
Arsenous. .
Boric ....

1 : 800

B. tvph. 1 : 500
Choi. spir. 1 : 1,000

Putrefactive Bac¬
teria in Bouillon.

1 : 6,000
1 :200
1 : 100

Alkalies
Potass, hydrox .

Ammon, hydrox.

Calcium hydrox.
Salts

Copper sulphate
Ferric sulphate
Mercuric chlorid .
Silver nitrate . . .

Potass, perman.

1 : 700

1 : 100,000

1 : 60,000

1 : 1,000

Choi. spir. 1 : 400
B. typh. 1 : 400
Choi. spir. 1 : 500
B. typh. 1 : 500
Choi. spir. 1 : 1,100
B. typh. 1 : 1,100

B. typhosus 1 : 60,000
Choi. spir.

B. typhosus 1 : 50,000

1 : 1,000
1 : 90
1 : 20,000

1 : 500

Halogens and Compounds

Chlorin .

Bromin .

Iodin .

Potass, iodid .

Sodium chlor .

1 : 1,500
1 : 1,500
1 : 5,000

1 : 60

Organic Compounds

Ethyl alcohol . . . . . . 1 : 12

Acetic and oxalic acids .

Carbolic acid . 1 . 800

B. diph. 1 : 500
B. typh. 1 : 400
Choi. spir. 1 : 600

Benzoic acid . .
Salicylic acid . .
Formalin (4%
hyde) .

1 : 1,000

1 : 1,500

formalde-

Chol. spir. 1 : 20,000
Staphylo. 1 : 5,000

Camphor .

Thymol .

Oil mentha pip .

Oil of terebinth .

Peroxide of hydrogen

1 : 1,000
1 : 10,000
1 : 3,000
1 : 8,000

1 : 4,000
1 : 2,000
1 : 5,000

1:7

1 : 10
1 : 400

1 : 1,000

1 : 3,500

1 : 2,000

THE DESTRUCTION OF BACTERIA

BACTERICIDAL STRENGTHS OF COMMON DISINFECTANTS.
Adapted from FlAgge, Leipzig, 1902.

Acids

Sulphuric . . .
Hydrochloric
Sulphurous .

Sulphurous

Boric .

Alkalies
Potass, hydrox. .
Ammon, hydrox.
Calcium .

Strepto- and
Staphylo¬

cocci.

5 Minutes.

1 : 10
1 : 10

1:5

Salts

Copper sulphate
Mercuric chlor.

Silver nitrate . .
Potass, permang.
u Calc, chlorid ”

Halogens and Com¬
pounds

Chlorin .

Tri chlorid of iodin . . .

Organic Compounds

Ethyl alcohol .• .

Acetic and oxalic acids

Carbolic acid

Lysol .

Creolin .

Salicylic acid .

Formalin (40% for¬
maldehyde) .

Peroxide of hydrogen .

1 : 10,000 to
1,000
1 : 1,000
1 : 200

1 per cent.
1 : 200

70%-15

minutes

1 : 60

1 : 300

1 : 1,000

1 : 10
Cone.

Anthrax and Typhoid Bacilli.
Cholera Spirillum.

5 Minutes.

1 : 100
1 : 100

1 : 300
1 : 300
1 : 1,000

1 : 2,000

1 : 500

.1 per cent.

1 : 1,000

70%-10mins

Cholera 1:200
Typh. 1 : 50
1 : 300
1 : 100

1 : 20
1 : 200

2-24 Hours.

1 : 1,500
1 : 1,500
(Typhoid
1 : 700)

1 : 300 (Gas
10 vol. %)
1 : 30

1 : 10,000

1 : 4,000

1 : 2-300

1 . 300

1 : 3,000

1 : 1,000

1 : 500

Anthrax Spores.

1 : 50 in 10 days
1 : 50 in 10 days

Cone. sol. incomplete
disinfection

1 : 20 (5 days)

1 : 2,000 (26 hours)

1 : 20 (1 day)

1 : 20 (1 hour)

2 per cent (in 1 hr.)

1 : 1,000 (in 12 hrs.)

Alcol. 50% for 4
months without

killing spores.

Koch.1

1 : 20 (4-45 days)

(at 40° in 3 hrs.)

(10% in 5 hrs.)

1 : 20 (in 6 hrs.)

1 : 100 (in 1 hr.)
3 : 100 (in 1 hr.)

1 Koch, Arb. a. d. kais. Gesundheitsamt, 1, 1881.

BIOLOGY AND TECHNIQUE

are made to determine the presence of living bacteria and to distinguish
between inhibition or antisepsis and bacterial death or disinfection.

The determination of the bactericidal or disinfectant value of a
chemical substance upon spores may be carried out by a variety of
methods. Koch,1 using anthrax spores as the indicator, dried the spores
upon previously sterilized threads of silk. These were exposed to the
disinfectant at a definite temperature for varying times, the disinfect¬
ant was then removed by washing in sterile water, and the threads
planted upon gelatin or blood serum media and incubated. A serious
objection to this method was pointed out by Geppert,2 who maintains
that it is impossible by simple washing to remove completely the disin¬
fectant in which the thread has been soaked. This author suggests that,
whenever possible, the disinfectant, at the end of the time of exposure,
should be removed by chemical means. In the case of bichloride of mer¬
cury Geppert exposes emulsions of the bacteria to aqueous solutions of
the disinfectant, and at the end of exposure precipitates out the bichlor¬
ide of mercury with ammonium sulphide. In the case of a large number
of disinfectants, however, this is not possible, and, when the thread
method is used, removal of the chemical agent by washing must be
practised. Complete removal of the disinfectant is especially desirable,
since spores previously exposed to these substances are more easily in¬
hibited by dilute solutions than are normal spores. The spores may be
dried upon the end of a glass rod, which, after exposure, is washed in
distilled water or salt solution and then immersed in sterile broth.3

A simple method is that in which graded percentages of the disin¬
fectant are added to the menstruum, blood, blood serum, broth, etc., in
which the disinfectant is to be tested, and equal quantities of bacteria
thoroughly emulsified in water or salt solution are added. Loopfuls of
these mixtures are then planted from time to time in agar or gelatin
plates upon which colony counts can afterward be made.

In all such tests it is important to remember that the presence of
organic fluids, blood serum, mucus, etc., considerably alters the efficiency
of germicides, and whenever practical deductions are made, experimental
imitation of the actual conditions should be attempted.

Practical Disinfection. — In practical disinfection with chemical
agents, the disinfectant must be chosen to a certain extent in accordance
with the material to be disinfected.

1 Koch, Arb. a. d. kais. Gesundheitsamt, 1, 1881.

2 Geppert, Berl. klin. Woch., xxvi, 1889.

3 Hill , Rep. Am. Pub. Health Assn., xxiv, 1898.

THE DESTRUCTION OF BACTERIA

Sputum is a substance extremely difficult to disinfect because the
bacteria present are surrounded by dense envelopes of mucus, through
which disinfectants do not easily diffuse. For sputum disinfection, es¬
pecially tuberculous sputum, carbolic acid — 5 per cent solution — or
any of the phenol derivatives in similar concentration, may be used.
Bichloride of mercury is of very little use in sputum disinfection be¬
cause of the dense protective layers of albuminated mercury which form
about the microorganisms. Sputum should always be received into
cups containing the disinfectant, and contaminated handkerchiefs
should be soaked in the solution.

Feces from typhoid, dysentery, and cholera patients should be steril¬
ized by burning, if possible, or by thoroughly mixing with large quan¬
tities of boiling water; but if chemical disinfectants are to be used, five
per cent carbolic acid or dilute formalin are convenient. Milk of lime
and chloride of lime are useful, though somewhat inconvenient. Bichlo¬
ride of mercury is of little value in this case for the same reason
that it is valueless in sputum disinfection. In all cases of feces dis¬
infection it is extremely important that the chemical agent should be
added in large quantities and thoroughly mixed with the discharge.

Linen , napkins, and other cloth materials which have come into con¬
tact with patients should be soaked for one or two hours in one per cent
formaldehyde, five per cent carbolic acid, or 1 : 5,000 or 1 : 10,000
bichloride of mercury. After this, the material may be taken from the
sick-room and boiled. It is extremely important that cloth material
should never be removed from the sick-room in a dry state.

Urine may be easily disinfected by the addition in proper con¬
centration of any of the disinfectants named above.

The methods for sterilization of surgical instruments and the prepara¬
tion of the skin of the patient for operation are subject to so many local
variations that it is hardly within the scope of a text-book on bacteriology
to mention them. Metal instruments are usually sterilized by boiling
in soda solution and may be subsequently immersed in five per cent car¬
bolic acid solution. Catgut may be sterilized by boiling in alcohol or by
subjecting it to temperatures of 140° C. and over, for several hours in
oils (albolin).

The disinfection of the hands is also a matter of much variation.
Two methods frequently quoted are those of Welch and of Furbringer.

In Welch’s method the hands are brushed with green soap in water
as hot as it can be borne for at least five minutes. They are then rinsed
and immersed for two minutes in a warm saturated solution of perman-

BIOLOGY AND TECHNIQUE

ganate of potash in which they are rubbed with a sponge or sterile
cotton. They are then transferred to a saturated solution of oxalic acid,
until the red color has entirely disappeared. Following this, they are
rinsed in sterile water and then immersed in a 1 : 500 bichloride of
mercury solution for one to two minutes.

According to Fiirbringer’s method, the finger nails are carefully
cleaned with an orange-wood stick or nail file; the hands are then thor¬
oughly brushed with a nail brush in green soap and hot water for five
minutes. Following this they are immersed in 60 per cent alcohol for
one minute, then in 3 per cent carbolic acid solution for one minute;
after which they are rinsed in sterile water and dried.

Rooms, closets, and other closed spaces which are contaminated, must
be disinfected largely by gaseous disinfectants. After such disinfection
in the case of cellars, privies, and other places where feasible, walls and
ceilings should be whitewashed.

Gaseous Disinfectants for Purposes of Fumigation. — There are a
large number of gaseous agents which are harmful to bacteria. Only a
few, however, are of sufficient bactericidal strength to be of practical
importance.

Oxygen, especially in the nascent state, may exert distinct bacteri¬
cidal action upon some bacteria. That strictly anaerobic strains are
inhibited by its presence has already been mentioned.

Ozone was shown by Ransome and Fullerton 1 to exert considerable
germicidal power when passed through a liquid medium in which bac¬
teria were suspended, but was absolutely without activity when em¬
ployed in the dry state.

Chlorine because of its powerful germicidal action was once looked
upon with favor, but has been found quite inadequate from a practical
point of view because of its injurious action upon materials, and its
irregularity of action. Chlorine, too, is but weakly efficient unless in
the presence of moisture.2

Sulphur dioxide or sulphurous anhydrid (S02), which was formerly
much used for room disinfection, is no longer regarded as uniformly ef¬
ficient for general use. The gas is produced by burning ordinary roll
sulphur. In order that it shall be at all effective, water should be
vaporized at the same time, since the disinfectant action is dependent
upon the formation of sulphurous acid. The concentration of the gas
should be at least 8 per cent of the volume of air in the room. For this

1 Ransome and Fullerton, Rep. Public Health, July, 1901.

2 Fischer und Proskauer, Mitt. a. d. kais. Gesundheitsamt, x, 11, 1882.

THE DESTRUCTION OF BACTERIA

purpose about three pounds of sulphur should be burned for every
thousand cubic feet of space. It should be allowed to act for not less
than twenty-four hours. The researches both of Wolff htigel 1 and of
Koch 2 have shown that the gas is not sufficient for the destruction of
spores, under the best circumstances, probably because of its lack of pen¬
etrating power. Park 3 believes that sulphur dioxide used in quantities
of four pounds of sulphur to 1,000 cubic feet is of practical value for
fumigating purposes in cases of diphtheria and the exanthemata.

Of all known gaseous disinfectants by far the most reliable is form¬
aldehyde. There are many methods of generating this gas, and many
devices for its practical use have been introduced. In all cases where
formaldehyde fumigation is intended, clothing, bed-linen, and fabrics
should be spread out, cupboards and drawers freely opened. The
cracks of windows and doors should be hermetically sealed with paper
strips or by calking with cotton. The generation of gas may be carried
out in an apparatus left within the room or it may be generated outside
and the gas introduced by a tube passed within the keyhole. In all
cases moisture should be provided for, either in the generating appa¬
ratus or by a separate boiler.

The first of the methods of generating formaldehyde for fumigation
purposes was that of Trillat,4 who devised a lamp in which formaldehyde
was produced by the incomplete combustion of methyl alcohol. This
method has proved expensive because of the complete oxidation of a
large percentage of the alcohol.

Direct evaporation of formaldehyde from formalin solutions has been
the principle underlying some other devices. If such evaporation is
attempted from an open vessel, however, polymerization of formal¬
dehyde to the solid trioxymethylene occurs. To prevent this, Trillat 5
and others have constructed special autoclaves in which 20 per cent of
calcium chloride is added to formalin which is then vaporized under
pressure. By this means polymerization is practically eliminated.

The Trillat autoclave, as well as others constructed on the same
principle, consists of a strong copper chamber of a capacity of about a
gallon, fitted with a cover which can be tightly screwed into place.
The cover is perforated by an outlet vent, a pressure gauge, and a

1 Woljjhugel , Mitt. a. d. kais. Gesundheitsamt, i, 1881.

2 Koch, Mitt. a. d. kais. Gesundheitsamt, i, 1881.

3 Park, ‘‘Pathogen. Bact.,” N. Y., 1908.

4 Trillat, Compt. rend, de l’acad. des sc., 1892.

6 Trillat, Compt. rend, de l’acad. des sc., 1896.

BIOLOGY AND TECHNIQUE

thermometer. The whole apparatus is adjusted upon a stand and set
over a kerosene lamp. Into the chamber is put about one-half to three-
quarters its capacity of 40 per cent formaldehyde (commercial formalin)
containing 15-20 per cent calcium chloride. The solution used should
be free from methyl alcohol, since this leads to the formation, with
formaldehyde, of methylal, which is absolutely without germicidal
action. The flame is lighted and the exit tube kept closed until the
pressure gauge indicates a pressure of three atmospheres. Then the
vapor is allowed to escape through the tube. For a room of about 3,000
cubic feet Trillat advises the continuance of the gas flow for about an
hour. The method is not uniformly reliable.

A method which has found much favor is that in which glycerin—
usually in a concentration of 10 per cent — is added to formalin. Ac¬
cording to Schlossmann 1 the presence of
glycerin hinders polymerization. An appa¬
ratus in which this mixture is conveniently
utilized is that of Lentz (see Fig. 12). For¬
malin with 10 per cent glycerin is placed
in the copper tank and heated by a burner.
Formaldehyde leaves the nozzle (which can
be introduced through the keyhole) mixed
in a fine spray with steam. This apparatus
has been favorably endorsed by the War De¬
partment of the United States.

The so-called Breslau method of generat¬
ing formaldehyde depends upon the evapora-
Fig. 12. — Lentz Formalin tion of formaldehyde from dilute solutions, v.

Apparatus. Brunn 2 claims that where formalin in 30 to 40

per cent strength is evaporated, water vapor
is generated more rapidly than formaldehyde is liberated, and a
concentration leading to polymerization occurs. If, however, dilu¬
tion is carried out until the formaldehyde in the solution is not
more than 8 per cent, the generation of water vapor and formaldehyde
takes place at about equal speed and no concentration occurs. Schloss¬
mann 1 furthermore claims that polymerization in the vaporized formal¬
dehyde does not occur if sufficient water vapor is present — a principle
which may also contribute to the efficiency of the Breslau method.
In practice, the apparatus devised by v. Brunn (Fig. 13) consists of a

1 Schlossmann, Mlinch. meet. Woch., 45, 1898.

2 v. Brunn, Zeit. f. Hyg., xxx, 1899.

THE DESTRUCTION OF BACTERIA

strong copper kettle of about 34 cm. diameter by 7.5 cm. height.
This is completely closed except for two openings in the slightly
domed top, one of which is the exit vent, the other, laterally
placed, is for purposes of filling and is closed by a screw stopper.
The kettle is set up on a metal stand over an alcohol lamp, so arranged
with a double circle of burners that heating may be carried out rapidly.
The tank is filled with a solution of formalin of a strength of from 8 to
10 per cent (commercial formalin 1:4). The apparatus permits the
evaporation of large quantities of fluid in a short time (3 liters in one
hour) . When the lamp is left in a closed room care should be taken
to fill it with a quantity of alcohol proportionate to the amount of fluid
to be evaporated. This, according to v. Brunn, is about one-quarter
of the volume of formalin solu¬
tion used. By using 1.5 liters of
8 percent formalin for each 1,000
cubic feet of space, this apparatus
is said to yield a concentration
of formaldehyde of about 25
grams to the cubic meter, a
strength sufficient to complete
surface disinfection within seven
hours.

To do away with the use of
liquid formalin solutions, a meth¬
od has been devised which de¬
pends in principle upon the breaking up by heat of the solid polymer
of formaldehyde (trioxymethylene). The apparatus (trade name,
“Schering’s Paraform Lamp”) as described by Aronson 1 consists of a
cylindrical mantle of sheet-iron placed upon a stand and supplied
below with an alcohol lamp. Set into the top of the mantle is a small
chamber, into which 1 gram tablets of trioxymethylene are placed.
The alcohol lamp, so placed that the wicks project but slightly — to
avoid overheating — is lighted, and the formalin generated passes out
through slits in the upper case, mingling with the water vapor and other
gases liberated by the alcohol flame. The more modern devices have
water-boiler attachments to insure sufficient moisture. Two tablets
are sufficient for the fumigation of about thirty-five cubic feet, and
2 c.c. of alcohol are filled into the lamp for each tablet. One hundred
to one hundred and fifty tablets are usually enough for the ordinary

a b

Fig. 13. — Breslau Formaldehyde
Generator and Section of Same.
(After v. Brunn.) a , Inlet; b, Exit vent.

1 Aronson, Zeit. f. Hyg., xxv, 1897.

BIOLOGY AND TECHNIQUE

room. Modifications of this method are in common use, some well-
known firms preparing so-called “paraform candles,” in which para-
form, in the powdered state, is volatilized by heat.

A simple method of generating formaldehyde is that which is known
as the “lime method.” In a wide shallow pan 40 per cent formalde¬
hyde solution (commercial formalin) is poured over quicklime (CaO).
According to Park, the previous addition of concentrated sulphuric
acid to the formalin, in proportions of one to ten, increases the
speed of formalin liberation, and aids in limiting polymerization.
About one and one-half to two pounds (one-half to one kilogram) of
quicklime are used for every 500 c.c. of the formalin solution. The
heat generated in the slaking of the lime produces volatilization of
the formalin.

A modification of this method is that of Schering 1 in which tablets
of paraform and unslaked lime are together laid into a pan and warm
water is poured over them.

A highly efficient method, which has universal approval because of
its simplicity, is the potassium permanganate method of Evans and
Russell.2 This method depends upon the active reaction occurring when
formalin and potassium permanganate are mixed. In practice, about
300 grams of small crystals of potassium permanganate are poured into
a half liter of 40 per cent formalin. The mixture results in an active
evolution of heat and the evaporation of formalin together with water
vapor. Because of the active foaming which takes place, high cylin¬
drical vessels should be used, about one foot in height, preferably wfth a
funnel-like flare at the top. The yield of gas by this method is said
to be about 80 per cent of the amount present in the solution, and
within the first five minutes most of this is liberated.

Harrington 3 states that the equivalent of 110 c.c. for formalin
suffices to produce sterility within two and a half hours in a space of
one thousand cubic feet.

The room in which formaldehyde has been liberated is kept sealed,
in the manner already described, for at least twelve hours, after which
the windows and doors are opened and thorough airing practised. The
odor which remains after formaldehyde fumigation may be removed by
sprinkling with ammonia, or by the use of some one or another of the
various sorts of apparatus devised for the liberation of ammonia.

1 Schering, Hyg. Rundschau, 190Q.

2 Evans and Russell, Rep. State Bd. Health, Maine, 1904.

3 Harrington, “Practical Hygiene,” Phila., 1905.

CHAPTER VI

METHODS USED IN THE MICROSCOPIC STUDY AND STAINING

OF BACTERIA

MICROSCOPIC STUDY OF BACTERIA

Bacteria may be studied microscopically, in the living and un¬
stained state, and, after the application of dyes, in colored preparations.
For the manipulation of bacteria for such study, glass slides and cover-
slips of various design are used. These must be perfectly clean if the
preparations are to be of any value.1

The Study of Bacteria in the Living State. — Living bacteria may be
studied in what is spoken of as the “hanging-clrop” preparation.
For this purpose a so-called hollow slide is employed, in the center of
which there is a circular concavity about three-quarters of a centimeter
to one centimeter in diameter. The preparation is manipulated as
follows: If the bacteria are growing in a fluid medium a drop of the
culture fluid is transferred to the center of a cover-slip. If taken from
solid media, an emulsion may be made in broth or in physiological salt
solution, and a drop of this transferred to the cover-slip, or the bac¬
teria may be emulsified in a drop of salt solution, or broth, directly upon
the cover-slip. The concavity on the slide, having first been rimmed
with vaseline, by means of a small cameTs-hair brush, the cover-slip is
inverted over the slide in such a way that the drop hangs freely within
the hollow space. The preparation is then ready for examination under
the microscope.

1 Although the silicates of which glass is composed are extremely stable, never¬
theless alkaline silicates which are said to separate out on the surface, together with
grease and dirt left upon the glass by handling, during blowing and cutting, neces¬
sitate cleansing before use. This may be accomplished by a variety of methods. A
simple one suitable for general application is as follows: (1) The slides and cover-
slips are thrown singly into boiling water and left there for half an hour; (2) wash
in twenty-five per cent sulphuric acid; (3) rinse in distilled water; (4) wash in
alcohol; (5) wipe wdth a clean cloth and keep dry under a bell-jar. Another method
convenient for routine use is to immerse, after thorough washing in soap-suds
and acid, in ninety-five per cent alcohol and to leave in this until the time of use.

BIOLOGY AND TECHNIQUE

Another method, known as the 11 hanging block method/’ devised by
Hill/ for the study of living bacteria in solid media is carried out as fol¬
lows: Nutrient agar is poured into a Petri dish and allowed to solidify.
Out of this layer a piece about a quarter of an inch square is cut. This
is placed on a sterile slide. The upper surface of the agar block is then
inoculated with bacteria by surface smearing, and the preparation
covered with a sterile dish and allowed to dry for a few minutes in the
incubator. A sterile cover-slip is then dropped upon the surface of the

■ - ■ ' ~~ ; J

Fig. 14. — Hanging Drop Preparation.

block and sealed about the edges with agar. Block and cover-slip are
then taken from the slide and fastened over a moist chamber with paraf¬
fin. The entire preparation can be placed upon the stage of a microsocpe.
This method is especially designed for the study of cell-division.

Living bacteria may also be studied in stained preparations by the
so-called “intra vital” method of Nakanishi. Thoroughly cleaned slides
are covered with a saturated aqueous solution of methylene-blue. This
is spread over the slide in an even film and allowed to dry. -After drying
the slide should appear of a transparent sky-blue color. The micro¬
organisms which are to be examined are then emulsified in water, or are
taken from a fluid medium and placed upon a cover-slip. This is dropped,
face downward, upon the blue ground of the slide. In this way bacteria
may be stained without being subjected to the often destructive proc¬
esses of heat or chemical fixation. According to Nakanishi, cytoplasm
is stained blue, while nuclear material assumes a reddish or purplish
hue.

The Study of Bacteria in Fixed Preparations. — Stained preparations
of bacteria are best prepared upon cover-slips, the process consisting of
the following steps : (1) Spreading on cover-slip; (2) drying in air; (3)
fixing; (4) staining; (5) washing in water; (6) blotting; (7) mounting.

(1) Smearing. — Bacteria from a fluid medium are transferred in a
small drop of the fluid, with a platinum loop, to a cover-slip and care¬
fully spread over the surface in a thin film. If taken from a solid medium
a small drop of sterile water is first placed upon the cover-slip and the
bacteria are then in very small quantity carefully emulsified in this drop
with the platinum needle or loop and spread in an extremely thin film.

1 Hill, Jour, of Med. Research, vii, 1902.

MICROSCOPIC STUDY AND STAINING

(2) The film is allowed to dry in the air.

(3) When thoroughly dried, fixation is carried out by passing the
preparation, film side up, three times through a Bunsen flame, at about
the rate of a pendulum swing. Fixation by heat in this manner is most
convenient for routine work, but is not the most delicate method, in¬
asmuch as the degree of heat applied can not be accurately controlled.
The other methods which may be employed are immersion in methyl
alcohol, formalin, saturated aqueous bichloride of mercury, Zenker's
fluid, or acetic acid. If chemical fixatives are used, they must be re¬
moved by washing in water before the stain is applied. If a prepara¬
tion is made upon a slide instead of a cover-slip, passage through the
flame should be repeated eight or nine times.

(4) Staining . — The dyes used for the staining of bacteria are, for
the greater part, basic anilin dyes, such as methylene-blue, gentian-
violet, and fuchsin. These may be applied for simple staining in 5
per cent aqueous solutions made up from filtered saturated alcoholic
solutions, or directly by weight. They are conveniently kept in the
laboratory as saturated alcoholic solutions. The strengths of some
saturated solutions are as follows:

Saturated Solutions 1 (Stains Gruebler or Merck).

Fuchsin (aqueous), 1.5 per cent.

Fuchsin (alcohol 96 per cent), 3 per cent.

Gentian-violet (aqueous), 1.5 per cent.

Gentian-violet (alcohol 96 per cent), 4.8 per cent.

Methylene-blue (aqueous), 6.7 per cent.

Methylene-blue (alcohol 96 per cent), 7 per cent.

The staining solution, in simple routine staining, is left upon the fixed
bacterial film for from one-half to one and one-half minutes according to
the efficiency of the stain used. Methylene-blue is the weakest of the
three stains mentioned; gentian-violet the strongest.

(5) The excess stain is removed by washing with water.

(6) The preparation is thoroughly dried by a blotter or between
layers of absorbent paper.

(7) A small drop of Canada balsam is placed upon the film side of
the dry cover-slip, which is then inverted upon a slide. The prepara¬
tion is now ready for microscopical examination.

1 After Wood,H 1 Chemical and Microscopical Diagnosis/' Appendix. N. Y., 1909.

BIOLOGY AND TECHNIQUE

The chemical principles which underlie the staining process are still
more or less in doubt.1 Suffice it to say here that most of the dyes in
common use by bacteriologists and pathologists are coal-tar derivatives
belonging to the aromatic series, all of them containing at least one
“ benzolring ” combined with what Michaelis terms a “chromophore
group/’ chief among which are the nitro-group (N02), the nitroso-group
(NO), and the azo-group (N = N). Just what the actual process of stain¬
ing consists in, is a question about which various opinions are held, some
believing that the phenomenon is purely chemical, in which a salt is
formed by the combination of the dye and the protoplasm of the cells,
others that there is no such salt formation, and that the process takes
place by purely physical means. To support the latter view it is argued
that certain substances like cellulose are stainable without possessing
the property of salt formation, and that staining may often be accom¬
plished without there being a chemical disruption of the dye itself.
Michaelis sums up his views by stating that probably both processes
actually take place. A dye stuff, as a whole, may enter into and be de¬
posited upon a tissue or cell by a process which he speaks of as “ insorp¬
tion.” In such a case the coloring matter may be subsequently ex¬
tracted by any chemically indifferent solvent. On the other hand, a dye
after being thus deposited upon or within a cell, may become chemically
united to the protoplasm by the formation of a salt, and in such a case
the color can be removed only by agents which are capable of decom¬
posing salts, such as free acids.

The staining power of any solution may be intensified either by
heating while staining, by prolonging the staining process, or by the
addition of alkalies, acids, anilin oil, and other substances which will
be mentioned in the detailed descriptions of special staining methods.

One of the most common examples of such an intensified stain
is the so-called Loeffler’s alkaline methylene-blue. This is made up in
the following way:

Saturated alcoholic solution of methylene-blue, 30 c.c.

1 : 10,000 solution potassium hydrate in water, 100 c.c.

Another solution designed with a similar purpose is the Koch-Ehrlich
anilin-water solution. Anilin oil, one part, is shaken up with dis¬
tilled water, nine parts; after thorough shaking, the mixture is filtered

1 For comprehensive reviews of the subject, the reader is referred to dissertations
such as those of Mann (“ Physiol. Hist. Methods and Theory/’ Oxford, 1902) and
of Michaelis (“ Ein filming in die Farbstoffchemie,” etc., Berlin, 1902).

MICROSCOPIC STUDY AND STAINING

through a moist filter paper until perfectly clear. A saturated alco¬
holic solution of either fuchsin or gentian-violet is added to this anilin
water in proportions of about one to ten or until a slightly iridescent
pellicle appears upon the surface of the solution.

An extremely useful and very strong staining solution is the Ziehl
carbol- fuchsin solution , made up as follows : 1

Fuchsin (basic) . 1 gm.

Alcohol (absolute) . 10 c.c.

Five per cent carbolic acid . 100 c.c.

To make up this staining solution, mix 90 c.c. of a five per cent aque¬
ous solution of carbolic acid with 10 c.c. of saturated alcoholic basic
fuchsin.

It may also be made up as follows:

Weigh out

Basic fuchsin . 1 gram

Carbolic acid . 5 grams

Dissolve in

Distilled water . 100 c.c.

Filter and add

Absolute alcohol . 10 c.c.

SPECIAL STAINING METHODS

Spore Stains. — Abbott’s Method.2 — Cover-slips are smeared and
fixed by heat in the usual manner.

Cover with Loeffler’s alkaline methylene-blue and heat the stain
until it boils, repeat the heating at intervals but do not boil continuously.
Keep this up for one minute.

Rinse in water.

Decolorize with a mixture of alcohol eighty per cent 98 c.c. and nitric
acid 2 c.c., until all blue has disappeared.

Rinse in water.

Dip from three to five seconds in saturated alcoholic solution of
eosin 10 c.c., and water 90 c.c.

Rinse in water, blot, and mount in balsam.

By this method the spores are stained blue, the bodies of the bacilli
are stained pink.

1 Ziehl, Deut. med. Woch., 1882. 2 Abbott, “Prin. of Bact.,” Phila., 1905.

BIOLOGY AND TECHNIQUE

Moeller’s Method.1 — Cover-slips are prepared as usual and fixed
in the flame.

Wash in chloroform for two minutes.

Wash in water.

Cover with five per cent chromic acid one-half to two minutes.

Wash in water. Invert and float cover-slip on carbol-fuchsin solu-

tion in a small porcelain dish and heat gently with a flame until it steams;
continue this for three to five minutes. (This step can also be done by
covering the cover-glass with carbol-fuchsin and holding over flame.)

Decolorize with five per cent sulphuric acid five to ten seconds.

Wash in water.

Stain with aqueous methylene-blue one-half to on,e minute. By
this method spores will be stained red, the body blue.

Capsule Stains. — Welch’s Method.2 — Cover-slips are prepared as
usual and fixed by heat.

Cover with glacial acetic acid for a few seconds. Pour off acetic acid
and cover with anilin water gentian-violet, renewing stain repeatedly
until all acid is removed. This is done by pouring the stain on and off
three or four times and then finally leaving it on for about three minutes.

Wash in two per cent salt solution and examine in this solution.

Hiss’ Methods.3 — (1) Copper Sulphate Method. — Cover-slip prepara¬
tions are made by smearing the organisms in a drop of animal serum,
preferably beef-blood serum.

Dry in air and fix by heat.

Stain for a few seconds with —

Saturated alcoholic solution of fuchsin or gentian-violet 5 c.c., in
distilled water 95 c.c.

The cover-slip is flooded with the dye and the preparation held for a
second over a free flame until it steams.

Wash off dye with twenty per cent aqueous copper sulphate solution.

Blot (do not wash) .

Dry and mount.

By this method permanent preparations are obtained, the capsule
appearing as a faint blue halo around a dark purple cell body.

(2) Potassium Carbonate Method. — This method consists in using
as a dye a half-saturated solution of gentian-violet. Gentian-violet in

1 Moeller, Cent. f. Bakt., I, x, 1891.

2 Welch, Johns Hopkins Hosp. Bull., 1892.

3 Hiss, Cent. f. Bakt., xxxi, 1902; Jour. Exper. Med., vi, 1905.

MICROSCOPIC STUDY AND STAINING

substance is added in excess to distilled water and allowed to dissolve
to its full extent. The solution is then filtered and diluted to twice its
volume.

Cover-glass preparations are made by spreading the bacteria on a
oover-slip in a drop of animal serum as in preceding method. They are
allowed to dry in the air and fixed by heat as usual. The dye is then
poured upon the preparation and allowed to remain for a few seconds.
It is then washed off with a twenty-five-hundredth per cent solution
of potassium carbonate in water, and studied in this solution. The
cover-slip inverted on a slide may be rimmed with vaseline to prevent
evaporation.

Buerger's Method.1 — Cover-slip preparations are made by smear¬
ing in serum as in Hiss' method.

As the edges of the smear begin to dry, pour over it Zenker's fluid
(without acetic acid) and warm in flame for three seconds.

(Zenker's fluid is composed of potassium bichromate 2.5 gm.,
sodium sulphate 1 gm., water 100 c.c., saturated with bichloride of
mercury.)

Wash in water.

Flush with ninety-five per cent alcohol.

Cover with tincture of iodin, U. S. P., one to three minutes.

Wash with ninety-five per cent alcohol.

Dry in the air.

Stain with anilin water gentian-violet two to five seconds.

Wash with two per cent salt solution.

Mount and examine in salt solution.

Wadsworth's Method.2 * * — Wadsworth has devised a method of
staining capsules which depends upon the fixation of smears with forma¬
lin. After such fixation capsules may be demonstrated both with simple
stains and by Gram's method. The technique is as follows:

Smear preparations, made as usual, are treated as follows:

1. Formalin, 40 per cent, two to five minutes.

2. Wash in water, five seconds.

Simple Stain. , Differential Stain (Gram’s Method).

3. Ten per cent aqueous gentian-violet. 3. Anilin gentian-violet, two minutes.

4. Wash water, five seconds. 4. Iodin solution, two minutes.

5. Dry, mount in balsam. 5. Alcohol, 95 per cent, decolorize.

6. Fuchsin, dilute aqueous solution.

7. Wash water, two seconds.

8. Dry, mount in balsarq

1 Buerger, Med. News, Dec., 1904 2 Wadsworth, Jour. Inf Pis., i :)0(!.

^ • 3 ' ’U ’ ’> -IT ’) »• *>

' ' 5 > ' ■ •

> JO 3> )

100

BIOLOGY AND TECHNIQUE

It is important that the formalin be fresh and the exposure to
water momentary. When decolorizing in the Gram method, strong
alcohol only should be used. Wadsworth also found that encapsulated
pneumococci could be demonstrated in celloidin sections of pneumonic
lesions hardened in strong formalin. The lungs should be distended with
the formalin or the lesions cut in very thin bits, hardened, dehydrated,
embedded, and cut in the usual way. The celloidin sections may be fixed
on the slides by partially dissolving the celloidin in alcohol and ether
and setting the celloidin quickly in water before staining. Failure to
obtain pneumococci encapsulated in such sections is usually due to
improper or inadequate fixation in the formalin.

The differential method employed by Wadsworth for tissue staining
is as follows:

1. Fix in formalin forty per cent, two to five minutes.

2. Wash in water.

3. Anilin gentian-violet, two minutes.

4. Iodin solution, two minutes.

5. Alcohol, ninety-five per cent, decolorize.

6. Eosin alcohol, counterstain.

7. Clear in oil of origanum.

8. Mount in balsam.

Flagella Stains. — All flagella stains, in order to be successful, neces¬
sitate particularly clean cover-slip preparations, best made from young
agar cultures emulsified in sterile salt solution. Scrupulous care should
be exercised in cleaning the glassware used.

Loeffler’s Method.1 — The preparation is dried in the air and fixed
by heat. It is then treated with the following mordant solution :

Twenty per cent aqueous tannic acid . 10 parts.

* Ferrous sulphate aq. sol. saturated at room temperature . 5 parts.

Saturated alcoholic fuchsin solution . . 1 part.

This solution, which should be freshly filtered before using, is
poured over the cover-glass and allowed to remain there for one-half
to one minute, during which time it should be gently heated, but not
allowed to boil.

Wash thoroughly in water.

Stain with five per cent anilin water fuchsin or anilin water gen-

1 Loeffler, Cent. f. Bakt., I, vi, 1889.

MICROSCOPIC STUDY AND STAINING

101

tian-violet made slightly alkaline by the addition of one-tenth per cent
sodium hydrate.

The stain should be filtered directly upon the cover-slip. Warm
gently and leave on for one to two minutes. Wash in water. Mount in
balsam.

Van Ermengem’s Method.1 — This method requires the preparation
of three solutions.

(1) Twenty per cent tannic acid solution . 60 c.c.

Two per cent osmic acid solution . 30 c.c.

Glacial acetic acid . .' . 4-5 drops.

The cover-slip carrying the fixed preparation is placed in this solu¬
tion for one hour at room temperature, or for five minutes at 100° C.
(boiling) .

Wash in water.

Wash in absolute alcohol.

Immerse the cover-slip for one to three seconds in

(2) Silver nitrate, 0.25-0.5 per cent solution.

Without washing, transfer to

(3) Gallic acid . . 5 gm.

Tannic acid . 3 “

Fused potassium acetate . 10 “

Distilled water . 350 c.c.

Immerse in this for a few minutes, moving the cover-slip about.

Return to the silver nitrate solution until the preparation turns
black.

Wash thoroughly in water.

Blot and mount.

Smith’s Modification of Pitfield’s Method.2 — A saturated solu¬
tion of bichloride of mercury is boiled and is poured while still hot into a
bottle in which crystals of ammonia alum have been placed in quantity
more than sufficient to saturate the fluid. The bottle is then shaken and
allowed to cool. Ten c.c. of this solution are added to 10 c.c. of freshly
prepared ten per cent tannic acid solution. To this add 5 c.c. carbol-
fuchsin solution. Mix and filter.

To stain, filter the above mordant directly upon the fixed cover-slip

1 Van Ermengem, Cent. f. Bakt., I, xv, 1894.

2 Smith, Brit. Med. Jour., I, 1901, p. 205.

102

BIOLOGY AND TECHNIQUE

preparation. Heat gently for three minutes, but do not allow to boil.
Wash in water and stain with the following solution:

Saturated alcoholic solution gentian-violet . 1 c.c.

Saturated solution ammonia alum . 10 c.c.

Filter the stain directly upon the preparation and heat for three or four
minutes. Wash in water, dry, and mount in balsam.

Differential Stains. — Gram’s Method.1 — By this method of staining,
which is extremely important in bacterial differentiation, bacteria are
divided into those which retain the initial stain and those which are
subsequently decolorized and take the counterstain. The former are
often spoken of as the Gram -positive, the latter as Gram-negative
bacteria.

Preparations are made on cover-slips or slides in the usual way.

The preparation is then covered with an anilin gentian-violet solu¬
tion which is best made up freshly before use.

The staining fluid is made up, according to Gram’s original direc¬
tions,2 as follows:

Five c.c. of anilin oil are shaken up thoroughly with 125 c.c. of dis¬
tilled water. This solution is then filtered through a moist filter paper.

To 108 c.c. of this anilin water, add 12 c.c. of a saturated alcoholic
solution of gentian-violet. The stain acts best when twelve to twenty-
four hours old, but may be used at once. It lasts, if well stoppered, for
three to five days. A more convenient and simple method of making up
the stain is as follows:

To 10 c.c. of distilled water in a test tube add anilin oil until on
shaking the emulsion is opaque; roughly, one to ten. Filter this through
a wet paper until the filtrate is clear. To this add saturated alcoholic
solution of gentian-violet until the mixture is no longer transparent,
and a metallic film on the surface indicates saturation. One part of
alcoholic saturated gentian-violet to nine parts of the anilin water
will give this result. This mixture may be used immediately and lasts
two to five days if kept in a stoppered bottle.

Cover the preparation with this; leave on for 5 minutes. Pour off
excess stain and cover with Gram’s iodin solution for 2 to 3 minutes.

Iodin . . lgm.

Potassium iodid . . . 2 gm.

Distilled water . 300 c.c.

1 Gram} Fortschr. d. Med., ii, 1884.

2 Gram , loc. cit.

MICROSCOPIC STUDY AND STAINING

103

Decolorize with ninety-seven per cent alcohol until no further
traces of the stain can be washed out of the preparation. This takes
usually thirty seconds to two minutes, according to thinness of prepara¬
tion.

Wash in water.

Counterstain with an aqueous contrast stain, preferably Bismarck
brown.1

Paltauf’s Modification of Gram’s Stain.2 — The staining fluid as
prepared by this modification possesses the advantage of retaining its
staining power for a longer period than does the anilin-water-gentian-
violet described in the original method.

The staining fluid is prepared as follows:

3-5 c.c. anilin oil are added to
90 c.c. distilled water and
7 c.c. absolute alcohol.

This mixture is thoroughly shaken and filtered through a moist
filter paper until clear. Then add:

Gruebler’s gentian- violet 2 gm.

The fluid should stand twenty-four hours, during which a precipi¬
tate forms. This is filtered before use.

This gentian-violet solution retains its staining power for from four
to six weeks. It is good only when a metallic luster develops on the
surface.

It is used in the following way: Spreads on cover-slips or slides are
dried and fixed as usual.

Then apply: —

Anilin water gentian-violet (as above), three minutes.

Gram’s iodin solution, two minutes.

Absolute alcohol (with stirring), thirty seconds.

Counterstain, without washing in water, in aqueous fuchsin or in
weak carbol-fuchsin.

1 To make up Bismarck brown solution, prepare a saturated aqueous solution of
the powdered dye by heating. Allow it to cool, and filter. Dilute one to ten with
distilled water.

2 Sharnosky, Proc. N. Y. Pathol. Soc., Oct., 1909, n. s., ix, 5.

104

BIOLOGY AND TECHNIQUE

Classification of the Most Important Pathogenic Bacteria

According to Gram’s Stain.

Gram-positive.

(. Retain the Gentian-violet.)

Micrococcus pyogenes aureus
Micrococcus pyogenes albus
Streptococcus pyogenes
Micrococcus tetragenus
Pneumococcus
Bacillus subtilis
Bacillus anthracis
Bacillus diphtheria
Bacillus tetanus
Bacillus tuberculosis and other
acid-fast bacilli
Bacillus aerogenes capsulatus
Bacillus botulinus

Gram-negative.

( Take Counter stain.)

Meningococcus

Gonococcus

Micrococcus catarrhalis
Bacillus coli
Bacillus dysenteriae
Bacillus typhosus
Bacillus paratyphosus
Bacillus fecalis alkaligenes
Bacillus enteriticlis
Bacillus proteus (proteus)
Bacillus mallei
Bacillus pyocyaneus
Bacillus influenzae
Bacillus mucosus capsulatus
Bacillus pestis
Bacillus maligni cedematis
Spirillum cholerae
Bacillus Koch-Weeks
Bacillus Morax-Axenfeld

Stains for Acid-Fast Bacteria. — These methods of staining are chiefly
useful in the demonstration of tubercle bacilli. These bacteria because
of their waxy cell membranes are not easily stained by any but the most
intensified dyes, but when once stained, retain the color in spite of ener¬
getic decolorization with acid. For this reason they are known as acid-
fast bacilli. The first method devised for the staining of tubercle
and allied bacilli was that of Ehrlich.

Ehrlich Method.1 — This method is now rarely used. Cover-slip
preparations are prepared as usual and fixed by heat.

Stain with anilin water gentian -violet, hot, three to five minutes,
or twenty-four hours at room temperature.

1 Ehrlich, Deut. med. Woch., 1882.

MICROSCOPIC STUDY AND STAINING

105

Decolorize with thirty-three per cent nitric acid one-half to one
minute.

Treat with sixty per cent alcohol, until no color can be seen to come
off.

Counterstain with aqueous methylene-blue.

Rinse in water, dry, and mount.

Ziehl-Neelson Method.1 — Thin smears are made upon cover-
slips or slides.

Fix by heat.

Stain in carbol-fuchsin solution as given on page 97. The slide
or cover-slip may be flooded with the stain, and this gently heated with
the flame until it steams, or else the cover-slip may be inverted upon
the surface of the staining fluid, in a porcelain dish or watch-glass, and
this heated until it steams. This is continued for three to five min¬
utes. Decolorize with either five per cent nitric acid, five per cent
sulphuric acid, or one per cent hydrochloric acid for three to five
seconds. The treatment with the acid is continued until subsequent
washing with water will give only a faint pink color to the preparation.

Wash with ninety per cent alcohol until no further color can be re¬
moved. If, after prolonged washing with alcohol, a red color still re¬
mains in very thick places upon the smear, while the thin areas appear
entirely decolorized, this may be disregarded.

Wash in water and counterstain in aqueous methylene-blue for
one-half to one minute.

Rinse n water, dry, and mount.

By this method the tubercle bacilli are colored red, other bacteria
and cellular elements which may be present are stained blue.

Gabbet’s Method.2 — Gabbet has devised a rapid method in which
the decolorization and counterstaining are accomplished by one solu¬
tion. The specimen is prepared and stained with carbol-fuchsin as in
the preceding method. It is then immersed for one minute directly in

the following solution:

Methylene-blue . 2 gms.

Sulphuric acid 25 per cent (sp. gr. 1018) . 100 c.c.

Then rinse in water, dry, and mount.

This method, while rapid and very convenient, is not so reliable as
the Ziehl-Neelson method.

1 Ziehl, Deut. med. Woch., 1882; Neelson, Deut. med. Woch., 1883.

2 Gabbet, Lancet, 1887.

106

BIOLOGY AND TECHNIQUE

Pappenheim’s Method.1 — The method of Pappenheim is devised
for the purpose of differentiating between the tubercle bacillus and the
smegma bacillus. Confusion may occasionally arise between these two
microorganisms, especially in the examination of urine where smegma
bacilli are derived from the genitals, and less frequently in the examina¬
tion of sputum where smegma bacilli may occasionally be mixed with
the secretions of the pharynx and throat.

Preparations are smeared and fixed by heat in the usual way.

Stain with hot carbol-fuchsin solution for two minutes.

Pour off dye without washing and cover with the following mixture:

Corallin (rosolic acid) . 1 gm.

Absolute alcohol . 100 c.c.

Methylene-blue added to saturation

Add glycerin 2 . 20 c.c.

This mixture is poured on and drained off slowly, the procedure being
repeated four or five times, and finally the preparation is washed in
water. The combination of alcohol and rosolic acid decolorizes the
smegma bacilli, but leaves the tubercle bacilli stained bright red.

Bunge and Trautenroth Method.3 — This method is designed to
differentiate between the tubercle and smegma bacilli.

Smear and fix by heat in the usual way.

Wash with absolute alcohol to remove fat.

Treat with five per cent chromic acid for fifteen minutes.

Wash in several changes of water.

Stain with hot carbol-fuchsin for five minutes.

Decolorize with sixteen per cent sulphuric acid for three minutes.

Counterstain with alcoholic methylene-blue for five minutes.

Wash in water, dry, and mount.

By this method the tubercle bacillus remains red, the smegma bacil- •
lus is decolorized.

Baumgarten’s Method.4 — This method is recommended by the
author for differentiation between the bacillus of tuberculosis and the
bacillus of leprosy and depends upon the fact that the tubercle bacillus
is less easily stained than Bacillus leprse.

Smears are prepared and fixed by heat in the usual way.

1 Pappenheim, Berl. klin. Woch., 1898.

2 The glycerin is added after the other constituents have been mixed.

3 Bunge und Trautenroth, Fortschr. d. Med., xiv, 1896.

5 Baumgarten, Zeit. f. wissensch. Mikrosk., 1, 1884.

MICROSCOPIC STUDY AND STAINING

107

Stain in dilute alcoholic fuchsin for five minutes.

Decolorize for twenty seconds in alcohol, ninety-five per cent, ten
parts, nitric acid one part.

Wash in water.

Counterstain in methylene-blue.

Wash in water, dry, and mount.

The tubercle bacillus should be blue and the bacillus of leprosy red.

Special Stains for Polar Bodies. — These staining methods are designed
to bring into view polar bodies as found, for instance, in the bacilli of
diphtheria and plague.

Neisser’s Method.1 — Smear and fix in the usual manner.

Stain for two to five seconds in the following solution:

Methylene-blue . 1 gm<

Absolute alcohol . 20 c.c.

Glacial acetic acid . 50 c.c.

Distilled water . 1,000 c.c.

Wash in water.

Counterstain in two per cent aqueous Bismarck brown solution for
five seconds.

By this method polar bodies are stained blue, while the bacillary
bodies are stained brown.

Roux’s Method.2 — Two solutions are necessary.

(1) Dahlia violet . . . 1 gm.

Alcohol 90 per cent . 10 c.c.

Aqua destillata ad . 100 c.c.

(2) Methyl-green . 1 gm.

Alcohol 90 per cent . 10 c.c.

Aqua destillata ad . 100 c.c.

Before use, one part of solution No. 1 is mixed with three parts of
solution No. 2. The preparation is stained with the mixture for two
minutes in the cold.

Polychrome Stains. — The various polychrome stains are of value to
the bacteriologist chiefly for the staining of pus and exudates where the
relation of bacteria to cellular elements is to be demonstrated. They
are also extremely useful in the study of fixed specimens of protozoan
parasites. There is a large number of these stains in use; a few only,

1 Neisser, Zeit. f. Hyg., xxiv, 1897.

2 Roux and Yersin, Annal. de Tinst. Past., 1890.

108

BIOLOGY AND TECHNIQUE

however, can be given here. In principle, all these stains depend upon a
combination of eosin and methylene-blue, these elements staining not
only as units, but acting together in combination. One and the same
solution, therefore, contains at least three elements which color the
various structures of the preparation selectively.

Jenner’s Method.1 — This stain, because of its simplicity, is useful
for routine use. It is made up as follows : Equal parts of eosin (Gruebler,
“ W. G.”) one and two-tenths per cent aqueous solution, and methylene-
blue (medicinal, Gruebler) one per cent aqueous solution, are mixed and
allowed to stand for twenty-four hours. A coarse granular precipitate
is formed which appears dark, with a metallic luster on its surface. This
is separated by filtration and washed with distilled water until the fil¬
trate appears almost clear.

To make up the stain 0.5 gram of the dry precipitate is dissolved
in 100 c.c. of methyl alcohol.

In using the stain, preparations are not fixed, but simply dried in
the air and immersed in the stain for one to two minutes. After this,
wash in distilled water and examine.

Wright’s Modification of Leishman’s Method.2 — A one per cent
solution of methylene-blue (Gruebler) in five-tenths per cent solution of
sodium bicarbonate in distilled water is steamed in a sterilizer at 100°
C. for one hour. After this has cooled, a one-tenth per cent aqueous
solution of eosin (Gruebler, W. G.) is added until a metallic scum ap¬
pears on the surface of the mixture. (About five parts of eosin solution
to one of methylene-blue is necessary.) The precipitate which forms is
collected by filtration, dried, and a saturated solution then made in
methyl alcohol. This is filtered and diluted with one-quarter its bulk
of methyl alcohol.

To stain, cover the dried preparation with the stain for one to
one and one-half minutes. Dilute by dropping upon the stain distilled
water from a pipette until a metallic film appears upon the top. Leave
this on for three to fifteen minutes. Wash in distilled water.

Giemsa’s Method.3 — The method of Giemsa is really a modification
of the Romanowsky method. It is widely applicable, being of great
value in the staining of the Spirochsete pallida, Vincent’s spirilla, pro¬
tozoa, and Negri bodies. The stain has been modified several times by

1 Jenner, Lancet, i, 1889.

2 Wright, Jour. Med. Research, ii, 1902.

3 Giemsa, Cent. f. Bakt., I, xxxvii, 1904.

MICROSCOPIC STUDY AND STAINING

109

its originator, the following being the formula given by him in 1904:
The substance referred to as azur II and purchasable under that name,
consists of pure methylenazur chloralhydrate combined with an equal
quantity of methylene-blue chloralhydrate. The substance referred to
as azur II-eosin is a combination of this substance with eosin.

The staining fluid is made up as follows:1

Azur II-eosin . 3 gms.

Azur II . 8 gms.

This mixture is thoroughly dried over sulphuric acid in a desiccator,
fineLy powdered, and rubbed through a fine sieve. It is then dissolved in
250 gms. of C. P. glycerin (Merck), at 60° C. To this is added methyl
alcohol (Kahlbaum) 250 c.c., previously warmed to 60° C. This mix¬
ture is well shaken and allowed to stand at room temperature for
twenty-four hours. The mixture is now ready for use.

For use 10 c.c. of distilled water are poured into a test tube and
one to two drops of a one per cent potassium carbonate solution are
added. Ten drops of the staining solution described above (one drop to
the c.c.) are mixed with this slightly alkaline water. The preparation
which is to be stained is fixed in methyl alcohol, dried, and covered with
the diluted staining solution. For the staining of protozoa and ex¬
udates containing bacteria, ten to fifteen minutes are sufficient. For
the staining of Negri bodies or Spirochsete pallida, one or more hours
of staining should be employed. After staining, wash in running tap
water and blot.

Wood’s Method.2 — Wood has devised a simple staining method
based on the principles of the Giemsa stain, in which azur II and eosin
may be used in separate solutions. Preparations are fixed in strong
methyl alcohol for five minutes and are then stained in a 0.1 per cent
aqueous solution of eosin until the preparation is pink. The eosin is
then poured off and the preparation is covered with a 0.25 per cent
aqueous solution of azur II for one-half to two minutes. Following this,
it is washed in tap water and dried by blotting.

When an intense stain is desired, the solution of eosin and azur II
may be flooded over the preparation together, using an excess of azur
II. They are then left on from five to ten minutes. At the end of this
time washing and drying as before completes the process.

1 It is best not to attempt to make up the undiluted staining fluid, since this is
purchasable under the name of “ Giemsa Losung fur Romano wsky Farbung.”

2 Wood, Med. News, 83, 1903.

110

BIOLOGY AND TECHNIQUE

The Staining of Bacteria in Tissues. — The preparation of tissue for
bacterial staining is, in general, the same as that employed for purposes
of cellular studies, in histology. For bacteriological studies the most
useful fixative is alcohol; other fixations, such as that by formalin,
Zenker’s fluid, or Mueller’s fluid, give less satisfaction. In other respects
the details of dehydration and embedding are the same as those used in
histological studies, except that it is desirable that the tissues should be
handled rather more carefully than is necessary for ordinary patholog¬
ical work, and the changes from the weaker to the stronger alcohols
should be made less abruptly.1

Embedding in paraffin is preferable to celloidin, although the latter
method is not unsuccessful if carefully carried out. The chief disadvan¬
tages of celloidin are the retention of color by the celloidin itself and the
consequent unclearness of differentiation. It is also easier to cut thin
sections from paraffin blocks than from those prepared with celloidin.

When staining tissue sections for bacteria, it is most convenient
to carry out the process with the section attached to a slide. For cel¬
loidin sections this may be accomplished by means of ether vapor. For
paraffin sections it is necessary to cover the slide with an extremely thin
layer of a filtered mixture of equal quantities of egg albumin and glycerin,
to which a small crystal of camphor or a drop or two of carbolic acid
has been added. The sections are then floated upon a slide so prepared,
and set away in the thermostat for four or five hours.

Loeffler’s Method.2 — Stain in alcoholic methylene-blue solution
five to fifteen minutes, or in Loeffler’s alkaline methylene-blue solution
one to twenty-four hours.

Wash in one to one-thousand acetic acid solution for about ten
seconds.

Treat with absolute alcohol by pouring the alcohol over the prepara¬
tion for ten to twenty seconds.

Clear with xylol.

Mount in balsam.

When celloidin sections are stained in this way ninety-five per cent
alcohol should be substituted for the absolute. A number of other
staining solutions may be used in the same way, aqueous fuchsin or
aqueous gentian-violet yielding good result.

1 For details of such work reference should be had to the standard textbooks on
pathological technique, notably the very excellent one of Mallory and Wright.

2 Loeffler, Mitt. a. d. kais, Gesundheitsamt, ii, 1884.

MICROSCOPIC STUDY AND STAINING

111

Nicolle advises the use of a ten per cent aqueous solution of tannic
acid for a few seconds after washing with the acetic acid. This fixes
the stain and prevents a too vigorous decolorization during the process
of dehydration.

Method of Staining Gram-Positive Bacteria in Tissue Sections.
— Celloidin Sections. — After fixing section to the slide by pressure with
a filter paper or by ether vapor, cover with anilin-water gentian-violet
five minutes.

Pour off excess of stain and cover with Gram's iodin solution for
two minutes.

Decolorize with ninety-five per cent alcohol until no more color
comes out.

Stain quickly with eosin-alcohol (ninety-five per cent alcohol to
which enough eosin has been added to give a transparent pink color;
about 1 : 15). Clear in eosin-oil of origanum (oil of origanum, 25 c.c.
and eosin alcohol, as above, about 3 c.c.).

Blot and mount in balsam.

Paraffin Sections. — Stain with anilin-water gentian-violet five to ten
minutes.

Wash in water.

Cover with Gram's iodin solution one minute.

Wash in water.

Decolorize with absolute alcohol until no more color comes out.

Clear in xylol.

Mount in balsam.

Gram-W eigert Method} — (For celloidin sections.) — Stain for one-half
hour in the following freshly filtered solution:

Carmine . 3-5 grams.

Saturated aqueous solution of lithium carbonate. . . . 100 c.c.

Dehydrate in ninety-five per cent alcohol.

Stick section to slide with ether vapor.

Stain in anilin-water gentian-violet for five to fifteen minutes (or
n a saturated solution of aqueous crystal violet diluted with water
one to ten, five to fifteen minutes).

Wash in physiological salt solution.

Cover with Gram's iodin solution one to two minutes.

Wash in water and blot.

1 W eigert, Fortschr. d. Med., v, 1887.

112

BIOLOGY AND TECHNIQUE

Decolorize with anilin oil until no more color comes off.

This both decolorizes and dehydrates.

Treat with xylol. Mount in balsam.

Method of Staining for Tubercle Bacilli in Sections.1 —
Paraffin Sections. — Stain in carbol-fuchsin solution hot for five minutes
(or better cold, for twenty-four hours).

Wash in water.

Decolorize and counterstain in Gabbet’s methylene-blue sulphuric
acid mixture for one minute.

Wash in water.

Dehydrate in absolute alcohol.

Clear in xylol.

Mount in balsam.

Celloidin Sections .2 — Stain lightly in alum hematoxylin.

Wash in water.

Dehydrate in ninety-five per cent alcohol.

Attach the slide by ether vapor.

Stain with steaming carbol-fuchsin two to five minutes.

Wash in water.

Wash with Orth’s acid alcohol (alcohol ninety per cent., 99 c.c.;
cone. HC1, 1 c.c.) one-half to one minute.

Wash in water several changes.

Treat with ninety-five per cent alcohol until red color is entirely
gone.

Blot and cover with xylol until clear. Mount in balsam.

Method of Staining Actinomyces in Sections. — Mallory’s Method 3.
— 1. Stain deeply in saturated aqueous eosin ten minutes.

2. Wash in water.

3. Anilin gentian -violet two to five minutes.

4. Wash in normal saline solution.

5. Weigert’s iodin solution (iodin 1, KI 2, and water 100 parts)
one minute.

6. Wash in water and blot.

7. Clear in anilin oil.

8. Xylol several changes.

9. Mount in balsam.

1 Mallory and Wright, “ Pathol. Tech.,” p. 413.

2 After Mallory and Wright.

3 Mallory and Wright, “ Pathol. Tech.,” 1904.

CHAPTER VII

THE PREPARATION OF CULTURE MEDIA

GENERAL TECHNIQUE

The successful cultivation of bacteria upon artificial media requires
the establishment, of an environment which shall be suitable in regard to
the presence of assimilable nutritive material, moisture, and osmotic
relations. These requirements are fulfilled in the composition of the
nutrient media described in another section, media which are to some
extent varied according to the special requirements of the bacteria
which are to be; cultivated. If cultivation, furthermore, is to have any
value for scientific study of individual species, it is necessary to ob¬
tain these species free from other varieties of microorganisms, that is,
in pure culture, and to protect such cultures continuously from con¬
tamination with the other innumerable species which are everywhere
present.

The technique which is employed for these purposes has been gradu¬
ally evolved from the methods originally devised by Pasteur, Koch.
Cohn, and others.

Bacterial cultivation is carried out in glassware of varied construc¬
tion, the forms most commonly employed being test tubes of various
sizes, Erlenmeyer flasks, the common Florence flasks, and Petri dishes.
All glassware, of course, must be thoroughly cleansed before being used.

Preparation of Glassware. — The cleansing of glassware may be ac¬
complished by any one of a number of methods. New glassware may
be immersed in a one per cent solution of hydrochloric or nitric acid in
order to remove the free alkali which is occasionally present on such glass.
It is then transferred to a one per cent sodium hydrate solution for a
few hours, and following this is washed in hot running water.

In the case of old glassware which has contained culture media,
sterilization in the autoclave is first carried out, then the glassware is
boiled in five per cent soda solution or in soapsuds. After this, thorough
mechanical cleansing is practiced, and the glassware may be treated by
acid and alkali followed by running water, as given above. These last
9 113

114

BIOLOGY AND TECHNIQUE

steps, however, are not essential, thorough washing in hot water after
the soapsuds or soda solution being usually sufficient to yield good
results. Other workers have recommended immersion of the glassware
after mechanical cleansing in five per cent to ten per cent potassium
bichromate solution in twenty-five per cent sulphuric acid. This is
followed by thorough washing in hot running water, and drying.

Clean flasks and test tubes are then stoppered with cotton, which has
been found to be a convenient and efficient seal against the bacteria
of the air, catching them in the meshes of the fibers as in a filter. The
technique of the stoppering or plugging of glass receptacles is important,

Fig. 15. — Florence Flask.

in that, when poorly plugged, sterility is not safeguarded, and the pur¬
pose of culture study is defeated.

In almost all laboratories in this country non-absorbent cotton or
“ cotton batting ” is used for the plug. In a few of the German labora¬
tories the absorbent variety is employed. The disadvantages of the
latter, especially in the case of fluid media, are obvious. The plugs
should fit snugly, but not so tightly that force is necessary to remove
them. Care should be taken, furthermore, that no creases are left be¬
tween the surface of the glass and the periphery of the plug; for these,
if present, may serve as channels for the entrance of bacteria. Fig.
18, accompanying, will illustrate some of the more common and un¬
desirable defects in poorly made plugs. The plugging itself is carried
out by tearing a small piece of cotton, about 2X2 inches, from the roll,

THE PREPARATION OF CULTURE MEDIA

115

folding over one of its comers, and, applying the smooth end of a glass
rod to the folded portion, gently pushing it into the mouth of the tube.

After plugging and before media are introduced into the tubes and
flasks, these should be sterilized. This is best done in one of the “ hot¬
air sterilizers” (see Fig. 8, p. 69), by exposing the tubes for one hour
to a temperature of 150° C. If greater speed is desired exposure to 180°
to 190° C. for half an hour is usually safe. If by mistake, however, the
temperature is allowed to rise above 200° C., a browning of the cotton
plugs occurs and the glassware is apt to be stained by the burning of
the fat and other organic material derived from the cotton. Petri dishes

ftn . . Jiil

Fig. 17. — Petri Dish.

after cleansing are fitted together in the manner shown in Fig. 17,
and are sterilized in the hot-air chamber at 150° C. for one hour.

Glassware so prepared is ready for the reception of media.

Ingredients of Culture Media. — The food requirements of bacteria
have been discussed in another section. From what has there been
said, it is apparent that artificial culture media must, to a certain extent,
be adjusted to the peculiarities of individual bacteria. In the cases of
the more strictly parasitic microorganisms growth can be obtained only
by the most rigid observance of special requirements. For the large
majority of pathogenic bacteria, however, routine or standard media
may be employed, which, while slightly more favorable for one species
than for another, are sufficiently general in their composition to per¬
mit the growth of all but the most fastidious varieties.

The basis of many of our common media is formed by the soluble
constituents of meat. These substances are best obtained by macerating
500 grams of lean beef in 1,000 c.c. of distilled water, The mixture is

116

BIOLOGY AND TECHNIQUE

;\\A
V >\s
Vv v \.\

allowed to infuse in the ice chest over night, and then strained through
cheese-cloth. To this infusion are added the other required constituents
in the manner given in the detailed instructions below. The soluble
constituents of meat, however, may also be procured in a simpler way

by the use of the commercial meat extracts,
such as that of Liebig. These extracts are
dissolved in quantities of five grams to the
liter, and other constituents are added to
this nutrient basis.

Though simpler to make, the meat-ex¬
tract media are less favorable for the culti¬
vation of the more delicate organisms than
are the media made directly from fresh meat.
Nevertheless, they suffice for the cultivation
of the large majority of the more saprophytic
pathogenic microorganisms and hold an im¬
portant place in laboratory technique.

The ingredients and methods used in va¬
rious laboratories in the preparation of such
standard media should be, as much as pos¬
sible, uniform, in order that confusion in re¬
sults may be avoided; for, as is well known,
the biological characteristics of one and the
same bacterial species may vary considerably
if grown on media differing in their compo¬
sition.

A committee of the American PubUc
Health Association,1 appointed in 1897 for
the sake of standardizing the methods of preparation of media, recom¬
mended that the following rules should govern the choice of ingredients:

1. Distilled water should be used in all cases.

2. The meat used should be fresh, lean beef (when veal or chicken
is substituted the change should be stated).

3. The pepton used should be Witte’s pepton, dry, made from meat.

4. Only C. P. NaCl should be used.

5. For alkalinizing C. P. sodium hydrate should be used in normal
solutions.

Fig. 18. — Test Tube (a)
incorrectly stoppered; ( b )
correctly stoppered.

1 Rep. Com. of Amer. Bact. to Com. of Amer. Pub. Health Assn. Meeting,
Philadelphia, Sept., 1897.

THE PREPARATION OF CULTURE MEDIA

117

6. For acidification C. P. hydrochloric acid in normal solution should
be used.

7. When glycerin is used, this should be of the redistilled variety.

8. The agar-agar employed should be of the finest grade of commer¬
cial thread agar.

9. The gelatin should be the commercial sheet gelatin washed as
free as possible of acid and impurities.

10. Chemicals and carbohydrates which are used should be as
nearly chemically pure as possible.

Titration of Media. — Next in
importance to the actual composi¬
tion of media is the adjustment of
their reaction. Bacteria are highly
susceptible to variations in the
acidity and alkalinity of media,
excessive degress of either mav
completely inhibit development or
moderate variations may lead to
marked modifications of cultural
characteristics. It is necessary,
therefore, to adjust the reaction
both for the sake of favoring
growth and in order to insure uni¬
formity of growth characters. This
is accomplished by titration which
is best carried out according to the
recommendations of the committee
mentioned above.

The color indicator employed for
the titration is a five-tenths per cent
solution of phenolphthalein in fifty
per cent alcohol. The chief aclvan-
tage of this indicator over others is
due to the fact that it indicates the
presence of organic acid and acid
compounds in its reaction. For

actual titration — (J-. normal) solutions of sodium hydrate or of hy¬
drochloric acid are used. Since media in the process of preparation
are usually acid, the NaOH solution is the one most frequently needed.
Five c.c. of the medium to be tested is measured accurately in a care-

Fig. 19.-

-Burette for Titrating
Media.

118

BIOLOGY AND TECHNIQUE

fully washed pipette and transferred into a porcelain evaporating dish.
To this are added 45 c.c. of distilled water. The mixture is thoroughly
boiled for three minutes over a free flame. The boiling drives off C02,
giving the true neutral point, and approximates the conditions prevailing

during the further sterilization of
the medium from which the 5 c.c.
have been taken. After boiling, 1
c.c. of the phenolphthalein is added.
If the medium is acid, no color is
present; if alkaline, a pink or red
color appears. The alkali or
acid solution is allowed to drop
into the dish from a graduated
burette. When the neutral point
is approached in an acid solution,
each drop of sodium hydrate added
brings forth at first a deep red,
which, however, upon slight stir¬
ring with a clean rod, completely
disappears.1 The end reaction is
reached when a faint but clear and
distinct pink color remains in the
fluid after stirring.

When titrating alkaline media,
the addition of the phenolphthalein
produces a red color in the hot
Fig. 20— Tubing Media. medium which gradually fades upon

the addition of HC1, becoming-
colorless at the end point of titration. Titration should be done
quickly and in a hot solution. From the result of the titration the
computation for the neutralization of the entire bulk of the medium
can be made by a simple arithmetical process as illustrated in the
following example:

Let us suppose that we have used :

2.5 c.c. of NaOH to neutralize 5 c.c. of the medium,

then 2.5 c.c. of y NaOH will neutralize 100 c.c. “ “

and 25 c.c. of ^ NaOH will neutralize 1,000 c.c., or one liter.

1 See standard textbooks on volumetric analysis.

THE PREPARATION OF CULTURE MEDIA

119

The adjustment of the reaction of media is largely determined by the
particular uses for which the media are designed. For examinations in
the practice of sanitation, such as analyses of water, ice, and milk, etc.,
the American Public Health Association recommends a standard reac¬
tion of + 1 per cent (the plus sign is used to indicate acidity, the minus
alkalinity; + 1 per cent is the expression used to indicate that one per
per cent of ^ sodium hydrate solution would be required to neutralize
the medium or 10 c.c. to the liter). For general work with pathogenic
bacteria, the most favorable reaction for routine media is slight alka¬
linity, neutrality, or an acidity not exceeding + 1 per cent.

Methods of Clearing Media. — Clearing with Eggs. — When culture
media are prepared from substances containing no coagulable proteid,
it is often necessary, for purposes of clearing, to add the whites of eggs,
and then to. heat for forty -five min¬
utes in the Arnold sterilizer. In
the following detailed descriptions,
the direction “ clear with egg ” has
been given whenever such a step is
deemed necessary. The exact tech¬
nique of such a procedure is as
follows :

In a small pot or pan, the
whites of several eggs (one or two
eggs to each liter of medium) are
beaten up thoroughly with a little
water (20 c.c.). This egg white is
then poured into the medium,
which, if hot, as in the case of
melted agar or gelatin, must first
be cooled to about 50° to 55° C.

The mixture is then thoroughly
shaken and steamed in the Arnold
sterilizer for thirty minutes. At
the end of this time the flask con¬
taining the medium is removed from
the sterilizer and thoroughly shaken
so as completely to break up the coagulum which has formed. It is
then replaced and allowed to steam for another fifteen minutes. At
the end of this time the medium between the coagula should be clear.
It is now ready for filtration through cotton.

j •

I I

v yf

■ A,

Fig. 21. — Media in Tubes: a, broth;
b, agar slant; c, potato.

120

BIOLOGY AND TECHNIQUE

Filtering Media through Cotton. — The filtration of media after
clearing, either by the addition of eggs or by. the coagulation of the pro-
teids originally contained in it, is best done through absorbent cotton.
A small spiral, improvised of copper wire, is placed as a support in the
bottom of a large glass funnel. A square piece of absorbent cotton is

Fig. 22. — Berkefeld Filter.

then split horizonta y. giving two squares of equal size. Ragged edges
and incisures shorn d be avoided. These two layers of cotton are then
placed in the funnel, one piece above the other in such a way that the
direction of the fibers of the two layers is at right angles one to the other.
They are then gently depressed into the filter with the closed fist. The

THE PREPARATION OF CULTURE MEDIA

121

edges of the cotton are made to adhere to the sides of the funnel by
allowing a thin stream of tap water to run over them, while smoothing
them against the glass with the hand.

The medium, when poured into such a filter, should be poured along
a glass rod at first, to avoid running down the sides or bursting the filter.
After filtration has begun, the filter should
be kept as full as possible. The first liter
or so which comes through may not be clear,
but the filter gains in efficiency as the coag-
ulum settles into the fibers of the cotton,
and the first yield may be sent through a
second time. Filtration of agar or gelatin is
best done in a warm room with windows
and doors closed, and the filter covered with
a lid, to avoid too rapid cooling. The funnel
and filter should be warmed just before use.

j Filtering through Paper. — Many media
may be efficiently cleared by filtration
through close filter paper without the aid of
coagula.

The Tubing of Media.— Most of the media
described in the foregoing section are used
in test tubes. In order to fill these tubes,
the media are best poured into a large glass
funnel to which a glass discharging tube has
been fitted by means of a short piece of
rubber tubing (see Fig. 20). Upon this is
placed a thumb cock. The plug is then re¬
moved from the test tube by catching it be¬
tween the small and ring fingers of the right
hand and the glass outlet is thrust deeply
into the test tube, in order to prevent the
medium from touching the upper portion
of the test tube where the cotton plug
will be lodged. About 7 to 8 c.c. is put in each test tube.

Sterilization of Media. — By Heat. — Media which contain neither
sugars, gelatin, glycerin, nor animal serum may be sterilized in the auto¬
clave at fifteen pounds pressure for fifteen minutes to half an hour.
Media which contain these or other substances subject to injury from
the high temperature, must be sterilized by the fractional method,

122

BIOLOGY AND TECHNIQUE

i.e., by twenty minutes’ exposure in the live steam sterilizer (Arnold,
Fig. 9, p. 70) on each of three consecutive days. During the intervals
between sterilizations, they should be kept at room temperature or in the
incubator, to permit the germination of spores which may be present.
Media containing animal serum or other albuminous solutions which
are to be sterilized without coagulation, may be sterilized in wate
baths, or in hot-air chambers (Fig. 10, p. 71), at temperatures varying

Fig. 24. — Reichel Filter.

from 60° to 70° C., by the fractional method. In such cases five or
six exposures of one hour on succeeding days should be employed.

By Filtration. — It is often desirable in bacteriological work to free
fluid from bacteria. This is frequently necessary for the sterilization
of blood-serum or exudate fluids, or for obtaining toxins free from bac¬
teria. For these purposes a large variety of filters are in use. Those
most commonly employed are of the Chamberland1 or Berkefeld type,
which consist of hollow candles made of unglazed porcelain or dia-
tomaceous earth. Both these types are made in various grades of fine¬
ness, upon which depend both the speed of filtration and the efficiency.
They are made in various forms and models, some of which are shown

1 Pasteur and Chamberland, Compt. rend, de Facad. des sci., 1884.

THE PREPARATION OF CULTURE MEDIA

123

in the accompanying figures. In most of the methods of filtration
commonly employed the fluid which is to be filtered is sucked through
the walls of the filter, either by a hand suction-pump or by some form
of vacuum-pump attached to an ordinary water-tap.

The hollow candle-filter may either be firmly fitted into a cylin¬
drical glass chimney and surrounded by the
fluid which is to be filtered, or else the candle
may be connected to the collecting flask
with sterile rubber tubing and suspended
freely in the fluid. Perfect filters of these
types will hold back any of the bacteria
known to us at present.

Filters before use must be sterilized.

The candles themselves are subjected to
150° C. in the hot-air sterilizer for one hour.

The glassware and washers necessary for
setting up the apparatus may be sterilized
by boiling. In order that filters may be re¬
peatedly used with good result, it is neces¬
sary that they should be carefully cleaned
from time to time. This is best done in the
following way:

Filters through which fluids from living
cultures have passed are first sterilized in
the Arnold steam sterilizer. Their exterior
is then carefully cleaned with a fine brush.

Following this a five-tenths per cent solu¬
tion of potassium permanganate is passed
through them and this again removed by
sucking through a five per cent solution of

bisulphite of soda. This last is washed out pIG 25. _ Kitasato Filter.

by sending a considerable quantity of dis¬
tilled water through the filter, which is then dried and sterilized by
heat.

The suction necessary for filtration through these filters is usually
applied by means of the ordinary suction-pump attached to a running
faucet.

Slanting of Media. — Solid media which are to be used in slanted form
in test tubes should be inclined on a ledge (easily improvised of glass
tubing) at the proper slant, after the last sterilization. Agar, the medium

124

BIOLOGY AND TECHNIQUE

most frequently employed in this way, should be left in this position
for two or three days. (See Fig. 21, b.)

ACTUAL STEPS IN THE PREPARATION OF NUTRIENT MEDIA

Broth. — Meat Extract Broth. — 1. To 1,000 c.c. of distilled or clear
tap water add 5 gms. Liebig’s meat extract, 10 gms. Witte’s pepton,

and 5 gms. common salt (NaCl).

2. Weigh solution with containing
vessel (any suitable agate-ware vessel
or glass flask will do).

3. Heat, over free flame until thor¬
oughly dissolved, stirring constantly.

4. Weigh again and make up loss
by evaporation.

5. Determine volume.

6. Titrate and adjust to required
reaction, heating over free flame for
five minutes.

7. Filter through paper until clear.

8. Sterilize.

If medium can not be cleared by
filtering through paper, clearing by
white of egg ma}^ be resorted to and
the medium filtered through cotton.

Meat Infusion Broth. — 1. Infuse
500 gms.1 of lean meat, twelve to
twenty-four hours, with 1,000 c.c. of
distilled water in refrigerator.

2. Strain through wet cotton flan¬
nel or wet cheese-cloth and make up
volume to 1,000 c.c.

3. Add 5 gms. common salt and
10 gms. Witte’s pepton.

4. Weigh with containing vessel. 1

5. Warm over flame or water bath, stirring until pepton is dissolved,
not allowing temperature to rise above 50° C.

6. Determine volume.

Fig. 26. — Maassen Filter, for
small Quantities of Fluid,

1 Roughly, 1 pound (11 lb.).

THE PREPARATION OF CULTURE MEDIA

125

7. Titrate and adjust reaction to neutral.

8. Heat in Arnold sterilizer for thirty minutes; shake or stir well
and heat again for fifteen minutes.

9. Determine weight and restore loss by evaporation.

10. Determine volume, titrate, and adjust reaction to desired point
(usually one per cent acid).

11. Heat again for five minutes if adjustment of reaction has been
necessary.1

12. Filter through absorbent cotton, passing the filtrate through
the same filter until clear.

13. Titrate and record the final reaction.

Place in cotton-plugged sterile flasks or plugged sterile test tubes,
and sterilize for thirty minutes in the Arnold sterilizer on three suc¬
cessive days, leaving at room temperature in the intervals.

Sugar-Free Broth. — 1. Make 1 liter of meat infusion broth, following
steps 1, 2, 3, 4, 5, 6, 7, and 82; then filter through thin cotton filter to
remove gross particles — total clearing is not necessary.

2. Put the broth in a flask and cool. Then add 10 c.c. of a twenty-
four-hour broth culture of B. coli communis.

3. Place the flask, stoppered with cotton, in the incubator at 37° C.
for eighteen hours. (The bacteria will ferment and thus destroy any
sugar [monosaccharid] which may be present in the broth, and thus
render the broth sugar-free and acid.)

4. Heat thoroughly to kill the bacteria.

5. Determine weight and bring to 1,015 gms. Then determine
volume and titrate, and adjust to neutral. Heat thoroughly again.

6. Filter through filter paper until clear.

7. The pure sugars, dextrose, lactose, saccharose, etc., are then added
to separate portions (250 c.c.) of the broth in the proportion of one
per cent.

8. When the sugars are dissolved, tube the broth immediately in
fermentation tubes, and sterilize by discontinuous sterilization, never
heating over twenty minutes at a time, as heat tends to destroy or
change the sugars.

Glycerin Broth. — To ordinary, slightly acid or neutral meat infusion
broth, add six per cent of C. P. glycerin. Sterilize by fractional method.

1 Media become more acid on boiling, probably because of a driving out of C02,
and a second titration therefore becomes necessary.

2 These steps refer to the regular directions for making infusion broth. One
liter of previously made infusion broth may be used instead.

126

BIOLOGY AND TECHNIQUE

Calcium Carbonate Broth. — This medium is designed for obtaining
mass cultures of pneumococcus or streptococcus for purposes of im¬
munization or agglutination.

To 100 c.c. of meat infusion broth in small flasks, add one per cent
of powdered calcium carbonate, and one per cent of glucose. It is a
wise precaution to sterilize the dried calcium carbonate in the hot-air
chamber before using. Small pieces of marble may be used as sug¬
gested by Bolduan.

Pepton-Salt Solution (Dunham’s solution) :

1. Distilled water . 1,000 c.c.

Pepton (Witte) . 10 gms.

NaCl . 5 “

2. Heat until ingredients are thoroughly dissolved.

3. Filter through filter paper until perfectly clear.

4. Tube twenty-five tubes, and store remainder in 250 c.c. flasks.

Sterilize by discontinuous method.

Nitrate Solution. —

1. Distilled water . 1,000 c.c.

Pepton . 10 gms.

Potassium nitrate . 0.2 “

2. Heat until ingredients are thoroughly dissolved.

3. Filter through filter paper until perfectly clear.

4. Tube twenty-five tubes, and store remainder in 250 c.c. flasks.
Sterilize by discontinuous sterilization.

Uschinsky’ s Proteid-Free Medium.1 — To one liter of distilled water add :

Asparagin . 3.4 grams.

Ammonium lactate . 10

Sodium chloride . 5

Magnesium sulphate . 0.2

Calcium chloride . 0.1

Potassium phosphate . 1.0 “

When these substances are thoroughly dissolved, add 40 c.c. of glycerin
Tube and sterilize.

Gelatin. — Meat-Extract Gelatin. — 1. To 1,000 c.c. of distilled water
add Liebig’s extract 5 gms., pepton 10 gms., NaCl 5 gms., and 120
gms. of the finest French sheet gelatin.2

1 Uschinsky, Cent. f. Bakt., 1, xiv, 1893.

2 The acidity and consistence of the different commercial gelatins vary con¬
siderably and care should be taken in selecting a uniform and suitable brand, such as
Hesterberg’s gold label gelatin. It is advisable, when working during the summer
or in hot climates, to add 130 instead of 120 grams.

THE PREPARATION OF CULTURE MEDIA

127

2. Weigh with containing vessel.

3. Dissolve by warming.

4. Adjust weight, determine volume, titrate, and adjust reaction.

5. Cool to 60° C., add whites of two eggs, and stir thoroughly.

6. Heat for thirty minutes, stir thoroughly, and heat for fifteen
minutes.

7. Adjust weight.

8. Filter through cotton.

9. Sterilize.

Meat-Infusion Gelatin. — 1. Infuse 500 gms. lean meat twelve to
twenty-four hours with 1,000 c.c. of distilled water in refrigerator.

2. Strain through wet cotton flannel or wet cheese-cloth and make
up volume to 1,000 c.c.

3. Add 5 gms. common salt, 10 gms. Witte’s pepton, and 120 gms.
of the finest French sheet gelatin.

4. Weigh with containing vessel.

5. Warm over flame or water bath, stirring till pepton and gelatin
are dissolved and not allowing temperature to rise above 50° C.

6. Determine volume.

7. Titrate and adjust reaction to neutral.

8. Heat in Arnold sterilizer for thirty minutes; shake or stir well
and heat again for fifteen minutes.

9. Determine weight and restore loss by evaporation.

10. Determine volume, titrate, and adjust reaction to desired point,
if necessary (one per cent acid).

11. Heat five minutes over free flame, constantly stirring, if ad¬
justment of reaction has been necessary.

12. Filter through absorbent cotton, passing the filtrate through
the same filter until clear.

13. Titrate and record the final reaction.

Place gelatin in cotton-plugged sterile 250 c.c. flasks or about 8 c.c.
in plugged sterile test tubes and sterilize for thirty minutes in the Arnold
sterilizer on three successive days, leaving at room temperature in the in¬
tervals. Never heat the gelatin for longer than is necessary to comply
with directions, or it may not be solid enough for use. With some
brands of gelatin it may be necessary to add thirteen per cent in order
to obtain sufficient stiffness.

Agar. — Meat-Extract Agar. — 1. To 1,000 c.c. of distilled water (or
tap water) add 15 gms. of thread agar, 10 gms. of Witte's pepton, and
5 gms. of Liebig’s meat extract, and 5 gms. of common salt.

128

BIOLOGY AND TECHNIQUE

2. Weigh with containing vessel.

3. Heat over free flame until agar is dissolved, thirty to forty-five
minutes. (Great care should be exercised in determining that agar is
completely in solution.)

4. Determine weight and make up loss by evaporation.

5. Determine volume, titrate, and adjust to desired reaction.

6. Cool to 60° C.

7. Add whites of two eggs and stir thoroughly.

8. Heat in Arnold sterilizer thirty minutes, stir, and reheat fifteen
minutes.

9. Weigh and make up loss by evaporation.

10. Determine volume, titrate, and correct reaction if necessary.1

11. Heat for five minutes, if reaction is corrected.

12. Filter through cotton, tube, and sterilize.

Meat-Infusion Agar.2 — (A) 1. Infuse 500 gms. lean meat twelve to
twenty-four hours in 500 c.c. of distilled water in refrigerator.

2. Strain through wet cotton flannel or wet cheese-cloth, and make
up volume to. 500 c.c.

3. Add 10 gms. of Witte’s pepton and 5 gms. of common salt.

4. Weigh solution and containing vessel.

5. Warm over free flame or water bath till pepton and salt are dis¬
solved, not allowing temperature to rise above 50° C.

6. Determine volume, titrate, and neutralize.

(B) 7. Add 15 gms. of thread agar to 600 c.c. of distilled water and
boil over free flame for thirty to forty-five minutes, watching and stirring
constantly till agar is completely dissolved. This will lose weight by
evaporation; final weight should be 515 gms.

8. Cool this to about 60° C.

10. Heat for thirty minutes in Arnold sterilizer. Shake or stir
thoroughly, and heat fifteen minutes more. Adjust weight by adding
water.

11. Determine volume, titrate, and adjust reaction to plus one per
cent acid or any desired reaction.

12. Boil for two minutes over free flame, constantly stirring.

1 While titrating, care should be taken that medium does not solidify along sides
of vessel. Agar may be made more quickly and successfully in autoclave.

2 Glycerin agar is made by adding 6 per cent of C. P. glycerin to meat-extract
or meat-infusion agar.

THE PREPARATION OF CULTURE MEDIA

129

13. Filter through absorbent cotton, passing the filtrate through the
same filter until clear.

14. Titrate and record final reaction.

Place agar in cotton-plugged sterile flasks or plugged sterile test
tubes and sterilize for thirty minutes on three successive days.

Lactose-Litmus-Agar (Wurtz). — 1. Put 1,500 c.c. distilled water in
previously weighed agate-ware vessel.

2. Add 15 gms. thread agar and boil over free flame for thirty to
forty-five minutes, watching and stirring constantly till the agar is
completely dissolved.

3. Add 5 gms. Liebig's extract of meat, 5 gms. NaCl, 10 gms. Witte’s
pepton, and dissolve completely.

4. Restore loss by evaporation to 1,035 gms.

5. Determine volume, titrate, and adjust reaction to one per cent
acid.

6. Place in a flask and cool to 60° C.

7. Add the whites of two eggs beaten up in 50 c.c. of water and mix
thoroughly.

8. Heat for thirty minutes in Arnold sterilizer, shake thoroughly,
and heat again for fifteen minutes.

9. Adjust weight.

10. Filter through absorbent cotton to clear.

11. Add two per cent pure lactose (milk sugar).1

12. Add enough pure five per cent litmus solution 2 to bring to
purple color when cold.

13. Tube and sterilize.

Welch’s Modification of Guarnieri’s Medium .3 — This medium is made
on a meat-infusion basis, according to the directions given for the prep¬
aration of meat-infusion agar. It contains 5 grams of agar, 80 grams of
gelatin, 5 grams of NaCl, and 10 grams of pepton to one liter. It should

1 Add lactose and litmus to 250 c.c. for 25 tubes; keep the remainder, with¬
out lactose, stored in small sterile flasks for further use.

2 The litmus solutions used in the preparation of media are best made up as fol¬
lows: Litmus in substance — Merck’s purified, or Kaulbaum’s — is dissolved in water
to the extent of 5 per cent. The solution is made by heating in an Arnold sterilizer
for about one to two hours, shaking occasionally. The solution is then filtered through
paper and sterilized. It should be kept sterile, as molds will grow in it otherwise.

A standard litmus solution,, which is marketed for laboratory purposes, known
as “Kubel and Tiemann’s” solution, may be used.

3 Welch, Bull. Johns Hopkins Hosp.

130

BIOLOGY AND TECHNIQUE

be adjusted to a neutral reaction. It is used for stab cultures and is
designed chiefly for pneumococcus cultivation and storage.

Dorsett Egg Medium. — This medium is chiefly useful for the culti¬
vation of tubercle bacilli.

1. Carefully break eggs and drop the contents into a wide-moutheci
flask. Break up the yolk with a sterile platinum wire, and shake up
the flask until the whites and yolks are thoroughly mixed.

2. Add 25 c.c. of distilled water to every four eggs; strain through
sterile cloth.

3. Pour 10 c.c. each into sterile test tubes and slant in an inspissa-
tor and expose to 73° C. for four to five hours on two days.

4. On the third day, raise the temperature to 76° C.

5. The sterilization may be finished by a single exposure to 100°
C. in the Arnold sterilizer for fifteen minutes. Before inoculation, add
two or three drops of sterile water to each tube.

Potato Media. — Large potatoes are selected, carefully washed in
hot water, and scrubbed with a nail brush. They are then peeled,
considerably more than the cuticle being removed. The peeled potatoes
are again washed in running water for a short time, following which
cylindrical pieces are removed from them with a large apple corer. The
cylinders are cut into wedges by oblique cuts.

Since the reaction of the potato is normally acid, this should be cor¬
rected by washing the pieces in running water over night, or, better,
by immersing them in a one per cent solution of sodium carbonate for
half an hour.

The pieces are then inserted into the large variety of test tubes
known as “potato tubes. ” (See Fig. 21, c.) In the bottom of the
tubes a small amout of water (about 1 c.c.) or a small quantity of
moist absorbent cotton should be placed in order to retard drying out
of the potato. The tubes are sterilized by fractional sterilization,
twenty minutes to half an hour in the Arnold sterilizer on three
successive days.

Glycerin Potato. — In preparing glycerin potato the potato wedges
are treated as above, and are then soaked in a ten to twenty-five per
cent aqueous glycerin solution for one to three hours. A small quantity
of a ten per cent glycerin solution should be left in the tubes. In steril¬
izing these tubes, thirty minutes a day in the Arnold after heating of
the sterilizer should be regarded as sufficient, to avoid changes in the
glycerin.

Milk Media. — Fresh milk is procured and is heated in a flask for

THE PREPARATION OF CULTURE MEDIA

131

fifteen minutes in an Arnold sterilizer. It is then set away in the ice
chest for about twelve hours in order to allow the cream to rise. Milk
and cream are then separated by siphoning the milk into another
flask. It is rarely necessary to adjust the reaction of milk prepared in
this way, since, if acid at all, it is usually but slightly so. If, however,
it should prove more than 1.5 per cent acid, it should be discarded
or neutralized with sodium hydrate. The milk may then be poured
into test tubes without further additions, or litmus solution may be
added in a quantity sufficient to give a purplish blue color. The
tubes are sterilized by fractional sterilization in the Arnold sterilizer for
thirty minutes on three successive days.

Serum Media. — Loeffler's Medium. — Beef blood is collected at the
slaughter house in high cylindrical jars holding two quarts or more.
It is desirable that attempts should be made to avoid contamination
as much as is feasible by previously sterilizing the jars, keeping them
covered, and exercising care in the collection of the blood.

The blood is allowed to coagulate in the jars, and should not be
moved from the slaughter house until coagulated. All unnecessary
shaking of jars should be avoided. As soon as the coagulum is fully
formed, adhesions between the clot and the sides of the jar should be
carefully separated with a sterile glass rod or wire. The jars are then
set away in the ice chest for 24 to 36 hours. At the end of this time
clear serum will be found over the top of the clot, and between the clot
and the jar. This should be pipetted off, preferably with a large pipette
of 50 to 100 c.c. capacity, or siphoned off with sterile glass tubing, and
transferred to sterile flasks.

To three parts of the clear serum is then added one part of a one
per cent glucose beef infusion or veal infusion bouillon. The mixture is
filled into tubes, perferably the short test tubes commonly used for
diagnostic diphtheria cultures. The tubes are then placed in a slanting
position in the apparatus known as an inspissator (see p. 71). This is
a double-walled copper box covered by a glass lid, cased in asbestos,
and surrounded by a water jacket. It is heated below by a Bunsen
flame. Together with the tubes a small open vessel containing water
should be placed in the inspissator to insure sufficient moisture. The
temperature of the inspissator is now raised to 70°-75° C., care being
taken that the rise of temperature takes place slowly. The temperature
is maintained at this point for two hours, and the process is repeated, for
the same length of time, at the same temperature, on six successive
days, preferably without removing the tubes from the inspissator at

132

BIOLOGY AND TECHNIQUE

any time. It is also possible, though less regularly yielding good results,
to sterilize in the inspissator for one day, following this on the second
and third days by exposure for thirty minutes to 100° C. in the Arnold
steam sterilizer. In doing this, the Arnold should be very gradually
heated, at first without outer jacket, this being lowered only after
thorough heating has taken place.

Serum-Water Media for Fermentation Tests. — For the deter¬
mination of the fermentative powers of various microorganisms
for purposes of differentiation, Hiss has devised the following media
in which the cleavage of any given carbohydrate is indicated,
not only by the production of an acid reaction, but by the coagulation
of the serum proteids.

Obtain clear beef serum by pipetting from clotted blood in the same
way as this is obtained for the preparation of Loeffler’s blood-serum
medium. Add to this two or three times its bulk of distilled water,
making a mixture of serum and water in proportions of one to two or
three. Heat the mixture for fifteen minutes in an Arnold sterilizer at
100° C. to destroy any diastatic ferments present in the serum. Add
one per cent of a five per cent aqueous litmus solution (the varia¬
tion in the different litmus preparations as obtained in laboratories
necessitates a careful addition of an aqueous litmus solution until
the proper color, a deep transparent blue, is obtained, rather than
rigid adherence to any quantitative directions). Add to the various
fractions of the medium thus made one per cent respectively of the
sugars which are to be used for the tests.

For the preparation of inulin medium, made in this way for pneu¬
mococcus-streptococcus differentiation, it is necessary to sterilize the
inulin dissolved in the water to be added to the serum in an autoclave
at high temperature (15 pounds for 15 minutes) in order to kill spores
before mixing with the serum. The serum-water media are sterilized by
the fractional method at 100° C., at which temperature they remain fluid.

Special Media for Colon-Typhoid Differentiation.1 — Hiss’ Plating
Medium .2 — The composition of this medium is as follows:

Agar . 15 gms.

Gelatin . 15 “

Liebig’s meat extract . 5 “

Sodium chloride . 5 “

Dextrose . 10 “

Distilled water . 1,000 c.c.

1 For details of use of these special media see also chapter on Bacillus typhosus.

2 Hiss, Jour, Exp. Med., ii, 1897 ; Jour. Med. Research, viii, 1902,

THE PREPARATION OF CULTURE MEDIA

133

The agar is thoroughly dissolved in 1,000 c.c. of distilled water. When
the agar is melted, the gelatin, meat extract, and salt are added and dis¬
solved by further heating. Any loss in weight is then adjusted by the
addition of water. No titration or adjustment of reaction is necessary.
The medium should be cleared with the whites of two eggs, and filtered
through cotton. To the cleared medium is added one per cent of
dextrose, and the medium tubed, about 8 c.c. to each tube, and sterilized.
Hiss’ Tube Medium. — The composition is as follows:

Agar . . . 5 gms.

Gelatin . 80 “

Liebig’s meat extract. . . 5 “

Sodium chloride . 5 “

Dextrose. . 10 “

Distilled water . 1,000 c.c.

The method of preparation is the same as for the plating medium.
The agar is thoroughly dissolved, and then the gelatin, meat extract,
and salt are added and dissolved. After adjusting the loss in weight,
the volume should be determined, a careful titration made, and the re¬
action adjusted to one and five-tenths per cent acid by the addition of
Y HC1 solution. The medium is then cleared with the whites of eggs,
filtered, and one per cent dextrose added. It is then tubed and sterilized.

Hesse’s Medium J — The medium devised by Hesse for typhoid-colon
differentiation depends for its usefulness, as does the Hiss tube medium,
upon the great motility of the typhoid bacillus. It may be used directly
for the examination of feces or, as suggested by Jackson and Melia,1 2
after preliminary enrichment of the material to be examined by the
use of the lactose-bile medium of Jackson. (See p. 138.)

The Hesse medium is made up as follows:

Agar . .TT . 5 gms. (4.5 gms. absolutely dry.)

Pepton (Witte) . 10 “

Liebig’s beef extract . 5 “

Sodium chloride . 8.5 “

Distilled water . 1,000 c.c.

Jackson and Melia, in studying this medium, have found that
complete drying of the agar and the use of 4.5 gms. of this dried prepara¬
tion give more uniform results, since the amount of moisture in com-

1 Hesse , Zeit. f. Hyg., lviii, 1908.

2 Jackson and Melia, Jour, of Inf. Dis., vi, 1909.

134

BIOLOGY AND TECHNIQUE

mercial agar varies considerably. The preparation of the medium is
as follows:

Dissolve 4.5 gms. of dry agar in 500 c.c. of distilled water over a
free flame, making up for loss by evaporation. In another vessel 10
gms. of pepton, 5 gms. of beef extract, and 8.5 gms. of salt are dis¬
solved in 500 c.c. of distilled water. This may be heated until com¬
plete solution has taken place and the loss by evaporation made up.

The two solutions are then mixed and heated for thirty minutes;
loss by evaporation is then made up with distilled water and the solu¬
tion is filtered through cotton until clear. The reaction is then adjusted
to one per cent acidity and the medium tubed — 10 c.c. to each tube.
Sterilize in autoclave, cool, and store in ice chest.

The typhoid bacillus is characteristic on the Hesse medium only
when the dilution poured in the plates is so high that only a few colonies
appear. The typhoid colonies are much larger than are the colon colonies
and may often be several centimeters in diameter.

Piorkowski’s Urine Gelatin ? — Normal urine of a specific gravity of
about 1.020 is collected for several days. At the end of this time, when
its reaction has become alkaline, pepton 5 per cent and gelatin 3.3
per cent are added. This mixture is heated upon a water bath for one
hour, filtered, and tubed. The tubes are sterilized by the fractional
method.

In using this medium for the isolation of typhoid bacilli from the
feces, two loopfuls of feces are placed into a tube of the melted urine
gelatin, and from this dilutions are made into other tubes, taking four
loopfuls from the first into the second and six to eight from the second
to the third. Plates are then poured and kept at about 20° C. The
typhoid colonies show fine processes or filaments, while the colon
colonies are quite compact.

Capaldi’s Medium .1 2 — The composition of this medium is as follows:

Distilled water . 1,000 c.c.

Pepton (Witte) . 20 gms.

Gelatin . . 10 “

Agar . 20 “

Dextrose (or preferably mannit) . 10 “

NaCl . 5 “

KC1 . 5 “

It is advisable to dissolve the agar in 500 c.c. of water, making up

1 Piorkowski, Deut. med. Woch., vol. 25, 1899.

2 Capaldi, Zeit. f. Hyg., xxiii, 1896.

THE PREPARATION OF CULTURE MEDIA

135

the loss by evaporation with distilled water, and to dissolve the other
ingredients in a similar quantity of water, finally mixing the solutions.
The mixture is rendered alkaline by the addition of 10 c.c. of normal
NaOH, and is cleared, filtered, and filled into test tubes.

Plates are made with this medium and surface smears made of the
suspected material. B. typhosus grows in small, glistening, bluish
translucent colonies. Colonies of B. coli are larger, more opaque, and
show a brownish tinge.

Conradi-Drigalski Medium } — The following are the directions given
by the originators of this medium for its preparation, (a) Three pounds
of meat are infused in two liters of water for twelve hours or more.
After straining, boil for one hour and add 20 gms. of Witte’s pepton, 20
gms. of nutrose, 10 gms. of NaCl; boil one hour and filter. To the filtrate
add 60 gms. of agar. Boil for three hours (or one hour in an autoclave)
until agar is dissolved. Render weakly alkaline to litmus paper, filter,
and boil for half an hour more.

(b) Litmus solution: Two hundred and sixty c.c. of litmus solution
are boiled for ten minutes. (The litmus solution used by Conradi and
Drigalski is the very sensitive aqueous litmus recommended by Kubel
and Tiemann, and purchasable under the name.) After boiling, 30
grams of chemically pure lactose are added to the litmus solution.
The mixture is then boiled for fifteen minutes, and, if a sediment has
formed, is carefully decanted.

(c) Add the hot lactose mixture to the hot fluid agar solution; mix
well and, if necessary, again adjust to a weakly alkaline reaction, litmus
paper being used as an indicator. To this mixture add 4 c.c. of a hot,
sterile, ten per cent solution of sodium carbonate, in order to render it
alkaline, and 20 c.c. of a freshly made solution of crystal violet (c. p.
Hochst), 0.1 gram in 100 c.c of sterile distilled water.

The medium contains thirteen per cent of litmus solution and one
one-thousandth per cent of crystal violet.

(The plates used by Conradi and Drigalski are large plates 15 to
20 cm. in diameter.) Surface smears are made upon the medium after
solidification. These are incubated twenty-four hours. Typhoid colonies
are small, blue, and transparent. Colon colonies are large, red, and
opaque.

Endows Medium.1 2 — 1. Prepare one liter of meat infusion three per
cent agar, containing 10 grams of pepton and 5 grams of NaCl.

1 Conradi-Drigalski , Zeit. f. Hyg., xxxix, 1902.

2 Endo, Cent. f. Bakt., xxxv, 1904.

136

BIOLOGY AND TECHNIQUE

2. Neutralize and clear by filtration.

3. Add 10 c.c. of ten per cent sodium carbonate solution in order to
render it alkaline.

4. Add 10 grams of chemically pure lactose.

5. Add 5 c.c. of alcoholic fuchsin solution, filtered before using.
(Endo in his original contribution does not mention the strength of
this fuchsin solution, which, however, should be saturated.)

This colors the medium red.

6. Add 25 c.c. of a ten per cent sodium sulphite solution. This
again decolorizes the medium, the color not entirely disappearing, how¬
ever, until the agar is cooled.

7. Put into test tubes, 15 c.c. each, and sterilize.

The medium should be kept in the dark. For use, plates are poured
and surface smears of stools made. Endo claims that upon this medium
the typhoid bacillus outgrows the colon bacillus and its colonies remain
colorless, while those of bacilli coli become red.

The preparation of Endo’s medium presents certain difficulties
which arise largely from the varying purity of the sodium sulphite.
Kastle and Elvove 1 accordingly recommend the use of anhydrous sodium
sulphite instead of the crystallized variety which is hydrated. Harding
and Ostenberg 2 recommended the following method of preparing Endo’s
medium which we believe to be excellent. They adopted the method
largely because Na2S03 is easily oxidized and therefore varies in S02
content. They add sodium sulphite solution to a measured amount of
.5 per cent fuchsin solution until they determine the proportions which
give the greatest delicacy of reaction as tested with formaldehyde. The
proportions so determined are then added to the hot 3 per cent agar.
This insures a delicate medium.

Although Endo described his medium as dependent upon the forma¬
tion of acid by the bacteria, this is not so. Acids give no coloration of
the sulphite-fuchsin mixture. Indeed this mixture is used by chemists
under the name of Schiff’s reagent as a test for aldehydes. Acids decolor¬
ize the red caused by aldehydes, and this accounts for the frequent late
discoloration of red colon colonies on prolonged cultivation. The
medium is red when hot, and colorless when cold, because the compound
between sulphite and fuchsin dissociates in the hot solution.

Malachite-Green Media.3— The principle of these media is that mal-

1 Kastle and Elvove, Jour, of Inf. Dis., xvi, 1909.

2 Harding and Ostenberg, Jour, of Inf. Dis., xi, 1, 1909.

3 Loeffler, Deut. med. Woch., 32, 1906.

THE PREPARATION OF CULTURE MEDIA

137

achite green inhibits the growth of the colon bacillus without exerting
any such influence upon the typhoid bacillus. To make one liter:

1. Prepare a neutral, one-half strength, meat-infusion bouillon
(500 grams of meat to 2 liters of water) by the usual technique.

2. Acidify this with 7.5 c.c. of normal hydrochloric acid (to facilitate
the solution of agar).

3. Dissolve in this 30 grams of agar (three per cent) by boiling.

4. Neutralize with 7 c.c. normal KOH or NaOH (until neutral to
litmus) .

5. Add 5 c.c. of normal sodium carbonate solution to make it alka¬
line and heat in Arnold sterilizer for several hours.

6. Add 100 c.c. of a ten per cent nutrose solution (one per cent).
This agar may be sterilized and stored in quantities of 100 c.c. without
further manipulation.

7. Before use, redissolve, and to 100 c.c. add 2 to 2.9 c.c. of a two
per cent solution of malachite green (trade mark, “Hochst 120”)-
This solution is made in sterilized water, but is not boiled.

8. Fifteen to twenty c.c. of this medium are poured into Petri
dishes, allowed to cool, and inoculated by surface smears.

Malachite-Green Bouillon (Peabody and Pratt).1 — To 100 c.c. of
beef infusion broth add 10 c.c. of one per cent solution of malachite
green, Hochst 120, made with sterile water. This is tubed, 10 to 15
c.c. being put in each tube.

This medium is used merely as an enriching fluid. One drop of the
suspected material (emulsified stool) is added to each tube and after
incubation for eighteen to twenty-four hours inoculations may be made
upon Conradi-Drigalski plates or other media.

Peabody and Pratt found a reaction of .5 per cent acidity to phenol-
phthalein most favorable.

Bile Medium.2 — (Recommended for blood cultures by Buxton and
Coleman.) The medium is prepared as follows:

Ox-bile . 900 c.c.

Glycerin . 100 c.c.

Pepton . 20 grams

Put into small flasks containing quantities of about 100 c.c. each and
sterilized by fractional sterilization.

1 Peabody and Pratt, Boston Med. and Surg. Jour., clviii, 7, 1908.

2 Conradi, Deut. med. Woch., 32, 1906.

138

BIOLOGY AND TECHNIQUE

Jackson’s Lactose-Bile Medium.1 — Jackson has devised a medium
now in general use by water-analysts, which is of great use in isolating
B. typhosus and B. coli from water, and serves as a valuable enriching
medium in isolating these organisms from other sources, such as feces.
Jackson and Melia 2 have found that in this medium B. typhosus and B.
coli outgrow all other microorganisms and that eventually B. typhosus
will even outgrow B. coli.

It consists of sterilized undiluted ox-bile (or an eleven per cent
solution of dry, fresh ox-bile) to which have been added one per cent
pepton and one per cent lactose. It is filled into fermentation tubes
holding 40 c.c., and sterilized by the fractional method.

MacConkey’s Bile-Bait Agar. —

Sodium glycocholate

Pepton .

Lactose .

Agar .

Tap water .

per

cent

1.5

3.5

1.5

q.s.

The agar and pepton are dissolved and cleared and the lactose and
sodium glycocholate added before tubing. In this medium the B.
typhosus produces no change; B. coli, by producing acid from the
lactose, causes precipitation of the bile salts.

Neutral-Red Medium. — To 100 c.c. of a one or two per cent glucose
agar add 1 c.c. of a saturated aqueous solution of a neutral-red.

The medium is used in tubes, stab or shake cultures. The typhoid
bacillus produces no change, while members of the colon group render
the medium colorless by reduction of the neutral-red and produce gas
by fermentation of the sugar.

Barsiekow’s Medium .3 — To 200 c.c. of cold water, add 10 grams of
nutrose and allow to soak for one-half to one hour. Pour this into 800
c.c. of boiling water, and heat for twenty minutes in an Arnold sterilizer
at 100° C. Filter through cotton and to the opalescent solution of
nutrose add 5 grams of NaCl, 10 grams of lactose, and sufficient aqueous
litmus solution to give a pale blue color.4

Dieudonne’s Selective Medium for cholera spirillum. See page 584.

Enriching Substances Used in Media. — For the cultivation of micro-

1 Jackson, "Biol. Studies of Pupils of W. T. Sedgwick,” 1906, Univ. Chicago
Press.

2 Jackson and Melia, Jour. Inf. Dis., vi, 1909.

3 Barsiekow, Wien. klin. Rund., xliv, 1901.

4 Filtration may be done through paper, but takes a long time.

THE PREPARATION OF CULTURE MEDIA

139

organisms which are sensitive to their food environment, it is often neces¬
sary or advisable to add to the ordinary media enriching substances,
which empirical study has shown to favor the growth of the organism
in question. The substances most commonly used for such enrichment
are glucose, nutrose (sodium caseinate), glycerin, sodium formate, and
unsolidified animal proteids. As animal and blood serum and whole
blood must frequently be used in this way, an understanding of the
methods employed in obtaining these substances is necessary.

Method of Obtaining Blood and Blood Media. — Blood serum from
beef and sheep may be collected in the manner recommended for the
collection of such serum in the preparation of Loeffler’s medium, pipetted
into test tubes, and sterilized in the fluid state by exposure to tempera¬
tures ranging from 60° to 65° C., for one hour upon six consecutive days.
It is not a simple matter to sterilize serum in this way and requires much
time and care.

The method most commonly employed, in laboratories which have
access to hospitals, for obtaining clear serum depends upon the collection
of exudate or transudate fluids by sterile methods directly from the
pleural cavity, the abdominal cavity, or the hydrocele cavity. Sterile
flasks or test tubes are prepared and the fluid is allowed to flow directly
out of the cannula into these. It is necessary to avoid carbolic acid or
other disinfectants in sterilizing instruments and rubber tubing used
during the operation. These should be brought into the ward in the
water in which they have been boiled and not in strong antiseptic solu¬
tions, as is frequently done. The fluid so obtained may be incubated
and the contaminated tubes discarded. The serum may then be added,
in proportions of one to three, to sterile broth or melted agar.

Agar thus used is melted and cooled to 60° C., or below. One-third
of its volume of warmed exudate fluid is added, and the plates are
poured.

Serum may be rendered free of bacteria by filtration through a
Berkefeld or Pasteur-Chamberland filter. This is an effectual method,
but requires much time and care.

Whole blood may be obtained for cultural purposes by bleeding
rabbits or dogs or other animals directly from a blood-vessel into tubes of
melted agar. In the case of a rabbit, after the administration of an anes¬
thetic (ether), an incision is made directly over the trachea, and, by
careful section, the carotid artery is isolated, lying close to the side of
the trachea.

140

BIOLOGY AND TECHNIQUE

THE INFLUENCE OF DYE STUFFS UPON BACTERIAL GROWTH,
AND AS ADDITIONS TO SELECTIVE MEDIA

In describing the selective media for typhoid bacilli we have seen
that malachite green and crystal violet have been found to exert a
certain amount of selective action upon the typhoid and colon groups.
The selective influence of various dyes has been recently again studied
by Churchman. Churchman 1 found that the addition of gentian
violet in dilutions of 1 : 100,000, to media, inhibited some bacteria, while
others grew luxuriantly in its presence. Extremely interesting, both
practically and theoretically, is his observation that upon such gentian
violet media bacteria fall into two groups. Those which grow on
gentian violet correspond in a general way to the Gram-negative bacteria;
those which fail to develop on these media correspond roughly with the
Gram-positive species. One strain of the enteritidis group could not
be cultivated on gentian violet, and this was found to differ from the
others also in its agglutination tests.

Signorelli 2 claims that dahlia is useful in differentiating true cholera
strains from similar spirilla. The true cholera strains grew with colored
colonies, while others remain colorless, in his experiments.

Krumwiede and Pratt 3 were unable recently to confirm the claims
of Signorelli. However they fully confirm the findings of Churchman
both as to the selective action of gentian violet and in regard to the
classification of bacteria into two groups corresponding to their reaction
to the Gram stain. They state that among Gram-negative bacteria a
strain is occasionally found which will not grow on the gentian violet
media, differing in this respect from other members of the same species.
They find also that the reaction is quantitative.

The streptococcus-pneumococcus group, according to their investi¬
gations, differs from other bacteria in being able to grow in the presence
of quantities of violet which inhibit other Gram-positive species. Dys¬
entery bacilli show variations. Other dyes which they investigated
showed no specific inhibitory properties which could be utilized for
classification.

1 Churchman , Jour. Exp. Med., 16, 1912; also Churchman and Michael , ibid.

2 Signorelli, Centralbl. f. Bakt., Orig. 56, 1912.

3 Krumwiede and Pratt , Centralbl. f. Bakt., Orig. 68, 1913; and Proc. N. Y.
Path. Soc., xiii, 1913.

CHAPTER VIII

METHODS USED IN THE CULTIVATION OF BACTERIA

INOCULATION OF MEDIA

The transference of bacteria from pathological material to media,
or from medium to medium, for purposes of cultivation, is usually ac¬
complished by means of a platinum wire or loop. The platinum wire
used should be thin and yet possess a certain amount of stiffness and be
about two to three inches in length. This is fused into the end of a glass
rod six to eight inches long. It is an advantage, though not necessary,
to use rods of so-called “ sealing-in 11 glass which, having the same co¬
efficient of expansion as platinum, does not crack during sterilization.
For work with fluid media, the wire should be bent at its free end
so as to form a small loop which will pick up a drop of the liquid. For
the inoculation of solid media and the making of stab cultures, a straight
“ needle” or wire should be used. Other shapes of these wires and spat-
ulse from heavy wire have been devised for various purposes and are
easily improvised as occasion demands. (See Fig. 27.)

When making a transfer from one test tube to another, the tubes
should be held between the thumb and first and second fingers of the
left hand, as shown in Fig. 28. The plugs are then removed by grasping
them between the small and ring fingers and ring and middle fingers of
the right hand, first loosening any possible adhesions between glass and
plugs b}^ a slight twisting motion. The platinum wire is held meanwhile
by the thumb and index fingers of the right hand in the manner of a pen.
The wire is heated red hot in a Bunsen flame, and is then passed into the
culture tube without being allowed to touch the glass. It is held sus¬
pended within the tube for a few seconds to permit of cooling before
touching the bacterial growth. The wire is then allowed to touch lightly
the surface of the growth and a small amount is picked up. (See Fig.
29.) It is then removed from the tube without allowing it to touch the
sides of the glass, and is passed into the tube which is to be inoculated.
If the tube contains a slanted medium, such as agar, a light stroking
motion from the bottom of the slant to its apex will deposit the bacteria

141

142

BIOLOGY AND TECHNIQUE

upon the medium evenly along a central line. The needle may also be
plunged downward into the substance of the nutritive material so
that in the same tube both surface growth and deep growth may be
observed. If a stab culture is to be made in unslanted agar or in gelatin,

the needle is simply plunged straight
downward as nearly as possible along
the axis of the medium. If a fluid
medium is being inoculated, the wire
should be introduced only into the
upper part of the liquid and the bac¬
teria gently rubbed into emulsion
against the side of the glass. The
needle is then removed from the tube,
the stopper carefully replaced, and the
platinum wire immediately sterilized in
the flame. This sterilization of plati¬
num needles after they have been in
contact with bacteria should become
second nature to those working with
bacteria, since an infraction against
this rule may give rise to serious and
widespread consequences. In burning
off platinum needles it is well to re¬
member that a part of the glass rod,
as well as the wire itself, is introduced
into the tubes and may become con¬
taminated, and for this reason the
rod itself, at least in its lower two or
three inches, should be passed through
Fig. 27. — Platinum Wires. the flame as well as the wire. As an

extra precaution against contamina¬
tion, the lips of test tubes and flasks and the protruding edges of cotton
plugs may be passed through the flame and singed.

THE ISOLATION OF BACTERIA IN PURE CULTURE

It is obvious that in many cases where bacteria are cultivated from
water, milk, pathological material, or other sources, many species may
be present in the same specimen. It is likewise obvious that scientific
bacteriological study of any bacterium can be made only if we obtain

METHODS USED IN CULTIVATION OF BACTERIA

143

this particular species entirely apart from others, in what is known as
“pure culture. ” The earliest methods for accomplishing this were the
methods of Pasteur and of Cohn who depended upon the power of one
species to outgrow all others, if cultivated for a sufficient length of time
in fluid media. This method, of course, was inadequate in that it was
often purely a matter of chance which one of the mixture of species
was finally obtained by itself. A later method, by Klebs, depends
upon serial dilution, in test tubes of fluid media, by which the eventual
transference of one germ only, to the last tube was attempted. Such
methods, none of them of great practical value, have been entirely dis-

Fig. 28. — Taking Plugs from Tubes before Inoculation.

placed by those made possible by Koch's introduction of solid media
which may be rendered fluid by heat.

The methods now employed for the isolation of bacteria depend upon
the inoculation of gelatin or agar, when in the melted state, the thorough
distribution of the bacteria in these liquids by mixing, and the sub¬
sequent congealing of these media in thin layers. By this means the in¬
dividual bacteria, distributed in the medium when liquid, are held apart
and separate when the medium becomes stiff. The masses of growth
or “colonies" which develop from these single isolated microorganisms
are discrete and are descendants of a single organism, and can be trans¬
ferred, by means of a process known as “colony-fishing," to fresh sterile
culture media.

144

BIOLOGY AND TECHNIQUE

Plaitng.— The first method employed by Koch for bacterial
isolations was one that consisted in the use of simple plates of glass,
about 3x4 inches in size, mounted upon a leveling stand. A wooden
triangle, supported upon three adjustable screw-feet, formed the base of
this apparatus. Upon this was set a covered crystallizing dish which
could be filled with ice water. Upon the top of this rested the sterilized
plates under a bell j ar. By screwing up or down upon the supports of the
triangle, leveling of the plate could be achieved and controlled by a spirit-
level placed at its side. The inoculated gelatin was poured upon the

Fig. 29. — Inoculating.

plate and spread and rapidly cooled and hardened by the cold water
contained in the crystallizing dish.

The original method of Koch has been modified considerably and the
method universally employed at present depends upon the use of circular
covered dishes, the so-called Petri dishes. These obviate the necessity of
a leveling stand and prevent contamination of the plate when once
poured. Each Petri dish plate consists of two circular glass dishes; the
smaller and bottom dish has an area of 63.6 square centimeters; the
larger is used as a cover for the smaller, and forms a loosely fitting lid.

METHODS USED IN CULTIVATION OF BACTERIA

145

The plates when fitted together are sterilized and thus form a closed
cell which, if properly handled, may remain sterile indefinitely.

The technique for making a pour plate for the purpose of isolating
bacteria from mixed culture is as follows:

The actual “ pouring ” of plates is preceded by the preparation of
usually three graded dilutions of the material to be examined. For this
purpose three agar or gelatin tubes are melted and, in the case of the agar,
are cooled to a temperature of about 42° C. in a water bath. A platinum
loopful of the material to be examined is transferred to one of these tubes.
The bacteria are then thoroughly distributed throughout the melted

Fig. 30. — Pouring Inoculated Medium into Petri Plate.

gelatin or agar b}^ alternately depressing and raising the plugged end
of the tube, giving it a rotary motion at the same time. This thoroughly
distributes the bacteria throughout the medium without allowing the
formation of air-bubbles. Two loopfuls of this mixture are then trans¬
ferred to the second tube and a similar mixing process is repeated.
This second tube contains the bacteria in much greater dilution than
the first and the colonies which will form in the plate poured from this
tube will be farther apart. A third dilution is then made by transferring
five loopfuls of the mixture in the second tube to the third. This again
is mixed as before. The contents of the tubes are then poured into three
11

146

BIOLOGY AND TECHNIQUE

sterile Petri dishes. The pouring should be done with great care.
The cover of the dish is raised along one margin simply far enough to
permit the insertion of the end of the test tube, the plug of which has
been removed and the lips passed, with a rotary movement, through the
flame. The medium, is poured into the dish without the lips of the
tube being allowed to touch either the bottom or the cover of the dish.
The cover is then replaced and the medium allowed to harden.

When agar has been used, the dishes may be placed in an incubator
at 37° C. It is well to place the plates upside down in the incubator.
This prevents the condensation water, squeezed out of the agar dur¬
ing hardening, from collecting on its surface, and forming channels for
the diffuse spreading of bacteria. The same end m'ay be attained by the
use of Petri plates provided with porous earthenware lids, as suggested
by Hill. Simple inversion of the plates, however, usually suffices. When
gelatin has been used, the plates are allowed to remain in a dark place at
room temperature or in a special thermostat kept at 22°-25° C.

Colonies in agar, kept at 37.5° C. , usually develop in eighteen to twenty-
four hours; those in gelatin or agar at room temperature in from twenty-
four to forty-eight hours, depending upon the species of bacteria which
are being studied. Often in the second dilution, more frequently in
the third, the colonies will be found well apart and can then be “fished.”
The process of “ colony-fishing ” is one which requires practice and should
always be done with care, for upon its success depends the purity of the
sub-culture obtained. Colonies should never be fished under the naked
eye, no matter how far apart and discrete they may appear, since not
infrequently close to the edge of or just beneath a larger colony there
may be a minute colony of another species which may be too small to be
visible to the naked eye, but which, nevertheless, if touched by accident
will contaminate the sub-culture.

For proper “fishing,” the Petri plate with cover removed, should be
placed upon the stage of the microscope and examined with a low power
objective, such as Leitz No. 2 or Zeiss AA. The sterilized platinum
needle, held in the right hand, is then carefully directed into the line
of focus of the lens, while the small finger of the hand is steadied upon
the edge of the microscope stage. When the point of the needle is
clearly visible through the microscope, it is gently depressed until it
is seen to touch the colony and to carry away a portion of it. The
needle is then withdrawn without again touching the nutrient medium
or the edges of the glass or the lens, and transferred to a tube of what¬
ever medium is desired. In this way, individuals of one colony, de-

METHODS USED IN CULTIVATION OF BACTERIA

147

scendants of a single bacterium of the original mixture, — are carried
over to the fresh medium.

Esmarch Roll Tubes.1 — A simple method of obtaining separate colo¬
nies is that devised by von Esmarch and known as “ roll-tube ” cultiva¬
tion. Tubes of melted gelatin are inoculated with various dilutions of
the bacterial mixture and, while still liquid, are laid in an almost horizon-

Fig. 31. — Streak Plate.

tal position upon a block of ice, which has been grooved slightly by
means of a test tube filled with hot water. The test tube containing the
gelatin, after being placed in this groove, is rapidly revolved by passing
the fingers of the right hand across it, while its base is carefully
steadied with the left hand. If the revolving is carried out with

1 Esmarch } Zeit. f. Hyg., i, 1886.

148

BIOLOGY AND TECHNIQUE

sufficient speed, the gelatin will harden in a thin layer on the inner
surface of the tube. The colonies will develop in this layer and may be
“ fished ” by means of a platinum wire with bent point introduced into the
tube. This method is useful for certain purposes, but is too inconvenient
for routine work. It is now rarely used.

Separation of Bacteria by Surface Streaking.— When it is necessary to

isolate bacteria like the gonococcus, Bacillus influ¬
enzae, the pneumococcus, and others, which, because
of great sensitiveness to environment and possibly a
preference for free oxygen, are not readily grown
in pour plates, it is often advantageous first to
pour plates of suitable media, allow them to
harden, and then gently smear over their surfaces
dilutions of the infectious material, usually in three
or four parallel streaks. (See Fig. 31.)

Upon such plates, if dilutions have been prop¬
erly made, and this is only a question of judgment
based upon an estimation of the numbers of bac¬
teria in the original material, discrete colonies of the
microorganisms sought for may develop, and can be
“ fished ” in the usual manner.

The media most favorable for the cultivation of
various microorganisms will be discussed in the
sections dealing with the individual species.

■ V ^

ANAEROBIC METHODS

We have seen in a preceding chapter (p. 26)

pIG gg _ Deep ^h-8! many bacteria, the so-called anaerobes, will

Stae Cultivation develop only in an environment from which free
of Anaerobic oxygen has been excluded.

Bacteria. The exclusion of oxygen for purposes of anaero¬

bic cultivation may be accomplished by a variety of
methods, depending upon a few simple principles which have been
applied, either separately or in combination, by many workers.

The earliest methods depended upon the simple exclusion of air Uy
mechanical devices. In other methods, the oxygen of the air is displaced
by inert gases (hydrogen), and others again depend upon the oxygen-
absorbing qualities of alkaline solutions of pyrogallol.

Cultivation by the Mechanical Exclusion of Air. — Koch succeeded in

METHODS USED IN CULTIVATION OF BACTERIA

149

growing anaerobic bacteria upon plates by simply dropping upon the
surface of the inoculated agar or gelatin a flat piece of sterile mica. This
method, however, rarely succeeds in sufficiently excluding the air.

Liborius’ Method.1 — This method consists in the use of deeply filled
tubes of agar or gelatin, from which all oxygen has been removed by
boiling for fifteen minutes or more. It is advantageous, as has been
pointed out in the section on anaerobiosis, that
media used for this purpose should contain carbo¬
hydrates in some form, perferably glucose. After
boiling, the tubes are rapidly transferred to ice
water so that as little oxygen as possible may be
absorbed during the hardening of the medium.

The tubes are then inoculated by deep stabs. After
inoculation, the medium may be covered with a
thin layer of agar, gelatin, or oil (albolin), and
further sealed with sealing-wax to prevent oxygen-
absorption.

This method may be utilized for the isolation
of anaerobes (as in the original method of Libo-
rius) by inoculating the medium just before it
solidifies. The tubes may be gently shaken in
order to distribute the bacteria throughout the
medium and then rapidly cooled. In this case
colonies which develop may be scattered through¬
out the deeper layers of the agar or gelatin, and
may be “fished” after breaking the tube.

Esmarch’s Method.2 — Von Esmarch has applied
the principles of his roll-tube to the cultivation of
anaerobic bacteria. Gelatin tubes are inoculated
as above and roll-tubes prepared. The tubes are
then set into cold water to prevent melting of the
thin gelatin layer and the interior of the tube is
filled with melted gelatin.

Roux’s Method.3 — Anaerobic bacteria are culti¬
vated by sucking the inoculated gelatin or agar into narrow tubes,
which are then closed at both ends by fusing in the flame. After
growth has taken place the tubes are broken and the organism re¬
covered by “fishing.”

11 Liborius, Zeit. f. Hyg., i, 1886. 2 Von Esmarch, loc. cit.

3 Roux, Ann. Past., i, 1887.

Fig. 33. — Deep
Stab Cultiva¬
tion of Anaero¬
bic Bacteria.

150

BIOLOGY AND TECHNIQUE

Fluid Media Covered with Oil. — Erl enmeyer flasks or other vessels
are partially filled with glucose-bouillon over which a thin layer of al-
bolin or other oil is allowed to flow. The oxygen is driven out of the
liquid by vigorous boiling for fifteen minutes or more.

It should be remembered whenever using this or similar methods that
a layer of fluid oil does not form an impermeable seal. By covering an

alkaline pyrogallol solution with oil it
can easily be shown that oxygen slowly
diffuses through the oil into the medi¬
um below.

The simple exclusion of air, also,
is the principle underlying the culti¬
vation of anaerobic bacteria in the
closed arm of a Smith fermentation
tube.

Wright’s Method.1 — Wright has
described a simple and excellent
method for the cultivation of anaero¬
bic bacteria in fluid media. The ap¬
paratus necessary is easily improvised
with the materials at hand in any
laboratory. A short piece of glass
tubing, constricted at both ends and
fitted at each end with a small piece of
soft-rubber tubing, is inserted into a
test tube containing nutrient broth.
The upper end of the inserted glass
tubing is connected by the rubber
with a pipette passed through the
cotton plug in the tube. The entire
apparatus, plus broth, may be steril¬
ized after being put together. When
a cultivation is made, the fluid in the test tube is inoculated as usual. The
fluid is then sucked up into the glass tubing until this is completely filled.
A downflow of the fluid is then prevented by placing the finger over the
pipette through which the suction has been made or by constricting a
small piece of rubber tubing attached to the upper end of the pipette.
The entire system of tubes is then pushed downward in such a way that
both pieces of rubber tubing, attached to the ends of the little glass

■

Fig. 34.

Anaerobes

Albolin.

— Cultivation op
in Fluid under

1 Wright , J. H. Quoted from Mallory and Wright, “Path. Technique,” Phila., 1904.

METHODS USED IN CULTIVATION OF BACTERIA

151

chamber, are kinked. The entire apparatus may then be incubated.
Growth of anaerobic bacteria takes place within the air-tight chamber
formed by the short glass tubing within the test tube. The fluid in
the test tube, outside of this chamber, usually remains clear.

When cultivation has been successful, the bacteria may be obtained
either for morphological study or for further
cultivation, by simply allowing the fluid to
flow out of the little air-tight chamber back
into the test tube. The method is simple and
usually successful.

Methods Based upon the Displacement of Air
by Hydrogen. — The principle of air-displace¬
ment by hydrogen, first utilized by Hauser,1
has been widely applied to the cultivation of
anaerobic bacteria. In substance it consists
of the conduction of a stream of hydrogen
through an air-tight chamber in which plates
or tubes containing inoculated media have been
placed.

For the production of the hydrogen, the
most convenient apparatus is the Kipp hydro¬
gen generator or some modification of it.

Hydrogen is generated from zinc and sul¬
phuric acid and this may be passed through
a series of Woulfe-bottles, containing solu¬
tions of lead acetate and of pyrogallic acid,
to remove traces of sulphuretted hydrogen
and of oxygen, respectively. Some authors
recommend also the interpolation of a bottle
containing Lugol’s solution to absorb traces
of acid vapor, and of one with a silver-
nitrate solution to take up any hydrogen
arsenide which may be derived from impurities
contained in the zinc.

For anaerobic cultivation upon solid media,
the inoculated tubes or plates are placed in an apparatus such as
the Novy jar. This is connected with the hydrogen apparatus and
hydrogen allowed to flow through it for five or ten minutes, and the
stop-cocks then closed.

Fig. 35. — Wright’s
Method of Anaerobic
Cultivation in Fluid
Media.

1 Hauser, 11 Ueber Faulnissbakterien,” 1885.

152

BIOLOGY AND TECHNIQUE

In applying the hydrogen method to fluid media, flasks containing
the broth are fitted with sterile, tightly fitting rubber stoppers per¬
forated by two holes, through which glass tubes are passed. One of
these tubes, the inlet, passes below the surface of the liquid. The other
one, the outlet, extends only a short distance below the stopper and is
always kept above the surface of the medium. The flasks are inoculated
and hydrogen is passed through the medium so that it enters the
long tube, bubbles up through the fluid, and leaves by the short tube.

The broth may be covered with a
thin layer of liquid paraffin or
albolin.

The Use ot Pyrogallic Acid
Dissolved in Alkaline Solutions
for Oxygen Absorption. — Buchner1
has applied the principle of
chemical absorption for the re¬
moval of oxygen to the cultiva¬
tion of anaerobic bacteria. This
has been made use of in a
number of different ways. The
method is based upon the fact
that alkaline solutions of pyro-
gallol possess the power of ab¬
sorbing large quantities of free
oxygen. At first such solutions
are of a light straw-color, which
becomes dark brown as oxygen
is absorbed. The absorption of
all the oxygen in the environ¬
ment may be assumed when
there is no further deepening of
the brown color.

Buchner first utilized this
principle by placing a small wire or glass holder within a large test
tube, dropping dry pyrogallol (pyrogallic acid) into the bottom of this
tube, then running a five per cent sodium hydrate solution into it,
and inserting within this large tube a smaller test tube containing the
inoculated culture medium. The large tube was then tightly closed

Fig 36. — Novy Jar.

1 Buchner, Cent. f. Bakt., I, iv, 1888.

METHODS USED IN CULTIVATION OF BACTERIA 153

with a rubber stopper. In this way, the air space surrounding the
smaller tube was rendered oxygen free.

A simple modification of the preceding method of Buchner has
been devised by Wright.1 Stab-cultures of gelatin or agar in test
tubes are made in the usual way. The cotton stopper closing the tube
is then thrust into the tube to such a depth that its upper end lies at
least 1 cm. below the mouth of the tube.

A small quantity of sodium or potassium
hydrate solution in which some pyrogallic
acid has been dissolved, is then allowed to
flow on to the cotton of the plug and the
mouth of the tube is immediately sealed by
a tightly fitting rubber stopper. The cotton
stopper in these cases must be made of ab¬
sorbent cotton; 1.5 to 2.5 c.c. of the pyro¬
gallic acid solution is usually sufficient for
test tubes of ordinary size.

For cultivation of anaerobic bacteria
upon agar slants, a simple technique may be
applied and easily improvised in the labora¬
tory as follows: The tube of slant agar is
inoculated with the infectious material in the
usual way. It is then, with stopper removed,
inverted into a tumbler or beaker containing
about a gram of dry pyrogallic acid. A
small quantity of a five per cent or three
per cent sodium hydrate solution is then run
into the tumbler and this is covered with a
thin layer of liquid paraffin or albolin before
the pyrogallic acid has been completely dis¬
solved. In this way, completely anaerobic
conditions are obtained in the tube and the
growth of anaerobes takes place upon the
surface of the slant.

For the cultivation of anaerobes in Petri
dishes, for purposes of separation, a combination of the pyrogallic
acid method and the hydrogen displacement methods is often em¬
ployed. For this purpose the jars devised by Novy and by Bulloch
are extremely convenient.

Fig. 37. — Wright’s Meth¬
od of Anaerobic Cultiva¬
tion by the Use of Pyro¬
gallic Acid Solution.

1 Wright, Jour, of the Boston Soc. of Med. Sci., Dec., 1900.

154

BIOLOGY AND TECHNIQUE

In using the Novy jar, the inoculated plates are set upon a wire
frame resting about an inch above the bottom of the jar. The cover
is then tightly set in place and the air in the j ar exhausted by means of a
suction pump. The arrangement of the double stop-cock in the top
renders it possible now, by simply turning this, to admit hydrogen from
a Kipp generator into the j ar. The process of alternate exhaustion and

admission of hydrogen may be
several times repeated.

A combination of air exhaus¬
tion, oxygen absorption, and hy¬
drogen replacement may be prac¬
ticed in jars such as that shown
in Fig. 39. Tubes or plates after
inoculation are placed in this jar,
on a raised wire frame. Dry py-
rogallic acid is placed in the
bottom of the jar and the cover
tightly fitted. An opening in
the side of the jar connects its
interior with a bottle containing
sodium or potassium hydrate so¬
lution. Through the stopper of
this bottle pass two glass tubes,
one of them of such length that
it can be pushed down into the
alkaline solution, or pulled up¬
ward above the level of the fluid.
This tube connects the jar with
the bottle. The other glass tube
is short, passing just through the
stopper and at the top made in
the form of a T, one arm of the T
being connected with a Kipp hydrogen generator, the other with a
suction-pump.

After the jar has been sealed, the glass tube connecting the jar and
the bottle is raised above the level of the fluid in the bottle and, the con¬
nection to the hydrogen generator being closed, the air in the jar is
exhausted with the suction-pump. Connection to the suction may then
be closed, and the other arm of the T being open, hydrogen is allowed to
flow into the jar. Alternate suction and hydrogen replacement may be

Fig. 38. — Jar for Anaerobic Cul¬
tivation.

METHODS USED IN CULTIVATION OF BACTERIA

155

carried out two or three times. After the last exhaustion, the glass tube
in the bottle connecting it with the jar is pushed down into the fluid,
and the vacuum will draw the sodium hydrate solution into the bottom
of the jar, dissolving the pyrogallol, which will then absorb any traces of
free oxygen remaining in the jar. Hydrogen is again introduced and the
jar closed. If exhaustion of oxygen has been sufficiently thorough,
the pyrogallic solution in the bottom of the jar will remain light brown.

A simple method for the separation of anaerobes in plates without the
use of hydrogen or of specially constructed jars, may be carried out as
follows l: The apparatus used consists of two circular glass dishes, fitting
one into the other as do the halves of a Petri dish, and similar to these
in every respect except that they are higher, and that a slightly greater
space is left between their sides when they are placed together. The

Fig. 39. — Apparatus for Combining the Methods of Exhaustion, Hydrogen,

Replacement, and Oxygen Absorption.

dishes should be about three-fourths to one inch in height, they need
be of no particular diameter, although those of about the same size as
the usual Petri dish are most convenient. An important requirement
necessary for the success of the method is that the trough left between the
two plates, when put together, shall not be too broad, a quarter of an
inch being the most favorable.

Into the smaller of these plates the inoculated agar is poured exactly
as this is done into a Petri dish in the ordinary aerobic work. Pro¬
longed boiling of the agar before plating is not essential. When the agar
film has become sufficiently hard on the bottom of the smaller dish, the
entire apparatus is inverted. The smaller dish is now lifted out of the

1 Zinsser, Jour. Exp. Med., viii, 1906.

156

BIOLOGY AND TECHNIQUE

larger, and placed, still inverted, over a moist surface — a towel or the
wet surface of the table — to prevent contamination. Into the bottom
of the larger dish, which now stands open, there is placed a quantity
(about 3 grams) of dry pyrogallic acid. Into this, over the pyrogallic
acid, the smaller dish, still inverted, is then placed. A five per cent solu¬
tion of sodium hydrate is poured into the space left between the sides of
the two dishes, in quantity sufficient to fill the receiving dish one-half
full. While this is gradually dissolving the pyrogallic acid, albolin,
or any other oil (and this is the only step that requires speed), is
dropped from a pipette, previously filled and placed in readiness, into

Fig. 40. — Simple Apparatus for Plate Cultivation of Anaerobic Bacteria,

(Zinsser.)

the same space, thus completely sealing the chamber formed by the two
dishes.

If these steps have been performed successfully, the pyrogallic solu¬
tion will at this time appear of a light brown color, and the smaller plate,
with its agar film, will float unsteadily above the other. Very rapidly,
as the pyrogallic acid absorbs the free oxygen in the chamber, this plate
is drawn down close to the other, and the acid assumes a darker hue,
which remains without further deepening even after three or four days’
incubation.

The Use of Fresh Sterile Tissue as an Aid to Anaerobic Cultivation.

— The addition of small pieces of fresh sterile tissue (rabbit or guinea-
pig) to culture tubes, either solid or fluid, greatly favors the growth of
anaerobic bacteria. By such a method anaerobes can be made to de¬
velop even when other precautions for the establishment of anaerobiosis

METHODS USED IN CULTIVATION OF BACTERIA

157

are imperfectly observed. This was noticed first by Theobald Smith and
by Tarozzi and has become an extremely useful reenforcement to other
methods. It has been utilized most extensively by Noguchi of recent

Fig. 41. — Incubator.

years in his technique for the cultivation of various treponemata.
The simplest way to apply this method is to place a piece of freshly
excised rabbit kidney, testicle, or spleen into the bottom of a high test
tube (20 cm.) and then pouring over it the culture fluid. Kidney or

158

BIOLOGY AND TECHNIQUE

other tissues are more suitable for this purpose than liver tissue since
the latter is not easily obtained in a sterile condition, bacteria
often getting into it during life through the portal circulation. The
action of the tissues depends probably upon its great reducing
power.

THE INCUBATION OF CULTURES

After bacteria have been transferred to suitable culture media, it
is necessary to expose them to a temperature favorable to their develop¬
ment. In the case of many saprophytes,
the ordinary room temperature is suffi¬
ciently near the optimum to obviate the
use of any special apparatus for maintain¬
ing a suitable temperature; in the case
of most pathogenic bacteria, however,
the body temperature of man, 37.5°
C., is either a necessary requirement
for their growth, or at any rate
favors speedy and characteristic develop¬
ment.

For the purpose of obtaining a uniform
temperature of any required degree, the
apparatus in general use is the so-called
incubator or thermostat. This may be
adjusted for gelatin cultivation at 20 to
22° C., or for agar, broth, or other media
at 37.5° C.

Incubators, while varying in detail,
are all constructed upon the same prin¬
ciples. They consist of double-walled copper chambers, which are
fitted with a set of double doors, the outer being made of asbestos-
covered metal, the inner of glass. (See Fig. 41.) The space be¬
tween the two walls is filled with water, which, being a poor
heat conductor, tends to prevent rapid changes of temperature
within the chamber as the result of changes in the external
environment. Both walls are perforated above by openings to
admit thermometers into the interior and one wall is perforated
so that a thermo-regulator may be inserted into the water
jacket. The under surface of the chamber is heated by a gas

Fig. 42.

Fig. 43.

Fig. 42. — Thermo-regulator.
(Lautenschlager . )

Fig. 43. — Thermo-regulator.
(Reichert.)

METHODS USED IN CULTIVATION OF BACTERIA

159

flame, the size of which is automatically regulated by the thermo¬
regulator.

A number of thermo-regulators are on the market, all of them con¬
structed upon modifications of the same principle. One of the most
efficient of those in common use is that shown in Fig. 42. This con¬
sists of a long tube of glass fitted with a metal cap through which an in¬
let tube (A) projects into the interior. Slightly below the middle of the
tube there is a glass diaphragm separating its interior into two com¬
partments. In the middle of the diaphragm an aperture leads into a
spiral of glass which projects into the lower compartment. The lower
compartment is filled with ether and mercury. The lower end of the inlet
tube ( A ) has a wedge-shaped slit. The gas from the supply pipe
passing through the tube (A) is conducted through the slit-like opening
in its lower end into the inner chamber and passes out to the burner
through the elbow ( B ). When the temperature is raised, the ether and
mercury in the lower chamber expand and the mercury rises in the
upper chamber, gradually restric ting the opening through the V-shaped
slit in the inlet tube. Thus the gas supplied to the burner is
diminished, the flame reduced, and the temperature again falls. The
temperature can be arbitrarily adjusted by raising or lowering
the inlet tube. A scale at the upper end of the inlet tube allows
exact adjustment. Complete shutting off of the gas is prevented by a
small circular opening placed in the inlet tube just above the slit.

Another cheaper and simpler them o-regulator is shown in
Fig. 43. This consists of a long tube open at the top and fitted
about 1§ inches from the top with two hollow glass elbows. One of these
elbows remains open, the other, situated on a slightly lower level, is closed
by a brass screw-cap. The tube is filled with mercury to a point slightly
above the level of the elbow containing the screwr-cap. The height of
the mercury can thus be increased or decreased by screwing in or out
upon the cap. Into the upper end of the tube there is fitted another
device which consists of a T-shaped system of glass tubes, one arm
of the T being open and the other closed, the perpendicular leg of the
T tapering to a minute opening at the bottom. The gas passes into
one arm of the T down through the tapering leg and into the space
immediately above the mercury. It then passes out through the open
elbow of the main tube. As the mercury rises, it gradually diminishes
the space between its surface and the small opening in the tapering tube
above it, finally completely shutting off the gas from this source. Gas can
now pass only through a minute hole perforating the vertical leg of the

160

BIOLOGY AND TECHNIQUE

T an inch above its end. The flame decreases and the temperature
again sinks.

Since gas pressure in laboratories is apt to vary, it is convenient to
interpose between the gas supply and thermo-regulator some one of the
various forms of gas-pressure regulators. The use of these is not ab¬
solutely necessary but aids considerably in the maintenance of a con¬
stant temperature. The one most commonly employed is the so-called
Moitessier apparatus. This consists of a cylindrical metal chamber
within which there is fitted an inverted metal bell. Glycerin is poured
into the cylinder to the depth of about two inches. An inlet pipe con-

Fig. 44. — Moitessier Gas-Pressure Regulator.

ducts gas into the open space between the top of the glycerin and the bell.
From the top of the bell is suspended a conical piece of metal which hangs
free in the outlet pipe. As the gas pressure under the bell increases,
this is raised and the opening of the outlet pipe is gradually diminished
by the cone. Thus the relation between the pressure in the inlet pipe
and the actual quantity of gas passing through is equalized. A cup con¬
nected to the top of the bell through the roof of the cylinder by a bar can
be filled with birdshot and the pressure against the gas can thus be
modified to conform with existing conditions.

METHODS USED IN CULTIVATION OF BACTERIA 161

Colony Study. — Cultures are usually incubated for from twelve to
forty-eight hours. Considerable aid to the recognition of species is
derived from the observation of both the speed of growth and the ap¬
pearance of the colonies. It is therefore necessary to proceed in the
study of developed co’onies in a systematic way. The development of
colonies should be observed in all cases both upon gelatin and upon agar.
In forming any judgment about colonies, the acidity or alkalinity, and
the special constitution of the media should be taken into account.
The colonies are carefully examined with a hand lens and with the low
power (Leitz No. 2, Zeiss AA, Ocular No. 2) of the microscope. The
colonies should be observed as to size, outline, transparency, texture,
color, and elevation from the surface of the media. Much information,

Fig. 45. — Variations in the Conformation of the Borders of Bacterial

Colonies. (After Chester.)

also, can be obtained by observing whether a colony appears dry,
mucoid, or glistening, like a drop of moisture. By a careful obser¬
vation of these points, definite differentiation, of course, can not usu¬
ally be made, but much corroborative evidence can be obtained which
may guide us in the methods to be adopted for further identification
and for a final summing up of species characteristic as a whole.

The Counting of Bacteria. — It is often necessary to determine the
number of bacteria per c.c. contained in water, milk, or other substances.
For this purpose definite quantities of the material to be analyzed are
mixed with gelatin or agar and poured into Petri plates. The exact
dilutions of the suspected material must largely depend upon the number
of germs which one expects to find in it. The plates, if prepared with
gelatin, are allowed to develop at room temperature for twenty-four to
12

162

BIOLOGY AND TECHNIQUE

forty-eight hours. If agar has been used, they are usually placed in the
incubator at 37.5° C. At the end of this time, the colonies which have
developed are enumerated. For this purpose, a Petri dish is placed upon a
Wolffhiigel plate. This plate consists of a disk or square of glass which is
divided into small squares of one square centimeter each. Diagonal lines
of these squares running at right angles to each other are subdivided into
nine divisions each in order to facilitate counting when the colonies are
unusually abundant. The Petri dish is placed upon the plate in such a
way that the center of the dish corresponds to the center of the plate.

Fig. 46. — Wolffhugel Counting Plate.

The colonies in a definite number of squares are then counted. The
greater the number of squares that are counted the more accurate the
estimation will be. When the growth is so abundant that only a limited
number of squares can be counted, these should be chosen as much as
possible from different parts of the plate, and in practice one counts
usually six squares in one direction and six at right angles to these, so
as to preclude errors arising from unequal distribution. The final calcu¬
lation is then made by ascertaining the average number of colonies con¬
tained in each square centimeter. If standard Petri dishes have been

METHODS USED IN CULTIVATION OF BACTERIA

163

used, this is multiplied by 63.6, the number of squares in the area of the
dish, and then by the dilution originally used.

Thus if twelve squares have been counted with a total number of
one hundred and forty-four colonies — the average for each square is
twelve. Twelve times 63.6 is 763.2, which represents the total number
of colonies in the plate. Now if 0.1 c.c. of the original material
(water or milk) has been plated, this material may be assumed to have
contained 10 X 763.2, or 7,632 bacteria to each cubic centimeter.

If dishes of an unusual size are employed, the square area must be
ascertained by measuring the radius and multiplying its square by n
(7i x R2 = area) ( ?r = 3.141592).

CHAPTER IX

METHODS OF DETERMINING BIOLOGICAL ACTIVITIES OF BACTERIA

ANIMAL EXPERIMENTATION

Gas Formation. — Bacteria of many varieties produce gas from the
proteid and the carbohydrate constituents of their environment.

Gas formation can be observed in a very simple manner by making
stab cultures in gelatin or agar containing the fermentable nutrient
substances. In such cultures bubbles of gas will form along the track
of the inoculation, or, in the case of such semisolid media as the tube
medium of Hiss, will spread throughout the tube. In the case of some
anaerobes gas formation in stab cultures will occur to such an extent that
the medium will split and break. It should be borne in mind in carrying
out such methods that air is readily carried into the medium with
the inoculating needle or loop by splitting of the medium, also that
media which have been stored in the cold may absorb air. Expansion
of the air in such tubes may simulate small amounts of gas formation
and lead to error. It is advisable, therefore, whenever making stab
inoculations with the above purpose, to heat the media and rapidly
cool them before use.

A more accurate method of gas determination is by the use of fer¬
mentation tubes, such as those devised by Smith. The gas which is
formed collects in the closed arm of the fermentation tube and may be
quantitatively estimated. The fermentation, with gas production, of
certain substances such as carbohydrates, may be determined by adding
these materials in a pure state to the media before inoculation with
organisms.

In the case of carbohydrates this method has proved of great differ¬
ential value, since the power of splitting specific carbohydrates with gas
production is a species characteristic of great constancy for many forms
of bacteria.

Analysis of Gas Formed by Bacteria. — Carbon Dioxide. — For
the estimation both qualitatively and roughly quantitatively of carbon
dioxide produced by bacteria, cultures are grown in fermentation tubes
containing sugar-free broth (see page 125) to which one per cent of pure
dextrose, lactose, saccharose, or other sugars has been added. The tubes
are incubated until the column of gas formed in the closed arm no longer

164

DETERMINING BIOLOGICAL ACTIVITIES OF BACTERIA 165

increases (twenty-four to forty-eight hours). The level of the fluid
in the closed arm is then accurately marked and the column of gas
measured.

The bulb of the fermentation tube is then completely filled with
f NaOH solution, the mouth closed with a clean rubber stopper, and
the bulb inverted several times in order to mix the gas with the soda
solution. The tube is then again placed in the upright position, allow¬
ing the gas remaining to collect in the closed arm. The gas lost may
be roughly estimated as consisting of C02.

Hydrogen.— The gas remaining, after removal of the 00 2 in the pre¬
ceding experiment, at least when working with carbohydrate solutions,

Fig. 47. — Types of Fermentation Tubes.

may be estimated as hydrogen. When allowed to collect near the mouth,
further evidence of its being hydrogen may be gained by exploding it
with a lighted match.

Hydrogen Sulphide (H2 S, Sulphuretted hydrogen). — In alkaline
media sulphuretted hydrogen, if formed, will not collect as gas, but
will form a sulphide with any alkali in the solution. For the estimation
of the formation of hydrogen sulphide, bacteria are cultivated in a strong
pepton solution to which 0.1 c.c. of a one per cent solution of ferric
tartrate or lead acetate has been added. The addition of these substances
gives rise to a yellowish precipitate in the bottom of the tubes. If, on

166

BIOLOGY AND TECHNIQUE

subsequent inoculation, the bacteria produce H._, S, this precipitate will
turn black. The solution recommended by Pake for this test is prepared
as follows:

1. Weigh out 30 grams of pepton and emulsify in 200 c.c. of tap water at 60° C.

2. Wash into a liter flask with 80 c.c. tap water.

3. Add sodium chloride 5 grams and sodium phosphate 3 grams.

4. Heat at 100° C. for 30 minutes, to dissolve pepton.

5. Filter through paper.

6. Fill into tubes, 10 c.c. each, and to each tube add 0.1 c.c. of a one per cent
solution of ferric tartrate or lead acetate. These solutions should be neutral.

7. Sterilize.1

Accurate quantitative gas analyses of bacterial cultures can be
made only by the more complicated methods used in chemical labora¬
tories for quantitative gas analysis. The gas, in such cases, is collected
in a bell jar mounted over mercury, and subjected to analysis by the
usual method described in works on analytical chemistry.

Acid and Alkali Formation by Bacteria. — Many bacteria produce acid
or alkaline reactions in culture media, their activity in this respect
depending to a large extent upon the nature of the nutrient material.
Many organisms which on carbohydrate media produce acid will give
rise to alkali if cultivated upon media containing only proteids.

Information as to the production of acid or alkali can be obtained
by the addition of one of a variety of indicators to neutral media. The
indicators most often employed for this purpose are litmus or neutral
red. Changes in the color of these indicators show whether acids or
alkalis have been produced.

Great help in differentiation is obtained by adding chemically pure
carbohydrates to media to which litmus has been added and then de¬
termining whether or not acid is formed from the substances by the
microorganisms. These tests have been of special importance in the
differentiation of the typhoid-colon groups of bacilli.

Quantitative estimation of the degree of acidity or alkalinity pro¬
duced by bacteria may be made by careful titration of definite volumes
of the medium before and after bacterial growth has taken place.

The variety of acid formed by bacteria depends largely upon the
nature of the nutrient medium. The acids most commonly resulting
from bacterial growth are lactic, acetic, oxalic, formic, and hippuric
acids. Qualitative and quantitative estimation of these acids may be
made by any of the methods employed by analytical chemists.

1 Quoted from Eyre, “ Bact. Technique,” Phila., 1903.

DETERMINING BIOLOGICAL ACTIVITIES OF RACTERIA 167

Indol Production by Bacteria. — Many bacteria possess the power of
producing indol. Though formerly regarded as a regular accompani¬
ment of proteid decomposition, later researches have shown that indol
production is not always coexistent with putrefaction processes and
occurs only when pepton is present in the pabulum.

Indol formation by bacteria is determined by the so-called nitroso-
indol reaction. Organisms are grown in sugar-free pepton broth or in the
pepton-salt bouillon of Dunham. (See page 126.) Media containing
fermentable substances are not favorable for indol production since acids
interfere with its formation. The cultures are usually incubated for three
or four days at 37° C. At the end of this time, ten drops of con¬
centrated sulphuric acid are run into each tube. If a pink color
appears, indol is present, and we gather the additional information
that the microorganism in question has been able to form nitrites
by reduction (e.g., cholera spirillum). If the pink color does not
appear after the addition of the sulphuric acid alone, nitrites must
be supplied. This is done by adding to the fluid about 1 c.c. of a 0.01
per cent aqueous solution of sodium nitrite. The sodium nitrite solu¬
tion does not keep for any length of time and should be freshly made up
at short intervals.

Phenol Production by Bacteria. — Phenol is often a by-product in the
course of proteid cleavage by bacteria. To determine its presence in
cultures, bacteria are cultivated in flasks containing about 50-100 c.c.
of nutrient broth. After three to four days’ growth at 37° C., 5 c.c. of
concentrated HC1 are added to the culture, the flask is connected with a
condenser, and about 10-20 c.c. are distilled over.

To the distillate may be added 0.5 c.c. of Millon’s reagent (solution of
mercurous nitrate in nitric acid), when a red color will indicate phenol;
or 0.5 c.c. of a ferric chloride solution, which will give a violet color if
phenol is present.

Reducing Powers of Bacteria. — The power of reduction, possessed by
many bacteria, is shown by their ability to form nitrites from nitrates.
This is easily demonstrated by growing bacteria upon nitrate broth
(see page 126). Bacteria are transferred to test tubes containing this
solution and allowed to grow in the incubator for four or five days.
The presence of nitrites is then chemically determined.* 1

1 We are indebted to Dr. J. P. Mitchell, of Stanford University, for the following
technique for nitrite tests:

I. Sulphanilic Acid. — Dissolve 0.5 g. in 150 c.c. of acetic acid of Sp. Gr. 1.04.

168

BIOLOGY AND TECHNIQUE

In bacteriological work 4 c.c. of the culture fluid is poured into a
clean test tube, and to it are gradually added 2 c.c. of the mixed test
solutions. A pink color indicates the presence of nitrites, the intensity
of the color being proportionate to the amount of nitrite present.

The reducing powers of bacteria may also be shown by their ability
to decolorize litmus, methylene-blue, and some other anilin dyes,
which on abstraction of oxygen form colorless leukobases.

Enzyme Action. — The action of the enzymes produced by bacteria
may be demonstrated by bringing the bacteria, or their isolated fer¬
ments, into contact with the proper substances and observing both the
physical and chemical changes produced. In obtaining enzymes free
from living bacteria, it is convenient to kill the cultures by the addition
either of toluol or of chloroform. Both of these substances will
destroy the bacteria without injuring the enzymes. Enzymes may also
be obtained separate from the bodies of the bacteria by filtration.

Proteolytic Enzymes. — The most common evidences of proteolytic
enzyme action observed in bacteriology are the liquefaction of gelatin,
fibrin or coagulated blood-serum, and the peptonization of milk. This
may be observed both by allowing the proper bacteria to grow upon
these media, or by mixing sterilized cultures with small quantities of
these substances.1 The products of such a reaction may be separated
from the bacteria by filtration and then tested for pepton by the biuret
reaction.

Proteolytic 2 enzymes may also be determined by growing the bac¬
teria upon fluid media containing albumin solutions, blood serum, or
milk serum, then precipitating the proteids by the addition of ammonium
sulphate (about 30 grams to 20 c.c. of the culture fluid) and warming
between 50 to 60° C. for thirty minutes. The precipitate is then filtered
off, the filtrate made strongly alkaline with NaOH, and a few drops
of copper sulphate solution added. A violet color indicates the pres¬
ence of pepton — proving proteolysis of the original albumin.

■ ; <J ...

(Acetic acid of 1.04 prepared by diluting 400 c.c. of cone, of Sp. Gr. 1.75 with 700
c.c. of water.)

II. A-Naphthylamin. — Dissolve 0.1 g. in 20 c.c. of water, boil, filter (if necessary),
and to clear filtrate add 180 c.c. of acetic acid, Sp. Gr. 1.04.

The solutions are kept separate and mixed in equal parts just before use.

In carrying out the test, put 2 c.c. of each reagent in a test tube and add substance
to be tested. (In ordinary water analysis use 100 c.c.) Cover tube with watch
glass and set in warm water for 20 minutes. Observe presence or absence of pink
color promptly. Always run a blank on the distilled water used for rinsing to avoid
errors due to nitrites in the water, or in the air of the laboratory.

1 Bitter, Archiv f . Hyg., v. 1886.

2 Hankin and Wesbrook, Ann. Past., vi., 1892.

DETERMINING BIOLOGICAL ACTIVITIES OE BACTERIA 109

Diastatic Enzymes. — The presence of diastatic ferments may be
determined by mixing broth cultures of the bacteria with thin starch
paste. It is necessary that both the cultures and the starch paste be
absolutely free from sugar. After remaining in the incubator for five or
six hours, the fluid is filtered and the filtrate tested by methods used for
determining the presence of sugars.

Inverting Ferments. — Inverting ferments are determined by a pro¬
cedure similar to the above in principle. Dilute solutions of cane sugar
are mixed with old cultures or
culture filtrates of the respective
bacteria and the mixture allowed
to stand. It is then filtered,
and the filtrate tested for glucose,
preferably by Fehling’s solution.

ANIMAL EXPERIMENTATION

In the study of pathogenic
microorganisms, animal experi¬
mentation is essential in many
instances. The virulence of anv
given organism for a definite ani¬
mal species and the nature of the
lesions produced are character¬
istics often of great value in
differentiation. Isolation, more¬
over, of many bacteria is greatly
facilitated by the inoculation of
susceptible animals and recovery
of the pathogenic organism from

the heart’s blood or from the lesions produced in various organs. That
investigations into the phenomena of immunity would be absolutely
impossible without the use of animal inoculation is, of course, self-
evident, for by this method only can the action of bacteria in relation to
living tissues, cells, and body-fluids be observed .

The animals most commonly employed for such observations are
guinea-pigs, white mice, white rats, and rabbits. The method of
inoculation may be either subcutaneous, intrapleural, intraperi-
toneal, intravenous, or subdural, etc. It must be borne in mind
always that the mode of inoculation may influence the course of an

Fig. 48. — Types of Gelatin Liquefac¬
tion by Bacteria.

170

BIOLOGY AND TECHNIQUE

infection no less than does the virulence of the microorganism or the
size of the dose.

Inoculations are made with some form of hypodermic needle fitted to

Fig. 49. — Intraperitoneal Inoculation of Rabbit.

Fig. 50. — Intravenous Inoculation of Rabbit.

a syringe. The most convenient syringes are the all-glass Luer or the
Debove syringes, which, however, are expensive. Any form of steriliz-
able syringe may be used. In making inoculations the hair of the

DETERMINING BIOLOGICAL ACTIVITIES OF BACTERIA 171

animal should be clipped and the skin disinfected with carbolic acid
or alcohol.

Subcutaneous inoculations are most conveniently made in the abdom •

Fig. 52. — Guinea-pig Cage.

inal wall, where the skin is thin. After clipping and sterilizing, the
skin is raised between the fingers of the left hand and the needle plunged

172

BIOLOGY AND TECHNIQUE

in obliquely so as to avoid penetrating the abdominal wall and entering
the peritoneum.

In making intraperitoneal inoculations, great care must be exercised
not to puncture the gut. This can be avoided by passing the needle
first through the skin in an oblique direction, then turning it into a posi¬
tion more vertical to the abdomen and perforating the muscles and perito¬
neum by a very short and carefully executed stab.

Intravenous inocidations in rabbits are made into the veins running
along the outer margins of the ears. The hair over the ear is clipped and
the animal held for a short time head downward so that the vessels
of the head may fill with blood. An assistant holds the animal firmly in

Fig. 53. — Rabbit Cage.

a horizontal position, the operator grasps the tip of the ears with the
left hand, and carefully passes his needle into the vein in the direction
as nearly as possible parallel to its course. (See Fig. 50.)

Mice are usually inoculated under the skin near the base of the tail.
They may be placed in a jar over which a cover of stiff wire-gauze is
held. They are then grasped by the tail, by which they are drawn up
between the side of the jar and the edge of the wire cover, so that the
lower end of the back shall be easily accessible. The skin is then wiped
with a piece of cotton dipped in carbolic solution and the needle is in¬
serted. Great care must be exercised to avoid passing the needle too
close to the vertebral column. Mice are extremely delicate, and any
injury to the spine usually causes immediate death.

DETERMINING BIOLOGICAL ACTIVITIES OF BACTERIA 173

With proper care mice or rats may be easily injected intravenously
if a sufficiently fine needle is used. There are four superficially placed
veins running along the tail, which stand out prominently when rubbed
with cotton moistened with xylol. Into these the injections are made.

When inoculating rats or guinea-pigs with bacillus pestis the Kolle
vaccination method is used. The skin is merely shaved and a loopful
of the culture vigorously rubbed into the shaven area.

The various forms of animal holders which have been devised are
rarely necessary in bacteriological work unless working unassisted, im¬
mobilization of the animals being easily accomplished by the hands of a
skilled assistant.

Autopsies upon infected animals must be carefully made. The ani¬
mals are tied, back down, upon pans fitted in the corners with clamps for
the strings. They are then moistened either with hot water or with a
weak solution of carbolic acid, so that contamination by hair may be
avoided. A median cut is made, the skin is carefully dissected back,
and the body cavities are opened with sterile instruments. Cultures
may then be taken from exudates, blood, or organs under precautions
similar to those recommended below for similar procedures at autopsy
upon man.

Inoculated animals should be, if possible, kept separate from healthy
animals. Rabbits and guinea-pigs are best kept in galvanized iron-wire
cages, which are fitted with floor-pans that can be taken out and cleaned
and sterilized. Mice may be kept in battery jars fitted with perforated
metal covers. The mice should be supplied with large pieces of cotton
upon batting since they are delicately susceptible to cold.

CHAPTER X

THE BACTERIOLOGICAL EXAMINATION OF MATERIAL FROM

PATIENTS

In making bacteriological examinations of material taken from
living patients, or at autopsy, the validity of result is as fully dependent
upon the technique by which the material is collected, as upon proper
manipulation in the later stages of examination.

Material taken at autopsy should be, if possible, directly transferred
from the cadaver to the proper culture media. If cultures are to be taken
from the liver, spleen, or other organs, the surface of the organ should
first be seared with a hot scalpel and an incision made through the cap¬
sule of the organ in the seared area, with the same instrument. The
platinum needle can then be plunged through this incision and material
for cultivation be taken with little chance of surface contamination.
When blood is to be transferred from the heart, the heart muscle may be
incised with a hot knife, or else the needle of a hypodermic syringe may
be plunged through the previously seared heart muscle and the blood
aspirated. The same end can be accomplished by means of a pointed,
freshly prepared Pasteur pipette. In taking specimens of blood at au¬
topsy it is safer to take them from the arm or leg, by allowing the blood
to flow into a broad, deep cut made through the sterilized skin, than from
the heart, since it has been found that post-mortem contamination of
the heart's blood takes place rapidly, probably through the large veins
from the lungs. Exudates from the pleural cavities, the pericardium,
or the peritoneum may be taken with a sterilized syringe or pipette.

Materials collected at the bedside or in the operating-room should
be transferred directly to the proper media or else into sterile test tubes
and so sent to the laborator}^. When the material is scanty, it may be
collected upon a sterile cotton swab, which should be immediately re¬
placed in the sterilized containing tube and sent to the laboratory.

Syringes, when used for the collection of exudates or blood, should
be of some variety which is easily sterilizable by dry heat, or boiling.
Most convenient of the forms in common use are the all-glass “ Luer ”
syringe, or the cheaper “Sub-Q” model. Instruments which can be
sterilized only by chemical disinfectants should not be used. When

174

EXAMINATION OF MATERIAL FROM PATIENTS

175

fluids are collected for bacteriological examination, such as spinal fluid,
paracentesis fluid, or pleural exudate, it is convenient to have them taken
directly into sterilized centrifuge tubes, since it is often necessary to
concentrate cellular elements by centrifugalization. By immediate col¬
lection in these tubes, the danger of contamination is avoided.

Examination of Exudates. — Pus. — Pus should first be examined
morphologically by some simple stain, such as gentian-violet, and by
the Gram stain. It is convenient, also, to stain a specimen by Jenner’s
stain, in order to show clearly the relation of bacteria to the cells.
Such morphological examination not only furnishes a guide to future
manipulation, but supplies a control for the results obtained by cultural
methods. Specimens of the pus are then transferred to the proper
media, and pour-plates made or streaks made upon the surface of
previously prepared agar or serum-agar plates.

A guide to the choice of media is often found in the result of the
morphological examination. In most cases, it is well also to make
anaerobic cultures by some simpler method. (See page 148 et seq.)

The colonies which develop upon the plates should be studied under the
microscope, and specimens from the colonies transferred to cover-glasses
and slides for morphological examination and to the various media for
further growth and identification. Animal inoculation and agglutination
tests must often also be resorted to. A knowledge of the source of the
material may furnish considerable aid in making a bacteriological diag¬
nosis, though great caution in depending upon such aid is recommended.

In the examination of peritoneal, pericardial, or pleural exudates it is
often advantageous to use the sediment obtained by centrifugalization.
A differential count of the cells present may be of aid in confirming the
bacteriological findings. Morphological examination and cultural exam¬
ination are made as in the case of pus. Specimens should also in these
cases be stained for tubercle bacilli. Whenever morphological exami¬
nations of such fluids are negative, no bacteria being found, and especially
when among the cellular elements the lymphocytes preponderate, the
search for tubercle bacilli should be continued by means of animal inocu¬
lation. Guinea-pigs should be inoculated intraperitoneally from speci¬
mens of the fluid. The animals will usually die within six to eight weeks,
but can be killed and examined at the end of about six weeks if they
remain alive. The chances for a positive result are considerably
increased if the fluid is set away in the ice-chest until a clot has formed
and the animals are inoculated with the material from the broken-up
clot

176

BIOLOGY AND TECHNIQUE

The routine examination of spinal fluid is best made upon the sedi¬
ment of centrifugalized specimens. The microorganisms with which we
deal most frequently in this fluid are the meningococcus, the pneumococ¬
cus, the streptococcus, and the tubercle bacillus. If morphological ex¬
amination reveals bacteria resembling the first three of these in appear¬
ance and staining-reaction, surface smears should preferably be made
upon plates of serum agar, blood agar, or upon tubes of Loeffler’s co¬
agulated blood-serum. Failure to find organisms morphologically does
not exclude their presence and careful cultivation should be done in all
cases. When organisms are not found by simple morphological examina¬
tion and the fluid and sediment are scanty, specimens should be stained
by the Ziehl-Neelson method for tubercle bacilli. In such cases it is
often of advantage to set away the specimen until a thin thread-like
clot of fibrin has formed in the bottom of the tube. In smears of such
a clot, tubercle bacilli are found with far greater ease than they are found
in centrifugalized specimens. If these examinations are without result,
inoculation of guinea-pigs should be resorted to.

Examination of Urine. — Bacteriological examination of the urine is
of value only when specimens have been taken with sterile catheters,
and care has been exercised in the disinfection of the external genitals.
Many of the numerous finds of bacillus coli in urine are unquestionably
due to defective methods of collecting material. Urine should be cen¬
trifugalized and the sediment examined morphologically and pour-
plates made and surface smears made upon the proper media. If
necessary, animal inoculation may be done. In examining urine for
tubercle bacilli, special care should be taken in staining methods so
as to differentiate from Bacillus smegmatis.

Examination of Feces. — Human feces contain an enormous num¬
ber of bacteria of many varieties. Klein,1 by special methods, es¬
timated that there were about 75,000,000 bacteria in one milligram
of feces. It has been a noticeable result of all the investigations upon
the feces, that although enormous numbers can be counted in morpho¬
logical specimens, only a disproportionately smaller number can be
cultivated from the same specimen. This is explicable upon the ground
that special culture media are necessary for many of the species found
in intestinal contents and upon the consideration that many of the
bacteria which are present in the morphological specimen are dead, show¬
ing that there are bactericidal processes going on in some parts of the

1 Klein, Ref. Cent. f. Bakt., I, xxx, 1901.

EXAMINATION OF MATERIAL FROM PATIENTS 177

intestinal tract, possibly through the agency of intestinal secretions,
bile, and the action of the products of metabolism of the hardier species
present. By far the greater part of the intestinal flora consists of mem¬
bers of the colon group, bacilli of the lactis aerogenes group, Bacillus
fsecalis alkaligenes, Bacillus mesentericus, and relatively smaller num¬
bers of streptococci, staphylococci, and Gram -positive anaerobes. Many
other species, however, may be present without being necessarily con¬
sidered of pathological significance. Certain writers have recently laid
much stress upon a preponderance of Gram-positive bacteria in speci¬
mens of feces, claiming that such preponderance signifies some form
of intestinal disturbance. Herter 1 has recently advanced the opinion
that the presence of Bacillus aerogenes capsulatus in the intestinal canal
is definitely associated with pernicious anemia. The determination of
these bacilli in the stools is made both by morphological examination
by means of Gram stain and by isolation of the bacteria. Such isola¬
tion is easily done by the method of Welch and Nuttal.2 A suspension
of small quantities of the feces in salt solution is made and 1 c.c. of the
filtered suspension is injected into the ear vein of a rabbit. After a few
minutes the rabbit is killed and placed in the incubator. After five hours
of incubation, the rabbit is dissected, and if the Welch bacillus has
been present in the feces, small bubbles of gas will have appeared in
the liver from which the bacilli may be cultivated in anaerobic stab-
cultures.

Bacteriological examination of feces is most often undertaken for
the isolation of Bacillus typhosus. This is accomplished with a great
deal of difficulty because of the overwhelming numbers of colon bacilli
which easily outgrow the typhoid germs, and because of the similarity
of their colonies in most media. Many methods have been devised for
this purpose, all of which depend upon the use of special media aimed
at the inhibition of colon and other bacilli and the production of recog¬
nizable differences in the colonies of typhoid and colon bacilli. Such
media are those of Eisner, Hiss, Conradi-Drigalski, Loeffler, Hesse, and
others, which are described in the section upon special media. (See page
133.) The methods of using these media will be found described in the
chapter on Bacillus typhosus (p. 399.)

Cholera spirilla may be recognized in and isolated from the stools of
patients by morphological examination, and by cultivation. (See
section on Sp. cholerse.)

1 Herter, “ Common Bacterial Infections of the Digestive Tract, ” N. Y., 1907.

2 Welch and Nuttal. See ref. p. 469.

178

BIOLOGY AND TECHNIQUE

For the isolation of dysentery bacilli from feces, no satisfactory
special methods have as yet been devised, Here we can depend only up¬
on careful plating upon agar and gelatin and extended colony “fishing/’
and the study of pure cultures. The complete absence of motility of
these bacteria is of much aid in such identification.

The determination of tubercle bacilli in stools is difficult and of
questionable significance, in that they may be present in people suffer¬
ing from pulmonary tuberculosis as a consequence of swallowing sputum
or infected food, and in that there may be other acid-fast bacilli, such
as the timothy bacillus, present.

Blood Cultures. — The diagnosis of septicemia can be positively made
during life only by the isolation of bacteria from the blood. Such exam¬
inations are of much value and are usually successful if the technique
is properly carried out. A large number of methods are recommended,
the writers giving, however, only the one which they have found
successful and simple for general use.

The blood is taken by preference from the median basilic vein of the
arm. If, for some reason (both forearms having been used for saline
infusion), these veins are unavailable, blood may be taken from the
internal saphenous vein as it turns over the internal malleolus of the
ankle joint. The skin over the vein should be prepared, preferably an
hour before the specimen is taken, with green soap, alcohol, and bichlo-
rid of mercury, as for a surgical operation. The syringe which is used
should be of some sterilizable variety (the most convenient the Luer
model), which is easily manipulated and does not draw with a jerky,
irregular motion. Its capacity should be at least 10 c.c. It may be
sterilized by boiling for half an hour, or preferably, when all-glass syringes
are used, they may be inserted into potato-tubes and sterilized at high
temperature in the hot-air chamber. Before drawing the blood, a linen
bandage is wound tightly about the upper arm of the patient in order to
cause the veins to stand out prominently. When the veins are plainly
in view, the needle may be plunged through the skin into the vein in a
direction parallel to the vessel and in the direction of the blood-stream.
After perforation of the skin, while the needle is groping for the vein,
gentle suction may be exerted with the piston. Great care should be ex¬
ercised, however, that the piston is not allowed to slip back, and air be,
by accident, forced into the vessel. In most cases no suction is necessary,
the pressure of the blood being sufficient to push up the piston. After the
blood has been drawn, it should be immediately transferred to the proper
media. Epstein has recently recommended the mixture of the blood

EXAMINATION OF MATERIAL FROM PATIENTS

179

with sterile two per cent ammonium oxalate solution in test tubes, by
which means the clotting is prevented, and transfers can be made more
leisurely to culture media. While this method is convenient in cases
where blood must be taken at some distance from the laboratory, it is

Fig. 54.— Blood-Culture Plate Showing Streptococcus Colonies. Note

halo of hemolysis about each colony.

preferable, whenever possible, to make cultures from the blood im¬
mediately at the bedside.

The choice of culture media for blood cultures should, to a certain
extent, be adapted to each individual case. For routine work, it is best
to employ glucose-meat-infusion agar and glucose-meat-infusion broth.
At least six glucose-agar tubes should be melted and immersed in water
at 45° C. Before the blood is mixed with the medium, the agar should be

180

BIOLOGY AND TECHNIQUE

cooled to 41° in order that bacteria, if present, may not be injured by
the heat. The blood is added to the tubes in varying quantities, ranging
from 0.25 to 1 c.c. each, in order that different degrees of concentration
may be obtained. Mixing is accomplished by the usual dipping and
rotary motion, the formation of air-bubbles being thus avoided. The
mouth of each test tube should be passed through the flame before pour¬
ing the contents into the plates. Three flasks of glucose broth, contain¬
ing 100 to 150 c.c. of fluid each, should be inoculated with varying
quantities of blood — at least one of the flasks containing the blood in
high dilution. The most stringent care in the withdrawal and replace¬
ment of the cotton stoppers should be exercised.1 The writers have
found it convenient to use, in place of one of these flasks, one containing,
in addition to the glucose, 1 gm. of powdered calcium carbonate.
This insures neutrality, permitting pneumococci or streptococci, which
are sensitive to acid, to develop and retain their vitality.

In making blood cultures from typhoid patients, various methods
have been recommended. Buxton and Coleman 2 have obtained excel¬
lent results by the use of pure ox-bile containing ten per cent of glycerin
and two per cent of peptone in flasks. The writers have had no difficulty
in obtaining typhoid cultures by the use of slightly acid meat-extract
broth in flasks containing 200 or more c.c. to which comparatively
little blood has been transferred in order to insure high dilution.

In estimating the results of a blood culture, the exclusion of contami¬
nation usually offers little difficulty. If the same microorganism ap¬
pears in several of the plates and flasks, if colonies upon the plates are
well distributed within the center and under the surface of the medium,
and if the microorganisms themselves belong to species which commonly
cause septicemia, such as streptococcus and pneumococcus, it is usually
safe to assume that they have emanated from the patient’s circulation.
When colonies are present in one plate or in one flask only, when they
are situated only near the edges of a plate or upon the surface of the
medium, and when they belong to varieties which are often found sapro¬
phytic upon the skin or in the air, they must be looked upon with ex¬
treme suspicion. It is a good rule to look upon all staphylococcus blood
cultures skeptically, discarding Staphylococcus albus as a contamina¬
tion, and taking, if possible, another corroborative culture when the
organism is Staphylococcus pyogenes aureus.

1 Small Florence flasks are preferable to the Erlenmeyer flasks usually employed.

2 Buxton and Coleman, Am. Jour, of Med. Sci., 1907.

SECTION II

INFECTION AND IMMUNITY

CHAPTER XI

FUNDAMENTAL FACTORS OF PATHOGENICITY AND INFECTION

When microorganisms gain entrance to the animal or human body,
and give rise to disease, the process is spoken of as infection.

Bacteria are ever present in the environment of animals and human
beings and some find constant lodgment on various parts of the body.
The mouth, the nasal passages, the skin, the upper respiratory tract, the
conjunctive, the ducts of the genital system, and the intestines are
invariably inhabited by numerous species of bacteria, which, while sub¬
ject to no absolute constancy, conform to more or less definite charac¬
teristics of species distribution for each locality. Thus the colon organ¬
isms are invariably present in the normal bowel, Doderlein’s bacillus
in the vagina, Bacillus xerosis in many normal conjunctive, and staphy¬
lococcus, streptococcus, various spirilla, and pneumococcus in the mouth.
In contact, therefore, with the bodies of animals and man, there is a large
flora of microorganisms, some as constant parasites, others as transient
invaders; some harmless saprophytes and others capable of becoming
pathogenic. It is evident, therefore, that the production of an infection
must depend upon other influences than the mere presence of the micro¬
organisms and their contact with the body, and that the occurrence of
the reaction — for the phenomena of infection are in truth reactions be¬
tween the germ and the body defenses — is governed by a number of
important secondary factors.

In order to cause infection, it is necessary that the bacteria shall gain
entrance to the body by a path adapted to their own respective cultural
requirements, and shall be permitted to proliferate after gaining a foot¬
hold. Some of the bacteria then cause disease by rapid multiplication,
progressively invading more and more extensive areas of the animal
tissues, wThile others may remain localized at the point of invasion and

181

182

INFECTION AND IMMUNITY

exert their harmful action chiefly by local growth and the elaboration of
specific poisons.

The inciting or inhibiting factors which permit or prohibit an in¬
fection are dependent in part upon the nature of the invading germ and
in part upon the conditions of the defensive mechanism of the subject
attacked.

Bacteria are roughly divided into two classes, saprophytes and
parasites. The saprophytes are those bacteria which thrive best on
dead organic matter and fulfill the enormously important function in
nature of reducing by their physiological activities the excreta and
dead bodies of more highly organized forms into those simple chemical
substances which may again be utilized by the plants in their con¬
structive processes. The saprophytes, thus, are of extreme importance
in maintaining the chemical balance between the animal and plant
kingdoms. Parasites, on the other hand, find the most favorable
conditions for their development upon the living bodies of higher forms.

While a strict separation of the two divisions can not be made, nu¬
merous species forming transitions between the two, it may be said
that the latter class comprises most of the so-called pathogenic or
disease-producing bacteria. Strict saprophytes may cause disease,
but only in cases where other factors have brought about the death
of some part of the tissues, and the bacteria invade the necrotic
areas and break down the proteids into poisonous chemical sub¬
stances such as ptomains, or through their own destruction give
rise to the liberation of toxic constituents of their bodies. It is
necessary, therefore, that bacteria, in order to incite disease,
should belong strictly or facultatively to the class known as para¬
sitic. It must not be forgotten, however, that the terms are relative,
and that bacteria ordinarily saprophytic may develop parasitic
and pathogenic powers when the resisting forces of the invaded
subject are reduced to a minimum by chronic constitutional disease
or other causes.

Organisms that are parasitic, however, are not necessarily pathogenic,
and there are certain more or less fundamental requirements which
experience has taught us must be met by an organism in order that it
may be infectious (or pathogenic) for any given animal; and by infec¬
tiousness is meant the ability of an organism to live and multiply in the
animal fluids and tissues. For instance, an organism which is shown
not to grow at the body temperature of warm-blooded animals may
safely be assumed not to be infectious for such animals; and experience is

FACTORS OF PATHOGENICITY AND INFECTION

183

gradually teaching us that strictly aerobic organisms, those thriving
only in the presence of free oxygen and not able to obtain this gas in
available combination from carbohydrates, can also be safely excluded
from the infectious class. We have also learned that anaerobic organ¬
isms, although infectious when gaining entrance to tissues not abun¬
dantly supplied with blood, are practically unable to multiply in the
blood stream and give rise to generalized infection.

The pathogenic microorganisms differ very much among themselves
in the degree of their disease-inciting power. Such power is known as
virulence. Variations in virulence occur, not only among different
species of pathogenic bacteria, but may occur within the same species.
Pneumococci, for instance, which have been kept upon artificial media
or in other unfavorable environment for some time, exhibit less viru¬
lence than when freshly isolated from the bodies of man or ani¬
mals. It is necessary, therefore, in order to produce infection, that
the particular bacterium involved shall possess sufficient virulence.

Whether or not infection occurs depends also upon the number of
bacteria which gain entrance to the animal tissues. A small number of
bacteria, even though of proper species and of sufficient virulence, may
easily be overcome by the first onslaught of the defensive forces of the
body. Bacteria, therefore, must be in sufficient number to overcome local
defenses and to gain a definite foothold and carry on their life processes,
before they can give rise to an infection. The more virulent the germ,
other conditions being equal, the smaller the number necessary for the
production of disease. The introduction of a single individual of the
anthrax species, it is claimed, is often sufficient to cause fatal infection;
while forms less well adapted to the parasitic mode of life will gain a
foothold in the animal body only after the introduction of large numbers.

The Path of Infection. — The portal by which bacteria gain entrance
to the human body is of great importance in determining whether or not
disease shall occur. Typhoid bacilli rubbed into the abraded skin may
give rise to no reaction of importance, while the same microorganism,
if swallowed, may cause fatal infection. Conversely, virulent strepto¬
cocci, when swallowed, may cause no harmful effects, while the same
bacteria rubbed into the skin may give rise to a severe reaction.

Animals and man are protected against invasion by bacteria in
various ways. Externally the body is guarded by its coverings of skin
and mucous membranes. When these are healthy and undisturbed,
microorganisms are usually held at bay. While this is true in a gen¬
eral way bacteria may in occasional cases pass through uninjured

184

INFECTION AND IMMUNITY

skin and mucosa. Thus the Austrian Plague Commission found that
guinea-pigs could be infected when plague bacilli were rubbed into the
shaven skin, and there can hardly be much doubt of the fact that
tubercle bacilli may occasionally pass through the intestinal mucosa into
the lymphatics without causing local lesions.

Even after bacteria of a pathogenic species, in large numbers and of
adequate virulence, have passed through a locally undefended area in
the skin or mucosa of an animal or a human being by a path most favor¬
ably adapted to them, it is by no means certain that an infection will
take place. The bodies of animals and of man have, as we shall see, at
their disposal certain general, systemic weapons of defense, both in the
blood serum and the cellular elements of blood and tissues which, if
normally vigorous and active, will usually overcome a certain number
of the invading bacteria. If these defenses are abnormally depressed,
or the invading microorganisms are disproportionately virulent or plen¬
tiful, infection takes place.

Bacteria, after gaining an entrance to the body, may give rise merely
to local inflammation, necrosis, and abscess formation. They may, on
the other hand, from the local lesion, gain entrance into the lymphatics
and blood-vessels and be carried freely into the circulation, where, if they
survive, the resulting condition is known as bacteriemia or septicemia.
Carried by the blood to other parts of the body, they may, under favor¬
able circumstances, gain foothold in various organs and give rise to
secondary foci of inflammation, necrosis, and abscess formation. Such
a condition is known as pyemia. The disease processes arising as the
result of bacterial invasion may depend wholly or in part upon the
mechanical injury produced by the process of inflammation, the dis¬
turbance of function caused by the presence of the bacteria in the capil¬
laries and tissue spaces, and the absorption of the necrotic products
resulting from the reaction between the body cells and the micro¬
organisms. To a large extent, however, infectious diseases are char¬
acterized by the symptoms resulting from the absorption or diffusion
of the poisons produced by the bacteria themselves.

Bacterial Poisons. — It was plain, even to the earliest students of this
subject, that mere mechanical capillary obstruction or the absorption
of the products of a local inflammation were insufficient to explain the
profound systemic disturbances which accompany many bacterial in¬
fections. The very nature of bacterial disease, therefore, suggested the
presence of poisons.

It was in his investigations into the nature of these poisons that

FACTORS OF PATHOGENICITY AND INFECTION

185

Brieger 1 was led to the discovery of the ptomains. These bodies, first
isolated by him from decomposing beef, fish, and human cadavers, have
found more extended discussion in another section. Accurately classified,
they are not true bacterial poisons in the sense in which the term is
now employed. Although it is true that they are produced from pro-
teid material by bacterial action, they are cleavage products derived
from the culture medium upon the composition of which their nature
intimately depends. The bacterial poisons proper, on the other hand,
are specific products of the bacteria themselves, dependent upon the
nature of the medium only as it favors or retards the full development
of the physiological functions of the microorganisms. The poisons, pro¬
duced to a greater or lesser extent by all pathogenic microorganisms,
may be of several kinds. The true toxins, in the specialized meaning
which the term has acquired, are soluble, truly secretory products of
the bacterial cells, passing from them into the culture medium during
their life. They may be obtained free from the bacteria by filtration and
in a purer state from the filtrates by chemical precipitation and a vari¬
ety of other methods. The most important examples of such poisons
are those elaborated by Bacillus diphtherise and Bacillus tetani. If
cultures of these bacteria or of others of this class are grown in fluid
media for several days and the medium is then filtered through porce¬
lain candles, the filtrate will be found toxic often to a high degree, while
the residue will be either inactive or comparatively weak. Moreover,
if the residue possesses any toxicity at all, the symptoms evidencing
this will be different from those produced by the filtrate.

There are other microorganisms, however, notably the cholera
spirillum and the typhoid bacillus, which act in an almost diametrically
opposed manner. If these bacteria are cultivated and separated from
the culture fluid by filtration in the method described above, the fluid
filtrate will be toxic to only a very slight degree, whereas the residue may
prove very poisonous. In these cases, we are dealing, evidently, with
poisons not secreted into the medium by the bacteria, but rather at¬
tached more or less firmly to the bacterial body. Such poisons, separable
from the bacteria only after death by some method of extraction, or by
autolysis, are termed endotoxins. The greater number of the patho¬
genic bacteria seem to act chiefly by means of poisons of this class.
The first to call attention to the existence of such intracellular poisons
was Buchner, who formulated his conclusions from the results of ex-

1 Brieger, “Die Ptomaine/’ Berlin, 1885 and 1886.

186

INFECTION AND IMMUNITY

periments made with a number of microorganisms, notably the Fried-
lander bacillus and Staphylococcus pyogenes aureus, with dead cultures
of which he induced the formation of sterile abcesses in animals and
symptoms of toxemia. The conception of “endotoxins,” subsequently,
however, received its clearest and most definite expression in the work
of Pfeiffer 1 on cholera poison.

Some clarity of conception, based on visual perception, may possibly
be gained by comparing some of the products of pathogenic bacteria
with bacterial pigments and with insoluble interstitial . or intercellular
substance, which may be seen accompanying bacteria in cover-glass
preparations. Soluble toxic secretions are to be compared to such pig¬
ments as the pyocyanin of Bacillus pyocyaneus, which is so readily
soluble in culture media; endotoxins proper, to pigments confined to
the bacterial cell, or at least, when secreted, being insoluble in culture
media, such for instance as the well-known red pigment of Bacillus pro-
digiosus, which may often be seen free among the bacteria in irregular
red granules like carmine powder. That bodies such as this latter might
be extruded from pathogenic bacteria and not be soluble in the usual cul¬
ture fluids, is not improbable, and the fact that more or less insoluble
interstitial substances are not infrequent among bacteria is well known.

In all bacterial bodies, after removal of toxins and endotoxins, a
certain proteid residue remains which, if injected into animals, may
give rise to localized lesions such as abscesses or merely slight temporary
inflammations. The nature of this residue has been carefully studied,
especially by Buchner, who has named it bacterial protein and he
believes the substance to be approximately the same in all bacteria,
without specific toxic action, but with a general ability to exert a positive
chemotactic effect on the white blood cells, thereby causing the forma¬
tion of pus. The nature of the bacterial proteins is by no means clear,
and it is still in doubt whether the separation of these substances from
the endotoxins can be upheld.

A number of bacteria may give rise to both varieties of poisons.
Thus, recently, Kraus has claimed the discovery of a soluble toxin for
the cholera spirillum and Doerr for the dysentery bacillus, both of which
microorganisms were regarded as being purely of the endotoxin-pro¬
ducing type.

It is plain, moreover, that occasionally it may be very difficult to
distinguish between a soluble toxin and an endotoxin. In the filtration

1 Pfeiffer, Zeit. f. Hyg., xl, 1892.

FACTORS OF PATHOGENICITY AND INFECTION

187

experiment recorded above, it might well be claimed that the toxicity
of the filtrate, when not very strong, may depend upon an extraction
of endotoxins from the bodies of the bacteria by the medium. The
final test, in such instances, lies in the power of true toxins to stimu¬
late in animals the production of antitoxins; for, as we shall see later,
the injection of true soluble toxins into animals gives rise to antitoxins,
whereas the formation of such neutralizing bodies in the serum or plasma
does not, it is claimed, follow the injection of endotoxins. This distinc¬
tion will become clearer as we proceed in the discussion of immunity. It
must not be forgotten, however, that our knowledge of bacterial poisons
is by no means complete, and that sharp distinctions as those given above
must be regarded to a certain extent as tentative.

In resistance to chemical action and heat, the various poisons show
widely divergent properties. As a general rule, most true soluble
toxins are delicately thermolabile, they are destroyed by moderate
heating, and deteriorate easily on standing. Their chemical nature is
by no means clear, but, on precipitation of toxic solutions with mag¬
nesium sulphate, these poisons come down together with the globulins.
The nature of the endotoxins is still less clearly understood. Most of
them, while less labile than the extracellular poisons, are, nevertheless,
destroyed by exposure to 70° C. On the other hand, certain specific and
powerful intracellular poisons, like those of the Gartner bacillus of meat
poisoning, may undergo exposure to even 100° C. and still retain their
toxic properties. The nature of each individual poison will be discussed
in connection with its microorganism.

The Mode of Action of Bacterial Poisons. — Close study of the toxic
products of various microorganisms has shown that many of the bac¬
terial poisons possess a more or less definite selective action upon special
tissues and organs. Thus, certain soluble toxins of the tetanus bacillus
and Bacillus botulinus attack specifically the nervous system. Again,
certain poisons elaborated by the staphylococci, the tetanus bacillus,
the streptococci, and other germs, the so-called “ hemolysins/ ’ attack
primarily the red blood corpuscles. Other poisons again act on the
white blood corpuscles; in short, the characteristic affinity of specific
bacterial poisons for certain organs is a widely recognized fact.

In explanation of this, behavior, much aid has been given by the
researches of Meyer,1 Overton,2 Ehrlich,3 and others upon the causes for

1 Meyer, Arch. f. exper. Pathol., 1899, 1901.

2 Overton, “Studien lib. d. Narkose,” Jena, 1901.

3 Ehrlich, “Sauerstoffs-Bediirfniss des Organiemus,” Berlin, 1885.

188

IMMUNITY AND INFECTION

the analogous selective behavior of various narcotics and alkaloids.
It seems probable, from the researches of these men, that the selective
action of poisons depends upon the ability, chemical or physical or both,
of the poisons to enter into combination with the specifically affected
cells. From the nature of the combinations formed, it seems not unlikely
that the physical factors, such as solubility in the cell plasma, may also
play an important part.

Observations of a more purely bacteriological nature have tended
to bear out these conclusions. Wassermann and Takaki,1 for instance,
have shown that tetanus toxin, which specifically attacks the nervous
system, may be removed from solution by the addition of brain sub¬
stance. Removal of the brain tissue by centrifugation leaves the solu¬
tion free from toxin. In the same way it has been shown that hemo¬
lytic poisons can be removed from solutions by contact with red
blood cells, but only when the red blood cells of susceptible species are
employed.

Similar observations have been made in the case of leukocidin, a
bacterial poison acting upon the white blood cells specifically.2

That bacterial poisons injected into susceptible animals rapidly
disappear from the circulation is a fact which bears out the view that
a combination between affected tissue and toxin must take place.
Donitz,3 for instance, has shown that within four to eight minutes after
the injections of certain toxins, considerable quantities will have dis¬
appeared from the circulation. Conversely, Metchnikoff 4 has ob¬
served that tetanus toxin injected into insusceptible animals (lizards)
may be detected in the blood stream for as long as two months after
administration.

1 Wassermann unci Takaki, Berl. klin. Woch., 1898.

2 Sachs, Hofmeister’s Beitrage, 11, 1902.

3 Donitz, Deut. med. Woch., 1897.

* Metchnikoff , “L’immunite dans les malad. infect.”

CHAPTER XII

DEFENSIVE FACTORS OF THE ANIMAL ORGANISM

GENERAL CONSIDERATIONS

We have seen that the mere entrance of a pathogenic microorganism
into the human or animal body through a breach in the continuity of
the mechanical defenses of skin or mucosa does not necessarily lead to
the development of an infection. The opportunities for such an invasion
are so numerous, and the contact of members of the animal kingdom with
the germs of disease is so constant, that if this were the case, sooner or
later all would succumb. It is plain, therefore, that the animal body
must possess mor subtle means of defense, by virtue of which pathogenic
germs are, even after their entrance into the tissues and fluids, dis¬
posed of, or at least prevented from proliferating and elaborating their
poisons. The power which enables the body to accomplish this is spoken
of as resistance. When this resistance, which in some degree is com¬
mon to all members of the animal kingdom, is especially marked, it is
spoken of as “immunity.”

From this it follows naturally that the terms resistance and immunity,
as well as their converse, susceptibility, are relative and not absolute
terms. Degrees of resistance exist, which are determined to a certain
extent by individual, racial, or species peculiarities; and persons or
animals are spoken of as immune when they are unaffected by an ex¬
posure or an inoculation to which the normal average individual of the
same species would ordinarily succumb. The word does not imply,
however, that these individuals could not be infected with unusually
virulent or large doses, or under particularly unfavorable circumstances.
Thus, birds, while immune against the ordinary dangers of tetanus bacilli,
may be killed by experimental inoculations with very large doses of
tetanus toxin.1 Similarly, Pasteur rendered naturally immune hens
susceptible to anthrax by cooling them to a subnormal temperature, and
Canalis and Morpurgo did the same with doves by subjecting them to
starvation.

1 Quoted from Abel, Kolle und Wassermann, “Handbuch,” etc.

189

190

INFECTION AND IMMUNITY

Absolute immunity is exceedingly rare. The entire insusceptibility
of cold-blooded animals (frogs and turtles) under normal conditions to
inoculation with even the largest doses of many of the bacteria patho¬
genic for warm-blooded animals, and the immunity of all the lower
animals against leprosy, are among the few instances of absolute immu¬
nity known.1 Apart from such exceptional cases, however, resistance,
immunity, and susceptibility must be regarded as purely relative terms.

The power of resisting any specific infection may be the natural
heritage of a race or species, and is then spoken of as natural immunity.
It may, on the other hand, be acquired either accidentally or artificially
by a member of an ordinarily susceptible species, and is then called
acquired immunity.

Natural Immunity. — Species Immunity. — It is well known that many
of the infectious diseases which commonly affect man, do not, so far
as we know, occur spontaneously in animals. Thus, infection with B.
typhosus, the vibrio of cholera, or the meningococcus occurs in ani¬
mals only after experimental inoculation. Gonorrheal and syphilitic
infection, furthermore, not only does not occur spontaneously, but is
produced experimentally in animals with the greatest difficulty — the
consequent diseases being incomparably milder than those occurring in
man. Other diseases, like leprosy, influenza, and the exanthemata,2
have never been successfully transmitted to animals.

Conversely, there are diseases among animals which do not spon¬
taneously attack man. Thus, human beings enjoy immunity against
Rinderpest, and, to a lesser degree, against chicken cholera.

Among animal species themselves great differences in susceptibility
and resistance toward the various infections exist. Often-quoted ex¬
amples of this are the remarkable resistance to anthrax of rats and dogs,
and the immunity of the common fowl against tetanus.

The factors which determine these differences of susceptibility and
resistance among the various species are not clearly understood. It
has been suggested that diet in some instances may influence these re¬
lations, inasmuch as carnivorous animals are often highly resistant to
glanders, anthrax, and even tuberculous infections, to which herbiv¬
orous animals are markedly susceptible.3 It is likely, too, that the great
differences between animals of various species in their metabolism,
temperature, etc., may call for special cultural adaptation on the part

Lubarsch, Zeit. f. klin. Mediz., xix.

With the possible exception of smallpox.

3 Hahn, in Kolle und Wassermann, vol. iv.

DEFENSIVE FACTORS OF THE ANIMAL ORGANISM

191

of the bacteria. The fact that the bacillus of avian tuberculosis —
whose natural host has a normal body temperature of 40° C. and above
— will grow on culture media at 40 to 50° C., wherOas B. tuberculosis
of man can not be cultivated at a temperature above 40° C., would
seem to lend some support to this view. The difference between warm-
and cold-blooded animals has already been noted. The necessity for
cultural adaptation, too, would seem to be borne out by the great
enhancement observed in the virulence of certain microorganisms for
a given species after repeated passage through individuals of this species.

Racial Immunity. — Just as differences in susceptibility and im¬
munity exist among the various animal species, so the separate races or
varieties within the same species may display differences in their reac¬
tions toward pathogenic germs. Algerian sheep, for instance, show
a much higher resistance to anthrax than do our own domestic sheep,
and the various races of mice differ in their susceptibility to anthrax
and to glanders.

Similar racial differences are common among human beings. As a
general rule, it may be said that a race among whom a certain disease
has been endemic for many ages is less susceptible to this disease than
are other races among whom it has been more recently introduced. The
appalling ravages of tuberculosis among negroes, American Indians, and
Esquimaux, bear striking witness to this fact. Conversely, the compar¬
ative immunity of the negro from yellow fever, a disease of the greatest
virulence for Caucasians, furnishes further evidence in favor of this
opinion. It must not be forgotten, however, in judging of these rela¬
tions, that the great differences in the customs of personal and social
hygiene existing among the various races may considerably affect the
transmission of disease and lead to false conclusions.

In so far as the statement made above is true, however, it seems to
indicate that the endemic diseases have carried in their train a certain
degree of inherited immunity.

In other cases * — as in the instance of the malaria-immunity of
negroes — the resistance seems to be acquired rather than inherited, for,
as Hirsch was first to note, death from this disease occurred frequently
among the children, while adult negroes w7ere rarely attacked.

Differences in Individual Resistance. — In bacteriological ex¬
perimentation with smaller test animals, a direct ratio may often exist
between body weight and dosage in determining the outcome of an

3 Hahn, in Kolle und Wassermann, loc. cit.

192

INFECTION AND IMMUNITY

infection, provided the mode of inoculation has been the same and the
virulence of the germ not excessive. It would seem, therefore, that
among these animals the difference in resistance in the face of an arti¬
ficial infection between individuals of the same race is very slight.

In higher animals, however, especially in the case of man, the ex¬
istence of such apparent individual differences is a well-established fact,
although in judging of them we must not forget that the conditions of
infection are not subject to the uniformity and control which animal
experimentation permits. Of a number of persons exposed to any
given infection there are always some who are entirely unaffected and
there are great variations in the severity of the disease in those who
are attacked. In the absence of positive evidence in support of the
direct inheritance of this individual immunity, the most reasonable
explanation for such differences in resistance seems to lie in attrib¬
uting them to individual variations in metabolism or body chem¬
istry. Depressions, for instance, in the acidity of the gastric secretion
would predispose to certain infections of gastro-intestinal origin. Ana¬
tomical differences, too, may possibly influence resistance. Thus,
Birch-Hirschfeld believed that certain anomalous arrangements of the
bronchial tubes predisposed to tuberculosis.

Instances of transient susceptibility induced by physical or mental
overwork, starvation, etc., should hardly be classified under this head¬
ing, since the conditions in such cases correspond simply to experi¬
mental depression of natural species or race resistance.

Acquired Immunity. — It is a matter of common experience that many
of the infectious diseases occur but once in the same individual. This
is notably the case with typhoid fever, yellow fever, and most of the
exanthemata, and is too general an observation to require extensive
illustration. A single attack of any of the diseases of this class alters in
some way the resistance of the individual so that further exposure to
the infective agent is usually without menace, either for a limited period
after the attack, or for life. Resistance acquired in this way is often
spoken of as acquired immunity.

The protection conferred by certain diseases against further attack
was recognized many centuries ago, and there are records which show
that attempts were made in ancient China and India to inoculate healthy
individuals with pus from small-pox pustules in the hope of producing
by this process a mild form of the disease and its consequent immunity.

Pasteur, before all others, thought philosophically about the phenom¬
ena of acquired immunity, and, with adequate knowledge, realized the

DEFENSIVE FACTORS OF THE ANIMAL ORGANISM

193

possibility of artifically bestowing immunity without inflicting the
dangers of the fully potent infective agent. The first observation which,
made by him purely accidentally, inspired the hope of the achievement
of such a result, occurred during his experiments with chicken cholera.
The failure of animals to die after inoculation with an old culture of the
bacilli of chicken cholera, fully potent but a few weeks previously,
pointed to the attenuation of these bacilli by their prolonged cultivation
without transplantation. With this observation as a point of departure
he carried out a series of investigations with the purpose of discovering
a method of so weakening or attenuating various incitants of disease
that they could be introduced into susceptible individuals without en¬
dangering life and yet without losing their property of conferring pro¬
tection. The brilliant results achieved by Jenner, many years before,
in protecting against smallpox by inoculating with the entirely innocu¬
ous products of the pustules of cowpox furnished an analogy which
gave much encouraging support to this prospect.

The experimental work which Pasteur carried out to solve this prob¬
lem not only reaped a rich harvest of facts, but gave to science the first
and brilliant examples of the application of exact laboratory methods to
problems of immunity.

ACTIVE IMMUNITY

Active Artificial Immunity. — The process of conferring protection
by treatment with either an attenuated form or a sublethal quantity
of the infectious agent of a disease, or its products, is spoken of as “ active
immunization.”

Whatever the method employed, the immunized individuals gain
their power of resistance by the unaided reactions of their own tissues.
They themselves take an active physiological part in the acquisition of
this new property of immunity. For this reason, Ehrlich has aptly
termed these processes “active immunization.”

There are various methods by which this can be accomplished, all
of which were, in actual application or in principle, discovered by
Pasteur and his associates, and can be best reviewed by a study of their
work.

Active Immunization with Attenuated Cultures. — In the course
of his experiments upon chicken cholera, as mentioned above, Pasteur 1

1 Pasteur, Compt. rend, de l’acad. des sci., 1880, t. xc.

194

INFECTION AND IMMUNITY

accidentally discovered that the virulence of the bacilli of this disease
was greatly reduced by prolonged cultivation upon artificial media.
This was especially noticeable in broth cultures which had been stored
for long periods without transplantation. By repeated injections of such
cultures into fowl, he succeeded in rendering the animals immune against
subsequent inoculations with lethal doses of fully virulent strains.

During the same year, 1880, in which Pasteur published his observa¬
tions on chicken cholera, Toussaint 1 succeeded in immunizing sheep
against anthrax by inoculating them with blood from infected animals,
defibrinated and heated to 55° C. for ten minutes. Toussaint wrongly
believed, however, that the blood which had been used in his immuniza¬
tions was free from living bacteria. In repeating this work Pasteur-
showed that the protection in ToussainUs cases was conferred by living
bacteria, the virulence of which had been reduced by their subjection to
heat.

In following out the suggestions offered by these experiments,
Pasteur2 3 discovered that he could reduce the virulence of anthrax
bacilli much more reliably than by Toussaint’s method, by cultivating
the organisms at increased temperatures (42° to 43° C.) . By this process
of attenuation he was able to produce “ vaccines ” of roughly measurable
strength, with which he succeeded in immunizing sheep and cattle.
A successful demonstration of his discovery was made by him at Pouilly-
le-Fort, soon after, upon a large number of animals and before a commis¬
sion of professional men.

It is a fact well known to bacteriologists that certain of the pathogenic
microorganisms, when passed through several individuals of the same
animal species, become gradually more virulent for this species. In his
studies on the bacillus of hog cholera, Pasteur observed that when this
microorganism was passed through the bodies of several rabbits it gained
in virulence for rabbits, but became less potent against hogs. He suc¬
ceeded, subsequently, in protecting hogs against fully virulent cultures
by treating them with strains which had been attenuated by their
passage through rabbits.

A further principle of attenuation for purposes of immunization was,
at about this time, contributed by Chamberland and Roux,4 who re-

1 Toussaint, Compt. rend, de Facad. des sci., 1880, t. xci.

2 Pasteur, Chamberland et Roux, Compt. rend, de Facad. des sci., 1881, t. xcii.

3 Pasteur, Compt. rend, de Facad. des sci., 1882, t. xcv.

4 Chamberland et Roux, Compt. rend, de Facad. des sci., 1882, t. xcvi.

DEFENSIVE FACTORS OF THE ANIMAL ORGANISM

195

duced the virulence of anthrax cultures by growing them in the presence
of weak antiseptics (carbolic acid 1 : 600, potassium bichromate 1 : 5,000,
or sulphuric acid 1 : 200) . Cultivated under such conditions the bacilli
lost their ability to form spores and became entirely avirulent for sheen
within ten days. A similar result was later obtained by Behring 1 when
attenuating B. diphtheria cultures by the addition of terchlorid of
iodin.

Active Immunization with Sublethal Doses of Fully Virulent
Bacteria. — The use of fully virulent microorganisms in minute
quantities for purposes of immunization was first suggested by Chau-
veau,2 and is naturally inapplicable to extremely virulent organisms
like B. anthracis. The principle, however, is perfectly valid, and has
been experimentally applied by many observers, notably by Ferran 3
in the case of cholera. A similar method proved of practical value in
the hands of Theobald Smith and Kilborne 4 in prophylaxis against the
protozoan disease, Texas fever.

Active Immunization with Dead Bacteria. — Suggested by Ohau-
veau, the method of active immunization with gradually increasing doses
of dead microorganisms has been successfully employed by various ob¬
servers, chief among whom are Pfeiffer, Brieger, Wright, and Wasser-
mann. The method is especially useful against that class of bacteria
in which the cell bodies (endotoxins) have been found to be incomparably
more poisonous than their extracellular products (toxins). From a
practical point of view, the method is of the greatest importance in
routine laboratory immunization against B. typhosus, Vibrio cholerse
asiaticae, B. pestis, and a number of other bacteria. In the therapy
of human disease, this method has recently come into great prominence,
chiefly through the work of Wright, whose investigations will be more
fully discussed in a subsequent section.

Active Immunization with Bacterial Products. — Many bacteria
when grown in fluid media produce extracellular, soluble poisons which
remain in the medium after the microorganisms have been removed by
filtration or centrifugalization. Since the diseases caused by such
microorganisms are, to a large extent, due to the soluble poisons excreted
by them, animals can be actively immunized against this class of bac-

1 Behring, Zeit. f. Hyg., xii, 1892.

2 Chauveau, Compt. rend, de l’acad. des sci., 1881, t. xeii.

3 Ferran, Compt. rend, de l’acad. des sci., 1895, t. ci.

4 Th. Smith and Kilborne, U, S, Dept, of Agri., Bureau of Ani. Indust., Wash.,
1893.

196

INFECTION AND IMMUNITY

teria by the inoculation of gradually increasing doses of the specific
poison or toxin. This method is naturally most successful against those
microorganisms which possess the power of toxin formation to a highly
developed degree. Most important among these are B. diphtherise
and B. tetani. The first successful application of this principle of active
immunization, however, was made by Salmon and Smith1 in the case of
hog cholera.

PASSIVE IMMUNITY

In Pasteur’s basic experiments, as in those of the other scientists who
followed in his footsteps, the methods of immunization were based upon
the development of a high resistance in the treated subject by virtue of
its own physiological activities. This process we have spoken of as
“ active immunization ” and it is self-evident that a method of this kind
can, in the treatment of disease, be employed prophylactically only
against possible infection, or in localized acute infections, or at the
beginning of a long period of incubation before actual symptoms have
appeared, as in rabies or in chronic conditions in which the infection is
not of a severe or acute nature.

A new and therapeutically more hopeful direction was given to the
study of immunity when, in 1890 and 1892, v. Behring and his collabora¬
tors discovered that the sera of animals immunized against the toxins
of tetanus 2 and of diphtheria 3 bacilli would protect normal animals
against the harmful action of these poisons. The animals thus pro¬
tected obviously had taken no active part in their own defense, but
were protected from the action of the poison by the substances trans¬
ferred to them in the sera of the actively immunized animals. Such
immunity or protection, therefore, is a purely passive phenomenon
so far as the treated animal is concerned, and the process is for this
reason spoken of as “passive immunization.”

Passive immunization of this description is practically applicable
chiefly against diseases caused by bacteria which produce powerful
toxins, and the sera of animals actively immunized against such toxins
are called antitoxic sera. In the treatment of the two diseases men¬
tioned above, diphtheria and tetanus, the respective antitoxic sera have

1 Salmon and Smith, Rep. of Com. of Agri., Wash., 1885 and 1886.

2 v. Behring and Kitasato, Deut. med. Woch., 49, 1890.

s v. Behring and Wernicke, Zeit, f. Hyg., 1892.

DEFENSIVE FACTORS OF THE ANIMAL ORGANISM

197

reached broad and beneficial therapeutic application, and innumerable
lives have been saved by their use.

Passive immunization against microorganisms not characterized by
marked toxin formation was attempted, even before Behring’s dis¬
covery, by Richet and Hericourt,1 experimenting with cocci, and by
Babes,2 in the case of rabies ; and the underlying thought had been the
basis of Toussaint’s work upon anthrax. Microorganisms, however,
which exert their harmful action rather by the contents of the bacterial
cells than by secreted, soluble toxins, do not, so far as is known, pro¬
duce antitoxins in the sera of immunized animals. The substances
which they call forth in the process are directed against the invading
organisms themselves in that they possess the power of destroying or
of causing dissolution of the specific germs used in their production.

Such antibacterial sera are extensively used in the laboratory in the
passive immunization of animals against a large number of germs, and
are fairly effectual when used before, at the same time with, or soon after,
infection. Their therapeutic use in human disease, however, has, up
to the present time, been disappointing and their prophylactic and cura¬
tive action has been almost invariably ineffectual or feeble at best, ex¬
cept when the antibacterial sera could be brought in direct contact
with the germs, in closed cavities or localized lesions. Thus, in epidemic
meningitis, such sera have proved extremely useful in the hands of
Flexner, when injected directly into the spinal canal.

ANTIBODIES AND THE SUBSTANCES GIVING RISE TO THEM

In the foregoing sections we have seen that the process of active
immunization so changes the animal body that it becomes highly
resistant against an infection to which it had formerly in many in¬
stances been delicately susceptible. In the absence of visible anatomical
or histological changes accompanying the acquisition of this new power,
investigators, in order to account for it, were led to examine the physio-
logical properties of the body cells and fluids of immunized subjects.
While it was reasonable to suppose that all the cells and tissues were
affected by, or might have taken part in, a physiological change so
profoundly influencing the individual, the blood, because of its unques¬
tionably close relation to inflammatory reactions, and because of the

1 Richet et Hericourt, Compt. rend, de Pacad. des sci., 1888.

2 Babes et Lepp, Ann. de Pinst. Pasteur, 1889.

198

INFECTION AND IMMUNITY

ease with which it could be obtained and studied, claimed the first
and closest attention. The bactericidal properties of normal blood
serum noted in 1886 by Nuttall,1 v. Fodor,2 and Flugge, moreover, aided
in pointing to this tissue as primarily the seat of the immunizing
agents. It is an interesting historical fact, that, long before this time,
the English physician Hunter had noted that blood did not decompose
so rapidly as other animal tissues.

The study of the blood serum of immunized animals as to simple
changes in chemical composition or physical properties has shed little
light upon the subject. Beljaeff 3 in a recent investigation found little
or no alteration from the normal in the blood sera of immunized animals
as to index of refraction, specific gravity, and alkalinity. Joachim 4 and
Moll agree in stating that immune blood serum is comparatively richer
in globulin than normal serum. Similar observations had been made
by Hiss and Atkinson 5 and others. Important and significant as these
purely chemical observations are, they have helped little in explaining
the nature of the processes going on in immune sera. The first actual
light was thrown upon the mysterious phenomena of immunity by
the investigations of Nuttall,6 v. Fodor, Buchner, and others, who not
only demonstrated the power of normal blood serum to destroy bacteria,
but also showed that this property of blood serum became diminished
with age and was destroyed completely by heating to 56° C. The
thermolabile substance of the blood serum possessing this power was
called by Buchner,7 alexin.

Soon after this work, Behring, in collaboration with Kitasato 8 and
Wernicke,9 in 1890 and 1892, made further important advances in the
elucidation of the immunizing processes by showing that the blood sera
of animals actively immunized against the toxins of diphtheria and tet¬
anus would protect normal animals against the poisons of these diseases.
He believed, at the time of discovery, that such sera contained substances
which had the power of destroying the specific toxins which had been

1 Nuttall, Zeit. f. Hyg., i, 1886.

2 v. Fodor, Deut. med. Woch., 1886.

3 Beljaeff, Cent. f. Bakt., xxxiii.

4 Joachim, Pfliigers Archiv, xciii.

6 Hiss and Atkinson, Jour. Exper. Med., v, 1900.

6 Nuttall, Zeit. f. Hyg., 1886.

7 Buchner, Cent. f. Bakt., i, 1889.

8 Behring und Kitasato, Deut. med. Woch., 1890, No. 49.

9 Behring und Wernicke, Zeit. f. Hyg., 1892.

DEFENSIVE FACTORS OF THE ANIMAL ORGANISM

199

used in the immunization. He called these bodies antitoxins. While
Behring’s first conception of actual toxin destruction soon proved to
be erroneous, his. discovery of the presence in immune sera of bodies
specifically antagonistic to toxins was soon confirmed and extended,
and stands to-day as an established fact.

Ehrlich,1 soon after Behring’s announcement, showed that specific
antitoxins could also be produced against the poisons of some of the
higher plants (antiricin, antikrotin, antirobin) , and Calmette 2 produced
similar' antitoxins against snake poison (antivenin) . Stimulated by these
researches, other observers have, since then, added extensively to the
list of poisons against which antitoxins can be produced. Kempner 3
has produced antitoxin against the poison of Bacillus botulinus, and
Wassermann,4 against that of Bacillus pyocyaneus. Antitoxin has been
produced by Calmette 5 against the poison of the scorpion, and by Sachs 6
against that of the spider. Thus a large number of poisons of animal,
plant, or bacterial origin have been found capable of causing the pro¬
duction of specific antibodies in the sera of animals into which they are
injected.

The formation of antitoxins directed against soluble poisons, how¬
ever, did not explain the immunity acquired by animals against bacteria
like Bacillus anthracis, the cholera vibrio, and others which, unlike diph¬
theria and tetanus, produced little or no soluble toxin. It was evident
that the antitoxic property of immune blood serum was by no means
the sole expression of its protective powers. Much light was shed upon
this phase of the subject by the discoveries of Pfeiffer in 1894, who
worked along the lines suggested by the investigations of Nuttall and
Buchner. Pfeiffer 7 showed that when cholera spirilla were injected into
the peritoneal cavity of cholera-immune guinea-pigs, the microorganisms
rapidly swelled up, became granular, and often underwent complete
solution. The same phenomenon could be observed when the bacteria
were injected into a normal animal together with a sufficient quantity
of cholera-immune 8 serum.

1 Ehrlich, Deut. med. Woch., 1891.

2 Calmette, Compt. rend, de la soc. de biol., 1894.

8 Kempner, Zeit. f. Hyg., 1897.

4 Wassermann, Zeit. f. Hyg., xxii.

5 Calmette, Ann. de l’inst. Pasteur, 1898.

6 Sachs, Hofm. Beit., 1902.

» Pfeiffer, Zeit. f. Hyg., xviii, 1894.

8 Pfeiffer und Isaeff, ibid.

200

INFECTION AND IMMUNITY

This process he observed microscopically by abstracting, from time
to time, a small quantity of the peritoneal exudate and studying it in
hanging-drop preparations. The reaction was specific in that the de¬
structive process took place to any marked extent only in the case of the
bacteria employed in the immunization.

Metchnikoff,1 Bordet, and others not only confirmed Pfeiffer's obser¬
vation, but were able to show that the lytic process would take place
in vitro , as well as in the animal body. The existence of a specific
destructive process in immune serum was thus established for the vibrio
of cholera and soon extended to other microorganisms. The constitu¬
ents of the blood serum which gave rise to this destructive phenomenon
were spoken of as bacteriolysins.

Following closely upon the heels of Pfeiffer's observation came the
discovery of another specific property of immune serum by Gruber and
Durham.2 These workers noticed that certain bacteria, when brought
into contact with the serum of an animal immunized against them,
were clumped together, deprived of motility, and firmly agglutinated.
They spoke of the phenomenon as agglutination and of the substances
in the serum giving rise to it as agglutinins.

The list of antibodies was again enlarged by Kraus,3 who in 1897
showed that precipitates were formed when filtrates of cultures of
cholera, typhoid, and plague bacilli were mixed with their specific
immune sera. He called the substances which bestowed this property
upon the sera precipitins.

The treatment of the animal body, therefore, with bacteria or their
products gives rise to a variety of reactions which result in the presence
of the “ antibodies " described above. Extensive investigation has shown,
however, that the power of stimulating antibody production is a phe¬
nomenon not limited to bacteria and their products alone. Antitoxins,
we have already seen, may be produced with a variety of poisons of
plant and animal origin. Lysins, agglutinins, and precipitins, likewise
may be produced by the use of a large number of different substances.
Chief among these, because of the great aid they have given to the theo¬
retical investigation of the phenomena of immunity, are the red blood
cells. Bordet 4 and, independently of him, Belfanti and Carbone 5 showed

1 Metchnikoff, Ann. de l’inst. Pasteur, 1895.

2 Gruber und Durham, Munch, med. Woch., 1896.

3 Kraus, U., Wien. klin. Woch., 32, 1897.

4 Bordet, Ann. de Finst. Pasteur, 1898.

5 Belfanti et Carbone, Giornale della R. Acad, di Torino, July, 1898.

DEFENSIVE FACTORS OF THE ANIMAL ORGANISM

201

in 1898 that the serum of animals repeatedly injected with the defibri-
nated blood of another species exhibited the specific power of dissolving
the . red blood corpuscles of this species. This was the first demonstration
of “ hemolysis” — a phenomenon which, because of the ease with which
it can be observed in vitro, has much facilitated investigation.

The knowledge that specific 11 cytotoxins ” or cell-destroying anti¬
bodies could be produced by injection of red blood cells naturally sug¬
gested the possibility of analogous reactions for other tissue cells. It
was not long, therefore, before Metchnikoff 1 and, independently of
him, Landsteiner 2 succeeded, by repeated injections of spermatozoa, in
producing a serum which would seriously injure these specialized cells.
Von Dungern 3 obtained similar results with the ciliated epithelium of
the trachea. Since then a host of cytotoxins have been produced with
the cells of various organs and tissues. Thus, Neisser and Wechsberg 4
produced leucotoxin (leucocytes) ; Delezenne,5 neurotoxin and hepa-
totoxin; Surmont, 6 pancreas cytotoxin; and Bogart and Bernard,7 su¬
prarenal cytotoxin.

One of the most interesting of the cytotoxins, moreover, is nephro-
toxin — produced by the treatment of animals with injections of emul¬
sions of kidney tissue.

In all cases it was supposed by those first working with these bodies,
that the injection of the sera of animals previously treated with any
particular tissue substance would produce specific injury upon the or¬
gans homologous to the ones used in immunization. It need hardly be
pointed out how very important such phenomena would be in throwing
light upon the degenerative pathological lesions occurring in disease.
As a matter of fact, however, sera so produced have been shown to be
specific for certain organs in a limited sense only. The question of
specific cytotoxins has been of especial importance in the case of
nephritis, where Ascoli and Figari 8 and others have suggested an
autonephrotoxin as the basis of the pathology of this disease. In
the hands of Pearce and others, however, the strict specificity of

1 Metchnikoff, Ann. de Tinst. Pasteur, 1898.

2 Landsteiner, Cent. f. Bakt., i, 25, 1899.

3 v. Dungern, Munch, med. Woch., 1899.

4 Neisser und Wechsberg, Zeit. f. Hyg., xxxvi, 1901.

5 Delezenne, Ann. de l’inst. Past. 1900; Compt. rend, de l’acad. des sci. 1900.

6 Surmont, Compt. rend, de la soc. de biol., 1901.

7 Bogart et Bernard, ibid., 1891.

8 Ascoli and Figari, Berl. klin. Woch., 1902.

202

INFECTION AND IMMUNITY

nephrotoxin could not be upheld and the subject is still in the ex¬
perimental stage.

Recent experiments by Pearce 1 suggest that at least a part of the
local injury to organs exerted by such u cytotoxic ” sera may not be
due to a specific action upon the organ cells so much as upon the
hemagglutinatmg action of the sera causing embolism and necrosis.

It is a fact also that most cytotoxic sera are usually hemolytic as
well. It is not easy to decide, therefore, how much of the action upon
the organs is due to their true cytotoxic properties and how much is
attributable to the concomitant action upon blood cells. The extrav¬
agant hopes at first based upon cytotoxin investigation, especially in
regard to the problem of malignant tumors, have been disappointed,
and much is still obscure in regard to the cytotoxins which calls for
further research.

The many points of similarity existing between bacterial toxins and
digestive ferments, by animal inoculation, suggested to several observ¬
ers the possibility of producing antibodies against the latter. As a
result, a number of antiferments have been obtained, chief among which
are antilab (Morgenroth 2) , antipepsin (Sachs 3) , antisteapsin (Schutze 4) ,
and antilactase (Schutze).

The stimulation of antibody formation in the sera of animals is a
consequence, therefore, of the injection of a large variety of substances —
some of them poisonous, some of them entirely innocuous. The sub¬
stances possessing this power have been conveniently named antigens or
antibody-producers by German writers. The term antigen — though ety¬
mologically wrong, nevertheless is convenient and has crept into general
usage. It signifies simply a substance which can stimulate the pro¬
duction or formation of an antibody. Such substances, so far as is
known, belong to the group of proteids and are derivatives of animal or
plant tissues. Being proteids, all antigens are colloids. Recently, how¬
ever, some crystalloidal substances have been described as possessing
antigenic properties.

1 Pearce, Jour. Exper. Med., viii, 1906.

2 Morgenroth, Cent. f. Bakt., 1899.

3 Sachs, Fort. d. Med., 1902.

4 Schutze, Deut. med. Woch., 1904; Zeit. f. Hyg., 1905.

CHAPTER XIII

TOXINS AND ANTITOXINS

The Toxin-Antitoxin Reaction. — Apart from the therapeutic possi¬
bilities disclosed by the discovery of antitoxins, new light of inestimable
value was thrown by these observations upon the biological processes
involved in immunization. The most vital problem, of course, which
immediately thrust itself upon all workers in this field was the question
as to the nature of the reaction in which toxin was rendered innocuous
by antitoxin.

The simplest conception of this process would be an actual destruction
of the toxin by its specific antitoxin, and it is not unnatural, therefore,
that this was the view which, for a short time, found favor with some
observers. Roux, and more particularly Buchner,1 however, under the
sway of cellular pathology, advanced the opinion that the antitoxins
in some way influenced the tissue cells, rendering them more resistant
against the toxins. Antitoxin, according to this theory, did not act
directly upon toxin, but affected it indirectly through the mediation
of tissue cells. Ehrlich,2 on the other hand, conceived that the reac¬
tion of toxin and antitoxin was a direct union, analogous to the chem¬
ical neutralization of an acid by a base — an opinion in which Behring
soon joined him.

The conception of toxin destruction received unanswerable refuta¬
tion by the experiments of Calmette.3 This observer, working with snake
poison, found that the poison itself (unlike most other toxins) possessed
the property of resisting heat even to 100° C., while its specific anti¬
toxin, like other antitoxins, was delicately thermolabile. He noted,
furthermore, that non-toxic mixtures of the two substances, when sub¬
jected to heat, regained their toxic properties. The natural inference
from these observations could only be that the toxin in the original mix¬
ture had not been destroyed, but had been merely inactivated by the

1 Buchner, “ Schutzimpfung,” etc., in Penzoldt u. Stinzing, “Handbuch d. spez.
Therap. d. Infektkrank.,” 1894.

2 Ehrlich, Deut. med. Woch., 1891. 3 Calmette, Ann. de Tinst. Past., 1895.

203

204

INFECTION AND IMMUNITY

presence of the antitoxin, and again set free after destruction of the
antitoxin by heat. A similar observation, made soon after by Wasser-
mann 1 in the case of pyocyaneus toxin and antitoxin, fully supported
the results of Calmette.

An ingenious proof of the direct action of antitoxin upon toxin
was obtained by Martin and Cherry.2 It was found by them that very
dense filters, the pores of which had been filled with gelatin, permitted
toxin to pass through under high pressure, while the presumably larger
antitoxin molecule was held back. Through such filters they forced
toxin-antitoxin mixtures, under a pressure of fifty atmospheres, at vary¬
ing intervals after mixing. They found that, if filtered immediately,
all the toxin in the mixtures came through, but that, as the interval
elapsing between mixing and filtration was prolonged, less and less toxin
appeared in the filtrate, until, finally, two hours after mixing, no toxin
whatever passed through the filter. Besides demonstrating the direct
action of antitoxin upon toxin, this work of Martin and Cherry showed
that the element of time entered into the toxin-antitoxin reaction, just
as it enters into reactions of known chemical nature. The absolute non¬
participation of the living tissue cells in these reactions was demonstrated
by Ehrlich himself. Robert and Stillmarck 3 had shown that ricin pos¬
sessed the power of causing the red blood cells of defibrinated blood to
agglutinate in solid clumps, a reaction which could easily be observed
in vitro. Ehrlich,4 who had obtained antiricin in 1891 by injecting
rabbits with increasing doses of ricin, found that this antibody pos¬
sessed the power of preventing the hemagglutinating action of ricin
in the test tube. By a series of quantitatively graded mixtures of ricin
and antiricin, with red blood cells as the indicator for the reaction, he
succeeded in proving not only that the toxin-antitoxin neutralization
was in no way dependent upon the living animal body, but that definite
quantitative relations existed between the two substances entirely
analogous to those which, according to the law of multiple proportions,
govern reactions between different substances of known chemical
nature. Similar quantitative results were subsequently obtained by
Stephens and Myers 5 for cobra poison and its antitoxin, by Kossel fl

1 Wassermann, Zeit. f. Hyg., xxii, 1896.

2 Martin and Cherry, Proc. Royal Soc., London, lxiii, 1898.

3 Robert und Stillmarck, Arb. d. phar. Inst. Dorpat, 1889.

4 Ehrlich, Fort. d. Med., 1897.

6 Stephens and Myers, Jour, of Path, and Bact., 1898.

0 Kossel, Berl. klin. Woch., 1898.

TOXINS AND ANTITOXINS

205

for the toxic eel blood serum, and by Ehrlich 1 for the hemolytic tetanus
poison known as tetanolysin.

The introduction of the test-tube experiment into the investigation
of these reactions permitted of much more exact observations, and by
this means, as well as by careful, quantitatively graded, animal experi¬
ments, the further facts were ascertained that toxin and antitoxin com¬
bined more speedily in concentrated than in dilute solutions, and that
warmth hastened, while cold retarded, the reaction — observations 2
which in every way seem to bear out Ehrlich’s conception of the chemi¬
cal nature of the process.

Ehrlich’s Analysis of Diphtheria Toxin. — Shortly after the discovery
and therapeutic application of diphtheria antitoxin, it became apparent
that no two sera, though similarly produced, could have exactly the
same protective value. It was necessary, therefore, to establish some
measure or standard by which the approximate strength of a given anti¬
toxin could be estimated. Von Behring 3 attempted to do this for
both tetanus and diphtheria antitoxins by determining the quantity of
immune sera which, in each case, was needed to protect a guinea-pig of
known w7eight against a definite dose of a standard poison. He ascer¬
tained the quantity of standard toxin-bouillon which would suffice to kill
a guinea-pig of 250 grams, and called this quantity the “toxin unit.”
This unit was later more exactl}r limited by Ehrlich, who, considering
the element of time, stated it as the quantity sufficient to kill a guinea-
pig of the given weight in from four to five days.

Appropriating the terminology of chemical titration, v. Behring
spoke of a toxin-bouillon which contained one hundred such toxin units
in a cubic centimeter, as a “normal toxin solution” (“ DTN1 M250 ”),
and designated as “ normal antitoxin ” a serum capable of neutraliz¬
ing, cubic centimeter for cubic centimeter, the normal poison.4 A cubic
centimeter of such an antitoxic serum was sufficient, therefore, to neu¬
tralize one hundred toxin units, and was spoken of as an “antitoxin
unit.” In the experiments of v. Behring, toxin and antitoxin had been
separately injected. Ehrlich 5 6 improved upon this method by mixing
toxin and antitoxin before injection, thereby obviating errors arising

» Ehrlich, Berl. klin. Woch., 1898.

* Knorr, Fort. d. Med., 1897.

8 v. Behring, Deut. med. Woch., 1893.

* DTN 1 M250 signifies: D, Diphtheria; TN1, Normal Toxin solution; M250, Meer-

schweinchen or guinea-pig weighing 250 grams.

6 Ehrlich, Kossel und Wassermann, Deut. med. Woch., 1894.

206

INFECTION AND IMMUNITY

from differences which may have existed in the depth of injection or
rapidity of absorption.

In order, however, that any such method of standardization of an¬
titoxin may be practically applicable, it is necessary to produce either
a stable toxin or an unchangeable antitoxin. This Ehrlich achieved for
antitoxin by drying antitoxic serum in vacuo and preserving it in the
dark, at a low temperature and in the presence of anhydrous phosphoric
acid. By the use of such a stable antitoxin, various toxins may be
measured and other antitoxic sera estimated against these.

Given thus a constant antitoxin, the standardization of toxins would
be a comparatively simple matter were the poison obtainable in a per¬
fectly pure state. Unfortunately for the ease of measurement, how¬
ever, this is not the case. The problem is rendered difficult by a number
of complicating factors, many of which have been brought to light by
Ehrlich 1 in his laborious researches into the quantitative relationship
between the two reacting bodies.

As previously stated, it had been noted by Ehrlich and others that
toxin solutions would deteriorate with time; that is, a toxin-bouillon

Fig. 55. — Toxin and Body Cell.

which was found soon after production to contain, say, eighty toxin
units in each cubic centimeter, would, after four or five months, be found
to contain but forty units in the same gross quantity. It had lost, there¬
fore, in this case, just one-half of its toxic power. In spite of this loss,
however, Ehrlich found that such bouillon had retained its full original
power of neutralizing antitoxin. If the reaction was purely one of
chemical neutralization, there seemed to<be but one explanation of this.
The toxin molecule must contain two separate atom groups. One of
these must possess the power of binding antitoxin and be stable; this

1 Ehrlich, Klin. Jahrbuch, vi, 1897; Deut. med. Woch., 1898.

TOXINS AND ANTITOXINS

207

he designates as the “haptophore” or “anchoring” group. The other,
the one by which the foxin molecule exerts its deleterious action, must
be more easily changed or destroyed; this he calls the “toxophore”
or “ poison ” group. In the altered toxin-bouillon in which a part of
the poisonous action has been lost while the antitoxin-neutralizing power
is intact, the toxophore group of some of the toxin must have been
changed or destroyed. Such altered toxin he speaks of as “toxoid.”

In support of this hypothesis and for the purpose of perfecting the
methods of standardization, Ehrlich was led to determine, for a large
variety of specimens of diphtheria toxin, the precise quantity, in
cubic centimeters, which was necessary to neutralize exactly one unit
of his standard antitoxin. This he accomplished by making a series of
toxin-antitoxin mixtures, in each of which the quantity of antitoxin
was exactly one unit, while the amount of toxin was gradually increased.
These mixtures were injected into guinea-pigs of 250 grams weight.
It is self-evident that in such an experiment the mixtures containing
the smaller quantities of toxin would have no effect upon the guinea-
pigs. Soon, however, a mixture would be reached in which toxin would
be sufficiently in excess of antitoxin to produce the symptoms of slight
poisoning, as evidenced in local edema, rise of temperature, etc. The
largest quantity of toxin which could be added without producing such
symptoms was then regarded as exactly neutralizing one antitoxin unit.
This quantity of toxin Ehrlich speaks of as “ Limes zero ” (Limes =
threshold) or, briefly, “ L0.”

For instance:

One antitoxin unit + 0.6 c.c. toxin . No symptoms of poisoning.

“ “ “ 0.8 c.c .

“ “ “ 0.9 c.c . “ “ “ “

“ “ “ 1. c.c . “ “ “ “

“ “ “ 1.1 c.c . Local edema. Paralysis in 30 days.

“ “ “ 1.2 c.c . Death in 10 days.

In this example, L0, therefore, equals 1 c.c.

It is obvious, however, that because of the great difficulty in esti¬
mating the very slightest evidences of toxic action in guinea-pigs, a
more exact method of standardizing the poisons against antitoxin
would be to determine how much toxin would be required to neu¬
tralize one antitoxin unit and still be sufficiently in excess to cause
the death of a guinea-pig of 250 grams in four to five days . This would
then correspond to the action of one toxin unit, unmixed with antitoxin.
A 'priori it would seem that this value (expressed by Ehrlich as “ Limes

208

INFECTION AND IMMUNITY

death ” or “ L+) must simply be L0 plus one toxin unit. This, however,
was found not to be the case. Thus, in the example given, in which T
(the toxin unit — the quantity of the bouillon killing a guinea-pig of 250
grams in four to five days) was equal to O.Olc.c., L0 (the quantity of
toxin completely neutralizing one antitoxin unit) was found to be 1 c.c.
or 100 T. In this same poison, however, L+ (the quantity of toxin neces¬
sary both to neutralize one antitoxin unit and yet to be sufficiently in
excess of neutralization to kill a guinea-pig of 250 grams in four or five
days) was not found to be merely L0 + IT; but on actual experi¬
ment proved to be L0 + 101 T.

Expressed graphically, the conditions may be stated as follows:

.01 c.c. of the toxin bouillon.

L + (neutral, of 1 antitox. unit yet killing 1 pig) = 2.01 c.c. or 201 T.

Lq (complete neutral, of 1 antitox. unit) = 1. c.c. or 100 T.

Difference = 1.01 c.c. or 101 T.

Ehrlich, at first, endeavored to explain this surprising phenomenon
on the basis of toxoids. He argued that the toxoids formed by de¬
terioration of toxin might be conceived as possessing three different
degrees of affinity for antitoxin. If their affinity for antitoxin were
equal to, or more marked than, that of the toxin itself, they could have
no influence upon the dose L+ . If, however, their affinity for antitoxin
were weaker than that of toNin, each fresh toxin unit added to the dose
L0 would, first uniting with antitoxin, replace a corresponding quan¬
tity of these nontoxic substances of weaker affinity, and L+ would
be reached only after all of these “ epitoxoids,” as Ehrlich called them,
had been replaced, and toxin became free in the mixture.

Thus, in analyzing our example, we have:

100 tox.-antitox. + 100 epitox.-antitox. = L0 ;
add 1 T, and we have 101 tox.-antitox. + 99 epitoxoid-antitoxin + 1 epitoxoid free;
add 101 T and we have 200 toxin-antitoxin + 100 epitoxoid free +1 T free = L + .

Two facts, however, led Ehrlich to abandon the opinion that epi¬
toxoid was merely a variety of toxoid. He found, in the first place,
that the stated relations between L0 and L+ were true for perfectly,
fresh toxin-bouillon in which little or no deterioration had taken place.
He observed, furthermore, that in old, altered toxin bouillon, while
T was very much affected, the quantity needed to kill a pig con¬
stantly increasing, and the number of actual fatal doses in Lp con-

TOXINS AND ANTITOXINS

209

stantly decreasing (by reason of toxoid formation), L+ remained
practically unchanged.

Simply stated, this means that the epitoxoids or substances which
have weaker affinity for antitoxin than toxin itself are already present
in fresh bouillon and are not increased with time. For this reason,
Ehrlich has separated these substances from toxoids. He calls them “ tox¬
on” and believes them to be, like toxin, primary secretory products of
the diphtheria bacilli. The toxoids themselves, Ehrlich believes, are of
two kinds, those with a stronger affinity for antitoxin than toxin it¬
self (protoxoids), and those whose affinity for antitoxin is equal to that
of toxin. These latter he calls “syntoxoids.”

The toxon (epitoxoid originally), as Ehrlich believes, has a hapto-
phore or “ binding” group similar to that of toxin, but a different
toxophore or 11 poisoning” group. Qualitatively it has been shown
to differ from toxin in that, lacking the power to produce acute symp¬
toms, it causes gradual emaciation and paresis in animals.

That this difference in the poisonous action of toxin and toxon
is not merely a quantitative difference, referable to small quantities of
toxin, was proved by Dreyer and Madsen,1 who showed that if they made
a toxin-antitoxin mixture in which after injection the only evidence of
incomplete neutralization lay in the emaciation and final paralysis of
the test animals, the quantity of such a mixture could be increased
five- and tenfold, without producing the true toxin symptoms in ani¬
mals. These authors, too, claim to have been able to immunize against
toxin with such mixtures, thereby proving the identity of the haptophore
groups of the two substances. The importance of this observation will
become more evident in connection with the section on the “side-
chain theory/’

Method of Partial Absorption of Toxin. — Ehrlich 2 has gathered
more exact data in support of his views from what he terms the “ Method
of Partial Absorption ” of toxin by antitoxin.

In order to understand this method clearly, it is necessary to re¬
member that Ehrlich 3 believes the union of toxin with antitoxin to
take place according to the chemical laws of valency. Just as in H20
oxygen has an atomic valency of 2 for hydrogen, so, in the fully
neutralized toxin-antitoxin compound, he believes antitoxin to have a

1 Dreyer und Madsen, Zeit. f. Hyg. , xxxvii , 1901.

2 Ehrlich, “ Gesammelte Arbeiten zur Immunitatsforsch.,” Berlin, 1904.

3 Ehrlich, Deut. med. Woch., 1898.

210

INFECTION AND IMMUNITY

valency of 200 for toxin. It would require, according to this, 200 T
or toxin molecules to satisfy the affinities of one antitoxin molecule.1

This belief is based upon the following consideration : In determining
the L0 dose, or fully neutralized toxin-antitoxin union, Ehrlich, as well
as Madsen, found that the number of T units contained in such a
dose was almost regularly a factor of one hundred, recurring again
and again as 25, 33, 50, 75, etc. This pointed to more or less regularity
in the deterioration of toxin into toxoid, and to a more or less regular
relation of toxin to toxon. Now, as we have seen before, if we could
procure a perfectly pure toxin, the L0 dose plus one toxin unit would
give us the L+ dose; that is, one toxin unit in excess of full neutraliza¬
tion would suffice to kill a guinea-pig of 250 grams in four to five days.
Since a perfectly pure toxin, however, has not been obtainable up to the
present time, it is clear that the number of pure toxin bonds contained
in L+ must be less than the actual number of neutralizing units in the
combination, a part of the antitoxin being bound by toxon and toxoid.
The actual values obtained for the number of T units in L+ has
never exceeded 200, and has usually been more than 100, the highest
value ascertained by Madsen being 160. Given, therefore, a combining
value which, being a multiple of one hundred, is often more than one
hundred, but in an obviously impure state has never reached 200, it is
most likely that 200 represents the actual value sought for.

Assuming, therefore, upon the foregoing considerations, that the
valency of antitoxin for toxin is 200, Ehrlich carries out his experi¬
ments in the following way:

Given a toxin, the unit (T) of which is 0.024 c.c., he first deter¬
mines the L+ dose which, tested against the standard antitoxin unit, in
this case is 2.05 c.c. But 2.05 c.c. = 85 T. (or 2.05 -r- .024) units.
By mixing the L+ dose of toxin and antitoxin in such a way that the
quantity of antitoxin is gradually increased, while the toxin remains
always L + , and determining upon animals the amount of free toxin
contained in each mixture, the following table may be constructed:2

0 anti tox. unit representing 0 valencies + L + = 85 free T units.

+ L + = 85 “

.25

+ L + = 60 “

160

+ L+ = 10 “

180

+ L + = 3.5 “

1 Ehrlich, “ Schlussbetrachtungen,” Nothnagel’s System.

2 Example taken from Ehrlich, Deut. med. Woch., 1898.

TOXINS AND ANTITOXINS

211

It is plain that the substances with the strongest affinity for antitoxin
must be bound first by the antitoxin. This does not diminish the toxic
value of the mixture; and these are the protoxoids. Next are bound
syntoxoids and toxins, and, finally, the toxons. It is plain that, by
this method, the constitution of any given toxin may be ascertained,
and Ehrlich has constructed, on the basis of these observations, what he
terms his toxin spectrum. Minor differences of toxicity and affinity for
the antibody have caused him, by the partial saturation method de¬
scribed, still further to divide toxin into proto-, deutero-, and trito-toxin.

His spectra graphically describe the constitution of any given toxic
bouillon and trace its deterioration as follows:

Fty. /

Toxons

160 170160 100! 00

Figr.2.

Toxons

toxins

Deutero -

Prototoxoids or toxoids oc TYitotoxoids or

Toxons

Prototoxins /? Deutero - Trito toxins
toxins/3

Ehrlich’s opinion as to the constitution of toxin is by no means
fully accepted. Arrhenius,1 the great physical chemist, and Madsen,

1 Arrhenius Madsen, Zeit. f. physik.Chem., 1902; Festschrift, Kopenhagen, 1902.

Modification (Toxoids/

111!

catooti////////

mwm.

- '

212

INFECTION AND IMMUNITY

a bacteriologist who was once a pupil of Ehrlich, have recently opposed
Ehrlich’s theory on grounds of physical chemistry.

Modern theories of solution maintain that substances in solution
are broken up into their atoms or atom-groups, known as ions. Thus,
NaCl in solution would be “dissociated” into its Na ion and its Cl ion,
the completeness of the dissociation depending upon the concentra¬
tion of the solution. A solution of NaCl, therefore, contains, according
to this view, three substances, NaCl undissociated and free ions of Na
and Cl, the relative quantities of the three present in any given solution
being calculable, and depend upon a law known as the law of mass-
action of Guldberg and Waage. These free ions are the elements, there¬
fore, which are active in the formation of further chemical combination.
When a strong acid, in solution, acts upon a base, say HC1 upon am¬
monia (NH3), strong acid having the property of quite complete dis¬
sociation in relatively concentrated solutions, little or no ammonia
would remain unbound. A weak acid, like boric acid, however, not being
as completely dissociated, would leave some ammonia uncombined even
after more than quantitatively sufficient boric acid had been added.
Arrhenius and Madsen, on the basis of careful researches into the re¬
action between tetanolysin and its antibody, believe that toxin and anti¬
toxin possess weak chemical avidity for each other, their interaction
being comparable to that taking place between a weak acid and a base.
Toxin-antitoxin solutions, therefore, would contain the neutral com¬
pound, but at the same time uncombined toxin and antitoxin. The
qualities which Ehrlich ascribes to toxon, they believe, are due to
the unbound toxin present in such mixtures. In careful studies
in which they inhibited the hemolytic action of ammonia by gradual
addition of boric acid, they were able to show complete parallelism
between the conditions governing this neutralization and those con¬
cerned in their tetanus experiments. Their explanation has the
advantage of extreme simplicity over that of Ehrlich, but since the
differences of opinion are now the subject of active experimental
controversy, a critical discussion must rest until further facts are
revealed.

The Side-Chain Theory. — We have seen that the extensive researches
of Ehrlich into the nature of the toxin-antitoxin reaction led him to
believe that the two bodies underwent chemical union, forming a neu¬
tral compound. The strictly specific character of such reactions, further¬
more, diphtheria antitoxin binding only diphtheria toxin, tetanus
antitoxin only tetanus toxin, etc., led him to assume that the chemical

TOXINS AND ANTITOXINS

213

affinity between each antibody and its respective antigen depended
upon definite atom groups contained in each.

Ehrlich 1 had, in 1885, published a treatise in which he discussed
the manner of cell-nutrition and advanced the opinion that in order to
nourish a cell, the nutritive substance must enter directly into chemical
combination with some elements of the cell protoplasm. The great
number and variety of chemical substances which act as nutriment led
him to believe that the highly complex protoplasmic molecules of
cells were made up of a central atom-group (Leistungs-Kern) upon
which depended the specialized activities of the cell, and a multi¬
plicity of side chains (a term borrowed from the chemistry of the
benzol group) , by means of which the cell entered into chemical relation
with food and other substances brought to it by the circulation. If
we illustrate graphically by the chemical conception from which the
term side chain was borrowed, in salicylic acid, the formula given, the

H— C C— COOH

H— C C— H

benzol ring represents the “ Leistungs-Kern,” or radicle, while OOOH
and OH are side chains by means of which a variety of other substances
may be brought into relation with the “radicle,” for instance, as in
methyl salicylate.

co2chs

Just as nutritious substances are thus brought into workable re¬
lation with the cell by means of the atom-groups corresponding to side
chains, so Ehrlich believes toxins exert their deleterious action only
because the cells possess side chains by means of which the toxin can be
chemically bound. These side chains, Ehrlich in his later work calls
“receptors.” The receptors or side chains present in the cells and

1 Ehrlich, “ Das Sauerstoffbediirfniss des Organismus,” Berlin, 1885.

214

INFECTION AND IMMUNITY

possessing by chance specific affinity for a given toxin, are, by their
union with toxin, rendered useless for their normal physiological func¬
tion. By the normal reparative mechanism of the body these recep¬
tors are probably cast off and regenerated. Regenerative processes of
the body, however, do not, as a rule, stop at simple replacement of lost
elements, but, according to the hypothesis of Weigert,1 usually tend to

overcompensation. The receptors eliminated
by toxin absorption are not, therefore, simply
reproduced in the same quantity in which
they are lost, but are reproduced in excess of
the simple physiological needs of the cell.
Continuous and increasing dosage with the
poison, consequently, soon leads to such
excessive production of the particular re¬
ceptive atom-groups that the cells involved
in the process become overstocked and cast
them off to circulate freely in the blood. These freely circulating re¬
ceptors — atom-groups with specific affinity for the toxins used in their
production — represent the antitoxins. These, by uniting with the
poison before it can reach the sensitive cells, prevent its deleterious
action. (Fig. 56.)

The theory of Ehrlich, in brief, then, depends upon the assumptions
that toxin and antitoxin enter into chemical union, that each toxin
possesses a specific atom-group by means of which it is bound to a pre¬
existing side chain of the affected cell, and that these side chains, in ac¬
cordance with Weigert’s law, under the influence of repeated toxin stimu¬
lation, are eventually overproduced and cast off by the cell into the
circulation.

It stands to reason that this theoretical conception would be vastly
strengthened were it possible to show that such receptors or toxin¬
binding atom-groups actually pre-existed in the animal body, and such
support was indeed given by the experiments of Wassermann and Taka-
ki.2 These observers succeeded in showing that tetanus toxin could be
rendered innocuous if, before injection into animals, it was thoroughly
mixed with a sufficient quantity of the fresh brain substance of guinea-
pigs. Similar observations were independently made by Asakawa,3 and

Toxin.

-AnUioxin

Fig. 56. — Toxin and
Antitoxin.

1 Weigert, Verhandl. d. Ges. Deutsch. Naturf. u. Aerzte, Frankfurt, 1896.

2 Wassermann und Takaki, Berl. klin. Woch., 1898.

s Asakawa, Cent. f. Bakt., 1898.

TOXINS AND ANTITOXINS

215

variously confirmed. Kempner and Schepilewsky 1 showed a similar
relation to exist between brain tissue and botulismus toxin, and Myers 2
brought proof of analogous conditions in the case of suprarenal tissue
and cobra poison.

In the discussion of Ehrlich’s toxin analysis, we have seen that he
accounted for variations in the quantitative relations by the existence
of toxoids and toxons. He explained the striking fact that toxoids had
lost their poisonous nature and yet retained full powers to neutralize
antitoxin by the assumption that toxin was made up of two separate
atom-groups; one, the haptophore group which possessed the specific
affinity for the antitoxin or cell receptor; the other, the toxophore
group by means of which the actually harmful effects were produced.
The haptophore groups of all three of these substances, toxin, toxoid,
and toxon, by virtue of their antitoxin-binding power, he assumed to be
alike; in toxoid, the toxophore group has been destroyed or altered;
in toxon, the toxophore group is qualitatively different from that of
toxin. The haptophore group, however, being alone concerned in neu¬
tralizing receptors, all three of these substances should, if Ehrlich’s
theory is to be tenable, produce antitoxin. Dreyer and Madsen,3 ac¬
cordingly, actually succeeded in producing diphtheria antitoxin by im¬
munization with toxon. Attempts to produce antitoxin with toxoids
have succeeded in the hands of Ehrlich and others. Such experiments
have not, however, been always successful, a notable failure being that
of Bruck.4 On the basis of such negative results the theory was advanced
by Wassermann that overproduction of receptors was stimulated by
the irritation (Zellreiz) produced by the toxophore group — a stimula¬
tion not present in the case of toxoids.

1 Kempner und Schepilewsky, Zeit. f. Hyg., 1898.

2 Myers, Cent. f. Bakt., i, 1899. 3 Dreyer und Madsen, Zeit. f. Hyg., 1901.

4 Bruck, Zeit. f. Hyg., 1904.

CHAPTER XIV

PRODUCTION AND TESTING OF ANTITOXINS

DIPHTHERIA ANTITOXIN

In spite of the great advances in our theoretical knowledge of anti¬
bodies, gained during the last three decades, extensive therapeutic
application has been made of the antitoxins only. Pre-eminent among
these from a practical point of view are the antitoxins against diphtheria
and tetanus toxin. For diphtheria, careful statistical studies have
demonstrated, beyond doubt, the therapeutic value of the serum
treatment. Biggs and Guerard, in a general statistical review, ar¬
rived at the conclusion that the death rate of diphtheria had been re¬
duced fifty per cent by the use of antitoxin. Approximately the same
estimate is made by Dieudonne1 who studied almost 10,000 treated cases.

Production of Diphtheria Antitoxin. — The methods for producing
diphtheria antitoxin vary only in minor technical details. The first
requisite for successful antitoxin production is the possession of a strong
toxin. The various means of obtaining this are outlined in the section
on diphtheria toxin. The toxin used should be of such potency that
less than 0.01. cc. will kill a guinea-pig of 250 grams weight in four
to five days.2

For experimental purposes, goats or sheep may be used for immuniza¬
tion; for antitoxin production on a large scale, horses have been found
to be the most useful animals. The horses should be young, four to
six years old, vigorous, and healthy. It is advisable that they be sub¬
jected to the mallein test to exclude possible infection with glanders.

The toxin injections are made subcutaneously. Because of the dif¬
ferences in susceptibility noted in various horses, it is advisable that the
first doses of toxin should be either very small or weakened by chemicals
or heat, or combined with antitoxin. In the Pasteur Institute in Paris,
the small initial dose of toxin (0.5 c.c.) is mixed before injection with
an equal quantity of Lugol’s solution (iodin-potassium iodicl solution).

1 Dieudonne, Arb. a. d. kais. Gesundheitsamt, 1895 and 1897.

2 Park, “ Pathog. Bacteria and Protozoa,” N. Y., 1908.

216

PRODUCTION AND TESTING OF ANTITOXINS

217

Park 1 advises an initial dose of 5,000 toxin units (about 20 c.c. of
a strong toxin) combined with 100 units of antitoxin. The same
amount is given with the second and third doses of toxin. The intervals
between injections are from five days to a week, depending upon the
time necessary for complete subsidence of the reaction (temperature).
The doses of toxin are gradually increased until, at the end of two or
three months, more than ten times the original dose is given (50,000
units) .

Horses vary greatly in the strength of antitoxin which they will pro¬
duce. At the end of three or four months in favorable animals one
cubic centimeter of serum may contain 250 to 800 antitoxin units. Fur¬
ther immunization will often increase the antitoxin output to 1,000 and
more units to the cubic centimeter of serum. Park states that none of
the horses used by him has ever yielded 2,000 units to the cubic centi¬
meter. The same writer advises a three months7 period of rest from
immunization at the end of every nine months. Given such resting
periods, some horses have continued to furnish high-grade antitoxin for
from two to four years.

In order to obtain serum from horses, a sharp cannula is introduced
into the jugular vein. After leading the horse into a specially con¬
structed stall, its head is slightly deflected and pressure is made upon the
jugular vein below the point into which the needle is to be plunged.
Compression can also be made by surrounding the neck of the horse close
to the shoulders with a leather strap over a pad laid directly upon
the vein. The vein becomes visible along the lower margin of the neck
in a line running from the angle of the jaw to the edge of the scapula.
The skin, of course, is previously shaved and sterilized. The cannula
is then plunged into the vein, either with or without previous incision
through the skin, and, through a sterile rubber tube, the blood is
allowed to flow into high glass cylinders or slanted Erlenmeyer flasks.
In this way, large quantities of blood may be obtained and, according
to Kretz,2 * as much as six liters may be taken at a time at intervals of
a month, without injuring the animal. Ligature of the vein after
bleeding is unnecessary.

The cylinders and flasks are allowed to remain standing for two or
three days in a cool place, preferably at or below 10° C. At the end
of this time, the serum may be pipetted or siphoned away from the

1 Park, loc. cit., p. 212.

2 Kretz, in “ Handb. der Techn. u. Meth. d. Immun./’ Kraus and Levaditi, vol. ii,

1908.

218

INFECTION AND IMMUNITY

clot and stored in the refrigerator. In order to diminish the chances of
contamination, five-tenths per cent of carbolic acid or four-tenths per
cent of tri-cresol may be added.

Antitoxin is fairly stable and if kept in a cool, dark place, may re¬
main active, with but slight deterioration, for as long as a year. Kept
in a dry state, in vacuo , over anhydrous phosphoric acid, by the method
of Ehrlich, it retains its strength indefinitely.

Standardization. — Since antitoxin units are measured in terms of
toxin, it is obvious that uniformity of measurement necessitates the
possession by the various laboratories of a uniform toxin. Antitoxin
being more stable than toxin, uniformity of toxin is obtained by means
of a standard antitoxin distributed from a central laboratory. This was
first done by Ehrlich in Germany, and is now done for the United States
by the Public Health and Marine Hospital Service laboratories. Bottles
of the distributed antitoxin are marked with the number of units con¬
tained in each cubic centimeter. Dilutions of this antitoxin are mixed
with varying quantities of the toxin to be tested, the mixtures are al¬
lowed to stand for fifteen minutes to permit union of the two elements,
and injections into guinea-pigs of 250 grams weight are made. In this
way, the L+ dose of the toxin is determined. (The L+ dose, as we have
seen in a previous section [p. 208], is the quantity of poison not only suf¬
ficient to neutralize one antitoxin unit,1 but to contain an excess beyond
this sufficient to kill a guinea-pig of 250 grams in four to five days.
L+ is chosen rather than L0, the simple neutralizing dose, because of
the difference between toxins in their contents of toxoid and toxon.2)

The L+ dose of the toxin having thus been determined, this quantity
is mixed with varying dilutions of the unknown antitoxin.3 Thus,
given an antitoxin in which 300 to 400 units to the cubic centimeter
are suspected, dilutions of 1 : 200, 1 : 250, 1 :300, etc., are made. One
cubic centimeter of each of these is mixed with the L+ dose of the toxin,
and the mixtures are injected into guinea-pigs of about 250 grams. If
the guinea-pig receiving L+ plus the 1 : 250 dilution lives and the one re¬
ceiving L+ plus the 1 : 300 dilution dies in the given time, we know that
the unit sought must lie between these two values, and further similar
experiments will easily limit it more exactly. The possibility of error in

1 A unit of diphtheria antitoxin is a quantity of antitoxin sufficient to protect a
guinea-pig of 250 grams against 100 times the fatal dose of diphtheria toxin.

2 Madsen, in Kraus u. Levaditi, “ Handbuch,” etc., 1907.

3 Donitz, “ Die Werthbem. der Heilsera,” in Kolle u. Wassermann, “ Hand¬
buch, ” etc.

PRODUCTION AND TESTING OF ANTITOXINS

219

measurement is much diminished by the use of larger quantities of dilu¬
tions higher than those given. ' Four c.c. is the volume usually injected.

Since 1902, the production and sale of diphtheria antitoxin has been
regulated by law in the United States. From time to time, antitoxin is
bought in the open market and examined at the hygienic laboratories of
the United States Public Health and Marine Hospital Service. Anti¬
toxic serum which contains less than two hundred units to each cubic
centimeter is not permitted upon the market.

In a previous section we have seen that Hiss and Atkinson 1 and
others have shown an increase in the globulin contents of blood serum
of immunized animals. It has been shown, furthermore, that the pre¬
cipitation of such serum with ammonium sulphate carried down in the
globulin precipitate all the antitoxic substances contained in the serum.
Upon a basis of globulin precipitation, Gibson 2 has recently perfected a
method of concentrating and purifying diphtheria antitoxin for thera¬
peutic use. This procedure, as carried out at the New York Depart¬
ment of Health, is, in principle, as follows:

The serum, as taken from the horse, is heated to 56° C. for twelve
hours. This converts about half of the pseudoglobulin into euglobulin,
the antitoxin remaining in the pseudogiobulin fraction.3 It is then 4
precipitated with an equal volume of a saturated ammonium sulphate
solution. After two hours, the precipitate is caught in a filter and
redissolved in a quantity of water corresponding to the original quantity
of serum. After filtration, this solution is again precipitated with
saturated ammonium sulphate solution and the precipitate again fil¬
tered off. The precipitate is then treated with a saturated solution of
sodium chloride of double the volume of the original serum. This is
allowed to stand for about twelve hours. At the end of this time the
antitoxin-containing globulin is in solution and is pipetted away from
the precipitate and filtered. This salt-solution extract is then pre¬
cipitated with twenty-five hundredths per cent acetic acid. The re¬
sulting precipitate of globulin is thoroughly dried by pressure between
filter papers and placed in a parchment dialyzer. Dialysis with run¬
ning water is continued for seven to eight days, after neutralization with
sodium carbonate, in order to remove the sodium chloride. At the
end of this time, the globulin solution remaining in the dialyzer is fil-

1 Hiss and Atkinson, Jour. Exper. Med., v, 1900.

2 Gibson, Jour, of Biol. Chem., i, 1906.

3 Dr. Banzhaf, personal communication.

4 Gibson and Collins, Jour, of Biol. Chem., iii, 1907.

220

INFECTION AND IMMUNITY

tered through a Berkefeld candle for the purpose of sterilization, after
the addition of 0.8 per cent sodium chlorid. According to Gibson, this
method produces a yield of antitoxin which equals about four-fifths
of the original quantity but is concentrated five- to seven-fold. The
method has more recently been modified as follows:

After heating to 56° C., as above, and cooling, ammonium sulphate is
added to the serum to thirty per cent saturation. This brings down all
the euglobulins. This is then filtered and the filtrate, which contains
the pseudoglobulins with the antitoxin, is again precipitated with
ammonium sulphate in a concentration of fifty-four per cent of satura¬
tion. The precipitate is then separated on a paper, pressed to dryness,
and directly dialyzed.1

Park and Thorne2 have found that the use of such concentrated
antitoxin is, therapeutically, equally efficient as the unconcentrated,
and possesses the advantage of less frequently giving rise to the sec¬
ondary reactions in skin and mucous membranes occasionally noticed
after the use of ordinary antitoxin, and referable, probably, to some
other constituent of the horse serum.

Diphtheria antitoxin is therapeutically used in doses ranging from
3,000 to 20,000 units. For prophylactic immunization of healthy
individuals, about 500 units should be used.

TETANUS ANTITOXIN

Production of Tetanus Antitoxin. — The production of tetanus anti¬
toxin is, in every way, analogous to that of diphtheria antitoxin. It
is necessary in the first place to produce a powerful tetanus toxin. The
methods of procuring this will be discussed in the section upon tet¬
anus toxin, page 458. Suffice it to say here that the most satisfactory
method of obtaining toxins consists in cultivating the bacilli upon veal
broth containing five-tenths per cent to two per cent sodium chlorid
and one per cent pepton. It has been advised, also, that the broth should
be neutralized by means of magnesium carbonate rather than with
sodium hydrate. The bacilli are cultivated for eight to ten days at incu¬
bator temperature and the broth filtered rapidly through Berkefeld
filters. The toxin may be preserved in the liquid form with the ad¬
dition of five-tenths per cent carbolic acid, or may be preserved in the
dry state after precipitation with ammonium sulphate.

1 Dr. Banzhaf, personal communication.

2 Park and Throne, Amer. Jour. Med. Sci., Nov., 1906.

PRODUCTION AND TESTING 01’ ANTITOXINS

221

It is necessary to determine the strength of the poison. This is done
according to Behring 1 by determining the smallest amount of toxin
which will kill a white mouse of twenty grams weight within four days.
This is most easily done by making dilutions of the toxin ranging from
1 : 100 to 1 : 1,000, and then injecting quantities of 0.1 c.c. of each of
these dilutions subcutaneously into white mice. In this way, the mini¬
mal lethal dose is ascertained.

For the actual production of antitoxin, horses have been generally
found to be the most favorable animals. The horses should be healthy
and from five to seven years old. The first injection of toxin admin¬
istered to these animals should be attenuated in some way. Vari¬
ous methods for accomplishing this have been in use. In America,
the first injection of about ten to twenty thousand minimal lethal doses 2
(for mice of twenty grams weight) is usually made subcutaneously to¬
gether with sufficient antitoxin to neutralize this quantity. In Germany,
v. Behring uses, for his first injection, a much larger dose of toxin to
which about 0.25 per cent of terchlorid of iodin has been added.
Immediately after an injection, the animals will usually show a reac¬
tion expressed by a rise of temperature, refusal of food, and some¬
times muscular twitching. A second injection should never be given
until all such symptoms have completely subsided. This being the case,
after five to eight days double the original dose is given together with
a neutralizing amount of antitoxin or with the addition of terchlorid of
iodin. Again after five to eight days, a larger dose is given and there¬
after, at similar intervals, the quantity of toxin is rapidly increased. In
America the neutralizing antitoxin is omitted after the third or fourth
injection; in v. Behring’s laboratory the quantity of terchlorid of iodin
is gradually diminished. The increase of dosage is often controlled by
the determination of the antitoxin contents of the animal’s blood serum.
The immunization is increased until enormous doses (500 c.c.) of a
toxin in which the minimal lethal dose for mice is represented by
0.0001 c.c., or less, is borne by the horse without apparent harm.

The antitoxic serum is then obtained by bleeding from the jugular
vein, as in the case of diphtheria antitoxin. It may be preserved in the
liquid state by the addition of five-tenths per cent of carbolic acid or
four-tenths per cent of tricresol.

1 v. Behring, Zeit. f. Hyg., xii, 1892 ; Dent. med. Woch., 1900.

2 According to Park the “ horses receive 5 c.c. as the initial dose of a toxin
of which 1 c.c. kills 250,000 grams of guinea-pig, and along with this a sufficient
amount of antitoxin to neutralize it.”

222

INFECTION AND IMMUNITY

Standardization. — The universal prophylactic use of tetanus antitoxin
has, as in the case of diphtheria antitoxin, necessitated its standardiza¬
tion. A variety of methods are in use in different parts of the world.
In the following description the American method only will be consid¬
ered as laid down under the law of July, 1908, and based upon the work
of Rosenau and Anderson 1 at the United States Hygienic Laboratories
at Washington.

In conjunction with a committee of the Society of American Bac¬
teriologists, these authors have defined the unit of tetanus antitoxin as
follows:

The unit shall be ten times the least amount of serum necessary to
save the life of a 350 gram guinea-pig for ninety-six hours against the
official test dose of standard toxin. The test dose consists of 100 minimal
lethal doses of a precipitated toxin preserved under special conditions
at the hygienic laboratory of the Public Health and Marine Hospital
Service. (The minimal lethal dose is in this case, unlike Behring’s
minimal lethal dose, measured not against 20 gram mice, but against
350 gram guinea-pigs.)

In the actual standardization of tetanus antitoxin, as in that of diph¬
theria antitoxin, the L+ dose of toxin is employed. The L+ dose is,
however, in this case, defined as the smallest quantity of tetanus toxin
that will neutralize one-tenth of an immunity unit, plus a quantity of
toxin sufficient to kill a 350 gram guinea-pig in just four days. At the
Hygienic Laboratory at Washington, a standard toxin and antitoxin
are preserved under special conditions, and standard toxin and anti¬
toxin, arbitrary in their first establishment, are kept constant by being
measured against each other from time to time. In measuring the anti¬
toxic serum thus preserved, at the Hygienic Laboratory, a mixture of
one-tenth of a unit of antitoxin and 100 minimal lethal doses of the
standard toxin must contain just enough free poison to kill the guinea-
pig in four days. This L+ dose of the standard toxin is given out to
those interested commercially or otherwise in the production of antitoxin.

In measuring an unknown antitoxic serum against this L+ dose of
toxin, a large number of mixtures are made, each containing the
L+ dose of the toxin and varying quantities of the antitoxin. Dilu¬
tions must always be made with 0.85 per cent salt solution and the
total quantity injected into the animals should always be brought up to

1 Rosenau and Anderson, Pub. Health and Mar. Hosp. Serv. U. S., Hyg. Lab. Bull.
43, 1908.

PRODUCTION AND TESTING OF ANTITOXINS

223

4 c.c. with salt solution in order to equalize the conditions of concen¬
tration and pressure. The mixtures are then kept for one hour at
room temperature in diffused light. After this they are subcutaneously
injected into a series of guinea-pigs weighing from 300 to 400 grams.
The following example of a test is taken from the article by Rosenau
and Anderson quoted above.

No. of
Guinea-

Weight of
Guinea-pig
(Grams.)

Subcutaneous Injection of a
Mixture of

■

Time of Death.

pig-

Toxin

(Test Dose).

Antitoxin.

1 .

360

Gram.

0.0006

c.c.

0.001

2 days 4 hours

2 .

350

.0006

.0015

4 days 1 hour

3 .

350

.0006

.002

Symptoms

4 .

360

.0006

.0025

Slight symptoms

5 .

350

.0006

.003

No symptoms

In this series the guinea-pig, receiving 0.0015 c.c. of the antitoxin, died
in approximately four days; 0.0015 c.c. therefore represents one-tenth
of an immunity unit.

In therapeutically employing antitoxin for prophylactic purposes,
about 1,500 units should be employed.

CHAPTER XV

LYSINS, AGGLUTININS, PRECIPITINS, AND OTHER ANTIBODIES

LYSINS

In the immediately preceding sections, we have dealt solely with
immunity as it occurs where soluble toxins play an important part
and in which antitoxins are developed in the immunized subject. There
are many species of pathogenic bacteria, however, which stimulate
the production of little or no antitoxic substance when introduced into
animals, and the resistance of the immunized animal can not, therefore,
be explained by the presence of antitoxin in the blood.

v. Fodor,1 Nuttall,2 Buchner,3 and others had in 1886 and the years
following carried on investigations which showed that normal blood
serum possessed the power of killing certain of the pathogenic bacteria.
Nuttall, working under the direction of Fliigge, made the important dis¬
covery that this bactericidal power became gradually diminished with
time, and could be experimentally destroyed by exposure of the serum
to a temperature of 56° C. for one-half hour. Buchner, who confirmed
and extended the observations of Nuttall, called this thermolabile sub¬
stance upon which the bactericidal character of the serum seemed to
depend “ alexin.’7

Our knowledge of the bactericidal action of serum was, soon there¬
after, extensively increased by the discovery, by Pfeiffer and Isaeff,4
that cholera spirilla injected into the peritoneal cavity of a cholera-
immune guinea-pig were promptly killed and almost completely dis¬
solved. The same phenomenon could be observed when the spirilla,
mixed with fresh immune serum, were injected into the peritoneum of a
normal guinea-pig.

The processes observed by Pfeiffer as taking place intraperitoneally
were soon shown by Metchnikoff,5 Bordet,6 and others to take place,
though to a lesser extent, in vitro. Bordet, furthermore, observed that

1 v. Fodor, Deut. med. Woch., 1886. 2 Nuttall, Zeit. f. Hyg., 1886.

3 Buchner, Cent. f. Bakt., 1889. 4 Pfeiffer undlsaeff, Zeit. f. Hyg., 1894.

5 Metchnikoff, Ann. de l’inst. Pasteur, 1895. 6 Bordet, ibid., 1895.

224

LYSINS, AGGLUTININS, PRECIPITINS, ETC.

225

the bacteriolytic digestive power of such immune serum, when destroyed
by heating, or after being attenuated by time, could be restored by the
addition to it of small quantities of normal blood serum. It could, in
other words, be “reactivated” by normal serum. From this obser¬
vation Bordet drew the conclusion that the bactericidal or bacteriolytic
action of the serum depended upon two distinct substances. The one
present in normal serum and thermolabile, he conceived to be identical
with Buchner’s alexin. The other, more stable, produced or at least
increased in the serum by the process of immunization, he called the
“sensitizing substance.” This substance, he believed, acting upon
the bacterial cells, rendered them vulnerable to the action of the alexin.
Without the previous preparatory action of the “sensitizing substance”
the alexin was unable to act. Without the co-operation of alexin, the
“sensitizing substance ” produced no visible effects.

Bordet’s interpretation of the phenomenon of lysis differs essentially
from that of Ehrlich, in that both active serum components are con¬
ceived by him, though independent, to act directly upon the bacterial
cell. A few years later, Bordet was able to show that exactly analogous
conditions governed the phenomenon known as “hemolysis” or dis¬
integration of red blood cells.

It had been known for many years that in the transfusion of blood
from an animal of one species into an animal of another species, in¬
jury was done to the red corpuscles which were introduced. Observed
in the test tube, the red cells in the heterologous serum were seen to
give up their hemoglobin in the fluid, the mixture taking on the red
transparency characteristic of what is known as “laked” blood. Buch¬
ner,1 in his alexin studies, had shown that the blood-cell destroying
action of the normal serum was subject to the same laws as the bac¬
tericidal power of similar serum, in that it was destroyed by heating,
and he assumed that both the bacteriolytic and the hemolytic action
of normal serum were due to the same “ alexin.” Metchnikoff,2 more¬
over, had pointed out the striking analogy between the two phenomena
as early as 1889.

Bordet 3 now observed that the blood serum of guinea-pigs previously
treated with the defibrinated blood of rabbits developed marked powers
of dissolving rabbits’ corpuscles, and that this hemolytic action could

1 Buchner, Arch. f. Hyg., xvii, 1893; Waremberg, Arch, d. med, exper., 1891.

2 Metchnikoff, Ann. de Tinst. Pasteur, 1889.

3 Bordet, Ann, de Tinst, Pasteur, t, xii, 1898,

226

INFECTION AND IMMUNITY

be destroyed by heating to 56° C., but “ reactivated ” by the addition of
fresh normal serum. He had thus produced an immune hemolysin,
just as Pfeiffer had produced immune bacteriolysin, and had demon¬
strated the complete parallelism which existed between the two phe¬
nomena.

A practical test-tube method was thus given for the investigation of
the lysins, just as a practical test-tube method for antitoxin researches
had been developed by Ehrlich in his ricin-antiricin experiments.

The path of investigation thus pointed out by Bordet was soon ex¬
plored in greater detail by Ehrlich and Morgenroth.1 The reasoning
which Ehrlich had applied in explaining the production of antitoxins
was thought, by these observers, to be equally applicable to the phe¬
nomena of bacteriolysis and hemolysis.

Since the thermolabile substance or alexin, renamed by Ehrlich
“ complement/’ was already present in normal serum and had been shown
to be little, if at all, increased during the process of immunization,
this substance could have but little relation to the changes taking place
in the animal body as immunity was acquired. The more stable serum-
component, however, the “ substance sensibilisatrice ” of Bordet, or,
as Ehrlich now called it, the "immune body,” was the one which seemed
specifically called forth by the process of active immunization. Ehrlich
argued, therefore, that when bacteria or blood cells were injected into
the animal, certain atom-groups or chemical components of the injected
substances were united to other atom-groups or “side chains” of the
protoplasm of the tissue cells. These “ side chains ” or receptors, then
reproduced in excess and finally thrown free into the circulation, con¬
stituted the “immune body.” The immune body, therefore, he con¬
cluded, must possess atom complexes which endow* it with specific
chemical affinity for the bacteria or red blood cells used in its produc¬
tion. This contention was supported by Ehrlich and Morgenroth by
an ingenious series of experiments.

Having in their possession, at that time, the blood serum of a goat
immunized against the red blood cells of a sheep, they inactivated it
(destroyed the complement or alexin) by heating to 56° C. The serum
then contained only the “ substance sensibilisatrice ” or immune body.
To this inactivated serum they added sheep’s red corpuscles, without
obtaining hemolysis. Having left the inactive serum and the sheep’s
corpuscles in contact with each other for some time, they separated

1 EhrUch und Morgenroth . Berl. klin. Woch., 1, 1899.

LYSINS, AGGLUTININS, PRECIPITINS, ETC.

227

them by centrifugalization. To the supernatant fluid, they now added
sheep-blood corpuscles and normal goat serum (complement) and found
that no hemolysis took place. The immune, body had apparently gone
out of the serum. The red cells which had been in contact with the serum
and separated by the centrifuge were then washed in salt solution and
to them complement was added in the form of fresh normal serum.

Fig. 57. — Ehrlich’s Conception of Cell-Receptors, Giving Rise to Lytic
Immune Bodies (Haptines of the Third Order).

Hemolysis occurred. It was plain, therefore, that the immune body
of the inactivated serum had gone out of solution and had become at¬
tached to the red blood cells, or, as Ehrlich expressed it, the immune
body by means of its “ haptophore ” atom-group had become united
to the corpuscles. In contrast to this, if normal goat serum (containing

Cell use d for immuni

Fig. 58. — Complement, Amboceptor or Immune Body, and Antigen Orv

Immunizing Substance.

complement only) was added to sheep corpuscles and separated again
by centrifugalization, the supernatant fluid was found to be still capable
of reactivating inactivated serum (immune body) . This he interpreted
as proving that the complement was not bound to the corpuscles directly.

If the three factors concerned — corpuscles, immune body, and
complement — were mixed and the mixture kept at 0° C., no hemolysis

228

INFECTION AND IMMUNITY

occurred; yet, centrifugalized at this temperature, immune body was
found to have become bound to the corpuscles, the complement re¬
maining free in the supernatant fluid. If the same mixture, however,
was exposed to 37° C., hemolysis promptly occurred.

From these facts, Ehrlich concluded that complement did not direct¬
ly combine with the corpuscles, but did so through the intervention of
the immune body. This immune body, he reasoned, possessed two
distinct atom-groups or haptophores; one, the cytophile haptophore
group, possessing strong chemical affinity for the red blood cell; the
other, or complementophile haptophore group, with weaker avidity
for the complement. Because of this double combining power, Ehrlich
speaks of the immune body as “ amboceptor.” His views as to the nature
and action of immune body and complement are graphically represented
in Figs. 57 and 58 (p. 227).

From what has been said before, it will be seen that the fundamental
difference between the conceptions of the mechanism of the lytic proc¬
esses as held by Bordet and by Ehrlich lies in the ability of the alexin
or complement to act directly upon the antigen, as claimed by Bor¬
det, or, as Ehrlich holds, only through the intermediation of the im¬
mune body. Bordet’s views,1 by no means disproved and still held by
many bacteriologists, may be summed up in his own words as follows:
“Neither immune body nor antigen (bacterium, blood cell, etc.) alone
has any manifest affinity for alexin (complement) ; but, united, they
form a complex which can absorb alexin.” The absorption of comple¬
ment is thus conceived as a property of the immune body or amboceptor
(or, in Bordet’s language, sensitizer) plus its specific antigen — acting as a
complex and not through a complementophile group of the immune body.

AGGLUTININS

Although Metchnikoff 2 and Gharrin and Roger 3 had noticed pecul¬
iarities in the growth of bacteria when cultivated in immune sera, which
were unquestionably due to agglutination, the first recognition of the
agglutination reaction as a separate function of immune sera was the
achievement of Gruber and Durham. While investigating the Pfeiffer
reaction with B. coli and the cholera vibrio, Gruber and Durham 4

1 Bordet, A Resume of Immunity in “ Studies in Immunity.” Transl. by Gay,
Wiley & Son, 1909.

2 Metchnikoff, “ Etudes sur l’immunite,” IV Memoir, 1891.

3 Charrin et Roger, Compt. rend, de la soc. de biol., 1889.

4 Gruber und Durham , Munch, med. Woch., 1896.

LYSINS, AGGLUTININS, PRECIPITINS, ETC,

229

noticed that if the respective immune sera were added to bouillon cul¬
tures of these two species, the cultures would lose their turbidity and
flake-like clumps would sink to the bottom of the tube, the supernatant
fluid becoming clear. Gruber, at the same time, called attention to the
fact that immune sera would affect in this way not only the microor¬
ganism used in their production, but, to a less energetic extent, other
closely related bacteria as well.

Widal, very soon after Gruber and Durham's announcement, ap¬
plied the agglutination reaction to the practical diagnosis of typhoid
fever, finding that the serum of patients afflicted with this disease
showed agglutinating power over the typhoid bacillus at early stages
in the course of the fever. The reaction, thus practically applied to
clinical diagnosis, was soon shown to be of great importance in its

Fig. 59— Microscopic Agglutination Reaction.

bearing on bacteriological species differentiation. Since animals im¬
munized against a definite species of bacteria acquire in their sera
specific agglutinating powers for these bacteria and at best only slight
agglutinating powers for other species, immune sera can be used ex¬
tensively in differentiating between bacterial varieties.

Agglutination may be observed microscopically or macroscopically.
Bacteria brought into contact with agglutinating serum in the
hanging drop rapidly lose their motility, if motile, as in the case of
typhoid bacilli, and gather together in small clumps or masses. The
microscopic picture is striking and easily recognized and the reaction
takes place with varying speed and completeness, according to the
strength of the agglutinating serum.

As the reaction approaches completeness, the clumps grow larger,

230

INFECTION AND IMMUNITY

individual microorganisms become more and more scarce, finally
leaving the medium between clumps entirely clear. While the clumping
of a motile organism suggests that motility has something to do with the
coming together in clumps, it nevertheless has no relation whatever
to agglutination, motile and non-motile organisms alike being subject
to the reaction.

Macroscopically observed, in small test tubes or capillary tubes, ag¬
glutination evidences itself by the formation of flake-like masses which

Fig. 60. — Macroscopic Agglutination. Dilutions from 1 in 10 to 1 in 1,000.
The first tube contains a 1 : 20 control with the bacteria and normal serum.
Agglutination complete in the tubes marked 10, 20, 50, 100.

settle into irregular heaps at the bottom, leaving the supernatant
fluid clear, in distinct contrast to the even flat sediment and the clouded
supernatant fluid of the control. Macroscopically, too, agglutination
is evidenced when bacteria are grown in broth to which immune serum
has been added. Instead of evenly clouding the broth, the micro¬
organisms develop in clumps or chains.

Another phenomenon probably produced by agglutinins is the so-

LYSINS, AGGLUTININS, PRECIPITINS, ETC.

231

called “ thread-reaction ” of Pfaundler.1 This consists in the forma¬
tion of long convoluted threads of bacterial growth in the hanging drop
of dilute immune serum after twenty-four hours. Very strict speci¬
ficity is attributed to this reaction by Pfaundler.

Agglutinins act upon dead as well as upon living bacteria. For the
microscopic tests bacterial emulsions killed by formalin were intro¬
duced by Neisser.

Ficker 2 has recently succeeded in preparing an emulsion of typhoid
bacilli, which is permanent and may be kept indefinitely, and may
be employed for macroscopic agglutinations.3

Attention has been called by various workers to a source of error in
all these methods, known as pseudo-clumping.4 The causes for such
clumping not due to agglutinins seem to lie in the presence of blood cells
in the serum or excessive alkalinity of the culture medium.5

While the microscopic methods are more suitable for clinical-diag¬
nostic purposes, because of the smaller amounts of blood required, the
macroscopic tests are far preferable for the purposes of bacterial differen¬
tiation and research. Greater exactitude of dilution is possible when
dealing with larger quantities; microscopic unevenness in the bacterial
emulsion does not become a source of error; and positive and negative
reactions are more sharply defined.

Nature of Agglutinins. — Gruber and Durham,6 the discoverers of ag¬
glutinins, at first advanced the opinion that the agglutinins were identical
with the immune body concerned in the Pfeiffer reaction, which by in¬
juring the bacteria rendered them susceptible to the alexins. Pfeiffer 7
and Kolle 8 soon showed, however, that by the addition of cholera vibrio
to immune serum, the agglutinins could be completely absorbed, or used
up, while bacteriolytic substances still remained. The same authors
demonstrated that immune serum, preserved for several months, would
lose its agglutinins without a corresponding loss of bacteriolytic power.
It has been variously shown since then, by these and other authors,
that the agglutinins and the bactericidal substances are in no way parallel

1 Pfaundler , Cent. f. Bakt., xxiii, 1898.

2 Ficker, Berl. klin. Woch., 1903.

3 The exact method of production of “Ficker’s Diagnosticum ” is a proprietary
secret.

* Savage, Jour, of Path, and Bact., 1901.

5 Biggs and Park, Amer. Jour, of Med. Sci., 1897; Block, Brit. Med. Jour., 1897.

6 Loc. cit.

7 Pfeiffer, Deut. med. Woch., 1896.

8 Pfeiffer und Kolle, Cent. f. Bakt., xx, 1896.

232

INFECTION AND IMMUNITY

in their development, and that strongly agglutinating sera may be ex¬
tremely weak in bactericidal substances and vice versa , the relative quan¬
tity of either of these substances depending to a certain extent upon
the method of immunization. Whether or not agglutinins possess
any direct protective function can not at present be stated with certainty.
Metchnikoff 1 assigns to them a purely secondary role. As a matter of
fact, agglutinated bacteria 2 are not killed by the act of agglutination
and are often as virulent as non-agglutinated cultures.

The agglutinins, furthermore, unlike the bactericidal substances in
sera, remain active after exposure to temperatures of over 55° C., some
of them withstanding even 65° to 70°, and can not be reactivated by the
subsequent addition of normal serum. These facts definitely preclude
the participation in the reaction of the alexin or complement and have
an important bearing upon Ehrlich’s views of their structure 3 (see
page 238).

As a result of these and a multitude of other studies, the agglu¬
tinins have come to be regarded as separate antibodies, closely related
to the precipitins.

The agglutinins may be chemically precipitated out of serum together
with the globulins. They do not dialyze. Bordet 4 made the observa¬
tion that agglutinins do not act in the absence of NaCl. Whether the
presence of the salt aids the reaction in a chemical or purely physical
way, as Bordet supposed, is uncertain.

Production of Agglutinins. — Just as normal sera contain small quan¬
tities of bactericidal substances, so do they contain agglutinins in small
amount. In a general way these “normal agglutinins” have the same
nature as the immune agglutinins, and it is probable that their presence
is traceable to the various microorganisms parasitic upon the human and
animal body.

As a matter of fact, the blood serum of new-born guinea-pigs hardly
ever contains agglutinin for B. coli, while that of adults acts upon these
bacilli in dilutions of 1 : 20. 5 Similarly, infants show lower normal ag¬
glutinating values than adults.6

1 Metchnikoff, “ L’immunite,” etc., 1901, p. 214.

2 Mesnil, Ann. de Finst.- Pasteur, 1898.

3 Pane, Cent. f. Bakt., 1897; Trumpp, Arch. f. Hyg., 1898; Forster, Zeit. f. Hyg.,
xxiv.

4 Bordet, Ann. de l’inst. Pasteur, 1899.

5 Kraus und Low, Gesell. d. Aerzte, Wien, 1899.

6 Pfaundler, Jahrb. f. Kinderheilk., Bd. 50.

LYSINS, AGGLUTININS, PRECIPITINS, ETC.

233

Agglutinins may be produced in the sera of animals by the intro¬
duction of microorganisms subcutaneously, intravenously, or intraperi-
toneally. The intravenous method seems to give the most abundant and
speedy results.1 The formation of agglutinins is a reaction to the body-
substances of the bacteria themselves, rather than to their toxic prod¬
ucts. Thus agglutinins are produced in response to the introduction
of dead bacteria and soluble extracts of cultures. Pathogenicity 2 does
not influence agglutinin formation to any great extent, non-pathogenic
as well as pathogenic giving rise to these substances in serum. As a
rule, however, agglutinins are more easily produced against avirulent
than against fully virulent strains of bacteria of the same species.

While agglutinins can be produced with almost all the known bac¬
teria, there are great differences between various species in the quantity
and speed of production, and Nicolle and Thenel 3 have classified bac¬
teria in three groups according to their power of stimulating the pro¬
duction of agglutinins in immunized animals. As a rule, the agglutinins
appear in the blood of animals three to six days after the introduction of
bacteria. From the third to the sixth day they rapidly increase to a
maximum at the seventh to thirteenth day. They then fall off rapidly
until they reach a level at which they remain for a long period without
very considerable change. Curves to illustrate these phases have been
constructed by Jorgensen and Madsen.4

The Reaction between Agglutinin and Agglutinin-Stimulating Sub¬
stances {Agglutinogen) . — The fact that agglutinin can be removed from,
or absorbed out of, serum by the specific bacilli which have led to its
formation indicates that there is in the act of agglutination a com¬
bination between the agglutinin and the agglutinin-stimulating sub¬
stance (agglutinogen) . It is likely that this combination is of a chemical
nature, since', as we have mentioned, agglutinins result from the in¬
jection of bacterial extracts as well as from the introduction of living
bacteria. The probability that the process follows chemical laws of
combination is furthermore strengthened by the work of Joos5 and
others, who have demonstrated that definite quantitative relations ex¬
ist between the agglutinin-stimulating substances and the agglutinins.
Every agglutination reaction, therefore, will vary in its degree of com-

1 Hoffmann, Hyg. Rundschau, 1903.

2 Nicolle, Ann. de Pinst. Pasteur, 1898.

3 Nicolle et Thenel, Ann. de Pinst. Pasteur, 1902.

4 Jorgensen and Madsen, Festschrift, Kopenhagen, 1902,

5 Joos, Zeits, f, Hyg-, xxxvi, 1901.

234

INFECTION AND IMMUNITY

pleteness with the quantities of agglutinin and agglutinogen, a fact
which makes it necessary, especially for clinical tests, to preserve a
certain uniformity in the quantity and density of the bacterial culture
or emulsion employed.

Specificity. — From the very beginning, Gruber and Durham 1 had
claimed specificity for the agglutination reaction, and in this sense it was
clinically utilized by Widal for the diagnosis of typhoid fever. It was
noticed, however, even by these earliest workers, that the serum of an
animal immunized against one microorganism would often agglutinate,
to a less potent degree, other closely related species. Thus, the serum
of a typhoid-immune animal may agglutinate the typhoid bacillus in
dilutions of 1 : 1,000, and the colon bacillus in dilutions as high as 1 : 200;
while the agglutinating power of normal serum for the colon bacillus
rarely exceeds 1 : 20. The specificity of the reaction for practical pur¬
poses, thus, is not destroyed if proper dilution is carried out, the degree
of agglutinin formation being always far higher for the specific organism
used in immunization than it is for allied organisms. The specific
immune-agglutinin in such experiments is spoken of as the chief ag¬
glutinin (hauptagglutinin) , and the agglutinins formed parallel with it,
as the partial agglutinin (metagglutinin) , terms introduced by Wasser-
mann. Hiss has spoken of these as major and minor agglutinins. The
relative quantities of the specific chief agglutinin and partial agglutinins
present in any immune serum depend upon the individual cultures used
for immunization, and the phenomenon is probably dependent upon
the fact that certain elements in the complicated bacterial cell-body
may be common to several species and find common receptors in the
animal body. Whenever an immune serum agglutinates a number of
members of the group related to the specific organism used for its produc¬
tion, the reaction is spoken of “ group agglutination.”

The partial agglutinins (metagglutinins) have been extensively
studied by Castellani 2 and others, by a method spoken of as the “ ab¬
sorption method.” This consists in the separate addition of bacterial
emulsions (agglutinogens) of the various species concerned in a group
agglutination, to the agglutinating serum. In this way, specific and par¬
tial agglutinins can be separately removed from the immune serum by
absorption — each by its corresponding agglutinogen. In such experi¬
ments all agglutinins will lie removed by the organisms used for im-

1 Gruber und Durham, loc. cit.

2 Castrtlani, Zeits. f. Hyg., xl, 1902.

LYSINS, AGGLUTININS, PRECIPITINS, ETC.

235

munization, a partial removal only resulting from the addition of allied
strains. This method has thrown much light upon the intimate relations
existing between members of various bacterial species, and has been
particularly valuable in the study of the typhoid-colon-dysentery
group. It is important to mention, however, that “groups” as de¬
termined by agglutination tests do not always correspond to classi¬
fications depending upon morphological and cultural characteristics.

An interesting phenomenon of great practical importance, which
has been noticed by a number of observers, and which may often be
encountered in routine agglutination tests, is the frequent failure of a
strongly agglutinating serum to produce agglutination if used in concen¬
tration, while in dilutions it produces a characteristic reaction. This has
been explained theoretically by what is known as the “ proagglutinoid
zone.” It is assumed that agglutinins may deteriorate as do toxins and
be converted into substances which are capable of combining with agglu¬
tinogen without causing agglutination. Such substances, as we will
see in discussing Ehrlich’s views on the structure of agglutinins, may
have a stronger affinity for agglutinogen than the agglutinins them¬
selves, and are, therefore, termed “proagglutinoids.” In strongly
agglutinating sera these proagglutinoids may be present in considerable
quantities and prevent the combination of agglutinin with agglutinogen.
In dilution, this proagglutinoid action would naturally become weaker
and of no actual significance in obscuring the reaction.

Agglutination, like other immune phenomena, is a manifestation of
broad biological laws and not limited to bacteria. Thus, as hemolysins
are produced by the injection of red blood cells, so hemagglutinins, or
substances which clump together red blood cells, are similarly formed.

PRECIPITINS

(' Coagulins )

In the year 1897, R. Kraus,1 of Vienna, demonstrated that the sera
of animals immunized against B. pestis, B. typhosus, and Vibrio
cholerse, when mixed with the clear filtrate of bouillon cultures of the
respective organisms, gave rise to macroscopically visible precipitates.
The precipitates occurred only when filtrate and immune serum were
homologous, that is to say, the animal from which the serum was ob¬
tained was immunized by the same species of microorganism as that
used in the test; and for this reason Kraus spoke of them as “specific

1 Kraus , Wien. klin. Woch., 1897.

236

INFECTION AND IMMUNITY

precipitates.’’ It was evident, therefore, that during the process of active
immunization with these organisms, a specific antibody had been pro¬
duced in the serum of the treated animal, which, because of its precipitat¬
ing quality, was named “ precipitin.” This peculiar reaction was soon
found to hold good, not only for the bacteria used by Kraus, but also for
other bacteria, few failing to stimulate the production of specific precipi-
tins in the sera of immunized animals. The phenomenon of precipitation,
however, is not limited to bacterial immunization, but has been found,
like the phenomena of agglutination and lysis, to depend upon biolog¬
ical laws of broad application. Thus, Bordet 1 found that the blood
serum of rabbits treated with the serum of the chicken gave a specific
precipitate when mixed with chicken serum. Tchistovitch 2 demon¬
strated a similar reaction with the sera of rabbits treated with horse and
eel sera. By the injection of milk, Wassermann,3 Schiitze,4 and others
produced an antibody which precipitated the casein of the particular
variety of milk employed for immunization. The reaction was thus
applicable to many albuminous substances. These substances, because
of their precipitin-stimulating quality, are called “ precipitinogens.”

Nature of Precipitins. — The precipitins, like the agglutinins, may be
inactivated by heating to from 60° to 70° C., and can not be reactivated
by the addition of normal serum or by any other known method.
Such inactivated precipitin, however, while unable to produce precipi¬
tates, has not lost its power of binding the precipitinogen. This is
shown by the fact that the inactivated precipitin, when mixed with pre¬
cipitinogen, will prevent subsequently added fresh precipitin from caus¬
ing a reaction. From these facts the conclusion has been drawn that
precipitin, like toxin, is built up of two atom groups,5 a stable hap-
tophore and a labile precipitophore group. By the destruction of the
latter, an inactive, yet neutralizing substance is produced which is
spoken of as “precipitoid.” The precipitoids, like protoxoids, have
a higher affinity for precipitinogen than the unchanged precipitin, and
thus are able to prevent the action of these.

Our own opinion would rather incline toward regarding the pre¬
cipitins as identical in structure with sensitizer or amboceptor — being
in fact “ album inoly sins” in the sense of Gengou. This problem is too

1 Bordet, Ann. de l’inst. Pasteur, 1899.

2 Tchistovitch, Ann. de l’inst. Pasteur, 1899.

3 Wassermann, Deut. med. Woch., 29, 1900.

4 Schiitze, Zeit. f. Hyg., 1901.

6 Kraus und v. Pirquet, Cent. f. Bakt., Orig. Bd. xxxii.

LYSINS, AGGLUTININS, PRECIPITINS, ETC.

237

complex to be discussed in detail in a summary of immunity as brief
as the one here presented.

Specificity. — The specificity of precipitins is a question of the
greatest importance, since, as we shall see, these bodies have been used
extensively for the differentiation of animal proteids. In regard to
bacterial precipitins it may be said that, just as in agglutination, there
is in precipitation a certain degree of “group reaction.” The pre¬
cipitin obtained with a colon bacillus, for instance, will cause precipita¬
tion with culture-filtrates of closely allied organisms, though in a less
marked degree. According to Kraus, such confusion may be easily
overcome by the proper use of dilution and quantitative adjustment,
similar to that used in agglutination tests. Norris1 found that the
precipitates given by immune sera with the filtrates of the homologous
bacteria were invariably heavier than those given with allied strains
and that the latter could be eliminated entirely by sufficient dilution.

Specificity becomes of still greater importance in the forensic use
of the precipitin reaction introduced by Uhlenhuth,2 Wassermann and
Schutze,3 and Stern.4 These authors found that the precipitin reaction
furnished a means of distinguishing the blood of one species from that
of another. Thus, blood spots, dissolved out in normal salt solution,
could be recognized by this reaction as originating from man or from
an animal, even after months of drying and in dilutions as high as 1 : 50,-
000. Since the value of this test depends entirely upon the strict
specificity of the reaction, this question has been studied with especial
care, notably by Nuttall.5 All who have investigated the subject find
the only important source of confusion in the blood of the anthropoid
apes. The specificity of the reaction, too, has been found to depend very
closely upon the amount of precipitin in the serum employed. If a
highly immune serum is insufficiently diluted, the reaction loses much
of its specific value.6 This source of error is easily eliminated in practice
by careful control and titration of the sera used for the tests.

Unlike agglutinins, precipitins have, so far, not been demonstrated
in normal sera.7

1 Norris, Jour. Inf. Dis., i, 3, 1904.

2 Uhlenhuth, Deut. med. Woch., xlvi, 1900; vi and xvii, 1901.

3 Wassermann und Schutze, Berl. klin. Woch., vi, 1901.

4 Stern, Deut. med. Woch., 1901.

5 Nuttall, Brit. Med. Jour., i, 1901; ii, 1902.

6 Kister und Wolff, Zeit. f . Medizinal-Beamte, 1902.

7 Kraus, loc. cit., and Norris, loc. cit.

238

INFECTION AND IMMUNITY

Theoretical Considerations Concerning Agglutinins and Precipitins.—

We have seen that Ehrlich evolved his theories of antibody forma¬
tion from his early views upon the absorption of nutritive substances by
the body cells, and we have followed, in more or less detail, the steps of
his reasoning as he developed his hypothesis in its application to the
antitoxic and the lytic substances. There still remained the agglutinins
and precipitins, bodies which because of their individual characteris¬
tics can be classed neither with the group of antitoxic, nor with that of
the lytic substances. These two antibodies, while by no means identical,
possess the common characteristics of being more thermostable than the
bacteriolytic substances, and of being insusceptible to reactivation by
normal serum. It is plain, therefore, that both agglutinating and
precipitating reactions take place without the co-operation of comple¬
ment. The substances which give rise to precipitins and agglutinins,

Fig. 61. — Ehrlich’s Conception of the Structure of Agglutinins and

Precipitins.

moreover, are not of the relatively simple soluble character of the toxins,
but are intrinsic portions of complex albuminous molecules, comparable
to and often identical with the true nutritive substances. For these
reasons Ehrlich believes that the cell-receptors for the various substances
which give rise to agglutinins and precipitins are neither of the simple
structure of the toxin receptor, nor of the double-haptophore nature of
the bacteriolytic receptors, but contain a single haptophore group for
the anchorage of the ingested material and at the same time a constantly
attached zymophore group or ferment by means of which the an¬
chored substance is transformed preparatory to its absorption by the
cell protoplasm. For the sake of clearness, this form of receptor may
be compared to a bacteriolytic or hemolytic amboceptor with a per¬
manently attached and inseparable complement.

Three forms of receptors, then, are proposed by Ehrlich in explana¬
tion of all known varieties of antibodies. The first, the simplest side

Body cell | Antitoxin

Antiferment

239

Fig. 62. _ The Structure of Cell-Receptors and Immune Bodies, According to Ehrlich s Conception,

(After Aschoff.)

240

INFECTION AND IMMUNITY

chains of the body cells, he calls “ receptors or haptines of the first order.”
These, overproduced and cast off, constitute the antitoxin and antifer¬
ments. Next “haptines of the second order” are the receptors planned
both for the anchorage and further digestion of antigens. These, free
in the circulation, are the precipitins and agglutinins. “Haptines or
receptors of the third order” are merely able to anchor a suitable sub¬
stance, but exert no further action upon it until re-enforced by the com¬
plement normally present in the serum. These, free in the circulation,
with a chemical group having avidity for the antigen, and another
complementophile group, are the amboceptors or immune bodies of
bacteriolytic, cytolytic, and hemolytic sera. (See Fig. 62.)

It is plain that all these receptors while still parts of their respec¬
tive cells, serve by their chemical affinity to attract and hold the foreign
substances injected; freely circulating, on the other hand, they serve
in preventing these substances from reaching the cells. As Behring
has aptly expressed it, the very elements which situated in the animal
cells render the body susceptible to toxic substances serve to protect
when circulating freely in the blood.

Bordet,1 at present the strongest antagonist of Ehrlich’s point of
view, claims that the conception of Ehrlich rests upon the basis of a
number of undemonstrated hypotheses. He asserts, and with justice,
that it has never been shown beyond question that the antibodies,
free in the serum, are identical with the receptors of the body cells
upon which the antigen originally acts.

In regard to agglutinins, Ehrlich, as we have seen, believes that it is
the agglutinin itself which, first uniting with its antigen by its hap-
tophore group, then causes clumping by its zymophore group. Now,
as a matter of fact, Bordet2 has shown that it is not the agglutinin itself
which agglutinates, but that agglutinin with its antigen forms a com¬
plex which is then agglutinable by the salt present in the solution. This
conclusion seems borne out by the later work of Gengou,3 Landsteiner
and Jagic,4 and others, who have shown that bacteria which have ab¬
sorbed other substances, such as uranium compounds, colloidal silicic
acid, etc., are subsequently agglutinable by salts. In consequence,
from these and other observations, Bordet concludes that it is neither
necessary nor accurate for the explanation of these phenomena, to

1 Bordet, Resume of Immunity in Bordet’s “ Studies in Immunity/’ transl. by Gay,
Wiley & Sons, 1909.

2 Bordet , Ann. de l’inst. Pasteur, 1899. 3 Gengou, Annal. Past.. 1904.

4 Landsteiner und Jagic. Wien. klin. Woch., iii, 1904.

LYSINS, AGGLUTININS, PRECIPITINS, ETC.

241

assume the conditions conceived by Ehrlich, but that the phenomenon
of agglutination consists primarily of the union of the antibody with its
antigen in a colloidal solution, and that the actual subsequent agglu¬
tination is a purely secondary phenomenon which depends possibly
upon a change in the physical properties of the emulsion — upon, as
he expresses it, its colloidal stability. A similar condition he assumes
for precipitins.

FURTHER FACTS AND THEORIES CONCERNING ANTIBODIES

Multiplicity of Amboceptors. — Fresh normal serum, as Nuttall 1 was
first to show, possesses moderate bactericidal powers which are lost
when the serum is subjected to heat. Since such inactivated normal
serum can be reactivated by the addition of fresh peritoneal exudates,
as the experiments of Moxter 2 have demonstrated, it is plain that the
bactericidal power of normal serum must depend, like that of immune
serum, upon amboceptor and complement. But normal serum often
exerts lytic powers upon several species of bacteria, or, in the case of
hemolytic tests, upon the red blood cells of several species of animals.
It is supposed that this multiplicity of action is due to the presence
in the normal serum of a variety of different amboceptors or immune
bodies. The method for proving this was devised by Ehrlich and
Morgenroth.3 They worked with normal goat’s serum, which has the
power of hemolyzing the red blood cells of guinea-pigs as well as
those of rabbits. Goat serum, inactivated by heat, was mixed with
rabbits’ corpuscles. After the mixture had been allowed to stand for a
short time, the corpuscles were removed by centrifugalization. The
serum was then reactivated and found still to possess its hemolytic
power for guinea-pigs’ blood, but to have lost this power for rabbits’
blood. By a similar technique, Pfeiffer and Friedberger 4 were able to
demonstrate the multiplicity of bactericidal immune bodies in normal
sera.

The immunity acquired by an animal as the result of treatment with
any of the various antigens is specific. An animal immunized against
the cholera vibrio, for instance, possesses marked bactericidal powers
for the cholera vibrio only.

1 Nuttall, loc. cit. 2 Moxter, Cent. f. Bakt., xxvi, 1896.

3 Ehrlich und Morgenroth, Berl. klin. Woch., 1901.

* Pfeiffer und Friedberger , Deut. med. Woch., 1901.

242

INFECTION AND IMMUNITY

According to Ehrlich’s views, the amboceptor or immune body atom
enters into direct relation with the substance used for immunization,
and it would seem natural therefore that the specificity of immune sera
depends entirely upon the increase of amboceptor or immune body.

Von Dungern,1 indeed, was able to show that the specific amboceptor
was increased as immunity was acquired, without there being a cor¬
responding enhancement of the complement. The chief difference be¬
tween a normal and an immune serum in this respect, therefore, con¬
sists in an enormous increase, in the latter, of the specific amboceptor.

Multiplicity of the Complement. — A number of very complicated ex¬
periments have been carried out by Ehrlich, Morgenroth,2 Sachs,3 and
others, which seem to show that the same serum may contain a variety
of complements. Similar conclusions have been drawn by Wechsberg 4
and by Wassermann,5 who demonstrated separate complements for bac¬
tericidal and hemolytic amboceptors in the same serum. Bordet 6 and
his school, on the other hand, deny the multiplicity of the complement,
and, basing their views upon numerous experimental data, contend that
any given serum contains but one alexin or complement. Buchner and
Gruber share the views of Bordet, and, in the light of recent work,
especially with complement fixation (see below), it seems more likely
that one and only one alexin exists in any given serum.

Anticomplements and Antiamboceptors. — Hemolytic sera, having
the power of destroying red blood cells, must necessarily prove in the
presence of sufficient complement to be powerful poisons when intro¬
duced into animals whose corpuscles they are able to injure. By care¬
ful and gradual dosage with such hemolytic sera, Ehrlich and Morgen¬
roth,7 as well as Bordet,8 have been able to produce immunity against
the hemolytic action. Thus antihemolytic sera have been produced,
the action of which may depend either upon the presence of anticomple¬
ment or of antiamboceptor. The presence of anticomplement in such
sera, it is believed, has been demonstrated by mixing inactivated
hemolytic serum with its respective red blood cells, then adding the

1 v. Dungern, Munch, med. Woch., xx, 1900.

2 Ehrlich und Morgenroth, Berl. klin. Woch., 1900.

3 Ehrlich und Sachs, Berl. klin. Woch., 1902.

4 W echsberg , Zeit. f. Hyg., 1902.

5 Wassermann , Zeit. f. Hyg., 1901.

6 Bordet, Ann. de Tinst. Pasteur, 1900 and 1901.

7 Ehrlich und Morgenroth, Berl. klin. Woch., xxxi, 1900.

8 Bordet, Ann. de Tinst. Pasteur, t. 14, 1900.

LYSINS, AGGLUTININS, PRECIPITINS, ETC.

243

antiserum and later complement. After centrifugalization and sepa¬
ration of the corpuscles, these may be dissolved by the addition of
fresh complement. This proves conclusively that there was no obstacle
in the original mixture to the absorption of the immune body by the
red blood cells, and that the antihemolytic properties of the serum
must be attributed to an anticomplement. This was the method of
experimentation employed by Ehrlich and Morgenroth.1 Antiambocep¬
tors have been produced by the same authors as well as by Bordet 2 and
Muller, 3 against hemolytic amboceptors.

Complementoids. — Ehrlich and Morgenroth and Muller have suc¬
ceeded in producing anticomplements by the treatment of animals with
normal heated serum. They explain this by assuming that the heating
has not entirely destroyed the complement in the normal serum, but that
this, analogous to toxin, possesses two groups, a haptophore and a zymo¬
phore group. Heating destroys the zymophore without affecting the
haptophore group. The resulting body, which corresponds to toxoid,
they call “ complementoid.”

Further evidence for the existence of such complementoids has been
claimed by Ehrlich and Sachs 4 in working with dog serum. Unheated
dog serum hemolyzes guinea-pig corpuscles. Heated to 52° C. for
thirty minutes, however, it no longer hemolyzes these corpuscles ow¬
ing go complement destruction. Such heated dog serum can be reacti¬
vated by fresh guinea-pig serum (fresh complement). If, however, the
corpuscles are left in contact with the heated dog blood for two hours,
reactivation by the guinea-pig serum no longer occurs — that is, the ad¬
dition of guinea-pig serum no longer causes hemolysis. They conclude
from this that the hemolytic amboceptor of the dog serum has been
attached by its complementophile group to complementoids produced
in the heating — leaving no point of attachment for the complement added
later. These experiments have failed of confirmation by Gay 5 — who
with Bordet denies the existence of complementoids.

Muir, on the other hand, claims to have demonstrated the existence
of complementoids by experiments too complicated to be detailed in
this place. The question of complementoids must be left undecided until
further work has been done.

1 Ehrlich and Morgenroth, loc. cit.

2 Bordet, loc. cit.

3 P. Th. Muller, Cent. f. Bakt., 1901.

* Ehrlich and Sachs, u Ehrlich Collected Studies on Immunity,” trans. by Boldnau.

6 Gay, Cent. f. Bakt., I, xxxix, 1905.

244

INFECTION AND IMMUNITY

Filtration of Immune Body and Complement. — Muir and Browning
have recently shown that, on the filtration of serum, amboceptor or
immune body will pass through the filter, whereas alexin or comple¬
ment is held back. The amboceptor filters equally well, whether or
not mixed with the complement.

The Fixation of Complement by Precipitates. — It has been found by
Gengou 1 and confirmed by Gay 2 and others, that when the serum of an
animal immunized with the serum of another species or with a foreign
albumin is mixed with a solution of the substance used in the immu¬
nization, the precipitate formed will remove complement from the mix¬
ture. In other words, precipitates formed by the reaction of precipitin
with its antigen will fix complement. This phenomenon is of great im¬
portance in complement-fixation tests such as those of Wassermann or
Noguchi (see below); for because of insufficient washing, the blood
cells used in producing the hemolytic amboceptor, may, from the
presence of serum, give rise to a precipitin as well as a hemolysin.
In the test done subsequently, a precipitin reacion may take place
and by thus removing complement may give a false result. The ab¬
sorption of complement by such precipitates takes place when the two
reacting factors, the precipitin and its antigen, are in extremely high
dilution — in fact, when a visible precipitate can not be observed.

Quantitative Relationship Between Amboceptor and Complement. — -
Morgenroth and Sachs,3 in studying the quantitative relationship ex¬
isting between hemolytic amboceptor and its complement, have suc¬
ceeded in showing that within certain limits an inverse relationship
exists between these two bodies. If for a given quantity of red blood cells
a certain quantity of amboceptor and complement suffices to produce
complete hemolysis, reduction of either the complement or the ambo¬
ceptor necessitates an increase of the other factor. As amboceptor is
increased, in other words, complement may be reduced and vice versa.
This result is of great importance in arguing against the original con¬
ception of Ehrlich in supposing these substances to act together mul¬
tiple for multiple as do compounds in chemical reactions.

Deviation of the Complement (Complement-Ablenkung) . — It was no¬
ticed by Neisser and Wechsberg 4 that in mixing together bacteria,

1 Gengou, Ann. Past., 1902.

2 Gay, Cent. f. Bakt., I, xxix, 1905.

3 Morgenroth und Sachs, “ Gesammel. Arb. fur Immunitatsforschung. ” Berlin,
Hirschwald, 1904.

4 Neisser und Wechsberg, Munch, med. Woch., xviii, 1901.

LYSINS, AGGLUTININS, PRECIPITINS, ETC.

245

inactivated bactericidal immune serum (immune body), and comple¬
ment in the test tube, a great excess of immune body hindered rather
than helped bactericidal action. As the amount of immune body in
the mixture was carried beyond the experimental optimum, bac¬
tericidal action became less and less pronounced, and was finally
completely suspended. They explain this by assuming that free im¬
mune body, uncombined with complement, has a greater affinity for the
bacterial receptor than the immune body combined with complement.
The complement is consequently diverted and prevented from activating
the amboceptor attached to the bacterial cell. Graphically, the con¬
ditions may be illustrated as follows:

Fig. 63. — Neisser and Wechsberg’s Conception of Complement

Deviation.

The above theory of Neisser and Wechsberg is here stated simply
because of the wide discussion it has aroused. In the light of our present
knowledge concerning the relations between antigen, amboceptor, and
complement, their conception is obviously erroneous.

246

INFECTION AND IMMUNITY

Fixation of the Complement. — Bordet and Gengou 1 in 1901, devised
an ingenious method of experimentation by which even very small
quantities of any given immune body (amboceptor) can be demon¬
strated in serum. The term “fixation of complement/ ’ by which their
method of investigation is now generally known, explains itself, as the
steps of experimentation are followed. They prepared the following
mixtures:

(a)

Bacteriolytic amboceptor
( Plague immune serum , heated )

Plague emulsion

Normal serum, heated

Plague emulsion

Complement Complement

( Fresh normal serum) ( Fresh normal serum)

To both of these after five hours was added
Hemolytic amboceptor
(Heated hemolytic serum)

Red blood cells

Results :

(a) showed no hemolysis.

(b) showed hemolysis + .

The conclusion to be drawn from this was that in (a) the presence
of immune body had led to absorption of all the complement. In (b) ,
there being no bacteriolytic immune body to sensitize the bacteria
and enable them to absorb complement, the latter substance was
left free to activate the subsequently added hemolytic ambocep¬
tors. The Bordet-Gengou phenomenon has been extensively used
by Wassermann and Bruck,1 Neisser and Sachs,2 and others to demon¬
strate the presence of immune bodies in various sera. (See p. 262.)

It should be noted that this method, if valid, must presuppose
the identity of the hemolytic and bactericidal complement in the
activating serum.

Complement fixation will be more extensively discussed in the
section dealing with the Wassermann reaction.

1 Bordet et Gengou, Ann. de Tinst. Pasteur, 1901.

1 Wassermann und Bruck, Med. Klin., 1905.

2 Neisser und Sachs, Berl. klin. Woch., xliv, 1905, and i, 1906.

LYSINS, AGGLUTININS, PRECIPITINS, ETC.

247

The Specificity of Hemolysins. — In the sections preceding we have
seen that the blood cells of one animal, injected into an animal of
another species, give rise to a hemolytic substance in the blood
serum of the second animal, which is strictly specific for the variety of
cells injected. Such hemolysins, when produced in one animal

1. ^j| t Complement

SijphiliUc
immune or*
anti body

Together at
37.50 C.
for one hour

-A.nH<$en,

_ Jtaemolytic
Amboceptor

- Red blood cell

If (2) present, no Haemolysi s .

If (2) not present, + Haemolysis.

Fig. 64. — Schematic Representation of Complement Fixation in the

Bordet-Gengou Reaction.

against blood cells of another species, are spoken of as heterolysins .
In studying the nature of hemolysis, Ehrlich and Morgenroth 3 now
discovered that hemolysins could also be produced if an animal was
injected with red blood cells of a member of its own species. Such
hemolytic substances they called isolysins. In their experiments they
injected goats with the washed red blood corpuscles of other goats
and found that the serum of the recipient developed the power of

3 Ehrlich und Morgenroth, Berliner klin. Woch., xxi, 1900.

248

INFECTION AND IMMUNITY

causing hemolysis of the red blood cells of the particular goat whose
blood had been used for injection. It did not, however, possess the
power of producing hemolysis in the blood of all goats, nor did it pro¬
duce hemolysis with the red corpuscles of its own blood. It is thus
shown that the specificity of the hemolysins extends even within the
limits of species, and is, to a certain extent at least, an individual
property.

The production of autolysins , that is, of substances in the blood serum
which will produce hemolysis of the individual's own corpuscles, has,
so far, been unsuccessful.

Ehrlich and Morgenroth, in the course of these experiments, further¬
more succeeded in showing that the injection of isolysins into animals
produced antiisolysins, and that these again were strictly specific.

The almost universal failure of autolysin production has found no
satisfactory explanation. It is supposed by Ehrlich and Morgenroth
that autolysins may be formed, but are probably speedily neutralized
by the production of antiautolysins.

The clinical significance of the presence of isolysins and possibly of
autolysins in human beings, is too evident to require much discussion.
A practical and extremely interesting result which these investigations
have yielded is that of Donath and Landsteiner,1 who discovered an
autolysin in the blood serum of patients suffering from paroxysmal
hemoglobinuria. In these cases the sensitizing substance or ambo¬
ceptor appeared to be absorbed by the red blood cells only at low tem¬
peratures — probably in the capillaries during exposure to the cold, and
hemolysis subsequently resulted in the blood stream by the action
of complement. These observations have been confirmed by other
writers, but the phenomenon is surely not present in all cases of paroxys¬
mal hemoglobinuria. The writers have had occasion to examine care¬
fully several clinically typical cases with negative results.

1 Donath und Landsteiner , Miinch. med. Woch., xxxvi, 1904.

CHAPTER XYI

THE TECHNIQUE OF SERUM REACTIONS

Obtaining Serum from Animals and Man. — To obtain blood serum
from man, the blood may be taken from the finger or the ear, either
into a sterile centrifuge tube or into a Wright capsule. (See section
on Opsonins, page 284.) When taken into a centrifuge tube, the
blood is allowed to clot and the serum separated from the coagulum by
a few revolutions of the centrifuge. When larger quantities of blood
are desired, it may be taken with a syringe from the median basilic
vein and either slanted in sterile test tubes in the ice chest or put into
centrifuge tubes and centrifugalized.

In bleeding small laboratory animals, a number of methods may be
employed, depending upon whether a large or small amount of serum is
required.

The animals most frequently used for laboratory purposes are
rabbits. To obtain small quantities of serum from rabbits, the animals
may be bled from the marginal vein of the ear. In doing this, a satis¬
factory yield of blood may be obtained by following a simple method
devised by Wadsworth. The animal is strapped upon a tray and under¬
neath it is placed a rubber bag filled with warm water. This keeps the
body temperature of the rabbit somewhat higher than normal, causes
dilatation of the vessels, and thus facilitates the flow of blood. The
tray is then placed upon a simply constructed easel so that the animaFs
head hangs downward. The skin over the ear vein is shaved and
sterilized, and an incision is made into the vein in its long axis with a
sharp knife. The blood is caught in test tubes or centrifuge tubes.

When larger quantities of blood are desired, the animal is strapped
down, anesthetized, and the neck shaved and sterilized. The carotid
artery is then isolated by dissection. In rabbits, the carotid artery may
be found lying just lateral to the trachea and deeply placed, and must be
carefully separated from the pneumogastric nerve by blunt dissection.
The distal end of the artery is then tied off and the proximal end tem¬
porarily closed with a small clamp. (This clamp should be rather weak
and not exert sufficient pressure to injury the artery and cause throm*

249

250

INFECTION AND IMMUNITY

bosis.) The artery is then raised out of the wound on a knife or forceps
handle and, with sharp-pointed scissors, a small incision is made into
but not through the vessel. A small glass cannula is now introduced into
the proximal end of the artery through the incision and tied into place
by a thread. To this cannula a small rubber tube fitted with a pinch-
cock should have been attached, the whole being sterilized. The clamp
on the artery is then released and the blood allowed to flow into sterile
test tubes which are slanted and placed in the cold for separation of the
serum. A larger yield of serum will be obtained if, after coagulation,
the clot is separated from the glass with a sterile platinum wire.

In obtaining blood from larger animals, horses, sheep, etc., a cannula
may be introduced into the jugular or internal saphenous veins. The
skin is shaved and sterilized and a rubber tourniquet placed about the
neck or thigh, as the case may be, in order to cause the vein to stand
out. A small incision may be made through the skin over the vein, but
is not necessary. A cannula, with rubber tubing attached, is then plunged
into the vein and the blood caught in sterile high cylindrical jars,
allowed to clot, and placed in the refrigerator. The serum is taken off
after twenty-four to forty-eight hours with sterile pipettes.

Agglutination Tests. — For the determination of the agglutinating
power of serum it is necessary to make suitable dilutions of the serum,
and to prepare an even emulsion of the microorganisms to be tested.
The test may be made microscopically or macroscopically. The micro¬
scopic test is the one in general use in the diagnosis of typhoid fever,
and is occasionally applied to some other diseases. In its application
to typhoid fever it is usually spoken of as the Gruber-Widal reaction.

Twelve- to eighteen-hour broth cultures of the typhoid bacillus,
grown at incubator temperature, may be used. It is preferable, how¬
ever, to use an emulsion of a twelve to twenty-four hour old agar culture
in physiological salt solution (0.85 per cent) . The salt-solution emulsion
is made by adding about 10 c.c. of normal salt solution to the fresh agar
slant culture, carefully detaching the culture from the surface of the
agar with a flexible platinum wire, and pipetting off the emulsion thus
made. With some microorganisms it is sufficient simply to allow the
larger clumps to settle and to pipette off the supernatant turbid emulsion.
With other microorganisms, the tendency to form clumps makes it
necessary to resort to further methods of securing an even distribution
of the bacteria. This may be done either by sucking the emulsion in and
out through a narrow pipette held perpendicularly against the bottom of
a watch glass, as in Wright’s technique for the opsonic test (see section

THE TECHNIQUE OF SERUM REACTIONS

251

on Opsonins, p. 285), or by carefully rubbing the clumps against the
watch glass with a stiff platinum wire. In the case of the tubercle ba¬
cillus not even this suffices, but it becomes necessary to grind the moist
bacillary masses in a mortar before emulsifying. With the tubercle bacil¬
lus, too, it is preferable to use salt solution at 1.5 per cent concentration.

In preparing cultures of streptococcus and pneumococcus for
agglutination tests, it has been found convenient by Hiss to grow
microorganisms for about four days in flasks of a one per cent glucose,
two per cent pepton meat-infusion broth, to which has been added one
per cent of calcium carbonate. (See page 126.) The insoluble calcium
carbonate sinks to the bottom, but by neutralizing the inhibiting acid
formed in the broth by the microorganisms, permits the development
of a mass culture. The flasks should be shaken thoroughly at least
once a day. The broth may be pipetted off and the clumps may be
removed by a few revolutions of a centrifuge. Without this technique
it is sometimes difficult to get sufficient growths of these bacteria for
any quantity of emulsion unless large surfaces of agar are employed in
special receptacles or by making many slant cultures.

The serum dilutions are obtained by first making a one to ten
dilution of serum with normal salt solution. The serum used for this
purpose may be cleared of red blood corpuscles by centrifugalization.
From the one to ten dilution any number of higher dilutions may be
made, simply by mixing given parts of the one to ten dilution with
normal salt solution; thus one part of a one to ten dilution plus an equal
quantity of salt solution gives a dilution of one to twenty. One part of
one to ten dilution plus two parts of normal salt solution gives one to
thirty, and one part of one to twenty dilution plus one part normal salt
solution gives one to forty, etc. It must not be forgotten that, when
equal parts of the serum and bacillary emulsion have been mixed, each
one of these dilutions is doubled.

In making the microscopic agglutination test , minute equal quanti¬
ties of serum dilution and bacterial emulsion are mixed upon the sur¬
face of a cover-slip. The mixture may be made either by measuring out a
drop of each substance with a standard platinum loop, depositing them
close together on the cover-slip, and mixing; or, more exactly, equal quan¬
tities may be sucked up, each to a given mark, in a capillary pipette,
mixed by suction in and out, and then deposited upon the cover-slip.
The cover-slip is inverted over a hollow glass slide, the rim of which has
been greased with vaseline. The drop is then observed, preferably
through a (Leitz) No. 7 lens, ocular No. 3.

252

INFECTION AND IMMUNITY

The macroscopic agglutination test, always preferable for exact
laboratory research, is made in narrow test tubes especially designed
for the purpose, measuring about 0.5 cm. in diameter and about 5 cm. in
length (Fig. 60, p. 230).

In these test tubes equal quantities, usually 1 c.c. each, of serum
dilution and emulsion are mixed. A series of tubes is prepared, in each
subsequent one of which the dilution is higher. These mixtures may
be placed in the incubator for a few hours and then kept at room tem¬
perature. It has been observed by Hiss that after removal from the in¬
cubator agglutination is in some instances hastened by transference to
the ice chest. When agglutination takes place in these tubes, clumps of
bacteria may be seen to form, which settle to the bottom of the tube, very
much like snow-flakes. The surface of the sediment is heaped up and
irregular. The supernatant fluid becomes entirely clear. When the
reaction does not occur the sediment is an even, granular one with a flat
surface, and the emulsion remains turbid.

Instead of using test tubes as described above, Wright has sug¬
gested the use of throttle pipettes of comparatively large diameter into
each of which at least three or four different dilutions can be sucked
with a nipple, a small air bubble being left between the mixtures. By
sealing the distal end of these pipettes in a flame the various dilutions
are kept at a distance from each other, and the pipettes may be set on
end in a tumbler and observed just as are the test tubes (Fig. 68, p. 285).

Precipitin Tests. — In an earlier section on precipitins we have seen
that precipitates are formed when clear filtrates of bacterial extracts
or of broth cultures are mixed with their specific immune sera.
Such precipitin reactions are not limited to the realm of bacteria, but
have a broad biological significance, in that specific precipitating sera
may be produced with proteids of varied source.

For actually carrying out a precipitin test, the following reagents are
required :

1 . A specific precipitating antiserum (antibacterial or antiproteid) ;

2. A bacterial filtrate or proteid solution.

The Production of Precipitating Antisera.1 — Antibacterial
precipitins may be produced in animals by any one of a variety of
methods. Animals, preferably rabbits, are injected either with broth
cultures or with salt solution emulsions of agar cultures of the bacteria,
in gradually increasing quantities. Five or six injections are given in-

1 R. Kraus, Wien. klin. Woch., 1897; Norris, Jour. Inf. Dis., 1 and 3, 1904.

THE TECHNIQUE OF SERUM REACTIONS

253

traperitoneally or intravenously, at intervals of from five to six days,
the dosage and mode of administration being adapted in each case to
the pathogenic properties of the microorganisms in question. It has
been asserted by Myers 1 that when pepton-broth cultures are used for
immunization a specific precipitin for pepton may be formed which by
giving a precipitate with a culture filtrate containing pepton may lead
to error. This observation could not be confirmed by Norris.2

The immunized animals should be bled about seven to twelve days
after the last injection of bacteria.

Specific precipitating antisera against proteid solutions are prepared
by methods analogous to those employed for the production of anti¬
bacterial sera. A variety of methods have been described. The sera
or proteid solutions used should be sterile. This may be accomplished
by filtration through small porcelain filters. The injections into animals
may be made subcutaneously, intraperitoneally, or intravenously. The
subcutaneous route has no advantages unless the substances to be used
are contaminated.

Nuttall advises the use of rabbits. The animals should be weighed
from time to time, and if considerable loss of weight ensues during im¬
munization, the intervals between injections should be increased.
Dosage should be carefully graded, beginning, in the case of an animal
serum, for instance, with 2 c.c. and increasing gradually through 3, 5,
and 8 c.c. to possibly 15 c.c. at the last injection. A single injection of
a large quantity has occasionally yielded a precipitating serum of con¬
siderable strength,3 but this method is not usually successful. Injec¬
tions are made at intervals of from five to seven days. Seven to twelve
days after the last injection the animals may be bled, and a preliminary
test made to ascertain the precipitating value of the serum. If this is
insufficient for the desired purposes, more injections may be made before
the animal is finally bled. Bleeding should be done seven to twelve
days after the last injection. Such sera may be preserved by sealing in
glass bulbs and keeping in the dark and at a low temperature. If a
preservative is to be added, Nuttall recommends chloroform, but dis¬
approves of the phenols, because of occasional turbidity produced by
these.

The precipitating antisera used for the tests should be absolutely

1 Myers, Lancet, ii, 1900.

2 Norris, loc. cit.

3 Michaelis, Deut. med. Woch., 1902.

254

INFECTION AND IMMUNITY

clear. If turbidity is present, the sera should be filtered through small
Berkefeld or porcelain candles.

Preparation of Bacterial Filtrates and Proteid Solutions
for Precipitin Tests. — Bacteria may be grown in broth made of
Liebig’s beef extract five-tenths per cent, pepton one per cent, NaCl five-
tenths per cent, and having an initial reaction of neutrality or five-tenths
per cent acidity to phenolphthalein. The cultures are incubated for
times varying from a week to several months, and are then filtered
through porcelain or Berkefeld candles until perfectly clear. Bacterial
extracts may also be made by emulsifying agar cultures in salt solution,
placing at 37.5° C. in the incubator for a week or longer, and filtering.
More rapid extraction of bacteria may be accomplished by repeated,
rapid freezing and thawing of salt-solution emulsions. This is easily
and simply done by placing the test tubes in battery jars filled with
brine and cracked ice.

Proteid solutions to be tested should be made in salt solution. When
dealing with blood stains, as is frequently the case in doing the test for
forensic purposes, the stains should be dissolved out in salt solution, an
approximate dilution of one in five hundred being aimed at. This solution
if turbid should be filtered through a small porcelain filter. Before use
it should be perfectly clear and colorless, should show a faint cloud on
boiling with dilute acetic acid, and, according to Muller, should show
distinct frothing when shaken.

When the reaction is to be done with the purpose of determining the
nature of meat (detection of horse-meat substitution for beef, etc.),
about 20 to 40 grams of the suspected meat are macerated by being
placed in a flask, and covered with 100 c.c. of physiological salt solution.
This mixture is allowed to infuse at room temperature for three to four
hours, and is then placed in the refrigerator for twelve hours or more.
At the end of this time 2 c.c. may be poured into a test tube and shaken.
If frothing 1 appears easily and profusely, the extract is ready for use.
It is then filtered clear, either through paper, or, if this is unsuccessful,
through infusorial earth in a Buchner or Nutsche filter. Berkefeld
filters may also be used, but their use is less simple. The clear solution
is then further diluted until the addition of concentrated HN03 produces
only a slight even turbidity. Before use, furthermore, the reaction of
the meat extract should be tested, and if necessary adjusted to neutrality
or slight acidity or alkalinity.

1 P. Th, Muller , “ Technik d. serodiagnos. Methoden.”

THE TECHNIQUE OF SERUM REACTIONS

255

In the actual test with bacterial filtrate, the procedure is as follows:
In a series of narrow test tubes, the following mixtures are made:

Tube 1. Antibacterial serum .5 c.c. + bacterial filtrate 1. c.c.

“ 2. Normal serum .5 c.c. + bacterial filtrate 1. c.c.

“ 3. Antibacterial serum .5 c.c. + salt solution 1. c.c.

“ 4. Salt solution .5 c.c. + bacterial filtrate 1. c.c.

Place the tubes in the incubator at 37.5° 0. In a positive test, tube
1 only should show a haziness which develops into a distinct cloudiness
or even a flocculent precipitate within one hour. Tubes 2, 3, and 4
should remain clear.

For the testing of an unknown proteid with the serum of an animal
immunized with the proteid sought for, the technique of the test is as
follows :

1. 0.1 c.c. immune serum + 2 c.c. unknown proteid solution.

2. 0.1 c.c. immune serum + 2 c.c. known proteid solution of variety suspected

(similarly diluted).

3. 0.1 c.c. immune serum + 2 c.c. proteid solution of different nature (similarly

diluted).

4. 0.1 c.c. immune serum + 2 c.c. salt solution.

5. 2 c.c. unknown proteid solution.

The test is positive when a precipitate appears in tube 1 and in tube
2, but not in any of the others. The precipitate should appear definitely
within fifteen to twenty minutes.

Bactericidal and Bacteriolytic Tests. — The bactericidal and bac¬
teriolytic powers of serum may be tested either in the animal body
or in the test tube. The most common bacteriolytic test, in vivo , is
that which is known as Pfeiffer’s test. This test depends upon the
fact considered in a previous section, that bacteria, when injected
into the peritoneal cavity of a guinea-pig, together with a homologous
immune serum, undergo dissolution.

As practiced in bacteriological work, the test finds a double appli¬
cation. It may be carried out either for the determination of the
specific bacteriolytic power of a given serum against a known micro¬
organism, or for the identification of a particular microorganism by
means of its susceptibility to lysis in a known immune serum.

1. Determination of the bacteriolytic power of serum against a
known microorganism in vivo : 1

i P. Th. Muller, “ Technik d. serodiagnos. Methoden,” Jena, 1909.

256

INFECTION AND IMMUNITY

A number of dilutions of the serum are made with sterile neutral
bouillon or salt solution, ranging from 1 in 20 to 1 in 500, or higher. It
is convenient to make a first solution of 1 in 20. One c.c. of this mixed
with 4 c.c. of broth will give 1 in 100. One c.c. of the 1 in 100 dilution
with 1 c.c. of broth, 2 c.c. of broth and 4 c.c. of broth will give 1 in 200,
1 in 300, and 1 in 500, respectively. Into one cubic centimeter of each
of these dilutions there is placed one platinum loopful of a twenty-four-
hour agar culture of the microorganism against which the serum is to
be tested. Into another test tube is placed 4 c.c. of broth, without
serum, and with one loopful of the microorganisms. The mixtures
are thoroughly emulsified in each case by rubbing the bacteria against
the sides of the tube with the platinum loop.

Intraperitoneal injections into guinea-pigs are then made of 1 c.c.
of each of the serum-dilution-bacterial-emulsions. A control guinea-pig
(better two or three) receives lc. c. of the broth emulsion — one-fourth
as many bacteria, therefore, as the animals receiving the serum
dilutions.

Before making the injections, areas on the lateral abdominal walls
of the guinea-pigs are shaved, and small incisions made through the

Fig. 65. — Capillary Pipette for Removal of Exudate in doing the

Pfeiffer Test.

skin, down to the muscular layers. The needle of the syringe is then
introduced perpendicular to the skin until it has penetrated the peri¬
toneum, and then carefully slanted to avoid puncturing the gut. The
animals need not be strapped down during this procedure and after¬
ward may be allowed to run about.

After one-half hour, and again after one hour has elapsed, a drop
of peritoneal exudate is removed from each guinea-pig and examined
in the hanging drop for granulation and swelling of the bacteria. The
method of obtaining the peritoneal exudate is as follows: Small glass
tubing is drawn out into capillary pipettes, the ends of the capillaries
being again drawn to fine points in a small yellow flame. A number of
such pipettes should be prepared before the test is begun. The guinea-
pig is then held down upon a table, either by an assistant or by the left
hand of the operator, and the point of the pipette pushed through the
cut in the abdominal wall into the peritoneum by a sharp, quick thrust-

THE TECHNIQUE OF SERUM REACTIONS

257

ing motion. A column of peritoneal fluid will run into the glass tubing
by capillary attraction; this can then be blown out upon a cover-slip
for hanging-drop examination or may be blown upon a slide, smeared,
and examined after staining. The reaction is regarded as positive if
within thirty minutes to an hour the peritoneal exudates of the animals
receiving immune sera contain only swollen or disintegrated microor¬
ganisms, while in that of the control animals only well-preserved and
undegenerated bacteria are found. In dealing with typhoid bacilli
and cholera spirilla, in connection with which the test is most often
used, active motility in the controls is of much help. Should there be
extensive degeneration of the bacteria in the exudate of the control
animals the test is of no value.

2. Identification of a microorganism by observing its a susceptibility to
lysis in a known immune serum in vivo:

The technique for this test is practically the same as that of the
preceding except that in this case we require a potent known immune
serum and normal serum for control. It is necessary, furthermore, that
by previous tests we should know the degree of dilution in which the
immune serum will cause complete bacteriolysis of the microorganism
used in its production. Thus, if we are employing a typhoid immune
serum and are about to test by this method an unknown Gram-negative
bacillus, we must know the titer of the serum for the typhoid bacillus
itself.

Mixtures are then made of dilutions of this serum and definite
quantities of the microorganism to be tested. It is best, always, to
employ from ten to one hundred times the amount of immune serum
which suffices to produce lysis with its homologous microorganism.
Thus, if the serum has been found to be active in dilutions of 1 : 1,000, it
is employed in the test in dilutions of 1 : 1,000, 1 : 100, and 1 : 10. These
dilutions are then injected into guinea-pigs in quantities of 1 c.c. together
with the bacteria to be tested, and control guinea-pigs are injected with
undiluted normal serum mixed with the bacteria and with salt solution
and the bacteria. The exudates are then observed in the same way as
in the preceding experiment.

Bactericidal Reactions in the Test Tube— Bactericidal reactions
in the test tubes may be made by mixing in small sterile test tubes,
definite quantities of the bacteria with inactivated serum and com¬
plement, the latter in the form of unheated normal serum. The
mixtures, diluted with equal volumes of neutral broth or salt solution,
are set away for a definite time three to four hours in an incubator at

258

INFECTION AND IMMUNITY

37.5° C., and equal quantities from all the tubes are then inoculated
into melted agar at 40° C., and plates are poured. Control plates
must be made in each case with mixtures of similar quantities of
bacteria in salt solution, and similar quantities of bacteria in normal
serum. By colony counting after the plates have developed, it is then
possible to estimate the degree of bacterial destruction in any of the
given dilutions.

In actually carrying out the test, dilutions of the inactivated serum
are first made, ranging from 1 : 10 to 1 : 1,000 and over. An emulsion
of bacteria from a twenty-four-hour agar slant is then made in salt solu¬
tion, or a twenty-four-hour broth culture properly diluted may be used.
Complement is obtained by taking fresh normal rabbit serum and dilut¬
ing it with salt solution 1 : 10 or 1 : 15. Into a series of test tubes, then,
1 c.c. of each of the serum dilutions is placed, and to each tube is added
0.5 c.c. of the diluted fresh normal rabbit serum (complement). To these
mixtures the bacteria are then added. In adding the bacterial emulsion
to these tubes, the writers have found it more accurate to discard the
use of the platinum loop and to measure the bacterial emulsion in a
marked capillary pipette such as that used in the opsonin test. (See page
285, Fig. 68.) The controls are set up in a similar way, all of them con¬
taining a similar quantity of bacterial emulsion, one control containing
1.5 c.c. of salt solution, another control containing 1 c.c. of salt solution +
0.5 c.c. of the diluted complement, and the third control containing in¬
activated normal serum 1 c.c. +0.5 c.c. of diluted complement. Defi¬
nite quantities of these mixtures, taken with a standard loop, or
preferably with a capillary pipette, are plated in agar immediately
after mixing.

BACTERICIDAL TEST IN VITRO

(To Determine the Bactericidal Power of a Typhoid Immune Serum

against Typhoid Bacilli).

Plates
Poured
After 3 Hrs.
at 37° C.

1 C.(

4-0.5

1 “

“ “ “ 1:400 +0.5

44 44 44

+ 0.5

4 4

1 “

“ “ “ 1:800 +0.5

44 44 44

+ 0.5

1 “

“ “ “ 1:1600 +0.5

44 44 44

+ 0.5

1 “

“ “ “ 1:3200 +0.5

44 44 44

+ 0.5

1 “

“ “ “ 1:6400 +0.5

44 44 44

+ 0.5

1 “

“ “ “ 1:12800 + 0.5

44 44 44

+ 0.5

4 4

1 “

“ “ “ 1:25600 + 0.5

44 44 44

Controls

+ 0.5

4 4

la i

1.5 c.c. NaCl + 0.5 Typh. Emulsion

Plated

+ 0.5
+ 0.5

after 3 hrs. >

a 4 4. 4 1 I

Colonies.

100-1,000

Colonies

More than
10,000
Colonies

+ 0.5 c.c. Rab. Ser. 1:15

THE TECHNIQUE OF SERUM REACTIONS

259

After incubation for two or three hours similar quantities are again
measured into tubes of melted agar with the capillary pipette. With a
little practice, great accuracy in these measurements can be acquired.
The inoculated agar tubes are very thoroughly mixed, and plates are
poured. At the end of twenty-four hours’ incubation, an enumeration
of the colonies in the various plates is made and the results are compared.

The in vitro bactericidal tests have been employed, practically, chiefly
in the diagnosis of typhoid fever by Stern and Korte.1 While the serum
of normal individuals shows practically no bactericidal power for
typhoid bacilli, the sera of typhoid patients may be actively bacteri¬
cidal in dilutions as high as 1 : 50,000.

Hemolytic Tests. — Determination of the hemolytic action of blood
serum, bacterial filtrates, and of a variety of other substances, such as
tissue extracts and animal and plant poisons, is frequently made in
bacteriological laboratories. Familiarity with the methods of carrying
out such tests is especially essential since hemolytic tests are also em¬
ployed in determining other serum reactions, such as the “ complement-
fixation tests ” discussed in another section.

For these tests it is necessary to prepare washed red corpuscles
of the species of animal against which the hemolysins are to be
tested, and to obtain these, blood may be taken in one of the
following ways :

A. If small quantities of blood corpuscles are desired, the blood may
be received into a sterile test tube into which a copper or other wire bent
into a loop at the lower end has been introduced. This is used to
prevent clotting and to remove the fibrin. Immediately after receiving
the blood into this tube, the wire is twirled between the fingers so that
the blood is beaten by the wire as by an egg-beater. At the end of five
minutes of continuous agitation, the fibrin adhering in a mass to the wire
may be lifted out. The corpuscles are then washed and centrifugalized
in several changes of salt solution to remove all traces of serum, and
are finally emulsified in salt solution.

B. The blood may be taken into a centrifuge tube and immediately
centrifugalized before clotting has taken place. The plasma is then
poured off and the corpuscles are washed with salt solution, as before,
to remove the serum.

C. The blood may be taken directly into a solution containing
five-tenths per cent sodium chlorid and one per cent sodium citrate.

1 Stern und Korte, Berl. klin. Woch., 1904.

260

INFECTION AND IMMUNITY

The corpuscles are concentrated by centrifugalization, the citrate solu¬
tion is decanted, and corpuscles are washed with salt solution, as before,
to remove the serum.

D. When large 'quantities of blood are desired, either from man or
from an animal, the blood may be received directly into a flask into
which a dozen or more glass beads or short pieces of glass tubing have
been placed. The flask is shaken for five or ten minutes, immediately
after the blood has been taken and, in this way, defibrination is accom¬
plished.

Since, for comparative tests, it is necessary to establish some stand¬
ard concentration of red blood cells, it is customary in these tests to
employ a five per cent emulsion of corpuscles in salt solution. To
obtain this, the thoroughly washed corpuscles from one volume of the
original blood are mixed with nineteen parts of 0.85 per cent salt solu¬
tion.1 Such an emulsion, if kept sterile and in the refrigerator, will
serve for hemolytic tests for from one to three days. An emulsion
should not be used if the supernatant salt solution shows any transpa¬
rent redness, as this indicates hemolysis.

If the substance in which hemolysins are to be determined is serum,
this may be used either as such or it may be inactivated by exposure to
56° C. in a water bath, and to each test, complement may be added in
the form of fresh guinea-pig or rabbit’s serum. No absolute rule for
the quantity of complement to be used in these tests can be given. As
a starting-point, however, when 1 c.c. of a 5 per cent emulsion of red
corpuscles is used, it is best to use about 0.1 to 0.2 c.c. of fresh guinea-
pig serum as complement.

In the actual test, mixtures are made of the corpuscle emulsion, the
inactivated immune serum, and complement in small test tubes and the
volumes of the various tubes made equal by the addition of definite
quantities of salt solution. The contents of the tubes are thoroughly
mixed and the tubes put in the incubator or in a water bath at
37.5° C. If complete hemolysis occurs, the fluid in the tube will as¬
sume a deep Burgundy red. If no hemolysis occurs, the fluid will
remain uncolored and the corpuscles will settle out. Incomplete hemo¬
lysis will be evidenced by a lighter tinge of red in the tube and by the
settling out of a varying quantity of blood corpuscles.

1 The method here given was formerly much employed. It is now the general
practice, however, to use one volume of the actual sediment to nineteen volumes of
salt solution.

THE TECHNIQUE OF SERUM REACTIONS

261

In all hemolytic tests the time element is important. No hemo¬
lysis should be adjudged as incomplete unless at least one hour has
elapsed.

It is often necessary to carry out hemolytic tests on the blood
corpuscles of one human being with the serum of another in order to
determine the advisability of performing transfusion. In this case, the
serum of the recipient is mixed with a corpuscle emulsion of the cells of
the donor, and vice versa. Since it is often difficult to obtain much
blood for these tests, the writers have found it convenient to make the
test in throttle pipettes, instead of in test tubes. By this technique, ten
or fifteen drops of blood and a very small amount of serum will suffice.
It should be stated, however, that whenever sufficient quantities of
serum can be obtained this technique should not be employed.

The Determination of Antibodies in Sera by Complement Fixation. —
The principle of complement fixation, discovered by Bordet and Gengou1
in 1901, has been utilized both in bacteriological investigations, and in
practical diagnosis for the determination in serum of the presence of
specific antibodies. Although spoken of in another section of this book,
it may be well to review, briefly, the principles of the Bordet-Gengou
phenomenon. The reaction depends upon the fact that when an antigen,
i.e., a substance capable of stimulating the formation of antibodies in
animals or man, is mixed with its inactivated antiserum, in the presence
of complement, the complement is firmly fixed by the combined immune
body and antigen in such a way that it can no longer be found free in
the mixture. If such a mixture is allowed to stand at a suitable tem¬
perature for an hour or more, and to it is then added an emulsion of red
blood cells together with a suitable quantity of inactivated hemolytic
serum, no hemolysis will take place, since there is no free complement
available to complete the hemolytic system. If, on the other hand, the
original mixture contains no antibody for the antigen used, the comple¬
ment present is not fixed and is available for the activation of the
hemolytic serum later added.

The reaction thus depends, in principle, intimately upon the fact
that neither antigen 2 alone, nor amboceptor (antibody) alone, can fix
complement, but that this fixation is carried out only by the combina¬
tion of antigen plus amboceptor. Any specific amboceptor can be deter¬
mined by this method, provided the homologous or stimulating antigen

1 Bordet and Gengou, Ann. de l’inst. Pasteur, xv, 1901.

2 Bordet and Gay, Ann. de Tinst. Pasteur, xx, 1906.

262

INFECTION AND IMMUNITY

is used; and vice versa , by the use of a known antibody a suspected
antigen may be determined.

When testing immune sera for amboceptors given rise to in man or
animals by microorganisms which can be cultivated, either the whole
bacteria or extracts of the bacteria may be used as an antigen.

For the diagnosis of syphilis by this method, in the so-called “ Wasser-
mann reaction,” the antigen employed was originally obtained by the
extraction of syphilitic organs, in which free syphilitic antigens, i.e.,
uncombined products of Spirochscte pallida, were assumed to be present.
As this reaction has recently become prominent and has proven of no
inconsiderable diagnostic value, the technique given below for immune-
body determination by complement fixation will be that utilized in
the Wassermann test for syphilis.

The reader will, however, bear in mind that the test may be
applied to other diseases simply by the substitution of the suitable,
specific antigen. Thus, when cultivatable bacteria are used as antigens,
Bordet and Gengou make use of a thick salt-solution emulsion of a
twenty-four-hour agar-slant, culture of the microorganisms. In the
case of the tubercle bacilli, these authors emulsify 80 milligrams of
the bacilli in 1 c.c. of the salt solution. Wassermann and Brack,1 on
the other hand, prepare their bacterial antigen in the following way:
The growths of about ten agar slant cultures are emulsified in 10 c.c.
of sterile, distilled water. This emulsion is shaken for twenty-four
hours in a shaking apparatus. At the end of this time 0.5 per cent of
carbolic acid is added and the fluid cleared by centrifugalization. These
antigens become slightly weaker during the first ten or fourteen days,
but after that remain fairly constant. For the determination of tuber¬
culosis antibody, these authors make use of either old tuberculin
or the new tuberculins aTR” or “Bazillen Emulsion.”'

The Wassermann Test for the Diagnosis of Syphilis.2 — The sub¬
stances for the test are the following:

I. The Antigen. — In their original experiments, Wassermann and
his collaborators made use of salt-solution extracts of the organs (chiefly
of the spleen) of a syphilitic fetus. The tissue substance was cut in¬
to small pieces and to one part by weight of this substance, four parts of
normal salt solution and 0.5 per cent of carbolic acid were added. This

1 Wassermann und Bruch, Med. Klinik, 55, 1905, and Deut. med. Woch., xii,
1906.

2 Wassermann, Neisser und Bruch, Deut. med. Woch., xix, 1906; Wassermann,
Neisser, Bruch und Schucht. Zeit. f. Hyg., lv, 1906.

THE TECHNIQUE OF SERUM REACTIONS

263

mixture was shaken in a shaking apparatus for twenty-four hours, and
after this the coarser particles were removed by centrifugalization.
The reddish supernatant fluid was used as the antigen and could be
preserved for a long time in dark bottles in the ice chest.

Michaelis 1 obtained the antigen in the following way : The liver of
a syphilitic fetus was preserved in a frozen state and from time to time
small quantities of extract were prepared for the purpose of obtaining
antigen. This was obtained by thoroughly grinding up a small piece
of the liver in a mortar and adding five parts of salt solution and about
0.5 per cent of carbolic acid. This mixture was shaken in a shaking
apparatus for several hours and was then allowed to stand at a tem¬
perature slightly above 0° C. for several days. Finally it was cleared
by filtration or centrifugalization.

Alcoholic extracts of syphilitic organs have been used by a number
of authors. Porges and Meier 2 3 extract the chopped-up syphilitic liver
for twenty-four hours with five times the volume of absolute alcohol.
This is then filtered through paper and the alcohol evaporated in vacuo
at a temperature not above 40° C. The greenish sticky residue should,
have an alkaline or neutral reaction. About 1 gram of this material
is then emulsified in 100 c.c. of salt solution to which 0.5 per cent
of carbolic acid has been added. The fine emulsion which results is
filtered through thin paper and the filtrate used as the antigen.

Porges and Meier, as well as a number of others, have discovered that
in actual practice it is not necessary to make use of syphilitic organs in
order to obtain an antigen which will combine with syphilitic immune
body. This fact, of course, has thrown much suspicion upon the
specificity of the phenomenon. In practice, however, it appears as a
purely empirical fact that many of the non-specific antigens, neverthe¬
less, give reasonably reliable results. The authors mentioned above
have found that a 1 percent emulsion of commercial lecithin (Kahlbaum)
in carbolized salt solution furnishes a suitable antigen. This has not
been universally confirmed. The same authors have obtained good
results by extracting a normal fetal liver by alcohol in the same way as
they extracted the syphilitic organ. Landsteiner, Muller, and Poetzl J
have successfully employed an alcoholic extract of the heart substance
of a guinea-pig.

1 Michaelis, Berl. klin. Wocli., 1907.

2 Porges und Meier , Berl. klin. Woch., xv, 1908,

3 Landsteiner, M idler und Poetzl, Wien, klin. Wocli., 50, 1907.

264

INFECTION AND IMMUNITY

Similar alcoholic extracts of normal human spleen or of normal
rabbit's liver may be employed. Although often claimed that the anti¬
gen in such extracts is furnished by the lipoids, as a matter of fact it is
at the present day unknown to which ingredient the immune-body
binding power is to be attributed.

The antigen used in several hundred reactions by the writers with
satisfactory result is one prepared according to the method of
Noguchi/ as follows:

Fresh normal liver or spleen is covered and thoroughly macerated
with five times its volume of absolute alcohol. This is allowed to extract
in the incubator for six to eight days, being thoroughly stirred up at
least once a day. It is then pressed through cheese-cloth and filtered
through paper. This alcoholic extract is evaporated to dryness at room
temperature with the aid of a wind fan. The sticky, brownish residue
resulting is taken up in a small quantity of ether and the solution
poured into four times its volume of C. P. acetone. A heavy flocculent
precipitate forms which settles to the bottom as a sticky brown mass.
This is retained as antigen and may be preserved under acetone. The
acetone-soluble fraction is thrown away. For use, about 0.2 gram of
the sticky paste is dissolved in about 5 c.c. of ether and 100 c.c. of
salt solution added. This is shaken until the ether has evaporated.
The resultant antigen, ready for use, is a slightly opalescent greenish
fluid from which nothing settles out on standing.

Before an antigen can be used for the actual test, it is necessary to
determine the quantity which will furnish a valid result. The substances
which are used as antigens often have the power, if used in too large
quantity, of themselves binding complement. It is necessary, there¬
fore, to determine the largest quantity of each given antigen which
may be used without exerting an anti-complementary action, i.e., which
will not inhibit in the presence of normal serum but which will at the
same time inhibit hemolysis when syphilitic serum is used. This is done
by mixing graded quantities of the antigen with a constant quantity
of complement (0.1 c.c. of fresh guinea-pig serum), in duplicate sets,
adding to each tube of one set 0.2 c.c. of a normalserum, and to the other
0.2 c.c. of a known syphilitic serum. These substances are allowed to
remain together for one hour and then red blood corpuscles and inac¬
tivated hemolytic serum are added. The quantity which has given*
complete inhibition with the syphilitic serum, but absolutely no inhibi-

> Noguchi, Personal communication.

THE TECHNIQUE OF SERUM REACTIONS

265

tion with normal serum, is the one to be employed in subsequent re¬
actions. Before actual use, it is convenient to make a dilution of antigen
in salt solution in such a way that 1 c.c. shall contain the amount re¬
quired. Thus if 0.05 c.c. is wanted, mix 0.5 c.c. with 9.5 c.c. salt solu¬
tion. Then 1 c.c. of this can be added to each tube in the test.

II. The Hemolytic Serum. — The hemolytic amboceptor, for the
reaction, is obtained by injecting into rabbits the washed red blood
corpuscles of a sheep. A 5 per cent emulsion of the corpuscles is made
and of this 5 c.c., 10 c.c., 15 c.c., etc., are injected at intervals of five
or six days. Three or four graded injections of this kind are usually
sufficient to furnish a serum of adequate hemolytic power. The injec¬
tions may be made intraperitoneally or intravenously. About nine or
ten days after the last injection of corpuscles, the rabbit is bled from the
carotid artery and the serum obtained by pipetting it from the clot.

It is best to have a hemolytic serum of high potency in order that the
quantities used for the reaction may be as small as possible. This is
desirable because of the fact that the serum may contain small amounts
of precipitins for sheep’s serum, due to insufficient washing of the cor¬
puscles employed in the immunization. If such precipitins should be
present in any quantity in the serum used for the reaction, precipitates
might be formed, and these, as we know, have a tendency to carry down
complement from a mixture.

While the quantitative relations of the complement and antigen in
the Wassermann reaction are important, they are vastly more so in the
case of the hemolytic amboceptor. For the actual reaction most
observers make use of two hemolytic units. A hemolytic unit is the
quantity of inactivated immune serum which, in the presence of com¬
plement, suffices to cause complete hemolysis in 1 c,c. of a 5 per cent
emulsion of washed blood corpuscles. Noguchi 1 has pointed out very
clearly the dangers of not delicately adjusting the quantity of ambo¬
ceptor used in the reaction. In a recent communication upon the
subject, he has called attention to the experiments of Morgenroth and
Sachs 2 who have shown that the relationship between complement and
amboceptor necessary for hemolytic reactions is one of inverse propor¬
tions. To state it more clearly, in their own words, "in the presence of
larger quantities of amboceptor, smaller quantities of complement suf¬
fice,” and vice versa. Noguchi, in his work, has found that, while, in the

1 Noguchi, Proc. Soc. for Exper. Biol, and Med., VI., 3, 1909.

2 Morgenroth und Sachs, in Ehrlich’s “ Gesammelte Arbeiten,” etc., Berlin, 1904.

266

INFECTION AND IMMUNITY

presence of one unit of amboceptor, 0.1 c.c. of guinea-pig’s complement
is required to produce hemolysis, by using four, eight, and twenty units
of amboceptor, complete hemolysis is obtainable with one-third, one-
fifth, and one-tenth of the 0.1 c.c. of complement, respectively. For
this reason an excess of amboceptor might result in complete hemolysis
in a test, if a small fraction of the complement were left unfixed by
the syphilitic antibody. Another result of an excess of amboceptor
would consist in a partial dissociation of the complement from its com¬
bination with the antigen-antibody compound. As Noguchi puts it,
“a quantity of syphilitic antibody just sufficient to fix 0.1 c.c. of the
complement against two units of the amboceptor is no longer efficient
in holding back the complement from partial liberation against the
influence exerted by more than four units of the amboceptor.”

From these considerations it follows that the serum from rabbits
immunized against sheep corpuscles must, in each case, be titrated in
order to determine the hemolytic unit. For this purpose a number of
mixtures are made in test tubes, containing each 0.1 c.c. of complement
(fresh guinea-pig serum), 1 c.c. of a 5 per cent emulsion of sheep’s cor¬
puscles, and diminishing quantities of the inactivated hemolytic serum,
thus :

.1 c.c. of ''i
complement j
fresh |
guinea-pig
serum.

1. c.c.

+ i

of 5 per
cent
emul¬
sion
sheep's
corpus¬
cles.

Inac¬
tivated
+ herno-
I lytic
l serum.

^ .01 c.c. = complete hemolysis.

.009 c.c. = complete hemolysis.

.005 c.c. — complete hemolysis.

.003 c.c. = complete hemolysis.

.001 c.c. = complete hemolysis.

.0009 c.c. = partial hemolysis.
.0005 c.c. = no hemolysis.

.0003 c.c. = no hemolysis.1

In the given case, 0.001 c.c. of the serum represents one unit, and
0.002 c.c., two units, is the quantity to be used for each test.

III. The Complement. — The complement for the Wassermann re¬
action is used in the form of fresh guinea-pig serum. This may be
obtained in one of the following ways: A guinea-pig may be killed by
an incision in the throat and the blood allowed to flow into a large
Petri dish. This is set away in the ice chest until clear beads of serum
have formed upon the surface, and these are then carefully removed
with a pipette. The writers have found it convenient, however, to
anesthetize the guinea-pigs, then, by a longitudinal incision into the

per

In each tube the volume of the mixture should be made up to 5 c.c. with 0.85
cent salt solution.

THE TECHNIQUE OF SERUM REACTIONS

267

neck to lay bare the carotid artery and, severing this, to allow the blood
to flow into a sterile centrifuge tube. When clotting has occurred, the
clot is loosened from the glass with a platinum needle and the serum
separated by centrifugalization. Such serum should be used for no
longer than three days after being taken and should be kept, except
when in actual use, at a low temperature. The complement in
guinea-pig serum is sufficiently constant in quantity for practical
purposes.

IV. The Sheep Corpuscles . — The sheep corpuscles for the actual
reaction are obtained by receiving the blood of a sheep in a small flask
containing a sterile solution of a 0.5 per cent sodium citrate and 0.85
per cent sodium chloride, or into one containing glass beads or short
pieces of glass tubing. In the former case, the citrate solution prevents
clotting and the corpuscles may be washed free from the citrate solution
and emulsified in salt solution before use in the test. In the latter case,
it is necessary to shake the blood in the flask immediately after taking,
and to continue the shaking motion for about ten minutes. At the end
of this time, the blood will be defibrinated and the corpuscles are washed
free from serum by centrifugalization in salt solution. A 5 per cent
emulsion of the corpuscles in salt solution is employed for the test,
made by measuring the bulk of centrifugalized corpuscles and adding
nineteen parts of sterile salt solution. Thorough washing of the cor¬
puscles is essential both in order to preclude the occurrence of pre¬
cipitates and to remove any traces of complement present in the serum.

V. The Serum to be Tested for Syphilitic Antibody. — The serum of
the patient upon whom the test is to be made is best obtained in the
same way that blood is obtained for blood cultures. After surgical
precautions as to sterilization, a needle is plunged into the median
basilic vein and 3 or 4 c.c. of blood are removed. Whenever circum¬
stances do not permit such procedure, blood may be obtained from the
finger or the ear, always in sufficient quantity to furnish at least 1 c.c.
of clear serum. Before use for the test, the patient’s serum must be
inactivated by heating in a water bath to 56° C. for twenty minutes
to half an hour. As, according to some observers, 56° C. destroys
the syphilitic antibody in part, Noguchi advises inactivation at
54° C.

The Test. — The actual test for antibody in a suspected serum is
carried out in the following way: In a test-tube of suitable size, 0.1 c.c.
of complement, 0.2 c.c. of the inactivated suspected serum, and the

antigen, in quantity determined by titration, are mixed, and the total
18

268

INFECTION AND IMMUNITY

volume brought up to 3 c.c. with normal salt solution. This mixture is
thoroughly shaken, and placed for one hour in a water bath or in the
incubator at 37.5° C. At the end of this time, there is added .1 c.c. of
a 5 per cent emulsion of sheep’s corpuscles, and two units of hemolytic
amboceptor, determined by a titration of the inactivated hemolytic
rabbit serum, as described above. This mixture is again placed at 37.5°
C. for one to two hours. If the antibody is present in the suspected
serum, no hemolysis takes place. If absent, haemolysis is complete.

No test is of use unless suitable controls are made. The controls
set up should be as follows:

Control 1. For each serum tested the mixture described above,
omitting antigen.

Controls 2 and 3 . The mixture made as in the test but with known
syphilitic serum (2) with and (3) without antigen.

Controls 4 and 5. The mixture made as in the test, but with
normal serum (4) with and (5) without antigen.

Control 6. Antigen and complement alone, left together for an hour
before the addition of blood cells and amboceptor in order to preclude
the possibility of the antigen itself fixing complement. When working
with a well-controlled antigen this control may be omitted.

Controls 7 and 8. The hemolytic system, complement, blood cells
and amboceptor, set up in order to show that the system is in working
order (7) with and (8) without antigen. It is convenient to set the
tubes in two rows in a rack, the front row containing antigen, the
back row containing the same mixture without antigen.

In a positive test, the test itself, and Control 2, alone, should show
inhibited hemolysis. The other tubes should show complete solution
of the hemoglobin. (See scheme, p. 259.)

Modifications of the Wassermann Test. — Bauer’s Modification. —
Bauer1 utilizes the fact that normal human serum contains a certain
amount of hemolytic amboceptor for sheep’s corpuscles. In consequence
he omits in his reaction the use of specifically immunized hemolytic
rabbit serum. In carrying out the test he uses but four tubes:

1. Contains 0.1 c.c. of complement, the titrated amount of antigen,
and 0.2 c.c. of the inactivated serum to be tested.

2. Contains the same mixture without antigen.

3. Is like one, except that normal serum is substituted for that of the
patient.

1 Bauer, Deut. med. Woch., xii, 1908, and Berl. klin. Woch., xvii, 1908.

THE TECHNIQUE OF SERUM REACTIONS

269

4. Is like three, except that antigen is omitted.

If this modification is used at all (and its value is by no means
established), a fifth control should be added in which known syphilitic
serum is used.

These tubes are exposed to 37.5° C. for one hour, at the end of which
time 1 c.c. of a 5 per cent emulsion of sheep’s corpuscles are added.

If the test is positive, tube one should be without hemolysis, as well
as the fifth control with known syphilitic serum. Tubes two, three,

SCHEME FOR WASSERMANN TEST.

ADAPTED TO ORIGINAL WASSERMANN SYSTEM AFTER SCHEME OF NOGUCHI.

Test with Unknown
Serum.

Test with Known
Positive Syphilic
Serum.

Test with Known
Negative Normal
Serum.

Test without Serum
to Control Efficiency
of Hemolytic
System.

Serum .2 c.c.

. <D

o -g

Q Complement

0 Complement

Q Complement

.1 c.c.

• H
£

Salt sol. 3. c.c.

E-i

Serum .2 c.c.

£ a

o a>

Q Complement

£.«>

.1 c.c.

>
t <

■+J

.1 c.c.

.1 c.c

<2 ^

Antigen

(required amount
in 1 c.c. salt sol.).

Salt sol. 2. c.c.

Antigen

Salt sol. 2. c.c.

Antigen

Salt sol. 2. c.c.

Antigen

Salt. sol. 2. c.c.

O = test tube.

Place in water bath at 40° C. for one hour, then add to all tubes red blood cells and
amboceptor. These are previously mixed so that 2 c.c. contains the equivalents of
1 c.c. of a 5 per cent emulsion of sheep corpuscles and 2 units of amboceptor. Again
expose to 40° C. If the serum tested is positive, tubes 1 and 3 should show no
hemolysis, all the other tubes showing complete hemolysis in one hour.

and four, on the other hand, should show complete hemolysis. This
modification of the Wassermann test has not found universal adoption
and is little used at present.

270

INFECTION AND IMMUNITY

Noguchi's Modification. — Noguchi1 has much simplified the test by
making use of an anti-human hemolytic amboceptor instead of an anti¬
sheep amboceptor. In this way, he avoids the necessity of procuring
fresh sheep corpuscles for each test by using the corpuscles of the patient
himself. He has determined empirically that human serum contains,
normally, no amboceptor active against the human red corpuscles.
This fact is extremely important and has a decided advantage over
the original Wassermann test, in that in any reaction in which sheep
corpuscles are used as an indicator with human serum, the actual amount
of hemolytic amboceptor used in the test is uncertain. For, as we have
mentioned above, human serum, normally, may contain a variable
quantity of amboceptor for sheep corpuscles. In Noguchi’s test, there¬
fore, the actual quantity of amboceptor is exactly known by previous
titration. The hemolytic amboceptor for Noguchi’s test is obtained by
four or five injections of washed human corpuscles into rabbits. These
corpuscles may be obtained from the heart’s blood at autopsies, or
better, if possible, from placentae at childbirth. The unit for this
amboceptor is obtained by titration as in the case of the sheep-blood
amboceptor for the original Wassermann test. In setting up Noguchi’s
test, the following substances are used: —

(а) Patient’s serum. Obtained in small glass capsule. About 2
c.c. should be taken.

(б) Complement. Fresh guinea-pig serum: 0.1 c.c. of a forty per
cent, fresh, guinea-pig serum in salt solution is used in the test. Obtain
by adding 1 part of guinea-pig serum to one and one-half parts of salt
solution.

(d) Human corpuscles. Normal corpuscles or those of the patient
himself may be employed. If the patient’s red cells are chosen, these
should not be used for other tests than that on the patient’s own serum ;
1 c.c. of a one per cent emulsion of washed corpuscles is used for the
test.

(e) Anti-human amboceptor prepared by the injection of washed
human corpuscles into rabbits and titrated against human corpuscles.
Two units are used in the test.

The test itself is set up as follows:

1 Noguchi , Jour, of Exper. Medicine, 1909.

THE TECHNIQUE OF SERUM REACTIONS

271

Tube 1. 1 drop patient’s serum + complement (.1 c.c. of 40 per cent guinea-
pig serum + antigen.

Tube 2. 1 drop patient’s serum + complement (no antigen).

Tube 3. 1 drop known syphilitic serum + complement + antigen.

Tube 4. 1 drop known syphilitic serum + complement (no antigen).

Tube 5, 1 drop known normal serum + complement -f antigen.

Tube 6. 1 drop known normal serum + complement (no antigen).

Tube 7. Complement alone (for hemolytic system control).

To each tube then add 1 c.c. of the one per cent emulsion of human
corpuscles. Shake mixtures thoroughly and incubate or place in water
bath at 38-40° C. for one hour. Then add to each tube two units of
amboceptor and replace in water bath for one hour. At the end of this
time in a positive test there will be no hemolysis in tubes one and three
while all the other tubes will show hemolysis.

Noguchi has simplified the technique of complement fixation
further by drying measured amounts of antigen and amboceptor upon
small squares of blotting paper. These may be dropped into the tubes
directly, obviating the necessity of preparing fresh dilutions of the con¬
centrated substances for each test. The substances in the dried state,
moreover, may be preserved for longer periods than when kept in the
liquid form.

The Determination of Antigen by Complement Fixation. — The prin¬
ciples underlying the preceding tests for the determination of sus¬
pected antibodies may be equally applied to the determination of
suspected antigen. In the former case it was necessary to bring the
serum to be tested into contact with the antigen specific for the suspected
antibody, in the presence of complement, and at a suitable tempera¬
ture. At the end of an hour the mixture was tested for free comple
ment by the addition of hemolytic amboceptor and red blood cells.
In testing for antigen, the procedure is reversed, in that the serum or
other substance (bacterial extract) to be tested is brought into contact
with an antibody specific for the antigen, in the presence of complement;
and at the end of an hour at suitable temperature, free complement
is again determined by hemolytic reaction as before.

When dealing with bacterial antigen, it is necessary, therefore, to
prepare a highly potent immune serum against the bacteria which
contain the specific antigen which is sought.

Thus in testing for typhoid-bacillus antigen in the serum of a patient,
the substances required are as follows :

1. Complement: obtained from fresh guinea-pig serum. It is best
to titrate the complement when possible, using for the test double the

272

INFECTION AND IMMUNITY

quantity necessary to produce complete hemolysis of 1 c.c. of a five
per cent emulsion of blood cells, in the presence of two units of ambo¬
ceptor. When titration is omitted 0.1 c.c. may be used in routine work,
and is sufficiently accurate.

2. Hemolytic amboceptor: rabbit serum hemolytic for sheep
corpuscles. Inactivated and titrated as for Wassermann test. Two
units are used in the test.

3. A five-per-cent emulsion of sheep corpuscles in salt solution, pre¬
pared as for Wassermann test.

4. A highly potent typhoid antiserum obtained from an immunized
rabbit. In this case the smallest quantity, of the immune serum which
will cause the fixation of complement in the presence of an emulsion or ex¬
tract of typhoid bacilli is determined by experiment. The bacillary emul
sion is prepared by scraping the growth from twenty-four-hour agar slant
cultures, drying it, and macerating in a mortar with salt solution until
a slightly opalescent emulsion is formed. A series of tubes is prepared
into each of which is placed 0.1 c.c. of the emulsion of bacteria, 0.1 c.c.
of fresh guinea-pig serum, as complement, and gradually diminishing
quantities of the inactivated specific immune serum, ranging from 0.1 c.c.
downward. These tubes are left for one hour at 38° to 40° C., and,
following this, there are added the required quantities of red blood
cells and hemolytic immune serum. The smallest quantity of im¬
mune serum which has completely r inhibited hemolysis is the unit
and a quantity slightly greater than this should be used in the actual
test.1

5. Serum from the patient, inactivated at 56° C. for twenty
minutes.

In the actual test a series of tubes are prepared each of which con¬
tains: j. ) • ',!■

1. Complement, the determined quantity or 0.1 c.c.

2. Antiserum, the determined quantity.

3. Diminishing quantities of the serum to be tested for antigen be¬
ginning with 1 c.c.

Salt solution is added for dilution to 3 c.c.

These substances are left together at 40° C. for one hour and then
the required quantities of amboceptor and red cells are added. The
reaction is controlled by tubes containing the same ingredients without

1 Miiller, “ Technik d. serodiagnos. Methoden,” Jena, 1909; Leidke, “ Zur Kennt-
niss d. Komplemente,” Wurzburg, 1908.

THE TECHNIQUE OF SERUM REACTIONS

273

the typhoid antiserum. In a positive test there will be no hemolysis
in the tubes containing the patient's serum.

Proteid Differentiation by Complement Fixation. — That the technique
of complement fixation was applicable to the determination of specific
proteid antigen — such as human or animal blood — was shown by Gen-
gou 1 in 1902. The principles worked out by him have been practically
applied by Neisser and Sachs 2 and others to the forensic differentiation
of animal proteids and these tests are said to be more delicate and
reliable than precipitation tests made for the same purpose.

The substances necessary for the reaction are as follows:

1. Complement, titrated as above.

2. Hemolytic amboceptor as above.

3. A five-per-cent emulsion of sheep corpuscles as above.

4. Specific antiserum.

This is obtained from a rabbit immunized with the proteid for which
the test is to be made; viz. : human or animal blood serum. This must
be titrated. In order to do this, diminishing quantities of the antiserum
are mixed in a series of tubes with the determined quantity of comple¬
ment, and the antigen which is to be tested for, i.e., the homologous
serum with which the antiserum has been produced. Since the test
should be sufficiently delicate to determine 0.0001 c.c. of the antigen,
this quantity is added to each tube. The actual titration is as
follows:3

1. Antiserum, undiluted .1 + homologous serum .0001 + complement.

2. Antiserum diluted by 10 .75 + homologous serum .0001 + complement.

3. Antiserum diluted by 10 .75 + homologous serum .0001 + complement.

4. Antiserum diluted by 10 .3 + homologous serum .0001 + complement.

etc., down to .1

These tubes are incubated for one hour and hemolytic amboceptor
and red blood cells are added. The smallest quantity of antiserum
which has completely inhibited hemolysis is the “unit” and one and a
half to two times this quantity is used for the test.

5. A solution of the blood spot or other material to be tested pre¬
pared as for precipitin test. (See page 254.)

1 Gengou, Ann. de l’inst. Pasteur, 1902.

2 Neisser und Sachs, Berl. klin. Woch., 1905 and 1906. See also Citron „ in Kraus
and Levaditi “ Handbuch,” etc.

3 Citron, loc. cit.

274

INFECTION AND IMMUNITY

For the actual test the following mixtures are made in a series of
tubes, each of which contains:

1. Complement ) , , . . ,.

2 Antiserum \ (Iuan^1^ determined by titration.

3. Diminishing quantities of the substance in which the antigen is suspected ,
ranging from 0.1 c.c. downward to 0.0001 c.c.

Salt solution is added as a diluent up to 3 c.c. and the tubes are
placed in the incubator or water-bath at 37.5° to 40° C. At the end
of this time red blood cells and amboceptor are added as before.

The tubes are controlled by a series containing all the above ingredr
ents except the antiserum.

CHAPTER XVII
PHAGOCYTOSIS

The studies on immunity which we have outlined in the preceding
sections have dealt entirely with the phenomena occurring in the re¬
action between bacteria or bacterial products and the body fluids. These
studies, we have seen, have formed the basis of a theoretical conception
of immunity formulated chiefly by the German school of bacteriologists
under the leadership of Ehrlich, Pfeiffer, Kruse, and others. Parallel
with these developments, however, investigations on immunity have
been carried on which have brought to light many important facts con¬
cerning the participation of the cellular elements of the body in its
resistance to infectious germs.

The inspiration for this work and the greater part of the theoretical
considerations which have been based upon it have emanated from
Metchnikoff1 and his numerous pupils at the Pasteur Institute in Paris.
The phenomenon which these observers have studied in great detail
and upon the occurrence of which they have based their conceptions of
immunity, is known as phagocytosis.

It is well known that among the lowest unicellular animals the
nutritive process consists in the ingestion of minute particles of organic
matter by the cell. The rhizopods, which may be found and studied
in water from stagnant pools or infusions, when observed under the
microscope, may be seen to send out short protoplasmic processes, the
pseudopodia, by means of which they gradually flow about any foreign
particle with which they come in contact. If the ingested particle is
of an inorganic nature and indigestible, it will be again extruded after a
varying period. If, however, the ingested substance is of a nature which
can be utilized in the nutrition of the protozoon, it is rapidly surrounded
by a small vacuole within which it is gradually dissolved and becomes a
part of the cellular protoplasm. This digestion within the unicellular
organism is probably due to a proteolytic enzyme 2 which acts in the

Metchnikoff, “ LTmmunite dans les maladies infectueuses.”
a Mouton, Ann. de l’inst. Pasteur, xvi, 1902.

275

276

INFECTION AND IMMUNITY

presence of a weakly alkaline reaction. This has been shown by the
actual extraction, from amebae, of a trypsin-like ferment.

As we proceed higher in the scale of the animal kingdom, we find that
this power of intracellular digestion, while not uniformly an attribute of
all the body cells, is still well developed and a necessary physiological
function of certain cells which have retained primitive characters. In
animals like the coelenterata, in which there are two cell layers, an
entoderm and an ectoderm, the ectodermal cells have lost the power of
intracellular digestion, while the entodermal cells are still able to ingest
and digest suitable foreign particles. It is only as we proceed to animals
of a much higher organization that the function of cell ingestion of crude
food is entirely removed from the process of general nutrition. Never¬
theless, in these animals also, the actual cell ingestion of foreign particles
occurs, but it is now limited entirely to a definite group of cells. In the
higher animals and in man, this function of phagocytosis is limited
to the white blood cells of the circulation, or leucocytes, to certain large
endothelial cells lining the serous cavities and blood-vessels, and to cells
of a rather obscure origin which contribute to the formation of giant
cells within the tissues. A convenient division of these phagocytic cells
is that into “ wandering cells” and “ fixed cells.” The wandering cells
are the polymorphonuclear leucocytes, called “ microphages ” by
Metchnikoff, and certain large mononuclear elements or “ macrophages.”
Fixed cells, also called macrophages by Metchnikoff and possessing the
power of ameboid motion, include the cells lining the serous cavities,
and the blood and lymph spaces. The small lymphocytes, so far as we
know, have no phagocytic functions.

In studying the cellular activities which come into play whenever
foreign material of any description gains entrance into the animal body,
a definite reaction on the part of the phagocytic cells may be observed.
When we inject into the peritoneal cavity of a guinea-pig a small quan¬
tity of nutrient broth, and examine the exudate within the cavity from
time to time, we can observe at first a diminution from the normal of
the cells present in the peritoneal fluid. This may be due either to an
injury of the leucocytes by the injected substance, or to an actual repel¬
lent influence which the injected foreign material exerts upon the wan¬
dering cells.1 Very soon after this, however, the exudate becomes ex¬
tremely rich in leucocytes, chiefly of the polymorphonuclear variety, the
maximum of the reaction being reached about eighteen to twenty-four

i pierrallini, Ann. de Finst. Pasteur, 1897.

PHAGOCYTOSIS

277

hours after the injection. . After this, there is a gradual diminution in
the leucocytic elements until the fluid in the peritoneal cavity again
reaches its normal condition. It is plain, therefore, that the presence
of the foreign material in the peritoneal cavity has, after a primary
repellent action upon the phagocytes, attracted them in large numbers
to the site of the foreign substance. Such repelling or attracting in¬
fluences upon the leucocytes are spoken of as negative or positive
chemotaxis. The reasons for chemotaxis are not well understood. In
the case of bacteria, which chiefly interest us in the present connection,
chemotactic attraction or repulsion is intimately dependent upon the
nature of the microorganism, and very probably has a definite relation¬
ship to its virulence. Whether or not the principles of chemotaxis may
serve to explain the hypo- and hyper-leucocytoses, observed and diag¬
nostically utilized in clinical medicine, is by no means positive. It is
likely, however, that the two phenomena are closely associated. Leva-
diti 1 believed that he obtained some evidence that negative chemotaxis
may take place within the blood-vessels when he noticed that the intra¬
venous injection of cholera spirilla into immunized guinea-pigs resulted
in an immediate disappearance of leucocytes from the circulating blood,
and their accumulation in the internal organs. On the other hand, this
may possibly be more logically explained by a concentration of both
bacteria and leucocytes in the capillary system of such an organ as the
liver, as it is known that injected bacteria rapidly disappear from the
general circulation, but may be demonstrated in the various organs for
some time after injection.

We have seen, therefore, that the invasion of the animal body by
foreign material, living or dead, is followed by a prompt response on the
part of the phagocytic cells. In the case of bacteria, when these are
deposited in the subcutaneous areolar tissues, the inflammatory reaction
which follows brings with it an emigration of microphages (polynuclear
leucocytes) from the blood-vessels — and these are the so-called pus
cells. When the injection of bacteria is intraperitoneal, after a primary
diminution, there is an increase of leucocytes in the peritoneal cavity
which soon results in the formation of a copious turbid exudate. If the
pus of an abscess or the exudate from an infected peritoneum is ex¬
amined microscopically, it will be seen that many of the microphages
have taken bacteria into their cytoplasm. That fully virulent living
bacteria can be so taken up has been variously proven. The phago-

lLevaditi, Presse med., 1900.

278

INFECTION AND IMMUNITY

cytosis is, therefore, not simply a removal of the dead bodies of bacteria
previously killed by the body-fluids, but represents an actual attack upon
living and fully virulent microorganisms. That the ingested bacteria are
often alive after ingestion is proved by the fact that the injection of exu¬
date containing, so far as can be determined, only intracellular bacteria,
has, in several instances, been found to give rise to infection.

After the bacteria have remained for some time within the cytoplasm
of the leucocyte, vacuoles may be seen to form about them, similar to
those mentioned in discussing the- digestive processes of amebae. If the
preparations are, at this stage or later, stained with a one-per-cent
solution of neutral red, it will be found that the bacteria, colorless under
normal conditions, will be stained pink, an evidence of their beginning
disintegration. At a later stage in the process of intracellular digestion,
the bacteria will lose their form, and appear swollen, granular, and
vacuolated, and finally will be no longer distinguishable. If, on the
other hand, the ingestion of bacteria brings about the death of a leucocyte,
the neutral red will not stain the bacteria, the digestive vacuoles will not
form, and the leucocyte itself will disintegrate.

It must not be forgotten, however, that not all microorganisms are
equally susceptible to phagocytosis. Some may resist ingestion more
energetically than others by agencies not fully understood. Others
again, like the tubercle bacillus and the anthrax bacillus for instance,
may, after ingestion, oppose great difficulties to intracellular digestion.

To a certain extent, moreover, the variety of the bacterium deter¬
mines the variety of phagocyte attracted to the point of invasion. In
the cases of most of the bacteria of acute diseases, the microphages or
polymorphonuclear leucocytes are the ones upon which the brunt of
the battle devolves. Other invaders, like the Bacillus tuberculosis,
blastomyces, and others, find themselves opposed chiefly by the macro¬
phages. Cells of animal origin, such as the dead or injured cells of the
animals’ own body or the cells of other animals artificially introduced,
are ingested by macrophages. This is true also of many parasites of
animal nature.

It is clear, thus, that the process of phagocytosis is a universal re¬
sponse on the part of the body to the invasion of foreign particles of
dead material, of alien cells, and of living microorganisms. It remains
to be shown upon what basis this process may be regarded as an essential
feature in protecting the body against infection.

The numerous researches of Metchnikoff have brought out the
important fact that phagocytosis is regularly more active in cases in

PHAGOCYTOSIS

279

which the infected animal or human being eventually recovers. In
animals, furthermore, which show a high natural resistance against any
given microorganism, phagocytosis is decidedly more energetic than it
is in animals more susceptible to the same incitant. Thus, experiment¬
ing with anthrax infection in rats, Metchnikoff was able to show that, in
these animals, a decidedly more rapid and extensive phagocytosis of
anthrax bacilli takes place than in rabbits and guinea-pigs and other
animals which are delicately susceptible to this infection. While
different interpretations have been attached to this phenomenon, its
actual occurrence may be accepted as a proven fact.

In his later investigations, furthermore, Metchnikoff was able to
show that a direct parallelism existed between the development of
immunity in an artificially immunized animal and the phagocytic powers
of its white cells. He showed that rabbits artificially immunized to
anthrax, responded to anthrax infection by a far more active phagocy¬
tosis than did normal, fully susceptible animals of the same species.

It is quite impossible, in the space allotted, to recount the many
similar experiments by which the accuracy of these observations has been
confirmed. While few bacteriologists at the present day harbor any
doubt as to the truth of these contentions, the fundamental differences
between the conclusions drawn from these various phenomena by the
school of Metchnikoff and by that of the German workers may be
clearly stated as follows: Metchnikoff believes that phagocytosis is
the cardinal factor which determines immunity, while Pfeiffer and
others maintain that the determining factors upon which recovery or
lethal outcome depends, lie in the fluids of the body, the serous exudate
and its contents of immune body and complement, while the phagocy¬
tosis occurring coincidently, is merely a means of removal of the bacteria
after the outcome has already been decided.

In the further developments of his theory, Metchnikoff has claimed
that the immune body and complement — the presence of which in
blood serum and exudates he by no means overlooks — are derivatives
of the leucocvtes.

The immune body or “ fixator,” as Metchnikoff has named it, has
been shown by Wassermann and Takaki1 to be most plentiful in the
spleen, lymph nodes, and bone marrow of animals — all of them organs in
which large collections of leucocytic elements are found. Metchnikoff s
opinions as to the leucocytic origin of the complement, or “cytase,”

1 Wassermann und Takaki , Berl. klin. Woch., 1898.

280

INFECTION AND IMMUNITY

have found support in the experiments of Levaditi,1 who was able to
demonstrate the absence of complement in blood plasma, — i.e., where no
destruction of leucocytes had taken place — and in those of Cantacuzene,2
who showed that cholera-immune guinea-pigs would succumb to intra-
peritoneal injection of these bacteria when the diapedesis of leucocytes
had been prevented by the administration of opium.

The chapter of phagocytosis in its relation to bacterial immunity is
by no means closed. The problems involved in it are intricate and will
require much further study. The subsequent sections upon opsonins,
aggressins, and upon leucocyte extract, incorporate the more recent
studies which may be said to have followed logically in the footsteps
of MetchnikofFs work.

1 Levaditi, Presse med., 1900.

2 Cantacuzene , Ann. de Tinst. Pasteur, 1897

CHAPTER XVIII

OPSONINS, LEUCOCYTE EXTRACT, AND AGGRESSINS

OPSONINS

ALTHough the theories of immunity are, as we have stated, generally
classified as the humoral and the cellular or phagocytic theories, the
separation has never, even in the minds of the warmest partisans, been
an absolute one. Thus, Buchner and his successors looked for the
origin, first, of alexin, then of complement, in the leucocytes, and
Metchnikoff attributed to immune serum the quality of stimulating the
leucocytes (stimulins) to increased phagocytosis. The serum, accord¬
ing to Metchnikoff, acted, not directly upon the bacteria, in the nature
of bactericidal or lytic substances, but rather upon the leucocytes, pre¬
paring or arming these for the fray. Denys and Leclef 1 were the first
definitely to oppose this view. These authors, on the basis of ex<
periments done upon streptococcus immunity in rabbits, came to the
conclusion that the serum aided phagocytosis rather by its action upon
the bacteria than by its influence upon the leucocytes.

Wright2 in 1903 and 1904 undertook a systematic study of the re¬
lation of the blood serum to phagocytosis, in a series of careful experi¬
ments. Using his own modifications of the technique of Leishman,3
he first determined the direct dependence of phagocytosis upon some
substance contained in the blood serum. He further proved conclu¬
sively that this serum component acts upon the bacteria directly and
not upon the leucocytes, is bound by the bacteria, and renders them
subject to phagocytosis. The presence of these substances in sera,
furthermore, which appear entirely free from bactericidal or lytic
bodies, and the thermolabile character of the substances (60° for ten
or fifteen minutes destroys them) seemed to exclude their identity
with the immune bodies of other authors.

1 Denys et Leclef, La cellule, xi, 1895.

2 Wright and Douglas, Proc. Royal Soc. London, lxxii, 1904.

3 Leishman, Brit. Med. Jour., i, 1902.

281

282

INFECTION AND IMMUNITY

Because of their action in preparing the bacteria for ingestion by the
leucocytes, he named these bodies “opsonins” {pfnuviu), to prepare food).

Neufeld and Rimpau1 soon after, and independently of Wright,
described similar substances in the blood serum of streptococcus and
pneumococcus immune animals, which they called bacteriotropins.
Because of their greater thermostability it is not yet possible to identify
these bacteriotropins absolutely with the opsonins.

The importance of these opsonic substances in immunity was shown
by Wright 2 in a series of experiments in which he determined that in
persons ill with staphylococcus or tubercle-bacillus infections, the phago¬
cytic powers were relatively diminished toward these microorganisms,
but could be specifically increased upon active immunization with dead
bacteria or bacterial products.

The results of Wright have been confirmed and elaborated by nu¬
merous workers.

The diminished power of leucocytes to take up bacteria without the
co-operation of serum was demonstrated, after Wright, by Hektoen and
Ruediger,3 who worked with gradually increasing dilutions of serum.
The contention of the Wright school, however, that leucocytes are en¬
tirely impotent for phagocytosis without the aid of serum, can not be
regarded as proven, in face of the work of Lohlein 4 and others who
have observed phagocytosis on the part of washed leucocytes.

The specificity of opsonins and their multiplicity in a given serum
were shown mainly by the work of Bullock and Atkin,5 Hektoen and
Ruediger,6 and Bullock and Western.7 These authors showed that the
opsonic substances in sera could be absorbed out of the sera, one by one,
by treatment with various species of bacteria, a procedure analogous to
the method of absorption used in the study of agglutinins.

The increase of phagocytic power demonstrated by Wright in immune
sera naturally led to the question whether this depended merely upon
an increase of the normal opsonins or whether the newly formed immune
opsonins were entirely different substances. The greater thermosta¬
bility of the opsonins in immune sera seemed, at first, to support the

1 Neufeld und Rimpau, Deut. med. Woch., xl, 1904.

2 Wright and Douglas, Proc. Roy. Soc., London, lxxiv, 1905

3 Hektoen and Ruediger, Jour. Inf. Dis., ii, 1905.

4 Lohlein, Ann. de l’inst. Pasteur, 1905 and 1906.

6 Bullock and Atkin, Proc. Roy. Soc., London, lxxiv, 1905.

6 Hektoen and Ruediger, loc. cit.

1 Bullock and Western. Proc. Roy. Soc., loc. cit.

OPSONINS

283

latter view. Dean/ however, showed that not all of the normal opsonins
are thermolabile and that, by absorption experiments, bacteria treated
with normal sera could be prevented from taking up opsonins from
immune sera. These facts seem to point strongly toward the identity of
normal and immune opsonic substances.

Further study of the opsonins has led to numerous other questions
regarding their structure, their relation to other immune bodies, etc.,
which are largely still in the stage of controversy, and for which the
original monographs must be consulted.

The controversial questions may be briefly reviewed as follows:

As stated above, Wright believed originally that the bodies dis¬
covered by him in normal sera, the “ normal opsonins/’ in other words,
were distinct bodies that could not be identified with either the comple¬
ment or antibodies present in serum. Neufeld and Hiine,1 2 Levaditi and
Inmann,3 and others, on the other hand, maintain that the opsonic
action of normal serum, at least, is intimately related to the complement
contents of such serum.

They base this contention not only upon the thermolability of normal
opsonins, but also upon the fact that opsonin may be removed from
normal serum at the same time as complement by the method of com¬
plement fixation, detailed in another section (see pp. 245 and 261). 4

The contention of Wright that the thermostable opsonic substances
of immune serum are distinct bodies, not identical with the ambocep¬
tors, is supported by the work of Hektoen,5 6 * Neufeld and Topfer,8
and others. The problem, however, can by no means be regarded as
finally settled, since other workers, notably Levaditi, are inclined to
identify the immune opsonins with lytic amboceptors.

As to the structure of the opsonic substances, moreover, dif¬
ferences of opinion still exist. Hektoen and Ruediger 7 who have
investigated the question attribute to opsonins a complex constitution.
They believe them to possess a thermostable haptonhore group
and a thermolabile 11 opsonophore ” group and that heating beyond
a definite temperature converts the opsonins into opsonoids by

1 Dean, Proc. Roy. Soc., London, Ixxvi, 1905.

2 Neufeld and Hune, Arb. a. d. kais. Gesundheitsamt, xxv.

3 Levaditi and Inmann, Compt. rend, de la soc. de biol., 62, 1907.

* Levaditi, Presse medicale, 70, 1907. /:

5 Hektoen, Jour, of Inf. Dis., iii, 1906.

6 Neufeld und Topfer, Cent. f. Bakt., xxxviii/ 4905.

» Hektoen and Ruediger, Jour, of Inf. Dis., ii, 1905.

284

INFECTION AND IMMUNITY

Fig

destruction or alteration of the “ opsonophore ” group. This view is
not shared by all workers and has been disputed by Bullock and Atkin.1

The Technique of Wright. — The three factors necessary for the per¬
formance of an opsonic test are (1) the blood serum to be tested; (2)
an even emulsion of bacteria, and (3) leucocytes.

(1) Blood serum is obtained by bleeding from the finger and receiving
the blood into glass capsules (Fig. 66). These are sealed at both ends;
the blood is allowed to clot; and the separation of serum is hastened by
a few revolutions of a centrifuge.

(2) The bacterial emulsion is obtained by rubbing up a few
loopfuls of a twenty-four-hour slant agar culture with a little physio¬
logical salt solution (0.85 per cent) in a watch glass. A very small

amount of salt solution is used at first
and more is gradually added, drop by
drop, as the emulsion becomes more
even. The final breaking up of the
smaller clumps is best accomplished by
cutting off very squarely the end of a
capillary pipette, placing it perpen¬
dicularly against the bottom of the
watch glass, and sucking the emulsion in and out through the narrow
chink thus formed. (Fig. 67.)

Emulsions of tubercle bacilli are more difficult to make. The bacilli
filtered off in the manufacture of old tuberculin are commonly used.
These are washed in salt solution on the filter, and are then scraped off
and sterilized. They are then, in a moist condition, placed in a mortar
and thoroughly ground into a paste. While grinding, salt solution
1.5 cent) is gradually added until a thick emulsion appears. This
emulsion may be diluted and larger clumps separated by centri-
fugalization.

(3) The leucocytes are obtained by bleeding from the ear or finger
directly into a solution containing eighty-five hundredths per cent to one
per cent of sodium chlorid and five-tenths to one and five-tenths per cent
of sodium citrate. Ten or fifteen drops of blood to 5 or 6 c.c. of the
solution will furnish sufficient leucocytes for a dozen tests. This
mixture is then centrifugalized at moderate speed for five to six minutes.
At the end of this time, the corpuscles at the bottom of the tube
will be covered by a thin grayish pellicle, the buffy coat, consisting

66. — Wright’s Capsule for
Collecting Blood.

Bullock and Atkin, Proc. Royal Soc., lxxiv, 1905.

OPS'ONINS

285

chiefly of leucocytes. These are pipetted off with a capillary pipette
(by careful superficial scratching movements over the surface of the
buffy coat).

There being, of course, no absolute scale for phagocytosis, whenever
an opsonin determination is made upon an unknown serum, a parallel
control test must be made upon a normal serum. This normal is best
obtained by a “pool” or mixture of the sera of five or six supposedly
normal individuals.

The three ingredients— serum, bacterial emulsion, and leucocvtes —
having thus been prepared, the actual test is carried out as follows:

Fig. 67. — Pipette for Opsonic Work.

Capillary pipettes of about six or seven inches in length and of nearly
even diameter throughout, are made. These are fitted with a nipple
and a mark is made upon them with a grease-pencil about 2 to 3 cm.
from the end (Fig. 68). Corpuscles, bacteria, and serum are then
successively, in the order named, sucked into the pipette up to
the mark, being separated from each other by small air-bubbles. Equal
quantities of each having thus been secured, they are mixed thoroughly
by repeatedly drawing them in and out of the pipette upon a slide.
The mixture is then drawn into the pipette; the end is sealed; and
incubation at 37.5° is carried on for an arbitrary time, usually fifteen
to thirty minutes.1 The control with normal serum is treated in exactly

Fig. 68. — Pipette with three Substances, Corpuscles, Bacteria, and Serum,

AS FIRST TAKEN UP.

the same way. After incubation the end of the pipette is broken off,
the contents are again mixed, and smears are made upon glass slides in
the ordinary manner of blood smearing. Staining may be done by
Wright’s modification of Leishman’s stain, by Jenner’s, or by any other
of the usual blood stains. In these smears, then, the number of bacteria
contained in each leucocyte is counted. The contents of about eighty

1 For the purpose of incubation, specially constructed water baths, marketed under
the name of “opsonizers/’ may be used.

286

INFECTION AND IMMUNITY

to one hundred cells are usually counted and an average is taken.
This average number of bacteria in such leucocytes is spoken of as the
“phagocytic index.” The phagocytic index of the tested serum, divided
by that of the “normal pool” (control) serum, gives the “opsonic
index.”

Another method of estimating the opsonic content of a given blood
serum has been contributed by Simon, Lamar, and Bispham.1 These
authors employed dilutions both of the patient’s serum and of normal
serum ranging from one in ten to one in one hundred. With these
dilutions, they carry out opsonic experiments with bacterial emulsions
and washed leucocytes in the same way as this is done in the Wright
method, except that they recommend the employment of thinner bac¬
terial emulsions than are usually employed in the former method. In
examining their slides, they do not estimate the number of bacteria
found within the leucocytes, but rather the percentage of leucocytes
which actually take part in the phagocytic process,2 i.e., which con¬
tain bacteria.

By the same method of dilution, they determine what they have
called “the opsonic coefficient of extinction,” a phrase which is used to
express the degree of dilution of the serum at which no further phagocy¬
tosis takes place. They claim for their methods the more delicate de¬
termination of variations in opsonic power. The method has not been
sufficiently used to permit the expression of an opinion as to its value.

The Vaccine Therapy of Wright. — In connection with his more
theoretical work upon opsonins, Wright has laid much stress upon the
value of active immunization in the treatment of infectious diseases.
Beginning his work with staphylococcus and tubercle-bacillus infections,
he has extended his methods, with the aid of many collaborators, to
gonococcus, streptococcus, pneumococcus, and a number of other
bacterial infections. In all these cases, when possible, he uses for
therapeutic purposes a so-called “autogenous vaccine” which is made
with the bacteria isolated from the patient himself. In the case of
tubercle-bacillus infections, he uses for treatment the new-tuberculin-
bacillary-emulsion of Koch. The production of vaccine is, according
to Wright, as follows:

Production of Vaccines. — After isolation of the organisms from the
patient, cultures are made with a view of obtaining considerable amounts

1 Simon, Lamar, and Bispham, Jour. Exp. Med., viii, 1906.
8 Simon and Laipar, Johns Hopkins Hosp. Bull., xvii, 1906.

OPSONINS

287

of bacterial growth. In making vaccines with poorly growing organisms,
large surfaces must be inoculated. Organisms are best grown for this
purpose upon the surface of agar or glucose agar (the enrichment of the
agar with sugar or acetic fluid, etc., depending upon the cultural require¬
ments of the organism in question), in square eight-ounce medicine
bottles laid upon their sides. This furnishes a large area for inoculation.
After sufficient growth has taken place upon the agar, two or three cubic
centimeters of sterile normal salt solution are introduced into the
bottles with a sterile pipette. With this the growth is gently washed
off the surface of the agar, more salt solution gradually being added as
necessary. The emulsification may be facilitated by gently scraping
the growth off the medium by means of a flexible platinum loop. This
thick bacterial emulsion is then pipetted out of the bottles, during which
process an equalization of the emulsion can be attained by repeated
sucking in and out with the pipette. The emulsion is then placed in a
sterile test tube which may then be drawn out at its open end into a
capillary opening. It is a point of practical importance that, in pre¬
paring such capsules out of a test tube, a few inches of air space should
be left above the surface of the emulsion, so that expansion during
heating may not blow out the top of the glass tube. A dozen or so of
sterile glass beads may be put into these tubes in order to aid in emul¬
sification. Shaking the beads in such a tube will help in breaking up
small clumps of bacteria.

The emulsion is then standardized; that is, a numerical estimation
of bacteria per cubic centimeter must be made. This standardization
is best done before sterilization, because during the latter process a
number of bacteria may be broken up, and, while unrecognizable
morphologically, are, nevertheless, represented in the emulsion by their
products. The^standardization may be accomplished by highly diluting
a definite volume of the emulsion, planting plates with definite quan¬
tities of the dilution, and counting colonies. Wright prefers, as more
exact, an enumeration of the bacteria against red blood cells. This is
done in the following way:

A little of the emulsion is placed in a watch glass and from it, with a
pipette as used in the estimation of the opsonic index, one volume is
taken and is mixed with an equal volume of blood from the finger and
two or three volumes of salt solution. The salt solution is added in
order to dilute the red cells so that they can be conveniently counted
and to prevent clotting. These substances are thoroughly mixed in a
pipette and spread upon a slide as in making a blood smear, and as even

288

INFECTION AND IMMUNITY

and uniform a smear as possible should be made. They are then stained
either by Jenner’s or Wright’s blood stain.

The preparations are examined with an oil-immersion lens. In
order to limit a definite microscopic field, it is convenient, to use an
Ehrlich diaphragm, or else, in lieu of this, to mark a circle with a blue
pencil upon the lens of the eye-piece. The red blood cells and bacteria,
in a number of these fields, are counted and the ratio between them is
estimated. Knowing the number of red blood cells to the cubic milli¬
meter in the particular blood employed, by previous blood count,
and knowing that equal volumes of blood and of bacterial emulsion
have been used in the mixture, it is easy from this ratio to ascertain
the number of bacteria contained in a cubic millimeter of the original
emulsion. Thus, for instance, if in an average of twenty fields bacteria
are to red blood cells as two is to one, and the blood employed con¬
tains five million red blood cells to each cubic millimeter, then a cubic
millimeter of our emulsion contained ten million bacteria, and a cubic
centimeter one thousand times as many.

The vaccine, thus produced and standardized, is sterilized by sus¬
pension in a Avater bath at 60° 0. for one hour on each of five or six
consecutive days. Its sterility is then controlled by culture.

From this stock emulsion small quantities may be drawn off and
diluted for therapeutic use.

The initial dose given by Wright in staphylococcus infections, in
which the method has been most frequently employed, varies from
fifty to one hundred millions of bacteria. In working with the tubercle
bacillus, the ordinary tuberculin dosage is adhered to.

Wright, in his work, makes use of the opsonic index in order to
estimate changes in the resistance of the patient against the given in¬
fection. In other words, he bases his judgment as to whether the
patient is improving or not, upon the opsonic power of the patient’s
serum. In following the opsonic index of a patient during systematic
treatment Avith vaccine, Wright has found definite changes upon the
basis of which he constructs a curve of opsonic power. Immediately
after the injection of vaccine, he finds that there is a brief period during
which the opsonic power of the patient is depressed below its original
state. This he calls the negatKe phase. The length of time occupied
by this negative phase depends both upon the condition of the patient
and upon the size of the dose given. It is usually completed within
twenty-four hours. After this, there is a gradual rise in the opsonic poAver,
at first rapid, later more slow, until a maximum is reached after a vary-

LEUCOCYTE EXTRACT

289

mg number of days. This period of rise represents the positive phase.
The second inoculation with vaccine should, according to Wright, be
made when the opsonic power is again beginning to sink after the highest
point of the positive phase.

The facts of Wright’s investigations have been given in the preceding
pages, purposely without critical considerations. The existence of
opsonic or phagocytosis stimulating substances in blood serum may be
accepted as fact. It is also of unquestionable value to the science of
immunity that renewed vigor has been infused into the investiga¬
tion of active therapeutic immunization. The far-reaching claims of
therapeutic benefit, which have been made by Wright and his school,
however, have not yet received sufficient support by clinical observa¬
tion to be fully accepted, although intelligent application of this treat¬
ment in suitable cases has undoubtedly proved its therapeutic value.

LEUCOCYTE EXTRACT

In the foregoing sections upon Phagocytosis and Opsonins, we have
discussed the protective action exerted by the living leucocytes against
bacterial infection and the relation of these cells to the blood serum.
We have seen, furthermore, that, while our knowledge of the blood
serum, as developed at present, shows that phagocytes may be aided
by this in the ingestion of bacteria, the subsequent digestion of the
germs, and possibly the neutralization or destruction of their intracel¬
lular poisons, is, as far as we know, largely accomplished by the unaided
phagocytic cell. It is an obvious thought, therefore, that, in the struggle
with bacterial invaders, the leucocytic defenders might be considerably
re-enforced if they were furnished, as directly as possible, with a further
supply of the very weapons which they were using in the fight with
the microorganisms. With this thought as a point of departure, Hiss 1
conceived the plan of injecting into infected subjects the substances
composing the chief cells or all the cells usually found in exudates, in
the most diffusible form and as little changed by manipulation as possi¬
ble; and he also assumed that extracts would be more efficacious than
living leucocytes themselves, since if diffusible they would be distrib¬
uted impartially to all parts of the body by the circulatory mechan¬
ism. They would then, as quickly as absorption would permit, relieve
the fatigued leucocyte and also protect by any toxin-neutralizing or
other power they might possess, the cells of highly specialized functions.

1 Hiss, Jour. Med. Res., N. S., xiv, 3, 1908.

290

INFECTION AND IMMUNITY

The method of obtaining these substances as used both in animal
experiments and in the treatment of human subjects is at present as
follows:

Rabbits, preferably of 1,500 grams weight or heavier, receive intra¬
pleural injections of aleuronat. This is prepared by making a three
per cent solution of starch in meat-extract broth, without heating,
and adding to this, after the starch has gone into thorough emulsion,
five per cent of powdered aleuronat. This is thoroughly mixed, boiled
for five minutes, and filled into sterile potato tubes, 20 c.c. into each tube.
Final sterilization is done preferably in an autoclave. The rabbit in¬
jections are carried out by injecting 10 c.c. into each pleural cavity
in the intercostal spaces at the level of the end of the sternum, in the
anterior axillary line, great care being exerted to avoid puncturing of
the lungs. The rabbits are left for twenty-four hours, at the end of
which time a copious and very cellular exudate will have accumulated
in the pleural cavities. This is removed, after killing the animals with
chloroform, by opening the anterior chest wall under rigid precautions
of sterility, and pipetting the exudate into sterile centrifuge tubes.
Immediate centrifugalization before clotting can take place then per¬
mits the decanting of the supernatant exudate fluid. To the leuco¬
cytic sediment is then added about 2 c.c. of sterile distilled water, and
the emulsion is thoroughly beaten up with a stiff bent platinum spatula.
Smears are now made on slides, stained by Jenner’s blood stain, and ex¬
amined for possible bacterial contamination. It is well also to take
cultures. Sterile distilled water is then added to each tube, about twenty
volumes to one volume of sediment, and the tubes are set away in the
incubator for eight hours. At the end of this time the sterility is again
controlled as above, and further extraction in the refrigerator continued
until the extract is used.

In experimenting upon animals, Hiss 1 observed that pneumococcus,
staphylococcus, streptococcus, meningococcus, and typhoid, dysentery,
and cholera infections in rabbits and guinea-pigs were profoundly modi¬
fied when injections of leucocyte extracts, prepared as above, were ad¬
ministered intraperitoneally or subcutaneously during the course of the
infection. In many cases animals were saved by these substances from
infections which proved rapidly fatal in untreated control animals, even
when the protective injections were made as late as twenty-four hours
after intravenous infection.

1 Hiss, Jour. Med. Res., N. S., xiv, 3, 1908.

AGGRESSINS

291

In applying this method of treatment, by subcutaneous injections,
to infections in man, Hiss and Zinsser observed distinctly beneficial
results in cases of epidemic cerebrospinal meningitis, in lobar pneumonia,
in staphylococcus infections, and in erysipelas.1

In experimenting with the leucocyte extracts in vitro the same au¬
thors were able to show that precipitates occurred when clear leucocyte,
extract and the clear extract of various bacteria were mixed.2

Further experiments, carried out both in animals and in the test tube;
showed that while the leucocytic extracts possessed slight bactericidal
powers for a variety of microorganisms, these attributes did not seem
sufficient to explain the profound, modifying influences exerted , upon
bacterial infections by these extracts. Experiments have also shown
that the leucocyte extracts possess some distinct power of neutralizing
or destroying the poisonous products of typhoid and dysentery bacilli.
Whether or not the final explanation of the action of these extracts will
be found to lie in these endotoxin-neutralizing properties of the leuco¬
cytic substances, can not as yet be determined, and this problem must
be left for further research to decide.

That bactericidal substances can be extracted from leucocytes by
various methods has been repeatedly shown by Schattenfroh, Petter-
son, Korschun, and others.3 The researches of Petterson as well as,
more recently, the work of Zinsser, have shown that these “endolysins,”
as Petterson has called them, have a structure quite different from that
of the serum bacteriolysins in that they are not rendered inactive by
temperatures under 80° C., but, when once destroyed by higher tem¬
peratures, can not be reactivated either by the addition of fresh serum
or of unheatecl leucocyte extracts. The last-named authors, moreover,
have shown that these endocellular bactericidal substances are not
increased by immunization, the quantity present in each leucocyte being
probably at all times simply sufficient for the digestion of the limited
number of bacteria which can be taken up by the individual leucocyte.

AGGRESSINS

An extremely obscure chapter in our knowledge of the reaction of
animals and man against infection is the one dealing with the questions

1 Hiss and Zinsser, Jour. Med. Res., N. S., xiv, 3, 1908; ibid., xv, 3, 1909.

2 Hiss and Zinsser, ibid., xiv, 3, 1908.

3 Schattenfroh, Arch. f. Hyg., 1897; Petterson, Cent. f. Bakt., I, xxxix, 1905, and
ibid., xlvi, 1908; Korschun, Ann. de l’inst. Pasteur, xxii, 1908; Zinsser, Jour. Med.
Res., xxii, 3, 1910.

292

INFECTION AND IMMUNITY

of varying pathogenicity between different bacterial species and between
different races of the same microorganism. We know that certain bac¬
teria may be injected into an animal or human being in considerable
quantities, without producing anything more than the temporary local
disturbance following the subcutaneous administration of any innocuous
material. Other bacteria, on the other hand, such as the bacillus of an¬
thrax or the bacillus of chicken cholera, injected in the most minute
dosage, may give rise to a rapidly fatal septicemia. Within the same
species, furthermore, fluctuations in virulence may take place which
may depend upon a variety of influences which have been discussed in
another section and need not be recapitulated. Suffice it to say that
variations in the susceptibility of inoculated subjects do not, in any
way, furnish a sufficient explanation for these phenomena and we are
forced to seek for the key to the problem in the activities of the bacteria
themselves.

In an effort to cast light upon this subject, Bail, following in the
footsteps of his predecessors, Kruse,1 Deutsch and Feistmantel,2 has
formulated his so-called “ aggressin-theory.” In its reasoning, this
theory is indirectly an offspring of MetchnikofUs phagocytic theory
and is, in many of its phases, antagonistic to the purely humoral con¬
ception of immunity.

Bail 3 was first led to the formulation of his theory by extensive re¬
searches which he had made in conjunction with Petterson 4 into an¬
thrax immunity. He had noted, as others before him had, that animals,
highly susceptible to anthrax, often possessed marked bactericidal
powers against this bacillus. When such animals, whose serum should
surely be capable of bringing about the death of, at least, a few hundred
anthrax bacilli, were injected with doses far less than this number they
nevertheless succumbed rapidly and the bacilli multiplied enormously
in their bodies. He argued from this that the injected microorganisms
must possess some weapon whereby they were enabled to counteract
the protective forces of the animal organism. In an anthrax-immune
animal, as a matter of fact, no proliferation of bacteria took place and
the injected germs were rapidly disposed of by the protective forces,
foremost of which was phagocytosis.

1 Kruse, Ziegler’s Beitrage, xii, 1893.

2 Deutsch und Feistmantel, “ Die Impfstoffe in Sera,” Leipzig, 1903.

3 Bail, Cent. f. Bakt. I, xxvii, 1900, and xxxiii, 1902.

4 Bail und Petterson, Cent. f. Bakt., I, xxxiv, 1903; xxxv, 1904; xxxvi, 1904.

AGGRESSINS

293

The theory of Bail 1 as eventually formulated, after extended in¬
vestigations which need not be outlined, contains the following basic
principles :2

Pathogenic bacteria differ fundamentally from non-pathogenic
bacteria in their power to overcome the protective mechanism of the
animal body, and to proliferate within it. They accomplish this by
virtue of definite substances given off by them, probably in the nature
of a secretion, which acts primarily by protecting them against phagocy¬
tosis. These substances (referred to by Kruse as “ Lysins ”) were named
by Bail, “Aggressins.” The production of aggressins by pathogenic
germs is probably absent in test-tube cultures, or, at any rate, is
greatly depressed under such conditions, but is called forth in the animal
body by the onslaught of the germicidal or other influences encountered
after inoculation.

These aggressins can be found, according to Bail, in the exudates
occurring about the site of inoculation in rapidly fatal infections. He
obtained them, separate from the bacteria themselves, by the prolonged
centrifugation and subsequent decanting of edema fluid, and pleural
and peritoneal exudates.

Two fundamental experimental observations are brought forward
by Bail in support of the truth of his contentions. In the first place, he
was able to show that fatal infection could be produced in animals by
the injection of sublethal doses of bacteria, when, together with the
germs, there was administered a small quantity of “ aggressin.” He
inferred from this experiment that the injected aggressin had served in
paralyzing the onslaught of phagocytic and other protective agencies,
and had thus made it possible for the bacteria to gain a foothold and
to proliferate.

The second experimental support upon which Bail’s theory is
founded consists in the successful immunization of animals with aggres¬
sin. Animals were treated with aggressive exudates, from which all bac¬
teria had been removed by prolonged centrifugalization and which had
been rendered entirely sterile by three hours’ heating to 60° C. and by the
addition of five-tenths per cent of phenol. Animals so treated were not
only immune themselves, but contained a substance in their serum
which permitted the passive immunization of other untreated animals.
Bail explained this by assuming the production of antiaggressins in the

1 Bail, Arch. f. Hyg., lii, 1905; liii, 1905; Wien. klin. Woch., xvii, 1905.

2 Bail und Weil, Wien. klin. Woch., ix, 1906; Cent. f. Bakt., I, xl, 1906; xlii,
1906.

294

INFECTION AND IMMUNITY

treated subjects. His experiments and those of his pupils were con¬
ducted with a large variety of microorganisms, among which were the
typhoid and dysentery bacilli, the bacilli of chicken cholera and of plague,
the cholera spirillum, and various micrococci. According to whether a
microorganism is capable of producing an aggressin and consequently
of invading the animal body, he divides bacteria into “pure parasites/’
“half parasites/’ and “saprophytes.”

The theory of Bail has been extensively attacked by a number of
authors, chief among whom are Wassermann and Citron,1 Wolff,2 and
Sauerbeck.3 The criticism which these investigators make of Bail’s
views is based upon laborious experimentation and has succeeded in
placing the “aggressin” theory upon a very precarious footing. It is
claimed by them, in the first place, that much of the “aggressive”
character of Bail’s exudates is due to their containing liberated bacterial
poisons (endotoxins). This they have maintained both because the
sterile “aggressin” exudates could be shown to possess a considerable
degree of independent toxicity and because the aggressive action of such
an exudate could be duplicated by aqueous extracts of bacteria. Citron,4
furthermore, was able to show, by the Bordet-Gengou method of com¬
plement fixation, that the exudates of Bail contained considerable
quantities of free bacterial receptors, which, in taking up bacteriolytic
immune body, would neutralize any lytic power on the part of the in¬
fected animal. By this antilytic action, he believes, Bail’s first conten¬
tion, the virulence-enhancing action of the exudates, can be explained.
The nature of the immunity produced in animals by Bail’s method of
treatment is less easily explained and less exposed to adverse criticism.
Bacteriolytic immunity alone probably can not account for the high
degree of resistance imparted by a few injections of the aggressins.
On the other hand, the establishment of an antiaggressive immunity
has not been sufficiently supported to stand as a proven fact. Final
judgment must be postponed until further investigation shall have
brought a better understanding of the phenomenon.

1 Wassermann and Citron, Deut. med. Woch., xxviii, 1905.

2 Wolff, Cent. f. Bakt., I, xxxviii, 1906.

3 Sauerbeck, Zeit. f. Hyg., lvi, 1907.

* Citron, Cent. f. Bakt., I, xl, 1905; xli, 1906; and Zeit. f. Hyg., lii, 1905.

CHAPTER XIX

ANAPHYLAXIS OR HYPERSUSCEPTIBILITY

PHENOMENA OF ANAPHYLAXIS

The phenomena now grouped together under the heading of anaphy¬
laxis and hypersusceptibility have but recently become the subject of
systematic experimentation. Nevertheless, manifestations now recog¬
nized as belonging to this category, had not escaped the attention of a
number of the earlier workers in immunity.

By anaphylaxis is meant the following train of phenomena: When
a foreign proteid is introduced by subcutaneous, intraperitoneal, intra¬
venous, or subdural injection (or in some cases by feeding) into the
animal body, after a time there will appear a specific hypersuscepti¬
bility of the animal for this proteid. After a definite interval, a second
injection of the same substance, harmless in itself, will produce violent
symptoms of illness and often rapid death in an animal so prepared.
The phenomena are not limited to any given class of proteids, but are
manifest in the case of animal, vegetable, and bacterial proteids, and
within certain limits are specific.

As early as 1893, Behring1 2 and his pupils 3 had noticed that animals,
highly immunized against diphtheria toxin, with high antitoxin content
of the blood, would occasionally show marked susceptibility to injections
of small doses of the toxin.

The phenomena observed by them was interpreted as an increased
tissue susceptibility to the toxin, and Wassermann, reasoning on the
basis of Ehrlich’s side-chain theory, formulated the conception that the
increased susceptibility was due to toxin receptors, increased in number
by immunization, but not yet separated from the cells that had produced
them; the cells thereby becoming more vulnerable to the poison. In
the same category belongs the observation of Kretz, who noticed that
normal guinea-pigs did not show any reaction after injections of innocuous

1 Behring, Deut. med. Woch., 1893.

2Knorry Dissert., Marburg, 1895; Behring und Kitashina, Berl. klin. Woch.,

1901.

295

296

INFECTION AND IMMUNITY

toxin-antitoxin mixtures, but that marked symptoms of illness often
followed such injections when made into immunized guinea-pigs. Other
phenomena which are now regarded, a posteriori, as probably depending
upon the principles involved in anaphylaxis, are the tuberculin and
mallein reactions, fully described in another place, and the adverse
effects often following the injections of antitoxins in human beings,
conditions spoken of under the heading of “ serum sickness. ” The last-
named condition has been made the subject of an exhaustive study by
v. Pirquet and Schick.1

That the injection of diphtheria antitoxin in human beings is often
followed, after an incubation time of from three to ten days, by ex¬
anthematous eruptions, urticaria, swelling of the lymph glands, and
often albuminuria and mild pulmonary inflammations, has been noticed
by many clinicians, who have made extensive therapeutic use of anti¬
toxin. It was recognized early that such symptoms were entirely inde¬
pendent of the antitoxic nature of the serum, but depended upon other
constituents or properties peculiar to the antitoxic serum. Moreover,
symptoms of this description were by no means regular in patients in¬
jected for the first time, but seemed to depend upon an individual pre¬
disposition, or idiosyncrasy, v. Pirquet and Schick, however, noticed
that in those injected a second time, after intervals of weeks or months,
the consequent evil effects were rapid in development, severe, and
occurred with greater regularity. Many of the phases of such “ serum
sickness7’ are still obscure, since experimental conditions can not be
controlled, and many modifying factors can not be excluded in observa¬
tions made upon human beings, and the grouping of the above conditions
with the phenomena of anaphylaxis is still tentative.

The fundamental observations from which our present knowledge of
anaphylaxis takes its origin are those made in 1898 by Hericourt and
Richet,2 who observed that repeated injections of eel serum into dogs
gave rise to an increased susceptibility toward this substance instead
of immunizing the dogs against it. Following up the lines of thought
suggested by this phenomenon, Portier and Richet3 * later made an in¬
teresting observation while working with actino-congestin — a toxic
substance which they extracted from the tentacles of Actinia. This

lv. Pirquet and Schick, “ Die Serum Krankheit,” monograph, Leipzig and Wien,
1905.

2 Hericourt and Richet, Compt. rend, de la soc. de biol., 53, 1898.

3 Portier and Richet, Compt. rend, de la soc. de biol., 1902; Richet, Ann. de

l’inst. Pasteur, 1907 and 1908.

ANAPHYLAXIS OR HYPERSUSCEPTIBILITY

297

substance in doses of 0.042 grams per kilogram produced vomiting,
diarrhea, collapse, and death in dogs. If doses considerably smaller
than this were given in quantities sufficient to cause only temporary
illness, and several days allowed to elapse, a second injection of a
quantity less than one-quarter or one-fifth of the ordinary lethal dose
would cause rapid and severe symptoms and often death. Similar
observations were made soon after this by Richet with mytilo-conges-
tin, a toxic substance isolated from mussels. In these experiments there
remained little doubt as to the fact that the first injection had given rise
to a well-marked increased susceptibility of the dogs for the poison used.

It was Richet who first applied to this phenomenon the term “ ana¬
phylaxis ” (o-vd against, yoAdAf? protection), to distinguish it from
immunization or prophylaxis.

Soon after Richet’s earlier experiments, and simultaneously with his
later work, Arthus1 made an observation which plainly confirmed
Richet’s observations, though in a somewhat different field. The ob¬
servation of Arthus is universally spoken of as the “ phenomenon of
Arthus.”

He noticed that the injection of rabbits with horse serum (a sub¬
stance in itself without toxic properties for normal rabbits) rendered
the rabbits delicately susceptible to a second injection made after an
interval of six or seven days. The second injection — even of small doses
—regularly produced severe symptoms and often death in these animals.

An observation very similar to that of Arthus was made by Theobald
Smith2 in 1904. Smith observed that guinea-pigs injected with diph¬
theria toxin- antitoxin mixtures in the course of antitoxin standardiza¬
tion, would be killed if after a short interval they were given a subcu¬
taneous injection of normal horse serum.

The fundamental facts of hypersusceptibility had thus been observed,
and Otto,3 working directly upon the basis of Smith’s observation,
carried on an elaborate inquiry into the phenomenon. Almost simul¬
taneously with Otto’s publication there appeared a thorough study of
the condition by Rosenau and Anderson.4

The researches of Otto, and Rosenau and Anderson, besides con¬
firming the observations of previous workers, brought out a large number

1 Arthus, Compt. rend, de la soc. de biol., 55, 1903.

2 Th. Smith, Jour. Med. Res., 1904.

3 Otto, “Leuthold Gedenkschrift,” 1905.

4 Rosenau and Anderson, Hyg. Lab. U. S. Pub. Health and Marine Hosp. Serv.
Bull, 29, 36, 1906, 1907.

298

INFECTION AND IMMUNITY

of new facts. They showed conclusively that the action of the horse
serum had no relationship to its toxin or to its antitoxin constituents,
that the “ sensitization” of the guinea-pigs by the first injection became
most marked after a definite incubation time of about ten days. Sen¬
sitization was accomplished by extremely small doses (one one-millionth
in one case, usual doses ywv ’to 1 c.c.). Rosenau and Anderson, further¬
more, excluded hemolysin or precipitin action as explanations of the
phenomena, and proved that hypersusceptibility was transmissible from
mother to offspring, and that it was specific — animals sensitized with
horse serum not being sensitive to subsequent injections of other pro-
teids. These authors, Vaughan1 and Wheeler, Nicolle,2 and others,
furthermore, showed that the reaction was by no means limited to animal
sera, but was elicited by proteids in general, pepton, egg albumin, milk,
the extract of peas, and bacterial extracts.

The typical anaphylactic reaction, then, is obtained when animals,
preferably guinea-pigs, are injected with a small quantity of a given
proteid, and ten or fifteen days subsequently given a second injection
of the same substance employed for the first or sensitizing inoculation.
The quantity used for the second injection should be considerably
larger than that used for sensitization when the injection is made
intraperitoneally or subcutaneously. When given intravenously, intra-
cranially, or intracardially, amounts as small as 0.25 to 0.008 c.c. may
suffice. The time at which a second injection gives rise to the most
violent symptoms, moreover, is to a large extent dependent upon the
size of the sensitizing dose.3 After extremely small initial quantities
(0.005-0.002 c.c.), the anaphylactic state is usually well developed, ac¬
cording to Rosenau and Anderson,4 after twelve or fourteen days. After
larger doses 5 the time required for the development of anaphylaxis is
usually longer — extending often over weeks, or even months.

While the sensitizing or first dose may be given subcutaneously,
intravenously, intraperitoneally, or intracardially with equal success,
Besredka and Steinhardt maintain that no anaphylaxis results if the
first dose is given intracranially. This statement, however, has found
contradiction in the work of Rosenau and Anderson. The time required
for full sensitization, furthermore, depends, according to the last-named

1 Vaughan, Assn. Am. Phys., May, 1907.

2 Nicolle, Ann. de l’inst. Pasteur, 2, 1903.

3 Besredka, Ann. de l’inst. Pasteur, 1907.

4 Rosenau and Anderson, loc. cit.

sOtto, Miinch. med. Woch., 1907.

ANAPHYLAXIS OR HYPERSUSCEPTIBILITY

299

authors, also upon the mode of injection of the first dose; on this point,
however, no conclusions are, at present, justified.

At reinjection, the symptoms are more prompt in developing and
more severe when the injection is made intraperitoneally, intracardially,
or intracerebrally than when the subcutaneous route is chosen.

The symptoms occurring in sensitized animals after the second
or anaphylactic injection are usually well-marked and unmistakable.
The animals move about restlessly, breathe rapidly, and may cough.
Often they stagger about or fall upon one side, and die frequently in
convulsions within a time ranging from five minutes to one hour after
the injection. During thi's time there is a rapid fall of temperature and
frequently defecation and urination. Animals that recover from the
condition after such symptoms, return to normal within a remarkably
short time — twelve to twenty -four hours.

Animals dead of anaphylaxis, according to Gay and Southard,1 show
congestion of the serous membranes of peritoneum, pleura, and pericar¬
dium, with small hemorrhagic spots on the heart and lungs and the
pleura. In some cases there is fatty degeneration in the parenchyma
cells of the heart, the muscles, and in the nervous system. Such lesions,
however, could not be found by Otto, but were found in some cases by
Doerr.2

When sensitized animals recover from the second injections, they are
thereafter immune— that is, they do not react to subsequent injections
of the same substance.

This immunity or “ antianaphylaxis ” as Besredka 3 and Steinhardt
have called it, appears immediately after recovery from the second in¬
jection. Antianaphylaxis may also be produced if animals which have
received the first or sensitizing dose are injected with comparatively
large quantities of the same substance during the preanaphylactic period
— or, as it is sometimes spoken of, during the anaphylactic incubation
time. This injection should not be done too soon after the first dose, but
rather toward the middle or end of the preanaphylactic period.

If given within one or two days after the sensitizing injection, ana¬
phylaxis will develop, nevertheless. Whether or not the antianaphylactic
condition is transitory or permanent is not yet fully shown. Besredka
and Steinhardt believe that it lasts a long time, while Otto found guinea-

1 Gay and Southard, Jour. Med. Res., May, 1907.

2 Doerr, in Krauss and Levaditi, “Handbuch,” etc.; 2, Die Antikorper.
s Besredka and Steinhardt, Ann. de l’inst. Pasteur, 1907.

300

INFECTION AND IMMUNITY

pigs immunized in the above manner to lose their antianaphylaxis within
three weeks.

An important development of our knowledge of the phenomena of
anaphylaxis was achieved when Nicolle, Otto/ Gay and Southard,1 2 and
others 3 succeeded in showing that the hypersusceptible state could be
passively transferred to normal animals by injecting them with the
serum of anaphylactic animals. In such experiments the serum of the
anaphylactic animal is first injected in quantities of 0.5 c.c. or preferably
more, and twenty-four hours later an injection of the specific antigen — •
that is, the proteid used for sensitization — is given. The animals so
treated show typical symptoms of hypersusceptibility and often die.

Simultaneous inoculation of the two substances, either mixed or
injected separately, does not, according to Rosenau and Anderson, pro¬
duce the same effect. On this point, however, there is not complete
unanimity, since Weill-Halle and Lemaire 4 report aphylactic symp¬
toms in guinea-pigs injected simultaneously with horse serum, and the
serum of guinea-pigs hypersusceptible to horse serum. Reversal of the
procedure originally described, however, may be successfully practiced.
Thus Pick and Y^amanouchi 5 have recently succeeded in producing
anaphylactic symptoms by injecting rabbits first with beef serum, and
some time later with anti-beef serum from rabbits. Their experiments,
however, are not entirely analogous to those given above, since the anti¬
serum used by them for reinjection was actually a precipitating immune
serum. A remarkable fact, observed by Otto, is that the serum of
guinea-pigs who have been given the sensitizing or first injection will
confer passive anaphylaxis on the eighth or tenth day after injection,
before the animals themselves show evidences of being actively hyper-
sensitized. It is also true that occasionally the serum of antianaphy-
lactic animals will possess the power of conferring passive anaphylaxis
upon other normal animals.

Anaphylaxis may also be passively transmitted by inheritance.
Thus, according to Rosenau and Anderson, the young of anaphylactic
guinea-pigs show hypersusceptibility, irrespective of whether the mother
became hypersusceptible before or after the beginning of pregnancy.

1 Otto, loc. cit.

2 Gay and Southard, loc. cit.

3 Nicolle, Ann. de Tinst. Pasteur, 1907, 1908.

4 Weill-Halle and Lemaire, Compt. rend, de la soc. de biol., 1907.

5 Pick and Yamanouchi, Wien. klin. Woch., xliv, 1908.

ANAPHYLAXIS OR HYPERSUSCEPTIBILITY

301

Such anaphylaxis has no. reference whatever to the condition of the
father, and is not transmitted by the milk.

THEORIES CONCERNING ANAPHYLAXIS

Our present knowledge of anaphylaxis is largely empirical. We are
not yet in a position to correlate the many data gained experimentally
into a theory which offers anything like a satisfactory explanation for
all the phenomena observed. Nevertheless, a number of hypotheses
have been advanced which deserve serious consideration, since they are
experimentally supported and may serve as points of departure for
future research.

One of the earliest ideas advanced was based upon the Ehrlich theory
of receptor overproduction by tissue cells during immunization. It was
suggested that hypersusceptibility might well be due to the stimulation
of new specific receptors which as yet remained sessile upon the body
cells instead of having been thrown off into the blood stream. As a
consequence, the cells, having an affinity for more of the toxic substance
of the antigen than they possessed normally, had become more vulnerable.
This opinion, however, is hardly tenable, in face of the facts of passive
anaphylaxis, in which, we have seen, the hypersusceptibility may be
transmitted with the serum of the sensitized individual.

v. Pirquet and Schick,1 as well as many other observers, have re¬
garded the anaphylactic process as analogous to other immune reactions,
and believe that an antigen in the serum first injected produces a specific
antibody. The reaction between these two substances following the
second injection gives rise to the anaphylactic symptoms. The essen¬
tial feature of this opinion is the assumption that the substance which
sensitizes after the first injection is identical with that which exerts the
anaphylactic injury after reinjection.

Wolff-Eisner2 has expressed a belief which has found much experi¬
mental support in the hands of Vaughan and Wheeler.2 Wolff-Eisner
holds that all cells and proteids contain a toxic substance which is
characterized by its inability to produce a neutralizing antibody when
injected into animals. The first injection produces a lysin for the
proteid injected, which possesses the power of liberating such poisons
from the complex molecule. A second injection is followed, conse-

1 v. Pirquet und Schick, loc. cit.

2 Wolf -Eisner, Berl. klin, Woch., 1904,

302

INFECTION AND IMMUNITY

quently, by a rapid liberation of the toxic fraction, and injury to the
animal results. This view has been expressed in slightly different form
by Richet 1 and has been more clearly formulated and experimentally
supported by V aughan and Wheeler 2 who were actually able to extract
from various proteids toxic substances which gave rise in animals to a
symptom complex not unlike that of typical anaphylaxis. (Extrac¬
tion with alkalinized seventy-per-cent alcohol.)

Not entirely unlike these views is the hypothesis advanced by Gay
and Southard,3 who assume that a part of the proteid introduced on first
injection is assimilated and removed, but that another part, unassimi-
lable, remains in the circulation and exerts a constant irritation upon
the tissue cells, rendering them abnormally susceptible to reinjections
of the same substance. They speak of this toxic or irritating, non-
assimilable substance as “ anaphylactin,” and believe that in passive
sensitization it is the transference of this element which renders the
recipient anaphylactic.

Opposed to the opinions of most other workers is that expressed
by Besredka.4 Besredka believes that the substance which produces
sensitization is not identical with that which gives rise to the symptoms
on reinjection. According to his conception of the process, the sensitiz¬
ing injection contains an active element (called by him “ sensibilisino-
gen”), which gives rise in the injected animal to a specific antibody
(“sensibilisin”). This sensibilisin circulates in the blood and is stored
by the cells of the central nervous system. On reinjection of the same
proteid, a reaction takes place between the anchored sensibilisin and a
third substance, present in the proteid and not identical with sensibili-
sinogen, which acts typically upon the nerve cells and gives rise to the
symptoms. This third substance he speaks of as “ antisens ibilisin.”
That the process of anaphylaxis takes place probably in the nervous
system is rendered plausible by the fact that ether narcosis seems to
prevent its occurrence (a fact maintained by Besredka but contradicted
by Rosenau and Anderson). Furthermore, direct introduction of the
second dose into the brain gives rise to more rapid and violent anaphy¬
laxis than when any other route is chosen. Besredka calls attention to
the fact, furthermore, that no anaphylactic symptoms occur when the
serum of a sensitive animal is introduced into another animal simul-

1 Richet, Ann. de l’inst. Pasteur, xxi, 1907.

2 Vaughan and Wheeler, Jour. Infect. Dis., iv, 1907.

* Gay and Southard, loc. cit.

* Besredka , Ann. de l’inst. Pasteur, 1907 and 1908.

ANAPHYLAXIS OR HYPERSUSCEPTIBTLITY

303

taneously with its specific proteid, but only when the serum of the
sensitized animal precedes the injection of its antigen. This, he argues,
points to an anchorage of the sensitizing elements to the body cells
before an anaphylactic injury can occur. If the two substancse,
sensibilisin and antisensibilisin, meet in the blood stream, no harm
results, the elements neutralize each other and the animal is anti-ana¬
phylactic. His contention that there are two separate elements,
sensibilisinogen and antisensibilisin, in the original proteid, is based on
the fact that sensitization can be accomplished by sera heated to from
100° to 120° C., but that, after sensitization, no anaphylaxis results if
the injected serum be exposed previously to temperatures of 100° C.,
and the reaction is rendered much less violent even by exposure of sera
to temperature of 50 to 60° C.

Recently an attempt has been made to associate the phenomena of
anaphylaxis with the formation of precipitates. Hamburger1 early ex¬
pressed the opinion that anaphylaxis may be nothing more than the for¬
mation of emboli by serum precipitates. This view, however, has found
few adherents in face of the facts that we have no positive evidence
of the actual occurrence of precipitates in the blood streams of living
animals, and that it has been shown by Friedemann2 that the precipitates
produced in vitro will, when injected intravenously in animals, pass
through the capillaries without harmful effects.

Doerr and Russ,3 on the other hand, have recently studied carefully
the relationship between anaphylaxis and the precipitin reaction and
have shown a close parallelism between the two. These observers in¬
jected rabbits with precipitating antisera and twenty-four hours later
treated the same animals with the antigen employed for the production
of these precipitating sera. They found that, in such experiments, the
regularity of occurrence and degree of anaphylaxis which ensued, were
directly proportionate to the precipitating powers of the serum first-
injected . They claim, from such results, that precipitable antigen and
anaphylactic antigen are identical substances. They conceive the phe¬
nomenon of anaphylaxis as a reaction between precipitins, attached
to the tissue cells, and the precipitable antigen. In other words, the
anaphylactic shock is looked upon as an intracellular precipitin reaction.

Friedberger and Hartoch4 have recently called attention to another

1 Hamburger, quoted by U. Friedemann, Zeit. f. Immunitatsforschung, ii, 1909.

2 Friedemann, loc. cit.

* Doerr and Russ, Zeit. f. Immunitatsforschung, iii, 1909.

* Friedberger und Hartoch , Zeit. f. Immunitatsforschung, iii, 1909.

304

INFECTION AND IMMUNITY

factor which may possibly lead to further elucidation of phenomena of
hypersusceptibility. They have shown that, in passive anaphylaxis at
least, simultaneously with the occurrence of symptoms, there is a marked
diminution of complement in the serum of the treated animal. Intra¬
venous injection of substances which prevent complement absorption in
vitro — concentrated salt solution, for instance — prevented anaphylaxis
in both actively and passively sensitized animals. They suggest that
sudden removal of complement from the circulation has a definite causal
relationship to anaphylaxis.

Friedberger’s more recent work has shown that the action of com¬
plement in vitro, both upon bacteria and upon the precipitates formed
when a dissolved antigen is mixed with its antiserum, will produce
poisons which kill guinea-pigs in typical anaphylactic shock. His
results, much confirmed by his own work and that of others, seem to
indicate that anaphylactic shock may be due to a poison, “anaphyla-
toxin,” which is formed by the proteolytic action of the complement
upon the foreign protein which is injected into the animal, and which is
sensitized to the action of the complement by the antibody formed in
response to the first injection.

The toxic effects of Friedberger’s “ anaphylatoxins " are in many
ways similar to those of Vaughan’s toxic-protein-split products, and the
subsequent development of his theory of anaphylaxis and infectious
disease may be logically regarded as a further elaboration of Vaughan’s
views, though approached from a different point of departure.

CHAPTER XX

FACTS AND PROBLEMS OF IMMUNITY IN THEIR BEARING UPON
THE TREATMENT OF INFECTIOUS DISEASES

While the various facts and theories of immunity and infection have
been given in the preceding sections, no systematic attempts have been
made to correlate the facts presented, or to determine their bearing on
the most vital problem of all — the treatment of infectious diseases.

To understand more fully this point of view, it is necessary briefly
to recall certain of the facts which are known about the physiology,
metabolism, and composition of the bacteria, and of their ability to
neutralize directly or to respond adaptively to the agents directed against
them by the invaded animal. Some of these facts are so well understood
that passing mention here is sufficient: such, for instance, is the fact
that certain microorganisms, especially the bacilli of diphtheria and tet¬
anus, secrete soluble poisons both during artificial cultivation and dur¬
ing their life in the animal body, which poisons are eminently toxic.
These poisons are true secretions and are largely independent of the
composition of the surrounding medium so long as this favors the physi¬
ologic activities and growth of the germs. Such germs, then, once having
gained even an insecure foothold in the animal body, by no matter what
favoring circumstances, are possessed of a powerful weapon of offense
against the sensitive physiologic bases of the host and, possibly, of de¬
fense against its more immediate and mobile means of combating the
germs themselves. In the case, however, of most other pathogenic
bacteria, the secretion, at least in artificial media, of such highly soluble
and potent poisons has not been demonstrated satisfactorily, although
certain investigations point fairly conclusively to the production of
some minor bodies which have been shown to act deleteriously on the red
blood cells and on the leucocytes — the hemolytic, leucocidic, and leu-
colytic substances which are looked on as probably true soluble toxins,
like the toxins of diphtheria and tetanus, which give rise in the animal
body to the production of true antitoxins: i.e., are neutralized by
their antisera, unit for unit, according to the law of multiples.

305

306

INFECTION AND IMMUNITY

Other minor poisons may in some instances be demonstrated in
culture media, and also may possibly be formed in the animal body by
the metabolic activities of the germs. These are either simply waste
products of metabolism or bodies due to the decomposition of the
nutrient media in which the germs are gowing. These bodies are usually
referred to as ptomains, and differ entirely from the true secreted toxins,
both in their chemical composition and in their physiologic action, re¬
sembling in both of these the alkaloids. They are not known to give
rise to antibodies of any kind in animals.

Apart from all the poisons just mentioned; i.e., the toxins, hemoly¬
sins, leucocidins, and ptomains, there is supposed to exist a most vit lly
important and interesting group of poisonous substances, the so-called
endotoxins. These, so far as our knowledge goes, are poisons rather
firmly seated in the bacterial cell, which are not secreted in our ordinary
cultural media, and are supposed by most observers not to be separable
in the animal fluids and tissues from the intact bacterial cell. These
poisons may be demonstrated in old cultures, in which the bacteria
are dead and disintegrating or undergoing autolysis — although Pfeiffer
does not consider autolytic products necessarily similar to endotoxins
— or they may be obtained by destroying the bacteria mechanically by
pressure and grinding, or by breaking them while frozen. In the animal
body they are said to become free when the bacteria die and decompose
or are disintegrated by the digestive bodies by which they have been
attacked. These endotoxins are recognized by the fact that the}"
do not call out true antitoxins which become free in the plasma and
serum, but do, nevertheless, lead to the formation of digestive antibodies,
these not following, however, the “law of multiples” in protecting in¬
fected animals from the poisons. The liberation of these poisons by the
destruction of bacteria in the animal body is best illustrated by the so-
called phenomenon of Pfeiffer which takes place when cholera vibrios
and immune cholera serum are introduced into the peritoneal cavity
of a guinea-pig. If specimens are withdrawn from time to time from
the peritoneal cavity of an animal so treated, a rapid swelling up,
disintegration, and disappearance of the vibrios can readily be demon¬
strated. The organisms apparently do not multiply in the animal body
under these conditions and are almost immediately destroyed. This
disintegrating power is also claimed for the body fluids of normal
animals and is supposed to be demonstrated by the following experi¬
ment. When graded quantities of a fresh cholera culture are introduced
into the peritoneal cavity of normal guinea-pigs of equal weight, the

FACTS AND PROBLEMS OF IMMUNITY

307

following phenomena can be regularly observed: Minimal doses of the
culture produce a febrile condition which continues for a few hours with
no serious symptoms. Slightly larger doses give rise, after a short
interval, during which there is fever, to a marked drop in tempera¬
ture and definite symptoms of cholera poisoning — muscular weakness,
twitching, and general prostration. These symptoms of poisoning then
gradually disappear, and after twenty-four hours the guinea-pigs are
again normal. If the quantity of cholera culture injected is carefully
increased up to the minimal lethal dose, the animal dies with all the
symptoms of cholera intoxication, but on autopsy the peritoneum is
found to be entirely sterile, or only a few isolated cholera spirilla are
found, usually inclosed in pus cells. Finally, if larger quantities of
living cholera spirilla are injected, the peritoneal cavity shows a profuse,
serous, sometimes hemorrhagic exudate, which contains innumerable
actively motile microorganisms. The point of interest in this experi¬
ment is the demonstration of the fact that the normal guinea-pigs
which receive enough of the cholera vibrios to prove fatal have de¬
stroyed the vibrios and presumably died from the poison thus liberated,
and not from poisons secreted by living vibrios, or from an overcoming
of their systems by the rapid multiplication of the organisms. It is only
when the animal system is previously flooded with an overwhelming dose
that the vibrios are found alive and multiplying even locally in the peri¬
toneum after death. This does not mean, however, that no multiplica-
cation ever goes on hand in hand with the destruction of the germs in the
infected animal; on the contrary, such a multiplication is probably the
rule rather than the exception, as has been shown fairly conclusively
by the experiments of Radziewsky, and was beautifully illustrated by
an experiment of Pfeiffer and Wassermann, who after having shown that
the blood serum of human beings who have recovered from Asiatic chol¬
era has the power to protect guinea-pigs from ordinarily fatal doses
of cholera spirilla, even when used in high dilutions, then proved that
this protective power is not an antitoxic one, but depends largely, if
not entirely, on the ability of the serum to aid in the immediate dissolu¬
tion of the vibrios. Thus animals which received only a fraction of a
milligram of such a serum were able to bear the injection of a loopful of
virulent cholera vibrios, practically without reaction, while control
animals succumbed to one-fourth of the dose with typical symptoms.
Now, however, if the dose was increased to three or five loopfuls, not
even ten thousand times the original amount of the serum would protect
the animals against the inoculation. The toxic effects may, in fact, as

308

INFECTION AND IMMUNITY

shown by Pfeiffer, appear with extraordinary rapidity, so that in these
animals the temperature may show the lethal drop within two hours
after inoculation, while control animals which have received the
same quantity of cholera germs without the serum may not show a
similar lethal drop in temperature for four to five hours.

An explanation of the results of this experiment is found, probably,
in the fact that guinea-pigs are able to withstand a certain quantity of
the intracellular cholera poison (endotoxin) which may be represented by
one loopful of a fresh culture. If the animals are given smaller quantities
without the serum, say one-fourth to one-half loopful, the bacteria may
increase for a time without producing marked symptoms. Parallel
with the increase, however, the phenomenon of germ destruction is
going on and characteristic symptoms of intoxication appear at the
moment when the number of vibrios destroyed has become so large that
it corresponds to more than one loopful of the cholera culture. An
animal will thus withstand a culture of any size when mixed with im¬
mune serum, if the dose does not exceed the limit of intoxication before
it is entirely destroyed. On the other hand, when guinea-pigs receive
the larger dose of three to five loopfuls, the serum, not being anti¬
toxic, is not able to counteract the fatal effects of the liberated
cholera poisons, but, on the other hand, enormously increases the rate
of destruction of the vibrios, and hence intoxication appears earlier
in such treated animals than in the controls receiving the organisms
alone.

This classic cholera experiment has been selected because it illus¬
trates the most extreme limit of the endotoxin point of view, and, further,
because the cholera organism, standing at one end of the scale, is the
most extreme example of pathogenicity by virtue of its own destruction,
while the diphtheria bacillus at the other end, as we have seen, is one
of the classic examples of pathogenicity by virtue of secreted toxins.
Neither of these organisms is truly invasive or highly parasitic, and both
are harmful usually by the action of their poisons alone and acting, as it
were, from a base of supply on the periphery of the animal system. Be¬
tween these two extremes stand all grades of pathogenic and infective
germs.

These two organisms are typical examples of their kind, but there
are few organisms which secrete such highly toxic soluble bodies as do
diphtheria bacilli, and there are few so susceptible as the cholera organ¬
ism to disintegration within the animal body; and yet there are many
germs which are extremely pathogenic, and in many cases capable of

FACTS AND PROBLEMS OF IMMUNITY

309

severely and detrimentally infeeting the animal body. In view of this
unquestioned fact, the teaching which considers all poisonings as due
either to true soluble secreted poisons, or to true endotoxins liberated
only on disintegration of the bacterial cell, is probably too narrow; and it
would seem not unlikely that many organisms, possibly all, secrete bodies
which are not soluble in their condition at secretion in culture media or
in the body fluids, but which are susceptible to digestion in the animal
body, and may thus become soluble and assimilable, and when toxic act
harmfully on the body cells. This question is an important one and
will be considered later. Besides these actively poisonous bodies which
we have been considering, there are probably bodies such as some at
least of the substances called aggressins by Bail, which, while not being
toxic in themselves for the animal body, nevertheless are active defen¬
sive agents of the bacteria, probably neutralizing certain bodies of the
animal economy, which are indirectly injurious to the bacteria. Further
than this, certain bacteria may be furnished with envelopes, capable
possibly of protecting them either chemically or physically from harm¬
ful influences.

Some clarity of conception may, as we have suggested, be gained
by comparing some of the products of pathogenic bacteria with bacterial
pigments and with insoluble interstitial or intercellular substance,
which may be seen accompanying bacteria in cover-glass prepara¬
tions. Soluble toxic secretions are to be compared to such pigments
as the pyocyanin of Bacillus pyocy emeus , which is so readily soluble in
culture media; endotoxins proper, to pigments confined to the bac¬
terial cell or, at least when secreted, being insoluble in culture media,
such, for instance, as the well-known red pigment of Bacillus prodigiosus,
which may often be seen free among the bacteria in irregular red gran¬
ules like carmine powder. That bodies such as this latter might be
extruded from pathogenic bacteria, and not be soluble in the usual cul¬
ture fluids, is not improbable, and the fact that more or less- insoluble
interstitial substances are not infrequent among bacteria is well known.
Among pathogenic germs these characters are often more marked in
freshly isolated cultures. The sticky, almost slimy character of cul¬
tures of meningococcus may be recalled, a character which tends to
disappear after a few generations of artificial cultivation, and the highly
mucinous capsule of the Streptococcus mucosus which tends to decrease
under artificial cultivation, as do also the capsules of pneumococci and
streptococci.

Now, it seems — and this view has been supported by Walker, Deutsch,

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INFECTION AND IMMUNITY

Welch, and Eisenberg, and is, in fact, but an axiom which would be
recognized immediately by any trained biologist — that all micro¬
organisms will adapt themselves so far as is permitted by their physio¬
logic peculiarities to the stress of the environment, the exact direction
which this adaptation will take being determined by the character of
the environment, chemical and physical, and the physical, chemical, and
physiologic characteristics of the germ involved.

Thus far, in considering the means of offense and defense at the com¬
mand of the bacteria, we have largely left out of consideration the ani¬
mal organism against which these are directed, or by the changes in
whose functions, metabolism, tissues, cells, and fluids, we are largely
made aware of their existence.

The internal defenses of the animal body — and with these alone we
are concerned — have largely been elucidated, as we have seen, through
morphologic investigation of cellular activities taking place in the ani¬
mal body or under controlled conditions in the test tube, and by visible
reactions taking place in test tubes between the fluids of normal or im¬
munized animals and the bacteria and their products, and, finally, by
the more purely physiologic tests of the protecting power and mechan¬
ism of action of animal fluids or extracts when introduced into another
animal of the same or different species, along with the bacteria or their
products.

Such studies have, as is well known, afforded a vast amount of in¬
formation. Through them the soluble secreted bacterial poisons have
been demonstrated and have been found to stimulate the production of
neutralizing bodies, the antitoxins; bacteria and their culture filtrates
have been shown to call forth bodies which are present in the serum of
animals treated with them, and which cause a precipitation of certain
bacterial constituents of the filtrate — the precipitins; and injections of
animals with bacteria or their products have been found to cause the
production of bodies which are present in the serum and which have
the power of agglutinating the bacteria when brought into contact with
them — the agglutinins; and other bodies are likewise produced which
are capable under proper conditions of killing the bacteria — the bac¬
tericidal substances — or even of dissolving them as we have seen in
some instances — the bacteriolytic substances. All of these bodies may
be demonstrated in the serum of certain normal animals and may be
shown to be increased during the immunization of these animals with
bacteria or their products. The complementing body, however, which
is necessary for the activation of the bactericidal and bacteriolytic

FACTS AND PROBLEMS OF IMMUNITY

311

bodies is not known to be increased during immunization, at least so
far as its presence in the serum is concerned.

These facts we have learned from the study of the serum; on the
other hand, the morphologic investigations instigated and carried on
largely by Metchnikoff and his followers have taught us the great part
which the formed elements of the blood and lymph play in the protec¬
tion against and cure of germ diseases, and the importance of the poly¬
morphonuclear and large mononuclear leucocytes as phagocytes is now
widely recognized.

Of these cells, the polymorphonuclear leucocytes take a very active
part in the ingestion and destruction of bacteria, while the large mono¬
nuclear leucocytes and endothelial cells, especially those lining the blood
vessels and body cavities, although also able to ingest bacteria directly,
are chiefly active in taking up cells of animal origin, principally those
which necessarily, in the normal course of events, belong to the same
animal and have probably become injured or have suffered death.

It does not seem, in this connection, a far-fetched idea to suppose
that phagocytic cells may use naturally other cells and bacteria as a
part of their regular food supply. The polymorphonuclear leucocytes
may thus depend to some extent on the ever-entering bacteria and their
remains; for, as we know, bacteria are constantly entering along the
regular channels of absorption; and it is just as obvious that numbers
of blood and tissue cells are constantly dying out and must be disposed
of, for such processes are always in evidence in the spleen, and the inges¬
tion of polymorphonuclear leucocytes by the large 'mononuclears can be
observed wherever leucocytes are collected in exudates, due either to
infections, poisons, or supposedly benign irritants. The simple fact
that these cells retain the basic physiologic activities and an ability to
ingest and digest food in its crudest form, which ability was the heritage
of their free-swimming ancestors, and that they have not suffered the
total specialization and physiologic degeneracy of the fixed tissue cells,
seems sufficient evidence to warrant the conclusion that they are most
active factors in the protection of the specialized internal tissue cells,
which control the general metabolism and higher functions of the
animal body. It seems worth mentioning that the leucocytes, alone
probably of all the true cells of the body, are entirely independent of the
nerve control, and are subject only to the stimulation of their chemical
and physical environment, and are thus susceptible of adaptation to
and capable of subserving various purposes which would be fatal to the
duties of cells controlled by the nerve mechanism for the special func-

312

INFECTION AND IMMUNITY

tions of the organism at large. Further than this the death of leuco¬
cytes does not matter, as would the death of specialized and nerve-
controlled cells, for no special metabolic or functional derangement
occurs from their destruction.

In considering this independence of the leucocytes it must not be
assumed that they have not varied from primitive ameboid cells, for un¬
doubtedly their life and proper functioning are largely determined by the
special plasma in which they live, and it may be that their food, although
at times crude compared with that of the other body cells, is never¬
theless usually prepared for them by processes going on in the plasma.

Questions relating to the independence and to the interrelation of
the plasma and leucocytes in their action on invading microorganisms
and the action of plasma as compared with serum have been ground for
scientific strife for many years, one side contending for the activity of
the plasma, the other for the activity of the phagocytes; the humoralist
at first neglecting, if not absolutely forgetting, that a fluid can not be
self-replenishing, while the supporters of phagocytosis largely over¬
looked the fact that plasma is not necessarily an inert menstruum such
as salt solution.

While these differences have been to some extent adjusted by the
theory and work of Ehrlich, an immediate point of contention is still the
question of the similarity of action of plasma and serum. The humoral
school contends that the alexin of Buchner — complement of later
writers — is secreted into the plasma, while the Metchnikoff school
claims that it is only given up from injured leucocytes in the body,
and to the serum by destruction of leucocytes during coagulation. The
Metchnikoff school admits, however, that the amboceptors necessary
for bactericidal and bacteriolytic action are formed in excess in the
phagocytes, and given off from these to the plasma, yet asserts that
they are inactive for lack of the complement which is normally retained
in the leucocytes, and that they simply prepare the bacteria for com¬
plete digestion in the leucocytes. The relation of the bacteriolytic ambo¬
ceptors to intracellular digestion is not settled, although it seems illogical
for a digestive body to be produced in excess that has not arisen from
cells by the stimulation of its use, and, as the leucocytes take up the
bacteria, they are the most likely producers and users of this body.

In 1894 a further adjustment of differences took place, when certain
phenomena observed by Denys and his pupil Leclef demonstrated that
the act of phagocytosis when performed in serum, in some instances
at least, was dependent on the presence of certain substances in the

FACTS AND PROBLEMS OF IMMUNITY

313

serum. Thus, they were able to show that leucocytes removed from
normal blood and placed with bacteria in immune serum enulfed
the bacteria actively, while leucocytes from immunized animals mixed
with bacteria in normal serum took up the organisms no more actively
than the normal leucocytes. The bodies inciting the phagocytosis must
obviously, then, they concluded, be in the serum. Whether these bodies
acted on the leucocyte or on the bacteria was not then determined,
but Denys concluded, in 1898, that the bacteria were directly affected.
The fact that the action is exerted on the bacteria was recently de¬
termined positively by Wright for normal serum, and by Neufeld and
Rimpau, independently of Wright, for immune serum. These bodies
have been called ops.onins by Wright, and bacteriotropins by Neufeld,
and have been shown to attach themselves to the bacteria and thus
prepare them for ingestion by the phagocytes. It has also been shown
by various observers that the more virulent the germ, the less susceptible
it is to phagocytosis and the more potent the antisera must be to permit
of the ingestion by the cells.

If now, for clarity of conception, we summarize briefly the disease-
producing agents possessed by the bacteria and the opposing substances
of the serum and processes of the animal body, we find the true toxins,
including probably leucocidins and hemolysins, opposed by antitoxins
which become free in the plasma; the bacterial bodies and probably
the endotoxins opposed by leucocytes, and possibly directly in the
plasma by lytic substances formed of amboceptor and complement,
which either kill or dissolve the bacteria and free the endotoxins, but
do not neutralize them; and, third, we have probably certain secretions
which oppose the opsonins, and thus prevent phagocytosis — antiopsonins
— bodies which may possibly be the so-called aggressins of Bail, and
which are p, resent in exudates and, although not toxic in themselves, in¬
crease the infectiousness of the bacteria with which they are injected;
and, finally, opposed to bacteria and their broth filtrates we have the
agglutinins and precipitins, the activities of which are manifest in
serum, but whose relation to immunity is not altogether obvious, as
they have not been shown satisfactorily to bring about agglutination or
precipitation in the animal body.

While all of these different functions and chemical substances are
possessed by animals as a class, it is becoming more and more obvious
that these are not always present or active in the same degree, and that
there are recognizable differences in the protective mechanism of dif¬
ferent animal species — in species, in fact, not far removed from each

314

INFECTION AND IMMUNITY

other in the natural classification. An explanation of reactions to a
given infection which applies in the case of one species is not, therefore,
obviously applicable in the case of another species. -This is true not only
of the mechanism of protection as it takes place in the serum of dif¬
ferent animals and in their plasma, but also of phagocytosis and phagocy¬
tic digestion and the factors which contribute to the perfection of these
processes. The constant stumbling-block in the way of a correct in¬
terpretation of processes going on in the animal body is our inability, as
we have seen, to argue from serum phenomena to phenomena occurring
in the plasma. A failure to keep this in mind, although it is fully recog¬
nized, has undoubtedly led to many hasty conclusions, particularly
connected with the theory of lytic immunity. This may be illustrated
by a well-known example: Fresh rabbit serum is actively germicidal for
anthrax bacilli, dog serum is not; yet rabbits are extremely sensitive
to a true anthrax infection, while dogs are very resistant. Experiment
has shown that there are lytic amboceptors in the sera of both these
animals, but that the dog’s serum does not contain the complement
necessary for their action on the bacilli; the complement presumably
has remained in the body cells, whereas in the case of the rabbit it
has possibly been liberated from the leucocytes during clotting. The
reason the dog is insusceptible is, then, not because of a more active
plasma destruction of the invading anthrax germs, but probably
because of a more perfect adjustment of the cellular mechanism to the
infection, although if we simply followed the theory of the bactericidal
action of serum and plasma as being coextensive, and the active pro¬
tective mechanism, the rabbit should have been protected, while the dog
should have succumbed. The difference here probably depends upon
the possession of all requisites for the perfect performance of phagocy¬
tosis, and the complete digestion of the bacteria by the phagocytes of
of the dog, while in the rabbit either the mechanism of ingestion is in¬
complete or the cells fail to cope successfully with their contents after
ingestion. This example has been selected because anthrax bacilli have
been shown to contain less toxic intracellular bodies — endotoxins —
than many other infectious germs, and the likelihood of the rabbit being
poisoned by any primary plasma disintegration of the bacilli is not very
great, so that if the plasma mechanism had corresponded to that of the
serum the animal should have been saved. The validity of such an
argument would not have been so apparent if we had substituted
cholera vibrios for anthrax bacilli in rabbits, for the bodies liberated
from cholera bacilli at their disintegration are very toxic.

FACTS AND PROBLEMS OF IMMUNITY

315

Even if the evidence so far in our possession warranted the conclu¬
sion that the bactericidal and bacteriolytic bodies which are present
in the sera of various animals are present and active against certain
microorganisms in the same manner in their plasma, we should, never¬
theless, still have a number of microorganisms which are singularly
insusceptible to such action of the serum or plasma, even of animals
highly immunized against them. The method of resistance against
these would have to be explained by a different mechanism, and if this
death and destruction are not accomplished in the plasma, then we must
look largely to the activities of the leucocytes for their accomplishment.

Now, not only the serum substances which further leucocytosis have,
as we have seen, received much attention of late, but the bodies an¬
tagonistic to the bacteria which are supposed to be contained in the leu¬
cocytes have also been extensively investigated.

Experiments bearing on these questions make it appear extremely
probable that bactericidal and bacteriolytic action depend on two
processes; one of these is the bacteriolytic action of the serum and
plasma, the other the bactericidal action of substances retained in the
leucocytes. As an example of the type supposed to depend solely on
the bactericidal substances of the serum or plasma, the mechanism of
the natural and artificial immunity of guinea-pigs to typhoid and cholera
may be cited, since in these animals no one has as yet succeeded in de¬
monstrating that substances derived from the leucocytes by extrac¬
tion have any bactericidal action on the organisms of these two diseases.
This does not mean, however, that the bactericidal action takes place
naturally outside of the leucocytes, for the bacteria loaded with am¬
boceptors are probably taken into the leucocyte and there digested.
As examples of immunity depending on substances within the leucocytes,
the natural and artificial immunity of dogs and cats to anthrax, and the
immunity of guinea-pigs to certain strains of proteus, may be cited,
for in these cases the leucocyte extract is germicidal, while the serum is
not.

Stated impartially, then, our knowledge of immune bodies and proc¬
esses stands somewhat thus: Bodies which are bactericidal and bac¬
teriolytic may be present in the plasma, and certainly in the serum,
wherever this is formed in a pathologic process, which when supplied
with complement, either normally present in the plasma or derived from
injured leucocytes or other sources, may be active against microorgan¬
isms, either killing them or actually breaking them up in some cases and
liberating bodies which are then directly poisonous or become so by fur-
21

316

INFECTION AND IMMUNITY

ther digestion. Besides these germicidal bodies, there are other bodies
which, while not directly harmful to the bacteria, render them powerless
against the phagocyting power of the leucocytes. These bodies are
probably present in the plasma, certainly in the serum. They are the
opsonins or bacteriotropins.

After phagocytosis has taken place, the germs may be killed and
digested. Some of the bactericidal bodies of the phagocytes are bodies
differing in character from the lytic bodies of the serum, and are either
not given off to the serum or are not active in it; but there is no proof
that the lytic amboceptors present in the serum are not normally,
in part at least, derived from the leucocytes, and active in intracellular
digestion when activated by complement. This is supported by the sup¬
position that guinea-pig leucocyte extracts are not germicidal for
cholera and typhoid organisms. Nevertheless, intracellular digestion
of these germs does go on; it is possible, therefore, that the ambo¬
ceptors present in the plasma, whatever their source, attach themselves
to the germs and aid in intracellular digestion.

None of the processes just mentioned leads to the formation of anti¬
toxins which become free in the plasma or serum. Now, in view of these
facts and suppositions, it may possibly be logical to conceive that nearly
all pathogenic germs secrete bodies which are not readily soluble in cul¬
ture fluids or in the fluids of the animal body ; that these bodies are not
readily, if at all, assimilable by non-phagocyting cells. These bodies may,
however, be broken up by digestive bodies present in the serum, and
from them may thus be liberated a poisonous substance, which may
then be assimilated by the higher cells of the body, and, when in suffi¬
cient quantity, cause death. The more rapid the process of liberation
the more quickly death ensues. This plasma digestion is, then, according
to this conception, a mechanism which is faulty when applied to bacteria
and their products, and if this conception is correct the fault may occur
somewhat as follows: Bacteria and their insoluble or non-assimilable
products when taken into the phagocyte are subjected to two processes,
a primary bactericidal and coagulating one, and then a more leisurely
lytic or disintegrating action, during which poisonous products are
probably liberated, but slowly enough to be taken care of by destroy¬
ing or neutralizing bodies. Even if the leucocyte dies, it is usually taken
up by a mononuclear cell, and the poisons do not become free in the
fluids. Now, in this process the only bodies which are produced in excess
and at the same time are capable of escaping from the leucocytes are the
f}Tic bodies; neither the toxin-neutralizing body nor the coagulating

FACTS AND PROBLEMS OF IMMUNITY

317

body are secreted or given off from the cell. Such lytic amboceptors,
then, when present in the plasma and activated by complement, may
thus become an active agent for harm by liberating poisonous sub¬
stances from the bodies of germs which are susceptible to such action,
or from the insoluble or non-assimilable products of these or more lysis-
resisting members of the invasive organisms; and by the action of these
poisons, phagocytosis may be hindered and the specialized cells poisoned.
Since the neutralizing or poison-destroying bodies are not present in the
plasma, the leucocytes are then poisoned from without, just as are the
specialized cells, and the more active the plasma digestion, the more
deranged the true protective mechanism becomes.

These are some of the problems of immunity, particularly those
relating to the microorganisms which are harmful to the animal body,
not so much through their ability to secrete harmful soluble poisons, as
through their insistently invasive character, or by the liberation of the
toxic products resulting from the destruction of their secretions or of
their own bodies. It is the diseases caused by these organisms on which
the attention of bacteriologists is now chiefly centered.

The organisms of these diseases undoubtedly belong to two or more
classes, in one of which may be placed the typical septicemia producers
— anthrax, pneumococcus, streptococcus, etc. — in the other the less in¬
vasive organisms, typified by cholera and to some degree b}^ typhoid.
Between these two extremes there are all grades.

If the data amassed in the study of these types of microorganisms,
and of the processes supposed to be involved in meeting infection and
establishing cure and immunity from them, have been made clear, it
may be easier to comprehend some of the problems which daily face
investigators in their struggle to arrive at a rational method of biologic
treatment, and to realize more fully, in the light of this knowledge, why
disappointment has so persistently followed in the wake of serum therapy
as applied to these infectious diseases. For, in spite of the most persist¬
ent attempts to produce curative sera, the results have not been satis¬
factory and have not led, except in rare instances, to the practical use of
such sera in the treatment of disease in man.

The sera, thus produced, have not, except in a very minor way,
been antitoxic in the usually accepted sense, and depend, as we have
seen, probably, for any protective value they may possess, on their germi¬
cidal and bacteriolytic power and on the opsonins they may carry, and
thus facilitate phagocytosis. These sera are capable of protecting an
animal from an infecting organism, when mixed with it in surprisingly

318

INFECTION AND IMMUNITY

minute quantities; but that consistent curative effects, other than
merely local, have been definitely determined as due to their action,
after an infection has once been established, is open to serious
doubt.

On the other hand, indeed, test and experiment have shown that
animals and man suffering from a true infection may and often do
themselves furnish sera capable of strong bactericidal and bacteriolytic
action (when combined with normal sera containing complement), and
yet in spite of this, they succumb or may be subject to severe relapses.

In the light of these and other facts which have been cited, it seems
that one might well refrain from attempts to produce beneficial effects by
injecting still further amounts of bacteriolytic or similar bodies, and seek
further for an explanation of the exact methods and processes of the
cure effected in those animals and man who do survive an infection.

Failure to solve these problems on lines hitherto followed should not
discourage us, however, while we know that animals and man do re¬
cover naturally from such infections. The conclusion that this power
must reside in increased digestive and neutralizing or poison-destroying
powers of the animal organisms can not well be avoided, and these
functions of the animal mechanism will probably be found to take place
largely in some group of cells.

The animal body, then, ideally protected in the time of bacterial in¬
vasion, may well be one in which some set of cells — phagocytes — are
immediately ready and able to take up the bacterial invaders and de¬
stroy them, and within their own bodies to neutralize any poisons se¬
creted by such invaders or arising from their destruction by digestion,
and this without serious harm to the ingesting cells; or — failing this
full immunity from serious harm — it may be that these ingesting cells
are, in their turn, taken up and, with their noxious contents, digested by
other scavenging cells, with a minimum liberation of the substances
which could injure the body cells dedicated to specialized functions.
The whole struggle of the infected organism may be summed up as a con¬
flict between the leucocytes and the germs, and that it is an attempt to
bring the invading germs within the leucocytes, and is a process with
which the system at large often has little or nothing to do, except as an
innocent and injured bystander, and that extracellular destruction of
bacteria and toxicogenic bodies is an untoward event after the thorough
establishment of infection often leading to dire consequences, and
depending on the chance occurrence of suitable digestive bodies in the
gerum which have been thrown off in excess from the cells, and which

FACTS AND PROBLEMS OF IMMUNITY

319

may thus become a menace to the system at large by liberating poison¬
ous bodies from comparatively harmless compounds.

Thus, in many instances, it seems we are probably dealing with an
immunity, a large part of the mechanism of which is intracellular, not
only in the sense of phagocytosis and digestion, but in the neutralization
or destruction of poisons which arise from the disintegration of the bac¬
teria and their products — a mechanism in which the protecting cells
must intervene and, largely unaided by antitoxic bodies in the plasma,
neutralize or destroy within themselves the poisonous products of the in¬
vading microorganisms.

It was this thought which suggested the idea of treating infections
with the extract of leucocytes, and thus aiding the phagocytes by
furnishing them as directly as possible with the weapons which they
use in their fight with invading microorganisms, and also to protect
them and the cells of specialized function from destruction and give
them an opportunity to recuperate and carry on successfully their
struggle against the invading germs.

The treatment of infections with vaccines also is based upon the
recognition of the necessity of the direct intervention of phagocytes
in the cure of certain bacterial diseases and is, as we have seen, an
endeavor to stimulate the production of substances facilitating the
ingestion of the organisms by the phagocytes.

Finally, it must be remembered that while animal experiments are
necessary, and often extremely instructive, one can, nevertheless, not
always argue directly from these to occurrences in man. An injection
disease is not an injections disease , and we are dealing usually with con¬
ditions in man which are at least not entirely analogous to artificial in¬
fections in animals. Artificial infections are usually accomplished by an
abrupt introduction of a large quantity of infecting germs and their
products; the animal powers of resistance are often immediately and se¬
verely taxed ; the incubation period thus artificially shortened ; and the
germs themselves, being present in large numbers, are not subjected to
such a searching elimination as is usually the case with the few organisms
gaining a foothold by the natural channels of infection. This difference
is most marked in septicemias, in which, in animal experiments, the or¬
ganisms have been introduced directly into the circulation in quantities
sufficient to bring about a very rapid poisoning and overwhelming of the
animal, with probably only a very partial adaptation of the bacteria
to the animal agents of resistance. On the other hand, the septicemic
invasion in man most often follows the adaptation of the germs in some

320

INFECTION AND IMMUNITY

more favorable ?iidus, and probably has to do with an evolution in the
bacterial resistance to the protective powers, rather than a decrease in
protective strength on the part of man. Indeed, both of these processes
may increase hand in hand, and we may have septicemias extending over
weeks, months, and even years. We may have, iu fact, an “ armed
peace77 and the prepared bacterial army is not to be routed by the
application of means which under other circumstances might prove effi¬
cacious, for we have seen how the bacteria may possibly become resist¬
ant to the protective agents of the animal body, and may continue to
survive attacks which might well prove fatal to less well-adapted
members of their species.

Theoretically, then, we are safe in assuming that the infections in
man which most closely simulate the usual artificial infection in animals
are fresh local infections, and infections of any character, in their earliest
stages, before the bacteria have been adapted to carry on their fight
with the powers of the infected body.

The point which should be made clear is that the outcome of our
attempts to treat infectious diseases is, if we have the real means in our
hands, probably more dependent on the degree of adaptation of the
germs than the actual powers of resistance of the patient. These latter,
of course, determine largely the picture of the disease, but give little
information as to the power of the invader. This has been forced on us
by the fact that, although we are able to cure positively acute septice¬
mias in animals, the more subacute septicemias of man do not yield
readily to our present modes of treatment, whereas even extremely
severe acute and chronic localized diseases, due to the same organisms,
respond to treatment.

SECTION III

PATHOGENIC MICROORGANISMS

CHAPTER XXI

THE STAPHYLOCOCCI (MICROCOCCI)

The power to incite purulent and sero-purulent inflammations and
localized abscesses in man and animals is possessed by a large variety of
pathogenic bacteria. Most infections, in fact, in which the relative
virulence of the incitant and the resistance of the infected subject are
so balanced that temporary or permanent localization of the infec¬
tious process takes place are apt to be accompanied by the formation of
pus. The large majority of acute and subacute purulent processes,
however, are caused by the members of a well-defined group of bacteria
spoken of as the pyogenic cocci. Among these, pre-eminent in import¬
ance, are the “ staphylococci ” or “ micrococci

Many of the earlier investigators of surgical infections had seen small
round bodies in the pus discharged from abscesses and sinuses and had
given them a variety of names. Careful bacteriological studies, how¬
ever, were not made until 1879 and the years immediately following,
when Koch, Pasteur, Ogston,1 and others not only described morphologi¬
cally, but cultivated the cocci from surgical lesions of animals and man.
Of fundamental importance are the studies published by Rosenbach 2
in 1884, in which the technical methods of modern bacteriology were
brought to bear upon this subject for the first time. The group of
staphylococci — so named from their growth in irregular, grape-like
clusters — is made up of several members, by far the most important
of which, pathologically, is the Staphylococcus pyogenes aureus.

i Ogston , Brit. Med. Jour., 1881.

2 Rosenbach, “ Microorganismen bei Wundinfektion,” 1884.

321

322

PATHOGENIC MICROORGANISMS

STAPHYLOCOCCUS PYOGENES AUREUS

Morphology and Staining. — This microorganism, the most frequent
cause of abscesses, boils, and many surgical suppurations, is a spherical
coccus having an average diameter of about 0.8 micra, but varying
within the extreme limits of 0.4 to 1.2 micra. Any considerable variation
from the average size, however, is rare. The perfectly spherical charac¬
ter may not develop, whenever, as is usually the case, two or more are

Fig. 69. — Staphylococcus pyogenes aureus. (After Gunther.)

grouped together, unseparated after cell cleavage. In this case, adj acent
cocci are slightly flattened along their contiguous surfaces.

Examined in smears from cultures or pus, the staphylococci may
appear as single individuals, in pairs, or, most frequently, in irregular
grape-like clusters. Occasionally, short chains of three or four may be
seen. In very young cultures in fluid media, the diplococcus form may
predominate.

The staphylococci stain with all the usual basic aqueous anilin dyes,
and, less intensely, with some of the acid dyes. Stained by the method
of Gram, they retain the anilin-gentian- violet. Gram’s method of
staining is excellently adapted for demonstration of these cocci in
tissue sections.

STAPHYLOCOCCUS PYOGENES AUREUS

323

Although exhibiting marked Brownian movements in the hanging
drop, staphylococci are non-motile and possess no flagella. They are
non-sporogenous and form no capsules.

Cultural Characters. — Staphylococci grow readily upon the usual
laboratory media. The simpler media, made of meat extract, are quite
as efficient for their cultivation as are the freshly made meat-infusion

Fig. 70. — Staphylococcus Colonies

products. The optimum temperature for staphylococcus cultivation
lies at or about 30° C., though growth readily takes place at tempera¬
tures as low as 15° C., and as high as 40° C. Slow but definite growth
has been observed at a temperature as low as 10° C.

While development is most characteristic and luxuriant under
aerobic conditions, staphylococci are facultatively anaerobic on suitable
media. They grow readily in an atmosphere of hydrogen.

324

PATHOGENIC MICROORGANISMS

As to the reaction of media, staphylococcus develops most favorably
upon those having a slightly alkaline titer. Moderately increased
alkalinity or even moderate acidity of media does not inhibit growth.

On gelatin plates, growth occurs readily at room temperature, form¬
ing within thirty-six to forty-eight hours, small, shining, pin-head
shaped colonies, appearing, at first, grayish-white, and later assuming a
yellowish hue, which intensifies into a light brown and often a bronze
color as the colony grows older. The intensity of the color differs con¬
siderably in different races of staphylococci. Liquefaction of the gelatin
occurs, and, shallow, saucer-shaped depressions are formed about the
colonies after forty-eight hours or more. These zones of fluidification
grow larger as the colonies grow, finally becoming confluent. Micro¬
scopically, the colonies themselves, before liquefaction has destroyed
their outline, are round, rather finely granular, with smooth edges.
They are not flat, but rise from the surface of the medium as the seg¬
ment of a sphere. In gelatin stab cultures in tubes, fluidification leads
to the formation of a funnel-shaped depression, with, finally, complete
liquefaction of the medium and sedimentation of the bacteria. Lique¬
faction of gelatin by the staphylococcus is due to a ferment-like body
elaborated by it, which is spoken of as “gelatinase.” This substance
can be obtained apart from the cocci by the filtration of cultures.1 It
is an extremely thermolabile body.

On agar plates the characteristics of the growth, barring liquefaction,
are much like those on gelatin. Colonies do not show a tendency toward
confluence, remaining discrete, and show a rather remarkable differ¬
ence in the size of the colonies occurring upon the same plate. Upon
slanted agar in tubes, rapid growth occurs, at first grayish- white, but
soon covering the surface of the slant as a glistening, golden-brown
layer.

In broth, growth is rapid, leading to a general, even clouding of the
medium, and giving rise, after forty-eight or more hours, to the formation
of a thin surface pellicle. As growth increases, the bacteria sink to the
bottom, forming a heavy, mucoid sediment. The odor of old cultures
is often peculiarly acrid, not unlike weak butyric acid.

In milk, staphylococcus causes coagulation usually within three or
four days, with the formation of lactic and butyric acids.

On potato, growth is abundant, rather dry and usually deeply pig¬
mented.

1 Loeb, Cent. f. Bakt., xxxii, 1902.

STAPHYLOCOCCUS PYOGENES AUREUS

325

Upon coagulated animal sera, rapid growth takes place and eventually
slight liquefaction of the medium is said to occur.

In nitrate solutions, reduction of the nitrates to nitrites is caused.

In Dunham’s broth, indol is formed.

In media containing the carbohydrates — dextrose, lactose, or sac-
charose— acidification takes place with the formation chiefly of lac¬
tic, butyric, and formic acids. There is no gas formation, however.
In proteid media free from sugars, the staphylococcus produces
alkali.

The reducing action of staphylococcus is shown by decolorization
in cultures of litmus, methylene-blue, and rosanilin.1

Pigment Formation. — Differentiation between the various members
of the staphylococcus group is based largely upon the formation of
pigments. These pigments, so far as we know, seem to be species
characteristics. Thus, Staphylococcus pyogenes aureus is recognized
primarily by its production of a yellowish-brown pigment, varying in
different strains from a pale brown hue to a deep golden yellow. Pro¬
longed cultivation upon artificial media may lead to a diminution in the
depth of color produced.2 It appears only when cultivation is carried
on under freely aerobic conditions, anaerobic cultivation resulting in
unpigmented colonies. The coloring matter is insoluble in water but
soluble in alcohol, chloroform, ether, and benzol.3 According to Schnei¬
der,4 the pigment belongs to the class of 11 lipochromes ” or fatty pig¬
ments, and is probably composed of carbon, oxygen, and hydrogen,
without nitrogen. Treatment with concentrated sulphuric acid changes
it to a green or greenish-blue.5

Resistance. — Although not spore formers, staphylococci are more
resistant to heat than many other purely vegetative forms. The thermal
death point given for Staphylococcus pyogenes aureus by Sternberg 6
lies between 56° and 58° C., the time of exposure being ten minutes.
The same author states that, when in a completely dried state, the coccus
is still more resistant, a temperature of from 90° to 100° C. being re¬
quired for its destruction. Against low temperatures, staphylococci are
extremely resistant, repeated freezing often failing to sterilize cultures.

1 Fr. Muller, Cent. f. Bakt., xxvi, 1899.

2 Fliigge, “Die Microorg.,” etc.

3 Migula, “System d. Bakt.,” Jena, 1897.

4 Schneider, Arb. a. d. bakt. Inst., Karlsruhe, 1, vol. i, 1894.

5 Fischer, “Vorles. iiber die Bakt.,” Jena, 1903.

6 Sternberg, “Textbook,” etc., N. Y., 1901, p. 375.

326

PATHOGENIC MICROORGANISMS

Desiccation is usually well borne, staphylococci remaining alive for
six to fourteen weeks when dried upon paper or cloth.1 On slant agar,
staphylococci may be safely left for three or four months without trans¬
plantation, and remain alive.2

The resistance of staphylococci to chemicals, a question of great
surgical importance, has been made the subject of extensive researches,
notably by Liibbert,3 Abbott,4 Franzott,5 and many others. According
to Liibbert, inhibition of staphylococcus growth is attained by the use of
boric acid 1 in 327, salicylic acid 1 in 650, corrosive sublimate 1 in
80,000, carbolic acid 1 in 800, thymol 1 in 11,000. Staphylococci are
killed by corrosive sublimate 1 in 1,000 in ten minutes, by carbolic acid
1 per cent in 35 minutes, 3 per cent in 2 minutes (Franzott). Ethyl
alcohol,6 even when absolute, is not very efficient as a disinfectant.
Nascent iodin, as split off from iodoform in wounds, is extremely power¬
ful in destroying staphylococci.

Pathogenicity. — Separate strains of Staphylococcus pyogenes aureus
show wide variations in relative virulence. The most highly virulent
are usually those recently isolated from human suppurative lesions,
but no definite rule can be formulated in this respect. The virulence
of a given strain, furthermore, may be occasionally enhanced by re¬
peated passages through the body of a susceptible animal. Prolonged
cultivation upon artificial media is liable to decrease the virulence of any
given strain, though this is not regularly the case. There are, more¬
over, unquestionably, many staphylococci constantly present in the air,
dust, and water, which although morphologically and culturally not
unlike the pathogenically important species, may be regarded as
harmless saprophytes.

The susceptibility of animals to staphylococcus infection is,
likewise, subject to extreme variations, depending both upon differ¬
ences between species and upon fortuitous individual differences
in susceptibility among animals within the same species. Animals
on the whole are less susceptible to staphylococcus than is man.
Among the ordinary laboratory animals, rabbits are most sus¬
ceptible to this microorganism. Mice, and especially the white

1 Deslongchamps , Paris, 1897.

2 Passet, Fort. d. Med., 2 and 3, 1885.

3 Liibbert, “Biol. Untersuch.,” Wurzburg, 1886.

4 Abbott, Medical News, Phila., 1886.

5 Franzott, Zeit. f. Hyg., 1893.

6 Hanel, Beit. z. klin. Chir., xxvi.

STAPHYLOCOCCUS PYOGENES AUREUS

327

Japanese mice, show considerable susceptibility. Guinea-pigs possess
a relatively higher resistance.1

Subcutaneous or intramuscular inoculation of a susceptible animal
usually results in the formation of a localized abscess with much pus
formation and eventual recovery. Intraperitoneal inoculation is more
often fatal. Intravenous inoculation of doses of 0.5 c.c., or more, of
fresh broth cultures of virulent staphylococci usually leads to pyemia
with the production of secondary abscesses, located chiefly in the kid¬
neys and the heart and voluntary muscles, but not infrequently in
other organs as well. In the kidney they occur as small foci, situated
most often in the cortex, composed of a central, necrotic pus cavity,
surrounded by a zone of acute inflammatory exudation. Staphylo¬
coccus lesions form histologically the typical “acute abscess.” Not
infrequently the pyemic condition is accompanied by suppurative
lesions in the joints. Intravenous injections of virulent staphylococci
preceded by injury to a bone is often followed by the development of
osteomyelitis. Mechanical or chemical injury of the heart valves
preceding intravascular staphylococcus inoculation may result in
localization of the infection on or about the heart valves, leading
to “malignant endocarditis.” The pyemic conditions following staphy¬
lococcus inoculation usually lead to chronic emaciation and death
after an interval dependent upon the relative virulence of the micro¬
organism, the amount injected, and the resistance of the infected
subject. Large doses of unusually virulent cultures cause death within
twenty-four hours, or even less, without abscess formation.

As above stated, the susceptibility of man to spontaneous staphy¬
lococcus infection is decidedly more marked than is that of animals.
The form of infection most frequently observed is the common boil
or furuncle. As Garre,2 Biidinger,3 Schimmelbusch,4 and others have
demonstrated by experiments upon their own bodies, energetic rubbing
of the skin with virulent staphylococcus cultures may often be followed
by the development of a furuncle. Subcutaneous inoculation of the
human subject invariably gives rise to an abscess. The pathological
lesions which may be produced in man by virulent staphylococci are
naturally of great variety, depending upon the mode of inoculation, and

1 Terin, Ref. in Lubarsch und Ostertag, Ergebnisse, 1896; Lingelsheim, “Aetiol.
d. Staph. Inf.,” etc., Wien, 1900.

2 Garre, Beit. z. klin. Chir., x, 1893.

3 Biidinger, Lubarsch und Ostertag, Ergebnisse, etc., 1896.

4 Schimmelbusch, Ref. by Biidinger.

328

PATHOGENIC MICROORGANISMS

upon the relation between the virulence of the incitant and the resist¬
ance of the subject. Apart from the formation of 'localized abscesses,
staphylococci are common as the incitants of surgical suppurations
and wound infections. The large majority of acute suppurative in¬
flammations of bone (osteomyelitis) are caused by staphylococci. Ab¬
scesses of the brain, of the liver, and of the lung may be due to this
microorganism. It may give rise to ascending infections of the genito¬
urinary tract, leading to pyelonephritis. Empyema or peritonitis may
be caused by its entrance into the serous cavities from the lung or
bowel. When gaining access to the circulation from some localized
focus, it gives rise to septicemia and may lead to malignant endocarditis
and, by secondary localization in the viscera, to general pyemia. As
the incitant of septicemia it can frequently be found by blood culture
during the life of the patient. Puerperal sepsis is not infrequently a
staphylococcus disease. Of recent years several authors have claimed
direct etiological relationship for the Staphylococcus pyogenes aureus
with acute articular rheumatism.1 While not unlikely, this claim is
not, at present, substantiated by sufficiently exact evidence.

Apart from the local inflammatory reactions called forth by
staphylococcus invasion, all such infections, if severe or prolonged,
give rise to profound toxic manifestations evidenced by characteris¬
tically irregular temperature (the so-called “ septic type”), by head¬
ache, nausea, and general malaise, and not infrequently by chills.
Prolonged chronic infection with staphylococci may give rise to the
so-called amyloid changes in liver, spleen, and kidneys.

Toxic Products. — Endotoxins. — The dead bodies of staphylococci
injected into animals may occasionally give rise to abscess formation,
and,2 if in sufficient quantity, may cause death. To obtain the latter
result, however, large quantities are necessary, the endotoxic substances
within the dead cell body of these microorganisms being probably neither
very poisonous nor abundant.3

That dead cultures of Staphylococcus aureus exert a strong positive
chemotaxis for leucocytes was shown beyond question by the experi¬
ments of Borissow.4

Hemolysins. — In 1900 Kraus5 noticed the hemolytic action of

1 A. H. Weis, Inaug.-Diss., Berlin, 1901.

2 Schattenfroh, Arch. f. Hyg., xxxi, 1887.

3 v. Lingelsheim, “ Aetiol. u. Therap. d. Staph. Krank.,” Wien, 1900.

* Borissow Zieglers Beitr., xvi, 1894.

5 Kraus, Wien. klin. Woch., iii, 1900.

STAPHYLOCOCCUS PYOGENES AUREUS

329

staphylococci growing upon blood-agar plate cultures. Neisser and
Wechsberg1 then showed that this hemolytic substance, secreted by the
staphylococcus, could be demonstrated in filtrates- of bouillon cultures.
Such hemolysins are produced by Staphylococcus aureus, and, to a
lesser degree, by Staphylococcus albus. The quantity produced varies
enormously with different strains and seems to be roughly proportionate
to the virulence of the particular microorganism, though exceptions to
this rule are not uncommon. Absolutely avirulent races do not, so
far as we know, produce hemolysins. The culture medium most favor¬
able to the formation of these substances is, according to Neisser and
Wechsberg, a moderately alkaline beef bouillon. Cultivated at 37.5° C.,
the bouillon contains the maximum amount of hemolytic substance be¬
tween the eighth and fourteenth day, and this may be separated from
the bacteria by filtration through Berkefeld or Chamberland filters.

The hemolytic action may be observed by the general technique for
determining hemolysis (given on page 259). It is important to wash
the red blood corpuscles used for the experiments, since many animals
normally possess small quantities of antihemolysin in their blood-sera
(man and horse especially).2 The red blood corpuscles of rabbits, dogs,
and guinea-pigs are extremely susceptible to the action of the staphylo-
hemolysin. Those of man are less easily injured by it. The hemolytic
action takes place, as Todd3 and others4 have shown, not only in
vitro, but in the living animal as well.

The staphylo-hemolysin is comparatively thermolabile. According
to Neisser and Wechsberg, heating it to 56° C. for twenty minutes de¬
stroys it. According to some other authors, however, higher tempera¬
tures (60° to 80° C.) are required. Reactivation of a destroyed staphylo-
hemolysin has so far been unsuccessful. The fact that antistaphylolysin
is occasionally present in normal sera has been mentioned above. This
antibody is most abundant in the blood of horses and of man. Arti¬
ficially antistaphylolysin formation is easily induced by subcutaneous
inoculation of staphylolysin into rabbits.

Leucocidin. — In 1894, Van de Velde 5 discovered that the pleural
exudate of rabbits following the injection of virulent staphylococci
showed marked evidences of leucocyte destruction. He was subse-

1 Neisser und Wechsberg, Zeit. f. Hyg., xxxvi, 1901.

2 Neisser, Deut. med. Woch., 1900.

3 Todd, Trans. London Path. Soc., 1902.

* Kraus, Wien. klin. Woch., 1902.

5 Van de Velde, La Cellule, x, 1894.

330

PATHOGENIC MICROORGANISMS

quently able to show that the substance causing the death and partial
solution of the leucocytes was a soluble toxin formed by the staphylo¬
coccus, not only in vivo, but in vitro as well; for cultures of Staphylo¬
coccus pyogenes aureus, grown in mixtures of bouillon and blood
serum, contained, within forty-eight hours, marked quantities of this
“leucocidin.”

Other workers since Van de Yelde have evolved various methods for
obtaining potent leucocidin. Bail1 obtained it by growing virulent
staphylococcus in mixtures of one-per-cent glycerin solutions and rab¬
bit serum. Neisser and Wechsberg2 advise the use of a carefully titrated
alkaline bouillon. To obtain the leucocidin free from bacteria, the
cultures are passed through Chamberland or Berkefeld filters, after
about eight to eleven days’ growth at 37° C., at which time the con¬
tents in leucocidin are usually at their highest point.

The action of leucocidin upon leucocytes may be observed in vivo
by the simple method of Van de Velde, of injecting virulent staphylo¬
cocci intrapleurally into rabbits and examining the exudate. Bail
advises the production of leucocytic intrapleural exudates by the use
of aleuronat and following this after twenty-four hours by an injection
of leucocidin-filtrate. In vitro the phenomenon may be observed by
direct examination of mixtures of leucocytes and leucocidin in the
hanging drop on a warmed stage, or by the “ methylene-blue method”
of Neisser and Wechsberg. This method is based upon the fact that
living leucocytes will reduce methylene-blue solutions and render them
colorless, while dead leucocytes have lost this power. Leucocidin and
leucocytes are allowed to remain in contact for a given time and to them
is then added an extremely dilute solution of methylene-blue. If the
leucocytes have been actively attacked by leucocidin, no reduction takes
place. This method is particularly adapted for quantitative tests.

All staphylococcus strains do not produce leucocidin to the same
degree. Almost all true Staphylococcus pyogenes aureus cultures
produce some of this toxin, but one strain may produce fifty- and a
hundred-fold the quantity produced by another. Staphylococcus
pyogenes albus gives rise to this substance but rarely, and then in small
quantity.

Leucocidin seems to be similar to the soluble toxins of other bacteria.
It is rapidly destroyed by heat at 58° C., and deteriorates quickly in

1 Bail, Arch. f. Hyg., xxxii, 1898.

2 Neisser und Wechsberg, Zeit. f. Hyg., xxxvi, 1901.

STAPHYLOCOCCUS PYOGENES AUREUS

331

culture fluids at incubator temperatures. It is distinct from staphylo-
hemolysin as shown by differences in thermostability.

Soon after Van de Velde’s discovery of leucocidin, Denys and Van
de Velde1 produced an antileucocidin by treating rabbits with pleural
exudate containing leucocidin. Neisser and Wechsberg 2 later confirmed
these results and showed that among staphylococci, leucocidin is not
specific, the toxin of all strains of Staphylococcus aureus and albus
examined being neutralizable by the same antileucocidin. Antileuco¬
cidin is often found in the normal sera of horses and man.3

Leucocidin should not be confounded with “leucotoxin,” a substance
obtained in serum by treatment of animals with leucocytes, a true
“eytotoxin,” having no connection whatever with the staphylococcus.

Staphylococci, besides the toxic substances already mentioned, give
rise to gelatinase, spoken of in the section upon cultivation, and to a
proteolytic ferment by means of which albuminous media (Loeffler’s
serum) may be slightly digested.

Immunization.— Animals can be rendered actively immune by re¬
peated inoculations with carefully graded doses of living or dead
staphylococcus cultures.4 The production of antistaphylolysin and of
antileucocidin in the sera of animals so treated, has been alluded to
in the preceding sections. The sera of such actively immunized animals
possess distinct protective power when administered to other animals,
slightly before or at the same time with an inoculation of staphylo¬
cocci. They do not, however, exhibit very high bactericidal value
in vitro. The use of immune sera to combat staphylococcus infection
has not so far given very encouraging results.5

Agglutinins have been demonstrated in staphylococcus immune sera
by a number of authors, and have been shown to be of value in differ¬
entiating between the several groups of staphylococci.6 A rather sur¬
prising result of these researches has been the recognition that immune
sera obtained with pathogenic staphylococci will agglutinate other
pathogenic staphylococci, whether belonging to the group of Staphy¬
lococcus pyogenes aureus or that of Staphylococcus pyogenes albus,

1 Denys et Van de Velde, La Cellule, xi, 1895.

2 Loc. cit.

3 Van de Velde, Presse medicale, i, 1900.

4 Richet et Hericourt, Compt. rend, de l’acad. des sci., cvii, 1888.

5 Kolle und Otto, Zeit. f. Hyg., xli, 1902.

6 Proscher, Cent. f. Bakt., xxxiv, 1903; v. Lingelsheim, “ Aetiol. u. Therap. d
Staphyl.,” etc., Wien, 1900.

332

PATHOGENIC MICROORGANISMS

but will not agglutinate any of the non-pathogenic members of
either group. 1

Active immunization of human beings suffering from staphylococcus
infections has been extensively practiced by Wright, in connection with
his work on opsonins. There can be no question about the fact that the
opsonic substances in the blood are increased by the injection of dead
staphylococci. The procedure is of therapeutic value in subacute and
chronic cases. The work of Hiss on the use of leucocyte extracts in
animals infected with Staphylococcus pyogenes aureus has given en¬
couragement for such treatment in human beings. A number of
staphylococcus infections in man have been successfully treated with
leucocyte extract by Hiss and Zinsser.

STAPHYLOCOCCUS PYOGENES ALBUS

This coccus differs from Staphylococcus pyogenes aureus simply in
the absence of the golden yellow coloration of its cultures. Morpho¬
logically, culturally, and pathogenically, it is in every way identical
with the staphylococcus described in the preceding section, but its
toxin- and enzyme-producing powers in general are less developed than
those of the aureus variety. Its close biological relationship to aureus
is furthermore demonstrated by its agglutination in Staphylococcus
pyogenes aureus immune sera.

STAPHYLOCOCCUS EPIDERMIDIS ALBUS

The Staphylococcus epidermidis albus described by Welch is merely,
one of the non-pathogenic varieties of Staphylococcus pyogenes albus
and possibly does not deserve separate classification. It may give rise
to unimportant stitch abscesses.

STAPHYLOCOCCUS PYOGENES CITREUS

Staphylococcus pyogenes citreus produces a bright yellow or lemon-
colored pigment of distinctly different hue from that of Staphylococcus
pyogenes aureus. It may be pyogenic and in every way similar to
Staphylococcus pyogenes aureus, but is less often found in con¬
nection with pathological lesions than either of the preceding staphy¬
lococci.

1 Proscher, Dent. med. Woch., xi, 1903.

STAPHYLOCOCCUS, PYOGENES CITREUS

333

A large number of staphylococci, differing from those described
above in one or another detail, have been observed. They are of com¬
mon occurrence and are met with chiefly as contaminations in the course
of bacteriological work. Few of these have any pathological significance
and none of them are toxin-producers, so far as we know. Many of them
differ, furthermore, in their inability to liquefy gelatin. All of them
belong more strictly to the field of botany than to that of patho¬
logical bacteriology.

Atypical pathogenic staphylococci have been described by a number
of observers. Thus Weichselbaum 1 isolated a staphylococcus from a
case of malignant endocarditis which could not be cultivated at room
temperature, and grew only in very delicate colonies. Veillon,2 moreover,
has described a strictly anaerobic staphylococcus cultivated from the
pus of an intra-abdominal abscess.

MICROCOCCUS TETRAGENUS

In 1881, Gaffky 3 discovered a micrococcus which occurs regularly
in groups of four or tetrads. He first isolated it from the pus discharged
by tuberculous patients with pulmonary lesions. Observed in smear
preparations from pus, the tetrads are slightly larger in size than the
ordinary staphylococcus, flattened along their adjacent surfaces, and
surrounded by a thick halo-like capsule. Preparations from cultures
often lack these capsules. The micrococcus is easily stained by the
usual basic anilin dyes. Stained by Gram’s method, it is not decolor¬
ized, retaining the gentian-violet.

Cultivation. — Micrococcus tetragenus grows on the ordinary labora¬
tory media, showing a rather more delicate growth than do the staphy¬
lococci.

On agar, the colonies are at first transparent, later they become
grayish-white, but are always more transparent than are staphylococcus
cultures.

On gelatin, growth is rather slow and no liquefaction takes place.

Broth is evenly clouded.

On potato there is a white, moist growth which shows a tendency to
confluence.

1 Weichselbaum, Baumgarten Jahresb., 1899, Ref.

2 Veillon, Compt. rend. soc. de biol., 1893.

3 Gaffky, Mitteil. a. d. kais. Gesundheitsamt, i, 1881.

334

PATHOGENIC MICROORQANISMS

Milk is coagulated and litmus milk indicates acid formation.

Pathogenicity. — Micrococcus tetragenus is especially pathogenic
for Japanese mice, which succumb within three or four days to subcuta¬
neous inoculation.1 Gray mice, rats, guinea-pigs, and rabbits are less
susceptible, showing only a localized reaction at the point of inoculation.

Fig. 71. — Micrococcus Tetragenus. (In spleen of infected mouse.)

The organism has occasionally been isolated from spontaneous abscesses
observed in domestic animals.

In man, this microorganism is usually found without any particular
pathogenic significance, in sputum or saliva. In isolated cases, how¬
ever, it has been described as the sole incitant of abscesses.

Bezangon 2 has isolated Micrococcus tetragenus from a case of menin¬
gitis. A single case of tetragenus septicemia is on record, reported in
1905 by Forneaca.3

In America, this microorganism has not been frequently observed in
connection With disease. It is often found, however, in considerable
numbers* in smears of sputum which are being examined for pneumo¬
cocci or tubercle bacilli.

1 Muller, Wien. klin. Woch., 17, 1904.

2 Bezangon, Semaine med., 1898.

3 Forneaca, Rif. med., 1903.

CHAPTER XXII
THE STREPTOCOCCI

Among the pyogenic cocci, there is a large and important group of
organisms which multiply by division in one plane of space only, and
thus give rise to appearances not unlike chains or strings of beads.
The term streptococcus or chain-coccus is, therefore, a purely morpho¬
logical one which includes within its limits microorganisms which may
differ from each other considerably, both as to cultural and pathogenic
properties. Thus, cocci which form chains may be isolated from water,
milk, dust, and the feces of animals and man. These may have little
but their morphological appearance in common with the pyogenic
streptococci which are so important as the incitants of disease. The
interrelationship between streptococci from different sources, how¬
ever, is by no means fully understood, and we are forced at present to
content ourselves with the recognition of a large morphological group,
in no individual case taking the pathogenic or more special cultural
characteristics for granted.

STREPTOCOCCUS PYOGENES

Of paramount importance among the streptococci are those which
possess the power of giving rise to disease processes in animals and in
man, and which, because of their frequent association with suppura¬
tive inflammations, are roughly grouped under the heading of Strep¬
tococcus pyogenes.

The same researches upon surgical infections which led to the dis¬
covery of the staphylococci, laid the basis for our knowledge of the
streptococci. The fundamental studies of Pasteur and Koch1 were fol¬
lowed, in 1881, by the work of Ogston,2 who was the first to
differentiate between the irregularly grouped staphylococci and the
chain-cocci.

1 Koch, “ Untersuch. liber Wundinfektion,” etc., 1878.

2 Ogston, Brit. Med. Jour., 1881.

335

336

PATHOGENIC ORGANISMS

Pure cultures of streptococci were first obtained by Fehleisen1 in 1883
and by Rosenbach2 in 1884. The thorough and systematic researches of
the last-named authors, together with those of Passet,3 were of special

Fig. 72. — Streptococcus pyogenes.

influence in placing our knowledge of the pathogenic properties of
streptococci upon a scientific basis.

Morphology and Staining. — The individual streptococcus is a spherical
microorganism measuring from 0.5 micron to 1 micron in diameter.
Since the line of cleavage of cocci, when in chains, is perpendicular to the

1 Fehleisen, “ Aetiol. d. Erysipelas,” Berlin, 1883.

2 Rosenbach, “ Mikroorg. bei Wundinfektion,” etc., Wiesbaden, 1884.

3 Passet, “ Untersuch. tiber die eitrigen Phlegm.,” etc., Berlin, 1885.

STREPTOCOCCUS PYOGENES

337

long axis of the chain, adjacent cocci often show slight flattening of the
contiguous surfaces, forming, as it were, a series of diplococci arranged
end to end. As a general rule the streptococci pathogenic for man,
when grown upon favorable media, have a tendency to form chains
made up of at least eight or more individuals, while the more saprophy¬
tic, less pathogenic varieties are apt to be united in shorter groups.
Upon this basis a rough morphological distinction has been made by v.
Lingelsheim,1 who first employed the terms Streptococcus “longus”
and “brevis.” A differentiation of this kind can hardly be re¬
lied upon, however, since the length of chains is to some degree de--
pendent upon cultural and other environmental conditions. Species
which exhibit long and tortuous chains, when grown upon suitably
alkaline bouillon, or ascitic broth, may appear in short groups of three or
four, or even in the diplo form, when cultivated upon solid media or
unfavorable fluid media. Stained specimens often show swelling and
enlargement of individual cocci, giving the chains an irregularly beaded
appearance. These swollen individuals are probably to be interpreted as
involution forms and are seen with especial frequency in old cultures.
Streptococci do not form spores, are non-motile, and do not possess
flagella. There can be no doubt that certain species of true streptococci
may possess capsules, though these are not so regularly demonstrable
and are more delicately dependent upon cultural conditions than are the
capsules of the pneumococci.2 The capsulated streptococci will be dis¬
cussed more comprehensively in the section upon the differentiation of
pneumococcus from streptococcus (page 367).

Streptococci are easily stained by the usual anilin dyes. Stained
by the method of Gram, the pyogenic streptococci are not decolorized
and invariably retain the gentian- violet. Certain species found in stools
and described as Gram-negative, are rare and are non-pathogenic.
Others of the “Streptococcus brevis” variety, and purely saprophytic,
may stain irregularly by the Gram method.

Cultivation. — The pyogenic streptococci are easily cultivated upon
all the richer artificial media. While meat extract-pep ton media may
suffice for certain strains, it is usually better to employ those
media which have the beef or veal infusion for a basis. For the
cultivation of more delicate strains of streptococci, especially when

1 v. Lingelsheim, “ Aetiol. u. Therap. d. Streptok. Infek.” Beit. z. Exp. Ther.,
Hft. 1, 1899.

2 Pasquale, Zieglers Beit., xii; Bordet, Ann. de l’inst. Pasteur, 1887; Schottmiiller,
Munch, med. Woch., xx, 1903; Hiss , Jour. Exp. Med., vi, 1905.

338

PATHOGENIC ORGANISMS

taken directly from the animal or human body, it is well to add to the
media animal albumin in the form of whole blood, blood serum, or asci¬
tic or pleural transudates. Glucose, added in proportions of one to two
per cent, likewise renders media more favorable for streptococcus culti¬
vation. Prolonged cultivation of all races upon artificial media renders
them less fastidious as to cultural requirements. The most favorable
reaction of media for streptococcus cultivation is moderate alkalinity
(two-tenths to five-tenths per cent alkalinity to phenolphthalein) .
Growth may be readily obtained, however, in neutral media or even in
those slightly acid. The optimum temperature for growth is at or about
37.5° C. Above 43° to 45° C., development ceases. At from 15° to
20° C., growth, while not energetic, still takes place, an important point
in the differentiation of these microorganisms from pneumococci. While
the free access of oxygen furnishes the most suitable environment
for most races of streptococci, complete anaerobiosis does not pre¬
vent development in favorable media. Strictly anaerobic streptococci
have been cultivated from the human intestinal tract by Perrone1 and
others.

In alkaline bouillon at 37.5° C., pyogenic streptococci grow rapidly,
form long and tortuous chains, and have a tendency to form flakes which
rapidly sink to the bottom. Diffuse clouding occurs rarely and is a
characteristic rather of the shorter so-called Streptococcus brevis.
When sugar has been added to the broth the rapid formation of lactic
acid soon interferes with extensive development. This may be obviated,
especially when mass cultures are desired, without sacrifice of the growth-
increasing influence of the glucose, by adding to the sugar-broth one per
cent of sterile powdered CaC03.2

In milk, Streptococcus pyogenes grows readily with the formation
of acid, followed, in most cases, by coagulation of the medium.

On agar-plates at 37.5° C., growth appears within eighteen to twenty-
four hours. The colonies are small, grayish, and delicately opalescent.
They are round with smooth or very slightly corrugated or lace-like
edges, and rise from the surface of the medium in regular arcs, like
small droplets of fluid. Microscopically they appear finely granular and
occasionally, under high magnification, may be seen to be composed of
long intertwining loops of streptococcus chains, which form the lace-like
edges. When ascitic fluid or blood serum has been added to agar,
growth is more energetic and the colonies correspondingly more rapid in

1 Perrone, Ann. de l’inst. Pasteur, xix, 1905.

2 Hiss, Jour. Exp. Med., vi, 1905.

STREPTOCOCCUS PYOGENES

339

appearance and luxuriant in development. In glucose-ascitic-agar,
acid formation from the sugar causes coagulation of albumin with
the consequent formation of flaky white precipitates throughout the
medium.1 2

In gelatin stab-cultures growth takes place slowly, appearing after
twenty-four to thirty-six hours as a very thin white line, or as discon¬
nected little spheres along the line of the
stab. The colonies on gelatin plates are
similar in form to those on agar, but are
usually more opaque and more distinctly
white. The gelatin is not liquefied by the
pyogenic streptococci, though certain of
the more saprophytic forms may occa¬
sionally bring about slow fluidification.

On Loeffler’s coagulated blood serum,
growth is rapid and luxuriant, and may
show a slight tendency to confluence if
the medium is very moist. Good chain
formation takes place on this medium.

Upon potatoes, growth is said not to Fig. 73— Streptococcus Col-
take place 2 onies, on Serum Agar.

On media containing red blood cells,
most pathogenic streptococci cause hemolysis and decolorization (see
Fig. 74, p. 345). It is useful to remember this when examining
blood-culture plates, for here the yellow transparent halo of hemo¬
lysis and decolorization surrounding the colonies may aid in differenti¬
ating these bacteria from pneumococci. This is of especial importance,
since many streptococci, when cultivated directly out of the human
blood, do not exhibit chain formation, but appear as diplococci.

In the inulin-serum media of Hiss,3 streptococci do not produce
acid and coagulation. The so-called Streptococcus mucosus, a capsule¬
bearing, inulin-fermenting microorganism, is very probably a sub-species
of the pneumococcus (see later section).

Resistance. — Streptococci on the ordinary culture media, without
transplantation and kept at room temperature, usually die out within
ten days or two weeks. They may be kept alive for much longer periods
by the use of the calcium-carbonate-glucose bouillon, if the cultures are

1 Libman, Medical Record, lvii, 1900.

2 Frosch und Kolle, in Fliigge, “Die Mikroorganismen,” 1891.

3 Hiss, Jour. Exp. Med., vi, 1905.

340

PATHOGENIC ORGANISMS

thoroughly shaken and the powdered marble thoroughly mixed with
the bouillon from time to time.1 Preservation at low temperatures
(1° to 2° C.), in the ice chest, considerably prolongs the life of cul¬
tures. Virulence is preserved longest by frequent transplantation
upon albuminous media. In sputum or animal excreta, streptococci
may remain alive for several weeks.

Streptococci are killed by exposure to a temperature of 54° C . for
ten minutes.2 Low temperatures, and even freezing, do not destroy some
races.

The action of various chemical disinfectants has been thoroughly
investigated by v. Lingelsheim,3 who reports among others the following
results: Carbolic acid 1 : 200 kills streptococci in fifteen minutes. In
the same time, bichloride of mercury is efficient in a dilution of 1 : 1,500,
lysol in a dilution of 1 : 200, peroxide of hydrogen 1 : 35, sulphuric acid
1 : 150, and hydrochloric acid 1 : 150. Inhibition is exerted by car¬
bolic acid 1 : 550, and by bichloride of mercury 1 : 65,000. Exposure
to direct sunlight kills streptococci in a few hours.

Virulence and Pathogenicity. — Different races of pyogenic strepto¬
cocci show considerable variations in virulence, and there are few organ¬
isms, pathogenic both for animals and man, which show such peculiari¬
ties in virulence. The character or severity of the lesion in man gives
little evidence as to the virulence of the organism for animals. Such
differences are, to a certain extent, dependent upon inherent individual
characteristics, but are rather more likely to be the consequences of pre¬
vious environment or habitat. Prolonged cultivation upon artificial
media usually results in the reduction of the virulence of a streptococcus,
while an originally low or reduced virulence may often be much en¬
hanced by repeated passage of the streptococci through animals. It is
noteworthy, however, that while the passage of a streptococcus through
rabbits will usually enhance its virulence for susceptible animals in
general, repeated passages through mice may increase the virulence
for these animals only, even occasionally depressing the virulence
for rabbits.4

Among the domestic animals, those most susceptible to experimental
streptococcus infection are white mice and rabbits. Guinea-pigs and

1 Hiss, loc. cit.

2 Sternberg, “Textbook of Bact.,” 2d ed., 1901; Hartmann, Arch. f. Hyg., vii.

3 v. Lingelsheim, “Aetiol. u. Therap. d. Streptoc. Inf.,” etc., Beit. z. Exper.
Therap., Hft. 1, 1899.

4 Knorr, Zeit. f . Hyg., xiii.

STREPTOCOCCUS PYOGENES

341

rats are less easily ‘ infected, and the larger domestic animals, cattle,
horses, goats, cats, and dogs, are extremely refractory. Almost complete
immunity toward streptococcus infections prevails among birds.

The nature of the lesions following animal inoculation depends upon
the manner of inoculation, the size of the dose given, and most of all
upon the grade of virulence of the inoculated germ. Subcutaneous
inoculations, according to the virulence of the inoculated material, may
result in a simple localized abscess, differing from a staphylococcus
abscess only in the more serous nature of the exudate and the frequent
occurrence of edema, or in a severe general septicemia with a hardly
noticeable local lesion. Subcutaneous inoculation of mice results almost
invariably in general sepsis followed by death within thirty-six to forty-
eight hours, or less, and the presence of streptococci in the heart’s blood
and the viscera. Intrapleural or intraperitoneal inoculation of suscep¬
tible animals with virulent streptococci leads usually to a peculiarly
hemorrhagic form of exudate, due both to the diapedesis caused by the
violent inflammatory process, and to the hemolysis of the red cells by the
streptococcic hemolysins. Inoculation of rabbits at the base of the ear
with virulent streptococci may result in the formation of a lesion
indistinguishable histologically from erysipelas in man.1 Marbaix 2 has
shown that such erysipeloid lesions could be produced in rabbits by
streptococci from various and indifferent sources, provided that the
virulence of each strain could be sufficiently enhanced. This marked
variability of the resulting lesion as determined by the degree of virulence
of the incitant, whatever its original source, forms a strong argument in
favor of the opinion that all the pyogenic streptococci are members of a
single species.

Intravenous inoculation of rabbits with virulent cultures usually
results in a rapidly fatal septicemia. An animal which has died of a
streptococcus infection usually shows serosanguineous edema about the
point of inoculation, multiple hemorrhagic spots upon the serous mem¬
branes, and congestion of the viscera. The microorganisms can almost
invariably be found in the heart’s blood, in the spleen, and in the exudate
about the inoculated area. Microscopically, when the process has lasted
sufficiently long, parenchymatous degeneration of all the organs may be
observed. In the more chronic infections articular and periarticular
lesions may occur.3

1 Fehleisen, loc. cit. ; Frankel, Cent. f. Bakt., vi.

2 Marbaix, La Cellule, 1892.

3 Schiitz, Zeit. f. Hyg., iii; Hiss, Jour. Med. Res., xix, 1908.

342

PATHOGENIC MICROORGANISMS

Spontaneous streptococcus disease seems to occur among some of
the larger domestic animals. Thus, a contagious form of inflammation
of the respiratory passages of horses has been attributed to streptococcus
infection.1 Among cattle these microorganisms have been found to
produce purulent inflammation of the udder and occasionally post¬
partum uterine inflammation in cows. Among the smaller labora¬
tory animals, occasional streptococcus infections may be observed in
rabbits. Recently an epidemic disease among white mice due to strep¬
tococcus was studied by Kutscher.2 As a rule, however, streptococcus
disease is by far more rare among animals than it is among human beings.

In man, a large variety of pathological processes may be caused by
streptococci and here again the nature of the infection, whether definitely
localized or generally distributed, depends upon the relationship existing
between the virulence of the incitant and the resistance of the subject.

The first cultivation of streptococcus from human lesions was made
by Fehleisen,3 who obtained them from cases of erysipelas. It was
long believed that the so-called Streptococcus erysipelatis was a
similar but essentially different species from the common Streptococcus
pyogenes. The production of erysipelas in animals with streptococci
from other sources, however, has shown definitely that the two groups
can not be separated.4 Superficial cutaneous infections are frequently
caused by streptococci and these in the milder cases may be similar
to the localized abscesses caused by staphylococci. In severe cases,
however, infection is followed by rapidly spreading edema, lymph¬
angitis, and severe systemic manifestations with the development of a
grave cellulitis, often threatening life and requiring energetic surgical
interference. Invasion of the respiratory organs by streptococci is not
rare, and may lead to bronchitis, pneumonia, or empyema. They are
frequently present also as secondary invaders in pulmonary tubercu¬
losis.5 Streptococcus infections of the lungs and pleura not infrequently
lead to pericardial involvement.

Suppurations of bone may be caused by streptococci, and constitute
a severe form of osteomyelitis. Such lesions when occurring in the
mastoid bone are not infrequently secondary to streptococcus otitis and
may lead to a form of meningitis which is in most cases fatal. In the

1 Van de Velde, Monatsheft Bakt., Thierheilk., ii.

2 Kutscher, Cent. f. Bakt., xlvi.

3 Fehleisen, loc. cit.

4 Marbaix, La Cellule, 1892; Petruschky, Zeit. f. Hyg., xxiii.

5 Cornet, “Die Tuberkulose,” Wien, 1899.

STREPTOCOCCUS PYOGENES

343

mouth and throat streptococci may give rise to pharyngitis and are one
of the most frequent causes of a form of tonsillitis often clinically indis¬
tinguishable from diphtheria. The throat inflammation accompanying
scarlatina is, almost without exception, referable to streptococcus infec¬
tion.1 The occasional presence of the streptococcus in the blood of
scarlatina patients, moreover, has led some authors to suggest a pos¬
sible etiological connection between this microorganism and the disease.2
This, however, is at present merely conjectural.

In diphtheric inflammations of the throat, a secondary streptococcus
infection is a frequent and serious complication. As incitants of disease
of the intestines, streptococci have been found in appendicular abscesses 3
and have been described as the cause of some forms of infantile diarrhea.4
From any of the local processes streptococci may pass into the circulation,
causing sepsis. The septicemia occurring during the puerperium is most
often caused by this microorganism.

Secondary foci in the viscera may be established, leading to pyemia,5
or, if these localizations occur upon the heart valves, septic endocarditis
may ensue. All such forms of general streptococcus infection, whether
running acute or chronic courses, present a high rate of mortality. The
diagnosis in these cases is usually easy if blood cultures are taken upon
suitable media.

Streptococcus throat infections have recently appeared in fulmi¬
nating epidemics. Several small epidemics were described in England,
and three extensive outbreaks have occurred in this country; one in
Boston of 1,400 cases; a second in Baltimore of about 1,000 cases, and
a third in Chicago of about 10,000 cases. These outbreaks were studied
by Winslow, Stokes, Davis,6 and by Rosenow.7 In each case the major¬
ity of infections were traced to a single milk supply, though secondary
cases doubtless occurred by contact. Severe complications such as
suppurative adenitis, otitis, erysipelas, peritonitis, and septicemia
were not uncommon. A similar organism — a capsulated, hemolytic
streptococcus — was found in each epidemic.

1 Baginsky, Deut. med. Zeit., 1900.

2 Baginsky und Sommerfeld, Berl. klin. Woch., xxvii, 1900.

3 Kelly, “Pathogenesis of Appendicitis.’ ’

4 Lanz and Tavel, Rev. de Chir., 1904; Perrone, Ann. de l’inst. Pasteur, 1905;
Escherich, Jahrb. f. Kinderheilkunde, 1899.

5 Libman, Cent. f. Bakt., xxii.

6 Cited from Capps, Jour. A. M. A., 1912, p. 1848.

7 Rosenow, Jour, of Inf. Dis., 1912.

344

PATHOGENIC MICROORGANISMS

Toxic Products. — In spite of extensive researches by many inves¬
tigators upon the nature of the poisons produced by streptococci,
our understanding of these substances is still very incomplete. The
grave systemic symptoms so often accompanying comparatively
slight streptococcus lesions argue strongly for the production by these
microorganisms of a powerful diffusible poison. Toxic filtrates of
streptococcus cultures have indeed been obtained by Roger,1 Marmier,2
Baginsky and Sommerfeld,3 Marmorek,4 and many others; but these
have in no case been comparable in potency to the soluble toxins of
diphtheria or of tetanus. When injected into young guinea-pigs in
sufficient quantity, these filtrates produce rapid collapse and death.
The inability to produce strong toxins is generally attributed to the
difficulty of obtaining very abundant growth of these bacteria upon
fluid media, development being self -limiting, either because of the ex¬
haustion of specific nutritive material (Marmorek 5), or, more probably,
because of the inhibitory effects of the products of growth, chiefly acid
formation. This last factor can be partially overcome by the use of the
glucose-calcium-carbonate broth mentioned above, in which acid neutral¬
ization is constantly taking place. For toxin production, Baginsky and
Sommerfeld 6 advise a strongly alkaline reaction of the media; Mar¬
morek 7 has used human blood-serum-bouillon with success. The
toxins so produced are relatively thermostable. According to v.
Lingelsheim, heating to 60° or 70° C. destroys them in part only.
The endotoxins contained within the cell-bodies of streptococci them¬
selves have been found to possess but slight toxic qualities.

Apart from these substances, some streptococci produce a hemolysin
which has the power of bringing about destruction of red blood cor¬
puscles. The observation of this phenomenon for streptococci was first
made by Marmorek 8 in 1895. According to this author, there is a
direct relationship between virulence and hemolytic power. Other
investigators, however, notably Schottmuller,9 believe the hemolytic
power to be a constant characteristic of’ certain strains unchangeable by

1 Roger , Rev. de med., 1892.

2 Marmier , Ann. de l’inst. Pasteur, ix, 1895, p. 533.

3 Baginsky und Sommerfeld, Berl. klin. Woch., 1900.

4 Marmorek, Bed. klin. Woch., 1902.

6 Marmorek, Berl. klin. Woch., xiv, 1902.

6 Loc. cit.

7 Marmorek , Ann. de l’inst. Pasteur, 1895.

8 Marmorek, Ann. de l’inst. Pasteur, 1895.

9 Schottmuller, Munch, med, Woch., 1903.

STREPTOCOCCUS PYOGENES

345

experimental enhancement or reduction of the virulence. Streptococcus
hemolysins may be conveniently observed by cultivation of the organ¬
isms upon blood-agar plates. They may be produced in alkaline pepton-
broth and obtained separate from the bacteria by filtration — a procedure,
however, in which the quantities obtained are never large. Besredka 1
and Schlesinger 2 believe, for this
reason, that the hemolytic sub¬
stances are closely attached to the
bacterial bodies. The last-named
author, furthermore, has deter¬
mined that, in contradistinction to
the other toxic substances, strepto¬
coccus hemolysins are extremely
labile, disappearing from culture
fluids after standing for from five
to seven days at ordinary room
temperature.

Immunization. — For reasons not
wholly understood at present, re¬
covery from streptococcus infection
does not to any marked degree
produce immunity against these
bacteria. Active immunity may,

however, be produced in rabbits, goats, horses, and other domestic
animals by treatment with gradually increasing doses of streptococcus
cultures.3

In carrying out such immunizations it is necessary to use for the first
injection attenuated or dead bacteria. Attenuation may be accom¬
plished by moderate heating or by the addition of chemicals (terchloride
of iodin). Neufeld 4 advises, for the first injection in immunizing
rabbits, the use of ascitic-broth cultures killed by heating to 70° C. This
is followed, after ten days, by a second injection of a small quantity of
fully virulent cocci. Following this, injections are made at intervals
of ten days with constantly increasing doses. Modifications of these
general principles are employed in most laboratories.

The sera of animals so treated contain no demonstrable antitoxic or

1 Besredka, Ann. de l’inst. Pasteur, xv, 1901, p. 880.

2 Schlesinger , Zeit. f. Hyg., xxiv, 1903.

3 Koch und Petruschky, Zeit. f. Hyg., xxiii, 1896.

4 Neufeld, Zeit. f. Hyg., xliv, 1903.

Fig. 74. — Streptococcus Colonies
from Blood Culture on Blood-
Agar Plate. Showing areas of hemol¬
ysis about colonies.

346

PATHOGENIC MICROORGANISMS

antihemolytic substances.1 They exert, however, demonstrable bacteri¬
cidal power both in vivo and in vitro and distinctly enhance phagocytosis
when brought into contact with leucocytes and streptococci. This
“ opsonic” power has been noticed both intraperitoneally (Bordet2)
and in vitro (Denys and Leclef 3).

The protective value of streptococcus immune sera for infected
animals is considerable, reaching often a potency hardly explicable by
the demonstrable bactericidal or opsonic power, and thereby suggesting
some other active factor not understood as yet.4 Aronson 5 has produced
immune sera by the treatment of horses with a streptococcus derived
from a case of scarlatina, 0.0004 c.c. of which sufficed to protect mice
from ten times the fatal dose of a streptococcus culture. These high
protective values, however, are obtained only when the serum injections
are given simultaneously with the bacteria. Given four or six hours
after infection, much higher dosage must be employed and protective
results are much less regular in occurrence.6 Other antistreptococcic
sera have been produced by Denys, Menger, Tavel, and others, all show¬
ing more or less marked potency in protecting animals.7

Since these sera, while in a general way potent against all streptococci,
have been found protective chiefly against the specific microorganism em¬
ployed for their production, Van de Velde,8 Denys, Aronson, and others
have advised the immunization of the animal with a large variety of
streptococcus races, derived from many different human sources. The
resulting “polyvalent” serum is more apt to exert equally high protective
powers against all streptococcus infections. The therapeutic value of
such sera in the treatment of human infections is still sub judice. Un¬
deniably favorable reports are published each year in increasing number,
but are by no means regular or comparable to results such as those ob¬
tained in diphtheria with diphtheria antitoxin. Nevertheless, in mild
cases or in those in which the lesions have been distinctly localized, the

1 Lingelsheim, Zeit. f. Hyg., x, 1891.

2 Bordet, Ann. de l’inst. Pasteur, 1897.

3 Denys et Leclef, Cellule, t. ix.

4 Denys et Marchand, “Mecanisme de l’immunite,” etc., Brussels, 1896.

5 Aronson, Berl. klin. Woch., xxxii, 1896; ibid., xlii and xliii, 1902; ibid., viii and
ix, 1905.

6 Denys, “Le Serum antistreptoc.,” Louvain, 1896; Van de Velde, Ann. de l’inst.
Pasteur, 1896.

7 Denys et Marchand, Bull, de l’acad. roy. de med. de Belgique, 1898; Menger,
Berl. klin. Woch., 1902; Tavel, Corr.-Bl. f. Schw. Aerzte.

8 Van de Velde, Arch, de med. exper., 1897.

STREPTOCOCCUS PYOGENES

347

sera have seemed to be sufficiently useful to justify their use and to
necessitate their standardization.

Standardization is accomplished by the methods first devised by
Marx 1 for the standardization of swine-plague serum, and depends upon
the ability of the serum to protect animals against a measured dose of
virulent streptococci. Aronson 2 designates as a “normal serum” one
of which 0.01 c.c. will protect a mouse against ten to one hundred
times the fatal dose of virulent streptococci. One cubic centimeter
of this serum equals one serum unit. Comparisons by animal experi¬
ment with this standard serum approximately determine the value of
other sera.

Leucocyte extracts have been employed by the writers and others,
as advised by Hiss,3 in various forms of streptococcus infections of man,
with success in many cases. Very uniformly favorable results have
been obtained with these extracts in cases of erysipelas.

The agglutinins found in streptococcus immune sera are usually most
active toward the race of bacteria employed in the immunization. Other
streptococci, however, are also agglutinated, but in relatively higher
concentration of the serum. Thus, while a specific group reaction is
extremely useful in differentiating streptococci from other species, such as
pneumococci, agglutination can not be relied upon to differentiate in¬
dividual streptococci from one another (Hiss). It has even been found
that a serum produced with a streptococcus from one source con¬
tained a higher agglutinating value for some other streptococcus than
for the one employed in its production. Agglutinins may be produced by
treating animals with dead as well as with the living virulent streptococci.
While the technique of the streptococcus agglutination tests is not
difficult when we are dealing with strains which grow diffusely and with
even clouding in fluid media, the frequency with which these micro¬
organisms clump spontaneously in broth cultures necessitates the use of
a special technique. The most simple of these methods, and possibly
the best, is the one in which calcium-carbonate-glucose broth is used
for cultivation.4 Growing in this medium and thoroughly shaken once a
day, the streptococci are usually found evenly divided in the supernatant
fluid after the settling out of the heavier calcium-carbonate powder.

1 Marx, Deutsche thierarzt. Woch., vi, 1901.

2 Aronson, Berl. klin. Woch., xliii, 1902; Otto, Arb. a. d. konigl. Inst., etc., Frank¬
furt a. M., Heft 2, 1906.

3 Hiss, Jour. Med. Res., xix, 1908.

4 Hiss, Jour. Exp. Med., vii, 1905.

348

PATHOGENIC MICROORGANISMS

Precipitins have been found by Aronson 1 in streptococcus immune
horse serum. Special methods of extracting the bacteria were em¬
ployed.

Classification. — Frequently observed differences in the minor cul¬
tural characteristics and in the virulence of streptococci obtained from
various sources have given rise to much discussion as to the identity of
all races of streptococci. The earliest observers were forced to abandon
their separation of the streptococci of erysipelas from other streptococci
because of the work of Marbaix 2 and others, who produced erysipelas
in rabbits with streptococci from non-erysipelatous lesions, after en¬
hancement of their virulence. V. Lingelsheim 3 has proposed a purely
morphological differentiation of “longus” and “brevis”; the former
class including the streptococci most usually found in pyogenic le¬
sions and having a tendency to form chains of six or more links, the
latter designating the short-chained varieties, including, as a rule,
the less virulent streptococci. This classification, however, is not
scientifically tenable because of the considerable dependence of chain
formation upon reaction, consistency, and nutritive qualities of the
media employed for cultivation, and upon the influence of animal fluids
if the microorganisms are taken direct from lesions. Schottmiiller,4
who has made a careful study of streptococci, in 1903 proposed a classi¬
fication based both upon morphology and the appearance of cultures
upon human blood agar. By this method he divided streptococci into
two main groups as follows: I. Streptococcus longus seu erysipelatos,
consisting of the most virulent varieties, having a tendency to form long
chains, and regularly producing hemolysis upon blood media. II.
Streptococcus mitior seu viridans , including less virulent strains, with
usually shorter chain-formation, and producing green, non-hemolyzing
colonies upon blood media. These are the streptococci which he
usually obtained from milder or more chronic lesions. A third group
which he adds, Streptococcus mucosus, will receive special considera¬
tion in a separate section, and is probably more closely related to the
pneumococci than to the streptococcus groups.

Attempts to separate the streptococci into subdivisions by their
powers to ferment various carbohydrates have been made by Hiss,
Gordon, and others. These attempts have, so far, been without practical

1 Aronson, Deut. med. Woch., 25, 1903.

2 Marbaix, loc. cit.

3 v. Lingelsheim, “Aetiol. u. Therap. d. Streptokok. Krankh.,” etc., Berlin, 1899.

4 Schottmiiller, Munch, med. Woch., 1903.

STREPTOCOCCUS PYOGENES

349

result. Hiss 1 indicated a tentative division of streptococci into those
which fermented monosaccharids alone, those which were also able
to ferment disaccharids, and those in which the fermentative powers
were extended to the polysaccharids, starch, dextrin, and glycogen.

Gordon,2 after a thorough study of many strains upon seven carbo¬
hydrates, found ten different fermentation reactions among twenty
pyogenic streptococci examined, and forty-eight different fermentation
reactions among two hundred streptococci isolated from saliva. Other
work by Andrewes and Horder and by Buerger 3 confirms the irregu¬
larity of the fermentation reactions within this group.

Andrewes and Horder suggest the following classification:

(1) Streptococcus pyogenes. A type which grows in long chains
and which ferments lactose, saccharose, and salicin, but does not coag¬
ulate milk. Most of the streptococci which cause suppurative lesions or
severe systemic infections belong to this group.

(2) Streptococcus mitis. A saprophytic type found frequently in
the mouth which shows the same cultural characteristics as the strep¬
tococcus pyogenes, but grows in short chains.

(3) Streptococcus anginosus. A type found frequently in the throats
of scarlet-fever patients which differs from the pyogenes only in coag¬
ulating milk.

(4) Streptococcus salivarius. A short-chain type which ferments
lactose, saccharose, and raffinose, and coagulates milk. Streptococci of
this type are found frequently in the mouth, but are rarely pathogenic.

(5) Streptococcus fecalis. A short-chain type which ferments lac¬

tose, saccharose, and mannite. This type is found normally in the
intestine, and is occasionally pathogenic. ' J \ - r ■, A\v&

(6) Streptococcus equinus. A short-chain type which does not
ferment lactose. It is found normally in horse dung and is never
pathogenic.

Quantitative determinations of the amount of acid formed in
various sugars by different races have also been made by Winslow
and Palmer 4 and others, but have led to no satisfactory classification.

Studies by Hopkins and Lang seem to show that the streptococci
found in most human infections may be differentiated from the ordinary

1 Hiss, Cent. f. Bakt., xxxi, 1902; Jour. Exp. Med., vi, 1905.

2 Gordon , Annual Report, Local Govern. Board, 33, London, 1903.

3 Andrewes and Horder, Lancet, 1906; Buerger , Jour. Exp. Med., ix, 1907.

4 Jour, of Inf. Dis., No. viii, 1910, 1,

350

PATHOGENIC MICROORGANISMS

saprophytic types by the fact that they ferment lactose and salicin,
but fail to ferment raffinose, inulin, or mannite. According to their re¬
sults, the usual saprophytic types found in the mouth either fail to fer¬
ment salicin or ferment raffinose or inulin, whereas the usual fecal types
ferment mannite. They also found in infection mannite fermenters
which were apparently of fecal origin. Streptococci which gave the
same fermentative reaction as the mouth saprophytes were, however,
frequently found in malignant endocarditis.

Probably the most reliable method of determining the interrelation¬
ships existing between bacteria, not only within this group, but in all
bacterial classes, is that depending upon their reactions to immune
sera. The work of Aronson,1 Marmorek,2 and others has shown that
streptococcus immune sera produced with any one race of pyogenic
streptococci exerted considerable, though variable, protective action
against many other strains of streptococci. The same authors, as well
as many others, working with the agglutination reaction, have shown
that the agglutinins produced with one streptococcus strain were active
against many other streptococci. While most active usually against
the particular microorganism with which they were produced, this was
by no means the rule, a serum produced with a streptococcus from a case
of sepsis, in one case, agglutinating a streptococcus from a case of
scarlatina more highly than its own microorganism. As with other
“ group agglutinations,” the more highly immune the serum is, the
more general is the agglutinating power over the whole group. Thus,
while agglutination is practically useless in separating streptococci
from one another, it is highly useful in differentiating these organisms
from allied groups, such as the pneumococci. The immune reactions,
therefore, seem to indicate a very close relationship between strepto¬
cocci as a class.

Streptococcus mucosus. — This microorganism was first definitely
described by Howard and Perkins 3 in 1901, and was subsequently care¬
fully studied by Schottmuller,4 who isolated it from cases of parame¬
tritis, peritonitis, meningitis, and phlebitis. The organism has since
been described by many observers as the incitant of a variety of
lesions and as an apparently harmless inhabitant of the normal mouth.

1 Aronson, Berl. klin. Woch., 1902; ibid., 1903.

2 Marmorek, Berl. klin. Woch., 1902.

3 Howard and Perkins, Jour. Med. Res., 1901, N. S., i.

4 Schottmuller, Munch, med. Woch., xxi, 1903.

STREPTOCOCCUS MUCOSUS

351

Morphologically, though showing a marked tendency to form chains, on
solid media it often appears in the diplococcus form. It is enclosed in
an extensive capsule, which appears with much regularity and persist¬
ence. Though very similar in appearance, therefore, to pneumococci,
these bacteria do' not appear in the typical lancet shape. Upon solid
media they show a tendency to grow in transparent moist masses. The
regularity with which this microorganism ferments inulin medium, and
its agglutinative characters, make it probable that it is more accurate to
place it with the group of pneumococci than with that of streptococci.1
(For agglutinations see section on pneumococcus agglutination, p. 364.)

STREPTOCOCCI AND RHEUMATISM

In 1910 Poynton and Paine 2 described a diplococcus which they
obtained from eight cases of acute rheumatic fever and with which
they were able to produce lesions in rabbits which they considered
typical of rheumatism. The organism was recovered from the blood,
the pericardial fluid, or the tonsil of their patients. They described a
minute Gram-negative diplococcus growing best in acid media and
under anaerobic conditions, but capable of growth on the surface of ordi¬
nary media. Many investigators have attempted to confirm their work,
but with negative results for the most part, though some have found
streptococci and diplococci from rheumatic lesions. Recently Rosenow 3
has reported the isolation of a streptococcus from the joints of seven
cases of articular rheumatism. He was also able to produce non¬
suppurative arthritis, endocarditis, and pericarditis in rabbits with
these cultures. He describes them as intermediate in character between
the streptococcus viridans and streptococcus hemolyticus.

More recently Rosenow 4 has reported the production of gastric
ulcers in rabbits and dogs with streptococci of a certain grade of viru¬
lence. He has also obtained streptococci from human peptic ulcers
which showed a remarkable “ affinity ” for the gastric mucous membranes
of experimental animals.

1 Hiss, Jour. Exp. Med., 1905; Buerger, Cent. f. Bakt., I, xli, 1906.

2 Poynton and Paine, Lancet, 1900, ii, 861, 932.

3 Rosenow, Jour. A. M. A., 1913, lx, 1223.

4 Rosenow , Jour. A. M. A., 1913, lxi, 1947, 2007.

CHAPTER XXIII

DIPLOCOCCUS PNEUMONIAE

( Pneumococcus , Diplococcus lanceolatus)

The opinion that lobar pneumonia is an infectious disease was held
by many far-sighted clinicians long before the actual bacteriological
facts had been ascertained. This idea,, so well founded upon the nature
of the clinical course of the disease, with its violent onset and equally
rapid defervescence, led many of the earlier bacteriologists to make it the
subject of their investigations — a subject made doubly difficult by the
abundant bacterial flora found normally in the upper respiratory pas¬
sages, and by the fact, which is now recognized, that lobar and other
pneumonias are by no means always caused by one and the same micro¬
organisms.

Cocci of various descriptions and cultural characteristics were isolated
from pneumonia cases by Klebs,1 Koch,2 Giinther,3 Talamon,4 and many
others, which, however, owing to the insufficient differential methods at
the command of these investigators, can not positively be identified
with the microorganism now known to us as Diplococcus pneumoniae
or the pneumococcus. Although thus unsuccessful as to their initial
object, these early investigations were by no means futile, in that they
gave valuable information regarding the manifold bacterial factors
involved in acute pulmonary disease and incidentally led to the dis¬
covery by Friedlander5 of B. mucosus capsulatus.

Communications upon lance-shaped cocci found in saliva, and
capable of producing septicemia in rabbits, were published almost simul¬
taneously by Sternberg 6 and by Pasteur 7 in 1880. These workers

1 Klebs, Arch. f. exp. Path., 1873.

2 Koch, Mitt. a. d. kais. Gesundheitsamt, Bd. 1.

3 Gunther, Deut. med. Woch., 1882.

4 Talamon, Progr. med., 1883.

5 Friedlander , Virchow’s Arch., lxxxvii.

6 Sternberg , Nat. Board of Health Bull., 1881.

7 Pasteur, Bull, de l’acad. de med., 1881.

352

DIPLOCOCCUS PNEUMONIA

353

beyond reasonable doubt were dealing with the true pneumococcus, but
did not in any way associate the microorganisms they described with
lobar pneumonia. The solution of this problem was reserved for the
labors of A. Frankel 1 and Weichselbaum 2 who published their results,
independently of each other, in *1886, demonstrating beyond question
that the pneumococcus is the etiological factor in a large majority of
cases of lobar pneumonia.

Morphology and Staining. — The morphology of the pneumococcus is,
in general, one of the most valuable guides to its identity.

When typical, the pneumococcus is a rather large, lancet-shaped coc¬
cus, occurring in pairs, and surrounded by a definite and often wide
capsule, which usually includes the two approximated cocci without a
definite indentation opposite their lines of division. The pneumococci
may, however, occur singly or in short chains, and even fairly long
chains are not infrequently met with under artificial cultural conditions.
This may be chiefly due to the cultural conditions or may be a promi¬
nent characteristic of certain strains. Apparently the capsules of or¬
ganisms making up the chains are continuous; wavy indentations are
usually present, however, in the capsule of chains, and at times distinct
divisions are observed.

The chief variations from the typical morphology consist either in
the assumption of a more distinctly spherical coccus type, or in an
elongation approximating the bacillary form. Under certain conditions
of artificial cultivation a distinct flattening of the organisms, particularly
of those making up chains, may be seen, and even the impression of a
longitudinal line of division, characteristic of many streptococcus
cultures, is not infrequently gained.

The capsules under certain conditions, especially in artificial media,
may be absent or not demonstrable, and in certain strains capsules ap¬
parently may not be present under any conditions. Practically any of
the described variations may dominate one and the same culture under
different or even apparently the same conditions of cultivation, and all
grades may occur in capsule development, from its typical formation
through all variations, to its total and apparently permanent absence.

The presence or absence of capsules depends, to a large extent, upon
the previous environment of the pneumococci under observation. The
most favorable conditions for the development or preservation of the
pneumococcus capsule are found in the body fluids of man and animals

1 A. Frankel, Zeit. f. klin. Med., x, 1886.

2 Weichselbaum, Med. Jahrbucher, Wien, 1886.

354

PATHOGENIC MICROORGANISMS

suffering from pneumococcus infection. For instance, capsules may be
demonstrated with ease by the usual capsule-staining methods in the
blood, serum, and inflammatory exudate of the infected rabbit and
white mouse. Capsules may be equally well marked in the fresh sputum
of pneumonia patients, especially in the early stages of the disease and
in the exudate accompanying such pneumococcus infections as menin¬
gitis, otitis media, and empyema. In sputum and the exudates of
various localized infections, the organisms are, however, frequently
degenerated or under chemical conditions unfavorable for capsule
staining, and satisfactory results are not then easily obtained. The

Fig. 75. — Pneumococci, Grown on
Loeio ler’s Serum. (Capsule stain
by gentian- violet-potassium-carbonate
method.)

Fig. 76. — Pneumococci, from Rab¬
bit’s Heart Blood. (Capsule stain by
copper-sulphate method.)

same is often true of the scrapings from lungs of patients dead of
pneumonia, even in the stage of red hepatization.

In artificial cultivation, if the nutrient medium is not milk or does not
contain serum, capsules can not usually be demonstrated by the ordinary
methods of preparing and staining. Capsules may, however, with much
regularity be demonstrated on pneumococci, in agar, broth, or on almost
all, if not all, artificial media, irrespective of the length of time the organ¬
isms have been under artificial cultivation if beef or rabbit serum is used
as the diluent, when they are spread on the cover-glass for staining.1

The pneumococcus is non-motile and possesses no flagella. Spores
are not formed. Swollen and irregular involution forms are common
in cultures more than a day old.

1 Hiss , Cent. f. Bakt., xxxi, 1902; Jour. Exp. Med., vi, 1905.

DIPLOCOCCUS PNEUMONIAE

355

The pneumococcus is stained readily with all the usual aqueous
anilin dyes. Stained by the method of Gram, it is not decolorized.
Special methods of staining have been devised for demonstra¬
tion of the capsule. The ones most generally used are the glacial
acetic-acid method of Welch1 and the copper-sulphate method of Hiss.2
More recently Buerger 3 has devised a more complicated method for
staining capsules, for which he claims differential value. (For methods
see section on Technique, p. 98.)

For simple staining of pneumococci in tissue sections, the Gram-
Weigert technique is excellent. For demonstration of the capsules
in tissue sections, Wadsworth 4 has described a simple method.

Cultivation and Isolation. — The pneumococcus being more strictly
parasitic than many other bacteria, presents greater difficulties in its
cultivation. On meat-extract media growth does not take place with
regularity. On those media, however, which have beef or veal infusion
for their basis, growth can be obtained with considerable regularity,
although such growth may be sparse and delicate.

Growth takes place most regularly at a temperature of 37.5° C.
Development does not usually occur below 25° nor above 41° C.5 At
ordinary room temperature, 18-22° C., the temperature used for gelatin
cultivation, growth either does not take place at all or is exceedingly
slow and unenergetic. Aerobic and anaerobic conditions are equally
favorable for pneumococcus cultivation, there being very little difference
in speed or extent of growth along the course of deep stab cultures in
favorable media. The most favorable reaction of media for the culti¬
vation of this microorganism is neutrality or moderate alkalinity (two-
tenths to eight-tenths per cent alkalinity to phenolphthalein) . Slight
acidity, however, if not exceeding eight-tenths per cent, does not
materially hamper development.

The growth of pneumococci on all media may be considerably
enhanced by the addition to these media of animal or human serum or
whole blood. Additional substances which, among others, unquestion¬
ably have a favorable influence upon pneumococcus growth, are glucose,
nutrose, and glycerin. The addition of the latter substances to the
media, however, probably because of acid formation, hastens the death

1 Welch, Johns Hopk. Hosp. Bull., xiii, 1892.

2 Hiss, Cent. f. Bakt., xxxi, 1902; Jour. Exp. Med., vi, 1905.

3 Buerger, Medical News, lxxxviii, 1904.

4 Wadsworth, “ Studies by the Pupils of W. T. Sedgwick,” Chicago, 1896.

6 A. Frankel , Dent. med. Woch., xiii, 1886.

356

PATHOGENIC MICROORGANISMS

of pneumococcus cultures. An increase of the amount of pepton
used for the preparation of media is desirable for the cultivation of
this microorganism; two to four per cent of pepton may be found
’ advantageous.

In suitably alkaline, nutrient broth, growth is rapid, and within
twenty-four hours leads to slight clouding of the fluid. This clouding,
as a rule, eventually disappears as the microorganisms, sinking to
the bottom of the tube or disintegrating, leave the fluid more or
less clear. In broth, pneumococci have a tendency to form short
chains. When glucose has been added to the broth, growth is more
rapid and profuse, but considerable acid formation causes the cultures
to die out rapidly. It is possible, however, to employ glucose as a
growth-enhancing element in broth cultures without interfering with the
viability of the cultures by adding small quantities (one per cent) of
sterile, powdered calcium carbonate. This method of cultivation in
broth is especially adapted to the production of mass cultures for purposes
of immunization or agglutination.1 The addition of ascitic fluid or blood
serum to broth, in the proportion of one to three, makes an extremely
favorable medium in which growth is rapid and profuse.

Upon agar plates, pneumococcus growth is not unlike that of strepto¬
coccus. The colonies are small, round, and slightly more transparent
than those of the streptococci. They appear more moist than strepto¬
coccus colonies and often are more flat. Microscopically examined, the
colonies are finely granular, with dark centers and slightly corrugated
lighter-colored peripheral areas. Under high magnification no such in¬
tertwining convolutions can be seen as those noticed under similar
magnification in streptococcus cultures. The addition of animal albu¬
min to agar results in the more rapid development, larger size, and deeper
opacity of the colonies.

Agar stab cultures show growth within twenty-four to thirty-six
hours, which takes place with equal thickness along the entire course of
the stab. There is nothing distinctive in these cultures to differentiate
them from similar streptococcus cultures.

I a gelatin plate and stab cultures at 22° C., growth, as a rule, does not
take place. This, however, is not true of all races of pneumococci.
Occasionally strains are met with which will grow fairly abundantly in
gelatin at a temperature of 22° C. When the gelatin is rendered suffi¬
ciently firm to bear 25° to 26° C. without melting, growth appears

1 Hiss, Jour. Exp. Med., vii, 1905.

DIPLOCOCCUS PNEUMONIAE

357

slowly and sparsely as minute, grayish-white, transparent colonies.
The gelatin is not liquefied by the organisms.

Growth upon milk is rapid and profuse, resulting usually in the
production of acid and consequent coagulation of the medium. Ex¬
ceptionally, races are encountered in which this function is suppressed
and coagulation in milk is absent or long delayed.

Upon potato, a thin, grayish, moist growth occurs, hardly visible to
the naked eye, and often indistinguishable from an increased moisture
on the surface of the medium.

Upon Loeffler’s coagulated hlood serum, the pneumococcus develops
into moist, watery, discrete colonies which tend to disappear by a
drying out of the colonies after some days, differing in this from strep¬
tococcus colonies, which, though also discrete, are usually more opaque
and whiter in appearance than those of the pneumococcus and remain
unchanged for a longer time. This medium, as. will be seen, is use¬
ful in differentiating pneumococci from the so-called Streptococcus
mucosus.

Upon a medium made up of mixtures of whole rabbit’s blood and
agar, the pneumococcus grows with considerable luxuriance, and forms,
after four or five days or longer, thick black surface colonies, not unlike
small sun blisters on red paint. These colonies are easily distinguished
from the hemolyzing colonies of most streptococci, and are in this
respect of considerable differential value.1

Special media of various descriptions have been devised for pneu¬
mococcus cultivation. Thus, Guarnieri 2 3 has recommended a medium
with a pepton-beef-infusion basis rendered semisolid by mixtures of
agar and high percentages of gelatin. A modification of this medium
has been described by Welch and has been much employed in work
with the pneumococcus. Cultivation within eggs and upon egg media4
has been advised and used by various observers. Wadsworth 5 has
recommended a medium composed of ascitic fluid to which three-tenths
per cent agar has been added — sufficient to give a soft jelly-like con¬
sistency to the medium. He observed prolonged viability and the
preservation of the virulence in this medium.

For the purpose of differentiating pneumococci from streptococci,

1 Hiss, loc. cit.

2 Guarnieri, Att. dell’ Acad, di Roma, 1883.

3 Welch, Johns Hopk. Hosp. Bull., iii, 1892.

4 Sclavo, Riv. d’lgiene, 1894.

6 Wadsworth, Proc. N. Y. Path. Soc., 1903.

358

PATHOGENIC MICROORGANISMS

Hiss 1 devised a medium composed of beef serum one part, and dis¬
tilled water two parts, to which is added one per cent of inulin (c. p.)7
and enough litmus to render the medium a clear, transparent blue.
By fermentation of the inulin the pneumococcus acidifies this mixture,
rendering the litmus red and causing coagulation of the serum. Strep¬
tococci do not ferment inulin and the medium remains blue and fluid.
(For the preparation of special media, see section on Media, p. 132.)

For the isolation of pneumococci from mixed cultures or from
material containing other species, such as sputum, the most reliable
method is to make surface smears of the material containing the bac¬
teria upon plates of neutral glucose-agar or preferably of glucose-serum-
agar. According to the number of bacteria present in the infected ma¬
terial from which the isolation is to be made, it may be smeared
directly upon the plate, or diluted with sterile broth or salt solution
before planting. After incubation for twenty-four hours, the pneumo¬
coccus colonies are easily differentiated from all but those of strepto¬
coccus. With practice, however, they may be distinguished from these
also, by their smoother edges and greater transparency and flat¬
ness. Pour-plates, prepared in the usual way, can also be made but
are less useful since deep colonies of pneumococci show no distinctive
features.

Another method for pneumococcus isolation, useful to eliminate
other bacteria, is that of animal inoculation. White mice are inoc¬
ulated with 0.5 to 1 c.c. of the infectious material by subcutaneous
injection, made most easily at the base of the tail. If virulent pneumo¬
cocci are present in the inoculated material, death from septicemia
usually occurs within twenty-four to forty-eight hours. Surface smears
should be made on glucose-agar plates with the heart’s blood. By
this method pure cultures may usually be obtained directly from the
mouse blood.

Resistance. — Kept upon artificial media, the viability of the pneu¬
mococcus is not great. Cultures upon agar or bouillon should, to be
kept alive, be transplanted every third or fourth day, if the cultures are
kept at incubator temperatures. In all media in which rapid acid
formation takes place, such as glucose media, the death of cultures may
occur even more rapidly. In media containing albumin and of a proper
degree of alkalinity, preservation for one or even two weeks is possible.
The longer the particular race has been kept upon artificial media, the

Hiss, Jour. Exp. Med., vi, 1905.

DIPLOCOCCUS PNEUMONIAE

359

more profuse is its growth, and the greater its viability, both qualities
going hand in hand with its diminishing parasitism. The length of life
of these bacteria may be much increased by their preservation at a low
temperature, in the dark, and by the exclusion of air. By far the best
medium for keeping pneumococci alive is the previously mentioned
calcium-carbonate-infusion broth. Grown in this medium and kept in
the ice-chest, cultures may often remain alive for months.

In sputum the viability of pneumococci seems far to exceed that
observed upon culture media. The studies of Guarnieri,1 Bordoni-
Uffreduzzi,2 and others have shown that pneumococci slowly dried in
sputum may remain not only alive but virulent, after from one to four
months, when protected from light; and as long as nineteen days
when exposed to diffused light at room temperature. Experiments by
Ottolenghi 3 have, in the main, confirmed these results; the virulence
seems, in Ottolenghi’s experiments, to have become considerably attenu¬
ated before the death of the cocci occurred. More recent studies by
Wood,4 whose attention was focused chiefly upon pneumococcus viabil¬
ity in finely divided sputum — in a condition, in other words, in which in¬
halation transmission would be possible — have shown that pneumococci
in finely sprayed sputum survive for only about one and one-half hours,
under ordinary conditions of light and temperature. Exposed to strong
sunlight pneumococci die off within an hour, often within a few minutes.

Low temperatures are well borne by pneumococci, temperatures
slightly above zero being even conducive to the prolongation of life and
the preservation of virulence.

The resistance of the pneumococcus to heat, on the other hand, is
low, 52° C. destroying it within ten minutes.5 To germicidal agents,
carbolic acid, bichlorid of mercury, permanganate of potassium, etc.,
the pneumococcus is extremely sensitive, being destroyed by weak solu¬
tions after short exposures.

The disinfection of sputum, offering considerable difficulties because
of the protective coating of the secretions about the bacteria, has been
recently made the subject of a spec al study by Wadsworth.6 The con¬
clusions reached by this writer indicate that pneumococci in exudates

1 Guarnieri, Att. della R. Acad. Med. di Roma, iv, 1888.

2 Bordoni-Uffreduzzi, Arch. p. 1. sc. med., xv, 1891.

3 Ottolenghi, Cent. f. Bakt., xxv, 1889.

* Wood, Jour. Exp. Med., vii, 1905.

6 Sternberg, Cent. f. Bakt., xii, 1891.

« Wadsworth, Jour. Inf. Diseases, iii, 1906.

3 GO

PATHOGENIC MICROORGANISMS

are most rapidly destroyed by twenty per cent alcohol, other and
stronger disinfectants being less efficient, probably because of slighter
powers of diffusion.

Virulence and Pathogenicity. — The virulence of pneumococci is
subject to much variation, depending largely upon the length of time
during which the microorganism has been cultivated under artificial
conditions. It has been mentioned above that under certain conditions
— such as those prevailing in dried sputum or blood 1 2 — the virulence of
pneumococci may be preserved for several weeks. Ordinarily, however,
the virulence diminishes gradually as the cocci adapt themselves more
saprophytically to life upon artificial media. Upon media containing
animal albumin, such as ascitic fluid or blood agar, this attenuation is
less rapid than upon the simple meat-infusion preparations.

In the blood of rabbits dead of a pneumococcus infection, taken
directly into sterilized tubes, sealed and kept in the dark, Foa3 has been
able to preserve the virulence of pneumococci for as long as forty-five
days. Whether or not the virulence of pneumococci is attenuated by
sojourn within the human body during disease is a question much dis¬
cussed but hardly settled. It is a matter of fact, however, that many
pneumococci obtained by blood culture from more or less chronic cases
of pneumococcus septicemia fail to kill susceptible test animals, even
when injected in considerable doses. The attenuation of virulent
pneumococci on artificial media may be hastened, according to Frankel,3
by cultivation of the organism at or above a temperature of 41° C.

Freshly isolated from the human saliva or pneumonic lesions, the
differences in virulence between various strains of pneumococci are not
very marked, almost all such strains showing considerable pathogenic
powers toward the usual test animals.

The virulence of attenuated cultures may be rapidly enhanced by
the passage of the organisms through the bodies of susceptible animals.
The extreme virulence of some of these pneumococcus strains may be
illustrated by citing the experiments of Eyre and Washburn4 who
possessed cultures of which one millionth of a loopful would kill a mouse
within four days.

Among the domestic animals those most susceptible to pneumococcus
infection are white mice and rabbits. Guinea-pigs, dogs, rats, and cats

1 Guarnieri, loc. cit.

2 Foa, Zeit. f. Hyg., iv, 1888.

3 Frankel, Deut. med. Woch., 13, 1886.

4 Eyre and Washburn , Jour, of Path, and Bac., v.

DIPLOCOCCUS PNEUMONIAE

361

are more resistant, but still may be infected with large doses. Young
animals are usually more susceptible than adults. Birds are practically
immune.

The results of pneumococcus inoculation into susceptible animals
vary according to the size of the dose, the virulence of the introduced
bacteria, the mode of administration, and the susceptibility of the
subject of the inoculation. Subcutaneous inoculation of virulent
pneumococci into mice and rabbits usually results in an edematous,
often fibrinous exudation at the point of inoculation, which, in all cases
n which the dose given has not been extremely small, leads to septicemia
and death within twenty-four to seventy-two or more hours. When
the dose has been extremely small or the culture unusually attenuated,
a localized abscess may be the only result. Intravenous inoculation is
usually more rapidly fatal in these animals than the subcutaneous
method. Intraperitoneal inoculation in rabbits results in the formation
of a rapidly spreading peritonitis in which the inflammatory exudate in
many cases exhibits differences from similar exudates produced by the
streptococcus. Pneumococcus exudates are apt to be thicker, to be
accompanied by a deposit of fibrin, and to lack the transparent red color
so often caused by the hemolyzing streptococci. With very virulent
strains, these differences are less marked. In almost all of these infec¬
tions death is preceded by septicemia and the microorganisms can be
recovered from the heart’s blood of the victims.

After such infections, the animals exhibit a rise of temperature, at
times visible depression, and, rarely, diarrhea. General hyperemia of
the organs with secondary effusions in the pleural cavities and often
hemorrhages upon the serous surfaces may be found at autopsy.

The production in animals of lesions comparable to the lobar pneu¬
monia of human subjects has been the aim of many investigators.
Wadsworth,1 recognizing that such lesions probably depended upon the
partial immunity which enabled the infected subjects to localize the
pneumococcus processes in the lungs after infection by way of the
respiratory passages, succeeded in producing typical lobar pneumonia
in rabbits by partially immunizing these animals and inoculating them
intratracheally with pneumococci of varying virulence. By this method
he actually carried out, for the first time, Koch’s postulates in regard to
lobar pneumonia.

In man, the most frequent lesion produced by the pneumococcus is

1 Wadsworth, Amer. Jour. Med. Sci., May, 1904.

362

PATHOGENIC MICROORGANISMS

acute lobar pneumonia. About ninety per cent of all cases of this
disease are caused by the pneumococcus/ the remainder being due to
streptococci, influenza bacilli, Friedlander’s bacilli, and exceptionally
to other microorganisms. Lobular pneumonia is caused by the pneu¬
mococcus with almost equal regularity. During the course of these
diseases the cocci are found in large numbers within the pulmonary
alveoli, and in the capillaries and lymph vessels of the lung. Whether
or not the pneumococci enter the blood stream in all these cases is a
question not yet definitely settled. Frankel1 2 states it as his belief that
in most, if not in all, cases, the diplococci at some time during the disease
could be found in the circulating blood. Prochaska in a study of ten
unselected cases obtained positive blood cultures in every one of them.
A review of the literature upon the question indicates positive blood-
culture findings in certainly over twenty-five per cent of the cases.

In complications of pneumonia, pneumococci are found usually in
the pleura where they may cause a simple dry pleurisy or even empy¬
ema. Less frequently they may cause pericarditis and endocarditis.
Meningitis may be caused by pneumococci, either secondarily to pneu¬
monia or independently. Such cases are extremely grave, almost
invariably ending in death. Other lesions which may be caused by
pneumococci, either as post-pneumonic processes or without previous
pneumonia, are otitis media, osteomyelitis, and arthritis. Cases of
pneumococcus peritonitis occur sometimes secondary to appendicular
inflammations, occasionally without traceable portal of entry. Severe
catarrhal conjunctivitis may be caused by these diplococci, usually
during the course of a pneumonia. Ulcerative endocarditis with pneu¬
mococcus septicemia, apparently independent of a pulmonary lesion, is
not infrequent.

Toxic Products of the Pneumococcus. — Our knowledge of pneumococcus
poisons is still very imperfect. Attempts to obtain soluble toxins by
the filtration of cultures have been practically unsuccessful in the hands
of many careful workers. G. and F. Klemperer,3 Mennes,4 Pane,5 Foa
and Carbone,6 and others failed to obtain pneumococcus filtrates of
any marked degree of toxicity, though working with highly virulent

1 Netter, Compt. rend, de la soc. de biol., 1890.

2 Frankel, “v. Leyden Festschr.,” 1902. »

3 G. and F. Klemperer, Berl. klin. Woch., xxxiv and xxxv, 1891.

4 Mennes, Zeit. f. Hyg., xxv, 1897.

6 Pane, Rif. med., xxi, 1898.

e Foa und Carbone, Cent. f. Bakt., x, 1899.

DIPLOCOCCUS PNEUMONIAE

363

strains. Attempts to demonstrate by the production of antitoxin
the specific nature of the feeble poisons obtained have also met with
failure. Isaeff,1 though confirming the feeble toxicity of fluid cultures,
made the interesting observation that a filtrate of the blood of pneumo¬
coccus-infected rabbits contained a poison often more potent than that
obtained in culture filtrates. Carnot and Fournier 2 obtained a poison
of distinct though feeble potency by dialysis of pneumococcus cultures.

The general failure, however, to procure strong soluble poisons from
cultures, gives weight to the assumption that the most potent toxic
products of pneumococci are in the nature of endotoxins and closely
bound to the cell-bodies themselves. This assumption is borne out by
the more recent experiments of Macfadyen.3 This author obtained
acutely poisonous substances from pneumococci by trituration of the
organisms after freezing, and extracting them with a one 1 : 1,000
caustic potash solution. With the filtrates of these extracts he was able
to cause rapid death in rabbits and guinea-pigs by the use of doses not
exceeding 0.5 to 1 c.c. He found, furthermore, a striking parallelism
between the degree of toxicity and the virulence of the extracted culture.

Immunization. — Recovery from a spontaneous pneumococcus in¬
fection confers immunity for only a short period. Two and three
attacks of lobar pneumonia in the same individual are not unusual,
and it is uncertain whether even a temporary immunity is acquired
in such infections. Active immunization of laboratory animals may
be carried out by various methods. The method usually followed is
to begin by injecting attenuated 4 or dead bacteria or bacterial ex¬
tracts. Subsequent injections are then made with gradually increas¬
ing doses of living, virulent microorganisms. Great care in increasing
the dosage should be exercised since the loss of an animal after two or
three weeks’ treatment by a carelessly high dose of pneumococci is not
unusual. Wadsworth has recommended the following method for
preparing pneumococci for the first injections in immunizing rabbits.
Freshly grown pneumococcus cultures are centrifugalized, and the
supernatant bouillon is thoroughly decanted. To the pneumococcic
sediment a definite quantity of concentrated salt solution is added, and
the mixture is allowed to stand over night. At the end of this time, the
pneumococci are dead and considerable destruction of the cell-bodies

1 Isaeff, Ann. de l’inst. Pasteur, vii, 1893.

2 Carnot et Fournier, Arch, de med. exper., 1900.

3 Macfadyen, Brit. Med. Jour., ii, 1906.

* Radziewsky, Zeit. f. Hyg., xxxvii, 1901; Neufeld, Zeit. f. Hyg., xi, 1902.

364

PATHOGENIC MICROORGANISMS

has taken place. Dilution with water until the solution equals 0.85
per cent NaCl now prepares the emulsion for inoculation. Whichever
of the various methods is adopted, the intervals of injection should not
be shorter than a week, preferably ten days. The animals so immunized
will at the end of six or more weeks withstand an inoculation with many
times the fatal dose of virulent pneumococci. The sera of animals
immunized with pneumococci contain active bacteriolytic and bacteri¬
cidal substances, easily demonstrable in vivo and in vitro.

Specific agglutinins in pneumococcus immune sera were first thoroughly
studied by Neufeld 1 and since then have been made the subject of ex¬
tensive studies by Wadsworth,2 Hiss,3 and many others. In the sera of
normal animals and man, pneumococci are rarely agglutinated in dilu¬
tions higher than one in ten. In the serum of patients suffering from
lobar pneumonia, pneumococci agglutinate in dilutions ranging any¬
where from one in ten to one in fifty. In the sera of immunized rab¬
bits, readings up to one in 800 are not rare. Such specific agglutinating
sera are most reliable in differentiating between pneumococci and
closely allied bacteria and in identifying all pneumococci.

The table on page 365 illustrates this uniformly high agglutinative
power of various pneumococcus-immune sera upon several races of this
microorganism, and shows the value of such sera for biological differenti¬
ation. The table, furthermore, records the peculiar fact that pneu¬
mococci are agglutinated in high dilution by sera obtained by immu¬
nization with Streptococcus mucosus, a fact which argues strongly
in favor of classifying Streptococcus mucosus more intimately with
the pneumococci than with the Streptococci of the pyogenes group.

To overcome the difficulties often attending agglutination tests
with pneumococci, Wadsworth 4 has proposed centrifugalizing young
broth cultures and shaking up the sediment with small quantities
of isotonic salt solution. Hiss recommends 5 cultivation in glucose-
calcium-carbonate broth in small flasks containing 100 to 150 c.c each.
After three or four days at 37° C., the growth is usually at its optimum
for agglutination work. The flasks should be thoroughly shaken at
least once in twenty-four hours. About one hour before use the flasks
are again shaken and the calcium carbonate and larger clumps are
allowed to settle.

1 Neufeld, loc. cit.

2 Wadsworth, loc. cit.

3 Hiss, Jour. Exp. Med., vii, 1905.

4 Wadsworth, Jour. Med. Res., x, 1905.

6 Hiss , loc. cit.

DIPLOCOCCUS PNEUMONIAE

365

Precipitins have been demonstrated in pneumococcus immune sera
by Neufeld,1 Wadsworth,2 and others. Neufeld obtained precipitates
with pneumococcus cultures in which lysis had been produced by the
addition of bile. He found that normal rabbit’s bile added to pneu-

IMMUNE SERA3

Organism.

Pneum.

23.

Strepto¬

coccus

mucosus

Strepto¬

coccus

mucosus

7 a.

Strepto¬

coccus

pyo¬

genes.

Pneumo. 1. . . .

400-800

200-800

400-800

200-800

400

0-100

Pneumo. 3. . . .

400-800

200-800

0-100

Pneumo. 23. . .

100-800

200-800

100-200

Pneumo. 45. . .

400-800

600-200

200-800

100-400

Pneum. El...

100-800

100-200

Pneum. E 32. .

200-800

100 +

Pneum. E 55. .

100-400

400-800

200-400

Pneum. N 7. . .

200-800

800

200-800

Pneum. N 17 .

200-800

800

200-800

Streptococcus

pvog:. 1 . . . .

200-800

800-6400

IT *7

Streptococcus

mucos. 7. . .

0-10

10-100

10-200

0-50

Streptococcus

mucos. 22. .

0-10

10-50

0-50

mococcus cultures (one drop to 1 c.c. of culture) caused the cultures to
become perfectly clear and transparent, and no longer contain demon¬
strable pneumococcus cell bodies. The addition of pneumococcus im¬
mune sera to such cultures produced precipitates. Wadsworth ob-

1 Neufeld, Zeit. f. Hyg., xi, 1902. 2 Wadsworth, loc. cit.

3 Hiss, Jour. Exp. Med., vii, 1905, p. 564.

366

PATHOGENIC MICROORGANISMS

ta'ned similar precipitates with pneumococcus cultures treated with
concentrated salt solution as described above (see p. 363).

Pneumococcus immune sera also contain specific phagocytosis-
stimulating substances. The first investigators to describe these sub¬
stances for pneumococcus sera, Neufeld and Rimpau,1 separated them
from the opsonins on the basis of their greater thermo-stability and
named them bacteriotropins. It is doubtful whether such differentia¬
tion is tenable. Great importance for pneumococcus immunity is
attributed to these bodies by some authors. This question has been
studied more recently by Park and Williams,2 however, who were un¬
able to find distinct parallelism between opsonic power and the protec¬
tive value of a serum.

Passive immunization with pneumococcus immune sera has been
extensively attempted. Washburn,3 Mennes,4 Pane,5 and many others
have succeeded in protecting subsequently infected animals by treatment
with such sera. Neufeld and Haendel in Germany, and in this
country Cole, have recently used pneumococcus immune sera exten¬
sively in the treatment of man. The serum, given intravenously,
seems to exert a favorable influence, and while unfinished, the work
is exceedingly encouraging. Encouraging results were obtained by
Hiss in treatment of pneumococcus infection in animals and by Hiss
and Zinsser 6 in treatment of pneumonia in man with aqueous leuco¬
cyte extracts.

Experiments both with passive immunization and with agglutina¬
tion show that all pneumococci do not react alike. Sera which will
protect white mice against the homologous strain react similarly
to some other strains, but not to all. Neufeld and Haendel 7 as a result
of protection experiments concluded that the majority of pneumococci
belonged to one type, but that a number of other types could be recog¬
nized. On the basis of agglutination and protection tests Dochez and
Gillespie 8 describe three distinct races of pneumococci and a fourth
group of heterogeneous strains. One of these definite types occurred
in nearly half of their sixty-two cases of lobar pneumonia.

1 Neufeld und Rimpau, Deut. med. Woch., 1904. .

2 Park and Williams, Jour. Exp. Med., vii, 1905.

3 Washburn, Brit. Med. Jour., 1897.

4 Mennes, Zeit. f. Hyg., 1897.

5 Pane, Rif. med., 1897.

6 Hiss and Zinsser, Jour. Med. Res., xix, 1908.

7 Neufeld and Haendel, Arb. aus dem Ivais. Gesunds., 1910, xxxiv, 293.

8 Dochez and Gillespie, J. A. M. A., 1913, lxi, 727.

DIPLOCOCCUS PNEUMONIAE

367

Differentiation of Pneumococcus from Streptococcus. — Pneumococci
and streptococci which do not differ in morphology from their classic
types can usually be differentiated from each other and identified by
their morphological characters without difficulty; but it is equally true
that certain cultures of these organisms, either at the time of their
isolation or after cultivation on artificial media, approach the type of the
other so closely that it may be impossible to identify them by their mor¬
phology alone. When such morphological variations occur there are no
constant and distinctive cultural or pathogenic characters as yet de¬
monstrated which can with certainty be depended upon as distinguish¬
ing marks between these organisms.

This lack of distinct cultural differences between pneumococci and
streptococci has not infrequently led to confusion, and that uncertainty
should exist and mistakes be made in identification is not surprising
when one considers the characters usually depended upon to distinguish
pneumococci from streptococci. Chief among these, as has just been
implied, are the morphological features which are, in the case of pneu¬
mococci, a slightly lancet or elongated form rather than the more typical
coccus form characteristic of the streptococci, and an arrangement of
such cocci in pairs rather than in chains; added to these features is the
possession of a more or less well-defined capsule. All of these char¬
acters are subject to variation or may be absent. Compared with the
morphological, the cultural characters are of minor importance and are
variable. They consist in a more moist and flatter appearance of the
pneumococcus colonies on coagulated blood serum and on agar, and
in the usual inability of the freshly isolated pneumococcus to develop
readily or at all on gelatin at temperatures below 22° C.

The distinctness of the capsule of the pneumococcus in the body
fluids of man and animals, and at times when this organism is culti¬
vated artificially on blood serum, milk, or serum agar, has really been
depended upon as the chief distinguishing and diagnostic character.

Nevertheless, from time to time, instances have been reported of
distinct capsule formation by organisms which had either been pre¬
viously identified as Streptococcus pyogenes, or at the time of their
isolation could not be definitely identified by their discoverers as be¬
longing to either this group or to the pneumococci, but were considered
intermediate in their character.1

1 Brief Description of Organisms Reported as Capsulated Streptococci. — Bordet
( Bordet , Ann. de l’inst. Pasteur, 1897, xi, p. 177), working with an organism previously

368

PATHOGENIC MICROORGANISMS

There are occasions, then, both within the animal body and in arti¬
ficial cultivations, when it is practically impossible to distinguish defi¬
nitely between some races of pneumococci and races of streptococci.
This difficulty is especially heightened when the pneumococcus has
become non-virulent, and at the same time no very typical morphology
or capsule formation is to be determined and a tendency to chain-forma¬
tion is marked. Cultures of pneumococci in such condition can not
readily be distinguished morphologically from streptococcus cultures.

Under these circumstances recourse must be had to a careful bio¬
logical study of the organism in question. The following are the criteria
mainly relied upon at present for the differentiation of these two groups.

identified as Streptococcus pyogenes, described such capsule formation occurring in
the peritoneal exudate of infected rabbits.

Schuetz’ ( Schuetz , Cent. f. Bakt., Ref. 1, 1887, p. 393) Diplokokkus der Brustseuche
der Pferde, Poels and Nolen’s ( Poets und Nolen, Fort. d. Med., iv, 1886, p. 217)
streptococcus of contagious pneumonia of cattle, and especially the organism de¬
scribed by Bonome ( Bonome , Ziegler’s Beit., viii, 1890, p. 377) as Streptococcus
der meningitis cerebrospinalis epidemica, may all be looked upon as organisms
differentiated on insecure grounds from either pneumococcus or streptococcus. The
first two of these organisms, however, are said to be decolorized by Gram’s method,
and as suggested by Frosch and Kolle ( Frosch und Kolle, Flugge’s “ Mikro-
organis.,” ii, 1896, p. 161), in the case of Schuetz’ organism may belong to a group
intermediate between Fraenkel’s diplococcus and the chicken-cholera group.

Tavel and Krumbein ( Tavel und Krumbein, Cent. f. Bakt., xviii, 1895, p. 547)
describe a streptococcus with a capsule, which was isolated from a small abscess on
the finger of a child. Capsules were also present in the artificial cultures, and
although ordinarily remaining uncolored, could be stained by Loeffler’s flagella stain.
This organism was said to be differentiated from Fraenkel’s diplococcus and also in
general from streptococcus (pyogenes) by a rapid and rich growth on gelatin, agar,
and potato. A pellicle was formed on broth. The organisms forming this pellicle
possessed capsules, but those in the deeper portions of the broth generally lacked
the capsule.

In 1897, Binaghi ( Binaghi , Cent. f. Bakt., xxii, 1897, p. 273) described a
capsulated streptococcus isolated from a guinea-pig dead of a spontaneous peribron¬
chitis and multiple pulmonary abscesses. In the pus were found some diplococci and
short chains (four to six) surrounded by a capsule, which could be made evident by
staining with carbol fuchsin. This organism he proposes to call Streptococcus
capsulatus.

Le Roy des Barres and Weinberg in 1899 {Le Roy des Barres et Weinberg, Arch,
d. med. exper., xi, 1899, p. 399) published an account of a streptococcus with a
capsule. This was isolated from a man who had apparently been infected from a
horse which had died of an acute intestinal disorder. The patient neglected the
infection and died. Diplococci and short chains furnished with a capsule were
found in the subcutaneous tissue at the area of infection. The blood, liver, and

DIPLOCOCCUS PNEUMONIA

369

Pneumococci ferment inulin, if cultivated in inulin-serum-water
medium. Acid formation from the inulin results within two days
or more in coagulation of the serum and reddening of the litmus.
Streptococci because of their inability to attack the inulin leave the
medium unchanged.1

Cultivated on whole-blood-agar, streptococci usually cause hemo¬
lysis, pneumococci usually do not.2 In contradistinction to Streptococ¬
cus viridans which does not hemolyze, pneumococci have a tendency on
these media to form the black, dry, paint-blister colonies.3

Neufeld,4 in 1900, noticed that normal rabbits’ bile added in quan-

spleen also contained these organisms. The capsule in all the preparations remained
uncolored, but the authors say that its existence was not to be doubted. Ascitic
broth inoculated from the peritoneal exudate of a rabbit dying from the infection
gave streptococci in extremely long chains and surrounded by capsules. These were
not so distinct as in the case of the organisms in the original smear preparations.
All fluid media (bouillon, milk, and ascitic broth) were said to be strongly acid after
twenty-four hours. These authors report that Achard and Marmorek have assured
them that they have seen capsulated streptococci, and that Marmorek showed them
some preparations in which one of his streptococci presented the same characters as
that isolated by them.

Although Le Roy des Barres and Weinberg have used the term encapsulated,
they believe that it would perhaps be more prudent to call their organism strepto-
coque aureole, since they were not able to define this capsule by staining it.

Howard and Perkins ( Howard and Perkins, Jour. Med. Res., 1901, iv, p. 163)
have lately described an organism, probably of the foregoing type, which was present
in a tubo-ovarian abscess and in the peritoneal exudate, the blood, and some of the
organs of a woman dying in the Lakeside Hospital, Cleveland, Ohio. The organisms
were biscuit-shaped cocci in pairs, usually arranged in chains of four, six, eight,
or twenty elements, and surrounded by a wide and sharply staining capsule. In the
artificial cultures special capsule stains, it was noted, failed to stain any definite area,
but numerous small deeply stained granules were to be seen within the halo, espe¬
cially near its outer border. Howard and Perkins propose for the group composed of
the streptococci of Bonome, Binaghi, and their own organism, the name Strepto¬
coccus mucosus. Streptococci isolated from cases of epidemic sore-throat have also
shown capsules (p. 343).

Reference to the original descriptions of these various capsulated streptococci
will show that, with the exception of a rather poorly staining capsule, the majority of
these organisms are separated from the typical Streptococcus pyogenes or from the
pneumococcus by exceedingly slight and unstable morphological and cultural charac¬
ters. The same is true of the difference observed in their pathogenic action in
animals.

1 Hiss, Cent. f. Bakt., xxxi, 1902; Jour. Exp. Med., vi, 1905.

2 Schottmuller, Miinch. med. Woch.

3 Hiss, Jour. Exp. Med., vii, 1905.

4 Neufeld, Zeit. f. Hyg., 1901.

370

PATHOGENIC MICROORGANISMS

tities of 0.1 c.c. to each one or two cubic centimeters of a pneumococcus
broth culture caused lysis of the bacteria, rendering the culture fluid
transparent and clear. This phenomenon does not occur with strep¬
tococci, and has been used to differentiate the two species. According
to the recent studies of Libman and Rosenthal,1 great reliance may be
placed upon this method.

The most convenient reagent for use in the Neufeld bile test is a
10 per cent solution of sodium taurocholate in physiological salt solution.
This should be sterilized or kept on ice. One-tenth volume of such a
solution produces prompt lysis in a broth culture of pneumococci.

Decisive differential importance may be attached to the agglutina¬
tions of these microorganisms in immune sera (see p. 364).

The permanency of the various types in the pneumococcus-strepto¬
coccus group is still open to question. E. C. Rosenow 2 has recently re¬
ported that he has transmuted typical pneumococci into typical hemo¬
lytic streptococci by methods which he has not as yet fully described, but
among which were animal passage, growth in symbiosis with bacillus
subtilis, and growth in an atmosphere of oxygen. The pneumococci
when first altered took on the characteristics of the streptococcus
viridans, later of the so-called streptococcus rheumaticus, and finally
of streptococcus hemolyticus. Together with cultural characteristics
the pathogenicity of these various strains for rabbits changed. The
pneumococcus produced acute sepsis, the streptococcus viridans caused
endocarditis, the streptococcus rheumaticus periarticular or serous
arthritis, and hemolyticus suppurative arthritis. An intermediate stage
was found in which the organisms quite regularly produced myositis.
Although he was able to transmute these types one into the other in
both directions, Rosenow believes that the cultural characteristics
of each type correspond to a fairly definite type of pathogenicity both
in animals and man. This work has not as yet appeared in detail and
has not been confirmed.

1 Libman and Rosenthal , Proc. N. Y. Path. Soc., March, 1908.

2 Rosenow, J. A. M. A., 1913, lxi, 2007.

CHAPTER XXIV

MICROCOCCUS INTRACELLULARIS MENINGITIDIS

(MENINGOCOCCUS)

Infectious processes in the meninges may be caused by many dif¬
ferent microorganisms.

Meningitis may be primary or secondary. Secondary meningitis
may often occur during the course of pneumonia, when pneumococci,
carried to the meninges by the blood stream, give rise to a usually fatal
form of the disease. More rarely a similar process may occur as a
secondary manifestation of typhoid fever or influenza. Meningitis may
also result secondarily by direct extension from suppurative lesions about
the skull, such as those occurring in diseases of the middle ear or frontal
sinuses or after compound fractures. In such cases the invading or¬
ganisms are usually staphylococci, streptococci, or pneumococci.

Isolated cases of meningeal infection with B. coli, B. paratyphosus,
Bacillus pestis, and Bacillus mallei have been reported. A frequent,
more chronic form of the disease is caused by Bacillus tuberculosis.

Primary acute meningeal infection, however, is due chiefly to two
microorganisms, Micrococcus intracellularis meningitidis, and the pneu¬
mococcus.

A tabulation of the comparative frequency with which the various
microorganisms are found in the meninges has been attempted by
Marschal.1 This author estimates that about 69.2 per cent of all
acute cases are due to the meningococcus, 20.8 per cent to Diplococcus
pneumoniae, and the remaining 10 per cent to the other bacteria
mentioned.

The cases caused by the pneumococcus and the other less frequent
incitants usually occur sporadically. When the disease occurs in epi¬
demic form, it is almost always due to the meningococcus.

Diplococcus intracellularis meningitidis was first seen in menin¬
geal exudates by Marchiafava and Celli 2 in 1884. These authors not
only described accurately the morphological characteristics now recog-

1 Marschal, Diss. Strassburg, 1901, Quoted from Weichselbaum, in Kolle und
Wassermann, “ Handbuch.”

2 Marchiafava and Celli, Gaz. degli ospedali, 8, 1884.

371

372

PATHOGENIC MICROORGANISMS

nized, but also called attention to the intracellular position of the micro¬
organism and to its gonococcus-like appearance. They failed, however,
to cultivate it.

Observations confirmatory of the Italian authors were, soon after,
made by Leichtenstern.1 Cultivation and positive identification as a
separate species was not accomplished, however, until Weichselbaum,2
in 1887, reported his observations upon six cases of epidemic cerebro-

Fig. 77. — Meningococcus, Pure Culture. (Very highly magnified.)

spinal meningitis in which he not only found the cocci morphologically,
but was able to study their biological characteristics in pure culture.
The researches of Weichselbaum were soon confirmed and extended
by elaborate studies 3 which left no doubt as to the specific relationship
between the microorganism cultivated by him and the clinical condition.

1 Leichtenstern, Deut. med. Woch., 1885.

2 Weichselbaum, Fort. d. Med., 1887.

3 Councilman, Mallory, and Wright, Special Rep. Mass. Board of Health, 1898;
Albrecht und Ghon, Wien. klin. Woch., 1901.

MICROCOCCUS INTRACELLULARIS MENINGITIDIS

373

Morphology and Staining. — Stained in the spinal fluid from an in¬
fected patient, the meningococcus bears a striking similarity to the gon¬
ococcus. The microorganisms appear intra- and extracellularly, usually
in diplococcus groups, sometimes as tetrads, or even in larger agglomer¬
ations. The individual diplo-forms are flattened on the sides facing each
other, presenting somewhat the biscuit-form of the gonococcus. The
variation in size of the cocci in the same smear is a noticeable feature

Fig. 78. — Meningococcus in Spinal Fluid.

and of some diagnostic importance. This dissimilarity in size is notice¬
able also in cultures, which, especially when older than twenty -four-
hours, contain forms double or even triple the size of the average coccus.
These may possibly be involution forms.

The meningococcus is non-motile and non-spore forming. It
stains easily with all the usual aqueous anitin dyes. Its behavior
toward Gram's stain was long a subject of controversy, owing to the
error of Jaeger,1 who claimed to have found it Gram-positive. There

1 Jaeger, Zeit. f. Hyg., xix, 1895.

374

PATHOGENIC MICROORGANISMS

is no question now, however, that the cocci decolorize by Gram’s method
when this is carefully carried out.

In spinal fluid very beautiful preparations may be obtained
by staining in Jenner’s blood stain. Councilman, Mallory, and
Wright 1 were the first to notice that, when stained with Loeffler’s
methylene-blue, meningococcus stains irregularly, showing metachro-
matic granules in the center of the cell bodies. These granules can be
demonstrated more clearly with the Neisser stain employed for similar
demonstration in the case of B. diphtherise (see p. 107) and have some
value in differentiating meningococcus from gonococcus.

Cultivation. — Micrococcus intracellularis meningitidis grows readily
upon all the meat-infusion culture-unedia. It may even be culti¬
vated upon meat-extract media, but growth upon these is not profuse.
Upon agar , colonies appear within eighteen to twenty-four hours as
grayish, glistening spots with smooth edges and raised granular centers.
These show a tendency to enlargement and eventual confluence.

Growth is more luxuriant and rapid upon media to which animal
proteid in the form of blood serum or ascitic fluid has been added. Co¬
agulated serum is not liquefied. For cultivation of the meningococcus
directly from the human body it is wise to use the richer serum or blood
media, ability to grow easily upon simple agar being occasionally acquired
only after previous cultivation upon richer media. Agar to which whole
rabbit’s blood has been added forms an excellent medium, both for cul¬
tivation and for keeping the organism alive. Loeffler’s blood serum
is also very favorable. It is advisable, too, when cultivating directly
from spinal fluid, to plant rather large quantities (1 to 2 c.c.), since
many of the cocci in the exudate will fail to develop colonies, possibly
because of their prolonged exposure either to the body fluids or to their
own products in a closed space.

Upon broth , growth is slow and takes place chiefly upon the surface,
the sediment consisting mainly of dead bacteria. Glucose added to agar
or to broth renders the medium more favorable for rapid growth, but,
owing to acid formation, tends to cause a more rapid death of the culture.
In flasks of broth containing glucose one per cent, and CaC03 one per
cent, however, cultures have been kept alive for as long as fourteen
months (Hiss). On milk, growth takes place without coagulation
of the casein. Potatoes are not a favorable medium, though growth
occasionally takes place.

1 Councilman, Mallory, and Wright, Rep. Mass. State Bd. of Health, 1898.

MICROCOCCUS INTRACELLULARIS MENINGITIDIS

375

While slight alkalinity or acidity does not inhibit, the most favor¬
able reaction of media is neutrality.

Oxygen is necessary for development. Complete anaerobiosis, while
not absolutely inhibitory, is extremely unfavorable, unless proper
carbohydrates be present.

While growth may take place at temperatures ranging from 25°

Fig. 79. — Meningococcus Culture. Streak culture from spinal fluid on

serum agar-plate.

to 42° C., the optimum is 37.5° C. Apart from the remarkable viability
displayed upon calcium-carbonate broth, the average length of time
during which the meningococcus will remain alive without transplanta¬
tion is rather short. Recently isolated cultures grown on agar or serum-
agar may die within two or three days. Accustomed to artificial cul¬
tivation through a number of generations, however, the cultures become

376

PATHOGENIC MICROORGANISMS

more hardy and transplantation may safely be delayed for a week or
even longer. Albrecht and Ghon 1 have kept a culture alive on agar
for one hundred and eighty-five days. It is a strange fact that after
prolonged artificial cultivation some strains of meningococcus may
gradually lose their growth energy and finally be lost because of their
refusal to develop in fresh transplants. Storage is best carried out at
incubator temperatures. At room temperatures or in the ice chest,
the diplococcus dies rapidly.2

Resistance. — The meningococcus is killed by exposure to sunlight or
to drying within twenty-four hours.3 It is extremely sensitive to heat
and cold and by the common disinfectants is killed in high dilutions
and by short exposures. At 0° C. it usually dies within two or three
days.

Pathogenicity. — As stated above, the form of meningitis caused by
the diplococcus of Weichselbaum occurs usually in epidemics, though
isolated sporadic cases are seen from time to time in all crowded com¬
munities. Epidemics have been numerous and widespread, and their
records far antedate the discovery of their causative agent. As a rule,
these epidemics have occurred during the winter and spring months,
and have attacked chiefly that part of the population which is forced by
poverty to live in crowded unhygienic surroundings. The manner in
which the microorganism enters the human body is still a subject for
investigation. Weichselbaum,4 Ghon and Pfeiffer,5 and, more recently,
Goodwin and v. Sholly 6 of the New York Department of Health, have
succeeded in demonstrating culturally the presence of the meningococ¬
cus in the nasal cavities, not only of patients suffering from the disease,
but occasionally in those of healthy subjects as well. Similar findings
have been reported by many others; but in many cases morphological
examination only was made, which, owing to the danger of confusion
with Micrococcus catarrhalis, a frequent inhabitant of the nose, renders
such reports valueless. The careful work of the writers mentioned, how¬
ever, has given ground for the theory that meningeal infection, which is

1 Albrecht und Ghon, Wien. klin. Woch., 1901.

2 A very thorough biological study of meningococcus and related organisms has
recently been made by Elser and H untoon (Jour. Med. Res., N. S. vol. xv, 1999),
which may be consulted for a more detailed description of cultural characteristics.

3 Councilman, Mallory, and Wright, Boston, 1898; Albrecht and Ghon, loc. cit.

4 Weichselbaum, Fort. d. Med., 1887.

5 Ghon und Pfeiffer, Zeit. f. klin. Med., xliv, 1901.

Goodwin und v. Sholly, Jour. Inf. Dis., Suppl. 2, Feb., 1906.

MICROCOCCUS INTRACELLULARIS MENINGITIDIS

377

often preceded by nasal catarrh, may take place along the paths of the
lymphatics, passing out of the nose and its accessory cavities toward the
base of the skull. These facts, together with the low resistance shown
by the meningococcus against drying, and the general failure so far to
demonstrate it in air, dust, or fomites, would seem to indicate that trans¬
mission usually occurs directly from one human being to another.

The disease produced in man consists anatomically in a suppurative
lesion of the meninges, involving the base and cortex of the brain and the
surface of the spinal cord. The nature of the exudate may vary from a
slightly turbid serous fluid to that of a thick fibrinous exudate. In
chronic cases encephalitis and dilatation of the ventricles may take
place. Apart from their presence in the meninges and in the naso¬
pharynx, meningococci have not been satisfactorily demonstrated in
any of the complicating lesions of the disease. Reports of their presence
in the conjunctivse, in the bronchial secretions from broncho- or lobar
pneumonia, and in otitis media, have usually been based upon insuf¬
ficient bacteriological evidence.

The occurrence of this microorganism in the circulating blood of men¬
ingitis cases has been definitely proved by Elser,1 who found it in ten
cases.

Animals are not very susceptible to infection with Diplococcus
meningitidis. Subcutaneous inoculation is rarely followed by more
than a local reaction unless large quantities are used. White mice are
rather more susceptible than other species. Intraperitoneal and intra¬
venous inoculation of sufficient quantities usually results in the death
of mice, rabbits, guinea-pigs, and dogs. Occasional strains have been
found to possess a not inconsiderable degree of toxicity for rabbits,
grave symptoms or even death following intravenous injection of but
moderate quantities without any traceable development of the micro¬
organisms in the organs of the animals.

Similar observations have been made by Albrecht and Ghon,2 who
succeeded in killing white mice with dead cultures. It would seem
therefore that the effect of this coccus upon animals depends chiefly
upon the poisonous substances contained in the bacterial bodies (endo¬
toxins). Lepierre 3 has obtained the meningococcus toxin by alcohol
precipitation of broth cultures.

Weichselbaum himself succeeded in producing meningeal suppura-

1 Elser, Jour. Med. Res., xiv, 1906.

2 Albrecht und Ghon., loc. cit.

3 Lejpierre, Jour, de phys. et de path, gen., v, No. 3.

378

PATHOGENIC MICROORGANISMS

tion and, in one case, brain abscess, by subdural inoculation of dogs.
Councilman, Mallory, and Wright 1 produced a disease in many re¬
spects similar to the human disease by intraspinous inoculation of a
goat. Recently, Flexner 2 has succeeded in producing in monkeys a
condition entirely analogous to that occurring in human beings.

Agglutination. — Immunization of animals by repeated inoculations
of meningococcus 3 results in the formation in the blood serum of
agglutinins. Kolle and Wassermann4 obtained from horses a serum
which had an agglutinating value of 1 : 3,000 for the homologous
strain, and of as much as 1 : 500 for other true meningococcus
strains. Similar experiments by Dunham 5 and others have proved the
unquestionable value of agglutination for species identification of this
group. Great differences may, however, exist between individual
races in their agglutinability in the same immune serum.

Kutscher has recently called attention to the fact that strains
which can not be agglutinated in specific sera at 37° C. will often yield
positive results when subjected to 55° C., a fact of some practical im¬
portance if confirmed.

Elser and Huntoon 6 have shown that in the serum of infected human
subjects agglutination of some strains takes place in dilutions as high
as 1 : 400.

Serum Therapy of Meningitis. — During recent years, many attempts
have been made to treat epidemic cerebrospinal meningitis by injec¬
tions, subcutaneous and intraspinous, of meningococcus-immune serum.
Wassermann,7 in 1907, reported the results obtained by such treatment
in one hundred and two patients, with a recovery of 32.7 per cent.
The serum, as manufactured by Wassermann and his associates, was
obtained from horses immunized with pure cultures of meningococcus
and with toxic meningococcus extracts. More recently Flexner and

Jobling 8 have used a similar serum in the United States with appa-

rently excellent results. The serum, in Flexner’s cases, is injected
intraspinously after a quantity of spinal fluid had been withdrawn.
The cases treated by Flexner and Jobling’s method have now reached

1 Councilman, Mallory, and Wright, loc. cit.

2 Flexner, Jour. Exp. Med., 1906.

3 Albrecht and Ghon, Wien. klin. Woch., 1901.

* Kolle und Wassermann, Deut. med. Woch., 15, 1906.

6 Dunham, Jour. Inf. Dis., 11, 1907.

0 Elser and Huntoon, loc. cit.

7 Wassermann, Deut. med. Woch., 39, 1907.

8 Flexner and Jobling, Jour. Exper. Med., x, 1908.

MICROCOCCUS INTRACELLULARIS MENINGITIDIS

379

large numbers, both in this and foreign countries and the value of the
serum as a therapeutic agent seems firmly established.

Hiss and Zinsser 1 have treated a number of meningitis patients with
subcutaneous injections of leucocyte extracts and believe that they have
favorably influenced the course of the disease.

Pseudomeningococcus. — Elser and Huntoon 2 have described a diplo-
coccus very similar to the meningococcus which they differentiated
from it only by serum reactions. This diplococcus could be identified
only by agglutinin absorption tests. They named it pseudomeningo¬
coccus.

1 Hiss and Zinsser, Jour. Med. Res., Nov., 1908.

2 Elser and Huntoon, Jour. Med. Res., xxi, 1909.

CHAPTER XXV

DIPLOCOCCUS GONORRHCE7E (GONOCOCCUS), MICROCOCCUS
CATARRHALIS, AND OTHER GRAM-NEGATIVE COCCI

DIPLOCOCCUS GONORRHOEA

Neisser,1 in 1879, described diplococci which he had found regularly
in the purulent secretions of acute cases of urethritis and vaginitis and
in the acute conjunctivitis of the new-born. His researches were purely
morphological, as were the numerous confirmatory investigations which
rapidly followed his announcement.

Cultivation of this diplococcus, now usually spoken of as gonococcus,
was not definitely successful until 1885, when Bumm 2 obtained growth
upon tubes of coagulated human blood serum. Bumm was not only
able to keep the organisms alive by transplantation in pure culture, but
produced the disease by inoculation of his cultures upon the healthy
urethra.

Morphology and Staining. — The gonococcus is usually seen in the
diplococcus form, the pairs being characteristically flattened along the
surfaces facing each other. This gives the cocci a peculiar coffee-bean
or biscuit shape. The size of the diploforms is about 1.6 micra in the
long diameter, about 0.8 micron in width. Stained directly in gonorrheal
pus from acute cases, the microorganisms are found both intra- and
extracellularly, a large number of them crowded characteristically
within the leucocytes. They are never found within the nucleus. The
phagocytosis which produces this picture has been shown by Scholtz 3
and others to take place in the free secretions, not in the depth of the
tissues. The intracellular position, which is of considerable diagnostic
importance, is lost to a great extent in secretions from chronic cases.
In smears made from pure cultures the arrangement in groups of two
may often be less marked than in pus, clusters of eight or more being
common.

1 Neisser, Cent. f. d. med. Wiss., 1879.

2 Bumm, “ Beitr. z. Kenntniss des Gonococcus, ” Wiesbaden, 1885.

3 Scholtz, Arch. f. Dermat., 1899.

380

DIPLOCOCCUS GONORRHOEA

381

The gonococcus is non-motile and does not form spores. It is easily
stained with the usual aqueous anilin dyes. Methylene-blue alone,
or eosin followed by methylene-blue, or the neutral red stain of Plato,1
gives good results. Gram’s method of staining, however, is the only
one of differential value. With this method the gonococcus is rapidly
decolorized and can be counterstained with fuchsin or Bismarck brown.
The Gram stain applied to pus from the male urethra, while not
absolutely reliable, is, for practical purposes, sufficiently so to make
a diagnosis. In exudates from the vagina or from the eye the mor¬
phological picture is not so reliable, owing to the frequent presence

Fig. 80. — Gonorrheal Pus from Urethra, showing the Cocci within a

Leucocyte.

in these regions of other Gram-negative cocci. The great scarcity of
gonococci in very chronic discharges necessitates thorough cultural
investigation; negative morphological examination in such cases can
not be regarded as conclusive.2

Cultivation. — The gonococcus is extremely delicate and is difficult
to cultivate. After many failures to grow it upon the ordinary media,
Bumm 3 obtained his first growths upon human blood serum which
had been heated to partial coagulation.

The medium most commonly used at the present day was introduced

1 Plato , Bed. klin. Woch., 1894. 2 Heiman, Medical Record, 1896,

s Bumm, Deut. med. Woch., 1885.

382

PATHOGENIC MICROORGANISMS

by Wertheim,1 and consists of a mixture of two or three parts of meat
infusion-agar with one part of uncoagulated human ascitic fluid,
hydrocele fluid, or blood serum. The agar is melted and cooled to 45°
before the serum is added. The mixture may then be slanted in the test
tube or poured into a Petri plate. This medium may be improved by
the addition to the agar of six per cent of glycerin or one per cent of

Fig. 81. — Gonococcus. Smear from pure culture.

glucose. Cultures in fluid media may be obtained by similar addi¬
tions of serum to meat-infusion-pepton-broth (pepton, one to two
per cent) . While human sera may be replaced by animal sera, these
in general are not so favorable for growth of the gonococcus. They
are useful chiefly in cases where it is difficult to obtain the
human serum. Whole rabbit’s blood added to agar, or the swine-

Wertheim, Arch. f. Gynakol., 1892.

DIPLOCOCCUS GONORRHCEiE

383

serum-nutrose medium of Wassermann 1 may occasionally be used
with success.

Plates may also be made by smearing for enrichment a drop of blood
from the finger over the surface of agar in the manner of Pfeiffer’s
method for influenza-bacillus cultivation. Inoculations from gonorrheal
material are best made by surface smearing upon plates, since the
gonococcus grows best in the presence of free oxygen. Growth is more
easily obtained and becomes more luxuriant after prolonged culti-

Fig. 82. — Gonococcus Colony. Low power of magnification. (After Mallory

and Wright.)

vation upon artificial media. The most favorable reaction of media is
neutrality or slight acidity.

Whenever the gonococcus has been successfully cultivated from pus
upon media without serum additions, the success has probably been due
to the substances carried over in the pus.

The gonococcus will develop a sparse growth under anaerobic con¬
ditions, but displays a very marked preference for aerobiosis. The op¬
timum temperature for growth is 37.5° C. Growth ceases above 38.5°
and below 30°.

' Wassermann, Berl. klin. Woch., 1897.

(Fifteen c.c. swine-serum, 35 c.c. of water, 3 c.c. glycerin, with two per cent
nutrose. The nutrose is dissolved by boiling and the solution sterilized. This is then
added to agar, in equal parts, and used in plates.)

384

PATHOGENIC MICROORGANISMS

Upon suitable media colonies appear as extremely delicate, grayish,
opalescent spots, at the end of twenty to twenty-four hours. The sepa¬
rate colonies do not tend to confluence and have slightly undulated
margins. Touched with a platinum loop their consistency is found to
be slimy or sticky. In fluid media, growth takes place chiefly at the
surface and there is no general clouding.

Resistance. — Cultures of gonococcus, if not transplanted, usually
die out within five or six days at incubator temperature. At room
temperature they die more rapidly. In the ice chest they may be kept
alive for somewhat longer periods. In this they differ from meningo¬
cocci, which are always killed by temperatures approximating zero C.
In these respects, however, individual strains show much variation, some
cultures dying out after but a few hours’ removal from the incubator.

The resistance of the gonococcus to light and heat is very slight.
A temperature of 41° to 42° kills it after a brief exposure. Complete
drying destroys it in a short time. Incompletely dried, however, and
protected from light (gonorrheal pus) it may live, on sheets and cloth¬
ing, for as long as eighteen to twenty-four hours.1

It is easily killed by most disinfectant solutions 2 even when these
are highly diluted and seems to be almost specifically sensitive to the
various silver salts, a fact of therapeutic importance.

Pathogenicity. — Gonorrheal infection occurs spontaneously only in
man. True gonorrheal urethritis has never been experimentally pro¬
duced in animals. In human beings, apart from the common seats of
the infection in the male and female genital tracts, and in the conjunc¬
tive, the gonococcus may produce cystitis, proctitis, and stomatitis.
It may enter the general circulation, giving rise to septicemia 3 and,
secondarily, to endocarditis and arthritis. Isolated cases of gonorrheal
periostitis and osteomyelitis have been reported.4

The common acute infections of the genito-urinary passages in man
are often followed by an indefinitely prolonged chronic infection, which,
though quiescent, may for many years be a source of social danger.
In children, especially females, the infection is not rare, and may
assume epidemic characters, traveling from bed to bed in institu¬
tions. Such hospital epidemics can be stopped only by the most
rigid isolation. It is advisable, in view of this danger, to examine all

1 Heiman, Medical Record, 1896.

2 Schaeffer und Steinschneider , Kong. Deut. Dermat. Gesells., Breslau, 1894.

3 Review of cases of Gon. Septicemia, Faur e-Beaulieu, Thesis, Paris, 1906.

4 Ullmann, Wien. med. Presse, 1900.

MICROCOCCUS CATARRHAL IS

385

female children applying for admission to a hospital, by vaginal
smear and, if possible, to keep them in a receiving ward for twenty-
four hours in order that the examination may be repeated before
admission to the general wards. In the best-equipped institutions,
furthermore, separate thermometers, bed linen, and diapers are set
aside for each child in order to preclude any possibility of accidental
transmission from cases which may have escaped detection by smear
examination.

While inoculation of animals has never resulted in active prolifera¬
tion of the gonococcus upon the new host, local necrosis, suppuration,
and temporary systemic reactions have been produced by subcutaneous
and intraperitoneal inoculation. These are probably referable to the
endotoxin contained in the bodies of the gonococci. This toxin has been
isolated by Nikolaysen1 by extraction from the bacterial bodies with
distilled water or sodium hydrate solutions. It was found to be resist¬
ant to a temperature of 120° and to remain potent after complete drying.
The same author found that the isolated toxin and dead cultures were
fully as toxic for animals as living cultures, 0.01 gram killing a white
mouse.

Specific injury to the nervous system by injections of gonococcus
toxin has been reported by Moltschanoff.2

The secretion of a true soluble toxin by the gonococcus, asserted
by Christmas,3 is denied by Wassermann,4 Nikolaysen,5 and others.
The two authors last named, furthermore, do not believe that a general
immunity is developed in subjects infected with gonococcus. Christ¬
mas 6 on the other hand, and, more recently, Torrey,7 have reported
successful active immunization of animals by repeated injections of
whole bacteria. Torrey and others apparently have successfully treated
human cases by injections of the serum of immunized animals.

MICROCOCCUS CATARRHALIS

Micrococcus catarrhalis is a diplococcus described first by R. Pfeiffer,8
who found it in the sputum of patients suffering from catarrhal in-

1 Nikolaysen, Cent. f. Bakt., 1897.

2 Moltschanoff, Munch, med. Woch., 1899.

3 Christmas, Ann. de l’inst. Pasteur, 1897.

4 Wassermann, Zeit. f. Hyg., xxvii, 1897.

5 Nikolaysen, Fort. d. Med., xxi, 1897.

6 Christmas, loc. cit.

v Torrey, Jour. Amer. Med. Assn., xlvi, 1906.

8 Fliiqge, “ Die Mikroorg.,” 3d ed., 1896.

386

PATHOGENIC MICROORGANISMS

flammations of the upper respiratory tract. It was subsequently care¬
fully studied by Ghon and H. Pfeiffer.1 According to these authors
the pathogenic significance of the micrococcus is slight, though occasion¬
ally it may be regarded as the causative factor in catarrhal inflammations.
Its chief claim to attention, however, lies in its similarity to the meningo¬
coccus and the gonococcus, from neither of which it can be morphologi¬
cally distinguished. It is decolorized by Grands stain, appears often in
the diplococcus form, and has a tendency, in exudates, to be located
intracellularly. Not unlike the two microorganisms mentioned, too, it
shows but slight pathogenicity for animals.

Differentiation from gonococcus is extremely simple in that Micro¬
coccus catarrhalis grows easily on simple culture media and shows
none of the fastidious cultural requirements of the gonococcus.

From meningococcus the differentiation is less simple and, because
of the presence of both microorganisms in the nose, is of great impor¬
tance.

Distinction between the two its made entirely upon cultural charac¬
teristics and agglutination reactions. Culturally, Micrococcus catar¬
rhalis grows more heavily than meningococcus upon the ordinary
culture media. The colonies of Micrococcus catarrhalis are coarsely
granular and distinctly white in contradistinction to the finely granu¬
lar, grayish meningococcus colonies.2 Micrococcus catarrhalis will
develop at temperatures below 20° C., while meningococcus will not
grow at temperatures below 25° C.3

Dunham,4 who has recently made a comparative study of meningo¬
coccus and other Gram-negative diplococci from the nose and throat,
states that while some of the supposed Micrococcus catarrhalis cul¬
tures are easily distinguished from meningococcus simply by the char¬
acteristics of their growths upon two-per-cent glucose agar, others offer
great difficulties to differentiation. He recommends as a differential
medium a mixture of sheep serum and bouillon containing one per
cent of glucose. Upon this medium all true meningococci produce
acid, but no coagulation, with twenty-four hours. Cultures from the
nose and throat, however, produce acid and coagulation, or else pro¬
duce an alkaline reaction.

1 Ghon und H. Pfeiffer, Zeit. f. klin. Med., 1902.

2 Ghon und Pfeiffer, loc. cit.

3 Weichselbaum, in Kolle und Wassermann, Bd. iii, p. 269.

* Dunham, Jour. Inf. Dis., 1907.

GRAM-NEGATIVE COCCI

387

OTHER GRAM-NEGATIVE COCCI

Micrococcus pharyngis siccus. — This organism was first described by
von Lingelsheim 1 in 1906. It is described by Elser and Huntoon as
readily differentiable from meningococcus and other Gram-negative
cocci by the firm adherence and dryness of its colonies. It is similar to
Micrococcus catarrhalis from which it may, however, be differentiated
by fermentation tests.

Diplococcus mucosus. — This organism was also described by von
Lingelsheim together with the preceding one. Its colony formation is
similar to that of meningococcus, but slightly more sticky and mucoid.
Stained by the capsule methods, it is seen to possess a distinct capsule.

Chromogenic Gram -negative Cocci. — These microorganisms all pro¬
duce a greenish-yellow pigment on the ordinary culture media. When
pigment is absent, as is frequently the case when grown upon sugar-free
media, these microorganisms can be distinguished from meningococcus
only by sugar fermentation and serum reactions.

An exhaustive study of Gram-negative micrococci has recently
been made by Elser and Huntoon.2 These authors, in studying
the differential value of sugar fermentation in the diagnosis of these
bacteria, have constructed the following table:

Strains Tested.

Strains.

Dextrose.

Maltose.

Levulose.

Saccharose.

Lactose.

Galactose.

Meningococcus .

200

Pseudomeningococcus .

Gonococcus .

Micrococcus catarrhalis .

Micrococcus pharyngis siccus .

Chromogenic group I .

Chromogenic group II .

Chromogenic group III .

Jaeger meningococcus, Krai .

Diplococcus crassus, Krai .

1 v. Lingelsheim, Klin. Jahrb., 15, 1906.

2 Elser and Huntoon, loc. cit.

CHAPTER XXVI

BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP

The bacilli belonging to this group of microorganisms, while present¬
ing great differences in their pathogenic characteristics, possess many
points of morphological and biological similarity which have made their
differentiation extremely difficult. Among pathogenic bacilli, they are
probably the ones most commonly encountered and because of the fact
that some of them are specifically pathogenic, while others are essen¬
tially saprophytic and are pathogenic only under exceptional conditions,
the necessity of accurate differentiation is a daily occurrence in bacteri¬
ological laboratories. It has been through the study of this group par¬
ticularly that many of the modern differential methods of bacteriology
have been developed.

The group includes the colon bacillus and its allies, the typhoid
bacillus, the paratyphoid organisms, the several varieties of dysentery
bacillus and numerous closely related species, and Bacillus fecalis alka-
ligenes. Closely related to the group though not properly within it,
are Bacillus lactis aerogenes, bacilli of the Friedlander or mucosus
capsulatus group, and a number of less important subdivisions of this
last group.

All bacilli of the group possess morphological characteristics which,
although exhibiting slight differences, are insufficient to permit accurate
morphological diagnosis. They are none of them spore-bearing. Stained
by Grands method they are decolorized.

Cultivated upon artificial media, they grow readily both at room and
at incubator temperatures. None of them liquefies gelatin. Though
showing, often, distinct differences in the speed and luxuriance of growth
upon ordinary media, these differences are, nevertheless, too slight to
become the basis of differentiation.

In order to distinguish between the individual members of this
group, therefore, we are forced to a careful biological and cultural
study. This is carried out by the observation of the cultural character¬
istics upon special media and by the study of serum reactions in speci¬
fic immune sera. Our mainstays in the accurate differentiation of these

388

BACILLUS COLI COMMUNIS

389

bacilli are the observation of their fermentative action upon carbohy¬
drate media, and their agglutinating reactions in immune sera. These
points will be alluded to in the description of the individual microor¬
ganisms, and will be again summarized in the differential tables given
at the end of the chapters dealing with this group.

BACILLUS COLI COMMUNIS AND MEMBERS OF THE COLON

BACILLUS GROUP

Under the name of “ colon bacilli” are grouped a number of bac¬
terial varieties differing from one another somewhat in minor character¬
istics, but corresponding in certain cardinal points which stamp them
as close relatives and amply warrant their consideration under one
heading. While usually living as harmless parasites upon the animal
and human body, and capable of leading a purely saprophytic existence,
they may, nevertheless, under certain circumstances become pathogenic
and thus, both culturally and in their pathological significance, form a
link between pure saprophytes like Bacillus lactis aerogenes, on the
one hand, and the more strictly pathogenic Gram-negative bacilli of
the paratyphoid, typhoid, and dysentery groups, on the other. As a
type of the group we may consider in detail its most prominent and
thoroughly studied member, Bacillus coli communis.

BACILLUS COLI COMMUNIS

This microorganism was seen and described by Buchner 1 in 1885.
It was thoroughly studied in the years immediately followihg, especially
by Escherich,2 in connection with the intestinal contents of infants.

Morphology. — Bacillus coli communis is a short, plump rod about
1-3 micra long, and varying in thickness from one-third to one-fifth
of its length. Under varying conditions of cultivation, it may appear
to be more slender than this or shorter and even coccoid in form. In
stained preparations, it usually appears singly, but occasionally may be
seen in short chains. It stains readily with the usual anilin dyes and
decolorizes by Gram’s method. Spores are not formed. It is motile, and
flagella staining reveals eight or more flagella peripherally arranged. Its
motility is subject to wide variations. Young cultures, in the first gen-

1 Buchner, Arch. f. Hyg., 3, 1885.

2 Escherich, “Die Darmbakt. des Sauglings,” Stuttgart, 1886; Cent. f. Bakt., 1,

1887.

390

PATHOGENIC MICROORGANISMS

eration, after isolation from the body, may be extremely motile, while old
laboratory strains may show almost no motility. Independent of these
modifying conditions, however, separate races may show individual
characteristics as to motility, varying in range between a motility
hardly distinguishable from Brownian movement and one which is so
active as to be but little less than that of the typhoid bacillus. Ordi¬
narily, the colon bacillus possesses a motility intermediate between
these two extremes.

Cultivation. — The bacillus is an aerobe capable of anaerobic growth
under suitable cultural conditions. It grows well on the simplest media

Pig. 83.- — Bacillus coli communis.

at temperatures ranging from 20° to 40° C., but finds its optimum
growth at about 37.5° C. Upon broth it grows rapidly, giving rise to
general clouding; later to a pellicle and a light, slightly slimy sediment.
Within moderate ranges, it is not delicately susceptible to reaction,
growing equally well on media slightly acid and on those of a moderate
alkalinity.

Upon agar, it forms grayish colonies which become visible within
twelve to eighteen hours, gradually becoming more and more opaque
as they grow older. The deep colonies are dense, evenly .granular, oval,

BACILLUS COLI COMMUNIS

391

or round. Surface colonies often show a characteristic grape-leaf
structure, or may be round and flat, and show a definitely raised, glisten¬
ing surface. Upon agar slants, growth occurs in a uniform layer.

On gelatin the colon bacillus grows rapidly, causing no liquefaction.
Surface colonies are apt to show the typical grape-leaf formation. Deep
colonies are round, oblong, and glistening. In gelatin stabs growth takes
place along the entire line of inoculation, spreading in a thin layer over
the surface of the medium.

On potato , growth is abundant and easily visible within eighteen
to twenty-four hours, as a grayish-white, glistening layer which later
turns to a yellowish-brown, and in old cultures often to a dirty green¬
ish-brown color.

In pepton solution indol is formed. In milk there is acidity and co¬
agulation. In lactose-litmus-agar acid is formed, the medium becom¬
ing red, and gas-bubbles appear along the line of the stab inoculation.

In carbohydrate broth , gas is formed in dextrose, lactose, and mannit,
but not in saccharose. Levulose, galactose, and maltose are also fer¬
mented with the formation of acid and gas.

Cultures of the colon bacillus are characterized by a peculiar fetid
odor which is not unlike that of diluted feces. The acids formed by the
colon bacillus from sugars are chiefly lactic, acetic, and formic acids.
The gas it produces consists chiefly of C02 and hydrogen. The bacillus
grows well on media containing urine and on those containing bile.
Upon the latter fact some methods for the isolation of the colon
bacillus from water and feces have been based.

Isolation of the colon bacillus from mixed cultures is most easily
accomplished by plating upon lactose-litmus-agar, the Conradi-Drigal-
ski medium, or the Endo medium after preliminary enrichment of the
specimen to be tested in bile or malachite-green broth. (In the case
of feces such enrichment is superfluous.)

Distribution. — The colon bacillus is a constant inhabitant of the
intestinal canal of human beings and animals. It is also found occasion¬
ally in soil, in air, in water, and in milk and is practically ubiquitous in
all neighborhoods which are thickly inhabited. When found in nature
its presence is generally taken to be an indication of contamination from
human or animal sources. Thus, when found in water or milk, much
hygienic importance is attached to it. Recently, Papasotiriu 1 and,
independently of him, Prescott,2 have reported finding bacilli apparently

1 Papasotiriu, Arch. f. Hyg., xli. 2 Prescott, Cent. f. Bakt., Ref., xxxiii, 1903,

392

PATHOGENIC MICROORGANISMS

identical with Bacillus coli upon rye, barley, and other grains. They
believe, upon the basis of this discovery, that Bacillus coli is widely
distributed in nature and that its presence, unless it appears in large
numbers, does not necessarily indicate recent fecal contamination.
These reports, however, have not found confirmation by the work of
others, and can not, therefore, be as yet accepted.

In man, Bacillus coli appears in the intestine normally soon after
birth, at about the time of taking the first nourishment.1 From this time
on, throughout life, the bacillus is a constant intestinal inhabitant ap¬
parently without dependence upon the diet. Its distribution within the
intestine, according to Cushing and Livingood,2 is not uniform, it being
found in the greatest numbers at or about the ileocecal valve, diminish¬
ing from this point upward to the duodenum and downward as far as
the rectum. Adami 3 and others claim that, under normal conditions,
the bacillus may invade the portal circulation, possibly by the inter¬
mediation of leucocytic emigration during digestion. After death, at
autopsy, Bacillus coli is often found in the tissues and the blood with¬
out there being visible lesions of the intestinal mucous membrane.4 It
is probable, also, that it may enter and live in the circulation a few
hours before death during the agonal stages.

Extensive investigations have been carried out to determine wheth¬
er or not the constant presence of this microorganism in the intestinal
tract is an indication of its possessing a definite physiological function of
advantage to its host. It has been argued that it may aid in the fermen¬
tation of carbohydrates. The question has been approached experiment¬
ally by a number of investigators. Nuttall and Thierf elder 5 delivered
guinea-pigs from the mother by Cesarean section and succeeded in
keeping them without infection of the intestinal canal for thirteen days.
Although no microorganisms of any kind were found in the feces of
these animals, no harm seemed to accrue to them, and some of them
even gained in weight. Schottelius,6 on the other hand, obtained con¬
tradictory results when working with chicks. Allowing eggs to hatch in
an especially constructed glass compartment, he succeeded in keeping the

1 Schild, Zeit. f. Hyg., xix, 1895; Lembke, Arch. f. Hyg., xxvi, 1896.

2 Cushing and Livingood, “ Contributions to Med. Sci. by Pupils of Wm. Welch,”
Johns Hopk. Press, 1900.

3 Adami, Jour, of Amer. Med. Assn., Dec., 1899.

4 Birch-Hirschfeld, Ziegler's Beitr., 24, 1898.

5 Nuttall und Thierfelder, Zeit. f. Physiol. Chemie, xxi and xxii,

Schottelius, Arch. f. Hyg., xxxiv, 1889,

BACILLUS COL I COMMUNIS

393

chicks and their entire environment sterile for seventeen days. During
this time they lost weight, did not thrive, and some of them were mori¬
bund at the end of the second week, in marked contrast to the healthy,
well-nourished controls, fed in the same way, but under ordinary
environmental conditions. Although insufficient work has been done
upon this important question, and no definite statement can be made,
it is more than likely that the function of the Bacillus coli in the intes¬
tine is not inconsiderable if only because of its possible antagonism to
certain putrefactive bacteria, a fact which has been demonstrated in
interesting studies by Bienstock 1 and others.2

Pathogenicity. — The pathogenicity of the colon bacillus for animals
is slight and varies greatly with different strains. Intraperitoneal in¬
jections of 1 c.c. or more of a broth culture will often cause death in
guinea-pigs. Large doses intravenously administered to rabbits may
frequently cause a rapid sinking of the temperature and death with
symptoms of violent intoxication within twenty-four to forty-eight hours.
Subcutaneous inoculation of moderate doses usually results in nothing
more than a localized abscess from which the animals recover. It is likely
that, even in fatal cases, death results chiefly from the action of poisons
liberated from the disintegrating bacteria and not from the multipli¬
cation of the bacilli themselves, for often no living organism can be
found unless large doses are given.

In man, a large variety of lesions produced by Bacillus coli have
been described. It is a surprising fact that disease should be caused
at all, in man, by a microorganism which is so constantly present in
large numbers in the intestine and against which, therefore, it is to be
expected that a certain amount of immunity should be developed. A
number of explanations for this state of affairs have been advanced,
none of them entirely satisfactory. It is probable that none of the poi¬
sonous products of the colon bacillus is absorbed unchanged by the
healthy unbroken mucosa and that, therefore, the microorganisms are,
strictly speaking, at all times, outside of the body proper. Under these
circumstances, no process of immunization would be anticipated. It
is also possible that, whenever an infection with Bacillus coli does occur,
the infecting organism is one which has been recently acquired from
another host, having no specific adaptation to the infected body. Viru¬
lence may possibly be enhanced by inflammatory processes caused by
other organisms. Considering the subject from another point of view,

1 Bienstock, Arch. f. Hyg., xxxix, 1901.

2 Tissier and Martelly, Ann. de linst. Pasteur, 1902.

394

PATHOGENIC MICROORGANISMS

colon-bacillus infection may possibly take place simply because of unu¬
sual temporary reduction of the resistance of the host. Whether or not
altered cultural conditions in the intestine may lead to marked enhance¬
ment in the virulence of the colon bacilli can not at present be decided.
The opinion has been frequently advanced, however, without adequate
experimental support.

Septicemia, due to the colon bacillus, has been described by a large
number of observers. It is doubtful, however, whether many of these
cases represent an actual primary invasion of the circulation by the
bacilli, or whether their entrance was not simply a secondary phenomenon
occurring during the agonal stages of another condition. A few unques¬
tionable cases, however, have been reported, and there can be no doubt
about the occurrence of the condition, although it is probably less
frequent than formerly supposed. The writers have observed it on
two occasions in cases during the lethal stages of severe systemic
disease due to other causes. An extremely interesting group of such
cases are those occurring in new-born infants, in which generalized
colon-bacillus infection may lead to a fatal condition known as
WinckeFs disease or hemorrhagic septicemia.1 Prominent among
disease processes attributed to these microorganisms are various diar¬
rheal conditions, such as cholera nostras and cholera infantum. The
relation of these maladies to the colon bacillus has been studied es¬
pecially by Escherich,2 but satisfactory evidence that these bacilli may
specifically cause such conditions has not been brought. While it is not
unlikely that under conditions of an excessive carbohydrate diet, colon
bacilli may aggravate morbid processes by a voluminous formation of
gas, they do not, of themselves, take part in actual putrefactive proc¬
esses. It is likely, therefore, that in most of the intestinal diseases
formerly attributed purely to bacilli of the colon group, these micro¬
organisms actually play but a secondary part.3

It is equally difficult to decide whether or not these bacilli may be
regarded as the primary cause of peritonitis following perforation of
the gut. Although regularly found in such conditions, they are hardly
ever found in pure culture, being accompanied usually by staphylococci,
streptococci, or other microorganisms, whose relationship to disease is
far more definitely established. Isolated cases have been reported,
however, one of them by Welch, in which Bacillus coli was present in

1 Kamen, Ziegler’s Beitr., 14, 1896.

2 Escherich , loc. cit.

3 Herter, “ Bact. Infec. of Digest. Tract,” N. Y., 1907.

BACILLUS COL I COMMUNIS

395

the peritoneum in pure culture without there having been any intestinal
perforation.1 Granting that the bacillus is able to proliferate within
the peritoneum, there is no reason for doubting its ability of giving rise
to a mild suppurative process.

Inflammatory conditions in the liver and gall-bladder have fre¬
quently been attributed to the colon bacillus. It has been isolated from
liver abscesses, from the bile, and from the center of gall-stones. Welch
has reported a case of acute hemorrhagic pancreatitis in which the
bacillus was isolated from the gall-bladder and from the pancreas.

In the bladder, Bacillus coli frequently gives rise to cystitis and oc¬
casionally to ascending pyonephrosis. No other microorganism, in fact,
is found so frequently in the urine as this one. It may be present, often,
in individuals in whom all morbid processes are absent. The condition
is frequently observed during the convalescence from typhoid fever.
It may disappear spontaneously, or cystitis, usually of a mild, chronic
variety, may supervene.

Localized suppurations due to this bacillus may take place in all
parts of the body. They are most frequently localized about the anus
and the genitals. They are usually mild and easily amenable to the
simplest surgical treatment.

Poisonous Products of the Colon Bacillus. — The colon bacillus belongs
essentially to that group of bacteria whose toxic action is supposed to
be due to the poisonous substances contained within the bacillary body.
Culture filtrates of the colon bacillus show very little toxicity when in¬
jected into animals; whereas the injection of dead bacilli produces
symptoms almost equal in severity to those induced by injection of the
live microorganisms. Corroborative of the assumption of this endo toxic
nature of the colon-bacillus poison is the fact that, so far, no antitoxic
bodies have been demonstrated in serum as resulting from immuniza¬
tion.

Immunization with the Colon Bacillus. — The injection into animals of
gradually increasing doses of living or dead colon bacilli gives rise to
specific bacteriolytic, agglutinating, and precipitating substances.

The bacteriolytic substances may be easily demonstrated by the
technique of the Pfeiffer reaction. In vitro bacteriolysis is less marked
than in the case of some other microorganisms such as the cholera spiril¬
lum or the typhoid bacillus. Owing probably to the habitual presence
of colon bacilli in the intestinal tracts of animals and man, considerable

1 Welch, Med. News, 59, 1891.

396

PATHOGENIC MICROORGANISMS

bacteriolysis may occasionally be demonstrated in the serum of normal
individuals.

Agglutinins for the colon bacillus have often been produced in the
sera of immunized animals in concentration sufficient to be active in dilu¬
tions of 1 : 5,000 and over. The agglutinins are produced equally well
by the injection of live cultures and of those killed by heat, if the tem¬
perature used for sterilization does not exceed 100° C. It is1 a notice-

1 2 3

Fig. 84. — Bacillus coli communis. Grown in: 1. Dextrose, 2. Lactose, 3.
Saccharose broth. The bacillus forms acid and gas from dextrose and lactose,
not from saccharose. Note the absence of growth in the closed arm of the sac¬
charose tube, in which no acid or gas is formed.

able fact that the injection of any specific race of colon bacilli
produces, in the immunized animal, high agglutination values only for
the individual culture used for immunization, while other strains of
colon bacilli, although agglutinated by the serum in higher dilution
than are paratyphoid or typhoid bacilli, require much higher concen¬
tration than does the original strain. The subject has been extensively
studied by a number of observers and illustrates the extreme individual

1 Wolff, Cent. f. Bakt., xxv, 1899.

BACILLUS COLI COMMUNIS

397

1 Kraus und Low, Wien. klin. Woch., 1899.

12 3

Fig. 85. — Bacillus coli communior. Grown in: 1. Dextrose, 2. Lactose, 3.

Saccharose broth.

in the serum of patients convalescing from typhoid fever or dysentery
is probably to be explained, partly by the increase of the group
agglutinins produced by the specific infecting agent, and partly by the
invasion of colon bacilli, or the absorption of its products induced by
the diseased state of the intestinal mucous membrane.

Varieties of the Colon Bacillus. — During the earlier days of bacteriolog¬
ical investigations, a large number of distinct varieties of colon bacilli
were described, many of which may now be dismissed as based simply

specificity of the agglutination reaction. Thu^ a serum which will
agglutinate its homologous strains in dilutions of one 1: 1,000 will often
fail to agglutinate other races of Bacillus coli in dilutions of 1 : 500
or 1 : 600.

The normal serum of adult animals and man will often agglutinate
this bacillus in dilutions as high as 1 : 10 or 1 : 20 — a phenomenon pos¬
sibly referable to its habitual presence within the body. Corrobo¬
rating this assumption is the observation of Kraus and Low/
that the serum of new-born animals possesses no such agglutinating
powers. The fact that agglutinins for the colon bacillus are increased

398

PATHOGENIC MICROORGANISMS

upon a temporary depression of one or another cultural characteristic of
Bacillus coli communis, while others can be definitely included within
other closely related, but distinct groups.

That secondary features, such as dimensions, motility, and luxuri¬
ance of growth upon various media, may be markedly altered by arti¬
ficial cultivation is a common observation. It has not, however, been
satisfactorily shown that cardinal characteristics, such as the forma¬
tion of indol from pepton, or the power to produce gas from dextrose
and lactose, can be permanently suppressed without actual injury or
inhibition of the normal vitality of the microorganism. Such alter¬
ation is, in fact, contrary to experience, which demonstrates that
whenever such changes do occur, they are purely temporary and a few
generations of cultivation under favorable environmental conditions
will regularly restore the organism to its normal activity.

Bacillus coli communior. — Distinct and constant varieties of Bacil¬
lus coli, however, do occur. The most common of these is one which
Dunham has named Bacillus coli communior , because of the fact that
he believes it to be more abundant in the human and animal intestine
than is coli communis itself. This bacillus possesses all the cardinal
characteristics of the colon group. It is a Gram-negative bacillus,
moderately motile, non-sporulating, and morphologically indistinguish¬
able from the communis variety. It does not liquefy gelatin, it
produces indol from pepton, coagulates and acidifies milk, and grows
characteristically upon agar and potato. It differs from Bacillus coli
communis in that it produces acid and gas from saccharose as well as
from dextrose and lactose, whereas the former does not form acid or
gas from saccharose.

CHAPTER XXVII

BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP

( Continued )

THE BACILLUS OF TYPHOID FEVER

( Bacillus typhosus, Bacillus typhi abdominalis )

Typhoid fever, because of its wide distribution and almost con¬
stant presence in most communities, has from the earliest days been the
subject of much etiological inquiry. A definite conception as to its
infectiousness and transmission from case to case was formed as early
as 1856 by Budd.1

But it was not until 1880 that Eberth 2 discovered in the spleen and
mesenteric glands of typhoid-fever patients who had come to autopsy,
a bacillus which we now know to be the cause of the disease. Final
proof of such an etiological connection was then brought by Gaffky,3
who not only saw the bacteria referred to by Eberth, but succeeded in
obtaining them in pure culture and studying their growth characteristics.

Morphology and Staining. — The typhoid bacillus is a short rod from
1-3.5 u in length with a varying width of from .5 to .8 y. In appear¬
ance it has nothing absolutely distinctive which could serve to differen¬
tiate it from other bacilli of the typhoid-colon group, except that it has
a general tendency to greater slenderness. Its ends are rounded without
ever being club-shaped. Contrary to the descriptions of the earliest
observers, typhoid bacilli do not form spores. They are actively motile
and have twelve or more flagella peripherally arranged.

' The bacilli stain readily with the usual anilin dyes. Stained by
Gram’s method, they are decolorized.

Cultivation. — Bacillus typhosus is easily cultivated upon the usual
laboratory media. It is not delicately susceptible to reaction, but will
grow well upon media moderately alkaline or acid. It is an aerobic and
facultative anaerobic organism, when the proper nutriment is present.
Upon agar plates growth appears within eighteen to twenty-four hours

1 Budd, “ Intestinal Fever,” Lancet, 1856.

2 Eberth, Virch. Archiv, 81, 1880, and 83, 1881.

3 Gaffky, Mitt. a. d. kais. Gesundheitsamt, 2, 1884.

399

400

PATHOGENIC MICROORGANISMS

as small grayish colonies at first transparent; later opaque. Upon agar
slants growth takes place in a uniform layer. There is nothing charac¬
teristic about this growth to aid in differentiation.

In broth , the typhoid bacillus grows rapidly, giving rise to an even
clouding, rarely to a pellicle.

Upon gelatin, the typhoid bacillus grows readily and does not
liquefy the medium. In stabs, growth takes place along the entire extent
of the stab and over the surface of the gelatin in a thin layer. In gela¬
tin plates the growth may show some distinction from that of other mem¬
bers of this group, and this medium was formerly much used for isolation

FrG. 86.— Bacillus typhosus, from twenty-four-hour culture on agar.

of the bacillus from mixed cultures. Growth appears within twenty-
four hours as small, transparent, oval, round, or occasionally leaf-shaped
colonies which are smaller, more delicate, and more transparent than
contemporary colonies of the colon bacillus. They do not, however,
show any reliable differential features from bacilli of the dysentery
group. As the colonies grow older they grow heavier, more opaque, and
lose much of their early differential value.

On potato the growth of typhoid bacilli is distinctive, and this medium
was recommended by Gaffky 1 in his early researches for purposes of

1 Gaffky, loc. cit.

BACILLUS OF TYPHOID FEVER

401

identification. On it typhoid bacilli, after twenty-four to forty-eight
hours, produce a hardly visible growth, evident to the naked eye
only by a slight moist glistening, an appearance which is in marked
contrast to the grayish-yellow or even brown and abundant growth
of the colon bacilli. If the potato medium is rendered neutral or
alkaline, this distinction disappears, the typhoid bacillus growing
more abundantly.

In milk, typhoid bacilli do not produce coagulation. In litmus-milk,
during the first twenty-four hours, the color is changed to a reddish or
violet tinge by the formation of acid from the small quantities of mono-

Fig. 87. — Bacillus typhosus, showing flagella. (After Frankel and Pfeiffer.)

saccharid present. Later the color becomes deep blue from the forma¬
tion of alkali.

In Dunham’s pepton solution no indol is produced. According to
Peckham, however, continuous cultivation in rich pepton media may
lead to eventual indol formation by typhoid bacilli. This fact appears
to have no bearing on the value of the indol test, as indol is never formed
under the usual cultural conditions.

In dextrose , mannite, lactose , and saccharose broth , the typhoid bacil¬
lus produces no gas. A comparative summary of the action of other
bacilli of this group in these sugar media will be given in the final dif¬
ferential table on page 443.

402

PATHOGENIC MICROORGANISMS

Tested for its power to form acid from sugars commonly used in
differential tests, typhoid bacilli give the following reactions:

Acid formation
Dextrose +

Levulose +

Galactose +

Mannit +

Acid formation
Maltose +
Lactose —
Saccharose —
Dextrin +

In the Hiss tube medium (see section on Media, page 133), the
typhoid bacillus within eighteen to twenty -four hours produces an even
clouding by virtue of its motility, but does not form gas. In contradis¬
tinction to this, dysentery bacilli grow only along the line of inocula-

Fig. 88. — Surface Colony of Bacillus typhosus on Gelatin. (After Heim.)

tion, while bacilli of the colon group move in irregular sky-rocket-like
figures away from the stab, at the same time breaking up the medium
by the formation of gas-bubbles. Some actively motile colon bacilli
cloud the medium, but the ruptures caused by the gas are always
evident.

The differentiation of the typhoid bacillus in pure culture from similar
microorganisms by means of its growth upon media has been the sub¬
ject of many investigations. It is neither practicable nor desirable to
enumerate all the various media which have been devised and reported.

BACILLUS OF TYPHOID FEVER

403

The aim has been chiefly the differentiation of typhoid bacilli from the
bacilli of the colon group, and most of the media have been devised with
this end in view. (See section on Media.)

Rothberger 1 has devised a mixture of glucose agar to which is added
one per cent of a saturated aqueous solution of neutral-red. Shake-cul¬
tures or stab-cultures are made in tubes of this medium. The typhoid
bacillus causes no changes in it, while members of the colon group, by
reduction of the neutral-red, decolorize the medium and produce gas by
fermentation of the sugar.

Utilizing the fact that bile-salts are precipitated in the presence of
acids, Macconkey devised a medium composed of sodium glycocholate,
pepton, lactose, and agar (the composition of this medium is given on
page 138), in which Bacillus typhosus grows without causing much
change, but distinct clouding results from the growth of the colon bacillus
which, producing acid from the lactose, causes precipitation of the bile-
salts.

On Wurtz’s lactose-litmus-agar (see page 129) the typhoid bacillus
produces no acid, but eventually deepens the purple color to blue;
the colon bacillus produces acid and in stab-cultures gas bubbles and
the color changes to red.

In Barsiekow’s (see page 139) lactose-nutrose-litmus mixture the
typhoid bacillus causes no change, while the colon bacillus produces
coagulation and an acid reaction.

Especially designed for the isolation of typhoid bacilli from the
feces, are the media of Drigalski and Conradi, the agar-gelatin media
of Hiss, the medium of Hesse, the fuchsin medium of Endo, and the
malachite-green media of Loeffler, and others. These media have all
been described in detail in the section on the preparation of media, pages
133-138.

Biological Considerations. — The typhoid bacillus is an aerobic and
facultatively anaerobic organism growing well both in the presence and in
the absence of oxygen when certain sugars are present, showing a slight
preference, however, for well aerated conditions. It grows most luxu¬
riantly at temperatures about 37.5° C., but continues to grow within a
range of temperature lying between 15° and 41° C. Its thermal death
point, according to Sternberg, is 56° C. in ten minutes. It remains alive
in artificial cultures for several months or even years if moisture is sup¬
plied. In carefully sealed agar tubes Hiss has found the organisms

1 Rothberger , Cent. f. Bakt., xxiv, 1898.

404

PATHOGENIC MICROORGANISMS

alive after thirteen years. In natural waters it may remain alive as
long as thirty-six days, according to Klein.1 In ice, according to Prud-
den,2 it may remain alive for three months or over. Against the ordi¬
nary disinfectants, the typhoid bacillus is comparatively more resistant
than some other vegetative forms. It is killed, however, by 1 : 500
bichlorid or five-per-cent carbolic acid within five minutes.

Pathogenicity. — In animals, some early investigators to the contrary,
typhoidal infection does not occur spontaneously and artificial inocula¬
tion with the typhoid bacillus does not produce a disease analogous to
typhoid fever in the human being. Frankel 3 was able to produce intes¬
tinal lesions in guinea-pigs by injection of the bacilli into the duodenum,
and recovered the bacteria from the spleens of the animals after death,
but the disease produced was in no other respect analogous to typhoid
fever in the human being. It is probable that typhoid bacilli injected
into animals do not multiply extensively and that most of the symp¬
toms produced are due to the endotoxins liberated from the dead bac¬
teria. In corroboration of this view is the observation that inoculation
with dead cultures is followed by essentially the same train of symp¬
toms as inoculation with live cultures.4 The injection of large doses into
rabbits or guinea-pigs intravenously or intraperitoneally is usually
followed by a rapid drop in temperature, often by respiratory em¬
barrassment and diarrhea. Occasionally blood may be present in
the stools. According to the size of the dose or the weight of the ani¬
mal, death may ensue within a few hours, or, with progressive emacia¬
tion, after a number of days, or the animal may gradually recover.
Welch and Blachstein 5 have shown that typhoid bacilli injected into
the ear vein of a rabbit appear in the bile and may persist in the gall¬
bladder for weeks. Typhoid bacilli isolated from different sources may
show considerable variations in virulence and toxicity.

Doerr,6 Koch,7 Morgan,8 and more recently Johnston9 have all
confirmed this, the last named showing that the typhoid bacillus could

1 Klein , Med. Officers’ Report, Local Govern. Bd., London, 1894.

2 Prudden, Med. Rec., 1887.

3 Frankel, Cent. f. klin./Med., 10, 1886.

4 Petruschky, Zeit. f. Hyg., xii, 1892.

5 Welch and Blachstein , Bull. Johns Hop. Hosp., ii, 1891.

6 Doerr, Centralbl. f. 'jBakt., 1905.

7 Koch, Zeitschr. f. H}Kg., 1909.

8 Morgan, Jour, of Hyg.y 1911.

9 Johnston, Jour, of Med. Res., xxvii, 1912.

BACILLUS OF TYPHOID FEVER

405

not only remain latent for a long time in the gall-bladder of rabbits,
but would appear in the blood stream with considerable regularity
after the seventh or ninth day, and persist for as long as 125 days.
Gay and Claypole 1 have been able to produce the carrier state in
rabbits with great regularity by growing the typhoid cultures used for
inoculation upon agar containing 10 per cent defibrinated rabbit’s
blood. Such cultures are not as readily agglutinated by immune serum
as are those grown on plain agar, and it may well be that they have
acquired a certain degree of resistance to the serum antibodies which
renders them more competent to survive in the body of the rabbit.
Gay has used rabbits inoculated with such cultures for the determination
of the efficacy of his sensitized vaccines.

In man the overwhelming majority of typhoid infections take the
form of the disease clinically known as typhoid fever. For a descrip¬
tion of the clinical course and pathological lesions of the disease, the
reader is referred to the standard text-books of medicine and pathology.
During the course of the disease, and during convalescence, the bacilli
may be cultivated from the circulating blood, the rose spots, the feces,
the urine, and in exceptional cases from the sputum. At autopsy the
bacilli may be obtained from these sources as well as from the lesions in
the intestine, the spleen, and often from the liver, kidneys, and from the
gall-bladder.

Though formerly regarded as primarily an intestinal disease, recent
investigations have brought convincing proof that the disease is in its
inception actually a bacteriemia. It is not unlikely that the intestinal
lesions are largely the result of toxic products which are excreted
through the intestinal wall.

Typhoid Bacilli in the Blood duriny the Disease . — The investigations
of many workers have shown that typhoid bacilli are present in the
circulating blood of practically all patients during the early weeks of the
disease. Series of cases have been studied by Castellani,2 Schottmul-
ler,3 and many others. More recently Coleman and Buxton4 have
reported their researches upon 123 cases, and have at the same time
analyzed all cases previously reported. Their analysis of blood cultures
taken at different stages in the disease is as follows:

1 Gay and Claypole, Arch, of Inf. Med., Dec., 1913.

2 Castellani, Riforma medica, 1900.

3 Schottmueller, Deut. med. Woch., xxxii, 1900, and Zeit. f. Hyg., xxxvi, 1901.

4 Coleman and Buxton, Am. Jour, of Med. Sci., 133, 1907.

406

PATHOGENIC MICROORGANISMS

Of 224 cases during first week, 89 per cent were positive.

Of 484 cases during second week, 73 per cent were positive.

Of 268 cases during third week, 60 per cent were positive.

Of 103 cases during fourth week, 38 per cent were positive.

Of 58 cases after fourth week, 26 per cent were positive.

The technique recommended by Coleman and Buxton for obtaining
blood cultures is that recommended by Conradi,1 slightly modified. The
blood is taken into flasks each containing about 20 c.c. of the following
mixture :

Ox-bile . 900 c.c.

Glycerin . 100 c.c.

Pepton . 20 grams.

About 3 c.c. of blood are put into each flask. The ox-bile, besides
preventing coagulation, may possibly neutralize the bactericidal sub¬
stances present in the drawn blood. The flasks are incubated for eigh¬
teen to twenty-four hours, at the end of which time streaks are made
upon plates of lactose-litmus-agar and the organisms identified by
agglutination or by cultural tests.

European workers have generally preferred to make high dilution of
the blood in flasks of bouillon, small quantities of blood, 1 to 2 c.c., being
mixed with 100 to 150 c.c. of nutrient broth.

Epstein 2 has reported excellent results from mixing the blood in
considerable concentration with two-per-cent glucose agar and pouring
plates.

The writers in hospital work have had equally good results with the
bile medium and with broth in flasks, rather less uniform but still satis¬
factory results with the plating method. In general it may be said that
any one of these methods carried out with reasonable accuracy may be
satisfactorily employed.

Typhoid Bacilli in the Stools. — The examination of the stools for
typhoid bacillus is performed for diagnostic purposes chiefly in obscure
cases. It may, furthermore, furnish information of extreme hygi¬
enic importance. Thus Drigalski 3 and Conradi have succeeded in
isolating typhoid bacilli from the stools o' ambulant cases so mild that
they were not clinically suspected. It la oy means of such examina¬
tions that the so-called typhoid-carriers are detected, cases which,

1 Conradi, Deut. med. Woch., xxxii, 1906.

2 Epstein, Proc. N. Y. Path. Soc., N. S., vi, 1906.

3 Drigalski and Conradi, Zeit. f. Hyg., xxxix, 1902.

BACILLUS OF TYPHOID FEVER

407

though perfectly well themselves, may be a means of spreading the
disease. Such cases have been known to harbor the bacilli for periods
as long as several years.

The examination itself is fraught with great difficulties, owing to
the preponderating numbers of colon bacilli found in all feces and the
difficulty of isolating the typhoid bacilli from such mixtures.

Reviewing the data collected by a number of investigators, it
seems probable that the bacilli do not appear in the stools, at least
in numbers sufficient for recognition, much before the middle of the
second week, or, in other words, as pointed out by Hiss, about the
time that the intestinal lesions are well ad¬
vanced and ulceration is occurring. Thus
Wiltschour 1 could not determine their pres¬
ence before the tenth day; Redtenbacher,2
in reviewing the statistics, states that in a
majority of cases the bacilli first appear
toward the end of the second week, and
Horton-Smith 3 could not find the bacilli be¬
fore the eleventh day. Hiss,4 in an investi¬
gation of the same subject, obtained the
following results:

First to tenth day, inclusive, twenty-
eight cases examined; typhoid bacilli isolated
from three; percentage of positive cases
10.7 per cent.

Eleventh to twentieth day, inclusive, forty-four cases examined; ty¬
phoid bacilli from twenty-two; percentage of positive cases 50 per cent.

Twenty-first day to convalescence, sixteen cases examined; typhoid
bacilli isolated from thirteen; percentage of positive cases 81.2 per
cent.

The difficulties encountered in such examinations have led to the
development of a large number of methods. The first method which
yielded successful results was that of Eisner,5 who employed a potato-
extract gelatin containing one per cent of potassium iodid, a medium
which prevented the growth of many intestinal bacteria, allowing only

1 Wiltschour, Cent. f. Bakt., 1890.

2 Redtenbacher, Zeit. f. klin. Med., xix, 1891.

3 Horton-Smith, Lancet, May, 1899.

4 Hiss, Med. News, May, 1901.

6 Eisner, Zeit. f. Hyg., xxi, 1895.

Fig. 89. — Bacillus coli.
Deep colonies on Hiss plate
medium.

408

PATHOGENIC MICROORGANISMS

colon, typhoid, and a few others to develop. This medium is at present
rarely used.

Hiss 1 has employed with success an agar-gelatin mixture containing
one per cent of glucose, the preparation of which has been described in
detail in the section on media. The actual technique of the test is as
follows: One to two loopfuls of feces are transferred to a tube of broth,
making the broth fairly cloudy. From this emulsion five or six plates
are made by transferring in series one to five loopfuls of the emulsion
to tubes containing the melted plate medium, and then pouring the con¬
tents of these tubes into Petri dishes. These dishes, after the medium

Fig. 90. — Bacillus typhosus. Deep colonies in Hiss plate medium.

has hardened, are placed in an incubator at 37° C., and allowed to re¬
main for eighteen to twenty-four hours, when they are ready for examina¬
tion. If typhoid bacilli are present they will be found as small, usually
glistening colonies with a fringe of threads growing out like flagella from
their peripheries (see Figs. 90 and 91). These colonies are smaller and
quite distinct from those of colon bacilli, which are heavier and darker
and do not display the fringing threads. Suspicious colonies may be
fished and transferred to the Hiss tube medium (see page 133) or iden¬
tified by other reliable methods.

A method which has been found useful, especially in Europe,
is that in which smears of diluted feces are made upon large plates
of the Conradi-Drigalski medium. (For preparation see page 135.)
The principles underlying the use of this medium are the formation
of acid from the lactose by the colon bacilli and the inhibition of cocci
and many other bacteria by the crystal-violet. In practice, an emul-

1 Hiss, Jour, of Exp, Med., ii, 1897; Med. News, May, 1901; and Jour. Med. Res.,
N. S., iii, 1902.

BACILLUS OF TYPHOID FEVER

409

sion is made of a loopful of feces in a tube of broth. Into this
is dipped a bent glass smearing rod, the excess of fluid is allowed to
drip off, and smears are made upon plates of the medium, several
plates being smeared without redipping the rod. Colonies of the
colon bacillus on these plates will appear opaque, comparatively large,
and will produce an acid reaction with consequent reddening of the
medium. Typhoid colonies will be smaller, transparent, and without
acid formation. These colonies are fished and the microorganisms may

Fig. 91. — Bacillus typhosus.. Colony in Hiss plate medium, highly magnified.

be identified by agglutination or by stab cultures in the Hiss tube
medium.

The malachite-green media of Loeffler and others have found
less general use than was originally expected, because of the
difficulty in obtaining uniform preparations of malachite-green.
Peabody and Pratt 1 have applied the principle of colon-bacillus in¬
hibition by malachite-green, by adding this dye to broth in the
manner described in the section on media (page 137), planting the
feces directly into this broth, and, after incubation for several hours,
making smears from these tubes upon plates of the Conradi-Drigalski
medium.

Marked success has been reported in the isolation of typhoid bacilli

1 Peabody and Pratt , Boston Med. and Surg. Jour., 1908.

410

PATHOGENIC MICROORGANISMS

from the feces by the use of the Endo fuchsin-agar. Emulsions of feces
are made in tubes of ordinary broth in the manner described in the Con-
radi-Drigalski method, and smears of this emulsion are made upon
plates of the fuchsin-agar by means of a glass smearing rod. The
colonies of Bacillus coli, after eighteen or more hours of incubation, will
be found to have brought back a deep red color to the medium, whereas
the typhoid colonies are smaller, more transparent, and have left the
medium uncolored.

In all cases where plates are prepared from broth emulsions of feces,
it is desirable to allow the emulsion to stand at incubator temperature
for several hours, or, better, to centrifugalize the emulsion and then allow
it to stand without agitation. Subsequent removal of fluid from the

Fig. 92. — Colon and Typhoid Colonies in Hiss Plate Medium. (Planted
from stool. Note the small thread-forming typhoid colonies.)

upper layers of the medium is likely to bring away a comparatively
larger number of the motile organisms.

The methods of isolating typhoid bacilli given above do not ex¬
haust the records of work done upon this problem. Other methods have
been devised, but those given are the ones most generally in use. It is
not satisfactory to compare any two of these methods as to practical
value, since all of them require a considerable amount of working famil¬
iarity with organisms and media. In fact, it may be said that all of the
methods given are satisfactory if consistently employed by a worker
who has become thoroughly accustomed to the peculiarities and
variations of the typhoid colonies upon the medium with which he
is working.

BACILLUS OF TYPHOID FEVER

411

Typhoid Bacilli in the Urine. — Careful investigation by a number of
workers has revealed typhoid bacilli in the urine in about twenty-five
per cent of all patients. Neumann1 discovered the bacilli in eleven out
of forty-six cases and Karlinski 2 in twenty-one out of forty-four cases.
Investigations by Petruschy,3 Richardson,4 Horton-Smith,5 Hiss,6 and
others have confirmed these results. In general the bacilli have not
been found before the fifteenth day of the disease, and examination of
the urine, therefore, can be of little early diagnostic value. A series of
seventy-five cases examined by Hiss before the fourteenth day of the
disease did not once reveal typhoid bacilli in the urine. On the other
hand, they have been found to be present for weeks, months, and, in
isolated cases, for years after convalescence, the examination thus hav¬
ing much hygienic importance. They are probably present in about
twelve per cent of cases during the early days of convalescence. In
most of these cases where typhoid bacilli are found, albumin is present
in the urine in considerable quantities. The bacilli usually appear and
disappear with the albuminuria.

It is not infrequent that an obstinate cystitis caused by typhoid ba¬
cilli may follow in the path of typhoid fever. Such cases have been re¬
ported by Blumer,7 Richardson,8 and others. Suppurative processes in
the kidneys are less frequent. It is noteworthy, also, that in the course
of, and following, typhoid fever there often occurs voiding of Bacillus
coli with the urine. This may obstinately persist for considerabe periods
after convalescence. The reasons for this are not entirely clear.

Typhoid Bacilli in the Gall-Bladder. — Typhoid bacilli have been
frequently observed in the gall-bladder at autopsy. They have also
been found present in this organ, at operations for cholecystitis, months
and years after the occurrence of typhoid fever. Miller 9 has reported a
case in which typhoid bacilli were present in the gall-bladder seven
years after the disease; v. Dungern 10 has cultivated them from an in¬
flamed gall-bladder fifteen years after the disease. Zinsser has had

1 Neumann, Berl. klin. Woch., xxvii, 1890.

2 Karlinsky, Prag. med. Woch., xv, 1890.

3 Petruschy, Cent. f. Hyg., xxiii, 1898.

* Richardson, Jour. Exp. Med., 3, 1898.

5 Horton-Smith, Lancet, May, 1899.

6 Hiss, Med. News, May, 1901.

7 Blumer, Johns Hopkins Hosp. Reports, 5, 1895.

8 Richardson, loc. cit.

9 Miller, Johns Hopkins Hosp. Bull., 1898.

10 v. Dungern, Munch, med. Woch., 1897.

412

PATHOGENIC MICROORGANISMS

occasion1 to observe a case in which an operation for gall-stotie seven¬
teen years after the occurrence of typhoid fever revealed the pres¬
ence of the bacilli in the gall-bladder. In such cases typhoid bacilli
may be constantly discharged from the intestine with the feces and prove
a menace to the health of the community. An extremely interesting-
example of such a typhoid carrier has been carefully studied and re¬
ported by Park.2

Typhoid Bacilli in the Rose Spots. — A number of observers have
succeeded in isolating typhoid bacilli from the rose spots. Neufeld,3 who
made an extensive investigation of this question, obtained positive
results in thirteen out of fourteen cases. According to his researches
and those of Frankel,4 the bacilli are localized not in the blood, which
is taken when the rose spots are incised, but are crowded in large
numbers within the lymph spaces.

Typhoid Bacilli in the Sputum. — In rare cases typhoid bacilli have
been found in the sputum of cases complicated by bronchitis, broncho¬
pneumonia, and pleurisy. Such cases have been reported by Chantemesse
and Widal,5 Frankel,6 and a number of others. Empyema, when it
occurs in connection with such cases, is usually accompanied by a mixed
infection. From a hygienic point of view the spread of typhoid fever
by means of the sputum must be considered, but is probably of rare
occurrence.

Suppurative Lesions Due to Typhoid Bacillus. — In the course of typhoid
convalescence or during the latter weeks of the disease, suppurative
lesions may occur in various parts of the body. The most frequent locali¬
zation of these is in the periosteum, especially of the long bones, and in
the joints. A large number of such lesions have been described by Welch,
Richardson,7 and others. They usually take the form of periosteal ab¬
scesses, often located upon the tibia, occurring either late in the disease
or even months after convalescence, and are characterized by very
severe pain. Osteomyelitis may also occur, but is comparatively rare.
Subcutaneous abscesses and deep abscesses in the muscles, due to this
bacillus, have been described by Pratt.8 Synovitis may also occur.

1 Zinsser, Proc. N. Y. Pathol. Soc., 1908.

2 Park, “ Pathogenic Bacteria,” N. Y., 1908.

8 Neufeld, Zeit. f. Hyg., xxx, 1899.

4 Frankel, Zeit. f. Hyg., xxxiv, 1900.

5 Chantemesse and Widal, Arch, de physiol, norm, et path., 1887.

6 Frankel, Deut. med. Woch., xv and xvi, 1899.

7 Richardson, Jour. Boston Soc. Med. Sci., 5, 1900.

8 Pratt, Jour. Boston Soc. Med. Sci., 3, 1899.

BACILLUS OF TYPHOID FEVER

413

Meningitis, due to the typhoid bacillus, occurs not infrequently,
usually during convalescence from typhoid fever. A case of primary
typhoid meningitis has been reported by Farnet.1

Peritoneal abscesses, due to the typhoid bacillus, have been re¬
ported. Zinsser 2 has reported a case in which typhoid bacilli were
found free in the peritoneal cavity during typhoid fever without per¬
foration of the gut.

Isolated instances of typhoid bacilli in abscesses of the thyroid and
parotid glands and in brain abscesses have been observed.

Typhoid Fever without Intestinal Lesions.— A considerable number
of cases have been reported in which typhoid bacilli have been isolated
from the organs after death or from the secretions during life of pa¬
tients in whom the characteristic lesions of typhoid fever have been lack¬
ing. Most of these cases must be regarded as true typhoid septicemias.
In some cases the bacilli were isolated from the spleen, liver, or kidneys;
in others, from the urine or the gall-bladder. In a case observed by
Zinsser the bacilli were isolated from an infarct of the kidney removed
by operation. In this case the clinical course of the disease had pointed
only toward the existence of an indefinite fever accompanied by symp¬
tom’s referable to the kidneys. The Widal test, however, was positive.
An excellent summary of such cases, together with several personally
observed, has been given by Flexner.3

Hygienic Considerations. — Although typhoid fever is frequently
spoken of as an epidemic disease, it is, more truly, endemic in character
in almost all parts of the world, but subject to occasional epidemic ex¬
acerbations. In the larger communities of the temperate zones these
epidemic increases take place chiefly in the autumn and, unlike epidemics
of diseases such as influenza, are usually distinctly circumscribed —
limited usually by the distribution of a particular water-supply.

Since the disease never occurs except by transmission, directly or
indirectly, from a previous case, it is amenable more than most other
maladies to sanitary regulation, and it may be said without exaggera¬
tion, in the light of our present knowledge, that any extensive prevalence
of typhoid fever in a large community is a direct consequence of some
defect in the system of sanitation. The disease is acquired by ingestion
of the specific bacteria. Infection by any other channel than that of the
alimentary tract has not, so far, been satisfactorily demonstrated.

1 Farnet, Bull, de la soc. med. des hop. de P., 3, 1891.

2 Zinsser, Proc. N. Y. Path. Soc., 1907.

^ Flexner , Johns Hopkins, Rep., 5, 1896.

414

PATHOGENIC MICROORGANISMS

Prophylactic measures in typhoid fever, therefore, should begin
with the isolation of the patient and the disinfection of excreta, dis¬
charges, linen, and all utensils which have been in contact with the
patient. The bacilli leave the body chiefly in the feces and the urine
and the dangers of contamination, by these substances, of all objects in
immediate contact with the patient are considerable. Excreta should
therefore be either mixed with boiling water or chemically disinfected,
preferably by means of thoroughly mixing with carbolic acid, lysol, or a
solution of freshly slaked lime, and, if possible, destroyed by burning.
Linen, tableware, and eating utensils should be soaked in similar
solutions and boiled. The observance of such measures, furthermore,
should not be discontinued until bacteriological examination has
demonstrated the absence of the bacilli from feces and urine. Disre¬
gard of this last precaution may well be one of the main causes of the
endemic persistence of the disease in large cities — especially considered
in the light of our recent knowledge of “typhoid carriers” in whom
chronic infection of the gall-bladder leads to the discharge of the bacilli
in the feces for months and even years after the cessation of symptoms.

It can hardly be doubted, at the present day, that typhoid fever, in
the large majority of cases, is transmitted by the agency of water. In
an analysis of six hundred and fifty typhoid epidemics Schtider 1 found
four hundred and sixty-two reported, upon reasonable evidence, as orig¬
inating from water. The technical difficulties attending the isolation
of typhoid bacilli from contaminated water have prevented actual
bacteriological proof in most epidemics; nevertheless, indirect evi¬
dence of pollution of the suspected water-supply, correspondence of the
distribution of this supply with that of the disease, and reduction of
typhoid morbidity upon the substitution of an uncontaminated supply
are sufficiently convincing to remove reasonable doubt. Added to this is
our knowledge, from the experiments of Jordan, Russell, and Zeit 2 and
others, that typhoid bacilli may remain alive in natural waters for as
long as five days. That the bacilli may survive freezing for as long as
three months has been demonstrated by Prudden, and dangers of in¬
fection from this source are therefore considerable.

Next to water, the most important source of typhoid fever is found
in contaminated milk. In the statistical summary by Schiider,3 quoted
above, one hundred and ten of the four hundred and sixty epidemics

1 Schiider, Zeit. f. Hyg., xxxviii, 1901.

2 Jordan, Russell, and Zeit, Jour, of Inf. Dis., 1, 1904.

3 Schiider, loc. cit,

BACILLUS OF TYPHOID FEVER

415

recorded, were attributable to milk. Actual discovery of Bacillus ty¬
phosus in milk by Vaughan, Conradi, and others has been discussed in
another section (see page 685). The fact that this bacillus causes no
visible modifications in milk makes this source especially insidious.
When contamination of milk has occurred, it has often been traceable
to the water used in washing the cans or to attendants employed at
the dairies, who had been in contact with typhoid cases, or who are
convalescing from, or actually suffering from, the infection them¬
selves.

Excluding water and milk, all remaining causes of typhoid dissemi¬
nation constitute about twelve per cent and are found chiefly in the
use of vegetables contaminated from infected soil, and other food prod¬
ucts. Recently Conn has called attention to the fact that oysters
grown in waters close to sewage discharges may be the means of typhoid
transmission. An epidemic occurring at Wesleyan University was at¬
tributed by him to this cause. Experiments by Foote 1 2 have actually
demonstrated that typhoid bacilli may be found alive within oysters
for three weeks or more after they have disappeared from the sur¬
rounding water.

Indirect contamination of food and water by the intermediation of
flies and other insects has been emphasized by Veeder 3 as one of the
methods of typhoid transmission. This observer called attention to
the fact that in camps during the Spanish-American War flies in large
numbers traveled to and fro between the sinks and the cook-tents, and
it is not unlikely that at least some of the typhoid fever occurring at
that time may have been caused in this way.

Poisons of the Typhoid Bacillus. — The investigation of the toxic
products of the typhoid bacillus has occupied the attention of a large
number of workers. The first to do experimental work upon the sub¬
ject was Brieger 4 soon after the discovery and cultivation of the micro¬
organism. That toxic substances can be obtained from typhoid cultures
is beyond question. There is, however, a definite difference of opinion
as to whether these poisons are so-called endotoxins only, or whether
they are in part composed of soluble toxins comparable to those of
diphtheria and tetanus, following the injection of which antitoxic sub¬
stances may be formed.

The evidence so far seems to bear out the original contention of

1 Conn, Med. Record, Dec., 1894. 2 Foote, Med. News, 1895.

a Veeder, Med. Record, 45, 1898. 4 Brieger, Deut. med. Woch., xxvii, 1902.

416

PATHOGENIC MICROORGANISMS

Pfeiffer/ who first advanced the opinion that the poisonous substances
are products of the bacterial body set free by destruction of the bacteria
by the lytic substances of the invaded animal or human being. These
poisons, when injected into animals for purposes of immunization, in
Pfeiffer’s experiments, did not incite the production of neutralizing or
antitoxic bodies, but of bactericidal and lytic substances. That these
endotoxins constitute by far the greater part of the toxic products of
the typhoid bacillus can be easily demonstrated in the laboratory, by
the simple experiment of filtering a young typhoid culture (eight or
nine days old) and injecting into separate animals the residue of bacilli
and the clear filtrate respectively. In such an experiment there will be
little question as to the overwhelmingly greater toxicity of the bacillary
bodies as compared with that of the culture filtrate. On the other
hand, if such cultures, especially in alkaline media, are allowed to
stand for several months and the bacilli thus thoroughly extracted by
the broth, the toxicity of the filtrate is found to be greatly increased.

Nevertheless, more recent experiments by Besredka,1 2 Macfadyen,3
Kraus and Stenitzer,4 and others have tended to show that, together
with such endotoxic substances, typhoid bacilli may produce a true
toxin which is not only obtainable by proper methods from compara¬
tively young typhoid cultures, but which fulfils the necessary require¬
ment of this class of poisons by producing in treated animals a true
antitoxic neutralizing body.

The typhoid endotoxins may be obtained by a variety of methods.
Hahn 5 has obtained what he calls “ typhoplasmin ” by subjecting them
to a pressure of about four hundred atmospheres in a Buchner press.
The cell juices so obtained are cleared by filtration. Macfadyen has
obtained typhoid endotoxins by triturating the bacilli after freezing
them with liquid air and extracting in 1 : 1,000 potassium hydrate.
Besredka obtained toxic substances by emulsifying agar cultures of
bacilli in salt solution, sterilizing them by heating to 60° C. for about
one hour, and drying in vacuo. The dried bacillary mass was then
ground in a mortar and washed in sterile salt solution which was
again heated to 60° C. for two hours. The remnants of the bacterial

1 Pfeiffer, Deut. med.Woch., xlviii, 1894; Pfeiffer und Kolle, Zeit. f. Hyg., xxi, 1896.

2 Besredka, Ann. de l’inst. Pasteur, 1895, 1896.

3 Macfadyen and Rowland, Cent. f. Bakt., I, xxx, 1901; Macfadyen, Cent. f„
Bakt., I, 1906.

4 Kraus und Stenitzer, Quoted from “Handb. d. Tech.,” etc., 1, Fischer, Jena, 1907.

5 Hahn, Munch, med. Woch., xxiii, 1906.

BACILLUS OF TYPHOID FEVER

417

bodies settle out and the slightly turbid supernatant fluid contains the
toxic substances.

Vaughan 1 has obtained poisons from typhoid bacilli by extracting
at 78° C. with a two-per-cent solution of sodium hydrate in absolute
alcohol. In this way he claims to separate by hydrolysis a poisonous
and a non-poisonous fraction. He claims, moreover, that this poison¬
ous fraction is similar to the poisons obtained in the same way from
Bacillus coli and the tubercle bacillus, and other proteicl substances,
believing that the specific nature of such proteids depends upon the
non-toxic fraction.

A simple method of obtaining toxins from typhoid bacilli is carried
out by cultivating the microorganisms in meat-infusion broth, rendered
alkaline with sodium hydrate to the extent of about one per cent.
The cultures are allowed to grow for two or three weeks and then steril¬
ized by heating to 60° C. for one hour, and allowed to stand for three
or four weeks at room temperature. At the end of this time the cul¬
tures may be filtered, through a Berkefeld or Pasteur-Chamberland filter
and will be found to contain strong toxic substances.

The accounts concerning the thermostability of the various toxins
obtained are considerably at variance. In general, corresponding with
other endotoxins, observers agree in considering them moderately re¬
sistant to heat, rarely being destroyed at temperatures below 70° C.

Intravenous inoculation of rabbits with typhoid endotoxins, if in
sufficient quantity, produces, usually within a few hours, a very marked
drop in temperature, diarrhea, respiratory embarrassment, and death.
If given in smaller doses or by other methods of inoculation —
subcutaneous or intraperitoneal — rabbits are rendered extremely ill,
with a primary drop in temperature, but may live for a week or ten days,
and die with marked progressive emaciation, or may survive. Guinea-
pigs and mice are susceptible to the endotoxins, though somewhat
less so than rabbits.

Immunity in Typhoid Fever. — As a rule, one attack of typhoid fever
protects against subsequent ones. Although exceptions to this rule
may occur, they are so rare that the history of a previous attack of this
disease practically excludes its consideration in the diagnosis of any
obscure condition.

Animals may be actively immunized by the injection of typhoid
bacilli in gradually increasing doses. In actual practice, this is best

1 Vaughan, Am. Jour, of Med. Sci., 136, No. 3, 1908.

418

PATHOGENIC MICROORGANISMS

accomplished by beginning with an injection of about 1 c.c. of broth
culture heated for ten minutes at 60° in order to kill the bacilli. After
five or six days, a second injection of a larger dose of dead bacilli is
administered; at similar intervals, gradually increasing doses of dead
bacilli are given and finally considerable quantities of a living and fully
virulent culture may be injected without serious consequences to the
animal. While this method is convenient and usually successful, it
is also possible to obtain satisfactory immunization by beginning with
very small doses of living microorganisms, according to the early
method of Chantemesse and Widal,1 and others.

Such active immunization, successfully carried out upon rabbits and
guinea-pigs, within a short time after the discovery of the typhoid bacil¬
lus, was believed to depend upon the development of antitoxic sub¬
stances in immunized animals. This point of view, however, was not
long tenable, and was definitely disproven by the investigations of Pfeif¬
fer and Kolle 2 in 1896. These investigators, as well as a large number of
others working subsequently, have shown satisfactorily that there are
present in the blood serum of typhoid-immune animals and human
beings, bacteriolytic, bactericidal, and agglutinating substances, and to
a lesser extent, precipitating and opsonic bodies.

Bactericidal and Bacteriolytic Substances. — The bacteriolytic sub¬
stances in typhoid-immune serum may be demonstrated either by the
intraperitoneal technique of Pfeiffer or in vitro. In the former experi¬
ment a small quantity of a fresh culture of typhoid bacilli is mixed
with the diluted immune serum and the emulsion injected into the
peritoneal cavity of a guinea-pig. Removal of peritoneal exudate with
a capillary pipette and examination in the hanging drop will reveal,
within a short time, a swelling and granulation of the bacteria — the
so-called Pfeiffer phenomenon. The test in vitro , as recommended by
Stern and Korte,3 may be carried out by adding definite quantities of
a fresh agar culture of typhoid bacilli to progressively increasing dilu¬
tions of inactivated immune serum together with definite quantities of
complement in the form of fresh normal rabbit or guinea-pig serum.
At the end of several hours’ incubation at 37.5° C. definite quantities
of the fluid from the various tubes are inoculated into melted agar
and plates are poured to determine the bactericidal action. Careful
colony counting in these plates and comparison with proper controls

1 Chantemesse and Widal, Ann. de Tinst. Pasteur, 1892.

2 Pfeiffer und Kolle, Zeit. f. Hyg., xxi, 1896.

3 Stern und Korte, Berl. klin. Woch., x., 1904.

BACILLUS OF TYPHOID FEVER

419

will not only definitely demonstrate the presence of bactericidal sub¬
stances in the immune serum, but will furnish a reasonably accurate
quantitative estimation. (For these tests see p. 255.)

Although normal human serum contains in small quantity substances
bactericidal to typhoid bacilli, moderate dilution, 1 : 10 or 1 : 20, of such
serum will usually suffice to eliminate any appreciable bactericidal action.
The bactericidal powers of immune serum, on the other hand, are often
active, according to Stern and Korte, in dilutions of over 1 : 4,000 and in
one case even of 1 : 4,000,000. The specificity of such reactions gives
them a considerable degree of practical value, both in the biological
identification of a suspected typhoid bacillus in known serum and in the
diagnosis of typhoid fever in the human patient by the action of the
patient’s serum on known typhoid bacilli. In the publication of Stern
and Korte, quoted above, it was found that typhoid patients during the
second week often possess a bactericidal power exceeding 1 : 1,000,
whereas the blood of normal human beings was rarely active in dilu¬
tions exceeding 1 : 50 or 1 : 100. While scientifically accurate, the prac¬
tical application of bactericidal determinations for diagnosis presents
considerable technical difficulties, and gives way to the no less accurate
method of agglutination.

Agglutinins. — Agglutinins are formed in animals and man inoculated
with typhoid bacilli, and in the course of typhoid fever. It was, in fact,
while studying the typhoid bacillus that the agglutinins were first dis¬
covered by Gruber and Durham.

In animals, by careful immunization, specific typhoid agglutinins
may easily be produced in sufficient quantity to be active in dilution
of 1 : 10,000, and occasionally even 1 : 50,000 or over. In the blood
of typhoid patients, the agglutinins may often be found in dilu¬
tions of 1 : 100 and over. It is interesting to note that irrespec¬
tive of the agglutinin contents of any given serum, there may
occasionally be noted differences in the agglutinability of various
typhoid cultures, a point which is practically important in the choice
of a typhoid culture for routine diagnosis work. Weeny 1 has
called attention to the fact that bacilli which do not readily agglutinate
when directly cultivated from the body, may often be rendered more
sensitive to this reaction by several generations of cultivation upon
artificial media. Walker has noted 2 a loss of agglutinability if the bacilli

1 Weeny, Brit. Med. Jour., 1889.

2 Walker, Jour, of Path, and Bact., 1892; Totsuka, Zeit. f. Hyg., xlv, 1903.

420

PATHOGENIC MICROORGANISMS

are cultivated in immune serum. A similar alteration in the agglutin-
ability of typhoid bacilli was noted by Eisenberg and Volk 1 when they
subjected the microorganism to moderate heat or to weak acids such as

? HCL

The practical application of agglutination to bacteriological work is
found, as in the case of the bactericidal substances, in the identification
of suspected typhoid bacilli, and in the diagnosis of typhoid fever.

When it is desired to determine by means of agglutination whether
or not a given bacillus is a typhoid bacillus, mixtures may be made
of young broth cultures, or preferably of emulsions of young agar cul¬
tures in salt solution, with dilutions of immune serum. The tests are
made microscopically in the hanging-drop preparation or, preferably,
macroscopically in small test tubes. In all cases it is desirable first to
determine the agglutinating power of the serum when tested against
a known typhoid culture. (For detailed technique, see chapter on
Technique of Serum Reactions, p. 250.)

In scientific investigations, specific agglutinations in high dilutions
of immune serum constitute very strong proof of the species of the micro¬
organism and may often furnish much information as to the biological
relationships between similar species. It is found in immunizing ani¬
mals with any given strain of typhoid bacilli, that there are formed
the “chief” or “major” agglutinins which are specific and active
against the species used in immunization, and the “group ” or “ minor ”
agglutinins, active also against closely related microorganisms. The
following extract from a table will serve to illustrate this point in the
case of typhoid and allied bacilli.

Highly Immune Typhoid Serum.

1 : 100

1 : 250

1 : 500

1 : 1,000

1 : 2,500

B. typh .

B. paratypli. (Schottmtiller) .

—

B. enteritidis .

—

B. coli communis .

—

The sera of most adult normal animals and human beings usually
contain a small amount of agglutinin for these bacilli. Immuniza¬
tion with the typhoid bacillus, while increasing chiefly the agglutinin

1 Eisenberg und Volk , Zeit. f. Hyg., xlv, 1903.

BACILLUS OF TYPHOID FEVER

421

for this bacillus itself, also to a slighter extent increases the group ag¬
glutinins for other closely allied species. That these group agglutinins
are separate substances and not merely a weaker manifestation of the
action of the typhoid agglutinin itself upon these other microorganisms,
may be demonstrated by the experiments of agglutinin absorption.
(See section on Agglutinins, page 234.)

Immune serum obtained by immunization with one particular ty¬
phoid culture usually agglutinates this culture in higher dilutions than
it will agglutinate other typhoid strains. This has been noticed in a
large number of investigations, but is not always the case.

In the clinical diagnosis of typhoid fever, the phenomenon of agglu¬
tination was first utilized by Widal.1 This observer called attention to
the fact that during the last part of the first or the earlier days of the
second week of typhoid fever, as well as later in the disease and in con¬
valescence, the blood serum of patients would cause agglutination of
typhoid bacilli in dilutions of 1 : 10, or over, whereas the serum of
normal individuals usually exerted no such influence. Upon this basis
he recommended, for the diagnosis of the disease, the employment of a
microscopic agglutination test carried out by the usual hanging-drop
technique. The reaction of Widal is, at present, widely depended upon
for diagnostic purposes and although not universally successful, owing
to irregularities in agglutinin formation in some patients, and because of
differences in agglutinability of the cultures employed, it is nevertheless
of much value. The original conclusions as to the dilutions of the
serum which must be employed, have, however, necessarily been modi¬
fied. Owing to the fact that Gruber,2 Stern,3 Frankel,4 and a number of
others have found that occasionally normal serum will give rise to ag¬
glutination of typhoid bacilli in dilutions exceeding I : 10, it has been
found necessary, whenever making a diagnostic test, to make several
dilutions, the ones most commonly employed being 1 : 20, 1 : 40, 1 : 60,
and 1 : 80. The wide application of the method has given rise to the
development of a number of technical procedures, all of them devised
with a view toward simplification. In ordinary hospital work, it is most
convenient to keep on hand upon slant agar, a stock typhoid culture, the
agglutinability of which is well known. From this stock culture, fresh

1 Widal, Bull, de la soc. med. des hopit., vi, 1896; Widal et Sicard, Ann. de
l’inst. Pasteur, xi, 1897.

2 Gruber, Verhand. Congr. f. inn. Med., Wiesbaden, 1896.

3 Stern, Cent. f. inn. Med., xlix, 1896.

4 Frankel, Deut. med. Woch., ii, 1897.

422

PATHOGENIC MIRCOORGANISMS

inoculations upon neutral bouillon should be made each day, so that a
young broth culture may always be on hand to furnish actively motile,
evenly distributed bacteria. These bouillon cultures may be grown
for from six to eight hours at incubator temperature or for from twelve
to eighteen hours at room temperature. The temperatures at which
the broth cultures are kept must depend, to a certain extent, upon the
peculiarities of the typhoid bacillus employed, since some strains are
rather more actively motile and furnish a more suitable emulsion if kept
at a temperature lower than 37.5° C. A false clumping in the broth
cultures due to a too high acidity of the bouillon or a too prolonged
incubation, must be carefully guarded against. It is also possible to
use for this test an emulsion of typhoid bacilli prepared by rubbing up a
small quantity of a young agar culture in salt solution. Uniformity
in the preparation of broth cultures or of emulsions should be observed,
since the quantitative relationship between typhoid bacilli and agglu¬
tinins will markedly affect the completeness or incompleteness of the
reaction. In high dilutions an excess of typhoid bacilli may bring about
complete absorption of all the agglutinins present, without agglutinat¬
ing all the microorganisms.

The blood of the patient to be used for a Widal test may be obtained
in a number of ways. The most convenient method is to bleed the pa¬
tient from the ear or finger into a small glass capsule, in the form of that
used in obtaining blood for the opsonin test, or into a small centrifuge
tube. About 0.5 to 1 c.c. is amply sufficient. These capsules or tubes,
after clotting of the blood, may be placed in the centrifuge which in a
few revolutions will separate clear serum from clot. The dilutions of
the serum are then made. It is best to use sterile physiological salt solu¬
tion as a diluent, but neutral broth may be used. The dilutions may be
made either by means of an ordinary blood-counting pipette or by means
of a capillary pipette upon which a mark with a grease pencil, made
about an inch from the tip, furnishes a unit of measure, and upon
which suction is made by means of a rubber nipple. It is convenient
to have at hand a small porcelain palette such as that used by painters,
in which the various cup-like impressions may be utilized to contain the
various dilutions. Dilutions of the serum are made, ranging from 1 : 10
to 1 : 50. A drop of each of these dilutions is mixed with a drop of the
typhoid culture or emulsion upon the center of a cover-slip and the cover-
slip inverted over a hollow slide. A control with normal serum and
the same culture should always be made and also one with the culture
alone to exclude the possibility of spontaneous clumping. Mixture

BACILLUS OF TYPHOID FEVER

423

with the typhoid culture, of course, each time doubles the dilutions
so that, for instance, a drop of serum dilution 1 : 10, plus a drop of
the typhoid culture, gives the final dilution of 1 : 20. The prepara¬
tions may be examined with a high power dry lens or an oil im¬
mersion lens. In a positive reaction, the bacilli, which at first swim
about actively, singly or in short chains, soon begin to gather in small
groups and lose much of their activity. Within one-half to one hour,
they will be gathered in dense clumps between which the fluid is clear
and free from bacteria, and only upon the edges of the agglutinated
masses may slight motility be observed. The degree of dilution and
the time of exposure at which such a reaction may be regarded as of
specific diagnostic value, have been largely a matter of empirical de¬
termination. It is generally accepted at present that complete agglu¬
tination within one hour in dilutions from 1 : 40 to 1 : 60 is definite
proof of the existence of typhoid infection. Exceptions, however, to
this rule may occur. Agglutinations of typhoid bacilli in dilutions of
1 : 40, and over, have occasionally been observed in cases of jaundice
and of tuberculosis, and these conditions must occasionally be consid¬
ered, though their importance was formerly exaggerated.

The method of making the Widal test from a drop of whole blood,
dried upon a slide, is not to be recommended, as accuracy in dilution
by this method is practically impossible.

As stated above, the agglutinin reaction rarely appears in typhoid
fever before the beginning of the second week. It may continue during
convalescence for as long as six to eight weeks and occasionally, in cases
where there is a chronic infection of the gall-bladder, a Widal reac¬
tion may be present for years after an attack.

For very exact work, even in clinical cases, the microscopic agglu¬
tination method may be replaced by macroscopic agglutination, ac¬
cording to the technique described in another section (page 229) .

In order to avoid both the necessity of keeping alive typhoid cultures
for routine agglutination tests and also to preclude the danger of in¬
fection by the use of living culture, Ficker 1 has recommended the use
of typhoid bacilli killed by formalin. This method has no advan¬
tages for practical purposes and in scientific bacteriological work it is,
of course, not to be considered in comparison with the other exact
methods.

Precipitins. — The investigations of Kraus 2 in 1897, by which the

1 Ficker, Berl. klin. Woch., xlviii, 1903, * Kraus, Wien. klin. Woch., xxxii, 1897.

424

PATHOGENIC MICROORGANISMS

precipitins were discovered, revealed specific precipitating substances,
among others, also in typhoid immune sera. Since Kraus’ original in¬
vestigation, these substances have been studied by Norris 1 and others.2

Opsonins. — A number of observers have shown that opsonins specific
for the typhoid bacillus are formed in animals immunized with these
organisms. Opsonins are formed also in patients suffering from typhoid
fever, but exact opsonic estimations in all these cases are extremely
difficult because of the rapid lysis which these bacteria may undergo
both in the serum, and intracelluiarly after ingestion by the leucocytes.
Klein 3 has attempted in part to overcome this difficulty by working
with dilutions of serum and at the same time using comparatively thick
bacterial emulsions and exposures to the phagocytic action not exceed¬
ing ten minutes. Chantemesse 4 has claimed that the opsonic index of
typhoid patients was increased after treatment with a serum obtained
by him from immunized horses, and Harrison 5 has reported similar
results in patients treated by a modification of Wright’s method of
active immunization. Klein claims to have demonstrated that in
typhoid-immune rabbits, after five injections, the opsonic contents of
the blood were increased to an equal extent with the bactericidal sub¬
stances. He concludes from this interesting observation that it may
well be that the opsonins are quite as important in typhoid immunity
as are the latter substances.

For diagnostic purposes in typhoid fever the estimation of the opsonic
index, so far, has not been proven to be of great value.

Specific Therapy in Typhoid Fever. — The failure to produce a soluble
toxin from typhoid cultures has naturally so far precluded the possibility
of an antitoxic therapy, such as that which has been successful in diph¬
theria. In the light of our present knowledge of the poisonous products
of the typhoid bacillus it seems but natural that attempts by earlier
investigators to apply the principles of Behring’s work to typhoid fever
were doomed to fail. Attempts to employ specific bactericidal and bac¬
teriolytic sera for therapeutic purposes in this disease have also been
without favorable result.

Active Immunization. — We have seen that work by Pfeiffer and Kolle
and subsequently by a large number of others has shown that it is com-

1 Norris, Jour, of Inf. Dis., I, 3, 1904.

2 Barker and Cole, 22d Ann. Session, Assn, of Amer. Phys., Wash., 1897.

3 Klein, Bull. Johns Hopkins Hosp., 1907.

4 Chantemesse, 14th Internatl. Cong, for Hyg., Berlin, 1907.

3 Harrison, Jour. Royal Army Med. Corps, 8, 1907,

BACILLUS OF TYPHOID FEVER

425

paratively easy to immunize animals actively against typhoid infection
by the systematic injection of graded doses, at first of dead bacilli, later
of fully virulent live cultures. Attempts to apply these principles pro-
phylactically have been made recently on a large scale by Wright and
his associates upon English soldiers in South Africa, and by German
observers in German East Africa.

The first recorded experiment of this sort which was done upon human
beings was that of Pfeiffer and Kolle,1 who in 1896 treated two in¬
dividuals with subcutaneous injections of an agar culture of typhoid
bacilli which had been sterilized at 56° C. The first injection was made
with two milligrams of this culture. Three or four hours after the in¬
jection the patient suffered from a chill, his temperature gradually rose
to 105° F., and there was great prostration and headache, but within
twenty-four hours the temperature had returned to normal.

This experiment showed that such injections could be practiced upon
human beings without great danger.

Simultaneously with the work of Pfeiffer and Kolle, Wright 2 con¬
ducted similar experiments on officers and privates in the English army.

The actual number of persons treated directly or indirectly under
Wright’s 3 supervision in an investigation covering a period of over four
years comprised almost one hundred thousand cases. The methods
employed by Wright have been modified several times by him and his
collaborators in minor details; the principles, however, have remained
consistently the same. In the first experiments Wright employed an
agar culture three weeks old, grown at 37° C., then sterilized at a tem¬
perature below 60° C., and protected from contamination by the ad¬
dition of five-tenths per cent of carbolic acid. Later, Wright 4 5 employed
bacilli grown in a neutral one-per-cent pepton bouillon in shallow layers
or flasks. Great importance is attached both to the virulence of the ty¬
phoid strain, which may to a moderate extent be standardized by pas¬
sage through guinea-pigs, and to care in using low temperatures
for final sterilization. The temperature recommended by Harrison,0
working with Wright’s method, is 52° C., after which the cultures are
carbolized. For the first dose in a human being, Wright recommends

1 Pfe^ffer und Kolle, Deut. med. Woch., xxii, 1896; xxiv, 1898.

2 Wright, Lancet, Sept., 1896.

3 Wright and Semple, Brit. Med. Jour., 1897; Wright and Leishman, Brit. Med.
Jour., Jan., 1900.

* Wright, Brit. Med. Jour., 1901; Lancet, Sept., 1902; Brit. Med. Jour., Oct., 1903.

5 Harrison, Jour. Royal Army Medical Corps, 1907.

426

PATHOGENIC MICROORGANISMS

the quantity of bacilli fatal for 100 grams of guinea-pig. The dose may
also, according to Wright, be regulated by making numerical counts of
the emulsions used, by his usual method of counting against red blood
corpuscles, and using for the first injection 750 to 1,000 millions of dead
bacteria. The second injection, given after eleven days, should be double
this quantity. Usually the first dose is followed by local inflammatory
symptoms and the general systemic symptoms of toxemia. These,
however, usually disappear after forty-eight hours.

Although the observations of Wright are extensive, it is nevertheless
extremely difficult to tabulate satisfactory statistics from a mass of
experiments which must of necessity be observed by a large number
of individuals, in all of whom the personal equation modifies the results
of the observations. On the whole, however, it seems fair to state that
distinctly advantageous results followed the active immunization prac¬
ticed by Wright. Wright’s own estimation, in a careful attempt to
present the subject fairly, gives a reduction of the morbidity from
typhoid fever in the British army of fifty per cent, and a reduction of
the mortality of those who became infected in spite of inoculations of
fifty per cent also. Combining these two results, the actual reduction
of the death rate by the method of vaccination would appear to
amount to at least seventy-five per cent.

The method of Pfeiffer and Kolle, originally used by them in their
experiments, has been extensively carried out by German observers
upon the army taking part in the late East African campaign.

Roughly, the method consists in the injection of salt-solution emul¬
sions of fresh agar cultures sterilized at 60° C. The results reported
from a large material were in general favorable, indicating that the
morbidity of all the troops taking part was reduced by the inoculation
and that the death rate among the inoculated persons was lower than
that among normal individuals.

Recent extensive tests in the United States Army, carefully observed
by Russell1 and others, seem to have removed any doubt which may have
existed as to the efficacy of prophylactic typhoid vaccination. How¬
ever, another point of importance in this connection has recently been
raised by Metchnikoff and Besredka2. They vaccinated chimpanzees
with typhoid bacilli and found that when emulsions of the clear bac¬
teria were used, protection was only slight. Better results were ob-

1 Russell, Am. Jour, of Med. Sc., cxlvi., 1913.

2 Metchnikoff and Besredka , Am. de Tlnst. Past., 1911.

BACILLUS OF TYPHOID FEVER

427

tained — that is, apparently complete protection within 8 to 10 days,
when living sensitized bacteria were injected. (Bacteria which had
been exposed to the action of inactivated immune serum.) Broughton1
has applied this method to human beings. Gay 2 has also prepared a
sensitized dead typhoid vaccine which he has already used in a consid¬
erable number of cases. It will take some time, however, before a
statistical estimation of the superiority of this method over the older
vaccination with dead bacteria will be possible.

BACILLUS FECALIS ALKALIGENES

In 1896 Petruschky 3 described a bacillus which is a not infrequent
inhabitant of the human intestine, being found chiefly in the lower
part of the small intestine and the large intestine. This organism,
which he called Bacillus fecalis alkaligenes, is of little pathogenic im¬
portance, although Neufeld states that he has seen a case of severe
gastroenteritis in which the watery defecations contained this bacillus
in almost pure culture. As a rule, however, this organism can not be
regarded as pathogenic, and is important chiefly because of the ease
with which it may be mistaken for Bacillus typhosus.

Bacillus fecalis alkaligenes is an actively motile, Gram-negative
bacillus, possessing, like the typhoid bacillus, numerous peritrichal fla¬
gella. On the ordinary culture media it grows like the typhoid bacillus.
It does not coagulate milk. It produces no indol, and on sugar media
in fermentation tubes produces no acid or gas. On potato, its growth,
while somewhat heavier than that of the typhoid bacillus, is not suf¬
ficiently so to permit easy differentiation. It differs from Bacillus
typhosus in that it produces no acid on any of the sugar media, and is
therefore easily differentiated by cultivation upon Hiss-serum-water
media or on pepton waters containing sugars. On the Hiss semi¬
solid tube-medium Bacillus fecalis alkaligenes, while clouding the
medium throughout, grows most heavily on the surface where, eventu¬
ally, it forms a pellicle.

1 Broughton, C. R. de 1’Acad. des Sc., cliv, 1911.

2 Gay, Arch, of Int. Med., 1914.

3 Petruschky, Cent. f. Bakt., I, xix, 1896.

CHAPTER XXVIII

BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP

(< Continued )

BACILLI INTERMEDIATE BETWEEN THE TYPHOID AND COLON

ORGANISMS

( [Bacilli of Meat Poisoning and Paratyphoid Fever)

There is an extensive group of Gram-negative bacilli which be¬
cause of their morphology, cultural behavior, and pathogenic properties,
are classified as intermediate between the colon and the typhoid types.
The microorganisms belonging to this group have been described, most
of them, within the last fifteen years, but few of them have been fully
identified with one another. They have been variously designated as the
“hog-cholera group,” “the enteritidis group,” the “paracolon group”
or “paratyphoid group,” because of the pathological conditions with
which the chief members under investigation have been found associated.

Attempts to systematize the group by the comparative study of a
large number of its members have been made, notably by Buxton1
and by Durham,2 and the work of these writers, based on cultural and
agglutinative studies, has added materially to our knowledge of these
organisms.

The microorganisms of this group are morphologically indistinguish¬
able from the colon and typhoid bacilli. They are Gram-negative and
possess flagella. Their motility is variable, but usually approaches
that of the typhoid bacilli in activity. They correspond, furthermore,
to the two other groups in their cultural characteristics upon broth, agar,
and gelatin. On potato, they vary, some of them approaching in deli¬
cacy the typhoid growth upon this medium, others more closely
approximating the heavy brownish growth of B. coli. Indol is rarely
formed by them, though this has not been absolutely constant in all
descriptions. As a group, they are easily distinguished from Bacillus

1 Buxton, Jour. Med. Res., N. S., iii, 1900. 2 Durham, Jour. Exper. Med., v, 1901.

428

BACILLI BETWEEN TYPHOID AND COLON ORGANISMS 429

typhosus on the one hand, and from Bacillus coli on the other, by the
following simple reactions tabulated by Buxton.1

B. coli.

Intermediates.

B. typhosus.

Coagulation of milk .

Production of indol .

—

Fermentation of lactose with gas .

—

Fermentation of dextrose with gas . .

—

Agglutination in typhoid- immune serum ....

—

The characteristics of the three groups as shown by fermentation tests
may be tabulated as follows:

Gas upon
Dextrose.

Gas upon
Lactose.

Gas upon
Saccharose.

B. typhosus . .

—

Intermediates .

—

B. coli communis .

—

B. coli communior .

Pathogenically, the bacilli of this “intermediate group” have attracted
attention chiefly in connection with meat poisoning, and with protracted
fevers indistinguishable from mild typhoidal infections.

In 1888, Gartner 2 described a bacillus which he isolated from the
meat of a cow, the ingestion of which had produced the symptoms of
acute gastrointestinal catarrh in fifty-seven people. One of these died
of the disease and the bacilli could be demonstrated in the spleen and in
the blood of the patient.

This bacillus, called Bacillus enteritidis by Gartner, was actively
motile, formed no indol, but produced gas in dextrose media. Acute
gastrointestinal symptoms could be induced by feeding the organisms
to mice, guinea-pigs, rabbits, and sheep, and the bacilli could be re¬
covered from the infected animals. An interesting observation, which
has since become important in characterizing the group of these bacilli
concerned in meat poisoning, was the fact that the bacterial bodies
themselves were found by Gartner to be extremely toxic, containing a
poison which, in contradistinction to the endotoxins of many other
microorganisms, was extremely resistant to heat. Sterilized cultures
showed the same pathogenic effects as the living bacilli. Epidemics

1 Buxton, loc. cit.

2 Gartner, Corresp. BL d. Aerzt. Vereins, Turingen, 1888,

430

PATHOGENIC MICROORGANISMS

of meat poisoning similar to the one described by Gartner, in which
similar bacteria were isolated, were those described by Van Ermengem,1
occurring at Morseele in 1891, the one described by Holst,2 the Rotter¬
dam epidemic described by Poels and Dhont,3 the one described by
Basenau, and many others.

Bacillus Morseele of Van Ermengem, Bacillus bovis morbificans of
Basenau,4 and the bacilli isolated in similar epidemics by other ob¬
servers, are, except for slight differences in minor characteristics,
almost identical with Gartner’s microorganism.

In 1893, Theobald Smith and Moore,5 studying the diseases of swine,
noted a great similarity between the so-called hog-cholera bacillus, the
bacilli of the Gartner group, and Bacillus typhi murium isolated by
Loeffler. These observers first used the term “ hog-cholera ” group for
the organisms under discussion.

In 1899 Reed and Carroll 6 called attention to the fact that Bacillus
icteroides, associated by Sanarelli with yellow fever, was culturally
closely similar to the bacillus of hog cholera.

Meanwhile, other observers had been isolating bacilli, similar to
those spoken of above, from cases of protracted fevers in human beings,
often closely simulating typhoid infections. The first cases of this
kind on record were those of Achard and Bensaude.7

In 1897, Widal and Nobecourt 8 described a bacillus which they had
isolated from an esophageal abscess following typhoid fever, which
closely resembled Bacillus psittacosis of Nocard,9 and which, following
a nomenclature previously suggested by Gilbert,10 they designated the
paracolon bacillus. This microorganism, as well as Bacillus psittacosis ,
isolated from a parrot by Nocard, showed a close resemblance to bacilli
of the Gartner group.

In 1898, Gwyn 11 reported a case occurring at the Johns Hopkins

1 Van Ermengem, Bull. Acad. d. med. de Belgique, 1892; “ Tray, de lab. de
Puniv. de Gand,” 1892.

2 Holst, Ref. Cent. f. Bakt., xvii, 1895.

3 Poels und Dhont, Holland Zeit. f. Tierheilkunde, xxiii, 1894.

4 Basenau, Arch. f. Hyg., xx, 1894.

5 Th. Smith and Moore, U. S. Bureau of Animal Industry Bull, vi, 1894.

6 Reed and Carroll, Medical News, lxxiv, 1899.

7 Achard and Bensaude, Bull, de la soc. d. hopitaux de Paris, Nov., 1906.

8 Widal et Nobecourt, Semaine med., Aug., 1897.

9 Nocard, Ref. BaumgarteiPs Jahresb., 1896.

10 Gilbert, Semaine med., 1895.

11 Gwyn, Johns Hopkins Hosp. Bull, 1898,

BACILLI BETWEEN TYPHOID AND COLON ORGANISMS 431

Hospital, which presented all the symptoms of typhoid fever, but lacked
serum agglutinating power for Bacillus typhosus. From the blood of
the patient, Gwyn isolated an organism, with cultural characteristics
similar to those of the Gartner bacillus, which he called a “paracolon
bacillus.” This bacillus was agglutinated specifically by the serum of
the patient.

Cushing,1 in 1900, isolated a similar microorganism from a cos¬
tochondral abscess, appearing during convalescence from typhoid
fever.

In the same year, Schottmiiller 2 reported five cases from which bacilli
similar to those previously described were isolated. Careful cultural
and agglutination studies of the microorganisms obtained from these
cases showed that they could be divided into two similar, yet distinctly
different types, one of them, the “Muller” organism, approaching
closely to the typhoid type, especially in its growth upon potato; the
other, the “Seeman” type, corresponding more closely to the Gartner
enteritidis bacilli. Similar cases were soon after reported by Kurth,3
Buxton and Coleman,4 5 Libman,0 and others.

The two types of organisms, paratyphoid A and B, described by
Schottmiiller and studied by many other observers, can be culturally
differentiated though not without difficulty.

Type A is more delicate in its growth on various media than B,
growing with almost invisible growth on potato, and differing from
typhoid in its gas formation on dextrose broth only. Milk is not co¬
agulated, but remains turbid, not being finally cleared by solution of
the casein as in similar cultures of type B. Lactose whey is acidified
and remains acid. This organism is not very important as a causative
agent of human disease, and has been isolated from themormal intestines
of animals by Morgan.6 Kutscher for this reason suggests that essen¬
tially and except in rare instances this organism is a non-pathogenic
saprophyte.

Type B grows more heavily on all media than A, especially on
potato (though this is not universally reported). Milk is slightly acid-

1 Cushing , Johns Hopkins Hosp. Bull., 1900.

2 Schottmiiller, Deut. med. Woch., 1900; Zeit. f. Hyg., xxvi.

3 Kurth, Deut. med. Woch., 1901.

4 Buxton and Coleman, Proc. N. Y. Pathol. Soc., Feb., 1902.

5 Libman, Jour. Med. Res., N. S., iii, 1902.

6 Morgan, cited from Kutscher, Kolle und Wassermann, Handbiich.

Erganzungs, I.

432

PATHOGENIC MICROORGANISMS

ified at first, but eventually is rendered strongly alkaline and cleared
up, possibly by casein solution. On lactose whey is alkalined and
becomes strongly blue.

Eventual differentiation in doubtful cases must be made by agglu¬
tination. Infection with type B is not uncommon and far outstrips
that with type A in importance.

Clinically, the diseases caused by the bacteria of this class may be
divided into two main groups.

I. Those which fall into the category of meat poisoning, having a
sudden and violent onset of gastroenteric symptoms directly following
the ingestion of meat, and characterized by profound toxemia; and

II. Those in which the disease simulates a mild form of typhoid fever,
differing from this only by the absence of the specific agglutination re¬
action for typhoid bacilli.

The differential diagnosis between the second type of case and
true typhoid fever may be extremely difficult. However, careful
studies by Lentz1 and others have revealed certain differences which
though not conclusive are at least of some aid in determining the nature
of the disease. In contradistinction to true typhoid the temperature
reaction of this case may set in more abruptly and remain more
irregular throughout the disease. Gastric symptoms, vomiting, and
nausea are often more prominent than in typhoid fever and enlargement
of the spleen is less regularly present than in the latter. Owing to the
low mortality of paratyphoid fever (in 120 cases observed by Lentz
less than 4 per cent, and in many other smaller epidemics no deaths
have occurred), we have remained relatively ignorant concerning the
pathologic anatomy of the disease. Longcope 2 observed a case which
was fatal after two weeks of illness in which there was no enlargement of
Peyer’s patches and no sign of even beginning ulceration. This seems to
have been the experience of most other observers who have found less
involvement of the lymphatics of the bowel than is found in typhoid
fever. During the disease the bacteria can often be cultivated from
the blood, and the serum of the patient may agglutinate specifically
paratyphoid strains. In this way the diagnosis can often be made.
Libmann 3 has isolated the organism from the fluid aspirated from the
gall bladder in a case operated on fpr cholecystitis.

Most of these microorganisms possess pathogenicity for mice, guinea-

1 Lentz, Klin. Jahrb. xiv, 1914.

2 Longcope, Amer. Jour, of Med. Sciences, cxxiv, 1902.

3 Libmann, Jour, of Med. Res., viii, 1902.

BACILLI BETWEEN TYPHOID AND COLON ORGANISMS 433

pigs, and rabbits, which exceeds that of the colon or typhoid bacilli.
A number of the bacilli of this group, furthermore, especially those most
closely similar to the original B. enteritidis of Gartner, contain an endo¬
toxin which shows a high resistance to heat, which may explain the fact
that illness has occasionally followed the ingestion of infected meat even
after preparation by cooking.

Bacteriological correlation of these bacilli has been attempted, as
stated above, by Durham and by Buxton, and more recently by
Kutscher and Meinicke.1 The subject is a difficult one and for ultimate
clearness will require much further work.

Harding and Ostenberg 2 have examined a series of organisms of the
intermediate group on various sugars, and find that by the use of
xylose and arabinose three definite groups can be established.

I. Those making aldehyd (red) on fuchsin-sulphite agar with both
arabinose and xylose — both Schottmiiller types A and B and strains
of Bacillus enteritidis.

II. Red on arabinose and not on xylose— typhi murium, para¬
typhoid Gwyn, paratyphoid Loomis, and three others.

III. Red on xylose and not on arabinose — B. hog cholera.

This work was carefully carried out and may possibly point toward
an ultimate classification. However, the strains employed were too
few to permit definite conclusions at present.

Durham,3 on the basis of cultural and agglutinative studies, has
formulated a classification of the Gram-negative bacilli of the typhoid-
colon and allied groups, which, though hardly final, aids considerably
in throwing light upon the interrelationships of the various species.
Durham’s divisions are as follows:

Division I. Typhoid-like Morphology (motile).

A. No sugars fermented. Type B. fecalis alkaligenes.

B. Acid in dextrose, but no gas. Type B. typhosus. Agglutination
in typhoid serum.

C. Acid in dextrose, but gas only when other constituents are favor¬
able. No acid or gas from lactose or saccharose. No agglutination in
typhoid serum. Includes Bacillus “Gwyn” and Bacillus “0” of
Cushing.

D. Acid and gas from dextrose. No acid or gas from lactose or

1 Kutscher und Meinicke, Zeit. f. Hyg., lii, 1906.

2 Harding and Ostenberg, Jour, of Inf. Dis., ii, 1912.

3 Durham, loc. cit.

434

PATHOGENIC MICROORGANISMS

saccharose. Grows more rapidly than typhoid. No agglutination in
colon-immune serum. Slight reaction with some typhoid sera. Includes
Gartner’s B. enteritidis, B. Morseele, Gunther’s meat-poisoning bacillus,
hog cholera bacillus, B. psittacosis, B. morbificans bovis, Durham’s
Bacillus “A,” B. typhi murium.

Division II. Colon-like Morphology (motile).

E. Acid and gas from dextrose, none from lactose or saccharose.
Rate of growth and colony appearance more like colon than typhoid.

F. Acid and gas from dextrose, and no gas from lactose. Types
isolated by Durham.

G. Acid and gas from dextrose; acid, no gas, from lactose. Differ
from F in serum reactions.

H. B. coli communis. Acid and gas from dextrose and lactose; none
from saccharose.

I. B. coli communior. Acid and gas from dextrose, lactose, and
saccharose.

Division III. N on-motile. Polysaccharide splitters (starch). Type
B. lactis aerogenes. Includes bacilli of mucosus capsulatus group, and
Friedlander’s bacillus.

CHAPTER XXIX

BACILLI OF THE COLON-TYPHOID-DYSENTERY GROUP

(< Continued )

THE DYSENTERY BACILLI

Although acute dysentery has been an extremely prevalent disease,
occurring almost annually in epidemic form in some of the Eastern coun¬
tries and appearing sporadically all over the world, its etiology was
obscure until 1898 when Shiga 1 described a bacillus which he isolated
from the stools of patients suffering from this disease in Japan, and es¬
tablished with scientific accuracy its etiological significance. Since the
discovery of Shiga’s bacillus a number of other bacilli have been de¬
scribed by various workers, all of which, while showing slight biological
differences from Shiga’s microorganism, are sufficiently similar to it
culturally and pathogenically to warrant their being classified together
with it in a definite group under the heading of the “ dysentery bacilli.”

The manner in which Shiga made his discovery furnishes an in¬
structive example of the successful application of modern bacteriological
methods to etiological investigation. Many workers preceding Shiga
had attempted to throw light upon this subject by isolations of bacilli
from dysenteric stools, and by extensive animal inoculation. Shiga,
following a suggestion made by Kitasato, approached the problem by
searching for a microorganism in the stools of dysentery patients which
would specifically agglutinate with the serum of these patients. His
labors were crowned with success in that he found, in thirty-six cases,
one and the same microorganism which showed uniform serum agglu¬
tinations. Further, he found that this bacillus was not present in the
dejections of patients suffering from other diseases nor in those of normal
men, and that when tested against the blood serum of such people it
was not agglutinated.

Morphology. — Shiga’s bacillus is a short rod, rounded at the ends,

1 Shiga, Cent. f. Bakt., xxiii, 1898; ibid., xxiv, 1898; Deut. med. Woch., xliii,
xliv, and xlv, 1901.

435

436

PATHOGENIC MICROORGANISMS

morphologically very similar to the typhoid bacillus, and, like it,
inclined to involution forms. The organism generally occurs
singly, more seldom in pairs. It is decolorized by Gram's
method of staining. With the ordinary anilin dyes it stains easily,
showing a tendency to stain with slightly greater intensity at
the ends. The organism is an aerobe and facultative anaerobe.
Although described at first by Shiga as being motile, its motility
has not been satisfactorily proven, and most observers agree in
denying the presence of flagella and affirming the complete absence
of motility.

Cultural Characteristics. — On agar the colonies are not characteristic,
resembling those of the typhoid bacillus.

On gelatin, the colonies appear very much like typhoid colonies and
the gelatin is not liquefied.

On potato, the growth, like that of typhoid, is at first not visible, but
after about a week turns reddish brown.

In broth, there is clouding, with moderate deposits after some days.
No pellicle is formed.

Milk is not coagulated. Litmus milk shows a slight primary acidity,
later again becoming alkaline and taking on a progressively deeper blue
color.

Indol is not formed in pepton water by all varieties.

No gas is formed in media containing dextrose, lactose, saccharose,
or other carbohydrate.

While not delicately susceptible to reaction, the bacillus prefers
slightly alkaline media.

Shiga differentiated his organism from the typhoid bacillus chiefly
by supposed differences in colony characters and by the agglutination
reaction.

Following the work of Shiga, a large number of investigators turned
their attention to the subject of dysentery, with the result that many
new forms were discovered and at first a considerable amount of con¬
fusion prevailed.

Flexner 1 in 1899 investigated dysentery in the Philippines, and
isolated a bacillus which, he considered, corresponded to Shiga's
organism.

Strong and Musgrave 2 in 1900 described a bacillus isolated from

1 Flexner , Phila. Med. Jour., vi, 1900, and Bull. Johns Hopkins Hosp., xi, 1900.

* Strong and Musgrave, Report Surg. Gen. of Army, Washington, 1900.

THE DYSENTERY BACILLI

437

dysentery cases in the Philippines which was essentially like that of
Flexner.

Nearly simultaneously with the papers of Flexner and of Strong and
Musgrave, Kruse 1 published investigations of an epidemic of dysentery
occurring in Germany. His observations were of the greatest importance
and largely formed the starting point of the further advances which
have been made in the etiology of dysentery.

Kruse’s organism was described as forming colonies on gelatin and
agar, practically like those of Bacillus typhosus. Like this bacillus, no
gas was formed from grape sugar, and the growth in milk and on potato,
and even in Piorkowski’s urine gelatin, resembled that of Bacillus
typhosus. According to Kruse, this organism was absolutely with¬
out motility.

In 1901 Kruse 2 contributed a second paper. In this, besides con¬
firming his previous observations, he described another class of organ¬
ism coming from cases which he designated as “pseudo-dysentery of
insane asylums.” In the case of one patient, and at two autopsies, he
isolated organisms ‘which he could not distinguish morphologically or cul¬
turally from the true dysentery bacillus, but which showed differences in
their serum reaction. By careful study of the behavior of these bacilli
in the serum of patients and in immune serum from animals, he not
only showed that they were different from his original cultures from
cases of epidemic dysentery which, no matter what their source, were
found to be alike, but that they showed differences among themselves
and apparently fell into two or more varieties. One of these organisms
culturally and by its serum reactions showed itself practically identical
with one of the cultures he had received from Flexner.

Spronck 3 in 1901 described an organism isolated in Utrecht from
dysentery cases, which showed great similarity to the Shiga- Kruse
organism; but, when tested in the serum of a horse immunized against
true dysentery bacillus, showed practically no agglutination. He placed
this organism in the group designated by Kruse as the “ pseudo-dysentery
bacilli.” His communication is of importance, since it is the first re¬
ported instance in which any investigator had recognized and associated
the so-called pseudo-dysentery bacilli with dysentery approaching the
acute epidemic form in type.

Following this work a number of investigators, including Vedder

1 Kruse, Deut. med. Woch., xxvi, 1900.

2 Kruse, Deut. med. Woch.; xxvii, 1901.

3 Spronck, Ref. Baumgarten’s Jahresber., 1901.

438

PATHOGENIC MICROORGANISMS

and Duval/ Flexner, and Shiga 1 2 himself, published communications in
which they claimed identity for the various forms previously described.

In 1902 Park 3 and Dunham described an organism which they
found in a small outbreak of dysentery occurring in Maine. This
organism differed from most of those previously described in that it
was found to produce indol in pepton solutions.

In the same year Martini 4 and Lentz published an article in which
they attempted to differentiate various dysentery bacilli by means of
agglutination. This research is of importance in that it supported the
work of Kruse and of Spronck, indicating a difference between the ag¬
glutinative character of the Kruse organism and the so-called “ pseudo-
dysentery” type, in which Flexner’s organisms were included. It is of
further interest, since it indicated a marked difference between Flexner’s
Philippine cultures and the Philippine culture of Strong, the Strong
organism refusing to agglutinate not only in “Shiga” immune serum,
but also in “Flexner” immune serum.

Simultaneously with this article Lentz 5 published the results of com¬
parative cultural researches with dysentery and “pseudo-dysentery”
bacilli, in which he made the important observation that the original
Shiga-Kruse bacilli did not affect mannit, while the “pseudo-dysentery ”
bacilli, including Flexner’s and Strong’s Philippine cultures, fermented
mannit, giving rise to a distinct acid reaction in the medium. The
Flexner organisms and others of the “pseudo-dysentery” bacilli, how¬
ever, fermented maltose, while the Shiga-Kruse type, as well as Strong’s
bacillus, left it unchanged at the end of forty-eight hours.

In January, 1903, Hiss and Russell 6 described a bacillus (“ Y”) from
a case of fatal diarrhea in a child, which by ordinary cultural test and
absence of motility was found to resemble the Shiga-Kruse and Flexner
bacilli. Immediately upon its isolation, it was found, however, to differ
from the Kruse culture by its ability to ferment mannit. This observa¬
tion was made independently of Lentz’s work, which, at that time, had
not become known in America. In the comparative study of Hiss and
Russell on the fermentative abilities of various dysentery cultures, the
serum water media (described on page 132) were used. By the use of

1 Vedder and Duval, Jour. Exp. Med., vi, 1902.

2 Shiga, Zeit. f. Hyg., 41, 1902.

3 Park and Dunham, N. Y. Univ. Bull, of Med. Sci., 1902.

* Martini und Lentz, Zeit. f. Hyg., xli, 1902.

5 Lentz, Zeit. f. Hyg., xli, 1902.

8 Hiss and Russell, Med. News, Feb., 1903.

THE DYSENTERY BACILLI

439

these media, it was found that the Kruse culture, a culture of Flexner’s
bacillus from the Philippines, and Duval’s “New Haven” culture fer¬
mented dextrose with the production of a solid acid coagulum, but did
not affect mannit, maltose, saccharose, or dextrin. The culture of Hiss
and Russell, on the other hand, fermented not only dextrose but also
mannit with the production of acid and coagulation of the medium.
Maltose, saccharose, and dextrin were not fermented. The “ Y ” bacillus,
furthermore, was shown to differ entirely from the cultures of Shiga,
Kruse, and “New Haven” in the serum of immunized animals. This
serum had for bacillus “ Y” a titer of 1 : 500 while the three other above-
named organisms did not agglutinate in it at any dilution. In normal
beef serum, the Hiss-Russell organism was found to agglutinate as highly
at 1 : 320, while the other three cultures gave no reaction in dilutions of
over 1 : 10 or 20.

Park and Carey,* 1 in March, 1903, described an epidemic of dysen¬
tery occurring in the town of Tuckahoe, near New York City, and
isolated an organism which resembled the Shiga-Kruse bacilli in not
fermenting mannit, but produced indol in pepton solution after five
days. It corresponded in agglutination with the cultures “ New Haven ”
and “Shiga” when tested in the serum of a goat immunized against
the mannit-fermenting culture “Baltimore,” i.e., did not react at 1:50,
whereas Flexner’s “Manila” and “Baltimore” cultures, Park and Dun¬
ham’s “Seal Harbor” culture, and some New York cultures, all fer¬
menting mannit, agglutinated up to two thousand dilution in the “ Bal¬
timore” serum.

The preceding review of a part of the literature, by which our knowl¬
edge of the dysentery bacilli was developed, demonstrates sufficiently
that we have to deal in this group with a number of different micro¬
organisms. This, as we have seen, was a fact first recognized by Kruse
when he spoke of his true dysentery and his pseudo-dysentery strains.
In spite of much confusion at first, the careful study of fermentation
phenomena, of specific agglutinations, and, more recently, by Ohno 2
and others, of the bacteriolytic phenomena in immune sera, has made
it possible to distinguish sharply between a number of groups.

Basing the grouping of these microorganisms upon a careful study
of fermentations, Hiss 3 has divided them as follows:

1 Park and Carey, Jour. Med. Res., ix, 1903.

1 Ohno, Philippine Jour, of Sci., 1, ix., 1906.

s Hiss, Jour. Med. Res., N, S., viii, 1904,

PATHOGENIC MICROORGANISMS

440

“ Shiga ” )

“Kruse,” > Ferment dextrose. Group I.

“New Haven”

“Y” (Hiss and Russell type)
“ Seal Harbor”

“ Diamond ”

“Ferra ”

“ Strong” (type)

“Harris” (type)

“Gray”

“Baltimore ”

“ Wollstein”

Ferment dextrose and mannit. Group II.

Ferments dextrose, mannit, saccharose
Group III.

Ferment dextrose, mannit, maltose, saccha¬
rose, dextrin. Group IV.

It was noticed, it should be mentioned, however, that in the case of the
“ Y,” “Diamond/’ and “Ferra” there was usually delayed acid fermen¬
tation of maltose, never any of dextrin.

In studying the agglutinative characters of these groups, furthermore,
it was found that fermentation tests and agglutinations went hand in
hand. The following table will illustrate this point:1

Serum of Rabbit immunized against Group I. (Shiga’s culture).

Bacilli of Group I.:

“Shiga” (homologous) . 20,000

“Kruse” . ... 20,000

“New Haven” . 20,000

Bacilli of Group II.:

“Y” . 200

“Ferra” . 200

“Seal Harbor” . 200

Bacilli of Group IV.:

“Baltimore” . 800

“Harris” . 800

“Gray” . 800

“Wollstein” . 800

Serum of Rabbit immunized against Group II. (“Y” culture,
Hiss and Russell).

Bacilli of Group I.:

“ Shiga ” . less than 100

“Kruse” . 100

“New Haven” . 100

1 Hiss, Jour, of Med, Research, 13, N. S., viii, 1904.

THE DYSENTERY BACILLI

441

Bacilli of Group II.:

“Y” (homologous) . 6,400

“Ferra” . •. . 6,400

“Seal Harbor . 6,400

Bacilli of Group IV.:

“ Baltimore ” . 1,600

“Gray” . 1,600

“Harris” . 1,600

“Wollstein” . 1,600

Serum of Rabbit immunized against Group IV. (“ Baltimore ”
culture) .

Bacilli of Group I.:

“Shiga” . less than 100

“Kruse” . 100

“New Haven” . 100

Bacilli of Group II.:

“Y” . 400

“Ferra” . 400

“Seal Harbor ” . 400

Bacilli of Group IV.:

“ Baltimore ” (homologous) . 3,200

“Harris” . 3,200

“Gray” . 3,200

“Wollstein” . 3,200

In common, all these groups possess an identical morphology, the
Gram-negative staining characteristics, the lack of motility with close
adherence to the line of inoculation in the Hiss tube medium, the in¬
ability to liquefy gelatin, the inability to form acid from lactose, and
the inability to produce gas from any carbohydrate media.

Biological Considerations. — The dysentery bacilli in neutral broth
or upon agar slants may remain alive without transplantation for
periods of several months. They are aerobes and facultative anaerobes
when proper sugars are present, preferring, however, the aerobic environ¬
ment. They are easily destroyed by heat, an exposure to 60° C. killing
them usually in a short time (ten minutes). Against cold they show
considerable resistance, surviving freezing for a period of several weeks.
They show little resistance to the usual strengths of the common chem¬
ical disinfectants.

Pathogenicity. — There is practically no doubt at the present time as to
the etiological connection between the bacilli of this group and the dis¬
eases clinically classified as acute dysentery. A more chronic form of

442

PATHOGENIC MICROORGANISMS

dysentery due to a protozoan, the Amoeba coli, though presenting much
clinical resemblance to the bacillary dysenteries is, nevertheless, an
entirely distinct disease.

Infection takes place, probably, entirely by ingestion of the bacteria
with infected water or food contaminated from the feces of dysentery
patients. A small epidemic occurring in a hospital in New York City
and caused by the bacillus “Y” of Hiss and Russell was indirectly
traced to milk by Zinsser.1

Endemic in a large part of the world, especially in the warmer
climates, the disease most frequently occurs in epidemics of more or
less definite localization, usually under conditions which accompany
the massing of a large number of human beings in one place, such as
those which occur in the crowded quarters of unsanitary towns, in insti¬
tutions such as insane asylums, or in military camps. The mortality of
such epidemics may be very large. According to Shiga,2 the disease in
Japan frequently shows a mortality of over twenty per cent.

The disease in human beings usually begins as an acute gastro¬
enteritis which is accompanied by abdominal pain and diarrhea. As
it becomes more severe, the colicky pains and diarrhea increase, the
stools lose their fecal character, becoming small in quantity and filled
with mucus and flakes of blood. There is often severe tenesmus at
this stage, and the bacilli are present in large numbers in the dejecta.
Owing to the absorption of toxic products, symptoms referable to the
nervous system, such as muscular twitching, may supervene, and if the
disease is at all prolonged, there are marked inanition and prostration.

At autopsy in early stages there may be found only a severe catar¬
rhal inflammation of the mucous membrane of the large intestine. In
the later stages there are extensive ulcerations, and the bacteria are
histologically found lodged within the depths of the mucosa and sub¬
mucosa. Occasionally they may penetrate to the mesenteric glands, but
as far as we know there is no penetration into the general circulation.

Poisonous Products of the Dysentery Bacilli. — The separate types of
dysentery bacilli vary exceedingly in. their powers to pro Luce toxic
substances. Of all the various types which have been described, the
strongest poisons have been produced with bacilli of the Shiga-Kruse
variety, less regularly active ones with bacilli of the Flexner and of the
“ Y ” tyPe- In fact, investigations carried out with the Shiga bacillus
have tended to show that the disease itself is probably a true toxemia,

j Zinsser , Proc. N. Y. Path. Soc., 1907. 2 Shiga, Cent. f. Bakt., xxiii, 1898.

THE DYSENTERY BACILLI

443

its symptoms being referable almost entirely to the absorption of the
poisonous products of the bacillus from the intestine.

The earliest investigations, carried on chiefly upon rabbits, which
are more susceptible to this poison than any other animals, showed that
even small doses of cultures of this bacillus administered intravenously
or subcutaneously would produce death within a very short time.
Conradi,1 Vaillard2 and Dopter, and others, finding that toxic symptoms
were almost as pronounced when dead cultures were given as when the
living bacilli were administered, came to the conclusion that the poisons
of this bacillus wTere chiefly of the endotoxin type. More recently Todd,3
Kraus,4 and Rosenthal5 have claimed independently that they were
able to demonstrate strong soluble toxins, similar in every way to diph¬
theria toxin. Kraus and Doerr,6 moreover, claim to have further cor¬
roborated this by producing specific antitoxins with these substances.

It is easy to obtain poisonous substances from dysentery cultures
in considerable strength, both by extracting the bacilli themselves
and by filtration of properly prepared cultures. It is therefore not
unlikely that both types of poison are produced by the bacilli.
Neisser and Shiga 7 obtained toxins by emulsifying agar cultures in
sterile salt solution, killing the bacilli at 60° C., and allowing them to
extract at 37.5° C. for three days or more. The filtrates from such emul¬
sions were extremely toxic. The simplest method of obtaining poisons
from these bacilli is to cultivate them for a week or longer upon moder¬
ately alkaline meat-infusion broth. At the end of this time, the micro¬
organisms themselves may be killed by heating to 60° and the cultures
filtered. According to Doerr,8 the toxins may be obtained in the dry
state by precipitation with ammonium sulphate and re-solution of the
precipitate in water.

The action of the dysentery toxin upon animals is extremely
characteristic and throws much light upon the disease in man. The
injection of a large dose intravenously into rabbits causes a rapid
fall in temperature, marked respiratory embarrassment, and a violent

1 Conradi, Deut. med. Woch., 1903.

2 Vaillard et Dopter, Ann. de l’inst. Pasteur, 1903.

3 Todd, Brit. Med. Jour., Dec., 1903, and Jour, of Hyg., 4, 1904.

< Kraus, Monatschr. f. Gesundheit, Suppl. 11, 1904.

5 Rosenthal , Deut. med. Woch., 1904.

■* Kraus und Doerr, Wien. klin. Woch., xlii, 1905.

7 Neisser and Shiga, Deut. med. Woch., 1903.

8 Doerr, “ Das Dysenterietoxin,” Jena, 1907.

444

PATHOGENIC MICROORGANISMS

diarrhea. This is at first watery, later contains large amounts of blood.
If the animals live a sufficient length of time, paralysis may occur, the
animal may fall to one side or may drag its posterior extremities. It is
a remarkable fact that intravenous inoculation gives rise to intestinal
inflammation of a severe nature, unquestionably due to the excretion
of the poison by the intestinal mucosa and limited, usually, to the ce¬
cum and colon, rarely attacking the small intestine. Flexner,1 who has
experimented extensively upon this question, believes it probable that
most of the pathological lesions occurring in the intestinal canal of dysen¬
tery patients are referable to this excretion of dysentery toxin, rather
than to the direct local action of the bacilli.

Toxins from the Shiga-Kruse type are the most potent and those
which cause paralysis.

Immunization with Dysentery Bacilli. — The immunization of small
animals, such as rabbits and guinea-pigs, against dysentery bacilli,
especially those of the Shiga type, is attended with much difficulty,
owing to the great toxicity of the cultures. Nevertheless, successful
results may be accomplished by the administration of extremely small
doses of living or dead bacilli, increased very gradually and at sufficient
intervals. Horses may be more easily immunized. The serum of such
actively immunized animals contains agglutinins in considerable con¬
centration and of a specificity sufficiently illustrated in the preceding
section dealing with the identification of the various species. For
diagnostic purposes in human beings, the agglutination reaction, accord¬
ing to the technique of the Widal reaction for typhoid fever, has been
utilized by Kruse 2 and others. According to most observers, normal
human serum never agglutinates dysentery bacilli in dilutions greater
than one in twenty, while the serum of' dysentery patients will often be
active in dilutions as high as one in fifty.

Bactericidal substances have been demonstrated in the serum of im¬
munized animals as well as in the serum of diseased human beings.
These have been determined, in vitro , by Shiga,3 and by the intraperito-
neal technique of Pfeiffer by Kruse.4 Bacteriolysis may take place in
high dilutions of the serum, and has recently been used for the differen¬
tiation of the types of the dysentery bacilli by Ohno.5

True antitoxins in immune sera have been recently described by
Kraus and Doerr.6

1 Flexner, Jour. Exp. Med., 8, 1906. 2 Kruse, Deut. med. Woch., 1901.

3 Shiga, Zeit. f. Hyg., xli. 4 Kruse, Deut. med. Woch., 1903,

5 Ohno, Philippine Jour, of Sci., vol. i, 1906. 6 Krcms und Doerr, loc. cit.

Fig. 93. — Scheme of the Fermentations of the Dysentery-Typhoid-Colon Group of Bacilli in

THE DYSENTERY BACILLI

445

446

PATHOGENIC MICROORGANISMS

Passive immunization of animals and human beings with the serum
of highly immunized horses has been variously attempted by Shiga/
Kraus/ Gay/ and others. All these observers have reported distinct
benefit to the patients and a reduction of the mortality by the use of
such sera. Striking and rapid reductions of temperature and rapid con¬
valescence, after a single injection, have occasionally been observed.
The earlier workers were inclined to attribute the beneficial results of
these sera entirely to their bactericidal value.

Todd has recently demonstrated that the mixture of such an immune
serum with solutions of toxin and exposure of the mixture at 37.5° C,
for a half hour would produce almost complete neutralization of the
poison, thus demonstrating that at least a large part of the beneficial
action of the immune sera was due to a true antitoxic process. Be¬
cause of the different varieties of dysentery bacilli, polyvalent serum has
been recommended. Prophylactic vaccination of human begins with
dead dysentery cultures has, so far, led to no practical result.

Shiga, Deut. med. Woch., 1901. 2 Kraus, loc. cit.

3 Gay, Penn. Med. Bull., 1902.

CHAPTER XXX

BACILLUS MUCOSUS CAPSULATUS, BACILLUS LACTIS AEROGENES,

BACILLUS PROTEUS

BACILLUS MUCOSUS CAPSULATUS

(. Bacterium pneumoniae, Friedldnder’s bacillus, Pneumobacillus )

In 1882, Friedlander 1 announced the discovery of a microorganism
which he believed to be the incitant of lobar pneumonia and which, in
his original communications, he described as a “ micrococcus.”

A superficial morphological resemblance between Friedlander’s
microorganism and Diplococcus lanceolatus, now recognized as the most
frequent cause of lobar pneumonia, led, at first, to much confusion, and
it was not until several years later, owing to the careful researches of
Frankel 2 and of Weichselbaum,3 that the “micrococcus” of Friedlander
was recognized as a short, encapsulated bacillus which occurred in
lobar pneumonia exceptionally only. Similar bacilli were subsequently
found by other observers, bacilli which, mainly upon morphological
grounds, are classified together as the “Friedlander group,” or the
“group of Bacillus mucosus capsulatus.”

Morphology and Staining. — The Friedlander bacillus is a short, plump
bacillus with rounded ends, subject to great individual variations as to
size. Its average measurements are from 0.5 to 1.5 micra in width and
0.6 to 5 micra in length. Forms approaching both extremes may be met
with in one and the same culture. The short, thick forms, frequently
found in animal and human lesions, are almost coccoid and account for
Friedlander’ s error in first describing the bacillus as a micrococcus.
The bacilli may be single, in diplo-form, or in short chains. They are
non-motile and possess no flagella. Spores are not formed.

The bacillus' is characteristically surrounded by a well-developed
capsule which is most perfectly demonstrated in preparations taken
directly from some animal fluid, such as the secretion or exudate
from infected areas. It is also seen, however, in smears made from agar

1 Friedlander, Virchow’s Arch., lxxxvii, 1882; Fort. d. Med., i, 1883; ibid.,
ii, 1884.

2 Frankel, Zeit. f. klin. Med., x, 1886.

? Weichselbaum, Med. Jahrb., Wien, 1886.

447

448

PATHOGENIC MICROORGANISMS

or gelatin cultures. The capsule is usually large, twice or three times
the size of the bacillus itself. When seen in chains or in groups, several
bacilli may appear to be inclosed in one capsule. Prolonged cultivation
on agar or gelatin may result in disappearance of the capsule. The bacil¬
lus is easily stained with the ordinary dyes, but is decolorized when
stained by the Gram-method. Capsules may often be seen when the
more intense anilin dyes are employed. They are brought out with much
regularity by any of the usual capsule stains.

Cultivation. — B. mucosus capsulatus is easily cultivated. It grows

Fig. 94. — Bacillus mucosus capsulatus.

readily on all the usual culture media, both on those having a meat-
infusion basis and on those made with meat extract. Growth takes
place at room temperature (18° to 20°) and more rapidly at 37.5° C. A
temperature of 60° C. and over kills the bacilli in a short time. The ther¬
mal death-point according to Sternberg is 56° C. Growth ceases below
10° to 12° C. Kept at room temperature and protected from drying,
the bacillus may remain alive, in cultures, for several months. \

The bacillus is not very fastidious as to reaction of media, growing

BACILLUS MUCOSUS CAPSULATUS

449

equally well on moderately alkaline or acid media. It is aerobic and
facultatively anaerobic; growth under anaerobic conditions, however,
is not luxuriant.

On agar, growth appears in the form of grayish-white mucus-like
colonies, having a characteristically slimy and semi-fluid appearance.
Colonies have a tendency to confluence, so that on plates, after three or
four days, a large part of the surface appears as if covered with a film of
glistening, sticky exudate, which, if fished, comes off in a tenacious,
stringy manner. It is often possible to make a tentative diagnosis of
the bacillus from the appearance of this growth.

In broth , there is rapid and abundant growth, with the formation
of a pellicle, general clouding, and later the development of a profuse,
stringy sediment.

Stab cultures in gelatin show, at first, a white, thin line of growth
along the course of the puncture. Soon, however, rapid growth at
the top results in the formation of a grayish mucoid droplet on the
surface, which, enlarging, gives the growth a nail-like appearance. This
nail-shape was originally described by Friedlander and regarded as diag¬
nostic for the bacillus. The gelatin is not fluidified. As the culture grows
older the entire surface of the gelatin tube may be covered with growth,
flowing out from the edges of the nail-head. The gelatin acquires a darker
color and there may be a few gas bubbles below the surface. Micro¬
scopically, colonies on gelatin plates have a smooth outline and a finely
granular or even homogeneous consistency.

On blood serum , a confluent mucus-like growth appears.

On potato , abundant growth appears, slightly more brownish in color
than that on other media.

In pepto?i solutions, there is no indol formation.

In milk, there is abundant growth and marked capsule develop¬
ment. Coagulation occurs irregularly.

In considering the general cultural characteristics of the Fried¬
lander bacillus, it must not be forgotten that we are dealing with a
rather heterogeneous group, the individuals of which are subject to
many minor variations. Capsule development, lack of motility, in¬
ability to fluidify gelatin, failure to form indol, and absence of spores, are
, characteristics common to all. In size, general appearance, gas forma¬
tion, and pathogenicity, individual strains may vary much, one from
the other. Strong 1 has studied various races as to, gas formation and

1 Strong, Cent. f. Bakt., xxv, 1899.

450

PATHOGENIC MICROORGANISMS

concludes that most strains form gas from dextrose and levulose, but
that lactose is fermented by some only. About two-thirds of the gas
formed is hydrogen, the rest C02. Acid formation, according to Strong,
is also subject to much variation among different races. Similar studies
by Perkins 1 show that most of the ordinary cultural characteristics of
bacilli of this group are extremely variable and can not serve as a basis
for differentiation. Reactions on sugars, however, are more constant.
Perkins suggests the following tentative division classes on this basis:

I. All carbohydrates fermented with the formation of gas.

II. All carbohydrates, except lactose, fermented with the formation
of gas.

III. All carbohydrates, except saccharose, fermented with the
formation of gas.

Type I. corresponds to B. aerogenes (Migula), Type II. to B.
Friedlander or Bacterium pneumoniae (Migula), and Type III. to
Bacillus lactis aerogenes.

Differentiation by means of serum reactions has not proved satis¬
factory.2

Pathogenicity. — When Friedlander first described this microorganism,
he assumed it to be the incitant of lobar pneumonia. Subsequent re¬
searches by Weichselbaum 3 and others have shown it to be etiologically
associated with pneumonia in about seven or eight per cent of all cases.
The percentage in this country is probably lower. Such cases can often
be diagnosed by the presence of the bacilli in the sputum, which is pecul¬
iarly sticky and stringy. Cases of Friedlander pneumonia are extremely
severe and usually fatal. The bacillus has been found in cases of ulcer¬
ative stomatitis and nasal catarrh; in two cases of severe tonsillitis in
children (Zinsser) ; in the pus from suppurations in the antrum of High-
more and the nasal sinuses (Frankel and others), and in cases of fetid
coryza (ozena), of which disease it is supposed by Abel 4 and others to
be the specific cause. Whether the ozena bacillus represents a separate

1 Perkins, Jour, of Infect. Dis., I, No. 2, 1904.

2 J. G. Fitzgerald, who has recently made a careful study of the mucosus cap-
sulatus group has concluded that present methods do not permit a subdivision of
these organisms into separate species. He offers the following “ tentative suggestion ” :
It is conceivable that mutations based on the necessity of maintaining a parasitic
existence have caused Gram-negative bacilli found normally in the body elsewhere
than in the intestinal tract to develop capsules for protection and a new group has
arisen which we designate B. mucosus capsulatus; and the varieties B. aerogenes
and B. acidi lactici connect the group with the non-encapsulated colon group.”

3 Weichselbaum, loc. cit. 4 Abel, Zeit. f. Hyg., xxi.

BACILLUS OF RHINOSCLEROMA

451

species or not, can not at present be decided. The bacillus of Fried-
lander has been found in empyema fluid, in pericardial exudate (after
pneumonia), and in spinal fluid.1 Isolated cases of Friedlander bacillus
septicemia have been described.2 Being occasionally a saprophytic
inhabitant of the normal intestine, it has been believed to be etiologic-
ally associated with some forms of diarrheal enteritis.

B. mucosus capsulatus is pathogenic for mice and guinea-pigs, less so
for rabbits. Inoculation of susceptible animals is followed by local in¬
flammation and death by septicemia. If inoculation is intraperitoneal,
there is formed a characteristically mucoid, stringy exudate.

The question of immunization against bacilli of the Friedlander
group is still in the stage of experimentation. Immunization with care¬
fully graded doses of dead bacilli has been successful in isolated cases.
Specific agglutinins in immune serum have been found by Clairmont,3
but irregularly and potent only against the particular strain used for
the immunization.

OTHER BACILLI OF THE FRIEDLANDER GROUP

Bacillus of Rhinoscleroma. — This bacillus, discovered by v. Frisch 4
in 1882, is a plump, short rod, with rounded ends/ morphologically
almost identical with Friedlander’s bacillus; it is non-motile and pos¬
sesses a distinct capsule. Although at first described as Gram-positive,
it has been shown to be decolorized with this method of staining. Cul¬
turally it is almost identical with B. mucosus capsulatus. It forms
slimy colonies, has a nail-like appearance in gelatin stab cultures, and in
pepton solutions produces no indol. It differs from B. mucosus cap¬
sulatus (Wilde 5) in forming no gas in dextrose bouillon, in producing
no acid in lactose bouillon, and in never coagulating milk.

Pathogenicity. — The bacillus of rhinoscleroma is but moderately
pathogenic for animals delicately susceptible to the bacillus of Fried¬
lander. Rhinoscleroma, the disease produced by this bacillus in man,
consists of a slowly growing granulomatous inflammation, located usu¬
ally at the external nares or upon the mucosa of the nose, mouth,
pharynx, or larynx. It is composed of a number of chronic, hard,
nodular swellings, which, on histological examination, show granulation
tissue and productive inflammation. In the meshes of the abundant

1 Jager, Zeit. f. Hyg., xix. 2 Howard, Johns Hopkins Hosp. Bull, 1899.

3 Clairmont, Zeit. f. Hyg., xxxix. 4 v. Frisch, Wien. med. Woch., 1882.

5 Wilde, Cent. f. Bakt., xx, 1896.

452

PATHOGENIC MICROORGANISMS

connective tissue lie many large swollen cells, the so-called Mikulicz
cells." 1 The rhinoscleroma bacilli lie within these cells and in the
intercellular spaces. They can be demonstrated in histological sections
and can be cultivated from the lesions, usually in pure culture. Rhino-
scleroma is rare in America. It is most prevalent in Southeastern
Europe. The disease is slowly progressive and comparatively intract¬
able to surgical treatment, but hardly ever affects the general health
unless by mechanical obstruction of the air passages.

B. Ozaenae. — The work of Abel 2 and others has shown that ozena, or

Fig. 95. — Bacillus of Rhinoscleroma. Section of tissue showing the micro¬
organisms within Mikulicz cells. (After Frankel and Pfeiffer.)

fetid nasal catarrh, is almost always associated with a bacillus morpho¬
logically and culturally almost identical with B. mucosus capsulatus.
The bacillus can not be definitely separated from the latter. According
to Wilde 3 it forms no gas in dextrose bouillon and is less pathogenic
for mice than B. Friedlander. Whether it is a separate species, or
merely an atypical form changed by environment, can not be stated
at present.

1 Mikulicz, Arch. f. Chir., xx, 1876.

3 Wilde, loc. cit.

2 Abel, Zeit. f. Hyg., xxi

BACILLUS LACTIS AEROGENES

453

BACILLUS LACTIS AEROGENES

Bacillus lactis aerogenes is the type of a group which is closely
similar to the colon group and often distinguished from it with difficulty.
It was first described by Escherich 1 in 1885 who isolated it from the
feces of infants. Since then it has been learned that this bacillus is almost
constantly present in milk, and, together with one or two other micro¬
organisms, is the chief cause of the ordinary souring of milk. Apart from
its occurrence in milk, moreover, the bacillus is widely distributed in
nature, being found in feces, in water, and in sewage. It is distinguish¬
able from the colon bacillus chiefly by the fact that it is non-motile,
possesses no flagella, hardly ever forms chains, and, when cultivated upon
suitable media, especially milk, it possesses a distinct capsule. It differs
from the colon bacillus, furthermore, in that it is capable of fermenting
polysaccharids, such as starch, and does not form indol upon pep-
ton media. It is distinguishable from the bacillus of Friedlander
(B. mucosus capsulatus), according to Wilde,3 by its more energetic
gas formation in dextrose broth, its ability to produce acid on lactose
media, and its invariable coagulation of milk.

The bacillus is about 0.5 to 1 micron in width and 2 to 4 micra in
length. It grows easily upon the simplest media, is a facultative anae¬
robe, and grows most abundantly at a temperature between 25° and
30° C.

Upon agar and gelatin it grows readily with a heavy white growth,
the colonies of which have a tendency to confluence and are distinctly
more mucoid in appearance than are those of Bacillus coli.

In broth, it causes a general clouding and a pellicle. The cultures
have a slightly sour or cheesy odor.

On potato, the growth is heavy and gas is formed.

On milk, there is rapid coagulation and acid formation. It is charac¬
teristic of this bacillus that it is capable of producing a large amount of
acid, chiefly lactic, and of being able to withstand these large amounts
of acid without being injured by them.

The pathogenicity of Bacillus lactis aerogenes for man is slight.
Its chief claims to importance lie in its milk-coagulating properties
and its almost constant presence in the human intestine. In infants, it
may give rise to flatulence and it has been occasionally observed as the

1 Escherich, Fort. d. Med., 16, 17, 1885. 2 Wilde, Cent. f. Bakt., xx, 1896.

454

PATHOGENIC MICROORGANISMS

sole incitant of cystitis. Among such cases rare instances have been
observed in which it has formed gas in the bladder (pneumaturia) .
When this occurs the urine is not ammoniacal but remains acid.

Different strains of this bacillus vary much in their pathogenicity
for animals. Wilde claims that it is more pathogenic for white mice and
guinea-pigs than is the bacillus of Friedlander. He speaks of it as the
most virulent member of this group. Kraus, writing in Flueggs's
u Mikroorganismen,” rates its pathogenicity less high.

Closely related to this bacillus, as well as to those of the Friedlander
group, is an encapsulated bacillus isolated from a case of broncho¬
pneumonia by Mallory and Wright,1 2 which is strongly pathogenic foi
mice, guinea-pigs, and rabbits.

BACILLI OF THE PROTEUS GROUP

The bacilli of this group have little pathological interest, but are im¬
portant because of the frequency with which they are encountered in
routine bacteriological work. They may confuse the inexperienced
because of a superficial similarity to bacilli of the colon-tvphoid group.
In form they may be short and plump or long and slender, staining easily
with anilin dyes and decolorizing with Gram’s method. They are
actively motile and possess many flagella. The individuals stain irreg¬
ularly, often showing unstained areas near the center. The type of the
group is found in the so-called Bacillus proteus vulgaris described by
Hauser 3 in 1885.

Bacilli of this group are widely distributed, being found in water,
soil, air, and wherever putrefaction takes place. In fact, proteus is one
of the true putrefactive bacteria possessing the power to cause the cleav¬
age of proteids into their simplest radicles.

Bacillus proteus vulgaris grows best at temperatures at or about
25° C. and develops upon the simplest media. It is a facultative anae¬
robe and forms no spores.

In broth, it produces rapid clouding with a pellicle and the forma¬
tion of a mucoid sediment.

In gelatin , the colonies are characteristically irregular, giving the
name to this group.

Gelatin is rapidly liquefied. Liquefaction, however, is diminished
or even inhibited under anaerobic conditions.

1 Mallory and Wright, Zeit. f. Hyg., xx, 1895.

2 Hauser, “Ueber Faulniss-Bakt.,” Leipzig, 1885.

BACILLUS PROTEUS

455

On agar and other solid media, as well as upon gelatin before lique¬
faction has taken place, characteristic colonies are produced. From the
central flat, grayish-white colony nucleus, numerous irregular streamers
grow out over the surrounding media, giving the colony a stellate
appearance.

On potato , it forms a dirty, yellowish growth.

In milk, there is coagulation and an acid reaction at first; later the
casein is redissolved by proteolysis.

Blood serum is often liquefied, but not by all races.

The pathogenic powers of proteus bacilli are usually slight. Large
doses injected into animals may give rise to localized abscesses. In man
proteus infections have been described as occurring in the bladder; in
most cases, however, in combination with some other microorganism.
The so-called Urobacillus liquefaciens septicus described by Krogius
was probably a variety of this group. Epidemics 1 of meat poisoning-
have been attributed to members of the proteus family by some ob¬
servers. Thus Wesenberg 2 was able to cultivate a proteus bacillus
from putrid meat which had caused acute gastroenteritis in sixty -three
individuals. Similar epidemics have been reported by Silberschmidt,3
Pfuhl,4 and others. In some of these the bacilli proved to be unusually
toxic when injected into animals, but could not be recovered from the
organs after death.

1 Schnitzler, Cent. f. Bakt., viii, 1890. 2 Wesenberg, Zeit. f. Hyg., xxviii, 1898.

3 Silberschmidt, Zeit. f. Hyg.; xxx, 1899. 4 Pfuhl, Zeit. f. Hyg.; xxxv; 1900.

CHAPTER XXXI

BACILLUS TETANI

Lockjaw or tetanus, though a comparatively infrequent disease,
has been recognized as a distinct clinical entity for many centuries.
The infectious nature of the disease, however, was not demonstrated
until 1884, when Carlo 1 and Rattone succeeded in producing tetanus in
rabbits by the inoculation of pus from the cutaneous lesion of a human
case. Nicolaier,2 not long after, succeeded in producing tetanic symp¬
toms in mice and rabbits by inoculating them with soil. In connec¬
tion with the lesions produced at the point of inoculation, Nicolaier
described a bacillus which may have been Bacillus tetani, but which he
was unable to cultivate in pure culture. Kitasato,3 in 1889, definitely
solved the etiological problem by obtaining from cases of tetanus pure
cultures of bacilli with which he was able again to produce the disease
in animals.

Kitasato succeeded where others had failed because of his use of
anaerobic methods and his elimination of non-spore-bearing con¬
taminating organisms by means of heat. His method of isolation
was as follows: The material containing tetanus bacilli was smeared
upon the surface of agar slants. These were permitted to develop at
incubator temperature for twenty-four to forty-eight hours. At the end
of this time the cultures were subjected to a temperature of 80° C. for
one hour. The purpose of this was to destroy all non-sporulating
bacteria, as well as aerobic spore-bearers which had developed into
the vegetative form. Agar plates were then inoculated from the slants
and exposed to an atmosphere from which oxygen had been com¬
pletely eliminated and hydrogen substituted. On these plates colonies
of tetanus bacilli developed.

Morphology and Staining. — The bacillus of tetanus is a slender bacil¬
lus, 2 to 5 micra in length, and 0.3 to 0.8 in breadth. The vegetative
forms which occur chiefly in young cultures are slightly motile and are

1 Carlo e Rattone, Giornale d. R. Acad. d. Torino, 1884.

2 Nicolaier, Inaug. Diss., Gottingen, 1885.

3 Kitasato, Deut. med. Woch., No. xxxi, 1889.

456

BACILLUS TETANI

457

seen to possess 1 numerous peritrichal flagella, when stained by special
methods. After twenty-four to forty-eight hours of incubation, the
length of time depending somewhat on the nature of the medium and
the degree of anaerobiosis, the bacilli develop spores which are char¬
acteristically located at one end, giving the bacterium the diagnostic
drumstick appearance.

As the cultures grow older the spore-bearing forms completely super-

Fig. 96. — Bacillus tetani. Spore stain.

sede the vegetative ones. Very old cultures contain spore-bearing bacilli
and spores only.

The tetanus bacillus is easily stained by the usual anilin dyes, and
reacts positively to Gram’s stain. Flagella staining is successful only
when very young cultures are employed.

Distribution. — In nature, the tetanus bacillus has been found by
Nicolaier and others to occur in the superficial layers of the soil. The

1 Vottaler, Zeit. f. Hyg., xxvii.

458

PATHOGENIC MICROORGANISMS

earth of cultivated and manured fields seems to harbor this organism
with especial frequency, probably because of its presence in the dejecta
of some of the domestic animals.

Biological Characteristics. — The bacillus of tetanus is generally de¬
scribed as an obligatory anaerobe. While it is unquestionably true
that growth is ordinarily obtained only in the complete absence of

oxygen, various observers, notably Ferran 1 and
Belfanti,2 have successfully habituated the bacillus
to aerobic conditions by the gradual increase of
oxygen in cultures. Habituation to aerobic condi¬
tions has usually been accompanied by diminution
or loss of pathogenicity and toxin-formation.
Anaerobic conditions may likewise be dispensed
with if tetanus bacilli be grown in symbiosis with
some of the aerobic bacteria. The addition to
culture media of suitable carbohydrates, and of
fresh sterile liver tissue, has also been found to
render it less exacting as to absolute anaerobiosis.3

Anaerobically cultivated, Bacillus tetani grows
readily upon meat-infusion broth , which it clouds
within twenty-four to thirty-six hours. Anaerobic
broth cultures may be simply made by covering the
surface of the medium with a layer of albolin or
any other oil, and removing the air by boiling.

Upon meat-infusion gelatin at 20° to 22° C. the
tetanus bacillus grows readily, growth becoming
visible during the second or third day. There is
slow fluidification of the gelatin.

On agar , at 37.5° C., growth appears within forty-
eight hours. Colonies on agar plates present a rather
characteristic appearance, consisting of a compact
center surrounded by a loose meshwork of fine fila¬
ments, not unlike the medusa-head appearance of subtilis colonies.
In agar stabs, fine radiating processes growing out in all directions
from the central stab tend to give the culture the appearance of a fluff
of cotton. Milk is a favorable culture medium and is not coagulated.
On potato , growth is delicate and hardly visible.

Fig. 97. — Young
Tetanus Culture
in Glucose Agar.

1 Ferran , Cent. f. Bakt., xxiv, No. 1.

2 Belfanti, Arch, per le sci. med., xvi.

3 Th. Smith, Brown, and Walker, Jour, Med. Res., N, S., ix, 1906.

BACILLUS TETANI

459

The most favorable temperature for the growth of this bacillus is
37.5° C. Slight alkalinity or neutrality of the culture media is most ad¬
vantageous, though moderate acidity does not altogether inhibit growth.
All the media named may be rendered more favorable still by the ad¬
dition of one or two per cent of glucose, maltose, or sodium formate.1
In media containing certain carbohydrates, tetanus bacilli produce acid.
In gelatin and agar, moderate amounts of gas are
produced, consisting chiefly of 002, but with the
admixtures of other volatile substances which give
rise to a characteristically unpleasant odor, not unlike
that of putrefying organic matter. This odor is due
largely to H2 S and methylmercaptan.

The vegetative forms of the tetanus bacillus are
not more resistant against heat or chemical agents
than the vegetative forms of other microorganisms.

Tetanus spores, however, will resist dry heat at
80° C. for about one hour, live steam for about
five minutes; five per cent carbolic acid kills them
in twelve to fifteen hours; one per cent of bichlo-
rid of mercury in two or three hours. Direct
sunlight diminishes their virulence and eventually
destroys them.2 Protected from sunlight and other
deleterious influences, tetanus spores may remain
viable and virulent for many years. Henrijean 3
has reported her success in producing tetanus with
bacilli from a splinter of wood infected eleven
years before.

Pathogenicity. — The comparative infrequency of
tetanus infection is in marked contrast to the wide
distribution of the bacilli in nature. Introduced into
the animal body as spores, and free from toxin, they
may often fail to incite disease, easily falling prey to
phagocytosis and other protective agencies before the vegetative forms
develop and toxin is formed. The protective importance of phagocyto¬
sis was demonstrated by Vaillard and Rouget,4 who introduced tetanus
spores inclosed in paper sacs into the animal body. By the paper cap-

Fig. 98. — Older
Tetanus Culture
in Glucose Agar.

1 Kitasato, Zeit. f. Hyg., 1891.

2 v. Eisler und Pribram, in Levaditi, “Handbuch,” etc., Jena, 1907.

3 Henrijean, Ann. de la soc. med. chir. de Liege, 1891.

4 Vaillard et Rouget, Ann. de Finst. Pasteur, 1892.

460

PATHOGENIC MICROORGANISMS

sules the spores were protected from the leucocytes, not from the body
fluids. Nevertheless, tetanus developed in the animals. The nature of
the wound and the simultaneous presence of other microorganisms seem
to be important factors in determining whether or not the tetanus bacilli
shall be enabled to proliferate. Deep, lacerated wounds, in which there
has been considerable tissue destruction, and in which chips of glass,
wood splinters, or grains of dirt have become embedded, are particularly
favorable for the development of these germs. The injuries of compound
fractures and of gunshot wounds are especially liable to supply these
conditions, and the presence in such wounds of the common pus cocci,
or of other more harmless parasites, may aid materially in furnishing an
environment suitable for the growth of the tetanus bacilli. Apart from
its occurrence following trauma, tetanus has been not infrequently ob¬
served after childbirth,1 and isolated cases have been reported in which
it has followed diphtheria and ulcerative lesions of the throat.2

A definite period of incubation elapses between the time of infection
with tetanus bacilli and the development of the first symptoms. In
man this may last from five to seven days in acute cases, to from four
to five weeks in the more chronic ones. Experimental inoculation of
guinea-pigs is followed usually in from one to three days by rigidity of
the muscles nearest the point of infection. This spastic condition rapidly
extends to other parts and finally leads to death, which occurs within
four or five days after infection.

Autopsies upon human beings or animals dead of tetanus reveal few
and insignificant lesions. The initial point of infection, if at all evident,
is apt to be small and innocent in appearance. Further than a general
and moderate congestion, the organs show no pathological changes.
Bacilli are found sparsely even at the point of infection, and have been
but rarely demonstrated in the blood or viscera. Nicolaier succeeded
in producing tetanus with the organs of infected animals in but eleven
out of fifty-two cases. More recently, Tizzoni 3 and Creite 4 have suc¬
ceeded in cultivating tetanus bacilli out of the spleen and heart's blood
of infected human beings.

The researches of Tarozzi 5 and of Canfora 6 have shown also that
spores may be transported from the site of inoculation to the liver,
spleen, and other organs, and there lie dormant for as long as fifty-one
days. If injury of the organ is experimentally practised and dead tissue

1 Baginsky, Deut. med. Woch., 1893. 4 Creite, Cent. f. Bakt., xxxvii.

2 Foges, Wien. med. Woch., 1895. 5 Tarozzi, Cent. f. Bakt. Orig. xxxviii.

3 Tizzoni , Ziegler’s Beit., vii. 6 Canfora, Cent. f. Bakt. Orig. xlv.

BACILLUS TETANI

461

or blood clot produced, the spores may develop and tetanus ensue.
These experiments may explain cases of so-called cryptogenic tetanus.

Tetanus Toxin. — The pathogenicity of the tetanus bacillus depends
entirely upon the soluble toxin which it produces. This toxin is produced
in suitable media by all strains of virulent tetanus bacilli, individual
strains showing less variation in this respect than do the separate strains
of diphtheria bacilli. While partial aerobiosis does not completely elimi¬
nate toxin formation, anaerobic conditions are by far more favorable for
its development.

The medium most frequently employed for the production of tetanus
toxin is neutral or slightly alkaline beef -infusion bouillon containing five-
tenths per cent NaCl and one per cent pepton. Glucose, sodium formate,
or tincture of litmus may be added, but while these substances increase
the speed of growth of the bacilli they do not seem to enhance the de¬
gree of toxicity of the cultures. Glucose is said even to be unfavorable for
strong toxin development. It is important, too, that the bouillon shall
be freshly prepared.1 There does not seem to be any direct relationship
between the amount of growth and the degree of toxicity of the cultures.
Under anaerobic conditions in suitable bouillon and grown at 37.5° C.,
the maximum toxin content of the cultures is reached in from ten days
to two weeks. After this time the toxin deteriorates rapidly.

Tetanus toxin has been produced without resort to anaerobic
methods by several observers, notably by Debrand,2 by cultivating the
bacilli in bouillon in symbiosis with Bacillus subtilis. By this method,
Debrand claims to have produced toxin which was fully as potent as
that produced by anaerobic cultivation.

The tetanus toxin, in solution in the bouillon cultures, may be sepa¬
rated from the bacteria by filtration through Berkefeld or Chamberland
filters. Since the poison in such filtrates deteriorates very rapidly,
much more rapidly even than diphtheria toxin, various methods have
been devised to obtain the toxin in the solid state. The most useful of
these is precipitation of the poison out of solution by oversaturation
with ammonium sulphate.3 Very little of the toxin is lost by this method
and, thoroughly dried and stocked in vacuum tubes, together with an¬
hydrous phosphoric acid, it may be preserved indefinitely without dete¬
rioration. The precipitate thus formed is easily soluble in water or

1 Vaillard et Vincent, Ann. de l’inst. Pasteur, 1891.

2 Debrand, Ann. de l’inst. Pasteur, 1890, 1902.

3 Brieger und Cohn, Zeit. f. Hyg., xv.

462

PATHOGENIC MICROORGANISMS

salt solution, and therefore permits of the preparation of uniform solu¬
tions for purposes of standardization.

Brieger and Boer 1 have also succeeded in precipitating the toxin
out of broth solution with zinc chloride. Vaillard and Vincent 2 have
procured it in the dry state by evaporation in vacuo.

Brieger and Cohn,3 Brieger and Boer,4 and others have attempted
to isolate tetanus poison, removing the proteids from the ammonium sul¬
phate precipitate by various chemical methods. The purest preparations
obtained have been in the form of fine yellowish flakes, soluble in water,
insoluble in alcohol and ether. Solutions of this substance have failed
to give the usual proteid reactions.

The toxin When in solution is extremely sensitive to heat. Kita-
sato 5 states that exposure to 68° C. for five minutes destroys it com¬
pletely. Dry toxin is more resistant,6 often withstanding temperatures
of 120° C. for more than fifteen minutes. Exposure to direct sunlight
destroys the poison in fifteen to eighteen hours.7

Interesting experiments as to the action of eosin upon tetanus toxin
have been carried out by various observers. Flexner and Noguchi 8
found that five per cent eosin added to the toxin would destroy it
within one hour. This action is ascribed to the photodynamic power
of the eosin.

The toxin exerts an extreme^ low osmotic pressure and is easily
destroyed by electric currents.

Tetanus toxin is one of the most powerful poisons known to us.
Filtrates of broth cultures, in quantities of 0.000,005 c.c., will often prove
fatal to mice of ten grams weight. Dry toxin obtained by ammo¬
nium sulphate precipitation 9 is quantitatively even stronger, values of
0.000,001 grams as a lethal dose for a mouse of the given weight not
being uncommon. Brieger and Cohn10 succeeded in producing a dry
toxin capable of killing mice in doses of 0.000,000,05 gram.

Different species of animals show great variation in their suscepti-

1 Brieger und Boer, Zeit. f. Hyg., xxi.

2 Vaillard et Vincent, Ann. de l’inst. Pasteur, 1891.

3 Brieger und Cohn, loc. cit.

4 Brieger und Boer, Zeit. f. Hyg., xxi.

5 Kitasato, Zeit. f . Hyg., x.

6 Morax et Marie, Ann. de l’inst. Pasteur, 1902.

7 Fermi und Pernossi, Cent. f. Bakt., xv.

8 Flexner and Noguchi, “Studies from Rockefeller Inst.,” v., 1905.

9 Brieger und Cohn., loc. cit.

10 Brieger und Cohn., Zeit. f . Hyg., xv.

BACILLUS TETANI

463

bility to tetanus toxin. Human beings and horses are probably the most
susceptible species in proportion to their body weight. The common
domestic fowls are extremely resistant. Calculated for grams of body
weight, the horse is twelve times as susceptible as the mouse, the guinea-
pig six times as susceptible as the mouse. The hen, on the other hand,
is 200,000 times more resistant than the mouse.

After the inoculation of an animal with tetanus toxin there is always
a definite period of incubation before the toxic spasms set in. This
period may be shortened by increase of the dose, but never entirely
eliminated.1 When the toxin is injected subcutaneously, spasms begin
first in the muscles nearest the point of inoculation. Intravenous
inoculation,2 on the other hand, usually results in general tetanus of
all the muscles. The feeding of toxin does not produce disease, the
poison being passed through the bowel unaltered.

The harmful action of tetanus toxin is generally attributed to its
affinity for the central nervous system. Wassermann and Takaki 3
show that tetanus toxin was fully neutralized when mixed with brain
substance. Other organs — liver and spleen, for instance — showed no such
neutralizing power. The central origin of the tetanic contractions was
made very evident by the work of Gumprecht,4 who succeeded in stop¬
ping the spasms in a given region by division of the supplying motor
nerves.

The manner in which the toxin reaches the central nervous system
has been extensively investigated, chiefly by Meyer and Ransom, and
Marie and Morax. Meyer and Ransom 5 from a series of careful experi¬
ments reached the conclusion that the toxin is conducted to the nerve
centers along the paths of the motor nerves. Injected into the circu¬
lation,6 the toxin reaches simultaneously all the motor nerve endings,
producing general tetanus. In this case too, therefore, the poison from
the blood can not pass directly into the central nervous system, but
must follow the route of nerve tracts.

These observations have been of great practical value in that they
pointed to the desirability of the injection of tetanus antitoxin directly
into the nerves and the central nervous system in active cases.

1 Courmont et Doyen , Arch, de phys., 1893.

2 Ransom, Deut. med. Woch., 1893.

3 Wassermann und Takaki, Berl. klin. Woch., 1898.

4 Gumprecht, Pfluger’s Arch., 1895.

5 Meyer und Ransom, Arch. f. exp. Pharm. u. Path., xlix.

6 Marie et Morax, Ann. de l’inst. Pasteur, 1902.

464

PATHOGENIC MICROORGANISMS

Tetanolysin. — Tetanus bouillon contains, besides the “tetano-
spasmin” described above which produces the familiar symptoms of the
disease, another substance discovered by Ehrlich 1 and named by him
“ tetanolysin.” Tetanolysin has the power of causing hemolysis of
the red blood corpuscles of various animals, and is an entirely separate
substance from tetanospasmin. It may be removed from toxic broth
by admixture of red blood cells, is more thermolabile than the tetano¬
spasmin, and gives rise to an antihemolysin when injected into animals.
For the production, standardization, and use of tetanus antitoxin, see
p. 220 et seq.

1 Ehrlich, Berl. klin. Woch., 1898.

CHAPTER XXXII

BACILLUS OF SYMPTOMATIC ANTHRAX, BACILLUS OF MALIGNANT
EDEMA, BACILLUS AEROGENES CAPSULATUS, BACILLUS

BOTULINUS

BACILLUS OF SYMPTOMATIC ANTHRAX

{Bacillus anthracis symptomatici, Rauschbrand, Charbon symptomatique,

Sarcophysematos bovis )

Symptomatic anthrax is an infectious disease occurring chiefly
among sheep, cattle, and goats. It is colloquially spoken of as “ quarter-
evil77 or “blackleg.77 The disease has never been observed in man. It
was formerly, and is often to-day, confused with true anthrax, largely
because of a superficial similarity between the clinical symptoms of
the two maladies. Bacteriologically, the two microorganisms are in
entirely different classes.

Geographically, symptomatic anthrax is of wide distribution and
infection is usually through the agency of the soil in which the bacillus
is present, probably in the form of spores which may retain viability
and pathogenicity for several years.

Morphology and Staining. — The bacillus of symptomatic anthrax is
a bacillus with rounded ends, somewhat shorter and relatively thicker
than the bacillus of malignant edema, being about four to six micra
long, and five-tenths to six-tenths micra wide. It is usually seen singly
and never forms long chains. The bacillus in its vegetative form is
actively motile and possesses numerous flagella placed about its
periphery. In artificial media it forms spores which are oval, broader
than the rod itself, and placed near, though never actually at, the
end of the bacillary body. This gives the bacillus a racket-shaped
appearance.

It is readily stained with the usual anilin dyes, but is decolorized by
Gram’s method of staining.

Cultivation. — The bacillus is a strict anaerobe. It was obtained in
pure culture first by Kitasato.1 hinder anaerobic conditions it is easily

1 Kitasato, Woch. f. Hyg., 1889.
465

466

PATHOGENIC MICROORGANISMS

cultivated upon the usual laboratory media, all of which are more
favorable after the addition of glucose, glycerin, or nutrose. In all
media there is active gas formation, which, owing to an admixture of
butyric acid, is of a foul, sour odor. The bacillus is not very delicate
in its requirements of a special reaction of media, growing equally well
on those slightly acid or slightly alkaline.

On gelatin 'plates , at 20° C., colonies appear in about twenty-four
hours, usually round or oval, with a compact center about which fine
radiating filaments form an opaque halo. The gelatin is fluidified.

Fig. 99. — Bacillus of Symptomatic Anthrax. (After Zettnow.)

Surface colonies upon agar plates are circular and made up of a
slightly granular compact center, from which a thinner peripheral zone
emanates, containing microscopically a tangle of fine threads.

In agar stabs , at 37.5° C., growth appears within eighteen hours,
rapidly spreading from the line of stab as a diffuse, fine cloud. Gas
formation, especially near the bottom of the tube, rapidly leads to the
formation of bubbles and later to extensive splitting of the medium.
In gelatin stab cultures growth is similar to that in agar stabs, though
'less rapid.

Pathogenicity. — Symptomatic anthrax bacilli are pathogenic for
cattle, sheep, and goats. By far the largest number of cases, possibly
the only spontaneous ones, appear among cattle. Guinea-pigs are very
susceptible to experimental inoculation. Horses are very little suscep-

BACILLUS OF SYMPTOMATIC ANTHRAX

467

tible. Dogs, cats, rabbits, and birds are immune. Man also appears
to be absolutely immune. Spontaneous infection occurs by the en¬
trance of infected soil into abrasions or wounds, usually of the lower
extremities. Infection depends to some extent upon the relative de¬
gree of virulence of the bacillus — a variable factor in this species.
Twelve to twenty-four hours after inoculation there appears at the
point of entrance a soft, puffy swelling, which on
palpation is found to emit an emphysematous crack¬
ling. The emphysema spreads rapidly, often reaching
the abdomen and chest within a day. The course
of the disease is extremely acute, the fever high,
the general prostration extreme. Death may result
within three or four days after inoculation.

At autopsy the swollen area is found to be
infiltrated with a thick exudate, blood-tinged and
foamy. Subcutaneous tissue and muscles are
edematous and crackle with gas. The internal
organs show parenchymatous degeneration and
hemorrhagic areas. The bacilli, immediately after
death, are found but sparsely distributed in the
blood and internal organs, but are demonstrable in
enormous numbers in the edema surrounding the
central focus.

If carcasses are allowed to lie unburied for some
time, the bacilli will attain a general distribution,
and the entire body will be found bloated with gas,
the organs filled with bubbles. Practically identical
conditions are found after experimental inocula¬
tion.

Toxins. — According to the investigations of Le-
clainche and Vallee,1 the bacillus of symptomatic
anthrax produces a soluble toxin. It is not formed
to any extent in ordinary broth, but is formed in
considerable quantities in broth containing blood or albuminous ani¬
mal fluids.

The best medium for obtaining toxin, according to the same authors,
is the bouillon of Martin,2 made up of equal parts of veal infusion and a

Fig. 100. — Ba¬
cillus of Symp¬
tomatic Anthrax.
Culture in glucose
agar.

1 Leclainche et Vallee, Ann. de Finst. Pasteur, 1900.

2 Martin, Ann. de Finst. Pasteur, 1898.

408

PATHOGENIC MICROORGANISMS

pepton solution obtained from the macerated tissues of the stomachs of
pigs. The toxin contained in filtrates of such cultures is quite resistant
to heat, but rapidly deteriorates if free access of air is allowed.

Immunity. — Active immunization against the bacillus of symptom¬
atic anthrax was first accomplished by Arloing 1 and his collaborators
by the subcutaneous inoculation of cattle with tissue-extracts of in¬
fected animals. The work of these authors resulted in a practical
method of immunization which is carried out as follows:

Two vaccines are prepared. Vaccine I consists of the juice of in¬
fected meat, dried and heated to 100° C. for six hours. Vaccine II is a
similar meat-juice heated to 90° C., for the same length of time. By
the heating, the spores contained in the vaccines are attenuated to
relatively different degrees. Vaccine I in quantities of 0.01 to 0.02
c.c. is emulsified in sterile salt solutions and injected near the end of
the tail of the animal to be protected. A similar quantity of Vaccine II
is injected in the same way fourteen days later.

This method has been retained in principle, but largely modified in
detail by various workers. Kitt 2 introduced the use of the dried and
powdered whole meat instead of the meat juice, and made only one
vaccine, heated to 94° C., for six hours. This method has been largely
used in this country.3 Passive immunization with the serum 4 of
actively immunized sheep and goats has been used in combination with
the methods of active immunization.

BACILLUS OF MALIGNANT EDEMA

(. Bacillus oedematis maligni , Vibrion septique)

In 1877, Pasteur 5 described a bacillus which he had found in guinea-
pigs and rabbits experimentally inoculated with putrefying animal
tissues. This bacillus, which he named “Vibrion septique,” he suc¬
ceeded in cultivating only under anaerobic conditions and in an impure
state, and described as its pathognomonic characteristics the formation
of an extensive edema in and about the point of inoculation.

1 Arloing, Cornevin, et Thomas, 11 Le Charbon Sympt.,” etc., Paris, 1887. Ref.
from Grassberger und Schattenfroh, Kraus und Levaditi, “ Handbuch,” etc., vol.
i, pt. 2.

2 Kitt, Ref. from Grassberger und Schattenfroh, loc. cit.

3 Report of Bureau of Animal Ind., Wash., 1902.

4 Arloing, Leclainche, et Vallee, loc. cit.

6 Pasteur, Bull, de Pacad. de med., 1877, p. 793.

BACILLUS OF MALIGNANT EDEMA

469

Koch/ who studied this infection in connection with his work upon
anthrax in 1881, called attention to the fact that the bacillus described
by Pasteur did not produce a true septicemia, and suggested the term
“ Bacillus of malignant edema/’ which is now in general use.

Gaffky 1 2 found that, apart from its presence in putrid material, the
bacillus occurred in the upper layers of garden soil and in dust. It
has since been found to be widely distributed in nature and in the
intestines of animals and of man. Its wide distribution is unques¬
tionably due to the great resistance of its spores.

Morphology and Staining.— The bacillus of malignant edema is a

Fig. 101. — Bacillus of Malignant Edema. (After Frankel and Pfeiffer.)

long slender rod, not unlike the anthrax bacillus, but decidedly more
slender. Its average measurements are 1 micron in thickness and 3
to 8 micra in length. It usually occurs as single rods, but frequently
appears in long threads showing irregular subdivisions. Often no sub¬
divisions can be seen and the threads appear as long, homogeneous
filaments. These threads are less frequently seen in preparations from
solid media than in those from bouillon or edema fluid. The bacilli
are motile and possess numerous laterally placed flagella. Their motil¬
ity is never very marked and is often entirely absent. The bacillus

1 Koch, Mitt. a. d. kais. Gesundheitsamt, i, 1881, p. 52 et seq.

2 Gaffky, Mitt. a. d. kais. Gesundheitsamt, 1881.

470

PATHOGENIC MICROORGANISMS

produces spores at temperatures above 20° C., which are oval, irregularly
placed either in the center or slightly nearer one or the other end, and
cause a bulging of the bacillary body.

It is readily stained by any of the usual anilin dyes. Stained by
Gram’s method it is decolorized.

Cultivation. — Bacillus oedematis maligni is strictly anaerobic.

Under anaerobic conditions it develops readily upon
any of the usual artificial media. The bacillus is not
very sensitive to the reaction of media and grows
more luxuriantly in all media to which glucose has
been added. In all media it forms, by the cleavage of
proteids, putridly offensive gases.

In gelatin at room temperature, colonies develop in
about three days as small grayish spherical growths,
which microscopically show an arrangement in radial
filaments. The gelatin is fluidified.

In gelatin stab cultures growth begins as a white
column extending to within a centimeter of the top
of the medium. Soon irregularly radiating processes
develop laterally and gas bubbles appear, breaking up
the medium.

Stab cultures in agar show growth within twenty-
four to thirty-six hours at 37.5° C., appearing at first
as a white line, but soon showing a cloud-like lateral
extension along the entire line of the stab. If sugar
is present bubbles appear throughout the medium.

In broth there is general clouding and a granular
sediment; no pellicle is formed. Milk is slowly coag¬
ulated. On blood serum growth is very luxuriant.
On potato , a medium used in the earliest studies of
the bacillus by Gaffky, the bacillus grows readily.

Isolation may be accomplished by any of the
ordinary anaerobic plating methods. The bacillus
can usually be obtained for subsequent isolation by
injection of a susceptible animal with soil, especially that of gardens or
manured fields.

Pathogenicity. — The bacillus is pathogenic for mice, guinea-pigs,
rabbits, horses, dogs, sheep, pigs, some birds, and man. Cattle were
formerly regarded as immune, an opinion which has since been found to
be erroneous.

Fig. 102. — Ba¬
cillus of Ma¬
lignant Edema.
Culture in glu¬
cose agar.

BACILLUS AEROGENES CAPSULATUS

471

Subcutaneous inoculation of pure culture into a susceptible subject
produces, within twenty-four to thirty-six hours, an acute edematous
inflammation about the point of inoculation. The edema extends
throughout the subcuticular and deeper layers, and consists of thin,
slightly bloody fluid. Neighboring lymph nodes become swollen and
hemorrhagic. In the mixed infections of accidental inoculation, but
more rapidly in experimental inoculations with pure cultures, gas is
formed and consequent subcutaneous emphysema. Together with this
there are symptoms of general toxemia. In the smaller test animals this
disease is usually fatal. At autopsy the bacilli are found in the edema
fluid about the local lesion. At autopsies done soon after death, the
organisms are not found in the blood or internal organs. Later they
may be generally distributed throughout the body. In mice only may
the bacilli enter the blood stream before death. The internal organs
of animals dead of this infection usually show parenchymatous degen¬
eration and occasionally hemorrhages.

Malignant edema is not a frequent disease. It has been occasionally
observed in horses, in cattle, and in sheep. In man the infection usually
appears after traumatism or secondarily after compound fractures or
upon the site of suppurating wounds. Isolated cases have been de¬
scribed as arising after hypodermic injections. One case has been
reported as arising in the uterus after instrumental abortion.

Immunity. — Recovery from an infection with the bacillus of malig¬
nant edema produces immunity against subsequent infections.1 The
bacillus in fluid media produces small amounts of a soluble toxin which
in bacteria-free filtrates is capable of killing guinea-pigs. Relatively
large quantities of filtrate must be employed. Roux and Chamberland,2 3
the first to work upon these toxins, were able, by means of them, to
immunize guinea-pigs. Similar immunity could be produced by treat¬
ment with the toxic, filtered sera of animals dead of the diseased

BACILLUS AEROGENES CAPSULATUS

Bacillus aerogenes capsulatus was first observed by Welch and fully
described by Welch and Nuttall 4 in 1892. It is identical with a bacillus

1 Arloing et Chauveau, Ann. de med. vet., 1884.

2 Roux et Chamberland, Ann. de l’inst. Pasteur, 1887.

3 Sanfelice, Zeit. f. Hyg., xiv.

* Welch and Nuttall, Bull. Johns Hopkins Hosp., iii, 1892, p. 81.

472

PATHOGENIC MICROORGANISMS

later described by Frankel,1 and named by him Bacillus phlegmonis
emphy sematosse .

Similar, probably identical, bacilli have been found and reported sub¬
sequently by other observers, ignorant of the work of Welch and Nuttall.
Such are the B. perfringens (Veillon and Zuber), and B. emphysematis
vaginae (Lindenthal, Wien. klin. Woch., 1897), and others.

Welch 2 first obtained the bacillus from the intravascular blood of a
case of ruptured aortic aneurysm, autopsiecl six hours after death, his
attention being called to the blood particularly by the existence of air
bubbles throughout the vessels.

Apart from its occurrence in infected subjects, the bacillus finds wide
distribution in nature, being found in soil, dust, and brackish water,
and in the normal intestinal tracts of human beings and mammals.3

Morphology and Staining. — Bacillus aerogenes capsulatus appears
usually as a straight rod, not unlike the anthrax bacillus, but more
variable in length and somewhat thicker in proportion than the latter.
Occasionally bacilli are seen which are slightly curved, but these are
rare. The bacillus averages 3 to 6 micra in length, but may be three
or four times longer than this. More rarely the bacillus may appear so
short as to be almost coccoid. In artificial cultures it is usually thicker
and shorter than it is in animal tissues. The bacilli are generally single,
but are often seen in short chains. Their ends are usually slightly
rounded but, especially when in chains, may be almost square. Chain-
formation seems to occur chiefly in the blood, long chains never occurring
in artificial media. This characteristic is regarded by Welch as a dis¬
tinguishing feature in differentiating this bacillus morphologically from
anthrax. In their first publication, Welch and Nuttall did not describe
spores as appearing in these bacilli. Dunham,4 however, in 1895, found
spores in cultures grown upon blood serum. The spores seem to be
formed only upon special media, rarely upon plain agar, never in the
animal body. The spores are oval, and may be placed centrally or
toward one end, one in each bacillus.

The bacillus is non-motile, and does not possess flagella. It pos-

1 Frankel, Cent. f. Bakt., xiii, 1893, p. 13.

2 Welch, Bull. Johns Hopkins Hosp., xi, 1900, p. 185.

3 Although B. aerogenes capsulatus and B. phlegmonis emphysematosae are
separately described in many books, notably Migula’s “ System d. Bakteriologie,”
the microorganisms have been shown beyond question to be identical and are
acknowledged to be so by Frankel himself.

4 Dunham, Johns Hopkins Hosp. Bull., Vlii, 1897, p. 68.

BACILLUS AEROGENES CAPSULATUS

473

sesses a capsule which, however, can not be constantly demonstrated.
The capsules are best seen when preparations are made from animal
fluids, but can often be demonstrated in those stained from artificial
cultures. They are demonstrated best by one or the other of the
ordinary capsule stains.

The bacillus is stained easily by the usual anilin dyes. In tissue
preparations, the bacilli regularly retain the gentian-violet when stained
by Gram’s method. In smears from artificial culture media, while
most of the bacilli stain by Gram, many will be seen wholly or partially
decolorized, owing probably to the rapid production of involution forms.

Cultivation. — Bacillus aerogenes capsulatus is an obligatory anaerobe.
The first cultivations by Welch and Nuttall were made in deep agar
stabs. It grows well upon all the usual media, preferring a neutral or
slightly alkaline reaction. All media are improved for the cultivation
of this bacillus by the addition of glucose, lactose, or some other easily
fermented carbohydrate.

Upon agar or gelatin plates, growth appears at 37.5° C. within
twenty-four hours, as a flat, grayish translucent round disk. The
margins of colonies are slightly irregular and fringed. Gelatin is slowly
liquefied by the large majority of cultures, but Welch states that occa¬
sionally liquefaction does not occur.

In deep agar stabs or in agar slant cultures, especially in those con¬
taining a carbohydrate, there is a rapid formation of gas bubbles, a
characteristic which is especially well developed and lends the cultures
of this bacillus their chief diagnostic feature.

In broth , growth is heavy and abundant. At first there is general
clouding. Within forty-eight hours, however, a heavy, white, flocculent
sediment is formed. Owing to the formation of gas, broth tubes if
undisturbed usually show a light froth of bubbles on the surface.

On potato, growth is scanty and the medium possesses no advantages
either for cultivation or diagnosis. On coagulated blood serum, growth
is heavy and rapid and this medium is especially adapted for spore
formation. There is slight peptonization of the blood serum. In milk,
there is rapid coagulation, rapid acidification, and gas formation.

The carbohydrates, glucose, lactose, and saccharose, are fermented
by this bacillus. Mannit is apparently not fermented.

Welch and Nuttall state that the bacillus is capable of producing gas
from proteid matter. The gas formed, according to Dunham,1 consists of

1 Dunham ,, loc. cit.

474

PATHOGENIC MICROORGANISMS

64 per cent of hydrogen, 28 per cent of C02, and 8 per cent of a mix¬
ture of gases, chiefly nitrogen. The gas from the infected animal body
is ignitable and burns with a bluish hydrogen flame.

Biological Considerations. — The bacillus, as stated, is anaerobic. Its
anaerobic requirements, however, are less exacting than those of some
other anaerobes, and in stab cultures it will often grow up to the surface
of the stab. It grows best at 37.5° C., but will also develop at room
temperature (20° to 22° C.).

Isolation. — The bacillus may, of course, be isolated by anaerobic
plating methods. It is best isolated, however, from mixed cultures by
animal inoculation. If, for instance, it is desired to obtain it from a
mixed culture or from feces, a suspension of about 1 c.c. of the
suspected material is made in 5 c.c. of sterile salt solution. This
is thoroughly emulsified and filtered through a sterile paper. One
to two c.c. of this suspension is then injected into the ear vein
of a rabbit. After four or five minutes the rabbit is killed. It
is then placed in the incubator for five to eight hours. At the
end of this time, the animal is usually found tensely distended with
gas. At autopsy, gas bubbles will be found distributed through¬
out the organs, most characteristically in the liver, where isolated
bubbles are found covering the surface. From these bubbles cul¬
tures or smears may be taken for identification. Identification is
easily made from its morphology, its capsule, lack of motility, and
gas formation.

Pathogenicity. — Bacillus aerogenes capsulatus is highly pathogenic
for guinea-pigs, but very slightly for rabbits. Its virulence is subject
to great variations, however, some strains showing little if any pathogen¬
icity even for guinea-pigs. In general, its pathogenicity for the ordi¬
nary laboratory animals may be regarded as slight. In man,1 the
bacillus has been isolated from numerous cases of so-called “emphyse¬
matous gangrene”2 (gangrene foudroyant). The infection usually
occurs upon the extremities and is characterized by a ' rapidly necro¬
tizing inflammation, with which there occurs extensive subcutaneous
emphysema. The infection usually follows traumatism,3 especially
compound fractures, and is extremely grave. The bacillus has also
been found in the uterus in puerperal infection,4 and in the fetus

1 Welch and Flexner, Jour. Exp. Med., 1, 1896.

2 Mann, Ann. of Surgery, xix, 1894.

3 Blooclgood, Progressive Med., 1899.

4 Dobbin, Johns Hopkins Hosp. Bull., viii, 1897,

BACILLUS BOTULINUS

475

dead in utero. It has been found in the blood before death, by Gwyn,1
in a case of chorea. The bacillus has also been found in infectious
processes of various other parts of the body, such as the gastrointes¬
tinal and biliary tracts, the lungs, the pleura, and the meninges.

As stated above, this bacillus is frequently present in the normal
intestinal contents. Its presence in abnormally high proportions, as
indicated by a Gram stain of a smear of the feces, has been associated
by a number of observers with various pathological conditions. Herter 2
has recently studied this subject and believes that the abnormal prolif¬
eration of the bacillus in the gastrointestinal tract has in some way
(probably by toxin absorption) an etiological relationship to pernicious
anemia. This assertion, however, can in no way be regarded as
conclusively proven.

BACILLUS BOTULINUS

Meat poisoning was formerly regarded as universally dependent upon
putrefactive changes taking place in infected meat, resulting in the
production of ptomains or other harmful products of bacterial putre¬
faction. It was not until 1888 that certain of these cases were definitely
recognized as true bacterial infections, in which the preformed poison
probably aided only in establishing the infection. Gartner, in that
year, discovered the Bacillus enteritidis, a microorganism belonging to
the group of the paracolon bacilli, and demonstrated its presence both
in the infecting meat and in the intestinal tracts of patients. The char¬
acteristics of this type of meat poisoning have been discussed more
particularly in the section describing the bacillus of Gartner and its
allied forms.

There is another type of meat poisoning, however, which is not only
much more severe (ending fatally in almost 25 per cent of the cases) , but
is characterized by a clinical picture more significant of a profound
systemic toxemia than of a mere gastroenteric irritation. The etio¬
logical factor underlying this type of infection was first demonstrated
by van Ermengem,3 in 1896, and named Bacillus botulinus. van
Ermengem isolated the bacillus from a ham, the ingestion of which
had caused disease in a large number of persons. Of the thirty-four
individuals who had eaten of it, all were attacked, about ten of them

1 Gwyn, Johns Hopkins Hosp. Bull., x, 1899.

2 Herter, “ Bacterial Infection of the Intestinal Tract,” New York, Macmillan, 1907.

s van Ermengem, Cent. f. Bakt., xix, 1896; Zeit. f. Hyg., xxvi, 1897.

476

PATHOGENIC MICROORGANISMS

very severely, van Ermengem found the bacilli in large numbers lying
between the muscle fibers in the ham, and was able to cultivate the same
microorganism from the stomach and spleen of one of those who died of
the infection.

The results of van Ermengem have been confirmed by Romer,1 and
others.

Morphology and Staining. — Bacillus botulinus is a large, straight rod
with rounded ends, 4 to 6 micra in length by 0.9 to 1.2 micra in thickness.
The bacilli are either single or grouped in very short chains. Involu¬
tion forms are numerous on artificial media. The bacillus is slightly
motile and possesses from four to eight undulated flagella, peripherally
arranged. Spores are formed in suitable media, most regularly in
glucose-gelatin of a distinctly alkaline titer. The spores are oval and
usually situated near the end of the bacillus, rarely in its center. Spores
are formed most abundantly when cultivation is carried out at 20°
to 25° C., and are usually absent when higher temperatures are em¬
ployed.

The bacillus is easily stained by the usual aqueous anilin dyes, and
retains the anilin-gentian-violet when stained by Gram. It is necessary,
however, in carrying out Grands stain to decolorize carefully with alco¬
hol since overdecolorization is easily accomplished, leaving the result
doubtful.

Cultivation. — The bacillus is a strict anaerobe. In anaerobic en¬
vironment it is easily cultivated on the usual meat-infusion media. It
grows most readily at temperatures about 25° C., less luxuriantly at
temperatures of 35° C. and over.

The bacillus is delicately susceptible to the reaction of media,
growing only in those which are neutral or moderately alkaline.

In deep stab cultures in one per cent glucose agar, growth is at first
noticed as a thin, white column, not reaching to the surface of the
medium. Soon the medium is cracked and split by the abundant
formation of gas. On agar plates, the colonies are yellowish, opalescent,
and round, and show a finely fringed periphery.

On gelatin, at 20° to 25° C., growth is rapid and abundant, and
differs little from that on agar, except that, besides the formation of
gas, there is energetic fluidification of the medium. On glucose-gelatin
plates, van Ermengem describes the colonies as round, yellowish,
transparent, and composed of coarse granules which, along the periphery

1 Romer, Cent. f. Bakt., xxvii, 1900.

BACILLUS BOTULINUS

477

in the zone of fluidification, show constant motion. The appearance of
the surface colonies on glucose-gelatin plates is regarded by the discov¬
erer as diagnostically characteristic.

In glucose broth there is general clouding and large quantities of
gas are formed. At 35° C. and over, the gas formation ceases after four
or five days, the broth becoming clear with a yellowish-white flocculent
sediment. At lower temperatures this does not occur.

Milk is not coagulated and disaccharids and polysaccharids are not
fermented.

The gas formed in cultures consists chiefly of hydrogen and methane.
All cultures have a sour odor, like butyric acid, but this is not so offensive
as that of some of the other anaerobic organisms.

The bacillus lives longest in gelatin cultures, but even upon these,
transplantations should be done every four to six weeks, since the
spores of this bacillus show less viability and resistance than do those of
most spore-formers.

Pathogenicity. — Botulism or allantiasis, as noticed in human beings,
is, as far as we know, always due to ingestion of infected meat, usually
of ham, canned meats, or sausages (botulus = sausage). Symptoms
appear only after a definite period of incubation which varies from
twenty-four to forty-eight hours. The first definite symptoms are
chilliness, trembling, and giddiness. These manifestations are soon
followed by headache, occasionally by vomiting. In contradistinction
to the meat poisonings caused by other microorganisms, those due to
Bacillus botulinus may show few or no symptoms directly referable to
the intestinal tract. The chief diagnostic characteristics of the disease
are a group of symptoms referable to toxic interference with the cranial
nerves. Loss of accommodation, dilated pupils, ptosis, aphonia, and
dysphagia may occur. Fever is usually absent. Consciousness is rarely
lost. The characteristic symptoms may be produced in various animals
by injection of living cultures or culture filtrates, i.e., toxins. The most
susceptible animals are guinea-pigs. These may be killed by the injec¬
tion of minute quantities of bouillon cultures or of toxin. Preceding
death, which occurs within twenty-four to thirty-six hours, there may
be general motor paralysis, dyspnea, and hypersecretion of mucus from
nose and mouth. Guinea-pigs may be infected per os as well as by
hypodermic injections. Cats, mice, and monkeys are highly susceptible;
rabbits are less so, but still favorable subjects for experimental studies.
Birds, especially pigeons, are highly resistant, but react typically to
large doses. Autopsies upon man or animals dead of botulism show
31

478

PATHOGENIC MICROORGANISMS

general hyperemia of the organs with much parenchymatous degenera¬
tion and many minute hemorrhages.

The bacilli have been found in the spleen after death/ but van
Ermengem does not believe that they are generally distributed during
the course of the disease. It is believed by most of those who have
studied this disease that poisoning in the human subject is due to the
toxins preformed in the infected meat by this bacillus. Experiments
have shown that little or no poison is produced by the bacilli after gain¬
ing entrance to the human or animal body.

The Toxin of B. botulinus. — Bacillus botulinus produces disease
chiefly by means of a strong soluble toxin secreted by it, and absorbed
by the infected subject. This toxin is active in animals and presumably
in man, not only when injected subcutaneously, but also when intro¬
duced through the gastrointestinal canal. The poison has been par¬
ticularly studied by Brieger and his collaborators. It is obtained in
filtrates of alkaline bouillon cultures. It has been precipitated out of
the filtrate by Brieger and Kempner 1 2 by means of a three per cent zinc
chlorid solution (2 volumes of 3 per cent ZnCl2). The toxin thus
obtained was sufficiently powerful to kill a 250-gram guinea-pig in fifty
hours.

Specific action of the toxin for the nerve-cells of the spinal ganglia
has been shown by Marinesco.3 A specific antitoxin has been produced
by Kempner and Pollack.4

1 Stricht, Quoted from van Ermengem, in Kolle und Wassermann.

2 Brieger und Kempner, Deut. med. Woch., xxxiii, 1897.

3 Marinesco, Compt. rend, de l’acad. des sci., Nov., 1896.

4 Kempner und Pollack, Deut. med. Woch., xxxii, 1897.

CHAPTER XXXIII

THE TUBERCLE BACILLUS

In view of the clinical manifestations of tuberculosis, it is not sur¬
prising that the infectious nature of the disease has been suspected for
many centuries. Transmission by means of tuberculous material was
first successfully accomplished by Klencke, in 1843, and, more elabo¬
rately, by Villemin,1 in 1865. It was not until 1882, however, that
Koch 2 succeeded in isolating and cultivating the tubercle bacillus.
Baumgarten 3 had previously seen the bacillus in tissue sections, but his
researches were limited to purely morphological observations. Koch,
in addition to demonstrating the bacillus in tuberculous tissues from
various sources, produced characteristic lesions in guinea-pigs and other
animals by infecting them with pure cultures, and established beyond
doubt the etiological relationship of the bacillus to the disease.

Morphology. — Tubercle bacilli appear as slender rods, 2 to 4 micra
in length, 0.2 to 0.5 micra in width. Their ends are usually rounded.
The rods may be straight or slightly curved; their diameters may be
uniform throughout; more often, however, they appear beaded and
irregularly stained. The beaded appearance is due to different causes.
Unstained spaces may occur along the body of the bacillus, especially
in old cultures. These are generally regarded as vacuoles. The bodies
of the bacilli, on the other hand, may bulge slightly here and there, often
in three or four places, showing oval or rounded knobs which stain with
great depth and are very resistant to decolorization. These thickenings
were formerly regarded as spores, but in view of the fact that the bacilli
are not more resistant against heat and disinfectants than other vegeta¬
tive forms, this interpretation is probably incorrect. The bacilli are said
to possess a cell membrane which confers upon them their resistance
against drying and entrance of stains. This membrane gives a cellulose
reaction and is believed to contain most of the waxy substances which
can be extracted from the cultures.

1 Villemin, Gaz. hebdom., 1865.

2 Koch, Berl. klin. Woch., 1882; Mitt. a. d. kais. Gesundheitsamt, 1884,

3 Baumgarten, Virchow’s Arch., lxxxii.

479

480

PATHOGENIC MICROORGANISMS

Various observers, notably Nocard and Roux,1 Mafucci,2 and Klein,3
have demonstrated branched forms of the tubercle bacillus. These ob¬
servations, variously extended and confirmed, make it probable that
Bacillus tuberculosis is not a member of the family of schizomycetes,

Fig. 103. — Tubercle Bacilli in Sputum.

but belongs rather to the higher bacteria, closely related to the actino-
myces.

Staining. — Tubercle bacilli do not stain easily with the ordinary
anilin dyes; to these they are made permeable only by long exposure
or by heating of the staining solution. Once stained, however, the dye
is tenaciously retained in spite of treatment with alcohol and strong

1 Nocard et Roux, Ann. de l’inst. Pasteur, 1887.

3 Klein, Cent. f. Bakt., 1890.

2 Mafucci, Zeit. f. Hyg., ii.

THE TUBERCLE BACILLUS

481

acids. For this reason, this bacillus, together with some other bacteria
to be mentioned later, is spoken of as “ acid-fast." The acid-fast nature
of the bacillus seems to depend upon the fatty substances contained in
it,1 and has furnished the basis for differential staining methods. All
the staining methods devised for the recognition of the tubercle bacillus
thus depend upon the use of an intensely penetrating staining solution,
followed by vigorous decolorization which deprives all but the acid-fast
group of their color. Counterstains of any of the weaker dyes may
then be used to stain the decolorized elements. One of the first of the
staining solutions to be of practical use was the anilin-water-gentian-
violet solution of Ehrlich 2 (11 c.c. saturated alcoholic gentian-violet
to 89 c.c. 5 per cent anilin water). This dye, although of sufficient
penetrating power, has the disadvantage of deteriorating rapidly and
has in practice been almost entirely displaced by ZiehTs 3 carbol-fuchsin
solution. (Fuchsin 1 gm. in 10 c.c. alcohol absolute, added to 90 c.c.
5 per cent carbolic.) This staining solution is the one now in
general use and is employed as follows: Thin smears, on slides
or cover-slips, are covered with the dye and gently heated. In
the case of cover-glasses, these may be floated, face downward,
in staining dishes filled with the dye. The dye is allowed to act
for about three minutes, steaming but not allowed to boil. At the
end of this time the preparation is washed either with 5 per cent
nitric acid, 5 to 20 per cent sulphuric acid, or 1 per cent hydro¬
chloric acid, until most of the red color has disappeared (a few
seconds), and the preparation appears pale pink. This results in
decolorization of all microorganisms with the exception of members
of the acid-fast group. Thorough washing in 80 to 95 per cent alcohol
is now employed to complete the decolorization. The preparation
is then rinsed in water and counterstained with 1 per cent aqueous
methylene-blue .

Tubercle-bacillus staining has been further simplified by Gabbett,4
who combines decolorization and counterstaining. In this method
preparations are stained with ZiehTs carbol-fuchsin as in the preceding;
they are then rinsed in water and covered with a solution containing
methylene-blue 1 gram, concentrated sulphuric acid 25 grams, and
distilled water 100 c.c. This is allowed to act for from two to four

1 Bienstock, Fort. d. Med., 1886; Weyl, Deut. med. Woch., 1891.

2 Ehrlich, Deut. med. Woch., 1882; Weigert, Deut. med. Woch., 1885.

3 Ziehl, Deut. med. Woch., 1883; Neelsen, “ Lehrb. d. allg. Path./’ 1894

* Gabbett, Lancet, 1887.

482

PATHOGENIC MICROORGANISMS

minutes, at the end of which time all elements in the preparation except
the acid-fast bacilli will be decolorized and counterstained.

Tubercle bacilli in very young culture are often not acid-fast and it
is not always possible to demonstrate acid-fast bacilli in pus from cold
abscesses in sputum, in serous exudates, and in granulomatous lesions
of the lymph nodes which can be shown by animal inoculation to be
tuberculous. Much 1 demonstrated in such material Gram-positive
granules which lay singly in short chains or in irregular clumps, and
which he believed to be non-acid-fast tubercle bacilli. He found similar
granules in cultures of tubercle bacilli which showed on further incuba¬
tion numerous acid-fast bacillary forms. His work has been repeatedly
confirmed, and there seems little doubt but that these granules are really
tubercle bacilli. Their demonstration is not, however, of great diag¬
nostic value, as other bacilli form granules of the same appearance.
Small rods and splinters are also found which stain by Gram’s method,
but not by carbol-fuchsin.2

To find “ Much’s granules,” smears or sections are steamed in a
solution of methyl violet B.N. (10 c.c. of saturated alcoholic solution
of the dye in 100 c.c. of distilled water containing 2 per cent phenol).
They are then treated with Gram’s iodine solution 1-5 minutes; 5 per
cent nitric acid 1 minute; 3 per cent hydrochloric acid 10 seconds; ab¬
solute alcohol and acetone equal parts, until decolorized. The granules
may be stained by other modifications of Gram’s method. Weiss 3
has devised a combination stain. One part of Much’s methyl violet
is mixed with three parts of Ziehl’s carbol-fuchsin and filtered; slides
are stained for 24 to 48 hours in the mixture. They are then decolorized
as in Much’s method and counterstained with Bismarck brown or
safranin 1 per cent. Both acid-fast and Gram-positive forms are
stained by this method and in the red may be seen blue-black granules.

While the acid-fast group of bacteria is composed of a number of
organisms to be mentioned later, a few only of these offer difficulties of
differentiation from the tubercle bacillus. Those to be considered
practically are the bacillus of leprosy and that of smegma. The latter
bacillus, because of its distribution, is not infrequently found to con¬
taminate feces, urine, or even sputum, and it is important to apply to
suspected specimens one or the other of the stains devised for the
differentiation of the smegma bacillus from Bacillus tuberculosis. The

1 Much, Berl. klin. Woch., 1908, xlv, 700.

2 Liebermeister, Deutsche med. Woch., 1909, xxxv, 1324.

3 Weiss, Berl. klin. Woch., 1909, xlvi, 1797.

THE TUBERCLE BACILLUS

483

one most frequently employed is that of Pappenheim.1 The preparations
are stained in hot carbol-fuchsin as before; the carbol-fuchsin is then
poured off without washing and the preparation immersed in a solution
made by saturating a 1 per cent alcoholic solution of rosolic acid with
methylene-blue and adding 20 per cent of glycerin. In such prepara¬
tions tubercle bacilli remain red, smegma bacilli appear blue.

Stained by Gram, tubercle bacilli retain the gentian- violet.

When tubercle bacilli are very sparsely present in sputum and
other material it may be impossible to find them by direct examination,
and often the only method of finding them will be animal inoculation.
However, a number of methods have been devised by which the bacilli
may be concentrated in such a way that they may be found even when
a few only are present. One of these is to add peroxide of hydrogen to
the sputum. By this the mucus is dissolved out and the solid particles
settle or may be centrifugalized. A method very commonly employed
to-day is that which depends on the use of “antiformin.” This is a
preparation used extensively for the cleansing of vats in breweries.
It is described by Rosenau 2 as consisting of equal parts of liquor sodse
chlorinate and a 15 per cent solution of caustic soda. The formula for
liquor sodse chlorinate he gives as:

Sodium carbonate . 600

Chlorinated lime . 400

Distilled water . 4,000

If sputum is poured into a 10 to 15 per cent solution of antiformin
and allowed to stand for several hours, most of the other elements of
the sputum, cells, and bacteria, will dissolve out, and acid-fast bacilli
be left in the residue. Strangely enough they are not killed by this
process and if sufficiently washed may be cultivated or can produce
lesions in guinea-pigs.

Isolation and Cultivation. — Tubercle bacilli are not easily cultivated.
Their slowness of growth precludes their isolation by the usual plating
methods. The first isolations by Koch 3 were made upon coagulated
blood serum from bits of tuberculous tissue smeared over its surface.

Isolation from tuberculous material may be greatly aided by in¬
oculation into guinea-pigs. These animals will often withstand

1 Pappenheim, Berl. klin. Woch., 1898.

2 Rosenau, “Preventive Medicine and Hygiene/’ D. Appleton, N. Y., 1913;
Uhlenhuth , Berl. klin. Woch., No. 29, 1908.

3 Koch, loc. cit

484

PATHOGENIC MICROORGANISMS

the acute infection which may be produced by the contaminating
organisms and succumb at a later date (four to six weeks) to the
tuberculous infection. The bacilli may then be obtained, after sterile
dissection, by making cultivations from lymph nodes or other tubercu¬
lous foci which contain only tubercle bacilli. When isolation from
sputum is attempted, whether directly or by means of animal inocula¬
tion, the sputum may be rendered comparatively free from contaminat¬
ing bacteria by a process of washing devised by Koch. The sputum is
thoroughly rinsed in running water to free it from its outer covering of
pharyngeal mucus. It is then washed in eight or ten changes of sterile
water. The material selected for cultivation is taken from the center
of the washed mass, if possible from the small flakes of caseous material
often visible in such sputum.

On blood serum at 37.5° C., colonies usually become visible at the
end of eight to fourteen days. They appear at first as small, dry, gray¬
ish-white, scaly spots with corrugated surfaces. After three or four
weeks’ cultivation, these join together, covering the surface of the
medium as a dry, whitish, wrinkled membrane. Coagulated dog serum
is regarded by Theobald Smith 1 as one of the most favorable media

for the growth of tubercle bacilli.

Slants of agar, to which whole rabbit’s blood has been added in
quantities of from 1 to 2 c.c. to each tube, make an excellent medium for
this bacillus, both for isolation and continuous cultivation.

Cultivation methods were simplified by the discovery by Roux
and Nocard that growth can be obtained upon media to which glycerin
has been added. Upon glycerin-agar (glycerin 3 to 6 per cent), at
37.5° C., colonies become visible at the end of from ten days to two
weeks, at first as dry, white spots; later, as delicately corrugated
membranes.

Glycerin bouillon (made of beef or veal with pepton one per cent,
glycerin six per cent, and rendered slightly alkaline) is an extremely
favorable medium. It should be filled, in shallow layers, into wide¬
mouthed flasks, since free access of oxygen is essential for growth.
Transplants to this medium should be made by carefully floating flakes
of the culture upon the surface. In this medium the bacilli will spread
out upon the surface, at first as a thin, opaque, floating membrane.
This rapidly thickens into a white, wrinkled, or granular layer, spread¬
ing out in all directions, and covering the entire surface of the fluid in
from four to six weeks. Later, portions of the membrane sink to the

1 Th. Smith, Jour. Exp. Med., iii, 1898.

THE TUBERCLE BACILLUS

485

‘‘Nahrstoff Hey den” 2 . 10 grams

Sodium chloride . 5 “

Glycerin . 30 “

Agar . 10 “

Normal sodium solution. . . 5 c.c.

Aq. dest . 1,000 “

bottom. In old cultures, the membrane assumes a yellowish hue.
These cultures emit a peculiar aromatic odor.

Glycerin potato forms a favorable culture medium for the bacillus.

Hesse 1 has devised a medium containing a proprietary preparation
known as “Nahrstoff Hey den,” upon which tubercle bacilli are said to
proliferate more rapidly than other bacteria. His method has yielded
excellent results in the hands of other
observers, both in isolation and in
rapid cultivation. It is prepared as
follows:

A variety of other culture media
have been devised, none of them, how¬
ever, possessing any marked advan¬
tages over those given.

Biological Considerations. — The
tubercle bacillus is markedly depend¬
ent upon the free access of oxygen.
The optimum temperature for its de¬
velopment is 37.5° C. Temperatures
below 30° and above 42° C. inhibit
its growth. In fluid media, the bacilli
are killed by a temperature of 60° in
fifteen to twenty minutes, by one of 80°

in five minutes, by one of 90° in one

Fig. 104. — Culture of Bacillus
Tuberculosis in Flask of Glyc¬
erin Bouillon.

to two minutes. They will withstand

dry heat at 100° C. for one hour. They are resistant to cold. The
comparatively high powers of resistance of the bacillus are attributed
to the protective qualities of the waxy cell membrane.3

The natural life of cultures, kept in favorable environment, is from

1 Hesse, Zeit. f. Hyg., xxxi.

2 “Nahrstoff Heyden” is prepared in Germany. It is a white powder similar
to nutrose.

3 Th. Smith, Jour. Exper. Med., 1899; Grancher et Ledoux-Lebard, Arch, de med.
exper., 1892; Galtier, Compt. rend, de l’acad. des sci., 1887.

486

PATHOGENIC MICROORGANISMS

two to eight months, varying to some extent with the nature of the
culture medium. The viability of the bacilli in sputum is of great
hygienic importance. In most sputum they may remain alive and
virulent for as long as six weeks, in dried sputum for more than two
months.1

Five per cent carbolic acid kills the bacilli in a few minutes.2 If used
for sputum disinfection, however, where the bacilli are protected by
mucus, complete disinfection by this method requires five to six hours.
Bichloride of mercury is not very efficient for sputum disinfection be¬
cause of the formation of albuminate of mercury.

For room disinfection, formaldehyde gas if thoroughly employed is
efficient. Direct sunlight kills tubercle bacilli in a few hours.

Pathogenicity. — The tubercle bacillus gives rise in man and suscep¬
tible animals to specific phenomena of inflammation which are so
characteristic that a diagnosis of tuberculosis may usually be made on
the basis of the histological examination of material, even without the
finding of tubercle bacilli. The foci of inflammation known as tubercles
have been systematically studied by Baumgarten 3 and many others
and descriptions of them may be found in any text-book of pathological
anatomy.

In man, tuberculosis is by far the most common of diseases.
Naegeli,4 in a large series of autopsies, found lesions of healed or active
tuberculosis in an appalling percentage of cases. His figures are in¬
teresting in showing not only the frequency of the disease, but its rela¬
tion to age. Before one year of age, he finds it very rare. From the first
to the fifth year it is rare, but usually fatal when occurring. From the
fifth to the fourteenth year, one-third of his cases showed tuberculous
lesions; from the fourteenth to the eighteenth year, one-half of the
cases. Between the ages of eighteen and thirty, almost all the cases
examined showed some trace of tuberculous infection. Three-quarters
of these were active, one-quarter healed. Two-fifths of all deaths occur¬
ring at these ages were due to tuberculosis. After the age of thirty,
active lesions gradually diminished in number, healed lesions increased.

In 1900, at a public hearing of the New York Tenement House
Commission, Pryor 5 stated that the average yearly mortality from

1 Schell und Fischer , Mitt. a. d. kais. Gesundheitsamt, 1884.

2 De Toma , Ann. di med., 1886.

3 Baumgarten, Berl. klin. Woch., 1901.

4 Naegeli, Virchow’s Arch., cix, 1900, p. 462.

5 pryor > Med. News, lxxvii, 1900,

THE TUBERCLE BACILLUS

487

tuberculosis in New York amounted to 6,000, and that in Manhattan
alone there were constantly 20,000 persons suffering from the disease.
Cornet 1 estimates that in 1894 the deaths in Germany from all other
infectious diseases amounted to 116,705, while those from tuberculosis
alone amounted to 123,904. Similar statistics might be chosen at will
from the health reports of any large city. While the disease is less
common in rural districts than in large towns, the difference is not so
striking as is generally supposed.

In man, pulmonary infection is by far the commonest type. Be¬
sides this, however, tuberculous processes may be found in the skin, the
bones, the joints, the organs of special sense, and the abdominal viscera
and peritoneum. No part of the human body is exempt from the danger
of infection.

Infection in the human subject may take place by inhalation or
through the skin or the digestive apparatus. V. Behring 2 has within
recent years expressed the belief that a large percentage of all cases of
tuberculosis originate in childhood from infection by way of the intes¬
tinal tract. He determined, as have others since his publication, that
tubercle bacilli may penetrate the intestinal mucosa without causing
lesions. Behring’s contention has caused a great deal of discussion,
and the question he has raised is intimately bound up with the problem
of the virulence of bovine tubercle bacilli for human beings, as he
assumes that the infection is due to the use of infected milk.

The problem is plainly of the greatest importance hygienically, and
for this reason has been diligently investigated during the last few
years. The only reliable available method of approaching it has been
to isolate the tubercle bacilli from large series of diseased human beings
and determine for each case whether the guilty organism belonged to
the human or the bovine type. These types, as we shall see presently,
can be differentiated definitely by cultural characteristics and patho¬
genicity, and it is not likely, at least in the light of our present knowledge,
that the type changes during the sojourn in the human body. Granted
this permanence of type, it is naturally of much value in revealing the
source of an infection, to determine whether or not a human being is
harboring a bacillus of the human type or one of the bovine type.

One of the most valuable contributions made to this problem during
the last three years is that of Park and Krumwiede.3 The accompanying

1 Cornet, “Die Tuberculose,” Wien, 1899, p. 1.

2 v. Behring , Deut. med. Woch., 39, 1903

3 Park and Krumwiede, Jour, of Med. Res., Oct., 1910.

488

PATHOGENIC MICROORGANISMS

tabulation is taken from their paper and represents a summary of their
own cases and those reported by others.

Combined Tabulation, Cases Reported and Own Series of Cases.

(From Park and Krumwiede, loc. cit.)

Diagnosis.

Adults

16 Years
and Over.

Children

5 to 16 Years.

Children
Under 5

Y ears.

Human

Bovine

Human

Bovine

Human

Bovine

Pulmonary tuberculosis .

568

1 ?

—

Tuberculous adenitis, axillary or inguinal .

—

■ —

—

Tuberculous adenitis, cervical .

Abdominal tuberculosis .

Generalized tuberculosis alimentary origin ....

Generalized tuberculosis .

—

Generalized tuberculosis, including meninges,

alimentary origin .

—

Generalized tuberculosis, including meninges . .

—

Tuberculous meningitis .

—

Tuberculosis of bones and joints .

—

Genito-urinary tuberculosis .

—

Tuberculosis of skin .

—

Miscellaneous Cases:

Tuberculosis of tonsils .

—

Tuberculosis of mouth and cervical nodes .

—

Tuberculous sinus or abscesses .

—

Sepsis, latent bacilli .

—

• —

—

Totals .

677

161

Mixed or double infections, 4 cases.

THE TUBERCLE BACILLUS

489

From this table it is evident that out of a total of 1,042 cases, 101
only were bovine in origin and over 50 per cent of these occurred in
children under five years of age. Fifty-one out of the 59 cases occurring
in the 161 infants were directly or indirectly traced to alimentary
infection.

It seems reasonably accurate, therefore, to state the case as follows:
Human adults are relatively insusceptible to bovine infection. Such
infection can take place, but is unusual. Below 16 years of age the
human race is relatively more susceptible and up to this age the danger
of milk infection is unquestionably great, this source accounting for
about one-third of the cases. Below 5 years the danger is greatest.
This table alone should form sufficient evidence to silence absolutely any
doubts as to the dangers of milk infection and remove any objections
to the most rigid sanitary control of milk supplies.

On the other hand, it also shows that Behring’s original claims were
far too sweeping and can not be upheld.

Rosenberger 1 has recently reported finding tubercle bacilli in the
circulating blood of all cases of human tuberculosis which he examined.
This announcement aroused much interest and has led to many investiga¬
tions by other workers. Rosenberger’s results were obtained by morpho¬
logical examination of smears of citrated blood taken from the patients,
dried upon slides and iaked with distilled water. Many other observers
have failed to confirm Rosenberger’s results. Anderson 2 examined
47 cases in which tubercle bacilli were found in the sputum and one case
of joint tuberculosis. In none of these 48 cases was he able to obtain
tubercle bacilli, neither by morphological examination nor by guinea-pig
inoculation. Brem 3 subsequently found that laboratory distilled water
may frequently contain acid-fast saprophytes — a fact which may
account in many cases for errors when morphological examination alone
is relied upon and blood examined by the technique of Rosenberger.
This, too, is suggested by the finding of acid-fast bacilli in the blood of
perfectly healthy individuals. Therefore, although the bacilli may be
present in the blood in a certain number of cases it does not seem likely
that they are so distributed in anything like the high percentages found
by Rosenberger.4

Bacillus tuberculosis (typus humanus) is pathogenic for guinea-

1 Rosenberger, Am. Jour, of Med. Sc., cxxxvii, 1909.

2 Anderson, U. S. P. H. Service, Hygienic Lab., Bull. 57, 1909.

3 Brem, Jour. A. M. A., liii., 1909.

4 Suzuki and Takaki, Centralbl. f. Bakt., Ixi, 1911.

490

PATHOGENIC MICROORGANISMS

pigs, less markedly for rabbits, and still less so for dogs. It is
slightly pathogenic for cattle, a question spoken of more extensively
below.

Chemical Analysis of Tubercle Bacilli.1 — Diligent efforts by many
investigators to isolate the specific toxins which lend tubercle bacilli
their pathogenic properties have led to careful chemical analysis of the
organisms. About 85.9 per cent of the bacillus consists of water; 20
to 26 per cent of the residue can be extracted with ether and alcohol.
This material consists of fatty acids and waxy substances (fatty acids
in combination with the higher alcohols). The residue after alcohol-
ether extraction is composed chiefly of proteids. These can be extracted
with dilute alkaline solutions, and consist chiefly of nucleo-albumins.
A nuclein present in this fraction shows extremely high toxicity and
has,2 therefore, been suspected of being the pathogenic principle of the
bacillus. After these extractions the remainder contains “cellulose,”
supposed to represent the framework of the cell membrane, and an ash
rich in calcium and magnesium.

Toxins of the Tubercle Bacillus. — The Tuberculins. — Filtrates of
bouillon cultures of Bacillus tuberculosis 3 will occasionally produce
slight emaciation when injected into guinea-pigs, and when administered
to tuberculous subjects in sufficient quantity will give rise to marked
increase of temperature. It is likely, therefore, that the tubercle
bacillus actually secretes a soluble toxin.4

The chief toxic principles, however, of Bacillus tuberculosis are
probably endotoxins or bacterial proteins, bound during cell life to the
body of the bacillus. Dead bacilli will produce sterile abscesses when
injected into animals. Prudden and Hodenpyl,5 Straus and Gamaleia,6
and others,7 moreover, have shown that the injection of dead and care¬
fully washed cultures of this bacillus will produce lesions histologi¬
cally similar to those occurring after infection with the living germs,
and will often lead to marasmus and other systemic symptoms of
poisoning.

The hope of actively immunizing with substances obtained from

1 Hammer schlag, Cent. f. klin. Med., 1891; Weyl, Deut. med. Woch., 1891; De
Schweinitz and Dorset, Jour. Amer. Chem. Soc., 1895; Hammerschlag, loc. cit.

2 Behring, Berl. klin. Woch., 1899.

3 Straus and Gamaleia, Arch. med. exp., 1891.

4 Denys, “Le Bouillon Filtre,” Louvain, 1905.

5 Prudden and Hodenpyl, N. Y. Med. Jour., June, 1891; Prudden, ibid., Dec. 5.

6 Straus and Gamaleia, loc. cit.

7 Mafucci, Cent. f. allg. Path., 1890,

THE TUBERCLE BACILLUS

491

dead bacilli led Koch to employ various methods of extraction of cultures
for the manufacture of tuberculin.

“Old Tuberculin (Koch) (“T.A.K.”). — The first tuberculin made by
Koch is produced in the following manner: Tubercle bacilli are grown
in slightly alkaline 5 per cent glycerin-pep ton bouillon for six to eight
weeks. At the end of this time, growth ceases and the corrugated
pellicle of tubercle bacilli, which during growth has floated on the
surface, begins, here and there, to sink to the bottom. The entire
culture is then heated on a water-bath at about 80° C., until reduced to
one-tenth of its original volume. It is then filtered either through
sterile filter paper or through porcelain filters. The resulting filtrate is
a rich brown, syrupy fluid, containing the elements of the original cul¬
ture medium and a 50 per cent glycerin extract of the tubercle bacilli.
While the glycerin is of sufficient concentration to preserve it indef¬
initely, 0:5 per cent phenol may be added as an additional precaution.
Dilutions of this fluid are used for diagnostic and therapeutic purposes.

“New Tuberculin” * (Koch) (TA, TO, TR).— Koch believed that
the immunity resulting from treatment with the old tuberculin was
purely an antitoxic immunity, devoid of all antibacterial action. The
use of whole dead tubercle bacilli for immunization purposes, however,
was impracticable; because, injected subcutaneously, they were not
absorbed, and introduced intravenously they were deposited in the lungs
and gave rise to lesions. Koch was led, therefore, to resort to more
energetic extraction of the bacilli in the hope of procuring a substance
which could be easily absorbed and would at the same time give rise,
when injected, to antibodies more definitely bactericidal. By extract¬
ing tubercle bacilli with decinormal NaOH, for three days, filtering
through paper and neutralizing, he obtained his TA (alkaline tubercu¬
lin). This preparation seemed to fulfil some of the hopes of its dis¬
coverer, but had the disadvantage of often producing abscesses at the
points of injection. Koch then resorted to mechanical trituration of
the bacilli. The method he subsequently followed for tuberculin pro¬
duction is now extensively used, and is carried out as follows : 3

Virulent cultures of tubercle bacilli are dried in vacuo and thoroughly
ground in a mortar. Grinding is continued until stained preparations
reveal no intact bacilli. (This is done by machinery in all large manu¬
factories.) One gram of the dry mass is shaken up in 100 c.c. of sterile
distilled water. This mixture is then centrifugalized at high speed.

1 Koch, Cent. f. Bakt., 1890; Deut. med. Woch., 1891.

2 Koch, Deut. med. Woch., 14, 1897. 3 Ruppel, Lancet, March 28, 1908.

492

PATHOGENIC MICROORGANISMS

The supernatant fluid, known as TO (Tuberculin-Oberschicht) , contains
the water-soluble constituents of the bacillus, gives no precipitate on
the addition of 50 per cent glycerin, and has the same physiological
action as the old tuberculin. The residue TR (Tuberculin-Riickstand),
after pouring off TO, is again dried and ground up, and again shaken in
water and centrifugalized. This process is repeated several times,
and eventually, after three or four repetitions, all the TR goes into
emulsion. The total volume of water used for these TR extractions
should not exceed 100 c.c. All of the TR emulsions are then mixed to¬
gether. This gives TR a precipitate with 50 per cent of glycerin, and is
supposed by Koch to contain substances important in producing an
antibacterial immunity. For purposes of standardization, the amount
of solid substance in 5 c.c. of the TR is determined by evaporation in
vacuo and drying. To the rest are added a little glycerin and formalde¬
hyde and enough water to allow each cubic centimeter of the solution
to contain 0.002 grams of solid material. Thus the culture and the
medium remaining the same, fairly accurate standardization is possible.

“New Tuberculin-Bacillary Emulsion.” 1 — In 1901, Koch combined
“TO” and “TR” by putting forth a preparation referred to as
“Bazillenemulsion.” This consists of an emulsion of pulverized bacilli
1 : 100 in distilled water. After several days of sedimentation to re¬
move the coarser particles, the supernatant fluid is poured off and fifty
per cent volume of glycerin is added to it for purposes of preservation.
This preparation contains 5 milligrams of solid substance in each cubic
centimeter.

Bouillon Filtre ( Denys ).1 2 3 — This preparation consists of the filtrate
(through Chamberland filters) of 5 per cent glycerin-pepton-bouillon
cultures of Bacillus tuberculosis. Phenol 0.25 per cent is added to
insure sterility. The filtered bouillon corresponds to the unconcentrated
old tuberculin of Koch, but, not having been heated, is supposed by
Denys to contain important soluble and possibly thermolabile secretory
products of the bacillus.

Tuber culoplasmin (. Buchner and Hahn)} — Buchner and Hahn, by
crushing tubercle bacilli by subjecting them to a pressure of 400
atmospheres, obtained a cell-juice in the form of an amber fluid, to
which they attributed qualities closely analogous to those of TR.

1 Koch, Deut. med. Woch., 1901.

2 Denys, “Le Bouillon Filtre,” Louvain, 1905.

3 Buchner und Hahn, Munch, med. Woch., 1897; Hahn, ibid.

THE TUBERCLE BACILLUS

493

Other tuberculins are those of Beraneck,1 highly recommended
clinically by Sahli,2 that of Klebs,3 and the tuberculin produced from
bovine tubercle bacilli by Spengler.4

Diagnostic Use of Tuberculin. — Subcutaneous Use. — The preparation
usually employed for diagnostic purposes is Koch's “Old Tuberculin"
(Alttuberculin) . This preparation is administered by hypodermic injec¬
tion of small quantities obtained by means of dilutions. The dilutions
are best made with a 0.5 per cent aqueous carbolic acid solution. In
practice a 1 per cent solution is made by pipetting 0.1 c.c. of tuberculin
into 9.9 c.c. of the 0.5 per cent carbolic solution. A cubic centimeter
of this then contains 0.01 c.c. of tuberculin. One c.c. of this solution
added to 9 c.c. of 0.5 per cent carbolic acid gives a solution in which
each cubic centimeter contains 0.001 c.c., or 1 milligram of tuberculin.

The initial dosage in adults in Koch's 5 early work, and as used by
Beck 6 on a large number of patients, was 1 milligram. This, according
to present opinions, is too high, and most clinicians to-day prefer 0.1
to 0.2 of a milligram. If after three or four days no reaction has occurred,
a second dose of 1 milligram is given. In the absence of reaction after
three further days, a third dose of 5 mgm. may be given and, under
similar conditions, a fourth of 10 mgm. This is the largest dose which
should ever be given, and absence of a reaction to this dose may gener¬
ally be regarded as proof that the patient is free from tuberculosis.
Doses larger than 10 mgm. may give reactions in perfectly healthy
subjects. Increase in dosage should be carried out only when the
preceding dose has been entirely without reaction. In all cases it
should be remembered that absolute rules of dosage can not be made
and the condition and physique of each patient must be separately
judged.

The reaction itself is recognized chiefly by the changes in tem¬
perature. In a positive reaction the patient's temperature will begin
to increase within six to eight hours after injection, rising sharply within
a few hours to 0.5 or 1.5° higher than the temperature before injection.
It then sinks more gradually than it rose, the reaction usually being
complete within thirty to thirty-six hours. With the temperature there

1 Beraneck, Compt. rend, de l’acad. des sci., 1903.

2 Sahli, Corrbl. d. Schw. Aerzte, 1906.

3 Klebs , Cent. f. Bakt., 1896; Deut. med. Woch., 1907.

4 Spengler, Deut. med. Woch., xxxi, 1904; xxxi and xxxiv, 1905.

5 Koch, Deut. med. Woch., 1890.

6 Beck, Deut. med. Woch., 1899.

494

PATHOGENIC MICROORGANISMS

may be nausea, a chill, rapid pulse, and general malaise. Locally
visible tuberculous processes, such as lupus, lymph nodes, etc., may
become more tender or swollen, and if the tuberculosis is pulmonary,
there may be coughing and increased expectoration. The tempera¬
tures of persons subjected to the test should be taken regularly for
three or four days before tuberculin is used.

Ophthalmo-Tuberculin Reaction. — Wolff -Eisner 1 and, soon after
him, Calmette 2 proposed a method of using tuberculin for diagnostic
purposes by instillation into the conjunctival sac. In tuberculous
patients this process is followed by a sharp conjunctival congestion last¬
ing from one to several days.

The preparation used for this purpose is produced in the following
way:

“Old Tuberculin” is treated with double the quantity of 95 per
cent alcohol, and the precipitate allowed to settle and the alcohol then
filtered off through paper. The sediment is washed with 70 per cent
alcohol until the filtrate runs clear, then pressed between layers of
filter paper to remove excess of moisture, scraped into a dish, dried
in vacuo over H2S04, and broken up in a mortar under a hood.

Solutions of the powder are made in sterile normal salt solution, 1
per cent by weight, boiled and filtered. The solutions are used in
strengths of 0.5 to 1 per cent, a drop of which is instilled into the con¬
junctival sac.3

Cutaneous Tuberculin Reaction. — Von Pirquet 4 has suggested the
cutaneous use of tuberculin for diagnostic purposes. A 25 per cent
solution of “Old Tuberculin” is made in the following way:

Tuberculin . 1

Salt solution . 2

5 per cent carbolic acid in glycerin . 1

After sterilization of the patient’s forearm, two drops of this solution
are placed upon the skin about 6 cm. apart. Within each of these drops
scarification is done, and the skin between them is scarified as a con¬
trol. Within twenty-four to forty-eight hours, in tuberculous patients,
erythema, small papules, and herpetiform vesicles will appear. The
reaction is irregular and more reliable in children than in adults. Ac-

1 Wolf -Eisner, Berl. med. Gesell., May 15, 1907.

2 Calmette , Acad, des sci., June 17, 1907.

3 Method in use at Saranac and kindly communicated by Dr. Baldwin.

4v. Pirquet, Berl. klin. Woch., xx, 1907; Med. Klinik, xl, 1907.

THE TUBERCLE BACILLUS

495

cording to recent investigations, about 70 per cent of adults show a
positive reaction and in such cases it is probable that an old healed
tuberculosis may give rise to a positive test where absolutely no active
process exists.

Recently, v. Pirquet has modified his procedure by using instead of
the 25 per cent solution given above, the pure, undiluted “Old Tuber¬
culin.”

Moro 1 has modified this by simply making a 50 per cent ointment of
tuberculin in lanolin and rubbing it into the skin without scarification.

It is more simple and equally efficient to massage into the skin a
drop of undiluted “Old Tuberculin.”

The Tuberculin Test as Applied to Cattle. — In cattle, the symptoms
of tuberculosis are not easily detected by methods of physical diag¬
nosis until the disease has reached an advanced stage. In conse¬
quence, cows may be elements of danger without appearing in any
way diseased to those who handle them. In consequence, routine
examination of herds by the tuberculin test has become one of
the necessary measures in public sanitation. According to Mohler,2
an accurate diagnosis may be established in at least 97 per cent of the
cases. It is natural that a good deal of objection to the test is encoun¬
tered on the part of dairy farmers and cattle raisers, and recently it has
been publicly claimed that the cattle are injured by the test. There is,
however, no scientific basis for this belief, if the test is carried out care¬
fully and intelligently. As a matter of fact, the systematic use of the
test would eventually be distinctly advantageous to the owners of the
cattle themselves, since it has been shown that cows, even in the early
stages of the disease, may expel tubercle bacilli, either during respira¬
tion or in the feces, and thus become a menace to healthy members of
the herd.

The tuberculin test on cattle should be made as follows: (The
directions given below are taken directly from the circular sent out from
the Bureau of Animal Industry at Washington.)

1. Begin to take the rectal temperature at 6 a.m., and take it every
two hours thereafter until midnight.

2. Make the injection at midnight.

3. Begin to take the temperature next morning at 6 a.m., and con¬
tinue as on preceding day.

To those who have large herds to examine, or are unable to give the

1 Moro , Munch, med. Woch., 1906, p. 216.

2 Mohler, Pub. H. and Mar. Hosp. Serv. Bull. 41, 1908.

496

PATHOGENIC MICROORGANISMS

time required by the above directions, the following shortened course is
recommended :

1. Begin to take the temperature at 8 a.m., and continue every 2
hours until 10 p.m. (omitting at 8 p.m., if more convenient); or take the
temperature at 8 a.m., 12 m., and 10 p.m.

2. Make the injection at 10 p.m.

3. Take the temperature next morning at 6 or 8 a.m., and every 2
hours thereafter until 6 or 8 p.m.

Each adult animal should receive 2 c.c. of the tuberculin as it is sent
from the laboratory. (The tuberculin sent out from the central labora¬
tory at Washington is already diluted; 2 c.c. represents 0.25 c.c. of the
concentrated “Old Tuberculin” of Koch.) Yearlings and two-year-olds,
according to size, should receive from 1 to 1.5 cubic centimeters. Bulls
and very large animals may receive three cubic centimeters. The injec¬
tion should be made beneath the skin of the neck or shoulders behind
the scapula, after washing the area with a weak carbolic acid solution.

There is usually no marked local swelling at the seat of the injection.

There are now and then uneasiness, trembling, and the more fre¬
quent passage of softened dung. There may also be slight acceleration
of the pulse and of the breathing.

The febrile reaction in tuberculous cattle following the subcutaneous
injection of tuberculin begins from six to ten hours after the injection,
reaches the maximum nine to fifteen hours after the injection, and
returns to normal eighteen to twenty-six hours after the injection.

A rise of two or more degrees Fahrenheit above the maximum tem¬
perature observed on the previous day should be regarded as an indica¬
tion of tuberculosis. For any rise less than this a repetition of the
injection after four or six weeks is highly desirable.

It is hardly necessary to suggest that for the convenience of the one
making the test the animals should not be turned out, but fed and
watered in the stable. It is desirable to make note of the time of feed¬
ing and watering and of any temperature fall after watering.

The tuberculin should not be used later than six weeks after the
date on the bottle, nor if there is a decided clouding of the solution.

Therapeutic Uses of Tuberculin. — Tuberculin was first used therapeu¬
tically, shortly after its discovery, by Koch.1 Hailed with the most
optimistic enthusiasm, its possibilities were overestimated and hope¬
less cases were treated unskilfully, with unsuitable dosage. The conse¬
quence was that harm was done, the method was attacked by Virchow

1 Koch, Deut. med. Woch., iii, 1891.

THE TUBERCLE BACILLUS

497

and others and the new therapy fell into almost complete neglect. At
present, the use of tuberculin has again been revived, but with greater
caution and with a thorough understanding of its limitations. The
tendency has been toward smaller dosage and the limitation of the agent
to early cases. No two institutions use tuberculin in exactly the same
manner, and it is, therefore, impossible to do more than outline the
general scheme of treatment. It must never be forgotten, however,
that all forms of tuberculin treatment consist in an “active immuniza¬
tion” in which, for the time being, the toxemia of the patient is increased
rather than neutralized. It is obvious, therefore, that only such cases
are at all suitable for treatment in which the process is not a very acute
one. The general principle of modern tuberculin therapy seems to lie
in choosing doses so small that no marked general reaction shall follow.
The preparations most frequently employed are Koch’s “Alttuber-
culin,” his “TR,” his “Neu Tuberkulin-Bazillen Emulsion,” and the
Bouillon filtre of Denys. Initial doses of Alttuberculin range from 0.1
to 0.01 of a milligram. In case of successful avoidance of a reaction,
the injection may be repeated, gradually increasing, about twice a week.
The occurrence of a reaction should be the signal for a longer interval
and a slower advance in the size of the dose.

The initial dose of “TR” is, as advised by Koch,1 about 0.002
mgm. This usually causes no reaction. The dose is doubled, at reason¬
able intervals, up to 1 mgm. After this, further increase is care¬
fully gauged by the clinical indications. The maximum dose is about
20 mgm.

“Neu Tuberkulin-Bazillen Emulsion,” 2 is begun with a dose of 0.001
mgm. Gradual increase as with the other preparations is then prac¬
ticed. The maximum dose is about 10 mgm.

Bouillon filtre has been used chiefly by Denys3 and with apparently
excellent results. Denys is very emphatic in advising the absolute
avoidance of any reaction. He begins with a millionth or even the
tenth of a millionth of a cubic centimeter of the bouillon and in¬
creases with extreme caution. His dilutions are made with glycerin
broth.

Passive Immunization in Tuberculosis. — Numerous attempts have
been made to immunize tuberculous subjects with the sera of actively

1 Koch, Deut. med. Woch., xiv, 1897.

2 Bandelier und Roepke, “Lehrb. d. spezifisch. Tub. Ther.,” Wurzburg, 1908;

Koch, Deut. med. Woch., 1901.

3 Denys , “Le Bouillon filtre,” Louvain, 1905.

498

PATHOGENIC MICROORGANISMS

immune animals. The most widely used method of producing such
serum is that of Maragliano.

Maragliano’s Serum 4 — Maragliano believes that a toxalbumin is
present in tubercle-bacillus cultures which is destroyed by the heating
employed in the usual tuberculin production. He procures this sub¬
stance by filtration of unheated cultures and precipitation with alcohol
(tossina praecipitata) . He furthermore makes an aqueous extract of
the bacillary bodies. With these two substances he immunizes horses.
He draws blood from these after four to six months of treatment. The
serum is extensively used in Italy. Its value is, at present, very
doubtful.

Marmorek’ s Serum.1 2 — Marmorek claims that the poisons produced by
Bacillus tuberculosis depend largely upon the medium on which it is
grown. He advanced the view in 1903 that the substances obtained
in tuberculin were not the true toxins of the tubercle bacillus, that there
was a marked difference between these and the poisons elaborated by a
younger (primitive) phase of the bacillus as it occurs only within the
animal body or on media composed of animal tissue. He consequently
grows his cultures on a medium composed of a leucotoxic serum (pro¬
duced by inoculating calves with guinea-pig leucocytes) and liver tissue.
Such cultures, he claims, contain no tuberculin. To the sera produced
by immunization with these cultures he attributes high curative powers.

Bacilli Closely Related to the Tubercle Bacillus. — The Bacillus of
Bovine Tuberculosis. — Tuberculosis of cattle (Perlsucht) was studied
by Koch 3 in connection with his early work on human tuberculosis.
Koch did not fail to recognize differences between the reactions to in¬
fection in the bovine type of the disease and that of man. He attrib¬
uted these, however, to the nature of the infected subject rather than
to any differences in the infecting agents. This point of view met
with little authoritative contradiction, until Theobald Smith,4 in 1898,
made a systematic comparative study of bacilli isolated from man and
from cattle and pointed out differences between the two types. The
opinion of Smith was fully accepted by Koch 5 in 1901.

Since that time, the question, because of its great importance to
prophylaxis, has been the subject of many investigations, most of them

1 Maragliano , Berl. klin. Woch., 1899; Soc. de biol., 1897.

2 Marmorek , Berl. klin. Woch., 1903, p. 1108; Med. Klinik, 1906.

3 Koch, Arb. a. d. kais. Gesundheitsamt, 11, 1882.

4 Th. Smith, Jour. Exp. Med., Ill, 1898.

5 Koch, Deut. med. Woch., 1901.

THE TUBERCLE BACILLUS

499

confirming Smith’s original work. Morphologically, Smith 1 found that
the bovine bacilli were usually shorter than those of the human type and
grew less luxuriantly than these upon artificial media. He determined,
furthermore, that, grown upon slightly acid glycerin bouillon, the bovine
bacillus gradually reduces the acidity of the culture medium until the
reaction reaches neutrality or even slight alkalinity. Fluctuations,
after this, do not exceed 0.1 or 0.2 per cent on either side of neutrality.
In the case of the human bacillus, on the other hand, there is but slight
reduction of the acidity during the first weeks of growth; after this
acidity increases and, though subject to fluctuations, never reaches
neutrality. This behavior is probably due to action exerted upon the
glycerin, since on ordinary bouillon no such differences between the two
varieties can be noticed. These observations of Smith were confirmed
by Ravenel,2 Vagedes,3 and others.

The cultural differences between the two types have been studied
with especial care by Wolbach and Ernst,4 and Kossel, Weber, and
Heuss.5 All of these observers bear out Smith’s contention that
luxuriance and speed of growth are much more marked in the human
than in the bovine variety. Marked differences, furthermore, have been
shown to exist in the pathogenic qualities of these bacilli toward various
animal species.

Guinea-pigs inoculated with the bovine type 6 die more quickly and
show more extensive lesions than those infected with human bacilli.
The difference in the pathogenicity of the two organisms for rabbits is
sufficiently striking to be of diagnostic value. The bovine bacilli usually
kill a rabbit within two to five weeks; the human bacilli produce a mild
and slow disease, lasting often for six months, and occasionally fail to
kill the rabbits at all.

The practical importance of distinguishing between the two types,
of course, attaches to the question as to whether the bovine and the
human disease are mutually intercommunicable. Extensive attempts to
infect cattle with bacilli of the human type have been made,7 for the most
part with very little or no success. Infections of human beings with

1 Th. Smith , Jour. Exp. Med., 1905.

2 Ravenel, Lancet, 1901; Univ. Penn. Med. Bull., 1902.

3 Vagedes, Zeit. f. Hyg., 1898.

4 Wolbach and Ernst, “Studies from the Rockefeller Inst.,” 11, 1904.

5 Kossel, Weber, und Heuss, Arb. a. d. kais. Gesundheitsamt, 1904 and 1905.

6 Smith, loc. cit., and Medical News, 1902.

7 Beck, “Festsch. R. Koch,” 1902; Smith, loc. cit.

500

PATHOGENIC MICROORGANISMS

bovine bacilli, however, have been reported and proved beyond reason¬
able doubt, by Smith,1 Ravenel,2 Kossel, Weber, and Heuss,3 Park and
Krumwiede,4 and others. Most of these infections have been in children.
It is likely, therefore, that while cattle are to a considerable degree im¬
mune against the bacillus of the human type, human beings do not
enjoy the same safeguard in respect to the bovine bacillus. During adult
life, the danger of such infection, however, is far less than it is during
infancy and early youth. This question has been discussed on p. 487.

The Bacillus of Avian Tuberculosis. — A disease resembling in many
features the tuberculosis of man is not uncommon among chickens,
pigeons, and some other bird species. Koch was the first to discover in
the lesions of diseased fowl bacilli much resembling Bacillus tuberculosis.
It was soon shown, however, by the studies of Nocard and Roux,5
Mafucci,6 and others, that the bacillus of the avian disease represented
a definitely differentiable species.

Morphologically, and in staining characteristics, the bacillus is
almost identical with that of the human disease. In culture, however,
growth is more rapid and takes place at a temperature of 41° to 45° C.7
(the normal temperature of birds), while the human type is unable to
thrive at a temperature above 40°.

Guinea-pigs, very susceptible to human tuberculosis, are very
refractory to infection with the avian type; while, on the other hand,
rabbits which are resistant to the human type, succumb rapidly to in¬
fection with avian tuberculosis.8 Prolonged cultivation and passage
through the mammalian body is said to cause these bacilli to approach
more or less closely to the mammalian type. Conversely, Nocard 9
succeeded in rendering mammalian tubercle bacilli pathogenic for fowl
by keeping them in the peritoneal cavities of hens in celloidin sacs for
six months.

Recently Koch and Rabinovitsch 10 have isolated from the spleen of

1 Smith, Trans. Assn. Amer. Phys., 1903.

2 Ravenel, Univ. Penn. Med. Bull., 1902.

s Kossel, Weber, und Heuss, loc. cit.

4 Park and Krumwiede, Jour. Med. Res., 1910.

5 Nocard et Roux, Ann. de l’inst. Pasteur, 1887.

6 Mafucci, Zeit. f. Hyg., xi.

7 Mafucci, loc. cit.

8 Straus et Gamaleia, Arch, de med. exper., 1891; Courmont et Dor, Arch, de med.
exp., 1891.

9 Nocard, Ann. de l’inst. Pasteur, 1898.

10 Koch und Rabinovitsch, Virch. Arch., Beiheft to Bd. 190, 1907.

THE TUBERCLE BACILLUS

501

a young man dead of tuberculosis, a microorganism which, culturally,
morphologically, and in its pathogenic action upon birds, seemed to
belong to the avian type. Lowenstein1 describes a similar organism
cultivated from a human case which seems to be a transitional type.
Observations of this order are, however, too few at the present time to
be used as the basis of a definite opinion as to the relationship between
the two varieties.

Tuberculosis in Cold-blooded Animals. — The bacillus isolated by
Dubarre and Terre 2 resembles Bacillus tuberculosis in morphology and
in a certain degree of acid-fastness. It grows at low temperatures,
15° to 30° C. It is non-pathogenic for animals, but kills frogs within a
month. Except for the acid-fastness it has little in common with
Bacillus tuberculosis.

Similar acid-fast bacilli have been isolated from other cold-blooded
animals (carp, frogs, turtles, snakes) by many observers.

There have been many attempts to show a close relationship between
the tubercle bacilli of cold-blooded and those of warm-blooded animals.
Moeller, Hansemann, Friedmann, Weber, Kiister, and others have
given this subject particular attention and it has gained especial interest
because of the recent notorious claims of Friedmann that he has suc¬
ceeded in obtaining, from turtles, a strain of acid-fast bacilli which can
be successfully used in actively immunizing human beings. In 1903
Friedmann 3 described two cases of spontaneous infection of a salt-water
turtle (Chelone corticata) with acid-fast bacilli, presenting lesions in
the lungs which simulated pulmonary tuberculosis in the human being
(cavity formation and miliary nodules). The organisms cultivated from
these lesions presented much similarity to those of the human type and,
according to Friedmann,4 unlike other acid-fast bacilli of cold-blooded
animals, could be grown at 37.5° C. As a possible human origin for the
turtle infections Friedmann mentions that the attendant who fed these
turtles suffered from a double pulmonary tuberculosis.

Upon inoculation into guinea-pigs localized lesions only were pro¬
duced, and dogs, rats, and birds were immune. The implication of
Friedmann’s work is that his culture represents a human strain attenu-

1 Lowenstein, quoted from Koch and Rabinovitsch, loc. cit.

2 Dubarre et Terre, Compt. rend, de la soc. de biol., 1897.

3 Friedmann, D. Med. Woch., No. 2, Jan., 1903, 25.

4 Friedmann, D. Med. Woch., No. 26, 464, 1903, and Centralbl. f. Bakt., I, xxxiv,
1903, also Zeitschr. f. Tuberkulose, iv, Heft 5, 1903.

502

PATHOGENIC MICROORGANISMS

ated for man by passage through the turtle, although, as far as we are
aware, no definite statement as to this has been made.

Summarizing the work of many investigators (Weber, Taute,
Kiister, Allegri, Bertarelli, and others) Kiister 1 makes a statement
which is, in essence, as follows: In the carp, in snakes, turtles,
and frogs spontaneous tuberculosis may occur. The organisms which
cause these diseases are specific for cold-blooded animals, similar in
many respects to the tubercle bacillus of warm-blooded animals, but
in the latter do not produce progressive disease. Human, bovine, and
avian tubercle bacilli inoculated into cold-blooded animals can produce
lesions which histologically simulate tuberculosis. These micro¬
organisms can remain a year in cold-blooded animals without losing
their pathogenicity for guinea-pigs. Mutation of the tubercle bacillus
of warm-blooded animals into cold-blooded ones has not been proven.

For these reasons it is quite impossible to exclude, in the apparently
positive work of Friedmann and others, the isolation of a true “cold¬
blooded” type organism, rather than a mutation form originally of
the warm-blooded type. What Friedmann’s present claims in this
respect are for his culture has not been stated as far as we know. The
possibility of a positive immunizing value of organisms isolated from
cold-blooded animals in human beings, though remote, is not out of ques¬
tion. The problem is so serious and important, and the experience of
many workers is, so far, so inconclusive that the time has not come for
commercial exploitation and the cruel arousing of false hopes. The
subject, however, deserves carefully controlled further investigations.

Bacillus of Timothy. — Moeller isolated from timothy-grass and from
the dust in haylofts acid-fast bacilli, like Bacillus tuberculosis. They
grow rapidly on agar, soon showing a deep red or dark yellow color.

Bacillus hutyricus ( Butter Bacillus). — Slightly acid-fast bacilli re¬
sembling Bacillus tuberculosis have been isolated from milk and butter
by Petri,2 Rabinovitsch,3 Korn,4 and others.

These bacilli are easily differentiated from Bacillus tuberculosis cul¬
turally. They are slightly pathogenic for guinea-pigs, but not for man.

Bacillus smegmatis and the bacillus of leprosy will be discussed in
separate sections. The differentiation of these organisms by staining
reactions has been discussed in the section on staining methods.

1 Kolle und Wassermann’s Handbuch, 2d edition, v, 767.

2 Petri, Arb. a. d. kais. Gesundheitsamt, 1897.

3 Rabinovitsch , Zeit. f. Hyg., 1897.

4 Korn, Cent. f. Bakt., 1899.

CHAPTER XXXIV

THE SMEGMA BACILLUS AND THE BACILLUS OF LEPROSY

BACILLUS SMEGMATIS

In 1884, Lustgarten 1 announced that he had succeeded in demon¬
strating, in a number of syphilitic lesions, a characteristic bacillus,
which he declared to be the etiological factor in the disease. The great
importance of the subject of Lustgarten’s communication caused nu¬
merous investigators to take up the study of the microorganisms found
upon the genitals of normal and diseased individuals. As a result of
these researches the presence of the Lustgarten bacilli upon the genitals
of many syphilitics was confirmed; but at the same time bacilli, which
in all essential particulars were identical with them, were found in the
secretions about the genital organs and anus of many normal persons.
The first to throw doubt upon the etiological significance of Lustgarten’s
bacillus, and to describe in detail the microorganism now recognized as
Bacillus smegmatis, were Alvarez and Tavel.2 Similar studies were
made soon afterward by Klemperer,3 Bitter,4 and others.

The smegma bacilli are now known to occur as harmless sapro¬
phytes in the preputial secretions of the male, about the external genital
organs of the female, and within the folds of thighs and buttocks. They
are usually found, in these situations, in clumps upon the mucous mem¬
brane, and occasionally in the superficial layers of the epithelium, intra-
and extra-cellularly.

Morphology. — The smegma bacilli are very similar to tubercle bacilli,
but show greater variations in size and appearance than do the latter.
In length the individuals may vary from two to seven micra. They
are usually straight or slightly curved, but according to Alvarez and
Tavel may show great polymorphism, including short comma-like forms,
and occasional S-shaped spiral forms.

1 Lustgarten, Wien. med. Woch., 47, 1884.

2 Alvarez et Tavel, Arch. d. physiol, norm, et path., Oct., 1885.

3 Klemperer, Deut. med. Woch., xi, 1885.

4 Bitter j Virchow’s Arch., ciii.

503

504

PATHOGENIC MICROORGANISMS

They are not easily stained, and though less resistant in this respect
than the tubercle bacillus, they yet belong distinctly to the group of
acid-fast bacilli. Once stained by the stronger dyes, such as carbol-
fuchsin or anilin-water-gentian-violet, they are tenacious of the dye,
though less so than tubercle bacilli.

The identification of the smegma bacillus by staining methods has
become of great practical importance since Fraenkel,1 Muller,2 and
others have demonstrated the occasional presence of acid-fast bacilli,
probably of the smegma group, in sputum, and in secretions from the
tonsillar crypts and throat. The methods of differentiation which have
been found most practical are those which depend upon differences in
the retention of stain shown by these bacilli. While it may be stated
as a general rule that the smegma bacilli are more easily decolor¬
ized than tubercle bacilli, it is nevertheless important that a con¬
trol, as suggested by Wood, be made with known tubercle bacilli
whenever a slide of suspected smegma bacilli is examined. For
the actual differentiation an excellent method is that of Pappenheim,
described in detail in the section on Staining, page 106. This method
depends upon the fact that prolonged treatment with alcohol and rosolic
acid decolorizes the smegma bacilli but not the tubercle bacilli.
Coles 3 has stated that smegma bacilli will resist Pappenheinds
decolorizing agent for four hours at the most, while tubercle bacilli
will retain the stain, in spite of such treatment, for as long as twenty-
four hours.

Although minor differences between the smegma bacillus and that
of Lustgarten have been upheld by Doutrelepont 4 and others, never¬
theless, the etiological significance of Lustgarten’s bacillus in syphilis
has been finally discredited, and, if not identical with the smegma
bacillus, it at least belongs to the same group.

The smegma bacilli have no pathogenic significance. They are
found upon human beings as harmless saprophytes, and all attempts to
infect animals have so far been unsuccessful. They are cultivated
with great difficulty, first cultivations from man being successful only
upon the richer media containing human serum or hydrocele fluid.
After prolonged cultivation upon artificial media they may be kept
alive upon glucose agar or ascitic agar. Their growth is slow;

1 Fraenkel, Berl. klin. Woch., 1898.

2 Muller, Deut. med. Woch., 1898.

3 Coles, Jour, of State Med., 1904.

4 Doutrelepont, quoted from Klemperer, loc. cit.

BACILLUS LEPRAE AND LEPROSY

505

and the colonies, appearing within five or six days after inoculation,
are yellowish white, corrugated, and not unlike tubercle-bacillus
colonies.

BACILLUS LEPR-ffi AND LEPROSY

The bacillus of leprosy was first seen and correctly interpreted as
the etiological factor in the disease in 1879, by G. Armauer Hansen,1
a Norwegian observer. Hansen found the bacilli in the tissues of the
nodular lesions of patients, lying in small clumps, intra- and extra-
cellularly, as well as in the serum oozing from the tissue during its
removal. Hansen’s observation was the fruit of over six years of careful
study and as to his priority in making this great discovery, there can
be no doubt. Almost simultaneously with his publication, however,
Neisser 2 published similar results, obtained by him during a brief stay
at Bergen, during the preceding summer. The bacilli described by
these workers are now recognized as being unquestionably the cause
of the various forms of the disease known as leprosy.

Morphology and Staining. — The leprosy bacillus is a small rod
measuring about 5 to 7y in length and has a close morphological re¬
semblance to Bacillus tuberculosis, except in that it is less apt to display
the beaded appearance and is slightly less slender than the latter. It
is non-motile, possesses no flagella, and forms no spores.

Like tubercle bacilli, furthermore, the leprosy bacilli belong to the
class of so-called acid-fast bacteria, being stained with much difficulty;
but when once stained they are tenacious of the color, offering con¬
siderable resistance to the decolorizing action of acids. It is necessary
for differential diagnosis, however, to note that both the difficulty of
staining and the resistance to decolorization are less marked in the case
of this microorganism than in the case of Bacillus tuberculosis. It was
this peculiar behavior to stains that caused the delay of several years in
Hansen’s publications, since he failed in obtaining good morphological
specimens until the work of Koch upon bacterial staining had supplied
him with proper methods. The bacillus is stained most easily with
anilin-water-gentian-violet or with carbol-fuchsin solution. Stained by
Gram’s method, it is not decolorized and appears a deep blue. Differ¬
ential staining by the Ziehl-Neelsen method shows the bacillus stained
red unless decolorization by means of the acid and alcohol are prolonged

1 Hansen, Virch. Arch., 79, 1879.

2 Neisser, Breslauer arztl. Zeitschr., 20, 1879.

506

PATHOGENIC MICROORGANISMS

for an unusual time. A differentiation from tubercle bacilli by virtue of
greater ease of decolorization is of value only in the hands of those
having much experience with these bacilli, and follows no regular laws
of acid-strengths or time of application which can be generally applied
by the inexperienced. In tissues, the bacilli are easily stained by the
methods used for staining tubercle bacilli. The sections are left in the
Ziehl carbol-fuchsin solution either from two to twelve hours at incu¬
bator temperature or for twenty-four hours at room temperature.
Subsequent treatment is that employed in the case of tuberculous tissue
sections (see p. 112).

Cultivation. — Cultivation of the leprosy bacillus has not met with
success. Hansen and others who have approached the problem with
a thorough knowledge of the microorganism, combined with a com¬
petent bacteriological training, have failed in all their attempts.
Numerous positive results reported by observers have always lacked
adequate confirmation. Recently, Rost,1 of the British Army Medical
Corps, has claimed success in cultivation of leprosy bacilli upon salt-free
bouillon, his point of departure being the previous observation that
salt-free media favored the growth of tubercle bacilli. His results have
not been confirmed.

In 1909 Clegg 2 succeeded in growing an acid-fast bacillus from
leprous tissue, obtaining his results by inoculating leprous material
upon agar plates upon which ameba coli had been grown in symbiosis
with other bacteria. On such plates the acid-fast bacilli multiplied,
and, subsequently, pure cultures were obtained by heating the cultures
to 60° C., which destroyed the ameba coli and other bacteria. These
results were confirmed by other workers and, soon after that, Duval 3
not only succeeded in repeating Clegg’s experiments, but obtained cul¬
tures of an acid-fast bacillus directly from leprous lesions without the
aid of ameba. He first observed that the leprosy organism would multi¬
ply around a transplanted piece of leprous tissue upon ordinary blood
agar tubes upon which influenza bacilli and meningococci were grown.
He concluded that such growth depended upon chemical changes in
the media and believed the formation of amino-acids essential for the
initial growth. The method he subsequently described depended upon
supplying these substances either by adding tryptophan to nutrient
agar or by pouring egg albumen and human blood serum in Petri dishes,

1 Rost, Brit. Med. Jour., 1, 1905.

2 Clegg, Philippine Jour, of Sc., iv, 1909.

3 Duval, Jour. Exp. Med., xii, 1910, and ibid., 15, 1912.

BACILLUS LEPR43 AND LEPROSY

507

inspissating, at 70° C., for three hours and, after inoculating with
leprous tissue, adding a 1 per cent solution of trypsin. Indirectly the
same result was obtained by employing culture media containing albu¬
minous substances and inoculating with bacteria capable of producing
amino-acids from the medium. After leprosy bacilli had been grown
on this medium for several generations, they could easily be cultivated
on agar slants without special additions or preliminary treatment.

In spite of extensive work upon this very important problem
opinions are still divided as to the specific nature of the organisms cul¬
tivated by Clegg and by Duval. Animal experiments with these cultures
have remained inconclusive. The cultures after prolonged preservation
upon artificial media grow heavily, often lose their acid-fast charac¬
teristics, develop into streptothrix-like or diphtheroid forms and become
markedly chromogenic, all these characteristics suggesting saprophytism.

In a recent communication, Duval and Wellman 1 state their opinion
as follows: From 29 cases of leprosy, 22 successive cultivations of acid-
fast bacilli were made; in 14 of them a chromogenic organism, similar
to that of Clegg, was found. This grows either as a non-acid-fast strep-
tothrix in subsequent cultivations or as non-acid-fast diphtheroid forms.
From eight cases an organism distinctly different from the former was
cultivated which grows only on specific media and by serological tests
seems to give reaction which differentiates it from Clegg’s organism. Du¬
val believes that there is no reason to assume specific etiological relation¬
ship for the first organism mentioned. In the case of the second, he
admits that not sufficient proof has been brought, but states his belief
that its etiological significance is probable.

Pathogenicity. — Innumerable attempts to transmit leprosy to ani¬
mals by inoculation have been unsuccessful. Nicolle,2 however, has
recently claimed successful experiments upon monkeys (macacus) in
whom inoculation with tissue from infected human beings was followed,
in sixty-two days, by the development of a small nodule at the site of
inoculation, in which, upon excision, leprosy bacilli were found. In
most cases, however, inoculation has given rise merely to a transient
inflammatory reaction.

Among human beings, leprosy has been a widely spread disease since
the beginning of history, and much evidence is found in ancient lit¬
erature which testifies to a wide distribution of the disease long before
the Christian era and throughout the Middle Ages. At the present day,

1 Duval and Wellman, Jour, of Inf. Dis., xi, 1912.

2 Nicolle, Sem. medicale, 10, 1905.

508

PATHOGENIC MICROORGANISMS

leprosy is most common in the eastern countries, especially in India ana
China. In Europe the disease is found in Norway, in Russia, and in
Iceland. In other European countries, while the disease occurs, it is
not at all common. In the United States, there are, according to Osier,
three important centers of leprosy situated in Louisiana, in California,
and among the Norwegian settlers in Minnesota. The disease is also
present in several provinces of Canada. In all countries in which
segregation of lepers is rigidly practiced, the disease is diminishing. In
Norway, according to Hansen, proper sanitary measures have reduced
the number of lepers from 2,870 in 1856, to 577 in 1900.

Clinically, the disease appears in two chief varieties, tubercular
leprosy and the so-called anesthetic leprosy. In the former variety,
hard nodular swellings appear, usually in the face, but often on other
parts of the body as well. These lead to frightful disfigurement and
are accompanied by a falling-out of hair and a loss of sensation in the
affected areas. In the anesthetic form, there is usually at first pain in
definite areas of the extremities and the trunk, which is soon followed
by the formation of flat or slightly raised pigmented areas, within which
there is absolute anesthesia with, later, atrophy and often secondary
necrosis in the atrophied parts. The disease is usually chronic in its
course.

The bacilli are found in large numbers in the cutaneous lesions. In
the knobs of the nodular variety, they lie in clumps between the con¬
nective-tissue cells and within the large spheroidal cells which make up
the nodules. They are found, also, in advanced cases, in the liver and
in the spleen, lying within the cells, and, to a slighter extent, in the
intercellular spaces. They have also been found within the kidneys,
the endothelium of the blood-vessels, and in the testicles.1 In the blood,
the bacilli have frequently been demonstrated, especially during the
febrile attacks which occur during the disease. Westphal and Uhlen-
hut 2 have found the bacilli within the central nervous system,
and these observers, as well as others, have found them lying
within the substance of the peripheral nerves, thus explaining the
anesthesia. A fact of enormous importance to the question of
transmission is the observation made by various observers, more
especially by Sticker, that the bacilli are found with great regu¬
larity in considerable numbers in the nasal secretions of persons
suffering from the disease. Sticker is inclined to regard the nose

1 Sticker , Munch, med. Woch., 39, 1897.

2 Westphal und Uhlenhut, Klin. Jahrb., 1901.

BACILLUS LEPILE AND LEPROSY

509

as the primary path of infection. Whether or not this be true can
not, at present, be decided. As a source of infection, however, the
nasal mucus and, secondarily, the saliva, are certainly the vehicles
by which large numbers of the bacilli leave the infected patient, and,
therefore, tend to spread the disease.

The contagiousness of leprosy is far less than is that of most other
bacterial diseases. Physicians and others who come into direct contact
with large numbers of leprous patients, observing at the same time the
ordinary precautions of cleanliness, rarely contract the disease. On
the other hand, intimate contact with lepers without such precautions
is the only possible means of transmission. The demonstration of
leprosy bacilli in dust, soil, etc., must always be looked upon with sus¬
picion, since, apart from actual human inoculation, there is no method
of positively differentiating the bacilli from similar acid-fast organisms.
Instances of transmission by contact are on record, not the least famous
of which is the case of Father Damien, who contracted the disease while
taking care of the lepers upon the island of Molokai. Hansen states
that in his knowledge no case of leprosy can be found in which careful
examination of the past history will not reveal direct contact with a
previous case. Direct inoculation of the human being with material
from a leprous patient has been successfully carried out by Arning,1
upon a Hawaiian criminal. In this case a piece of a leprous nodule
was planted into the subcutaneous tissue of the left arm. One month
after the inoculation, pain appeared in the arm and shoulder, and four
and a half months later a typical leprosy nodule was formed. Four
years after the inoculation, the patient was a typical leper.

Although our inability to cultivate the leprosy bacillus, and the lack
of success attending animal inoculation, have made it impossible to study
more closely the toxic action of this microorganism, there is, neverthe¬
less, some evidence which points toward the production of a poisonous
substance of some kind by the bacillus. Rost,2 who claims to have
cultivated the bacillus, manufactured from his cultures, by the technique
for the production of “Old Tuberculin,” a substance which he called
“leprolin,” and which he employed therapeutically in the same manner
in which tuberculin is employed in tuberculosis. As stated before, the
results of Rost still lack confirmation. Of far greater importance, both
in demonstrating the probability of the existence of a definite toxin as
well as in indicating the close relationship between the leprosy bacillus

1 Arning , Vers. d. Naturfor. u. Aerzte, 1886.

2 Rost, loc. cit.

510

PATHOGENIC MICROORGANISMS

and the Bacillus tuberculosis, are the investigations upon the action
of tuberculin upon leprous patients. When tuberculin is adminis¬
tered to lepers, a febrile reaction occurs usually twenty-four or more
hours after the administration. The fever differs from that produced
by the use of the same substance in tuberculous patients in that it is of
late occurrence and lasts considerably longer. At the same time, there
may be marked redness and tenderness of the nodules. In isolated
cases, Babes 1 has noticed alarmingly high and prolonged fever together
with systemic symptoms such as nausea, headache, and even uncon¬
sciousness, following the injection of tuberculin. The same .writer
claims to have extracted from the organs of lepers, which contained
enormous numbers of bacilli, substances which showed an action similar
to that of the tuberculin.

RAT LEPROSY

Stefansky 2 first observed this disease among rats in Odessa, and
since then it has been observed in Berlin (Rabinovitsch 3) , in London
(Dean4), in New South Wales (Tidswell 5), and in San Francisco
(Wherry 6 and McCoy7). The disease occurs spontaneously among
house rats and is characterized by subcutaneous induration, swelling of
lymph nodes, with, later, falling out of the hair, emaciation, and some¬
times ulceration. Its course is protracted and rats may live with it
for six months or a year. When a rat suffering from this disease is dis¬
sected there is usually found, under the skin of the abdomen or flank, a
thickened area which has the appearance of adipose tissue except that
it is more nodular and gray and less shiny than fat. It is so like fat,
however, that it is often possible to overlook it as evidence of disease
by one unfamiliar with the condition. In this area acid-fast bacilli
looking like the Bacillus leprae are found in large numbers. These
bacilli are also found in the lymph nodes and sometimes in small nodules
which appear in the liver and lung.

1 Babes , in Kolle und Wassermann, “Handbuch,” etc., Erst. Erganz. Bd., 1907.

2 Stefansky, Centralbl. f. Bakt., xxxiii, 481.

3 Rabinovitsch , Centralbl. f. Bakt., xxxiii, 577.

4 Dean, Centralbl. f. Bakt., xxxiv, 222; Jour. Hyg., xcix.

6 Tidswell, cited by Brinkerhoff in “The Rat and Its Relation to Public Health,”
Treas. Dept., Wash., 1910.

6 Wherry, J. A. M. A., June 6, 1908, p. 1903; Jour. Inf. Dis., dvii, Rep. U. S.
P. H., and M. H. S., xxiii, 1841.

’’McCoy, Rep. U. S, P. H. and M, H. S-, xxiii, 981; Abstr. in J. A. M. A.,
Aug. 22, 1908, 690,

RAT LEPROSY

511

The disease can be transmitted experimentally from rat to rat and
probably is transmitted naturally from rat to rat by the agency of
fleas (Wherry, McCoy). Although clinically not exactly like human
leprosy the condition is sufficiently like it to arouse much hygienic
interest. The distribution of the disease in various parts of the world
does not correspond with the distribution of leprosy. A peculiar feature
of its distribution is the fact that in San Francisco, as the writer was
told by McCoy, almost all the rats that suffered from this disease came
from the district in which the retail meat business is located, known as
“But chert own.” The organisms were made to multiply in vitro by
Zinsser and Cary in plasma preparations of growing rat spleen. Chapin
has succeeded in cultivating them by a method analogous to the trypsin-
egg albumen method employed by Duval. In the experiments of Zinsser
and Cary it was found that although the organisms may retain their
acid-fast characteristics for many weeks within leucocytes they degen¬
erate rapidly within the spleen cells, a fact which seems to have some
bearing on the mechanism of resistance possessed by the body against
acid-fast organisms.

CHAPTER XXXV

BACILLUS DIPHTHERIA, BACILLUS HOFFMANNI, AND BACILLUS

XEROSIS

BACILLUS DIPHTHERIA

Since 1821, when Bretonneau of Tours published his observa¬
tions, diphtheria has been an accurately recognized clinical entity.
Our knowledge of the disease in the sense of modern bacteriology,
however, begins with the first description of Bacillus diphtherise by
Ivlebs in 1883. Klebs 1 had observed in the pseudomembranes from
diphtheritic throats, bacilli which in the light of more recent knowledge
we can hardly fail to recognize as the true diphtheria organism. His
work, however, was purely morphological and, therefore, inconclusive.
One year after this announcement, Loeffler 2 isolated and cultivated an
organism which corresponded in its morphological characters to the one
described by Klebs. He obtained it from thirteen clinically unques¬
tioned cases of diphtheria, and, by inoculating it upon the injured mucous
surfaces of animals, succeeded in producing lesions which resembled
closely the false membranes of the human disease. His failure to find
the bacillus in all the cases he examined, his finding it, in one instance,
in a normal throat, and his inability to explain to his own satisfaction
some of the systemic manifestations of the infection which we now
know to be due to the toxin, caused him to frame his conclusions in
a tone of the utmost conservatism. The second and third publications
of Loeffler,3 however, and the inquiry into the nature of the toxins
produced by the bacillus, published in 1888 by Roux and Yersin,4
eliminated all remaining doubt as to the etiological relationship existing
between this organism and the disease.

Innumerable observations, both clinical and bacteriological, by
other workers, have, since that time, confirmed the early investigations,

1 Klebs, Verh. d. 2. Kongr. f. inn. Medizin, Wiesbaden, 1883.

2 Loeffler, Mittheil. a. d. kais. Gesundheitsamt, 1884.

3 Loeffler, Cent. f. Bakt., 1887 and 1890.

i Roux and Yersin, Ann. de l’inst. Pasteur, 1888 and 1889.

BACILLUS DIPHTHERIA

513

and it is to-day a scientific necessity to find the bacillus of Klebs and
Loeffler in the lesion before a diagnosis of u diphtheria ” can properly
be made.

Morphology and Staining. — While Bacillus diphtherise presents
certain characteristic appearances which facilitate its recognition, it is,
at the same time, subject to a number of morphological variations with

Fig. 105. — Bacillus diphtheria.

all of which it is important to be familiar. These variations are, to a
limited extent, dependent upon the age of the culture and upon the
constitution of the medium on which it has been grown. These
factors, however, do not control the appearance of the organism with
any degree of regularity, and any or all of its various forms may occur
in one and the same culture. It is likely that these different appear¬
ances represent stages in the growth and degeneration of the indi¬
vidual bacilli, but there does not seem to be any just reason for
believing that, as several observers have stated, there is definite correla¬
tion between its microscopic form and its biological characteristics, such

as virulence, toxicity, etc.

514

PATHOGENIC MICROORGANISMS

The bacilli are slender, straight, or slightly curved rods. In length
they vary from 1.2 micra to 6.4 micra, in breadth from 0.3 to 1.1. As
seen most frequently when taken from the throat they are about 4 to
5 micra in length. They are rarely of uniform thickness throughout
their length, showing club-shaped thickening at one or both ends.
Occasionally they may be thickest at the center and taper toward the
extremities. When thickened at one end only, a slender wedge-shape
results. Such forms are usually straight, of smaller size than their
neighbors, and are more often stained with great uniformity. These
are spoken of by Beck1 as the “ground type,” and assumed, for in¬
sufficient reasons, to be the young individuals. Branched forms
have been described by some investigators. They are rare and
probably to be regarded as abnormal or involution forms due to un¬
favorable environment.

The organisms stain with the aqueous anilin dyes. A characteristic
irregularity of staining which is of great aid in diagnosis is best obtained
with Loeffler’s “alkaline methylene-blue.” (For preparation see section
on Staining, p. 96.) Stained with this solution for five to ten minutes
many of the bacilli appear traversed by unstained transverse bands
which give them a striped or beaded appearance. The longer indi¬
viduals often have a strong resemblance to short chains of strepto¬
cocci. Others may appear unevenly granular. In cultures which
are about eighteen hours old, many of the bacilli may show deeply
stained oval bodies situated most frequently at the ends. These are
the so-called “polar” or “Babes-Ernst” bodies.2 Special stains have
been devised for the demonstration of these appearances. One of these
was originated by Neisser,3 who claims for it differential value in
distinguishing these organisms from pseudodiphtheria and xerosis
bacilli.

His method requires two solutions:

1. Methylene blue (Griibler) . 1 gram.

Alcohol, 96 per cent . 20 c.c.

Glacial acetic acid . 50 “

Water . 950 “

2. Bismarck brown . 2 grams.

Water . 1,000 c.c.

1 Beck, in Kolle und Wassermann, ii, p. 773.

2 Babes, Zeit. f. Hyg., Bd. V, 1889.

3 Neisser, Zeit. f. Hyg., xxiv, 1897.

BACILLUS DIPHTHERIAS

515

The cover-slip preparation, after having been fixed, is stained with so¬
lution No. 1 for one to three seconds. It is then washed in water and
immersed for from three to five seconds in solution No. 2. With this
stain the bodies of the bacilli appear brown, the polar granules blue.

Another method which has been extensively used is that of Roux.
The solutions required for this are :

1. Dahlia violet . 1 gram.

Alcohol, 90 per cent . 10 c.c.

Aq. dest . ad 100

2. Methyl green . 1 gram.

Alcohol, 90 per cent . 10 c.c.

Aq. dest . ad 100

The two solutions are mixed, one part of 1 being added to three parts
of 2. Preparations are stained in this mixture for two minutes. The
polar bodies appear a dark violet. Other methods for the staining of
polar bodies have been recommended. There is very little advantage
in the use of these double stains and most bacteriologists employ for
routine work the simple stain with Loeffler’s alkaline methylene blue.

The significance of the polar bodies is not well understood. Their
discoverer, Ernst, regarded them as bodies analogous to the spores of
other organisms. The ease with which they are stained, however, and
the low temperatures to which the bacteria succumb make this appear
very unlikely. A more probable interpretation seems to be that of
Escherich 1 who regards them as chromatic granules.

Stained by Gram's method, the diphtheria bacilli retain the gentian-
violet. Care must be used in carrying out this method and strict timing
adhered to, since slight carelessness in this respect may lead to irregular
results.

In stained smears from the throat or from cultures a characteristic
grouping of the bacilli has been observed. They lie usually in small
clusters, four or five together, parallel to each other, or at sharp angles.
Two organisms may often be seen attached to each other by their cor¬
responding ends while their bodies diverge to form a “ V ” or “ Y” shape.

Biological Characteristics. — The diphtheria bacillus is a non-motile,
non-flagellated, non-spore-forming aerobe. Its preference for oxygen
is marked, but it will grow in anaerobic environment in the presence of
suitable carbohydrates. It does not liquefy gelatin. The bacillus grows
at temperatures varying between 19° C. and 42° C., the most favorable

1 Escherich, “ Aetiologie, etc., d. Diphth. , Wien, 1894,

516

PATHOGENIC MICROORGANISMS

temperature for its development being 37.5° C. Temperatures above
37.5°, while not entirely stopping its growth, impede the development
of its toxin.

Resistance. — The thermal death point of this organism is 58° C. for
ten minutes, according to Welch and Abbott. Boiling kills it in about
one minute. Low temperatures, and even freezing, are well borne.
Desiccation and exposure to light are not so fatal to this organism as
to most of the other pathogenic bacteria. Sternberg 1 has found it
alive in dried bits of the pseudomembrane after fourteen weeks. It is
easily killed by chemical disinfectants in the strengths customarily
employed. H202 seems especially efficacious in killing the organisms
rapidly.

Cultivation. — The diphtheria bacillus grows readily on most of the
richer laboratory media. It will grow upon media made of meat
extract, but develops more luxuriantly on all those which have a meat
infusion as their basis. While it will grow upon both acid and alkaline
media, it is sensitive to the extremes of both, the most favorable reaction
for its development being probably about 0.5 per cent alkalinity ex¬
pressed in terms of y NaOH. Animal proteids added to the media,
in the form of blood serum, ascitic fluid, or even whole blood, increase
greatly the rapidity and richness of its growth. Horse serum is sup¬
posed by some to be especially favorable.2

Loeffler’s Medium. — The most widely used medium for the cultiva¬
tion of this bacillus is the one devised by Loeffler. This consists of :

Beef blood serum . 3 parts

One per cent glucose meat-infusion bouillon . 1 part

The mixture is coagulated at 70° C. in slanted tubes and sterilized at
low temperatures by the fractional method. Upon this medium the
diphtheria bacillus in twelve to twenty-four hours develops minute,
grayish-white, glistening colonies. These enlarge rapidly, soon out¬
stripping the usually accompanying streptococci. The medium seems
to possess almost selective powers for the bacillus and, for this reason,
it is especially valuable for diagnostic purposes.

Meat-Infusion Agar. — Upon slightly alkaline meat-infusion agar
the bacillus develops readily, though less so than on Loeffler’s serum.
Organisms which have been on artificial media for one or more genera¬
tions may grow with speed and luxuriance upon this medium. When
planted directly from the human or animal body upon agar, however,

1 Sternberg, “ Manual Bac.,” p. 455.

2 Michel, Cent. f. Bakt., 1897.

BACILLUS DIPHTHERIAS

517

growth may occasionally be slow and extremely delicate. Colonies
on agar appear within twenty-four to thirty-six hours as small,
rather translucent, grayish specks. The appearance of these colonies
is quite characteristic and easily recognized by the practiced observer.
Surface colonies are irregularly round or oval, showing a dark,
heaped-up, nucleus-like center, fringed about by a loose, coarsely
granular disk. The edges of these colonies have a peculiarly irregular,
torn appearance which distinguishes them readily from the sharply
defined chain-fringed streptococcus colonies. For these reasons agar
is the medium most commonly used for purposes of isolation. The
deep colonies in this medium are dense and sharply outlined.

The addition of dextrose 1 per cent, nutrose 2 per cent, or glycerin
6 per cent, renders agar more favorable for rapid growth, but unfits it
for the preservation of cultures, the organism dying out more rapidly,
probably because of acid formation.

Meat-Infusion Broth. — Upon beef or veal broth the diphtheria bacil¬
lus grows rapidly, almost invariably forming a pellicle upon the surface,
— another expression of its desire for oxygen. The broth remains
clear. Broth tubes with such growth, therefore, have a character¬
istic appearance.

Meat-infusion gelatin is a favorable medium for the Klebs-Loeffler
bacillus, but growth takes place slowly because of the lowT temperature
at which this medium must be kept. Gelatin is not fluidified.

Milk is an excellent medium, and for this reason may even occa¬
sionally be a vehicle of transmission. There is no coagulation of the
milk.

Upon potato , B. diphtheria will grow only after neutralization of the
acid. It is, at best, however, a poor nutrient medium.

Upon the various pepton solutions the bacillus of diphtheria produces
no indol.

Many special media have been recommended for the cultivation of
this organism. The most important of these are the modification of
Loeffler’s serum devised by Beck,1 the horse-blood-fibrin cake used by
Escherich, and WassermamTs ascitic-fluid-nutrose-agar, called by him
“Nasgar.” None of these has sufficient advantages over the simpler
media, however, to make its substitution desirable.

Isolation. — Because of the comparative ease with which B. diph¬
theria is isolated from mixed cultures, it is not necessary to give in de-

1 M. Beck, Kolle und Wassermann; Brit. Med. Jour.

518

PATHOGENIC MICROORGANISMS

tail the different methods devised for this purpose. The one de¬
scribed below is the one most frequently employed, and is both simple
and reliable.

Cultures are taken from throats upon Loeffler’s blood serum.
These are permitted to grow at 37.5° C. for from eighteen to twenty-
four hours. At the end of this time about 5 c.c. of bouillon are
poured into the tubes and the growth is gently emulsified in the broth
with a platinum loop. Two or three loopfuls of this emulsion are
then streaked over the surface of glucose agar, serum agar, or nutrose

Fig. 106. — Colonies of Bacillus diphtheria on Glycerin Agar.

agar. After twenty -four hours7 incubation these plates show char¬
acteristic colonies which can be easily fished and again transferred
to Coefher tubes or any other suitable medium. The same method

BACILLUS DIPHTHERLE

519

is well adapted for the isolation of pseudodiphtheria and xerosis
bacilli.

Pathogenicity. — Unlike most other microorganisms, Bacillus diph¬
theria causes a more or less specific local reaction in mucous mem¬
branes, which results in the formation of the so-called “ pseudo-mem¬
branes.77 When these are characteristically present, infection with this
bacillus should always be suspected. The consequent disease depends,
in part, upon the mechanical disturbance caused by these false mem¬
branes and, in part, upon the systemic poisoning with the toxin which the
bacilli produce. Although the diphtheria bacillus has been found after
death in the spleen and liver, we have no data which would justify the
assumption that a true diphtheria-septicemia may occur during life.
It is probable that in those cases which Baginsky 1 has called the sep¬
ticemic form of diphtheria, Bacillus diphtheriae has merely opened a
path by which accompanying streptococci have gained access to the
lymphatics and the blood stream. The most frequent sites of diph¬
theritic inflammation are the mucous membranes of the throat, larynx,
and nose. They have also been found in the ear, upon the mucous
membrane of the stomach and the vulva, and upon the conjunctiva and
the skin. According to Loeffler, Strelitz,2 and others, the bacillus may,
by extension from the larynx, give rise to a true diphtheritic broncho¬
pneumonia.

For most of the usual laboratory animals the diphtheria bacillus is
very pathogenic. Dogs, cats, fowl, rabbits, and guinea-pigs are sus¬
ceptible. Rats and mice are resistant to all but extremely large doses.
False membranes, analogous in every way to those found in human
beings, have been produced in many animals of susceptible species, but
only after inoculation with the bacillus had been preceded by mechanical
injury of the mucosa. The lesions produced in animals by subcutane¬
ous inoculation present many characteristic features which facilitate the
bacteriological recognition of the diphtheria bacillus. Small quantities
(0.5 to 1 c.c.) of a virulent broth culture, given subcutaneously to a
guinea-pig, may produce the gravest symptoms and within six to eight
hours the animal may show signs of great discomfort. Death occurs
usually within thirty-six to seventy-two hours. Upon autopsy the point
of inoculation is soggy with serosanguineous exudate; neighboring
lymph-nodes are edematous. Lungs, liver, spleen, and kidneys are
congested. There may be pleuritic and peritoneal exudates. Charac-

1 Baginsky, “ Lehrbuch d. Kinderkrankheiten.”

2 Strelitz , Arch. f. Kinderheilk., 1891.

520

PATHOGENIC MICROORGANISMS

teristic, and almost pathognomonic, is a severe congestion of both
suprarenal bodies. The gastric ulcerations recently described by Rose-
nau and Anderson 1 may occur, but are by no means regularly found
(two out of fifty in our series 2) .

Diphtheria Toxin.3 — Animals and man infected with B. diphtherise
show evidences of severe systemic disturbances and even organic de¬
generations, while the microorganism itself can be found in the local
lesion only. This fact led even the earliest observers to suspect that,
in part at least, the harmful results of such an infection were attrib¬
utable to a soluble and diffusible poison elaborated by the bacillus. The
actual existence of such a poison or toxin was definitely proved by
Roux and Yersin 4 in 1889. They demonstrated that broth cultures in
which B. diphtherise had been grown for varying periods would remain
toxic for guinea-pigs after the organisms themselves had been re¬
moved from the culture fluid by filtration through a Chamberland filter.

Methods of Production of Diphtheria Toxin. — While toxin can
be produced with almost all of the virulent diphtheria bacilli, there
is great variation in the speed and degree of production, dependent
upon the strain of organisms employed and upon the ingredients and
reaction of the medium upon which they are grown. Most labora¬
tories possess one or several strains of bacilli which are empirically
known to be especially potent in this respect. One of the most ex¬
tensively used, not only in this country, but in Europe as well, is
the strain known as “ Culture Americana/’ or “Park- Williams Bacillus
No. 8,” an organism isolated by Dr. Anna Williams of the New York
Department of Health in 1894. Throughout more than ten years of cul¬
tivation this bacillus has retained its great power of toxin production.

Because of the severity of cases of diphtheria in which the diph¬
theria bacilli were associated with streptococci, many observers were
led to believe that the presence of streptococci tended to increase the
toxin-producing power of B. diphtherise. Experiments by Hilbert,5
Theobald Smith,6 and others seem to have given support to this view.

The medium most frequently employed for the production of toxin is
a beef-infusion broth. There are minor differences of opinion as to the

1 Rosenau and Anderson, Journ. Inf. Dis., iv, 1907.

2 Zinsser, Journ. Med. Res., xvii, 1907.

3 Loeffler , Cent. f. Bakt., 1887.

4 Roux and Yersin, loc. cit.

5 Hilbert, Zeit. f. Hyg., xxix, 1898.

6 Smith, Medical Rec., May, 1896.

521

BACILLUS DIPHTHERIAS

most favorable constitution of this medium for the production of toxin.
All agree, however, in recognizing the importance of pepton, without
which, according to Madsen,1 no satisfactory toxin has yet been pro¬
duced. This is added in proportions of from one to two per cent. The
presence of sugars in the medium is not desirable in that it leads to acid
production; L. Martin 2 removes the sugars from the meat by fermen¬
tation with yeast. Smith 3 accomplishes the same purpose with B.
coli. According to Park and Williams,4 however, this is superfluous,
the quantity of sugar present in ordinary butcher’s meat not being-
sufficient to exert unfavorable influence.

Experience has shown that a primary alkaline reaction offers the
most favorable conditions for toxin production. In all cultures of B.
diphtheria in non-sugar free broth, there is, at first, a production of
acid and, while this continues, there is, as Spronk 5 has shown, little
or no evidence of toxin elaboration. It is only after this initial acid¬
ity has given way to alkalinity that cultures become decidedly toxic.
Park and Williams,6 in an inquiry into the question of reaction, came
to the conclusion that the best results are obtained with a broth to
which, after neutralization to litmus, y NaOH is added in an amount
of 7 c.c. to the liter. In such a medium the largest yield of toxin is
obtained after about five to eight days’ growth at a temperature of
37.5° C. If left at this temperature for a longer period a diminution
in the strength of the toxin takes place.

Free access of oxygen to the culture medium during the growth
of the organisms has been found to be of great importance. Roux
obtained this by passing a stream of oxygen through the bouillon. The
supply is quite sufficient for practical purposes, however, if the medium
is distributed in thin layers in large-necked Erlenmeyer flasks.

Chemical Nature and Physical Properties of Diphtheria
Toxin. — The chemical composition of diphtheria toxin is not known.
Brieger and Frankel,7 by repeated precipitation with alcohol, succeeded
in extracting from toxic bouillon a white, water-soluble powder which
possessed most of the poisonous proport ies of the broth itself. This, in

1 Madsen, Kraus und Levaditi, “ Handbuch d. Technic,” etc., 1907.

2 L. Martin, Ann. de Tinst. Pasteur, 1897.

3 Th. Smith, Jour. Exp. Med., iv, 1899.

4 Park and Williams . Journ. Exp. Med., 1897.

5 Spronk, Ann. de Tinst. Pasteur, 1895.

6 Park and Williams, Jour. Exp. Med., 1897.

7 Brieger und Frankel, Berl. klin. Woch., xi-xii, 1889.

522

PATHOGENIC MICROORGANISMS

solution, gave many of the usual proteid reactions, but differed from pro-
teids in failing to coagulate when boiled and in not giving precipitates
when treated with magnesium sulphate, sodium sulphate, or nitric acid.
It was believed by them to be closely related to the albumoses, bodies rep¬
resenting intermediate phases in the peptonization of albumins. Similar
results have been obtained by Wassermann and Proskauer,1 Brieger and
Boer,2 and others. Uschinsky,3 on the other hand, has disputed the
proteid nature of toxins in general and has succeeded in producing
diphtheria toxin by growing the organism upon a medium entirely free
from albuminous bodies. Uschinsky believes that the proteid reactions
observed by other workers may be due to ingredients of the precipitates
other than the toxin. It is not impossible, however, that the organ¬
isms may have produced proteid substances by synthesis from the
simpler substances in Uschinsky ’s medium. The production of toxin
from such a medium, therefore, is not a conclusive argument against the
proteid nature of toxins. Accurate chemical isolation and analysis of
diphtheria toxin have not yet been accomplished.

Diphtheria toxin is destroyed,4 when in the fluid form, by tem¬
peratures of 58° to 60° C. In the dry state, it may resist a temperature
of 70° C. and over, without noticeable change. Light and the free access
of air produce rapid deterioration. Sealed, protected from light, and
kept at almost freezing point, the toxin remains stable for very long
periods. Electrical currents passed through toxic broth have little or
no effect upon it.

Bacteria Similar to Bacillus Diphtheriae. — Bacillus Hoffmanni
(Pseudodiphtheria bacillus). — Hoffmann-Wellenhoff,5 in 1888, and, at
almost the same time, Loeffler,6 described bacilli which they had cul¬
tivated from the throats of normal persons and in several instances from
those of diphtheritic persons, which were in many respects similar to true
B. diphtheriae, but differed from this chiefly in being non-pathogenic for
guinea-pigs. These organisms were at first regarded by some observers
as merely attenuated diphtheria bacilli More recent investigations,
however, prove them to be unquestionably a separate species, easily
differentiable by proper methods. They differ from B. diphtheriae in so

1 Wassermann und Proskauer, Deut. med. Woch., 1891, p. 585.

2 Brieger und Boer, Deut. med. Woch., 1896, p. 783.

3 Uschinsky, Cent. f. Bakt., xxi, 1897.

4 Roux et Yersin, loc. cit.

5 Hoffmann-Wellenhoff, Wien. med. Woch., iii, 1888.

6 Loeffler, Cent. f. Bakt., ii, 1887.

BACILLUS DIPHTHERIA

523

many important features, moreover, that the term “pseudodiphtheria
bacillus” is hardly an appropriate one for them.

Morphology.— Bacillus Hoffmanni is shorter and thicker than
Bacillus diphtheria. It is usually straight and slightly clubbed at one
end, rarely at both. Stained with Loefheris blue it occasionally shows
unstained transverse bands; unlike B. diphtheria, however, these

Fig. 107. — Bacillus Hoffmanni.

bands hardly ever exceed one or two in number at most. In many
cultures the single transverse band gives the bacillus a diplococcoid
appearance.

Staining. — Stained by Neisseris or Roux’s method, no polar bodies
can be demonstrated. The bacillus forms no spores, is non-motile, and
possesses no flagella.

524

PATHOGENIC MICROORGANISMS

Cultivation. — On the usual culture media B. Hoffmanni grows
more luxuriantly than B. diphtheriae, developing even in first isola¬
tions from the human body upon the simple meat-extract media. On
agar plates its colonies are larger, less transparent, and whiter than are
those of true diphtheria bacilli. In fluid media there is even clouding

Fig. 108. — Colonies of Bacillus Hoffmanni on Agar.

and less tendency to the formation of a pellicle than with B. diphtheria}.
A positive means of distinction between the two is given by the inability
of B. Hoffmanni to form acid upon various sugar media. The differ¬
entiation on a basis of acid formation was first attempted by Cobbett 1

1 Cobbett, Cent. f. Bakt., 1898.

BACILLUS DIPHTHERIA

525

and has been recently worked out systematically by Knapp/ and con¬
firmed by various observers.1 2 The results of this work, carried out with
the serum-water media of Hiss to which various sugars were added,
show that B. Hoffmanni forms acid upon none of the sugars used, while
B. diphtherise acidifies and coagulates media containing monosaccharids

and several of the more complex sugars, as given in the diagram in the
section following, dealing with B. xerosis.

Differentiation can finally be made on the basis of animal pathogen¬
icity, B. Hoffmanni being entirely innocuous to the ordinary laboratory
animals. B. Hoffmanni forms no toxins, and animals immunized with it
do not possess increased resistance to B. diphtherise.

Bacillus xerosis. — In 1884, Ivutschert and Neisser 3 described a
bacillus which they had isolated from the eyes of patients suffering from

1 Knapp, Jour. Med. Res., vii, 1904.

2 Graham Smith, Jour, of Hyg., vi, 1906; Zinsser, Jour. Med. Res., xvii, 1907.

3 Kutschert und Neisser, Deut. med. Woch., xxiv, 1884.

526

PATHOGENIC MICROORGANISMS

a form of chronic conjunctivitis known as xerosis. This bacillus, which,
morphologically, is almost identical with B. diphtheria, they believed
to be the etiological factor of the disease. The frequency with which it
has been isolated from normal eyes, however, precludes this etiological
relationship, and it may safely be regarded as a harmless parasite which
may indeed be more abundant in the slightly inflamed than in the normal
conjunctiva.

Morphology. — B. xerosis resembles B. diphtheria closely. It is
occasionally shorter than this, but on the whole no absolute morphologi¬
cal differentiation between the two is possible. It forms no spores and
is non-motile. Polar bodies may occasionally be seen.

Cultivation. — On Loeffler’s blood serum , on agar , glycerin agar , and
in broth } its growth is very similar to that of B. diphtherial, but more
delicate throughout. It can not easily be cultivated upon the simple
meat-extract media, nor will it grow on gelatin at room temperature.
Its colonies on glycerin or glucose agar are microscopically identical
with those of B. diphtherise.

Differentiation. — It differs from B. diphtheria distinctly in its
acidifying action on sugar media. These relations were first worked
out by Knapp for various sugars and the alcohol mannit, and have been
extensively confirmed by others. The differentiations resulting may be
tabulated as follows:

Diphtheria.

Xerosis.

Hoffmanni.

Hiss serum- water media plus 1%

Dextrose .

Levulose .

—

Galactose .

—

Mannit .

—

Maltose .

—

Lactose .

—

Saccharose . .

—

Dextrin .

—

A reference to the table shows that differentiation may be made
by the use of two sugars — saccharose and dextrin. B. diphtherise
forms acid from dextrin, not from saccharose; B. xerosis from sac¬
charose, not from dextrin; B. Hoffmanni does not form acid from
either.

B. xerosis is non-pathogenic to animals and forms no toxin.

BACILLUS DIPHTHERIA

527

Other Bacilli Morphologically Resembling the Diphtheria Bacillus. —

Many bacilli have been described which have a slight morphological
resemblance to Bacillus diphtherise but which have little or no patho¬
logical significance. Such organisms are met with in milk, air, and
water, and as secondary invaders together with other bacteria in old
discharging wounds. These bacilli are usually larger than the diph¬
theria bacillus and, although transversely striped, rarely show polar
bodies. On the various media they grow heavily on even the simpler
nutrient substances, with heavy, usually white or yellowish-white colo¬
nies. Culturally they show all the qualities of saprophytes. Inoculated
into animals they produce at most a mild local reaction. In the litera¬
ture these organisms have often been loosely spoken of as “ Pseudodiph¬
theria ” bacilli, a term which is inappropriate since they have nothing
in common with the Klebs-Loeffler bacillus except a certain morpholog¬
ical resemblance. Differentiation is never difficult.

CHAPTER XXXVI

BACILLUS MALLEI
( Glanders Bacillus)

Glanders is an infectious disease prevalent chiefly among horses,
but transmitted occasionally to other domestic animals and to man.
The microorganism causing the disease, though seen and described by
several earlier authors, was first obtained in pure culture and accurately
studied by Loeffler and Schiitz 1 in 1882.

Morphology and Staining. — The glanders bacillus or B. mallei is a
rather small rod with rounded ends.2 Its length varies from 3 to 4
micra, its breadth from 0.5 to 0.75 micron. Variation in size be¬
tween separate individuals in the same culture is characteristic. The
rods are usually straight, but may show a slight curvature. The bacillus
is non-motile. There are no flagella and no spores are formed. The
grouping of the bacilli in smears shows nothing very characteristic.
Usually they appear as single bacilli lying irregularly parallel, often in
chains of two or more. In old cultures, involution forms appear which
are short, vacuolated, and almost coccoid.

While the glanders bacillus stains rather easily with the usual anilin
dyes, it is so easily decolorized that especial care in preparing specimens
must be observed. Stained in the usual manner with methylene-blue,
it shows marked irregularity in its staining qualities; granular, deeply
staining areas alternating with very faintly stained or entirely unstained
portions. This diagnostically helpful characteristic has been variously
interpreted as a mark of degeneration or a preparatory stage for sporula-
tion. It is probably neither of the two, but an inherent irregularity in
the normal protoplasmic composition of the bacillus, not unlike that
c f B. diphtherise. The bacillus is decolorized by Gram’s method of
staining.

Cultivation. — The glanders bacillus is easily grown on all of the

1 Loeffler und Schiitz, Deut. med. Woch., 1882.

2 Loeffler, Arb. a. d. kais. Gesundheitsamt, 1886.

528

BACILLUS MALLEI

529

usual meat-infusion media. It is practically indifferent to moderate
variations in reaction, growing equally well upon neutral, slightly acid,
or slightly alkaline culture media. Glycerin or small quantities of
glucose added to media seem to render them more favorable for the
cultivation of this bacillus.

Upon agar the colonies show little that is characteristic. They
appear after twenty-four hours at 37.5° C. as yellowish-white spots,
at first transparent, later more opaque. They are round, with an even

Fig. 110. — Glanders Bacillus. From potato culture. (After Zettnow.)

border, and microscopically appear finely granular. The older the cul¬
tures are, the more yellow do they appear.

On gelatin at room temperature, growth is slow, grayish-white, and
no liquefaction of the gelatin occurs. Growth upon this medium is
never abundant.

In broth, there is, at first, diffuse clouding, later a heavy, tough,
slimy sediment is formed. At the same time the surface is covered with
a similarly slimy pellicle. The broth gradually assumes a dark brown
color.

In milk, coagulation takes place slowly. In litmus milk, acidifica¬
tion is indicated.

The growth upon potato presents certain features which are diagnos¬
tically valuable. On potatoes which are not too acid growth is abundant
and within forty-eight hours covers the surface as a yellowish, trans-
35

530

PATHOGENIC MICROORGANISMS

parent, slimy layer. This gradually grows darker until it has assumed
a deep reddish-brown hue. In using this feature of the growth diagnos¬
tically, it must not be forgotten that a very similar appearance upon
potato occurs in the case of B. pyocyaneus.

Biological Considerations. — Bacillus mallei is aerobic.1 Growth under
anaerobic conditions may take place, but it is slow and impoverished.
The most favorable temperature for its cultivation is 37.5° C. It
fails to develop at temperatures below 22° C. or above 43° C. On
artificial media, if kept cool and in the dark, and in sealed tubes, the
glanders bacillus will retain its viability for months and years. On
gelatin and in bouillon, it lives for a longer time than on the other media.
Exposed to strong sunlight it is killed within twenty-four hours. Heat¬
ing to 60° C. kills it in two hours, to 75° C. within one hour. Thorough
drying kills the glanders bacillus in a short time. In water, under the
protected conditions that are apt to prevail in watering-troughs, the
bacillus may remain alive for over seventy days. The resistance to
chemical disinfectants is not very high.2 Carbolic acid, one per cent,
kills it in thirty minutes, bichlorid of mercury, 0.1 per cent, in fifteen
minutes.

Pathogenicity. — Spontaneous infection with the glanders bacillus
occurs most frequently in horses. It occurs also in asses, in cats, and,
more rarely, in dogs. In man the disease is not infrequent and is
usually contracted by those in habitual contact with horses. Experi¬
mental inoculation is successful in guinea-pigs and rabbits. Cattle,
hogs, rats, and birds are immune to experimental and spontaneous
infections alike.

Spontaneous infection takes place by entrance through the broken
skin, through the mucosa of the mouth or nasal passages. Infection in
horses not infrequently takes place through the digestive tract.3 In all
cases, so far as we know, previous injury to either the skin or to the
mucosa is necessary for penetration of the bacilli and the development
of the disease.

Glanders in horses may occur in an acute or chronic form, depending
upon the relative virulence of the infecting culture and the susceptibility
of the subject. The more acute form of the disease is usually limited
to the nasal mucosa and upper respiratory tract. The more chronic
type of the disease is often accompanied by multiple swellings of the

1 Loeffler, loc. cit. 2 Finger, Ziegler’s Beitr., vi, 1889.

3 Nocard, Bull, de la soc. centr. de med. vet., 1894.

BACILLUS MALLEI

531

skin and general lymphatic enlargement. This form is often spoken of
as “farcy.”

Acute glanders in the horse begins violently with fever and prostra¬
tion. After two or three days there is a nasal discharge, at first serous,
later seropurulent. At the same time there is ulceration of the nasal
mucosa and acute swelling of the neighboring lymph nodes. These
ma3r break down and form deep pus-discharging sinuses and ulcers.

Fig. 111. — Glanders Bacilli in Tissue. (From a drawing furnished by

Dr. James Ewing.)

Finally, there is involvement of the lungs and death within four to six
weeks.

When the disease takes the chronic form the onset is more gradual.
Concomitant with the nasal inflammation there is a formation of subcu¬
taneous swellings all over the body, some of which show a tendency to
break down and ulcerate. Together with this the lymphatics all over
the body become enlarged. The disease may last for several years, and
occasionally may end in complete cure. In horses the chronic form of

532

PATHOGENIC MICROORGANISMS

the disease is by far the more frequent. In man the disease is similar
to that of the horse except that the point of origin is more frequently
in some part of the skin rather than in the nasal mucosa, and the
clinical symptoms differ accordingly. The onset is usually violent,
with fever and systemic symptoms. At the point of infection a nodule
appears, surrounded by lymphangitis and swelling. A general papular
eruption may occur. The papules may become pustular, and the
clinical features may thus simulate variola. This type of the disease
usually ends fatally in eight to ten days. The chronic form of the
disease in man is much like that in the horse, but is more frequently
fatal.

The histological appearance of the glanders nodules is usually one of
diffuse leucocytic infiltration and the formation of young connective
tissue which preponderates more and more as the disease becomes
chronic. Virchow has classed these lesions with the granulomata.
From the center of such nodules B. mallei may often be obtained in pure
culture. The nodules may be generally distributed throughout the
internal organs. The bacilli themselves are found, apart from the
nodules, in the nasal secretions, and occasionally in the circulating
blood.1

The bacteriological diagnosis of glanders may be made by isolating
and identifying the bacilli from any of the above-mentioned sources.
When superficial nodules can be opened for the purpose of diagnosis this
may prove an easy task. The most diagnostically helpful medium in
such cases is potato. In a majority of cases, however, isolation is ex¬
tremely difficult and resort must be had to animal inoculation. The
most suitable animal for this purpose is the male guinea-pig. Intra-
peritoneal inoculation of such animals with material containing glanders
bacilli leads within two or three days to tumefaction and purulent
inflammation of the testicles. Such an experiment, spoken of as the
“ Strauss test ,” 2 should always be reinforced by cultural examination
of the testicular pus, the spleen, and the peritoneal exudate of the
animals employed.

Toxin of Bacillus mallei. — The toxin of B. mallei, or mallein, belongs
to the class of endotoxins. The toxic products have been invariably
obtained by extraction of dead bacilli.3 Mallein differs from many
other bacterial poisons in being extremely resistant. It withstands

1 Wassilieff, Deut. med. Woch., 1883.

2 Strauss, Arch, de med. exp., 1889.

3 Kresling, Arch. d. sci. bioh, 1892; Preuser, Berl. thierarzt. Woch., 1894.

BACILLUS MALLEI

533

temperatures of 120° C. and prolonged storage without noticeable loss of
strength.1 >

In its physiological action upon healthy animals, mallein is not a
powerful poison. It can be given in considerable doses without causing
death. Mallein may be obtained by a variety of methods. Helman
and Kalning, the discoverers of this toxin, used filtered aqueous and
glycerin extracts of potato cultures. Roux 2 cultivates virulent gland¬
ers bacilli in flasks containing 250 c.c. each of 5 per cent glycerin
bouillon. Growth is allowed to continue at 35° C. for one month. At
the end of this time, the cultures are sterilized at 100° for thirty min¬
utes, and evaporated on a water bath to one-tenth their original volume.
They are then filtered through paper. This concentrated poison is
diluted ten times with 0.5 per cent carbolic acid before use. Concen¬
tration is done merely for purposes of conservation. The diagnostic
dose of such mallein for a horse is 0.25 c.c. of the undiluted fluid.

At the Washington Bureau of Animal Industry, mallein is prepared
by growing the bacilli for five months at 37.5° C. in glycerin-bouillon.
This is then boiled for one hour and allowed to stand in a cool place
for one week. The supernatant fluid is then decanted and filtered
through clay filters by means of a vacuum pump. The filtrate is
evaporated to one-third its original volume on a water bath, and the
evaporated volume resupplied by a 1 per cent carbolic acid solution
containing about 10 per cent of glycerin.

Diagnostic Use of Mallein. — The injection of a proper dose of
mallein into a horse suffering from glanders is followed within six to
eight hours by a sharp rise of temperature, often reaching 104° to 106° F.
(40° C. T ) . The high temperature continues for several hours and then
begins gradually to fall. The normal is not usually regained for several
days. Locally, at the point of injection, there appears within a few
hours a firm, hot, diffuse swelling, which gradually extends until it may
cover areas of 20 to 30 centimeters in diameter. The swelling is in¬
tensely tender during the first twenty-four hours, and lasts for three to
nine days. Together with this there are marked symptoms of general
intoxication. In normal animals the rise of temperature following an
injection is trifling, and the local reaction is much smaller and more
transient. Injections are best made into the breast or the side of the

neck.

1 Wladimiroff, in Kraus und Levaditi, “ Handbuch,” etc., 1908.

2 Roux et Nocard, Bull. d. 1. soc. centr. vet., 1892.

534

PATHOGENIC MICROORGANISMS

The directions given by the United States Government for using
mallein for the diagnosis of glanders in horses are as follows:

“Make the test, if possible, with a healthy horse, as well as with
one or more affected or supposed to be affected with glanders. Take
the temperature of all these animals at least three times a day for one
or more days before making the injections.

“The injection is most conveniently made at 6 or 7 o’clock in the
morning, and the maximum temperature will then usually be reached
by or before 10 p.m. of the same day.

“Use for each horse one cubic centimeter of the mallein solution as
sent out, and make the injection beneath the skin of the middle of one
side of the neck, where the local swelling can be readily detected.

“Carefully sterilize the syringe after injecting each horse by flaming
the needle over an alcohol lamp or, better, use separate syringes for
healthy and suspected animals. If the same syringe is used, inject the
healthy animals first, and flame the needle of the syringe after each
injection.

“Take the temperature every two hours for at least eighteen hours
after the injection. Sterilize the thermometer in a 5 per cent solu¬
tion of carbolic acid, or a 0.2 per cent solution of corrosive sublimate,
after taking the temperature of each animal.

“The temperature, as a rule, will begin to rise from four to eight hours
after the injection, and reach its maximum from ten to sixteen hours
after injection. On the day succeeding the injection take the tempera¬
ture at least three times.

“ In addition to the febrile reaction, note the size, appearance, and
duration of any local swelling at the point of injection. Note the general
condition and symptoms of the animal, both before, during, and after
the test.

“Keep the solution in the sealed bottle and in a cool place, and do
not use it when it is clouded or if it is more than six weeks old • when it
leaves the laboratory of the Bureau it is sterile.”

If the result of first injection is doubtful, the horse should be
isolated and retested in from one to three months, when the slight
immunity conferred by the first injection will have disappeared.
The second injection into healthy horses usually shows no reaction
whatever.

Mallein may cause reactions in the presence of other diseases than
glanders, such as bronchitis, periostitis, and other inflammatory lesions
and is not so specifically valuable as tuberculin for diagnosis.

BACILLUS MALLEI

535

Immunity. — Recovery from a glanders infection does not confer
immunity against a second inoculation.1 Artificial active immunization
has been variously attempted by treatment with attenuated cultures,
with dead bacilli, and with mallein, but without convincing results.

The serum of subjects suffering from glanders contains specific
agglutinins.2 These are of great importance diagnostically if the tests
are made with dilutions of, at least, 1 in 500, since normal horse serum
may agglutinate B. mallei in dilutions lower than this.

1 Finger, Ziegler’s Beitrage, vi; 1899.

2 Galtier, Jour, de med. vet., 1901.

CHAPTER XXXVII

BACILLUS INFLUENZAE AND CLOSELY RELATED BACTERIA

There is no other epidemic disease which spreads over such enormous
territories, and with such speed, as influenza. Epidemics have been
numerous and reports of the disease, unquestionably recognizable, are
extant even from the most remote times. The last serious epidemic
occurred in the years 1889 to 1890, when the disease, spreading from
the East, traveled through Russia and, panclemically, attacked all of
Europe, then reached America, and eventually, having traveled east¬
ward as well as westward from its point of origin, became prevalent
in China, Japan, Australia, and Africa. Hundreds of thousands were
attacked and the mortality of this epidemic was high. Its enormous
scope and the rapidity of its spread were facilitated probably by the
activity of modern international commerce.

The character of the disease pointed so definitely to a bacterial
etiology that numerous attempts to isolate a specific microorganism
were, of course, made. Pfeiffer 1 finally, in 1892, described the bacillus
which is at present definitely recognized as the etiological factor of
influenza.

Morphology and Staining. — The bacillus of influenza (Pfeiffer bacillus)
is an extremely small organism, about 0.5 micron long by 0.2 to 0.3
micron in width. They are somewhat irregular in length, but show
rounded ends. They rarely form chains. They are non-motile, and
do not form spores.

Influenza bacilli stain less easily than do most other bacteria with
the usual anilin dyes, and are best demonstrated with 10 per cent
aqueous fuchsin (5 to 10 minutes), or with Loeffler’s methylene-blue
(5 minutes). They are Gram-negative, giving up the anilin-gentian-
violet stain upon decolorization. Occasionally slight polar staining
may be noticed. Grouping, especially in thin smears of bronchial
secretion, is characteristic, in that the bacilli very rarely form threads

1 PfeifFer> Deut. med. Woch., ii, 1892; Zeit. f. Hyg., xiii, 1892; Pfeiffer und Beck,
Deut. med. Woch., xxi, 1893.

536

BACILLUS INFLUENZAS

537

or chains, usually lying together in thick, irregular clusters without
definite parallelism.

Isolation and Cultivation. — Isolation of the influenza bacillus is
not easy. Pfeiffer 1 succeeded in growing the bacillus upon serum-agar
plates upon which he had smeared pus from the bronchial secretions of
patients. Failure of growth in attempted subcultures made upon agar

Fig. 112. — Bacillus influenzae. Smear from pure culture on blood agar.

and gelatin, however, soon taught him that the success of his first culti¬
vations depended upon the ingredients of the pus carried over from the
sputum. Further experimentation then showed that it was the blood,
and more particularly the hemoglobin, in the pus which had made growth
possible in the first cultures. Pfeiffer made his further cultivations

1 Pfeiffer, loc. cit.

538

PATHOGENIC MICROORGANISMS

upon agar, the surface of which had been smeared with a few drops of
blood taken sterile from the finger. Hemoglobin separated from the
red blood cells was found to be quite as efficient as whole blood. This
method of Pfeiffer is still the one most frequently employed for isolation
and cultivation. Whole blood taken from the finger may be either
smeared over the surface of slants or plates, or mixed with the melted
meat-infusion agar. In isolating from sputum, only that secretion should
be used which is coughed up from the bronchi and is uncontaminated
by microorganisms from the mouth. It may be washed in sterile water
or bouillon before transplantation, to remove the mouth flora adhe-

Fig. 113. — Bacillus influenzas. Smear from sputum. (After Heim.)

rent to the outer surface of the little clumps of pus. The blood of
pigeons or that of rabbits may be substituted for human blood. The
former seems to be the more favorable of the two and even more so than
human blood. Pigeons may be easily bled for this purpose from the
large veins under the wing. Huber 1 has succeeded in cultivating in¬
fluenza bacilli upon media containing a soluble hemoglobin derivative
known as hematogen. This substance, however, offers some difficulties
to sterilization and is not so favorable as whole blood. The absence
of oxyhemoglobin from the hematogen, however, is theoretically im¬
portant in that it shows that hemoglobin is suitable for the growth

1 Huber, Zeit. f. Hyg., xv, 1893.

BACILLUS INFLUENZAS

539

of this bacillus because of its nutrient qualities and not by virtue
of its oxygen-carrying properties. Although the presence of hemo¬
globin seems to be a necessity for the successful cultivation of the
bacillus, the quantity present need not be very large. Ghon and Preyss 1
showed that an amount too small to be demonstrated spectroscopically
sufficed for its growth.

Other substances which, added to neutral or slightly alkaline agar,
have been used for the cultivation of influenza bacilli are the yolk of
eggs 2 (not confirmed) and spermatic fluid.3 None of these, however,
is as useful as the blood media. Symbiosis with staphylococci,4 too,

Fig. 114.— Colonies of Influenza Bacillus on Blood Agar. (After Heim.)

has been found to create an environment favorable for theii develop¬
ment.

Influenza bacilli do not grow at room temperature. Upon suitable
media at 37.5° C. colonies appear at the end of eighteen to twenty-four
hours, as minute, colorless, transparent droplets, not unlike spots of
moisture. These never become confluent. The limits of giowth aie
reached in two or three days. To keep the cultures alive, tubes should

1 Ghon und Preyss, Cent. f. Bakt., xxxv, 1904.

2 Nastjukoff, Cent. f. Bakt., Ref., xix, 1896.

* Cantani, Cent. f. Bakt., xxii, 1897.

4 Grassberger, Zeit. f. Hyg., xxv, 1897.

540

PATHOGENIC MICROORGANISMS

be stored at room temperature and transplantations done at intervals
not longer than four or five days.

Biology— The bacillus is aerobic, growing in broth-blood mixtures
only upon the surface, hardly at all in agar stab cultures, and not at all
under completely anaerobic conditions.

As it does not form spores, it is exceedingly sensitive to heat, desicca¬
tion, and disinfectants.1 Heating to 60° C. kills the bacilli in a few min¬
utes. In dried sputum they die within one or two hours. They are
easily killed even by the weaker antiseptics. Upon culture media the
bacilli, if not transplanted, die within a week or less, the time depend¬
ing to some extent upon the medium used.

Pathogenicity. — The relationship between the clinical disease known
as influenza or grippe and the Pfeiffer bacillus has been definitely estab¬
lished by numerous investigators.2 During epidemics, the bacilli are
found with much regularity in the nasal passages and bronchial secre¬
tions of those afflicted with the malady. The organs most frequently
attacked in man are the upper respiratory passages and lungs. Here
the disease most frequently takes the form of a broncho- or lobular
pneumonia, and sections of the lung tissue of those who have died of the
infection show innumerable bacilli upon and within the mucosa of the
bronchioles. [Thin sections are stained for one-half to one hour in
dilute carbol fuchsin and are then dehydrated in slightly acid alcohol
(alcohol absolute 5 i, glacial acetic acid gtt. i-ij).]

Clinically, influenzal broncho-pneumonias are not essentially dif¬
ferent from those due to other microorganisms, and it must always
be left to the bacteriological examination to make the positive
diagnosis. Pulmonary influenzal infection is not infrequently followed
by abscess or gangrene of the lung, and occasionally develops into a
chronic interstitial process. The bacilli have also been found in the
middle ear,3 in the meninges,4 and in the brain and spinal cord. Bacilli
in the circulating blood have never been satisfactorily demonstrated,
although the general characters of the symptoms would suggest a septi¬
cemia. The short incubation period 5 of the disease was involuntarily
determined by Kretz, who fell ill twenty-four hours after accidentally
breaking an agar plate of a pure culture which he was photographing.

The bacilli are said to remain in the bronchial secretions of conval-

1 Kruse, in Fliigge, “Die Mikroorg.,” Leipzig, 1896.

2 Weichselbaum, Wien. klin. Woch., 32, 1892; Baumler, Munch, med. Woch.,
1894; Huber, loc. cit.

3 Kossell, Charite-Annalen, 1893. 4 Pfuhl, Berl. klin. Woch., xxxix, 1892.

5 Quoted from Tedesco, Cent. f. Bakt., xliii, 1907.

BACILLUS INFLUENZAE

541

escents or even of normal individuals for many years. They are found
for long periods in the lungs of those suffering from tuberculosis. To
such sources, probably, are attributable the sporadic cases developing
constantly in crowded communities. Occasional reactivation of the
influenzal infection may often aggravate the condition of phthisical
patients. Cases of influenza observed apart from the large epidemics
are rarely due to an unmixed Pfeiffer bacillus infection, but are usually
due to a mixed infection, including with this bacillus, pneumococci,
streptococci, and other secondary invaders.1 This may, in part, account
for the frequently atypical courses of such attacks.

Dr. Anna Williams 2 has recently studied hemoglobinophilic bacilli
isolated from the eye in cases of trachoma. She believes that trachoma
is probably caused by bacteria of this group. At first an acute infection
or acute conjunctivitis occurs. Later when chronic productive inflam¬
mation supervenes the clinical picture is that of trachoma.

Experimental infection of animals reveals susceptibility only in
monkeys. Pfeiffer and Beck3 produced influenza-like symptoms in mon¬
keys by rubbing a pure culture of the bacillus upon the unbroken nasal
mucosa. Intravenous inoculation in rabbits produced severe symptoms,
but the bacilli do not seem to proliferate in these animals, the reaction
probably being purely toxic. Cultures killed with chloroform may pro¬
duce severe transient toxic symptoms in rabbits.4 Immunity produced
by an attack of influenza, if present at all, is of very short duration.

Bacteria Closely Related to Influenza Bacillus. — Pseudo-influenza
Bacillus. — In the broncho-pneumonic processes of children, Pfeiffer 5
found small, non-motile, Gram-negative bacilli, which he was forced to
separate from true influenza bacilli because of their slightly greater size,
and their tendency to form threads and involution forms. These micro¬
organisms are strictly aerobic and grow, like true influenza bacilli, only
upon blood media. They are differentiated entirely by their morphology
upon twenty-four-hour-old blood-agar cultures. Wollstein,6 who has
made a careful study of influenza-like bacilli, both culturally and by
agglutination tests, has come to the conclusion that these bacilli are so
similar to the true influenza organisms that the term pseudo-influenza
should be discarded. Strains of similar bacilli isolated from cases of

1 Tedesco, loc. cit.

2 Dr. Anna Williams, Inter. Congress of Hygiene and Demography, Washington,

1912. 3 Pfeiffer und Beck, Deut. med. Woch., xxi, 1893.

4 Pfeiffer, loc. cit. 5 Pfeiffer, Zeit. f. Hyg., xiii, 1892.

6 Wollstein, Jour. Exp. Med., viii, 1906.

542

PATHOGENIC MICROORGANISMS

pertussis, while differing from the others in some of their characteristics,
could not properly be maintained as distinct species.

Koch-Weeks Bacillus. — Koch,1 in 1883, Weeks 2 and Kartulis, in
1887, described a small Gram-negative bacillus found in connection with
a form of acute conjunctival inflammation, which occurs epidemically.
The bacillus is morphologically similar to B. influenzae, but is generally
longer than this and more slender. The bacilli grow only at incubator
temperature, but, unlike influenza bacilli, can be cultivated upon media
of serum or ascitic fluid, without hemoglobin. In fact, growth upon
serum-agar is more active than upon hemoglobin media.3

Bacillus of Pleuro-Pneumonia of Rabbits. — This is a small

Fig. 115. — Koch-Weeks Bacillus.

Gram-negative bacillus, described by Beck, not unlike that of influenza.
These microorganisms are slightly larger than the Pfeiffer bacilli and
grow upon ordinary media even without animal sera or hemoglobin.

Bacillus murisepticus and Bacillus rhusiopathi^. — While mor¬
phologically similar to the microorganisms of this group, these bacilli
are culturally easily separated because of their luxuriant growth on
simple media. (The last two microorganisms are more closely related
to the groups of the bacilli of hemorrhagic septicemia. See page 551.)

1 Koch, Arb. a. d. kais. Gesundheitsamt, iii; Cent. f. Bakt., 1, 1887.

- Weeks, N. \ . Eye and Ear Infirmary Rep., 1895; Arch. f. Augenheilk., 1887.

3 Kamen, Cent. f. Bakt., xxv, 1899; W eichselbaum and Muller, Arch. f. Ophthalm.,
xlvii, 1899; Knapp, Studies from Dept, of Path., Coll, of P. and S., 1903.

CHAPTER XXXVIII

BORDET-GENGOU BACILLUS, MORAX-AXENFELD BACILLUS, ZUR
NEDDEN’S BACILLUS, DUCREY BACILLUS

BORDET-GENGOU BACILLUS

(“ Microbe de la Coqueluche,” Pertussis bacillus , Bacillus of whooping-

cough.)

In 1900 Bordet and Gengou 1 observed in the sputum of a child
suffering from pertussis a small ovoid bacillus which, though similar to
the influenza bacillus, showed a number of morphological characteristics
which led them to regard it as a distinct species. As they were at first
unable to cultivate this organism, their discovery remained ques¬
tionable until .1900, when cultivation succeeded and the biology of the
microorganism was more fully elucidated.

Morphology. — The morphology of this organism is described by
them as follows: The organism in the sputum, early in the disease, is
scattered in enormous numbers indiscriminately among the pus cells,
and at times within the cells. It is extremely small and ovoid, and
frequently is so short that it resembles a micrococcus. Often its poles
stain more deeply than the center. In general, the form of the or¬
ganisms is constant, though occasionally slightly larger individuals
are encountered. They are usually grouped separately, though occa¬
sionally in pairs, end to end.

Compared with the influenza bacillus in morphology, the bacillus
of pertussis is more regularly ovoid and somewhat larger. It has,
furthermore, less tendency to pleomorphism and involution.

Staining. — The Bordet-Gengou bacillus may be stained with alkaline
methylene-blue, dilute carbol-fuchsin, or aqueous fuchsin solutions.
Bordet and Gengou recommended as a staining-solution carbolated
toluidin-blue made up as follows:

Toluid in-blue . 5 gms.

Alcohol . 100 c.c.

Water . 500 c.c.

1 Bordet et Gengou, Ann. de Finst. Pasteur, 1906.

543

544

PATHOGENIC MICROORGANISMS

Allow to dissolve and add 500 c.c. of 5 per cent carbolic acid in water. Let this
stand one or two days and filter.

Stained by the method of Gram, the bacillus of Bordet and Gengou
is decolorized.

Cultivation. — Early attempts at cultivation made by the discov¬
erers upon ordinary ascitic agar or blood agar were unsuccessful. They
finally obtained successful cultures from sputum by the use of the
following medium:

One hundred grams of sliced potato are put into 200 c.c. of 4 per
cent glycerin in water. This is steamed in an autoclave and a glycerin
extract of potato obtained. To 50 c.c. of this extract 150 c.c. of 6-per¬
cent salt solution and 5 grams of agar are added. The mixture is melted
in the autoclave and the fluid filled into test tubes, 2 to 3 c.c. each, and

sterilized. To each tube, after sterilization,
is added an equal volume of sterile de-
fibrinated rabbit blood or preferably human
blood, the substances are mixed, and the
tubes slanted.

On such a medium, inoculated with
sputum, taken preferably during the par¬
oxysms of the first day of the disease,
colonies appear, which are barely visible
after twenty-four hours,1 plainly visible
after forty-eight hours. They are small,
grayish, and rather thick. After the first
generation the organisms grow with mark¬
edly greater luxuriance and speed. On the potato-blood medium,
after several generations of artificial cultivation, they form a grayish
glistening layer which, after a few days, becomes heavy and thick,
almost resembling the growth of typhoid bacilli. In these later
generations, also, they develop readily upon plain blood agar or
ascitic agar and in ascitic broth or broth to which blood has been
added. In the fluid media they form a viscid sediment, but no pellicle.

Culturally, the bacillus varies from B. influenzae in growing less
readily on hemoglobin media than the latter, on first cultivation from the
sputum. Later it grows much more heavily on such media and shows
less dependence upon the presence of hemoglobin than does B. influenzae.
It also grows rather more slowly than the influenza bacillus. It is

Fig. 116. — Bordet-Gengou
Bacillus.

1 Wollstein, Jour. Exp. Med., xi, 1909.

BORDET-GENGOU BACILLUS

545

strictly aerobic and in fluid cultures is best grown in wide flasks with
shallow layers of the medium.

The Bordet-Gengou bacillus grows moderately at temperatures about
37.5° C., but does not cease to grow at temperatures as low as 5° to 10° C.
On blood agar and in ascitic broth it may remain alive for as long as two
months (Wollstein).

Pathogenicity. — As regards the pathogenicity and etiological spe¬
cificity of this organism for whooping-cough, no positive statement
can as yet be made. The fact that it has been found in many cases in
almost pure cultures during the early paroxysms, renders it likely that
the organism is the specific cause of the disease. However, in early
cases true influenza bacilli have been often found, and these latter seem
to remain in the sputum of such patients for a longer period and in
larger numbers than the bacillus of Bordet and Gengou. Endotoxins
have been obtained from* the cultures of the bacilli by Bordet and Gengou
by the method of Besredka.1 The growth from slant cultures is washed
up in a little salt solution, dried in vacuo , and ground in a mortar with
a small, measured quantity of salt. Finally, enough distilled water is
added to bring the salt into a solution of 0.75 per cent and the mixture
is centrifugalized and decanted. One to two c.c. of such an extract will
usually kill a rabbit within twenty-four hours after intravenous inocula¬
tion. Subcutaneous inoculation produces non-suppurating necrosis and
ulceration without marked constitutional symptoms.

Inoculation of monkeys with the bacilli themselves by the respira¬
tory path has failed to produce the disease.

Specific agglutinins may be obtained in immunized animals which
prove absolutely the distinctness of this organism from Bacillus in¬
fluenzae.2 In the serum of afflicted children the agglutination is too
irregular to be of value.

Specific complement fixation with the serum of patients is reported
by Bordet and Gengou, but failed in the hands of Wollstein.

MORAX-AXENFELD BACILLUS

In 1896 Morax 3 described a diplo-bacillus, which he associated
etiologically with a type of chronic conjunctivitis to which he applied
the name “ conjondivite subaigue Soon after this, a similar micro¬
organism was found in cases corresponding to those of Morax by Axen-

1 Bordet, Bull, de la Soc. Roy. de Brux., 1907.

2 Wollstein, loc. cit. 3 Morax, Ann. de Tinst. Pasteur, 1896.

546

PATHOGENIC MICROORGANISMS

feld.1 The condition which these microorganisms characteristically
produce is a catarrhal conjunctivitis which usually attacks both eyes.
The inflammation is especially noticeable in the angles of the eye, most
severe at or about the caruncle. There is rarely much swelling of the
conjunctiva and hardly ever ulceration. The condition runs a subacute
or chronic course. Its diagnosis is easily made by smear preparations
of the pus which is formed with especial abundance during the night.

Morphology. — In smear preparations from the pus, the microorgan¬
isms appear as short, thick bacilli, usually in the form of two placed
end to end, but not infrequently singly or in short chains. Their ends

Fig. 117. — Morax-Axenfeld Diplo-Bacillus.

are distinctly rounded, their centers slightly bulging, giving the bacillus
an ovoid form. They are usually about two micra in length.

They are easily stained by the usual anilin dyes, and, stained by
the method of Gram, are completely decolorized.

Cultivation. The Morax-Axenfeld bacillus can be cultivated only
upon alkaline media containing blood or blood serum.

It grows poorly, or not at all, at room temperature.

Tpon Loeffler’s blood serum , colonies appear after twenty-four to
thirty-six hours as small indentations which indicate a liquefaction of
the medium. Axenfeld states that eventually the entire medium may

1 Axenfeld, Cent. f. Bakt., xxi, 1897.

ZUR NEDDEN’S BACILLUS

547

become liquefied. Upon serum agar delicate grayish drop-like colonies
are formed which are not unlike those of the gonococcus.

In ascitic bouillon general clouding occurs within twenty-four hours.

Pathogenicity. — Attempts to produce lesions in the lower animals
with this bacillus have been universally unsuccessful. Subacute con¬
junctivitis, however, has been produced in human beings by inocula¬
tions with pure cultures.

ZUR NEDDEN’S BACILLUS

In ulcerative conditions of the cornea, Zur Nedden has frequently
found a bacillus to which he attributes etiological importance.

The bacillus which he has described is small, usually less than
one micron in length, often slightly curved, and generally found singly.
It may be found in the diplo form but does not form chains. It is
stained by the usual dyes, often staining poorly at the ends. Stained
by Gram’s method it is decolorized. The bacillus is non-motile.

Cultivation. — It is easily cultivated upon the ordinary laboratory
media. Upon agar it forms, within twenty-four hours, transparent,
slightly fluorescent colonies which are round, raised, rather coarsely
granular, and show a tendency to confluence.

Gelatin is not liquefied.

Milk is coagulated.

Upon potato, there is a thick yellowish growth.

Upon dextrose media, there is acid formation, but no gas.

The bacillus forms no indol in pepton solutions.

Pathogenicity. — Corneal ulcers have been produced by inoculation
of guinea-pigs.

BACILLUS OF DUCREY

The soft chancre, or chancroid, is an acute inflammatory, destructive
lesion which occurs usually upon the genitals or the skin surrounding
the genitals. The infection is conveyed from one individual to an¬
other by direct contact. It may, however, under conditions of surgical
manipulation, be transmitted indirectly by means of dressings, towels,
or instruments.

The lesion begins usually as a small pustule which rapidly ruptures,
leaving an irregular ulcer with undermined edges and a necrotic floor
which spreads rapidly. It differs clinically from the true or syphilitic
chancre in the lack of induration and in its violent inflammatory

548

PATHOGENIC MICROORGANISMS

nature. Usually it leads to lymphatic swellings in the groin which,
later, give rise to abscesses, commonly spoken of as “buboes.”

In the discharges from such lesions, Ducrey,1 in 1889, was able to
demonstrate minute bacilli to which he attributed an etiological rela¬
tionship to the disease, both because of the regularity of their presence
in the lesions and the successful transference of the disease by means
of pus containing the microorganisms.

Morphology and Staining. — The Ducrey bacillus is an extremely
small bacillus, measuring from one to two micra in length and about
half a micron in thickness. It has a tendency to appear in short
chains and in parallel rows, but many of the microorganisms may be
seen irregularly grouped. It is not motile, possesses no flagella, and
does not form spores.

Stained by the ordinary anilin dyes, it has a tendency to take the
color irregularly and to appear more deeply stained at the poles. By the
Gram method, it is decolorized. In tissue sections, it may be demon¬
strated by Loeffler’s methylene-blue method, and in such preparations
has been found within the granulation tissues forming the floor of the
ulcers. In pus, the bacilli are often found within leucocytes.

Cultivation and Isolation. — Early attempts at cultivation of this
bacillus were universally unsuccessful in spite of painstaking experi¬
ments with media prepared of human skin and blood serum. In 1900,
Besangon, Griffon, and Le Sourd 2 finally succeeded in obtaining growths
upon a medium containing agar to which human blood had been added.
They were equally successful when dog’s or rabbit’s blood was substi¬
tuted for that of man. Since the work by these authors, the cultiva¬
tion by similar methods has been carried out by a number of investiga¬
tors. Coagulated blood, which has been kept for several days in sterile
tubes, has been found to constitute a favorable medium. Freshly
clotted blood can not be employed, probably because of the bacteri¬
cidal action of the serum. Serum-agar has occasionally been used
with success, but does not give results as satisfactory as those obtained
by the use of the whole blood.

The best method of obtaining pure cultures upon such media con¬
sists in puncturing an unruptured bubo with a sterile hypodermic needle
and transferring the pus in considerable quantity directly to the agar.
If possible, the inoculation of the media should be made immediately

1 Ducrey, Monatschr. f. prakt. Dermat., 9, 1889.

2 Besangon, Griffon, et Le Sourd, Presse med., 1900.

BACILLUS OF DUCREY

549

before the pus has had a chance to cool off or to be exposed to light.
When buboes are not available, the primary lesion may be thoroughly
cleansed with sterile water or salt solution, and material scraped from
the bottom of the ulcer or from beneath its overhanging edges with a
stiff platinum loop. This material is then smeared over the surface of a
number of blood-agar plates.

Upon such plates, isolated colonies appear, usually after forty-eight
hours. They are small, transparent, and gray, and have a rather firm,
finely granular consistency. The colonies rarely grow larger than pin¬
head size, and have no tendency to coalesce. At room temperature,
the cultures die out rapidly. Kept in the incubator, however, they may
remain alive and virulent for a week or more.

On the simpler media, glucose-agar, broth, or gelatin, cultivation
is never successful. On moist blood-agar and in the condensation
water of such tubes, the bacilli have a tendency to grow out in long
chains. Upon media which are very dry, they appear singly or in
short chains.

Pathogenicity. — Besangon, Griffon, and Le Sourd, and others, have
succeeded in producing lesions in man by inoculation with pure cultures.
Inoculation of the lower animals has, so far, been entirely without result.

MICROCOCCUS MELITENSIS (MALTA FEVER)

(. Bacillus melitensis)

Malta fever is a disease occurring along the Mediterranean coast
and its islands. It has been recently found to occur also in South
America, South Africa, China, and in the West Indies. The disease
is not very unlike typhoid fever, though more irregular and with a
lower mortality. It is accompanied by joint pains, sweating, constipa¬
tion, and occasionally orchitis. The spleen is almost always enlarged.

Recent investigations into the manner in which this disease is con¬
veyed have revealed that it is primarily an infection of goats. A large
percentage of the goats on Malta were shown to be infected and passed
the organism with the milk. Forty per cent of the goats gave positive
agglutination tests and the organisms have been found in the milk in
about 10 per cent of the animals.

The most susceptible animals seem to be goats, but horses and cows
are also susceptible. In guinea-pigs and rabbits the disease can be ex¬
perimentally produced, but usually takes a protracted course. Monkeys

550

PATHOGENIC MICROORGANISMS

are susceptible, and the disease produced in these animals is in many
features identical with that of man.

Transmission seems to take place chiefly by the ingestion of infected
milk. Direct cutaneous infection or through mucous membranes may
also occur. In human beings, suffering from the disease, the organisms
may be isolated from the blood stream during the entire course of
the disease and as early as the second day. The disease is rarely
fatal, death occurring in less than 2 per cent of the cases (Eyre, loc. tit.)}

The microorganism causing the disease was isolated in 1887 by
Bruce,1 2 a British army surgeon.

Morphology. — Micrococcus melitensis is a minute bacterium ap¬
pearing coccoid in smears from agar cultures, in broth cultures assum¬
ing the form of a short, slightly wedge-shaped bacillus resembling B.
influenzae. Babes 3 regards it as unquestionably a bacillus. Eyre de¬
scribes it as a minute coccus, and believes the bacillus-like individuals
to represent involution forms. It appears in irregularly parallel groups,
and occasionally forms short chains.

It is easily stained with the ordinary dyes, and is decolorized by
Gram’s method.

Cultivation. — Micrococcus melitensis can usually be cultivated from
the spleens of those who have succumbed to the disease and from the
blood stream in active cases. It grows on nutrient agar at 37.5° C.,
forming small, pearly white colonies at the end of two or three days.
It grows easily on all of the ordinary laboratory media.

Both in patients and in injected animals, infection with this bacte¬
rium produces specific agglutinins which are of great practical aid in
diagnosis.4

1 British Commission Report cited from Eyre in Kolle und W assermann ,
Handbuch, etc., Erganzungsband, Heft 2.

2 Bruce , Practitioner, 1887.

3 Babes, Kolle und Wassermann, iii, p. 443.

4 Wright and Lamb, Jour. Path, and Bact., v, 1899.

CHAPTER XXXIX

THE BACILLI OF THE HEMORRHAGIC SEPTICEMIA GROUP

AND BACILLUS PESTIS.

In many of the lower animals there occur violently acute bacterial
infections characterized by general septicemia, usually with petechial
hemorrhages throughout the organs and serous membranes and severe
intestinal inflammations. These diseases, spoken of as the “ hemor¬
rhagic septicemias,” are caused by a group of closely allied bacilli, first
classified together by Hueppe 1 in 1886. Some confusion has existed
as to the forms which should be considered within Hueppe’s group of
“ hemorrhagic septicemia,” a number of bacteriologists including in
this class bacilli such as Loeffler’s Bacillus typhi murium, and Salmon
and Smith’s hog-cholera bacillus, microorganisms which, because of
their motility and cultural characteristics, belong more properly to the
“Gartner,” “enteritidis,” or “paratyphoid” group, intermediate be¬
tween colon and typhoid.

The organisms properly belonging to this group are short bacilli, more
plump than are those of the colon type, and showing a marked ten¬
dency to stain more deeply at the poles than at the center. They are
non-motile, possess no flagella, and do not form spores. They grow
readily upon simple media, but show a very marked preference for
oxygen, growing but slightly below the surface of media. By some
observers they are characterized as “obligatory aerobes,” but this is
undoubtedly a mistake.

While showing considerable variations in form and differences in
minor cultural characteristics, the species characteristics of polar stain¬
ing, decolorization by Gram, immobility, lack of gelatin liquefaction,
and great pathogenicity for animals, stamp alike all members of the
group. Its chief recognized representatives are the bacillus of chicken
cholera, the bacillus of swine-plague (Deutsche Schweineseuche),

1 Hueppe, Berl. klin. Wocfi., 1886.
551

552

PATHOGENIC MICROORGANISMS

and the Bacillus pleurosepticus which causes an acute disease in
cattle and often in wild game.

Because of certain cultural and pathogenic characteristics, it
seems best to consider the bacillus of bubonic plague with this group.

BACILLUS OF CHICKEN CHOLERA

(. Bacillus avisepticus )

The bacillus of chicken cholera was first carefully studied by Pas¬
teur 1 in 1880. It is a short, non-motile bacillus, measuring from 0.5 to
1 micron in length. Stained with the ordinary anilin dyes, it displays
marked polar staining qualities, which often give it the appearance of
being a diplococcus. It is decolorized by Gram’s method. It does not
form spores, but may occasionally form vacuolated degeneration forms,
not unlike those described for Bacillus pestis.

The bacillus is easily cultivated from the blood and organs of infected
animals, it grows well upon the simplest media at temperatures vary¬
ing from 25° to 40° C. In broth , it produces uniform clouding with
later a formation of a pellicle. Upon agar it forms, within twenty -four
to forty-eight hours, minute colonies, white or yellowish in color, which
are at first transparent, later opaque. Upon gelatin , it grows without
liquefaction. Upon milk, the growth is slow and does not produce co¬
agulation. According to Kruse,2 indol is formed from pepton bouillon.
Acid, but no gas, is formed in sugar broth.

Among barnyard fowl, this disease is widely prevalent, attacking
chickens, ducks, geese, and a large variety of smaller birds. The infection
is extremely acute, ending fatally within a few days. It is accompanied
b}^ diarrhea, often with bloody stools, great exhaustion, and, toward the
end, a drowsiness bordering on coma. Autopsy upon the animals re¬
veals hemorrhagic inflammation of the intestinal mucosa, enlargement
of the liver and spleen, and often bronchopneumonia.

The specific bacilli may be found in the blood, in the organs, in exu¬
dates, if these are present, and in large numbers in the dejecta. Infection
takes place probably through the food and water contaminated by the
discharges of diseased birds.3

Subcutaneous inoculation or feeding of such animals with pure
cultures, even in minute doses, gives rise to a quickly developing
septicemia which is uniformly fatal. The bacillus is extremely patho-

1 Pasteur, Comptes rend, de Pacad. des sci., 1880.

2 Kruse, in Fliigge's “ Die Mikroorganismen.”

3 Salmon, Rep. of the Com. of Agriculture, 1880, 1881, and 1882.

BACILLI OF HEMORRHAGIC SEPTICEMIA GROUP

553

genic for rabbits, less so for hogs, sheep, and horses, if infection is prac¬
ticed by subcutaneous inoculation. Infection by ingestion does not
seem to cause disease in these animals.

Historically, the bacillus of chicken cholera is extremely interesting,
since it was with this microorganism that Pasteur 1 carried out some of
his fundamental researches upon immunity, and succeeded in immu¬
nizing chickens with attenuated cultures. The first attenuation ex¬
periment made by Pasteur consisted in allowing the bacilli to remain in
a broth culture for a prolonged period without transplantation. With
minute doses of such a culture (vaccin I) he inoculated chickens, fol¬
lowing this, after ten days, with a small dose of a fully virulent culture.
Although enormously important in principle, the practical results from
this method, as applied to chicken cholera, have not been satisfactory.
It was with this bacillus, furthermore, that Pasteur was first able to
demonstrate the existence of a free toxin which could be separated
from the bacteria by filtration.

BACILLUS OF SWINE PLAGUE

(. Bacillus suisepticus , Schweineseuche)

This microorganism is almost identical in form and cultural charac¬
teristics with the bacillus of chicken cholera. It is non-motile, forms
no spores, is Gram-negative, and does not liquefy gelatin. The bacillus
causes an epidemic disease among hogs, which is characterized almost
regularly by a bronchopneumonia followed by general septicemia.
There is often a sero-sanguineous pleural exudate, a swelling of bronchial
lymph glands and of liver and spleen. The gastrointestinal tract is
rarely affected. The bacilli at autopsy may be found in the lungs,
in the exudates, in the liver and spleen, and in the blood. The disease
is rarely acute, but, in young pigs, almost uniformly fatal.

It is probable that spontaneous infection usually occurs by inhala¬
tion. Experimental inoculation is successful in pigs, both when given
subcutaneously and when administered by the inhalation method.
Mice, guinea-pigs, and rabbits are also susceptible, dying within three
or four days after subcutaneous inoculation of small doses.

Active and passive immunization of animals against Bacillus suisep¬
ticus has been attempted by various observers. Active immunization,
if carried out with care, may be successfully done in the laboratory.

1 Pasteur, loc. cit.

554

PATHOGENIC MICROORGANISMS

Passive immunization of animals with the serum of actively immunized
horses has been practiced by Kitt and Mayr,1 Schreiber,2 and Wasser-
mann and Ostertag. The last-named observers, working with a poly¬
valent serum produced with a number of different strains of the bacillus,
have obtained results of considerable practical value. The researches
of Kitt and Mayr have revealed a fact pointing to the interrelationship
of the bacilli of the “ hemorrhagic septicemia” group. They were able
to show that the serum of horses immunized with chicken cholera
bacilli was able to protect, somewhat, against Bacillus suisepticus.

Infection with the bacillus of swine plague, in hogs, is often ac¬
companied by an infection with the hog-cholera bacillus (Schweinepest) .
The latter, as we have seen, is a microorganism belonging to the enteri-
tidis group, intermediate between Bacillus coli and Bacillus typhosus, and
differing from suisepticus in being actively motile, possessing flagella,
not showing the polar staining, having a more slender morphology, and
producing gas upon dextrose broth. A confusion between the two
bacilli frequently occurs because of their nomenclature. Bacteriologic-
ally and pathogenically, they are quite distinct. Bacillus suisepticus
produces an acute septicemia, accompanied by bronchopneumonia and
usually not affecting the gastro-intestinal canal. The bacillus of hog
cholera produces an infection localized in the intestinal canal.

BACILLUS PESTIS

{Bacillus of Bubonic Plague)

The history of epidemic diseases has no more terrifying chapter
than that of plague.3 Sweeping, time and again, over large areas of
the civilized world, its scope and mortality were often so great that
all forms of human activity were temporarily paralyzed. In the
reign of Justinian almost fifty per cent of the entire population of
the Roman Empire perished from the disease. The “ Black Death ”
which swept over Europe during the fourteenth century killed about
twenty-five million people. Smaller epidemics, appearing in numerous
parts of the world during the sixteenth, seventeenth, and eighteenth
centuries, have claimed innumerable victims. In 1893, plague appeared
in Hong Kong. During the epidemic which followed, Bacillus pestis,
now recognized as the etiological factor of the disease, was discovered by

1 Kitt und Mayr, Monatsh. f. prakt. Thierheilk., 8, 1897.

2 Schreiber, Berl. tierarztl. Woch., 10, 1899.

3 Hirsch, “ Handb. d. histor.-geogr. Path.,” 1881.

BACILLUS PESTIS

555

Kitasato 1 and by Yersin,2 independently of each other. By both ob¬
servers the bacillus could invariably be found in the pus from the buboes
of afflicted persons. It could be demonstrated in enormous numbers
in the cadavers of victims. The constancy of the occurrence of the
bacillus in patients, shown in the innumerable researches of many
bacteriologists, would alone be sufficient evidence of its etiological
relationship to the disease. This evidence is strengthened, moreover,
by accidental infections which occurred in Vienna in 1898, with labora¬
tory cultures.

Morphology and Staining. — Bacillus pestis is a short, thick bacillus
with well-rounded ends. Its length is barely two or two and a half times

Fig. 118. — Bacillus pestis. (After Mallory and Wright.)

its breadth (1.5 to 1.75 micra by 0.5 to 0.7 micron). The bacilli appear
singly, in pairs, or, more rarely, in short chains of three or more. They
show distinct polar staining. In size and shape these bacilli are sub¬
ject to a greater degree of variation than are most other microorganisms.
In old lesions or in old cultures the bacilli show involution forms which
may appear either as swollen coccoid forms or as longer, club-shaped,
diphtheroid bacilli. Degenerating individuals appear often as swollen,
oval vacuoles. All these involution forms, by their very irregularity,
are of diagnostic importance. They appear more numerous in artificial
cultures than in human lesions.

According to Albrecht and Ghon,3 the plague bacillus may, by

i Kitasato, Lancet, 1894. 2 Yersin, Ann. de Finst. Pasteur, 1894.

3 Albrecht und Ghon, Wien, 1898.

556

PATHOGENIC MICROORGANISMS

special methods, be shown to possess a gelatinous capsule. It does
not possess flagella and does not form spores.

The plague bacillus is easily stained with all the usual anilin
dyes. Diluted aqueous fuchsin and methylene-blue are most frequently
employed. With these stains the characteristically deeper staining
of the polar portions of the bacillus is usually easy to demonstrate.
Special polar stains have been devised by various observers. Most of
these depend upon avoidance of the usual heat fixation of the prepara¬
tions, which, in some way, seems to interfere with good polar staining.
Fixation of the dried smears with absolute alcohol is, therefore, prefer¬
able. The bacillus is decolorized by Gram's method.

Fig. 119. — Bacillus festis, Involution Forms. (After Zettnow.)

Isolation and Cultivation. — The bacillus is easify isolated in pure
culture from the specific lesions of plague patients, during life or at
autopsy. It grows readily and luxuriantly upon the meat-infusion
media. The optimum temperature for its cultivation is about 30° C.
Below 20° C. and above 38° C., growth is sparse and delayed, though it
is not entirely inhibited until exposed to temperatures below 12° C.,
or above 40° C. The most favorable reaction of culture media is neu¬
trality or moderate alkalinity, though slight acidity does not prevent
development.

On agar, growth appears within twenty-four hours as minute
colonies with a compact small center surrounded by a broad, irregularly
indented, granular margin.

BACILLUS PESTIS

557

On gelatin, similar colonies appear after two or three days at 20°
to 22° C. The gelatin is not liquefied.

In bouillon, the plague bacilli grow slowly. They usually sink to
the bottom or adhere to the walls of the tube as a granular deposit and
may occasionally form a delicate pellicle. Chain-formation is not un¬
common. In broth cultures, moreover, a peculiar stalactite-like growth
is often seen, when the culture fluid is covered with a layer of oil.
Delicate threads of growth hang down from the surface of the medium
into its depths like stalactites. Characteristic involution forms are
brought out best when the bacilli are grown upon agar containing 3
per cent NaCl.

Milk is not coagulated. In litmus-milk there is slight acid forma¬
tion. On potato and on blood serum the growth shows nothing char¬
acteristic or of differential value. On pepton media no indol is formed.

Biological Considerations. — Bacillus pestis is aerobic. Absence of
free oxygen is said to prevent its growth, at least under certain condi¬
tions of artificial cultivation. It is non-motile. Outside of the animal
body the bacilli may retain viability for months and even years if
preserved in the dark and in a moist environment. In cadavers they
may live for weeks and months if protected from dryness. In pus or
sputum from patients they may live eight to fourteen days. These
facts are of great hygienic importance.

Complete drying in the air kills the bacilli within two or three days.1
Thoroughly dried by artificial means they die within four or five hours.

Dry heat at 100° C. kills the bacillus in one hour.2 Live steam or boil¬
ing water is effectual in a few minutes. The bacilli possess great resist¬
ance against cold, surviving a temperature of 0° C. for as many as
forty days.

Direct sunlight destroys them within four or five hours. The common
disinfectants are effectual in the following strengths: carbolic acid, one
per cent kills them in two hours, five per cent in ten minutes; bichloride
of mercury 1 : 1,000 is effectual in ten minutes.

In a recent communication to the New York Pathological Society,
Dr. Wilson reported that plague cultures which he had kept sealed for as
long as ten years in the ice chest were found living and virulent at the
end of this time. This ability to go into a quasi latent stage under
suitable conditions is of the greatest importance in connection with
the problem of prevention.

1 Kitasato, Lancet, 1894.

2 Abel, Cent. f. Bakt., xxi, 1897.

558

PATHOGENIC MICROORGANISMS

Pathogenicity. — In man, plague is acquired1 by entrance of the bacil¬
lus either through the skin or by the respiratory tract. The period of
incubation is about three to seven days. Two distinct clinical types
of the disease occur, depending upon the mode of infection. When
cutaneous infection has occurred the disease is first localized in the
lymph nodes nearest the point of inoculation. If the respiratory tract
has been the portal of entrance the disease primarily takes the form of a
pneumonia.

Infection may take place through the most minute lesions, hardly
visible to the naked eye. Even the unbroken skin may admit the
microorganisms if these are rubbed in with sufficient energy. From the
primary lymphatic swellings, the bacilli enter the blood and may pro¬
duce secondary foci.

The pneumonic form of plague usually begins with symptoms not
unlike a typical pneumonia and is usually fatal within four or five or
even fewer days. This form of the disease is especially menacing as a
means of dissemination, because of the enormous numbers of plague
bacilli in the sputum.

One of the chief characteristics of the general systemic plague infec¬
tion is the very marked cardiac depression.

The bacteriological diagnosis during life may be made by finding the
bacilli in the sputum or in aspiration fluid from a bubo. The micro¬
organisms are identified morphologically, culturally, by animal experi¬
ment, and by agglutination reaction. Blood cultures from plague pa¬
tients often yield positive results, especially when the blood is well
diluted in neutral broth to prevent any inhibiting action of the anti¬
bodies in the serum.

At autopsy, in man, the bacilli are found in the primary lesions, in
the blood, and in the spleen, the liver, and the lymphatics. There may
be hemorrhages into the serous cavities. When pneumonia exists, it
usually takes the form of a bronchopneumonia with extensive swelling
of the bronchial lymph nodes.

In cases in which the disease is prolonged, there are often tubercle¬
like foci in the spleen, liver, and lungs. Histologically these foci show
central necrosis surrounded by the usual inflammatory cell reactions.
In more chronic cases connective-tissue encapsulation may appear.

Bacillus pestis is extremely pathogenic for rats, mice, guinea-pigs,
rabbits, and monkeys. The most susceptible of these animals are rats

1 Gottischlich, Zeit. f. Hyg., xxxv, 1900.

BACILLUS PESTIS

559

and guinea-pigs, in whom mere rubbing of plague bacilli into the un¬
broken skin will often produce the disease. This method of experimen¬
tal infection of guinea-pigs is of great service in isolating the plague
bacillus from material contaminated with other microorganisms. For
the same purpose, infection of rats subcutaneously at the root of
the tail may be employed. Such inoculation in rats is invariably
fatal.

The studies of McCoy 1 upon guinea-pigs and white rats show that
individual plague cultures may vary considerably in virulence. The
size of the dose, always excepting enormous quantities such as a whole
agar culture, seems to make little difference in the speed with which
the animals die. There may be considerable variation in the suscep¬
tibility of individual animals. Prolonged cultivation on artificial media
may gradually reduce the virulence of plague bacilli, though, as stated
above, this has not been the experience of all observers.

In rats, spontaneous infection with plague is common and plays an
important role in the spread of the disease. Rats become infected from
the cadavers of plague victims or by gnawing the dead bodies of other
rats dead of the disease. The pneumonic type of the disease is common
in these animals and has been produced in them by inhalation experi¬
ments. During every well-observed plague epidemic, marked mortality
among the domestic rats has been noticed.

Since the examination of rats for plague is an important phase of
the study of epidemics, it may be well to review the typical lesions in
these animals as described by an experienced American student of plague,
George W. McCoy.2 McCoy, agreeing with the Indian Plague Com¬
mission, states that the naked eye is superior to the microscopical ex¬
amination. There is engorgement of the subcutaneous vessels and a
pink coloration of the muscles. The bubo when present is sufficient
for diagnosis. Marked injection surrounds it and sometimes there is
hemorrhagic infiltration. The gland itself is firm but usually caseous or
occasionally hemorrhagic. In the liver there is apparent fatty change,
but this is due to necrosis. Pin-point spots give it a stippled appear¬
ance as though it had been dusted with pepper. Pleural effusion is an
important sign. The spleen is large, friable, and often presents pin¬
point granules on the surface. One or two per cent of rats may present
no gross lesions. Cultures should of course be made. The method
of examination consists in immersing the rat in any convenient antiseptic

1 McCoy, Jour, of Inf. Dis., vi, 1909.

2 George W. McCoy, Public Health Reports, July, 1912.

560

PATHOGENIC MICROORGANISMS

to kill fleas and other ectoparasites. The rats are nailed by their feet
to a shingle and the skin is reflected from the whole front of the body and
neck so as to expose the cervical, axillary, and inguinal regions. The
thoracic and abdominal cavities are then opened and examined.

Wherry,1 McCoy,2 and others have found that the California ground
squirrel was infected with plague, during the recent occurrence of
plague on the Pacific coast, and several cases of plague in man were
traced to this source. In studying these and other American ro¬
dents McCoy found that ground squirrels as a species were highly
susceptible, never showing natural immunity. Field mice were but
moderately susceptible. Gophers were highly resistant. McCoy has
also described a case of spontaneous infection in a brush rat (Neo-
toma fuscipes). Rock squirrels were found by McCoy to be readily
infected.

Wu Lion Teh (G. L. Tuck) 3 has recently found that the Manchurian
tarbagan or marmot (Arctomys bobac), an animal trapped for its fur,
occasionally suffers from plague. The disease is never extensive and the
animal of much less importance in spreading the disease than is the rat.

Plague is transmitted either by direct contact or inhalation, or in¬
directly by clothing, linen, and other objects worn or handled. The
role in the transmission of the disease played by rats is probably of
great importance. The animals vomit, defecate, and die in cellars,
storerooms, etc., and thus set free vast numbers of plague bacilli for
indirect accidental transmission to human beings. The actual mode in
which this transmission takes place is by no means certain. The fact
that in countries where plague is prevalent many of the natives go
insufficiently shod or barefooted, may account for many infections.

Simond 4 lays great stress upon transmission to man by means of
fleas, the Indian rat-flea often being parasitic upon man. His conclusions
are probably too far-reaching, though the possibility of such infection
can not be denied.5

It is a curious fact observed by various bacteriologists that plague
bacilli isolated from pneumonic cases are particularly apt to cause
pneumonic lesions, having, as it were, acquired a selective pathogenicity
for the lung. A most valuable contribution to our knowledge of pneu-

1 Wherry, Jour. Inf. Dis., v, 1908.

2 McCoy, Jour. Inf. Dis., vi, 1909; vii, 1910.

3 Wu Lien Teh, Jour, of Hyg., xiii, 1913.

4 Simond, Ann. de l’inst. Pasteur, 1893.

5 Nuttall, Cent. f. Bakt., xxii, 1897; Nuttall, Hyg. Rund., ix, 1899.

BACILLUS PESTIS

561

monic plague has recently been made by Strong, Teague, and Barber 1
in their report of the American Red Cross Expedition to Manchuria
during the plague epidemic of 1910-11. Their investigations were made
with remarkable courage and skill under difficult conditions.

The chief points of interest in their reports may be summarized as
follows: Expired air of plague patients rarely contains the bacilli; these
are thrown out in coughing or hawking. Transmission is, in this form,
direct from patient to patient and not indirect through animals. The
first localization (Strong, Teague, and Crowell) is in the bronchi from
which extension takes place. Septicemia soon follows the pneumonic
process. Spreading occurs most likely in the wet and cold of winter,
since the bacteria are rapidly destroyed by drying.

Toxin Formation. — The systemic symptoms of plague are largely due
to the absorption of poisonous products of the bacteria. Albrecht and
Ghon,2 Wernicke,3 and others were unable to obtain any toxic action
with broth-culture filtrates and concluded that the poisons of B. pestis
were chiefly endotoxins, firmly attached to the bacterial body. Kossel
and Overbeck,4 however, on the basis of a careful investigation, came
to the conclusion that, in addition to the endotoxin, there is formed in
older broth cultures a definite and important true, soluble toxin.

Immunization. — A single attack of plague usually protects human
beings from reinfection. A second attack in the same individual is
extremely rare. Immunization in animals produces specific agglutinat¬
ing and bacteriolytic substances which are of great importance in the
bacteriological diagnosis of the bacillus. The agglutinating action of the
serum of patients is clinically important in the diagnosis of the disease,
even in dilutions of one in ten, since undiluted normal human serum
has no agglutinating effect upon plague bacilli.

Active immunization of animals 5 is accomplished by inoculation of
the whole dead bacteria. Haffkine has attempted active immunization
in human beings by subcutaneous treatment with sterilized broth cul¬
tures of B. pestis. Gaffky 6 and his collaborators recommend, for similar
purposes, forty-eight-hour agar cultures of a bacillus of standard viru¬
lence, emulsified in bouillon and sterilized at 65° C.

1 Strong, Teague, and Barber, Philippine Jour, of Sc., Sect. B, vii, 1912, No. 3.

2 Albrecht und Ghon, loc. cit.

3 Wernicke, Cent. f. Bakt., Ref., xxiv, 1898.

4 Kossel und Overbeck, Arb. a. d. Gesundh., xviii, 1901.

5 Yersin, Calmette, et Roux, Ann. de l’inst. Pasteur, 1895.

6 Gaffky, Pfeiffer , Sticker , und Dieudonne, Arb . a. d. kais. Gesundheitsamt, xvi,
1899.

562

PATHOGENIC MICROORGANISMS

The curative plague serum prepared by Yersin and others by the
immunization of horses with plague cultures has been extensively used
in practice and though often disappointing, a definitely beneficial in¬
fluence on the milder cases has been noted. The sera are standardized
by their protective power as measured in white rats.

THE PLAGUE-LIKE DISEASE OF RODENTS (McCOY) 1

Bacterium Tularense (McCoy and Chapin) 2

McCoy has described a disease occurring in Californian ground
squirrels (Citellus beechyi) which presents lesions very similar to those
of plague in these animals. In fact the disease was noticed in the
course of the systematic examination of rodents by McCoy at the
Federal Laboratory in San Francisco. Although McCoy was able to
transmit the disease to guinea-pigs, mice, rabbits, monkeys, and gophers,
and plague-like lesions could be produced in most of the animals, he was
at first entirely unable to cultivate any organism from these lesions.
In 1912 McCoy and Chapin finally succeeded in growing the specific
bacterium on an egg medium made entirely of the yolk. Mor¬
phologically it is a very small rod, 0.3 to 0.7 micron in length and often
capsulated. The rods stain poorly with methylene blue, better with
carbol fuchsin or gentian violet. They are found in large numbers in
the spleens of animals dead of the disease.

1 McCoy, U. S. Public Health Bull. 43, 1911.

2 McCoy and Chapin, Jour, of Inf. Dis., x, 1912.

CHAPTER XL

BACILLUS ANTHRACIS AND ANTHRAX
(Milzbrand, Charbon )

Anthrax is primarily a disease of the herbivora, attacking especially
cattle and sheep. Infection not infrequently occurs in horses, hogs, and
goats. In other domestic animals it is exceptional. Man is susceptible to
the disease and contracts it either directly from the living animals or
from the hides, wool, or other parts of the cadaver used in the industries.

The history of the disease dates back to the most ancient periods and
anthrax has, at all times, been a severe scourge upon cattle- and sheep¬
raising communities. Of all infections attacking the domestic animals
no other has claimed so many victims as anthrax. In Russia, where
the disease is most common, 72,000 horses are said to have succumbed
in one year (1864).1

In Austro-Hungary, Germany, France, and the Eastern countries,
each year thousands of animals and numerous human beings perish of
anthrax. In England and America the disease is relatively infrequent.
No quarter of the globe, however, is entirely free from it.

Especial historical interest attaches to the anthrax bacillus in that
it was the first microorganism proved definitely to bear a specific etio¬
logical relationship to an infectious disease. The discovery of the an¬
thrax bacillus, therefore, laid, as it were, the cornerstone of modern
bacteriology. The bacillus was first observed in the blood of infected
animals by Pollender in 1849, and, independently, by Brauell in 1857.
Davaine,2 3 * however, in 1863, was the first one to produce experimental
infection in animals with blood containing the bacilli and to suggest
a direct etiological relationship between the two. Final and absolute
proof of the justice of Davaine's contentions, however, was not brought
until the further development of bacteriological technique, by Koch,5
had made it possible for this last observer to isolate the bacillus upon

1 Quoted from Sobernheim, Kolle und Wassermann., vol. ii.

2 Davaine, Comptes rend, de l’acad. des sci., lvii, 1863.

3 Koch , Cohn’s “ Beitr. z. Biol. d. Pflanzen,” ii, 1876,

563

PATHOGENIC MICROORGANISMS

564

artificial media and to reproduce the disease experimentally by inocu¬
lation with pure cultures.

Morphology and Staining. — The anthrax bacillus is a straight rod,
5 to 10 micra in length, 1 to 3 micra in width. It is non-motile.
In preparations made from the blood of an infected animal, the bacilli
are usually single or in pairs. Grown on artificial media they form
tangles of long threads. Their ends are cut off squarely, in sharp con-

Fig. 120. — Bacillus anthracis. From pure culture on agar.

trast to the rounded ends of many other bacilli. The corners are often
sharp and the ends of bacilli in contact in a chain often touch each other
only at these points, leaving in consequence an oval chink between the
ends of the organisms. The appearance of a chain of anthrax bacilli,
therefore, lias been not inaptly compared to a rod of bamboo. On
artificial media the anthrax bacillus forms spores. Oxygen is necessary
lor the formation of these spores and they are consequently not found

BACILLUS ANTHRACIS AND ANTHRAX

565

in the blood of infected subjects. The spores are located in the middle
of the bacilli and are distinctly oval. They are difficult to stain, but
may be demonstrated by any of the usual spore-staining procedures,
such as Moller’s or Novy’s methods. The bacilli themselves are easily
stained by the usual anilin dyes, and gentian-violet or fuchsin in aque¬
ous solution may be conveniently employed. They are not decolorized
by Gram’s method.

In preparations from animal tissues or blood, stained by special pro-

Fig. 121. — Bacillus anthracis. In section of kidney of animal dead of

anthrax.

cedures, the anthrax bacillus may occasionally be seen to possess a cap¬
sule. The capsule is never seen in preparations from the ordinary
artificial media. Some observers have demonstrated them in cultures
grown in fluid blood serum. In chains of anthrax bacilli, the capsule
when present seems to envelop the entire chain and not the individual
bacteria separately.

Isolation. — Isolation of the anthrax bacillus from infected material

566

PATHOGENIC MICROORGANISMS

is comparatively simple, both because of the ease of its cultivation and
because of the sharply characteristic features of its morphological and
cultural appearance.

Cultivation. — The anthrax bacillus is an aerobic, facultatively anaero¬
bic bacillus. While it may develop slowly and sparsely under anaerobic
conditions, free oxygen is required to permit its luxuriant and charac¬
teristic growth.

The optimum temperature for its cultivation ranges about 37.5° C.
It is not, however, delicately susceptible to moderate variations of tem-

Fig. 122. — Bacillus anthracis. In smear of spleen of animal dead of anthrax.

perature and growth does not cease until temperatures as low as 12° C.
or as high as 45° C. are reached. By continuous cultivation at some of
the temperatures near either the higher or the lower of these limits, the
bacillus may become well adapted to the new environment and attain
luxuriant growth.1

The anthrax bacillus may be cultivated on all of the usual artificial

media, growing upon the meat-extract as well as upon the meat-infusion
media.

1 Dieudonne, Arb. a. d. kais. Gesundheitsamt, 1894.

BACILLUS ANTHRACIS AND ANTHRAX

567

It may be cultivated also upon hay infusion, various other vegetable
media, sugar solutions, and urine. While moderate acidity of the
medium does not prevent the growth of this bacillus, the most favorable
reaction for media is neutrality or slight alkalinity.

Cn gelatin plates, colonies develop within twenty-four to forty -eight
hours as opaque, white disks, pin-head in size, irregularly round and
rather flat. As the colonies increase in size their outlines become less
regular and under the microscope they are seen to be made up of a
hair-like tangle of threads spreading in thin wavy layers from a more
compact central knot. The microscopic appearance of these colonies
has been aptly described as resembling a Medusa head. Fragments of a

Fig. 123. — Anthrax Colony on Gelatin. (After Gunther.)

colony examined on a slide with a higher power show the individual
threads to be made up of parallel chains of bacilli.

After a day or two of further growth, the gelatin about the colonies
becomes fluidified.

In gelatin stab cultures, growth appears at first as a thin white line
along the course of the puncture. From this, growth proceeds in thin
spicules or filaments diverging from the stab, more abundantly near the
top than near the bottom of the stab, owing to more active growth in
well oxygenated environment. The resulting picture is that of a small
inverted “ Christmas tree/7 Fluidification begins at the top, at first a
shallow depression filled with an opaque mixture of bacilli and fluid.

568

PATHOGENIC MICROORGANISMS

Later the bacilli sink to the bottom of the flat depression, leaving a clear
supernatant fluid of peptonized gelatin.

In broth, growth takes place rapidly, but does not lead to an even,
general clouding. There is usually an initial pellicle formation at the
top where the oxygen supply is greatest. Simultaneously with this a
slimy mass appears at the bottom of the tube, owing to the sinking of

Fig. 124. — Anthrax Colony on Agar.

bacilli to the bottom. Apart from isolated flakes and threads the inter¬
vening broth is clear. Shaken up, the tube shows a tough, stringy mass,
not unlike a small cotton fluff, and general clouding is produced only
by vigorous mixing.

Lpon agar plates, growth at 37.5° C. is vigorous and colonies appear

BACILLUS ANTHRACIS AND ANTHRAX

569

within twelve to twenty-four hours. They are irregular in outline,
slightly wrinkled, and show under the microscope the characteristic
tangled-thread appearance seen on gelatin, except that they are more
compact than upon the former medium. The colonies are slightly glisten¬
ing and tough in consistency.

On agar slants, the colonies usually become confluent, the entire
surface soon being covered by a grayish, tough pellicle which, if fished,
has a tendency to come away in thin strips or strands.

On potato, growth is rapid, white, and rather dry. Sporulation upon
potato is rapid and marked, and the medium is favorable for the study
of this phase of development.

Milk is slowly acidified and slowly coagulated. This action is chiefly
upon the casein; very few, if any, changes being produced either in the
sugars or in the fats of the milk. The acids formed are, according to
Iwanow,1 chiefly formic, acetic, and caproic acids.

Biological Considerations. — The anthrax bacillus is aerobic and facul¬
tatively anaerobic. It is non-motile and possesses no flagella. In the
animal body it occasionally forms capsules. In artificial cultures in
the presence of oxygen, it sooner or later invariably forms spores. The
spores appear after the culture has reached its maximum of develop¬
ment. Sporulation never occurs in the animal body, probably because
of the absence of sufficient free oxygen. Spores are formed most exten¬
sively 2 at temperatures ranging from 20° C. to 30° C. Spore formation
ceases below 18° C. and above 42° C. For different strains these figures
may vary slightly, as has been shown from the results of various
observers. Spores appear most rapidly and regularly upon agar and
potato media.

The spore — one in each bacillus — appears as a small, highly retractile
spot in the center of the individual bacterium. As this enlarges, the
body of the bacillus around it gradually undergoes granular degenera¬
tion and loses its staining capacity.3

If anthrax bacilli are cultivated for prolonged periods upon media
containing hydrochloric or rosolic acid or weak solutions of carbolic
acid,4 cultures may be obtained which do not sporulate and which seem
permanently to have lost this power, without losing their virulence to
the same degree. Similar results may be obtained by continuous cul-

1 Iwanow, Ann. de Tinst. Pasteur, 1892.

2 Koch, loc. cit.

3 Behring, Zeit. f. Hyg., vi and vii, 1889; Deut. med. Woch., 1889.

4 Chamberland et Roux, Comptes rend, de Pacad. des sci., xcvi, 1882.

570

PATHOGENIC MICROORGANISMS

tivation at temperatures above 42° C. By this procedure, however,
virulence, too, is considerably diminished.

Resistance. — Because of its property of spore formation, the anthrax
bacillus is extremely resistant toward chemical and physical environ¬
ment. The vegetative forms themselves are not more resistant than
most other non-sporulating bacteria, being destroyed by a temperature
of 54° C. in ten minutes. Anthrax spores may be kept in a dry state
for many years without losing their viability.1 While different strains
of anthrax spores show some variation in their powers of resistance,
all races show an extremely high resistance to heat. Dry heat at 140°
C. kills them only after three hours.2 Live steam at 100° kills them in
five to ten minutes. Boiling in water destroys them in about ten min¬
utes. Low temperatures have but little effect upon them. Ravenel 3
found that, frozen by liquid air, they were still viable after three hours.

The variability shown by different strains of spores in their resistance
to heat is even more marked in their behavior toward chemicals.4 Some
strains will retain their viability after exposure to five-per-cent carbolic
acid for forty days,5 6 while others are destroyed by the same solution in
two days. Corrosive sublimate, 1 : 2,000, kills most strains of anthrax
in forty minutes.

Direct sunlight destroys anthrax spores within six to twelve hours. (>

Pathogenicity. — The anthrax bacillus is pathogenic for cattle,
sheep, guinea-pigs, rabbits, rats, and mice. The degrees of susceptibil¬
ity of these animals differ greatly, variations in this respect existing even
among different members of the same species. Thus, the long-haired
Algerian sheep show a high resistance, while the European variety are
highly susceptible; and, similarly, the gray rat is much more resistant
than the white rat. Dogs, hogs, cats, birds, and the cold-blooded ani¬
mals are relatively insusceptible. For man the bacillus is definitely
pathogenic, though less so than for some of the animals mentioned
above.

While separate races of anthrax bacilli may vary much in their de¬
gree of virulence, a single individual strain remains fairly constant in
this respect if preserved, dried upon threads or kept in sealed tubes, in

1 Surmont et Arnould, Ann. de Tinst. Pasteur, 1894.

2 Koch und Wolff hugel, Mitt. a. d. kais. Gesundheitsamt, 1881.

3 Ravenel, Medical News, vii, 1899.

4 Frankel, Zeit. f. Hyg., vi, 1889.

5 Koch, loc. cit.

6 Momont, Ann. de Tinst. Pasteur, 1892.

BACILLUS ANTHRACIS AND ANTHRAX

571

a cold, dark place. Virulence may be reduced 1 by various attenuating
laboratory procedures which are of importance in that they have made
possible prophylactic immunization. Heating the bacilli to 55° C. for
ten minutes considerably reduces their virulence. Similar results are
obtained by prolonged cultivation at temperatures of 42° to 43° C.,
or by the addition of weak disinfectants to the culture fluids.2 Once
reduced, the new grade of virulence remains fairly constant. Increase
of virulence may be artificially produced by passage through animals.

Experimental infections in susceptible animals are most easily accom¬
plished by subcutaneous inoculations. The inoculation is followed, at
first, by no morbid symptoms, and some animals may appear perfectly
well and comfortable until within a few hours or even moments before
death, when they suddenly become visibly very ill, rapidly go into
collapse, and die. The length of the disease depends to some extent,
of course, upon the resistance of the infected subject, being in guinea-
pigs and mice from twenty-four to forty-eight hours. The quantity of
infectious material introduced, on the other hand, has little bearing
upon the final outcome, a few bacilli, or even a single bacillus, often
sufficing to bring about a fatal infection. Although the bacilli are not
demonstrable in the blood until just before death, they nevertheless
invade the blood and lymph streams immediately after inoculation,
and are conveyed by these to all the organs. This has been demonstrated
clearly by experiments where inoculations into the tail or ear were im¬
mediately followed by amputation of the inoculated parts without pre¬
vention of the fatal general infection. The bacilli are probably not at
first able to multiply in the blood. At the place of inoculation and
probably in the organs they proliferate, until the resistance of the in¬
fected subject is entirely overcome. At this stage of the disease, no
longer held at bay by any antagonistic qualities of the blood, they enter
the circulation and multiply within it. Autopsy upon such animals
reveals an edematous hemorrhagic infiltration at the point of inocu¬
lation. The spleen is enlarged and congested. The kidneys are con¬
gested, and there may be hemorrhagic spots upon the serous mem¬
branes. The bacilli are found in large numbers in the blood and in the
capillaries of all the organs.

The mode of action of Bacillus anthracis is as yet an unsettled point.
It is probable that death is brought about to a large extent by purely

1 Toussaint, Comptes rend, de Pa cad. des sci., xci, 1880; Pasteur, Chamberland
et Roux, Comptes rend, de Pacad. des sci., xcii, 1881.

2 Chamberland et Roux, ibid., xcvir 1882.

572

PATHOGENIC MICROORGANISMS

mechanical means, such as capillary obstruction. Neither a true
secretory toxin nor an endotoxin has been demonstrated for the anthrax
bacillus. The decidedly toxemic clinical picture of the disease, however,
in some animals and in man, precludes our definitely concluding that
such poisons do not exist. It is a matter of fact, however, that neither
culture filtrates nor dead bacilli have any noticeable toxic effect upon
test animals, and exert no appreciable immunizing action.

Spontaneous infection of animals takes place largely by way of the
alimentary canal, the bacilli being taken in with the food. The bacteria
are swallowed as spores, and therefore resist the acid gastric juice. In
the intestines they develop into the vegetative forms, increase, and
gradually invade the system. The large majority of cattle infections
are of this type. Direct subcutaneous infection may also occur sponta¬
neously when small punctures and abrasions about the mouth are made
by the sharp spicules of the hay, straw, or other varieties of fodder.

When infection upon a visible part occurs, there is formed a diffuse,
tense local swelling, not unlike a large carbuncle. The center of this may
be marked by a black, necrotic slough, or may contain a pustular de¬
pression.

Infection by inhalation is probably rare among animals. Trans¬
mission among animals is usually by the agency of the excreta or un¬
burned carcasses of infected animals. The bacilli escaping from the
body are deposited upon the earth together with animal and vegetable
matter, which forms a suitable medium for sporulation. The spores
may then remain in the immediate vicinity, or may be scattered by
rain and wind over considerable areas. The danger from buried car¬
casses, at first suspected by Pasteur, is probably very slight, owing to the
fact that the bacilli can not sporulate in the anaerobic environment to
which the burying-process subjects them. The disease, in infected cattle
and sheep, is usually acute, killing within one or two days. The mortality
is extremely high, fluctuating about eighty per cent.

In man the disease is usually acquired by cutaneous inoculation. It
may also occur by inhalation and through the alimentary tract.

Cutaneous inoculation occurs usually through small abrasions or
scratches upon the skin in men who habitually handle live-stock, and
in butchers, or tanners of hides. Infection occurs most frequently upon
the hands and forearms. The primary lesion, often spoken of as “ malig¬
nant pustule,” appears within twelve to twenty -four hours after inocula¬
tion, and resembles, at first, an ordinary small furuncle. Soon, however,
its center will show a vesicle filled with sero-sanguineous, later sero-

BACILLUS ANTHRACIS AND ANTHRAX

573

purulent fluid. This may change into a black central necrosis sur¬
rounded by an angry red edematous areola. Occasionally local gangrene
and general systemic infection may lead to death within five or six days.
More frequently ; however, especially if prompt excision is practiced, the
patient recovers. The early diagnosis of the condition is best made
bacteriologically by finding the bacilli in the local discharge.

The pulmonary infection, known as “wool-sorter's disease,” occurs
in persons who handle raw wool, hides, or horse hair, by the inhala¬
tion or by the swallowing of spores. The disease is fortunately rare in
this country. The spores, once inhaled, develop into the vegetative
forms 1 and these travel along the lymphatics into the lungs and
pleura. The disease manifests itself as a violent, irregular pneumonia,
which, in the majority of cases, leads to death. The bacilli in these
cases can often be found in the sputum before death.

Infection through the alimentary canal may occasionally, though
rarely, occur in man, the source of infection being usually ingestion of
the uncooked meat of infected animals. This form of infection is rare,
because in many cases the bacilli have not sporulatecl in the animal
and the ingested vegetative forms are injured or destroyed by the acid
gastric juice. When viable spores enter the gut, however, infection may
take place, the initial lesion being localized usually in the small intes¬
tine. The clinical picture that follows is one of violent enteritis with
bloody stools and great prostration. Death is the rule. The diagnosis
is made by the discovery of the bacilli in the feces.

General hygienic prophylaxis against anthrax consists chiefly in the
destruction of infected animals, in the burying of cadavers, and in the
disinfection of stables, etc. The practical impossibility of destroying
the anthrax spores in infected pastures, etc., makes it necessary to re¬
sort to prophylactic immunization of cattle and sheep.

Immunity against Anthrax. — Minute quantities of virulent anthrax
cultures usually suffice to produce death in susceptible animals. Dead
cultures are inefficient in calling forth any immunity in treated subjects.
It is necessary, therefore, for the production of active immunity to
resort to attenuated cultures. The safest way to accomplish such at¬
tenuation is the one originated by Pasteur,2 consisting in prolonged
cultivation of the bacillus at 42° to 43° C. in broth. Non-spore-forming
races are thus evolved.

The longer the bacilli are grown at the above temperature the greater

1 Eppinger, Wien, med, Woch., 1888.

2 Pasteur, loc. cit.

574

PATHOGENIC MICROORGANISMS

is the reduction in their virulence. Koch, Gaffky, and Loeffler/ utilizing
the variations in susceptibilities of different species of animals, devised a
method by means of which the relative attenuation of a given culture may
be estimated and standardized. Rabbits are less susceptible than guinea-
pigs, and virulent anthrax cultures, grown for two or three days under the
stated conditions, lose their power to kill rabbits, but are less virulent for
guinea-pigs. After ten to twenty days of further cultivation at 42° C.
the virulence for the guinea-pig disappears, but the culture is potent
against the still more susceptible mouse. Even the virulence for mice
may be entirely eliminated by further cultivation at this temperature.

The method of active immunization first practiced by Pasteur, and
still used extensively, is carried out as follows: Two anthrax cultures
of varying degrees of attenuation are used as vaccins. The premier
vaccin is a culture which has lost its virulence for guinea-pigs and
rabbits, and is potent only against mice. The deuxieme vaccin is a cul¬
ture which is still definitely virulent for mice and guinea-pigs, but not
potent for rabbits. Forty-eight-hour broth cultures of these strains,
grown at 37.5° C., form the vaccin actually employed. Vaccin I is
subcutaneously injected into cattle in doses of 0.25 c.c., sheep receiving
about half this quantity. After twelve days have elapsed similar quan¬
tities of Vaccin II are injected.

Pasteur’s method has given excellent results and confers an im¬
munity which lasts about a year.

Chauveau 1 2 has modified Pasteur’s method by growing the bacilli
in bouillon at 38° to 39° C., at a pressure of eight atmospheres. Cul¬
tures are then made of races attenuated in this way, upon chicken
bouillon and allowed to develop for thirty days. Single injections of
0.1 c.c. each of such cultures are said to protect cattle.

Active immunization of small laboratory animals is very difficult,
but can be accomplished by careful treatment with extremely attenu¬
ated cultures.

Passive immunization by means of the serum of actively immune
animals was first successfully accomplished by Sclavo.3

The subject of passive immunization has been especially investigated
and practically applied by Sobernheim.4 The serum used is produced by
actively immunizing sheep. It is necessary to carry immunization to an

1 Koch, Gaffky, und Loeffler , Mitt. a. d. kais. Gesundheitsamt, 1884.

2 Chauveau, Comptes rend, de Facado des sci., 1884.

3 Sclavo, Cent. f. Bakt., xviii, 1895.

* Sobernheim , Zeit. f. Ilyg., xxv, 1897; xxxi, 1899.

BACILLUS ANTHRACIS AND ANTHRAX

575

extremely high degree in order to obtain any appreciable protective
power in the serum. This is accomplished by preliminary treatment
with Pasteur’s or other attenuated vaccines, followed by gradually
increasing doses of fully virulent cultures. Treatment continued at
intervals of two weeks, for two or three months, usually produces an
effective serum. Horses and cattle may also be used for the process, but
they are believed by Sobernheim to give less active sera than sheep.
Bleeding is done about three weeks after the last injection. The sera
are stable and easily preserved.

Injections of 20 to 25 c.c. of such a serum have been found to protect
animals effectually from anthrax and to confer an immunity lasting
often as long as two months. Animals already infected are said to be
saved b}^ treatment with 25 to 100 c.c. of the serum.

Neither specific bactericidal nor bacteriolytic properties have, so
far, been demonstrated in these immune sera. In fact, these properties
are distinctly more pronounced against Bacillus anthracis in the normal
sera of rats and dogs. Agglutinins have not been satisfactorily demon¬
strated in sera, partly because of the great technical difficulties en¬
countered in the active chain-formation of the bacillus in fluid media.
An increase of opsonic power of such serum over normal serum has
not been satisfactorily demonstrated.

Bacteria Closely Resembling Bacillus anthracis. — In most laboratory
collections there are strains of true anthrax bacilli so attenuated that
they are practically non-pathogenic. These do not differ from the
virulent strains in any morphological or cultural characteristics.
Besides such strains there are numerous non-virulent bacteria culturally
not identical with Bacillus anthracis, but resembling it very closely.

B. anthracoides ( Hueppe and Wood *). — A Gram-positive bacillus,
morphologically different from B. anthracis in that the ends are more
rounded. Culturally, somewhat more rapid in growth and more rapid
in gelatin fluidification. Non-pathogenic. Otherwise indistinguishable
from B. anthracis.

B. radicosus (Wurzel Bacillus) . — Cultivated from water — city water
supplies. Morphologically somewhat larger than Bacillus anthracis, and
the individual bacilli more irregular in size. Very rapid fluidification of
gelatin and growth most active at room temperature. Non-pathogenic.

B. subtilis ( Hay Bacillus). — Although not very closely related to
the anthrax group, this bacillus is somewhat similar and conveniently

1 Hueppe und Wood, Berl. klin. Woch., xvi, 1889.

576

PATHOGENIC MICROORGANISMS

described in this connection. It is of importance to workers with patho¬
genic bacteria, because of the frequency with which it is found as a
saprophyte or secondary invader in chronic suppurative lesions.

Morphology. — Straight rod, 2 to 8 micra long, 0.7 micron wide. Spores
formed usually slightly nearer one pole than the other. Grows in long
chains and only in such chains are spores found. It does not decolor¬
ize by Gram’s method. Is actively motile in young cultures in which

Fig. 125. — Bacillus Subtilis. (Hay Bacillus.)

the bacilli are single or in pairs. In older cultures chains are formed
and the bacilli become motionless. Gelatin is liquefied. On gelatin
and agar the bacilli grow as a dry corrugated pellicle. Microscopically,
the colonies are made up of interlacing threads, being irregularly round
with fringed edges. There is a tendency to confluence. The bacillus
is found in brackish water, infusions of vegetable matter, etc., and is
practically non-pathogenic, occurring only occasionally as a saprophyte
in old sinuses and infected wounds.

CHAPTER XLI

BACILLUS PYOCYANEUS

It is a matter of common surgical experience that many suppurating
wounds, especially sinuses of long standing, discharge pus which is of a
bright green color. The fact that this peculiar type of purulent inflam¬
mation is due to a specific chromogenic microorganism was first demon¬
strated by Gessard 1 in 1882. The bacillus which was described by Ges-
sard has since become the subject of much careful research and has been
shown to hold a not unimportant place among pathogenic bacteria.2

Morphology and Staining. — Bacillus pyocyaneus is a short rod, usu¬
ally straight, occasionally slightly curved, measuring, according to
Fliigge, about 1 to 2 micra in length by about 0.3 of a micron in thickness.
The bacilli are thus small and slender, but are subject to considerable
variation from the measurements given, even in one and the same cul¬
ture. While ordinarily single, the bacilli may be arranged end to end in
short chains of two and three. Longer chains may exceptionally be
formed upon media which are especially unfavorable for its growth, such
as very acid media or those containing antiseptics.

Spores are not found. The bacilli are actively motile and possess
each a single flagellum placed at one end.

Bacillus pyocyaneus is stained easily with all the usual dyes, but is
decolorized by Gram’s method. Irregular staining of the bacillary body
is common, but is always an indication of degeneration, and not a
normal characteristic, as, for instance, in the diphtheria group.

Cultivation. — The pyocyaneus bacillus is aerobic and facultatively
anaerobic. It can be adapted to absolutely anaerobic environments, but
does not produce its characteristic pigment without the free access of
oxygen. The bacillus grows readily upon the usual laboratory media
and is not very sensitive to reaction, growing equally well upon moder¬
ately alkaline or acid media. Development takes place at temperatures
as low as 18° to 20° C., more rapidly and luxuriantly at 37.5° C.

1 Gessard, These de Paris, 1882.

2 Charrin, “ La maladie pyocyanique,” Paris, 1889.

38 577

578

PATHOGENIC MICROORGANISMS

On agar slants, growth is abundant and confluent, the surface of the
agar being covered by a moist, grayish or yellowish, glistening, even layer.
The pigment which begins to become visible after about eighteen hours
soon penetrates the agar itself and becomes diffused throughout it,
giving the medium a bright green fluorescent appearance, which grows
darker as the age of the culture increases.

In gelatin stabs, growth takes place much more rapidly upon the
surface than in the depths. A rapid liquefaction of the gelatin takes
place, causing a saucer-shaped depression. As this deepens, pigment
begins to form in the upper layers, often visible as a greenish pellicle.

In gelatin plates, the colonies have a characteristic appearance. They
are round and are composed of a central dense zone, and a peripheral,
loosely granular zone, which extends outward into the peripheral fluidi¬
fied area in a fringe of fine filaments. When first appearing, they are
grayish yellow, later assuming the characteristic greenish hue.

In broth, growth is rapid and chiefly at the surface, forming a thick
pellicle. Below this, there is moderate clouding. The pigment is formed
chiefly at the top. In old cultures there is a heavy flocculent precipitate.
In fluid media containing albuminous material, strong alkalinity is
produced.

On potato, growth develops readily and a deep brownish pigment ap¬
pears, which is not unlike that produced by B. mallei upon the same
medium.

Milk is coagulated by precipitation of casein and assumes a yellowish-
green hue. In older cultures the casein may again be digested and liquefied

The pigment of Bacillus pyocyaneus has been the subject of much
investigation. It was shown by Charrin 1 and others that this pigment
had no relation to the pathogenic properties of the bacillus. It is found
in cultures as a colorless leukobase which assumes a green color on the
addition of oxygen. Conversely, the typical green “pyocyanin,” as
the pigment is called, may be decolorized by reducing substances. This
explains the fact that it is not found in cultures sealed from the air. Pyo-
cyanin may be extracted from cultures with chloroform and crystallized
out of such solution in the form of blue stellate crystals. These, on
chemical analysis, have been found to belong to the group of aromatic
compounds, with a formula, according to Ledderhose,2 of C14H14N20.

Besides pyocyanin, Bacillus pyocyaneus produces another pig-

1 Charrin, loc. cit.

3 Ledderhose, quoted from Boland, Cent. f. Bakt., xxv, 1889.

BACILLUS PYOCYANEUS

579

ment which is fluorescent and insoluble in chloroform, but soluble in
water.1 This pigment is common to other fluorescent bacteria, and not
peculiar to Bacillus pyocyaneus. The reddish-brown color seen in old
cultures 2 and supposed by some writers to be a third pigment, is probably
a derivative from pyocyanin by chemical change.

Chloroform extraction of pyocyanin from cultures may serve oc¬
casionally to distinguish the pyocyaneus bacilli from other similar
fluorescent bacteria. Ernst has claimed that there are two types of B.
pyocyaneus, an a-type which produces only the fluorescent, water-
soluble pigment, and a /5-type which produces both this and pyocyanin.3

Pathogenicity. — Bacillus pyocyaneus is one of the less virulent patho¬
genic bacteria. It is widely distributed in nature and may be found
frequently as a harmless parasite upon the skin or in the upper respira¬
tory tracts of animals and men. It has, however, occasionally been
found in connection with suppurative lesions of various parts of
the body, often as a mere secondary invader in the wake of another
incitant, or even as the primary cause of the inflammation. In most
cases where true pyocyaneus infection has taken place, the subject is
usually one whose general condition and resistance are abnormally low.4
Thus pyocyaneus may be the cause of chronic otitis media in ill-nour¬
ished children. It has been cultivated out of the stools of children suf¬
fering from diarrhea, and has been found at autopsy generalfy distributed
throughout the organs of children dead of gastro-enteritis.5 It has been
cultivated from the spleen at autopsy from a case of general sepsis
following mastoid operation. The bacillus has been found, further¬
more, during life in pericardial exudate and in pus from liver abscesses.6

Brill and Libman,7 as well as Finkelstein,8 have cultivated
B. pyocyaneus from the blood of patients suffering from general sepsis.
Wassermann 9 showed the bacillus to have been the etiological factor in
an epidemic of umbilical infections in new-born children. Similar exam¬
ples of B. pyocyaneus infection in human beings might be enumerated in
large numbers, and there is no good reason to doubt that, under given

1 Boland, loc. cit.

2Gessard, Ann. de l’inst. Pasteur, 1890, 1891, and 1892.

3 Ernst, Zeit. f. Hyg., ii, 1887.

* Rohner, Cent. f. Bakt., xi, 1892.

s Neumann, Jahrb. f. Kinderheilk., 1890.

* Kraunhals, Zeit. f. Chir., xxxvii, 1893.

7 Brill and Libman, Amer. Jour. Med. Sci., 1899.

8 Finkelstein, Cent. f. Bakt., 1899.

* Wassermann, Virchow’s Arch., elxv, 1901.

580

PATHOGENIC MICROORGANISMS

conditions, fatal infections may occur. Such cases, however, are still to
be regarded as depending more upon the low resistance of the individual
attacked than upon the great pathogenicity of B. pyocyaneus.

Many domestic animals are susceptible to experimental pyocyaneus
infection, chief among these being rabbits, goats, mice, and guinea-
pigs. Guinea-pigs are killed by this bacillus with especial ease. Intra-
peritoneal inoculation with a loopful of a culture of average virulence
usually leads to the death of a young guinea-pig within three or four days.

Toxins and Immunization. — Emmerich and Low have shown that
filtrates of old broth cultures of B. pyocyaneus contain a ferment-like
substance which possesses the power to destroy some other bacteria,
apparently by lysis. They have called this substance “ pyocyanase ” and
claim that, with it, they have succeeded in protecting animals from
anthrax infection. During recent years pyocyanase has been employed
locally for the removal of diphtheria bacilli from the throats of convales¬
cent cases. Broth-culture filtrates evaporated to one-tenth their volume
in vacuo are used for this purpose.

Pyocyanase is exceedingly thermostable, resisting boiling for several
hours, and is probably not identical with any of the other toxins or
peptonizing ferments produced by B. pyocyaneus.

The toxins proper of B. pyocyaneus have been the subject of much
investigation, chiefly by Wassermann.1 Wassermann found that filtrates
of old cultures were far more poisonous for guinea-pigs than extracts
made of dead bacteria. He concludes from this and other observations
that B. pyocyaneus produces both an endotoxin and a soluble secreted
toxin. The toxin is comparatively thermostable, resisting 100° C. for a
short time. Animals actively immunized with living cultures of B. pyo¬
cyaneus give rise in their blood serum to bacteriolytic antibodies only.
Immunized with filtrates from old cultures, on the other hand, their
serum will contain both bacteriolytic and antitoxic substances. The
true toxin of B. pyocyaneus never approaches in strength that of diph¬
theria or of tetanus. Active immunization of animals must be done
carefully if it is desired to produce an immune serum, since repeated
injections cause great emaciation and general loss of strength. Specific
agglutinins have been found in immune sera by Wassermann 2 and
others. Eisenberg 3 claims that such agglutinins are active also against
some of the fluorescent intestinal bacteria.

1 Wassermann, Zeit. f. Hyg., xxii, 1896. 2 Wassermann, Zeit. f. Hyg., 1902.

3 Eisenberg, Cent. f. Bakt., 1903.

BACILLUS PYOCYANEUS

581

Bulloch and Hunter 1 have recently been able to show that old
broth cultures of B. pyocyaneus contain a substance capable of
hemolyzing the red blood corpuscles of dogs, rabbits, and sheep.
This “ pyocyanolysin ” seems intimately attached to the bacterial
body. Prolonged heating of cultures does not destroy it. Heating of
hemolytic filtrates, however, destroys it in a short time. The filtration
of young cultures yields very little pyocyanolysin in the filtrate. In
old cultures, however, a considerable amount passes into the filtrate.
Whether or not the hemolytic power is due to a specific bacterial
product or is dependent upon changes in the culture fluid, such as
alkalinization, etc., can not yet be regarded as certain.

Gheorghiewski 2 claims to have found a leucocyte-destroying ferment
in pyocyaneus cultures.

1 Bulloch und Hunter, Cent. f. Bakt., xxviii, 1900.

2 Gheorghiewski, Ann. de Tinst. Pasteur, xiii, 1899.

CHAPTER XLTT

ASIATIC CHOLERA AND THE CHOLERA ORGANISM
(; Spirillum, cholerce asiaticw, Comma Bacillus)

The organism of Asiatic cholera was unknown until 1883. In this
year, Koch/ at the head of a commission established by the German
government to study the disease in Egypt and India, discovered the
“comma bacillus” in the defecations of patients, and satisfactorily de¬
termined its etiological significance.

Koch’s investigations were carried out on a large number of cases
and many investigations have since then corroborated his results.
The numerous morphologically similar spirilla which were later found
in normal individuals and in connection with other conditions, have
been shown by accurate bacteriological methods to be closely related,
but not identical.

Apart from the evidence of the constant association of the cholera
vibrio with the disease, the etiological relationship has been clearly
demonstrated b}^ several accurately recorded accidental infections oc¬
curring in bacteriological workers, and by the famous experiment of
Pettenkofer and Emmerich, who purposely drank water containing
cholera bacilli. Both observers became seriously ill with typical clini¬
cal symptoms of cholera, and one of them narrowly escaped death.

Morphology and Staining. — The vibrio or spirillum of cholera is a small
curved rod, varying from one to two micra in length. The degree of
curvature may vary from the slightly bent, comma-like form to a
more or less distinct spiral with one or two turns. The spirals do not
lie in the same plane, being arranged in corkscrew fashion in three
dimensions. The spirillum is actively motile and owes its motility
to a single polar flagellum, best demonstrated by Van Ermengem’s
flagella stain. Spores are not found. In young cultures the comma
shapes predominate, in older growths the longer forms are more nu¬
merous. Strains which have been cultivated artificially for prolonged

1 Koch , Deut. med. Woch., 1883 and 1884.

582

ASIATIC CHOLERA AND THE CHOLERA ORGANISM

583

periods without passage through the animal body have a tendency to
lose the curve, assuming a more bacillus-like appearance. The spirilla
are stained with all the usual aqueous anilin dyes. They are decolor¬
ized by Gram’s method. In histological section they are less easily
stained, but may be demonstrated by staining with alkaline methylene
blue.

Cultivation. — The cholera spirillum grows easily upon all the usual
culture media, thriving upon meat-extract as well as upon meat-infusion

Fig. 126. — Cholera Spirillum. (After Frankel and Pfeiffer.)

media. Moderate alkalinity of the media is preferable, though slight
acidity does not prevent growth.

In gelatin plates growth appears at room temperature within twenty-
four hours as small, strongly refracting yellowish -gray, pin-head colonies.
As growth increases the gelatin is fluidified. Under magnification these
colonies "appear coarsely granular with margins irregular because of
the liquefaction. Liquefaction, too, causes a rapid development in
such colonies of separate concentric zones of varying refractive power.
Old strains, artificially cultivated for long periods, lose much of their
liquefying power.

In gelatin stab cultures fluidification begins at the surface, rapidly
giving rise to the familiar funnel-shaped excavation.

Upon agar plates, within eighteen to twenty-four hours, grayish,
opalescent colonies appear, which are easily differentiated by their

584

PATHOGENIC MICROORGANISMS

transparency from the other bacteria apt to appear in feces. Agar
plates, therefore, are important in the isolation of these organisms.

Coagulated blood serum is fluidified by the cholera vibrio. On
potato, growth is profuse and appears as a brownish coarse layer. In
milk, growth is rapid and without coagulation. In broth, general
clouding and the formation of a pellicle result. The rapidity and luxuri¬
ance of growth of the cholera spirillum upon alkaline pepton solutions
render such solutions peculiarly useful as enriching media in isolating
this microorganism from the stools of patients. In pepton solution,
too, the cholera spirillum gives rise to abundant indol, demonstrated
in the so-called “ cholera-red ” reaction. This reaction has a distinct
diagnostic value, but is by no means specific.1 In the case of the cholera
vibrio the mere addition of strong sulphuric acid suffices to bring out
the color reaction. This is due to the fact that, unlike some other indol-
producing bacteria, the cholera organism is able to reduce the nitrates
present in the medium to nitrites, thus itself furnishing the nitrite
necessary for the color reaction. The medium which is most suitable
for this test is that proposed by Dunham,2 consisting of a solution
of 1 per cent of pure pepton and .5 per cent NaCl in water.

Dieudonne3 has recommended a selective medium upon which
cholera spirilla will grow well, but upon which the colon bacillus will
grow either very sparsely or not at all. Its preparation is very simple.
To 70 parts of ordinary 3 per cent agar, neutralized to litmus, there are
added 30 parts of a sterile mixture of defibrinated beef blood and normal
sodium hydrate.

The latter is sterilized by steam before being added to the agar.
This pure alkali agar is poured out in plates and allowed to dry several
days at 37° or 5 minutes at 60°. The material to be examined is smeared
upon the surface of these plates with a glass rod.

The principle of this medium is that cholera will grow in the presence
of an amount of alkali which inhibits other fecal bacteria. The medium
has been studied by Krumwiede, Pratt, and Grund,4 who have recom¬
mended a modification. They find the following combination sat¬
isfactory and an improvement upon Dieudonnd’s medium because
transparent and more easily prepared. They prepare the following
mixtures :

1 See indol reaction, p. 167. 2 Dunham, Zeit. f. Hyg., ii, 1887.

3 Dieudonne , A., Cent. Bakt., 1., orig., 1909.

4 Krumwiede, Pratt, and Grund, Jour, of Inf. Dis., x, 1912.

ASIATIC CHOLERA AND THE CHOLERA ORGANISM

585

Egg-White Medium.

A. White of egg and water a. a.

Sodium carbonate cryst. 12 per cent.

Mix in equal parts, steam in Arnold sterilizer for 20 minutes.

B. Meat pepton 3 per cent agar, neutral to litmus.

30 parts of A are added to 70 parts of B.

Another modification recommended by them is as follows:

Whole-Egg Medium.

A. Whole egg and water a.a.

Sodium carbonate 12 to 13.5 per cent.

Mix in equal parts, steam for 20 minutes.

B. Meat free agar, viz., pepton, salt, and 3 per cent agar.

30 parts of A are mixed with 70 parts of B while the agar is boiling hot as
above.

The medium is poured on the plates in a thick layer and allowed
to stand open for 20 to 30 minutes and then the inoculation is carried
out by surface streaking.

Isolation. — Isolation of the cholera vibrio from the feces, while
easy in many cases, is occasionally attended with some difficulty
owing to the large number of other bacteria present. The most
satisfactory method of procedure is to inoculate a set of gelatin
plates, another of agar plates, and a number of Dunham’s pepton-
broth tubes, with small quantities of the suspicious material. When
the spirilla are numerous they can frequently be fished directly from sus¬
picious colonies in the plates and isolated for further identification.
When less numerous, they can usually be found in relatively increased
numbers after eight or ten hours at 37.5° C., in the topmost layers of
the Dunham broth, which is an almost selectively favorable medium for
these organisms. They collect at the surface where free oxygen is
readily obtained. From the pepton broth, plate dilutions can then be
prepared and colonies fished.1 Once isolated, the spirilla are identified
by their morphology, by the appearance of their colonies, by their
manner of growth upon gelatin stabs, by the cholera-red reaction,
and, finally, by agglutinative and bacteriolytic tests in immune sera.
Owing to the existence of other spirilla morphologically and cultu¬
rally similar, the serum reactions are the only absolutely positive dif¬
ferential criteria.

1 Abel und Claussen, Cent. f. Bakt., xvii, 1895.

586

PATHOGENIC MICROORGANISMS

For isolation of the bacteria from water, it is, of course, necessary
to use comparatively large quantities. Fliigge 1 and Bitter advise the
distribution of about a liter of water in ten or twelve Erlenmeyer flasks.
To each of these they add 10 c.c. of sterile pepton-salt solution (pepton
ten per cent, NaCl five per cent). After eighteen hours at 37.5° C. the
surface growths in these flasks are examined both microscopically and
culturally as before.

Biological Considerations. — The cholera spirillum is aerobic and
facultatively anaerobic. It does not form spores. The optimum tem¬
perature for its growth is about 37.5° C. It grows easily, however, at a
temperature of 22° C. and does not cease to grow at temperatures as
high as 40°. Frozen in ice, these bacteria may live for about three
or four days. Boiling destroys them immediately. A temperature of

Fig. 127. Fig. 128.

Fig. 127. — Cholera Spirillum. Stab Culture in Gelatin, three days old.

Fig. 128. — Cholera Spirillum. Stab Culture in Gelatin, six days old. (After
Frankel and Pfeiffer.)

60° C. kills them in an hour. In impure water, in moist linen, and in
food stuffs, they may live for many days. Associated with sapro¬
phytes in feces and other putrefying material, and wherever active
acid formation is taking place, they are destroyed within several days.
Complete drying kills them in a short time. The common disin¬
fectants destroy them in weak solutions and after short exposures
(carbolic acid, five-tenths per cent in one-half hour; bichlorid of
mercury, 1 : 100,000 in ten minutes; mineral acids, 1 : 5,000 or 10,000
in a few minutes).2

Pathogenicity.— Cholera is essentially a disease of man. Endemic in
India and other Eastern countries, it has from time to time epidemically
invaded large territories of Europe and Asia, not infrequently assuming

1 Fliigge , Zeit. f. Hyg., xiv, 1893.

2 F orster, Hyg. Rundschau, 1893.

ASIATIC CHOLERA AND THE CHOLERA ORGANISM

587

pandemic proportions and sweeping over almost the entire earth.1 Five
separate cholera epidemics of appalling magnitude occurred during
the nineteenth century alone; several of these, spreading from India to
Asia Minor, Egypt, Russia, and the countries of Central Europe, reached
even to North and South America. The last great epidemic began about
1883, traveled gradually westward, and in 1892 reached Germany where
it appeared with especial virulence in Hamburg, and thence, fol¬
lowing the highways of ocean commerce, entered America and Africa.
During this epidemic in Russia alone 800,000 people fell victims to the
disease.

In man the disease is contracted by ingestion of cholera organisms
with water, food, or any contaminated material. The disease is essen¬
tially an intestinal one. The bacteria, very sensitive to an acid reaction,
may often, if in small numbers, be checked by the normal gastric secre¬
tions. Having once passed into the intestine, however, they proliferate
rapidly, often completely outgrowing the normal intestinal flora. Fatal
cases, at autopsy, show extreme congestion of the intestinal walls.
Occasionally ecchymosis and localized necrosis of the mucosa may be
present and swelling of the solitary lymph-follicles and Peyer’s
patches. Microscopically the cholera spirilla may be seen to have
penetrated the mucosa and to lie within its deepest layers close
to the submucosa. The most marked changes usually take place
in the lower half of the small intestine. The intestines are filled
with the characteristically fluid, slightly bloody, or “rice-water”
stools, from which often pure cultures of the cholera vibrio can
be grown. The microorganisms can be cultivated only from the
intestines and their contents, and the parenchymatous degenera¬
tions taking place in other organs must be interpreted as being
purely of toxic origin.

In animals, cholera never appears as a spontaneous disease. Nikati
and Rietsch 2 have succeeded in producing a fatal disease in guinea-pigs
by opening the peritoneum and injecting cholera spirilla directly into
the duodenum. Koch 3 succeeded in producing a fatal cholera-like
disease in animals by introducing infected water into the stomach
through a catheter after neutralization of the gastric juice with sodium
carbonate. At the same time, he administered opium to prevent active
peristalsis. A method of infection more closely analogous to the infec-

1 Hirsch, “Handb. d. histor.-geogr. Path./’ 1881.

2 Nikati und Rietsch, Deut. med. Woch., 1884.

3 Koch, Deut. med. Woch., 1885.

588

PATHOGENIC MICROORGANISMS

tion in man was followed by Metchnikoff,1 who successfully produced
fatal disease in young suckling rabbits by contaminating the maternal
teat.

Subcutaneous inoculation of moderate quantities of cholera spirilla
into rabbits and guinea-pigs rarely produces more than a temporary
illness. Intraperitoneal inoculation, if in proper quantities, generally
leads to death. It will be remembered that when working with intra¬
peritoneal cholera inoculations the phenomenon of bacteriolysis was
discovered by Pfeiffer.2

Different strains of cholera spirilla vary greatly in their virulence.
The virulence of most of them, however, can be enhanced by repeated
passages through animals. Most of our domestic animals enjoy consid¬
erable resistance against cholera infection, though under experimental
conditions successful inoculations upon dogs, cats, and mice have been
reported. Doves are entirely insusceptible.3

Hygienic Considerations. — The cholera spirillum leaves the body of
the infected subject with the defecations only. Infection takes place,
so far as we know, only by way of the mouth. From these two facts it
follows that the chief source of danger for a community lies in infection
of its water supply. As a matter of fact the bacteria have been fre¬
quently found in the wells, lakes, rivers, and harbors of afflicted terri¬
tories, and in several cases it has been possible to define the limits of
an epidemic almost precisely by the distribution of the contaminated
water supply. A classic example of this is that of the Hamburg epi¬
demic, during which Altona, a town as close to Hamburg as Brooklyn
is to New York, with unrestricted interurban traffic but with separate
water supply, was almost spared, while Hamburg itself was undergoing
one of the most virulent epidemics of its history. It has been statistically
noted, moreover, chiefly by Koch, that cholera in its spread not infre¬
quently follows the water courses. Apart from infection through the
water supply, cholera may be transmitted directly or indirectly by con¬
tact with contaminated linen, bedclothes, etc., the organism being con¬
veyed to the mouth by the fingers, or by infected food. Epidemics due
to this mode of infection alone, however, are apt to be more narrowly
localized and more sporadic in their manifestations. It is probable that
this mode of infection is of great importance in countries where the disease

1 Metchnikoff, Ann. d. l’inst. Pasteur, 1894 and 1896.

2 Pfeiffer, loc. cit.

3 Pfeiffer und Nocht, Zeit. f. Hyg., vii, 1889.

ASIATIC CHOLERA AND THE CHOLERA ORGANISM

589

is endemic, but its significance in producing epidemics is limited owing to
the fortunately low resistance of the spirillum to desiccation. The
sudden appearance of cholera in a place far distant from the seat of a
prevalent epidemic may be explained by the occasional presence of
cholera spirilla in the dejecta of convalescents as late as two or three
weeks after apparent recovery from the disease and consequent release
from quarantine.

Cholera Toxin. — The absence of the cholera spirilla from the in¬
ternal organs of fatal cases, in spite of the severe general symptoms
of the disease, points distinctly to the existence of a strong poison pro¬
duced in the intestine by the microorganisms and absorbed by the
patient. It was in this sense, indeed, that Koch first interpreted the
clinical picture of cholera. Numerous investigations into the nature of
these toxins have been made, the earlier ones defective in that definite
identification of the cultures used for experimentation were not carried
out.

Pfeiffer,1 in 1892, was able to show that filtrates of young bouillon
cultures of cholera spirilla were but slightly toxic, whereas the dead
bodies of carefully killed agar cultures were fatal to guinea-pigs even in
small quantities. In consequence, he regarded the cholera poison as
consisting chiefly of an endotoxin.2 The opinion as to the endotoxic
nature of the cholera poison is not, however, shared by all workers.
Metchnikoff, Roux, and Salimbeni,3 in 1896, succeeded in producing
death in guinea-pigs by introduction into their peritoneal cavities of
cholera cultures enclosed in celloidin sacs. Brau and Denier,4 and,
more recently, Kraus,5 claim that they have succeeded not only in
demonstrating a soluble toxin in alkaline broth cultures of cholera
spirilla, but in producing true antitoxins by immunization with such
cultures. It appears, therefore, that the poisonous action of the cholera
organisms may depend both upon the formation of true secretory toxins
and upon endotoxins. Which of these is paramount in the produc¬
tion of the disease can not be at present definitely stated. In favor
of the great importance of the endotoxic elements is the failure,
thus far, to obtain successful therapeutic results with supposedly
antitoxic sera.

1 Pfeiffer, Zeit. f. Hyg., xi, 1892.

2 Pfeiffer und Wassermann, Zeit. f. Hyg., xiv, 1893.

3 Metchnikoff, Roux, et Salimbeni, Ann. de l’inst. Pasteur, 1896.

4 Brau et Denier, Comptes rend, de l’acad. des sci., 1906.

5 R, Kraus, Cent. f. Bakt., 1906.

590

PATHOGENIC MICROORGANISMS

Cholera Immunization. — One attack of cholera confers protection
against subsequent infection. Active immunization of animals may
be accomplished by inoculation of dead cultures, or of small doses of
living bacteria. In the serum of immunized animals specific bacterio¬
lytic and agglutinating substances are found. The discovery of bacte¬
riolytic immune bodies, in fact, was made by means of cholera spirilla.
Both the bacteriolysins and the agglutinins, because of their specificity,
are of great importance in making a bacteriological diagnosis of true
cholera organisms.

Protective inoculation of man has been variously attempted by
Ferran 1 and others. Experiments on a large scale were done, more re¬
cently, by Haffkine,2 who succeeded in producing an apparently dis¬
tinct prophylactic immunization by the subcutaneous inoculation of
dead cholera cultures. Similar immunization with bacterial filtrates
has been attempted by Bertarelli.3

CHOLERA-LIKE SPIRILLA

The biological group of the vibriones, to which the cholera spirillum
belongs, is a large one, numbering probably over a hundred separate
species. Most of these are of bacteriological importance chiefly because
of the difficulties which they add to the task of differentiation, for while
some of them simply bear a morphological resemblance to the true
cholera vibrio, others can be distinguished only by their serum reac¬
tions and pathogenicity for various animals. Additional difficulty,
too, is contributed by the fact that within the group of true cholera
organisms occasional variations in agglutinability and bacteriolytic
reactions may exist. Certain strains, too, the six El Tor cultures
isolated by Gottschlich, while in every respect similar to true cholera
spirilla, are considered as a separate sub-species by Kraus,4 because of
their ability to produce hemolytic substances, a function lacking in
other cholera strains.

Spirillum Metchnikovi. — This spirillum was discovered by Gamaleia 5
in the feces and blood of domestic fowl, in which it had caused an in¬
testinal disease. Morphologically and in staining reactions it is identical

1 Ferran, Comptes rend, de l’acad. des sciences, 1885.

2 Haffkine, Bull, med., 1892.

3 Bertarelli, Deut. med. Woch., 33, 1904.

4 Kraus, Kraus und Levaditi, “Handbuch,” vol. i, p. 186.

6 Gamaleia, Ann. de l’inst. Pasteur, 1883,

ASIATIC CHOLERA AND THE CHOLERA ORGANISM

591

with Spirillum cholerae asiaticse. It possesses a single polar flagellum,
and is actively motile. Culturally it is identical with Vibrio cholerse
except for slightly more luxuriant growth and more rapid fluidification
of gelatin. It gives the cholera-red reaction in pepton media.

It is differentiated from the cholera vibrio by its power to produce
a rapidly fatal septicemia in pigeons after subcutaneous inoculation of
minute quantities.1 It is much more pathogenic for guinea-pigs than
the cholera vibrio. It is not subject to lysis or agglutinated by cholera
immune sera.

Spirillum Massaua. — This organism was isolated at Massaua by
Pasquale 2 in 1891 from the feces of a clinically doubtful case of cholera.
Culturally and morphologically it is much like the true cholera vibrio,
but in pathogenicity is closer to Spirillum Metchnikovi, in that small
quantities produce septicemia in birds. It possesses four flagella.
It does not give a specific serum reaction with cholera immune
serum.

Spirillum of Finkler-Prior.3 — Isolated by Finkler and Prior from the
feces of a case of cholera nostras. Morphologically it is like the true
cholera spirillum, though slightly larger and less uniformly curved.
Culturally it is much like the cholera vibrio, but grows more rapidly
and thickly upon the usual media. It does not give the cholera-red
reaction, nor does it give specific serum reactions with cholera im¬
mune serum.

Spirillum Deneke.4 — A vibrio isolated by Deneke from butter. Much
like that of Finkler-Prior. It does not give the cholera-red reaction.

1 Pfeiffer und Nocht, Zeit. f. Hyg., vii, 1889.

2 Pasquale , Giorn. med. de r. eserc. ed. R. Marina, Roma, 1891.

3 Finkler und Prior, Erganz. Hefte, Cent. f. allg. ges. Phys., 1884.

4 Deneke, Deut. med. Woch., iii, 1885.

CHAPTER XLIII

DISEASES CAUSED BY SPIROCHETES (TREPONEMATA)

The microorganisms known as spirochsetes are slender, undulating,
corkscrew-like threads which show definite variations both structurally
and culturally from the bacteria as a class. Most important among
them are the spirochsete of relapsing fever, Spirochsete pallida of
syphilis, the spirillum of Vincent, Spirochsete refringens, Spirillum
gallinarum, a microorganism which causes disease in chickens,
Spirochsete anserina, which causes a similar condition in geese, and
several species which have been found as parasites, both in animals
and in man, without having definite etiological connection with disease.

The classification of these various species in one group is rather more
a matter of convenience than one of scientific accuracy, since our knowl¬
edge of them is not far advanced, and our inability to cultivate almost
all of them has not permitted their detailed biological study. Formerly
many of these organisms were regarded as bacteria belonging to the gen¬
eral group of the spirilla. Recently Schaudinn,1 the discoverer of the
syphilis spirochsete,has claimed, upon the basis of a careful morphological
study, that many of these forms are actually protozoa. He based this
claim upon the observation that stained preparations often showed undu¬
lating membranes extending along the long axis of the microorganisms
and that definite nuclear structures were demonstrable. This observer
also claimed that many of the spiral forms reproduce by cleavage along
the longitudinal axis. Other observers have not agreed with this view,
Laveran,2 Novy and Knapp,3 and others asserting that their own obser¬
vations indicate a close relationship of these microorganisms to the true
bacteria. Whatever the final conclusion may be, the question is more
or less an academic one, in that our ideas as to the exact line of division
between the unicellular animals and the unicellular plants is not by any
means founded upon a sound basis. In common with the bacteria, most

1 Schaudinn, Arb. a. d. kais. Gesundheitsamt, 1904.

2 Laveran, Comptes rend, de Tacad. des sci., 1902 and 1903.

3 Novy and Knapp, Jour, of Infec. Dis., 3, 1906.

592

DISEASES CAUSED BY SPIROCHETES

593

of these microorganisms have the power of multiplication by transverse
fission. They possess flagella and, in the case of some of them at least,
definite immune bodies can be demonstrated in the serum of infected
subjects similar to those produced by bacteria during infection. The
undulating membranes and the definite differentiation between nucleus
and cytoplasm claimed for them by some observers have not been uni¬
formly confirmed, and their similarity to the trypanosomes has not
therefore been established. On the other hand, none of these micro¬
organisms has so far been successfully cultivated upon artificial media,
with the exception of the spirilla which occur in Vincent’s angina. For
some of the diseases caused by this class of parasites, moreover, trans¬
mission by an intermediate host, in which the spirilla undergo multipli¬
cation, has been definitely shown, a fact which corresponds with the
conditions observed in many protozoan infections. Upon a careful re¬
view of these various data, it seems to be fully justified, on the basis
of our present knowledge, to group these microorganisms, as Kolle and
Hetsch 1 have done, in a class between bacteria and protozoa.

The terms spirochsete and spirillum have been indiscriminately used.
In the original classification of Migula the difference between the two
groups was based upon the rigidity of the cell body in the case of the
spirilla and the sinuous or flexible nature of the cell in the case of the
spirochaetse. Although the term spirillum is still colloquially used for
some members of this group, merely because of past usage, it would be
better to speak of all the microorganisms here grouped together by the
term “spirochsetes.”

SYPHILIS AND SPIROCH^ITA PALLIDA

(: Treponema pallidum)

The peculiar manifestations of syphilis, its mode of transmission,
and the fact that its primary lesion was always located at the point
of contact with a preceding case, have always stamped it as unques¬
tionably infectious in nature. Until very recently the microorgan¬
ism which gives rise to syphilis was unknown. Many bacteriologists
had attacked the problem and many microorganisms for which defi¬
nite etiological importance was claimed had been described. Most of
these announcements, however, aroused little more than a sensational
interest and received no satisfactory confirmation. A bacillus described

1 Kolle und Hetsch , “Die experimentelle Bakt.,” Berlin, 1906.
39

594

PATHOGENIC MICROORGANISMS

by Lustgarten 1 in 1884 seemed, for a time, to have solved the mystery.
The Lustgarten bacillus was an acid-fast organism very similar to
Bacillus tuberculosis, and found by its discoverer in a large num¬
ber of syphilitic lesions. The observation, at first, aroused much interest
and received some confirmation. Later extensive investigations, how¬
ever, failed to uphold the etiological relationship of this bacillus to the
disease and practically identified it with the smegma bacillus, so often
a saprophyte upon the mucous membranes of the normal genitals.

In 1905, Schaudinn,2 a German zoologist, working in collaboration
with Hoffmann, investigated a number of primary syphilitic indurations
and secondarily enlarged lymph nodes, and in both lesions discovered a
spirochsete similar to, but easily distinguished from, the spirochsetes

Fig. 129. — Spiroch^eta pallida. Smear preparation from chancre stained

by the india-ink method.

already known. He failed to find similar microorganisms in uninfected
human beings.

The microorganism described by him as “Spirochseta pallida” is an
extremely delicate undulating filament measuring from four to ten micra
in length, with an average of seven micra, and varying in thickness from
an immeasurable delicacy to about 0.5 of a micron. It is thus distinctly
smaller and more delicate than the spirochsete of relapsing fever. Ex¬
amined in fresh preparations it is distinctly motile, its movements con¬
sisting in a rotation about the long axis, gliding movements backward
and forward, and, occasionally, a bending of the whole body. Its con¬
volutions, as counted by Schaudinn, vary from 3 to 12 and differ from
those observed in many other spirochsetes by being extremely steep, or,
in other words, by forming acute, rather than obtuse, angles. The ends
of the microorganism are delicately tapering and come to a point. In

1 Lustgarten, Wien. med. Woch., xxxiv, 1884.

2 Schaudinn und Hoffmann , Arb. a. d. kais. Gesundheitsamt, 22, 1905.

DISEASES CAUSED BY SPIROCHETES

595

his first investigations, Schaudinn was unable to discover flagella and
believed that he saw a marginal undulating membrane similar to that
noticed in the trypanosomes. Later observations by this observer, as
well as by others, revealed a delicate flagellum at each end, but left the
existence of an undulating membrane in doubt. Uncertain, in his later
investigations, whether the microorganisms described by him could
scientifically be classified with the spirochaete proper, Schaudinn sug¬
gested the name of “Treponema pallidum.”

In the same preparations in which Spirochseta pallida was first
seen, other spirochaetes were present, which were easily distinguished
from the former by their coarser contours, their flatter and fewer undula¬
tions, their more highly retractile cell bodies, and, in stained prepara¬
tions, their deeper color. These microorganisms were not found
regularly, and were interpreted merely as fortuitous and unimportant
companions. To them Schaudinn gave the name of “Spirochseta re-
fr ingens.”

The epoch-making discovery of Schaudinn and Hoffmann was soon
confirmed by many observers, and the etiological relationship of Spiro¬
chseta pallida to syphilis may now be regarded as an accepted fact.
Although our inability to cultivate the microorganism has made it
impossible to carry out Koch’s postulates, nevertheless indirect evi¬
dence of such a convincing nature has accumulated that no reasonable
doubt as to its caustive importance can be retained. The spirochaetes
have been found constantly present in the primary and secondary
lesions of all carefully investigated cases, and, so far, have invariably
been absent in subjects not afflicted with syphilis.

Schaudinn himself, not long after his original communication, was
able to report seventy cases of primary and secondary syphilis in which
these microorganisms were found. Spitzer 1 found them constantly
present in a large number of similar cases. Sobernheim and Tomas-
czewski2 found the spirochaetes in fifty cases of primary and secondary
syphilis, but failed to find them in eight tertiary cases. Mulzer,3 who
found the microorganisms invariably in twenty cases of clinical syphilis,
failed to find them in fifty-six carefully investigated non-syphilitic sub¬
jects. The voluminous confirmatory literature which has accumulated
upon the subject can not here be reviewed. The presence of these
spirochaetes in the blood at certain stages of the disease has been demon-

1 Spitzer, Wien. klin. Woch., 1905.

2 Sobernheim und Tomasczewski, Munch, med. Woch., 1905.

3 Mulzer, Berl. klin. Woch-, 1905, and Archiv f, Dermat. u. Syph,, 79, 1906.

596

PATHOGENIC MICROORGANISMS

strated by Bandi and Simonelli1 who found them in the blood taken
from the roseola spots, and by Levaditi and Petresco 2 who found them
in the fluid of blisters produced upon the skin.

In tertiary lesions the spirochetes have been found less regularly
than in the primary and secondary lesions, but positive evidence of their
presence has been brought by Tomasczewski,3 Ewing,4 and others who
succeeded in demonstrating them in gummata. Noguchi and Moore5
have recently found the Spirochseta pallida in the brain of patients dead
of general paresis.

In congenital syphilis, many observers have found Spirochseta
pallida in the lungs, liver, spleen, pancreas, and kidneys, and, in isolated
cases, in the heart muscle. The organisms were always present in large
numbers and practically in pure culture. These results more than any
others seem to furnish positive proof of the etiological relationship be¬
tween the spirochaete and the disease.

Demonstration of Spirochseta pallida. — In the living state the
spirochsetes have been observed in the hanging drop or under a cover-
slip rimmed with vaseline. It is extremely important, in preparing such
specimens from primary lesions or from lymph glands, to obtain the
material from the deeper tissues, and thus as uncontaminated as possible
by the secondary infecting agents present upon the surface of an ulcer,
and also as free from blood as possible. An ordinary microscope
and condenser may be used, provided that the light is cut down con¬
siderably by means of the iris diaphragm. This method is, however,
difficult and uncertain. It is better to employ a special device known
as a “ condenser for dark-field illumination” (Dunkel-Kammer-
Beleuchtung) . This apparatus is screwed into the place of the Abbe
condenser. The preparation is made upon a slide and covered with a
cover-slip as usual. A drop of oil is then placed upon the upper sur¬
face of the dark chamber and the slide laid upon it so that an even
layer of oil, without air-bubbles, intervenes between the top of the dark
chamber and the bottom of the slide. The preparation is then best
examined with a high-power dry lens. An arc light furnishes the most
favorable illumination. In such preparations the highly refractive cell-

1 Bandi und Simonelli , Cent. f. Bakt., 40, 1905.

2 Levaditi et Petresco , Presse med., 1905.

3 Tomasczewski, Munch, med. Woch., 1906.

4 Ewing, Proc. N. Y. Path. Soc., N. S., 5, 1905.

5 Noguchi and Moore, Jour. Exp. Med., xvii, 1913.

DISEASES CAUSED BY SPIROCHETES

597

bodies stand out against the black background, and the motility of the
organisms may be observed.1

The dark-field condenser is without question the easiest method of
finding the Spirochseta pallida. Its use is easily learned and the appara¬
tus is sufficiently cheap so that it lends itself to the use of the clinic and
the office. With very little practice it is possible to detect the spiro¬
chete in suspension if care is taken that not too much blood or other
solid particles are mixed with the preparation. Should it be impossible
to obtain the material scraped from syphilitic lesions in a sufficiently
dilute condition it is best to emulsify it in a drop or two of human
ascitic fluid.

Examination in Smears. — The Spirochaeta pallida can not be
stained with the weaker anilin dyes, and even more powerful dyes, such
as carbol-fuchsin and gentian-violet, give but a pale and unsatisfactory
preparation. The staining method most commonly used is the one
originally recommended by Schaudinn and Hoffmann. This depends
upon the use of Giemsa’s azur-eosin stain employed in various modi¬
fications. The most satisfactory method of applying this solution is
as follows:

Make smears upon slides or cover-slips, if possible from the depth of
the lesions, as free as possible from blood.

Fix in methyl alcohol for ten to twenty minutes and dry.

Cover the preparation with a solution freshly prepared as follows:

Distilled water . 10 c.c.

Potassium carbonate 1 : 1,000 . 5-10 gtt.

Add to this:

Giemsa’s solution ( fur Romanowski Fdrbung ) . 10-12 gtt.

This staining fluid is left on for one to four hours, preferably in a
moist chamber.

Wash in running water.

Blot.

By this method Spirochseta pallida is stained characteristically
with a violet or reddish tinge.

A rapid and convenient method for staining such smears consists in
the use of azur I and eosin in aqueous solutions as recommended by

1 For a critical summary of the various methods of dark-field illumination, the
reader is referred to an article by Siedentopf, Zeit. f. wiss. Mikrosc., xxv, 1908.

598

PATHOGENIC MICROORGANISMS

Wood (see section on Staining, page 109). The smears are fixed in
methyl alcohol as before and are then flooded with the azur I solution.
The eosin solution is then dropped on the preparation until an iridescent
pellicle begins to form. Satisfactory preparations may be obtained by
this method after ten or fifteen minutes of staining.

Goldhorn1 has prepared a stain which gives excellent results and is
extremely rapid. He describes the preparation of his staining fluid as
follows:

One gram of lithium carbonate is dissolved in 200 c.c. of water.
To this are added 2 grams of methylene-blue and the mixture is care¬
fully heated, filtered, and divided into two equal parts. To one of
these parts is added 5 per cent acetic acid until acid to litmus. The
two parts are then mixed, and a weak solution of eosin is added until
a pale blue color is obtained. The fluid is then allowed to stand for
a day and the precipitate which is formed is filtered off and al¬
lowed to dry without heat. One gram of this precipitate is dissolved
in 100 c.c. of methyl alcohol. This stain is applied for five minutes or
longer after methyl-alcohol fixation. Excellent results are usually
obtained with this stain, but variations due to the difficulty of manu¬
facturing it make it less reliable than the two methods previously
mentioned.

Recently a rapid and extremely simple and reliable method for the
demonstration of Spirochseta pallida in smears, by the use of India ink,
has been described.

Smears are prepared in the following way: A drop of the fluid
squeezed out of the syphilitic lesion, as free as possible from blood cells,
is mixed, on a slide, with a drop of India ink (best variety is “ Chin chin ”
Gunther- Wagner Liquid Pearl ink), and the mixture smeared with the
edge of another slide as in making blood smears. When the smear dries,
which takes about a minute, it may be immediately examined with an
oil-immersion lens. The organisms are seen unstained on a black back¬
ground. (See Fig. 129, p. 594.)

Demonstration of Spirochetes in Tissues. — Ordinary his¬
tological staining methods do not reveal the spirochsetes in tissue
sections. It is customary, therefore, to employ some modification of
Cajafis silver impregnation. The technique most commonly employed
is that known as Levaditi’s method ,2 which is carried out as follows:

1 Goldhorn, Proc. N. Y. Path. Soc., N. S., 5, 1905.

2 Levaditi, Comptes rend, de la soc. de biol., 59, 1905.

DISEASES CAUSED BY SPIROCHETES

599

The fresh tissue is cut into small pieces which should not be thicker
than 2 to 4 millimeters.

Fix in ten-per-cent formalin (four-per-cent formaldehyde) for twenty-
four hours.

Wash in water.

Dehydrate in 96-per-cent alcohol twenty-four hours.

Wash in water.

Place in a 3-per-cent silver-nitrate solution at incubator temperature
(37.5° C.) and in the dark for three to five days.

Wash in water for a short time.

Place in the following solution (freshly prepared) :

Pyrogallic acid . 2-4 grams.

Formalin . 5 CiC<

Distilled water . 100 “

Leave in this for twenty-four to forty-eight hours at room tem¬
perature.

Wash in water.

Dehydrate in graded alcohols.

Embed in paraffin and cut thin sections.

The sections may be examined without further staining, or, if de¬
sired, may be weakly counterstained with Giemsa’s solution or hema¬
toxylin.

A modification of this method which has been much recommended is
that of Levaditi and Manouelian.1 The directions given by these
authors are as follows:

Fix in formalin as in previous method.

Dehydrate in 96-per-cent alcohol twelve to twenty-four hours.

Wash in distilled water.

Place in a 1 -per-cent silver-nitrate solution to which 10 per cent of
pyridin has been added just before use.

Leave in this solution for two to three hours at room temperature
and from four to six hours at 50° C. approximately.

Wash rapidly in 10-per-cent pyridin.

Place in a solution containing 4 per cent of pyrogallic acid to which 10
per cent of C. P. acetone, and 15 per cent (per volume) of pyridin
have been added just before use. Leave in this solution two to three
hours.

1 Levaditi et Manouelian , Comptes rend, de la soc. de biol., 60, 1906.

600

PATHOGENIC MICROORGANISMS

Wash in water, dehydrate in graded alcohols, and embed in paraffin
by the usual technique.

Examined after treatment by either of these methods, the spiro-
chsetes appear as black, untransparent bodies lying chiefly extracellu-
larly. They are characteristically massed about the blood-vessels of the
organs and only exceptionally seem to penetrate into the interior of
the parenchyma cells.

Attempts at cultivating Spirochseta pallida were at first unsuccessful.
Recently Schereschewsky 1 has reported that he has succeeded in ob¬
taining multiplication of the organisms on artificial media as

follows : Sterile horse serum in centri¬

fuge tubes was coagulated at 60° C.
until it assumed a jelly-like consist¬
ency. It was then placed in the in¬
cubator at 37.5° C. for three days be¬
fore being used. The cultures were
planted by snipping off a small piece of
tissue from a syphilitic lesion, dropping
it into such a tube, and causing it to
sink to the bottom by means of centri-
fugalization. The tube was then tightly
stoppered with a cork. In such an¬
aerobic serum cultures Schereschewsky
claims to have grown the organisms for several generations, though
not in pure culture.

Miihlens also obtained growth of Spirochseta pallida in horse serum
agar by a method which is very similar to that of Schereschewsky.
None of these observers, however, succeeded in carrying out Koch’s
postulates with the cultures they obtained. This has recently been
done in the splendid investigations of Noguchi. Noguchi2 began his
work upon the Spirochseta pallida in 1910 and 1911. His first success¬
ful cultivations were made from the syphilis-infected testicles of
rabbits, and after many unsuccessful attempts, with slightly varying
media and technique, he finally succeeded in the following way: He
prepared tubes (20 cm. high and 1.5 cm. wide), containing 10 c.c. of a
serum-water made of distilled water, three parts; and horse, sheep, or
rabbit serum, one part. These were sterilized by the fractional method
in the usual way (15 minutes each day). Into them was then placed a

Fig. 130. — Spirochseta pal¬
lida. Spleen, congenital syphilis.
(Levaditi method.)

1 Schereschewsky, Deut. med. Woch., N. S., xix and xxix, 1909.

2 Noguchi, Jour. Exp. Med., xiv, 1911; xvii, 1913.

DISEASES CAUSED BY SPIROCHETES

601

small piece of sterile rabbit kidney or testicle and a bit of the testicle of a
syphilitic rabbit, in which many spirochaetes were present. The fluid
was then covered with sterile paraffin oil and placed in an anaerobic jar.
After 10 days at 33.5° C. the spirochaetes had multiplied considerably, in
all but one case, together with bacteria. He obtained pure cultures
from these initial cultivations after much difficulty, by a number of
methods. At first he succeeded only by allowing the spirochaetes to
grow through Berkefeld filters, which they did on the fifth day. A
better method more recently adopted by him consists in preparing
high tubes of three parts of very slightly alkaline or neutral agar to which
a piece of sterile tissue has been added. These tubes are then in¬
oculated from the impure cultures with a long pipette. Close to the

Fig. 131. — Spiroch^eta pallida. Liver, congenital syphilis.

(Levaditi method.)

tissue and along the stab the spirochaetes and bacteria will grow and,
after about ten days to two weeks, the spirochaetes will have wandered
away from the stab and will be visible as hazy colonies. They can
then be fished, after cutting the tubes, and directly transplanted to
other serum-agar-tissue tubes prepared as before, and eventually will
grow in pure culture. By this method Noguchi has also cultivated
pure cultures from lesions in monkeys, and has produced lesions both in
rabbits and monkeys with his pure cultures. He has thus for the first
time carried out Koch’s postulates with syphilis and established beyond
the shadow of a doubt the etiological significance of Spirochaeta pallida
in syphilis.

Animal Pathogenicity. — Until very recently, all experimental inocu¬
lation of animals was unsuccessful. During the year 1903 Metchnikoff
and Roux1 finally succeeded in transmitting the disease to monkeys.
The monkey first used by these observers was a female chimpanzee.
At the point of inoculation, the clitoris, there appeared, twenty-six days

1 Metchnikoff et Roux, Ann. de l’Inst. Pasteur, 1903, 1904, and 1905.

602

PATHOGENIC MICROORGANISMS

after inoculation, a typical indurated chancre, which was soon followed
by swelling of the inguinal glands. Fifty-six days after the inoculation
there appeared a typical secondary eruption, together with swelling of
the spleen and of the lymph nodes. Similar successful experiments were
made soon after this by Lassar.1 Soon after the experiments of Metch-
nikoff and Roux, successful inoculations upon lower monkeys (maca-
cus) were carried out by Nicolle.2 Since that time, it has been found
by various observers that almost all species of monkeys are susceptible.
Simple subcutaneous injection is not sufficient to produce a lesion.
The technique which has given the most satisfactory results consists
in the cutaneous implantation of small quantities of syphilitic tissue
obtained by excision or curetting of primary and secondary lesions. A
small pocket is made under the mucous membrane of the genitals or of
the eyebrows and the tissue placed in this under aseptic precautions.
The inoculation may be made directly from the human being, but can
also be successfully carried out from monkey to monkey for many
generations. Attempts at transmission from tertiary lesions have so far
been unsuccessful. The spirochsetes can be demonstrated both in the
primary lesions of the inoculated animal and in the secondarily enlarged
glands. The successful inoculation of rabbits with syphilis has been
recently performed by Bertarelli.3 He obtained ulcerative lesions by
inoculation upon the* cornea and into the anterior chamber of the eye
and was able to prove the syphilitic nature of these lesions by finding
the spirochsete within the tissue. In these animals, as well as in the
lower monkeys, the disease usually remains localized.

In 1907, Parodi showed that syphilitic lesions could be produced by
direct inoculation into the testicles of rabbits. This method of inocula¬
tion has been subsequently studied by many investigators, especially
by Uhlenhuth and Mulzer.4 It is the easiest method of obtaining
spirochsete in any quantity from lesions in man. The spirochsete-con-
taining lesions may be either excised or scraped as conditions permit
and rubbed up in a mortar with sterile sand, in a few centimeters of
sterile human ascitic fluid. This emulsion is then injected directly
into the substance of rabbit testicles. A swelling supervenes which
is often noticeable after two weeks, and is usually at its height in 5 to
7 weeks. At this time the testicle is much larger than normal, some-

1 Lassar, Berl. klin. Woch., xl, 1903.

2 Nicolle, Ann. de l’inst. Pasteur, 1903.

3 Bertarelli, Cent. f. Bakt., xli, 1906.

4 Uhlenhuth und Mulzer, Arb. a. d. k. Gsndhtsamte., xxxiii, 1909.

DISEASES CAUSED BY SPIROCHETES

603

times evenly swollen and sometimes nodular, and of a firm elastic con¬
sistency. When taken out at castration it oozes a sticky fluid, both
from testicle and the tunica, which is rich in actively motile spirochsetes.
By continuous transinoculation from one rabbit to another such a strain
can be indefinitely carried along. It can be inoculated from rabbits
to monkeys and vice versa. This method as well as Noguchi’s cul¬
tivations have opened a new era of spirochsete investigation. It is
stated by some observers that intravenous inoculation of rabbits may
be followed by localization in the testis and occasionally gummatous
infections in other parts of the body have been induced after such
inoculation by Uhlenhuth, Mulzer, and others.

Immunization in Syphilis. — It is a well-known fact observed by
clinicians that one attack of syphilis usually protects the infected in¬
dividual at least from the development of another chancrous lesion.
That this immunity develops quite rapidly was shown by Metchnikoff
and Roux, who found that reinfection of a monkey was possible if
attempted within two weeks of the first inoculation, but was unsuccess¬
ful if delayed beyond this period.

On the basis of this knowledge as to the actual development of an
immunity, Metchnikoff,1 Finger and Landsteiner,2 and others have
made extensive attempts to devise some method of active immunization.
Working along the line of Pasteur’s original attenuation of virus, these
observers attempted to attenuate the syphilitic virus by repeated pas¬
sage through monkeys. These experiments were entirely without suc¬
cess, the last-mentioned observers finding absolutely no attenuation
after twelve generations of monkey inoculation.

The study of rabbits has permitted a little more definite formulation
of our ideas on syphilis immunization. Bertarelli and others have
shown that the production of a syphilitic lesion on the cornea of
one eye does not protect against an inoculation subsequently done
on the other eye. Apparently rabbits that have been inoculated
with spirochsete material and that have not developed syphilitic
disease can be successfully inoculated on subsequent attempts. The
offspring of female rabbits with syphilis of the cornea are, according
to Muhlens, not immune.

There is no evidence so far that specific therapy or treatment with
spirochsete material has had favorable influence upon the disease except

1 Metchnikoff, Arch. gen. de med., 1905.

2 Finger und Landsteiner, Sitzungsber. d. Wien. Akad. d. Wiss., 1905.

604

PATHOGENIC MICROORGANISMS

in isolated cases reported by Uhlenhuth and Mulzer. Chemotherapy
has had results analogous to those obtained in man.1

Attempts at passive immunization have been entirely without
success.

The occurrence of a Wassermann reaction was formerly supposed to
indicate the existence of specific syphilitic antibodies in the serum of
patients. Our more recent information regarding this reaction seems to
show that it depends upon the presence in the serum of syphilitic
patients of substances produced indirectly because of the presence of
syphilitic infection. It may be a relative increase of globulins or, as
Schmidt has suggested, a change in the physical state of the globulins
or other substances present in the serum. At any rate it has been found
that the fixation of complement in the Wassermann reaction does not
depend upon the occurrence of a specific antigen-antibody reaction.
In the first place the antigens most commonly used, and successfully
so, in the Wassermann reactions, are non-specific lipoidal extracts of
normal organs. This alone would show that the specific spirochsetal
substance has no relation to the reaction. Again it has been demon¬
strated that extracts of cultures of the Spirochseta pallida as well as ex¬
tractions from the testes of syphilitic rabbits do not furnish an antigen
suitable for the Wassermann reaction. This has followed especially
from the work of Noguchi,2 of Craig and Nichols,3 and of others. This
forms a corollary to the other experiments previously mentioned and
shows that, whatever the Wassermann reaction may be (and space does
not permit us to review the theories, especially since none are definitely
proven), it is not a specific complement fixation in the sense of Bordet
and Gengou. It must be admitted, therefore, that our knowledge of
syphilis immunity is in its infancy and that we know very little about
the systemic reactions which follow infection with the Spirochseta
pallida.

The fact that the syphilitic virus does not pass through a filter has
been demonstrated by Klingmuller and Baermann,4 who inoculated
themselves with filtrates from syphilitic material.

1 Von Prowazek, “Handbuch der pathogenen Protozoen,” i, 1912, Leipzig, Bartsch.

2 Noguchi , Jour. Am. Med. Assoc., 1912.

3 Craig and Nichols, Jour. Exp. Med., xvi, 1912.

4 Klingmuller und Baermann, Deut. med. Woch., 1904.

DISEASES CAUSED BY SPIROCHETES

605

THE SPIROCHETES OF RELAPSING FEVER

The microorganisms causing relapsing fever were first observed in
1873, by Obermeier,1 who demonstrated them in the blood of patients
suffering from this distinct type of fever. Since his time extensive

Fig. 132. — Spirochete of Relapsing Fever. (After Norris, Pappenheimer,

and Flournoy.)

studies by many other observers have proven beyond question the
etiological connection between the disease and the organisms.

Morphology and Staining. — The spirochsete of Obermeier is a delicate
spiral thread measuring from 7 to 9 micra in length (Novy), and about
1 micron in thickness. While this is its average size, it may, according
to some observers, be considerably longer than this; its undulations
varying from 4 to 10 or more in number. Compared with the red blood

1 Obermeier, Cent. f. d. med. Wiss., 11, 1873.

606

PATHOGENIC MICROORGANISMS

cells among which they are seen, the microorganisms may vary from
one-half to 9 or 10 times the diameter of a corpuscle. In fresh prepara¬
tions of the blood, very active corkscrew-like motility and definite lateral
oscillation are observed. In stained preparations no definite cellular
structure can be made out, the cell body appearing homogeneous, except
in degenerated individuals, in which irregular granulation or beading
has been observed. Flagella have been described by various observers.

Fig. 133. — Spirochete of Relapsing Fever. Citrated normal rat blood.
(After Norris, Pappenheimer, and Flournoy.)

Novy and Knapp1 believe that the organisms possess only one terminal
flagellum. Zettnow,2 on the other hand, claims to have demonstrated
lateral flagella by special methods of staining. Norris, Pappenheimer,
and Flournoy,3 in smears stained by polychrome methods, have described
long, filamentous tapering ends which they interpreted as bipolar,
terminal flagella, never observing more than one at each end. Spores
are not found.

Cultivation. — Innumerable attempts to induce these microorganisms
to multiply upon artificial media have been made. Novy and Knapp
succeeded in keeping the microorganisms alive and virulent in the

1 Novy and Knapp, Jour, of Infec. Dis., 3, 1906.

2 Zettnow , Deut. med. Woch., 32, 1906.

3 Norris, Pappenheimer, and Flournoy, Jour, of Inf. Dis., 3, 1906.

DISEASES CAUSED BY SPIROCHETES

607

original blood for as long as forty days, and call attention to the fact
that the length of time for which they may be kept alive depends to a
great extent upon the stage of fever at which the blood is removed from
the patient. They do not, however, believe that extensive multiplica¬
tion, or, in other words, actual cultivation, had taken place in their
experiments. Norris, Pappenheimer, and Flournoy, on the other hand,
have obtained positive evidence of multiplication of the spirochetes' in
fluid media. They obtained their cultures by inoculating a few drops
of spirochetal rat blood into 3 to 5 c.c. of citrated human or rat blood.
Smears made from these tubes, after preservation for twenty-four hours
at room temperature, showed the microorganisms in greater number

Fig. 134. — Spirochete of Relapsing Fever. (From preparation furnished

by Dr. G. N. Calkins.)

than in the original infected blood. A similar multiplication could be
observed in transfers made from these “first-generation” tubes to other
tubes of citrated blood. Attempts at cultivation for a third generation,
however, failed.

Noguchi 1 has lately successfully cultivated the spirochsete of Ober-
meier in ascitic fluid containing a piece of sterile rabbit's kidney and a
few drops of citrated blood under anaerobic conditions.

Four different, probably distinct varieties of spirochsete have been
described in connection with relapsing fever, all of which have been
cultivated by Noguchi by means of this method. The first is known as
the spirochsete of Obermeier mentioned above. Probably distinct

1 Noguchi , Jour. Exp. Med., xvii, 1913.

608

PATHOGENIC MICROORGANISMS

are the Spirochseta Duttoni, described by Dutton and Todd1 in 1905, the
Spirochseta Kochi, and the Spirochseta Novyi,2 the organism studied by
Norris and Flournoy and Pappenheimer, and regarded as a different
species by them.

Pathogenicity. — Inoculation with blood containing these spirochsetes
produces disease in monkeys, rats, and mice. Attempts to transmit
the disease experimentally to dogs, rabbits, and guinea-pigs have so
far been unsuccessful. The subcutaneous inoculation of monkeys is
followed after from two to four days by a rise of temperature which
occurs abruptly as is the case in the disease in man and which may last
several days. During this time the spirochsetes can be found in the
blood of the animals just as it is found in that of infected human beings.
The temperature subsides after a day or more, when it again rapidly
returns to normal. As a rule, the paroxysms are not repeated. Occa¬
sionally, however, two or three attacks may supervene before immunity
is established. In rats, an incubation time of from two to five days
occurs. At the end of this time the spirochsetes may be found in large
numbers in the blood, and the animals show symptoms of a severe
systemic infection. The attack lasts from four to five days, at the ehd
of which time the microorganisms again disappear. Occasionally even
in these animals relapses have been observed. Gross pathological
changes are not found, with the exception of an enlargement of the
spleen.

In man the disease caused by the spirochsete of Obermeier, commonly
known as relapsing fever, is common in India, Africa, and most of the
warmer countries. It has, from time to time, been observed epidemically
in Europe, especially in Russia, and a few epidemics have occurred in
the Southern United States. The disease comes on abruptly, beginning
usually with a chill accompanied by a sharp rise of temperature and gen¬
eralized pains. Together with the rise of temperature, which often ex¬
ceeds 104° F., there are great prostration and occasionally delirium. Early
in the disease the spleen becomes palpable and jaundice may appear.
The spirochetes are easily detected in the blood during the persistence
of the fever, which lasts usually from three to ten days. At the end
of this time the temperature usually drops as suddenly as it rose, and
the general symptoms rapidly disappear. After a free interval of
from one to three weeks a relapse may occur, which is usually less
severe and of shorter duration than the original attack. Two, three, or

1 Dutton and Todd , Brit. Med. Jour., 1905.

2 Novy and Fraenkel, cited from Noguchi.

DISEASES CAUSED BY SPIROCHETES

609

even four attacks may occur, but the disease is not very often fatal.
When patients do succumb, however, the autopsy findings are not
particularly characteristic. Apart from the marked enlargement of
the spleen, which histologically shows the changes indicating simple
hyperplasia, and a slight enlargement of the liver, no lesions are found.
The diagnosis is easily made during the febrile stage by examination of
a small quantity of blood under a cover-slip or in the hanging-drop
preparation.

Several types of relapsing fever have been described. In Africa the
disease has long been prevalent in many regions and the investigations
of Ross and Milne,1 Koch,2 Dutton and Todd,3 and others have brought

Fig. 135. — Spirochete of Dutton, African Tick Fever. (From prepara¬
tion furnished by Dr. G. N. Calkins.)

to light that many conditions occurring among the natives, formerly
regarded as malarial, are caused by a species of spirochete. Whether
or not the microorganisms observed in the African disease are exactly
identical with the spirochete observed by Obermeier is yet a question
about which several opinions are held. Dutton and Todd believe that
the same microorganism is responsible for both diseases. Koch, on the
other hand, believes that the slightly smaller size of the African spiro-
chsete and the milder course of the clinical symptoms indicate a defi¬
nite difference between the two. Animal experiments made with the
African organism, furthermore, usually show a much more severe in¬
fection than do similar inoculations with the European variety. The

1 Ross and Milne, Brit. Med. Jour., 1904.

2 Koch, Deut. med. Woch., xxxi, 1905.

3 Dutton and Todd, Lancet, 1905, and Jour, of Jrop. Med., 1905.

610

PATHOGENIC MICROORGANISMS

spirochsete found in the African disease is usually spoken of at present
as “Spirochseta Duttoni.” Novy and Knapp/ after extensive studies
with the microorganisms from various sources, have come to the conclu¬
sion that, although closely related, definite species differences exist be¬
tween the two types mentioned above, and that these again are definitely
distinguished from similar organisms described by Turnbull2 as occurring
in a similar disease observed in India.

The mode of transmission of this disease is not clear for all types.
Dutton and Todd, however, were able to show satisfactorily that, in the
case of the African disease at least, transmission occurs through the
intermediation of a species of tick. The conditions under which such
intermediation occurs have been carefully studied by Koch.3 The
tick (Ornithodorus moubata) infects itself when sucking blood from
an infected human being. The spirochsete may remain alive and
demonstrable within the body of the tick for as long as three days.
Koch has shown, furthernore, that they may be found also within the
eggs laid by an infected female tick. He succeeded in producing experi¬
mental infection in monkeys by subjecting the animals to the bites
of the infected insects. For the European variety of the disease no
such intermediate host has as yet been demonstrated.

Immunity. — It has long been a well-known fact that recovery from
an attack of relapsing fever usually results in a more or less definite
immunity. The blood of human beings, monkeys, and rats which have
recovered from an attack of this disease show definite and specific
bactericidal and agglutinating substances, and Novy and Knapp have
demonstrated that the blood serum of such animals may be used to
ponfer passive immunity upon others.

VINCENT’S ANGINA

The condition known as Vincent’s angina consists of an inflamma¬
tory lesion in the mouth, pharynx, or throat, situated most frequently
upon the tonsils. The disease usually begins as an acute stomatitis,
pharyngitis, or tonsillitis, which soon leads to the formation of a pseudo¬
membrane, which, at this stage, has a great deal of resemblance to that
caused by the diphtheria bacillus. At later stages of the disease there
may be distinct ulceration, the ulcers having a well-defined margin

1 Novy and Knapp, loc. cit. 2 Turnbull, Indian Med. Gaz., 1905.

3 Koch, Berl. med. Woch., 1906.

DISEASES CAUSED BY SPIROCHETES

611

and “punched-out” appearance, so that clinically they have often been
erroneously diagnosed as syphilis. Apart from the localized pain, the
disease is usually mild, but occasionally moderate fever and systemic
disturbances have been observed. Unlike diphtheria and syphilis, this
peculiar form of angina usually yields, without difficulty, to local treat¬
ment.

The nature of lesions of this peculiar kind was not clear until Pkaut,,1

Fig. 136. — Smear from the throat of a Case of Vincent’s Angina.

Giemsa Stain.

Vincent,2 and others reported uniform bacteriological findings in cases
of this description. These observers have been able to demonstrate
in smears from the lesions a spindle-shaped or fusiform bacillus, to¬
gether with which there is usually found a spirillum not unlike the
spirillum of relapsing fever. The two microorganisms are almost

1 Plaut, Deut. med. Woch., xlix, 1894.

2 Vincent, Ann. de l’inst. Pasteur, 1896, and Bull, et mem. de la soc. med. des
hop. de P., 1898,

612

PATHOGENIC MICROORGANISMS

always found together in this form of disease and were regarded by
the first observers as representing two distinct forms dwelling in sym¬
biosis. More recently Tunnicliff,1 on the basis of experimental work,
has claimed identity for the two forms, believing that they represent
different developmental stages of the same organism.

The fusiform bacilli described by Vincent, Plaut, Babes, and others,
are from 3 to 10 micra in length, and have a thickness at the center
varying from 0.5 to 0.8 micron. From the center they taper gradually

Fig. 137. — Throat Smear. Vincent's Angina. Fusiform bacilli and spirilla.

toward the ends, ending in blunt or sharp points. The length of these
bacilli may vary greatly within one and the same smear preparation.
They are usually straight, sometimes slightly curved. They do not stain
very easily with the weaker anilin dyes, but are readily stained by
Loeffler’s methylene-blue, carbol-fuchsin, or better, by Giemsa’s stain.
Stained by Gram, they are usually decolorized, though in this respect the
writers have found them to vary. Stained preparations show a charac¬
teristic inequality in the intensity of the stain, the bacilli being more

1 Tunnicliff, Jour, of Infec, Dis., 3, 1906.

DISEASES CAUSED BY SPIROCHETES

613

deeply stained near the end, and showing a banded or striped alternation
of stained and unstained areas in the central body. Their staining
qualities in this respect are not unlike those of the diphtheria bacillus,
and according to Babes 1 the dark areas are to be interpreted as meta-
chromatic granules. The bacilli are not motile.

The spirilla found in Vincent’s angina are usually somewhat longer
than the fusiform bacilli, and are made up of a variable number of un¬
dulations, shallow and irregular in their curvatures, unlike the more
regularly steep waves of Spirochseta pallida. They are stained with
even more difficulty than are the bacilli and usually appear less distinct
in the preparations. The stain, however, is taken without irregu¬
larity, showing none of the apparent metachromatism observed in the
bacilli.

By the earlier observers cultivation of these microorganisms was
attempted without success. Recently, however, it has been shown that
cultivation could be carried out under anaerobic conditions. Tunni-
cliff 2 has cultivated the organisms anaerobically upon slants of ascitic
agar at 37.5° C. This observer found that in such cultures, before the
fifth day, bacilli only could be found, that after this time, however,
spirilla gradually appeared and finally constituted the majority of the
organisms in the culture. It appeared to Tunnicliff from this study
that the spirilla might be developed out of the fusiform microorgan¬
isms representing the adult form.

The microorganisms of Vincent’s angina, when occurring in the
throat, are rarely present alone, being usually accompanied by other
microorganisms, such as staphylococci, streptococci, and not infre¬
quently diphtheria bacilli. When occurring together with diphtheria,
they are said, by some German observers, to aggravate the latter
condition considerably. This frequent association with other micro¬
organisms renders it impossible to decide conclusively that the fusi¬
form bacilli and spirilla are the primary etiological factors in these
inflammations. It has been frequently suggested that they may be
present as secondary invaders upon the soil prepared for them by other
microorganisms.

Animal inoculation with these microorganisms has led to little result.

Fusiform Bacilli other than those in Vincent’s Angina. — Fusiform
bacilli morphologically indistinguishable from those found in the angina
of Vincent may frequently be found in smears taken from the gums,

1 Babes, in Kolle und Wassermann, 1. Erganzungsband, 1907.

2 Tunnicliff, Jour, of Infec. Dis., 3, 1908.

614

PATHOGENIC MICROORGAN ISMS

from carious teeth, and occasionally among the microorganisms in the
pus from old sinuses. Several varieties of these bacilli have been de¬
scribed in connection with definite pathological conditions.

Babes,1 in 1893, observed spindle-shaped bacilli not unlike those
described above, but somewhat shorter, in histological sections prepared
from tissues from the gums of individuals suffering from scurvy. He
found similar bacilli in rabbits intravenously inoculated with material
from the patients and was able to cultivate the bacilli for several genera¬
tions. His descriptions, however, of the microorganisms as found in the
secondary cultures vary considerably from those of the original findings
in the gums of the patients. His results are not convincing.

In noma, a gangrenous disease of the gums and cheeks, occurring
occasionally in individuals who have been severely run down by acute
infectious diseases or great hardship, Weaver and Tunnicliff have found
spirilla and fusiform bacilli in large numbers. The organisms were pres¬
ent not only in smears from the surface, but were also found by histo¬
logical methods, in large numbers, lying in the tissues beyond the
area of necrosis. Here again it is not entirely certain whether these
microorganisms were the primary etiological factors or whether
they are to be regarded merely as secondary invaders of a necrotic
focus.

Fusiform bacilli are cultivated with greater ease than formerly sup¬
posed; we have found it relatively simple to grow them together with
Gram positive cocci in symbiosis in simple broth tubes covered with
paraffin oil without the addition of any enriching substance and in
similar symbiotic conditions on infusion agar plates under incomplete
anaerobic conditions. In such plates they form curious colonies in
which the fusiform bacilli and micrococci are intimately commingled.
Krumwiede 2 has had no difficulty in cultivating them in pure culture
in anaerobic plates.

SPIROCILffiTA PERTENUIS

In a disease known as “Framboesia tropica,” or popularly “Yaws,”
occurring in tropical and subtropical countries and much resembling
syphilis, Castellani,3 in 1905, was able to demonstrate a species of
spirochsete which has a close morphological resemblance to Spirochseta
pallida. The microorganism was found in a large percentage of the cases

1 Babes, Deut. med. Woch., xliii, 1893.

2 Krumwiede, Jour. Inf. Dis., 1913.

3 Castellarii, Brit. Med. Jour., 1905, and Deut. med. Woch., 1906.

DISEASES CAUSED BY SPIROCHETES

615

examined both in the cutaneous papules and in ulcerations. Confirm¬
atory investigations on a larger series of cases were later carried out by
von dem Borne.1

The microorganism is from 7 to 20 micra in length with numerous
undulations and pointed ends. Examined in fresh preparations, it has
an active motility similar to that of Spirochseta pallida. In smears
it is easily stained by means of the Giemsa method.

Both the clinical similarity between yaws and syphilis, as well as
the similarity between the microorganisms causing the diseases, has
opened the question as to the identity of the two microorganisms.
According to most clinical observers, however, yaws, which is a disease
characterized chiefly by a generalized papular eruption, is unquestion¬
ably distinct, clinically, from lues, and experiments of Neisser, Baermann,
and Halberstadter,2as well as of Castellani 3 himself, have tended to show
that there is a distinct difference between the immunity produced by
attacks of the two diseases. The disease is transmissible to monkeys,
as is syphilis, but it has been satisfactorily shown that monkeys inocu¬
lated with syphilitic material, while no longer susceptible to infection
with Spirochseta pallida, may still be successfully inoculated with
Spirochseta pertenuis.

SPIROCH7ETA G ALLIN ARUM

An acute infectious disease occurring among chickens, chiefly in
South America, has been shown by Marchoux and Salimbeni 4 to be
caused by a spirochete which has much morphological similarity to the
spirochete of Obermeier.

The disease comes on rather suddenly with fever, diarrhea, and great
exhaustion, and often ends fatally. The spirochete is easily demon¬
strated in the circulating blood of the animals by staining blood-smears
with Giemsa’s stain or with dilute carbol-fuchsin.

Artificial cultivation of the microorganism has not yet been ac¬
complished. Experimental transmission from animal to animal is easily
carried out by the subcutaneous injection of blood. Other birds, such
as geese, ducks, and pigeons, are susceptible; mammals have, so far,
not been successfully inoculated. According to the investigations of

1 Von dem Borne, Jour. Trop. Med., 10, 1907.

2 Neisser, Baermann, und H alber stadter, Munch, med. Woch., xxviii, 1906.

3 Castellani, Jour, of Hyg., 7, 1907.

4 Marchoux et Salimbeni, Ann. de l’inst. Pasteur, 1903.

616

PATHOGENIC MICROORGANISMS

Levaditi and Manouelian,1 2 the spirochsetes are found not only in the
blood but thickly distributed throughout the various organs.

Under natural conditions, infection of chickens seems to depend
upon a species of tick which acts as an intermediate host and causes
infection by its bite. The spirochsete, according to Marchoux and Sal-

imbeni, may be found in the intes¬
tinal canal of the ticks for as long
as five months after their infection
from a diseased fowl.

In the blood of animals which
have survived an infection, agglutin¬
ating substances appear and active
immunization of animals may be
carried out by the injection of in¬
fected blood in which the spirochsetes
have been killed, either by moderate
heat or by preservation at room
temperature. The serum of immune
animals, furthermore, has a pro¬
tective action upon other birds.

It is not impossible that the Spiro-
chseta gallinarum may be identical
with the Spirochseta anserina previ¬
ously discovered by Sacharoff ? This
last-named microorganism causes a disease in geese, observed espe¬
cially in Russia and Northern Africa, which both clinically and in its
pathological lesions corresponds closely to the disease above described
as occurring in chickens. The spirochsete is found during the febrile
period of the disease in the circulating blood, is morphologically indis¬
tinguishable from the spirochsete of chickens, and can not be cultivated
artificially. The similarity is further strengthened by the fact that
Spirochseta anserina is pathogenic for other birds, but not for animals
of other genera. Noguchi has succeeded in cultivating Spirochseta
gallinarum by the same method by which he has cultivated the or¬
ganisms of relapsing fever. Ascitic fluid tubes with a piece of sterile
rabbit kidney were inoculated with a few drops of blood containing the
spirochsetes and cultivated at 37.5° C. under anaerobic conditions.

Spirochseta phagedenis. — This is an organism cultivated by Noguchi

1 Levaditi et Manouelian, Ann. de l’inst. Pasteur, 1906.

2 Sacharoff, Ann. de l’inst. Pasteur, 1891.

Fig. 138. — Spirochveta gallina¬
rum. (From preparation furnished
by Dr. G. N. Calkins.)

DISEASES CAUSED BY SPIROCHETES

617

by his ascitic-fluid-tissue method from phagedenic lesions on human
external genitals. It is probably a new species.

Spirochseta macrodentium. — Cultivated by Noguchi; 1 is believed by
him to be identical with the spirochsete found in Vincent’s angina.

Spirochseta microdentium. — A similar organism with wide con¬
volutions, cultivated by Noguchi from the tooth deposits chiefly in
children. It was grown on mixtures of sheep serum water and sterile
tissue in a way similar to that employed by him for other organisms of
this group.

Spirochseta calligyrum. — Cultivated by Noguchi2 from condylomata
— is probably a new species.

1 Noguchi, Jour. Exp. Med., xv, 1912.

2 Noguchi, Jour. Exp. Med., xvii, 1913.

CHAPTER XLIV

THE HIGHER BACTERIA
(' Chlamydobacteriacece , Trichomycetes )

Standing midway between the true bacteria and the more complex
molds or Hyphomycetes, there are a number of pathogenic micro¬
organisms which offer great difficulties to classification. In the classifi¬
cation of Migula most of these forms have been placed in a rather
heterogeneous group, the Chlamydobacteriacese. By other authors,
notably Lachner-Sandoval,1 Berestnew,2 and by Petruschky,3 the close
relationship of these forms to the higher hyphomycetes has been em¬
phasized and they have been grouped as a subdivision of the true molds
under the family name of Trichomycetes.

Petruschky 4 proposes the following clear schematization, which,
even though possibly defective from a purely botanical point of view,
is at least serviceable for the purposes of the bacteriologist.

Hyphomycetes

True molds

Trichomycetes

Leptothrix

Cladothrix

Streptothrix

Actinomyces

Leptoihrix is used to designate those forms which appear as simple
threads without branching.

Cladothrix is a thread-like form in which false branching may be
recognized. By false branching is meant an appearance resulting from
the fragmentation of threads. The terminal cell breaks away from the
main stem, is set at an angle by the elongation of the thread itself, and,

1 Lachner-Sandoval, “Ueber Strahlenpilze.” Diss. Strassburg, 1898.

2 Berestnew, Ref. Cent. f. Bakt., xxiv, 1898.

3 Petruschky, in Kolle und Wassermann, “Handbuch,” etc.

4 Petruschky, loc. cit.

618

THE HIGHER BACTERIA

619

as both continue dividing, the simulation of true branching is pro¬
duced.

Streptothrix denotes forms with numerous true branches and spores
which usually appear in chains.

Actinomyces is of more complicated structure, characterized by the
formation of club-shaped ends and the stellate arrangement of its
threads.

LEPTOTHRIX

Members of the leptothrix group have been observed in connection
with inflammations of the mouth and pharynx by Frankel,1 Michelson,2
Epstein,3 and others. In many of these cases the organism was identi¬
fied by morphology chiefly, pure cultures not having been obtained.
The disease in none of these cases was accompanied by severe systemic
symptoms and it is likely that when found in human beings the organ¬
isms may be regarded simply as comparatively harmless saprophytes
appearing in connection with some other specific inflammation.

Cultivation of the Leptothrices is not easy and has been successful
only in the hands of Vignal 4 and Arustamoff.5

CLADOTHRIX

Owing to much confusion in the differentiation of these forms from
the streptothrices, it is not possible to determine whether cases of true
cladothrix infection have been observed. It is likely that most cases
ascribed to microorganisms of this class have really been due to strep¬
tothrix infection. The deciding criterion is, of course, the formation of
branches and these seem to have been observed in most of the cases
described. A closer differentiation, in the future, between true and
false branching can alone determine whether or not cases of cladothrix
infection proper may occur.

STREPTOTHRIX

Reports of cases of streptothrix infection of various parts of the
body, in both animals and man, are abundant in the literature. The

1 Frankel, Eulenburg’s “ Realencycl. d. gesam. Heilkunde/’ 1882.

2 Michelson, Berl. klin. Woch., ix, 1889.

3 Epstein, Prag. med. Woch., 1900.

* Vignal, Ann. de phys., viii, 1886.

s Arustamoff, Quoted from Petruschky, loc. cit.

620

PATHOGENIC MICROORGANISMS

earliest observations were made upon microorganisms isolated from the
human conjunctiva. Nocard 1 in 1888 described a member of this
group as the etiological factor in a disease “ farcies du boeuf ” occurring
among cattle in Guadeloupe. Eppinger 2 found streptothrices in the pus
of a cerebral abscess. Petruschky,3 Berestneff,4 Flexner,5 Norris and

Pig 139. — Cladothrix. Showing False Branching.

Larkin,6 and a number of other observers have found these microor¬
ganisms in cases of pulmonary disease, simulating tuberculosis. Sup¬
purations of bone and of the skin and the intestinal canal have been
reported. The infection, therefore, is not very rare, but the diverse
experiences of workers who have attempted to cultivate these micro-

1 Nocard, Ann. de l’inst. Pasteur, ii, 1888.

2 Epjpinger, Wien. klin. Woch., 1890.

3 Petruschky , Verhandl. d. Kongr. f. innere Mediz., 1898.

14 Berestneff, Zeit. f. Hyg., xxix, 1898.

4 Flexner, Jour. Exp. Med., iii, 1896.

d Norris and Larkin, Proc. of N. Y. Path. Soc., March, 1899.

THE HIGHER BACTERIA

621

organisms seem to indicate that not all of the incitants described be¬
longed to one and the same variety, but that probably a number of
different types may exist.

Morphology. — Morphologically the streptothrices show considerable
variation. In material from infectious lesions they have most often
appeared as rods and filaments with well-marked branching. Occasion¬
ally the filaments are long and interwinecl, and branches have shown
bulbous or club-shaped ends. In Norris and Larkin's case, the young
cultures in the first generations seem to have consisted chiefly of rod¬
shaped forms not unlike bacilli of the diphtheria group, showing marked
metachromatism when stained with Loeffler’s methylene-blue. They are

Fig. 140. — Streptothrix, Showing True Branching.

easily stained with this dye or with aqueous fuchsin. In tissue sections
they may be demonstrated by the Gram-Weigert method.

Cultivation. — Direct cultivation upon agar and gelatin plates has
occasionally been successful. At the end of four or five days grayish-
white, glistening, flat colonies may appear which attain a diameter of
several millimeters within two weeks. The colonic later may take on a
yellowish hue and begin to liquefy the gelatin. In bouillon flocculent
precipitates and surface pellicles of interwined threads may form, with¬
out clouding of the medium. Norris and Larkin1 found much difficulty
in cultivating, but finally succeeded by making smears of the infectious
material upon fresh, sterile kidney-tissue of rabbits. The micro-

» Norris and Larkin, loc, tit,

622

PATHOGENIC MICROORGANISMS

organisms grew abundantly upon this, but failed to grow on any of the
other tissues. After growth of several generations upon this medium,
cultures were finally obtained upon agar plates and upon broth.

Inoculation of cultures into rabbits and guinea-pigs have given rise
to subcutaneous abscesses, bronchopneumonia, and suppuration, accord¬
ing to the mode of infection.

ACTINOMYCES

Among the diseases caused by the Trichomycetes or higher bacteria,
the most important is actinomycosis. Occurring chiefly in some of the
domestic animals, notably in cattle, the disease is observed in man with
sufficient frequency to make it of great clinical importance. In cattle
the specific microorganism which gives ries to the disease was first
observed by Bollinger 1 in 1877. In the following year Israel 2 dis¬
covered a similar microorganism in human cases.

The parasites appear in the pus from discharging lesions as small
granular bodies, plainly visible to the naked eye and somewhat resem¬
bling sulphur granules, of a grayish or of a pale yellow color. In size
they measure usually a fraction of a millimeter. Ordinarily they are
soft and easily crushed under a cover-slip, but occasionally, especially in
old lesions, they may be quite hard, owing to calcification.

Microscopically they are most easily recognized in fresh preparations
prepared by crushing the granules upon the slide under a cover-slip and
examining them without staining. They may be rendered more clearly
visible by the addition of a drop or two of 20 per cent potassium hydrate.
When the granules are calcareous, the addition of a drop of concentrated
acetic acid will facilitate examination. Fresh preparations may be
examined after staining with Gram's stain. Observed under the micro¬
scope, the granules appear as rosette-like masses, the centers of which
are quite opaque and dense, appearing to be made up of a closely meshed
network of filaments. Around the margins there are found radially
arranged striations which in many cases end in characteristically club-
shaped bodies. Inside of the central network there are often seen
coccoid or spore-like bodies which have been variously interpreted as
spores, as degeneration products, and as separate cocci fortuitously
found in symbiosis with the actinomyces. Individually considered,

? Bollinger , Deutsch. Zeit. f. Thiermed., iii, 1877.
58 Israel, Virch. A.rph,, 74, 1878, and 78, 1879.

THE HIGHER BACTERIA

623

the central filaments have approximately the thickness of an anthrax
bacillus and are, according to Babes,1 composed of a sheath within
which the protoplasm contains numerous and different sized granules.

About the periphery of the granules the free ends of the filaments
become gradually thickened to form the so-called actinomycosis “ clubs.”
These clubs, according to most observers, must be regarded as hyaline
thickenings of the sheaths of the threads and are believed to represent a
form of degeneration and not, as some of the earlier observers believed,
organs of reproduction. They are homogeneous, and in the smaller and
presumably younger granules are extremely fragile and soluble in water.
In older lesions, especially in those of cattle, the clubs are more re¬
sistant and less easily destroyed.

They appear only in the parasites taken from active lesions in animals
or man, or, as Wright 2 has found, from cultures to which animal serum
or whole blood has been added. In cultures from media to which no
animal fluids have been added, such as glucose agar or gelatin, no clubs
are found. In preparations stained by Gram's method the clubs give
up the gentian-violet and take counter-stains, such as eosin.

The coccus-like bodies found occasionally lying between the filaments
of the central mass, most observers now agree, do not represent any¬
thing comparable to the spores of the true hyphomycetes. In many
cases they are unquestionably contaminating cocci; in others again
they may represent the results of degeneration of the threads.

In tissue sections, the microorganisms may be demonstrated by
Gram's method of staining or by a special method devised by Mallory.3
This is as follows for paraffin sections:

1. Stain in saturated aqueous eosin 10 minutes.

2. Wash in water.

3. Anilin gentian- violet, 5 minutes.

4. Wash with normal salt solution.

5. Gram's iodin solution 1 minute.

6. Wash in water and blot.

7. Cover with anilin oil until section is clear.

8. Xylol, several changes.

9. Mount in balsam.

Cultivation. — The isolation of actinomyces from lesions may be
easy or difficult according to whether the pus is free from contamination

1 Babes, Virch. Arch., 105, 1886.

2 J. H. Wright, Jour. Med. Res., viii, 1905.

s Mallory, Method No. 1, Mallory and Wright, “ Path. Technique,” Phila., 1908.

624

PATHOGENIC MICROORGANISMS

or whether it contains large numbers of other bacteria. In the latter
case it may be almost impossible to obtain cultures. The descriptions
of methods of isolation and of cultural characteristics given by various
writers have shown considerable differences. The most extensive
cultural work has been done by Bostroem,1 Wolff and Israel, and by J.
H. Wright. Bostroem has described his cultures as aerobic, but Wolff
and Israel 2 and Wright 3 agree in finding that the microorganisms iso-

Fig. 141.— Actinomyces Granule Crushed Beneath a Cover-glass. Un¬
stained. Low power. Shows radial striations. (After Wright and Brown.)

lated by them from actinomycotic lesions grow but sparsely under aerobic
conditions and favor an environment which is entirely free from oxygen,
or at least contains it only in small quantities. The method for isolation
recommended by Wright is, briefly, as follows: Pus is obtained, if
possible, from a closed lesion and washed in sterile water or broth. The
granules are then crushed between two sterile slides and examined for

1 Bostroem, Beitr. z. path. Anat. u. z. allg. Path., ix, 1890.

2 Wolff und Israel, Yirch. Arch., 126, 1891.

3 J . H. Wright, Jour. Med. Res., viii, 1905.

THE HIGHER BACTERIA

625

the presence of filaments. If these are present in reasonable abundance,
the material is distributed in tubes of glucose agar, which are then
allowed to solidify. If these first cultivations show a large number of
contaminations, Wright recommends the preservation of other washed
granules in test tubes for several weeks, in the hope that contaminating-
microorganisms may thus be killed by drying
before the actinomyces lose their viability.

If cultivation is successful colonies will ap¬
pear, after two to four days at 37.5° C., as minute
white specks, which, in Wright's cultures, ap¬
peared most abundantly within a zone situated
5 to 10 millimeters below the surface of the
medium. Above and below this zone they are
less numerous, indicating that a small amount of
oxygen furnishes the best cultural environment.

Upon the surface of agar slants, growth, if it
takes place at all, is not luxuriant.

In alkaline meat-infusion broth growth
takes place in the form of heavy, flocculent
masses which appear at the bottom of the tubes.

Surface growth and clouding do not take place.

Milk and potato have been used as culture
media but are not particularly favorable.

Pathogenicity. — As stated above, actinomy¬
cosis occurs spontaneously most frequently
among cattle and human beings. It may also
occur in sheep, dogs, cats, and horses. Its loca¬
tions of predilection are the various parts
adjacent to the mouth and pharynx. It occurs
also, however, in the lungs, in the intestinal
canal, and upon the skin. When occurring in
its most frequent location, the lower jaw, the
disease presents, at first, a hard nodular swell¬
ing which later becomes soft because of central necrosis. It often
involves the bone, causing a rarefying osteitis. As the swellings
break down, sinuses are formed from which the granular pus is
discharged. The neighboring lymph nodes show painless, hard swell¬
ings. Histologically, about the filamentous knobs or granules, there is a
formation of epithelioid cells and a small round-cell infiltration. In
older cases there may be an encapsulation in connective tissue and a
41

Fig. 142. — Actino-
myces Granule
Crushed Beneath a
Cover-glass. Un¬
stained. The prepara¬
tion shows the margin
of the granule and the
“ clubs. ” (After Wright
and Brown.)

626

PATHOGENIC MICROORGANISMS

calcification of the necrotic masses, leading to spontaneous cure. As a
rule, this process is extremely chronic. Infection in the lungs or in the
intra-abdominal organs is, of course, far more serious. When death
occurs acutely, it is often due to secondary infection. The disease is
acquired probably by the agency of hay, straw, and grain. Berestnew 1
has succeeded in isolating actinomyces from straw and hay which he
covered with sterile water in a potato jar and placed in the incubator.
After a few days small white specks looking like chalk powder appeared
upon the stalks, which, upon further cultivation, he was able to identify
as the organism in question.

Animal inoculation, carried out extensively both with pus and with
pure cultures by several observers, has yielded little result. Progressive

Fig. 143. — Branching Filaments of Actinomyces. (After Wright and Brown.)

actinomycotic lesions were never obtained, although occasionally small
knobs containing colonies surrounded by epithelioid cells and connective
tissue were observed, showing that the invading microorganisms were
able to survive and grow for a short time, but were not sufficiently
virulent to give rise to an extensive disease process. Transmission from
animal to animal, or from animal to man directly, has not been satis¬
factorily proven.

Whether or not there are various forms of actinomyces must
as yet be regarded as an open question. The investigations of
Wolff and Israel, however, together with those of Wright, who alone
observed thirteen different strains, seem to indicate that most, if
not all, of the cases clinically observed are due to one and the same
microorganism.

1 Berestnew, Ref. Cent. f. Bakt., 24, 1898.

THE HIGHER BACTERIA

627

MYCETOMA (MADURA FOOT)

The disease known by this name is not unlike actinomycosis. It
is more or less strictly limited to warmer climates and was first recog¬
nized as a clinical entity, in India, by Carter.1 Clinically it consists
of a chronic productive inflammation most frequently attacking the
foot, less often the hand, very infrequently other parts of the body.
Nodular swellings occur, which break down in their centers, leading to
the formation of abscesses, later of sinuses. Often the bones are in¬
volved and a progressive rarefying osteitis results. From the sinuses a
purulent fluid exudes, in which are found characteristic granular bodies.
These may be hard, brittle, and black, resembling grains of gunpowder,
or may be grayish-white or yellow and soft and grumous. According to
the appearance of these granules, two varieties of the disease are dis¬
tinguished, the “melanoid” and the “ochroid.” Many observers
believe that the yellow or ochroid variety is, in fact, actinomycosis.
The black variety, which is certainly a distinct disease, is caused by a
member of the hyphomycetes group. The parasite has been carefully
studied by Wright,2 from whose description the following points are
taken :

The small, brittle granules observed under the microscope show a
dark, almost opaque center along the edges of which, filaments, or
hyphse, may be seen in a thickly matted mass. By crushing the granules
under a cover-slip in a drop of sodium hypochlorite or of strong sodium
hydrate, the black amorphous pigment is dissolved and the structural
elements of the fungus may be observed. They seem to be composed
of a dense meshwork of mycelial threads which are thick and often
swollen, and show many branches. Transverse partitions are placed
at short distances and the individual filaments may be very long.
Spores were not observed by Wright. In a series of over fifty cultiva¬
tions on artificial media from the original lesion, Wright obtained growth
in a large percentage.

In broth, he obtained at first a rapid growth of long hyphse which
eventually formed a structure which he compares in appearance to a
powder-puff.

On agar, growth appeared within less than a week and spread over
the surface of the medium as a thick meshwork of spreading hyphao

1 Carter on My-cetoma, etc., London, 1874.

2 Wright, Jonr. of Exper. Med., 3, 1898.

628

PATHOGENIC MICROORGANISMS

of a grayish color. In old cultures black granules appeared among
the mycelial meshes.

On potato, he observed a dense velvety membrane, centrally of a pale
brown, white at the periphery. Small brown droplets appeared on the
growth in old cultures.

Animal inoculation with this microorganism has so far been un¬
successful.

CHAPTER XLY

THE YEASTS

( Blastomycetes , Saccharomycetes)

The yeasts or blastomycetes form a distinctive family among uni¬
cellular microorganisms, characterized essentially by their method of
multiplication by budding. By this, they are sharply separated from
the bacteria. Their differentiation from the higher fungi, the hypho-
mycetes, however, is less definitely established, since the chief character¬
istic of this latter class, the formation of hyphae and mycelial threads, has
occasionally been described for some of the forms otherwise identified
with the yeasts. It is probable that a gradual transition between the
two families exists, represented by a number of connecting forms, some¬
times spoken of as oidia. For the practical purposes of the bacteriolo¬
gist, the yeast family is sufficiently distinct, both morphologically and
biologically, to make a separate classification extremely useful.

The yeast cell, as a rule, is oval, but among the wild yeasts, or
“torulae,” spherical forms are common. In size, great variations occur,
but in general the yeasts are much larger than bacteria, measuring usually
from 10 to 20 micra in length with a width of about one-half or two-thirds
of the long diameter. They possess a well-defined, doubly-contoured
cell-membrane, composed chiefly of cellulose, and their body protoplasm,
unlike that of the bacteria, shows definite structure. Within a mass
of finely granul arcytoplasm, a number of highly refractive globules and
vacuoles may be observed. Some of the globules have been interpreted
as fat-droplets. Other granules, revealed by special staining methods,
are interpreted as nuclear material.

• When budding takes place, the mother cell sends out a small,
globular evagination of the cell membrane into which maternal proto¬
plasm flows. This bud gradually enlarges until it has attained approxi¬
mately the same size as the original cell. Until that time, free inter¬
communication between the protoplasm of mother and daughter cell
exists. Finally, by gradual narrowing of the isthmus connecting the
two, the daughter cell becomes complete and free. By some observers

629

630

PATHOGENIC MICROORGANISMS

definite karyokinetic changes in the nuclear structures have been de¬
scribed as accompanying the budding process. This observation, how¬
ever, has not been generally confirmed. Under conditions of special
stress, such as unfavorable environment or lack of nutrition, most
yeasts possess the power of forming spores. These, called “ ascospores,”
are formed within the yeast cell itself, each spore forming a separate
membrane of its own, but all of them lying well protected within the
original cell-membrane. The number of ascospores formed is constant
for each species, and rarely exceeds four.

The yeasts have been studied most extensively in connection with
fermentation and are industrially of great importance in the production

Fig. 144. — Yeast Cells. Young culture unstained. (After Zettnow.)

of wine and beer. Although Schwann, as early as 1837, recognized the
fact that many fermentations could not occur without the presence of
yeast, it was not until considerably later that the study of yeast fermen¬
tation was put upon a scientific basis. The typical fermentative action
consists in the transformation of sugar into ethyl alcohol according to
the following formula:

C6 Hl2 06 = 2 C2 H5 OH + 2 C02

The enzyme by which this fermentation is produced is known as “zy¬
mase,” and is, according to Buchner, in most cases, an endo-enzyme
which may be procured from the cell by expression in a hydraulic
press. In addition to this, however, the various yeasts also produce

THE YEASTS

631

other ferments by means of which they may split higher carbohydrates,
such as saccharose, maltose, and even starch, and prepare them for
action of the zymase. The manner in which this is accomplished, and
the by-products which are formed during the process, vary among
different species, and it is for this reason that the employment of pure
cultures is of such great importance in the wine and beer industries
where differences in flavor and other qualities may be directly dependent
upon the particular species of yeast employed for the fermentation. It
is due to the work chiefly of Pasteur 1 and of Hansen 2 that the beer and
wine industries have been carried on along exact and scientific lines.

As the incitants of disease in man, the yeasts have been much studied
since 1894, when Busse 3 reported a case of fatal, generalized yeast in¬
fection, beginning from a tibial bone abscess. The microorganism which
was found in this case he named “ Saccharomyces hominis.” In morpho¬
logical and biological characters it appeared to be a typical yeast, grew
readily upon most artificial media, and produced active fermentation in
sugars. Mycelia were not observed. When inoculated into animals,
this yeast proved pathogenic for mice and rats. A peculiarity of
Busse’s culture, observed since then in the case of many pathogenic
yeasts, was the formation of gelatinous capsules, of varying thicknesses,
about the individual cells, developing with particular luxuriance in the
animal lesions.

In 1896, Gilchrist 4 described a peculiar skin disease, which he spoke
of as pseudo-lupus vulgaris, in the lesions of which he demonstrated a
large number of round, doubly-contoured bodies which, though differing
somewhat from Busse’s saccharomyces, were unquestionably members
of, or closely related to, the family of blastomycetes.

In the same year, Curtis,5 in France, isolated a similar form from a
myxoma of the leg. Ophuls 6 has described a number of fatal cases
occurring in California, which at first were wrongly interpreted as
protozoan in origin, but later were determined by him to be caused
by a species of blastomycetes. In a case observed by Zinsser 7 simi¬
lar microorganisms were isolated from an abscess of the back, which

1 Pasteur, “ Etudes sur la biere,” Paris, 1876.

2 Hansen, “ Prac. Studies in Fermentation,” London, 1896.

3 Busse, Cent. f. Bakt., I, xvi, 1894, and Virch. Arch., 140, 1895.

4 Gilchrist, Bull. Johns Hopkins Hosp., vii, 1896.

5 Curtis, Ann. de l’inst. Pasteur, 10, 1896.

6 Ophuls, Jour. Exp. Med., 6, 1901.

7 Zinsser, Proc. N. Y. Path. Soc., vii. 1907.

632

PATHOGENIC MICROORGANISMS

in almost all respects corresponded to Gilchrist’s cultures. Animal
inoculation in rabbits and guinea-pigs proved positive in this case and
the organism seemed to show selective action for the lungs and spleen.
In the lungs of the animals, especially, lesions were found with surpris¬
ing regularity even when the inoculation was made intraperitoneally.

Cases of blastomycotic infection in man have been reported in large
numbers and appear to be less rare than they were formerly believed to
be. The clinical course of the disease is by no means uniform. A well-
defined clinical picture seems to characterize the cases of blastomycotic
dermatitis first described by Gilchrist. The eruption is very chronic
and begins usually as a small pimple or papule with moderate induration
of the skin. Scabs and pustules then form, which discharge yellowish-
white pus. As the lesion slowly spreads, the older areas show a tendency
to spontaneous healing. In Gilchrist’s 1 case, it took four years for the
lesion to spread two inches. When not purely cutaneous, blastomycotic
infection takes the form of chronic abscess formation occurring in various
parts of the body. In the latter, metastatic lesions in the lungs have
been occasionally observed, and in one case cited by Ophuls, 2 the lung
seemed to have been the primary focus.

The fact that blastomycetes have frequently been found in tumor
tissue has led several Italian observers 3 to assume an etiological
relationship between these microorganisms and malignant growths.
Absolutely no satisfactory evidence in favor of such a belief has
been advanced, however, and the yeasts in these conditions must be
regarded as purely fortuitous findings.

In animals, diseases caused by members of the yeast family have
been reported by various observers. The most important communica¬
tion of this kind is by Tokishige,4 who found these microorganisms in a
nodular skin disease occurring among horses in Japan. Sanfelice 5
has isolated similar microorganisms from the lymph glands of a horse
which was apparently suffering from a primary carcinoma of the liver.
The same author has described a member of this group which he obtained
from a cheesy consolidation occurring in the lung of a hog.

Demonstration of the organisms offers little difficulty either in fresh
preparations of the pus under a cover-slip, or in smears stained with

1 Rixford and Gilchrist, Johns Hopkins Hosp. Rep., i, 1896.

2 Ophiils, loc. cit.

3 Sanfelice, Cent. f. Bakt., I, xxxi, 1902.

* Tokishige, Cent. f. Bakt., I, xix, 1896.

5 Sanfelice, Cent. f. Bakt. I, xviii, 1895, and Zeitschr. f. Hyg., xxi, 1895.

THE YEASTS

633

thionin, methylene-blue, or the polychrome stains. In fresh prepara¬
tions the addition of a little NaOH in weak solution facilitates the
search. When found, the organisms are easily recognized by their size,
their highly refractive doubly-contoured cell-membrane, their vacuolated
protoplasm, and, most important, by the discovery of budding forms.
In tissue sections they are recognizable by the ordinary hematoxylin-
eosin technique, but may be better demonstrated by the Loeffler methy¬
lene-blue method in use for bacterial tissue-staining. Excellent prep¬
arations are obtained by staining frozen sections with thionin, a method
well adapted for the demonstration of capsules.

The cultivation of the blastomycetes is comparatively easy after
they have once been obtained in pure culture. Their isolation, however,
usually is difficult when they occur in material contaminated with bac¬
teria. Growing more slowly than the bacteria, cultures taken from such
contaminated material usually show very few yeast colonies. No special
methods of facilitating the procedure have been devised, but success
will often attend painstaking and oft-repeated plating of the mixed
cultures in high dilution. The most favorable medium for this process is
glucose agar. When once obtained in pure culture, the blastomycetes
can readily be kept alive for indefinite periods by transplantation re¬
peated every two or three months. On agar or glucose agar , colonies
appear after about three or four days as minute, glistening, white
hemispherical spots which are not unlike colonies of staphylococcus
albus. Planted in agar stab cultures, the microorganisms indicate
their preference for a well-oxygenated environment by growing but
slightly along the course of the stab, but by heaping up in a thick,
creamy layer upon the surface of the medium. This layer in old
cultures may be a quarter of an inch high. At first white, the
growth, after three or four weeks, may turn distinctly yellow or even
brown. In broth , the microorganisms form a stringy, gelatinous, and
uneven cloud. On Loeffler’ s blood-serum media, and upon gelatin,
growth is easily obtained. The gelatin is not liquefied. Sugar media
are fermented by few of the pathogenic blastomycetes, a fact which
places them rather in distinct contrast with the fermenting yeasts used
in the industries.

Great and fundamental differences seem to exist between the patho¬
genic species described by various observers, and attempts at system¬
atizing the various members of the group, such as that by Rickets,1

* Rickets, Jour. Med. Res., 6, 1902.

634

PATHOGENIC MICROORGANISMS

have met with almost insurmountable obstacles. Some of the forms
described, like that of Busse, have fermented sugars and have not
formed mycelia, while organisms like that of Gilchrist did not cause
fermentation in carbohydrate media, but, by their formation of my-
celia under certain conditions, have indicated their close relationship
or possibly their identity wdth the oidia, transitional forms between
the yeasts and the hyphomycetes. It is thus, in the light of our pres¬
ent knowledge of these microorganisms, quite impossible to establish
within this group a distinct classification that is at all reliable.

In considering the possible origin of blastomycotic infection in
animals and man, it is, of course, important that we should have some
knowledge as to the pathogenic properties of the yeast met with and
handled in daily life. Rabinowitsch,1 with this in view, has investigated
the pathogenic properties of fifty different species of yeasts obtained
from fruit, manure, dough, and other sources, and among them found
only seven varieties that had any pathogenicity for rabbits, mice, or
guinea-pigs. In most of her successful inoculations, however, the
disease produced in the animals had but very little resemblance to the
blastomycotic conditions observed in man.

1 Rabinowitsch, Zeit. f. Hyg., xxi, 1895.

CHAPTER XL VI

HYPHOMYCETES
(. Eumycetes , Molds)

The hyphomycetes or molds interest the bacteriologist chiefly
because of the frequency with which they appear as contaminants
during bacterial cultivation, and because they play the role of incitants
in a few common diseases of the skin and mucous membranes.

Morphologically they are entirely distinct from and much more
complex than the bacteria. To the yeasts they are ' more closely
related, the gap between the two classes being bridged by certain
forms often spoken of as “ oidia ” which, though usually appearing in
the budding yeast-form, may occasionally grow out in mycelial threads.

The characteristic feature of the hyphomycetes as a class is the
formation of long, interlacing filaments or threads, known as mycelia.
From these, branches come off which are spoken of as “hyphse.” Each
mycelial thread possesses a well-marked, doubly-contoured sheath or
cell-wall and a finely granular protoplasmic cell-body, which, in some of
the forms, is multinucleated.

Two main classes of hyphomycetes are distinguished, the phycomy-
cetes, and the so-called higher forms, or mycomycetes. The former class
is characterized by the fact that no partitions exist within the mycelial
threads or hyphse, the entire meshwork of a single microorganism con¬
sisting of one multinucleated cell. This group, furthermore, possesses
the power of reproduction by both a sexual and an asexual process.
The second class, or the mycomycetes, possess a mycelial meshwork
which is divided into numerous partitions, and reproduces usually by
the asexual process onty.

The process of reproduction, upon the basis of which the separation
of groups within this class is determined, is best illustrated by citing a
common example of each of the main divisions.

As an example of the phycomycetes, the division most commonly
met with upon contaminated gelatin plates, or upon exposed and moist
organic matter of any description, is the one spoken of as the muco-

635

PATHOGENIC MICROORGANISMS

(j 30

rince. Most forms belonging to this division appear, grossly, as a
light, cotton-like fluff, spreading in a thin fur over the surface of the
culture medium. Examined with the low power of a microscope, there
may be seen a complicated network of fine mycelial threads , which show
no septa and from which delicate hyphal branches arise. In the forma¬
tion of the asexual spore organs near the tip of each hypha a septum
appears. The tip of the hypha then gradually enlarges and forms a
spherical capsule which is known as the sporangium. The unswollen

Fig. 145. — Mucor mucedo. Single-celled mycelium with three hyphsp and
one developed sporangium. (After Kny, from Tavel.)

portion of the hypha which projects into the sporangium is spoken of
as the columella. Within the sporangium, a large number of small,
round spores are formed. When these are ripe, the wall of the spor¬
angium bursts and the spores escape. Upon suitable media, then, new
my celia develop from these spores. The sexual reproduction, which oc¬
curs in this group, takes place in the following way: From two hyphse,
placed in close apposition, lateral branches grow toward each other.
These are spoken of as gametophores . The tips of the gametophores
soon come in contact and, for a time, their protoplasm freely inter¬
communicates. Septa are then formed which cut off from the original
hyphre a central cell, the zygospore. This zygospore gradually enlarges

HYPHOMYCETES

637

and, under favorable conditions, sends out a branch which terminates
in a non-sexual sporangium.

Among the higher molds, or mycomycetes, various methods of
sporulation occur, but sexual reproduction does not usually take place.

One of the most commonly found genera is that of Penicillium. In
this form the mycelial threads are partitioned off, by many transverse
septa, into a number of separate cells. From these, hyphse, also sep¬
tate, are given off. From the ends of these hyphae, germinating
branches arise which are known as conidiophores . These conidiophores
divide into two or three septate branches, the sterigmata. From the

Fig. 146. — Mucor mucedo. 1. Sporangium, c. columella, m. sporangium
capsule, sp. spores. 2. Columella, after bursting of sporangium. 3. Poorly de¬
veloped sporangia. 4. Germinating spore. 5. Emptying of sporangium. (After
Brefeld.)

ends of these, other sterigmata may be given off and from the tip of
each of these a single chain of spores or conidia are constricted off.
The result is an appearance not unlike a hand in which the wrist
represents the conidiophore; the metacarpal bones, the sterigmata; and
the fingers, the long streptococcus-like chains of conidia.

Similar to and even more common than the penicillia are the varieties
known as Aspergillus. These forms, like the preceding, form delicate
mycelial mesliworks from which branches or conidiophores about 3-10
mm. in length, arise. These develop club-shaped expansions at their free
ends and from these club-shaped expansions radially arranged sterig-

638

PATHOGENIC MICROORGANISMS

mata arise. On the ends of these sterigmata spores or conidia develop
similar to those developed by penicillium.

Other forms of sporulation occur within this group. Thus, tubular
spore capsules may be formed within the end segments of the hyphse,
known as ascospores. In other cases, within a mycelial thread, a
swelling may take place into which protoplasm flows from the neighbor¬
ing cells, at both ends. In this way, an oval spore case is developed

Fig. 147. — Mucor mucedo. Formation of zygospore. 1. Two branches coa¬
lescing. 2 and 3. Process of conjugation. 4. Ripe zygospore. 5. Germination
of zygospore. 6 and 7. Mucor erectus. Azygo sporulation. No two branches
meet, but form spores without conjugation. 8 and 9. Mucor tenuis. Azygo
sporulation. The spores grow out from side branches without sexual union. (1-5
after Brefeld; 6-9 after Bainier, from Tavel.)

within the course of the mycelial thread. This is known as a chlamy-
dospore. The segments on each side of the chlamyclospore die out
and the spore capsule is liberated from the mycelium.

The classification of the various divisions of the hyphomycetes is a
problem requiring much study and great botanical insight, and can
hardly be discussed in a general work on bacteriology.

Upon artificial media, the members of this group are not at all
fastidious, growing easily upon organic matter of all kinds, provided

HYPHOMYCETES

639

moisture is present. In laboratories they are frequently found as con¬
taminants, and in order to procure them for purposes of study it is only
necessary to expose agar or gelatin plates in a dusty, dark corner. In
households they are frequently found growing in store-rooms upon stale
bread, shoes, leather trunks, and on remnants of food. Most of them
prefer an acid environment and are dependent upon a free supply of
oxygen. At room temperature they grow more readily than at the
usual incubator temperature.

DISEASES CAUSED BY HYPHOMYCETES

Pityriasis versicolor ( Microsporon furfur). — Pityriasis is a disease
of the skin observed chiefly among persons living under conditions of
uncleanliness, or among those who combine these conditions with a
tendency to profuse perspiration. It begins
as a small, light brown or yellowish patch
upon the skin of the abdomen, breast, or
back, is flat and barely raised from the cuta¬
neous surface. It spreads and coalesces with
similar spots until the entire area resembles
strongly the figures of a map with irregular
continents and islands. The disease does
not penetrate into the skin itself, but consists,
as Plaut has pointed out, of a simple sapro-
phytism of the inciting agent upon the skin.

The condition is caused by a member of
the group of Hyphomycetes, discovered in
1846 by Eichstedt, and later named Micro¬
sporon furfur. The microorganism consists
of a dense meshwork of mycelial threads,
from which septate hyphae arise in large
numbers. From the ends of these, spores
arise in rows, after the manner depicted for
penicillium (Fig. 149). The hyphae, accord¬
ing to Unna,1 show a characteristic bending at right angles, due to
a slight flattening of their diameters. Characteristic of the micro¬
sporon proper, in preparations made from cutaneous scrapings, are
the fragments of bent hyphae and the large numbers of free spores.

1 Unna, “ Histopathol. of Skin,” transl., N. Y., Macmillan, 1896.

Fig. 148. — Mucor ramo-
sus. Ripe sporangia on
columellae. (After Lindt.)

640

PATHOGENIC MICROORGANISMS

Cultivation of Microsporon furfur has been successfully carried
out by many observers.1 Growth is particularly characteristic upon
potato, white or yellowish-white colonies appearing within four or five
days and rapidly spreading over the entire surface of the potato.

Thrush ( Soor or Muguet; O'idium albicans). — Thrush is a localized
disease of the mouth occurring most frequently in children suffering

from malnutrition or it occurs, under con¬
ditions of uncleanliness, upon an area of
catarrhal inflammation of the mucous mem¬
brane.

The microorganism which causes the
condition was first described by Langenbeck
in 1839, and has, since then, been studied
by many observers. It was successfully
cultivated by Grawitz2 in 1886 and recog¬
nized by him as belonging to the hypho-
mycetes. The most careful cultural studies
which have been made upon the Oidium
albicans are those of Linossier and Roux.3

Morphologically, the oidium consists of
budding cells, resembling those of yeast,
which, under certain conditions, can pro¬
duce mycelia and hyphse from which
spores are developed. Two main varieties
are described, that which produces large
spores and liquefies gelatin in culture, and
that which gives rise to smaller spores and
fails to liquefy gelatin. In many cases only
the yeast-like budding cells can be found;
these, however, when subjected to unfavor¬
able cultural conditions, may be induced to
send out hyphae and form spores. Like
yeasts, but to a lesser degree, the Oidium
albicans causes fermentation in sugars. It
develops best under slightly acid conditions
and in the presence of free oxygen, upon gelatin and agar.

Favus ( Achorion Schoenleinii) . — Favus is a disease attacking chiefly

glaucum. A . Showing septate
mycelia. B. End of a hypha
— branching into two conidio-
phores, from which are given
off the sterigmata. From the
ends of these are developed
the round conidia. (After
Zopf.)

1 Kotjar, Ref. Baumgarten’s Jahresbericht, 1892.

2 Grawitz , Virch. Arch., 1886.

3 Linossier et Roux, Comptes rend, de l’acad. des sci., 1.889.

HYPHOMYCETES

641

the hairy portions of the body of man and some domestic animals.
In man, it is found most frequently in undernourished children upon the
scalp. It is a disease of extremely chronic course and is very resistant
to treatment. Beginning as a small erythematous spot, it soon develops
into small sulphur-yellow cupped crusts, which are placed usually about
the base of a hair. These may spread and coalesce. The small inden-
tated crust is spoken of as a scutulum. When such a scutulum is re¬
moved and examined under a microscope, teased out in a few drops of
strong sodium hydrate solution (20 per cent), the incitant of the disease
may be easily recognized and studied. In such a preparation the cen-

r. Ascospore. ' p. Germinating conidium. A. Ascus. (After de Bary.)

ter of the scutulum is found to be made up chiefly of small doubly-
contoured spores which are irregularly oval or round, and may be ar¬
ranged in chains, lying scattered among an extremely dense meshwork
of fine mycelial threads. Toward the periphery of the scutulum, the
spores are less numerous and the looser arrangement of the meshwork
permits us to distinguish distinct filaments of mycelia which give
off hyphse, the ends of which are often swollen into small knobs. The
interior of the scutulum usually contains a pure culture of the fungus.

Many varieties of achorion have been described, but Plaut1 believes
that, at the present time, it is not possible to separate these from one

1 Plaut, in Kolle und Wassermann’s “Handbuch,” I.

642

PATHOGENIC MICROORGANISMS

another, owing to the fact that selective cultivation has succeeded in
altering many of the characteristics displayed by many of the strains.
The same observer recommends the following method for obtaining pure
cultures of this microorganism. As much of the material as can be
conveniently obtained is gently rubbed up in a sterile mortar with fine
sand or infusorial earth. The triturated material is then inoculated into
fluid agar and plates are poured.

Ordinary streaked plates upon agar may also be used with success
with material directly from the centers of scutula.

The achorion grows best upon acid agar at a temperature of 37.5° C.

Fig. 151. — Thrush. Oidium albicans, unstained. (After Zettnow.)

Growth appears within from forty-eight hours to three days as yellowish
disks, which occasionally may be slightly furred with aerial hyphse.

Ringworm ( Trichophyton tonsurans). — Ringworm, Tinea circinata,
or Herpes tonsurans, is a contagious disease of the skin and hair, occur¬
ring most often in children and appearing upon both the haired portions
of the body, as well as upon free skin. It is characterized by the forma¬
tion of circular scaly patches, within which the hairs fall out.

The disease is caused by several species of the trichophyton, a genus
of hyphomycetes. These microorganisms were first recognized as inci-
tants of the disease by Gruby1 in 1841, and were most thoroughly

1 Gruby, Comptes rend, de l’acad. des sci., 13, 1841.

HYPHOMYCETES

643

studied later, by Sabouraud.1 The fungi consist of finely interlaced
narrow septate my celia, within which characteristic swellings appear.
From these swellings, chlamydospores develop. Hyphse, both aerial and
deep, grow out of the mycelial threads, on the ends of which ascospores
may develop. In the diseased skin, the fungi grow chiefly within the
hair sheath, causing an area of secondary inflammation about the base
of the hair. The infection probably begins first in the epidermis sur¬
rounding the hairs, from which it then spreads into the hair bulb and .
thence grows up into the substance of the hair in which mycelial threads

Fig. 152. — Achorion Schoenleinii. Section of favus crust. Stained by

Gram. (After Fraenkel and Pfeiffer.)

and spores develop in large numbers. The demonstration of the micro¬
organism from a case can easily be accomplished by epilating an af¬
fected hair, making sure that the hair bulb has been removed. This
is then immersed under a cover-slip in a drop of sodium hydrate or
potassium hydrate solution and examined under the microscope. In
this way enormous numbers of short mycelial threads and spores may
be seen to lie within the bulb. Many varieties of these fungi have been
described from different cases. Their interrelationship is not entirely
clear. In general, a division is made between those which develop large
spores and a more common small-spored variety. _ __

1 Sabouraud, Ann. de dermat. et de syph., 3, 1892, and 4, 1893.

644

PATHOGENIC MICROORGANISMS

Cultivation is comparatively simple and is best carried out upon
acid glucose agar or gelatin. Upon such media, within five or six days,
mycelial threads, which are septate and form chlamydospores, may be
observed. Pigment, reddish or brown, is occasionally noted. Gelatin
is liquefied. The disease may be produced with such cultures upon
guinea-pigs. In man, the disease is usually acquired by infection from
patient to patient.

Other Diseases in which Hyphomycetes have been Found. — A

number of cases have been described in which members of this group
have been found at autopsy in the lungs of persons dying of broncho¬
pneumonia.1 In most of these cases, the fungus found belonged to the
aspergillus group and was regarded as a merely secondary invader.
A few cases, however, have been reported in which the fungus was re¬
garded as the primary cause of the disease. A single case is on record,
in which an intestinal infection with a member of the genus mucor
resulted in a generalized infection with pulmonary and secondary
cerebral abscesses. In birds, a disease of the lungs has long been
known to be due to various species of aspergillus. In many domestic
animals, diseases of the skin and hair occur which are caused by micro¬
organisms similar to, or identical with, those occurring in man.

SPOROTRICHOSIS

Parasites closely allied to the blastomyces are the sporotrices which
were first described by Schenck 2 in this country and have been very
thoroughly studied by De Beurmann and Gougerot. The parasites
belong to the Fungi imperfecti. They occur in lesions as oval or cigar¬
shaped spores (conidia) and grow in culture as branching septate
mycelium with clusters of oval or spherical conidia about the ends of
the hyphse. According to some observers the conidia are attached
to the mycelium by short pedicles. The conidia also occur along the
sides of the hyphse and are often grouped in whorls about the threads.
Chlamydospores are also found in some cultures. The organisms are
obligate aerobes and grow on all ordinary culture media, but better on
those containing carbohydrates and of slightly acid reaction. The
growth forms a thick, leathery, white coating on the surface of the
medium which in older cultures becomes coffee-colored, and in some
instances black.

1 Sticker, Nothnagel, “Spez. Path. u. Ther.,” 14, 1900.

2 Schenck , Johns Hopkins Hosp. Bull., 1898, 286.

HYPHOMYCETES

645

De Beurmann and Gougerot 1 have described a number of species
of sporotrices* which are differentiated by variations in pigment pro¬
duction, in optimum temperature, and in profusion and morphology of
the conidia in culture. Other observers believe all these organisms
belong to the same species. The diagnosis may be made in some cases
by finding conidia in the softened material from the lesions. These are
best demonstrated by Gram’s stain. In other cases it is necessary to
resort to cultural methods, as the conidia can not always be found on
direct examination.

Only a few cases of the disease have been reported in this country, but
it is apparently common in France and has been reported in nearly
every quarter of the globe. The lesions are usually subcutaneous, but
visceral forms have been described. Numerous types of lesions are
found. The commonest forms are disseminated nodules which re¬
semble gummata. In other cases there are scattered subcutaneous
abscesses which are usually associated with lymphangitis. There is also a
papulo-vesicular form which usually leads to ulceration. The lesions
are chronic in character and simulate the lesions of syphilis or tuberculosis,
for which conditions many cases of sporotrichosis have probably been
mistaken. Nodular lesions have also been found in the bones, in
lymph nodes, and in the lungs and kidney. The lesions consist of foci
of chronic granulation, the tissue containing numerous giant cells,
which later undergo separation. There is as a rule little systemic
disturbance associated with the disease. The lesions often heal spon¬
taneously, leaving dense scars, but clear up very rapidly under iodide
therapy.

The most susceptible laboratory animals are mice and rats which
show lesions resembling those in man associated with marked cachexia,
though the disease is seldom fatal. The disease has also been produced
in rabbits, guinea-pigs, and dogs, though these animals are not sus¬
ceptible to all strains. In making cultures De Beurmann and Gougerot
recommend the use of Sabouraud’s glucose pepton agar (water, 1,000 c.c.;
pepton, 10 gm. ; glucose, 40 gm. ; agar, 18 gm. ; not neutralized) . Taylor 2
recommends glycerin agar with the addition of dextrose and 1 per cent
acetic or citric acid as the most favorable medium for these organisms.
Tubes should be inoculated with large amounts of pus (1 c.c. if pos¬
sible), and should be incubated for several days at room temperature.

1 De Beurmann et Gougerot , “Traite des Sporotrichoses/’ Felix Alcan, Paris, 1912.

2 Taylor, Jour. A. M. A., 1913, lx, 1142.

SECTION IV

DISEASES OF UNKNOWN ETIOLOGY

CHAPTER XLVII
RABIES

{Hydrophobia, Rage, Lyssa, Hundswuth)

Rabies is primarily a disease of animals, infectious for practically
all the mammalia, but most prevalent among carnivora, dogs, cats, and
wolves. It is said also to occur spontaneously among skunks of the
southwestern United States, and is readily inoculable upon guinea-pigs,
rabbits, mice, rats, and certain birds, chickens and geese being especially
susceptible. Man is subject to the disease. Infection usually occurs as
a consequence of the saliva of rabid animals gaining entrance to wounds
from bites or scratches. The disease is prevalent to an alarming extent
in all civilized countries except England, where the careful supervision of
dogs, enforcement of muzzling laws, and rigid legislation regarding the
importation of dogs, have caused a practical eradication of the disease
in that country. A fair estimate of the prevalence of the disease may
be obtained from the statistics of animals dying or killed because of
rabies in different countries. In Germany, according to Kolle and
Hetsch, during the fifteen years ending in 1901, there were 9,069 dogs,
1,664 cattle, 191 sheep, 110 horses, 175 hogs, 79 cats, 16 goats, 1 mule,
and 1 fox affected with rabies. In the eastern United States the dis¬
ease is not uncommon. The statistics of the New York Department
of Health, fora period of six months ending December 31, 1907, show
74 cases of rabies among dogs in the city of New York and vicinity.
Among human beings the disease is no longer common in civilized
countries, since early preventive treatment is successfully applied in
almost all infected subjects.

Experimental infection in susceptible animals is best carried out by .
injections of a salt-solution emulsion of the brain or spinal cord of an

646

RABIES

647

afflicted animal, subdurally, through a trephined opening in the skull,
but may also be accomplished by injection into the peripheral nerves,
the spinal canal, or the anterior chamber of the eye. Intravenous and
intramuscular injections are also successful, though less regularly so.

The time of incubation after inoculation varies with the nature of the
virus used, the location of the injection, and the quantity injected. In
accidental infections of man and animals the incubation is shortest and
the disease most severe when the wounds are about the head, neck, and
upper extremities and are deeply lacerated. This is explained by the
fact that the poison is conveyed to the central nervous system chiefly
by the path of the nerve trunks. This has been experimentally shown
by di Vestea and Zagari 1 who inoculated animals by injection into
peripheral nerves, and showed that the nerve tissue near the point of
inoculation becomes infectious more quickly than the parts higher up;
thus the lumbar cord of an animal inoculated in the sciatic nerve is in¬
fectious several days before virus can be demonstrated in the medulla.

In man, infected with “street virus,” that is, with the virus of a dog
or other animal not experimentally inoculated, the incubation period
varies from about forty to sixty days. Isolated cases have been reported
in which this period was prolonged for several months beyond this.

The virulence of rabic virus may be markedly increased or diminished
by a number of methods. By repeated passage of the virus through
rabbits, Pasteur 2 was able to increase its virulence to a more or less
constant maximum. Such virus which had been brought to the
highest obtainable virulence, he designated as “virus fixe.” Inocu¬
lation of rabbits, dogs, guinea-pigs, rats, and mice with slich virus
usually results in symptoms within six to eight days. The same animals
inoculated with street virus may remain apparently healthy for two to
three weeks.

In dogs and guinea-pigs inoculation usually results first in a stage
of increased excitability, restlessness, and sometimes viciousness. This
is followed by depression, torpor, loss of appetite, inability to swallow,
and finally paralysis. In rabbits the disease usually takes the form of
what is known as “dumb rabies,” the animals gradually growing more
somnolent and weak, with tremors and gradual paralysis beginning in
the hind legs.

In experimentally infected birds the disease is slow in appearing and

1 di Vestea and Zagari, Ann. de Pinst. Pasteur, iii.

2 Pasteur’s work on rabies. Compt. rend, de l’acad. des sciences, 1881, 1882, 1884,
1885, 1886, and Ann. de l’inst. Pasteur, 1887 and 1888.

648

DISEASES OF UNKNOWN ETIOLOGY

may show a course of gradually increasing weakness and progressive
paralysis extending over a period of two weeks after the appearance of
the first symptoms.

In man, the disease begins usually with headaches and nervous de¬
pression. This is followed by difficulty in swallowing and spasms of the
respiratory muscles. These symptoms occur intermittently, the free
intervals being marked by attacks of terror and nervous depression.
Occasionally there are maniacal attacks in which the patient raves and
completely loses self-control. Finally, paralysis sets in, ending event¬
ually in death.

Pathological examination of the tissues of rabid animals and human
beings reveals macroscopically nothing but ecchymoses in some of
the mucous and serous membranes. Microscopically, however, many
abnormal changes have been observed and were formerly utilized in
histological diagnosis of the condition. Babes1 has described a disap¬
pearance of the chromatic element in the nerve cells of the spinal cord.
This observation has been confirmed by others,2 but is no longer regarded
as pathognomonic of rabies. The same observer has described a
marked leucocytic infiltration which occurs about the blood-vessels of
the brain and about the ganglia of the sympathetic system. These
changes are not found in animals infected with virus fixe and are present
only in animals and human beings inoculated with street virus.

In 1903, Negri3 of Pavia described peculiar structures which he
observed in the cells of the central nervous system of rabid dogs. While
present in all parts of the brain, these “Negri bodies ” are most regularly
present and numerous in the larger cells of the hippocampus major and
in the Purkinje cells of the cerebellum. The presence of these structures
in rabid animals and man has been confirmed by a large number of
workers in various parts of the world, and the specific association of these
bodies with the disease is now beyond doubt. In consequence, the
determination of “Negri bodies” in the brains of suspected animals has
become an extremely important method of diagnosis — more rapid and
accurate than the methods previously known.

The demonstration of Negri bodies in tissues is carried out as follows:
A small piece of tissue is taken from the cerebellum or from the center
of the hippocampus major (cornu ammonis), and is fixed for twelve
hours in Zenker’s fluid. It is then washed thoroughly in water and

1 Babes, Virch. Arch., 110, and Ann. de l’inst. Pasteur, 6, 1892.

2 Van Gehuchten, Bull, de l’acad. de med. et biol., 1900.

3 Negri, Zeit. f. Hyg., xliii and xliv.

RABIES

649

dehydrated as usual in graded alcohols, embedded in paraffin, and
sectioned. The sections are best stained by the method of Mann, as
follows:

The sections, attached to slides in the usual way, are immersed in the follow¬
ing solution for from twelve to twenty-four hours :

Methylene-blue (Gruebler 00), 1 per cent . 35 c.c.

Eosin (Gruebler BA), 1 per cent . 35 c.c.

Distilled water . 100 c.c.

They are then differentiated in:

Absolute alcohol . 30 c.c.

Sodium hydrate, 1 per cent in absolute alcohol . 5 c.c.

In this solution blue is given off and the sections become red. After about five
minutes the sections are removed from this solution, are washed in absolute alco¬
hol, and are placed in water where they again become faintly bluish. It is of ad¬
vantage to immerse them, now, in water slightly acidified with acetic acid. Follow¬
ing this they are dehydrated with absolute alcohol and cleared in xylol, as usual.

In preparations made in this way, the nerve cells are stained a pale
blue, and in their cytoplasm, lying either close to the nucleus or near the
root of the axis-cylinder process, are seen small oval bodies stained a deep
pink. The bodies are variable in size, measuring from 1 to 27 micra in
diameter. They are round or oval, show a more deeply stained periph¬
eral zone which has been interpreted as a cell membrane, and, in their
interior, often show small vacuole-like bodies. There may be more than
one, often as many as three or four, in a single cell.

The rapid demonstration of Negri bodies in smears of brain tissue
has recently been advocated by many observers and has been extensively
used for diagnosis. It is carried out, according to Van Gieson,1 in the
following way: A small pin-head-sized piece of brain tissue from the
regions indicated above, is placed on one end of a slide under a cover-
glass and the cover is gently squeezed with the finger until the tissue is
flattened out into a thin layer. The glass cover is then gently shifted
across the slide until the brain tissue is smeared along the entire surface.
These smears may be fixed in methyl alcohol and stained by the Giemsa
method, as described in the chapter on Spirochseta pallida (see page
592).

Stained in this way, the Negri bodies are stained light blue, in con¬
trast to the darker and more violet cell-bodies.

1 Van Gieson , Proc. of N. Y. Pathol. Soc., N. S., iv, 1906.

650

DISEASES OF UNKNOWN ETIOLOGY

The smears may also be stained by a method originated by Van
Gieson, which gives an excellent contrast stain and reveals more clearly
the inner structure of the Negri bodies. Van Gieson’s stain is prepared

as follows :

Distilled water . . 10 c.c.

Saturated alcoholic solution of rosanilin violet . 2 drops.

Saturated aqueous solution of methylene-blue diluted one-half

with water . 2 drops.

This method has been modified by Williams and Lowden,1 who add
to 10 c.c. of distilled water 3 drops of saturated alcoholic basic fuchsin
and 2 c.c. of Loeffier’s methylene-blue. The slides are fixed in methyl
alcohol, washed in water, and covered with the freshly prepared stain.
The slide is held over the flame until the solution steams and is then
rinsed in water and dried. The Negri bodies assume a brilliant hue and
contain in their interior darkly stained, irregular particles which have
been interpreted as chromatin bodies. As to the nature of the Negri
bodies opinions are still divided. Their constant presence in rabic
brain tissue is unquestioned and their diagnostic significance well
established. Cultivation experiments, however, have been uniformly
unsuccessful. A number of observers, Negri himself, Calkins,2 Williams
and Lowden,3 and others, believe these bodies to be protozoa. The
last-named authors base this opinion upon the definite morphology of
the bodies, and their staining properties, which in many respects are
similar to those of protozoa. These observers also claim that the mor¬
phology of the bodies shows a number of regular cyclic changes which
are found accompanying different stages of the disease; these changes
correspond, according to these workers, to similar cycles occurring
among known protozoa of the suborders of the class Sporozoa. Many
pathologists still look upon them as specific degenerations of the nerve
cells similar to the changes observed by Babes.

It is not possible to decide absolutely from the facts at present at our
disposal whether or not the Negri bodies should be regarded as parasites
or as specific degeneration products. Their constant presence in rabic
animals, and their apparent absence from normal brains and the brains
of animals dead of other diseases, would tend to favor the parasitic
view. To us it would seem that added to this the clear outlines, apparent
regularity of structure, and curiously consistent grouping of the darkly

1 Williams and Lowden, Jour. Inf. Dis., 3, 1906.

2 Calkins, Discussion, Proc. N. Y. Pathol. Soc., N. S., vol. vi, 1906.

3 Williams and Lowden, loc. cit.

RABIES

651

staining inclusions would add weight to such an assumption. We have
triturated rabic tissue and shaken it up in anti-formin and seen many
free Negri bodies apparently enucleated from the cells in consequence.
Such complete extrusion from the cell also is seen in the ordinary smear
preparations. It is at least unlikely that a cell-degeneration area would
be expelled from the cytoplasm in so clearly outlined and morphologically
unaltered a form. The fact that the virus is filtrable, as shown by
Remlinger,1 Poor and Steinhardt,2 and others, would on the other hand
seem to contradict the etiological importance of the Negri bodies unless,
with some of the observers named, we assumed them to represent a
large stage in the life-cycle of a protozoan parasite, which also occurred
in smaller forms. It is a curious fact, also, that Negri bodies are
scarce or absent in the spinal cord and cerebrum, though these areas are
as virulent or more so than the hippocampus and cerebellum. They
are small and hard to find in virus fixe , largest and most plentiful in cases
in which the incubation period has been prolonged — as with street-virus
infection. Much can be said on both sides, but in analyzing the present
experimental facts, it seems fair to say that neither point of view is cer¬
tain, though the parasitic nature of the Negri bodies seems very likely.

The cultivation of parasites from rabic tissues has of course been
attempted by most bacteriologists who have studied the disease since
Pasteur. In all attempts, until very recently, either no results were
obtained or else the parasites described could be shown to be pres¬
ent because of extraneous contamination. Recently Noguchi an¬
nounced that he has been able to cultivate the virus by employing a
technique similar to his methods of cultivating Treponema pallidum and
poliomyelitis virus. Into high tubes filled with ascitic fluid a bit of fresh
sterile rabbit kidney and a small piece of rabic virus were placed. The
ascitic fluid was covered with sterile oil and the tubes incubated at
37.5° C. After five days’ incubation cloudiness appeared and, with it,
minute globoid bodies not unlike those seen in poliomyelitis. After
several generations large highly retractile bodies with dark central
spots appeared in the cultures, and these Noguchi3 regards as possibly
the parasites and similar to Negri bodies. Opinions are still divided as
to the significance of Noguchi’s results. However, whatever may be
one’s opinion regarding the nature of the peculiar bodies visible in his
cultures, he has accomplished the feat of preserving the virulence of the

1 Remlinger, Ann. de l’inst. Past., xvii, 1903.

2 Poor and Steinhardt, Jour, of Inf. Dis., xii, 1913.

3 Noguchi, Jour. Exp. Med., xviii, 1913.

652

DISEASES OF UNKNOWN ETIOLOGY

virus through 21 generations on artificial media, a fact which alone would
seem to prove that he had successfully cultivated it, even though his
“nucleated bodies” do not eventually turn out to be anything more
than cell degenerations. The possibility that he may have carried
original virus through 21 generations and that it has remained virulent
for about 100 days at 37.5° C. can not be excluded as yet, but seems
very remote.

The Specific Therapy of Rabies. — The treatment which is now pro-
phylactically applied to patients infected with or suspected of infection
with rabies has been but little altered either in principle or in technical
detail since it was first worked out by Pasteur. In principle it con¬
sists of an active immunization with virus, attenuated by drying, admin¬
istered during the long incubation period in doses of progressively
increasing virulence.

By the repeated passage of street virus through rabbits, Pasteur
obtained a virus of maximum and approximately constant virulence
which he designated as virus fixe. By a series of painstaking experi¬
ments he then ascertained that such virus fixe could be gradually at¬
tenuated by drying over caustic potash at a temperature of about 25°
C., the degree of attenuation varying directly with the time of drying.
Thus, while fresh virus fixe regularly caused death in rabbits after six to
seven days, the incubation time following the inoculation of dried virus
grew longer and longer as the time of drying was increased, until finally
virus dried for eight days was no longer regularly infectious and that
dried for twelve to fourteen days had completely lost its virulence.

The method of active immunization, which Pasteur used, consisted
in injecting, subcutaneously, virus of progressively increasing viru¬
lence, beginning with that derived from cords dried for thirteen days
and gradually advancing to a strong virus. Thus the patient was im¬
munized to a potent virus several weeks before the incubation time of
his own infection had elapsed. Pasteur successfully proved the efficacy
of his method upon dogs and finally upon human beings, the first
human case being that of a nine-year-old child — Joseph Meister.

Technique of Rabies Therapy. — The technique developed by
Pasteur is still, in the main, followed by those who treat rabies to-day.

I. As a preliminary, it is necessary to prepare or obtain virus fixe.
This may generally be procured from an established laboratory or may
be prepared independently by passing street virus through a series of
young rabbits (weighing from 700 to 1,000 gms.). According to Hogyes,1

1Hogyes, quoted from Kraus and Levaditi, “Handb.,” etc., I.

RABIES

653

the passage of the virus through twenty-one to thirty rabbits, in this
way, will reduce its incubation time to seven or eight days. Babes
claims to obtain a virus fixe more rapidly by passing the virus alter¬
nately through rabbits and guinea-pigs.

For purposes of inoculation, virus is prepared by emulsifying in
sterile salt solution pieces of the medulla or cerebellum of animals dead of

Fig. 153. — Method of Drying Spinal Cord of Rabbit for Purposes

of Attenuation.

a previous inoculation. The brain tissue which is not emulsified may be
preserved under sterile glycerin in a dark and cool place for further use.

II. Rabbits are inoculated with virus fixe by intracranial injection.
A small incision is made in the shaved scalp in the median line, and the
skin is retracted. With a small trephine or a round chisel, an opening
is made in the skull in the angle between the coronary and sagittal su¬
tures. Through this opening about 0.2 to 0.3 c.c. of the virus fixe is in¬
jected, either directly into the brain substance or simply under the dura.

As soon as a rabbit so inoculated has died it is autopsied. The
animal before dissection should be washed in a disinfectant solution
— lysol or carbolic acid. The skin is then removed and the animal,
lying on its ventral surface, is fastened to a dissecting board. The
spinal canal is then laid open with a pair of curved scissors and
the spinal cord carefully removed. This is accomplished by cutting
across the cord in the lumbar region, and lifting this with a forceps
while the nerve roots are divided from below upward.

The cord is suspended by a sterile thread within a large bottle into
the bottom of which pieces of potassium hydrate have been placed.
The bottle is then set away in a dark room or closet, the temperature of

654

DISEASES OF UNKNOWN ETIOLOGY

which is regulated so as to vary little above 25° C. Bacteriological
controls as to the sterility of the cord should also be made.

After a suitable period of drying, pieces of the cord are prepared
for injection. This is done in various ways at different laboratories.
No attempt at exact dosage is made. At the New York Depart¬
ment’ of Health 1 cm. of the cord is emulsified in 3 c.c. of sterile
eight-tenths per cent salt solution, the dose for injection being usu¬
ally 2.5 c.c. Marx 1 emulsifies 1 cm. of the cord in 5 c.c. of sterile
bouillon or salt solution, using 1 to 3 c.c. of this for injection according
to the age of the cord. For shipment an addition of 20 per cent of
glycerin and 0.5 per cent of carbolic acid is made.

The scheme of treatment is also subject to variations according to
the individual customs of various laboratories. The following scheme
is the routine of the Pasteur Institute in Paris, as quoted in Kraus
and Levacliti, “Handbuch fiir Immunitatsforschung,” Yol. I, p. 713.

Day of
Treatment.

Mild Cases.

Dose.

Medium Cases.

Dose.

Severe Cases.

Dose.

Days of
Drying.

Days of Drying.

14+13

3 c.c.

14 + 13

3 c.c.

A.M.14+ 13 p.m.12 + 11

3 c.c.

12+11

3 c.c.

12+11

3 c.c.

A. M. 10 + 9 P.M. 8+ 7

3 c.c.

10 + 9

3 c.c.

10 + 9

3 c.c.

A.M. 7 P.M. 6

2 c.c.

8 + 7

3 c.c.

8 + 7

3 c.c.

2 c.c.

6 + 6

3 c.c.

6 + 6

3 c.c.

2 c.c.

1 c.c.

2 c.c.

1 c.c.

2 c.c.

1 c.c.

2 c.c.

1 c.c.

2 c.c.

1 Marx, Dent. med. Woch., 1899, 1900.

RABIES

655

The treatment at the New York Department of Health is as follows:1

Day of
Treatment.

Mild Cases.

Medium Cases.

Severe Cases.

Days of Drying.

Dose.

Days of Drying.

Dose.

Days of Drying.

Dose.

14 + 13

4 c.c.

a.m.10 + 9 p.m.10 + 9

4 c.c.

12+11

4 c.c.

a.m. 8 + 7 p.m. 8 + 7

4 C:C.

10 + 9

4 c.c.

8 + 7

4 c.c.

8 + 7

4 c.c.

2 c.c.

4 •

2 c.c.

The severity or mildness of cases is estimated from the depth and
degree of laceration of the wounds, also from their location — bites about
the face and upper extremities being the most dangerous.

During the course of such treatment patients may show troublesome
erythema about the point of injection and occasionally backache and
muscular pains. Treatment need not be omittted unless these symp¬
toms become excessive.

The efficiency of the Pasteur treatment in rabies is no longer prob¬
lematical. According to Hogyes, 50,000 people have been treated within
ten years, with an average mortality of 1 per cent.

1 Personal communication from Dr. Poor, of the New York Department of Health.

656

DISEASES OF UNKNOWN ETIOLOGY

Although the method described above is the one which is extensively
used in all established institutes for the treatment of rabies, other
methods have been elaborated and used to a slight extent. One of the
most important of these is the “dilution method” of Hogyes. This
method is carried out as follows : A definite quantity of the spinal cord
of a rabbit dead of virus fixe is emulsified in 100 c.c. of normal salt
solution. Dilutions of this emulsion are made and the patient is injected
at first with a dilution of 1 : 1,000, subsequent injections being made of
gradually increasing concentration until a concentration of 1 : 100 is
reached. This method, so far as it has been used, has been satisfactory,
but it has not yet found extensive application.

Attempts to treat active rabies with the sera of immunized animals
have so far been unsuccessful.

CHAPTER XLVIII

SMALLPOX

Smallpox or variola is one of the most virulent of infectious diseases.
Throughout history it has been a severe scourge of mankind, prevailing
in China and other Eastern countries many centuries before Christ
and sweeping through medieval Europe, especially at the time of the
Crusades, in a series of severe epidemics. All races of men are suscep¬
tible and no age from childhood to senility is exempt. In modern times
the disease is endemic in most uncivilized countries, especially those of
the East, and occurs sporadically in all parts of the globe. Owing to
rigid enforcement of vaccination and of quarantine laws, however, the
disease has been practically eradicated from civilized countries.

The etiological factor which causes smallpox is still unknown.
Numerous researches aimed at the discovery of cultivatable microorgan¬
isms in the lesions or blood of infected patients have met with uniform
failure. Streptococci, though often found in the smallpox vesicles
and pustules, and often undoubtedly contributing materially to the fatal
outcome of the disease, may be regarded as purely secondary in signifi¬
cance.

Communications which have claimed the discovery of a protozoan
incitant of the disease have, on the other hand, been numerous and, in
some cases, have seemed plausible. Yet absolute proof has always been
lacking. The literature on this question is extensive and some of the
earlier contributions, such as those of Griinhagen,1 of Van der Loeff,2
and of Pfeiffer,3 possess historical interest only. The work which, of re¬
cent years, has attracted the most serious attention to this subject is
that published by Guarnieri4 in 1892. This observer found, in the deeper
cells of the epithelium covering the pustules, both of smallpox lesions
and of vaccination lesions, small bodies which were easily stained by
hematoxylin, safranin, or carmin. Similar bodies could be observed in

1 Griinhagen, Arch. f. Dermat. u. Syph., 1892.

2 Van der Loeff, Monat. f. prakt. Dermat., iv.

3 L. Pfeiffer, Zeit. f. Hyg., xxiii.

« Guarnieri, Arch, perle sc. med., xxvi, 1892; Cent. f. Bakt., I, xvi, 1894.

43 657

658

DISEASES OF UNKNOWN ETIOLOGY

the cells of corneal lesions experimentally produced in rabbits. Guar-
nieri claimed that he distinguished both cytoplasm and nucleus in these
bodies and described both binary division and reproduction by sporu-
lation as in the sporozoa. He named the supposed protozoan “Cy-
toryctes variolse.” At about the same time Monti 1 described similar
bodies in the cells of the Malpighian layer of the skin covering smallpox
lesions and, a few years later, Clarke 2 confirmed the researches of
Guarnieri. Subsequently, many researches were carried out on the
same subject in this country, the most notable being those of Council¬
man,3 Magrath and Brinckerhoff, and of Calkins.4 The former authors
came to the distinct conclusion that the bodies seen by Guarnieri were
parasites, and the latter author even described a distinct life-cycle
for these parasites comparable to that of some protozoa.

These researches, however, are by no means absolutely convincing,
and Ewing,5 while admitting that the vaccine bodies are probably
specific for variola, calls attention to the fact that specific cell-degen¬
erations or inclusions are found in diphtheria, measles, glanders, rabies,
and other infectious processes, which can not be regarded as in any
way related to these diseases etiologically, and suggests the probability
of a similar interpretation for the vaccine bodies. Much has been said
on both sides of the question since that time, and the problem can not
be regarded as settled. The burden of proof, of course, rests upon
those who claim the discovery of a specific microorganism, and absolute
proof will probably be lacking until our experimental methods are such
as will permit of other than purely morphological demonstration.

Whatever the actual causative agent may be, it is certain that the
disease is transmitted with extreme ease — actual contact, direct or in¬
direct, with a patient being unnecessary for its transmission. For this
reason the disease is often spoken of as being “air borne.” While we
have no certain knowledge of the portal of entry through which the virus
invades the human body, many considerations have made it seem plau¬
sible that this may take place through the mucosa of the upper respira¬
tory tract.

Our knowledge of the means of defense against the malady is for¬
tunately more advanced than is that of its etiology. It has been known

1 Monti, Cent. f. Bakt., I, xvi.

2 Clarke, Brit. Med. Jour., 2, 1894.

3 Councilman, Magrath, and Brinckerhoff, Jour. Med. Res., xi, 1904.

* Calkins, Jour. Med. Res., xi, 1904.

6 Ewing, Jour. Med. Res., xiii, 1905.

SMALLPOX

659

for centuries that one attack of smallpox protects against subsequent
attacks. This knowledge was made use of by the physicians of ancient
China and India, who, during mild epidemics, exposed healthy children
to infection, hoping that mild attacks would result which would confer
immunity. While dangerous in the extreme, such “variolation,” never¬
theless, was not without some benefit and was even introduced into
Europe in the eighteenth century by Lady Mary Wortley Montagu.

Such practices, however, were made unnecessary by the classical
investigations of Jenner 1 published in 1798. Jenner, as a student,
had been impressed with the fact that country-people who had been
infected with a disease known as cowpox, were usually immune against
smallpox. His studies and observations came to a practical issue when,
in 1796, he inoculated a boy, James Phipps, with pus from a cowpox
lesion on the hand of an infected dairy-maid. Two months later
the same boy was inoculated with material from a smallpox pustule
without subsequent disease. With this experiment the principles of
vaccination as in use at the present time wTere founded.

The question as to the identity of cowpox and smallpox has been
the basis of a long controversy. Many observers claimed from the be¬
ginning that the two diseases, though closely related to each other, were
essentially different. Others, on the contrary, and this seems to be the
prevailing opinion among scientists at the present day, maintain that
cowpox or vaccinia, as it is called when inoculated into a human being,
represents merely an altered and attenuated variety of variola. This
latter view is based on the following considerations, which we take from
Haccius as quoted by Paul.2

1. Variola is invariably transmissible to cattle, when proper methods
of inoculation are employed.

2. Variola carried through several animals, in the above way, be¬
comes altered in character, approaching in nature typical vaccinia or
cowpox.

3. Such virus, reinoculated into man, gives rise to purely local lesions
which are mild and unlike smallpox.

4. Inoculation with such virus protects both man and animals against
subsequent inoculation with cowpox, and, in the case of man, against
smallpox as well.

It has been claimed, moreover, that cowpox originally was trans-

1 Jenner, “Inquiry into the Causes and Effects of the Variola- Vaccinae,”
London, 1798.

2 Paul, “ Vaccination Kraus and Levaditi, “Handbuch,” etc., I.

660

DISEASES OF UNKNOWN ETIOLOGY

mitted to cattle by human beings affected with smallpox. This seems
likely both because of the comparative rarity of the former disease
and because of its spontaneous occurrence almost invariably upon the
teats of cows, although both males and females are equally susceptible
to experimental inoculation.

The relationship of variola to chicken-pox or varicella has been more
easily determined. Chicken-pox does not protect against smallpox nor
is this the case vice versa. The two diseases are unquestionably quite
distinct.

The Production of Vaccine. — During the early days of vaccination,
it was customary to inoculate human beings with the matter obtained
from the pustules of those previously vaccinated. While this method
was perfectly satisfactory for the immediate purposes in view, practical
difficulties and the occasional accidental transmission of syphilis have
rendered this practice undesirable. In consequence, at all institutes
at which vaccine is produced for use upon man, the virus is obtained
from animals. Horses and mules, both extremely susceptible to vac¬
cine, have been employed, and goats have, at times, been chosen because
of their insusceptibility to tuberculosis. Rabbits have also been used
more recently by Calmette and Guerin.1

The animals almost exclusively employed at the present day, how¬
ever, are calves, preferably at ages of from six months to two years.
Very young suckling calves are unsuitable because of the great speed of
development and small size of the lesions produced. The animals should
be healthy and at some institutes (Dresden) are subjected, before use,
to the tuberculin test; although, according to Paul,2 this produces a
hypersusceptibility to the vaccine, and can be omitted without danger
when careful supervision is observed. Some observers prefer to use light-
colored animals rather than dark-skinned or black ones, both for reasons
of greater ease of cleanliness and because the former are supposed to be
more susceptible than the latter. This contention is denied by others.
The sex of the animals seems to be immaterial.

During the period of use, the calves are fed, according to age, with
either an exclusive milk diet, or they are given, in addition, fresh hay.
The greatest cleanliness in regard to the bedding and stalls must be
observed and separate stables should be available for the animals under
treatment and those under observation before treatment. These stables,
if possible, should be so built that they can be easily scoured and flushed

1 Calmette and Guerin , Ann. de 1’inst, Pasteur, 1902,

2 Paul, loc. cit.

SMALL POX

661

with water, and stalls should be disinfected after occupation. If possible,
stables should be artificially heated and a comfortable temperature
maintained. Halters and fastenings should be so arranged that the
animals can not lick the scarified surfaces. Careful veterinary control
before vaccination and during the period of treatment must be observed
in order to eliminate animals with systemic disease or other complica¬
tions.

The calves may be vaccinated with material taken from previously
vaccinated animals. They may, on the other hand, be inoculated with
“seed virus” obtained from the vesicles of human vaccinia. This
method of using humanized virus for the inoculation of calves for
vaccine production is preferred by many workers and is spoken of
as “ retro vaccination.”

Actual vaccination of the animals is done as follows : Calves which
have been kept under observation for at least a week are thoroughly
washed and cleaned and the abdomen is clipped and shaved over an area
extending from the ensiform cartilage to the pubic region, including
the entire width of the belly and the inner folds of the thighs. It is
best to shave the animal a day of two before vaccination so as to avoid
fresh scratches and excoriations. Just before actual operation the
animal is strapped to a specially constructed operating table in such a
way as to allow free access to the shaved area. This area is now thor¬
oughly washed with soap and water followed by alcohol, or, in some
institutes, by a weak solution of lysol. If the latter is used, the field
of operation must again be thoroughly rinsed with sterile water. About
a hundred small scarifications are made in this area, preferably by
crossed scratches, covering for each scarification an area of about 3-4
square centimeters. Into these areas the virus is rubbed, using for each
small area a quantity about sufficient to vaccinate three children. Two
to three centimeter spaces are left between the lesions. The lesions are
then allowed to dry and may be covered with sterile gauze or, as in
Vienna,1 with a paste made up of beeswax, gum arabic, zinc oxid,
water, and glycerin. In some institutes the lesions are left entirely
uncovered.

Ordinarily within about twenty-four hours after vaccination a narrow
pink areola appears about the scratches. Within forty-eight hours the
scratches themselves become slightly raised and papular, and within four
or six days typical vaccinia vesicles have usually developed.

1 Paul, loc. cit.

662

DISEASES OF UNKNOWN ETIOLOGY

To obtain the vaccine from such lesions, the entire operative field is
carefully washed with warm water and soap, followed by sterile water.
In some cases two per cent lysol is employed, but must again be thor¬
oughly removed by subsequent washing with sterile water. Crusts, if
present, are then carefully picked off and the entire contents of the vesi-
icle, sticky serum, and pulpy exudate removed by the single sweep of a
spoon-curette. The curetted masses are caught in sterile beakers or
tubes and to them is added four times their weight of a mixture of glyce¬
rin fifty parts, water forty-nine parts, and carbolic acid one part.1 Ger¬
man workers prefer a mixture of glycerin eighty parts, and water twenty
parts, omitting the use of carbolic acid. The glycerinated pulp is allowed
to stand for three or four weeks in order to allow bacteria, which are
invariably present, to die out. After preservation for such a length of
time, moreover, thorough emulsification is obtained more easily than
when this is attempted immediately after curettage. At the end of
three or four weeks, the glycerinated pulp is thoroughly triturated,
either with mortar and pestle or by means of specially constructed trit¬
urating devices. Pulp so prepared should remain active for at least
three months if properly preserved in sealed tubes in a dark and cool
place.

From the serum oozing from the bases of the lesions, after curettage,
bone or ivory slips may be charged for vaccination with dry virus. The
glycerinated pulp is put up in small capillary tubes, sealed at both ends,
and distributed in this form. Park states that a calf should yield
about 10 grams of pulp (which when made up should suffice to vac¬
cinate one thousand five hundred persons), and, in addition, about two
hundred charged bone slips.

The virus may be tested for its efficiency by a variety of methods.
Calmette and Guerin 2 inoculate rabbits upon the inner surfaces of the
ears and estimate the potency of the virus from the speed of develop¬
ment and extensiveness of the resulting lesions. Guerin 3 has estimated
the potency of virus quantitatively by a method depending upon the
inoculation of rabbits with a series of dilutions. Beginning with a mix¬
ture containing equal weights of glycerin and vaccine pulp, dilutions are
made with sterile water ranging from 1 in 10 to 1 in 100. Rabbits
are shaved over the skin of the back and 1 c.c. of each of these dilu¬
tions is rubbed into the shaved areas. Fully potent virus should cause

1 Huddleston, quoted in Park, “ Pathogenic Bacteria,” N. Y., 1908.

2 Calmette and Guerin, Ann. de l’inst. Pasteur, 1902.

* Guerin, Ann. de l’inst. Pasteur, 1905.

SMALL POX

663

closely approximated vesicles in a dilution of 1 in 500, and numerous
isolated vesicles in a dilution as high as 1 in 1,000.

Quantitative estimations of the bacteria in the glycerinated virus
should be made by the plating method and the vaccine used only when
after several weeks of preservation the numbers of the bacteria have
been greatly diminished. In glycerinated pulp the bacteria will often
disappear entirely in the course of a month. The vaccine should also
be tested for the possible presence of tetanus bacilli, by the inoculation
of white mice.1

Vaccination of human beings is performed by slightly scarifying the
skin of the arm or leg with a sharp sterile needle or lancet and rubbing
into the lesion potent vaccine virus. The virus was formerly dried upon
wood, bone, or ivory slips and moistened with sterile water before the
operation. At the present day the glycerinated pulp is almost univer¬
sally employed.

That vaccination is of incalculable benefit to the human race is no
longer a question of opinion, and opposition to the practice is explicable
only on the basis of ignorance. Statistical compilations upon this point
are very numerous. It may suffice to select from the voluminous
literature a single example, taken from Jiirgensen, which embodies the
statistics of death from smallpox in Sweden, during the periods immedi¬
ately preceding and following the introduction of vaccination. In that
country the first vaccination was done in 1801. By 1810 the practice
was generally in use but not enforced. In 1816 it was legally enforced.
The years from 1774 to 1855 can thus be divided into three periods.

1. Pre vaccinal period, 1774—1801 (25 years). Deaths smallpox per

million inhabitants . 2,050

2. Transitional period, 1801-1810 (9 years) . 680

3. Vaccination enforced, 1810-1855 (35 years) . 169

Prevaccinal period death rate 20.00 per mille.

Vaccinal period death rate 0.17 per mille.

In considering the benefit of vaccination it must not be forgotten
that revaccination is quite as important as the first vaccination, which
confers immunity only for from seven to ten years. A child should there¬
fore be vaccinated soon after birth or at least before the eighth month,
and the process should be repeated every seven years thereafter.

1 Paul, loc. cit.

CHAPTER XLIX

ACUTE ANTERIOR POLIOMYELITIS

The disease known as acute anterior poliomyelitis has long been
recognized as an acute infectious condition, both because of the charac¬
teristics of its clinical manifestations and of its epidemic occurrence.
For these reasons it was classified with acute infectious diseases by
Marie and by Strumpell long before any experimental evidence of in¬
fection was obtainable.

Its contagiousness, while not a proven fact, seemed very likely from
the evidence of its mode of spreading and has been removed from the
sphere of mere conjecture by the careful study of a Swedish epidemic,
comprising one thousand cases, made by Wickman.1

While acute anterior poliomyelitis is almost exclusively a disease of
childhood, it is assumed by clinicians that it is etiologically closely re¬
lated to, possibly identical with, certain diseases of the adult, character¬
ized by bulbar paralysis and acute encephalitis. Into this category,
also, some observers place the condition known as “ Landry’s paralysis.”
The basis for the identification of these conditions with poliomyelitis
lies chiefly in the similarity of the pathological lesions and upon the
fact that the last-named diseases occur most often during the course of
poliomyelitis epidemics.

In consequence of the emphatically expressed opinion as to the infec¬
tious nature of acute poliomyelitis, the efforts to isolate specific micro¬
organisms from cases have been many, and numerous microorganisms
have been described as the causative agents of this disease. The out¬
come of all these investigations has been purely negative and the infec¬
tious agent of acute poliomyelitis still remains undiscovered.2

An important advance in the study of this disease was made in 1908
when Landsteiner and Popper3 succeeded in transmitting it to two
monkeys (Cyanocephalus hamadryas and Macacus rhesus) . The trans-

1 Wickman, quoted from Landsteiner and Popper, Zeit. f. Immunitatsforch., ii,
1909.

2 For literature, see Landsteiner and Popper, loc. cit.

* Loc. cit.

664

ACUTE ANTERIOR POLIOMYELITIS

665

mission was accomplished by intraperitoneal injections of a saline emul¬
sion of the spinal cord of a child that had died during the fourth day
of an attack of infantile paralysis — during the stage of acute fever. The
first monkey injected became severely ill six days after the injection and
died on the eighth day. The second animal became paralyzed seventeen
days after the injection and was killed two days later. Cultural experi¬
ments with the substance injected were negative, as were also inocula¬
tion experiments carried out upon guinea-pigs, rabbits, and mice. The
histological lesions produced in the inoculated monkeys were similar
to those occurring in children afflicted with the disease.

An attempt to transmit the disease to another monkey with spinal-
cord substance of the animal that was killed resulted negatively.

Soon after the successful experiments of Landsteiner and Popper, a
similar result was recorded by Knoepfelmacher.1 An attempt to trans¬
mit the disease from monkey to monkey was again negative.

Similar positive inoculation results were published, a little later than
this, by Flexner and Lewis 2 in November, 1909, and by Strauss and
Huntoon 3 in January, 1910.

Flexner and Lewis, in their work, moreover, succeeded in trans¬
mitting the disease through several inoculation-generations of monkeys,
proving thereby that successful inoculation did not depend merely
upon the transfer of an unorganized toxic body, but was due to a true
infection. The same workers 4 have ascertained that inoculation may
be successfully applied not only by the intraperitoneal route but intra-
cerebrally, subcutaneously, intravenously, and by the path of the larger
nerves. They also proved that not only the brain and cord of afflicted
animals contains the virus, but that this may be found, during the
early days of the disease at least, in the spinal fluid, the blood, the
nasopharyngeal mucosa, and fymph nodes near the site of inoculation.

Landsteiner and Levaditi,5 meanwhile, experimenting with the virus
independently, succeeded in transferring the disease from one animal to
others, demonstrated that the virus could pass through the pores of a
Berkefeld filter, and showed that the virus was present in the salivary
glands — a fact which may prove of great importance in possibly estab-

1 Knoepfelmacher , Mediz. Klinik, v, 1909.

2 Flexner and Lewis, Jour. Am. Med. Assn., 53, 1909.

3 Strauss and Huntoon, N. Y. Med. Jour., Jan., 1910.

* Flexner and Lewis, Jour. Exp. Med., 12, 1909.

« Landsteiner and Levaditi, Comptes rend, de la soc. de biol., Nov., 1909, and
Dec., 1909.

666

DISEASES OF UNKNOWN ETIOLOGY

lishing a clew to the mode of contagion among human beings. The same
authors, as well as Flexner and Lewis, were able to show that the virus
was preservable under glycerin for as long as ten days and retained its
virulence for from seven to eleven days when dried.

According to Flexner and Lewis the virus remains active, when
frozen, for as long as forty days, but is extremely sensitive to heat,
being destroyed by a temperature of from 45° to 50° C. maintained for
thirty minutes.

Experiments aimed at the isolation or even morphological detection
of a parasite in the virulent material have been entirely without success
until recently. Bacteria which in the past have been isolated from
nerve substance and spinal fluid in cases of poliomyelitis can of course
be excluded from etiological significance by the recent determination of
the filtrability of the virus as determined by Flexner and Lewis, and
Landsteiner and Levaditi. Small coccoid forms in smears from the
nerve tissue recently described by Proescher1 are of very uncertain
significance. The clouding of ascitic fluid after an incubation with
poliomyelitis nerve substance has been found to be due to protein pre¬
cipitation. The most important contribution which has been made in
the solution of this problem is that of Flexner and Noguchi.2 These in¬
vestigators placed small bits and emulsions of the brain of monkeys,
dead of poliomyelitis, into high tubes containing human ascitic fluid
together with a piece of fresh sterile rabbit kidney. In all essentials the
method was that followed by Noguchi in his cultivation of Treponema
pallidum, except that in the case of poliomyelitis anaerobic pus was
unnecessary. It sufficed to cover the ascitic fluid with a layer of sterile
alboline. It was necessary to use fresh unheated ascitic fluid. Heat
sterilization rendered it unsuitable.

By this method, after five days opalescence appeared about the
pieces of tissue. This increased until the tenth day when sedimenta¬
tion began. Microscopical examination by Giemsa’s method of stain¬
ing revealed small globoid bodies measuring from 0.15 to 0.3 micron in
diameter, arranged in pairs, short chains, and masses. Similar bodies
could later be found in poliomyelitis tissues. Cultures were obtained
from glycerinated as well as from fresh virus and from the filtered as well
as the unfiltered material. Typical lesions and death have been pro¬
duced in monkeys with such cultures even after the eighteenth genera¬
tion on artificial media.

1 Proescher, N. Y. Med. Jour., 1913.

2 Flexner and Noguchi, Jour, of Exp. Med., xviii, 1913.

ACUTE ANTERIOR POLIOMYELITIS

667

We have few data which throw light upon possible immunity to the
disease. Repeated attacks of the disease in the same human being
have not been noted; but this, as Flexner and Lewis point out, may be
due to the fact that the epidemics are rare, and individuals once afflicted
have passed beyond the susceptible age by the time of the second
epidemic. As a matter of fact, however, these workers have not suc¬
ceeded in reinfecting monkeys that had recovered, and incline to the
belief that one attack protects against subsequent infections.

Up to the present time monkeys and rabbits only have responded to
experimental inoculation; numerous attempts made upon a variety of
other animals have been without success.

In chickens a disease has been observed similar in many ways to
poliomyelitis, but further study has shown this to be a polyneuritis of
entirely different nature from infantile paralysis.

Of other animals besides monkeys, rabbits only have been success¬
fully inoculated with this disease. Transmission to these animals was
first reported by Kraus and Meinicke 1 and later by Lentz and Hunte-
muller.2 Marks 3 has studied the disease in rabbits thoroughly, and con¬
cludes that there is no doubt that the virus can be cultivated through
a limited number of generations in rabbits. He was able to transmit
to monkeys from rabbit material. The disease, however, does not
resemble that of man or monkeys clinically and no definite lesions of
the central nervous system are present. The rabbits seem perfectly
well for six or seven days, when rapid weakness and death in convulsions
occur.

1 Kraus und Meinicke, Deut. med. Woch., xxxv, 1909.

2 Lentz und Huntemuller, Zeitschr. f. Hyg., lxvi, 1910.

3 Marks, Jour, of Exp. Med., xiv, 1911.

CHAPTER L

YELLOW FEVER

Yellow fever is an acute infectious disease which prevails endemi-
cally in the tropical countries of the Western Hemisphere, but occurs also
along the western coast of Africa and has exceptionally appeared, in
epidemic invasions, in the north temperate United States and Europe.
Guiteras, as quoted by Osier, classifies the distribution of the disease into
three areas of infection.

1. The area in which the disease is never absent, including tropical
South American ports and Havana.

2. The area of periodic epidemics, including sea-ports of the tropical
Atlantic in America and Africa.

3. The area of accidental epidemics, extending from parallel 45°
north latitude to 35° south latitude. In the United States severe epi¬
demics have frequently occurred in Louisiana, Mississippi, and Alabama,
and occasional but severe epidemics have occurred in Philadelphia and
Baltimore.

The disease occurs spontaneously only in man, and experimental
inoculation of lower animals has been successful only in the chimpanzee
in a single case reported by Thomas.1

In man afflicted with the malady the clinical picture is one of a rapidly
developing fever with severe gastrointestinal symptoms, vomiting of
blood, albuminuria, and often active delirium. The mortality is usually
high, often reaching eighty per cent or more in the severe epidemics.

Etiology and Method of Transmission. — The actual infective agent
which causes yellow fever is, as yet, unknown. Numerous researches
have been aimed at the elucidation of the problem, and microorganisms,
for which etiological significance was claimed, have been isolated from
the dejecta, the vomitus, and the secretions of afflicted patients. None
of these claims has been supported by convincing proof and none of
them has found subsequent confirmation.

A few of these claims only have historical importance because of the

1 Thomas, Brit. Med. Jour., 1, 1907.

668

YELLOW FEVER

669

widespread interest they aroused among bacteriologists. Cornil and
Babes/ in 1883, described chained cocci to which they attributed etio¬
logical significance, but their contentions have remained entirely un¬
confirmed. Sternberg,1 2 in 1897, described a colon-like organism, “bacil¬
lus X,” for which he made very conservative claims, which he himself,
later, withdrew.

The most active discussion was aroused by the announcement of
Sanarelli,3 in 1897, that he had discovered, in the blood and tissues of
patients dead of yellow fever, a Gram-negative bacillus, which he be¬
lieved to be the etiological agent of the disease. He based his contention
upon the facts that he had isolated the organism from seven cases of
yellow fever, had produced symptoms similar to the disease of the human
being by the inoculation of pure cultures into dogs, and had obtained
agglutination of the bacillus in the serum of convalescent patients. Later
he inoculated five human beings subcutaneously with sterilized cultures
of this “Bacillus icteroides,” and obtained symptoms which he believed
simulated closely those of yellow fever. The claims of Sanarelli at first
found much apparent confirmation, but later work by Durham and
Myers,4 Otto,5 Agramonte,6 and others has definitely refuted his original
claims, and there is to-day no scientific basis for the assumption that
the Bacillus icteroides has any etiological relationship to the disease.
Protozoan incitants, also, have been described by Klebs,7 Schuller,8
Thayer,9 and others, without bringing conviction or even justifying
extensive investigation of their claims.

While thus the causative agent of yellow fever remains undiscovered,
some of its biological properties are known. Reed, Carroll, Agramonte,
and Lazear10 were able to show that the infecting agent is present in
the blood serum of patients during the first three days of the disease
and that it could pass through the pores of Berkefeld filters. Such
filtered serum remained infectious for human beings — a fact which de¬
monstrates that the incitant is extremely small and possibly ultra-

1 Cornil and Babes, Comptes rend, de Facad. des sci., 1883.

2 Sternberg, Cent. f. Bakt., I, xxii, 1897.

3 Sanarelli, Ann. de V inst. Pasteur, 1897, and Cent. f. Bakt. , I, xxii, xxvii, and xxix.

4 Durham and Myers, Thompson Yates Laboratory Reports 3 1902.

5 Otto, Vierteljahrsch. f. gericht. Medizin, etc., 27, 1904.

6 Agramonte, N. Y. Med. News, 1900.

i Klebs, Jour. Am. Med. Assn., April, 1898.

8 Schuller, Berl. klin. Woch., 7, 1906.

9 Thayer, Med. Record, 1907.

10 Reed, Carroll, Agramonte, and Lazear , Phila. Med. Jour., 1900,

670

DISEASES OF UNKNOWN ETIOLOGY

microscopic. Blood serum, filtered or unfiltered, becomes non-infectious
when heated to 56° C. for ten minutes.

Mode of Transmission. — Until comparatively recent years the mode
of transmission of yellow fever was not understood and many erroneous
theories were prevalent. It was supposed that yellow fever was conta¬
gious, and transmitted from person to person by direct or indirect con¬
tact with those afflicted or by fomites. The first to make the definite
assertion that yellow fever was transmitted by the agency of mosquitoes
was Carlos Finlay. Finlay,1 as early as 1881, advanced the theory that
mosquitoes were responsible for the transmission of this disease and, fur¬
thermore, recognized “ Stegomyia fasciata” or “Stegomyia calopus” as
the guilty species. Finlay’s opinion, although later proved to be correct,
was at first based only upon such circumstantial evidence as the corre¬
spondence of the yellow-fever zones with the distribution of this species
of mosquito and the great prevalence of mosquitoes at times during
which epidemics occurred. His theory was, therefore, received with
much skepticism and was neglected by scientists until its revival in
1900, when the problem was extensively investigated by a commission
of American army surgeons.

Reed, Carroll, Agramonte, and Lazear were the members of this
commission. The courage, self-sacrifice, and scientific accuracy which
characterized the work of these men have made the chapter of yellow
fever one of the most brilliant in the annals of American scientific
achievement.

Their work was much facilitated by the experience of Gorgas 2 and
others, who had demonstrated the absolute failure of ordinary sanitary
regulations to limit the spread of yellow fever.

They began their researches by investigating carefully the validity
of Sanarelli’s claims as to the etiological significance of his “Bacillus
icteroides.” The results of this work yielded absolutely no basis for
confirmation.

They then proceeded to investigate the possibility of an intermediate
host.

In August, 1900, the commission began its work on this subject by
allowing mosquitoes,3 chiefly those of the stegomyia species, to suck

1 Finlay, Ann. Roy. Acad. d. Havana, 1881.

2 Gorgas, Jour, of Trop. Med., 1903.

3 Reed, Carroll, Agramonte, and Lazear, Phila. Med. Jour., Oct., 1900; also Am.
Pub. Health Assn: Rep., 1903; Agramonte, N. Y. Med. News, 1900; Reed, Jour, of
Hygv 1902; Reed, Carroll, and Agramonte, Am. Medicine, July, 1901. Boston Med.

YELLOW FEVER

671

blood from patients, later causing the same insects to feed upon normal
susceptible individuals. The first nine experiments were negative.
The tenth, of which Carroll was the subject, was successful. Four days
after being bitten by the infected insect Carroll became severely ill with
an attack of yellow fever, by which his life was endangered, and from
the effects of which he died several years later.

On the 13th of September, Lazear, while working in the yellow-fever
wards, noticed that a stegomyia had settled upon his hand, and deliber-

Fig. 155. — Stegomyia. Fasciata.

(6)

(a) Female. ( b ) Male. (After Carroll.)

ately allowed the insect to drink its fill. Five days later he became ill
with yellow fever and died after a violent and short illness.

With these experiences as a working basis, the commission now
decided upon a more systematic and thoroughly controlled plan of
experimentation.

In November of the same year, 1900, an experiment station, Camp
Lazear,” was established in the neighborhood of Havana, about a mile
from the town of Quemados. The camp was surrounded by the strictest
quarantine. Volunteers from the army of occupation were called for,
and twelve individuals were selected for the camp, three immunes and
nine non-immunes. Two of the latter were physicians. The immunes

and Surg. Jour., 14, 1901; Carroll , Jour. Am. Med. Assn., 40, 1903; Carrol , “ Yellov
Fever ” in Mense, “ Handbuch der Tropen-Krankheiten,” ii.

G72

DISEASES OF UNKNOWN ETIOLOGY

and the members of the commission only were allowed to go in and out.
All non-immunes who left the camp were prohibited from re-entering and
their places taken by other non-immune volunteers. During December,
five of the non-immune inmates were successfully inoculated with yel¬
low fever by means of infected mosquitoes. During January and Febru¬
ary five further successful experiments were made. Clinical observa¬
tions were made by experienced native physicians, Carlos Finlay among
them, and the patients, as soon as they were unquestionably ill with
yellow fever, were removed to a yellow-fever hospital. This was done
to prevent the possibility of the disease spreading within the camp it¬
self. The mosquitoes used for the experiments were all cultivated from
the larva and kept at a temperature of about 26.5° C.

A further important experiment was now made A small house was
erected and fitted with absolutely mosquito-proof windows and doors.
The interior was divided by wire mosquito netting into two spaces. With¬
in one of these spaces fifteen infected mosquitoes were liberated. Seven
of these had fed upon yellow-fever patients four days previously; four,
eight days previously; three, twelve days previously; and one, twenty-
four hours previously. A non-immune person then entered this room
and remained there about thirty minutes, allowing himself to be bitten
by seven mosquitoes. Twice after this the same person entered the
room, remaining in it altogether sixty -four minutes and being bitten fif¬
teen times. After four days this individual came down with yellow fever.

In the other room two non-immunes slept for thirteen nights with¬
out any evil results whatever.

It now remained to show that mosquitoes were the sole means of
transmission and to exclude the possibility of infection by contact with
excreta, vomitus, or fomites. For this purpose another mosquito-proof
house was constructed. By artificial heating its temperature was kept
above 32.2° C. and the air was kept moist by the evaporation of water.
Clothing and bedding, vessels, and eating utensils, soiled with vomitus,
blood, and feces of yellow-fever patients were placed in this house and
three non-immune persons inhabited it for twenty days. During this
time they were strictly quarantined and protected from mosquitoes.
Each evening, before going to bed, they unpacked and thoroughly
shook clothing and bedding of yellow-fever patients, and hung and
scattered these materials about their beds. They slept, moreover, in
contact with linen and blankets soiled by patients. None of these
persons contracted yellow fever. The same experiment was twice re¬
peated by other non-immunes, in both cases with like negative results.

YELLOW FEVER

673

All ol the non-immunes taking part in these experiments were
American soldiers. Four of them were later shown to be susceptible
to yellow fever by the agencies of mosquito infection or bloocl-injection.

The results obtained by the investigations of this commission may
be summarized, therefore, as follows:

Yellow fever is acquired spontaneously only by the bite of the
Stegomyia fasciata. It is necessary that the infecting insect shall have
sucked the blood of a yellow-fever patient during the first four or five
days of the disease, and that an interval of at least twelve days shall
have elapsed between the sucking of blood and the reinfection of an¬
other human being. Sucking of the blood of patients advanced beyond
the fifth day of the disease does not seem to render the mosquito infec¬
tious, and at least twelve days are apparently required to allow the para¬
site to develop within the infected mosquito to a stage at which rein¬
fection of the human being is possible.

The results of the American Commission were soon confirmed by
Guiteras 1 and by Marchoux, Salimbeni, and Simond.2 These latter ob¬
servers, moreover, confirmed the fact that infection could be experi¬
mentally produced by injections of blood or blood serum taken from
patients during the first three days of the disease. They showed that
blood taken after the fourth day was no longer infectious: that 0.1 c.c.
of serum sufficed for infection and finally that no infection could take
place through excoriations upon the skin. They furthermore confirmed
the observation of Carroll that the virus of the disease could pass through
the coarser Berkefeld and Chamberland filters,-— passing through a
Chamberland candle “ F ” but held back by the finer variety known as “ B.”

The fundamental factors of yellow-fever transmission thus discovered,
we are in possession of logical means of defense. The most important
feature of such preventive measures must naturally center upon the
extermination of the transmitting species of mosquito.

Stegomyia fasciata or calopus is a member of the group of “ Culi-
cidse.” It is more delicately built than most of the other members of
the group culicidae, is of a dark gray color, and has peculiar thorax-
markings which serve to distinguish it from other species. The moie
detailed points of differentiation upon which an exact zoological recog¬
nition depends are too technical to be entered into at this place.
Briefly described, they consist of lyre-like markings of the back,

1 Guiteras, Rev. d. med. trop., Jan., 1901, and Am. Med., 11, 1901.

2 Marchoux, Salimbeni. and Simond, Ann. de l’inst. Pasteur, 1908.

674

DISEASES OF UNKNOWN ETIOLOGY

unspotted wings, white stripes and spots on the abdomen, and band¬
like white markings about the metatarsi and tarsi of the third pair
of legs. The peculiar power of transmitting yellow fever possessed
by this species is explained by Marchoux and Simond 1 by the fact
that Stegomyia fasciata is unique among culicidse in that the female
lives for prolonged periods after sucking blood. Among other species
— Culex fatigans, Culex confirmatus, and most others — the female lays
its eggs within from two to eight days after feeding on blood and rarely
lives longer than the twelfth day — the time necessary for the develop¬
ment of the yellow-fever parasite.

The limitation of yellow fever to tropical countries 2 is explained by
the fact that stegomyia develops only in places where high tempera¬
tures prevail. The optimum temperature for this species lies between
26° and 32° C. At 17° C. it no longer feeds, and becomes practically
paralyzed at 15° C. In order to thrive, the species requires a temperature
never going below 22° C. at night and rising regularly above 25° C.
during the day. The females only are dangerous as sources of infection.
The insect, like Anopheles, has the peculiarity of feeding chiefly at night.

Experiments done by Reed, Carroll, Agramonte, and Lazear,3 to
ascertain whether the power of infecting was hereditarily transmissible
from the mosquito to following generations, were negative. A positive
result, however, has been reported by Marchoux and Simond.4 This
question must still await more extensive research.

Immunity. — Natural immunity against yellow fever was formerly
assumed to exist in the negro race. More recent investigations have
not borne out this assumption. The negro soldiers of the American
army in Cuba were afflicted equally with the white troops. The rela¬
tive immunity of dark-skinned races, however, is explained possibly
by the fact that the stegomyia prefers to attack light-colored surfaces.

A single attack seems to protect against subsequent infection
throughout life.

Artificial immunization has, so far, been unsuccessful. Relative
immunity was produced, however, by Marchoux, Salimbeni, and
Simond, by injections of the serum of convalescents, serum heated to
55° C., and of defibrinated blood preserved for eight days in vessels
sealed with vaseline.

1 Marchoux and Simond, Ann. de Tinst. Pasteur, 1906.

2 Otto, in Kolle und Wassermann, “Handbuch,” etc., 11, Erganzungsband.

8 Loc. cit.

4 Marchoux and Simond, Comptes rend, de la soc. de biol., 59, J905.

CHAPTER LI

MEASLES, SCARLET FEVER, TYPHUS FEVER, AND FOOT-

AND-MOUTH DISEASE

MEASLES

The causative agent of measles is unknown to the present day,
and it would be a thankless task to review the literature of the many
attempts to isolate microorganisms from this disease, none of which
has resulted in throwing any light on the etiology.

Attempts to produce the disease experimentally have frequently
been made, the earliest recorded being those of Home of Edinburgh,
published in 1759. 1 Home took blood from the arms of patients afflicted
with measles, and caught it upon cotton, and inoculated normal in¬
dividuals by placing this blood-stained cotton on to wounds made in
the arm. Home claimed that in this way he produced measles of a
modified and milder type in fifteen individuals. Home’s results, how¬
ever, while at first accepted, were assailed by many writers and it is
by no means certain that the disease produced by him was really measles.

A number of other observers after Home attempted experimental
inoculation of this disease, and positive results were reported by Stewart
of Rhode Island (1799), Speranza of Mantua (1822), Katowa of Hungary
(1842), and McGirr of Chicago (1850).

The experiments of all these early writers, however, are unsatisfac¬
tory, owing to the necessarily unreliable technique of their methods.

In 1905, Hektoen2 succeeded in experimentally producing the dis¬
ease in two medical students by subcutaneous injection of blood taken
from measles patients at the height of the disease (fourth day). The
experiments were carefully carried out and the symptoms in the sub¬
jects were unquestionable. They demonstrated beyond doubt that the
virus of the disease is present in the blood. Attempts at cultivation
carried out with the same blood were entirely negative. It was also
shown by Hektoen’s experiments that the virus of measles may be kept
alive for at least twenty-four hours when mixed with ascitic broth.

1 Home , “ Medical Facts and Experiments,” Edinburgh, 1759.

2 Hektoen , Jour. Inf. Dis., ii, 1905.

675

676

DISEASES OF UNKNOWN ETIOLOGY

SCARLET FEVER

{Scarlatina)

The etiology of scarlet fever, like that of measles, is still obscure.
Streptococci have been found with striking regularity in the throats of
scarlet-fever patients, and a large number of investigations have seemed
to furnish evidence for the etiological relationship of these microorgan¬
isms with the disease. According to von Lingelsheim, Crooke as early
as 1885 demonstrated the presence of streptococci in the cadavers of
scarlet-fever victims. Baginsky and Sommerfeld 1 in 1900 examined a
number of scarlatina cases with reference especially to streptococcus
infection, and reported the presence of streptococci in the heart’s
blood of eight patients who had died after a very acute and short
illness. They expressed the belief that the acuteness of the illness and
the rapidity of death in these cases precluded the possibility of the
streptococci being merely secondary invaders. A large number of
other observers have expressed similar opinions, but we can not, as
yet, justly conclude that streptococci are actually the etiological
agents in this disease.

Class 2 in 1899 described a diplococcus which he cultivated from a
large number of scarlatina patients and with which he was able to pro¬
duce exanthemata and acute fever in pigs. Subsequent investigations
seem to show that Class was really working with a streptococcus.

Moser,3 working in Escherich’s clinic, has recently reported the very
favorable influence upon the course of scarlet fever of polyvalent
streptococcus antisera. This is not really very strong evidence in favor
of the streptococcus etiology of the disease, since there is, of course, no
doubt that streptococcus infection complicates the disease, and it is
to be expected that antistreptococcus serum should, therefore, benefit
the patient’s condition by combating this complication.

Mallory 4 in 1904 published observations on four scarlatina cases on
which he bases the belief that scarlatina is caused by protozoa. In
the skin, between the epithelial cells, he found small bodies which were
easily stained with methylene-blue and which because of their arrange-

1 Baginsky and Sommerfeld , Berl. klin. Woch., 1900.

2 Class, Phila. Med. Jour., iii, 1899.

3 Moser, quoted by Escherich, Wien. klin. Woch., xxiii, 1903.

* Mallory, Jour. Med. Research, x, 1904,

TYPHUS FEVER

677

ment and form he interpreted as parasites not very unlike the plasmo-
dium of malaria. Subsequent investigations of Field 1 and others have
failed to substantiate Mallory’s conclusions.

TYPHUS FEVER

Typhus fever is an infectious disease which is characterized by an
incubation time of 5 days or more, high temperature, and a petechial
rash. It has been characterized as peculiarly a disease of filth and
has epidemically disappeared in most of the civilized countries, al¬
though it is still endemic in certain parts of Europe, North and South
America, and occurs epidemically in Mexico under the name of Tabar-
dillo. In New York it has recently been found to exist not infrequently.
It was described as a new clinical entity by Brill, and has been spoken
of as Brill’s disease, but the work of Anderson and Goldberger has
shown that Brill’s disease is identical with typhus fever. Great ad¬
vances have been made in the knowledge of the disease during the last
few years.

In 1909, Nicolle2 successfully inoculated an anthropoid ape, and
Anderson and Goldberger 3 in the same year succeeded in inoculating
lower monkeys, rhesus and capuchin. Similar successful monkey in¬
oculations were made by Ricketts and Wilder,4 by Gavino and Girard.5
In these animals inoculation with blood from active cases is followed
by a rapid rise of temperature after an incubation time of 5 days or
more, and the fever remains high for 3 to 5 days, after which it comes
down by lysis. Occasional recrudescences have been noticed in monkeys.
Goldberger and Anderson have had a mortality of 2 per cent in their
monkeys. The disease may be transmitted from monkey to monkey
with the blood, which is infectious during the febrile period and may be
so for as long as 32 hours after the temperature returns to normal.
Ricketts and Wilder have described short bacilli looking like organisms
of the haemorrhagic septicaemia group in smears from the blood, but
have not been able to cultivate them. Very recently Plotz 6 has reported

1 Field , Jour. Exper. Med., vii, 1905.

2 Nicolle, Compt. rend. Acad. d. Sc., 1909, p. 157; Ann. de Pinst. Past., 1910,
1911, 1912.

3 Anderson and Goldberger, Jour. A. M. A., 1912, p. 49; Jour. Med. Res., 1910,
p. 469; N. Y. Med. Jour., 1912, p. 976.

4 Ricketts and Wilder, Jour. A. M. A., Feb., 1910, p. 463; ibid., April 16, 1910,
p. 1304; ibid., April 23, 1910, p. 1373; ibid., July 23, 1910, p. 309.

5 Gavino and Girard, cited from Anderson and Goldberger.

6 Plotz, Jour, of the A. M. A., lxii, 1914, No. 20.

678

DISEASES OF UNKNOWN ETIOLOGY

the cultivation of a Gram-positive pleomorphic, anaerobic bacillus from
the blood of six vases of Brill’s disease and from an equal number of
typhus cases. Complement fixation was obtained, when this organism
was used as antigen, with the blood of typhus fever cases and it seems
not unlikely, at the present time, that Plotz’s bacillus may prove to be
the etiological factor of typhus. His detailed report has not yet ap¬
peared at the present writing. Cultivation experiments by other
writers have also been negative and the etiological significance of the
organisms of Ricketts and Wilder is very doubtful.

Filtration experiments carried out by Ricketts and Wilder and by
Anderson and Goldberger at first indicated that the virus did not go
through Berkefeld filters. Nicolle, Conor, and Conseil 1 noticed that
inoculation with the filtered blood rendered monkeys refractory, an
observation later recorded also by Wilder and Ricketts. Goldberger
and Anderson report similar results. By the French investigators this
fact has been interpreted as indicating that the virus is filtrable, and
Goldberger and Anderson admit this as a possibility. It is likely there¬
fore but not proven that the virus of typhus fever may have a filtrable
stage.

By the work of Nicolle and his associates, and of Ricketts and
Wilder,2 also of Anderson and Goldberger,3 it has been shown that the
virus can be transmitted from human being to human being by the
bites of the body louse (pediculus vestimenti) ; the flea and the bed bug
apparently do not transmit the disease. The head louse (pediculus
capitis) may possibly transmit it.

FOOT-AND-MOUTH DISEASE

This malady occurs chiefly in cattle, sheep, and goats, more rarely
in other domestic animals. It is characterized by the appearance of a
vesicular eruption localized upon the mucosa of the mouth and upon the
delicate skin between the hoofs. In the females similar eruptions may
appear upon the udders. With the onset of the eruption there may be
increased temperature, refusal of food, and general depression. Usually
the disease is mild; the vesicles go on to the formation of small ulcers
and pustules, and gradually heal with a disappearance of systemic

1 Nicolle, Conor , et Conseil, Compt. rend. Acad. d. Sc., Sept. 18, 1911.

2 Wilder, Jour. Inf. Dis., July, 1911, p. 9.

3 Goldberger and Anderson, Pub. Health Report, Wash., March, 1912; ibid.,
May 31, 1912.

FOOT-AND-MOUTH DISEASE

679

symptoms. Occasionally, however, the disease is complicated by ca¬
tarrhal gastroenteritis or an inflammation of the respiratory tract, and
death may ensue.

The disease is unquestionably transmitted from animal to animal
by means of virus contained in the vesicular contents. It is also held
that infection may take place through the agency of milk. It has been
claimed, moreover, though on the basis of insufficient proof, that infec¬
tion may take place through the air, without actual contact, direct
or indirect, with lesions.

On rare occasions the disease may be transmitted to man. Such
infection, when it does take place, occurs usually among the milkers and
attendants in dairies, and is transmitted by direct contact. The course
of the disease in man is usually very mild. Mohler states that the
disease may be transmitted to man through the consumption of milk
from infected animals. He 1 adds, however, that in the United States
the disease has been practically eradicated.

The causative agent of foot-and-mouth disease is unknown. A num¬
ber of organisms have been cultivated from the vesicles and mucous
membranes of afflicted animals, but none of these could be shown to
have etiological significance. Loeffler and Frosch,2 moreover, have
demonstrated that the virus contained in the vesicles may pass through
the pores of a Berkefeld filter. It must be assumed that the causative
agent of this disease is too small to be within the range of vision of our
microscopes.

The virus of the disease is easily destroyed by heating to 60° C. and
by complete desiccation.

It has been observed that one attack of foot-and-mouth disease pro¬
tects against subsequent attacks. This immunity in most cases lasts for
years, though rare cases of recurrence within a single year have been
reported. On the basis of such naturally acquired immunity, Loeffler
has actively immunized horses and cattle with graded doses of virus
obtained from vesicles and with the sera of such animals has produced
passive immunity in normal subjects.

1 Mohler, Buff. No. 41, U. S. Pub. Health and Mar. Hosp. Serv., Wash., 1908.

2 Loeffler und Frosch, Cent. f. Bakt., 1, 1908.

680

DISEASES OF UNKNOWN ETIOLOGY

FILTRABLE VIRUS

Recent investigations into the causation of disease have revealed
that a considerable number of infections may be caused by organisms
too small to be held back by filters through which even the smallest
bacteria cannot pass. The earliest observations of such “filtrable
virus” are probably those of Frosch (1898) in foot-and-mouth disease
and of Beijerinck in the mosaic disease of tobacco. Since then similar
investigations have shown that a large number of diseases are probably
caused by such minute organisms; their investigation, long delayed
by the belief in their invisibility by even the most powerful microscopic
aid, and by our inability to cultivate them, has taken new impetus
from the discovery of and the cultivation of minute globoid bodies from
the virus of poliomyelitis by Flexner and Noguchi (see below). The
following tabulation is based largely on the comprehensive summary
published by Wolbach.1

DISEASES CAUSED BY FILTRABLE VIRUS

Disease

Transmission

Occurrence

Direct

Indirect

Yellow fever .

Stegomya fasciata

Man

Molluscum contagiosum.

Direct contact

Man

Dengue fever .

Culex fatigans

Man

Verruca vulgaris filtra-
bility?

Man

Trachoma? filtrability . . .

Direct

Man

Poliomyelitis .

Unknown; proba¬
bly nasal, etc.,
discharges

Indirect by stable-
fly

Man

Measles filtrability
claimed Goldberger and
Anderson

Direct

Man

Typhus fever? Nicolle pos.
Ricketts neg .

Body louse (pedic-
ulus vestimenti)

Man

1 Wolbach, Jour, of Med. Res., xxvii, 1912.

FILTRABLE VIRUS

681

DISEASES CAUSED BY FILTRABLE VIRUS. — Continued

Disease

Transmission

Occurrence

Direct

Indirect

Scarlet fever? filtrability
claimed by Cantacuzene
and Bernhardt but
doubtful

Probably direct

Man

Chimpanzee

Foot-and-mouth disease .

Direct

Man, cattle,
and swine

Rabies .

Direct by bite with
saliva

Man and all
mammals;
birds can be
infected

Variola and vaccinia .

Direct

Man and cat¬
tle; can be
transmitted
to monkeys
and rabbits

Pleuro-pneumonia of
cattle

Direct

Bovine species

African horse-sickness . .

Probably insects,
mosquitoes

Horses

Direct

Sheep

UlltJvJp JdUA . .

Cattle plague .

Food contaminated
with excreta

Cattle

Direct

Hogs

Swamp fever of horses . .

Probably indirect
by insects

Horses

Agalactia of sheep and
goats

Contact

Sheep and goats

“Blue tongue” .

Sheep

Guinea-pig epizootic. . . .

Guinea-pigs

682

DISEASES OF UNKNOWN ETIOLOGY

DISEASES CAUSED BY FILTRABLE VIRUS. — Continued

Disease

Transmission

Occurrence

Direct

Indirect

Guinea-pig paralysis .

Guinea-pigs

Novy’s rat disease .

Rats

F owl pest . .

Feces

Pheasants,

sparrows,

geese

Fowl diphtheria .

Contact exudates,
etc.

Fowl

Rous’s chicken sarcoma. .

Chickens

SECTION y

BACTERIA IN AIR, SOIL, WATER, AND MILK

CHAPTER LII

BACTERIA IN THE AIR AND SOIL

BACTERIA IN THE AIR

Bacteria reach the air largely from the earth's surface, borne
aloft by currents of air sweeping over dry places. Their presence in
air, therefore, is largely dependent upon atmospheric conditions; humid¬
ity and a lack of wind decreasing their numbers, dryness and high
winds increasing them. Multiplication of bacteria during transit
through the air probably does not take place.

Apart from these considerations the presence of bacteria in air also
depends upon purely local conditions prevailing in different places.
They are most plentiful in densely populated areas and within buildings,
such as theaters, meeting halls, and other places where large numbers of
people congregate. On mountain tops, in deserts, over oceans, and in
other uninhabited regions, the air is comparatively free from bacteria.
A classical illustration of this fact is found in the experiments which
Pasteur carried out in his refutation of the doctrine of spontaneous
generation. Tyndall also, in working upon the same subject, demon¬
strated this fact. From the surface of the ground and other places
where bacteria have been deposited, they reach the air only after
complete drying. It is a fact of much importance, both in bacterio¬
logical work and in surgery, that bacteria do not rise from a moist
surface. From dry surfaces they may rise, but only when the air is
agitated either by wind or by air-currents produced in other ways.
In closed rooms, therefore, even when bacteria are plentiful and the walls
and floors are perfectly dry, there is little danger of the inhalation of
bacteria unless the air is agitated in some way. The most favorable
conditions for the occurrence of many bacteria in air are the existence
of a prolonged drought followed by a dry wind. Under such condi-

683

684

BACTERIA IN AIR, SOIL, WATER, AND MILK

tions, even the dark places and unlighted corners of streets and habita¬
tions are thoroughly dried out, and bacteria are taken up and carried
about together with particles of dust. At such times the dangers from
inhalation are much multiplied. By experiments made in balloons,
it has been found that bacteria are plentiful below altitudes of about
fifteen hundred feet and may be present, though much reduced in
numbers, as high up as a mile above the earth’s surface. The species
of bacteria found in the air are, of course, subject to great variation,
depending upon locality. Molds and spore-forming bacteria, being
more regularly resistant to the effects of sunlight and drying than
bacteria possessing only vegetative forms, are naturally more generally
distributed.

Out of air thus laden with bacteria, they may again settle when the
wind subsides and the air becomes quiescent. The process of settling,
however, is extremely slow, since the weight of a bacterium is probably
less than a billionth of a gram, and it may be held in suspension in air
for considerable periods. Rains, snow, or even the condensation of
moisture from a humid atmosphere, hastens this process considerably,
and large quantities of bacteria may settle out from air, in a com¬
paratively short time, in ice chests, in operating rooms, or in other places
in which much condensation of water vapor takes place.

The importance of the air as a means of conveying disease is still a
problem upon which much elucidation is needed. The importance of
this manner of conveyance in smallpox, in measles, in scarlet fever,
and in 'other exanthemata, can not be denied. As regards the dis¬
eases of known bacterial origin, conveyance by air is of importance
in the case of tuberculosis, where infection by inhalation may take
place, and in the case of anthrax, where inhaled anthrax spores may
give rise to the pulmonary form of the disease. The importance of air
conveyance for any great distance in pneumonia, in influenza, in diph¬
theria, and in meningitis is by no means clear and requires much fur¬
ther study. The expulsion of bacteria from the lungs and naso-pharynx
does not take place during simple expiration, since an air-current pass¬
ing over a moist surface is not sufficient to dislodge microorganisms.
Expulsion of bacteria in these conditions must take place together with
small particles of moisture carried out in sneezing, coughing, or any
forced expiration. The bacteria thus discharged are then subject to the
process of drying and often are exposed to direct sunlight for a con¬
siderable period before they are again taken up in the air.

The methods of estimating the bacterial contents of the air are not

BACTERIA IN THE AIR AND SOIL

685

entirely satisfactory. The simple exposure of uncovered gelatin or agar
plates for a definite length of time, and subsequent estimation of the
colonies upon the plates, yield a result which is variable according to the
air-currents and the degree of moisture in the atmosphere, and furnish
no volume standard for comparative results. The methods which
are in use at the present time depend upon the suction of a definite
quantity of air by means of a vacuum-pump through some substance
which will catch the bacteria. One of the first devices used for this pur¬
pose was that of Hesse, who sucked air through a piece of glass tubing,
about 70 cm. long and about 3.5 cm. in diameter, the inner surface of
which had been coated with gelatin in the manner of an Esmarch roll
tube. This method is not efficient, since a large number of the bacteria
may pass entirely through the tube without settling upon the gelatin.
One of the most satisfactory methods at present in use is that in which
definite volumes of air are sucked through a sand-filter. Within a
small glass tube, a layer of sterilized quartz sand, about 4 cm. in
depth, is placed. The sand is kept from being dislodged by a small
wire screen. After the air has been sucked through the filter the
sand is washed in a definite volume of sterile water or salt solution,
and measured fractions of this are planted in. agar or gelatin in Petri
plates. The colonies which develop are counted. Thus, if two liters
of air have been sucked through the filter, and the sand has been
washed in 10 c.c. of salt solution, and 1 c.c. of this is planted, with
the result of fifteen colonies, then the two liters of air have contained
one hundred and fifty bacteria.

BACTERIA IN SOIL

Besides the normal bacterial inhabitants of the soil, bacteria reach
the soil from the air, in contaminated waters, in the dejecta, excreta,
and dead bodies of animals and human beings, and in the substance of

decaying plants. It is self-evident, therefore, that the distribution of
bacteria in soil depends largely upon the density of population and the
use of the soil for agricultural or other purposes. Thus, bacteria aie most
plentiful in the neighborhood of cess-pools or in manured fields and gar¬
dens. Such conditions, however, may be regarded as abnormal. Even
in uncultivated fields there is a constant bacterial flora in the soil which
is of great importance in its participation in the nitrogen cycle, a phase
of the bacteriology of soil which has been discussed in detail in another

686

BACTERIA IN AIR, SOIL, WATER, AND MILK

section. (See page 40.) There are, thus, regular and normal inhabitants
of the soil which fulfil a definite function and may be found wherever
plant life flourishes. In addition to these, innumerable varieties of sapro¬
phytes and pathogenic germs may be present, which vary in species
and in number with local conditions. Numerous investigations into
the actual numerical contents of the soil have been made. Houston 1
found an average of 1,500,000 bacteria per gram in garden soil, and
about 100,000 bacteria per gram in the arid soil of uncultivated regions.
Fraenkel,2 in studying the horizontal distribution of bacteria in the earth,
has found that they are most numerous near the surface, a gradual
diminution occurring down to a depth of about two yards. Beyond
this, the soil may be often practically sterile.

Pathogenic bacteria may at times be found in the surface layers,
and these are often of the spore-bearing varieties. Most important
among them from the medical standpoint are the bacillus of tetanus, of
malignant edema, and the Welch bacillus. If a guinea-pig is inoculated
subcutaneously with an emulsion of garden soil, death will result almost
invariably with enormous bloating and swelling of the body due to gas
production. This is due to the fact that the spore-bearing, gas-producing
anaerobic bacilli are commonly present and are actively pathogenic for
these animals. The frequent occurrence of tetanus in persons sustain¬
ing wounds of the bare feet and hands in fields and excavations, is a
matter of common knowledge. Anthrax, also, may be easily conveyed
by soil in localities where animals are suffering from this infection. It
is not probable that pathogenic germs which are not spore-bearers sur¬
vive in the soil for any great length of time. Unless the soil is specially
prepared by the presence of defecations or other other organic material,
the nutrition at their disposal is not at all suitable for their needs,
since rapid decomposition of organic materials by saprophytes is always
going on in the upper layers. Furthermore, in the deeper layers the con¬
ditions of temperature and possibly oxygen supply are not at all favorable
for the growth of most pathogenic bacteria. Within a short distance from
the surface the temperature of the soil usually sinks below 14° or 15° C.
An interesting series of experiments by Fraenkel3 have demonstrated
this point. This investigator buried freshly inoculated agar and gelatin
cultures of cholera spirilla and of typhoid and anthrax bacilli at differ¬
ent levels, and examined them for growth after two weeks had elapsed.

1 Houston, Report Med. Officer, Local Govern. Bd., London, 1897.

2 Fraenkel, Zeit. f. Hyg., ii, 1887.

3 Fraenkel, Zeit. f. Hyg., xi, 1887.

BACTERIA IN THE AIR AND SOIL

687

The anthrax bacilli hardly ever showed growth at a depth below about
two yards, and cholera and typhoid developed colonies at these depths
only during the summer months. Under natural conditions it must be
remembered that, at these levels, suitable nutritive material is not
found.

A consideration of practical importance in this connection is the
possibility of infection by means of buried cadavers. An elaborate series
of experiments has been carried out upon this subject in Germany, with
results which demonstrate that the danger from the burial of persons
dead of infectious diseases was formerly much exaggerated. Experi¬
ments 1 usually failed to reveal the presence of cholera and typhoid
bacilli within two to three weeks after burial, and tubercle bacilli were
never found after three months had elapsed. It was only in the case
of sporulating microorganisms, such as the anthrax bacillus, that the
living incitants could be found for as long as two years after burial. The
dangers of infection of human beings through the agency of soil,
therefore, are chiefly those arising from the spore-bearing bacteria which
are able to remain alive in spite of the unfavorable cultural conditions.
It has been found by some observers,2 however, that, under special con¬
ditions, non-sporulating bacteria, more especially the typhoid bacillus,
may remain alive in soil for several months. Although these bacteria,
as well as those of cholera, diphtheria, etc., can not proliferate under the
conditions found in the soil, the fact that they can remain viable for such
prolonged periods in the upper layers suggests the possibility of danger
from the use of unwashed vegetables, such as lettuce or radishes or other
soil and sewage contaminated food products. The examination of soil
for colon bacilli, while demonstrating the presence or absence of manure
or sewage contamination, has no practical value, since colon bacilli are
found in the dejecta of animals.

Examination of specimens of soil for their numerical bacterial
contents is extremely unsatisfactory because the bacteria there found
can hardly ever all be cultivated together under one and the same
cultural environment. A large number are anaerobic, others again
thrive at low temperatures, while again another class may require un¬
usually high temperatures. When such examinations are made, how¬
ever, specimens of the soil from the surface layer may be taken in a
sterile platinum spoon. When taken from the lower levels, a drill,

1 Petri, Arb. a. d. kais. Gesundheitsamt, vii.

2 Firth and Horrocks, Brit. Med. Jour., Sept., 1902.

688

BACTERIA IN AIR, SOIL, WATER, AND MILK

such as that devised byFraenkel, maybe used. This consists of an iron
rod the lower end of which is pointed. Just above the point a movable
collar is fitted. This collar has a slit-like opening. The rod beneath the
collar has a deep longitudinal groove corresponding to the slit in the
collar. A flange on the collar permits opening and closing of the groove
while the instrument is below the ground. The drill is forced into the
earth to the desired depth, the groove is opened and earth is forced into
the chamber by twisting the rod. In the same manner the groove may
be closed. The soil obtained in this way is taken out of the chamber
and a definite quantity, say one gram, is dissolved and washed thor¬
oughly in a measured volume of sterile water or sterile salt solution.
Fractions of this are then mixed with the culture medium, plated, and
cultivated aerobically or anaerobically as desired.

CHAPTER LIII

BACTERIA IN WATER

All natural waters contain a more or less abundant bacterial flora.
This fact, combined with our knowledge that the incitants of several
epidemic diseases and a number of minor ailments of a diarrheal char¬
acter are water borne, gives the bacteriological investigation of water a
place of great importance in hygiene. In nature, there are very few
sources of water supply which do not contain bacteria of one or another
description. While pathogenic bacteria are usually not present except
in those waters which are directly contaminated from human sources, a
thorough understanding of the quantitative and qualitative bacterial
contents of all natural waters is necessary in order that we may in¬
telligently gather comparative data as to the fitness of any given water
for human consumption.

The gross appearance of water is rarely, if ever, an indication of its
danger. The turbid waters of running streams in sparsely populated
agricultural districts may be safe, while perfectly clear well waters
subjected to the dangers of contamination from neighboring sinks
or cess-pools may contain large numbers of pathogenic germs.

The diseases which are known to be more directly connected with
water supply are typhoid fever and cholera.

Typhoid germs discharged from the bowel or from the urine of
typhoid patients or convalescents may be carried by the sewage or from
the neighboring soil into a river or lake and lead to infection of the
population deriving its drinking water from this source. There are a
great many investigations on record in which severe typhoid epidemics
have been traced to such sources.

In the case of cholera, where the germs are discharged from the bowels
in enormous numbers, conveyance of the disease by water is even more
apparent, and the discoverers of the cholera germ themselves, in their
early work in Egypt and India, were able to isolate the bacteria from
contaminated water supplies.

In regard to the less clearly understood diarrheal diseases, dysen¬
tery, cholera infantum, etc., the direct relation to water supply has not
45 689

690

BACTERIA IN AIR, SOIL, WATER, AND MILK

been so definitely proven, and can be deduced only from the diminu¬
tion of such infections after the substitution of pure water for the pre¬
viously used impure supply. It is thus seen that water bacteriology is
one of the most important branches of the science of hygiene, and has
led, and is constantly leading, to enormous diminution of the death rate
in all communities where an intelligent study of the conditions has been
made.

The bacterial purity of natural waters, although dependent upon
special and local conditions in relation to possible contamination, differs
widely, according to the source from which such waters are derived.

Rain water and snow water are usually contaminated with bacteria
by the dust which they gather on their way to the ground, and are
especially rich in bacteria when taken during the first few hours of a
rain or snow storm when the air is still dusty and filled with floating
particles. During the later hours of prolonged storms, rain water and
snow water may be comparatively sterile. Miquel,1 who made exten¬
sive experiments in France on the bacterial contents of rain water,
found that in country districts, where the air is less dusty, rain water
contained an average of about 4.3 bacteria to the cubic centimeter.

The bacterial counts of snow water are usually somewhat higher than
those of rain.

The waters of streams, ponds, and lakes are usually spoken of as
surface waters, and these of all natural supplies contain the largest num¬
ber of bacteria. In each case, of course, the quantitative and quali¬
tative bacterial flora of such waters is intimately dependent upon the
conditions of the surrounding country, the density of the population,
and the relation of these waters to sewage. It is also, and to no less
important degree, dependent upon weather conditions, the influence of
light and temperature, and the food supply contained within the waters
in the form of decayed vegetation. In all such surface waters there is
constantly going on a process of self-purification. The chief factor in
this process is sedimentation. In stagnant ponds and lakes with but
sluggish currents there is a constant sedimentation of the heavier
particles, which gradually but steadily leads to a diminution of the
number of bacteria in the upper layers of the water. In rivers where
sedimentation is to a certain extent prevented by rapidity of current,
the effectiveness of such sedimentation is, of course, entirely dependent
upon the speed of the current.

1 Miquel, Revue d’hyg., viii, 1886.

BACTERIA IN WATER

691

The influence of light in purifying surface waters is important chiefly
in ponds, lakes, and sheets of water which expose a large surface to the
sunlight, and where the surroundings are such that the sun has free access
throughout the day. According to the researches of Buchner,1 the bac¬
tericidal effects of light penetrate through water to a depth of three feet.

The effects of temperature in purifying surface waters under natural
conditions are probably not great. There is, however, a general tendency
toward diminution of the bacterial flora as the temperature of such
waters becomes lower.

The presence of protozoa in natural waters as purifying agents has
recently been emphasized by Huntemuller,2 who claims that these organ¬
isms by phagocytosis greatly diminish the number of bacteria in any
given body of water. It is self-evident that the number of bacteria in
any of these waters is never constant, since all factors which tend to a
diminution or increase in volume, such as drying up of tributary streams
or the occurrence of heavy rains, would lead to differences of dilution
which would materially change numerical bacterial estimations. The
influence of rains, furthermore, may be a twofold one. On the one hand,
heavy rain-falls, by washing a large amount of dirt into the rivers and
lakes from the surrounding land, have a tendency to increase the
bacterial flora. This influence would be especially marked in all bodies
of water which are surrounded by cultivated land where manured fields
and grazing-meadows supply a plentiful source of bacteria. On the
other hand, in regions where arid, uninhabited lands surround any
given river or lake, the rain would carry with it very little living con¬
tamination and would act chiefly as a diluent and diminish the actual
proportion of bacteria in the water.

Another and extremely important source of water supply is that
spoken of technically as “ ground water.” The “ ground waters ” include
the shallow wells employed in the country districts, springs, and deep
or artesian wells. The shallow wells that form the water supply for a
large proportion of farms in the eastern United States are usually very
rich in bacteria and are by no means to be regarded as safe sources, ex¬
cept in cases where great care is observed as to cleanliness of the sur¬
roundings. In such wells the filtration of the water entering the well
may be subject to great variation according to the geological con¬
ditions of the surrounding ground. The proximity of barns and sinks
may lead to dangerous contamination of such waters.

i Buchner, Arch. f. Hyg., xvii, 1895.

2 Huntemuller , Arch. f. Hyg., liv, 1905.

692

BACTERIA IN AIR, SOIL, WATER, AND MILK

Examinations by various bacteriologists have shown that such wells
frequently contain as many as five hundred bacteria to the cubic centi¬
meter.

Perennial spring waters are usually pure. Examinations by the Mas¬
sachusetts State Board of Health 1 in 1901 showed an average of about
forty bacteria per cubic centimeter. As sources of water supply for
general consumption, however, springs can hardly be very important
because of the insignificant quantities usually derived from them.

Of much greater practical importance are deep artesian wells, which,
under ordinary conditions, are largely free from bacterial contamination.

Quantitative Estimations of Bacteria. — The quantitative estima¬
tion of bacteria in water is of necessity inexact, because of the difficulty
of always securing fair average samples from any large body of water,
and because of the large variations in cultural requirements of the
flora present in them. All these methods depend upon colony enumera¬
tion in plates of agar or gelatin, preferably of both. For the sake of
gaining some basis of comparison for results which, at best, can never be
entirely accurate, an attempt has been made by the American Public
Health Association 2 to standardize the methods of analysis.

Water for analysis should always be collected in clean, sterile bottles,
preferably holding more than 100 c.c. If water is to be taken from a
running faucet or a well supplied with piping, it is important that it
should be allowed to run for some time before the sample is taken, in
order that any change in bacterial content occurring inside of the pipes
may be excluded. It is obvious that in water pipes through which
the flow is not constant, bacteria may find favorable conditions for
growth and such a sample would not represent fairly the supply to
be tested.

When the water is taken from a pond, lake, or cess-pool, the bottle
may be lowered into the water by means of a weight, or may be plunged
in with the hand, great care being exercised not to permit contamina¬
tion from the fingers to occur. A number of devices for collecting water
have been originated, a very excellent one for small samples, by Stern¬
berg,3 consisting of a small glass bulb with a capillary neck which is
sealed while the bulb is hot. This is attached to a rod, and a wire noose
is fastened to the neck of the bulb. When immersed in water the neck

1 Mass. State Bd. of Health, 33d Annual Report for 1901.

2 Fuller, Trans. Amer. Public Health Assn., xxvii, 1902. Report of Com. on
Standard Methods of Water Analysis. Jour. Inf. Dis., Suppl. 1, 1905.

3 Sternberg, “Manual of Bact.”

BACTERIA IN WATER

693

may be broken off by means of the wire, and water will be forced into
the bulb to satisfy the vacuum.

After the water has been collected it is important to plate it before
the bacteria in it have a chance to increase. The changes taking place
during transportation, even when packing in ice has been resorted to,
have been found by Jordan and Irons 1 to be considerable. It is impera¬
tive, therefore, that plating of the water, if possible, shall not be delayed
for longer than one or two hours after collection.

Before plating, the bottle containing the sample should be shaken
at least twenty-five times in order to distribute the bacterial contents
evenly. The quantities to be plated will depend to a certain extent upon
the probability of there being a large or small number of bacteria.
If less than two hundred are suspected, 1 c.c. of the water should be
taken out of the bottle with a sterile pipette and placed in a sterile Petri
dish. To this is added 10 c.c. of gelatin at a temperature of about 30° C.,
or of agar at a temperature of not over 40° C. The water is thoroughly
mixed with the medium by repeated tilting of the plate, and finally al¬
lowed to harden in the regular way. If unusual pollution or other data
lead to a suspicion that the bacterial count is apt to be extremely high,
it is advisable to dilute the sample of water with sterile water before
plating. The reason for mixing water and medium in the Petri dish,
rather than in the tube, as was formerly done, is the fact that in the
pouring of the mixture from the tube a certain amount of residuum is
left which naturally leads to a diminution in the actual number of
colonies developing in the plate. The gelatin is incubated at 20° C..
the agar at 37.5° C.

Neither the gelatin nor the agar alone can give an accurate estima¬
tion of the total bacterial contents of water. A better estimate can of
course be made when both are used. However, since aerobic and
anaerobic bacteria may be present and since many water organisms may
require nutritive conditions and a reaction other than that of the stand¬
ard media, it is quite likely that none of the methods in use can give
an accurate total of organisms originally in the water. For sanitary
purposes, however, the agar counts, in which the plates are incubated at
37.5° C. or body temperature, are by far the most important since the
bacteria which grow at this temperature are the ones likely to possess
pathogenic significance, or at any rate to emanate from animal and
human sources. It has been suggested by Rosenau 2 and others that es-

1 Jordan and Irons, Reports of the Amer. Pub. Health Assn., xxv, 1889.

2 Rosenau, “Preventive Medicine and Hygiene,” 755. D. Appleton & Co., 1913.

694

BACTERIA IN AIR, SOIL, WATER, AND MILK

pecial value is attached to the organisms which grow at 40° C. since the
temperature excludes many non-pathogenic water bacteria, while it
permits organisms of the colon-typhoid group to grow.

The gelatin and agar which are used should be made according to the
standard methods recommended by the American Public Health Associa¬
tion. The gelatin should be made of meat infusion and not of meat ex¬
tract, and should contain one per cent of Witte’s pepton and ten per cent
of the best so-called French brand of gelatin. It should not soften when
kept at a temperature of 25° C. The agar medium should also be made
of meat infusion and of the highest grade of commercial thread agar.
For general purposes the standard reaction of media should be one
per cent acidity, but for long-continued work on water from the same
source the optimum reaction should be ascertained and adhered to,
and differences from the standard reaction should be mentioned in the
- report.

Incubation of the gelatin should be continued for forty-eight hours
in an atmosphere saturated with moisture. When agar is used, incuba¬
tion for twenty-four hours is usually sufficient, and it is advantageous to
employ Petri dishes supplied with porous earthenware covers.1 Simple
inversion of the Petri dishes when placed in the incubator obviates the
necessity of using the porous covers.

In counting, the ordinary counting plates divided into 1 cm. squares
may be used, but, whenever possible, all the colonies in a plate should be
counted.

The value of the quantitative estimation of bacteria in water is
only a comparative one, and no arbitrary standards can be established
for the purity of water on this basis. In general it may be said that
water containing one hundred bacteria to the cubic centimeter or less
is apt to be from a deep source and comparatively pure; that water
containing five hundred bacteria to the cubic centimeter or over is
open to suspicion, and that any water containing over one thousand
to the cubic centimeter is probably from a polluted source. At the
same time it is quite impossible to draw any direct conclusions from
numerical colony counts, and all such results must be carefully weighed
in the balance with qualitative analyses and chemical tests, and knowl¬
edge of environmental conditions.

Qualitative Bacterial Analyses of Water. — Of far greater importance
than quantitative analysis is the isolation of bacteria either distinctly
pathogenic, such as the cholera spirillum and the typhoid bacillus,

1 Hill, Jour. Med. Res., xiii, 1904.

BACTERIA IN WATER

695

or of other species probably emanating from contaminating sources,
such as a B. coli. Unfortunately there are no reliable methods by which
typhoid and cholera germs can be isolated from water with any degree
of regularity or certainty. Although frequently the isolation of such
organisms is possible, a negative result in these cases is by no means
eliminative of their presence.

The isolation of typhoid bacilli from water is very difficult, chiefly
because of the great dilution which contaminations undergo upon enter¬
ing any large body of water. The difficulty of isolating typhoid bacilli,
even from the stools of infected patients, makes it clear that such diffi¬
culties are enhanced when a considerable dilution of the excreta takes
place. Furthermore, water is by no means a favorable medium for the
typhoid bacillus. Russell and Fuller1 have shown that typhoid bacilli
may die in water within five days, and it is unquestionable that the
rate of increase of these bacteria is by no means equal to that of many
other microorganisms for which polluted water at the temperature en¬
countered in streams and lakes forms a much more favorable medium.

It is thus clear that even in infected waters the number of typhoid
bacilli encountered can never be very great.2

A large number of methods for the isolation of the typhoid bacillus
from water have been devised. Most of the media used are identical
with those employed for the isolation of these bacteria from the stools.
These media have been discussed in the chapter dealing with the typhoid
bacillus.

Drigalski 3 has reported a method for which he claims considerable
success in isolating typhoid bacilli from water, which depends upon the
motility of the organisms. One- to two-liter samples of water are taken
and allowed to stand at room temperature in high jars for one or two
days. Small quantities are then removed from the surface and planted
on Wurtz’s lactose litmus agar. The method depends upon the probabil¬
ity of the settling out of non-motile organisms and the possibility, there¬
fore, of getting motile organisms only in the plates.

Parietti4 and others have attempted to eliminate other organisms by
adding phenol and hydrochloric acid to neutral broth, in the hope that
the high acidity and the antiseptic qualities of the phenol will destroy
more delicate organisms than the typhoid bacillus. Inasmuch as B.

1 Russell and Fuller, Jour. Inf. Dis., Suppl. 2, 1908.

2 Laws and Anderson, Rep. of Med. Officer, London County Council, 1894.

3 Drigalski, Arb. a. d. kais. Gesundheitsamt, xxiv, 1906.

4 Parietti, Rev. d’igiene e san. pub., 1890.

696

BACTERIA IN AIR, SOIL, WATER, AND MILK

coli, however, withstands these reagents as well as B. typhosus, or even
better, these methods have not met with great success.

A method which has proved useful in the hands of Adami and Chapin1
is one which depends upon the phenomenon of agglutination. These
authors collect water in two-liter specimens and to each two liters add
20 c.c. of one per cent glucose broth. These samples are incubated at
37.5° C. for twenty-four hours, and at the end of this time quantities
of 10 c.c. are withdrawn and placed in test tubes. To each of these
tubes potent typhoid serum, preferably diluted 1 : 60, is added, and
whenever agglutination occurs the flocculi are washed and plated on
various media for identification.

Vallet and others have attempted to precipitate typhoid bacilli out
of water by chemical means. The purpose of these methods has been
to entangle the bacteria in an inert precipitate, and thus concentrate the
bacteria in water for purposes of cultivation. Vallet’s method is as
follows: To two liters of water add 20 c.c. of a 7.75 per cent solution
of sodium hyposulphite and 20 c.c. of a 10 per cent solution of lead
nitrate. When the precipitate has settled, the clear supernatant fluid
is decanted and the precipitate dissolved in a saturated sodium hypo¬
sulphite solution. This clear solution is then plated. Willson 2 has
modified this method by adding to the water 0.5 gm. of alum to each
liter. A precipitate is formed which may either be allowed to settle or
may be brought down by centrifugalization. The supernatant fluid is
removed and the precipitate plated.

In actual work many of the methods which are aimed purely at B.
coli may lead to success in the isolation of B. typhosus, because of
the similarity of the two organisms in their reaction to definite media.
Thus, the method of Jackson,3 who employs one per cent of lactose in
pure ox-bile for the isolation of B. coli, has occasionally led to the simul¬
taneous isolation of B. typhosus.

The isolation of the vibrio of cholera is less difficult than that of B.
typhosus, primarily because of the much greater numbers of these
microorganisms discharged into sewage. The number of cholera spirilla
in the excreta of cholera patients is enormously higher than is that of
B. typhosus in the stools of typhoid-fever patients. It is not infre¬
quent, therefore, that the source of a cholera infection may be directly
traced to the water supply. Koch,4 the discoverer of the cholera vibrio,

1 Adami and Chapin , Jour. Med. Res., xl, 1904.

2 W illson, Jour, of Hyg., v, 1905.

3 Jackson Journ. of Inf. Dis., Suppl. 2, 1907. 4 Koch , Zeit, f. Hyg., xiv, 1893.

BACTERIA IN WATER

697

has indicated a method which has frequently found successful applica¬
tion.

To 100 c.c. of the infected water are added one per cent of pepton
and one per cent of salt. This mixture is then incubated at 37.5° C.,
and after ten, fifteen, and twenty hours, specimens from the upper
layers are examined microsopically and are plated upon gelatin. Upon
the weak pepton solution cholera spirilla increase very rapidly at in¬
cubator temperatures, and then when plated in gelatin the detection of
characteristic colonies is comparatively easy.

Because of the great difficulties outlined above in isolating specific
pathogenic germs from polluted waters, bacteriologists have attempted
to form an approximate estimate of pollution by the detection of other
microorganisms which form the predominating flora of sewage. Chief
among these is B. coli. The isolation and numerical estimation
of B. coli in polluted water has been for a long time one of the criteria
of pollution. This so-called colon test, however, should always be ap¬
proached with conservatism and should never be carried out qualita¬
tively only. Careful quantitative estimation should be made in every
case.

B. coli in water is by no means always the result of human con¬
tamination, since this bacillus is found in great abundance in the in¬
testines of domestic animals. According to Poujol, B. coli does not
even always point to fecal contamination, since this author was able
to find the bacillus in the water of a number of wells where no possible
contamination of any sort could be traced. Prescott 1 explains this, as
well as similar cases, by the fact that organisms of the colon group
may occasionally be parasitic upon plants.

The opinions of hygienists are widely at variance as to the value
of the colon test. While the discovery of isolated bacilli of the colon
group may therefore be of little value, it is nevertheless safe to follow
the opinion of Houston,2 who states that the discovery of B. coli in
considerable numbers invariably points to sewage pollution, and that
the absolute absence of B. coli is, as a rule, reliable evidence of purity.

Rosenau states that a ground water should be condemned even if
only a few colon bacilli are found, for, as he puts it, “these bacteria have
no business in a soil-filtered and properly protected well or spring-
water.” Surface waters, however, may easily contain a few. colon
bacilli without necessarily having been exposed to contamination by

1 Prescott , Science, xv, 1903.

2 Houston, Rep. Medical Officer, Local Gov. Board, London, 1900.

698

BACTERIA IN AIR, SOIL, WATER, AND MILK

human forces. The limit of safety, Rosenau states, is one colon bacillus
per c.c. If more are present the water should be regarded-Jas sus¬
picious. If more than 10 per c.c. are found the water must be re¬
garded as dangerous and unqualifiedly condemned.

For the purpose of isolating B. coli from water, a large number
of methods have been devised. In examining sewage or other pol¬
luted waters in which the number of colon bacilli is comparatively
large, the direct use of lactose litmus agar plates yields excellent re¬
sults. Varying quantities of water may be added to this medium
and the development of red colonies at incubator temperatures usually
indicates the presence of bacilli of this species. These colonies may be
fished and further identified. Copeland 1 has proposed the addition of
.2 per cent of phenol to this medium in order to inhibit other bacteria.

In water less grossly polluted, some method of enrichment must be
employed in order to increase the number of bacteria so that they may
be found in plates. For this purpose glucose bouillon in fermentation
tubes, according to the method of Theobald Smith, may be employed.2
In this medium, at a temperature of 37.5° C., the colon bacilli grow with
great speed and transplants to plating media may be taken after eight
or more hours’ incubation.

A medium proposed by Jackson3 has been found successful. This
consists of undiluted ox-bile, to which has been added 1 per cent of lactose.

For quantitative estimation of colon bacilli in water, Theobald
Smith 4 has proposed the use of dextrose bouillon in fermentation tubes,
to which are added varying quantities of water, ranging from 0.001 to
1 c.c. The appearance of gas in any tube indicates the presence of
B. coli, and the number can be approximately computed from the
smallest quantity of water by which gas formation has been produced.

The presence of B. coli in such fermentation tubes may be deter¬
mined without isolation and cultivation, by estimating the comparative
amount of C02 in the gas.5 Whenever CO_> forms approximately 33
per cent of the gas present, Irons concludes that B. coli is present.

Jackson 6 believes when lactose ox-bile is used that 25 per cent of
gas within 72 hours may be regarded as positive for B. coli.

1 Copeland , Jour. Boston Soc. Med. Sciences, 1901.

2 Prescott, Science, xvi, 1902.

3 Jackson, Jour. Inf. Dis., Suppl. 2, 1907.

4 Th. Smith, 13th Ann. Rep. N. Y. S. Board of Health.

5 Irons, Trans. Amer. Pub. Health Assn., xxvi, 1900.

6 Jackson, “Biol. Studies of Pupils of W. T. Sedgwick,” Boston, 1906.

CHAPTER LIV

BACTERIA IN MILK AND MILK PRODUCTS, BACTERIA IN THE

INDUSTRIES

BACTERIA IN MILK

The universal use of cows’ milk as a food, especially for the nourish-
ment of infants, has necessitated its close study by bacteriologists and
hygienists. It furnishes an excellent culture medium for bacteria and
is, therefore, pre-eminently fitted to convey the germs of infectious dis¬
ease. The many changes which take place in milk, furthermore, and
which add or detract from its nutritive value, are due largely to bacterial
growth and have been elucidated by bacteriological methods.

Within the udder of the healthy cow, milk is sterile. If pyogenic or
systemic diseases of bacterial origin exist in the cow, the milk may,
under certain circumstances, be infected even within the mammary
glands. In the milk ducts and in the teats, however, even in perfectly
healthy animals, a certain number of bacteria may be found. For this
reason, even when all precautionary measures are followed, the milk
as received in the pail is usually contaminated. As a matter of fact,
the anatomical location of the udder and the mechanical difficulties of
milking make it practically impossible to collect milk under absolutely
aseptic conditions, and, under the best circumstances, from 100 to 500
microorganisms per c.c. may usually be found in freshly taken milk.
Withdrawn under conditions of ordinary cleanliness, the bacterial
contents of milk are considerably higher than this. After the proc¬
ess of milking, in spite of all practicable precautions, the chances
for the contamination of milk are considerable; but even could these
be eliminated, the bacterial contents of a given sample would ultimately
rapidly increase because of the rich culture medium which the milk
provides for bacteria. Whether large increases shall take place or not de¬
pends, in the first place, upon the temperature at which milk is kept, and,
in the second place, upon the length of time which intervenes before its
consumption. Though fresh milk possesses slight bactericidal powers,1

1 Rosenau and McCoy, Jour. Med. Res., 18, 1908.

699

700

BACTERIA IN AIR, SOIL, WATER, AND MILK

these are by no means sufficient to be of practical importance in the
inhibition of bacterial growth. Kept at or about freezing-point, the
bacterial contents of milk do not appreciably increase. At higher tem¬
peratures, however, a rapid propagation of bacteria takes place which,
especially during the summer months, speedily leads to enormous num¬
bers. In a case reported by Park,1 where milk, containing at the first
examination 30,000 microorganisms per cubic centimeter, was kept
at 30° C. (86° F.) for twenty-four hours, the count at the end of this
time yielded fourteen billions of bacteria for the same quantity.

It is of much importance, therefore, that the cleanliness of dairies,
of cattle, and in the handling of milk should be. reinforced by the utmost
care in chilling and icing during shipment and before sale.

Because of its great importance, especially for the health of the chil¬
dren in large cities during the summer months, the milk question has,
of recent years, received much attention from health officers. In the
city of New York, the question has been made the subject of many
careful studies by Park 2 and his associates. Commissions, working
in Chicago,3 Boston,4 and other large towns, have placed the sale of milk
under more or less exact bacteriological supervision. Park has de¬
termined that the milk, as sold in New York stores during the cold
weather, not infrequently averages seven hundred and fifty thousand
bacteria per cubic centimeter; during the hot summer months, the
bacterial contents of similar milk not infrequently average one million
and more, for the same quantity.5 In consequence of these and other
researches, large dairies have introduced bacteriological precautions
into their method of milk production. They have attempted the reduc¬
tion of the bacterial contents of milk by scrupulous cleanliness of the
barns and of the udders and teats of the cow, by the elimination of dis¬
eased cattle, by sterilization of the vessels in which the milk is received,
and of the hands of the milker; also by the immediate filtering and
cooling of the milk and the packing of the milk cans in ice, where
they remain until, delivered to the consumer. In consequence of such
measures, it is possible for cities to be supplied with milk containing no
more, and often less, than fifty thousand bacteria to the cubic centimeter.
A standard of cleanliness has been established in various towns by the

1 Park, W. H., “ Pathogenic Bacteria/’ New York, 1905, p. 463.

2 Park, Jour, of Hygiene, 1, 1901.

3 Jordan and Heinemann, Rep. of the Civic Federation of Chicago, 1904.

4 Sedgwick and Batchelder, Bost. Med. and Surg. Jour., 126, 1892.

5 Escherich, Fort. d. Medizin, 16 and 17, 1885.

BACTERIA IN MILK

701

introduction of the so-called “certified milk/’ which, by the New York
Milk Commission, is required to contain no more than thirty thousand
bacteria per cubic centimeter. Great stress is laid upon such numerical
counts simply in that they are approximate estimates of cleanliness.
Most of the bacteria, however, contained in milk are non-pathogenic,
and numbers much larger than the maximum required for certified milk
may be present without actual disease or harm following its consump¬
tion.

The various species of bacteria which may be found in milk include
almost all known varieties. Whether there are special, so-called milk
bacteria or not is a question about which investigators have expressed
widely differing opinions. It is probable that many of the species,
formerly regarded as specifically belonging to milk, are there simply by
virtue of their habitual presence in fodder, straw, or bedding, or upon
cattle. It is likely, furthermore, that some of these species are found
with great regularity because of their power to outgrow other species
under the cultural conditions offered them in milk.

Under normal conditions, milk always undergoes a process which is
popularly known as souring and curdling. This is due to the forma¬
tion of lactic acid from the milk sugar and is the result of the enzymatic
activities of several varieties of bacteria commonly found in milk. Most
common among these bacteria is the so-called Bacillus lactis aerogenes,
an encapsulated bacillus closely related to the colon-bacillus group. (See
page 451.) The transformation of the lactose into lactic acid may occur
either directly by hydrolytic cleavage:

Cu H22 Ou + H20 = 4 C3 H6 03;
or indirectly through a monosaccharid,

ca H22 On + H2 0 = 2 C8 H12 06 = 4 C3 H6 0,.

Other microorganisms which may cause lactic-acid fermentation in
milk are the so-called Streptococcus lacticus, the common pyogenic
streptococcus, the Staphylococcus aureus, Bacillus coli communis and
communior, and many other species. Most commonly concerned in lactic-
acid production, however, according to Heinemann,1 are the two first-
mentioned, Bacillus lactis aerogenes and Streptococcus lacticus. The
secret of the regularity with which some of these bacteria are present in
sour milk is probably found in the ability of these varieties to withstand
a much higher degree of acidity of the culture medium than othei species.

1 Heinemann, Jour, of Inf. Dis., 3, 1906.

702

BACTERIA IN AIR, SOIL, WATER, AND MILK

In consequence, they are able to persist and develop when cultural com
ditions are ' absolutely unsuited to other bacteria.

Consequent upon acidification of the milk by lactic-acid formation,
there is coagulation of casein. Casein precipitation, however, may
also be due to a non-acid coagulation caused by a bacterial ferment.
Casein precipitated in this way may be redissolved by a bacterial
trypsin or casease, produced by the same or other bacteria, the milk
again becoming entirely liquid, transparent, and of a yellowish color.
The casein precipitated by lactic-acid formation, however, is never
thus redissolved, because the high acidity does not permit the pro¬
teolytic ferments to act.1

Butyric-acid fermentation in milk, a common phenomenon, is also an
evidence of bacterial growth. As a rule, it is produced by the anaerobic
bacteria, and is a process developing much more slowly than other fer¬
mentations. A large number of bacteria have been described which
are capable of producing such changes, the chemical process by which
they are produced being, as yet, not entirely understood. It is probable
that the process takes place after hydrolysis of the disaccharid some¬
what according to the following formula:

C6 H12 06 = C4 H8 02 + 2 C02 + 2 Ha.

Special bacteria have been described in connection with this form of
milk fermentation,2 most of them non-pathogenic. It is unquestionable,
however, that many of the well-known pathogenic bacteria, such as
Bacillus aerogenes capsulatus, Bacillus cedematis maligni, possess the
power of similar butyric-acid formation. While less commonly observed
in milk, because milk is rarely kept long enough to permit of the action
or development of these enzymes, the butyric-acid fermentation is of
importance in connection with butter, where it is one of the causes pro¬
ducing rancidity.

Alcoholic fermentation may take place in milk as a result of the ac¬
tivities of certain yeasts. Upon the occurrence of such fermentations
depends the production of kefyr, koumys, and other beverages which
have been in common use for many years, especially in the region of the
Caucasus. The characteristic quality of these beverages is contrib¬
uted by the feeble alcoholic fermentation produced by members of the
saccharomyces group, but side by side with this process lactic-acid forma-

1 Conn, Exper. Stat. Rep., 1892.

2 Schattenfroh und Grasberger, Arch. f. Hyg., 37, 1900.

BACTERIA IN MILK

703

tion also takes place. Beijerinck,1 who has carefully studied the so-
called kefyr seeds, used for the production of kefyr in the East, has
isolated from them a form of yeast similar in many respects to the
ordinary beer yeast, and a large bacillus to which he attributes the
lactic-acid formation.

Occasional but uncommon changes which occur in milk lead to the
formation of the so-called “ slimy milk/' yellow and green milk, and
bitter milk. These may be due to a number of bacteria. A microorgan¬
ism which is commonly found in connection with the slimy changes in
milk is the so-called Bacillus lactis viscosus. According to the researches
of Ward,2 this microorganism is frequently derived from water and it is
the water supply which should attract attention whenever such trouble
occurs in dairies.

The so-called blue, green, and yellow changes are usually due to
chromogenic bacteria, such as Bacillus cyanogenes, Bacillus prodigiosus,
and others.

“ Bitter milk," a condition which has occasionally been observed epi¬
demically, is also the consequence of the growth of microorganisms.
Conn,3 in 1891, isolated from a specimen of bitter cream a diplococcus
which occasionally forms chains and which in sterilized milk develops
rapidly, producing an extremely bitter taste. The organism of Conn
differs from a similar diplococcus described by Wagmann4 in that it
possesses the ability of producing butyric acid.

Milk in Relation to Infectious Disease. — As a source of direct in¬
fection, milk is second only to water, and deserves close hygienic at¬
tention. A large number of infectious diseases have been traced to milk,
although the actual proof of the etiological part played by it in such cases
has often been difficult to adduce and has necessarily been indirect.
Nevertheless, even when indirect proof only has been brought, it has been
sufficiently convincing to necessitate the most careful investigation into
milk supplies whenever epidemics of certain infectious maladies occur.

Typhoid-fever epidemics have been frequently traced to milk in¬
fection, and, in this disease, milk is, next to water, the most frequent
etiological factor. Schiider,5 in an analysis of six hundred and fifty
typhoid epidemics, found four hundred and sixty-two attributed to

1 Beijerinck, Cent. f. Bakt., vi, 1889.

2 Ward, Bull. 165, Cornell Univ. Agri. Exp. Stat., 1899.

a Conn, Cent. f. Bakt., ix, 1891.

* Wagmann, Milchztg., 1890.

* Schiider , Zeit. f. Hyg., xxxviii, 1901.

704

BACTERIA IN AIR, SOIL, WATER, AND MILK

water, one hundred and ten to milk, and seventy-eight to all other
causes.

Trask 1 compiled statistics of one hundred and seventy-nine typhoid
epidemics supposed to have been caused by milk, in various parts of the
world. In all such epidemics the origin of infection was generally trace¬
able to diseased or convalescent persons employed in dairies, to con¬
taminated well water used in washing milk utensils, or to the use of cans
and bottles returned from dwellings where typhoid fever had existed.
Actual bacteriological proof of the infectiousness of milk by the isolation
of Bacillus typhosus is rare, but has been accomplished in isolated in¬
stances. In the case of one epidemic, Conradi 2 isolated the bacillus from
the milk on sale at a bakery at which a large number of the infected
individuals had purchased their milk. The examination of market milk
at Chicago, through a period of eight years, revealed the presence of
typhoid bacilli but three times.

In spite of the few cases in which actual bacteriological proof has been
brought, it is not unlikely that careful and systematic researches would
reveal a far greater number, since many writers have shown that typhoid
bacilli may remain alive in raw milk for as long as thirty days,3 and
may actively proliferate in the milk during this time. One peculiarity
of epidemics which may aid in arousing the suspicion that they have
originated in milk is that, in such cases, women and children are far
more frequently attacked than men.4 *

A feature which adds considerably to the dangers of milk infection
is the unfortunate absence of any gross changes, such as coagulation,
by the growth of typhoid bacilli.

Scarlet fever, ^ though as yet of unknown etiology, has in many cases
been traced indirectly to milk infection. Trask has collected fifty-one
epidemics of scarlet fever presumably due to milk. In one epidemic
occurring in Norwalk, Conn.,6 twenty-nine cases were distributed among
twenty-five families living in twenty-four different houses. The indi¬
viduals affected did not attend the same school, and were of entirely
different social standing, the only factor common to all of them being
the milk supply.

1 Trask, Bull. No. 41, U. S. Pub. Health and Mar. Hosp. Serv., Wash.

2 Conradi, Cent. f. Bakt., I, xl, 1905.

3 Heim, Arb. a. d. kais. Gesundheitsamt, v.

4 Wilckens, Zeit. f. Hyg., xxvii, 1898.

6 Trask, loc. cit.

R Herbert E. Smith, Rep. Conn. State Bd. of Health, 1897.

BACTERIA IN MILK

705

Diphtheria has been frequently traced to the use of infected milk.
In most of the epidemics reported as originating in this way, the proof
has been necessarily indirect. In two out of twenty-three epidemics
reported by Trask, however, Bacillus diphtherise was isolated from
the milk directly. The ability of the Klebs-Loeffler bacillus to proliferate
and remain alive for a long while in raw milk has been demonstrated
by Eyre 1 and others.

Whether or not cholera asiatica may be transmitted by means of
milk has been a disputed question. Hesse 2 claims that cholera spirilla die
out in raw milk within twelve hours. This statement, however, has not
been borne out by other observers.3 Unquestionable cases of direct
transmission of cholera by means of milk have been reported by a num¬
ber of writers, notably by Simpson.4

The relation of milk to the diarrheal diseases of infants has, of late
years, received a great deal of attention. In large cities, during the
summer months, numerous cases of infantile diarrhea among bottle-
fed babies occur, which, in many instances, are attributed to feeding with
contaminated milk. Park and Holt,5 who have made extensive re¬
searches upon this question in New York City, have come to the con¬
clusion that the harmful effects of contaminated milk upon babies
can not be ascribed to any given single microorganism in the milk.
Specifically toxic properties were found by these writers for none of the
one hundred and thirty-nine different species of bacteria isolated from
unsterilized milk. It is unlikely, therefore, that the diarrheal diseases
among babies have a uniform bacteriological cause. Whether or not
these diarrheal conditions depend entirely upon the bacterial contents
of milk or, in a large number of cases at least, upon the inability of the
child to digest cows' milk because of chemical conditions, must be left
undecided. Park and Holt, in analyzing their extensive data, conclude
that milk containing “ over one million bacteria to the cubic centimeter
is certainly harmful to the average infant."

The significance of the presence of streptococci in milk, as an element
of danger, has recently received much attention in the literature. Heine-
mann,6 who has made a careful comparison of Streptococcus lacticus

1 Eyre, Brit. Med. Jour., 1899.

* Hesse, Zeit. f. Hyg., xvii, 1894.

i Basenau, Arch. f. Hyg., xxiii, 1895.

* Simpson, Indian Med. Gaz., 1887.

6 Park and Holt, Arch, of Ped., Dec., 1903.
8 Heinemann, Jour. Inf. Dis., 3, 1906.

706

BACTERIA IN AIR, SOIL, WATER, AND MILK

(formerly spoken of as Bacillus acidi lactici [Kruse]), with other strep¬
tococci, has shown that, essentially, this streptococcus does not differ
from streptococci from other sources, and is practically indistinguish¬
able by cultural methods from Streptococcus pyogenes. Similar com¬
parisons made by Schottmuller,1 Muller, 2 and others have led to like re¬
sults. Since streptococci may be found in milk from perfectly normal
cows and are almost regularly associated with lactic-acid fermenta¬
tion, it is unlikely that these microorganisms hold ordinarily any
specific relationship to disease.

Recently, however, a number of epidemics of sore throat caused by
streptococci have been traced to milk upon reasonably reliable evidence.
Accounts of such epidemics in Chicago and in Baltimore have been
published by Capps and Miller 3 and by Hamburger.4

The presence of pus cells and leucocytes in milk, together with
streptococci, was also formerly regarded as of great importance.
Enumerations of leucocytes in milk were first made by Stokes and
Weggefarth.5 Their method of enumeration consisted in centrifugaliz-
ing a definite volume of milk, spreading the entire sediment over a
definite area on a slide, and counting the leucocytes found in a number of
fields. Calculations from this may then be made as to the number of
leucocytes per cubic centimeter. This method, and modifications of it,
have been used by a large number of observers, but the value of the con¬
clusions drawn from them has been much exaggerated. Normal milk
*

may contain leucocytes in moderate numbers, and importance may be
attached to such leucocyte counts only when their number largely ex¬
ceeds that present in other specimens of perfectly normal milk. When¬
ever such high leucocyte counts are found, of course, a careful veteri¬
nary inspection and examination for pyogenic disease should be made.

Foot-and-mouth disease, an infectious condition prevailing among
cattle, characterized by a vesicular rash on the mouth and about the
hoofs, has, in a number of cases, been definitely shown to be transmitted
to man through the agency of milk. Notter and Firth6 reported an
epidemic occurring among persons supplied with milk from a single dairy
in which foot-and-mouth disease prevailed among the cows. In this

1 Schottmuller , Munch, med. Woch., 1903.

2 Miiller , Arch. f. Hyg., lvi, 1906.

3 Capps and Miller , Jour. A. M. A., June, 1912, p. 1848.

4 Hamburger , Bull, of the Johns Hopk. Hosp., xxiv, Jan., 1913.

5 Stokes and Weggefarth, Med. News, 91, 1897.

G Notter and Firth, quoted from Harrington, “Theory and Practice of Hygiene.”

BACTERIA IN MILK

707

epidemic, two hundred and five individuals were affected with vesic-
lar eruptions of the throat, with tonsillitis and swellings of the cervical
lymph nodes. Similar cases have been reported by Pott.1

Although anthrax has never been definitely shown to have been
conveyed by milk, Boschetti 2 succeeded in isolating living anthrax
bacilli from a sample of milk two weeks after its withdrawal from the cow.

Milk and Tuberculosis. — The question of the conveyance of tuber¬
culosis by means of milk is a subject which, because of its great im¬
portance, has been extensively investigated by bacteriologists. A
large number of observers have succeeded in proving the presence of
tubercle bacilli in the milk of tuberculous cows by intraperitoneal in¬
oculation of rabbits and guinea-pigs with samples of milk. Such posi¬
tive results have been obtained by Bang,3 Hirschberger,4 Ernst,5 and
many others. A number of these observers, notably Ernst, have shown
that tubercle bacilli may be present in the milk without tuberculous dis¬
ease of the udders. In an examination of the milk supply of Washington,
D. C.,6 6.72 per cent of the samples contained tubercle bacilli.

The path of entrance of the bacilli from the cow into the milk has
long been a subject of controversy. That the bacilli may easily enter
the milk, when tuberculous disease of the udder is present, stands to
reason and is universally conceded. It is now believed also, on the
basis of much experimentation, that in systemically infected cows
tubercle bacilli may pass through the mammary glands into the milk,
without evidence of local disease in the secreting gland. An experi¬
ment performed by the Royal British Tuberculosis Commission 7 illus¬
trates this point. A cow, injected subcutaneously with tubercle bacilli
behind the shoulder, began to discharge tubercle bacilli in the milk
within seven days after inoculation and continued to do so until death
from generalized tuberculosis.

Milk may become indirectly contaminated, furthermore, with
tubercle bacilli emanating from the feces of cows. It has been shown
that tubercle bacilli are present in the feces of cattle so early in the
disease that diagnosis can be made only by a tuberculin test.8

Whether or not contaminated milk is common as an etiological

1 Pott, Munch, med. Woch., 1899. 2 Boschetti, Giorn. med. vet., 1891.

3 Bang, Deut. Zeit. f. Tierchem., xi, 1884.

4 Hirschberger, Deut. Arch. f. klin. Med., xliv, 1889.

5 Ernst, H. C., Amer. Jour. Med. Sci., xcviii, 1890.

6 Anderson, Bull. No. 41, U. S. Pub. Health and Mar. Hosp. Serv., Wash., 1908.

7 Quoted from Mohler, P. H., and Mar. Hosp. Serv. Bull. 41, 1908.

8 Schroeder and Cotton, Bull. Bureau Animal Industry, Wash., 1907.

708

BACTERIA IN AIR, SOIL, WATER, AND MILK

factor in human tuberculosis, must be considered at present as an un¬
settled question. Behring, at the Congress of Veterinary Medicine,
at Cassel, in 1903, advanced the view that pulmonary tuberculosis in
adults may be a late manifestation of a milk infection contracted dur¬
ing infancy. He stated as his own opinion, moreover, that most cases
of tuberculosis in man are traceable to this origin. The problem is as
difficult of solution as it is important. In bottle-fed infants, infection
by means of milk unquestionably occurs with considerable frequency.
Smith,1 Kossel, Weber, and Huess,2 and others, have isolated tubercle
bacilli of the bovine type from the mesenteric lymph nodes of many
infected children. Animal experimentation has, furthermore, revealed
that lesions in the mesenteric nodes, as well as later in the bronchial
lymph nodes, may occur as a consequence of feeding tubercle bacilli,
without any demonstrable lesions in the intestinal mucosa. It is thus
certain that infection by the ingestion of tuberculous milk may occur,
especially among young children who, as is well-known, are com¬
paratively susceptible to bacilli of the bovine type. Whether or not such
infection will account for many cases of tuberculosis in adults is a ques¬
tion which, for final solution, will require much more investigation. The
sole reliable method of approaching it lies in determining the type,
human or bovine, of the bacilli present in a large number of cases. Ex¬
perience thus far seems to indicate that the bovine type is comparatively
rare in the pulmonary disease of adults.

The value of the tuberculin reaction for diagnosis, and the elimination
of all cattle showing a positive reaction, for the prevention of tubercu¬
losis, can not be overestimated. The failure of the test in diseased
animals is rare, and an accurate diagnosis can be established in over
90 per cent of diseased animals.3 The assertion that the cattle are
permanently injured by tuberculin injections is without scientific basis.
If this test were conscientiously carried out, and infected cattle elim¬
inated, the dangers from bovine bacillus infection would be practically
eliminated, for there are but few instances in which science has been able
to furnish such definite information for absolute protection. It is need¬
less to say, however, that the carrying out of such precautions is
subject to great expense and great difficulties of organization.

Dairy inspection is practiced in the vicinity of many of our larger

1 Smith, Trans. Assn. Amer. Physic., 18, 1903.

2 Kossel, Weber, and Huess, Tuberkul. Arb. a. d. kais. Gesundheitsamt, 1904,
1905, Hft. 1 and 3.

3 Mohler, loc. cit.

BACTERIA IN MILK

709

cities, and the movement is daily gaining ground. Until fully estab¬
lished, however, upon a financial basis which brings the best products
within the means of the poorer classes, other inexpensive measures to
render milk safe must often be resorted to.

Sterilization by high temperatures is objected to by pediatricians
because of the physical and chemical changes produced in the milk
which are said to detract from its nutritive value.

The development of scurvy and rickets in infants has often been
attributed to the use of such milk. These objections, however, do not
apply to the use of milk which has been subjected to the process of
“pasteurization.” By this term is meant the heating of any substance
to 60° C. for twenty to thirty minutes. The process, first devised by
Pasteur for the purpose of destroying germs in wine and beer in which
excessive heating was supposed to injure flavor, brings about the death
of all microorganisms which do not form spores — in other words, of all
the bacteria likely to be found in milk which can give rise to infection
per os. At the same time the chemical and physical constitution of the
milk is not appreciably changed, at least not to an extent which renders
it less valuable as a food. Statistics by Park and Holt 1 have shown
strikingly the advantages of pasteurized over raw milk in infant feed¬
ing. Of fifty-one children fed with raw milk during the summer months,
thirty-three had diarrhea, two died, and only seventeen remained
entirely well. Of forty-one receiving pasteurized milk, but ten had
diarrhea, one died, and thirty-one remained entirely well throughout
the summer. The actual diminution of the living bacterial contents
of milk by pasteurization is enormous, the milk so treated often con¬
taining not more than one thousand, usually less than fifteen thou¬
sand, living bacteria to each cubic centimeter.

Methods of Estimating the Number of Bacteria in Milk. — In estimating
the number of bacteria in milk, colony counting in agar or gelatin plates
is resorted to. Great care must be exercised in obtaining the specimens.
If taken from a can, the contents of the can should be thoroughly mixed,
since the cream usually contains many more bacteria than the rest of
the milk. The specimen is then taken into a sterile test tube or flask.
If the milk is supplied in an ordinary milk bottle, this should be
thoroughly shaken before being opened, and the specimen for exam¬
ination taken out with a sterile pipette. Dilutions of the specimen can
then be made in sterile broth or salt solution. If an initial dilution

1 Park and Holt, loc. cit.

710

BACTERIA IN AIR, SOIL, WATER, AND MILK ^

of 1 : 100 is made, quantities ranging from 1 c.c. to 0.1 c.c. of this will
furnish 0.01 c.c. to 0.001 c.c. of the milk, respectively. Inoculation of
properly cooled tubes of melted neutral agar and gelatin, with varying
quantities of these dilutions, are then made and plates poured. After
twenty-four to forty-eight hours at room temperature or in the in¬
cubator, colony counting is done as described upon page 161, and the
proper calculation is made. In samples in which few bacteria are
expected, direct transference of 1/20 or l/40 of a c.c. of the whole
milk into the agar may be made. This saves time but is less accurate
than the method given above.

Bacteria and Butter. — Butter is made from cream separated from
milk either by standing or by centrifugalization. After this, the cream
is agitated by churning, which brings the small fat-globules into mutual
contact, allows them to adhere to each other and form clumps of butter.
It has been a matter of common experience, however, that unless the
cream is allowed to “ ripen” for a considerable period before churning,
the resulting butter lacks the particular quality of flavor which gives it
its market value. The interval of ripening, at first a necessity upon,
small farms where cream must be collected and allowed to accumulate,
has now been recognized as an essential for the production of the best
grades of butter, and it has been shown that the changes taking place
in the cream during this period are referable to the action of bacteria.
Cream, which before the ripening process contains but 50,000 bacteria
to each cubic centimeter, at the end of a period of “ripening ” will often
contain many millions of microorganisms. At the same time, the cream
becomes thick and often sour.

The species of bacteria which take part in this process and which,
therefore, must determine to a large extent the quality of the end prod¬
uct, are various and, as yet, incompletely known. Usually some variety
of lactic-acid bacilli is present and these, as in milk, outgrow other species
and, according to Conn,1 are probably essential for “ripening.”

It would be of great practical value, therefore, if definite pure
cultures of the bacteria which favor the production of agreeable flavors
could be distributed among dairies. In Denmark this has been attempt¬
ed by first pasteurizing the cream and then adding a culture of bacteria
isolated from “favorable” cream. These cultures, delivered to the
dairyman, are planted in sterilized milk, in order to increase their quan¬
tity, and this culture is then poured into the pasteurized cream. In

1 Conn, “Agricultural Bacteriology,’’ Phila., 1901,

BACTERIA IN MILK

711

most cases, these so-called “ starters ” are not pure cultures, but mixtures
of three or more species derived from the original cream.

Adverse accidents in the course of butter-making, such as “ souring ”
or “ bittering ” of butter, are due to the presence of contaminating,
probably proteolytic, microorganisms in the cream during the process of
“ripening.”

As a means of transmitting infectious disease, butter is of importance
only in relation to tuberculosis. Obermiiller,1 Rabinowitch,2 Boyce,3
and others, have repeatedly found tubercle bacilli in market butter, and
Mohler,4 Washburn, and Rogers have recently shown that these bacilli
could remain alive and virulent for as long as five months in butter kept
at refrigerator temperature. The acid-fast butter bacillus, described by
Rabinowitch as similar to the true Bacillus tuberculosis, shows decided
cultural and morphological differences from the latter.

Bacteria and Cheese. — The conversion of milk products into cheese
consists in a process of proteid decomposition which, by its end products,
leucin, tyrosin, and ammonia compounds, largely determines the cheese-
flavors. The production of cheese, therefore, is due to the action of
proteolytic bacterial enzymes 5 and the variety of a cheese is largely
determined by the microorganisms which are present and by the cultural
conditions prevailing. The sterilization of cream, or the addition of
antiseptics, absolutely prevents cheese production.

The organisms which are concerned in such processes have been
extensively studied and attempts have been made, with moderate
success, to produce a definite flavor with pure cultures.

In the production of cheese the two varieties, hard and soft cheeses,
depend not so much upon the bacterial varieties as upon the differ¬
ences in the treatment of the curds before bacterial action has begun.
In the former case, a complete freeing of the curds from the whey
furnishes a culture medium which is comparatively dry and of almost
exclusively proteid composition; in the latter, retention of whey gives
rise to cultural conditions in which more rapid and complete bacterial
action may take place. The holes, which are so often observed in some
of the hard cheeses, are due to gas production during the process of
“ripening.”

1 Obermuller, Hyg. Rundschau, 14, 1897.

2 Rabinowitch, Zeit. f. Hyg., xxvi, 1897.

3 Boyce and Woodhead, Brit. Med. Jour., 2, 1897.

4 Mohler, IT. S. P. Id. and Mar. Hosp. Serv. Bull. 41, 1908.

6 Freudenreich, Koch’s Jahresbericht, etc., 135, 1891,

712

BACTERIA IN AIR, SOIL, WATER, AND MILK

As to the varieties of microorganisms present in various cheeses, much
careful work has been done. Duclaux 1 attributed the “ ripening ” of
some of the soft cheeses to a microorganism closely related to Bacillus
subtilis. V. Freudenreich 2 in part substantiated this, but laid particular
stress upon the action of Oidium lactis, a mold, and upon several
varieties of yeast. Conn,3 more recently, in a bacteriological study of
Camembert cheese, has demonstrated that the production of this cheese
depends upon the united action of two microorganisms, one an oidium,
like the Oidium lactis of Freudenreich, which is found chiefly in the
interior softened areas, the other a mold belonging to the penicillium
variety, found in a matted felt-work over the surface and penetrating
but a short distance. In spite of the scientific basis upon which the
work of these men and of others has seemed to place cheese production,
attempts at uniformity in cheese production have met with almost
insuperable obstacles because of the presence of a variety of adventi¬
tious microorganisms which, depending in species and proportion upon
the local conditions under which the various cheeses have been produced,
have added minor characteristics of flavor which have determined
market value. Occasional failure of good results in cheese production 4
is due to contamination with other chromogenic or putrefactive bacteria.

In its relationship to the spread of infectious disease, cheese is
relatively unimportant except in regard to tuberculosis. Typhoid and
other non-spore forming pathogenic germs can not survive the condi¬
tions existing during cheese-ripening for any length of time. Tubercle
bacilli, both of the human and bovine types, have been found in cheese
by Harrison 5 and others, and Galtier has shown experimentally that
tubercle bacilli may remain alive and virulent in both salted and un¬
salted cheese for as long as ten days.

THE LACTIC-ACID BACILLI AND METCHNIKOFF’S BACTERIO-

THERAPY

A problem which has occupied clinical investigation for many years
is that of gastrointestinal autointoxication. There are a number of
conditions occurring in man, in which symptoms profoundly affecting

1 Duclaux, “Le Lait,” Paris, 1887.

2 V. Freudenreich, Cent. f. Bakt., II, i, 1895.

3 Conn, Bull. Statis. Agri. Exp. Stat. 35, 1905.

4 Beijerinck, Koch's Jahresber, etc., 82, 189.

5 Harrison and Galtier, quoted from Mahler, U. S. Pub. II. and Mar. IIosp. Serv.,
Hygiene Lab. Bull. 41, 1908.

LACTIC-ACID BACILLI

713

the nervous system, the circulation, and, in a variety of ways, the entire
body, can be clinically traced to the intestines, and can, in many cases,
be relieved by thorough purgation and careful diet. In some of these
conditions, specific microorganisms can be held accountable for the
diseases (B. enteritidis, B. botulinus, etc.). In other cases, however,
etiological investigations have met with but partial success because of
the large variety of microorganisms present in the intestinal tract and
because of the complicated symbiotic conditions thereby produced.
Intestinal putrefaction, recognized as the cardinal feature of such
maladies, has been attributed to Bacillus proteus vulgaris,1 to Bacillus
aerogenes capsulatus, to Bacillus putrificus,2 and to a number of other
bacteria, but definite and satisfactory proof as to the etiological im¬
portance of any of these germs has not yet been advanced. The fact
remains, however, that, whatever may be the specific cause, the disease
itself, a grave and often fatal affliction, may be clinically traced, in a
number of cases, to the absorption of poisons from the intestinal canal,
and it is more than likely that these poisons are the products of bacterial
activity. Reason dictates, furthermore, that the bacteria primarily
responsible for the production of these toxic substances do not belong to
the varieties which attack carbohydrates only, but must belong to that
class of aerobic and anaerobic germs which possess the power of breaking
up proteids — in other words, the bacteria of putrefaction.

On the basis of the mutual antagonism existing in culture between
many acid-producing bacteria and those of putrefaction — a phenomenon
recognized by some of the earliest workers in this field, many investigators
have suggested the possibility of combating intestinal putrefaction by
adding acid-forming bacteria together with carbohydrates to the diet
of patients suffering from this condition. The first to suggest this
therapy was Escherich 3 who proposed the use, in this way, of Bacillus
lactis aerogenes; with the same end in view, Quincke,4 a little later,
suggested the use of yeasts — Oidium lactis. The reasoning underlying
these attempts was meanwhile upheld by experiments carried out both
in vitro and upon the living patient. Thus Brudzinski 5 was able to
demonstrate that Bacillus lactis aerogenes, in culture, inhibited the
development of certain races of the proteus species and succeeded in

1 Lesage, Rev. de med., 1887.

2 Tissier, Ann. de Pinst. Pasteur, 1905.

3 Escherich, Therapeut. Monatshefte., Oct., 1887.

* Quincke, Verhandl. des Congress f. Inn. Med., Wiesbaden, 1898.

£ Brudzinski, Jahrbuch f. Kinderheilkunde, 52, 1900 (Erganzungsheft).

714 BACTERIA IN AIR, SOIL, WATER, AND MILK

obtaining markedly favorable results by feeding pure cultures of Bacillus
lactis aerogenes to infants suffering from fetid diarrhea. Similar ex¬
periments 1 carried out with the Welch bacillus (aerogenes capsulatus)
and Bacillus coli, however, had no such corroboratory results, since this
anaerobe possesses a considerable resistance against an acid reaction.
In considering the difficulties of the problems involved in this question,

Fig. 156. — Bacillus bulgaricus.

it occurred to Metchnikoff2 that much of the practical failure of therapy,
based upon the principles stated above, might be referred to insufficient
powers of acid production on the part of Bacillus coli, Bacillus lactis
aerogenes, and other germs previously used. In searching for more pow¬
erful acid producers, his attention was attracted to Bacillus bulgari-

1 Tissier and Martelly, Ann. de l’inst. Pasteur, 1906. L

2 Metchnikoff, “Prolongation of Life,” G. P. Putnam’s Sons, N. Y.; also in “Bac-
teriotherapie,” etc. “ Bibliotheque de therapeutique,” Gilbert and Carnot, Paris,
1909.

BACTERIA IN THE INDUSTRIES

715

cus, isolated from milk by Massol 1 and Cohendy2 in 1905. This bacillus,
according to the researches of Bertrand and Weisweiller,3 produces as
much as 25 grams of lactic acid per liter of milk. In addition to this,
it manufactures, from the same quantity of milk, about 50 centigrams
of acetic and succinic acids and exerts no putrefactive action upon pro-
teids. Added to these characters, it is especially adapted to therapeutic
application by its complete lack of pathogenicity.

The administration of the bacillus to patients suffering from intestinal
putrefaction, first suggested by Metchnikoff in 1906, has, since that time,
been extensively practiced and often with remarkable success. In
spite of sharp criticism, especially by Luersen and Kuhn, 4 who deny
mnch of the antiputref active activity of the bacillus, the treatment of
Metchnikoff has found many adherents, upon the basis of purely clinical
experiment. It is not possible to review completely the already ex¬
tensive literature. Among the more valuable contributions may be
mentioned the articles by Grekoff,5 by Wegele,6 and by Klotz.7 In
Metchnikoff s experiments and in the work of his immediate successors,
the bacillus was used either in milk culture or in broth in which it was
induced to grow in symbiosis with other microorganisms.

Recently, North 8 has suggested the use of Bacillus bulgaricus in
parts of the body other than the digestive tract. His work was made
feasible by the discovery that the bacillus could be cultivated in dex-
trose-pepton broth to which calcium carbonate has been added, after
the manner recommended by Hiss. With such cultures, applied in the
form of a spray, inflammations of the ear, nose, throat, genitourinary
tract, etc., have been treated, many of them with success.

BACTERIA IN THE INDUSTRIES

Bacteria and Tobacco. — In the manufacture of tobacco, the har¬
vested leaves are first dried and then heaped up in large masses in which
the tobacco undergoes fermentation. During this fermentation, which

1 Massol, Revue medieale de la Suisse romanae, 1905.

2 Cohendy, Comptes rend, de la soc. de biol., 60, 1906.

3 Bertrand and Weisweiller, Ann. de l’inst. Pasteur, 1906.

4 Luersen and Kuhn, Cent. f. Bakt., II, xx, 1908.

5 Grekoff, “ Observations cliniques sur Peffet du lact. agri.,” etc., St. Petersburg,
1907.

0 Wegele, Deut. med. Woch., xxxiv, 1908,

7 Klotz, Zentralbl. f. innere Med., 1908.

8 North, Med. Record, March, 1909,

716 BACTERIA IN AIR, SOIL, WATER, AND MILK

goes on at temperatures varying from 50° C. to 60° C., carbohydrates
are split up and much nicotin is destroyed.1 The end products consist
largely of C02 and various organic acids, butyric, formic, succinic, etc.
During the fermentation, bacteria of many varieties are found in the
heaps of tobacco leaves and many attempts have been made to deter¬
mine flavors artificially by inoculating tobacco leaves of a poorer quality
with cultures obtained from the finer Havana grades. Suchsland2 and
others, who have attempted this, claim to have obtained marked im¬
provements in domestic products by this method. The bacteria found
in tobacco fermentation belong to many varieties. Some of these are
closely related to the proteus and subtilis groups. Others are distinctly
thermophilic, an attribute required by the high temperatures attained
in the fermenting tobacco leaves. It is probable that the tobacco
flavors can not be regulated by bacteriological methods alone, since it
has been shown by Loew 3 that an important factor in the tobacco fer¬
mentation is contributed by the leaf-enzymes, which, of course, depend
intimately upon soil and climatic conditions.

Opium Productions. — In the preparation of opium for smoking pur¬
poses, the raw product is subjected to a prolonged period of fermentation
by which the carbohydrates in the material are destroyed. According
to various observers, the process is carried out in most cases by a species
of aspergillus.

Indigo Production. — Indigo, which is obtained from the plants
“ Isatis tinctoria ” and “ Indigofer tinctoria,” is not present, as such, in
the plants. In some of these it is found in the form of indican, in others,
as indoxyl. It has been shown by Alvarez and others that the oxida¬
tion of indican and indoxyl into indigo-blue is carried out largely by
bacterial oxydases. Sterilized indigo plants do not produce the blue
color. Alvarez 4 has isolated a bacillus closely related to the Bacillus
mucosus capsulatus group, to the action of which he attributes this
oxidation.

Bacteria in the Tanning of Hides. — Raw animal hides are subject
to decomposition until treated by a process known as tanning. This
consists first in the depilation of the dried and salted skins, either by
partial putrefaction in an atmosphere saturated with water vapor or
by chemical treatment with solutions of milk of lime. After this, the

1 Behrens, quoted from Flugge, “ Die Mikroorganismen,” Bd. 1, Leipzig, 1896.

2 Suchsland, Ber. der Deut. botan. Ges., ix.

3 Loew, Rep. U. S. Dep. Agriculture, 59, 1899.

i Alvarez , Comptes rend, de l’acad. des sci. vol. 105.

BACTERIA IN THE INDUSTRIES

717

tanning proper consists in subjecting the skins to prolonged immersion
in solutions made up according to a large variety of formulae — the
principle of all of which, however, seems to be found in the mixing of
various organic ingredients, such as bran-mash, oak-bark, and often dried
animal excreta, in which fermentation and acid production occurs.
According to ITaenlein 1 this acidification is the essential process by which
the leather is sterilized and rendered soft and pliable. This author has
described a microorganism, Bacillus corticalis, which he found regu¬
larly present in fir-tree bark and to which he ascribes the acid fermenta¬
tion occurring in tanning liquors in which this ingredient is employed.
Wood,2 who has worked extensively upon the subject, has attempted
with some success to substitute pure cultures for the old uncertain
chance mixtures employed. In spite of these investigations, however,
while we must acknowledge the probable importance of bacteria in the
tanning process, the subject is by no means upon a scientific or exact
basis.

1 Haenlein, Cent. f. Bakt. II, i, 1895.

2 Wood, Jour. Soc. Chem. Industry, 1895, 1899.

INDEX OF AUTHORS

Abbott, 97, 326
Abel, 189, 450, 452, 557
Abel and Claussen, 585
Achard and Bensaude, 430
Adami, 392

Adami and Chapin, 696
Agramonte, 669, 670
Albrecht and Ghon, 376, 377, 378,
555, 561
Allegri, 502
Alvarez, 716
Alvarez and Tavel, 503
Anderson, J. G., 707
Anderson and Goldberger, 677, 678
Anderson and McClintic, 80
Andrewes and Horder, 349
Aristotle, 2
Arloing, 63

Arloing and Chauveau, 471
Arloing, Cornevin and Thomas, 468
Arloing, Leclainche, and Vallee, 468
Arning, 509

Aronson, 91, 346, 347, 348, 350
Arrhenius and Madsen, 211
Arthus, 297
Arustamoff, 619
Asakawa, 214
Ascoli and Figari, 201
Axenfeld, 546

Babes, 11, 510, 514, 550, 613, 614, 623,
648

Babes and Lepp, 197
B aginsky, 343, 460, 519
Baginsky and Sommerfeld, 343, 344,
676

Bail, 292, 293, 330
Bail and Petterson, 292
Bail and Weil, 293
Baldwin, 494

Bandelier and Roepke, 497

Bandi and Simonelli, 596
Bang, 707
Banzhaf, 219, 220
Barker and Cole, 424
Basenau, 430, 705
Bauer, 268

Baumgarten, 106, 479, 486
Baumler, 540
Beck, 493, 499, 514

Beck, M., Kolle, and Wassermann, 517
Beckmann, 78
Behrens, 716

v. Behring, 76, 77, 78, 79, 195, 205, 221,
295, 487, 490, 569

v. Behring and Kitasato, 196, 198
v. Behring and Kitashina, 295
v. Behring and Wernicke, 196, 198
Beijerinck, 26, 27, 703, 712
Belfanti, 458

Belfanti and Carbone, 200
Beljaeff, 198
Beraneck, 493
Berestnew, 618, 620, 626
Bertarelli, 501, 590, 602, 603
Berthelot, 54

Bertrand and Weisweiller, 715
Besredka, 298, 302, 345, 416
Besredka and Steinhardt, 299
Beuermann and Gougerot, 645
BEZANgoN, 334

Bezanqon, Griffon, and Le Sourd, 548
Bienstock, 393, 481
Biggs and Park, 231
Billroth, 7
Binaghi, 368

Birch-Hirschfeld, 192, 392
Bitter, 31, 168, 503
Blaise and Sambac, 64
Bloodgood, 474
Blumer, 411

Bogart and Bernard, 201
719

720

INDEX OF AUTHORS

Boland, 579
Bollinger, 622
Bolton, 24

Bordet, 200, 224, 225, 228, 232, 236,
240, 242, 243, 337, 346, 367, 545
Bordet and Gay, 261
Bordet and Gengou, 245, 261, 262, 543
Bordoni-Uffreduzzi, 359
Borissow, 328
Borsiekow, 139
Boschetti, 707
Bostroem, 624
Boyce and Woodhead, 711
Brau and Denier, 589
Brauell, 6
Brieger, 185, 195, 415
Brieger and Boer, 462, 522
Brieger and Cohn, 461, 462
Brieger and Frankel, 521
Brieger and Kempner, 478
Brill and Libman, 579
Broughton, 427
Bruce, 550
Bruck, 215
Brudzinski, 713
v. Brunn, 90

Buchner, 22, 63, 152, 186, 198, 203, 224,
225, 389, 691
Buchner and Hahn, 492
Buchner and Meisenheimer, 50
Budd, 399
Budinger, 327
Buerger, 99, 349, 351, 355
Buffon, 4

Bullock and Atkin, 282, 284
Bullock and Hunter, 581
Bullock and Western, 282
Bumm, 380, 381

Bunge and Trautenroth, 106

Burkholtz, 63

Busse, 631

Butschli, 11, 12

Buxton, 428, 429

Buxton and Coleman, 180, 431

Cagniard-Latour, 3
Calkins, 650, 658
Calmette, 199, 203, 494

Calmette and Guerin, 660, 662

Canalis, 189

Canfora, 460

Cantacuzene, 280

Cantani, 539

Capaldi, 134

Capps and Miller, 706

Carlo and Rattone, 456

Carnot and Fournier, 363

Carriere, 47

Carroll, 671

Carter, 627

Castellani, 234, 405, 614
Certes, 35

Chamberland and Roux, 194, 569, 571

Chantemesse, 412, 424

Chantemesse and Widal, 418

Chapin, 562

Charrin, 577, 578

Charrin and Roger, 228

Chauveau, 195, 574

Christen, 67, 69

Christmas, 385

Chudiakow, 27

Churchman, 140

Churchman and Michael, 140

Citron, 273, 294

Clairmont, 449

Clarke, 658

Class, 676

Clegg, 506

Cobbett, 524

Cohendy, 715

Cohn, 5, 36

Cole, 366

Coleman and Buxton, 405
Coles, 504

Conn, 415, 702, 703, 710, 712
Conor, 678

Conradi, 137, 405, 443, 704
CoNRADI AND DrIGALSKI, 135
Conseil, 678
Copeland, 698
Cornet, 342, 487
Cornil and Babes, 669
Councilman, 658

Councilman, Mallory, and Wright,
374, 376, 377

INDEX OF AUTHORS

721

COURMONT AND DoYEN, 463

Craig and Nichols, 604

Cramer, 21

Creite, 460

Crowell, 561

Curtis, 631

Cushing, 431

Cushing and Livingood, 392

D’Arsonville and Charrin, 65

Davaine, 6, 563

Davis, 343

Dean, 283, 510

Debrand, 461

Delezenne, 201

Deneke, 591

Denys, 346, 490, 492, 497

Denys and Leclef, 281, 346

Denys and Marchand, 346

Denys and Van de Velde, 331

De Schweinitz and Dorset, 22, 490

Deslongchamps, 326

De Toma, 486

Deutsch and Feistmantel, 292

Dieudonne, 64, 216, 566, 584

Dobbin, 474

Dochez, 366

Doerr, 186, 299, 443

Doerr and Russ, 303

Donath and Landsteiner, 248

Donitz, 188, 218

Doutrelepont, 504

Dreyer and Madsen, 209, 215

Drigalski, 695

Drigalski and Conradi, 406

Dubarre and Terre, 501

Duclaux, 712

Ducrey, 548

v. Dungern, 201, 242, 411
Dunham, 386, 472, 473, 584
Durham, 428, 434
Durham and Myers, 669
Dusch, 4

Dutton and Todd, 608
Duval, 506

Duval and Wellman, 506

Eberth, 399
47

Ehrenberg, 2

Ehrlich, 7, 104, 187, 193, 199, 203, 204,
205, 206, 209, 210, 213, 238, 464, 481
Ehrlich, Kossel, and Wassermann,
205

Ehrlich and Morgenroth, 226, 241,
242, 243, 246

Ehrlich and Sachs, 242, 243
Eiciistedt, 639
Eisenberg, 580
Eisenberg and Volk, 420
v. Eisler and Pribram, 459
Ejkmann, 47, 49
Elser, 377

Elser and Huntoon, 376, 378, 379, 387

Elsner, 408

Endo, 135

Engelmann, 27, 61

Eppinger, 573, 620

Epstein, 77, 178, 619

Ernst, 11, 579, 707

Escherich, 343, 389, 394, 453, 515, 700,
713

v. Esmarch, 68, 147, 149
Evans and Russell, 92
Ewing, 596, 658
Eyre, 166, 550, 705
Eyre and Washburn, 360

Farnet, 413
Faure-Beaulieu, 384
Fehleisen, 336, 341, 342
Fermi and Pernossi, 462
Ferran, 28, 195, 458, 590
Ferri and Celli, 64
Ficker, 62, 231, 423
Field, 678
Finger, 530, 535
Finger and Landsteiner., 603
Finkelstein, 579
Finkler and Prior, 591
Finlay, 670

Firth and Horrocks, 687
Fischer and Proskauer, 88
Fisher, 9, 56, 59, 325
Fisher, A., 10, 19, 23, 24
Fitzgerald, 450

Flexner, 197, 378, 413, 436, 444, 620

722

INDEX OF AUTHORS

Flexner and Jobling, 378
Flexner and Lewis, 665
Flexner and Noguchi, 462
Flugge, 84, 85, 198, 325, 385, 586
Foa, 360

Foa and Carbone, 362
v. Fodor, 198, 224
Foges, 460
Foote, 415
Forneaca, 334
Forster, F., 32, 586
Fracastor, 2

Frankel, A., 353, 355, 360, 362, 404.

412, 421, 447, 472, 504, 570, 619, 686
Franzott, 326
Freudenreich, 711, 712
Friedberger, 304
Friedberger and Hartoch, 303
Friedemann, 303
Friedlander, 352, 447
Friedmann, 501
y. Frisch, 451

Frosch and Kolle, 339, 368
Fuhrmann, 44
Fuller, 692
Furbringer, 88

Gabbet, 105, 481
Gaffky, 333, 399, 400, 469
Gaffky, Pfeiffer, Sticker, and Dieu-
donne, 561
Galtier, 485, 535
Gamaleia, 590
Garre, 327
Gartner, 429
Gavino and Girard, 677
Gay, 243, 244, 427, 446
Gay and Claypole, 405
Gay and Southard, 299, 300, 302
Gengou, 240, 244, 273
Geppert, 86
Gessard, 577, 579
Gheorghiewski, 581
Ghon and Pfeiffer, 376, 386
Ghon and Preyss, 539
Gibson, 219

Gibson and Collins, 219
Giemsa, 108

Gilbert, 430

Gilchrist, 631

Globig, 33

Goldberger, 677

Goldhorn, 598

Goodwin and y. Sholly, 376

Gordon, 350

Gorgas, 670

Gottschlich, 23, 557

Gougerot, 644

Gram, 102

Gran, 49

Grancher and Ledoux-Lebard, 485

Grassberger, 539

Grawitz, 640

Grekoff, 715

Gruber, 67, 78, 421

Gruber and Durham, 200, 228, 234

Gruby, 642

Grund, 584

Grunhagen, 657

Guarnieri, 357, 359, 360, 657

Guerin, 662

Guiteras, 673

Gumprecht, 463

Gunther, 352

Gwyn, 430, 475

Haas, 66
Haenlein, 717
Haffkine, 590
Hahn, 190, 191, 416, 492
Hallier, 7
Hamburger, 303
Hammarsten, 46
Hammerschlag, 22, 490
Hanel, 326

Hankin and Leumann, 20
Hankin and Wesbrook, 168
Hansemann, 501
Hansen, 505, 631

Harding and Ostenberg, 136, 433

Harrington, 91

Harrison, 425

Harrison and Galtier, 712

Hartman, 340

Hauser, 151, 454

Heim, 704

INDEX OF AUTHORS

723

Heiman, 381, 384
Heinemann, 701, 705
Hektoen, 283, 675
Hektoen and Ruediger, 282, 283
Hellmich, 22

Hellriegel and Wilfarth, 55, 56

Henle, 3

Henrijean, 459

Hericourt and Richet, 296

Herter, 177, 394, 475

Hesse, 27, 133, 485, 705

Hilbert, 31, 520

Hill, 86, 94, 694

Hirsch, 554, 587

Hirschberger, 707

Hiss, 13, 98, 132, 251, 289, 337, 338, 339,
340, 347, 349, 351, 354, 355, 356, 357,
358, 364, 365, 369, 407, 408, 411, 439,
440

Hiss and Atkinson, 198, 219
Hiss and Russell, 438
Hiss and Zinsser, 291, 366, 379
Hoffman, 233

Hoffmann-Wellenhoff, 522
Hogyes, 652, 655, 656
Holst, 430
Home, 675

Hopkins and Lang, 349
Horton-Smith, 407, 411
Houston, 686, 697
Howard, 451

Howard and Perkins, 350, 369
Huber, 538, 540
Huddleston, 662
Hueppe, 551
Hueppe and Wood, 575
Huntemuller, 691
Hunter, 198

Irons, 698
Isaeff, 363
Israel, 622
Iwanow, 569

Jackson, 138, 696, 698
Jackson and Melia, 133, 138
Jager, 373, 451
Jenner, 108, 193, 659

Joachim, 198
Johnston, 404
Jones, 75
Joos, 233

Jordan and Heinemann, 700
Jordan and Irons, 693
Jordan, Russell, and Zeit, 414
Jorgensen and Madsen, 233

Kamen, 394, 534

Kappes, 21

Karlinsky, 411

Kelly, 343

Kempner, 199

Kempner and Pollack, 478

Kempner and Schepilewsky, 215

Kircher, 1

Kister and Wolff, 237
Kitasato, 456, 459, 462, 465, 555, 559
Kitasato and Weyl, 27
Kitt, 468

Kitt and Mayr, 554
Klebs, 7, 352, 493, 512, 669
Klein, 176, 404, 424, 480
Klemperer, 503
Klemperer, G. and F., 362
Klingmuller and Baermann, 604
Klotz, 715
Knapp, 525, 542
Knoepfelmacher, 665
Ivnorr, 205, 295, 340
Kobert and Stillmarcic, 204
Koch, 7, 63, 73, 77, 84, 86, 89, 321, 335,
352, 404, 469, 479, 483, 490, 491, 492,
493, 496, 497, 498, 542, 563, 566, 569,
570, 582, 587, 609, 610, 696
Koch and Petruschky, 345
Koch and Rabinovitsch, 500
Koch and Wolffhugel, 65, 570
Koch, Gaffky, and Loeffler, 65, 564
Kolle and Hetsch, 593
Kolle and Otto, 331
Kolle and Wassermann, 189, 190, 191,
378, 502
Korn, 502
Korschun, 291
Kossel, 204, 532
Kossel and Overbeck, 551

724

INDEX OF AUTHORS

Kossel, Weber, and Heuss, 499, 500,
708

Kotjar, 628
Kraunhals, 569

Kraus, 186, 200, 235, 237, 328, 329, 423,
443, 446, 589, 590
Kraus and Doerr, 441, 444
Kraus and Low, 232, 397
Kraus and v. Pirquet, 236
Kraus and Stenitzer, 416
Kresling, 532
Kretz, 217

Kronig and Paul, 74, 77, 78, 80,
Krumwiede, 614
Krumwiede and Pratt, 140
Krumwiede, Pratt, and Grund, 584
Kruse, 292, 437, 444, 446, 540, 552
Kurth, 431
Kuster, 501
Kutscher, 342, 431
Kutscher and Meinjcke, 432
Kutschert and Neisser, 525

• Lachner-Sandoval, 618
Landsteiner, 201
Landsteiner and Jagic, 240
Landsteiner and Levaditi, 665
Landsteiner, Muller, and Poetzl, 263
Landsteiner and Popper, 664
Lang, 349

Lanz and Tavel, 343
Lassar, 602
Lave ran, 592
Laws and Anderson, 695
Leclainche and Vallee, 467
Ledderhose, 578
Leeuwenhoek, 1
Leichtenstern, 372
Leishman, 281
Lembke, 392
Lentz, 438
Lepierre, 377

Le Roy, Des Barres, and Weinberg,
368

Lesage, 713
Leuchs, 137
Levaditi, 277, 280, 283
Levaditi and Inmann, 283

Levaditi and Manouelian, 599, 616
Levaditi and Petresco, 596
Lewith, 66
Libman, 339, 343, 431
Libman and Rosenthal, 370
Liborius, 149

v. Lingelsheim, 328, 331, 337, 340, 346,
348, 387

Linossier and Roux, 640
Lister, 6
Loeb, 324

Loeffler, 14, 100, 110, 136, 512, 520,
522, 528, 530

Loeffler and Frosch, 678
Loeffler and Schutz, 528
Loew, 25, 716
Lohlein, 282
Longcope, 432
Lowenstein, 501
Lubarsch, 190
Lubbert, 326
Luersen and Kuhn, 715
Lustgarten, 503, 594

Maassen, 53
Macfadyen, 363
Macfadyen and Rowland, 416
Madsen, 218, 521
Mafucci, 480, 490, 500
Mallory, 623, 676

Mallory and Wright, 110, 112, 150,
454

Mann, 96, 474

Maragliano, 498

Marbaix, 341, 342, 348

Marchiafava and Celli, 371

Marchoux and Salimbeni, 615

Marchoux, Salimbeni, and Simond, 673

Marchoux and Simond, 674

Marie and Morax, 463

Marinesco, 478

Marmier, 344

Marmorek, 344, 498

Marschal, 371

Martin, L., 468, 521

Martin and Cherry, 204

Martini and Lentz, 438

Marx, 347

INDEX OF AUTHORS

725

Massol, 715
McClintic, 80
McCoy, 510, 559, 560, 562
McCoy and Chapin, 562
Mennes, 362, 366
Mesnil, 232
Messea, 15

Metchnikoff, 188, 200, 201, 224, 225,
228, 232, 275, 588, 603, 714
Metchnikoff and Besredka, 426
Metchnikoff and Roux, 601
Metchnikoff, Roux, and Salimbeni,
589

Meyer, 187

Meyer and Ransom, 463
Michaelis, 96, 253, 263
Michel, 516
Michelson, 619
Mignesco, 63
Migula, 12, 36, 325, 472
Mikulicz, 452
Miller, 411, 706
Miquel, 32, 690
Mitchell, J. P., 167
Moeller, 98, 501

Mohler, 495, 679, 707, 708, 711, 712

Moll, 198

Moltschanoff, 385

Momont, 570

Monti, 658

Morax, 545

Morax and Marie, 462

Morgan, 404, 431

Morgenroth, 202

Morgenroth and Sachs, 265

Moro, 492

Morpurgo, 189

Moser, 676

Mouton, 275

Moxter, 241

Much, 482

Muhlens, 600, 603

Muhlschlegel, 17

Muller, 334, 504, 706

Muller, Fr., 325

Muller, Otto Friedrich, 2

Muller, P. Th., 243, 254, 255, 272

Mulzer, 595, 602

Muntz, 25

Muntz and Schlossing, 57
Myers, 215, 253

Naegeli, 486
Nakanishi, 10, 11, 16, 17
Nastjukoff, 539
Needham, 4
Negri, 648

Neisser, 107, 329, 380, 505, 514
Neisser, Baermann, and Halber-
STADTER, 615

Neisser and Sachs, 246, 273
Neisser and Shiga, 443
Neisser and Wechsberg, 201, 244, 329,
330, 331

Nencki and Scheffer, 21
Neufeld, 345, 364, 365, 369, 412
Neufeld and Haendel, 366
Neufeld and Hune, 283
Neufeld and Rimpau, 282, 366
Neufeld and Topfer, 283
Neumann, 411, 579
Nichols, 604
Nicolaier, 456

Nicolle, 233, 298, 300, 506, 602

Nicolle, Conor, and Conseil, 678

Nicolle and Thenel, 233

Nielsen, 481

Nikati and Rietsch, 587

Nikola ysen, 385

Nissen, 76

Nocard, 430, 500, 530, 620
Nocard and Roux, 10, 480, 500
Noguchi, 264, 265, 267, 270, 600, 601,
604, 616, 617, 651
Norris, 237, 253, 424
Norris and Larkin, 620, 621
Norris, Pappenheimer, and Flour¬
noy, 606
North, 715

Notter and Firth, 706

Novy and Freer, 64

Novy and Knapp, 592, 606, 610

Nuttall, 198, 224, 237, 241, 253, 560

Nuttall and Thierfelder, 392

Obermeier, 6, 605

726

INDEX OF AUTHORS

Obermuller, 711
Ogston, 321, 335
Ohno, 439, 446
Omelianski, 49, 58
Omeltschenko, 79
Ophuls, 631, 632
Oppenheimer, 43, 47
OSTENBERG, 136
Otto, 297, 298, 300, 669, 674
Ottolenghi, 359
Overton, 187

Palmer, 349
Pane, 232, 362, 366
Papasotiriu, 391
Pappenheim, 106, 483
Parietti, 695

Park, 89, 216, 217, 412, 700
Park and Carey, 439
Park and Dunham, 438
Park and Holt, 705, 709
Park and Krumwiede, 487
Park and Throne, 220
Park and Williams, 366, 521
Parodi, 602
Pasquale, 338, 591
Passet, 326, 337

Pasteur, 5, 41, 189, 192, 193, 194, 196,
321, 352, 468, 552, 553, 573, 631, 647
Pasteur and Chamberland, 122
Pasteur, Chamberland, and Roux,
194, 571

Paul, 659, 660, 661, 663
Peabody and Pratt, 137, 409
Pearce, 202
Perkins, 448
Perrone, 339, 344
Petri, 502, 717

Petruschky, 342, 404, 411, 426, 618, 620
Petterson, 291
Pfaundler, 231, 232, 234
Pfeffer, 54, 56

Pfeiffer, 195, 199, 230, 255, 279, 416,
536, 537, 541, 588, 589, 657
Pfeiffer and Beck, 536, 541
Pfeiffer and Friedberger, 241
Pfeiffer and Isaeff, 199, 224
Pfeiffer and Kolle, 231, 418, 425

Pfeiffer and Nocht, 588, 591

Pfeiffer and Wassermann, 589

Pfluger, 59

Pfuhl, 455, 540

Pick and Yamanouchi, 300

PlERRALLINI, 276

PlORKOWSKI, 134

V. PlRQUET, 494

V. PlRQUET AND SCHICK, 296, 301

Pitt, 14

Plato, 381

Plaut, 611, 641

Plenciz, 2

Plotz, 677

PoELS AND DhONT, 430
Poels and Nolen, 368
Pollack, 427

PoLLENDER, 6

Poor, 651
Porges, 13

PORGES AND MEIER, 263
PORTIER AND RlCHET, 296
Pott, 706

Pratt, 140, 412, 584
Prescott, 391, 697, 698
Preuser, 532
Proscher, 331, 332
Proskauer and Beck, 29
Prudden, 404, 490
Prudden and Hodenpyl, 490
Pryor, 486

Quincke, 713

Rabinovitsch, 502, 510, 634, 711
Radziewsky, 363
Ransom, 463

Ransome and Fullerton, 88
Ravenel, 500, 570
v. Recklinghausen, 7
Redtenbacher, 407
Reed, 670

Reed and Carroll, 430
Reed, Carroll, and Agramonte, 670
Reed, Carroll, Agramonte, and La-
zear, 669, 670, 674
Remlinger, 651
Richardson, 64, 411, 412

INDEX OF AUTHORS

727

Richet, 296, 302

Richet and Hericourt, 197, 331

Ricketts, 633

Ricketts and Wilder, 677

Rideal and Walker, 80

Rieder, 64

Rindfleisch, 7

Rixford and Gilchrist, 632

Roger, 344

Rohner, 579

Romer, 476

Rosenau and Anderson, 222, 297, 298,
520

Rosenau and McCoy, 699

Rosenbach, 321, 337

Rosenberger, 489

Rosenthal, 441

Rosenow, 343, 370

Ross and Milne, 609

Rost, 506, 509

Rothberger, 403

Roux, 64, 149, 203

Roux and Chamberland, 471

Roux and Nocard, 533

Roux and Yersin, 107, 512, 520, 522

Rubner, 24

Ruppel, 22, 491

Russell, 426

Russell and Fuller, 695

Sabouraud, 643
Sacharoff, 616
Sachs, 188, 199, 202
Sachse, 54
Sahli, 493
Salmon, 552
Salmon and Smith, 196
Sanarelli, 669
Sanfelice, 471, 632
Sauerbeck, 294
Saul, 77
Savage, 231

Schaeffer and Steinschneider, 384
Schafer, 31

SCHATTENFROH, 291, 328
SCHATTENFROH AND GRASBERGER, 702
SCHAUDINN, 592

SCHAUDINN ANP HOFFMANN, 594

Schell and Fischer, 486
Schenck, 644
Schereschewsky, 600
ScHERING, 92

Scheuerlen and Spiro, 74, 78
Schild, 392
SCHIMMELBUSCH, 327
SCHLESINGER, 345
Schlossmann, 90, 91
Schneider, 325
ScHNITZLER, 455
Scholtz, 380
ScHOTTELIUS, 392

Schottmuller, 337, 344, 348, 350, 369,
405, 431, 706
SCHREIBER, 546
SCHROEDER, 4

SCHROEDER AND COLTON, 707

SCHROETER, 60

Schuder, 414, 703

Schuller, 669

Schulze, 4

Schutz, 341, 368

Schutze, 202, 236

Schwann, 3, 4

Sclavo, 357, 574

Sedgwick and Batchelder, 700

Sharnosky, 103

Shattock, 22

Shiga, 435, 438, 442, 444, 446
SlEDENTOPF, 597
Signorelli, 140
SlLBERSCHMIDT, 455
Simon and Lamar, 286
Simon, Lamar, and Bispham, 286
Simond, 560
Simpson, 705

Simpson and Hewlett, 80
Smith, 101, 499, 500, 708
Smith, Graham, 525
Smith, Herbert E., 704
Smith, Th., 27, 34, 297, 484, 485, 498,
499, 520, 521, 698
Smith, Th., and Kilbourne, 195
Smith, Th., and Moore, 430
Smith, Th., Brown, and Walker, 458
Sobernheim, 563, 574
SOBERNHEIM AND TOMASCZEWSKI, 595

728

INDEX OF AUTHORS

SOMMERVILLE, 80
Spallanazani, Abbe, 4
Spengler, 493
Spilker and Gottstein, 65
Spitzer, 595
Spronck, 437
Spronk, 521
Stefansky, 510
Steinhardt, Dr., 651
Stern, 237, 421
Stern and Korte, 259, 418
Sternberg, 34, 325, 340, 352, 359, 516,
669, 692

Stevens and Myers, 204
Sticker, 508, 644
Stokes, 343

Stokes and Weggefarth, 706
Strauss, 532

Strauss and Gamaleia, 490, 500

Strauss and Huntoon, 652

Strelitz, 519

Stricht, 478

Strong, L. W., 449, 561

Strong and Musgrave, 436

Strong, Teague, and Barber, 561

Strong, Teague, and Crowell, 561

Suchsland, 716

SURMONT, 201

SuRMONT AND ArNOULD, 570
Suzuki and Takaki, 489

Tacke, 54
Talamon, 353
Tarozzi, 460

Tavel and Krumbein, 368
Taylor, 645
Teague, 561
Tedesco, 540, 541
Terin, 327
Thayer, 669
Thomas, 668
Tidswell, 510
Tissier, 713

Tissier and Martelly, 392, 714
Tizzoni, 460
Todd, 329, 443
Tokishige, 632
Tomasczewski, 596

Torini, 47
Torrey, 385
Totsuka, 419
Toussaint, 194, 571
Trask, 704
Trillat, 89
Tschistovitch, 236
Tsiklinski, 32
Tunnicliff, 612, 613
Turnbull, 610

Uhlenhuth, 237
Uhlenhuth and Mulzer, 602
Ullmann, 384
Unna, 639

Uschinsky, 28, 126, 522
Vagedes, 499

Vaillard and Dopter, 443

Vaillard and Rouget, 459

Vaillard and Vincent, 462

Valleri-Radot, 4

Vallet, 696

Van der Loeff, 657

Van de Velde, 329, 330, 342, 346

Van Ermengem, 14, 101, 430, 475

Van Gehuchten, 648

Van Gieson, 649

Vaughan, 298, 304, 417

Vaughan and Wheeler, 302

Vedder and Duval, 438

Veeder, 415

Veillon, 333

Di Vestea and Zagari, 647
Vignal, 619
VlLLEMIN, 479
Vincent, 611
Voges, 29

Von dem Borne, 615
VoTTALER, 457

Wadsworth, 99, 249, 355, 357, 359, 361,
364, 365
Wagmann, 703
Waldeyer, 7
Walker, 419
Ward, 64, 703
Washburn, 366

INDEX OF AUTHORS

729

Wassermann, 195, 199, 204, 236, 242,
383, 385, 579, 580, 604
Wassermann and Bruck, 246, 262
Wassermann and Citron, 294
Wassermann, Neisser, and Bruck, 262
Wassermann and Proskauer, 522
Wassermann and Schutze, 237
Wassermann and Takaki, 188, 214,
279, 463

Wassilieff, 532
Weber, 501
Wechsberg, 242
Weeks, 542
Weeny, 419
Wegele, 715

Weichselbaum, 333, 353, 372, 376, 386,
447, 450, 540

Weichselbaum and Muller, 542
Weigert, 7, 111, 214, 481
Weill-Halle and Lemaire, 300
Weis, A. H., 328
Weiss, 482

Welch, 98, 129, 355, 357, 395, 472

Welch and Blachstein, 404

Welch and Flexner, 474

Welch and Nuttall, 177, 471

Wernicke, 561

Wertheim, 382

Wesenberg, 455

Westphal and Uhlenhuth, 508

Weyl, 22, 481, 490

Wherry, 510

Wickman, 664

Widal, 421

Widal and Nobecourt, 430
Widal and Sicard, 421

Wilckens, 704
Wilde, 451, 452, 453
Wilder, 677
Williams, 533
Williams and Lowden, 650
Wilson, 557
Willson, 696
Wiltschour, 407
Winogradsky, 14, 54, 57
Winslow, 343
Winslow and Palmer, 349
Wladimiroff, 533
Wolbach, 680
WOLBACH AND ERNST, 499
Wolff, 294, 396
Wolff and Israel, 624
Wolff-Eisner, 301, 494
W OLFFHU GEL, 89
Wollstein, 541, 544, 545
Wood, 95, 109, 359, 717
Woronin, 55

Wright, 108, 150, 153, 195, 286, 288,
425, 623, 624, 627
Wright and Douglas, 281, 282
Wright and Lamb, 550
Wu Lien Teh, 560

Yersin, 555

Yersin, Calmette, and Roux, 561
Zeit, 64

Zettnow, 10, 11, 12, 606
Ziehl, 97, 105, 481

Zinsser, 16, 155, 291, 412, 413, 442, 520,
525, 631

Zinsser and Cary, 511

INDEX OF SUBJECTS

Abbott’s method of staining spores,
97

Absorption method in study of agglu¬
tination reaction, 234
Achorion Schoenleinii, 640
cultivation of, 642
morphology of, 641
varieties of, 641

Acid formation by bacteria, 166
Acid-fast bacteria, stains for, 104
Acquired immunity, 192
definition of, 192
Actinomyces, 622
cultivation of, 623
discovery of, in cattle, 622
in man, 622

morphology of, 619, 622
pathogenicity of, 625
in animals, 625
in man, 625

parts of body infected in, 625
staining of, 623
varieties of, 626
Actinomycosis, 625

Active immunity. See under Immunity
Aerobic organisms, facultative, 26
obligatory, 25

non-infectiousness of, 183
respiratory processes of, 27
Agar for culture media, 127
lactose-litmus, 129
meat extract, 127
meat infusion, 128

Agar slants, cultivation of anaerobic
bacteria on, 153
Agglutination reaction, 228

between agglutinin and agglutinin-
stimulating substances, 233
clinical diagnosis by, in typhoid, 229
concentrated agglutinin in, 235

Agglutination reaction, differentiation
of bacterial species by, 229
diluted agglutinins in, 235
group agglutination in, 234
immune or chief agglutinin in, 234
macroscopical observation of, 230
for bacterial differentiation, 231
major agglutinin in, 234
microscopical observation of, 229
for clinical diagnosis, 231
minor agglutinins in, 234
nature of, 228
of capsulated bacteria, 13
partial agglutinins in, 234

absorption method in study of, 234
proagglutinoid zone in, 235
proagglutinoids in, 235
pseudo-clumping in, 231
specificity of, 234
“thread-reaction” in, 231
upon dead bacteria, 231
upon living bacteria, 231
Agglutination tests, technique of, 250
macroscopic, 252
microscopic, 251
Agglutinins, 200, 228
action of, 240

agglutinin-stimulating substances and,
233

quantitative relations between, 233
reaction between, 233
bactericidal substances compared
with, 231

cell- receptors in, 238
concentrated, failure of, to produce
agglutination, 235

diluted, agglutination reaction with,
235

effect of heat on, 232
experimentation with, 231

731

732

INDEX OF SUBJECTS

Agglutinins, in agglutination raction,
chief or immune, 234
major, 234
minor, 234
partial, 234

in serum of glanders, 535
in staphylococcus immune sera, 331
nature of, 231
normal, 232

partial absorption method in study
of, 234

production of, 232

in sera of animals, by injection of
bacteria, 233
of culture extracts, 233
time of, 233

reaction of. See Agglutination reaction
specificity of, 234
structure of (Ehrlich), 238
theoretical considerations concerning,
238

“thread-reaction” in, 231
Agglutinogen, 233
Aggressins (Bail’s theory), 291
action of, 293
immunization with, 293
nature of, 293
occurrence of, 293
opposition to Bail’s theory of, 294
Air, bacteria in, 673

dryness and high winds favorable
to increase of, 683
estimation of numbers of, 684
occurrence of, in inhabited places,
683

scarcity of, in places high above
earth, 684

settling of, with rain, snow, etc., 684
infectious material carried by, 684
Alcohol, as fixative in staining, 110
Alcoholic fermentation, 51
by yeasts, 52
in milk, 684
process of, 51

Alcohols as disinfectants, 77
Alexin, 198

action of, in blood serum, 224, 225
Alkali formation by bacteria, 166

Allantiasis, 477

Amboceptor and complement, quanti¬
tative relationship between, 244
Amboceptors, filtration of, 244
multiplicity of, in normal sera, 241
Amylase, 48
action of, 49
occurrence of, 49

Amylolytic ferment. See Amylase
Anaerobic cultivation of bacteria, 148
use of sterile tissue as an aid in, 156
See also under Cultivation of bacteria
Anaerobic organisms, facultative, 26
Gram-positive, in feces, 177
infectiousness of, 183
non-invasion of blood stream by, 183
obligatory, 26
respiratory processes of, 26
Anaphylactin, 302
Anaphylatoxin, 304
Anaphylaxis, 295

autopsy findings in, 299

definition of, 297

experimentation in, early, 295

immunity after, 299

in diphtheria antitoxin injections, 296

incubation during, 299

inherited, 300

observations in, fundamental, 296
by Arthus, 297
by Besredka, 298, 299
by Besredka and Steinhardt, 299
by Doerr, 299

by Gay and Southard, 299, 300
by Hericourt and Richet, 296
by Nicolle, 298, 300
by Otto, 297, 300
by Pick and Yamanouchi, 300
by Portier and Richet, 296
by Rosenau and Anderson, 297,
298, 300

by Th. Smith, 297
by Vaughan and Wheeler, 298
by Weill-HalU and Lemaire, 300
passive, 300
phenomena of, 295
“phenomenon of Arthus” in, 297
proteid injection^ in, 295

INDEX OF SUBJECTS

733

Anaphylaxis, proteid injections in, mode
of giving, 298
quantity of, 298
symptoms in, 299
theories concerning, 301

based on Ehrlich’s receptor over¬
production theory, 301
of Besredka, 302
of Doerr and Russ, 303
of Friedberger and Hartoch, 303
of Gay and Southard, 302
of Hamburger, 303
of v. Pirquet and Schick, 301
of Richet, 302
of Wolff-Eisner, 301

Anilin dyes, influence of, upon bacterial
growth, 140
Animal alkaloids, 45
Animal experimentation, 169
animals used in, 169
cages for, 173
autopsies in, 173
inoculations in, 170
Antagonism of bacteria, 31
Anthrax, 563

bacterial causation of, 6
occurrence of, 563
Anthrax bacillus, 563
action of, 571
bacilli resembling, 575
Bacillus anthracoides, 575
Bacillus radicosus, 575
Bacillus subtilis, 575
biology of, 569
cultivation of, 566
early investigation of, 563
experimental inoculation with, 571
immunization against, 573

active (Pasteur’s method), 574
attenuation in, 573
passive (Sobernheim’s method), 574
in milk, 707
infection with, 572
by inhalation, 572
cutaneous, 572
pulmonary, 573
spontaneous, 572
through alimentary canal, 573

Anthrax bacillus, isolation of, 565
morphology of, 564
pathogenicity of, 570
prophylaxis against, 573
resistance of, 570
staining of, 565

susceptibility of animals to, 570
virulence of, 570

Anthrax, symptomatic, bacillus of, 465
cultivation of, 465
immunization against, 468
vaccines used in, 468
morphology and staining of, 465
occurrence of, 465
pathogenicity of, 466
autopsy findings in, 467
toxins of, 467

Anthropoid apes, blood of, distinguished
from human, 237
Antiaggressins, 293
Antiamboceptors, 242
Antianaphylaxis, 299
Antibodies, 197

experimentation and discovery of, 197
agglutinins, 200
alexin, 198
antiferments, 202
antitoxin, 198-199
bacteriolysins, 200
cytotoxins, 201
precipitins, 200

facts and theories concerning, 241
in sera, determination of, by comple¬
ment fixation. See under Comple¬
ment fixation.

Anticomplements, 242
Antiferments, 202

Antiformin, formula for, as given by
Rosenau, 483

in examination of sputum for tubercle
bacilli, 483

Antigen, definition of, 202
Antilab, 202
Antilactase, 202
Antileucocidin, 331
Antipepsin, 202

Antiricin, discovery and experimenta¬
tion with, 204

734

INDEX OF SUBJECTS

“Antisensibilisin,” 302
Antiseptics, inhibition strengths of va¬
rious, 84

values of, determination of, 80
table of, 80
Antistaphylolysin, 331
Antisteapsin, 202
Antistreptococcic sera, 346
Antitoxic sera, 196
Antitoxin, 198-199

diphtheria. See Diphtheria anti¬
toxin

normal, 205

production of, a final test between
toxin and endotoxin, 187
stability of, 206

tetanus. See Tetanus antitoxin
unit of, 205

valency of, for toxin, 210
Antivenin, 198
Arthrospores, 16
Ascospores, 630, 638
Ash in bacterial cell, 23
Asiatic cholera. See Cholera
Aspergillus, reproduction in, 637
Attenuated cultures in active immuni¬
zation, 193
Autoclave, 71

technical details of, 72
Trillat’s, 87

Autointoxication, gastrointestinal, 712
bacteria causing, 713
experimental combating of, by
acid-producing bacilli, 713
Metchnikoff’s treatment of, by
means of Bacillus bulgaricus,
714-715
Autolysins, 248

Autopsies of infected animals, 173
Avian tuberculosis, bacillus of. See
Tubercle bacillus, bacilli related to

Babes-Ernst granules, 11
Bacilli intermediate between typhoid
and colon organisms, 428
bacterial correlation of, 431

bacilli of colon-like morphology in,
433

Bacilli intermediate between typhoid
and colon organisms, bacterial correla¬
tion of, bacilli of typhoid-like mor¬
phology in, 433

non-motile bacilli in, 433
classification of, 432
differentiation of, from tjphoid and
colon groups, 428
by cultural characteristics, 428
by morphology, 428
by motility, 428
hog-cholera bacilli in, 430
meat-poisoning bacilli in, 429
Bacillus enteritidis in, 429
Bacillus icteroides in, 430
Bacillus Morseele in, 430
paratyphoid bacilli in, 430
Bacillus psittacosis in, 430
“Muller” bacillus, 431
paracolon bacillus in, 430
“Seeman” bacillus, 431
pathogenicity of, 431
toxic products of, 431
Bacillus, 37

general description of, 9
Bacillus aerogenes capsulatus, 471
and pernicious anemia, 177
biological considerations of, 474
cultivation of, 473
isolation of, 474
morphology of, 472
occurrence of, 472, 475
plathogenicity of, 474
staining of, 473
Bacillus anthracoides, 575
Bacillus avisepticus, 552
Bacillus botulinus, 475
antitoxin for, 199
cultivation of, 473
morphology of, 476
pathogenicity of, 477
staining of, 476
toxins of, 478

Bacillus bulgaricus, use of, by Metch-
nikoff for treatment of gastrointes¬
tinal autointoxication, 715
Bacillus butyricus, 502
Bacillus coli communior, 398

INDEX OF SUBJECTS

735

Bacillus coli communis, 389
agglutinins for, 396
in immune serum, 396
in normal serum, 397
bladder diseases due to, 395
cholera infantum attributed to, 394
cholera nostras attributed to, 394
cultivation of, 390

differentiation of, from meat-poisoning
and paratyphoid bacilli, 428
distribution of, 391
in animals, 392
in feces, 177
in man, 392
in milk, 391
in nature, 391
in water, 697
immunization with, 395
inflammatory conditions of liver and
gall-bladder attributed to, 395
isolation of, 391
morphology of, 389
pathogenicity of, 393
peritonitis following perforation at¬
tributed to, 394
septicemia due to, 394
staining of, 389
toxic products of, 395
varieties of, 397

Winckel’s disease in the newborn due
to, 394

Bacillus diphtherise. See Diphtheria
bacillus

Bacillus enteritidis, discovery and char¬
acteristics of, 429
Bacillus fecalis alkaligenes, 426

differentiation of, from typhoid ba¬
cillus, 427
in feces, 177

Bacillus Hoffmanni. See Diphtheria
bacillus, bacilli similar to
Bacillus icteroides, 430
Bacillus influenzae. See Influenza bacillus
Bacillus lactis aerogenes, 453
cultivation of, 453
morphology of, 453
occurrence of, 453
in feces, 177

Bacillus lactis aerogenes, pathogenicity
of, 453

Bacillus leprae. See under Leprosy
Bacillus mallei, 520. See also Glanders
biological characteristics of, 530
cultivation of, 528
immunity against, 535
morphology of, 528
pathogenicity of, 530

bacteriological diagnosis in, 532
in horses, 530
in man, 532
nodules in, 532
spontaneous infection by, 530
staining of, 528
toxin of, 532
action of, 533
diagnostic use of, 533

directions of U. S. Government
for, 534

obtaining and preparation of, 533
Bacillus melitensis. See Micrococcus
melitensis

Bacillus mesentericus in feces, 177
Bacillus Morseele, discovery and charac¬
teristics of, 430

Bacillus mucosus capsulatus, 447
association of, with pneumonia, 450
with other diseases of mucous lin¬
ings, 450-451
cultivation of, 448
cultural characteristics of, 449
Fitzgerald’s work on classification of,
450

immunization against, 451
inoculation of animals with, 451
morphology of, 447
pathogenicity of, 450
staining of, 448
Bacillus murisepticus, 542
Bacillus ozaense, 452
Bacillus pestis. See Plague bacillus
Bacillus proteus vulgaris, 454
cultivation of, 454-455
Bacillus prodigiosus, quantitative chem¬
ical analysis of, 21
Bacillus psittacosis, 430
Bacillus pyocyaneus, 577

736

INDEX OF SUBJECTS

Bacillus pyocyaneous, antitoxin against
products of, 199
cultivation of, 577
favorable conditions for, 577
pigment in, 578
fluorescent variety of, 579
pyocyanin in, 578
immunization against, 580
filtrates of old cultures in, 580
pyocyanase in, 580
true toxin in, 580
morphology of, 577
occurrence of, in lesions and inflam¬
matory affections, 579
pathogenicity of, 579
staining of, 577

susceptibility of animals to, 580
toxins of, 580

leucocyte-destroying ferment in,
581

pyocyanase in, 580
pyocyanolysin in, 580
true, 580

from filtrates of old cultures, 580
virulence of, 579
Bacillus radicicola, 55
Bacillus radicosus, 575
Bacillus rhusiopathiae, 542
Bacillus smegmatis. See Smegma ba¬
cillus

Bacillus subtilis, 575
Bacillus suisepticus, 553
Bacillus tetani. See Tetanus, bacillus of
Bacillus tuberculosis. See Tubercle
bacillus

Bacillus typhi abdominalis, 399
Bacillus typhi murium, 430
Bacillus typhosus. See Typhoid fever,
bacillus of

Bacillus xerosis. See Bacillus diph-
therise, bacilli similar to
Bacteria (see also Bacterial cell):
acid and alkali formation by, 166
acid-fast stains for, 104
action of, in the body, 184
anabolic or synthetic activities of, 54
in root tubercles, 55
in soil, 54

Bacteria, animal experimentation with,
169

antagonism of, 31
biological activities of, 40, 164
chemical agents injurious to, 73. See
also Disinfectants
classes of, 182
bacilli, 9
cocci, 9
spirilla, 9

classification of, 35

based on organs of motility, 15
by Bail with regard to aggressins,
294

by Gram stain, 104
by Migula, 37
counting of, 161
cultivation of, 141
by anaerobic methods, 148. See
also under Cultivation of bacteria
inoculation of media in, 141
dead, in active immunization, 193
degenerative forms of, 20
denitrifying, 52

destruction of. See Destruction of
bacteria

differentiation of, by fermentation, 48
enzymes produced by, 168
diastatic, 169
inverting, 169
proteolytic, 168

1 n^as formation by, 164. See also Gas
formed by bacteria
Gram-negative, 104
Gram-positive, 104
in air. See Air, bacteria in
in industries, 715
in milk. See Milk, bacteria in
in soil. See Soil, bacteria in
in tissues, staining of, 110
in water. See Water, bacteria in
incubation of, 156. See also under In¬
cubation of cultures
indol production by, 167
isolation of, methods of, 142
katabolic activities of, 41-53

by bacterial enzymes or ferments, 42
varieties of, 48

INDEX OF SUBJECTS

737

Bacteria, katabolic activities of, by
denitrifying bacteria, 52
by fat-splitting enzymes, 47
by lab enzymes, 46
by proteolytic enzymes, 43
liberation of energy by, 58
light production of, 59
microscopic study of. See Microscopic
study

nitrifying. See Nitrifying bacteria
nutrition of, 25. See also under
Nutrition of bacteria
occurrence of, in the body, 181
parasitic, 29

definition of, 182
pathogenic, 182
phenol production by, 167
physical agents injurious to, 62
pigment formation by, 59
protozoa and, differentiation of, 1
putrefactive, quantitative chemical
analysis of, 21
reducing powers of, 167
relation of, to moisture, 35
to physical environment, 31
to pressure, 35

relationship of, to other plants, 35
reproduction of, 18
rate of, 18
varieties of, 18
saprophytic, 29
definition of, 182
size of, 9

staining of, methods of. See Staining
of bacteria

sulphur. See Sulphur bacteria
symbiosis of, 31
thermal death points of, 34
variations in forms of, 19
virulence of, and infectiousness, 183
virulent, sublethal doses of, in active
immunization, 195
Bacteriaceae, 37
Bacterial cell, ash in, 23
Babes-Ernst granules in, 11
capsule of, 12
carbohydrates in, 23
chemical consitutents of, 21
48

Bacterial cell, chemical constituents of,
quantitative analysis of, 21
varieties of, 21
fats in, 22
globuhn in, 22
membrane of, 12
metachromatic granules in, 11
morphology of, 100
motility of, 13
Brownian, 14
by flagella, 14
effect of temperature on, 15
molecular, 14
true, 14
nucleus in, 10
organs of locomotion of, 13

classification of bacteria based on,
15

osmotic properties of, 23
permeability of membrane of, 23
plasmolysis of, 23
plasmoptysis of, 24
polar bodies in, 11
proteids in, 22

refractive index of parts of, 24
specific gravity of forms of, 24
water in, 21

Bacterial enzymes or ferments, 42
action of, 42

environmental conditions on, 43
reversible, 43

similarity of, to ferments of special¬
ized cells of higher plants and ani¬
mals, 43

Bacterial forms, variations of, 19

Bacterial poisons, 184
action of, 187
ptomains and, 185

resistance of, to chemical action and
heat, 187
summary of, 305
varieties of, 185
endotoxins, 185
proteins, 186
true toxins, 185

Bacterial products in active immuniza¬
tion, 195

Bacterial proteins, 186

738

INDEX OF SUBJECTS

Bacterial spores, 15
formation of, 15
germination of, 17
position of, 17
varieties of, 15
arthrospores, 16
true, or endospores, 16
vegetative forms from, 17
Bactericidal action of blood serum, 224
Bactericidal strengths of common dis¬
infectants, 85

Bactericidal substances compared with
agglutinins, 231
Bactericidal tests, 257
in test tube, 257
technique of, 258
for typhoid fever, 258
in vivo, 255-7

Bacteriemia, definition of, 184
Bacteriological examination of blood
cultures, 178
choice of media for, 179
results of, estimation of, 180
technique of obtaining material for,
178

from typhoid patients, 180
of exudates, 175
of feces, 176

of material from patients, 174
technique of collecting, 174
of urine, 176

Bacteriology, development and scope of,
1-8

Bacteriolysins, 200
immune, 225
Bacteriolytic tests, 255
Pfeiffer’s test in, 255

determination of bacteriolytic power
of serum against a known micro¬
organism in vivo by, 255
identification of microorganism in
known immune serum in vivo by,
257

Bacteriotropins, 282
Bacterium, 27
Bacterium tularense, 562
Bail, aggressin theory of, 292
opposition to, 294

Barsiekow’s medium for colon-typhoid
differentiation, 139

Bauer’s modification of Wassermann
test for syphilis, 268
“Bazillenemulsion,” 492
Beggiatoa, genus, 38
Beggiatoacese, 38
Berkefeld filter, 120, 121
Bile medium for colon-typhoid differen¬
tiation, 138

Bile-salt agar, MacConkey’s, for colon-
typhoid differentiation, 138
Bitter milk, bacteria causing, 703
Black Death, 554

Bladder diseases due to colon bacillus,
395

Blastomycetes. See Yeasts and Yeast
cells

Blood, laked, 225

the seat of immunizing agencies, 198
Blood corpuscles, red, in Ehrlich’s the¬
ory of lytic process in blood serum,
227

Blood cultures, bacteriological examina¬
tion of, 178

results of, estimation of, 180
choice of media for, 179
technique of obtaining material for,
178

from typhoid patients, 180
Blood media, method of obtaining, 140
Blood serum, bactericidal action of, 224
Bordet’s interpretation of lytic proc¬
esses of, 225
immune, 198

reactivation of bactericidal power
of, by normal serum, 225
reactivation of bacteriolytic powers
of, by normal serum, 226
lytic processes of, 224
normal, 198

Bordet’s lytic theory of constituents
of, 225

alexin in, 225

“sensitizing substance” in, 225
obtaining of, from animals, 249
from man, 249

reactions with. See Serum reactions

INDEX OF SUBJECTS

739

Blood serum reactions, method of ob¬
taining, 139

Bollinger, discovery of actinomyces of
cattle by, 622

Bordet and Gengou, discovery of whoop¬
ing-cough bacillus by, 543
Bordet-Gengou bacillus, 543
cultivation of, 544

compared with that of influenza
bacillus, 544
technique of, 544-545
morphology of, 543

compared with that of influenza
bacillus, 544
pathogenicity of, 545
staining of, 543
toxins of, 545
Botulism, 477

Bouillon, malachite green, for colon-
typhoid differentiation, 137
Bouillon filtre (Denys), 492
Bovine tuberculosis, bacillus of. See
Tubercle bacillus, bacilli related to
Broth used for culture media, 124
calcium carbonate, 126
glycerin, 125
meat extract, 124
meat infusion, 124
nitrate, 126
pepton-salt, 126
sugar-free, 125]

Uschinsky’s proteid-free, 126
Bruce, discovery of Malta fever bacil¬
lus by, 550
Buboes, 548

Buchner, discovery of Bacillus coli com¬
munis by, 389

Buchner’s method of pyrogallic absorp¬
tion of oxygen in cultivation of
anaerobic bacteria, 152
Wright’s modification of, 153
Buerger’s method of staining capsules, 99
Butter bacillus, 502
Butter, making of, 710
bacteria aiding, 710
transmission of infection by, 711
tubercle bacilli in, 711
Butyric-acid fermentation in milk, 702

Cadaverin, 45

Cages for animals, 171, 172, 173
Calcium-carbonate broth, 126
Capaldi’s medium for colon-typhoid dif¬
ferentiation, 134

Capsule stains in staining of bacteria, 98
Carbohydrates in bacterial cell, 23
Carbolic acid as disinfectant, 77
Carbolic-acid coefficient, 80
Carbon dioxid formed by bacteria, 164
Carbon in nutrition of bacteria, 25
Casein, coagulation of, in milk, 702
Castelli, discovery of Spirochaeta per-
tenuis by, 614

Cell-receptors, three forms of, in expla¬
nation of all known antibodies
(Ehrlich):

haptines of the first order, 240
haptines of the second order, 240
haptines of the third order, 240
Cellulase, 49
Charbon. See Anthrax
Charbon symptomatique. See Anthrax,
symptomatic, bacillus of
Cheese, making of, 711

bacteria aiding, 711, 712
pathogenic organisms in, 712
Chemotaxis, negative and positive, defi¬
nition of, 277

Chicken cholera bacillus, 552
cultivation of, 552
immunization with, 553
morphology and staining of, 552
occurrence of, in animals, 552
pathogenicity of, 552
Chicken-pox, relation of, to smallpox, 660
Chlamydobacteriacese, 38, 618
classification of, 618
morphology of various forms of, 618
Chloride of lime as disinfectant, 76
Chlorine as disinfectant, 88
Cholera, Asiatic, 582
diagnosis of, 584
epidemics of, 586
immunization in, 590
active, 590

protective inoculation in, 590
in animals, 587

740

INDEX OF SUBJECTS

Cholera, Asiatic, infection in, 587
pathological findings in, 587
spirillum of, 582

biological considerations of, 586
cultivation of, 583
diagnosis of, by “cholera-red”
reaction, 584

Dieudonne’s selective medium for
cultivation of, 584
Dieudonne’s selective medium for
cultivation of, modified by
Krumwiede and Pratt, 584
hygienic considerations of, 588
in feces, 177
isolation of, 585
from feces, 585
from water, 586, 696
morphology of, 582
pathogenicity of, 586
in animals, 587
in man, 587
resistance of, 586
spirilla resembling, 590
Spirillum Deneke, 591
Spirillum Massaua, 591
Spirillum Metchnikovi, 590
Spirillum of Finkler-Prior, 591
staining of, 583
toxin of, 589
traced to milk, 705
Cholera, fowl, bacillus of, 7
Cholera infantum attributed to colon
bacillus, 394

Cholera nostras attributed to colon
bacillus, 394

Cholera-red reaction, 584
Chromo-bacteria, 59
Chromogenic Gram-negative cocci, 387
Cladothrix, 38, 619
morphology of, 618
Clearing of culture media, 119
by filtering, 120
with eggs, 119

Clostridium Pasteurianum, 54
Coagulins, 235

Cobra poison and its antitoxin, experi¬
mentation with, 204
Coccacese, 37

Cocci, description of, 9
Coefficient of inhibition, 80
Colon bacillus. See Bacillus coli com¬
munis

Colon bacillus group, 389
Colon test, for analysis of water, 697
Colon-typhoid differentiation, media for,
132

Barsiekow’s, 138
bile, 137
Capaldi’s, 134
Conradi-Drigalski, 135
Endo’s, 135
Hesse’s, 133
Hiss’ plating, 133
Hiss’ tube, 133
Jackson’s lactose-bile, 138
Loeffler’s malachite green, 136
MacConkey’s bile-salt, 138
malachite green bouillon, 137
neutral-red, 138
Piorkowski’s urine gelatin, 134
Colon-typhoid-dysentery group, bacilli
of, 388

characteristics of, 388
Colonies in agar, 146
Colony-fishing, 146
Colony study of bacteria, 161
Color indicator in titration, 117
jComma bacillus. See Spirillum cholerae
asiaticae

Complement, deviation of, 244
filtration of, 244

fixation of. See Complement fixation
in Ehrlich’s theory of lytic process in
blood serum, 226
in normal blood serum, 226
multiplicity of, in normal sera, 242
Complement fixation, action in, 245
by precipitates, 244
determination of antigen by, in serum
reactions, 271

of antibodies in sera by, 261
principles of, 261
reaction in, 261

Wassermann test for. See Was-
sermann test for diagnosis of
syphilis

INDEX OF SUBJECTS

741

Complement fixation, proteid differentia¬
tion by, 273
Complementoids, 243
Conjonctivite subaigue, 545
Conradi-Drigalski medium, for colon-
typhoid differentiation, 135
isolation of typhoid bacillus in stools
by, 408

Cotton used in filtering culture media, 120
Counting of bacteria, 161
Cowpox, relation of, to smallpox, 659
use of, in immunization against small¬
pox, 659
Crenothrix, 38
Creolin, 78

Cultivation of bacteria, anaerobic, 148
by absorption of oxygen by pyro-
gallic acid in alkaline solu¬
tions, 152

Buchner’s method, 152

Wright’s modification of, 153
combined with air exhaustion
and hydrogen replacement, 153
on agar slants, 153
without use of hydrogen, 155
by displacement of air with hydro¬
gen, 151

by mechanical exclusion of air, 148
Esmarch’s method, 149
fluid media covered with oil, 150
Liborius’ method, 149
Roux’s method, 149
Wright’s method, 150
Zinsser’s apparatus for, 156
colony study in, 161
counting of bacteria in, 161
incubation in, 158. See also under
Incubation of cultures
Culture media, 124
agar for, 127

lactose-litmus, 129
meat extract, 127
meat infusion, 128
blood for, 140
blood serum for, 139
broth for, 124

calcium carbonate, 126
glycerin, 125

Culture media, broth for, meat extract,
124

meat infusion, 124
nitrate, 126
pepton-salt, 126
sugar-free, 125
Uschinsky’s proteid-free, 126
clearing of, 119

by filtering through cotton, 120
through paper, 121
with eggs, 119
Dorsett egg, 130

enriching substances used in, 138,
139

fluid, covered with oil, in anaerobic
cultivation of bacteria, 150
for colon-typhoid differentiation, 132
glassware preparation in, 113
glycerin for, 126
ingredients of, 115
choice of, 116

lactose litmus-agar, 129, 135
litmus milk, 130
milk, 130
potato for, 130
glycerin, 130
preparation of, 113

general technique of, 113
process of, 124-40
reaction of, 119
serum, 131

Hiss’ serum water, for fermenta¬
tion tests, 132
Loeffler’s, 131
slanting of, 123
special, 132
sterilization of, 121
filtration in, 122
heat in, 121
titration of, 117

color indicator in, 117
process of, 117

for alkaline media, 118
reaction of, 117
adjustment of, 119
tubing of, 121

Cultures, attenuated, in active immu¬
nization, 193

742

INDEX OF SUBJECTS

Cultures, incubation of. See under In¬
cubation of cultures
Cytase, 279

Cytoryctes variolae, 658
Cytotoxins, 201

specific, injury to organs by, 201

Dark-field condenser, 597
Decay, action of, 44

Defensive factors of animal organism,
189

Degenerative forms of bacteria, 20
Denitrifying bacteria, 52
activities of, 53
occurrence of, 53
Destruction of bacteria, 62

by chemical agents, 73. See also Dis¬
infectants
gaseous, 88
in solution, 73
inorganic, 73
organic, 77

by physical agents, 62
drying, 62
electricity, 65
heat, 65
light, 63

Diarrheal diseases traced to milk, 705
Diastase. See Amylase
Differential stains in staining of bac¬
teria, 102

Diphtheria, tracing of, to milk, 705
Diphtheria antitoxin, 216

anaphylaxis in injections of, 296
normal, 205
production of, 216
horses used in, 216-17
technique of, 217
toxin for, 216
stable, 206

standardization of, 218

concentration and purifying in, 219
technique of, 218
unit for, 205
Diphtheria bacillus, 512
bacilli similar to, 522
Bacillus Hoffmanni, 522
cultivation of, 524

Diphtheria bacillus, bacilli similar to,
Bacillus Hoffmanni, mor¬
phology of, 523
staining of, 523
Bacillus xerosis, 525
cultivation of, 526
differentiation of, from other ba¬
cilli, 526

other bacilli, 527
biological characteristics of, 515
cultivation of, 516
degenerative forms of, 19
differentiation of, from other forms,
107, 522, et seq.
grouping of, 515
isolation of, 517
morphology of, 513
“ ground” type in, 514
occurrence of, in body, 518, 519
pathogenicity of, 518
in animals, 519
“pseudo-membranes” in, 518
resistance of, 516
staining of, 574

Gram’s method of, 507
polar or Babes-Ernst bodies in, 514
Neisser’s stain for, 514
Roux’s stain for, 515
toxin of, 520. See also Diphtheria
toxin

chemical and physical properties
of, 520

production of, 520

media employed in, 520
Park-Williams Bacillus No. 8 in,
520

resistance of, 522

Diphtheria toxin, analysis of (Ehrlich),
205-15

method of partial absorption in, 209
side-chain theory of, 212
summary of, 215
constitution of (Ehrlich), 210
graphic form of (Ehrlich), 211
views of Arrhenius and Madsen on,
212

epitoxoid in, 208

molecule of, haptophore group in, 207

INDEX OF SUBJECTS

743

Diphtheria toxin, molecule of, toxophore
group in, 207
normal solution of, 205
partial absorption of, 209
standardization of, Limes death in, 208
Limes zero in, 207
time changes in, 206
toxoid form of, 207
protoxoids in, 209
syntoxoids in, 209
toxon in, 209
unit for, 205

Diplococcus gonorrhoese, 380
cultivation of, 381

conditions favorable to, 383
Wertheim’s medium for, 381-2
differentiation of, from Micrococcus
catarrhalis, 386
early work in, 380
infection by, in man, 384
morphology of, 380
pathogenicity of, in animals, 385
in man, 384
resistance of, 384
staining of, 381

Diplococcus lanceolatus. See Diplococ¬
cus pneumoniae
Diplococcus mucosus, 387
Diplococcus pneumoniae, 352
cultivation of, 355

differentiation of, from streptococcus,
357, 367
cultural, 368
morphological, 367
by bile test, 369
by capsule, 367
by fermentations, 369
by grouping, 367
by growth in blood media, 367,
369

by shape, 367
immunization against, 363
active, methods of, 363

agglutinins in immune sera in, 364
table of, 365
technique of, 364
opsonins in immune sera in, 366
precipitins in immune sera in, 365

Diplococcus pneumoniae, immunization
against, passive, with immune sera,
366

isolation of, 358
modes of inoculation with, 361
morphology of, 553
capsules in, 353-4
lancet-shape of cocci in, 353
pairing of cocci in, 353
pathogenicity of, 361
pneumonic complications and, 362
resistance of, 358
staining of, 355

susceptibility of animals to, 370
toxic products of, 362
virulence of, 360
in animals, 360
in man, 361
Disinfectants, 73

bactericidal strengths of common, 85
gaseous, 88
chlorine, 88
formaldehyde, 89
oxygen, 88
ozone, 88

sulphur dioxide, 88
testing of efficiency of, 79

by U. S. Public Health Service
method, 80

carbolic-acid coefficient in, 80
determination of antiseptic values
in, 83

of disinfectant values in, 86
table of, 85
factors in, 79
used in solution, 73
inorganic, 73

acids, bases, and salts, 74
halogens and derivatives, 75
chlorid of lime, 76
tetrachlorid of iodin, 76
oxydizing agents, 76
hydrogen peroxid, 76
potassium permanganate, 76
organic, 77
alcohols, 77
carbolic acid, 77
Cresol group, 78

744

INDEX OF SUBJECTS

Disinfectants, used in solution, organic,
essential oils, 78
formaldehyde, 78
iodoform, 77

Disinfection, practical, 86
of feces, 87
of hands, 87
of instruments, 87
of linen, etc., 87
of rooms, etc., 88
of sputum, 87
of urine, 87

Dissociation, electrolytic, 74

relation between degree of, and
bactericidal powers of solutions,
74

Dorsett egg medium, 130
Drying in destruction of bacteria, re¬
sistance to, 62

DTN1^!250, definition of, 205
Ducrey bacillus, 547

cultivation and isolation of, 548
discovery of, 548
infection with, 547
pathogenicity of, 549
Dysentery, autopsy findings in, 442
occurrence of, 442
symptoms of, 442
Dysentery bacilli, 435

biological considerations of, 441
differentiation of, by Hiss, through
agglutination tests, 440
through fermentation tests, 439
immunization with, 444
active, 444

agglutinins in, 444
bactericidal substances in, 444
true toxins in, 445
passive, 445
in feces, 178

investigation of, by Flexner, 436
by Hiss and Russell, 438
by Kruse, 437
by Lentz, 438
by Martini and Lentz, 438
by Ohno, 439
by Park and Carey, 439
by Park and Dunham, 438

Dysentery bacilli, investigation of, by
Shiga, 435
by Spronck, 437
by Strong and Musgrave, 436
by Vedder and Duval, 437
pathogenicity of, 441
pseudo-dysentery bacillus and, 437,
438

Shiga's bacillus in, 435

cultural characteristics of, 436
morphology of, 435
toxic products of, 442
action of, on animals, 443
obtaining of, from cultures, 442
“Y” bacillus in, 438

Eberth, discovery of typhoid bacillus
by, 399

Edema, malignant, bacillus of. See
under Malignant edema
Eel-blood serum, toxic, 205
Eggs used in clearing culture media, 119
Ehrlich's analysis of diphtheria toxin,
205-215. See also under Diphtheria
toxin

Eichstedt, discovery of Microsporon fur¬
fur by, 639

Electricity in destruction of bacteria, 65
Electrolytic dissociation, 74

relation between degree of and bac¬
tericidal powers of solutions, 74
Elser and Huntoon, discovery of pseudo-
meningococcus by, 379
Eisner's potato-extract gelatin, isola¬
tion of typhoid bacillus in stools by,
407

Emphysematous gangrene, isolation of
Bacillus aerogenes capsulatus from, 474
Endolysins, 291

Endo's fuchsin-agar, isolation of ty¬
phoid bacillus in stools by, 409
Endo’s medium, for colon-typhoid dif¬
ferentiation, 135

Harding and Ostenberg’s method of
preparation of, 136
Endospores, 16
Endotoxins, 185, 306

compared with pigments, 186, 309

INDEX OF SUBJECTS

745

Endotoxins of Staphylococcus pyogenes
aureus, 328

of Streptococcus pyogenes, 345
summary of, 306
toxins distinguished from, 186
Environment and bacteria, 31
Enzymes, definition of, 42

katalyzers and, analogy between, 42
produced by bacteria, 168
diastatic, 169
inverting, 169
proteolytic, 168

varieties of. See under specific names
Epitoxoid in toxin, 208
Erlenmeyer flask, 114
Escherich, discovery of Bacillus lactis
aerogenes by, 453

Esmarch roll tubes in isolation of bac¬
teria, 147

Esmarch’s method of anaerobic culti¬
vation of bacteria, 149
Essential oils as disinfectants, 78
Exudates, bacteriological examination
of. See Bacteriological examination
Eumycetes. See Hyphomycetes

Facultative aerobes, 26
Facultative anaerobes, 26
Farcy, 531

Fat-splitting enzymes, 47

action of, method of investigating,
47

varieties of, 47
Fats in bacterial cell, 22
Favus, 640

Feces, bacteriological examination of,
176

disinfection of, 87
number of bacteria in, 176
varieties of bacteria in, 177
Fermentation, alcoholic, 51
in milk, 702

butyric-acid, in milk, 702
enzymes of, 48, 52
in development of bacteria, 26
inversion in, 42
lactic-acid, 50, 701
process of, 48

Fermentation tests, serum media for,
132

Filtering, in clearing of culture media,
102

through cotton, 120
through paper, 121
in sterilization of culture media, 122
Filters, varieties of :

Berkefeld, 120, 121
Kitasato, 123
Maassen, 124
Reichel, 122
Filtrable virus, 679

table compiled by Wolbach, 680
Filtration of immune body and comple¬
ment, 244

Fishing, colony, 146

Fixation of complement, action in 246
by precipitates, 244
“Fixator,” 279 ,

Flagella, arrangement of, 15
structure of, 14
varieties of, 14

Flagella stains in staining of bacteria, 100
Florence flask, 114

Fluid media covered with oil, used in
cultivation of bacteria, 150
Foot-and-mouth disease, 678
etiological factor of, 679
immunity in, 679
in milk, 706
pathology of, 678
transmission of, 679
Formaldehyde as disinfectant, 78, 89
generation of, by breaking up solid
polymer with heat, 91
by Breslau method, 90
by direct evaporation, 89
by glycerin addition, 90
by lime method, 92
by potassium-permanganate meth¬
od, 92

by Trillat method, 89
gauging amount of formalin in, 92
Trillat autoclave for, 89
Fractional sterilization, 70
at high temperatures, 70
at low temperatures, 71

746

INDEX OF SUBJECTS

Frambcesia tropica, 614
Friedlander, discovery of Bacillus mu-
cosus capsulatus by, 447
Friedlander bacillus. See Bacillus mu-
cosus capsulatus

v. Frisch, discovery of Bacillus of rhi-
noscleroma, 451
Fusiform bacilli, 614
from carious teeth, 613
from noma, 614
from scurvy, 614
of Vincent’s angina, 612

Gaffky, discovery of Micrococcus tetra-
genus by, 334

Gartner, discovery of Bacillus enteri-
tidis by, 429

Gas formed by bacteria, 164
analysis of, 164

carbon dioxide in, 164
hydrogen in, 165
hydrogen sulphide in, 165
quantitative, 166

Gas-pressure regulators for incubators,
160

Moitessier’s, 160

Gastrointestinal autointoxication, 712
bacteria causing, 713
experimental combating of, by acid-
producing bacilli, 713
Metchnikoff’s therapy of, by means of
Bacillus bulgaricus, 714-715
Gelase, 49

Gessard, discovery of Bacillus pyocyaneus
by, 578

Glanders, bacteriological diagnosis of, 532
immunity in, 535
in horses, 530
acute form, 530
chronic form, 531
in man, 532
nodules of, 532
spontaneous infection in, 530
Glanders bacillus. See Bacillus mallei
Globulin in bacterial cell, 22
Glycerin, use of, for culture media, 126
meat extract, 126
meat infusion, 127

Glycerin broth, 125
Glycerin potato, 130
Gonococcus. See Diplococcus gonor-
rhoeae

Gram-negative bacteria, 104
Gram-negative cocci, chromogenic, 387
Gram-negative micrococci, table of di¬
agnosis of, by differential value of
sugar fermentation, 387
Gram-positive bacteria, 104
in feces, 177

Gram’s method of staining bacteria, 102
classification of pathogenic bacteria
by, 104

Paltauf’s modification of, 103
Group agglutination, 234
Gruber- Widal reaction, 250
Gruby, discovery of Trichophyton ton¬
surans by, 642

Guarnieri’s medium, Welch’s modifica¬
tion of, 129

Halogens as disinfectants, 75
“Hanging block” method in study of
bacteria, 94

“Hanging drop” method in study of
bacteria, 93

Hansen, discovery of lepra bacillus by,
505

Haptophore group in toxin molecule, 207
in toxon molecule, 209
Haptophore groups in immune body, 228
complementophile, 228
cytophile, 228

Hauser, discovery of Bacillus proteus
vulgaris by, 454

Heat, in destruction of bacteria, 65
dry and moist, comparison of, 66
effect of various degrees of, 65
moist, advantages of, 66
penetrating power of, 67
in sterilization of culture media, 121
Heat sterilization, 68
dry, 68

by burning, 68
by hot air, 68
moist, 69

by boiling, 69

INDEX OF SUBJECTS

747

Heat sterilization, moist, by fractional
sterilization, 70
by live steam, 60
by steam under pressure, 71
Hektoen and Ruediger on structure of
opsonins, 283
Hemagglutinins, 235
Hematogen, 538
Hemolysin, immune, 226
Hemolysins of Staphylococcus pyogenes
aureus, 329

of Streptococcus pyogenes, 344
specificity of, 247
varieties of :
autolysins, 247
heterolysins, 247
isolysins, 247
Hemolysis, 201, 225
Hemolytic tests, 259
obtaining blood for, 259
in large quantities, 260
in small quantities, 259
standard concentration of red blood
cells for, 259

Hemolytic unit, definition of, 265
Hemorrhagic septicemia bacilli, 551
morphology of, 551
staining of, 551
varieties of :

bacillus of chicken cholera, 552
Bacillus pestis, 554
bacillus of swine plague, 553
Hepatotoxin, 201

Hesse’s medium for colon-typhoid differ¬
entiation, 133
Heterolysins, 247
Hides, tanning of, bacteria in, 716
Hiss’ agar-gelatin medium, isolation of
typhoid bacillus in stools by, 407
Hiss’ leucocyte extract theory and
therapy, 289

Hiss’ methods of staining capsules, 98
Hiss’ plating media for colon-typhoid
differentiation, 133

Hiss’ tube medium for colon-typhoid
differentiation, 133

Hiss’ serum water media for fermenta¬
tion tests; 132

“Hog-cholera” bacilli, 430
differentiation of, from swine plague
bacillus, 554

Hogyes, dilution method of, in treat¬
ment of rabies, 656

Horse meat, detection of, by precipitin
tests, 254

Horses used in production of diphtheria
antitoxin, 216-17
of tetanus antitoxin, 221
Howard and Perkins, discovery of Strep¬
tococcus mucosus by, 350
Hydrogen, formation of, by bacteria,
165

in nutrition of bacteria, 28
Hydrogen peroxid as disinfectant, 76
Hydrogen sulphid formed by bacteria,
165

Hydrophobia. See Rabies
Hypersusceptibility. See Anaphylaxis
Hyphomycetes, 635

conditions favorable to growth of, 638
diseases caused by, 639
favus, 640

pityriasis versicolor, 639
ringworm, 642
thrush, 640

diseases sometimes accompanied by,
644

morphology of, 635
reproduction of, 635
mycomycetes in, 636

Aspergillus variety in, 637
Penicillium variety in, 637
sporulation by other methods in,
637

phy corny cetes in, Mucorinae vari¬
ety of, 635

sexual reproduction of, 636
structural classification of, 635
varieties of, 635
mycomycetes, 635

morphology of (typical), 635, 636
ascospores in, 638
conidia (or spores) in, 638
conidiophores in, 637
hyphal branches (septate) in,
637

748

INDEX OF SUBJECTS

Hyphomycetes, varieties of, mycomy-
cetes, morphology of, my¬
celial threads (septate) in,
637

sterigmata (septate) in, 637
morphology of less frequent
forms of, 637
ascospores in, 637
chlamydospores in, 638
phycomycetes, 635

morphology of (typical), 635-636
columella in, 636
hyphal branches in, 636
mycelial threads (non-septate)
in, 636

sporangium in, 636
spores in, 636

morphology of sexual reproduc¬
tion forms of, 636
gametophores in, 636
zygospores in, 636

“ Immune body” in blood serum, 226
in Ehrlich’s theory of lytic process j!n
blood serum, 226
haptophore groups of, 228
complementophile, 228
cytophile, 228
Immunity, absolute, 190
acquired, 192

definition of, 190, 192
active, 193
artificial, 193
definition of, 193

experimentation with attenuated
cultures for, 193
with bacterial products for, 195
with dead bacteria for, 195
with sublethal doses of fully viru¬
lent bacteria for, 195
definition of, 189
natural, 190
individual, 191
of races, 191
of species, 190
passive, 196

relation of, to phagocytotic powers in
animals7 279

Immunization, blood the seat of, 198
Incubation of cultures, 156
incubators in, 158
gas-pressure regulators for, 160
thermo-regulators for, 159
Indigo production, bacteria in, 716
Individual immunity, 191
in higher animals, 192
in lower animals, 191
Indol production by bacteria, 167
Industries, bacteria in, 715
Infection, definition of, 181
fundamental factors in, 181
paths of, 183
inner, 184
outer, 183

Infectiousness, definition of, 182
due to number of bacteria, 182
due to variations in virulence, 183
Influenza, epidemic of, in 1889-90, 536
organs attacked in, 540
Influenza bacillus, 536
bacteria related to, 541
Bacillus murisepticus, 542
bacillus of pleuro-pneumonia, 542
Bacillus rhusiopathise, 542
Koch- Weeks bacillus, 542
pseudo-influenza bacillus, 541
biology of, 540

isolation and cultivation of, 537
on agar and gelatin, 537
blood added in, 537
hemoglobin added in, 537
hematogen added in, 538
morphology and staining of, 536
pathogenicity of, 540

in experimental inoculation of ani¬
mals, 541

organs attacked in, 540
susceptibility of animals to, 541
Inhibition, coefficient of, 80
Inhibition strengths of various antisep¬
tics, 84

Inoculation of animals, 170
intraperitoneal, 172
intravenous, 172
subcutaneous, 171
Inoculation of media, 141

INDEX OF SUBJECTS

749

Inoculation of media, technique of trans¬
ferring bacteria in, 141
virus used in transferring bacteria in,
141

Insusceptibility of cold-blooded animals,
190

Intra vital method of Nakanishi in
study of bacteria, 94
Inulin media, 132, 369
Invasion, paths of, 183
inner, 184
outer, 183

Inversion by fermentation, 42
Invertase, 49

Iodoform as disinfectant, 77
Iodine, tetrachloride of, as disinfectant,
76

tincture cf, for skin sterilization, 76
Iron compounds in nutrition of bacteria,
29

Isolation of bacteria, 142
early methods of, 143
present methods of, 143
Esmarch roll tubes in, 147
Koch’s plates in, 144
surface streaking in, 148
Isolysins, 247

Israel, discovery of actinomyces of man
by, 622

Jackson’s lactose-bile medium for colon-
typhoid differentiation, 139
Jenner’s discovery of immunization in
smallpox by vaccinia, 659

Katalyzers, definition of, 42

enzymes and, analogy between, 42
Kefyr, 702

Kitasato, discovery of Bacillus tetani by,
456

discovery of plague bacillus by, 554
Kitasato filter, 123

Klebs, discovery of diphtheria bacillus
by, 512

Koch, discovery of cholera spirillum by,
582

discovery of tubercle bacillus by, 479
Koch plates in isolation of bacteria, 144

Koch- Weeks bacillus, 542
Koumys, 702

L+, definition of, 208
L0, definition of, 207
Lab enzymes, 46
Lactase, 50

Lactic-acid bacilli in therapy of gastro¬
intestinal autointoxication, 714-715
Lactio-acid fermentation, 50
bacteria of, 50
in milk, 701

Lactose-bile medium, Jackson’s, for
colon-typhoid differentiation, 138
Lactose-litmus agar, 129
Laked blood, 225

Langenbeck, discovery of Oidium albi¬
cans by, 640

Lautenschlager’s thermo-regulator, 158,
159

Leprolin, 502
Leprosy, 505
bacillus of, 502, 505
cultivation of, 506
by Clegg, 506
by Duval, 506

differentiation of, from tubercle ba¬
cillus by staining, 106, 506
inoculation with, 507, 509
morphology of, 505
occurrence of, in body, 508
pathogenicity of, 507
relation of, to tubercle bacillus, 509
staining of, 505
toxic products of, 509
clinical varieties of, 508
contagiousness of, 509
in rats, 510
occurrence of, 507

tuberculin administered in, results of,
510

Leptothrix, 619
morphology of, 618

Leucocidin of Staphylococcus pyogenes
aureus, 329

action of, upon leucocytes, 330
discovery of, 329

leucotoxin differentiated from, 331

750

INDEX OF SUBJECTS

Leucocidin of Staphylococcus pyogenes
aureus, obtaining of, 330
Leucocyte extract, 289

effect of, upon infections in animals,
290

in man, 291

experimentation with, 291
obtaining of, 290
Leucotoxin, 201, 331
Liborius’ method of anaerobic cultiva¬
tion of bacteria, 149
Light in destruction of bacteria, 63
action of, 64
resistance to, 63
varieties of, 64

Lime, chloride of, as disinfectant, 76
Limes death, definition of, 208
Limes zero, 207
Linen, etc., disinfection of, 87
von Lingelsheim, discovery of Diplococ-
cus mucosus by, 387
discovery of Micrococcus pharyngis
siccus by, 387
Lipase, 47

Liver and gall-bladder, inflammatory-
conditions of, attributed to colon
bacillus, 395

Lobar pneumonia, infectiousness of,
352

Loeffler and Schiitz, discovery of glan¬
ders bacillus by, 528
Loeffler’s malachite-green media, isola¬
tion of typhoid bacillus in stools by,
409

Loeffler’s method of staining flagella,
100

Loeffler’s serum medium, 131
Lustgarten, discovery of smegma bacil¬
lus by, 503
Lysins, 224

action of, Bordet’s interpretation of,
225

compared with Ehrlich’s, 225, 228
summary of, 228
Ehrlich’s theory of, 226

compared with Bordet’s, 226
complement in, 226
immune body in, 226

Lysins, action of, Ehrlich’s theory of
immune body in, formation
of, 226

haptophore groups in, 228
complementophile, 228
cytophile, 228
red corpuscles in, 227
side chains or receptors in, 226
over-reproduction of, 226
in blood serum, 225
experimentation in, 224
in immune blood serum, reactivated
by normal serum, 226
in normal blood serum, Bordet’s theory
of, 225

alexin in, 225

sensitizing substance in, 225
investigation of, by Ehrlich, 226
Lysol, 78

Lyssa. See Rabies
Maassen filter, 124

MacConkey’s bile-salt agar, for colon-
typhoid differentiation, 138
Macrophages, definition of, 276
Madura foot. See Mycetoma
Malachite-green media, for colon-ty-
, phoid differentiation, 136
bouillon, 137

Loeffler’s, for isolation of typhoid
bacillus in stools, 409
Malignant edema, bacillus of, 468
cultivation of, 470
early investigation of, 469
immunity in, 471
morphology of, 469
pathogenicity of, 470
staining of, 470
Mallein, 532
action of, 533
diagnostic use of, 533
directions of U. S. government for,
534

obtaining and preparation of, 533
Malta fever, 549
in domestic animals, 549
Maltase, 50

Maragliano’s serum for tuberculosis, 498

INDEX OF SUBJECTS

751

Marchiafava and Celli, discovery of
meningococcus by, 371
Marmorek’s serum for tuberculosis,
498

Measles, 675

investigation for virus of, 675
by Hektoen, 675
by Home, 675

Meat, determination of nature of, by
precipitin tests, 254
used for culture media, 115
soluble, 116
Meat extract, 116
Meat-extract agar, 127
Meat-extract broth, 124
Meat-extract gelatin, 126
Meat-infusion agar, 128
Meat-infusion broth, 124
Meat-infusion gelatin, 127
Meat-poisoning bacilli, 429, 475
Meningitis, microorgailisms causing, 371
primary, 371
secondary, 371
serum therapy of, 378
Meningococcus. See Micrococcus intra-
cellularis meningitidis
Metachromatic granules, 11
Metacresol as disinfectant, 77
Metchnikoff’s therapy of gastrointestinal
auto-intoxication by means of Bacil¬
lus bulgaricus, 714-715
by means of lactic-acid bacilli, 715
Mice, method of injecting, intravenously,
173

“Microbe de la coqueluche,” 543
Micrococci, 321. See also Staphylo¬
cocci

Micrococcus, 37
Micrococcus catarrhalis, 385

differentiation of, from gonococcus, 386
from meningococcus, 386
Micrococcus intracellularis meningitidis,
371

cultivation of, 374
oxygen in, 375
viability of organism in, 375
differentiation of, from Micrococcus
catarrhalis, 386

Micrococcus intracellularis meningitidis,
early observation of, 371-2
immunization against, 378

agglutinins in immune sera in,
378

modes of inoculation of, 377
morphology of, 373
pathogenicity of, 376
in animals, 377
in man, 376

pseudomeningococcus diff erentiated

from, 379
resistance of, 376
staining of, 374

susceptibility of animals to, 377
viability of, 375
Micrococcus melitensis, 549
cultivation of, 550
morphology and staining of, 550
Micrococcus pharyngis siccus, 387
Micrococcus tetragenus, 333
cultivation of, 333
pathogenicity of, 333
Microorganisms, discovery of, 1
pathogenic, 321
Microphages, definition of, 276
Microscopic study of bacteria, 93
in fixed preparations, 94

process of, 94. See also under
Staining

in living state, 93

by hanging block method, 94
by hanging drop method, 93
by intravital method of Nakanishi,
94

Microspira, 37
Microsporon furfur, 639

clinical picture of infection of, 639
cultivation of, 640
morphology of, 639
Milk, alcoholic fermentation in, 702
anthrax bacilli in, 707
bacteria in, 699

butter a means of transmitting, 711
butter-making aided by, 710
cheese-making aided by, 711
numbers of, 700
estimating of, 709

752

INDEX OF SUBJECTS

Milk, bacteria in, propagation of, 700
sources of, 699

under ordinarily hygienic condi¬
tions, 699
varieties of, 701
“bitter,” 703

butyric-acid fermentation in, 702
certified, 701
cholera traced to, 705
coagulation of casein in, 702
color changes in, 703
diarrheal diseases traced to, 705
diphtheria traced to, 705
foot-and-mouth disease virus in, 706
lactic-acid fermentation in, 701
pasteurization of, 709
pus cells and leucocytes in, 706
relation of, to infectious diseases, 703
scarlet fever traced to, 704
“slimy,” 703
streptococci in, 705
streptococcus throat infections con¬
veyed by, 343, 706
supervision of supply of, 700
tubercle bacilli in, 707
infection from, 708
precautions against, 708
dairy inspection in, 708
Milk, tubercle bacilli in, precautions
against, tuberculin test of cows
in, 708

transmission of, from cow, 707
typhoid fever epidemics traced to,
703

Milk media, 130
Milzbrand, 563

Moeller’s method of staining spores, 98
Moitessier’s gas-pressure regulator, 169
Molds. See Hyphomycetes
Morax-Axenfeld bacillus, 545
cultivation of, 546
morphology and staining of, 546
pathogenicity of, 547
Motility of bacteria, 14
by flagella, 14
Brownian, 14

effect of temperature on, 15
molecular, 14

Motility of bacteria, organs of, 13
true, 14

Mucorinae, reproduction in, 635-636
Muguet. See Thrush
Multiplicity of amboceptors in normal
sera, 241

of complement in normal sera, 242
Mycetoma, clinical picture of, 627
granules in, 627
melanoid, 627

cultivation of, 627-628
morphology of, 627
ochroid, 627
Mycomycetes, 635

Nakanishi, “intravital” staining method
of, in study of bacteria, 94
Negri bodies in central nervous system in
rabies, 648

demonstration of, 648
diagnosis of rabies by, 650
explanation of, 650
significance of, 651
staining of, 108

Neisser, discovery of Diplococcus gon-
orrhoeae bv, 380

discovery of lepra bacillus by, 505
Nephrotoxin, 201

Neufeld and Rimpau’s discovery of
opsonic substances, 282
Neurotoxin, 201

Neutral-red medium for colon-typhoid
differentiation, 138
“New tuberculin” (Koch), 491
“New tuberculin-bacillary emulsion,”
492

Nitrate-solution broth, 126
Nitrifying bacteria, 57
action of, 58

agricultural importance of, 58
Nitrogen fixation by bacteria, 54
microorganism of, 54
in root tubercles, 55
experimentation on, 56
microorganism of, 55
process of, 56
in soil, 54

Nitrogen in nutrition of bacteria, 28

INDEX OF SUBJECTS

753

Nitrogen in nutrition of bacteria,
sources of supply of, 28
Noguchi’s modification of Wassermann
test for syphilis, 270
Novy jar, 154

Nucleus in bacterial cell, 10
Nutrient media. See Culture media
Nutrition of bacteria, 25
carbon in, 25
hydrogen in, 28
nitrogen in, 25
oxygen in, 25
salts in, 29

Obermeier, discovery of spirochaetes
of relapsing fever by, 605
Obligatory aerobes, 25
Obligatory anaerobes, 26
O'idium albicans, 640
discovery of, 640
morphology of, 640
varieties of, 640
“Old tuberculin” (Koch), 491
Opium production, bacteria in, 716
“Opsonic coefficenLpf^extinction,” 286
Opsonic index, finding of, 286
Opsonic test, Wright’s, 284

obtaining of bacterial emulsion for,
284

of blood serum for, 284
of leucocytes for, 284
opsonic index in, finding of, 286
parallel control test on normal
serum in, 285
“pool” in, 285
technique of, 284

Simon, Lamar, and Bispham’s tech¬
nique of, 286
dilutions in, 286

opsonic coefficient of extinction in,
286

Opsonins, 281

decrease of phagocytic power upon
introduction of bacteria without,
282

definition of, 282

increase of phagocytic power upon in¬
troduction of, 283
49

Opsonins, Neufeld and Rimpau’s dis¬
covery of, 282
normal and immune, 282-3
specificity of, 282

structure of, according to Hektoen and
Ruediger, 283

Wright’s test of. See Opsonic test
Wright’s theory of, 282
Orthocresol as disinfectant, 78
Osmotic properties of bacterial cell, 23
Oxydases, 50

Oxygen as disinfectant, 88

in development of bacteria, 25
free, 26

absence of, 26
indirect supply of, 26
in nutrition of bacteria, 25
Ozone as disinfectant, 88

Paltauf’s modification of Gram’s stain,
103

Pancreascytotoxin, 201
Paper used in filtering culture media,
121

Paracolon bacillus, 430
Paracresol as a disinfectant, 78
Parasites, bacterial, 29
facultative, 30
media for growth of, 29
definition of, 182
infectiousness of, 182
pathogenicity of, 182
Paratyphoid bacilli, differentiation of,
on sugar, 433
types A and B, 431

Paratyphoid fever, differentiation of,
from typhoid, 432

Passive immunity. See under Immunity
Passive immunization, definition of, 196
Pasteur, discovery of bacillus of chicken
cholera by, 552

discovery of bacillus of malignant
edema by, 468

discovery of Diplococcus pneumoniae
by, 353

technique of, in rabies therapy, 652
Pasteurization of milk, 709
Pathogenic bacteria, 182, 321

INDEX OF SUBJECTS

754

Pathogenicity, fundamental factors of,
181

of bacteria, 182

Penicillium, reproduction in, 637
Pepton-salt solution broth, 126
Pericardial exudates, bacteriological ex¬
amination of, 175

Peritoneal exudates, bacteriological ex¬
amination of, 175

Peritonitis following perforation attrib¬
uted to colon bacillus, 394
Perlsucht, 498

Permanganate of potassium as disin¬
fectant, 76

Pernicious anemia and Bacillus aerogenes
capsulatus, 177

Peroxid of hydrogen as disinfectant, 76
Petri dish, 115, 144

Petruschky, discovery of Bacillus fecalis
alkaligenes by, 427

Pfeiffer, discovery of influenza bacillus
by, 536

discovery of Micrococcus catarrhalis
by,. 385

discovery of pseudo-influenza bacil¬
lus by, 541

Phagocytic index, 286
Phagocytosis, 275
cells active in, 276
“fixed,” 276
macrophages, 276
microphages, 276
“wandering,” 276
cells of animal origin in, 278
chemotaxis in, 277
complement or “cytase” in, 279
definition of, 275

dependence of, on opsonins, 281-2
diminution of, upon introduction of
bacteria without opsonic serum,
282

immune body or “fixator” in, 279
immunity and, 279
in higher animals, 276
in protozoa, 275

increase of, upon the introduction
of opsonic substances in serum,
282

Phagocytosis, macrophages in, 276
Metchnikoff’s theory of, 276
opposition to, 279
microphages in, 276
process of, in the body upon introduc¬
tion of bacteria, 277
upon introduction of nutrient
broth, 276

susceptibility of various microorgan¬
isms to, 278

variety of phagocyte in, determined by
the bacterium, 278
Phenol production by bacteria, 167
Phosphates in the nutrition of bacteria,
29

Phosphorescence produced by bacteria,
59

Phragmidiothrix, 38
Phycomycetes, 635

Pigment, formation of, by bacteria, 59
chemical nature of, 59
cultural conditions on, 60
Piorkowski’s urine gelatin for colon-ty¬
phoid differentiation, 134
Pityriasis versicolor, 639
clinical picture of, 639
microorganism causing. See Micro-
sporon furfur
occurrence of, 639
Plague, bacillus of, 554
biology of, 557
degeneration forms of, 20
immunization against, 561
active, 561

involution forms of, on salt agar, 557
isolation and cultivation of, 556
lesions in animals produced by, 559
morphology of, 555
pathogenicity of, 558
resistance of, 557
staining of, 556
transmission of, 560
toxins of, 561

variations in virulence of, 559
viability of, 557
epidemics of, 554
in animals, 558

in California ground squirrels, 560

INDEX OF SUBJECTS

755

Plague, bacillus of, in animals, in Man¬
churian marmot, 560
inoculation in, 559
spontaneous infection in, 559
in man, 558

autopsy findings in, 558
bacteriological diagnosis of, in life,
558

infection in, 558
localized form of, 558
pneumonic form of, 558
transmission of, 560
Plague-like disease in rodents, 562
Planococcus, 37
Planosarcina, 37
Plasmolysis of bacterial cell, 23
Plasmoptysis of bacterial cell, 24
Plating in isolation of bacteria, 143
Pleural exudates, bacteriological exami¬
nation of, 175

Pleuro-pneumonia, organism of, 542
Pneumobacillus. See Bacillus mucosus
capsulatus

Pneumococcus, discovery of, 7
different types of, 336
Pneumococcus. See Diplococcus pneu¬
moniae

Pneumococcus-streptococcus group, mu¬
tation, 370

Pneumonia, complications of, 362
lobar, infectiousness of, 352
serum therapy for, 366
Poisons, bacterial. See Bacterial poi¬
sons

Polar bodies, 11

special stains for, 107
Poliomyelitis, acute anterior, 664
immunity in, 667
infectiousness of, 664
inoculation of animals with spinal
substance of, 664
by Flexner and Lewis, 665, 666
by Knoepfelmacher, 665
by Landsteiner and Levaditi, 665
by Landsteiner and Popper, 665
resistance of virus of, 666
Polychrome stains in staining of bac¬
teria, 107

Potassium permanganate as disinfect¬
ant, 76

Potato media, 130
glycerin, 130

Pour plate, technique of making, 145
Precipitin tests, 252 *

bacterial filtrates for, 254
technique of, 255

determining nature of meat by,
254

precipitating antisera for, 252
against albumin solutions, 253
technique of production of, 252
proteid solutions to be tested by,
254

Precipitins, 200, 235

agglutinins and, structure of (Ehr¬
lich), 238

cell-receptors in, 238
theoretical considerations concern¬
ing, 238

bacterial differentiation by, 237
differentiation of proteids by, 237,
254

distinguishing blood of animal species
by, 237

effect of heat on, 236
experimentation in, 235
group reaction of, 237
identity of, with sensitizers, 236
nature of, 236
specificity of, 237
Proagglutinoids, 235
Proteid differentiation by complement
fixation, 273

substances necessary for, 273
technique of, 274
by precipitins, 237, 254
Proteid injections, anaphylaxis in, 295.

See also Anaphylaxis
Proteids in bacterial cell, 22
Proteins, bacterial, 186
Proteolytic enzymes, 43
action of, 44

in breaking down animal excreta, 46
bacteria producing, 44
proteids necessary to, 44
ptomains produced by, 45

756

INDEX OF SUBJECTS

Proteus group, bacilli of, 454
cultivation of, 454
morphology and staining of, 454
occurrence of, 454
pathogenicity of, 455
Protozoa and bacteria, differentiation
of, 1

staining of, 108

Pseudo-dysentery bacillus, 437, 438
Pseudo-influenza bacillus, 541
Pseudo-membranes in diphtheria, 519
Pseudomeningococcus, 379
Pseudomonas, 37
Ptomains, 45, 185, 306
bacterial poisons and, 185
discovery of, 185
occurrence of, 185
toxins distinguished from, 45
varieties of, 45

Pus, bacteriological examination of, 175
Pus cells and leucocytes in milk, 706
Putrefaction, action of, 44
Putrefactive bacteria, quantitative an¬
alysis of, 21
Putrescin, 45
Pyemia, definition of, 184
Pyocyanase, 580

immunizing powers of, 580
Pyocyanin, 578
Pyocyanolysin, 581

Pyrogallic acid, use of, in cultivation of
anaerobic bacteria, 152

Rabies, 646
course of, 647
in animals, 647
in men, 648

cultivation of organism of, by Nogu¬
chi, 651

diagnosis of, by presence of Negri
bodies in central nervous system,
648-650

experimental infection of, 646
incubation in, 647

Negri bodies in central nervous sys¬
tem, 648

demonstration of, 648

by Van Gieson’s method, 649

(

Rabies, Negri bodies in central nervous
system, demonstration of, by
Williams and Lowden’s meth¬
od, 650

staining in, 649

Mann’s method of, 649
diagnosis by, 649-650
occurrence of, 646
pathology of, 648

specific therapy of (Pasteur’s tech¬
nique), 652

attenuation and preparation of virus
fixe in, 652

inoculation of rabbits with virus fixe
in, 652

spinal cord of inoculated rabbits in,
desiccation of, 653
emulsification of, 654
treatment of cases with injections
of spinal-cord solution in, 654
Hogyes dilution method in, 656
scheme of, used at Pasteur In¬
stitute, 654

used in New York Department
of Health, 655
virulence of virus of, 647
Racial immunity, 191
“Rage.” See Rabies.

Rat leprosy, 510

relation of, to human leprosy, 511
Rauschbrand. See Bacillus of sympto¬
matic anthrax

Receptors of toxin molecule in side-
chain theory, 213
chemical action of, 213
over-production of, 214
Red blood cells, antibodies produced by,
200

Reducing powers of bacteria, 167
Refractive index of parts of bacterial
cell, 24

Reichel filter, 122

Reichert’s thermo-regulator, 158, 159
Relapsing fever, 605
immunity in, 610
symptoms of, 608
transmission of, 610
varieties of, 609

INDEX OF SUBJECTS

757

Relapsing fever spirochaete, 605
cultivation of, 606
morphology and staining of, 605
pathogenicity of, 608
in animals, 608
in man, 608

symptoms of, 608
transmission of, 610
varieties of, 609
Reproduction of bacteria, 17
Resistance, definition of, 189
Rhinoscleroma, bacillus of, 451
Ricin, experimentation with, 204
Ringworm, 642
Root tubercles, 55

microorganism of nitrogen fixation in,
55

Roux’s method of anaerobic cultivation
of bacteria, 149

Saccharomycetes. See Yeasts and
Yeast cells

Salts in nutrition of bacteria, 29
Saprophytes, bacterial, 29, 30
definition of, 182
Sarcina, 37

Sarcophysematos bovis. See Bacillus of
symptomatic anthrax
Scarlatina. See Scarlet fever
Scarlet fever, 676

favorable influence of streptococcus
antisera in, 676
streptococci present in, 676
traced to milk, 704

Schaudinn and Hoffmann, discovery of
Spirochseta pallida by, 594
Schizomycetes, 37
Schweineseuche, 553
Scorpion poison, antitoxin for, 199
“Sensibilisin,” 302
‘ ‘ Sensibilisinogen, ’ ’ 302
Septicemia, definition of, 184

diagnosis of, by isolation of bacteria
from the blood, 178
due to colon-bacillus infection, 394
hemorrhagic. See Hemorrhagic sep¬
ticemia

Serum media, 131

Serum media, Loeffler’s, 131
Serum reactions, technique of, 249.
See also under individual tests
agglutination tests in, 250
antigen determined in, by comple¬
ment fixation, 271
for typhoid fever, 271

obtaining of material for, 271
test in, 272

bactericidal and bacteriolytic tests
in, 255

complement fixation in, for deter¬
mination of antibodies, 261
for determination of antigen, 271
for proteid differentiation, 273
hemolytic tests in, 259
precipitin tests in, 252
proteid differentiation by comple¬
ment fixation in, 273
substances necessary for, 273
test in, 274

Wassermann test in, 262
modifications of, 268
“Serum sickness,” 296
Serum water media for fermentation
tests, 132

Shiga, discovery of dysentery bacillus by,
435

Shiga’s bacillus, 435

cultural characteristics of, 436
morphology of, 435

Side-chain theory of toxin-antitoxin
reaction, 212
chemical action in, 213
elements of molecules in, 213
atom group, 213
side chains or receptors, 213
over-production of receptors in, 214
Side chains, action of, in Ehrlich’s theory
of lytic process in blood serum, 226
Slanting of culture media, 123
“Slimy” milk, bacteria causing, 703
Smallpox, 657
etiological factor of, 657
immunization in, 658
by vaccination, 659, 663
Jenner’s discovery of, 659
technique of, 663

758

INDEX OF SUBJECTS

Smallpox, immunization in, by vaccina¬
tion, value of, 663
production of vaccine for, 647. See
also under Vaccine
occurrence of, 657

protozoan incitant of, research for, 657
relation of chicken-pox to, 660
relation of cowpox to, 659
transmission of, 658
vaccine bodies in, discovery of, 657
explanations for, 658
Ewing’s, 658

Smegma bacillus, 502, 503, 594
cultivation of, 504
morphology of, 503
occurrence of, 503
staining of, 504

identification of bacillus by, 504
tubercle bacillus and, differentiation
between, by stains, 106
Smith’s modification of Pitfield’s method
of staining flagella, 101
Snake poison, antitoxin for, 199
Soil, bacteria in, 685

from burial of infected cadavers, 687
in agricultural regions, 685
numerical estimation of, 687
pathogenic, in surface layers, 686
Solutions, saturated, for staining of
bacteria, 95
staining-power of, 96
Soor. See Thrush
Species immunity, 190
differences in, 190

Specific gravity of forms of bacterial
cell, 24

‘‘Specific precipitates,” 235-236
Spider poison, antitoxin for, 199
Spinal fluid, bacteriological examination
of, 176

Spirillaceae, 37
Spirillum, 38
description of, 9

Spirillum cholerae asiaticae. See under
Cholera

Spirillum Deneke, 591
Spirillum of Finkler-Prior, 539
Spirillum Massaua, 591

Spirillum Metchnikovi, 590
Spirochaeta, genus, 38
Spirochaeta anserina, 616
Spirochaeta Calligyrum, 617
Spirochaeta Duttoni, 610
Spirochaeta gallinarum, 615
cultivation of, by Noguchi, 616
immunization against, 616
similarity of, to Spirochaeta anserina,
616

transmission of, 615, 616
Spirochaeta macrodentium, 617
Spirochaeta microdentium, 617
Spirochaeta pallida, 593

animal pathogenicity of, 601
cultivation of, 600
dark-field examination of, 597
by Miihlens, 600
by Noguchi, 600
demonstration of, 596
in living state, 596
in smears, 597

by Goldhorn’s method of stain¬
ing, 598

by India-ink preparation, 598
by Schaudinn and Hoffmann’s
method of staining, 597
by Wood’s method of staining,
597

in tissues, 598

by Levaditi’s method, 598
by Levaditi and Manouelian’s
method, 599

immunization against, 603
active, 603
passive, 603

infection of animals by, 602
of cornea of rabbits, 602
of testes of rabbits, 602
morphology of, 595
observation of, 596-597
occurrence of, in syphilis cases, 595-
596

staining of, 108
Spirochaeta pertenuis, 614
morphology of, 615
similarity of, to Spirochaeta pallida, 615
Spirochaeta phagedenis, 616

INDEX OF SUBJECTS

759

“Spirochseta refringens,” 595
Spirochsete of relapsing fever. See Re¬
lapsing fever spirochsete
Spirochsete of Vincent’s angina. See
Vincent’s angina, spirochsete of
Spirochaetes, cultivation of, 593
differentiation of, from spirilla, 593
diseases caused by, 592. See also
under specific names
reproduction in, 592
structure of, 592
Spirosoma, 37

Spore stains in staining of bacteria, 97
Spores, bacterial, 15
formation of, 15
germination of, 17
position of, 17
varieties of, 16
arthrospores, 16
true or endospores, 16
vegetative forms from, 17
Sporotrichosis, 644

Sporulation, physiological significance of,

process of, 16

Sputum, disinfection of, 87
Stable antitoxin, 206
Staining of bacteria, chemical principles
in process of, 96
acid-fast bacteria stains, 104
Baumgarten’s method, 106
Bunge and Trautenroth’s method,
106

Ehrlich’s method, 104
Gabbet’s method, 105
Pappenheim’s method, 106
Ziehl-Neelson method, 105
capsule stains, 98
Buerger’s method, 99
Hiss’ methods, 98
copper sulphate, 98
potassium carbonate, 98
Wadsworth’s method, 99
Welch’s method, 98
differential stains, 102
Gram’s method, 102
classification by, 104
Paltauf’s modification of, 103

Staining of bacteria, flagella stains, 100
Loeffler’s method, 100
Smith’s modification of Pitfield’s
method, 101

Van Ermengem’s method, 101
polychrome stains, 107
Giemsa’s method, 108
Jenner’s method, 108
Wood’s method, 109
Wright’s modification of Leish-
man’s method, 108
special stains for polar bodies, 107
Neisser’s method, 107
Roux’s method, 107
spore stains, 97
Abbott’s method, 97
Moeller’s method, 98
staining in tissues, 1 10

for actinomyces in sections, 112
for Gram-positive bacteria, 111
Gram-Weigert method, 111
in celloidin sections, 111
in paraffin sections, 111
for tubercle bacilli in sections, 112
in celloidin sections, 112
in paraffin sections, 112
Loeffler’s method, 112
saturated solutions used in, 95
staining solutions in, power of, 96
steps in process of:

(1) smearing, 94

(2) drying, 95

(3) fixing, 95

(4) staining, 95

(5) washing, 95

(6) blotting, 95

(7) mounting, 95

Standardization of diphtheria antitoxin,
218

of tetanus antitoxin, 221
Staphylococci, 321. See also under
individual staphylococci
definition of, 321
in feces, 177

Staphylococcus epidermidis albus, 332
Staphylococcus pyogenes albus, 332
Staphylococcus pyogenes aureus, 322
cultural characters of, 323

760

INDEX OF SUBJECTS

Staphylococcus pyogenes aureus, im¬
munization against, 331
active, 332
agglutinins in, 331
modes of inoculation with, 327
morphology of, 322
pathogenicity of, 326
in animals, 327
in man, 327

pigment formation of, 325
resistance of, 325
to chemicals, 326
to desiccation, 326
to heat and cold, 325
staining of, 322

susceptibility of animals to, 326
susceptibility of man to, 327
thermal death point of, 325
toxic products of, 328
endotoxins, 328
hemolysins, 328

leucocidin, 329. See also under
Leucocidin
virulence of, 326

Staphylococcus pyogenes citreus, 332
Steam in sterilization, 67
live, 69
saturated, 68
superheated, 68
Stegomyia fasciata, 673
Sterilization of culture media, 121
filtration in, 122
heat in, 121

Sternberg, discovery of Diplococcus
pneumoniae by, 353
Stimulins, 281

Streaking, surface, in isolation of bac¬
teria, 148

“ Street virus,” 647
Streptococci, 37, 335

capsulated, description of organisms
reported as, 367-369
classification of, 348

by Andrewes and Horder, 349
by carbohydrate fermentation pow¬
ers, 348

by reactions to immune sera, 350
morphological, 318

Streptococci, classification of, morpho¬
logical, Streptococcus longus
seu erysipelatos in, 348
Streptococcus minor seu viridans
in, 348

Streptococcus mucosus in, 350
definition of, 335

differentiation of, from pneumococci,
357, 367
cultural, 368
morphological, 367
epidemic throat infections by, 343
in feces, 177
in milk, 705
in milk epidemics, 343
preparation of, for agglutination test,
251

pyogenic. See Streptococcus pyo¬
genes

Streptococcus anginosus, 349
Streptococcus equinus, 349
Streptococcus erysipelatis, 342
Streptococcus fecalis, 349
Streptococcus longus seu erysipelatos,
348

Streptococcus mitior seu viridans, 348
Streptococcus mucosus, 350, 351
Streptococcus pyogenes, 335
brevis, 337, 338
cultivation of, 337
early experimentation with, 335
immunization against, 345

immune sera of infected animals in,
345

agglutinins in, 347
precipitins in, 348
specificity of, 347
standardization of, 347
leucocyte extracts in, 347
technique of, 346
longus, 337

modes of inoculation with, 341
in animals, 341
in man, 342
morphology of, 337
pathogenicity of, 340
in animals, 340
in man, 342

INDEX OF SUBJECTS

761

Streptococcus pyogenes, resistance of,
339

staining of, 337

susceptibility of animals to, 341
toxic products of, 344
endotoxins, 344
hemolysins, 344
virulence of, 340
Streptococcus salivarius, 349
Streptothrix, 38, 619
cultivation of, 621
morphology of, 619, 621
Sublethal doses of virulent bacteria in
active immunization, 195
Sugar-free broth, 125
Sulphates in nutrition of bacteria, 29
Sulphur bacteria, 60
physiology of, 61
spectroscopic examination of, 61
varieties of, 60

Sulphur dioxid as disinfectant, 86
Sulphuretted hydrogen. See Hydrogen
sulphid

Suprarenal cytotoxin, 201
Swine-plague bacillus, 553

differentiation of, from hog-cholera
bacillus, 554

immunization against, 553
morphology of, 553
pathogenicity of, 553
Symbiosis of bacteria, 31
Symptomatic anthrax, bacillus of. See
Anthrax, symptomatic
Syphilis, 593
in monkeys, 602
in rabbits, 602

microorganism of. See Spirochseta
pallida

Tanning of hides, bacteria in, 716
Temperature, attained by application
of various degrees of pressure, 72
effect of, on activity of bacteria, 15
high, 34
low, 34

relation of, to bacteria, 31
maximum, 32
minimum, 32

Temperature, relation of, to bacteria,
optimum, 32

to cultures with spores, 33
to vegetative forms, 33
“Tetanolysin,” 205, 464
“Tetanospasmin,” 463
Tetanus antitoxin, 220
production of, 220
horses used in, 221
technique of, 221
toxin for, 220
standardization of, 222
unit of (Society of American Bacteri¬
ologists), 222
Tetanus bacillus, 456

autopsy findings in infections of, 460
biological characteristics of, 458
cultivation of, 458
distribution of, 457
early observation of, 456
favorable conditions for growth of, 459
incubation of, 460
isolation of, by Kitasato, 456
morphology of, 456
pathogenicity of, 459
following wounds, 460
relation of spores to, 459
resistance of, 459
staining of, 457
toxin of, 460

central nervous system attacked by,
463

mode of reaching, 463
incubation period of, 463
isolation of, 461

by chemical reaction, 462
by filtration, 461
by precipitation, 461
production of, 461
resistance of, 462
strength of, 462

susceptibility of animals to, 462
Tetanus spores, transportation of, to
organs, 460

Thermal death points, 34
Thermo-regulators for incubators, 159
Lautenschlager’s, 158, 159
Reichert, 158, 159

762

INDEX OF SUBJECTS

Thiothrix, 38
Thrush, 640

microorganism causing, 640
Timothy, bacillus of, 502
Tissue sections, method of staining, 111
Gram-Weigert, 111
in celloidin sections, 111
in paraffin sections, 111
staining of bacteria in, 110
Titration of culture media, 117
color indicator in, 117
process of, 117

for alkaline media, 118
reaction of, 117
adjustment of, 119
Tobacco industry, bacteria in, 715
Torulse, 617

Toxin, constitution of (Ehrlich), 210
graphic form of (Ehrlich), 211
views of Arrhenius and Madsen on,
212

diphtheria. See under Diphtheria
toxin

endotoxin distinguished from, 186
epitoxoid in, 208

in side-chain theory, cell-nutrition in,
213

chemical action of, 213
elements of, 213
atom group, 213
side chains or receptors, 213
over-production of receptors in, 214
molecule of, haptophore group in,
207

toxophore group in, 207
partial absorption of, 209
standardization of, 207
Limes death in, 208
Limes zero in, 207
time changes in, 206
toxoid form of, 207
protoxoids in, 209
syntoxoids in, 209

toxon and, difference in action of, 209
toxon in, 209

used for production of diphtheria
antitoxin, 216

valency of antitoxin for, 210

Toxin-antitoxin reaction, 203
side-chain theory in, 212
summary of, 215
theories as to process of, 203

by destruction of toxin by its
specific antitoxin, 203
by direct union of toxin and anti¬
toxin, 203

through mediation of tissue cells,
203

time element in, 204
Toxin solution, normal, 205
Toxin unit, 205
Toxins, 185

compared with pigments, 186, 309
summary of, 305

Toxoid form of diphtheria toxin, 207
Toxoids, varieties of, 209
epitoxoid form in, 208
protoxoids, 209
syntoxoids, 209

Toxon, in diphtheria toxin, 209

toxin and, difference in action of, 209
Toxon molecule, 209

haptophore group in, 209
toxophore group in, 209
Toxophore group in toxin molecule, 207
in toxon molecule, 209
Trachoma, hemoglobinophilic bacilli in,
541

Trichomycetes. See Chlamydobacteri-
acese

Trichophyton tonsurans, 642
cultivation of, 644
demonstration of, 643
morphology of, 643
occurrence of, 642
Tricresol, 78
Trillat autoclave, 89
Tubercle bacillus, 479
bacilli related to, 498
Bacillus butyricus, 502
bacillus of avian tuberculosis, 500
cultivation of, 500
discovery of, 500
morphology and staining of, 500
susceptibility of animals to, 500
bacillus of bovine tuberculosis, 498

INDEX OF SUBJECTS

763

Tubercle bacillus, bacilli related to,
bacillus of bovine tuberculosis,
early investigation of, 498
cultivation of, 499
differentiation of, from human
type, 499

morphology of, 499
bacillus of fish tuberculosis, 495
bacillus of leprosy, 502, 509
bacillus of timothy, 502
bacillus of turtle tuberculosis, 501
Bacillus smegmatis, 502
biological considerations of, 485
chemical analysis of, 490
cultivation of, 483

“Nahrstoff Hey den” in, 485
media for, 484
discovery of, 7
early investigation of, 479
examination for, by animal inocula¬
tion, 175

by Ziehl-Neelson staining method,
176

in circulating blood, 489
in feces, 178
in milk, 709
isolation of, 483

leprosy bacillus and, differentiation
between, by stains, 106
methods of staining, 104, 105, 106
in sections, 112
celloidin, 112
paraffin, 112
morphology of, 479
Much granules, 482
pathogenicity of, 486
frequency in, 486
mode of infection in, 487
mortality in, 486

preparation of, for agglutination test,
251

quantitative analysis of, 22
smegma bacillus and, differentiation
between, by stains, 106
staining of, 480

differentiation of, from acid-fast
group by Pappenheim’s method
of, 482

Tubercle bacillus, staining of, Ehrlich’s
anilin-water-gentian- violet solu¬
tion in, 481

Gabbet’s decoloration and coun-
terstaining in, 481

Ziehl’s carbol-fuchsin solution in, 481
toxins of, 490

endotoxins in, 490
tuberculins in, 490

bouillon filtre (Denys), 492
“new tuberculin-bacillary emul¬
sion” (Koch), 492
“new tuberculin” (Koch), 491
original method of making of,
491

present method of making of,
491

“old tuberculin” (Koch), 491
1 1 tuberculoplasmin ’ ’ (Buchner

and Hahn), 492

use of antiformin in examination for,
483

Tuberculin. See under Tubercle bacillus,
toxins of

“Tuberculoplasmin” (Buchner and
Hahn), 492

Tuberculosis, frequency of, 486
immunization in, passive, 497
Maragliano’s serum in, 498
Marmorek’s serum in, 498
human and bovine types of bacilli in,
in infections of man, 488
mode of infection in, 487
mortality of, 486

tuberculin in, diagnostic use of, 493
cutaneous reaction in, 494
in cattle, 495

ophthalmo reaction in, 494
subcutaneous injection of, 493
dosage and reaction in, 493
therapeutic uses of, 496
original, 496
present, 497
dosage in, 497

preparations employed in, 497

Tubing of culture media, 121

Typhoid bacillus. See under Typhoid
fever

764

NDEX OF SUBJECTS

Typhoid carrier state in rabbits, 404
Typhoid fever, bacillus of, 399
bacteriemia in, 405
biological conditions favorable to,
403

cultivation of, 399
differentiation of, from Bacillus
fecalis alkaligenes, 427
from- meat-poisoning and para¬
typhoid bacilli, 428
discovery of, 7, 399
immunization against. See under
Typhoid fever, immunization in
in blood during disease, 405
obtaining cultures of, 405
in feces, 177
in gall-bladder, 411
in rose spots, 412
in sputum, 412
in stools, 406

examination in, 406
isolation of, 407

on Conradi-Drigalski medium,
408

on Eisner’s potato-extract gel¬
atin, 407

on Endo’s fuchsin-agar, 409
on Hiss’ agar-gelatin media, 407
on Loeffler’s malachite-green
media, 409

time of appearance in, 406
in urine, 411
in water, 694

inoculation of animals with, 404
with endotoxin of, 417
isolation of, 403
morphology of, 399
pathogenicity of, 404
in animals, 404
in man, 404
staining of, 339

suppurative lesions due to, 412
toxic products of, 415
obtaining of, 417
varieties of :

endotoxins, 415 '
true toxins, 416
typhoplasmin, 416

Typhoid fever, diagnosis in, by agglutin¬
ins in blood serum, 420
Widal test in, 421

obtaining blood for, 422
by bactericidal substances in blood
serum, 419

by bactericidal tests in vivo , 258
by opsonic index, 424
epidemics of, traced to milk, 703
hygienic considerations in, 413
immunization in, 417

by inoculation with typhoid bacilli,
417

active, 424

technique of Pfeiffer and Kolle
in, 425

of Wright in, 425
substances found in blood after,
418

agglutinins in, 419
chief or major, 420
group, 420
bactericidal, 419
bacteriolytic, 418
opsonins in, 424
precipitins in, 423
obtaining blood cultures in, 180
prophylactic measures in, 414
prophylactic vaccination in, 426
specific therapy in, 424
transmission of, 413, 414
by flies, 415
from milk, 414
from oysters, 415
from water supply, 414
without intestinal lesions, 413
“ Typhoplasmin,” 416
Typhus fever, bacillus of Plotz, 678

bacillus of Ricketts and Wilder,
677

distribution of, 677

identity of, with Brill’s disease, 677

inoculation of animals with, 677

Urine, bacteriological examination of,
176

Urobacillus liquefaciens, 455
Uschinsky’s proteid-free medium, 126

INDEX OF SUBJECTS

765

Vaccine production, for immunization
in smallpox, 660
animals used in, 660
calves used for, 660

cleanliness observed in stabling of,
660

material used for vaccination of, 661
vaccination in, 661

preparation of field in, 661
scarifications in, 661
vaccinia vesicles developed in, 661
obtaining of vaccine from, 662
by curettage, 662
by ivory tips, 662

testing of vaccine in, for bacteria, 663
for efficiency, 662
Vaccine therapy of Wright, 286
dosage for, 288
opsonic curve in, 288
production of vaccines in, 286
standardization of emulsion in, 287
enumeration of bacteria against red
blood cells in, 287-8
sterilization of vaccine in, 288
Van Ermengem, discovery of Bacillus
botulinus by, 475

Van Ermengem’ s method of staining
flagella, 101

Variola. See Smallpox.

Vegetative forms from bacterial spores, 17
“Vibrion septique.” See Malignant
edema, bacillus of
Vincent’s angina, 610
spirochaete of, 611
cultivation of, 613
fusiform variety of, 612

bacilli of other diseases resem¬
bling, 613. See also under Fu¬
siform bacilli

other bacilli accompanying, 613
spirillum variety of, 67o
symptoms of, 610
Vincent’s spirilla, staining of, 108
Virulence, definition of, 183
variations in, and infectiousness, 183
Virulent bacteria, sublethal doses of, in
immunization, 195

Virus fixe, in specific therapy of rabies, 652

Wadsworth’s method of staining cap¬
sules, 99

Wassermann test for diagnosis of syph¬
ilis, 262

antigen for, 262

determination of necessary quantity
of, 264

obtaining of, from alcoholic ex¬
tracts of syphilitic organs, 263
from alcoholic solution of normal
organs, 263

from salt solution of syphilitic
liver, 263

of syphilitic spleen, 262
preparation of, by Noguchi meth¬
od, 264

complement in, 266
hemolytic serum in, 265
obtaining of, 265
potency of, 265
quantity of, 265
unit in, definition of, 265
determination of, 266
modifications of, 268
Bauer’s, 268
Noguchi’s, 270

performed with Spirochaeta pallida
antigen, 604
preparation for, 262
serum to be tested for syphilitic anti¬
body in, 267
sheep corpuscles in, 267
technique of, 267
Water, bacteria in, 689
in ground waters, 691
in perennial springs, 692
in wells, 691
in rain and snow, 690
in surface waters, 690
influence of rain on, 690
light and temperature factors in
purification of, 691
self-purification in, 690
pathogenic, 689
of cholera, 689
of diarrheal diseases, 689
of typhoid fever, 689
qualitative analysis of, 694

766 *

INDEX OF SUBJECTS

Water, bacteria in, qualitative analysis of,
isolation of cholera vibrio in, 696
Koch’s method of, 696
isolation of colon bacillus in, 695
isolation of typhoid bacillus in, 695
Adami’s and Chapin’s method
of, 696

Drigalski’s method of, 695
Parietti’s method of, 695
Vallet’s method of, 696
quantitative estimations of, 692
collecting of specimens for, 692
colon bacilli in, 698
colon test in, 697

counting of bacilli in, 698
counting in, 694
incubation of specimens in, 694
plating of specimens in, 693
value of, 694
in bacterial cell, 21

Welch, discovery of Bacillus aerogenes
capsulatus by, 471

Welch’s method of staining capsules, 98
Welch’s modification of Guarnieri’s
medium, 129

Wertheim’s medium for cultivation of
gonococcus, 381-2

Winckel’s disease in the newborn due
to colon bacillus, 394
Wires used in transferring bacteria, 141
Wolff hiigel counting plate, 162
Wool-sorter’s disease, 573
Wright, method of, of anaerobic cultiva¬
tion of bacteria, 150
modification by, of Buchner’s pyrogal-
lic method of cultivation of anaerobic
bacteria, 153

theory of opsonins of, 282
vaccine therapy of, See Vaccine
therapy of Wright

Xerosis bacillus, 525

Yaws, 603

Yeast cells, cultivation of, 633
demonstration of, 632
morphology of, 629
reproduction in, by budding, 629
by spore formation, 630

Yeasts, 629

differentiation of, from other microor¬
ganisms, 629
fermentation by, 630
industrial employment of, for fermen¬
tative purposes, 52
infection of, in animals, 632
in man, 631

clinical picture of, 632
pathogenic varieties of, 633
Yellow fever, 668

clinical picture of, 668
distribution of, 668
etiology of, 668
immunity in, 674

investigation of, by Guiteras and Mar-
choux, Salimbeni and Simond,
673

results of, 673

by Reed, Carroll, Agramonte, and
Lazear, 670
results of, 673

microorganism of, biological proper¬
ties of, 669
research for, 668

by Cornil and Babes, 669
by Sanarelli, 669
by Sternberg, 669
Stegomyia fasciata in, 673
description of, 673
power of transmission of infection
by, reasons for, 674
tropical countries most favorable
for, 674

transmission of, 670
by mosquitoes, 670

discovery of, by Finlay, 670
investigation and confirmation of,
by United States Commission,

•a-

Yersin, discovery of plague bacillus by,
555

Zur Nedden’s bacillus, 547
cultivation of, 547
morphology and staining of, 547
pathogenicity of, 547
Zymase, 51, 630

(5)

TEXT-BOOK

BACTERIOLOGY

Hiss

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3 1 197 21096 6468

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