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MEDICAL

Gift of
Hahnemann Medical College

A TEXT-BOOK

BACTERIOLOGY

GEORGE M. STERNBERG, M.D., LL.D.

SURGEOX-GEXERAL U. S. ARMY

EX-PRESIDENT OF THE AMERICAN MEDICAL ASSOCIATION AND OF THE AMERICAN PUBLIC HEALTH

ASSOCIATION; HONORARY MEMBER OF THE EPIDEMIOLOGICAL SOCIETY OF LONDON, OP

THE ROYAL ACADEMY OF MEDICINE OF ROME, OF THE ACADEMY OF MEDICINE OF

RIO DE JANEIRO, OF THE SOCI^Tfi FRAN9AISE D'HYGIENE, ETC., ETC.

ILLUSTRATED BY HELIOTYPE AND CHROMO-LITHOGRAPHIC PLATES
AND TWO HUNDRED ENGRAVINGS.

Seconb IRevteet) Edition

NEW YOEK
WILLIAM WOOD AND COMPANY

MDCCCCI

1901.

PRESS OF

THE PUBLISHERS' PRINTING COM°ANV

32-34 LAFAYETTE PLACE

NEW YORK

• -V

• • • I

PREFACE.

writer's Manual of Bacteriology, published in 1892, has been
very favorably received both in this country and abroad, but
its usefulness has no doubt been to some extent restricted by the
size and expense of the volume. The following is an extract from
the preface of the Manual :

"A Manual of Bacteriology, therefore, which fairly represents the
present state of knowledge, will consist largely of a statement of facts
established by experimental data, and cannot fail to be of value to
physicians and to advanced students of bacteriology as a work of
reference. The present volume is an attempt to supply such a man-
ual, and at the same time a text-book of bacteriology for students
and guide for laboratory work. That portion of the book which is
printed in large type will, it is hoped, be found to give an accurate
and sufficiently extended account of the most important pathogenic
bacteria, and of bacteriological technology, to serve as a text-book for
medical students and others interested in this department of science.
The descriptions of non-pathogenic bacteria, and of the less important
or imperfectly described species of pathogenic bacteria, are given in
smaller type."

For the benefit of students of medicine and others who do not care
especially for the detailed descriptions of non-pathogenic bacteria and
the extensive bibliography contained in the Manual, this TEXT-BOOK
OF BACTERIOLOGY is now published. It comprises that portion of
the Manual above referred to as printed in large type, revised to in-
clude all important additions to our knowledge of the pathogenic
bacteria since the original date of publication.

1896.

2.01*78

PREFACE TO SECOND EDITION.

A T the request of the publishers the author has again under>taken

a revision of his Manual of Bacteriology, published in 1892:

This is practically a third edition of that work, although the title

was changed in 1896, and it now appears as a second edition of a

TEXT-BOOK OF BACTERIOLOGY.

Considerable additions have been made to the present edition, in-
cluding a section on " Protective Inoculations in Infectious Diseases,"
and one on the " Bacteria of Plant Diseases." In order that the size
of the work might not be materially increased, descriptions of species
imperfectly described, or of minor importance, have been omitted.
In the Manual of Bacteriology an attempt was made to include all
species or distinct varieties which had been described by competent
bacteriologists up to that date, and to give a very full bibliography of
the subject. It was found to be impracticable to follow this plan in
bringing out a second edition as it would have called for two large
volumes instead of one, and the limited demand for such a work
would probably have made it a losing venture for the publishers.
In the TEXT-BOOK, therefore, the bibliography and the descriptions
of many non-pathogenic species were omitted. The Manual is now.
out of print, and those who have use for a comprehensive work, in
which an attempt has been made to include all species described up
to date of publication, are referred to Migula's " System der Bak-
terien " (Gustav Fischer, Jena, 1900).
WASHINGTON, May 27th, 1901.

TABLE OF CONTEXTS.

PART FIRST.

CLASSIFICATION, MORPHOLOGY, AND GENERAL BACTERIOLOGICAL

TECHNOLOGY.

PAGE

I. HISTORICAL, . .3

II. CLASSIFICATION, . . .10

III. MORPHOLOGY, . .20

IV. STAINING METHODS, .... ... 25

V. CULTURP: MEDIA, . . 37

VI. STERILIZATION OF CULTURE MEDIA, 52

VII. CULTURES IN LIQUID MEDIA, 62

VIII. CULTURES IN SOLID MET>IA, ... ... 69

IX. CULTIVATION OF ANAEROBIC BACTERIA, 80

X. INCUBATING OVENS AND THERMO-REGULATORS, . . .88
XI. EXPERIMENTS UPON ANIMALS, ... ... 96

XII. PHOTOGRAPHING BACTERIA, ... . 103

PART SECOND.
GENERAL BIOLOGICAL CHARACTERS.

I. STRUCTURE, MOTIONS, REPRODUCTION 117

II. CONDITIONS OF GROWTH, 125

III. MODIFICATIONS OF BIOLOGICAL CHARACTERS, .... 129

IV. PRODUCTS OF VITAL ACTIVITY, 133

V. PTOMAINES AND TOXALBUMINS 146

VI. INFLUENCE OF PHYSICAL AGENTS, 153

VII. ANTISEPTICS AND DISINFECTANTS (GENERAL ACCOUNT OF THE

ACTION OF), 164

VIII. ACTION OF GASES AND OF THE HALOID ELEMENTS UPON BAC-
TERIA, 172

IX. ACTION OF ACIDS AND ALKALIES, 180

X. ACTION OF VARIOUS SALTS, 186

XI. ACTION OF COAL-TAR PRODUCTS, ESSENTIAL OILS, ETC., . 197

XII. ACTION OF BLOOD SERUM AND OTHER ORGANIC LIQUIDS, . 208

XIII. PRACTICAL DIRECTIONS FOR DISINFECTION, .... 214

VI TABLE OF CONTENTS.

PART THIRD.
PATHOGENIC BACTERIA.

PAGE

I. MODES OF ACTION, 221

II. CHANNELS OF INFECTION, ..... . 229

III. SUSCEPTIBILITY AND IMMUNITY, 233

IV. PROTECTIVE INOCULATIONS, . ... 272
V. PYOGENIC BACTERIA, . 371

VI. BACTERIA IN CROUPOUS PNEUMONIA, 396

VII. PATHOGENIC MICROCOCCI NOT DESCRIBED IN SECTIONS V.

AND VI., 410

VIII. THE BACILLUS OF ANTHRAX, 422

IX. THE BACILLUS OF TYPHOID FEVER, 431

X. BACTERIA IN DIPHTHERIA, 449

XI. BACTERIA IN INFLUENZA, 463

XII. BACILLI IN CHRONIC INFECTIOUS DISEASES, .... 467

XIII. BACILLI WHICH PRODUCE SEPTICAEMIA IN SUSCEPTIBLE ANI-

MALS, 498

XIV. PATHOGENIC AEROBIC BACILLI NOT DESCRIBED IN PREVIOUS

SECTIONS, 528

XV. BACTERIA IN PLANT DISEASES, . . .... 571

XVI. PATHOGENIC ANAEROBIC BACILLI, . . . . . . 578

XVII. PATHOGENIC SPIRILLA, . 590

PART FOURTH.
SAPROPHYTES.

I. BACTERIA IN THE ATR, 613

II. BACTERIA IN WATER, 626

III. BACTERIA IN THE SOIL, 642

IV. BACTERIA OF THE SURFACE OF THE BODY AND OF EXPOSED

Mucous MEMBRANES, 648

V. BACTERIA OF THE STOMACH AND INTESTINES, .... 658
VI. BACTERIA OF CADAVERS AND OF PUTREFYING MATERIAL FROM

VARIOUS SOURCES, 664

VII. BACTERIA IN ARTICLES OF FOOD, 667

INDEX. *...,.,, . .673

LIST OF ILLUSTRATIONS.

FIG.

PAGE

Staphylococci, . . . ....

ZodgloBa ............

Ascococcus, ... .....

Streptococci, ... . ...

Tetrads, . .

Packets — sarcina, ..........

Involution forms, ........

Chains formed by binary division,

10.

Spirilla, ...... .....

11.

Cladothrix ......

12.

Fla^ella, .

13.

Platinum wire in glass handle,

14.

Flask for drawing off blood serum,

15.

Method of forcing blood serum into test tube,

16.

Suction pipette, . ........

17.

Hot- water funnel, . ... ...

18.

Karlinski's agar filter;

20.

Glass dishes for preserving potato cultures,

21.

Test tube for sterilizing potato

22.

Shape of potato for test-tube culture;

23.

Hot air oven

24.

Koch's steam sterilizer,

25.

Koch's steam sterilizer,

26.

Arnold's steam sterilizer, ... ....

27.

Milncke's steam sterilizer,

28.

Koch's apparatus for coagulating blood serum, ....

29.

Miincke s steam sterilizer and coagulator

30.

Pasteur Chamberlain filter,

31.

Pasteur Chamberlain filter without metal case, ....

32.

Modified Pasteur-Chamberlain filter, ......

33.

Erlenmeyer flask

34.

Flask used by Pasteur, ,

35.

Platinum wire loop, *.

36.

Platinum needle,

37.

Sternberg's bulb,

38.

Fermentation tube, .

39.

Method of making stick culture,

Vlll LIST OF ILLUSTRATIONS.

FIG. PAGE

40. Sloping surface of culture medium, 70

40A. Growth of non-liquefying bacteria in gelatin stick cultures, . . 70

41. Growth of same along line of puncture, ... . . 71

42. Growth of liquefying bacilli, ... .... 72

43. Colonies of bacteria, 73

44. Apparatus for gelatin plates .75

45. Esmarch roll tube, 76

46. (See Fig. 15).

47. Mode of development of a facultative anaerobic bacillus, . . go
4S. Mode of development of strict anaerobic in long stick culture, . . 80

49. Exhausted-air flask for liquid media, ..... . 82

50. Method of displacing air with hydrogen, . . 82

51. Salomonson's tube, . . 82

5'3. Friinkel's method of cultivation, . . .83

53. Sternberg's method of cultivation, .... . 83

54. Sternberg's method of cultivation, ... .... 84

55. Buchner's method of cultivation, ..... 84

56. Hydrogen generator, 85

57. Hydrogen apparatus for plate cultures, ....... 86

58. Incubating oven, . 89

59. Thermo-regulator for gas, . 90

60. Moitessier's pressure regulator, . . . . . . 90

61 . Mica screen for flame, ... . .... 9 1

6.'. Koch's device for cutting off fli MC. . . 91

63. Reichert's thermo regulator. . 91

64. Bohr's thermo-regulator, . ... ... 91

65. Miincke's thermo-regulator ..... . 92

66. Sternberg's thermo-regulator, ......... 92

67. Gas valve for the same, . . . .... 92

68. D'Arsonval's incubating apparatus, ...... 0 93

69. Roux's incubating oven and thermo-regulator, .... 93

70. Roux's thermo-regulator, 94

71. Koch's syringe, 97

72. Sternberg's glass syringe, $7

73. Pringle's photomicrographic apparatus, . . . . . . 108

74. Sternberg's photomicrographic apparatus for gas, ..... HO

75. Spores of bacilli, 121

76. Method of germination of spores, ........ 122

77. Apparatus for cultivating anaerobic bacilli,

78. Bacillus of mouse septicaemia in leucocytes from blood of mouse, . 256

79. Staphylococcus pyogenes aureus, ........ 374

80. Gelatin culture of Staphylococcus pyogenes aureus, .... 375

81. Vertical section through a subcutaneous abscess caused by inoculation

with staphylococci in the rabbit, 377

82. Pus containing streptococci, . 383

83. Streptococcus of erysipelas in nutrient gelatin, 384

84. Section from margin of anerysipelatous inflammation, showing strepto-

cocci in lymph spaces, 385

85. Gouococci 391

86. Gonococcus in gouorrhceal pus, 392

87. Gonorrhoeal conjunctivitis, second day of sickness, '. . 394

LIST OF ILLUSTRATIONS. IX

FIG. PAGE

88. Friedlander's bacillus,

89. Friedlander's bacillus; stick culture in gelatin, . 39?

90. Micrococcus pneumoniae crouposae, .... . 401

91. Micrococcus pneumonias crouposae, . . . 401
9'2. Micrococcus pneumonias crouposae, . . . 401

93. Micrococcus pneumonias crouposae, showing capsule, . . . 402

94. Single colony of Micrococcus pneumonias crouposae upon agar plate, . 403

95. Micrococcus pneumonias crouposae in blood of rabbit inoculated with

sputum, .... 407

96. Micrococcus tetragenus, 411

97. Streptococcus of mastitis in cows, . 417

98. Bacillus anthracis, showing development of long threads in convoluted

bundles, 423

99. Bacillus authracis, showing formation of spores, 424

100. Culture of Bacillus authracis in nutrient gelatin, ..... '425

101. Colonies of Bacillus authracis upon gelatin plates, .... 426

102. Bacillus anthracis in liver of mouse, 428

103. Bacillus authracis in kidney of rabbit, 429

104. Bacillus of typhoid fever; colonies in stained sections of spleen, . . 434

105. Bacillus of typhoid fever; colonies in stained sections of spleen, . . 434

106. Bacillus typhi abdominalis, 437

107. Bacillus typhi abdominalis, ......... 437

108. Bacillus typhi abdominalis, showing flagella, 438

109. Single colony of Bacillus typhi abdominalis in nutrient gelatin, . . 438

110. Bacillus typhi abdominalis; stick culture in nutrient gelatin, . . 439

111. Section through wall of intestine, showing invasion by typhoid bacilli, 443

112. Bacillus diphtherias, 453

113. Colonies of Bacillus diphtherias in nutrient agar, 454

114. Bacillus tuberculosis, 469

115. Bacillus tuberculosis in sputum, 470

116. Section through a tuberculous nodule in the lung of a cow, showing

two giant cells. 472

117. Tubercle bacilli from surface of culture upon blood serum, . . . 475

118. Culture of tubercle bacillus upon glycerin-agar, 477

119. Limited epithelioid-celled tubercle of the iris, 480

120. Section of a recent lepra nodule of the skin, 486

121. Bacillus mallei 489"

122. Section of a glanders nodule, 489

123. Section through a glanders nodule in liver of field mouse, . . . 492

124. Migrating cell containing syphilis bacilli, 495

125. Pus from hard chancre containing syphilis bacilli, .... 495

126. Bacillus of rhinoscleroma in lymphatic vessels of the superficial part of

tumor, 496

127. Bacillus septicaemias haemorrhagicae in blood of a rabbit, . . . 499

128. Bacillus septicaemias haeinorrhagicae; stick culture in nutrient gelatin, . 501

129. ' Bacillus of Schweineseuche, 501

130. Colonies of bacillus of swine plague, 501

131. Bacillus of Schweineseuche in blood of rabbit, 502

132. Bacillus of hog cholera, ... ..-.'« . 504

133. Bacillus of mouse septicaemia in leucocytes from blood of mouse, . . 511

134. Bacillus of rouget 512

X LIST OF ILLUSTRATIONS.

FIG- PAGE

135. Bacillus of mouse septicaemia ; culture in nutrient gelatin, . . 513

136. Bacillus of mouse septicaemia; single colony in nutrient gelatin, . v 513

137. Section of diaphragm of a mouse dead from mouse septicaemia, . , 514

138. Bacillus cavicida Havaniensis, 51g

139. Bacillus crassus sputigenus, , 5^7

140. Proteus hominis cnpsulatus, 5^9

141. Bacillus capsulatus, . 523

142. Bacillus hydrophilus fuscus, 523

143. Culture of Bacillus hydrophilus fuscus in nutrient gelatin, . . , 533

144. Bacillus coli commuuis, 530

145. Bacillus coli communis in nutrient gelatin, .... . 533

146. A portion of the growth shown in Fig. 147, ...... 533

147. Bacillus lactis aerogenes, 535

148. Bacillus acidiformans, 537

149. Culture of Bacillus acidiformans in nutrient gelatin, .... 537

150. Bacillus cuniculicida Havaniensis, 533

151. Colonies of Bacillus cuniculicida Havaniensis, 539

152. Colonies of Bacillus cuniculicida Havaniensis, 539

153. Bacillus pyocyaneus, .......... 543

154. Proteus vulgaris 548

155. " Swarming islands " from a culture of Proteus mirabilis, . . . 553

156. Spiral zooglcea from a culture of Proteus mirabilis, .... 553

157. Bacillus gracilis cadaveris, 559

158. Colonies of Bacillus gracilis cadaveris, 559

159. Tetanus bacillus, 579

160. Tetanus bacillus 579

161. Culture of Bacillus tetani in nutrient gelatin, . . . . » 580
163. Bacillus oedematis maligni, 581

163. Bacillus oedematis maligni, , 581

164. Cultures of Bacillus oedematis maligni in nutrient gelatin, . . . 582

165. Bacillus cadaveris, 584

166. Bacillus cadaveris 584

167. Bacillus of symptomatic anthrax, 585

168. Bacillus of symptomatic anthrax, 585

169. Culture of bacillus of symptomatic anthrax, 586

170. Spirillum Obermeieri, 591

171. Spirillum Obermeieri, 591

172. Spirillum cholerae Asiaticae . 593

173. Spirillum cholerae Asiaticae, 593

174. Colonies of Spirillum cholerae Asiaticae 594

175. Spirillum cholerae Asiaticae, 594

176. Cultures of Spirillum cholerae Asiaticae in nutrient gelatin, . . . 595

177. Spirillum choleras Asiaticae, 595

178. Colonies in nutrient gelatin of Spirillum cholene Asiaticae, Spirillum

tyrogenum, and Spirillum of Finkler and Prior, .... 596

179. Section through mucous membrane of intestine from cholera cadaver, 599

180. Spirillum of Finkler and Prior, 602

181. Colonies of Spirillum of Finkler and Prior, 602

182. Spirillum of Finkler and Prior ; culture in gelatin, .... 602

183. Spirillum tyrogenum, . 603

184. Colonies of Spirillum tyrogenum, . ..... 604

LIST OF ILLUSTRATIONS. XI

FIG. PAGE

185. Spirillum Metsclmikovi, . 604

186. Penicillum ghiucum, ... • 614

187. Miquel's aeroscope, .... . . 615

188. Hesse's aeroscope • 617

189. Miquel's flask, 619

190. Straus and Wurtz's soluble filter, 619

191. Petri's sand filter, '. 620

192. Sugar filter, 621

193. Sedgwick and Tuckers apparatus, 621

194. Sternberg's vacuum tube, . 627

195. Lepsius' apparatus for collecting water at various depths, . . . 628

196. Koch's plate method, ... 629

197. Smear preparation from liver of yellow-fever cadaver, . . 665

198. Bacillus cadaveris grandis, . . 665

-PART FIRST.

CLASSIFICATION, MORPHOLOGY, AND GENERAL
BACTERIOLOGICAL TECHNOLOGY.

I. HISTORICAL. II. CLASSIFICATION. III. MORPHOLOGY. IV. STAINING
METHODS. V. CULTURE MEDIA. VI. STERILIZATION OF CULTURE
MEDIA. VII. CULTURES IN LIQUID MEDIA. VIII. CULTURES
IN SOLID MEDIA. IX. CULTIVATION OF ANAEROBIC BAC-
TERIA. X. INCUBATING OVENS AND THERMO REGU-
LATORS. XI. EXPERIMENTS UPON ANIMALS.
XII. PHOTOGRAPHING BACTERIA.

PART FIRST.

HISTORICAL.

IT is probable that Leeuwenhoeck, " the father of microscopy/'
observed some of the larger species of bacteria in faeces, putrid in-
fusions, etc., which he examined with his magnifying glasses (1675),
but it was nearly a century later before an attempt was made to de-
fine the characters of these minute organisms and to classify them
(0. F. Muller, 1773).

In the absence of any reliable methods for obtaining pure cultures,
it is not surprising that the earlier botanists, in their efforts to classify
microorganisms, fell into serious errors, one of which was to include
under the name of infusoria various motile bacteria. These are now
generally recognized as vegetable organisms, while the Infusoria are
unicellular animal organisms.

Ehrenberg (1838), under the general name of Vibrioniens, de-
scribes four genera of filamentous bacteria, as follows :

1. Bacterium — filaments linear and inflexible ; three species.

2. Vibrio — filaments linear, snake-like, flexible ; nine species.

3. Spirillum — filaments spiral, inflexible ; three species.

4. Spirochcete — filaments spiral, flexible ; one species.

These vibrioniens were described by Ehrenberg as " filiform ani-
mals, distinctly or apparently polygastric, naked, without external
organs, with the body uniform and united in chains or in filiform
series as a result of incomplete division/'

Dujajdin (1841) also placed the vibrioniens of Ehrenberg among
the infusoria, describing them as " filiform animals, extremely slen-
der, without appreciable organization, and without visible locomotive
organs."

Charles Robin (1853) suggested the relationship of Ehrenberg's
vibrioniens with the genus Leptothrix, which belongs to the algae ;
and Davaine (1859) insisted that the vibrioniens are vegetable organ-

4 HISTORICAL.

isms, nearly allied to the algae. His classification will be found
in the " Dictionnaire Eiicyclop. des Sciences Medicales," art. " Bac-
teries" (18G8). This view is also sustained by the German botanist
Colin and is now generally accepted.

Spallaiizani, in 1776, endeavored to show by experiment that the
generally received theory of the spontaneous generation of micro-
organisms in organic liquids was not true. This he did by boiling
putrescible liquids in carefully sealed flasks. The experiment was
not always successful, but in a certain number of instances the
liquids were sterilized and remained unchanged for an indefinite
period. The objection was raised to these experiments that the oxy-
gen of the air was excluded by hermetically sealing the flasks, and
it was claimed, in accordance with the views of Gay-Lussac, that
free admission of this gas was essential for the development of fer-
mentation.

This objection was met by Franz Schulze (1836), who admitted air
to boiled putrescible liquids by drawing it through strong sulphuric
acid, in which suspended microorganisms were destroyed. He thus
demonstrated that boiled solutions, which, when exposed to the air
without any precautions, quickly fell into putrefaction, remained un-
changed when freely supplied with air which had been passed through
an agent capable of quickly destroying all living organisms con-
tained in it.

Schwann (1839) demonstrated the same fact by another method.
Air was freely admitted to his boiled liquids through a tube which
was heated to a point which insured the destruction of suspended
microorganisms. The same author is entitled to the credit of hav-
ing first clearly stated the essential relation of the yeast plant—
SaccJiaromyces cerevisice — to the process of fermentation in saccha-
rine liquids, which results in the formation of alcohol and carbonic
acid.

Helmholtz, in 1843, repeated the experiments of Schwann with
calcined air, and arrived at similar results — i. e. , he found that the
free admission of calcined air to boiled organic infusions did not pro-
duce fermentation of any kind.

It was objected to these experiments that the air, having been
subjected to a high temperature, had perhaps undergone some chem-
ical change which prevented it from inaugurating processes of fer-
mentation.

This objection was met by Schroder and Von Dusch (1854) by a
very simple device which has since proved to be of inestimable value
in bacteriological researches. These observers showed that a loose
plug of cotton, through which free communication with the external
air is maintained, excludes all suspended microorganisms, and that

HISTORICAL. 5

air passed through such a filter does not cause the fermentation of
boiled organic liquids.

The experiments of Pasteur and of Hoffman, made a few years
later, showed that even without a cotton filter, when the neck of the
flask containing the boiled liquid is long drawn out and turned down-
ward, the contents may be preserved indefinitely without change.
In this case suspended particles do not reach the interior of the flask,
as there is no current of air to carry them upward through its long-
drawn-out neck, and they are prevented by the force of gravity from
ascending.

Tyndall showed at a later date that in a closed chamber, in which
the air is not disturbed by currents, all suspended particles settle to
the floor of the chamber, leaving the air optically pure, as is proved
by passing a beam of light through such a chamber.

Notwithstanding the fact that the experimenters mentioned had
succeeded in keeping boiled organic liquids sterile in flasks to which
the oxygen of the air had free access, the question of the possibility
of spontaneous generation — heterogetiesis — still remained unsettled,
inasmuch as occasionally a development of bacterial organisms did
occur in such boiled liquids.

This fact was explained by Pasteur (1860), who showed that the
generally received idea that the temperature of boiling water must
destroy all living organisms was a mistaken one, and that, especially
in alkaline liquids, a higher temperature was required to insure ster-
ilization. His experiments showed that a temperature of 110° to
112° C. (230° to 233.6° F.), which he obtained by boiling under a
pressure of one and a half atmospheres, was sufficient in every case.
These experiments, which have been repeated by numerous investi-
gators since, settled the spontaneous-generation controversy ; and it
is now generally admitted that no development of microorganisms
occurs in organic liquids, and no processes of putrefaction or fermen-
tation occur in such liquids, when they are completely sterilized and
guarded against the entrance of living germs from without.

Pasteur at a later date (1865) showed that the atmospheric or-
ganisms which resist the boiling temperature are in fact reproduc-
tive bodies, or spores, which he described under the name of " corpus-
cles ovoides "or " corpuscles brill ants." Spores had been previously
seen by Perty (1852) and Robin (1853), but it was not until 1876 that
the development of these reproductive bodies was studied with care
by Cohn and by Koch. The last-named observer determined the
conditions under which spores are formed by the anthrax bacillus.
Five years later (1881) Koch published his valuable researches relat-
ing to the resisting power of anthrax spores to heat and to chemical
agents.

6 HISTORICAL.

The development of our knowledge relating to the bacteria,
stimulated by the controversy relating to spontaneous generation
and by the demonstration that various processes of fermentation
and putrefaction are due to microorganisms of this class, has
depended largely upon improvements in methods of research.
Among the most important points in the development of bacterio-
logical technique we may mention, first, the use of a cotton air
filter (Schroder and Von Dusch, 1854) ; second, the sterilization of
culture fluids by heat (methods perfected by Pasteur, Koch, and
others) ; third, the use of the aniline dyes as staining agents (first
recommended by Weigert in 1877) ; fourth, the introduction of
solid culture media, and the ' ' plate method " for obtaining pure cul-
tures, by Koch in 1881.

The various improvements in methods of research, and espe-
cially the introduction of solid culture media and Koch's "plate
method" for isolating bacteria from mixed cultures, have placed
bacteriology upon a scientific basis, and have shown that many of
the observations and inferences of the earlier investigators were
erroneous owing to the imperfection of the methods employed.

Since it has been demonstrated that certain infectious diseases of
man and the lower animals are due to organisms of this class, phy-
sicians have been especially interested in bacteriological researches,
and the progress made during the past fifteen years has been largely
due to their investigations. It was a distinguished French physi-
cian, Davaine, who first demonstrated the etiological relation of a
microorganism of this class to a specific infectious disease. The an-
thrax bacillus had been seen in the blood of animals dying from this
disease by Pollender in 1849 and by Davaine in 1850, but it was sev-
eral years later (18G3) before the last-named observer claimed to
have demonstrated by inoculation experiments the causal relation of
the bacillus to the disease in question.

The experiments of Davaine were not generally accepted as con-
clusive, because in inoculating an animal with blood containing the
bacillus, from an infected animal which had succumbed to the
disease, the living microorganism was associated with material
from the body of the diseased animal. This objection was subse-
quently removed by the experiments of Pasteur, Koch, and many
others with pure cultures of the bacillus, which were shown to have
the same pathogenic effects as had been obtained in inoculation ex-
periments with the blood of an infected animal.

The next demonstration of the causal relation of a parasitic mi-
croorganism to an infectious malady was made by'Pasteur, who de-
voted several years to the study of an infectious disease of silkworms
which threatened to destroy the silk industry of France— pebrine.

HISTORICAL. 7

In 1873 Obermeier, a German physician, announced the discov-
ery, in the blood of patients suffering from relapsing fever, of a mi-
nute, spiral, actively motile microorganism — the Spirochcete Ober-
meier i — which is now generally recognized as the specific infectious
agent in this disease.

The very important work of Koch upon traumatic infectious
diseases was published in 1878.

In 1879 Hansen reported the discovery of bacilli in the cells of
leprous tubercles, and subsequent researches have shown that this
bacillus is constantly associated with leprosy and presumably bears
an etiological relation to the disease.

In the same year (1879) Neisser discovered the " gonococcus " in
gonorrhceal pus.

The bacillus of typhoid fever was first observed by Eberth, and
independently by Koch, in 1880, but it was not until 1884 that Gaff-
ky's important researches relating to this bacillus were published.

In 1880 Pasteur published his memoir upon fowl cholera, and the
same year appeared several important communications from this
pioneer in bacteriological research upon the " attenuation " of the
virus of anthrax and of fowl cholera and upon protective inocula-
tions in these diseases.

In 1880 the present writer discovered a pathogenic micrococcus,
which he subsequently named Micrococcus Pasteuri, and which is
now generally recognized as the usual agent in the production of
acute croupous pneumonia — commonly spoken of as the " diplococ-
cus pneumonise," but described in the present volume under the
name of Micrococcus pneumonice crouposce.

In 1881 several important papers by Koch and his colleagues ap-
peared in the first volume of the " Mittheilungen " published by the
Imperial Board of Health of Germany.

The following year (1882) Koch published his discovery of the
tubercle bacillus.

The same year Pasteur published his researches upon the disease
of swine, known in France as rouget.

The same investigator (Pasteur) also published in 1882 his first
communication upon the subject of rabies.

Another important discovery was made in 1882 by the German
physicians Lofner and Schiitz, viz., that of the bacillus of glan-
ders.

Koch published his discovery of the cholera spirillum — " comma
bacillus "—in 1884.

The same year (1884) Loftier discovered the diphtheria bacillus.

Another important publication during the same year was that of
Rosenbach, who, by the application of Koch's methods, fixed defi-

8 HISTORICAL.

nitely the characters of the various microorganisms found in pus
from acute abscesses, etc.

The tetanus bacillus was discovered in 1884 by Nicolaier, a stu-
dent in the laboratory of Prof. Fliigge, of Gottingen. That this
bacillus is the cause of tetanus in man has been demonstrated by the
subsequent researches of numerous investigators. For an exact knowl-
edge of its biological characters we are especially indebted to Kitasato.

So far as human pathology is concerned, no important pathogenic
microorganism was discovered after the year 1884 until the year 1892.
After numerous unsuccessful researches by competent bacteriologists,
a bacillus was discovered by Pfeiffer, of Berlin, and independently
by Canon, which is believed to be the specific cause of influenza.

In 1894 the distinguished Japanese bacteriologist, Kitasato, dur-
ing a visit to China made for the purpose, discovered the bacillus
of the bubonic plague of the Orient.

Finally, we may refer to the discovery of the antitoxins of diph-
theria and of tetanus as among the most important events in the
history of bacteriology and of scientific medicine. The name of Behr-
ing has the first place in connection with this discovery.

Having briefly passed in review some of the principal events in
the progress of our knowledge in this department of scientific investi-
gation, it will be of interest to students to know something more of
the literature of bacteriology. Important papers have appeared in
medical and scientific journals in all countries, and research work of
value has been done by enthusiastic investigators of nearly every
nation. The brilliant pioneer work done by Pasteur and by Koch has
attracted to them many pupils and has made France and Germany
the leading countries in this line of investigation. The very great
advantages of Koch's methods of research, introduced at the com-
mencement of the last decade, have attracted many students from
various parts of the world to Berlin, and to other cities of Germany
where instruction was to be obtained from some of Koch's earlier
pupils. But to-day bacteriological laboratories have been established
in all parts of the world, and it is no longer necessary to go to Ger-
many to obtain such instruction. The literature of the subject is.
however, largely in the German and French languages. We can
only refer here to such periodicals as are principally devoted to bac-
teriological research work.

The Zeitxchrift fiir Hygiene has been published since 1886, and
contains numerous valuable papers, contributed for the most part bv
the pupils of Koch and of Fliigge, who are the editors of the journal.

The Annales de VInstitut Pasteur is a monthly journal which
has been published since 1888. It is edited by Duclaux, and contains
many important papers and reviews. H- well as the statistics of the

HISTORICAL. 9

Pasteur Institute relating to preventive inoculations against hydro-
phobia.

The Annales de Micrographie is a monthly journal, published in
Paris. The principal editor is Miquel.

The Centralllalt jur Bakteriologie und Parasitenkunde is a
weekly journal which has been published by Gnstav Fischer, of Jena,
since 1887. The editors are Uhlworm, of Cassel; Loffler, at present
professor a,t Greifswald; and Leuckart, professor at Leipzig.

The Journal of Pathology and Bacteriology is published
monthly in Edinburgh and London. It dates from 1892.

A most important work for students of bacteriology is the Jalires-
bertchtot Baumgarten, which has been published since 1885 by Harald
Bruhn, Braunschweig, Germany. This gives a brief abstract of
nearly ever}7 paper of importance relating to the subject which has
been published during the year.

The Journal of Hygiene is a new quarterly, edited by Dr. George
H. F. Nuttall, and published in Cambridge, England. In the first
number (January 1st, 1901) the accomplished editor says: "The
Journal of Hygiene will fulfil a definite purpose by serving as a
focus to English-speaking investigators for work in Physics, Chemis-
try, Physiology, Pathology, Bacteriology, Parasitology, and Epi-
demiology, in relation to Hygiene and Preventive Medicine."

II.

CLASSIFICATION.

THE earlier naturalists — Ehrenberg (1838), Dujardin (1841) —
placed the bacteria among the infusoria; but they are now recog-
nized as vegetable microorganisms, differing essentially from the
infusoria, which are unicellular animal organisms. One of the prin-
cipal points in differentiating animal from vegetable organisms
among the lowest orders of living things is the fact that animal
organisms receive food particles into the interior of the body, assimi-
lating the nutritious portion and subsequently extruding the non-
nutritious residue ; vegetable organisms, on the other hand, are
nourished through the cell wall which encloses their protoplasm, by
organic or inorganic substances held in solution.

Ehrenberg (1838), under the name of vibrioiiiens, established four gen-
era, as follows :

1. Bacterium — filaments linear and inflexible.

2. Vibrio — filaments linear, snake-like, flexible.

3. Spirillum — filaments spiral, inflexible.

4. Spirochcete — filaments spiral, flexible.

Dujardin (1841) united the two genera Spirillum and Spirochcate of
Ehrenberg, and added to the description of the generic characters as fol-
lows:

1. Bacterium — filaments rigid, with a vacillating movement.

2. Vibrio — filaments flexible, with an undulatory movement.

3. Spirillum — filaments spiral, movement rotatory.

It will be seen that this classification leaves 110 place for the motionless
bacilli, such as the anthrax bacillus and many others, and does not include
the spherical bacteria, now called micrococci.

The classification of Davaine (18(38) provides for the motionless, fila-
mentous bacteria, but does not include the micrococci. This author first
insisted that the vibrioniens of Ehrenberg are truly vegetable organisms,
allied to the algae. He makes four genera, as follows:

Filaments straight or bent, L Moving spontane- ] Rigid Bacterium.
but not in a spiral, ously, } Flexible Vibrio.

( Motionless, . Bacteridium.
Filaments spiral, Spirillum.

Following Davaine, the French bacteriologists frequently speak of the
motionless anthrax bacillus as la bacteridie.

Hoffman in 1869 included in his classification the spherical bacteria,
and pointed out the fact that motility could not be taken as a generic char-
acter, as it was not constant in the same species and depended to some ex-
tent upon temperature conditions, etc.

CLASSIFICATION. 11

Having determined that the bacteria are truly vegetable organ-
isms, the attention of botanists has been given to the question as to
what class of vegetable organisms they are most nearly related to.
There are decided differences of opinion in this regard. While Da-
vaine, Rabenhorst, and Cohn insist upon their affinities with the
alga3, Robin, Nageli, and others consider them fungi. One of the
principal characters which distinguish the alga3 from the fungi is
the presence of chlorophyll in the former and its absence in the latter.
Now, the bacteria are destitute of chlorophyll, and in this regard
resemble the fungi; yet in others their affinities with the Palmellacece
and Oscillatoriacece are unmistakable. It is not necessary, how-
ever, that we should consider them as belonging to either of these
classes of the vegetable kingdom. By considering them a distinct
class of unicellular vegetable organisms, under the general name of
bacteria, we may avoid the difficulties into which the botanists have
fallen.

We must refer briefly, however, to the classification, proposed by some
of the leading- German botanists.

Nageli, placing1 the bacteria among1 the lower fungi, which give rise to
the decomposition of organic substances, divides these into three groups:

1. The Mucorini, or mould fungi.

2. The Saccharomycetes, or budding fungi, which produce alcoholic fer-
mentation in saccharine liquids.

3. The Schizomycetes, or fission fungi, which produce putrefactive pro-
cesses, etc.

Cohn, under the iiamo of Schizophytes, has grouped these low vegetable
organisms, whether provided or not with chlorophyll, into two tribes hav-
ing the following characters:

1. GLJEOGENES — cells free or united into glairy families by an intercel-
lular substance.

2. NEMA.TOGEXES — cells disposed in filaments.

In the first tribe he has placed the genera Micrococcus (Hallier), Bacte-
rium (Dujardin), Merismopedia (Meyer), Sarcina (Goodsir),and Ascococcus
(Billroth), with various genera of unicellular algae containing chlorophyll.

In the second tribe we have the genera Bacillus (Cohn), Leptothrix
(Kg.), Vibrio (Ehr.), Spirillum (Ehr.), Spirochcete (Ehr.), Streptococcus
(Billr.), Cladothrix (Cohn), and Streptothrix (Cohn), associated with gen-
era of green filamentous algae.

The German botanist Sachs unites the fungi and the algae into a single
group, the Thallophytes, in which he establishes two parallel series, one in-
cluding those containing chlorophyll, and the other without, as follows:

THALLOPHYTES.

Forms with chlorophyll. Forms without chlorophyll.

Class I. — Protophytes.

A. Cyanophyceae (Oscillatoria- A. Schizomycetes (Bacteria),
ceae, etc.).

B. Palmellaceae. B. Saccharomycetes.

12 CLASSIFICATION.

Zopf , Avho insists upon the polymorphism of these low organisms, divides
the bacteria into four groups:

Genera.
Streptococcus,

1. COCCOCE^: — Up to the pre- Merismopedia,
sent time, only known in the form of j Sarcina,
cocci. Micrococcus,

Ascococcus.

2. BACTERIACEJE. — Have for the ] Bacterium,
most part spherical, rod-like, and j Spirillum,
filamentous forms ; the first (cocci) ( Vibrio,
may be wanting ; the last are not \ Leuconostoc,
different at the two extremities; fila- Bacillus,
ments straight or spiral. Clostridium.

3. LEPTOTRICHE.E.-- Spherical, ] Crmnthri'r
rod-shaped, and filamentous forms; ^ l'Y™
the last show a difference between the

two extremities ; filaments straight | Levtothrix
orspiral ; spore formation not known. J

4. CLADOTRICHE^. -- Spherical, |
rod-shaped, filamentous, and spiral |

forms ; the filamentous form pre- \ Cladothrix.

sents pseudo-branches ; spore forma- |
tion not known.

The main objection to this classification is that it assumes a pleomorph-
ism for the bacteria of the second group — Bact^riaceoe — which has only been
established for a few species, and which appear? not to be general among the
rod shaped and spiral bacteria.

De Bary divides the bacteria into two principal groups, one including
those which form endospores, and the other those which are reproduced by
arthrospores. But our knowledge is yet too imperfect to make this classifi-
cation of value, and the same may be said of Hueppe's recent attempt at
classification, in which the mode of reproduction is a principal feature.

The classification of Baumgarten (1890) appears to us to have
more practical value, and, with slight modifications, we shall adopt
it in the present volume. This author divides the bacteria into two
principal groups, as follows :

GROUP I. Species relatively monomorphous.

GROUP II. Species pleomorphous.

The first group includes the micrococci, the bacilli, and the
spirilla; the second group the spirulina of Hueppe, leptotrichece
(Zopf), and cladotrichece.

The pleomorphous species described by Hauser under the generic
name Proteus are included in the second group among the spirulina.
In the present volume we have described these pleomorphous species
among the bacilli.

The Cocci, in the classification of Baumgarten, constitute a single
genus with the following subgenera : 1, Diplococcus j 2, Strepto-
coccus ; 3, Merismopedia (Zopf) — "Merista" (Hueppe); 4, Sar-
cina (Goodsir) ; 5, Micrococcus (" staphylococci").

The BACILLI are included in a single genus embracing all of

CLASSIFICATION. 13

those species which only form rod-shaped cells, and filaments com-
posed of rod-like segments ; or straight filaments not distinctly seg-
mented, which may be rigid or flexible.

The SPIRILLA are also included in a single genus, embracing all
of those species in which the filaments are spiral in form and the
segments more or less spiral or ''comma-shaped " —filaments either
rigid or flexible.

This simple morphological classification of the monomorphous
group of Baumgarten corresponds with the nomenclature now gene-
rally in use among bacteriologists. We speak of the spherical bac-
teria as cocci or as micrococci, of the rod-shaped bacteria as bacilli,
and of the spiral bacteria as spirilla.

It is true, however, that we are sometimes embarrassed to decide
whether a particular microorganism belongs to one or the other of
these morphological groups or so-called genera. Among the bacilli,
for example, we may have, in the same pure culture, rods of very
different lengths, some being so short that if alone they might be
taken for cocci, while others may have grown out into long fila-
ments. But if we are assured that the culture is pure the presence
of rod forms establishes the diagnosis, and usually the cocci-like
elements, when carefully observed, will be seen to be somewhat
longer in one diameter than in the other. The German bacterio-
logists generally insist upon placing among the bacilli all straight bac-
teria in which, as a rule, one diameter is perceptibly greater than
that transverse to it ; and several species of well-known bacteria
which were formerly classed as micrococci a-re now called bacilli —
e.g., Friedlander's bacillus (" pneumococcus"), Bacillus prodigio&us.

The distinction made by Colin and others between the genus
Bacterium (Duj.) and the genus Bacillus (Colin) cannot be main-
tained, inasmuch as we may have short rods and quite long fila-
ments in the same pure culture of a single species ; and, again, the
character upon which the genus Vibrio (Ehr.) was established —
viz., the fact that the filaments are flexible and the movements
sinuous — is not a sufficient generic or even specific character, for in
a pure culture there may be short rods which are rigid, and long
filaments which are flexible and hjave a sinuous movement. We
therefore to-day speak of all the elongated forms as bacilli, unless
they are spiral and have a corkscrew-like motion, in which case they
are known as spirilla.

f The bacteria are also classified according to their biological char-
acters, and it will be necessary to consider the various modes of
grouping them from different points of view other than that which
relates to their form. This is the more important inasmuch as we
are not able to differentiate species by morphological characters

14 CLASSIFICATION.

alone. Thus, for example, there are among the spherical bacteria, or
micrococci, numerous well-established species which the most expert
microscopist could not differentiate by the use of the microscope
alone ; the same is true of the rod-shaped bacteria. The assump-
tion often made by investigators who are riot sufficiently impressed
with this fact, that two microorganisms from different sources, or
even from the same source, are the same because stained prepara-
tions examined under the microscope look alike, has led to serious
errors and to much confusion. As an example of what is meant we
may refer to the pus organisms. Before the introduction of Koch's
"plate method" micrococci had been observed in the pus of acute
abscesses. Some of these were grouped in chains — streptococci—
and some were single, or in pairs, or in groups of four ; but whether
these were simply different modes of grouping in a single species, or
whether the chain micrococci represented a distinct species, was not
determined with certainty. That there were in fact four or more
distinct species to be found in the pus of acute abscesses was not
suspected until Rosenbach and Passet demonstrated that this is the
case, and showed that not only is the streptococcus a distinct species,
but that among the cocci not associated in chains there are three
species which are to be distinguished from each other by their color
when grown on the surface of a solid culture medium. One of these
has a milk-white color, one is of a lemon-yellow color, while the third
is a golden-yellow.

Those microorganisms which form pigment are called chromo-
genes, or chromogenic ; those which produce fermentations are
spoken of as zymogenes, or zymogenic ; those which give rise to dis-
ease processes in man or the lower animals are denominated patho-
genes, or pathogenic. We cannot, however, classify bacteria under
the three headings chromogenes, zymogenes, and pathogenes, for
some of the chromogenic species are also pathogenic, as are some
of the zymogenes. These characters must therefore be considered
separately as regards each species, and in studying its life history and
distinguishing characters we determine whether it is chromogenic
or non-chromogenic ; whether it produces special fermentations ;
and whether it is or is not pathogenic when inoculated into the
lower animals. In making the distinction between pathogenic
and non-pathogenic microorganisms we must remember that a
certain species may be pathogenic for one animal and not for an-
other. Thus the anthrax bacillus, which is fatal to cattle, sheep,
rabbits, guinea-pigs, and mice, does not kill white rats ; the bacillus
of mouse septicaemia kills house mice, but field mice are fully im-
mune from its pathogenic effects ; on the other hand, the bacillus of
glanders is fatal to field mice but not to house mice.

CLASSIFICATION. 15

Again, it must be remembered that pathogenic power also de-
pends, to a greater or less extent, upon the dose injected into an
animal as compared to its body weight. Some pathogenic organ-
isms in very minute doses give rise to a fatal infectious malady ;
others are only able to overcome the vital resisting power of the
tissues and fluids of the body when introduced into the circulation,
or into the subcutaneous tissue or abdominal cavity, in considerable
amounts. Some pathogenic bacteria invade the blood ; others mul-
tiply only in certain tissues of the body ; and others again multiply
in the intestine and by the formation of poisonous products which
are absorbed show their pathogenic power.

Another classification of the bacteria relates to the environment
favorable to their development. Thus we speak of saprophytic and
parasitic bacteria, or of SAPROPHYTES and PARASITES.

The saprophytes are such as exist independently of a living host,
obtaining their supply of nutriment from dead animal or vegetable
material and from water containing organic and inorganic matters
in solution. The strict parasites, on the other hand, depend upon
a living host, in the body of which they multiply, sometimes without
injury to the animal upon which they depend for their existence, but
frequently as harmful invaders giving rise to acute or chronic infec-
tious diseases. Microorganisms which ordinarily lead a saprophy-
tic existence, but which can also thrive within the body of a living
animal, are called facultative parasites. Thus the leprosy bacillus,
which is only found in leprous tissues, is a strict parasite ; while the
typhoid bacillus, the cholera spirillum, etc. , are facultative parasites,
inasmuch as they are capable of maintaining an independent exist-
ence, for a time at least, external to the bodies of living animals.

It seems probable that the pathogenic organisms which are only
known to us to-day as strict parasites were, at some time in the past,
saprophytes, which gradually became accustomed to a parasitic
mode of existence, and, under the changed conditions of their envi-
ronment, finally lost the power of living in association with other
saprophytes exposed to variations of temperature, etc. The tubercle
bacillus, for example, is known to us only as a parasite which has its
habitat in the lungs, lymphatic glands, etc. , of man and of certain
of the lower animals. But we are able to cultivate it in artificial
media external to the body ; and it is in accord with modern views
relating to the development of species to suppose that at some time
in the past it was able to lead a saprophytic existence. Not to admit
this forces us to the conclusion that, at some time subsequent to the
appearance of man and the lower animals in which it is now found
as a parasite, it was created with its present biological characters,
which restrict it to a parasitic existence in the bodies of these ani-

16 CLASSIFICATION.

mals, and that, consequently, the immense destruction of human life
which has resulted from its parasitic invasion of successive genera-
tions was designed when it was created. The opposite view is sup-
ported by numerous facts which show that these low organisms, like
those higher in the scale, are subject to modifications as a result of
changed conditions of environment, and that such modifications, in
the course of time, may become well-established specific characters.

Again, the bacteria may be grouped into aerobic and anaerobic
species. This is a very important distinction, which was first estab-
lished by Pasteur, who found that certain bacteria will only grow
when freely supplied with oxygen, while others absolutely decline to
grow in the presence of this gas. The latter, which are spoken of as
strict anaerobics, may be cultivated in a vacuum or in an atmo-
sphere of hydrogen. Those species which grow either in the pre-
sence of oxygen or when it is excluded are called facultative an-
aerobics.

Certain bacteria produce a peptonizing ferment which has the
power of liquefying gelatin. This has led to the classification of
those microorganisms of this class which grow in Koch's flesh-pep-
tone-gelatin as liquefying and non-liquefying bacteria.

Again, we speak of them as motile or non-motile.

It is evident that these biological characters, although all-im-
portant in the definition of species, cannot serve us in an attempt to
establish natural genera ; for the lines a?e not sharply drawn between
the saprophytes and the parasites, the aerobics and the anaerobics,
etc. , inasmuch as we have facultative parasites and facultative an-
aerobics which we cannot include in either class, and which yet do
not form a distinct class by themselves. We therefore adhere to the
morphological classification, although this is open to criticism. For
example, among the rod-shaped organisms which we call bacilli and
describe under the generic name Bacillus there are some which
multiply by binary division only, while others form endogenous re-
productive bodies known as spores. Certainly so important a differ-
ence in the mode of reproduction should be sufficient to separate
these rod-shaped organisms into two natural groups or genera.

As heretofore stated, the German bacteriologist Hueppe has at-
tempted a classification based upon the mode of reproduction, in
which he makes two groups, or "tribes/' one in which reproduction
occurs by the formation of endogenous spores — •" endospores " —the
other in which it occurs by the formation of " arthrospores." The
latter group includes all of those bacteria in which no other mode of
multiplication is known than that by binary division, which is com-
mon to all. In the present state of our knowledge this classification
• ' An account of this mode of reproduction is given on page 19.

CLASSIFICATION. 17

is scarcely to be considered of practical value, inasmuch as the ques-
tion of spore formation is still undetermined for a large number of
species.

In the following table we shall give the characters of the dif-
ferent genera which have been described by recent botanists and
bacteriologists, arranged under the three headings, MiCROCOCCi,
BACILLI, SPIRILLA. Where we doubt the propriety of maintaining
a distinct generic name upon the supposed distinguishing characters,
the description will be printed in small type.

MICROCOCCI.

General Characters. — Spherical bacteria which are reproduced
by binary division ; usually without spontaneous movements ; do not
form endogenous spores. (According to some authors, certain cells,
known as arthrospores, may be distinguished by their greater size
and refractive power, and these are supposed to have greater resist-
ance to desiccation than the ordinary cocci resulting from binary
division, and to serve as reproductive bodies. ) Some micrococci are
not precisely round, but are somewhat oval in form ; and when in
process of division the cocci, necessarily, are more or less elongated
in one diameter before a complete separation into two spherical ele-
ments has occurred.

MiCROCOCCUS. — Division in one direction ; cocci single, in pairs,
or accidentally associated in irregular groups ; sometimes held to-
gether in irregular masses by a transparent, glutinous, intercellular
substance. (Micrococci belonging to this genus are frequently de-
scribed as " staphylococci," and Staphylococcus is used by Rosen-
bach as a generic name for the pus cocci described by him, which
are solitary or associated in irregular groups, as above described. )

Ascococcus. — Cocci associated in globular or lobulated, zooglcea
masses by a rather firm intercellular substance.

LEUCOXOSTOC. — Cocci, solitary or in chains, surrounded by a
thick, gelatinous envelope and forming zoogloea of cartilaginous
consistence.

STREPTOCOCCUS. — Division in one direction only ; cocci associ-
ated in chains.

Diplococcus. — Division in one direction only ; cocci associated in pairs.

Association in pairs is common to all of the micrococci, inasmuch as
they multiply by binary division. When such association has rather a per-
manent character, it is customary to speak of the microorganism as a diplo-
coccus, but we doubt the propriety of recognizing- this mode of association
as a generic character.

MERISMOPEDIA. — Division in two directions, forming groups of
four, which remain associated in a single plane — "tetrads."

SARCINA. — Division in three directions, forming packets of eight
2

18 CLASSIFICATION.

or more elements, which remain associated in more or less regular
cubical masses.

BACILLI.

General Characters. — Rod-shaped and filamentous (not spiral)
bacteria in which there is no differentiation between the extremities
of the rods ; reproduction by binary division in a direction trans-
verse to the long axis of the rods, or by binary division and the for-
mation of endogenous spores ; rigid or flexible ; motile or non-motile.

BACILLUS. — Characters as given above.

Bacterium. — This genus, established by Dujardin, is now generally
abandoned, the species formerly included in it being transferred to the genus
Bacillus. As denned by Cohn, the generic characters were : Cells cylindri-
cal or elliptical, free or united in pairs during their division, rarely in
fours, never in chains, sometimes in zoogloea (differing from the zooglcea
of spherical bacteria by a more abundant and firmer intercelluar substance),
having spontaneous movements, oscillatory and very active, especially in
media rich in alimentary material and in presence of oxygen.

Clostridium. — Rod-shaped bacteria which form large, endogenous, and
usually oval spores ; these are centrally located, and during the stage of
spore formation the rods become fusiform.

SPIRILLA.

General Characters. — Curved rods or spiral filaments ; rigid or
flexible ; reproduction by binary division, or by binary division and
the formation of endogenous spores (or by arthrospores ?) ; move-
ments rotatory in the direction of the long axis of the filaments.

SPIRILLUM. — Characters as above.

Spirochcete. — Flexible, spiral filaments; movements rotatory.

Vibrio, — Filaments flexible, straight or sinuous; movements sinuous.

A considerable number of bacteria which are usually seen as short, curved
rods, but which may grow out into long, spiral filaments, are described by
some authors under the generic name Vibrio, e.g., the so-called "comma
bacillus'1 of Koch — " Spirillum cholerae Asiaticae"; the spirillum of Finkler
and Prior — " Vibrio proteus" ; the spirillum described by Gameleia — "Vibrio
Metschnikovi,"etc. These microorganisms have not the characters which
distinguished the genus Vibrio as established by Ehrenberg, and we prefer to
follow Fliigge in describing them under the generic name Spirillum.

The pathogenic bacteria now known belong to one or the other
of the above-described genera, and the attention of bacteriologists
has been given chiefly to the study of micrococci, bacilli, and spirilla.
But the botanists place among the bacteria certain other forms which
are found in water, and which, in a systematic account of this class
of microorganisms, demand brief attention at least. These are in-
cluded in Baumgarten's second group, which includes the pleomor-
phous bacteria.

SPIRULINA (Hueppe). — The vegetative cells are sometimes rod-
shaped and sometimes spiral ; in suitable media they may grow out

CLASSIFICATION. 19

into long, straight, wavy, or spiral filaments. These filaments may
break up into cocci-like reproductive elements — 4< arthrospores."

LEPTOTRICHE.E (Zopf). — The vegetative cells present rod-shaped
and spiral forms, and grow out into straight, wavy, or spiral fila-
ments ; these may show a difference between the two extremities,
of base and apex. Cocci-like reproductive bodies are formed by seg-
mentation of the rod-shaped elements in these filaments. In some
of the species the segments are enclosed in a common sheath. Sub-
genera: LEPTOTHRIX, BEGGIATOA, CREXOTHRIX, PHRAGMIDIO-
THRIX (for generic characters see page 12).

CLADOTRICHE^E (Zopf). — The vegetative cells are rod-shaped
or spiral, and grow out into straight or spiral filaments, which may
present pseudo-ramifications. A single genus, CLADOTHRIX (see
page 12).

The various methods of classification heretofore referred to must
all be considered provisional and unsatisfactory from a scientific
point of view. Thus Hueppe says : " The existence of rigid form
species, which not only the earlier observers, but even Cohn, §chroter,
and Koch assumed, can be upheld no longer. The adaptability of
bacterial forms to changing conditions of nutrition is not so bound-
less as Naegeli and Billroth supposed, but it is considerably greater
than was once held to be compatible with the conception of the ex-
istence of constant species."

A. Fischer has attempted to make use of the presence, number,
and mode of attachment of flagella as a means of classification. No
doubt this character and the presence or absence of spores should re-
ceive consideration in any attempt at a scientific classification of the
bacteria.

III.

MORPHOLOGY.

IN the present chapter we shall give a general account of the
morphology,, modes of grouping, and dimensions of the bacteria.

The standard of measurement used by bacteriologists is the micro-
millimetre, or the one-thousandth part of a millimetre. This is
represented by the Greek letter //. One ^ (micromillimetre) is equal
to about one-twenty-five-thousandth of an English inch.

The spherical bacteria, or micrococci, differ greatly in size, and
also in the mode of grouping when, as a result of binary division,
they remain associated one with another. The smallest may mea-
sure no more than 0. I//, while some of the larger species are from
one to two ^ in diameter. The enormous number of these minute
organisms which may be contained in a small drop of a pure culture
may be easily estimated in a rough way. Compare a single micro-
coccus, for example, with a sphere having a diameter of one-twenty-
fifth of an inch. If our micrococcus is one of the larger sort, having
a diameter of one //, it would take a chain of one thousand to reach
across the diameter of such a sphere, and its mass, as compared
to the larger sphere, would be as 1 to 523,600,000.

The number of cocci in a milligramme of a pure culture of Staphy-
lococcus pyogenes aureus has been estimated by Bujwid, by count-
ing, at 8,000,000,000.

Not only do different species differ in dimensions, but consider-
able differences in size may be recognized in the individual cocci in a
pure culture of the same species. On the other hand, there are
numerous species which so closely resemble each other in size and
mode of association that they cannot be differentiated b}^ a micro-
scopic examination alone, and we must depend upon other characters,
such as color, growth in various culture media, pathogenic power,
etc. , to decide the question of identity or non-identity.

When in active growth the micrococci necessarily depart from a
typical spherical form just before dividing, and under these circum-
stances may be of a short or long oval. When division has taken
place, if the two members of a pair remain associated they are often
more or less flattened at the point of contact (Fig. 1, a).

MORPHOLOGY.

When in a culture the cocci are for the most part associated in
pairs (Fig. 1, d), we speak of the organism as a diplococcus.

The staphylococci are characterized by the fact that, for the most
part, the individual cocci in a culture are solitary (Fig. 1, 6). But,
inasmuch as multiplication occurs by binary division, we also have
pairs and occasionally a group of four — probably from the accidental
apposition of two pairs (Fig. 1, c) ; or they may be associated in grape-

OQO

8°
b

$89

FIG. 1.

like bunches ; and after staining and mounting a preparation we find
the cells associated in irregular groups. This results from the fact
that they are surrounded by a glutinous material which causes them
to adhere to each other (Fig. 1, e). A mass of cocci held together in

FIG. 2.

FIG. 3.

FIG. 4.

this way by a transparent, glutinous, intercellular substance is spoken
of as a zoogloea (Fig. 2). In the genus Ascococcus the intercellar
substance is quite firm and the zooglcea are in the form of spherical
or irregularly lobulated masses surrounded by a resistant envelope of
jelly-like material (Fig, 3).

When, as a result of division in one direction only, the cocci

22 MORPHOLOGY.

remain united in chains (Fig. 4, a), they are described as streptococci,
and are sometimes spoken of as in chaplets or in torula chains. In
such chains we frequently find the evidence of recent division of the
cocci, as shown by the grouping of the elements of the chain into
pairs (Fig. 4, b).

When division occurs habitually in two directions, groups of four
result, which are spoken of as tetrads. This is the distinguishing
character of the genus Merismopedia. In these groups of four the
individual cocci are often flattened at the points of contact, as in
Fig. 5, b. We also find pairs and groups of three in pure cultures of
species belonging to this genus, as shown in Fig. 5, c. In these,
transverse division has not yet occurred in one or in both elements of
a pair. This association of micrococci in tetrads seems to be main-
tained, in some species at least, by the fact that each group of four is
enclosed in a jelly-like capsule. The extent of this capsule differs in
the same species under different circumstances; as a rule, it is most
apparent when a culture has been made in a liquid medium. Some of

CD S

FIG. 5.

FIG

the diplococci have a similar capsule. The jelly-like substance does
not stain well with the aniline colors and is seen as a transparent
halo around the stained cocci. Some authors (Frankel and Pfeiffer)
believe that this capsule is formed by the swelling up of the cell
membrane as a result of the imbibition of water.

When division occurs in three directions packets of eight or
more elements are formed. This mode of association characterizes
the genus Sarcina. The "packet form " is best seen in an un-
stained preparation from a fresh culture, in which a little material
suspended in water is examined under a comparatively low-power
objective — one-sixth (Fig. 6).

Among the bacilli there is room for a wider range of morphologi-
cal characters. They differ not only in dimensions and in modes of
grouping, but in form. The relation of the transverse to the longi-
tudinal diameters affords a great variety of forms, varying from a
short oval element to a slender rod or elongated filament. But it
must be remembered that we may have short rods and long filaments
in a pure culture of the same bacillus— the typhoid bacillus, for

MORPHOLOGY.

example. There are also considerable differences in the transverse
diameter of bacilli belonging to the same species when cultivated in
different media, or even . in the same medium, although, as a rule,
the transverse diameter is tolerably uniform in pure cultures.

Again, the form of the extremities of the rods is to be observed
(Fig. 7). This may be square, or the corners may be slightly
rounded, or the extremities may be quite round or lance-oval, or
the outlines of the rod may be spindle-shaped from the formation of

Fio.

a large central spore — "cloatridium" — or one end may be dilated
from the formation of a large terminal spore.

In old cultures we frequently find irregular forms due to swellings
and constrictions, which probably occur in bacilli which have but
little vitality or are already dead. These are spoken of as involution
forms (Fig. 8).

The bacilli multiply by binary division in a direction transverse
to the longitudinal axis, and, as a result of such binary division, long

FIG. 9.

chains in which the elements remain associated may be formed
(Fig. 9) ; or the rods may be for the most part solitary or united in
pairs. Like the micrococci, the bacilli are sometimes surrounded by
a gelatinous envelope or capsule. They may also be united by a
glutinous material into zoogloea masses.

Bacilli which under certain conditions are seen as short rods
may, under other circumstances, grow out into long filaments, and
these may be associated in bundles or in tangled masses.

The spirilla differ from the bacilli in the form of the rods and fila-

MORPHOLOGY.

ments, which are curved or spiral. The shorter elements in a pure
culture may be simply curved, as in a, Fig. 10, while the spiral form
becomes apparent in those which are longer, and we may have one
or several turns of the spiral (Fig. 10, b). The spiral form may be
but slightly marked (Fig. 10, c), or the turns may be close and deep
as in a corkscrew (Fig. 10, d). Again, the curved filaments may be
short and rigid, or long and flexible (Fig. 10, e).

In the genus Cladothrix, which is placed by botanists among
the bacteria, the filaments appear to branch ; but this branching is
only apparent, and there is no true dichotomous branching in this
class of microorganisms. The false branching of Cladothrix
dichotoma, Cohn, is shown in Fig. 11. The fact that some of the
larger species of bacilli and spirilla are provided with slender, whip-
like appendages called flagella has been known for many years, and
it has for some time been suspected that all of the motile organisms

FIG. 10.

FIG. 11.

FIG. 12.

of this class are provided with similar appendages and that these are
organs of locomotion. Recently, by improvements in methods of
staining, Loffler has demonstrated the presence of flagella in many
species in which they had heretofore escaped observation. They are
sometimes single, at the ends of the rods (Fig. 12, a); or there may
be several at the extremity of a single rod (Fig. 12, 6); again, they
are seen in considerable numbers around the periphery of the rod
(Fig. 12, c).

The bacilli and spirilla sometimes contain in the interior of the
cells granules of different kinds. These may appear like little oil
drops or they may be more opaque. In the genus Beggiatoa grains
of sulphur are found in the interior of the cells. Again, we may
find vacuoles in the protoplasm ; or, in stained preparations, deeply
stained granules, which are not spores, may be seen at the extremi-
ties of the rods — end-staining. The morphological characters de-
pending upon the formation of endogenous spores will be referred to
hereafter.

IV.

STAINING METHODS.

THE rapid development of our knowledge with reference to the
minute microorganisms under consideration depends very largely
upon the discovery that they may be stained by various dyes, and es-
pecially by the aniline colors. Weigert (1876) was the first to employ
these colors in studying the bacteria, and Koch at once recognized
the value of the method and made use of it in his researches.

The basic aniline colors are those employed, and among these the
most useful are fuchsin, methylene blue, gentian violet, Bismarck
brown, and vesuvin.

Staining upon the Cover Glass or Slide. — By a "cover-glass
preparation " we mean that material supposed to contain bacteria
has been spread out upon a thin glass cover, dried, and stained for
microscopical examination. A small drop of a liquid culture may, for

FIG. 13.

example, be spread upon a perfectly clean cover glass by means of a
platinum wire held in a glass handle (Fig. 13). Or we may place a
drop of water in the centre of the thin glass cover, and by means of
the same instrument take a little material from a culture made upon
the surface of a solid medium and distribute it through the drop.
In this case we must be careful to take very little of the material, as
the smallest quantity will contain an immense number of bacteria,
and for a satisfactory view of the individual cells it is necessary that
they be well separated from each other, in some parts of the prepa-
ration at least, and not massed together.

Where the object is to make a cabinet preparation for permanent
preservation, special care should be taken to distribute the bacteria
uniformly through the drop of water. The next step consists in eva-
porating the liquid so that the bacteria may remain attached to the
surface of the glass cover. This may be done by simple exposure to
fche air or by the application of gentle heat. When the bacteria are

•>(! STAINING METHODS.

suspended in an albuminous medium it will be necessary, after the
film is dry, to heat the preparation sufficiently to coagulate the albu-
rn an, in order that it may not be washed off in the subsequent stain-
ing process. This is best done, in accordance with Koch's directions
for the preparation of tuberculous sputuzn, by passing the cover
glass, held in slender forceps, rather quickly through the flame of an
alcohol lamp three times in succession. In this operation it must
be remembered that too much heat will destroy the preparation,
while too little will fail to accomplish the object in view — coagu-
lation of the albumen. In passing the cover glass through the
flame the smeared side is to be held upward. The time required
will be about three seconds for passing it three times as directed ;
but this will vary according to the intensity of the flame, and some
little experience is necessary in order to obtain tjie best results.

The operation of " fixing," or coagulating the albumen, may also
be effected by exposure in a dry-air oven, heated to 120° to 130° C.,
for a few minutes (two to ten minutes), as directed by Ehrlich.

Bacteria simply suspended in distilled water adhere very well to
the cover glass when treated as directed, but if they have been taken
from a liquefied gelatin culture the film is very apt to be washed
away during the staining process. This is best avoided by taking as
little as possible of the gelatin medium and suspending the bacteria
to be examined in a drop of water, which dilutes the gelatin and
washes it away from the surface of the cells.

Smear Preparations. — In various infectious diseases bacteria are
found in the blood and tissues of the body, and their presence may
be demonstrated by making what is called a smear preparation. A
little drop of blood may be spread upon the thin glass cover, or it
may be brought in contact with the freshly cut surface of one of the
vascular organs, as the liver or spleen. It is especially desirable that
the material used for such a preparation be small in amount and dis-
tributed evenly in a very thin layer. In Germany it is the custom,
in making smear preparations, to press the material between two glass
covers, which are then separated by sliding them apart, thus leaving
a thin layer upon each. This answers very well, but the writer pre-
fers to spread the material by drawing across the face of the cover
glass the end of a well-ground and polished glass slide. This method
is especially useful for spreading blood in a uniform layer, in which
the corpuscles are evenly distributed and retain their normal form.
A very small drop of blood is placed near one edge of the cover glass,
which is placed upon a smooth surface ; the glass slide is held at a
very acute angle and is gently drawn across the cover glass, without
any pressure.

Most bacteriologists make their preparations upon the cover glass,

STAINING METHODS. 27

as above described, but the writer has for a number of years made
his mounts of bacteria upon the glass slide, and believes that this
method has some advantages for every-day work. The thin glass
covers required when a preparation is to be examined with an im-
mersion objective of high power, are easily broken and often dropped
from the ringers or forceps. When the material to be examined is
spread and dried directly upon the glass slide, the operation is at-
tended with less difficulty and fewer accidents and the results are
quite as good. In this case the slide is held in the fingers during the
various steps in the operation of distributing, drying, and staining,
while the thin glass cover must be held in delicate forceps.

Contact Preparations. — When a dry and clean cover glass is
brought in contact with a colony or surface culture we may often
obtain a very pretty preparation, showing the bacteria in a single
layer, and preserving the arrangement, as regards growth, which
characterizes the species. Similar preparations may sometimes be
obtained from the surface of liquid cultures, when the bacteria grow
upon the surface as a thin film. The cover glass is to be gently
brought into contact with this surface growth, which adheres to it
and is dried and stained by the usual methods.

Staining of the dried film is quickly effected by using an aqueous
solution of one of the aniline colors above mentioned. For general
use the writer prefers a solution of f uchsin, on account of the prompt-
ness of its staining action, and because, in preparations for permanent
preservation, it is not as likely to fade as methyleiie blue or gentian
violet. It is also a better color than blue or violet in case a photo-
micrograph is to be made from the preparation.

It is best to keep on hand saturated alcoholic solutions of the
staining agents named, and to make an aqueous solution whenever
required by the addition of a few drops to a little water in a watch
glass or test tube ; for the aqueous solutions do not keep well on ac-
count of the precipitation of the dye as a fine powder, which ren-
ders the solution opaque. The addition of ten per cent of alcohol
to the aqueous solution will, however, prevent this precipitation ;
but, as a rule, freshly prepared solutions are the best. These should
be filtered before use. We may place a few drops of the filtered
solution upon the dried film on the slide or cover glass, or the thin
cover may be floated upon a little of the solution in a watch glass.
In some cases it is best to use heat to expedite the staining, and this
ma}' be done by holding the slide or the watch glass over the flame
of an alcohol lamp until steam commences to be given off. If the
heating is carried too far the preparation is likely to be spoiled by
the precipitation of the staining agent. As a rule, heating will not
be necessary, and when an aqueous solution of f uchsin (one part to

28 STAINING METHODS.

one hundred of water) is used most bacteria are stained within a
few seconds to a minute. At the end of this time the staining solu-
tion is to be washed away by means of a gentle stream of water, or
by moving the cover glass about in a vessel containing distilled
water.

Decolorization. — It often happens that the albuminous material
associated with the bacteria which we propose to examine is stained
so deeply as to obscure the view of these ; and, generally, we will
obtain more satisfactory preparations by the use of a decolorizing
agent, by which the background is cleared up and the outlines of the
cells more clearly defined. The agents chiefly used for this purpose
are alcohol, diluted acids, and solution of iodine with potassium
iodide (Gram's solution).

Koch recommends a solution containing sixty parts of alcohol to
forty parts of water. The cover glass is to be quickly passed
through this solution two or three times. Some bacteriologists pre-
fer to use absolute alcohol.

Or we may use dilute acetic acid (one-half to one per cent) or
very dilute hydrochloric acid (ten drops to half a litre of water).

For decolorizing preparations containing the tubercle bacillus
strong solutions of the mineral acids are employed (one part of ni-
tric or of sulphuric acid to three parts of water).

Gram's solution contains one part of iodine and two parts of
potassic iodide in three hundred parts of water. Special directions
will be given for the use of these agents when we give an account
of the staining methods most useful for the various pathogenic
organisms.

Double Staining. — After decolorizing the background of albu-
minous material we may again stain this with a contrast stain,
such as eosin or vesuvin. In mounts made from pure cultures,
either liquid or solid, a single stain, for the bacteria only, is all that
we require, and our aim is to have the background as free as possi-
ble from any material which would obscure the view.

After staining, decolorizing, and washing the preparation the
cover glass or slide is again dried by exposure to the air or gentle
heat, and is then ready for the permanent mounting in Canada bal-
sam. If the bacteria have been stained upon the slide, a small drop
of balsam dissolved in xylol is placed in the middle of the prepara-
tion and a clean, thin glass cover applied.

If it is the intention to make the microscopical examination with
an immersion objective of high power, or to make photomicro-
graphs from it, only the thinnest glass covers should be used — one-
two-hundredths of an inch or less.

If the preparation is not intended for permanent preservation,

STAINING METHODS. 29

the examination may be made without drying the surface upon
which the stained bacteria are spread, the water taking the place of
balsam in a permanent mount ; or we may dry the film and use a
drop of cedar oil between the slide and cover.

While simple aqueous solutions of the aniline colors, when
freshly prepared, will promptly stain most bacteria, certain agents
may be added to these which aid in the preservation of the solution,
or which act as mordants, and are useful in special cases.

We shall only give here a few of the standard solutions which
are most frequently employed by experienced bacteriologists :

1. Aniline- Gentian-Violet (Ehrlich).

Saturated alcoholic solution of gentian violet, . . 5 cc.

Aniline water, ....... 100 cc.

2. Aniline- Methyl- Violet (Ehrlich- Weigert).

Saturated alcoholic solution of methyl violet, . .11 cc.
Absolute alcohol, ...... 10 cc.

Aniline water, ....... 100 cc.

Aniline water for the above solutions is prepared by shaking in a
test tube one part of aniline oil with twenty parts of distilled water,
and, after allowing it to stand for a short time, filtering the saturated
aqueous solution through a moistened filter. If the solution is not
perfectly transparent it should be filtered a second time.

3. Carbol-Fuchsin (Ziehl's solution).

Fuchsin, ......... 1 gm.

Alcohol, . . . . . . . 10 cc.

Dissolve and add 100 cc. of a fi\ 3-per-cent solution of carbolic acid.

4. Alkaline Blue Solution (Loffler's solution).

Saturated solution of methyl ene blue, ... 30 cc.

Solution of caustic potash of 1:10,000, . . 100 cc.

These solutions keep better than the simple aqueous solutions,
but after having been kept for a time they are likely to lose their
staining power as a result of the precipitation of the aniline color.

The following special methods of staining cover-glass prepara-
tions will be found useful in certain cases:

Gram's Method. — The dried film upon a slide or cover glass is
stained with an aqueous solution of methyl violet or with aniline-
gentian-violet solution (No. 1); it is then placed in the iodine solution
for a minute or two (iodine one part, potassic iodide two parts, water

30 STAINING METHODS.

three hundred partis) ; then washed in alcohol, dried, and, if for per-
manent preservation, mounted in balsam.

METHODS OF STAINING THE TUBERCLE BACILLUS.— Numerous
methods of staining the tubercle bacillus in sputum dried upon a
cover glass have been proposed, but we shall only give here two or
three of the most approved methods, either one of which may be
relied upon for satisfactory results if carefully followed.

1. The Ehrlich-Weigert Method. — Place in a watch glass a little
of the aniline-methyl-violet solution (No. 2); float upon the surface
of this the cover glass with the dried film downward ; heat over a
small flame until it begins to steam, then allow it to stand for from
two to five minutes ; decolorize in a tray containing one part of nitric
acid to three parts of water — the cover glass, held in forceps, is gently
moved about in the decolorizing solution for a few seconds. It is
then washed off in sixty-per-<5ent alcohol to remove the remaining
blue color — this usually takes but a second or two — and then in water.
For a contrast stain a saturated aqueous solution of vesuvin may be
used, a few drops being left upon the cover glass for five minutes.
The stained preparation is then washed, dried, and mounted in
balsam.

2. The Ziehl-Neelson Method. — Float the cover glass upon the
carbol-f uchsin solution (No. 3) ; heat gently until steam commences
to rise — from three to five minutes' time will usually be sufficient ;
wash off in water, and decolorize in nitric or sulphuric acid, twenty-
five-per-cent solution, then in sixty-per-cent alcohol for a very short
time to remove remaining color from albuminous background; wash
well in water and mount in Canada balsam.

3. Friedldnder's Method. — Spread and dry the sputum upon
the slide ; fix by passing the slide three times through the flame of
an alcohol lamp or Bunsen burner ; place upon the dried film three or
four drops of carbol-fuchsin (No. 3); heat gently over a flame until
steam is given off ; wash in a dish of distilled water ; drain off excess
of water, and add a few drops of the following decolorizing solution :

Acid, nitric, pure, . . . . . 5 cc.

Alcohol (eighty per cent), . . . .to 100 cc.

- — usually the preparation will be decolorized in about half a minute ;
wash in water ; add a few drops of an aqueous solution of methylene
blue as a contrast stain ; allow the stain to act for about five minutes,
without heating ; wash again in water, dry, and mount in balsam,
or for a temporary mount use a drop of cedar oil.

4. Gabbett's Method. — This is a slight modification only of a
very useful method recommended by B. Frankel in 1884. The con-
trast stain is added to the decolorizing solution. After staining with

STAINING METHODS. 31

earbol-fuchsin solution (No. />) the cover glass is placed for one or
two minutes in a solution containing:

Sulphuric acid (t \venty-five-per-ceiit solution), . . 100 cc.

Melhylene blue, ...... 2 gms.

Wash, dry, and mount in cedar oil or balsam.

METHODS OF STAINING SPORES.— When preparations containing
the spores of bacilli are stained by any of the methods above given,
these remain unstained and appear as highly refractive bodies in the
interior of the rods or filaments in which they have been formed, or
scattered about in the field if they have been set free. Owing to
the contrast with the stained protoplasm of the rod or spore-'bearing
filament, they are especially well seen in recent cultures ; while in
older cultures the bacilli often do not stain well, or are entirely dis-
integrated and spores only are to be seen. The discovery was made
at about the same time by Buchner (1884) and by Hueppe that
spores may be stained if they are first exposed to an elevated tem-
perature for some time. This may be accomplished by placing the
slide or cover glass, upon which the spore-containing culture has
been dried, in a hot-air oven at a temperature of 120° C. for an
hour; or a higher temperature (180° C.) may be employed for a
shorter time (fifteen minutes) ; or the cover glass may be passed
through the flame of an alcohol lamp or Bunsen burner eight or ten
times, instead of three times as is customary when the object in
view is simply to coagulate the albumen and fix the film upon the
cover glass. After such treatment the spores may be stained with
an aqueous solution of one of the basic aniline colors — fuchsin,
methyl violet, etc. — but the bacilli no longer take the stain so well.

To obtain satisfactory double-stained preparations, showing
both spores and bacilli, a different method is employed.

The film upon the cover glass is passed through the flame three
times, as heretofore directed ; it is then floated upon aniline-fuchsin
solution in a watch glass, and this is heated to near the boiling point
for an hour — Neisser's method. The aniline-fuchsin solution is
prepared by shaking an excess of aniline oil in a test tube with dis-
tilled water, filtering the saturated solution into a watch glass, and
then adding a few drops of a saturated alcoholic solution of fuchsin.
After this prolonged action of the hot staining fluid the spores of
some bacilli are deeply stained, while others do not take the stain so
well. The cover glass is next washed in water and then placed in
a decolorizing solution containing twenty-five parts of hydrochloric
acid to seventy-five parts of alcohol. This removes the stain from
the bacilli, but, if not allowed to act too long, leaves the spores still
stained. The preparation is next stained in a saturated aqueous

32 STAINING METHODS.

solution of methylene blue; and if the operation has been successfully
carried out the spores will be stained red and the protoplasm of the
bacilli in which they are present will be blue.

Moller has (1891) published the following method of staining
spores :

The cover-glass preparation, dried in the air, is passed three times
through a flame or placed for two minutes in absolute alcohol ; it is
then placed in chloroform for two minutes and washed in water; it
is now immersed in a five-per-cent solution of chromic acid for from
half a minute to two minutes and again thoroughly washed in
water; next a solution of carbol-fuchsin is poured upon it and it
is heated over a flame until it commences to .boil, for sixty seconds ;
the carbol-fuchsin solution is then poured off and the cover glass is
immersed in a five-per-cent solution of sulphuric acid until the
film is decolorized, after which it is again thoroughly washed in
water. It is then placed for thirty seconds in an aqueous solution of
methylene blue or of malachite green, and again washed in water,
after which the preparation should be dried and mounted in balsam.
As a result of this procedure the spores are stained dark red and the
protoplasm of the bacilli blue or green.

Fiocca (1893) claims that better results are obtained by the follow-
ing method :

About twenty cc. of a ten-per-cent ammonia solution is placed in a
watch glass, and from ten to twenty drops of an alkaline solution of
an aniline color is added ; heat is applied until steam commences to be
given off, when the cover glass is placed in the hot solution for from
three to fifteen minutes. The cover glass is then quickly washed in
a twenty-per-cent solution of nitric or sulphuric acid to decolorize;
then it should be thoroughly washed in water, after which it may
be stained with a contrast color by the use of an aqueous solution of
one of the aniline dyes — preferably vesuvin, malachite green, or
safranin.

METHODS OF STAINING FLAGELLA. — Koch first succeeded in de-
monstrating the flagella of certain bacilli and spirilla by staining
them with an aqueous solution of haamatoxylon, and dilute chromic
acid as a mordant. Loftier (1889) has succeeded in demonstrating,
by an improved staining method, the presence of flagella in a consider-
able number of species in which they had not previously been seen,
although generally suspected to be present. His method is as follows :

Loffler\s Method. — The following solution is used as a mordant:

No, 1.

Solution of tannin of twenty per cent, . . .10 cc.

Saturated (cold) solution of ferrous sulphate, . . . 5 cc.

Aqueous or alcoholic solution of fuchsin, . . . 1 cc.

(Or one cubic centimetre alcoholic solution of methyl violet.)

STAINING METHODS. 33

No. 2..

A one-per-cent solution of caustic soda.

No. 3.

A solution of sulphuric acid of such strength that one cubic centimetre
is exactly neutralized by one cubic centimetre of the soda solution.

According to Loffler, solution No. 1 is just right for staining the
flagellum of Spirillum concentricum, but for certain other bacteria it
is necessary to add to this some of No. 2 or of No. 3. Thus, for the
cholera spirillum from half a drop to a drop of the acid solution is
added to sixteen cubic centimetres of No. 1. For the bacillus of
typhoid fever one cubic centimetre of No. 2 is added to sixteen cubic
centimetres of No. 1. Bacillus subtilis requires twenty-eight to
thirty drops of No. 2; the bacillus of malignant cedema thirty-six to
thirty-seven drops, etc.

This method has not been very successful in the hands of other
bacteriologists, and improvements in the technique have been made
since it was first published. Van Ermengem (1893) points out the
fact that a principal condition of success is that the cover glasses shall
be absolutely clean. He boils them in a mixture composed of potas-
sium bichromate, sixty grammes; concentrated sulphuric acid, sixty
grammes ; water, one hundred grammes. After coming from this they
are thoroughly washed in water, then in absolute alcohol, and then
dried in an upright position under a bell-jar. Recent agar cultures
(ten to eighteen hours) are preferred, and the suspension in water
should be very much diluted so that in the cover-glass preparation
the bacteria are well isolated. The cover glass, held between the
fingers, is passed three times through a flame. A drop of the follow-
ing solution is then placed upon it : Osmic acid two-per-cent solution,
one part ; solution of tannin (ten to twenty -five per cent) two parts.
This is allowed to act for about five minutes at a temperature of 50°
to 60° C. — or half an hour at the room temperature. After careful
wrashing with water and alcohol the cover glass is immersed for a
few seconds in a bath containing one-quarter to one-half per cent of
nitrate of silver. Then without washing it is placed for a short
time in the following: Gallic acid, five grammes; tannin, three
grammes; fused potassium acetate, ten grammes; distilled water,
three hundred and fifty grammes. It is then returned to the silver
bath and kept there, with constant movement of the bath, until this
commences to turn black. It is then thoroughly washed in water,
dried, and mounted in balsam.

Pitfield (1895) has devised a much simpler method which, as
modified by Muir, is as follows :

" Prepare the following solutions :
3

34 STAINING METHODS.

A. — THE MORDANT.

Tannic acid, ten-per-cent aqueous solution, filtered, . 10 c.c.

Corrosive sublimate, saturated aqueous solution, . 5 c.c.

Alum, saturated aqueous solution, . . . . 5 c.c.

Carbol-fuchsin solution,1 . . . . . 5 c.c.

''Mix thoroughly. A precipitate forms, which must be allowed to de-
posit, either by centrifugalizing or simply by allowing to stand. Remove
the clear fluid with a pipette and transfer to a clean bottle. The mordant
keeps well for one or two weeks.

B.— THE STAIN.

Alum, saturated watery solution, . . . .10 c.c.

Gentian violet, saturated alcoholic solution, . . 2 c.c.

" The stain should not be more than two or three days old when used. It
may be substituted in the mordant in place of the carbol fuchsin. The film
having been prepared as above described, pour over it as much of the mor-
dant as the cover glass will hold. Heat gently over a flame till steam begins
to rise, allow to steam for about a minute, and then wash well in a stream
of running water for about two minutes. Then dry carefully over the flame,
and when thoroughly dry pour on some of the stain. Heat as before, allow-
ing to steam for about a minute, wash well in water, dry and mount in a
drop of xylol balsam ?' (Muir and Ritchie).

METHODS OF STAINING BACTERIA IN TISSUES. — The solutions re-
commended for staining cover-glass preparations are also used in
staining bacteria in thin sections of the various organs, in which
they are found in certain infectious diseases; but, in general, a
longer time is required to stain sections, and it is best not to hasten
the process by the use of heat. To obtain good thin sections, the
material, cut in small cubes, must bs very thoroughly hardened in
absolute alcohol. The piece selected for cutting may be attached to
a cork by the use of melted glycerin jelly, which is hardened by
placing the cork and attached piece of tissue in alcohol. This an-
swers for well-hardened pieces of liver, kidney, etc. , but the hollow
viscera and tissues of loose structure will require embedding in
paraffin or celloidin. Any well-made sledge microtome will answer
for cutting the sections, if the knife is properly sharpened. The sec-
tions should, of course, be cut under alcohol, and they can scarcely
be too thin when the object is to demonstrate the presence or ab-
sence of bacteria. Very thin sections may be cut dry by embedding
in paraffin having a melting point of 50° C. In this case the knife
is set at a right angle to the material to be cut, and the sections
are spread out upon and attached to the glass slide for staining.

One of the most useful solutions for staining tissues is Loffler's
alkaline solution of methylene blue (No. 4). A freshly-prepared so-

1 Basic fuchsin, 1 part; absolute alcohol, 10 parts; solution of carbolic acid
(1 : 20), 100 parts.

STAINING METHODS. 35

lution will stain sections in four or five minutes. Superfluous color
is removed by immersing the sections in diluted alcohol or in a one-
half -per-cent solution of acetic acid for a few seconds. The sections
are dehydrated in absolute alcohol, cleared up with oil of cedar, and
mounted in a drop of cedar oil for examination, or in balsam if
they are to be preserved.

Grain's method may be used as directed for cover-glass prepara-
tions, the sections being first stained in aniline-gentian-violet solu-
tion (No. 1), then washed in water, or in aniline water as recently
(1892) recommended by Botkin, then decolorized in the iodine solu-
tion (see page 29). The sections when decolorized are again washed
in water, dehydrated in absolute alcohol, cleared in cedar oil, and
mounted in balsam.

Weigert's Method. — This is a modification of Gram's method in
which the sections are dehydrated by the use of aniline oil. The
stained section, after having been washed, is transferred to a clean
glass slide, the excess of water is removed by the use of filtering
paper, and the iodine solution is placed upon it in sufficient quantity
to cover the entire section. When sufficiently decolorized this is re-
moved in the same way. The section is then dehydrated by placing
a few drops of aniline oil upon it, removing this with filtering paper,
and repeating the operation once or twice. The aniline oil must
then be completely removed by the use of xylol, after which the sec-
tion is mounted in balsam.

Kiihne's Method. — The object of this method is to prevent the removal
of the color from stained bacteria in sections during the treatment which
such sections usually receive before they are ready for mounting — i.e.,
during the washing and dehydrating processes usually employed. For
staining, Kiihne prefers a methylene-blue solution prepared as follows:
Methylene blue, 1.5 parts; absolute alcohol, ten par-ts ; triturate in a watch
glass and add gradually one hundred parts of a solution of carbolic acid
containing five parts in one hundred of water. The section is placed in this
solution for about half an hour, then washed in water and decolorized in a
weak solution of hydrochloric acid — ten drops to five hundred grammes of
water. This part of the operation must be conducted very carefully, and
usually thin sections will only require to be dipped in the acid solution for an
instant, after which they must be at once immersed in a solution of lithium
— eight drops of a saturated solution of carbonate of lithium in ten grammes
of water. They are then allowed to remain in a bath of distilled water for
a few minutes, after which they are dipped into absolute alcohol, which
Kiihne colors by the addition of methyl en e blue. The sections are then
placed in aniline oil which contains a little methylene blue in solution,
where they are dehydrated without the color being extracted from the stained
bacteria present. The aniline-oil blue solution is prepared by adding an ex-
cess of dry methylene blue to a small quantity of clarified aniline oil. The
undissolved pigment settles to the bottom, and a few drops of the colored
solution are added to a little aniline oil in a watch glass to make the colored
dehydrating bath. The section is next washed out in pure aniline oil — not
colored — after which every trace of aniline oil is to be removed by the use
of xylol. The section is cleared up in turpentine and mounted in balsam.

36 STAINING METHODS.

Ziehl-Neelson Method, for the tubercle bacillus in tissues.—-
Leave the sections for fifteen minutes in carbol-fuchsin solution
(No. 3) ; decolorize in sulphuric or nitric acid, twenty-five-per-cent
solution; wash in sixty -per-cent alcohol; place in a saturated aque«
ous solution of methylene blue for contrast stain; wash, dehydrate,
and mount in balsam.

The following- method of staining1 sections for the purpose of demon-
strating bacteria present in. the tissues is recommended by Pregl (1891) as a
substitute for the method of Klihne. The results are said to be excellent,
and it is much simpler and more expeditious.

The sections are made from tissues embedded in paraffin, and are attached
to clean glass slides with albumen-glycerin. Or they may be attached to a
cover glass by the following method when not embedded in paraffin : The
sections, completely dehydrated, are taken out of absolute alcohol on a thin
glass cover, upon, which they are extended ; a piece of filter paper is applied
to the side of the cover glass to absorb the alcohol, and before the section is
completely dry a drop of acetoii-celloidin solution is placed upon it by means
of a glass rod. The cover glass is now moved about in the air to promote
rapid evaporation of the alcohol, and is then placed in water. The section
now remains attached to the cover glass during subsequent manipulations.
The aceto)i-celloidin solution referred to is prepared by adding celloidin in
small, dry pieces to acetoii until a concentrated solution is obtained. A
large drop of this added to five cubic centimetres of absolute alcohol makes
a suitable solution for use. This must be kept in a glass-stoppered bottle, and
will require to be frequently renewed, as it is not suitable for use after hav-
ing absorbed moisture from the air. The acetoii as obtained from dealers
contains considerable water and must be dehydrated by adding to it red-hot
sulphate of copper.

The sections, attached to a slide or cover glass by one of the methods
mentioned, are stained with Kuhne's carbol-methylene-blue solution, which
is dropped upon them from a pipette. Usually they will be sufficiently
stained at the end of half a minute to a minute, but in some cases a longer
time and the application of heat will be desirable. They are then washed in
water and immediately placed in fifty-per-cent alcohol, where they remain
until the sections have a pale-blue color with a greenish tinge. They are
now completely dehydrated in absolute alcohol and subsequently cleared up
in xylol.

STAINING SECTIONS OF GELATIN STICK CULTURES. — Fischl, Weigert,
and Neisser have given an account of methods for staining stick cultures in
gelatin of non-liquefying bacteria. The object of this is to show the mode
of growth and the association of individual cells in undisturbed cultures.
Neisser gives the following directions : The gelatin cultures are inoculated,
by several punctures, with the microorganism to be studied. When the
development is deemed sufficient the cylinder of gelatin is removed from the
test tube by gently warming its walls. It is then placed for several days-
one to eight, according to its size and thickness — in a one-per-cent solution of
bichromate of potassium. While in this solution it must be exposed to the
light, which causes a change in the gelatin, rendering it insoluble. The
gelatin cylinder is thoroughly washed and then hardened in alcohol, first of
seventy per cent, and then of ninety-six per cent. It is then cut into suit-
able pieces, and these are attached to a cork in the usual manner and placed
for twenty-four hours in absolute alcohol. Thin sections may now be made
with a microtome, and these are attached to a glass slide and stained by
Gram's or Weigert's method or by the use of Loffler's solution (No. 4). The
decolorization should be effected by the use of alcohol and not with an acid
solution. When Gram's method 'is used decolorize by the alternate use of
alcohol and oil of cloves. Clear the preparation with oil of bergamot.

V.
CULTURE MEDIA.

To obtain a satisfactory knowledge of the biological characters
of the different species of bacteria, it is necessary to isolate them in
" pure cultures " and to study their growth in various culture media.
By a pure culture we mean a cultivation containing a single species
only ; and to be absolutely sure that we have a pure culture it is
desirable that all of the bacteria in a culture shall be the progeny of
a single cell. The methods of obtaining pure cultures will be given
later. At present we propose to give an account of the various cul-
ture media commonly employed by bacteriologists, and the methods
of preparing them for use.

By a natural culture medium we mean one which, as obtained in
nature, contains the necessary pabulum for the development of one
or more species of bacteria. An artificial culture medium is one
which is prepared artificially by adding nutritive material to water.
A sterile medium is one which does not contain any living micro-
organisms. We may obtain natural media in a sterile condition, but
artificial media require sterilization, as they are infallibly contami-
nated with living " germs " from the atmosphere during the process
of preparing them. Sterilization is usually effected by heat. For-
ceps, glass tubes, etc. , may be sterilized by passing them through
the flame of an alcohol lamp or Bunsen burner.

NATURAL CULTURE MEDIA. — The most important natural cul-
ture medium is blood serum, which may be obtained from one of
the lower animals — preferably from oxen or calves. This is to be
collected in a sterilized jar, with every precaution to insure cleanli-
ness, at the moment of slaughtering the animal. Or the blood of a
calf, sheep, or dog may be collected at the laboratory by a carefully
conducted operation, in which the femoral or carotid artery is con-
nected with a sterilized glass tube leading into a sterilized receptacle,
such as a Woulf's bottle, into one neck of which a cotton plug has
been placed to permit the air to escape as the bottle fills with
blood through a tube which is secured in the other neck. When
blood is passed directly from an artery into a sterilized receptacle
the serum will not subsequently require sterilization. The writer is in

MEDIA.

the habit of collecting it in this way, and, after the serum has sepa-
rated, of drawing it off in little flasks having a long neck, as shown
in Fig. 14. The neck of the flask, previously sterilized by heat, is
slipped into the Woulf's bottle beside the cotton plug, the bulb (a)
having been previously gently heated to expand the contained air.
As the heated air cools a partial vacuum is formed and the clear
serum mounts into the little flask. One after another is filled in
this way, and each one is hermetically sealed in the flame of a lamp

-a

Fio. 14.

Fio. 15.

FIG. 18.

as soon as it is withdrawn. The sterile blood serum may be pre-
served indefinitely in this way, and may be used as a liquid culture
medium in the little flask, or it may be transferred to a test tube
and solidified by heat whenever a solid blood-serum medium is re-
quired. The advantage of preserving blood serum and other liquid
media in these little flasks is in the fact that they may be preserved
indefinitely without becoming contaminated or drying up, and that
they are easily transported, while a liquid medium in a test tube
must be kept upright. The contents of one of these flasks are readily

CULTURE MEDIA. 39

transferred to a test tube by breaking off the sealed extremity with
sterile forceps and slipping it past the cotton plug, which must be
partly withdrawn for the purpose. Upon applying gentle heat to
the bulb its contents are forced out into the test tube (Fig. 15).
Blood serum which is collected without these special precautions
will require sterilization by heat, for which directions will be given
later.

To obtain the clear serum from blood collected as above directed,
the jars containing it are set aside in a cool place in order that a firm
clot may form, care being taken not to shake them. After the clot
has formed they may be transported to the laboratory, where they
are placed in an ice box or in a cool cellar for from twenty-four to
forty-eight hours. By this time the serum has separated from the
clot, and it may be transferred to sterilized test tubes by means of a
suction pipatte (Fig. 16), or may be distributed in little flasks as
above directed

31 ilk is largely used as a culture medium, and is especially useful
in studying the biological characters of various microorganisms, as
shown by their causing coagulation of the casein, or otherwise ; or
an acid or alkaline reaction of the liquid ; or peptonization of the
precipitated casein, etc. In the udder of healthy cows milk is quite
sterile, and by proper precautions it may be drawn into sterilized
flasks without any contamination and kept indefinitely without un-
dergoing coagulation or any other change. But in practice it
is easier to sterilize it in test tubes or small flasks by the use of
heat than to obtain it in a sterile condition from the udder of the
cow.

[TrmeJaas been used to some extent as a culture medium, and
many bacteria multiply in it abundantly, although, on account of its
acid reaction, other species fail to grow in it. As contained in the
healthy bladder it is sterile, but the mucous membrane of the mea-
tus urinarius always contains numerous bacteria upon its surface, and
some of these are sure to be carried away with the current when
urine is passed.

A culture fluid which the writer has found extremely useful, in
tropical countries where it is to be obtained, is the transparent fluid
contained in the interior of unripe cocoanuts — called agua coco by
the Spaniards. In countries where the cocoanut is indigenous this
cocoanut water is largely used as a refreshing drink. It contains
about four per cent of glucose in solution, together with some vege-
table albumen and salts. Some microorganisms multiply in it with-
out appropriating the glucose, while others split this up, producing
an abundant evolution of carbon dioxide and giving to the fluid
a very acid reaction. The following are the results of an analysis

40 CULTURE MEDIA.

made for me by Dr. L. L. Van Slyke in the chemical laboratory of
Johns Hopkins University : The weight of the fluid obtained from
six nuts averaged 339.1 grammes. The specific gravity averaged
1.02285. The amount of water averaged 95 percent; the amount
of inorganic ash, 0.618 per cent; the amount of glucose, 3.97 per
cent ; the amount of fat, 0.119 per cent ; the amount of albuminoids,
0.133 percent.

As this fluid is contained in a germ-proof receptacle, no steriliza-
tion is required when it is drawn off with proper precautions in the
little flasks heretofore described.

Hydrocele fluid has been used as a culture medium, and many
bacteria multiply in it abundantly.

Other natural culture media are found in animal and vegetable
substances, which are used, either cooked or raw, as solid sub-
strata upon which bacteria may be cultivated. One of the most use-
ful of these is the potato, which is a favorable medium for the de-
velopment of numerous species, and upon which (cooked) many of
them present characters of growth which are so distinctive as to aid
greatly in the differentiation of species.

Other tubers, roots, or fruits may also be used as solid media, or
their juices extracted and employed as liquid media. Cooked fish
and meats of various kinds are also suitable media for certain spe-
cies— e.g., the phosphorescent bacteria grow very well upon the sur-
face of boiled fish, and in a dark room give off a bright, phosphores-
cent light.

Eggs, sterilized "by boiling, have been used by some bacteriolo-
gists, especially for the cultivation of anaerobic species.

ARTIFICIAL CULTURE MEDIA. — A great variety of liquid media
have been employed by bacteriologists, the most useful of which are
infusions of beef or mutton, with the addition of a little peptone.
But Pasteur has shown that some species of bacteria will grow in a
medium which does not contain any albuminous material, nitrogen
being obtained from salts containing ammonia.

Pasteur's solution, which is rarely used at present, contains :
Distilled water, one hundred parts ; cane sugar, ten parts ; tartrate
of ammonia, one part, with the addition of the ashes from one
gramme of yeast.

Cohn modified this by leaving out the cane sugar, which favors
the development of moulds. These fluids are not, however, in-
tended for general use in the cultivation of bacteria, but to demon-
strate certain facts relating to their physiology.

Infusions of meat, or " flesh water," are made by chopping fine
lean beef or mutton (one pound) and covering it with water (one
litre). This is placed in an ice chest for twenty-four hours, and the

CULTURE MEDIA. 41

aqueous extract is then obtained by filtration through muslin by
pressure. This extract is cooked, filtered, and carefully neutralized
by the addition of a solution of carbonate of sodium, which is added
drop by drop. Usually we add to this one-half per cent of chloride
of sodium. The addition of ten grammes of peptone to a litre of
this meat infusion constitutes the flesh-peptone solution which is
largely used in the preparation of solid culture media, to be described
hereafter.

The addition of five per cent of glycerin to the above infusion
makes a useful liquid medium for the cultivation of the tubercle ba-
cillus (Roux and Xocard). The liquid should be again neutralized
after adding the glycerin, which commonly has an acid reaction.

Dunham's Peptone Solution. — This is used principally for de-
termining whether bacteria under investigation are capable of pro-
ducing indol. One part of pure dried peptone is added to 100 parts
of distilled water, and to this is added one-half per cent of sodium
chloride. The addition of rosalic acid to this solution affords a
means of determining whether bacteria cultivated in it produce an
acid or an alkaline reaction of the medium. The pale rose color im-
parted to the peptone solution by the addition of rosalic acid becomes
more intense when the solution becomes alkaline, and it fades out en-
tirely when it becomes acid. To obtain this reaction add 2 parts
of the following solution to 100 parts of Dunham's peptone solution:
rosalic acid (corralline), 2 parts; alcohol (eighty per cent), 100 parts.

Bouillon is made by cooking the chopped meat — one pound in a
litre of water — for about half an hour in a large glass flask or an
enamelled iron kettle. The filtered bouillon is then carefully neu-
tralized with sodium carbonate, and again boiled for an hour to pre-
cipitate all coagulable albuminoids. It is again filtered and dis-
tributed in test tubes or small flasks, in which it is subsequently
sterilized. For certain pathogenic bacteria a bouillon made from the
flesh of a fowl or of a rabbit is preferable to beef bouillon.

Flesh infusion may also be made from one of the standard beef
extracts, such as Liebig's (five grammes to a litre of water).

Various vegetable infusions may also be used as culture media,
such as yeast water, potato water, infusion of hay, of barley, or of
wheat, of dried fruits, beer wort, etc.

SOLID CULTURE MEDIA.— The introduction of solid culture
media, and especially the use of gelatin and agar-agar, as first
recommended by Koch (1881), for the isolation and differentiation of
species, was a most important advance in bacteriological technology.
We are concerned here only with the composition and preparation
of these media.

Flesh-Pept one-Gelatin. — This is made by adding ten per cent

CULTURE MEDIA.

of the best French gelatin to the flesh-peptone solution above de-
scribed. This is the standard gelatin medium, but more or less
gelatin may be added to serve a special purpose. Thus, in Havana
during the summer months the writer used a medium containing
twenty per cent of gelatin, because when but ten per cent was used
the gelatin was liquefied by the normal temperature of the atmo-
sphere. Ten-per-cent gelatin, of good quality and carefully pre-
pared, will stand a temperature of 20° to 22° C. (68° to 71.6° F.)
without melting. When twenty per cent of gelatin is used the
melting point is about 8° C. higher. It must be remembered that
exposure to a boiling temperature reduces the melting point of gela-
tin. It is therefore desirable to accomplish the operations of cook-

FIG. 17.

ing and sterilizing in as short a time as is practicable. The French
gelatin used comes in thin sheets ; this is broken up and added to
the flesh-peptone solution.

Usually we prepare a litre of nutrient gelatin at one time, and for
this quantity one hundred grammes of gelatin will be required for the
standard preparation (ten per cent). It is well to allow it to soak for
a time in the liquid before applying heat for the purpose of dissolving
it. Then apply gentle heat until it is completely dissolved. The gela-
tin of commerce usually has an acid reaction, and it will be necessary
to carefully neutralize the medium after it has been added. A slightly
alkaline reaction is usually no disadvantage, but certain pathogenic
bacteria will not grow when there is a trace of acid present. The
next step consists in clarifying the nutrient medium. It is allowed

CULTURE MEDIA. 43

to cool to about 50° C., and an egg, previously broken into one
hundred grammes of water, is gradually added while stirring the
liquid with a glass rod. A whole egg is used for a litre of the solu-
tion. Heat is again applied and the solution is kept at the boiling
point for about ten minutes, during which time the egg albumen is
precipitated and carries down with it all insoluble particles, which
without this clarifying process would have interfered with the trans-
parency of the medium, even when carefully filtered. The hot
solution is then filtered. A hot- water funnel (Fig. 17) is usually
employed, as the gelatin solution does not pass through filtering
paper very rapidly, and when cooled to near the point of solidifying
ceases to pass.

The advantages of the gelatin medium are that it is perfectly
transparent, that it is easily melted for making " plates/'" and that
many bacteria exhibit in it special characters of growth by which they
may be differentiated from others which resemble them in form.
The principal disadvantage is the low melting point, which prevents
us from making use of this medium for cultivating bacteria in an in-
cubating oven at a higher temperature than about 22° C. for ten-per-
cent gelatin.

This disadvantage is overcome by using agar-agar instead of
gelatin. This is prepared in Japan and other Eastern countries
from certain species of gelatinous algse. It comes to us in the form
of bundles of dried strips, which form a stiff jelly when dissolved in
water in the proportion of one to two per cent. This jelly remains
solid at a temperature of 40° C. and above. It was first employed
by Hesse, one of Koch's collaborators in the office of the imperial
board of health of Berlin. Koch, who was in search of a trans-
parent jelly which would stand the temperature required for the cul-
tivation of certain pathogenic bacteria (37° to 38° C.), quickly recog-
nized its value and introduced it into general use.

The agar-agar jelly is more difficult to filter than the gelatin
medium, and some skill is required in order to obtain a transparent
solution. It will bear long boiling without losing its quality of
forming a stiff jelly. From ten to twenty grammes are added to a
litre of flesh infusion, or we may make a peptonized agar in accor-
dance with the following formula which is given by Salomonson :
Add to one litre of distilled water five grammes Liebig's extract,
thirty grammes peptone, five grammes cane sugar, fifteen grammes
agar. Cook for an hour, render slightly alkaline, and cool to below
60° C. Clarify and cook again for an hour or more.

Glycerin-cigar is made by adding five per cent of glycerin to
the peptonized agar made by the above formula or by the use of the
flesh-peptone infusion. This is a very favorable medium for the cul-
tivation of the tubercle bacillus — first used by Roux and Nocard.

44 CULTURE MEDIA.

Agar-gelatin, a medium which has recently come into favor and
is said to be very useful, as it resembles gelatin in transparency and
has a considerably higher melting point than ten-per-cent gelatin, is
made by adding fifty grammes of gelatin and 7. 5 grammes of agar
to a litre of flesh-peptone solution. Care should be taken not to cook
this longer than is necessary.

In making all of these agar culture media the main difficulties
encountered result from the difficulty of dissolving the agar and the
slowness with which the solution passes through filtering paper.
These difficulties are best met as follows : Break up the sticks of agar
into small fragments and allow them to soak in cold water for twenty-
four hours. Pour off the water and add the flesh-peptone solution.
Boil for several hours until the agar is completely dissolved. Neu-
tralize by adding gradually a solution of carbonate of soda (or render
slightly alkaline). Filter.

The last operation is the most troublesome, and various plans
have been proposed to avoid the tedious filtration through filtering
paper 'in a hot- water filter. A method which gives satisfactory re-
sults is to place the filter containing the hot agar solution, and the
flask which is to receive the filtrate, in a steam sterilizing apparatus,
where it is left in an atmosphere of streaming steam until the filtra-
tion is completed. Or the solution may be put in a tall jar and left
in the steam sterilizer for several hours until it is clear as a result of
sedimentation. The clear solution is then obtained by decantation.
Or by conducting the operation in a tall cylindrical vessel, and al-
lowing sedimentation to occur in the steam sterilizer and the agar
subsequently to solidify by cooling, the cylinder of jelly may be re-
moved from the jar and the part containing the sediment can be cut
away. The transparent portion is then melted again and distributed
in test tubes for use.

In the present volume we frequently refer to the nutrient medium
made by adding one to two per cent of agar-agar to the standard
flesh-peptone solution as " nutrient agar" or simply as "agar."

The following method of filtering agar has (1890) been proposed
by Karlinsky. It is a modification of the method previously de-
scribed by Jakobi and depends upon the use of pressure.

In Fig. 18, a is a cylindrical vessel of tin, which is closed above by
a perforated rubber cork, through which is passed a glass tube, b.
This is enclosed in a larger tin cylinder, c, which contains water,
which may be kept hot by placing an alcohol lamp under the pro-
jecting arm d. The central cylinder has a tube, e, passing through
the bottom of the hot- water cylinder, and which is provided with a
stopcock for drawing off the filtered solution. Before pouring the
hot agar solution into the cylinder a, a cotton filter about ten centi-

CULTURE MEDIA.

metres thick is placed at the bottom of this cylinder and hot water
is poured upon it while the stopcock of the outlet tube is open. This
washes out the cotton and prepares the filter for the agar solution.
The apparatus is supported upon a tripod, not shown in the figure.
Filtration is said to occur rapidly when the air in the central cylinder
is compressed by means of the hand bellows attached to the tube b.

FIG. 18.

Schultz* Rapid Method of Preparing Sutrient Agar- Agar. —
Place one thousand five hundred cubic centimetres of water in an en-
amelled iron pot; add eighteen grammes of agar-agar, broken in small
pieces, and place upon a gas stove; boil for half an hour; add while
boiling two grammes of Liebig's extract of beef ; remove from fire and
cool to 60° C. ; then add ten grammes of dry peptone, five grammes
of sodium chloride, and the contents of one egg beaten up in a
sufficient quantity of water to supply that lost by evaporation; neut-
ralize the mixture by the addition of dilute hydrochloric acid; boil
again for five or ten minutes; filter through white filter paper. If
the filtrate is not entirely clear add to it the albumen of a second
egg and boil until this is coagulated \ then filter again. Always mois-
ten the filter ivitJi water before filtering solutions containing
gelatin or agar-agar. When the process is completed the amount
of filtered culture medium should be about one thousand cubic centi-
metres.

4t> Ct'LTl'KE MEDIA.

According to Abbott the filtration of agar-agar does not require
the use of a hot-water funnel or any other device for maintaining the
temperature of the mass. He gives the following directions for its
preparation :

" Prepare the bouillon in the usual way. Agar-agar reacts neutral or
very slightly alkaline, so that the bouillon may be neutralized before the
agar-agar is added. Then add finely chopped or powdered agar-agar in the
proportion of one to 1.5 per cent. Place the mixture in a porcelain-lined iron
vessel, and on one side of the vessel make a mark at the height at which the
level of the fluid stands. If a litre of medium is being made, add about two
hundred and fifty to three hundred cubic centimetres more water, and allow
the mass to boil slowly, occasionally stirring, over a free flame, from one and
a half to two hours; or until the excess of water — i.e., the two hundred and
fifty or three hundred cubic centimetres that were added — has evaporated.
Care must be taken that the liquid does not boil over the sides of the vessel.
From time to time observe if the fluid has fallen below its original level ; if
it has, add water until its volume of one litre is restored. At the end of the
time given remove the tlameand place the vessel containing the mixture in a
large dish of cold water ; stir the agar-agar continuously until it has cooled
to about 68 to 70 C., and then add the white of one egg which has been
beaten up in about fifty cubic centimetres of water; or the ordinary dried al-
bumeiiof commerce maybe dissolved in cold water in the proportion of about
ten per cent and used — the results are equally as good as when eggs are em-
ployed. Mix this carefully throughout the agar-agar and allow the mass to
boil slowly for about another half-hour, observing a 11 the while the level of the
fluid, which should not fall below the litre mark. It is necessary to reduce the
temperature of the mass to the point given, 68" to 70 C. ; otherwise the co-
agulation of the albumen will occur suddenly in lumps and masses as soon
as it is added, and its clearing action will not be uniform. The process of
clarification with the egg is purely mechanical ; the fine particles, which
would otherwise pass through the pores of the filter, being taken up by the
albumen as it coagulates and being retained in the coagula. At the end of
one- half hour the boiling mass may be easily and quickly filtered through a
heavy-folded paper filter at the room temperature."

For special purposes various substances are added to the above-
described solid and liquid media. A favorable addition for the
growth of a considerable number of bacteria is from one to three per
cant of glucose. The phosphorescent bacteria grow best in a minlmm
containing two to three per cent of sodium chloride. The addition
of three to four per cent of itotitxxinin- n it rate is made in conducting
experiments designed to test the reducing power of certain bacteria,
by which this salt is decomposed with the production of nitrites.
Acids are also added in various proportion to test the ability of
bacteria under investigation to grow in an acid medium. From
1 : 2,000 to 1 : 500 of hydrochloric acid may be used for this purpose.
The addition of litmus to milk or other culture media is fre-
quently resorted to for the purpose of ascertaining whether acids or
alkalies are developed during the growth of bacteria under investi-
gation. The addition of an if in c culorx which are variously changed
by the products of growth of certain spiv'u's has also been resorttnl
to in the differentiation of species. Various disinfecting agents, such
1 Abbott's "Principles of Bacteriology " Fifth edition, pp. 100 and 101.

CULTURE MEDIA. 47

as carbolic acid, etc., have also been used for the same purpose, and
it has been shown by experiment that some bacteria will grow in a
medium containing such agents in a proportion which would entirely
restrain the development of others.

The soluble silicates which form a jelly-like mass have been
proposed as a culture medium for certain bacteria which do not grow
in the usual media. Kuhne (1890), Winogradsky (1891), and Sles-
kin (181)1) have made experiments which indicate that this medium
has considerable value.

Winogradsky uses in the preparation of his silicate jelly the
following salts :

Ammonium sulphate, .... 0.4 gramme.

Magnesium sulphate, .... 0.05

Potassium phosphate, .... 0.1

Calcium chloridf, .... a trace.

Sodium carbonate, . . . 0.6 to 0.9 gramme.

Distilled water, . . . . . 100 grammes.

To this he adds a solution of silicic acid. According to Kiihne, a
solution containing 3.4 per cent of silicic acid and having a specific
gravity of 1.02 may be preserved in a liquid condition. To this the
salts are added in greater or less amount, according to the consis-
tence desired.

Sleskiii states that a suitable jelly is formed by the addition of
1.15 to 1.45 per cent of the salts, and recommends that concentrated,
sterilized solutions be added to the acid. He dissolves separately, in
as little water as possible, the sulphates, the potassium phosphate
and sodium carbonate, and the calcium chloride.

The use of a culture medium containing an extract from the je-
quirity seeds has been recommended by Kaufmann (1891), who has
found, by experimenting upon various bacteria, that such a medium
is useful in differentiating species.

TliejeqHt'rity solution, which may be used as a liquid medium
or may be employed in the preparation of nutrient gelatin or agar, is
prepared as follows : Ten grammes of jequirity seeds are bruised in
a mortar and the shells removed ; they are then placed in one hun-
dred cubic centimetres of water and cooked for two hours in the steam
sterilizer ; after allowing the infusion to cool it is filtered. The fil-
tered liquid has a pale-yellow color and a neutral or slightly alkaline
reaction. Certain bacteria grow in this solution without producing
any change in its color ; others, which produce an acid reaction,
cause it to be decolorized ; others, which produce an alkaline reac-
tion of the medium, change the color to green.

Lactose Litmus- Agar. — Trfts medium is useful for the detection
of the typhoid bacillus in mixed cultures, e.g., in faeces. It is made
by adding to nutrient agar-agar, having a slightly alkaline reaction,

48 CULTURE MEDIA.

two or three per cent of lactose and enough tincture of litmus to give
the culture medium a pale blue color. Colonies of bacteria growing
in this medium which cause a fermentation of the lactose, with
formation of acid, have a pale pink color, extending to the surround-
ing medium. Colonies which do not give rise to acid production
are pale blue. Thus, colonies of the colon bacillus would be red and
colonies of the typhoid bacillus blue.

Blood-serum Mixture of Loffler. — This consists of three parts
blood serum and one part of neutral meat infusion, containing one per
cent of glucose. It is sterilized and solidified as directed for blood
serum, but a higher temperature is required for coagulation of the
mixture than for plain blood serum.

Cooked Potato. — Schroter first used cooked potato as a culture
medium for certain chromogenic bacteria (1872), and Koch subse-
quently called attention to the great value of potato cultures for
differentiating species. His plan of preparing potatoes is as follows :
Sound potatoes are chosen in which the epidermis is intact. These
are thoroughly washed and scrubbed with a brush to remove all
dirt. The " eyes" and any bruised or discolored spots are removed
with a sharp-pointed knife. They are again thoroughly washed in
water, and are then placed for an hour in a bath containing
mercuric chloride in the proportion of 1 : 500, to thoroughly disinfect
the surface. They are then placed in a steam sterilizer for about
three-quarters of an hour, and after an interval of twenty-four hours
are again steamed for fifteen minutes. It is well to wrap each
potato in tissue paper before placing it in the bichloride bath, and to
leave it in this protecting envelope until it is placed in the glass dish
in which it is preserved from contamination by atmospheric germs
after being inoculated with some particular microorganism. Just
before such inoculation the potato is cut in halves with a sterilized
(by heat) table knife. The bacteria to be cultivated are placed upon
the cut surface and the potato is preserved in a glass dish (Fig. 20).

A more convenient method, and one which secures the potato more
effectually from atmospheric organisms, is to cut a cylinder, about
an inch in diameter, from a sound potato, by means of a tin instru-
ment resembling a cork borer or apple corer. This cylinder is cut
obliquely into two pieces having the form shown in Fig. 22, and
each, piece is placed in a large test tube having a cotton air filter, in
which it is sterilized. This method, first employed by Boltoii, has
been slightly modified by Roux, who recommends that a receptacle
for catching the water which separates during the sterilizing process
be formed by making a constriction around the test tube an inch
above its lower extremity. This is done by the use of a blowpipe.

CULTURE MEDIA.

The cylinder of potato rests upon the constricted portion of the tube,
as shown in Fig. 21.

Sometimes a potato paste is employed. The potatoes are boiled
for an hour and the skins removed, after which they are mashed
with a little sterilized water, placed in suitable plates, and sterilized
by exposure for half an hour on three successive days in the steam
sterilizer. Bread paste may be made in the same way, and is a very
favorable medium for the growth of certain bacteria and also for the
common moulds.

FIG. 20.

FIG. 21.

FIG. 22.

Neutralization of Culture Media. — For ordinary purposes
neutralization of acid culture media is accomplished by the use of a
saturated solution of sodium carbonate, the reaction being tested with
strips of blue and red litmus paper. But for certain investigations
it is essential that a more sensitive and reliable indicator should be
used, and that an exact method of titration be employed. Schultz
(1891) recommends the use of phenolphthalein as an indicator and
titration with a solution of caustic soda (four-per-cent stock solution,
to be diluted to 0.4 per cent for use). One drop of phenolphthalein
solution, containing one gramme to three hundred cubic centimetres

50 CULTURE MEDIA.

of alcohol, should be added to one cubic centimetre of bouillon. The
beginning of an alkaline reaction is indicated by the appearance of a
faint rose color. Fuller (1895), who has made a careful investiga-
tion of this subject, recommends a modification of the method of
Schultz. He gives the following directions in his paper published
in the Journal of the American Public Health Association (Vol.
XX., p. 386):

This indicator is prepared by dissolving- five grammes of commercial
phenolphthalein in one litre of fifty-per-cent alcohol. It is not feasible to use
this indicator 011 strips of paper as the alcohol quickly evaporates, leaving the
powder. The colorless liquid, however, may be added in small quantities to
solutions of an acid or neutral nature without any change of color, but alka-
lies quickly change it to a purple red. This change from 110 color to one of
purple red makes the indicator a very satisfactory one, owing to the ease
with which the eye detects the so-called end- point.

For the determination of the degree of reaction of nutrient media it is
the custom to put five cubic centimetres (practically five drachms) of the solu-
tion into a six-inch porcelain evaporating dish, together with forty-five cubic
centimetres of distilled water. This liquid is boiled for three minutes, after
which is added one cubic centimetre of the phenolphthalein solution. While
the solution is still hot it is quickly titrated against a twentieth normal solu-
tion of caustic alkali.

As already mentioned, Dahmen stated that this indicator is useless, ow-
ing to the presence of carbonates, and of ammonia and its salts. On gen-
eral grounds the point that this indicator is inaccurate under such conditions
is well taken, but so far as its application to nutrient media is concerned it
has been found that by proper precautions these objections may be over-
come.

With regard to the amount of free and combined ammonia present in
ordinary nutrient media at the times when their reaction is determined, it
has been found that it does not exceed 0.003 per cent. Experiments show
that this quantity is less than one- tenth of that necessary to interfere with
the accuracy of the method. It may be added that the reason why no am-
monia is produced by the addition of alkali to the nitrogenous bodies is that
at no time during the preparation is there an appreciable excess of free alkali
present.

The chief point by which the presence of carbon dioxide is obviated is by
the use of caustic soda instead of sodium carbonate for neutralization, as re-
ferred to beyond. It has been learned by actual experiment that the carbon
dioxide is practically all removed by heat during the preparation of the
media and at the time of boiling just prior to the titration. In order to pre-
vent atmospheric absorption of this gas the titration should be made quickly
and in a hot solution.

The remaining precaution concerns the solution against which the nu-
trient media are titrated. All of the usual media react acid to phenolphtha-
lein ; hence the solution in question must be alkaline. Caustic soda serves
the purpose well, and the strength may be conveniently one-twentieth normal,
equal to two grammes per litre. For the sake of prevention of interference
from carbon dioxide in the standard solution as it meets the indicator, it is
well to add a small quantity of calcium hydrate in order to precipitate this
gas as calcium carbonate and allow it to settle out in the reagent bottle.
After this solution has been accurately prepared, care is necessary in order
to keep it of uniform strength and free from carbon dioxide. This is best
done by placing the stock solution bottle on a shelf from which the liquid
may be delivered into the burette by means of a siphon that is connected
tightly with the top of the burette. In the tightly fitting stopper of the bottle

CULTURE MEDIA. 51

are three perforations : one through which the siphon passes, and another
for a U tube filled with concentrated caustic soda in order to absorb the
carbon dioxide from all the air which enters the bottle. The third perfora-
tion is for a by-pass which connects with the siphon just above the top of the
burette and below the cock by which the flow from the bottle is regulated.
The object of this is to provide for the entrance into the burette, as the solu-
tion is allowed to run from it, of air that has passed through the U tube and
has had its carbon dioxide removed.

When the manipulation is carried out uniformly in the manner thus de-
scribed, and with the constant employment of an end-point which has the
same intensity of color, very satisfactory and closely agreeing results may
be obtained by this method.

VI.
STERILIZATION OF CULTURE MEDIA.

A MOST important part of bacteriological technology consists in
the sterilization of the various culture media employed. A sterile
medium is essential for maintaining a pure culture, and we can only
obtain an exact knowledge of the biological characters of a species
by studying its growth in various media, its physiological reactions,
its pathogenic power, etc., independently of all other microorgan-
isms— i. e. , in pure cultures.

We may sterilize a culture medium either by heat or by filtration
through a substance which does not permit bacteria to pass. The
last-mentioned method is useful for certain special purposes ; but, in
general, sterilization of culture media, and of the vessels in which
they are preserved, is effected by heat.

The scientific use of heat as an agent for sterilizing our culture
media depends upon a knowledge of the thermal death-point of the
various microorganisms which are liable to be present in them, and
upon various facts relating to the manner in which heat is applied.
All this has been determined by experiment, and before giving
practical directions for sterilization it will be well to consider the
experimental data upon which our methods are based.

As a rule, bacteria which do not form spores are killed at a com-
paratively low temperature. Thus, in a series of experiments made
by the writer upon the thermal death-point of various pathogenic
organisms, the pus cocci were found to be the most resistant, and all
of these were killed by exposure for ten minutes to a temperature
of 62° C. (143.6° F.). There are several species of bacteria known,
however, which not only are not killed by this temperature, but are
able to grow and multiply at a temperature of 65° to 70° C. (Miquel,
Van Tieghem, Globig). But it is safe to say that exposure to a
boiling temperature for a minute or two will infallibly destroy all
microorganisms in the absence of spores, when they are in a moist
condition or moist heat is used — i.e., when they are directly ex-
posed to the action of boiling water or of steam. The power of dry
heat to destroy microorganisms in a desiccated condition is a differ-
ent matter and will require special consideration.

STERILIZATION OF CULTURE MEDIA. 53

The spores of bacilli have a much greater resisting power, and
the vitality of some of these reproductive bodies, from known spe-
cies, is not destroyed by a boiling temperature maintained for sev-
eral hours. Thus Globig found that the spores of a certain bacillus
from the soil — his " red potato bacillus " —required six hours' exposure
to streaming steam in order to destroy it. Steam under pressure, at
a temperature of 115° C., killed it in half an hour ; at 125° C. in five
minutes. This extreme resisting power is exceptional, however,
and many spores are destroyed in a few minutes by the boiling tem-
perature of water.

In practice we assume that some of the more resistant spores,
which are frequently present in the atmosphere, may have fallen
into our culture material, and to insure its sterilization we subject it
to a temperature which can be depended upon to destroy these ; or
we resort to the method of discontinuous heating. This method
was first employed by Tyndall (1877), and is now in general use in
the bacteriological laboratories of Germany, having been adopted by
Koch and his pupils ; while in France a single sterilization by means
of steam under pressure, securing a higher temperature, is still the
favorite method with many.

In the method by discontinuous heating we subject the culture
material for a short time to the temperature of boiling water, thus
destroying all bacteria in the vegetative stage. After an interval,
usually of twenty-four hours, we repeat the operation for the pur-
pose of destroying those which in the meantime have developed
from spores which may have been present. Again the material is
put aside, and after twenty-four hours it is again heated to the
boiling point. This is usually repeated from three to five times.
The object in view is to kill the growing bacteria which are de-
veloped from spores which were present ; and, as a matter of expe-
rience, we find that this method of sterilization is more reliable than
a single prolonged boiling, unless this be effected at a higher tem-
perature than that of boiling water at the ordinary pressure of the
atmosphere. Discontinuous heating is especially useful for the sterili-
zation of liquids which would be injured by prolonged boiling — as is
the case with solutions of gelatin — or which are coagulated by the
boiling temperature. By means of a water bath, the temperature
of which is regulated automatically, we may conduct the operation
at any desired degree. Thus in sterilizing blood serum we use a
temperature a little below that at which coagulation occurs (about
70° C.).

Test tubes, flasks, and apparatus of various kinds are commonly
sterilized by dry heat in & hot-air oven. This is usually made of
sheet iron, with double walls, and shelves for supporting the articles

54 STERILIZATION OF CULTURE MEDIA.

to be sterilized. The form shown in Fig. 23 is commonly used in
bacteriological laboratories.

It must be remembered that a much higher temperature is re-
quired for the destruction of microorganisms when dry heat is em-
ployed than is the case with moist heat. The experiments of Koch
and Wolffhiigel (1881) show that a temperature of 120° to 128° C.
(348° to 262° F.) is required to destroy the spores of mould fungi, and
micrococci or bacilli in the absence of spores. For the spores of ba-
cilli a temperature of 140° C. (284° F.), maintained for three hours,
was required.

In practice we usually maintain a temperature of about 150° C.

FIG. 23.

(302° F.) for an hour or more; and it is customary to sterilize all
test tubes and flasks, which are to be used as receptacles for culture
media, in the hot-air sterilizer. This procedure could no doubt, how-
ever, be dispensed with in many cases and reliance be placed upon
the sterilization of the flask, together with its contents, in the steam
sterilizer, especially with such culture media as are not injured by
long exposure to a boiling temperature — e. g. , bouillon and agar-agar.
When we propose to cultivate aerobic bacteria, or such as require
oxygen for. their development, a cotton air filter is placed in the
mouth of each test tube and flask before it is sterilized in the hot-air
oven. This is a loose plug of cotton, pushed into the neck of the
flask for an inch or more, and projecting from its mouth for a short
distance. These cotton filters should fill the tube completely and

STERILIZATION OF CULTURE MEDIA.

uniformly, but should not be packed so closely that there is difficulty
is removing them.

Steam Sterilizers. — Steam at the ordinary pressure of the atmo-
sphere has the same temperature as boiling water, and in practice is
preferable to a water bath for several reasons. The form of steam
sterilizer adopted by Koch, after extensive experiments made in col-
laboration with Loffler and Gaff ky, is now generally used in bacte-
riological laboratories. This is shown in Fig. 24. It consists of a
cylindrical vessel of zinc which is covered with a jacket of felt.
The cover, also covered with non-conducting material, has an aper-
ture at the top for the escape of steam. A glass tube, which is in
communication with the interior of the vessel, serves to show the

FIG. 24.

FIG. 25.

height of the water when the apparatus is in use. The bottom of
the cylindrical vessel should be of copper. A Bunsen burner having
three jets will commonly be required to keep the water in ebullition
and the upper part of the steam sterilizer rilled with ' ' live steam,"
which should escape freely from the aperture in the cover to insure
a temperature of 100° C. in the steam chamber. A perforated zinc
or copper shelf in the interior of the cylinder serves to support the
flasks, etc., which are to be sterilized. Usually they are lowered
into the cylinder in a light wire basket, or tin pail with perforated
bottom, of proper diameter to slip easily into the sterilizer.

Fig. 25 is a sectional view of this sterilizer.

The steam sterilizer shown in Fig. 26 ' is an American invention,

1 The Arnold steam sterilizer, manufactured at Rochester, X. Y.

STERILIZATION OF CULTURE MEDIA.

which answers the purpose admirably, and which has the advantage
of getting up steam very quickly and also of using comparatively
little gas.

The use of steam under pressure, by which higher temperatures
are obtained, requires a more expensive apparatus, made on the
principle of Papin's digester. The form manufactured by Muncke
is one of the best. This is shown in Fig. 27. It is provided with a
pressure gauge and a safety valve. A single sterilization in this ap-
paratus, at a temperature of 115° C., for half an hour, will usually

FIG. 26.

FIG. 27.

suffice, and for liquid culture media or for agar-agar this method is
entirely satisfactory ; but a gelatin medium which is exposed to this
temperature loses its property of forming a jelly at 20° to 22° C., and
consequently its value as a solid culture medium. In practice the
simpler form of apparatus in which streaming steam is used will be
found ^to answer every requirement. To insure sterilization with
this it is customary to resort to discontinuous heating, as heretofore
described. The standard flesh-peptone-gelatin medium should, as
a rule, be subjected to a temperature of 100° C. for ten minutes, at
intervals of twenty-four hours, four days in succession. Bouillon,
flesh infusions, and agar-agar jelly may be steamed for an hour at a
time two or three days in succession.

STERILIZATION OF CULTURE MEDIA. 57

It is always advisable to test the sterilization of culture material
before making use of it. This is done by placing it for a few days
in an incubating oven at 30° to 35° 0. If a considerable quantity of
material in test tubes has been prepared at one time, it will be suffi-
cient to put a few tubes in the incubating oven to test sterilization.

Failure to make this test often leads to serious complications in
experimental investigations. A laboratory sometimes becomes in-
fected with resistant spores, which are not all destroyed by the usual
methods of sterilization, and these may not develop until some time
has elapsed after the supposed sterilization.

Sterilization of Blood Serum. — Blood serum which has been
collected in test tubes or small flasks, as heretofore directed, is

FIG. 28.

sterilized in a water bath at 60° C. (140° F.) by the method of dis-
continuous heating. It is usually left in the hot- water bath for
about an hour, and this is repeated, at intervals of twenty-four hours,
for five to seven days. This rather tedious process may be avoided
by collecting the serum in the first instance with proper precautions
to prevent it from becoming contaminated with atmospheric organ-
isms. A special apparatus was devised by Koch for sterilizing blood
serum, but an improvised hot- water bath which is regulated to a
temperature of 60° C. by an automatic thermo-regulator will answer
the purpose. After being sterilized, the serum is solidified by careful
exposure to a temperature of about 68° C., which causes it to CO'
agulate, forming a transparent, jelly-like mass. When coagulated
at a higher temperature it becomes opaque. The time required for
this operation varies from half an hour to an hour, and it is best to
remove the tubes from the receptacle in which they are exposed to

58 STERILIZATION OF CULTURE MEDIA.

heat as soon as the serum is solidified. Koch's apparatus for coagu-
lating blood serum is shown in Fig. 28. It is customary to place the
test tubes in an oblique position, so that a large surface may be ex-
posed upon which to cultivate the tubercle bacillus or whatever
microorganism may be under investigation. A form of apparatus
designed for both sterilizing and coagulating blood serum is shown
in Fig. 29. It is manufactured by Miincke in accordance with the
directions of Hueppe, and special precautions have been taken to se-
cure a uniform temperature in all parts of the air chamber. We

FIG. 2ft.

may remark that since it has been shown by Roux and Nocard that
the tubercle bacillus grows very well in agar-agar jelly to which
five per cent of glycerin has been added, blood serum is not so
largely used as a culture medium in bacteriological laboratories.

Sterilization by Filtration.-*-This method is especially useful
for separating the soluble substances contained in a liquid culture of
bacteria from the living cells. It has been demonstrated that several
of the most important pathogenic bacteria produce toxic substances
during their growth which may cause the death of susceptible ani-
mals independently of the living bacteria ; and this demonstration

STERILIZATION OF CULTURE MEDIA.

has been made either by sterilizing a pure culture by means of heat,
or by separating the bacteria from the culture "liquid by nitration.
Some of these toxic products of bacterial growth are destroyed by a
comparatively low temperature ; the method of sterilization by ni-
tration is therefore very important in researches relating to the
composition and pathogenic power of these soluble products. Pas-
teur, in his earlier experiments, used plaster of Paris as a filter, and

Fig. 30.

subsequently resorted to the use of unglazed porcelain, through
which a liquid may be forced by pressure, but which does not per-
mit of the passage of suspended particles, however small.

As the porcelain filter is the most reliable and convenient for
accomplishing the object in view, we shall not describe other methods
of filtration which have been proposed and successfully used. The
porcelain used is a very fine paste, manufactured at Sevres, which is
moulded into cylinders (bougies) of the form proposed by Chamber-
land and baked at a high temperature.

STERILIZATION OF CULTURE MEDIA.

In Fig. 30 the Pasteur-Chamberland filter is shown as arranged
for the filtration of -water. A is the hollow porcelain cylinder, which
is enclosed in a metal case, D. The metal case is tightly clamped
against a projecting shoulder at the lower part of the porcelain filter,
a ring of rubber being interposed to secure a tight joint. When
water under pressure is admitted to the space E, between the cylin-
der of porcelain and the metal case, it slowly filters through, and,
running down the inner wall of the filter, escapes at B into a recep-
tacle placed to receive it. If we fill the space E with a liquid cul-
ture of bacteria and apply sufficient pressure (one or two atmo-
spheres), a clear filtrate is obtained which is entirely sterile if the
porcelain filter is sound and made of proper material. After the

FIG. 31.

filter has been in use for some time, however, it may permit the pas-
sage "of bacteria, and it will be necessary to subject it to a high tem-
perature for the purpose of destroying all organic matter contained
in the porous porcelain.

We may use the Chamberland filter without a metal case by im-
mersing it in a cylindrical glass vessel containing the liquid to be fil-
tered, as shown in Fig. 31. The porcelain cylinder is connected with
an aspirator bottle, «, and a small Eiienmeyer flask, b, is interposed
to catch the filtrate when it overflows from the interior of the filter.
Of course all the necessary precautions must be taken with reference
to the sterilization of the interior of the bougie, of the flask b, and of
the rubber tube connecting the two.

Another arrangement of the Pasteur-Chamberland filter for labora-
tory purposes is shown in Fig. 32. In this form of apparatus a

STERILIZATION OF CULTURE MEDIA.

receptacle, R, is provided for the liquid to be filtered, and a pump for
compressing air is attached to it by a rubber tube. Instead of this
pump, water pressure may be used indirectly by attaching a strong
bottle to the water supply and allowing it to fill slowly with water,
and at the same time to force out the air through a tube connected
with the filtering apparatus. For this purpose the bottle, having a
capacity of a quart or more, should be provided with a rubber stop-
per through which two short tubes are passed. One of these is con-
nected with the water supply and the other with the filter. Of
course this is only practicable when a water supply with sufficient
pressure is available.

FIG. 32.

As a rule, filtration cannot be substituted with advantage for ster-
ilization by heat in the preparation of culture media. Albuminous
liquids pass through the filter with difficulty, and the process of
sterilization by discontinued heating will usually prove more satis-
factory than filtration, which requires extreme precautions to pre-
vent accidental contamination of the filtered liquid. Moreover, the
filter may change the composition of the medium passed through it
by preventing the passage of colloid and albuminous material in so-
lution. Thus, in an attempt to separate blood corpuscles from the
serum by filtration through a Chamberland filter, the writer obtained
a transparent liquid which did not coagulate by heat — i. e. , the albu-
minous constituents of the serum did not pass through the filter.

VII.
CULTURES IN LIQUID MEDIA.

PRIOR to the introduction of gelatinous media by Koch in 1881,
cultures were made in various organic liquids, and these are still
largely used, being for certain purposes preferable to solid media.
The method of preparing and sterilizing the flesh infusions and
other organic liquids commonly used has already been given. We
are here concerned with the various modes of using these nutritive
liquids in cultivating bacteria.

Flasks and tubes of various forms have been employed by differ-
ent investigators, but the most useful receptacle for liquid as well as
for solid culture media is the ordinary test tube. These are care-
fully cleaned, plugged with a cotton air filter, sterilized in the hot-air
oven at 150° C., and are then ready to receive the filtered liquid.
Usually the tube should not be filled to more than one-third to one-
half of its capacity. Sterilization of the culture liquid is then effected
by placing the tubes in the steam sterilizer for half an hour on three
successive days. Before using, the tubes should be placed for a few
days in an incubating oven at 30° to 35° C. to test the sterilization.
This is especially important with liquid media, for if a single living
spore is present it may give rise to an abundant progeny, which will
be distributed through the liquid in association with the species
which has been planted. In solid cultures, on the contrary, such a
spore would give rise to a colony, which by its locality and characters
of growth would probably be recognized as different from the species
planted, and consequently accidental. This is the great danger in
the use of liquid media ; imperfect sterilization, or accidental contami-
nation by atmospheric germs, may lead the inexperienced student
into serious errors resulting from the assumption that the micro-
organisms present in his cultures are all derived from the seed he
planted.

On the other hand, liquid media are more convenient than solid
when it is the intention to isolate by filtration the soluble products of
bacterial growth; for injection into animals to test pathogenic power;
for experiments on the germicidal or antiseptic power of chemical
agents, etc.

CULTURES IX LIQUID MEDIA. 63

For larger quantities of liquid than can be held in an ordinary
test tube the small flasks with a flat bottom, known as Erlenmeyer
flasks, are very convenient (Fig. 33).

In his earlier researches Pasteur used flasks and tubes of various
forms, which served a useful purpose, but have been displaced in his
laboratory by the simpler form of apparatus shown in Fig. 34.
This is a little flask having a cover which is ground to fit the neck.
This cover is drawn out above into a narrow tube which admits
oxygen to the flask through a cotton air filter. To obtain access
to the interior of the flask for the purpose of introducing bacteria
to start a culture, or to obtain material for microscopical examina-
tion, the cover is detached at the ground joint by a gentle twisting
motion.

There is much less danger that a sterile culture liquid will become

FIG. 33.

FIG. 34.

contaminated during the momentary removal of the cover from
one of these little flasks, or of the cotton plug from a test tube, than
is usually supposed. Abundant laboratory experience demonstrates
that such contamination by bacteria floating in the atmosphere rarely
occurs. The spores of mould fungi are commonly more abundant
in the air, but even these do not very frequently fall into the culture
liquid when the tube is opened to inoculate it with the bacteria it is
proposed to cultivate. This inoculation is best made with a platinum
wire, bent into a loop at the free extremity, and sealed fast into the
end of a glass rod (Fig. 35). This is sterilized in the flame of a
Bunsen burner or alcohol lamp by bringing the platinum wire to a
red heat and passing the end of the glass rod which carries it
through the flame several times. With this instrument we may
transfer a little drop from a culture to the sterile fluid in another

CULTURES IX LIQUID MEDIA.

tube for the purpose of starting a new culture. Or we may start a
pure culture from a drop of blood taken from the veins of an animal
which has been inoculated with anthrax, or any similar infectious
disease in which the blood is invaded by a bacterial parasite.

But if we have not a pure culture to start with our liquid media
do not afford us the means of obtaining one ; and if two or more
bacteria which resemble each other in their morphology are associated
in such a culture we cannot differentiate them, and are likely to infer
that we have a pure culture of a single microorganism when this is
not really the case.

But if we have pure stock to start with we may maintain pure
cultures in liquid media without any special difficulty.

Various characters of groivth, etc., are to be observed in culti-
vating different microorganisms in liquid media. Thus some grow
at the surface in the form of a thin film or membranous layer — " my-
coderma " —while others are distributed uniformly through the liquid,
rendering it opalescent or more or less milky and opaque ; others,
again, form little flocculi which are suspended in the transparent

FIG. 35.

fluid. Usually, when active growth has ceased, the bacteria fall
the bottom of the tube as a more or less abundant, white or colored,
pulverulent or glutinous deposit. In some cases the liquid is colored
with a soluble pigment formed during the growth of the bacteria,
and usually this is formed most abundantly at the surface, where
there is free access of oxygen. The reaction of the medium is often
changed as a result of the growth of bacteria in it. From being neu-
tral it may become decidedly alkaline or acid in its reaction. These
changes may be observed by adding a litmus solution before sterili-
zation of the culture medium, and observing the change of color
when an acid-producing bacterium is under cultivation. The rt
ducing power of bacteria upon various aniline colors may also be
studied ; also their power to break up various organic substances, as
shown by the evolution of gas or other volatile products which
may be collected, or by substances which remain in solution and
can be studied by ordinary chemical methods.

Drop Cultures. — When we desire to study the life history of
microorganism and to witness its development from spores, for ex-
ample, its motions, etc., the method of cultivation in a hanging dro]

CULTURES IX LIQUID MEDIA. 65

of culture fluid, attached to a thin glass cover and suspended over a
circular excavation ground out of a glass slide, is very useful.
Such a drop culture may be left under the microscope and kept
under observation for hours or days.

In making these drop cultures it is necessary to sterilize the glass
slides and thin glass covers by heat, and to take every precaution to
prevent the inoculation of the drop of culture liquid with any other
bacteria than those which are to be studied.

The simplest form of moist chamber for drop cultures consists of
an ordinary glass slide having a concave depression, about fifteen
millimetres in diameter, ground out in its centre. This and the thin
glass cover, having been sterilized by exposure in the hot-air oven at
150° C. for an hour or more, or by passing them through the flame
of an alcohol lamp, are ready for use. The cover glass is held in
sterile forceps, and a little drop of the culture fluid containing the
bacterium to be studied is transferred to its centre by means of the
platinum loop heretofore described. It is best to spread the drop
out as thin as possible, and it may be inoculated, from a pure cul-

FIG. 36.

ture, with a platinum needle (Fig. 36) after it has been placed upon
the cover. This is then inverted over the hollow place in the glass
slide, and it is customary to prevent the entrance of air and attach
the cover by spreading a little vaseline around the margin of the
excavation.

Another form of moist chamber is made by attaching a glass
ring, having parallel, ground surfaces, to the centre of a glass slide
by a suitable cement.

In Ranvier's moist chamber there is a central eminence sur-
rounded by a groove ground into the glass slide, and the drop of
culture fluid is in contact with a polished glass surface below as well
as above. This affords a more satisfactory view under the micro-
scope.

The Author's Culture Method. — In a paper read at the meeting
of the American Association for the Advancement of Science, in
August, 1881, the writer described a method of conducting culture
experiments which he has since used extensively and with very satis-
factory results. The liquid culture medium is preserved in little flasks
having a long neck which is hermetically sealed. The principal ad-
vantages connected with the use of these little flasks, or * ' Stern-
5

CULTURES IN LIQUID MEDIA.

berg's bulbs," as they are sometimes called, are that a culture me-
dium may be preserved in them indefinitely and that they are easily
transported from place to place; whereas test tubes, Pasteur's flasks,
and similar receptacles must be kept upright, and after a time the
culture liquid in them is changed in its composition by evaporation.
They are also liable to be contaminated by the entrance of mould
fungi when kept in a damp place. The spores of these fungi, falling
upon the surface of the cotton air filter, germinate, and the myce-
lium grows down through the cotton into the interior of the tube,
where a new crop of spores is quickly formed. It is, therefore, a
convenience to have sterile culture liquids always ready for use in
a receptacle which can be packed in a box and transported from
place to place ; but for every-day use in the laboratory the ordinary

FIG. 37,

test tube, with its cotton air filter, is the most economical and conve-
nient receptacle for culture liquids as well as for solid media. With
reference to the method of making and using these little flasks, I
quote from a paper published in the American Journal of the
Medical Sciences in 1883 :'

The culture flasks employed contain from one to four fluidrachms.
They are made from glass tubing- of three- or four tenths inch diameter, and
those which the writer has used in his numerous experiments have all been
" home-made." It is easier to make new flasks than to clean old ones, and
they are thrown away after being once used. Bellows operated by foot, and
a flame of considerable size — gas is preferable — will be required by one who
proposes to construct these little flasks for himself.'2 After a little practice
they are made rapidly; but as a large number are required, the time and
labor expended in their preparation are no slight matter. After blowing a
bulb at the extremity of a long glass tube, of the diameter mentioned, this
is provided with a slender neck, drawn out in the flame, and the end of this

1 " The Germicide Value of Certain Therapeutic Agents," op. cit., vol. clxx.
8 A glass-blower ought to make them for two or three dollars per hundred.

CULTURES IN LIQUID MEDIA. 67

is hermetically sealed. Thus one little flask after another is made from the
same piece of tubing until this becomes too short for further use. To intro-
duce a culture liquid into one of these little flasks, heat the bulb slightly,
break off the sealed extremity of the tube and plunge it beneath the surface
of the liquid (Fig. 37). The quantity which enters will of course depend
upon the heat employed and the consequent rarefaction of the enclosed air.
Ordinarily the bulb is filled to about one-third of its capacity with the cul
ture liquid, leaving it two thirds full of air for the use of the microscopic
plants which are to be cultivated in it. ... Sterilization is effected by heat
after the liquid has been introduced and the neck of the flask hermetically
sealed in the flame of an alcohol lamp.

Sterilization may be effected by boiling for an hour in a bath of paraffin
or of concentrated salt solution, by which a temperature considerably above
that of boiling water is secured. The writer is in the habit of preparing a
considerable number of these flasks at one time, and leaving them, in a suit-
able vessel filled with water, for twenty four hours or longer 011 the kitchen
stove.1

To inoculate the liquid contained in one of these little flasks with mi-
croorganisms from any source, the end of the tube is first heated to destroy
germs attached to the exterior; the extremity is then broken off with steril-
ized (by heat) forceps; the bulb is very gently heated, so as to force out a
little air, and the open end is plunged into the liquid containing the organ-
ism to be cultivated (or into a vein, or one of the solid viscera of an animal
dead from an infectious germ disease, such as anthrax).

Inoculation from one tube to another may also be effected by means of
the ordinary platinum wire needle.

Before the introduction of Koch's plate method for isolating bac-
teria in pure cultures, certain methods had been proposed, and em-
ployed to some extent, which at present have a historical value only.

Thus Klebs (1873) proposed to take from a first culture in which
two or more species were associated a minute quantity, by means of a
capillary tube, and with this to inoculate a second culture. By re-
peating this procedure several times he expected to exclude all except
the species which was present in the greatest abundance and which
multiplied most rapidly in the medium employed.

The method by dilution, first employed with precision by Brefeld
(1872) in obtaining pure cultures of mould fungi, and subsequently
by Lister for the isolation of bacteria, consists in so diluting a minute
quantity of the mixed culture that the number of bacteria in the dilu-
tion may be less than one for each drop of the liquid. If now a
single drop be added to each of a series of tubes containing a small
quantity of sterile bouillon, some of the inoculations made may give
a pure culture, as the drop may have contained but a single vege-
tative cell.

Another method of obtaining a pure culture in liquid media, when
several microorganisms are associated which have a different ther-

1 Where a steam sterilizer is at hand they will be most conveniently sterilized in
the usual way, by subjecting them to the boiling temperature for an hour at a time
on three successive days.

CULTURES IX LIQUID MEDIA.

mal death-point, consists in the application of heat and thus destroy-
ing all except the most resistant species. This method is especially
applicable when one of the species, only, forms spores. By subject-
ing the mixed culture to a temperature which is sufficient to destroy
all the vegetative cells in it, the more resistant spores are left and,

under favorable conditions, may subsequently
vegetate and give us a pure culture of the
species to which they belong.

Fermentation. — The development of certain
bacteria is attended with an evolution of gas,
especially in media containing grape sugar or
glycerin. For the determination of the quantity
and kind of gas produced by a given micro-
organism the fermentation tube recommended
by Theobald Smith has special advantages.
This is a bent tube (Eihorn's) supported upon
a glass base as shown in the accompanying
figure taken from the catalogue of Eimer &
Amend. The graduation shown upon the up-
right arm is not essential for ordinary labora-
tory work. A liquid culture medium containing
one to two per cent of grape sugar is usually
used. This is introduced into the upright arm
of the fermentation tube, where it is held by atmospheric pressure.
A cotton plug is placed in the opening of the short and bulbous arm
of the tube, which is intended as a receptacle for the culture liquid
when it is forced out of the closed arm by the accumulation of gas at
its upper extremity.

FIG. 38.

VIII.

CULTURES IX SOLID MEDIA.

THE introduction of solid culture media in 1881 by the famous
German bacteriologist, Robert Koch, inaugurated a new era in the
progress of our knowledge relating to the bacteria. His methods
enable us to obtain pure cultures with ease and certainty, and to
study the morphological and biological characters of each species
free from the complications which led to so much error and confusion
before these methods were introduced. We have already given an
account of the method of preparing and sterilizing the various solid
culture media, and are here concerned with the manner
in which they are used and the special advantages which
they afford.

Koch's flesh-peptone-gelatiii, which contains ten per
cent of gelatin, is a transparent jelly which liquefies at
from 22° to 24° C. It is a favorable culture medium for
a great number of bacteria, and many species show de-
finite characters of growth in this medium which serve to
differentiate them. One of the most prominent of these
characters depends upon the fact that some bacteria liquefy
gelatin and others do not. This is made apparent when
we make "stab cultures." This is the usual manner of
inoculating a solid culture medium, and is illustrated in
Fig. 39. A platinum needle, consisting of a piece of
platinum wire inserted into a glass rod which serves as a
handle, is passed through the flame of an alcohol lamp to
sterilize it. When cooled, which occurs very quickly, the
point is introduced into the material containing the bac-
teria to be planted in the gelatin medium. We may ob-
tain our seed for a pure culture from a single colony, from FIG. 39.
another stab culture, from the blood of an infected animal,
etc. The point of the needle is then carried into the sterilized jelly,
as shown in the figure, care being taken to introduce it in the central
line and in a direction parallel with the sides of the tube. It is best

CULTURES IN SOLID MEDIA.

always to hold the tube inverted during the inoculation, and not
remove the cotton air filter until we are ready to make it. The
cotton plug is then returned to its place and the platinum needle
again brought to a red heat to destroy any bacteria which remain
attached to it.

Sometimes it is an advantage to have the culture medium with a

FIG. 40.

sloping surface, as shown in Fig. 40. We may then draw the

die over the surface in a longitudinal direction, and by this means

distribute the seed in a line along which development will take place.

The characters of growth in these stab cultures in gelatin are

very various. Non-liquefying bacteria may grow only on the sui
face, as at a, Fig. 40A ; or both on the surface and along the line
of puncture, as at b; or only at the bottom, as at c. In the first
case the microorganism is aerobic — that is, it requires oxygen, and
grows only in the presence of this gas. In the second case it is
not strictly aerobic, but may grow either in the presence of oxygen

CULTURES IN SOLID MEDIA.

or in its absence — a facultative anaerobic. In the third case the
microorganism is an anaerobic, which cannot grow in the presence
of oxygen, and consequently does not grow upon the surface of the
culture medium or along the upper portion of the line of puncture.

Again, we have differences as to the character of growth upon the
surface or along the line of puncture. The surface growth may be
a little mass piled up at the point where the needle entered the gela-
tin ; or it may form a layer over the entire surface, and this may
be thin or thick, dry or moist, viscid or cream-like, and of various
colors — green, blue, red, or yellow, of different shades — or more fre-
quently of a milk-white color.

The growth along the line of puncture also differs greatly with
different species. We may have a number of scattered spherical
colonies (a, Fig. 41), and these may be translucent or opaque ; or we
may have little tufts, like moss, projecting from the line of puncture
(&, Fig. 41) ; or slender, filamentous branches may grow out into the
gelatin (c, Fig. 41).

The liquefying bacilli also present different characters of growth.
Thus liquefaction may take place all along the line of puncture,
forming a long and narrow funnel of liquefied gelatin (a, Fig. 42) ;
or we may have a broad funnel, as at b ; or a cup-shaped cavity, as
at c; or the upper liquefied portion may be separated from that
which is not liquefied by a horizontal plane surface, as at d.

CULTURES IN SOLID MEDIA.

The characters of growth in agar-agar jelly are not so varied,
but this medium possesses the advantage of not liquefying at a tem-
perature of 35° to 38° C., which is required for the development of
certain pathogenic bacteria. Variations in mode of growth are
also manifested in nutrient agar similar to those referred to as pro-
duced by non-liquefying bacteria in flesh-peptone-gelatin. These
relate to the surface growth and to growth along the line of punc-
ture. One character not heretofore mentioned consists in the for-
mation of gas bubbles in stab cultures either in gelatin or agar.

Colonies. — If we melt the gelatin or agar in a test tube, pour
the liquid medium into a shallow glass dish previously sterilized,

FIG. 42.

and allow it to cool while properly protected by a glass cover, we
will have a broad surface of sterile nutrient material. If now we ex-
pose it to the air for ten or fifteen minutes, and again cover it and
put it aside for two or three days at a favorable temperature, we can
scarcely fail to have a number of colonies upon the surface of the
culture medium, which have been developed from atmospheric germs
which were deposited upon it during the exposure. Each of these
colonies, as a rule, is developed from a single bacterium or spore,
and consequently the little mass, visible to the naked eye, which we
call a colony, is a pure culture of a particular species. In this ex-
periment we are more apt to have colonies of mould fungi than of
bacteria, but the principle is the same, viz. , that a colony developed
from a single germ is a pure culture. By touching our platinum

CULTURES IX SOLID MEDIA. 73

needle, then, to such a colony, which is quite independent of, and
well separated from, all others, we may make a stab culture in gela-
tin or agar, and preserve the pure culture for further study. This
is a most important advantage which pertains to the use of solid
culture media. It is a singular fact that, as a rule, colonies of bac-
teria which lie near each other do not grow together, but each re-
mains distinct. If there are but few colonies, each one, having1

7 " O

plenty of room, may grow to considerable size ; if there are many
and they are crowded, they remain small, but are still independent
colonies.

Now, these colonies differ greatly in their appearance and char-
acters of growth, according to the species (Fig. 43). Some are
spherical, and these may be translucent or opaque, or they may have
an opaque nucleus surrounded by a transparent zone. Again, the

FIG. 43.— Colonies of Bacteria.

outlines may be irregular, giving rise to amoeba-like forms, or to a
fringed or plaited margin, or the form may be that of a rosette, etc. ;
or the colony may appear to be made up of overlapping scales or
masses, or of tangled filaments ; or it may present a branching
growth. In the case of liquefying bacteria, when the colonies have
developed in a gelatin medium they commonly do not at once cause
liquefaction of the gelatin, but at the end of twenty-four hours or
more the gelatin about them commences to liquefy and they are
seen in a little funnel of transparent liquefied gelatin ; or in other
cases little opaque drops of liquefied gelatin are seen, which, as the
liquefaction extends, run together. All of these characters are best
studied under a low-power lens, with an amplification of five to
twenty diameters ; an:! by a careful observation of the differences in
the form and development of colonies we are greatly assisted in the
differentiation of species.

Single, isolated colonies do not always contain a single species,
for they are not always developed from a single cell. We may have

74 CULTURES IN SOLID MEDIA.

deposited upon our plate, exposed as above described, a little mass
of organic material containing two or more different bacteria, and
this would serve as the nucleus of a colony from which we could not
obtain a pure culture.

Koch's Plate Method. — In the experiment above described,
colonies were obtained from air-borne germs which were deposited
upon the surface of our gelatin medium. By Koch's famous " plate
method " we obtain colonies of any particular microorganism which
we desire to study, or of two or more associated bacteria which we
desire to study separately in pure cultures. Evidently, when we
have obtained separate colonies of different bacteria upon the sur-
face of a solid culture medium, we can easily obtain a pure culture
of each by inoculating stab cultures from single colonies.

To obtain separate colonies we resort to the ingenious method of
Koch. Three test tubes containing a small quantity of nutrient
gelatin (or of agar) are commonly employed. The tubes are num-
b3red 1, 2, and 3. The first step consists in liquefying the nutrient
jelly by heat, and it will be well for beginners to place the tubes in
a water bath having a temperature of about 40° C. (104° F.) for the
purpose of keeping the culture material liquid, and at the same time
at a temperature which is not high enough to destroy the vitality of
the bacteria which are to be planted. We next, by means of a
platinum- wire loop or the platinum needle used for stab cultures,
introduce into tube No. 1 a small amount of the culture, or material
from any source, containing the bacteria under investigation. Care
must be taken not to introduce too much of this material, and it
must be remembered that the smallest visible amount may contain
many millions of bacteria. The reason for using three tubes will
now be apparent. It is usually impossible to introduce a few bac-
teria into tube No. 1, but we effect our object by dilution, as follows :
With the platinum- wire loop we take up a minute drop of the fluid in
tube No. 1, through which the bacteria have been distributed by
stirring, and carry it over to tube No. 2. Washing off the drop by
stirring, we may repeat this a second or third time — this is a matter
of judgment and experience ; often it will suffice to carry over a
single ose (the German name for the platinum- wire loop). Next
we carry over one, or two, or three ose from tube No. 2 to tube No.
3. By this procedure we commonly succeed in so reducing the num-
ber of bacteria in tube No. 3 that only a few colonies will develop
upon the plate which we subsequently make from it; or it may happen
that the dilution has been carried too far and that no colonies de-
velop upon the plate made from this tube, in which case we are
likely to get what we want from tube No. 2. The next step is to
pour the liquid gelatin upon sterilized glass plates, which are num-

CULTURES IN SOLID MEDIA. 75

bered to correspond with the tubes. The plates used by Koch are
from eight to ten centimetres wide and ten to twelve centimetres
long. They must be carefully cleaned and sterilized in the hot-air
oven, at 150° C., for two hours. They may be wrapped in paper be-
fore sterilization, or placed in a metal box especially made for the
purpose. In order that the liquid gelatin may be evenly distributed
upon the plate the apparatus shown in Fig. 44: is used. This con-
sists of a glass plate, g, supported by a tripod having adjustable feet.
By means of the spirit level / the glass plate is adjusted to a hori-
zontal position. A sterilized glass plate is placed in the glass tray,
shown in the figure, and the gelatin from one of the tubes is care-
fully poured upon it and distributed upon its surface with a steril-
ized glass rod, care being taken not to bring it too near the edge of
the plate. The glass tray in then covered until the gelatin has
cooled sufficiently to become solid, after which plate No. 1 is re-
moved and plates Nos. 2 and 3 are made in the same way. In

FIG. 44.

order to save time it is customary to fill the glass tray shown in the
figure with ice water, to place a second glass support upon it, and
upon this the sterilized glass plate upon which the liquid gelatin is
poured. This is protected by a glass cover, as before, until the gela-
tin becomes solid.

The three plates, prepared as directed, are put aside in a glass
jar of the form shown in Fig. 44, one being supported above the
other by a bench of sheet zinc or glass.

Petri's Dishes. — A modification of the plate method of Koch,
which has some advantages, consists in the use of three small glass
dishes of the same form as the larger one used by Koch to contain
the plates. These dishes of Petri are about ten to twelve centime-
tres in diameter and one to 1.5 centimetres high, the cover being of
the same form as the dish into which the gelatin is poured. These
dishes take less room in the incubating oven than the larger glass
jar used in the plate method, and they do not require the use of a
levelling apparatus. The colonies also may be examined and
counted, if desired, without removing the cover, and consequently

CULTURES IX SOLID MEDIA.

without the exposure which occurs when a plate prepared by Koch's
method is under examination.

In agar-agar cultures or in gelatin cultures of non-liquefying
bacteria made in Petri's dishes, we may examine and count colonies,
without removing the cover, by inverting the dish.

In pouring the liquefied gelatin from the test tubes in which the
dilution has been made into sterilized Petri's dishes, care must be
taken to first sterilize the lip of the test tube by passing it through
the flame of a lamp. We may at the same time burn off the top of
the cotton plug, then remove the remaining portion with forceps,
when the lip has cooled, for the purpose of pouring the liquid into the
shallow dish.

Von Esmarch' s Roll Tubes. — Another very useful modification
of Koch's plate method is that of von Esmarch. Instead of pouring
the liquefied gelatin or agar medium upon plates or in shallow

FIG. 45.

dishes, it is distributed in a thin layer upon the walls of the test tube
containing it. This is done by rotating the tube upon a block of ice
or in iced water. Esmarch first used a tray containing iced water,
and to prevent the wetting of the cotton filter a cap of thin rubber
was placed over the end of the tube. It is more convenient to turn
the tubes upon a block of ice having a horizontal flat surface, in
which a shallow groove is first made by means of a test tube con-
taining hot water (Fig. 45). Or, in the winter, we may turn the
tube under a stream of cold water from the city supply — i. e. , from a
faucet in the laboratory. A little practice will enable the student to
distribute the culture medium in a uniform layer on the walls of the
test tube, and as soon as it is quite solidified these may be placed
aside for the development of colonies from the bacteria which had
been introduced. When roll tubes are made from the agar jelly it is
best to place the tubes in a nearly horizontal position, for if placed
upright at once the film of jelly is likely to slip from the walls of the

CULTURES IX SOLID MEDIA. « «

tube. This is due to the fact that a little fluid is pressed out of the
jelly, probably by a slight contraction while cooling. If the tubes
are slightly inclined from the horizontal the film does not slip and
the fluid accumulates at the bottom. After a day or two they may
be placed in an upright position.

These roll tubes possess several advantages. They are quickly
made and take but little space in the incubating oven, and the film
of jelly is protected from contamination by atmospheric germs.
When colonies have formed we may examine them through the thin
\valls of the tube, either with a pocket lens or a low-power objective.
In making a stab culture from a single colony in one of these roll
tubes, we invert the tube, remove the cotton air filter, and pass the
point of a sterilized platinum needle up to the selected colony. In
the same way we obtain material for microscopical examination.

Streak Cultures. — In his earlier experiments with solid culture
media Koch made " streak cultures " by drawing the point of a plati-
num needle, charged with bacteria, over the surface of a gelatin or
agar plate ; and this method is still useful in certain cases. If we
draw the needle over the moist surface several times in succession
the greater number of bacteria will be deposited in the first streak,
and in the second or third single cells are likely to be left at such
intervals from each other that each will develop an independent
colony. If the streaks were made with impure stock we may thus
succeed in getting separate colonies of the several bacteria contained
in it, so that this method may be employed for obtaining pure cul-
tures. But for this purpose it is much inferior to the plate method,
and it is chiefly used for observing the growth of bacteria on the sur-
face of solid culture media. Thus we commonly make a streak upon
the surface of cooked potato or solidified blood serum in studying the
development of various bacteria on these culture media.

Cultures upon Blood Serum. — The use of blood serum as a
solid medium is practically restricted to stab cultures and streak
cultures, for we cannot substitute it for the gelatin and agar media
in making plates and roll tubes. This is because it only becomes solid
at a temperature which would be fatal to most bacteria (70° C.), and
when once made solid by heat cannot again be liquefied. Its use is,
therefore, restricted mainly to the cultivation of bacteria for which
it is an especially favorable medium. It may be used, however, in
combination with a gelatin or agar medium. For this purpose it is
most conveniently kept in a fluid condition in the little flasks hereto-
fore described (" Sternberg's bulbs ").

The gelatin or agar jelly in test tubes is liquefied by heat and
cooled in a water bath to about 40° C. The desired amount of ste-
rile blood serum is then forced into each tube by passing the slender

rvo
< o

CULTURES IN SOLID MEDIA.

neck of the little flask along the side of the cotton filter (see Fig. 46)
and applying gentle heat to the bulb. The slender neck is first ste-
rilized by passing it through a flame, and the point is broken off
with sterile forceps. After inoculating the liquefied medium in the
test tubes in the usual manner we may make plates or roll tubes.

Cultures on Cooked Potato. — The method of preparing pota-
toes for surface cultures has already been given (page 48). It was
in using them that Koch first got his idea of the importance of solid
media, which led to his introduction of the use of gelatin and agar-
agar and the invention of the plate method. By means of streak

Fin. 46.

cultures upon potato he had succeeded in obtaining isolated colonies
and pure cultures. We now use the potato chiefly for the purpose
of differentiating species. Some bacteria grow on the surface of
cooked potato and some do not. Those which do present various
characters of growth. Thus we have differences as to color, as to
rapidity of growth, as to the character of the mass formed — thick
or thin, viscid, moist or dry, restricted to line of inoculation or ex-
tending over the entire surface, etc.

Instead of using a cut section of the potato in the manner here-
tofore described, we may make a puree by mashing the peeled and
cooked tubers and distributing the mass in Erlenmeyer flasks. After

CULTURES IN SOLID MEDIA. 79

thorough sterilization by steam the culture medium is ready for use.
In the same way other vegetables, or bread, etc. , may be used for
special purposes, and especially for cultures of the mould fungi.

Potatoes usually have a slightly acid reaction, and on this ac-
count certain bacteria will not grow upon them. This acid reaction
is not constant and differs in degree, and as a result we may have
decided differences in the growth of the same species upon different
potatoes. To overcome this objection the writer has sometimes neu-
tralized the cones of potato in test tubes (see Fig. 21, page 49) by
first boiling them in water containing a little carbonate of soda.
The liquid is poured off after they have been in the steam sterilizer
for half an hour, and they are returned for sterilization.

Salomonson's Method of cultivation in capillary tubes has a his-
torical value only since the introduction of Koch's plate method.

The following modifications of Koch's plate cultures have recently
been introduced:

Kruse (1894) pours the liquefied gelatin or agar into Petri dishes,
and after it is solidified brushes the surface with a sterilized camel's-
hair brush which has been dipped into water containing in suspen-
sion— properly diluted — the bacteria to be studied. By this procedure
surface colonies only are obtained. Von Freudenreich (1894) prefers
to pour the contents of the test tube upon the surface of the sterile
medium, in Petri dishes. The fluid is allowed to run off by placing
the Petri dish in a vertical position, and this is subsequently placed in
the incubating oven in an inverted position — i.e., with cover below.
To obtain satisfactory plates with well-separated, superficial colonies
it may be necessary to use two or three dilutions, made in sterilized
water in the usual way — i.e., from one tube to another, by means of
the platinum wire having a loop at its extremity.

IX.

CULTIVATION" OF ANAEROBIC BACTERIA.

PASTEUR (1861) .first pointed out the fact that certain species of
bacteria not only grow in the entire absence of oxygen, but that for
some no growth can occur in the presence of this gas. Such bacteria
are found in the soil, and in the intestines of man and the lower ani-
mals. The cultivation of ''strict anae'robics " calls for methods by
which oxygen is excluded. The "facultative anae'robics'' grow

FIG. 47

FIG. 48.

either in the presence or absence of oxygen. There are various gra-
dations in this regard, from the strictly aerobic species which re-
quire an abundance of oxygen and will not grow in its absence, to
the strictly anaerobic species which will not grow if there is a trace
of oxygen in the medium in which we propose to cultivate them.
Among the most interesting pathogenic bacteria which are strictly
anaerobic are the bacillus of tetanus, the bacillus of malignant
oedema, and the bacillus of symptomatic anthrax.

CULTIVATION OF ANAEROBIC BACTERIA. 81

If we make an inoculation of one of the species which is not
strictly anaerobic into a test tube containing nutrient gelatin or agar-
agar, we may have a development all along the line of puncture,
and this may be more abundant below, as in Fig. 47. But when we
make a long stab culture with a strict anaerobic the development
occurs only near the bottom of the line of puncture (Fig. 48).

We may then, if we have a pure culture to start with, propagate
these anaerobic bacilli in long stab cultures. It is best to use tubes
which have been recently sterilized, as boiling expels the air from
the culture medium ; and a very slender needle should be used in
making the inoculation. To prevent the absorption of oxygen a
layer of sterilized olive oil may be poured into the tube after the in-
oculating puncture has been made, or it may be filled up with agar
jelly which has been cooled to about 40° C. Roux has proposed to
prevent the absorption of oxygen by the culture medium by plant-
ing an aerobic bacterium— Bacillus subtilis — upon the surface, after
making a long stab culture with the anaerobic species. The agar
jelly is first boiled and quickly cooled ; the inoculation is then made
with a slender glass needle ; some sterile agar cooled to 40° C. is
poured into the tube, and when this is solid the aerobic species is
planted upon the surface. The top of the test tube is then closed
hermetically and it is placed in the incubating oven. The aerobic
species exhausts the oxygen in the upper part of the tube by its
growth on the surface of the culture medium, and the anaerobic
species grows at the bottom of the tube. To obtain material for a
new culture or for microscopical examination the test tube is broken
near its bottom.

Cultures in liquid media may be made by exhausting the air in
a suitable recaptacle or by displacing it with hydrogen gas. The
first-mentioned method has been largely used in Paste :ir's laboratory,
but methods in which hydrogen gas takes the place of atmospheric
air in the culture tube are more easily applied an -1 require simpler
apparatus. The flask shown in Fig. 49 may be u ;od in connection
with an air pump. The sterile culture liquid is firjt introduced into
a long-necked flask and inoculated with the anaerobic bacillus to be
cultivated. The neck of the flask is then drawn out in a flame at c.
The open end is then connected with a Sprengle's pump or some
other apparatus for exhausting the air. The flask is placed in a
water bath at 40° C., which causes ebullition at the diminished pres-
sure, and the exhaustion is continued for about half an hour. The
narrow neck is then sealed at c by the use of a blowpipe flame.

The flask shown in Fig. 49, which can be made from a test tube,
may also be used in connection with a hydrogen apparatus. In this
case a slender glass tube is passed into the flask, as shown in Fig.
6

82 CULTIVATION OF ANAEROBIC BACTERIA.

50, and this is connected with a hydrogen apparatus by a rubber
tube, The hydrogen is allowed to bubble through the culture
liquid in a full stream for ten to fifteen minutes, in order that all of
the oxygen in the flask may be removed by displacement. Then,
while the gas is still flowing, the flask is sealed at a with a blow-
pipe flame, the hydrogen tube being left in position and melted fast
to the flask. Some little skill is required in the successful perform-
ance of the last step in this procedure, and it will be easier for those

FIQ. 49.

FIG. 51.

who are not skilful in the use of the blowpipe to use Salomonson's
tube, shown in Fig. 51. In this, hydrogen is admitted through the
arm 6, and escapes through the cotton plug a. The vertical tube is
sealed at c while the gas is flowing, and then the horizontal tube at b.
Frankel's Method. — Instead of these tubes specially made for
the purpose, an ordinary test tube may be used, as recommended by
Frankel. This is closed by a soft rubber cork through which two
glass tubes pass — one, reaching nearly to the bottom of the test tube,

CULTIVATION OF ANAEROBIC BACTERIA.

for the admission of hydrogen, which passes through the liquefied
culture medium ; and the other a short tube for the escape of the gas.
The outlet tube is sealed in the flame of a lamp while the gas is
freely flowing, and after sufficient time has elapsed to insure the
complete expulsion of atmospheric oxygen — which, when the hydro-
gen flows freely, requires about four minutes (Frankel) — melted
paraffin is applied freely to the rubber stopper to prevent leakage of
the hydrogen and entrance of oxygen. A roll tube may then be
made after the manner of Esmarch, and, after colonies have de-
veloped, the anaerobic culture will appear as shown in Fig. 52.

To isolate anaerobic bacteria in pure cultures it is well to make a

-t

-a

FIG. 53.

series of dilutions as heretofore described for aerobic cultures ; we
will then usually obtain isolated colonies in tube No. 2 or No. 3 of a
series, and by removing the rubber stopper we may transplant bac-
teria from these colonies to deep stab cultures in nutrient gelatin or
agar.

The Writer's Method.— The following simple method has been
successfully employed by the writer:

Three Esmarch roll tubes are prepared as is usual for aerobic cul-
tures. The cotton air filter, or a portion of it, is then pushed down
the tubes for a short distance, as shown at a, Fig. 53. A section of
a soft rubber stopper carrying two glass tubes is then pushed into the

CULTIVATION OF ANAEROBIC BACTERIA.

test tube for about half an inch, as shown at b, Fig. 53. The space
above the cork is then filled with melted sealing wax, which I have
found to prevent leakage better than paraffin, which contracts upon
cooling. The test tube is inverted while hydrogen is passed through
the tube c, and by reason of its levity the gas quickly passes through
the cotton air filter and displaces the oxygen in the test tube (Fig.
54). After allowing the gas to flow for a few minutes the outlet
tube is first sealed in a flame and then the inlet tube. As the cotton
filter is interposed between the rubber stopper and the culture mate-
rial, no special precautions need be taken for the sterilization of the
rubber cork and the glass tubes which it carries.

FIG. 54.

FIG. 55.

This method is more convenient than that previously described,
and the only objection to it is that the oxygen is not completely re-
moved from the film of solid gelatin or agar attached to the walls of
the test tube. But by passing the hydrogen for a long time it would
seem that by diffusion the oxygen remaining in this thin layer
would be gotten rid of. At all events, this method will serve for all
except the very strict anaerobics.

Method of Es march.— The following method has been proposed
by Esmarch : Three roll tubes are made in the usual way, and into
these liquid gelatin, that is nearly cooled to the point of becoming
solid, is poured. This fills the tube without melting the layer of

CULTIVATION OF ANAEROBIC BACTERIA.

gelatin, previously cooled upon its walls, which contains the bacteria
under investigation. When the anaerobic colonies have developed
the test tube must be broken to get at them, or the cylinder of gela-
tin may be removed by first warming the walls of the tube.

Another method, recommended by Liborius, consists in distri-
buting the bacteria in test tubes nearly filled with nutrient gelatin or
agar which has been recently boiled to expel air. Colonies of anaero-
bic bacteria will develop near the bottom of such a tube, while the
aerobic species will only grow near the surface. The cylinder of
jelly is removed by heating the walls of the tube, and sections are

fr a

Fia. 56.

made with a sterilized knife for the purpose of 'obtaining material
from individual colonies for further cultures, etc.

Koch and his pupils are in the habit of testing the aerobic char-
acter of bacteria in plate cultures by covering the recently made
plates with a thin sheet of mica which has been sterilized by heat.
The strictly aerobic species do not grow under such a plate ; but,
according to Liborius, the exclusion of oxygen is not sufficiently
complete for the growth of strict anaerobics.

Buclmer's Method consists in the removal of oxygen by means
of pyrogallic acid. The anaerobic species under investigation is
planted in recently boiled agar jelly in a small test tube. This is
placed in a larger tube having a tightly fitting rubber stopper, as
shown in Fig. 55. The small tube is supported by a bent-wire

CULTIVATION OF ANAEROBIC BACTERIA.

stand, and in the lower part of the large tube are placed ten cubic
centimetres of a ten-per-ceiit solution of caustic potash, to which one
gramme of pyrogallic acid is added. The absorption of the oxygen
takes some time, but, according to Buchner, it is finally so complete
that strict anaerobics grow in the small tube.

In practice, cultivation in an atmosphere of hydrogen will be
found the most convenient method, and for this any form of hydro-
gen generator may be used. The writer is in the habit of using the
form shown in Fig. 56. A perforation a quarter of an inch in
diameter is drilled through the bottom of a wide-mouthed bottle.
Some fragments of broken glass are then put into the bottle, form-

FIG. 57.

ing a layer two or three inches thick. Upon this is placed a quan-
tity of granulated zinc. This bottle has a tightly fitting cork,
through which passes a metal tube having a stopcock. The bottle
is placed in a glass jar containing diluted sulphuric acid (one part
by weight of sulphuric acid to eight parts of water). The acid, ris-
ing through the perforation in the bottom of the bottle, when it
comes in contact with the zinc gives rise to an abundant evolution
of hydrogen, which escapes by the tube a when the stopcock is
open. When this is closed the gas forces the acid back from con-
tact with the zinc. To remove any trace of oxygen present the
gas may be passed through a solution of pyrogallic acid in caustic
potash.

Evidently plates prepared by Koch's method, or Esmarch roll

CULTIVATION OF ANAEROBIC BACTERIA. 87

tubes, may be placed in a suitable receiver and the air exhausted, or
hydrogen substituted for atmospheric air. Such an apparatus for
hydrogen has been devised by Bliicher and is shown in Fig. 57. A
glass dish, A, contains a smaller dish, B, which has a diameter of
about seven centimetres. The small dish is kept in its position in
the centre of the larger one by the wire ring, having three project-
ing arms, which is shown in the figure. The culture medium con-
taining the anaerobic bacteria to be cultivated is poured into the
small dish and the glass funnel D is put in position. This is held
in its place by a weight of lead which encircles the neck of the fun-
nel at F. A mixture of glycerin and water (twenty to twenty-five
per cent) is poured into the dish A to serve as a valve to shut off
the atmospheric air from the interior of the funnel D. Hydrogen
gas is introduced through the tube E, which is connected by a rub-
ber tube with a hydrogen apparatus.

A somewhat similar apparatus has been devised by Botkin, in
which the hydrogen is admitted beneath a bell jar covering small
glass dishes containing the culture medium. We believe that in
practice the writer's method (page 83), in which Esmarch roll tubes
are first made, will be found more convenient than either of the last-
mentioned methods of preserving plates in an atmosphere of hydro-
gen ; or roll tubes may be prepared in the way usually practised in
cultivating aerobic bacteria, and these may be placed in a suitable
receptacle which can be filled with hydrogen.

The addition of a reducing agent to the culture medium favors
the growth of anaerobic bacteria. Kitasato and Weil have recom-
mended formic acid or sodium formate, in the proportion of 0.3 to 0.5
per cent. Theobald Smith has found 0.3 to 0.5 per cent of glucose
to be a useful addition with the same object in view.

X.
INCUBATING OVENS AND THERMO-REGULATORS.

THE saprophytic bacteria generally, and many of the pathogenic
species, grow at the ordinary temperature of occupied apartments
(20° to 25° C. ) ; but some pathogenic species can only be cultivated
at a higher temperature, and many of those which grow at the
"room temperature" develop more rapidly and vigorously when
kept in an incubating oven at a temperature of 35° to 38° C. Every
bacteriological laboratory should therefore be provided with one or
more brood ovens provided with thermo-regulators to maintain a
constant temperature. These incubating ovens are made with dou-
ble walls surrounding an air chamber. The space between the dou-
ble walls is filled with water, which is usually heated by a small gas
flame. The gas passes through the thermo-regulator, and its flow
is automatically controlled for any temperature to which this is ad-
justed. The exterior of the incubating oven is covered with felt or
asbestos to prevent the loss of heat by radiation. A simple and
cheap form which answers every purpose is shown in Fig. 58. The
quadrangular box with double walls should be made of zinc or cop-
per. An outer metal door covered with non-conducting material,
and an inner door of glass, give access to the interior space ; and a
thermometer introduced through an aperture in the top (Fig. 58, b)
shows the temperature of this space when the door is closed. The
stopcock e permits the drawing off of the water from the space be-
tween the double walls, and the glass tube d shows the height of
the water, as it is connected with the space containing it. The
thermo-regulator passes through an aperture at one side of the oven
into the water, the temperature of which, controls the flow of gas.

The ordinary thermo-regulator is shown in Fig. 50 as manufac-
tured by Rohrbeck. A glass receptacle, shaped like an ordinary
test tube, has an arm, c, for the escape of the gas, which enters by
the bent tube a, which passes through a perforated cork and is ad-
justable up and down. Tube a is connected with the gas supply and
tube c with the burner by means of rubber tubing. A glass parti-
tion extending downward as a tube, g, makes an enclosed space in

INCUBATING OVENS AND THERMO-REGULATORS.

the lower part of the instrument, and this, when immersed in water,
acts as a thermometer bulb. This space contains mercury below
and air or the vapor of ether above. When the air is expanded by
heat the mercury is forced up the tube g until it meets the end of
the inlet tube for gas at h, and by shutting off the flow of gas pre-
vents the temperature from going any higher. A small opening in
the inlet tube at e permits a small amount of gas to flow, so that the
flame under the brood oven ( Fig. 58, /) may not be entirely extin-
guished. The lower end of the bout tube a is bevelled, so that a tri-
angular opening is formed, which is closed gradually by the rising

FIG. 58.

mercury, instead of abruptly as would be the case if the lower end
of the tube a were cut off square. To adjust the temperature in
the air space of the incubating oven when the thermo-regulator is in
position, a full flow of gas is admitted to the burner until the ther-
mometer (Fig. 58, b) shows the desired temperature ; then the bent
tube a is pushed down through the cork until its lower extremity
meets the mercury and the flame / is somewhat reduced. The ap-
paratus is then left for a time, to see whether the flame runs too high
or too low, and a further adjustment is made. When the changes
in the exterior temperature are slight and the gas pressure regular
the temperature in the air chamber is controlled with great precision.
But this is not the case under the reverse conditions. Changes in

INCUBATING OVENS AND THERMO-REGULATORS.

the pressure of gas, especially, interfere with the maintenance of a
constant temperature, and for this reason a pressure regulator will
be required when great precision is desired. That of Moitessier is
commonly used in bacteriological laboratories (Fig. 60). But for
most purposes variations of temperature of 1° to 2° C. are not of
great importance. For ordinary use a brood oven should be regu-
lated to about 35° to 37° C. It is best to have a little cylindrical
screen of mica around the gas jet beneath the incubating oven, for
the purpose of preventing the flame from being extinguished by cur-
rents of air (Fig. 61).

Koch's ingenious automatic device for shutting off the gas if the
flame is accidentally extinguished is shown in Fig. 62.

FIG. 59.

FIG. 60.

Another form of thermo-regulator, which answers very well, is
that of Reichert (Fig. 63). In this the gas enters at a and escapes
at c. The mercury, which fills the bulb, shuts off the gas at the
point for which the instrument is regulated. By means of the
screw d the height of the mercury in the tube may be very accu-
rately adjusted for any desired temperature.

The regulator of Bohr, shown in Fig. 64, is more sensitive than
that of Reichert, and rather simpler in construction than the usual
form shown in Fig. 59. The thermometer bulb a contains only air,
and the gas which passes through the tube / is shut off at the
proper temperature by the mercury in the U-shaped tube c. The
stppcock b is left open when the bulb a is immersed in the water

INCUBATING OVENS AND THERMO-REGULATORS.

bath, and when the proper temperature is reached is closed so as to
confine the air in the bulb. An increase of temperature now causes

FIG. 61. FIG. 62.

the air in the bulb to expand, the mercury in the U-tube is forced up

...a

FIG. 63.

FIG. 64.

and shuts off the gas flowing through the tube /at its lower ex-
tremity, d. A small opening, e, permits sufficient gas to pass to

92 INCUBATING OVENS AND THERMO-REGULATORS.

maintain a small flame which must not be sufficient by itself to keep
up the desired temperature in the water bath.

Altmann has recently (1891) described a thermo-regulator which
is made by Miincke, of Berlin, and which is shown in Fig. 65. This
is said to act with great precision. It is a modification of Reichert's

FIG. 65.

FIG. <>6.

regulator. Its mode of action will be readily understood by a refe-
rence to the figure.

A thermo-regulator which gives very accurate results, which are
not influenced by differences in pressure, is that invented by the

FIG. 67.

writer over thirty years ago. The regulating thermometer may
contain mercury only, or air and mercury, as shown in the thermo-
regulator for gas (Fig. 59). In the simplest form a large bulb con-
taining mercury is used, and a platinum wire is hermetically sealed
in the glass so as to have contact with the mercury (Fig. 66, a).

INCUBATING OVENS AND THERMO-REGULATORS. 93

Another platinum wire passes down the tube of the thermometer, b,
and is adjustable for any desired temperature. The gas passes
through a valve which is controlled by an electro-magnet. A
simple form of valve is shown in Fig. G7. The bent tube a is con-
nected with the gas supply by a piece of rubber tubing. The up-
right arm of this tube is enclosed in a larger tube, b, having an out-

FIG. 68. FIG. 69.

let, e, which is connected with the burner under the incubating
oven. The upper end of this larger tube is closed by means of a
piece of sheet rubber, which prevents the escape of gas. When this
is depressed by means of the lever c, the flow of gas through the
valve is arrested. The lever c has attached to it the armature d,
and is operated by an electro-magnet under the control of the regu-
lating thermometer.

INCUBATING OVENS AND THERMO-REGULATORS.

"When the thermometer is immersed in a water bath the tem-
perature of which it is desired to regulate, and the proper electric
connections are made, it acts as a circuit breaker. When the de-
sired temperature is reached the mercury in the tube of the ther-
mometer touches the wire b (Fig. 66), an electric circuit is com-
pleted, and the valve is closed, shutting off the gas supply and
preventing the temperature from going any higher. When contact
is broken in the thermometer tube the valve opens and permits the
gas to flow again. A small opening, o (Fig. 67), permits the con-
stant flow of a sufficient amount of gas to prevent the flame from
being extinguished. In practice, however, it is better to have a
small side jet of gas, quite independent of that which passes through
the valve, which burns constantly and relights the principal jet when

FIG. 70.

the valve is opened. This apparatus is very well adapted for regu-
lating the temperature of a water bath with precision, but for gene-
ral use in connection with incubating ovens the ordinary gas regu-
lator is preferable, on account of the trouble connected with keeping
a galvanic battery in order when it is required to act at frequent
intervals " on a closed circuit/' for weeks and months together.

The incubating apparatus of D'Arsoiival is shown in Fig. 68. It
is a cylindrical vessel of copper having double walls, and is provided
with the thermo-regulator of D7 Arson val, by which very accurate
regulation is maintained at any desired temperature. In its form
this apparatus is not as convenient as are the brood ovens made
in the form shown in Fig. 58, with a swinging door which gives
easy access to the interior, which is provided with one or more
shelves upon which the cultures are placed. Various modifications

INCUBATING OVENS AND THERMO-REGULATORS. 95

of this simple and convenient incubating- oven are manufactured
by Robrbeck and by Muncke, of Berlin. The apparatus of D'Ar-
sonval, and other forms in favor at the French capital, may be ob-
tained from Wiesnegg, of Paris. The last-named manufacturer
also supplies the incubating oven and thermo-regulator described by
Roux (1891). This is shown in Fig. 69. The regulator is formed of
two metallic bars, one of steel and the other of zinc ; these are
soldered together in the shape of a letter (J ; the regulator is seen in
position in the cut (Fig. G9). The most dilatable metal (zinc) is on
the outside. When the temperature is raised the arms of the (J ap-
proach each other, and the reverse when it falls. The method by
which regulation is effected is shown in Fig. 70. The U-shaped
regulator is placed vertically, and one of its branches, A, is firmly
fixed to the wall of the incubating oven ; the other, free arm car-
ries a horizontal bar which projects through the wall of the incu-
bator in an opening which permits it to move freely under the influ-
ence of a change in the temperature within. The end of this
projecting bar is turned up at a right angle and the screw p passes
through it ; this can be fixed at any desired point by means of the
nut e. The end of the screw p rests against the stem of a conical
brass valve which controls the flow of gas. The valve is closed by a
spiral spring and opened by the screw p under the control of the
thermo-regulator.

In the absence of gas incubating ovens may be heated by a small
petroleum lamp, and various devices have been invented for control-
ling the temperature. Reichenbach describes an apparatus for this
purpose in the Centralblatt fur Bakteriologie, Vol. XV., p. 847,
1894. Dr. Borden of the U. S. Army has also invented a thermo-
regalator to be used in connection with a petroleum lamp. In the
absence of any regulating apparatus an incubating oven may be kept
at a tolerably uniform temperature by personal supervision — adjusting
the flame of the lamp and its distance from the bottom of the oven ac-
cording to the changes in the external temperature. For most bac-
teria a variation of several degrees is not important, so long as the
temperature is not allowed to rise above 37° to 38° C. The typhoid
bacillus, the diphtheria bacillus, the anthrax bacillus, the pus cocci,
and most saprophytic bacteria grow at the ordinary room temperature,
ap<l may therefore be cultivated without any form of incubating oven
01 thermo-regulator.

EXPERIMENTS UPON ANIMALS.

THE pathogenic power of various bacteria has been demonstrated
by injecting pure cultures into susceptible animals. As a rule, the
herbivora are more susceptible than the carnivora, and this is per-
haps to be explained in accordance with the theory of natural selec-
tion. Carnivorous animals often feed upon the bodies of animals
which have succumbed to infectious diseases, and upon dead animals
in which putrefactive changes have commenced. In their struggles
with each other they are wounded by teeth and claws soiled with in-
fectious material which would cause a fatal disease if inoculated into
the more susceptible herbivorous animals. As this has been going
on for ages, we may suppose that, by survival of the fittest, a race
tolerance has been acquired. The lower animals have their own in-
fectious diseases, some of which are peculiar to certain species and
some common to several. As a rule, the specific infectious diseases
of man cannot be transmitted to lower animals, and man is not sub-
ject to the diseases of the same class which prevail among animals.
But certain diseases furnish an exception to this general rule. Thus
tuberculosis is common to man and several of the lower animals ;
relapsing fever may by inoculation be transmitted to monkeys ;
diphtheria may be transmitted to pigeons and guinea-pigs. On the
other hand, anthrax and glanders may be contracted by man as a
result of accidental inoculation or contact with an infected animal.

Nearly allied species sometimes present very remarkable differ-
ences as to susceptibility. Thus the bacillus of mouse septicaemia is
fatal to house mice but not to field mice, while, on the other hand,
field mice are killed by the bacillus of glanders and house mice are
immune from this pathogenic bacillus.

The animals most commonly used for testing the pathogenic
power of bacteria are the mouse, the guinea-pig, and the rabbit.
Domestic fowls and pigeons are also useful for certain experiments.
The dog and the rat are of comparatively little use on account of
their slight susceptibility.

EXPERIMENTS UPON ANIMALS.

Inoculations are made directly into the circulation through a
vein, into the subcutaneous connective tissue, or into one of the
serous cavities — usually the peritoneal.

The ordinary hypodermic syringe may be used in making injec-
tions, but this is difficult to sterilize on account of the leather piston,
and complications are liable to arise from its use which it is best to
avoid. The best way to sterilize a piston syringe is to wash it thor-
oughly with a solution of bichloride of mercury of 1 : 1,000, and then
to remove every trace of bichloride by washing in alcohol. But one
never feels quite sure that the most careful washing will insure steril-
ization, and it is best to use a syringe which may be sterilized by

FIG 71.

heat, such as that of Koch, shown in Fig. 71. In this the metal point
and glass tube are easily sterilized in a hot-air oven. Fluid is drawn
into the syringe and forced out of it by a rubber ball which has a
perforation to be covered by the finger.

The writer has for some years boen in the habit of making injec-
tions in animals with an improvised glass syringe. This is made
from a piece of glass tubing in the same form as the collecting tubes
heretofore described. A bulb is blown at one end of the tube, and
the other end is drawn out to form a slender tube which serves as the

FIG. 72.

needle of the syringe (Fig. 72). By gently heating the bulb in an
alcohol lamp and immersing the open end of the capillary tube in
the fluid to be injected, this rises into the syringe as the expanded air
cools. Having introduced the glass point beneath the skin or into
the cavity of the abdomen of the animal to be injected, the contents
of the tube are forced out by again heating the bulb by means of a
small alcohol lamp. The glass point is easily forced through the
thin skin of a mouse or of a young.rabbit ; but for animals with a
thicker skin it is necessary to cut through, or nearly through, the
skin with some other instrument. A small pair of curved scissors,
answers very well for this purpose.

98 EXPERIMENTS UPON ANIMALS.

Generally, in making injections into animals, it is customary to
remove the hair for some distance around the point of inoculation
with scissors and razor, and then to sterilize the surface by careful
washing with a solution of bichloride of mercury. This precaution
is necessary in researches in which pathogenic bacteria are being
tested, in order to remove any possibility of accidental inoculation
with germs other than those under investigation, and, as a conse-
quence, a mistaken inference as to the pathogenic action of the spe-
cies under investigation. But when we know the specific pathogenic
power of a certain microorganism it is hardly necessary to take this
precaution, as a few drops of culture will contain millions of the bac-
teria, while contamination, if it occurs from the surface of the body,
must be by a comparatively small number of bacteria, which are
likely to be of a harmless kind which will have no influence on the
result of the experiment.

Instead of sterilizing the surface, the writer usually clips away a
small portion of skin with curved scissors, not cutting deep enough
to draw blood, but leaving a bare surface through which the point of
the syringe can be introduced with very little danger of carrying bac-
teria into the connective tissue other than those contained in the
syringe. *

In making injections into the peritoneal cavity care must be taken
not to wound the liver or the distended stomach. The intestine is
not very likely to be wounded, as it slips out of the way. By seizing
a longitudinal fold of the abdominal wall and pushing the point of
the syringe quite through it, and then releasing the fold and care-
fully withdrawing the instrument until the point remains in the
cavity, the danger of wounding the intestine will be reduced to a
minimum.

Injections into the circulation are made by exposing a vein and
carefully introducing the needle of the syringe in the direction of
the blood current. Care must of course be taken not to inject air.
In the rabbit one of the large veins of the ear may be conveniently
penetrated by the point of a hypodermic syringe without any pre-
vious dissection. The ear is first washed with a solution of bichloride
of mercury or simply with warm water. The animal had better be
carefully wrapped in a towel to control its movements. The veins
are distended by compressing them near the base of the ear. When
the point of the needle has not been properly introduced, and the
fluid to be injected escapes in the surrounding connective tissue, it
will commonly be best to withdraw the syringe and make the
attempt upon another vein. As pointed out by Abbott, the needle
of the syringe should be ground flat at the point, and not curved as
is commonly the case.

EXPERIMENTS UPON ANIMALS. 99

Large quantities of fluid may be injected into the cavity of the
abdomen or into the circulation by slowly forcing the fluid through
a slender canula, properly introduced, which is coupled with a large
syringe by means of rubber tubing, or with a glass receptacle from
which the fluid is forced by the pressure of air pumped in with a
rubber hand ball.

Mice are usually injected subcutaneously near the tail. The
little animal is first seized by a long pair of forceps, or "mouse
tongs," and the hair is clipped away on the back just above the tail.
If solid material is to be introduced a little pocket is made with scis-
sors or with a lancet, into which the infectious material is carried by
means of a platinum needle or slender forceps. Liquids may be in-
jected by the little glass syringe heretofore described, the point of
which is easily forced through the skin.

Pasteur's method of inoculating rabbits with the virus of hydro-
phobia consists in trephining the skull and injecting the material
beneath the dura mater. An incision through the skin is first made
to one side of the median line a short distance back of the eyes.
The edges of the wound are separated, and a small trephine (five or
six millimetres in diameter) is used to remove a button of bone. The
emulsion of spinal cord from a hydrophobic animal is then carefully
injected beneath the dura mater — two or three drops will be sufficient.
The wound is washed out with a two-per-cent solution of carbolic
acid and closed with a couple of sutures.

Injections into the intestine are made by carefully opening the
abdomen with antiseptic precautions, gently seizing a loop of the in-
testine, and passing the point of the syringe through its walls ; the
loop is then returned and the incision in the walls of the abdomen
carefully closed with sutures and dressed antiseptically.

Inoculations into the anterior chamber of the eye of rabbits and
other animals have frequently been practised, and offer certain ad-
vantages in the study of the local effects of pathogenic microorgan-
isms. The animal should be fastened to an operating board, belly
down, and its head held by an assistant, who at the same time holds
the eyelids apart. The conjunctiva is seized with forceps to steady
the eye, and an incision about two millimetres long is made through
the cornea with a cataract knife. Through this opening a small
quantity of a liquid culture may be injected, or a bit of solid material
introduced with slender curved forceps.

Ordinary injections give but little pain and do not call for the use
of an anaesthetic. When anaesthesia is required ether will usually
be preferable to chloroform. Rabbits, especially, are very apt to die
from chloroform, no matter how carefully it may be administered.
Dogs, rats, and mice stand ether very well. The smaller animals

100 EXPERIMENTS UPON ANIMALS.

may be brought under the anaesthetic by placing them in a covered
jar into which a pledget of cotton wet with ether has been dropped.
Before making injections into the anterior chamber of the eye it is
well to use a two-per-cent solution of cocaine as a local anesthetic.

Mice which have been inoculated are usually kept in a glass jar
having a wire-gauze cover. A quantity of cotton is put into the jar
to serve as a shelter for th# little animal, and it is well to partly fill
the jar with dry sawdust. Larger animals are kept in suitable cages
of wire or wood, and, as a rule, each one should be kept in a separate
cage while under observation after an inoculation experiment.

In experimenting upon animals the following points should be
kept in view and noted :

(a) The age and weight of the animal. Young animals are, as
a rule, more susceptible than older ones, and with many pathogenic
bacteria the lethal dose of a culture bears some relation to the size
of the animal.

(b) The point of inoculation. Injections into the circulation
are generally more promptly fatal and require a smaller dose than
those into a serous cavity or into the connective tissue. Pathogenic
bacteria introduced into the abdominal cavity reach the circulation
more promptly than those injected subcutaneously. But certain
microorganisms owe their pathogenic power to the local effect about
the point of inoculation and the absorption of toxic products formed
in the limited area invaded, and do not enter the general circulation,
or at least do not multiply in the circulating fluid, and quickly dis-
appear from it.

(b) The age of the culture injected. Old cultures sometimes
have greater and sometimes less pathogenic potency than recent cul-
tures. Some kinds of virus become ' i attenuated " when kept. But
when the pathogenic power depends chiefly upon toxic products
formed during the growth of the bacteria, old cultures are, as a rule,
more potent than those recently made.

(d) The medium in which the pathogenic bacteria are sus-
pended. Cultures in albuminous media, like blood serum, are in
some cases more potent than bouillon cultures ; and the virulence of
several pathogenic bacteria is greatly intensified by successive cul-
tures— by inoculation — in the bodies of susceptible animals. Ogston
found that pus cocci cultivated in the interior of eggs had an in-
creased virulence. According to Arloing, Cornevin, and Thomas,
the activity of a culture of the bacillus of symptomatic anthrax is
doubled by adding one-five-hundredth part of lactic acid to the cul-
ture fluid.

(e) The quantity injected is evidently an essential point when
the result depends largely upon the toxic products formed in the cul-

EXPERIMENTS UPON ANIMALS. 101

tare medium. It is also an essential point when pathogenic bacteria
are injected which kill susceptible animals in very minute doses, for
it has been shown by the experiments of Watson Cheyne and others
that in the case of some of these, at least, there is a limit below
which infection does not occur.

Inoculated animals should be carefully observed, and a note
made of every symptom indicating a departure from the usual con-
dition of health, such as fever, less of activity, loss of appetite,
weakness, emaciation, diarrhoea, convulsions, dilated pupils, the for-
mation of an abscess or a diffuse cellulitis extending from the point
of inoculation, etc. The temperature is usually taken in the rectum.
The temperature of small animals, like rabbits and guinea-pigs, va-
ries considerably as a result of external conditions. In the rabbit
the normal temperature may be given as about 102° to 103° F. ; in
the guinea-pig it is a little lower.

In making a post-mortem examination of an inoculated animal it
is best to stretch it out on a board, belly up, by tying its legs to nails
or screws fastened in the margin of the board. When the abdomen
is dirty, as is usually the case, it should be carefully washed with a
disinfecting solution. An incision through the skin is then made in
the median line the full length of the body, and the skin is dis-
sected back so as to expose the anterior walls of the abdomen and
thorax. These cavities are then carefully opened with a sterilized
knife or scissors, and the various organs and viscera examined. At-
tention should also be given to the appearances at the point of in-
oculation. To ascertain whether the microorganism injected has
invaded the blood, smear preparations should be made with blood
obtained from a vein or from one of the cavities of the heart. It
will be well also to make a smear preparation from a cut surface of
the liver and spleen. In the various forms of acute septica3inia the
spleen is usually found to be enlarged. If but few microorganisms
are present in the blood and tissues they may escape observation in
^tained smear preparations, and it will be necessary to make cultures
to demonstrate their presence. A little blood from a vein or from
one of the cavities of the heart is transferred, by means of a plati-
num loop (ose) or a sterilized collecting tube (see page 38), to a
test tube containing liquefied nutrient gelatin or agar-agar, and an
Esmarch roll tube is made. This is put aside for the development of
colonies from any scattered bacteria which • may be present. As a
rule, it will be best to make agar cultures, as these can be placed in
the incubating oven at 35° to 38° C. Stab cultures may also be
made and will serve to show the presence of microorganisms, but
will not give information as to how numerous they may be. The
roll tube also has the advantage of showing whether there is a

102 EXPERIMENTS UPON ANIMALS.

mixed infection or whether a pure culture of a single microorganism
is obtained from the blood. In the same way cultures may be made
from material obtained from the liver or spleen, and it may happen
that one or both of these organs contain bacteria when none are
found in the blood. Before passing the platinum needle or collect-
ing tube into the organ, the surface, which has been more or less ex-
posed to contamination, should be sterilized by applying to it a hot
spatula ; then at the moment of lifting the spatula the sterilized
needle is introduced into the interior of the organ, and the blood and
crushed tissue adhering to it at once carried over to the culture me-
dium. Or blood obtained with proper precautions from a vein, a
cavity of the heart, or the interior of the spleen or liver, may be
used to inoculate another animal.

Animals are also sometimes inoculated by excoriating the cutis
as in vaccination. They may also, in rare cases, be infected by in-
troducing cultures into the stomach, either mixed with the food in-
gested or by injection through a tube. Infection by inhalation is
accomplished by causing the animal to respire an atmosphere, in a
properly enclosed space, in which the pathogenic organism is sus-
pended, by the use of a spray apparatus for liquid cultures, or
some form of powder blower for powders containing the bacteria in
a desiccated condition.

One method of obtaining a pure culture of pathogenic bacteria
consists in the inoculation of susceptible animals with material con-
taining a pathogenic species in association with others which are not.
When the blood is invaded by the pathogenic species and the animal
dies from an acute septicaemia, we may usually obtain a pure cul-
ture by inoculating a suitable culture medium with a minute drop of
blood taken from a vein or from one of the cavities of the heart.
Sometimes, however, a mixed infection occurs and some other mi-
croorganism is associated in the blood with that one which was the
immediate cause of the death of the animal.

XII.
PHOTOGRAPHING BACTERIA.

WELL-MADE photomicrographs are unquestionably superior to
drawings made by hand as a permanent record of morphological
characters. This being the case, bacteriologists would no doubt re-
sort to this method more generally but for the technical difficulties
and the time and patience required in overcoming these. Koch, in
his earlier studies, gave much time to photographing bacteria, and
with very remarkable success. In his work on "Traumatic Infec-
tive Diseases " (1878) he says :

"With respect to the illustrations accompanying this work, I
must here make a remark. In a former paper l on the examination
and photographing of bacteria I expressed the wish that observers
would photograph pathogenic bacteria in order that their representa-
tions of them might be as true to nature as possible. I thus felt
bound to photograph the bacteria discovered in the animal tissues in
traumatic infective diseases, and I have not spared trouble in the
attempt. The smallest, and in fact the most interesting bacteria,
however, can only be made visible in animal tissues by staining
them and by thus gaining the advantage of color. But in this case
the photographer has to deal with the same difficulties as are expe-
rienced in photographing colored objects — e.g., colored tapestry.
These have, as is well known, been overcome by the use of colored
collodion. This led me to use the same method for photographing
stained bacteria, and I have, in fact, succeeded, by the use of eosin-
collodion, and by shutting off portions of the spectrum by colored
glasses, in obtaining photographs of bacteria which had been stained
with blue and red aniline dyes. Nevertheless, from the long ex-
posure required and the unavoidable vibrations of the apparatus, the
picture does not have sharpness of outline sufficient to enable it to be
of use as a substitute for a drawing, or, indeed, even as evidence of
what one sees. For the present, therefore, I must abstain from pub-
lishing photographic representations ; but I hope, at a subsequent
period when improved methods allow a shorter exposure, to be able
to remedy this defect/'

1 The paper referred to is published in Cohn's "Beitrage zur Biologic d. Pflanzen."

104 PHOTOGRAPHING BACTERIA.

Since the above was written considerable progress has been made
in removing the technical difficulties, and many bacteriologists have
succeeded in making very satisfactory photomicrographs. As speci-
mens of what may be done with the best apparatus and the highest
degree of skill, we may call attention to the photomicrographs in
the Atlas der Bakterienkunde of Frankel and Pfeiffer, and those
of Roux in the Annales of the Pasteur Institute. The writer, also,
has devoted much time to making photomicrographs which have
served as illustrations for several of his published works.

Those who have had no practical experience in making photo-
micrographs are apt to expect too much and to underestimate the
technical difficulties. Objects which under the microscope give a
beautiful picture, which we desire to reproduce by photography, may
be entirely unsuited for the purpose. . In photographing with high
powers it is necessary that the objects to be photographed be in a
single plane and not crowded together or overlying each other.
For this reason photographing bacteria in sections presents special
difficulties, and satisfactory results can only be obtained when the
sections are extremely thin and the bacteria well stained. Even
with the best preparations of this kind much care must be taken in
selecting a field for photography. It must be remembered that the
expert microscopist, in examining a section with high powers, has
his finger on the fine adjustment screw and focuses up and down to
bring different planes into view. He is in the habit of fixing his at-
tention on that part of the field which is in the focus and disregard-
ing the rest. But in a photograph the part of the field not in focus
appears in a prominent way which mars the beauty of the picture.
In a cover-glass preparation made from a pure culture, when the
bacteria are well distributed, this difficulty does not present itself, as
the bacteria are all lying in a single plane; but the portion of the field
which can be shown at one time is limited by the spherical aberra-
tion of the objective, which the makers do not seem able to overcome
in high-power lenses of wide angle, at least not without loss of de-
fining power.

Usually preparations of bacteria are stained for photography,
but with some of the larger forms, such as the anthrax bacillus,
very satisfactory photomicrographs may be made from unstained
preparations. In this case a small quantity of a recent culture is
put upon a slide, covered with a thin cover glass, and placed at once
upon the stage of the microscope. The main difficulty to be encoun-
tered results from the change of location of the suspended bacteria
resulting from the pressure of the objective in focussing. Motile
bacteria, of course, cannot be photographed in this way without first
arresting their movements by means of some germicidal agent ;

PHOTOGRAPHING BACTERIA. 105

and in general it will be found more satisfactory to fix the micro-
organisms to be photographed to a slide or cover glass by desiccation
and heat, and to stain them with one of the aniline colors.

Objects which are opaque cannot be photographed by transmitted
light, and objects which have a deep orange or red color are practi-
cally opaque for the actinic rays which are at the violet end of the
spectrum. Such objects simply intercept the light, but this gives
the outlines, and, where there are no details of structure, is all that
is required to illustrate the form and mode of grouping. Softer and
more satisfactory photomicrographs of bacteria are made when the
staining is not such as to entirely arrest the actinic rays. Among
the aniline colors Bismarck brown and vesuvin are the most suitable,
care being taken, with the larger bacteria especially, not to make
the staining too intense. Objects which are transparent for the ac-
tinic rays, or nearly so, give a very feeble photographic image, or
none at all, on account of the want of contrast in the impression
made upon the sensitive plate. This is the case when we attempt to
photograph, by ordinary white light, objects which are stained violet
or blue. But this want of contrast in the negative can be overcome
by the use of specially prepared plates and colored screens of glass
interposed between the object and the source of light. The so-called
orthochromatic plates are more sensitive to the rays toward the red
end of the spectrum than ordinary plates. They are prepared by
treating the plates with a solution of eosin, of erythrosin, or of rose
bengal (Vogel), and may now be purchased in this country from
dealers in dry plates. If we shut off the violet rays by the use of a
yellow screen, objects having a yellow or orange color may be pho-
tographed upon orthochromatic plates, although the time of exposure
will be quite long owing to the comparatively feeble actinic power
of the yellow rays.

We may also make photomicrographs of objects stained with
methylene blue or with fuchsin, because objects stained with these
colors are opaque for the rays from the red end of the spectrum, and
sufficiently so with yellow light to give a good photographic con-
trast. Frankel and Pfeiffer recommend the use of a green light-fil-
ter (green glass screen) for all preparations stained with methyl vio-
let, fuchsin, or methylene blue; and for brown-stained preparations a
pure blue light. The writer has been in the habit of using a yellow
glass screen for fuchsin-stained preparations, and has had excellent
results, but the time of exposure is necessarily long. A yellow glass
screen may be prepared by dissolving tropa3olin in negative varnish,
and pouring this upon a clean glass slide, where it is permitted to
dry.

To show bacteria in photographs in a satisfactory manner we

106 PHOTOGRAPHING BACTERIA.

require an amplification of five hundred to one thousand diameters ;
and as it is often desirable to make comparisons as to the dimen-
sions of microorganisms which resemble each other in form, it is
best to adopt a standard amplification. The writer has himself
adopted, and would recommend to others, a standard amplification
of one thousand diameters. This is about as high a magnifying
power as we can get with satisfactory definition, or as we require,
and it is a convenient number when measurements are made from
the photograph. The beginner, after having put his apparatus in
position, should focus the lines of a stage micrometer upon the
screen with the optical apparatus which he proposes to use ; then by
moving the screen forward or back as required, and carefully focus-
sing the lines, he will ascertain what is the position of the screen for
exactly one thousand diameters. If the stage micrometer is ruled
with lines which are one one-thousandth of an inch apart, it is evi-
dent that when projected upon the screen they should be one inch
apart to make the amplification one thousand diameters. But it
must be remembered that any change in the position of the optical
combination will change the amplification. If, therefore, the cover
correction of the objective is changed, or the position of the eyepiace
— if one is used — it will be necessary to again adjust the distance of
the screen.

Apparatus required. — A first-class immersion objective of one-
twelfth of an inch or higher power, a substantial stand which can be
placed in a horizontal position, and a camera which can be coupled
with the microscope tube, are the essential pieces of apparatus. If
sunlight is to be used a heliostat will also be required.

The oil-immersion objectives of any good maker may be used,
but the apochromatic objectives and projection eyepieces of Carl
Zeiss, of Jena, are especially to be recommended. Indeed, those who
can afford it will do well to get Zeiss' complete apparatus, which
includes a stand having a mechanical stage and a camera mounted
upon a metal frame conveniently provided with focussing appliances,
etc. However, good work may be done with less expensive appa-
ratus.

The stand should be substantial and well made, with a delicate,
fine adjustment. A mechanical stage is not essential, but is a great
convenience in finding and adjusting to the centre of the screen a
satisfactory field to photograph. The substage should be provided
with a good apochromatic condenser, and with appliances for moving
the condensing lens forward and back and for centring it, with dia-
phragms, etc.

By the use of a high-power objective, like the one-eighteenth-inch
oil-immersion of Zeiss, the desired amplification may be obtained

PHOTOGRAPHING BACTERIA. 107

without the use of an eyepiece ; and, as a rule, it is best not to use
an ordinary eyepiece to secure increased amplification, as this is ob-
tained at the expense of definition. But an amplifier may be used in
the tube of the microscope, as first recommended by Woodward. In
this case the amplifier must be carefully adjusted with reference to
the distance of the screen, to secure the best possible definition.

The projection eyepieces of Zeiss are constructed especially for
photography and possess a decided advantage. By the use of his
three-millimetre apochromatic oil-immersion objective and projec-
tion eyepiece No. 3 we may obtain an amplification of one thousand
diameters with excellent definition.

Light. — Sunlight is in many respects the most satisfactory for
photography, but has the disadvantage that it is not always available.
In some sections of the country weeks may pass without a single
clear day suitable for making photomicrographs. In addition to the
uncertainty arising from cloudy weather, we have to contend with
the fact that the sun is only available for use with a heliostat for a
limited time during each day, and that this time is greatly restricted
in Northern latitudes during the winter months. When sunlight is
to be employed the microscope and camera must be set up in a room
having a southern exposure on a line corresponding with the true
meridian of the place. The heliostat is placed outside the window in
such a position that when properly adjusted the light of the sun will
fall upon the condenser attached to the substage of the microscope.
The condenser must be carefully centred, so that the circle of light
falling upon the screen shall be uniform in intensity and outline. .

The calcium, magnesium, or electric light may be used as a sub-
stitute for sunlight, but they are all rather expensive, unless, in the
case of the electric light, a suitable current is available without the
expense of generating it for the special purpose in view. The writer
has obtained very good results with the calcium light, but has no ex-
perience in the use of the electric light. Woodward, as a result of
extended experiments, arrived at the conclusion that "the electric
light is by far the best of all artificial lights for the production of
photomicrographs. " He used a Grove battery of fifty elements to
generate the current, and a Duboscq lamp. The current from a
dynamo would no doubt be much cheaper and more conveniently
used, if an electric-lighting plant was in the vicinity.

The apparatus shown in Fig. 73 was designed by Mr. Pringle for
the use of the calcium light. It will serve to illustrate the arrange-
ment of the microscope and camera in connection with any other
light as well. An oil lamp may be placed in the position of the oxy-
hydrogen burner ; or, if sunlight is to be employed, a heliostat will
be placed in the same position.

108

PHOTOGRAPHING BACTERIA.

When a colored screen is used this may be placed either before
or behind the condensing lens — we prefer to place it behind, although

Neuhauss has shown that it makes no difference in the length of the
exposure.

We cannot in the present volume give full details with reference

PHOTOGRAPHING BACTERIA. 109

to the technique of making photomicrographs, but append an account
of a form of apparatus which we have used with great satisfaction :

"Photomicrography by Gaslight. — Those who have had much experience
in making photomicrographs will agree with me that one of the most essen-
tial elements of success is the use of a suitable source of illumination.

" Without question the direct light of the sun, reflected in a right line by
the mirror of a heliostat, is the most economical and, in some respects, the
most satisfactory light that can be used. But we cannot command this light
at all times and places, and it often happens that, when we are ready to de-
vote a day to making photomicrographs, the sun is obscured by clouds or
the atmosphere is hazy. Indeed, in. some latitudes and at certain seasons of
the year a suitable day for the purpose is extremely rare. The use of sun-
light also requires a room having a southern exposure and elevated above all
surrounding buildings or other objects by which the direct rays of the sun
would be intercepted. For these reasons a satisfactory artificial light is ex-
tremely desirable.

" The oxyhydrogeii lime light, the magnesium light, and the electric arc
light have all been employed as a substitute for the light of the sun, and all
give satisfactory results. I have myself made rather extensive use of the
'limelight,' and think it the best substitute for solar light with which I
am familiar. But to use it continuously, day after day, is attended with
considerable expense, and the frequent renewal of the -supply of gas which
it calls for is an inconvenience which one would gladly dispense with.

" These considerations have led some microscopists to use an oil lamp as
the source of illumination, and very satisfactory photomicrographs with
comparatively high power have been made with this cheap and convenient
light. But in my experience the best illumination which I have been able
to secure with an oil lamp has called for very long exposures when working
with high powers, and, as most of my photomicrographs of bacteria are
made with an amplification of one thousand diameters, I require a more
powerful illumination than I have been able to secure in this way. And
especially so because of the fact that a colored screen must be interposed,
which shuts off a large portion of the actinic rays, on account of the staining
agent usually employed in making my mounts. The most satisfactory
staining agents for the bacteria are an aqueous solution of fuchsin, or of
methylene blue, or of gentian violet; and all of these colors are so nearly
transparent for the actinic rays at the violet end of the spectrum tbat a
satisfactory photographic contrast cannot be obtained unless we shut off
these rays by a colored screen.

" I am in the habit of using a yellow screen for rny preparations stained
with fuchsin or methylene blue, and have obtained very satisfactory results
with the orthochromatic plates manufactured by Carbutt, of Philadelphia,
and a glass screen coated with a solution of tropaeolin dissolved in gelatin.

" But with such a screen, which shuts off a large portion of the actinic
light and increases the time of exposure three- or fourfold, the use of an
oil lamp becomes impracticable with high powers, on account of the feeble^
ness of the illumination.

"These considerations have led me to experiment with gaslight, and the
simple form of apparatus which I am about to describe is the result of these
experiments. I have now had the apparatus in use for several months,
during which time I have made a large number of very satisfactory photo-
micrographs of bacteria from fuchsin-stained preparations with an amplifica-
tion of one thousand diameters. My photographs have been made with the
three-millimetre oil-immersion apochromatic objective of Zeiss and his pro-
jection eyepiece No. 3. I use a large Powell and Lealand stand, upon the
substage of which I have fitted an Abbe condenser. The arrangement of
the apparatus will be readily understood by reference to the accompanying
figure.

"A is the camera, which has a pyramidal bellows front supported by the

110

PHOTOGRAPHING BACTERIA.

heavy block of wood B ; this can be pushed back upon the baseboard which
supports it, so as to allow the operator to place his eye at the eyepiece of the
microscope. When it is brought forward an aperture of the proper size ad-
mits the outer extremity of the eyepiece and shuts off all light except that
coming through the objective. C is the microscope, and D the Abbe con-
denser, supported upon the substage. E is a thick asbestos screen for pro-
tecting the microscope from the heat given off by the battery of gas burners
F. This asbestos screen has an aperture of proper dimensions to admit the
light to the condensjr D. The gas burners are arranged in a series, with
the flat portion of the flame facing the aperture in the asbestos screen E.
The concave metallic mirror G- is properly placed to reflect the light in the
desired direction. I have not found any advantage in the use of a condens-
ing lens other than the Abbe condenser upon the substage of the microscope.
The focussing is accomplished by means of the rod I, which carries at one
extremity a grooved wheel, H, which is connected with the fine adjustment
screw of the microscope by means of a cord.

I' The focussing wheel J may be slipped along the rod I to any desired
position, and is retained in place by a set screw. The rod I is supported

FIG. 74.

above the camera by arms depending from the ceiling, or by upright arms
attached to the baseboard.

" I have lost many plates from a derangement of the focal adjustment
resulting from vibrations caused by the passing of loaded wagons in the
street adjoining the laboratory in which I work. This has been overcome
to a great degree by placing soft rubber cushions under the whole appa-
ratus. " l

I have recently (1895) seen a gaslight which I believe would prove
to be a valuable substitute for ordinary street gas, and I judge that,
owing to its superior brilliancy, a single jet would suffice to replace the
five burners in a linear series which are shown in the above figure.
The gas referred to is acetylene, which may now be obtained in a
liquid form in strong metal cylinders. Reference has already been
made to the use of an oil light, and for low powers an ordinary lamp
with a flat wick may be used. That bacteria may be successfully
photographed, with an amplification of one thousand diameters, by
means of an oil lamp is shown by the beautiful photomicrographs
made by Capt. VV. C. Borden, Assistant Surgeon U. S. Army.
At my request Dr. Borden has prepared the following detailed
account of his method :

JFrom Johns Hopkins University Circulars, vol. ix., No. 81, p. 72.

PHOTOGRAPHING BACTERIA. Ill

DESCRIPTION OF APPARATUS FOR PHOTOMICROGRAPHY BY OIL

LIGHT.

The apparatus consists of a camera, hung- in a vertical position, of a
microscope with substage condensers, suitable objectives and projection
oculars, and a Laverne tri-wick, oil stereopticon with the projection objec-
tive removed.

The Light. — After trying all kinds of lamps, I found that the best illu-
mination could be obtained by using a tri-wick stereopticon with the pro-
jection objective removed, the middle wick only being lighted. The large
four-inch condensers serve to concentrate the light, while the double lantern
body prevents the radiation of heat to the microscope and shuts off all radiat-
ing-light. Consequently the microscope does not become heated, and if the
room is darkened the absence of extraneous light greatly aids in focussing on
the camera screen. The oil light itself is quite yellow and so nearly mono-
chromatic that with orthochromatic plates a color screen is seldom, if ever,
required. After experimenting by taking photographs with and without a
screen, I have found no particular difference in result even when photo-
graphing difficult bacteria, and now seldom use one. If a screen is used a
solution of bichromate of potash and sulphate of copper in dilute ammonia
water placed in a trough between the lantern and microscope gives excellent
results and does not materially lengthen the time of exposure. The lantern
is placed about twelve inches in front of the microscope and with its central
long axis in a plane which extends through the centre of the microscope
mirror, the substage condenser, the objective, ocular and centre of camera.

Microscope. — The microscope is used in the upright position. I have
used this position rather than the horizontal for several reasons. The
microscope is used on the work-table in an upright position, and in working
when an object is found which it is desired to photograph, the microscope
without changing adjustments has only to be carried to the photomicro-
graphic apparatus, placed in position, correct adjustments of light made, the
camera racked into contact and the exposure made. With a conveniently
placed dark room the whole operation will occupy but a few minutes. The
upright position is necessitated when liquid preparations, as colonies of
bacteria floating on liquefied gelatin, are to be photographed, or when the
microscope is used for clinical photomicrography, as in photographing uri-
nary deposits in urine, blood corpuscles in Thoma blood counter, etc. In
bacteriological work where the bacteria are stained on the cover and after
mounting the balsam is not quite dry, the cover is apt to slip if the micro-
scope is used horizontally, but this does not occur with the microscope placed
vertically. The horizontal position and long extension of camera is neces-
sary for certain work, particularly where large pictures (i.e., over four
inches in diameter) have to be taken, or where it is desired to obtain high am-
plification by extension of camera rather than by high eyepiecing, or in
photographing test diatoms with very high amplifications. For practical
work, however, up to amplifications of one thousand diameters, and for
photographs for illustration or reproduction, which are seldom required of
over three and one-half or four inches in diameter, the upright position is
much to be preferred on account of its ease of application and its practical
advantages.

Camera. — The upright position of the microscope necessitates a similar
position for the camera. To allow easy working, the camera is hung on a
rack- work attached to a rigid upright. " The upright is placed to the right of
the microscope so that it will be out of the way while working.

Both the upper and the lower ends of the camera are movable on the
rack-work. The upper end, which carries the screen and plate-holder, is
movable, in order that different amplifications within limits can be gotten
with the same objective. The lower end is movable that it may be racked

112 PHOTOGRAPHING BACTERIA.

up and out of the \vay and allow the operator to manipulate the microscope
before attaching1 the camera. The bellows has an extension of two feet,
measured from the eyepiece of the microscope to the focussing screen. This,
with a two-millimeter objective and projection ocular 4, gives an amplifica-
tion of one thousand diameters. With less extension of bellows and lower
objectives amplifications ranging down to ten diameters may be obtained.
In focussing, the operator can, by standing1, observe the image on the screen
with a focussing1 glass and manipulate the fine adjustment of the microscope
with his hand without using- a focussing' rod, though a suitable focussing rod
can be easily fastened to the camera upright if desired.

Setting Up the Apparatus. — The camera being hung 011 the rack-work
the microscope is placed beneath it, a stage micrometer is placed on the stage
and a medium-power objective and eyepiece attached to the microscope.
Light is reflected from the lantern upon the object by the mirror of the
microscope, the observer accurately centres the micrometer, then removing
the working1 eyepiece a projection ocular is inserted, the camera racked
down, and with the image of the micrometer projected on the camera screen
the microscope is moved in such position that the centre of the micrometer
image is exactly in the centre of the screen. This position of the microscope
is marked once for all, and whenever afterward the microscope is placed in
the same place the centre of the object will be projected on the centre of the
screen. To correctly place the lantern, a lower-power objective is used, to-
gether with a high-power (Abbe) condenser. The objective is accurately
focussed on the lines of the stage micrometer; by adjusting the substage con-
denser a clear image of the lamp flame is projected on the plane of the ob-
ject (micrometer) and the lantern is moved to such position that the image
will be central. If the camera is attached, the image will appear central
on the focussing screen.

This position of the lantern, like that of the microscope, should be fixed.

To Photograph. — In photographing- by oil light with all but the lowest
powers some form of substage condenser is necessary. This is due to the fact
that the source of light must always be focussed on the object in order to give
proper definition. In working with the objectives of four millimetres or
lower, it will be found advantageous to use objectives of lower power as
substage condensers, for it will be found that if placed in the substage for
ordinary work they greatly improve the definition of objects. In fact it may
be laid down as a general rule that whatever with a given light gives the
best definition to the observer's eye will give the sharpest photographic image.
Consequently, in high-power work where a condenser is used it will seldom
be necessary to change the microscope attachments when a photograph has
to be taken ; for in bacteriological work the Abbe condenser which gives
good definition will, when properly adjusted, give good photographic defi-
nition also, statements to the contrary notwithstanding.

To photograph, place the microscope and lantern in position, light the
centre wick of the lamp, place a ground glass between the lamp and camera,
and focus the objective accurately on the object. The ground glass is used
only to reduce the light which might otherwise injure the observer's eye.

The ground glass is then removed, a fine wire screen placed close against
the front of the lantern condenser, and by means of the substage condenser
an image of the screen is projected accurately on the object. This is very
important, for it is necessary that the light should be accurately focussed on
the object in order to produce sharp definition. After focussing the light, the
screen is removed and an opal glass is put in its place. On looking through
the eyepiece a clear sharp image of the object will be seen. If an Abbe con-
denser is used the iris diaphragm of the condenser should now be carefully
opened and closed until such an aperture is obtained that to the observer's
eye the object appears to the best advantage. The opal glass is now removed,
the camera attached to the microscope, and the projected image focussed on
the camera screen, preparatory to exposure.

PHOTOGRAPHING BACTEPtlA. 113

Plate Used. — Orthochromatic plates only should be used. Of these I use
the Cramer rapid, isochromatic plate exclusively. With these when photo-
graphing bacteria and using an amplification of one thousand diameters the
exposure will vary from one and one-half to three minutes, two minutes
being about the average.

It is with these plates that I have found a color screen unnecessary, and
since using them I have had 110 difficulty in photographing bacteria, for they
are particularly sensitive to the yellow-colored oil light.

Possibly other makes of orthochromatic plates might be found to work
equally well, but the oil light works so very well with the Cramer isochro-
matic that I have had no desire to try others.

Development. — For development, I have obtained best results with for-
mulas in which hydrochinon either alone or with some other developing
agent is used. The following gives excellent results, and I prefer it to other
developers as it gives good clear negatives of sufficient contrast and
gradation :

No. i.

Water, ....... 10 ounces.

Sodium sulphite, . ... 1 ounce.

Potassium bromide, . . ... 10 grains.

Hydrochinon, . . ... 30 grains.

Metol, ... 40 grains.

No. 2.

Water, . ...... 10 ounces.

Sodium carbonate, ... . 300 grains.

Use equal parts of No. 1 and No. 2.

Development should be continued until sufficient density is obtained. In-
tensification should be rarely required, for with proper exposure and develop-
ment a good negative can usually be obtained. If intensification is neces-
sary, after fixing and washing the plate, I prefer to use a saturated aqueous
solution of bichloride of mercury, followed by washing, the application of
dilute ammonia water, and a final washing.

Students who desire to perfect themselves in the art of making
photomicrographs are advised to first make themselves familiar with
the technique of photography with a landscape or portrait camera,
and not to undertake the more difficult task of photographing bac-
teria until they know how to make a good negative and to judge
whether an exposure has been too long or too short, etc.
8

PLATE I.

PHOTOMICROGRAPHS OF BACTERIA MADE BY GASLIGHT,

FIG. 1. — Streptococcus cadaveris, from a culture in aguacoco; stained
with fuchsiu. x 1,000. (Sternberg.)

FIG. 2. — Streptococcus Havaniensis. X 1,000. From a photomicro-
graph. (Sternberg.)

FIG. 3. — Bacillus cuniculicida Havaniensis, from peritoneal cavity of
inoculated rabbit, showing leucocytes containing bacilli and free bacilli;
stained with fuchsin. X 1,000. (Sternberg.)

FIG. 4. — Bacillus cadaveris, smear preparation from yellow-fever
liver kept for forty-eight hours in an antiseptic wrapping (Havana,
1889); stained with fuchsin. x 1,000. (Sternberg.)

Note. — All of the above photomicrographs were made with the three-
millimetre apochromatic horn. ol. iin. objective and projection eye-piece
of Zeiss.

PLATE II.

PHOTOGRAPHS OF COLONIES (iN ESMARCH ROLL TUBES) AXD OF TEST-
TUBE CULTURES.

FIG. 1. — Colonies of Bacillus leporis lethalis, in gelatin roll tube,
end of forty-eight hours at room temperature. X 5. (Sternberg.)

FIG. 2. — Colonies of Bacillus coli similis in gelatin roll tube, end of
twenty-four hours at 22° C. x 10. (Sternberg.)

FIG. 3. — Stick culture of Bacillus coli similis in nutrient gelatin, end
of seven days at 20° C. (Sternberg.)

FIG. 4. — Stick culture of Bacillus intestinus motilis in nutrient gel-
atin, end of four days at 22° C. (Sternberg.)

FIG. 5. — Stick culture of Bacillus leporis lethalis in nutrient gelatin,
end of eight days at 22° C. (Sternberg.)

FIG. 6. — Stick culture of Micrococcus tetragenus versatilis in nutrient
gelatin, end of two weeks at 22° C. (Sternberg.)

FIG. 7. — Colonies of Bacillus cuniculicida Havaniensis in gelatin roll
tube, end of forty-eight hours at 21° C. X 10. (Sternberg.)

FIG. 8. — Colonies of Bacillus coli communis in gelatin roll tube, end
of forty-eight hours at 22° C. X 10. (Sternberg.)

PLATE I.

STERNBERG'S BACTERIOLOGY.

v v * v , V

X v .- T

T-
*

Pig. 1.

Pig. 2.

i* I •

X '-

'i**LV^ !'?»•>

Fig. 3.

_i f\ *\ s* I

/* *£%JV*f •

~x tifffy}

' — >^i O fif-

^-*T *•* LVLl^L*

Fig. 4.

PHOTOMICROGRAPHS BY GAS LIGHT.

PLATE II.

STERNBERG'S BACTERIOLOGY.

Figr. 1.

Figr. 3.

Fig;. 4.

Figr. 2.

Figr. 5.

Figr. e.

jjjg

•W rv

Pig. 7.

Fig;. 8.

COLONIES AND TEST TUBE CULTURES.

PART SECOND.

GENERAL BIOLOGICAL CHARACTERS:

INCLUDING AN ACCOUNT OF THE ACTION OF ANTISEPTICS

AND GERMICIDES.

I. STRUCTURE, MOTIONS, REPRODUCTION. II. CONDITIONS OF GROWTH.

III. MODIFICATIONS OF BIOLOGICAL CHARACTERS. IV. PRODUCTS OF

VITAL ACTIVITY. V. PTOMAINES AND TOXALBUMINS. VI. INFLUENCE

OF PHYSICAL AGENTS. VII. ANTISEPTICS AND DISINFECTANTS

—GENERAL ACCOUNT OF THE ACTION OF. VIII. ACTION OF

GASES AND OF THE HALOID ELEMENTS UPON BACTERIA.

IX. ACTION OF ACIDS AND ALKALIES. X. ACTION OF
VARIOUS SALTS. XI. ACTION OF COAL-TAR PRO-
DUCTS, ESSENTIAL OILS, ETC. XII. AC-
TION OF BLOOD SERUM AND OTHER OR-
GANIC LIQUIDS. XIII. PRACTICAL
DIRECTIONS FOR DISINFECTION.

PART SECOND.

I.
STRUCTURE, MOTIONS, REPRODUCTION.

THE bacteria are unicellular vegetable organisms, and consist of
a cell membrane enclosing transparent and apparently structureless
protoplasm. The very varied biological characters which distin-
guish different species make it evident, however, that there are es-
sential differences in the living cell contents, although these differ-
ences are not revealed by our optical appliances. And among the
bacteria, as in the cells of higher plants and animals, the peculiar
biological characters of a species are transmitted to the cellular pro-
geny of each individual cell. These characters are, however, sub-
ject to various modifications as a result of differing conditions of
environment, as is the case with plants and animals higher in the
scale of existence, and in this way more or less permanent varieties
are produced. It is probable that among these lowly plants species
are evolved more quickly, as a result of the laws of natural selec-
tion, in the struggle for existence, than among those of more com-
plex organization. Still, this has not been proved, and, on the other
hand, we have ample evidence that widely distributed species exist
having very definite morphological and biological characters which
enable us to recognize them wherever found.

It has generally been supposed that these simple vegetable cells
are destitute of a nucleus, but a recent author (Frankel) suggests
the probability that a nucleus may exist, although it has not been
demonstrated. This suggestion is based upon the fact that in stain-
ing bacteria very quickly it sometimes happens that a portion of the
protoplasm is sharply differentiated by taking the stain more deeply
than the remaining portion.

Sjobring in 1892 made an investigation for the purpose of
ascertaining the structure of bacterial cells. Various methods
were employed, but the most satisfactory results were obtained by
fixing with nitric acid, with or without alcohol, and without pre-

118 STRUCTURE, MOTIONS, REPRODUCTION.

vious drying ; the preparations were then stained with carbol-meth-
ylene-blue or carbol-fuchsin solution ; they were decolorized with
nitric acid and examined in glycerin or in water. By this procedure
the author named was able to demonstrate two kinds of corpuscles.
One of these may be seen just inside the cell wall ; it stains deeply
with the carbol-fuchsin solution. The other lies in a position analo-
gous to that occupied by the nucleus of vegetable cells higher in the
scale, and resembles this both in its resting condition and in the
process of indirect division.

In his addross bofore the International Medical Congress of Ber-
lin (1800) Koch says :

' We had not succeeded, in spite of the constantly improving
methods of staining and in spite of the use of objectives with con-
stantly increasing angles of aperture, in learning more with reference
to the interior structure of the bacteria than was shown by the origi-
nal methods of staining. Only very recently new methods of stain-
ing appear to give us further information upon the structure of the
bacteria, inasmuch as they serve to differentiate an interior portion
of the protoplasm, which should probably be regarded as a nucleus,
from an exterior protoplasmic envelope from which is given off the
organ of locomotion, the flagellum."

Although usually transparent, the protoplasm sometimes presents
a granular appearance. The botanist Van Tieghem claims to have
found chlorophyll grains in some water bacteria studied by him, and
in the genus Beggiatoa grains of sulphur are found embedded in the
protoplasm of certain species.

The granules in bacterial cells which may be demonstrated by
special methods of staining are of two kinds : metachromatic gran-
ules and polar granules. The former lie in the protoplasm, and
when properly stained may present the appearance of a short chain
of cocci lying in the bacterial cell. To demonstrate their presence
Ernst recommends the use of Loffler's solution of methylene blue.
This is placed upon a cover-glass preparation and heated over a
flame until steam begins to rise. After washing in water the cover
glass is placed for a minute or two in a watery solution of Bismarck
brown. This shows the granules stained blue and the surrounding
protoplasm brown. The polar granules are often seen in prepara-
tions stained in the usual way with an aniline staining solution.
Some observers have regarded these stained granules as spores, but
this has not been demonstrated, and cultures containing them show
no greater resistance to heat or to chemical agents than that estab-
lished for the vegetative cells of the particular species in which they
are found. It seems probable that the matachromatic granules re-
sult from degenerative changes rather than that they are reproduc-
tive bodies.

STRUCTURE, MOTIONS, REPRODUCTION. 119

The cell membrane in certain species appears to be very flexible,
as may be seen in those which have a sinuous motion. It is not
easily recognized under the microscope, but by the use of reagents
which cause the protoplasm to contract may be demonstrated — e.g.,
by iodine solution. Outside of the true cell membrane a gelatinous
envelope — so-called capsule — is sometimes seen. This may perhaps
be, as claimed by some authors, nothing more than a jelly-like thick-
ening of the outer layers of the cell wall. This jelly-like material
causes the cells to adhere to each other, forming zooglcea masses.
In some cases the growth upon the surface of a culture medium is
extremely viscid, and may be drawn out into long threads when
touched with a platinum needle, owing to the gelatinous intercellular
substance by which the cells are surrounded.

There is but little more to be said of the structure of these minute
organisms, except to mention the fact that the motile species are
provided with slender, whip-like appendages called flagella. The
micrococci in general are not endowed with the power of executing
spontaneous movements, and they are not provided with flagella.
Bat recently two motile species have been described, and in one of
these — Micrococcus agilis of Ali-Cohen — the presence of flagella has
been demonstrated.

Many of the bacilli and spirilla are actively motile, and the pre-
sence of flagella, which has long been suspected, has recently been
demonstrated for a considerable number of species by Loffler and
others.

It must be remembered that the molecular movement which is
common to all minute particles suspended in a fluid is a vibratory
motion in situ, which does not change the relative position of the
moving particles. This so-called Brownian movement has frequently
been mistaken for a vital motion, as has also the movement due to
currents in the liquid in which non-motile organisms are suspended.
The latter is to be distinguished by the fact that the microorganisms
are all carried in one direction. This movement due to a current, in
connection with the vibratory Brownian movement, is very deceptive,
and it is often hard for a beginner in bacteriological study to con-
vince himself that what he sees is not a vital movement. But in
true vital movements we have progression in different directions, and
the individual microorganisms approach and pass each other, often
in a most vigorous and active manner, passing entirely across the
field of view or changing direction in an abrupt way. Sometimes
the motion is slow and deliberate, the bacillus progressing with a to-
and-fro motion, as if propelled by a trailing flagellum ; or it may be
serpentine when the moving filament is flexible ; or again it is
a darting forward motion which is so rapid that the eye can scarcely
follow the moving body. The spirilla have a rotary movement as

120 STRUCTURE, MOTIONS, REPRODUCTION.

well as a progressive one, and this is often extremely rapid. Some-
times bacilli spin around with a rotatory motion, as if they were an-
chored fast to a fixed point, as they may be by the flagellum being
attached to the slide or cover glass. Frequently, in a pure culture,
the individual bacilli may be seen to come to rest, and, after an inter-
val of repose, to dart forward again in the most active way. Or we
may find, on examining the same culture at different times, that
upon one occasion there is no evidence of vital movements, and on
another all of the bacilli are actively motile. These differences de-
pend upon the age of the culture, temperature conditions, etc.

Reproduction by binary division is common to all of the bacte-
ria, and in many species this is the only mode of reproduction known.
When circumstances are favorable for rapid multiplication the indi-
vidual cells grow in length, and a constriction occurs in the middle
transverse to the long diameter. This becomes deeper, and after a
time the cell is completely divided into two equal portions, which
again divide in the same way. Separation may be complete, or the
cells may remain attached to each other, forming chains (strepto-
cocci) or articulated filaments (scheinfdden of the Germans).

The bacilli and spirilla divide only in a direction transverse to the
long diameter of the cells, but among the micrococci division may
occur either in one direction, forming chains ; or in two directions,
forming tetrads ; or in three directions, forming " packets "of eight
or more elements. The staphylococci, in which the cells do not re-
main associated, divide indifferently in any direction.

The rapidity of multiplication by binary division varies greatly in
different species, and in the same species depends upon conditions re-
lating to the culture medium, age of the culture, temperature, etc.
Under favorable conditions bacilli have been observed to divide in
twenty minutes, and it is a matter of common laboratory experience
that colonies of considerable size and containing millions of bacilli
may be developed from a single cell in twenty-four to forty-eight
hours. A simple calculation will show what an immense number of
cells may be produced in this time as a result of binary division oc-
curring, for example, every hour. The progeny of a single cell
would be at the end of twenty-four hours 16,777,220, and at the end
of forty-eight hours the number would be 281,500,000,000.

Some of the earlier observers have noted the presence of oval or
spherical refractive bodies in cultures containing bacilli ; but that
these were reproductive elements, although suspected, was not de- .
monstrated until a comparatively recent date. Pasteur was one of
the first to point out the fact that certain bacteria have two modes of
reproduction — by fission and by the formation of endogenous spores ;
but the first careful study of the last-mentioned method was made by

STRUCTURE, MOTIONS, REPRODUCTION. 121

Koch in his classical stud}' of the anthrax bacillus (1878), and by
Cohn, who studied the formation of spores in Bacillus subtilis.

These reproductive bodies serve the same purpose in the preserva-
tion of species as the seeds of higher plants. They resist desiccation
and may retain their vitality for months or years until circumstances
are favorable to their development, when, under the influence of heat
and moisture, they reproduce the vegetative form — bacillus or spiril-
lum— with all of its biological and morphological characters. They
are composed of condensed protoplasm which retains the vital char-
acters of the soft protoplasm of the mother cell from which it has
been separated ; and it is evident that whether reproduction occurs
by fission or by the formation of endogenous spores, the protoplasm
of the cells in a pure culture of any microorganism is simply a sepa-
rated portion of the protoplasm of the progenitors of these cells.

Some of the bacilli grow out into long filaments before the forma-
tion of spores occurs ; and these filaments may be associated in bun-
dles or intertwined in irregular masses. At first the protoplasm of the

FIG. 75.

filaments is homogeneous, but after a time it becomes segmented,
and later the protoplasm of each segment becomes condensed into
a spherical or oval refractive body, which is the spore. For a time
these are retained in a linear position by the cell membrane of the
filament (Fig. 75, a), but this is after a while dissolved or broken
up and the spores are set free. In liquid cultures they sink to the
bottom as a pulverulent precipitate, and upon the surface of a solid
medium they form a layer which is usually of a white or yellowish-
white color, and which, when examined under the microscope, in old
cultures is found to consist almost entirely of shining spherical or
oval bodies which do not stain, by the ordinary methods, with the
aniline colors. While many of the bacilli during the stage of spore
formation grow out into long filaments, others do not, and one or
more spores make their appearance in rods of the ordinary length
which characterizes the species. These may be located in the centre
of the rod or at one extremity (Fig. 75, b). It sometimes occurs
that when a single central spore is formed the rod becomes very
much enlarged in its central portion, assuming a spindle shape (Fig.

122 STRUCTURE, MOTIONS, REPRODUCTION.

75 c) ; or one extremity may be enlarged, producing forms such as
are shown in Fig. 75, d. Some of the smaller spherical spores meas-
ure less than 0. 5 //. in diameter, but they are, for the most part, oval
bodies having a short diameter of 0.5 to 1 //- and a long diameter of
one to two /^, or even more. They are enveloped in a cellular en-
velope which, according to some observers, consists of two layers—
an exosporium and an endosporium.

The mode of spore formation shown in Fig. 75, c and d, has been
adopted by some authors as a generic character. When the spores
are located in the central part of the rods, giving rise to a spindle-
shaped body, as at c, the bacilli are assigned to the genus Clos-
tridium; when located at one end, as at d, the bacilli are shaped
like a drumstick, and this mode of spore formation is used as the
distinguishing characteristic of the genus Plectridium. Hueppe
groups all rod-shaped bacteria which form endospores under the
generic name Bacillus, with three sub-genera : Bacillus, straight
rods; Clostridium, spindle-shaped rods; Plectridium, drumstick-
shaped rods.

The germination of spores has been studied by Prazmowski,
Brefeld, and others. The process is as follows : By the absorption
of water they become swollen and pale, losing their shining, refrac-
tive appearance. Later a little protuberance is seen upon one side
or at one extremity of the spore, and this rapidly grows out to form
a rod which consists of soft-growing protoplasm enveloped in a
membrane which is formed of the endosporium or inner layer of the
cellular envelope of the spore. The outer envelope, or exosporium,
is cast off and may be seen in the vicinity of the newly formed rod
(Fig. 76). Sometimes the vegetative cell emerges from one extrem-

ity of the oval spore, as shown at a, Fig. 76, and in other species the
exosporium is ruptured and the bacillus emerges from the side, as
seen at b.

The considerable resistance of these endogenous spores to desic-
cation, to heat, and to various chemical agents is an important fact
both from a biological and from a hygienic point of view, and will
be fully considered in a subsequent chapter. The fact that certain
bacilli and spirilla do not withstand a temperature of 80° to 90° C.,
which does not destroy the vitality of known spores, leads to the in-

STRUCTURE, MOTIONS, REPRODUCTION.

ference that they do not form similar reproductive bodies. But re-
productive elements of a different kind are described by some botan-
ists as being produced during the development of these bacteria, and
also of the micrococci. These are the so-called arthroxpores. In
the process of binary division certain cells in a chain ma}7 be observed
to be somewhat larger than others and to refract light more strongly.
The same may be true of certain cells in a culture in which the ele-
ments are not united in chains. These cells are believed by De Bary
and others to have greater resisting power to desiccation than the re-
maining cells in the culture, and to serve the purpose of reproductive
elements.

Hueppe groups all rod-shaped bacteria which do not form endo-
spores under the generic name Arthrobacterium. This author be-
lieves that, as a rule, bacilli which do not form endospores under
certain circumstances produce more resistant cells which "take
charge of the perpetuation of the species under the guise of a resting
stage or spore." According to Hueppe, true arthrospores are spheri-
cal in form.

It has generally been supposed that spore formation is most likely
to occur when the pabulum for supporting the growth of the vegeta-
tive form is nearly exhausted. But, as pointed out by Frankel, facts
do not support this view, as many species form spores when condi-
tions are most favorable for a continued development. An abundant
supply of oxygen favors the formation of spores in aerobic species,
and, in some instances at least, the temperature has an important in-
fluence upon spore formation. Thus the anthrax bacillus does not
form spores at temperatures below 20° C. or above 42° C.

The very interesting fact has been demonstrated by Lehman and
by Behring that a species which usually forms spores may be so
modified by certain influences that it is no longer capable of spore
production, and that such an asporogenous variety may be cultivated
for an indefinite time without showing any return to the stage of
spore formation. This was effected in Behring's experiments by
cultivating the anthrax bacillus in a medium containing some agent
detrimental to the vitality of the vegetative cells, but not in suffi-
cient quantity to restrain their development.

The pseudo-branching of the filaments in the genus Cladothrix
has been referred to in the chapter on Morphology. Recent re-
searches show that other bacteria heretofore included in the -genus
Bacillus may also present branching forms. This is especially true
of the tubercle bacillus, which when obtained from cases of fowl
tuberculosis not infrequently exhibits a sort of branching. Hueppe
and Fischel have also demonstrated the presence of branching forms
of the bacillus of mammalian tuberculosis, and as a result of his ob-

124 STRUCTURE, MOTIONS, REPRODUCTION.

servations Hueppe has "arrived at the definite opinion that the
tubercle bacillus is the parasitic growth- form of a pleomorphic mould,
and is not a true bacterium at all." Metschnikoff has reported his
observations of branching forms of the cholera spirillum, Frankel of
the diphtheria bacillus, and Semmer of the bacillus of glanders, but
whether these are examples of pseudo-branching, such as occurs in the
genus Cladothrix, or a veritable dichotomous growth such as occurs
in the mould fungi, has not been definitely determined.

The chemical composition of the bacterial cells has been inves-
tigated by Nencki, Brieger, and others. Putrefactive bacteria culti-
vated in a two-per-cent solution of gelatin, and which produced an
abundant intercellular substance connecting the cells in zoogloea
masses, were found by Xeiicki to have the following composition :
Water, 84.26 per cent; solids, 5.74 per cent, consisting of albumin
87.46 per cent, fat 6.41, ash 3.04, undetermined remnant 3.09.
The albuminous substance, according to Nencki, is not precipitated
by alcohol, and differs in its chemical composition from other known
substances of this class. He calls it wykoprotein and gives the fol-
lowing as its chemical composition : C, 52.32 percent; H, 7.55 per
cent ; N", 14. 75 per cent. It contains no sulphur and no phosphorus.
The spores of the anthrax bacillus, according to Nencki, do not con-
tain mykoprotein, but a peculiar albuminous substance which he
calls anthrax-protein. Brieger analyzed a gelatin culture of Fried-
lander's bacillus, with the following result : Water, 84.2 per cent ;
solids, 5.8 per cent, containing 1.74 per cent of fats. After removal
of the fat the solids gave an ash of 30.13 per cent ; this contains cal-
cium phosphate, magnesium phosphate, sodium sulphate, and sodium
chloride. The amount of nitrogen in the dried substance after re-
moval of the fat was 9. 75.

II.

CONDITIONS OF GROWTH.

BACTERIA only grow in presence of moisture, under certain condi-
tions of temperature, and when supplied with suitable pabulum. As
they 'do not contain chlorophyll, they cannot assimilate carbon diox-
ide, and light is not favorable to their development.

The aerobic species obtain oxygen from the air and cannot grow
unless supplied with it. The anaerobic species, on the other hand,
will not grow in the presence of oxygen, and must obtain this ele-
ment, as they do carbon and nitrogen, from the organic material
which serves them as food.

As a class the bacteria are supplied with nutriment by the higher
plants and animals, the dead tissues of which they appropriate, and
which it is their function to decompose, releasing the organic ele-
ments as simple compounds which may again be assimilated by the
chlorophyll-producing plants.

Water is essential for the development of bacteria, and many spe-
cies have their normal habitat in the waters of the ocean, of lajses,
and of running streams ; others thrive upon damp surfaces or in the
interior of moist masses of organic material. Many species grow in-
differently either in salt or fresh water, but it is probable that cer-
tain species will be found peculiar to the waters of the ocean. Some
of the water bacteria multiply in the presence of an exceedingly
minute amount of organic pabulum, or even in distilled water. This
is shown by the experiments of Bolton and others. The author
named tested two species of water bacteria (Micrococcus aquatilis
and Bacillus erythrosporus) in the following manner : Ten cubic
centimetres of distilled water in a test tube were infected with a small
quantity of a culture of one of these microorganisms. A drop from
this tube was transferred' to the same quantity of distilled water in
a second tube, and from this to a third. The number of bacteria in
this tube No. 3 was now ascertained by counting, and it was put
aside for two or three days, at the end of which time the number was
again estimated by counting. In every case there was an enormous
increase in the number of bacteria. In order to be sure that the dis-

126 CONDITIONS OF GROWTH.

tilled water was pure, it was distilled a second time in a clean glass
retort, but the result was the same. Bolton remarks, with reference
to these results: "If we seek to explain this remarkable fact we
must remember, in the first place, what an extremely small abso-
lute mass is represented by an enormous number of bacteria, and
what a minute amount of material is required for the formation of
this mass. In ten cubic centimetres of distilled water, in the experi-
ment last referred to, there were about twenty million bacteria (two
million per cubic centimetre). If we estimate the diameter of each
at one yu, with a specific weight of 1, the absolute weight would
be for the entire number one-one-hundredth of a milligramme—
that is to say, a quantity which cannot be determined by any of our
methods of weighing."

Bolton supposes that the small amount of organic pabulum re-
quired fell into the water in the shape of dust, or was attached to the
walls of the test tube in spite of all the precautions taken.

Nitrogen is chiefly obtained from albuminoid substances, but
Pasteur has shown that it may also be obtained from ammonia.
This is shown by cultivating bacteria in a medium containing an
ammonia salt, as in the following : *

PASTEUR'S SOLUTION.

Distilled water, ...... 100

Cane sugar, ...... 10

Tartrate of ammonia, ...... 1

Ashes of one gramme of yeast, .... 0.075

CORN'S SOLUTION.

'Distilled water, ...... 100

Tartrate of ammonia, ..... 1

Ashes of yeast, ....... 1

Many bacteria multiply abundantly in these solutions.

Carbon is obtained from the various organic substances contain-
ing it ; among others, from starch, sugars, glycerin, organic acids
and their salts, etc.

Temperature. — There are certain limits of temperature within
which development may take place, but these dift'er greatly with
different species. As a rule, growth is arrested when the tempera-
ture falls below 10° C. (50° F.), but some species multiply at a still
lower temperature. Thus Bolton observed a very decided increase
in certain water bacteria kept in an ice chest at 6° C., and other ob-
servers have witnessed development at the freezing temperature.

Most saprophytic bacteria grow within rather wide temperature
limits, but the rapidity of development is greatest at a certain favor-
able temperature, which is usually between 25° and 30° C. The

CONDITIONS OF GROWTH. 127

parasitic species have a more restricted range, which approaches the
normal temperature of the animals in which they habitually de-
velop. At 40° C. (104° F.) growth, as a rule, ceases, hut there are
some notable exceptions to this rule.

Miquel some years ago found a bacillus in the water of the Seine
which grew at a temperature of 69° to 70° C. ; Van Tieghem reports
having observed species in thermal waters capable of growth at a
still higher temperature (74° C.) ; and Globig has more recently ob-
tained from garden earth several species which multiplied at G5° C.
Some of the species found by the last-named observer were even
found to require a temperature of about 60° for their development ;
and yet this temperature is quickly fatal to a large number of the
best known species.

Loiv temperatures, while arresting the growth of bacteria, do not
destroy their vitality. This has been demonstrated by numerous ex-
periments, in which they have been exposed for hours in a refrigerat-
ing mixture at —18° C. Frisch has even subjected them to a tempe-
rature of — 87° C. by the evaporation of liquid carbon dioxide, and
found that they still grew when placed in favorable conditions.

Parasitism. — The strict parasites grow only in the bodies of liv-
ing animals, or in artificial media kept at a suitable temperature.
As examples we may mention the bacillus of tuberculosis, the bacil-
lus of leprosy, the micrococcus of gonorrhoea, the spirillum of re-
lapsing fever. There is also a large class of facultative para-
sites which, when introduced into the body of a susceptible animal,
multiply in it, and may continue to live as parasites so long as they
are transferred from one animal to another, but which are also able
to live as saprophytes independently of a living host. To this class
belong the pus cocci, the bacillus of typhoid fever, the spirillum of
cholera, and many others.

It seems extremely probable that the strict parasites were at one
time capable of living a saprophytic existence, and that their restric-
tion to a parasitic mode of life has been effected in course of time in
accordance with the laws of natural selection. This view is sup-
ported by the fact that the tubercle bacillus, which has been regarded
as a strict parasite, which can only be cultivated artificially under
very special conditions, has been shown co be capable of modification in
this regard to such an extent that when cultivated for a time in a favor-
able medium — bouillon with five per cent of glycerin — it will even grow
in ordinary bouillon made from the flesh of a calf or a fowl (Roux).

Reaction of Medium. — Some bacteria grow readily in a medium
having an acid reaction, while the slightest trace of acidity prevents
the development of others. As a rule, the pathogenic species require
a neutral or slightly alkaline culture medium.

128 CONDITIONS OF GROWTH.

While many species grow in various media and under various
conditions of temperature, etc., others are greatly restricted in this
regard ; thus Bumm only succeeded in cultivating the gonococcus
upon human blood serum, and even upon this was not able to
carry it through a series of successive cultures. It is very probable
that certain species can only grow in association with others which
elaborate products necessary for their development.

Substances favorable for the growth of a particular species may
restrain its development if present in too large an amount. Thus
the phosphorescent bacilli multiply abundantly in a nutrient solution
containing 2. 5 per cent of sodium chloride ; but this amount would
restrain the development of some other species, and a considerable
increase in the quantity of salt prevents the growth of all microor-
ganisms. In the same way the addition of two per cent of glucose
to culture solutions is favorable for the development of certain spe-
cies, and especially for the anaerobic bacteria ; but a concentrated
solution of the same substance prevents the growth of all bacteria.

The influence of one species upon the groivth of another has
been studied by various bacteriologists, and especially by Sirotinin
and by Freudenreich. When several species are associated in the
same culture one may take the precedence and the others may de-
velop later ; or two or more species may develop at the same time ;
or the growth of one species may prevent the development of an-
other, either (a) by exhausting the pabulum necessary for its growth
or (6) by producing substances which inhibit the development of an-
other species or destroy its vitality.

Freudenreich found, as a result of his numerous experiments,
that the following species cause a change in bouillon which renders
it unfit for the growth of other species : Bacillus pyocyaneus, Bacil-
lus cyanogenus, Bacterium phosphorescens, Bacillus prodigiosus, Spi-
rillum cholerse Asiaticse. The following species do not cause such a
change in bouillon as to render it unfit for the growth of other spe-
cies : Bacillus typhi abdominalis, Bacillus aiithracis, Bacillus septi-
ca3mise hsemorrhagica3, Spirillum tyrogenum. The following have a
decided antagonism : Bacillus pyogenes fcetidus prevents the growth
of Spirillum cholerse Asiaticse ; Micrococcus roseus prevents the
growth of Micrococcus tetragenus. The cholera spirillum will not
grow in sterilized cultures of Bacillus pyocyaneus, or in bouillon
which has served for a previous culture of the same microorganism
(Kitasato). Other bacteria which fail to grow in bouillon which
has already served for the cultivation of the same species are Bacil-
lus typhi abdominalis, Bacillus cyanogenus, Bacillus prodigiosus,
Micrococcus roseus, etc. (Freudenreich).

III.

MODIFICATIONS OF BIOLOGICAL CHARACTERS.

WE have already referred to the production of an asporogenous
variety of the anthrax bacillus. This was effected by Behring by
cultivation in media containing small amounts of hydrochloric acid,
caustic soda, methyl violet, malachite green, and various other
agents. This is only one of many instances of a change in biologi-
cal characters due to changed conditions of environment. We have
abundant experimental evidence that growth may occur under ad-
verse conditions when the species is gradually habituated to these
conditions. Thus the temperature limitations may be passed by suc-
cessive cultivations at temperatures approaching these limits, and
bacteria may grow in the presence of agents which in a given pro-
portion have a complete restraining influence upon their develop-
ment. For example, in the experiments of Kossiakoff, published in
the Annales of the Pasteur Institute (vol. i.), it was found that the
several species tested all became habituated to the presence of anti-
septic agents in proportions which at first completely restrained
their growth.

This modification of biological characters is well shown in the
case of the chromogenic bacteria, some of which only form pig-
ment under exceptionally favorable conditions of growth. It has
been shown by several observers that iion-chromogenic varieties
of some of the best known chromogenic species may be produced
by special methods of cultivation. Thus Wasserzug obtained a
non-chromogenic variety of the bacillus of green pus (Bacillus
pyocyaneus) by the action of time added to that of antiseptics. He
says: " These two actions combined have permitted me to obtain
cultures which remained without color in a durable way, and in
which, consequently, the chromogenic function was abolished by
heredity." In the case of a chromogenic bacillus obtained by the
writer in Havana (my Bacillus Havaniensis), a non-chromogenic vari-
ety was obtained from a culture on nutrient agar which had been kept
in a hermetically sealed glass tube for about a year. The variety
preserved the morphological characters of the original stock, but, al-
0

130 MODIFICATIONS OF BIOLOGICAL CHARACTERS.

though carried through successive cultures for a considerable period,
did not regain its power to produce the brilliant carmine color which
is the most striking character of the species. Katz, in cultivating
the phosphorescent bacilli isolated by him from sea water at New
South Wales, found that, after being propagated for some time in
artificial media, their power to give off a phosphorescent light was
diminished or temporarily lost. He also found that two species
which when first cultivated did not liquefy gelatin, subsequently,
after a year, caused liquefaction of the usual gelatin medium.

Modification shoivn in Cultures. — When bacteria have been
subjected to the action of heat or chemical agents, without having
their vitality completely destroyed, they often show diminished vigor
of growth. Cultures which would ordinarily show an abundant de-
velopment within twenty-four hours may not commence to grow for
several days. For this reason, in disinfection experiments, it is neces-
sary to test the question of destruction of vitality by leaving the cul-
tures for a week or -more under favorable conditions as to tempera-
ture. In plate cultures or Esmarch roll tubes a few colonies may
develop in this tardy way, showing that there was a difference in the
vital resisting power of the individual cells, some having survived
while the majority were killed. This is well illustrated by Abbott's
experiments upon the germicidal action of mercuric chloride as tested
upon Staphylococcus pyogenes aureus. Irregularities in the results in
experiments in which the conditions were identical having been no-
ticed, Abbott inferred that this was due to a difference in the resist-
ing power of individual cocci (arthrospores ?). By making cul-
tures from colonies which developed from these more resistant cocci,
and again exposing the micrococci in these cultures to mercuric chlo-
ride in the proportion of 1:1,000 for a longer time and making new
cultures from the surviving cocci, and so on, Abbott obtained cultures
in which a majority of the cells survived exposure to a solution of the
strength mentioned for ten to twenty minutes, whereas in his original
culture most of the cocci were killed by this solution in five minutes.

These changes in vital resisting power enable us to comprehend
other modifications which can only be detected by chemical or bio-
logical reactions. Thus the reducing power for various substances
may be modified by changes in the conditions of environment. And
among the pathogenic bacteria changes of a more or less permanent
nature may be induced, which are shown by a modified degree of
virulence when injected into susceptible animals.

Attenuation of Virulence may be effected by several methods,
all of which depend upon subjecting the cultures to prejudicial in-
fluences of one kind or another.

Pasteur first announced, in 1880, that the microbe of fowl cholera

MODIFICATIONS OF BIOLOGICAL CHARACTERS. 131

could be modified by special treatment in such a manner that it no
longer produced a fatal form of the disease. He found that the viru-
lence was greatest when cultures were made from fowls which had
died from a chronic form of the disease, and that this virulence was
not lost by successive cultivations in chicken bouillon, repeated at
short intervals. But when an interval of more than two months
was allowed to elapse without renewing the cultures, the virulenco
was diminished and fewer deaths occurred in fowls inoculated with
such cultures. This diminution of virulence became more marked
in proportion to the length of time during which a culture solution
containing the microbe remained exposed to the action of the atmo-
sphere, and at last all virulence was lost as a result of the death of
the pathogenic microorganism. When the virus was preserved in
hermetically sealed tubes it did not undergo this modification, but re-
tained its full virulence for many months. According to Pasteur,
the various degrees of modification of virulence resulting from pro-
longed exposure to the air may be preserved in successive cultures
made at short intervals. Subsequent experiments with cultures of
the anthrax bacillus gave similar results and enabled him to produce
an " attenuated virus " for his protective inoculations.

In the case of the anthrax bacillus it was- found that the spores
retain their full virulence for years, and that the production of an at-
tenuated virus required the exclusion of these reproductive elements.
Cultivations were consequently made at a temperature of 42° to 4o°
C., at which point this bacillus is incapable of producing spores.
Cultivation at this temperature for eight days gave an attenuated
virus suitable for use in protective inoculations.

Attenuation by Heat. — Toussaint has shown that a similar modi-
fication of virulence may be produced by exposure for a short time
to a temperature a little below that which destroys the vitality of the
pathogenic organism. This is best accomplished, according to Chau-
veau, in the case of the bacillus of anthrax, by exposure for eighteen
minutes to a temperature of 50° C. Exposure to this temperature for
twenty minutes is said to completely destroy the vitality of the bacillus.

Attenuation- IHJ Antiseptic Agents. — The writer, in -1880, ob-
tained evidence that attenuation of virulence may result from ex-
posure to the action of antiseptic agents. In a series of experiments
made to determine the comparative value of disinfectants, the blood
of a rabbit recently dead from a form of septica3inia induced by the
subcutaneous injection of my own saliva, and due to the presence of
a micrococcus (Micrococcus pneumonia crouposa3), was subjected to
the action of various chemical agents, a-nd subsequently injected
into a rabbit to test the destruction of virulence. In the published
report of these experiments the following statement is made :

132 MODIFICATIONS OF BIOLOGICAL CHARACTERS.

' The most important source of error, however, and one which
must be kept in view in future experiments, is the fact that a pro-
tective influence has been shown to result from the injection of virus
the virulence of which has been modified, without being entirely de-
stroyed, by the agent used as a disinfectant. "

"Sodium hyposulphite and alcohol were the chemical reagents
which produced the result noted in these experiments ; but it seems
probable that a variety of antiseptic substances will be found to be
equally effective when used in proper proportion. Subsequent ex-
periments have shown that neither of these agents is capable of de-
stroying the vitality of the septic micrococcus in the proportion used
(one per cent of sodium hyposulphite or one part of ninety -five-per-
cent alcohol to three parts of virus), and that both have a restraining
influence upon the development of this organism in culture fluids. "

Cultivation in' the Blood of an Immune Animal. — It has
been shown by the experiments of Ogata and Jasuhara that when
the anthrax bacillus is cultivated in the blood of an immune animal,
such as the dog or the white rat, its pathogenic power is modified
so that it no longer kills susceptible animals and may be used as a
vaccine.

Pasteur had previously shown (1882) that the virus of rouget can
be attenuated by passing it through rabbits.

Recovery of Virulence. — Pasteur has shown that when the viru-
lence of a pathogenic organism has been modified it may be re-
stored by successive inoculations into susceptible animals. Thus in
the case of the anthrax bacillus a culture which would not kill an
adult guinea-pig may be inoculated into a very young animal of the
same species with a fatal result ; and by inoculating the blood of
this animal into another, and so on, the original virulence may be
restored, so that a culture is obtained which will kill a sheep. In.
the same way the attenuated virus of fowl cholera may be restored
to full vigor by inoculating a small bird — sparrow or canary — to
which it is fatal. After several successive inoculations the virus
resumes its original activity.

In general, pathogenic virulence is increased by successive inocu-
lations into susceptible animals, and diminished by cultivation in arti-
ficial media under unfavorable conditions. Thus various pathogenic
bacteria which have been cultivated in laboratories for a length of
time are likely to disappoint the student if he makes inoculation ex-
periments for the purpose of demonstrating their specific action as
described in text books.

1 Quoted from " Bacteria," pages 207, 208, written in 1883.

IT.
PRODUCTS OF VITAL ACTIVITY.

ALL living cells, animal or vegetable, while in active growth,
appropriate certain elements for their nutrition from the pabulum
with which they are supplied, and at the same time excrete certain
products which, in some cases at least, it is their special function to
produce. In the higher plants and animals specialized cells excrete
substances which are injurious to the economy of the individual,
and secrete substances which are required to maintain its existence.
As an example in animals we may mention the excretion of urea by
the epithelium of the kidneys, the retention of which is fatal to the
individual, and the gastric secretion which is essential for its con-
tinued existence. Among the- higher plants we have an immense
variety of substances formed in the cell laboratories, some of which
are evidently useful for the preservation of the species, while others
are perhaps to be considered simply as excretory products. The
odorous volatile products given off by flowers are supposed. to be
useful to the plant in attracting insects by which cross-fertilization
is effected. The various poisonous substances stored up in leaves
and bark may serve to protect the plant from enemies, etc.

The minute plants with which we are especially concerned also
produce a great variety of substances, some of which may be useful
to the species in the struggle for existence. Thus the deadly pto-
maines produced by some of the pathogenic bacteria serve to para-
lyze the vital resisting power of living animals and enable the para-
sitic invader to thrive at the expense of its host. In the present
section we shall consider in a general way these various products of
bacterial growth.

Pigment Production. — A considerable number of species are
distinguished by the formation of pigment of various colors and
shades. We have all of the shades of the spectrum from violet to
red. The color, as a rule, is only produced in the presence of oxy-
gen, and when the pigment-producing microorganisms are massed
upon the surface of a solid culture medium the pigment production
is often limited to the superficial portion of the mass. In some
cases a soluble pigment is formed which is absorbed by the transpa-

134 PRODUCTS OF VITAL ACTIVITY.

rent culture medium, coloring especially the upper portion, in stab
cultures in nutrient gelatin or agar. This is the case with Bacillus
pyocyaneus, which produces a blue pigment which has been isolated
and carefully studied by Gessard and others. The pigment, which
is called pyocyaniii, is soluble in chloroform and crystallizes from a
pure solution in long blue needles. Acids change the blue color to
red, reducing substances to yellow. It resembles the ptomaines in
its chemical reactions, being precipitated by platinum chloride and
phosphomolybdic acid.

In some media the color produced by the Bacillus pyocyaneus
(bacillus of green pus) is a fluorescent green. The recent studies of
Gessard show that this is a different pigment. According to this
author, cultures in a two-per-cent solution of peptone give a beautiful
blue tint, the production of which is hastened by adding to the liquid
five per cent of glycerin. In nutrient gelatin and agar cultures a
fluorescent green color is developed, which, according to Gessard,
is due to the presence of albumin. Peptone and gelatin are said to
produce pyocyaniii without the fluorescent-green pigment, and cul-
tures in bouillon to give both this and pyocyanin. In milk the
fluorescent-green color is first seen, but subsequently, when the ca-
sein has been peptonized by a diastase produced in the culture, pyo-
cyanin is also formed. Several other microorganisms are known
which produce a fluorescent-green color, due probably to the same
pigment as is produced by the bacillus of green pus in albuminous
media.

Babes claims to have obtained two pigments from cultures of the
Bacillus pyocyaneus in addition to pyocyanin : one, soluble in alcohol,
has by transmitted light a chlorophyll-green color, by reflected light
it is blue; the other, insoluble in alcohol and chloroform, by trans-
mitted light is of a dark orange-red, by reflected light a greenish-
blue.

In Gessard's latest publication (1891) he shows that the produc-
tion of pyocyanin or of the fluorescent-green pigment does not de-
pend alone upon the culture medium, but that there are different
varieties of the Bacillus pyocyaneus. He has succeeded in producing
four . distinct varieties — one which produces both pyocyanin and
fluorescence, one which produces pyocyanin alone, one which pro-
duces the fluorescent-green pigment alone, and one which produces
no pigment. The last-mentioned non-chromogenic variety was pro-
duced by subjecting the second variety to the action of heat. A
temperature of 57° maintained for five minutes destroyed the power
to produce pigment without destroying the vitality of the bacillus,
which was propagated through successive cultures without regaining
tli is power.

STK; • s HACTKKIOLOGY.

Plal t- II

Pie 3.

Sarcina lutea,a£ar culture

iius prodieiosus.a^ar culture.
Bacillus procyanus, agaj1 culture.
Bacillus Havaniensis, potato culture.

PRODUCTS OF VITAL ACTIVITY. 135

The well-known Bacillus prodigiosus (also described as a micro-
coccus) produces a red pigment which is insoluble in water but solu-
ble in alcohol. By the addition of an acid the color becomes car-
mine and then violet, which is changed to yellow by an alkali. The
color is said by Schottelius to be diffused in the young cells, and
after the death of the cells to be present in their vicinity in the form
of granules. The same author has shown that by subjecting the
bacillus to special conditions a variety may be obtained which no
longer produces pigment.

The conditions which govern the formation of pigment in the
chromogenic bacteria are determined with comparative facility be-
cause the results of changed conditions are apparent to the eye ; in
the case of products which are not colored the difficulties attending
the study of these conditions are much greater, but the results are in
many instances more important. The following are among the best
known pigment-producing (chromogenic} bacteria :

Staphylqcoccns pyogenes aureus, Staphylococcus p}Togenes cit-
reus, Sarcina aurantiaca, Sarcina lutea, Bacillus cyanogenus, Bacillus
janthinus, Bacillus fluorescens liquefaciens, Bacillus indicus, Bacillus
pyocyaneus, Bacillus prodigiosus, Spirillum rubrum.

Liquefaction of Gelatin. — Many species of bacteria, when
planted in a medium containing gelatin, cause a liquefaction of the
gelatin in the immediate vicinity of the growing microorganisms,
while many others multiply abundantly in the same medium with-
out liquefying the gelatin. This character, as first shown by Koch,
is an important one in the differential diagnosis of species which re-
semble each other in form and in other respects. It has no relation
to pathogenic power, as some liquefying organisms are harmless sap-
rophytes and some deadly disease germs, while, on the other hand,
non-liquefying bacteria may be very pathogenic or quite innocent.

Liquefaction is produced by a soluble peptonizing ferment formed
during the growth of the cells. This is shown by the fact that if a
liquefying organism is cultivated in bouillon and the living cells re-
moved by filtration or killed by heat, the power of liquefying gelatin
remains in the culture fluid. This was first observed by Bitter (1886)
and independently by the writer in 1887. In experiments made to
determine the thermal death-point of various bacteria the writer
found that when cultures of liquefying species were subjected to a
temperature which killed the microorganisms, a few drops of the
culture added to nutrient gelatin which had been liquefied by heat
prevented it from subsequently forming a solid jelly when cold.

In a study of the ferments produced by bacteria which cause
liquefaction of gelatin — "tryptic enzymes" — made by Fermi, in the

136 PRODUCTS OF VITAL ACTIVITY.

laboratory of the Hygienic Institute of Munich (1891), the following
results were obtained :

The enzymes were not obtained pure, and their isolation from
other proteids present in the cultures was found to be attended with
great difficulties, but their ferment action was studied and was found
to be influenced by various conditions.

All were destroyed by a temperature of 70° C. , but the enzymes
produced by various liquefying bacteria differed considerably as to
the temperature which they were able to withstand. Some were de-
stroyed by a temperature of 50° to 55° C. — Bacillus megatherium,
Bacillus ramosus, Staphylococcus pyogenes aureus ; some by a tem-
perature of 55° to 60° C. — Bacillus subtilis, Bacillus pyocyaneus, Ba-
cillus fluorescens liquefacieris, Sarcina aurantiaca; some by 65° to
70° C. — Bacillus anthracis, Spirillum choleras Asiatics, Spirillum of
Finkler and Prioi\ Spirillum tyrogenum.

These enzymes, like the previously known pepsin, trypsin, and
invertin, do not dialyze.

Only a few of these bacteria enzymes acted upon fibrin, and no
action was observed upon casein or upon egg albumen.

Their liquefying action upon gelatin was prevented by the action
of sulphuric acid, and to a less degree by nitric acid, but was not in-
terfered with by acetic acid.

The liquefying bacteria, as a rule, only produce enzymes when
cultivated in a medium containing albumen.

These enzymes are not produced by a solution of the protoplasm
of dead bacterial cells, but are a product of the vital activity of liv-
ing cells.

Among the numerous liquefying bacteria known to bacteriolo-
gists we may mention the following species as deserving the student's
special attention: Staphylococcus pyogenes aureus, Staphylococcus
pyogenes albus, Sarcina lutea, Sarcina aurantiaca, Bacillus anthra-
cis, Bacillus pyocyaneus, Bacillus subtilis, Bacillus indicus, Bacillus
prodigiosus, Spirillum cholerse Asiaticse, Spirillum of Finkler and
Prior, Proteus vulgaris.

Fermentation. — The fermentation produced by various species of
bacteria in culture solutions containing saccharose, glucose, or lac-
tose constitutes a valuable character for the differentiation of species.
While some bacteria give rise to fermentation in solutions contain-
ing either of the carbohydrates above mentioned, others break up lac-
tose, but have no effect upon glucose or saccharose, and others again
are without any ferment action. The gases evolved are chiefly car-
bon dioxide and hydrogen. Ferment action may be tested by adding
one to two per cent of glucose to a solid culture medium — preferably
agar-agar. This is liquefied by heat in the test tube containing it and

PRODUCTS OF VITAL ACTIVITY. 137

a small quantity (one ose) of the microorganism to be tested is intro-
duced. The culture medium is then quickly solidified by placing the
test tube in iced water. It is then placed in the incubator, and when
colonies form bubbles of gas will be seen in their vicinity, if the bac-
terium under observation is able to cause fermentation of glucose.
For accurate observations as to the quantity and nature of the gases
produced the fermentation tube should be used, as recommended by
Theobald Smith (see Fig. 38).

Production of Acids. — Numerous bacteria give an acid reaction
to the media in which they are cultivated, and the acids produced
are various — lactic, acetic, butyric, propionic, succinic, etc.

The power to produce an acid is well shown by adding to neu-
tral or alkaline culture media a solution of litmus. The change in
color due to the formation of an acid may be followed by the eye,
and comparative tests may be made to aid in the differentiation of
similar bacteria.

A considerable number of bacteria are able to produce lactic
acid from milk sugar and other carbohydrates. One of these is
considered the special lactic-acid ferment — Bacillus acidi lactici — and
is the usual cause of the acid fermentation of milk. Pure cultures
of this bacillus introduced into sterilized milk or solutions of milk
sugar, cane sugar, dextrin, or mannite, give rise to the lactic-acid
fermentation, in which carbonic acid is also set free. The process
requires free access of oxygen, and progresses most favorably at a
temperature of 35° to 40° C., ceasing at about 45°. In milk, coagu-
lation of the casein occurs within fifteen to twenty-four hours after
adding a small quantity of a pure culture of the lactic-acid bacillus.
This is not due, however, to the acid fermentation, but to a ferment
resembling that of rennet, which is produced by many different
bacteria, some of which do not produce an acid reaction of the milk.
Among the bacteria which produce lactic acid from milk sugar we
may mention the staphylococci of pus, Bacillus lactis aerogenes, and
Bacillus coli communis.

The formula showing the transformation of sugar into lactic
acid is usually stated as follows : C6H12O6 — 2(HC3H5O3).

Acetic acid is also produced from dilute solutions of alcohol by
the action of a special bacterial ferment, which accumulates upon
the surface of the fluid as a mycoderma, consisting almost entirely
of the Bacillus aceticus (Mycoderma aceti). Free access of oxygen
is required, and a temperature of about 33° C. is most favorable to
the process. According to Duclaux, the " Mycoderma aceti " oxi-
dizes the alcohol, in solutions containing it, so long as any is present,
and when it is exhausted it oxidizes the acetic acid previously

138

PRODUCTS OF VITAL ACTIVITY.

formed by oxidation of the alcohol, producing from it carbon diox-
ide and water.

The formation of acetic acid from alcohol is shown by the follow-
ing formula : Ethyl alcohol CH3.CH2.OH + O2 = CH3.COOH + HaO.

Butyric acid is produced by a considerable number of bacteria,
one of which, named Bacillus butyricus, has received the special at-
tention of Prazmowski. This is strictly anaerobic. In solutions of
starch, dextrin, sugar, or salts of lactic acid, when oxygen is ex-
cluded it produces butyric acid in considerable quantity, and at the
same time carbon dioxide and hydrogen gas are set free. Duclaux
gives the f ollowing formula of a solution containing lactate of lime
in which the action of the butyric-acid ferment may be well studied :

Water,

Lactate of lime (pure),
Phosphate of ammonia,
Phosphate of potash,.
Sulphate of magnesa,
Sulphate of ammonia,

8 to 10 litres.

225 grammes.

0.75

0.4

0.2

FIG. 77.

This is introduced into a flask with two necks, such as is shown
in Fig. 77. Having filled the flask with the culture liquid, the bent
neck is dipped into a porcelain dish containing the same. Heat is
then applied both to flask and dish, and the liquid in each is kept in
ebullition for half an hour. By this means the air is completely
driven out of the flask. This is now allowed to cool, while the fluid
in the shallow dish is kept hot, so that the liquid mounting from it
into the flask shall be free from air. When the flask is full it is
transferred to an incubating oven heated to 25° to 30° C., and the bent
tube is immersed in a dish containing mercury. The little funnel

PRODUCTS OF VITAL ACTIVITY. 139

attached to the upright tube is then filled with carbon dioxide and a
culture of the butyric-acid bacillus is introduced into the funnel.
By turning the stopcock in the upright tube a little of the culture is
admitted to the flask without admitting any air. Fermentation com-
mences very soon, as is seen by the bubbles of gas given off. The
liquid loses its transparency and the lactic acid is gradually con-
sumed, butyrate of lime taking the place of the lactate.

Aerobic bacilli capable of producing butyric acid in culture solu-
tions containing grape sugar or milk sugar have also been described
by Liborius and by Hueppe.

Fitz has shown that in culture solutions containing glycerin the
Bacillus pyocyaneus produces butyric acid in addition to ethyl alcohol
and succinic acid. Bacillus Fitzianus also produces some butyric acid
in solutions containing glycerin, although the principal product of the
fermentation caused by this microorganism is, according to Fitz,
ethyl alcohol, twenty-nine grammes of which may be obtained from
one hundred grammes of glycerin.

Botkin (1892) has described a "Bacillus butyricus" (No. 46G)
which he has not been able to identify positively with the butyric-
acid ferment described by Prazmowski. It is a widely distributed
anaerobic bacillus, which he was able to obtain from milk or water
containing it by placing it in the steam sterilizer for half an hour.
The spores resisted this temperature and subsequently grew in anae-
robic cultures, in a suitable medium, while all other bacteria and
spores present were destroyed.

The writer has described a bacillus which causes active acid
fermentation in culture solutions containing glycerin. The acid
formed is volatile and is probably propionic acid — see Bacillus acidi-
formans.

The Caucasian milk ferment — Bacillus Kaukasicus — produces
a variety of products in the fermented milk which is a favorite
drink among the Caucasians. The principal ones are ethyl alcohol,
lactic acid, and carbon dioxide, but in addition to these small quanti-
ties of succinic, butyric, and acetic acids are formed. The inhabi-
tants of the Caucasian mountains prepare this fermented drink in a
very simple manner from the milk of cows or goats, to which they
add the dried ferment collected from a receptacle in which the fermen-
tation had previously taken place. Flugge gives the following di-
rections for the preparation of this drink :

" Two methods may be employed. In the first the dry brown kefir-kor-
ner of commerce are allowed to lie in water for five to six hours until they
swell; they are then carefully washed and placed in fresh milk, which
should be changed once or twice a day until the korner become pure white
in color and when placed in fresh milk quickly mount to the surface — in
twenty to thirty minutes. One litre of milk is then poured into a flask and a
full tablespoonful of the prepared korner added to it. It is allowed to stand

140 PRODUCTS OP VITAL ACTIVITY.

open for five to eight hours ; the llask is then closed and kept at 18° C. It
should be shaken every two hours. At the end of twenty four hours the
milk is poured through a fine sieve into another flask, which must not be
more than four-fifths full. This is corked and allowed to stand, being1
shaken from time to time. At the end of twenty-four hours a drink is ob-
tained which contains but little COa or alcohol. Usually it is not drunk
until the second day, when, upon standing, two layers are formed, the
lowrer milky, translucent, and the upper containing fine flakes of casein.
When shaken it has a cream- like consistence. On the third day it again
becomes thin and very acid.

" The second method is used when one has a good kefir of two or three
days to start with. Three or four parts of fresh cow's milk are added to one
part of this and poured into flasks which are allowed to stand for forty-
eight hours with occasional shaking When the drink is ready for use a
portion (one-fifth to one-third) is left in the flask as ferment fora fresh
quantity of milk. The temperature should be maintained at about 18° C. ;
but at the commencement a higher temperature is desirable. The Jcorner
should be carefully cleaned from time to time and broken up to the size of
peas. The cleaned korner may be dried upon blotting paper in the sun or
in the vicinity of a stove: when dried in the air they retain their power to
germinate for a long time."

Fermentation of urea. The alkaline- fermentation of urine is
effected by various microorganisms, but chiefly by the Micrococcus
urese, the ferment action of which has been carefully studied by Pas-
teur, Duclaux, and others. The change which occurs under the
action of the living ferment was determined by the chemist Dumas
as long ago as 1830, but it remained for Pasteur to show that this
change depends upon the presence and vital activity of a living
microorganism.

The transformation of urea into carbonate of ammonia is shown
by the following formula : COH4N2 + 2H2O = CO, + 2NH3 +
H20 = (NH4)2C08.

According to Van Tieghem, Micrococcus ureaB continues to grow
in a liquid containing as much as thirteen per cent of carbonate of
ammonia. It may be cultivated in an artificial solution of urea, with
the addition of some phosphates, as well as in urine.

The Bacillus urese of Miquel has also the power of producing the
alkaline fermentation of urine, but it does not thrive in so strong a
solution of carbonate of ammonia.

A different micrococcus — Micrococcus urese liquef aciens — nas also
been studied in Flugge's laboratory which possesses the same power.
According to Musculus, a soluble ferment may be isolated from urine
which has undergone alkaline fermentation, which changes urea into
carbonate of ammonia. He obtained it from urine containing con-
siderable mucus, in a case of catarrh of the bladder. But Leube has
shown that cultures of Micrococcus urese from which the micrococ-
cus was removed by filtration through clay do not induce alkaline
fermentation. The soluble ferment obtained by Musculus must
therefore be from some other source.

PRODUCTS OF VITAL ACTIVITY. 141

Miquel has given special attention to the study of bacteria which
produce alkaline fermentation in urine, and in addition to the spe-
cies above mentioned has described the following : Urobacillus Pas-
teuri, Urobacillus Duclauxi, Urobacillus Freudenreichi, Urobacillus
Maddoxi, Urobacillus Schutzenbergi.

Viscous fermentation. A special fermentation which occurs
sometimes in wines, and in the juices of bulbous roots containing
glucose, and in milk, is produced by various bacteria. One of these
is a micrococcus which has been described by Conn under the name
of Micrococcus lactis viscosus. The fermented juices become very
viscous, owing to the formation of a gum-like product resembling
dextrin; at the same time mannite and CO2 are produced. The
gum-like substance, called viscose by Bechamp, is soluble in cold
water and is precipitated by alcohol. Guillebeau (1892) has de-
scribed a micrococcus and a bacillus which produce viscous fer-
mentation ID milk — Micrococcus Freudenreichi and Bacillus Hessi.
A micrococcus producing viscous fermentation in milk has also
been described by Schmidt-Muhlheim, and a bacillus by Lomer.
Bacillus mesentericus vulgatus also produces a similar change in
milk.

Marsh gas, CH4, is produced by the fermentation of cellulose,
through the action of microorganisms the exact characters of which
have not yet been determined. According to Tappeiner, there are
two different fermentations of cellulose. The first occurs in a neu-
tral one-per-cent flesh extract solution to which cotton or -paper pulp
has been added. The gases given off are CO2 and CH4 and small
quantities of H2S. The second fermentation occurs when an alkaline
solution of flesh extract containing cellulose in suspension is used.
The gases formed are CO2 and H. In both cases small quantities of
aldehyde, isobutyric acid, and acetic acid are produced.

Hydrosulpliuric acid, H2S. This gas is produced during the
growth of certain bacteria. The conditions governing its develop-
ment have been studied by Holschewnikoff, who experimented with
two species, one isolated by himself and one by Lindenborn. named
respectively Bacterium sulfureum and Proteus sulfureus. The first-
mentioned bacterium, when inoculated into eggs, produced within
three or four days an abundant quantity of H,S ; the other did not.
Upon rawr albumin both species produced but little, and on the yolk
of egg a considerable amount of this gas. Upon cooked egg the
action was the reverse. In peptone-bouillon the evolution of H2S
was abundant : in the absence of peptone, ven~ slight.

Putrefactive fermentation. The putrefactive decomposition
of albuminous material of animal and vegetable origin is effected
by a great variety of microorganisms and gives rise to the forma-

142 PRODUCTS OF VITAL ACTIVITY.

tion of a great variety of products, some of which are volatile and
are characterized by their offensive odors. According to Fliigge, the
first change which occurs consists in the transformation of the albu-
mins into peptone, and this may be effected by a large number of
different bacteria. Among the products of putrefactive fermenta-
tion known to chemists are the following substances : Carbon diox-
ide, hydrogen, nitrogen, hydrosulphuric acid (H,S), phosphoretted
hydrogen (PH3), methane, formic acid, acetic acid, butyric acid,
valerianic acid, palmitic acid, crotonic acid, glycolic acid, oxalic
acid, succinic acid, propionic acid, lactic acid, amidostearic acid,
leucin, ammonia, ammonium carbonate, ammonium sulphide, tri-
methylamine, propylamine, indol, skatol, tyrosin,neuridin, cadaverin,
putrescin, cholin, neurin, peptotoxin, and various other volatile"
acids, ptomaines, etc.

The special products of putrefaction vary according to the nature
of the material, the conditions in which it is placed, and the micro-
organisms present. One or the other of the bacteria concerned will
take the precedence when circumstances favor its growth. Thus the
aerobic bacteria cannot grow unless the putrefying material is freely
exposed to atmospheric oxygen ; the anaerobic species require its
exclusion. Some saprophytic bacteria grow at a comparatively low
temperature, others take the precedence when the temperature is
high ; some, no doubt, thrive only in presence of products evolved
by other species, and are consequently associated with and depend-
ent upon these species ; some are restrained in their growth sooner
than others by the products evolved as a result of their own vital
activity or that of associated organisms ; some grow in the presence
of acids and give rise to an acid fermentation which wholly prevents
the development of other species.

At the outset putrefaction is often attended with the presence
of several species of micrococci and certain large bacilli, which are
displaced later by short motile bacteria belonging to a group which
includes several bacilli formerly described under the common name
of Bacterium termo.

The malodorous volatile products of putrefaction are to a consid-
erable extent produced by anaerobic species. For this reason these
odors are more pronounced when masses of albuminous material
undergo putrefaction in situations where the oxygen of the air has
not free access, or where it is displaced by carbon dioxide. The
body of a dead animal, although freely exposed to the air, furnishes
in its interior a suitable nidus for these anaerobic gas-forming spe-
cies, and they may give rise to products of one kind, while aerobic
species upon the surface of the mass induce different forms of putre-
factive fermentation. In the bodies of living animals these anaero-

PRODUCTS OF VITAL ACTIVITY. 143

bic microorganisms are constantl}7 present in the intestine, and after
death they quickly invade the body and multiply at its expense
under favorable conditions as to temperature. The surface decom-
position due to aerobic bacteria occurs later and is not attended
with the same putrefactive odors, the products evolved being of a
simpler chemical composition — CO2, HN3. No doubt these aerobic
bacteria, by consuming the oxygen and forming an atmosphere of
carbon dioxide, help to make the conditions favorable for the con-
tinued development of the anaerobics in the interior of the organic
mass ; at the same time they find a suitable pabulum in some of the
more complex products of decomposition occurring in the absence
of oxygen. The gases produced in the interior of a putrefying mass
are mainly CH4, H2S, and H.

Many of the bacteria of putrefaction are facultative anaerobics—
that is to say, they are able to multiply either in the presence of oxy-
gen or in its absence. The products evolved by these differ, no
doubt, according to whether they are or are not supplied with atmos-
pheric oxygen.

The anaerobic bacteria concerned in putrefaction have as yet
received comparatively little attention. Among the aerobics and
facultative anaerobics the following are best known: Micrococcus
fcetidus, Bacillus saprogenes I., II., and III., Bacillus coprogenes
foetidus, Bacillus putrificus coli, Proteus vulgaris, Proteus Zenkeri,
Proteus mirabilis, Bacillus pyogenes fcetidus, Bacillus fluorescens
liquefaciens, Bacillus pyocyaneus, Bacillus coli communis, Bacillus
janthinus.

Soluble Ferments. — Several species of bacteria produce soluble
ferments capable of changing starch into maltose, dextrin, etc.
Hueppe has shown that the lactic-acid bacillus produces a diastase,
and Miller obtained from the human intestine a species which dis-
solves starch. Marcano, by filtering cultures of species capable of
this ferment action through porcelain, was able to show that the
effect is due to a soluble ferment, which must have been produced
by the vital activity of the living microorganisms. Wortmann also
obtained a diastase from culture liquids which was precipitated by
alcohol and again dissolved in water ; in slightly acid solutions it
promptly converted starch into glucose. This is said to be produced
in culture liquids only when these do not contain albumin. In the
presence of albumin a peptoiiizing ferment was formed ; in its ab-
sence, a diastase by which starch was dissolved to serve as pabulum
for the bacteria present. These experiments were not made with
pure cultures, and more exact researches in this direction are de-
sirable.

144 PRODUCTS OF VITAL ACTIVITY.

A peptonizing ferment for gelatin is produced by a considerable
number of bacteria, as stated under the heading " Liquefaction of
Gelatin." The jellified albumin in cultures in blood serum is also
liquefied by a peptonizing ferment produced by certain species of bac-
teria.

Some authors also speak of a soluble ferment capable of inverting
cane sugar or milk sugar. According to Hueppe, such a ferment
is produced by the Bacillus acidi lactici. A soluble ferment for cel-
lulose is supposed by Fliigge to be produced by several species—
among others by Bacillus butyricus and by Vibrio rugula.

Several bacilli produce a soluble ferment capable of coagulating
the casein of milk.

Reduction of Nitrates, and Nitrification. — The researches of
Gayon, Dupettit, and others show that certain bacteria are able to
reduce nitrates with liberation of ammonia and free nitrogen. This
is effected in the absence of oxygen by anaerobic bacteria, and,
among others, by Bacillus butyricus. Certain aerobic bacteria also
accomplish the same result. Thus Heraaus obtained two species
from water which reduced nitrates in a very decided manner. On
the other hand, a number of species are known to oxidize ammonia,
producing nitric acid. Schlosing and Miinz, as a result of numerous
experiments, arrived at the conclusion that in the soil nitrification is
effected by a single species. But it is doubtful whether they worked
with pure cultures, and more recent researches show that several,
and probably many, different bacteria possess this power. Accord-
ing to Herseus, the following species, tested by him, oxidize am-
monia : Bacillus prodigiosus, the cheese spirillum of Deneke, the
Finkler-Prior spirillum, the typhoid bacillus, the anthrax bacillus,
the staphylococci of pus. The oxidation does riot always go to the
point of forming nitrates, but nitrites may be formed in the soil
(Duclaux). Warrington. states that certain bacteria which formed
nitrates in a suitable culture medium produced only nitrites when,
after an interval of four or five months, some of the culture was
transferred to a solution containing muriate of ammonia. The same
author states that the process of nitrification occurs only in the
dark.

The researches of Winogradsky, of the Franklands, and of Jor-
dan show that the failure of earlier investigators to obtain the nitri-
fying bacteria from the soil in pure cultures was due to the fact that
these bacteria do not grow in the usual culture media. By the use
of certain saline solutions the authors named have succeeded in iso-
lating nitrifying bacteria in pure cultures, or nearly so. It is still
uncertain whether these investigators have obtained the same bac-
teria, but the microorganisms described by them, and obtained from

PRODUCTS OF VITAL ACTIVITY. 145

widely distant sources, are similar in their morphological and bio-
logical characters, and at least belong to the same group. In a com-
munication made in 1891 Winogradsky arrives at the conclusion
that the ferments which cause the oxidation of ammonia and pro-
duction of nitrites are not capable of producing nitrates, but that
other microorganisms are concerned in the oxidation of nitrites.
In sterilized soil to which a pure culture of his nitromonas was
added nitrites only were produced, and the presence of various
microorganisms common in the soil did not result in the forma-
tion of nitrates so long as the specific ferment was absent to which
this second oxidation is ascribed (nitrifying bacillus of Winograd-
sky).

Phosphorescence. — Several different bacteria have been studied
which, in pure cultures, give rise to phosphorescence in the medi-
um in which they are cultivated. In gelatin cultures the light
is sufficient in some instances to enable one to tell the time by a
watch in a perfectly dark room, and such cultures have even been
photographed by their own light.

The phosphorescence is influenced by changes in the culture
medium and by conditions of temperature, but we have no exact
knowledge of the mode of its production. The Bacillus phosphores-
cens from sea water in the vicinity of the West Indies gives the
most striking results, especially when planted upon the surface of
cooked fish and placed in an incubating oven at 30° C. Two other
species have been studied by Fischer — one obtained from the water
of the harbor at Kiel, and the other a widely distributed species
called by Fischer Bacterium phosphorescens. Katz (1891) has de-
scribed several species obtained by him from sea water and from
phosphorescent fish in the markets at Sydney, New South Wales —
Bacillus smaragdino-phosphorescens, Bacillus argenteo-phosphores-
cens, Bacillus cyaneo-phosphorescens, Bacillus argenteo-phosphores-
cens liquefaciens.
10

PTOMAINES AND TOXALBUMINS.

VARIOUS basic substances containing nitrogen, and in chemical
constitution resembling the vegetable alkaloids, have been isolated
by chemists from putrefying material and from cultures of the bac-
teria concerned in putrefaction, and also from certain pathogenic
species. Some of these ptomaines are non-toxic, and others are
very poisonous in minute doses (toxines). The toxic substances
sometimes developed in milk, cheese, sausage, etc., are also of this
nature, and are doubtless produced by the action of microorganisms.
The pathogenic power of .the bacteria which cause various infectious
diseases in man and the lower animals has also been shown to result
from the production of toxic ptomaines or of toxalbumins. Selmi first
gave the name ptomaines to cadaveric alkaloids isolated by him, and
Panum subsequently called attention to the fact that poisonous basic
substances of this class are contained in putrefying material. Ex-
tended researches with reference to the ptomaines have since been
made by numerous chemists, the most important being those of Berg-
mann, Schmiedeberg, Zuelzer and Sonnenschein, Hager, Otto, Sel-
mi, Brieger, Gautier and Etard, and Vaughan.

For a full account of the history and chemical composition of the
ptomaines the reader is referred to the valuable work of Vaughan
and Novy (" Ptomaines and Leucomaines," Philadelphia, 1891). In
the present volume we shall give a brief account only of some of the
most important.

NON-TOXIC PTOMAINES.

Neuridin, C5HJ4N2. — This is one of the most common of the al-
kaloids of putrefaction and was isolated by Brieger in 1884. It is
obtained most abundantly from tissues containing gelatin. Very
soluble in water, but insoluble in ether and absolute alcohol. Has a
disagreeable odor.

Cadaverin, C5H14N2. — Isomeric with neuridin ; has a very dis-
agreeable odor ; forms a thick, transparent, syrupy liquid ; is vola-
tile, and can be distilled with steam without undergoing decomposi-
tion. When exposed to the air the base absorbs carbon dioxide and

PTOMAINES AND TOXALBUMINS. 147

forms a crystalline mass. Is produced in cultures of the cholera
spirillum and of the spirillum of Finkler and Prior which have been
kept for a month or more at 37° C.

Putrescin, C4H]2N2. — A base resembling cadaverin and com-
monly associated with it. Obtained by Brieger from various sources,
most abundantly from substances containing gelatin and in the
more advanced stages of putrefaction. It is obtained in the form of
a hydrate, which is a transparent liquid having a boiling point of
about 135°. With acids it forms crystalline salts.

Saprin, C5A16N2. — Resembles cadaverin and is commonly as-
sociated with it in putrefying material. Isolated by Brieger.

Methylamine, CH3.NH2. — Obtained by Brieger from putrefying
fish and from old cultures of the cholera spirillum.

Dimetliijlamine, (CHt),.NH. — Obtained by Brieger from putre-
fying gelatin and by Bocklisch from decomposing fish.

Trimethylamine, (CH3)3N. — Obtained from various sources, and
by Brieger from cultures of the cholera spirillum and of the strepto-
coccus of pus.

TOXIC PTOMAINES.

Neurin, C5H13NO. — First obtained by Liebreich in 1865 as a
decomposition product of protagon from the brain. Obtained by
Brieger from putrefying muscular tissue. When crystallized from
an aqueous solution it forms five- or six-sided plates ; from an alco-
holic solution it crystallizes in the form of needles (Liebreich). This
base is toxic in small doses. In frogs the injection of a few milli-
grammes produces paralysis of the extremities. Respiration is first
arrested and the heart stops in diastole. Atropine appears to be a
physiological antidote to the toxic effects of neurin. In rabbits it
produces profuse salivation. The pupil is contracted by the direct
application of a concentrated solution.

Cholin, C5H15NX)2. — First obtained from hog's bile by Strecker
in 1862. Has been obtained by Brieger from various sources, in-
cluding cultures of the cholera spirillum. It is also found widely
distributed in the vegetable kingdom. May be prepared from the
yolk of eggs by the method of Diakonow. Cholin is obtained in the
form of a syrupy, alkaline liquid which combines with acids to form
deliquescent salts. At first this base was not supposed to have toxic
properties, but more recent researches have shown that in compara-
tively large doses it produces symptoms resembling those caused by
minute doses of neurin.

Muscarin, C5H]5NO3. — This toxic principle of poisonous mush-
rooms has also been obtained by Brieger from putrefying fish. It may
be produced artificially by the oxidation of cholin. In small doses
it kills rabbits and frogs. In the rabbit it produces lacrymation and

148 PTOMAINKS AND TOXALBUMIXS.

salivation, the pupil is contracted, and the animal dies in convul-
sions. Frogs are completely paralyzed by the action of muscarin
and die with arrest of the heart's action in diastole.

Peptotoxin. — The exact composition of this ptomaine has not
been determined. Brieger obtained it during the early putrefac-
tion of proteid substances and also from the artificial digestion of
fibrin. It is very poisonous for frogs, which become paralyzed and
die within fifteen or twenty minutes after the subcutaneous injection
of a few drops of a dilute solution. Rabbits also are killed by doses
of half a gramme to a gramme, the symptoms being paralysis of the
posterior extremities and stupor. Peptotoxin is soluble in water,
but insoluble in ether or chloroform. It is not destroyed by boiling.

Tyrotoxicon. — First obtained by Yaughan in poisonous cheese,
and subsequently by the same chemist and others in poisonous milk
mid ice cream. Chemically tyrotoxicon is very unstable. It is de-
composed when heated with water to 90° C. It is insoluble in ether.
From sixteen kilogrammes of poisonous cheese Vaughan obtained
0. 5 gramme of the poison. The symptoms produced in man by eat-
ing cheese or milk containing tyrotoxicon are vertigo, nausea, vomit-
ing, and severe rigors, with pain in the epigastrium, cramps in the
legs, griping pain in the bowels attended with purging, numbness
and a pricking sensation in the limbs, and great prostration.

Methyl-guanidin, C2H7N3. — Obtained by Brieger from putrefy-
ing horseflesh which had been kept at a low temperature for several
months. This base was previously known to chemists, having been
obtained by the oxidation of creatin. By Bocklisch it has been ob-
tained from impure cultures of the Finkler-Prior spirillum which
had been kept for about a month. It is obtained as a colorless mass
having an alkaline reaction, and which is quite deliquescent. Brie-
ger gives the following account of the toxic action as . tested on
guinea-pigs in a dose of 0. 2 gramme : The respiration increases in
rapidity, the pupils dilate to the extreme limit, the animal has copi-
ous discharges of urine and faeces, the extremities become paralyzed,
and at the end of about twenty minutes death occurs in convulsions.

Mytilotoxin. — Obtained by Brieger from poisonous mussels.
The toxic action resembles that of curare.

Typhotoxin, C7H17NO2. — Obtained by Brieger from bouillon
cultures of the typhoid bacillus which had been kept for a week or
more at a temperature of about 37.5° C. In mice and guinea-pigs
this base produces salivation, rapid respiration, dilatation of the
pupils, diarrhoea, and death in from twenty-four to forty-eight hours.
It is believed by Brieger that the specific action of the typhoid bacil-
lus is due to the production of this ptomaine.

A base which is isomeric with typhotoxin has been obtained by

PTOMAINES AND TOXALBUMINS. 14!)

Brieger from putrefying horseflesh which was kept at a low tempe-
rature for several months. Unlike it, however, the free base has
an acid reaction, while typhotoxin is strongly alkaline. It differs also
in its physiological action, being more toxic and producing convul-
sions ; the heart is arrested in diastole. Typhotoxin, on the other
hand, does not induce convulsions and the heart is arrested in systole.

Tetanin, C13H30N2O4. — Obtained by Brieger from impure cul-
tures of the tetanus bacillus cultivated in bouillon in an atmosphere
of hydrogen. (The tetanus bacillus is a strict anaerobic. ) Obtained
subsequently by the same chemist from the amputated arm of a pa-
tient with tetanus. This base has been obtained, by crystallization
from hot alcohol, in clear yellow plates which are not very soluble in
water. The hydrochloride is a deliquescent salt which dissolves
readily in alcohol. When injected into guinea-pigs or mice in rather
large doses, tetanin first causes the animal to fall into a lethargic
condition, followed by increased rapidity of respiration and tetanic
convulsions. In guinea-pigs opisthotonos is induced, together with
the characteristic tetanic convulsions as seen in animals suffering from
tetanus. Three other toxic bases have been obtained by Brieger
from cultures of the tetanus bacillus, which cause similar symptoms.
One — tetanotoxin — is given by Brieger the formula C.HnN. A
second base, the composition of which has not been determined, is
called spasmotoxin.

Cholera Ptomaines. — Brieger has obtained from pure cultures
of the cholera spirillum several of the toxic ptomaines heretofore re-
ferred to — cadaverin, putrescin, cholin, methyl-guanidin. In addi-
tion to these he found two toxic substances which appear to be pe-
culiar products of this microorganism. One induces cramps and
muscular tremors in small animals, the other diarrhoea and symp-
toms of collapse.

Toxalbmnins. — Researches by Brieger and Frankel (1890) show
that very toxic substances of a different nature are present in cultures
of some of the pathogenic bacteria; these have been designated by the
authors named " toxalbumins."

Roux and Yersin had previously shown that filtered cultures of the
diphtheria bacillus contain a toxic substance which produces paralysis
and death in guinea-pigs and rabbits. This substance has now been
obtained in a pure state and its toxic action tested by the authors
first named. It is destroyed by a temperature of 60° C., but remains
in an active condition in cultures which have been sterilized by seve-
ral hours' exposure to a temperature of 50°, or in those which have
been passed through a clay filter. It is not volatile, and differs essen-
tially from the ptomaines and also from the soluble ferments. It
was obtained as a snow-white, amorphous mass which was ex-

150 PTOMAINES AND TOX ALBUMINS.

tremely toxic in its action upon small animals. When injected into
guinea-pigs in the proportion of two and one-half milligrammes to
one kilogramme of body weight, it caused death after a considerable
interval of time (from a few days to several weeks), during which
the animal became emaciated and spreading abscesses and necrosis
of the tissues occurred at the point of injection. This toxalbumin
was obtained in a pure state by repeated precipitation from an aque-
ous solution by means of alcohol. It is produced most abundantly
in cultures containing albumin, and old cultures are more toxic than
recent ones. Chemical analysis gave the following result : C 45.35,
H 7.13, N 16.33, S 1.39, O 20.80. The authors remark, however,
that the chemical characters have not yet been fully determined.

The same chemists have obtained toxic substances of a similar
nature from cultures of the bacillus of typhoid fever, of the tetanus
bacillus, of the Staphylococcus aureus, and of the cholera spirillum.
Hankin had previously obtained a toxic "albumose" from cultures
of the anthrax bacillus by precipitation with alcohol, drying, solu-
tion in water, and filtration through porcelain ; and Christmas had
obtained an albuminous substance from cultures of Staphylococcus
aureus which produced pus formation when injected beneath the
skin of rabbits or into the anterior chamber of the eye.

According to Brieger and Frankel, these toxalbumins are divided
into two principal groups, one of which is characterized by solubility
in water, as in that produced by the diphtheria bacillus ; and one in
which the albumin is insoluble or but slightly soluble, as is the case
with those obtained from cultures of the typhoid bacillus, the cholera
spirillum, and the Staphylococcus aureus.

The toxalbumin from cholera cultures, obtained as pure as pos-
sible and suspended in water, when injected under the skin of a
guinea-pig, caused its death in two or three days. It was not, how-
ever, toxic for rabbits, even when injected in considerable quantity.

On the contrary, the toxalbumin of the typhoid bacillus, which is
dissolved with difficulty in water, was more poisonous for rabbits
than for guinea-pigs. When injected subcutaneously into rabbits
death usually occurred in eight to ten days. No notable pathologi-
cal changes were observed at the autopsy.

The toxalbumin of Staphylococcus aureus killed rabbits and
guinea-pigs within a few days, and in some cases at the end of
twenty-four hours. The post-mortem appearances were necrosis or
purulent breaking down of the tissues at the point of injection, with
swelling and redness of the surrounding tissues and general inflam-
matory appearances. The toxalbumin of anthrax cultures resembles
that of the diphtheria bacillus in being soluble in water. It was
obtained by Brieger from the organs of animals recently dead from

PTOMAINES AND TOXALBUMINS.

151

anthrax. In a dry condition it has a grayish-white color and gives
the reactions of albumins.

The toxalbumin of the tetanus bacillus is also soluble in Avater.
It is best obtained in bouillon cultures containing glucose.

G. and F. Klemperer (1891) have announced their success in
obtaining a toxalbumin from cultures of Micrococcus pneumonia?
croupossB ('diplococcus pneumonia?') ; this they propose to call pneu-
motoxin.

Some recent authors prefer the name toxins for the poisonous
products of bacterial growth designated by Brieger and others as
"toxalbumins." This avoids any definite statement as to their
chemical composition, which appears to be still in doubt. The
poisonous precipitates obtained from cultures of the tetanus or the
diphtheria bacillus give the reactions of an albumin or albumose
(Martin), but it is possible that the toxic substance is simply as-
sociated with bodies of this class, and that they have not yet been
isolated in a pure state. These toxins in some cases are intimately
associated with the bacterial cell — intracellular toxins — and their
toxic effects are exhibited when small quantities of dead bacteria
are introduced into a susceptible animal. The extracellular toxins
are better known, and may be obtained from filtered culture solutions
by precipitation with strong alcohol. In this case they are associated
with the proteids which may have been present in the culture. The
fact that a considerable interval elapses — twenty-four hours to several
da}Ts — after the injection of these toxins into a rabbit or a guinea-pig
before death occurs, has given rise to the inference that these sub-
stances are of the nature of enzymes or ferments. This view is also
supported by the ver}" minute quantity required to produce a fatal
result. According to Vaillard a dose of 0.00025 gramme of the tet-
anus toxin is sufficient to kill a guinea-pig.

Indol Production. — Numerous species of bacteria, as a result of
their vital activity, give rise to the production of indol. This may
be detected by cultivation in " Dunham's solution " of peptone
(dried peptone, I part; sodium chloride, 0.5 per cent; distilled water,
100 parts). Upon adding a drop of yellow nitric acid to ten cubic
centimetres of a culture in this medium the presence of indol will be
revealed by the development of a rosy red color. The presence of
nitrous acid in the yellow nitric acid is essential for the reaction,
which, however, may be obtained with pure nitric or sulphuric acid
if a small quantity of potassium nitrate is added to the culture — one
cubic centimetre of a 0.2-per-cent solution.

"Koch's Tuberculin." -This is a glycerin extract of the toxic
substances present in cultures of the tubercle bacillus. Crude tu-
berculin is obtained from liquid cultures made in veal broth to which

152

PTOMAINES AND TOXALBUMINS.

one per cent of peptone and four to five per cent of glycerin have
been added. This culture liquid is placed in flasks and inoculated
upon the surface with small masses from a pure culture of the tu-
bercle bacillus. A tolerably thick and dry white layer is developed,
which after a time covers the entire surface. At the end of six to
eight weeks development ceases and the culture liquid is evaporated
over a water bath to one-tenth its volume ; this, after being filtered,
constitutes the crude tuberculin. By precipitation with sixty-per-
cent alcohol Koch has obtained from this a white precipitate which
has the active properties of the glycerin extract. This is soluble in
water and in glycerin, and has the chemical reactions of an albumi-
nous body.

Zuelzer has (1891) reported his success in isolating a toxic sub-
stance from tubercle cultures. The contents of tubes containing
pure cultures of the bacillus are first treated with hot water
acidulated with hydrochloric acid. This solution is filtered, evapo-
rated, and then several times precipitated with platinum chloride.
The double salt formed is decomposed by hydrosulphuric acid,
after which the liquid is filtered and evaporated to dryness. A
white, crystalline salt is thus obtained which is soluble in hot water.
This salt was toxic for rabbits and guinea-pigs in doses of from one
to three centigrammes. Death usually occurred in from two to four
days. In guinea-pigs one centigramme injected subcutaneously
caused, within a few minutes, a greatly increased frequency of respi-
ration, an elevation of temperature, and protrusion of the eyeballs.

Malleln. — Kalwing, Preusse, and Pearson have obtained from
cultures of the glanders bacillus a "lymph" which somewhat re-
sembles the crude tuberculin of Koch. This was obtained by
Preusse by treating old potato cultures of the glanders bacillus with
glycerin and water. The extract was filtered several times and then
sterilized in a steam sterilizer. This lymph injected into horses in-
fected with glanders gives rise to a very decided elevation of tempe-
rature, while in horses free from this disease no such result follows.

VI.
INFLUENCE OF PHYSICAL AGENTS.

Heat. — We have already seen (Section II., Part Second) that the
temperature favorable for the growth of most bacteria is between 20°
and 40° C. ; that some species are able to multiply at the freezing tem-
perature, and others at as high a temperature as 60° to 70° C. ; that,
as a rule, the parasitic species require a temperature of 35° to 40°;
and that low temperatures do not kill bacteria.

Frisch (1877) exposed various cultures to a temperature of —87° C.,
which he obtained by the evaporation of liquid CO2, and found that
micrococci and bacilli, after exposure to such a temperature, multi-
plied abundantly when again placed in favorable conditions. Prud-
den has also made extended experiments upon the influence of
freezing. He found that while certain species resisted the freezing
temperature for a long time, others failed to grow. Thus Bacillus
prodigiosus did not grow after being frozen for fifty-one days ; Pro-
teus vulgaris was killed in the same time, and a slender, liquefying
bacillus obtained from Croton aqueduct water was killed in seven
days. Staphylococcus pyogenes aureus withstood freezing for sixty-
six days, a fluorescent bacillus from Hudson River ice for seventy-
seven days, and the bacillus of typhoid fever for one hundred and
three days. Cultures made at intervals showed, however, a dimi-
nution in the number of bacteria. A similar diminution would per-
haps have occurred in old cultures in which the pabulum for growth
was exhausted, independently of freezing ; for bacteria, like higher
plants, die in time — which varies for different species — as a result of
degenerative changes in the living protoplasm of the cells, and con-
tinued vitality in a culture depends upon continued reproduction.

Repeated freezing and thawing was found by Prudden to be
more fatal to the typhoid bacillus than continuous freezing. Cul-
tures were sterilized by being thawed out at intervals of three days
and again ref rozen, after repeating the operation five times.

Cadeac and Malet kept portions of a tuberculous lung in a frozen
condition for four months, and found that at the end of this time
tuberculosis was still produced in guinea-pigs by injecting a small
quantity of this material.

154 INFLUENCE OF PHYSICAL AGENTS.

In considering the influence of high temperatures we must take
account of the very great difference in the resisting power of the
vegetative cells and the reproductive elements known as spores, also
of the fact as to whether dry or moist heat is used and the time of
exposure.

Dry Heat. — When microorganisms in a desiccated condition are
exposed to the action of heated dry air, the temperature required for
their destruction is much above that required when they are in a
moist condition or when they are exposed to the action of hot water
or steam. This was thoroughly demonstrated by the experiments of
Koch and Wolff hugel (1881). A large number of pathogenic and
non-pathogenic species were tested, with the following general result :
A temperature of 78° to 123° C. maintained for an hour and a half
(over 100° for an hour) failed to kill various non-pathogenic bacteria,
but was fatal to the bacillus of mouse septicaemia and that of rabbit
septicaemia. To insure the destruction of all the species tested, in
the absence of spores, a temperature of 120° to 128° C., maintained
for an hour and a half, was required.

The spores of Bacillus anthracis and of Bacillus subtilis resisted
this temperature and required to insure their destruction a tempera-
ture of 140° C. maintained for three hours. This temperature was
found to injure most objects requiring disinfection, such as clothing
and bedding. But the lower temperature which destroys micro-
organisms in the absence of spores (120° C. =. 248° F.) can be used
for disinfecting articles soiled with the discharges of patients with
cholera, typhoid fever, or diphtheria, as the specific germs of these
diseases do not form spores. It is probable also that it may be safely
used to disinfect the clothing of small-pox patients, for we have ex-
perimental evidence that a lower temperature destroys the virulence
of vaccine virus (90°-95° C. — Baxter).

In practical disinfection by means of dry heat it will be necessary
to remember that it has but little penetrating power. In the experi-
ments of Koch and Wolffhiigel it was found that registering ther-
mometers placed in the interior of folded blankets and packages of
various kinds did not show a temperature capable of killing bacteria
after three hours' exposure in a hot-air oven at 133° C. and above.

Moist Heat. — The thermal death-point of bacteria, in the ab-
sence of spores, is comparatively low when they are exposed to moist
heat. The results of the writer's experiments are given below :

" In my temperature experiments I have taken great pains to insure the
exposure of the test organisms to a uniform temperature, and have adopted
tt-n minutes as the standard time of exposure. The method employed
throughout has been as follows: From glass tubing having a diameter of
about three-sixteenths of an inch I draw out in the flame of a Bunsen burner
a number of capillary tubes, with an expanded extremity which serves as

INFLUENCE OF PHYSICAL AGENTS.

155

an air chamber. A little material from a pure culture of the test organ-
ism is drawn into each of these capillary tubes by immersing- the open
extremity in the culture, after having1 gently heated the expanded end. The
end of the tube is then hermetically sealed by heat These tubes are im-
mersed in a water bath maintained at the desired temperature for the stan-
dard time. The bath is kept at a uniform temperature by personal supervi-
sion. At the bottom of the vessel is a thick glass plate which prevents the
thermometer bulb and capillary tubes, which rest upon it, from being ex-
posed to heat transmitted directly from the bottom of the vessel To further
guard against this I am in the habit of applying the flame to the sides of the
vessel, and a uniform temperature throughout the bath is maintained by
frequent stirring1 with a glass rod. It is impossible to avoid slight variations,
but by keeping my eye upon the thermometer throughout the experiment
I have kept these within very narrow limits. . . . No attempt has been made
to fix the thermal death-point within narrower limits than 2° C., and in the
table the lowest temperature is given which has been found, in the experi-
ments made, to destroy all of the microorganisms in the material subjected
to the test. No doubt more extended experiments would result, in some in-
stances, in a reduction of the temperature given as the thermal death-point
for a degree or more. But the results as stated are sufficiently accurate for
all practical purposes." 1

The results obtained in these experiments, for non-sporebearing
bacteria, are given in the following table. The time of exposure
was ten minutes, except for the cholera spirillum and the cheese spi-
rillum of Deneke.

THERMAL DEATH-POINT OF BACTERIA.

Spirillum cholene Asiatic* 52°

Spirillum tyrogenum (cheese spirillum) 52

Spirillum Flakier- Prior 50

Bacillus typhi abdominalis 56

Bacillus of schweine-rothlauf (rouget) 58

Bacillus murisepticus 58

Bacillus Neapolitanus (Emmerich's bacillus) 62

Bacillus cavicida 62

Bacillus pneumoniae (Friedlander's) 56

Bacillus crassus sputigenus 54

Bacillus pyocyaueus 56

Bacillus iudicus 58

Bacillus prodigiosus 58

Bacillus cyanogenus 54

Bacillus fluorescens . . . . 54

Bacillus acidi lactici 56

Staphylococcus pyogenes aureus . . . 58

Staphylococcus pyogenes citreus 62

Staphylococcus pyogenes albus 62

Streptococcus pyogenes 54

Micrococcus tetragenus 58

Micrococcus Pasteuri 52

Sarcina lutea 64

Sarcina aurantiaca 62

Centigrade.

Fahrenheit.

125.6C

125.6

122.

138.8

136.4

143.6

132.8

129.2

132.8

136.4

129.2

132.8

136.4

143.6

1292

136.4

125.6

147.2

143.6

(4m.)
(4m.)

The following determinations of the thermal death-point of path-

1 Quoted from the Report of the Committee on Disinfectants of the American Pub-
lic Health Association, pages 136 and 152.

156 INFLUENCE OF PHYSICAL AGENTS.

ogenic organisms have been made by the authors named : Bacillus
anthracis (Chauveau), 54° C. ; Bacillus mallei — the bacillus of glan-
ders— (Loffler), 55° C., Bacillus gallinarum — micrococcus of fowl
cholera— (Salmon), 56° C. ; Bacillus of diphtheria (Loffler), 60° C.

In the writer's experiments the micrococcus of gonorrhoea was
apparently killed by exposure for ten minutes to a temperature of
60° C.

" Some gonorrhceal pus from a recent case which had not undergone
treatment was collected for me by my friend Dr. Rohe in the capillary
glass tubes heretofore described. A microscopical examination of stained
cover-glass preparations showed that this pus contained numerous ' gono-
cocci ' in the interior of the cells. Two of the capillary tubes were placed
in a water bath maintained at 60° C. for ten minutes. The pus was then
forced out upon two pledgets of cotton wet with distilled water. Two
healthy men had consented to submit to the experiment, and one of these
bits of cotton was introduced into the urethra of each and left in situ for
half an hour. As anticipated, the result WHS entirely negative. For obvi-
ous reasons no control experiment was made to fix the thermal death-point
within narrower limits.

" In connection with these experiments upon the thermal death -point of
known pathogenic organisms, it is of interest to inquire whether the viru-
lence of infectious material, in which it has not been demonstrated that this
virulence is due to a microorganism, is destroyed by a correspondingly low
temperature. Evidently, if this proves to be the case, it will be a strong
argument in favor of the view that we have to deal with a microorganism
in these diseases also. We have experimental proof that a large number of
pathogenic organisms are killed by exposure for ten minutes to a tempera-
ture of 55° to tfO° C. But, s ) far as I am aware, this low temperature would
not be likely to destroy any of the poisonous chemical products which might
be supposed to be the cause of infective virulence, leaving aside the fact that
such chemical products have no power of self-multiplication, and, there-
fore, could not be the independent cause of an infectious disease.1

" Vaccine Virus. — Carstens and Coert have experimented upon the tem-
perature required to destroy the potency of vaccine virus. In a paper read
at the International Medical Congress in 1879 they report, as a result of
their experiments, that the maximum degree of heat to which fresh vaccine
virus can be exposed without losing its virulence probably varies between
52° and 54° C. Fresh animal vaccine heated to 52° C. for thirty minutes
does not lose its virulence. Fresh animal vaccine heated to 54. 5 3 for thirty
minutes loses its virulence.

"Rinderpest. — According to Semmer and Raupach, exposure for ten
.minutes to a temperature of 55° C. destroys the virulence of the infectious
material in this disease.

'' Sheep-pox. — The authors last mentioned have also found that the same
temperature — 55° C. for ten minutes — destroys the virulence of the blood of
an animal dead from sheep-pox.

" Hydrophobia. — Desiring to fix the thermal death -point of the virus of
hydrophobia, I obtained, through the kindness of Dr. H. C. Ernst, a rabbit
which had been inoculated, by the method of trephining, with material
which came originally from Pasteur's laboratory. The rabbit sent me
showed the first symptom of paralytic rabies on the eighth day after inocu-
lation. It died on the eleventh day (March 2d, 1887), and I at once pro-
ceeded to make the following experiment :

" A portion of the medulla was removed and thoroughly mixed with

1 Since this was written Brieger has isolated a toxalbumin from cultures of the
diphtheria bacillus which is destroyed by u temperature of 60° C., but resists 50°.

INFLUENCE OF PHYSICAL AGENTS. 157

sterilized water. The milky emulsion was introduced into four capillary
tubes, such as had been used in my experiments heretofore recorded. Two
of these tubes were then placed for ten minutes in a water bath, the tem-
perature of which was maintained at 60° C. Four rabbits were now inocu-
lated by trephining1, two with the material exposed to 60° C. for ten min-
utes, and two with the same material from the capillary tube not so exposed.
The result was as definite and satisfactory as possible. The two control
rabbits were taken sick, one on March 10th and one on the llth ; both died
with the characteristic symptoms of paralytic rabies on the third day. The
two rabbits inoculated with material exposed to 00° C. remained in perfect
health. On the 26th of March one of these rabbits was again inoculated,
by trephining, with material from the medulla of a rabbit just dead from
hydrophobia. This rabbit died from paralytic rabies oil the 8th of April.
Its companion remains in perfect health.

"A second experiment was made in the same way on the 14th of March.
Two rabbits were inoculated with material exposed for ten minutes to a
temperature of 50° C. ; two with material exposed to 55° C. ; and two con-
trol rabbits with material not so exposed. One of the rabbits inoculated
with material exposed to 50° C., and one of the control rabbits, died on the
25th; the other rabbit inoculated with the material exposed to 50°, the other
control, and one inoculated with material exposed to 55°, on the 26th. The
second rabbit inoculated with material exposed to 55° died five days later
with the characteristic symptoms of the disease- These experiments show,
then, that the virus of hydrophobia is destroyed by a temperature of 603 C.,
and that 55° C. fails to destroy it, the time of exposure being ten minutes."1

The experimental data given show that the pathogenic bacteria
tested and different kinds of virus are all killed by a temperature of
60° C. or below ; some, like the cholera spirillum and Micrococcus
pneumonise crouposse, failing to grow after exposure to as low a tem-
perature as 52° C. for four minutes. By extending the time a still
lower temperature will effect the same result. Thus, according to
Chauveau, the anthrax bacillus is killed by twenty minutes' exposure
to a temperature of 50° C. ; and Brieger sterilizes cultures of the
diphtheria bacillus, to obtain the soluble toxalbumin produced in
them, by exposure for several hours to 50° C. A temperature of 60°
has been found to decompose the toxalbumin. The non-pathogenic
bacteria tested have, as a rule, a higher thermal death-point — 58° C.
for Bacillus prodigiosus, 64° C. for Sarcina lutea, etc.

It is a remarkable fact that certain bacteria not only are not de-
stroyed at higher temperatures than this, but are able to multiply at
a temperature of 65° to 70° C. Thus Miquel, in 1881, found in the
waters of the Seine a motionless bacillus which grew luxuriantly in
bouillon at a temperature of 69° to 70° C. Van Tieghem has also
cultivated several different species at about the same temperature,
and more recently Globig has obtained from the soil several species
which grow at temperatures ranging from 50° to 70° C.

The resisting power of spores to heat also varies in different spe-
cies ; but the spores of known pathogenic bacteria are quickly de-
stroyed by a temperature of 100° C. (212° F.). In the writer's experi-

1 Report of the Committee on Disinfectants (op. cit.), p. 147.

158 INFLUENCE OF PHYSICAL AGENTS.

ments the spores of Bacillus anthracis and of Bacillus alvei failed to
grow after exposure to a temperature of 100° C. for four minutes,
and only a few colonies developed after two minutes' exposure to this
temperature. The thermal death-point of spores of the " wurtzel ba-
cillus " and of Bacillus butyricus (of Hueppe) was the same — 100° C.
for four minutes.

Schill and Fischer, in 1884, made a number of experiments to de-
termine the thermal death-point of Bacillus tuberculosis. They
found that five minutes' exposure to a temperature of 100° C. in
steam destroyed the vitality of the bacillus in sputum in five min-
utes. When the time was reduced to two minutes a negative result
from inoculation was obtained in two guinea-pigs, but one inoculated
at the same time became tuberculous. My own experiments and
those of Yersin, made since, lead me to think that there may have
been some cause of error in this experiment of Schill and Fischer,
and that the thermal death-point of the spores of Bacillus tuber-
culosis is considerably below the boiling point of water. I inoculated
guinea-pigs with tuberculous sputum subjected for ten minutes to
the following temperatures : 50°, 60°, 70°, 80°, 90° C. The animal
inoculated with material exposed to 50° died from tuberculosis at the
end of seven weeks. None of the others developed tuberculosis.

Yersin exposed an old culture in glycerin bouillon, in which many
of the bacilli contained spores — "tres nettes" — to the following tem-
peratures : 55°, 60°, 65°, 70°, 75°, 80°, 85°, 90°, 100° C. ' ' At the end of
ten days the bacilli heated to 55° gave a culture in glycerin bouillon ;
those exposed to 60° grew after twenty-two days ; none of the
bacilli heated above 70° gave any development. This experiment,
repeated a great number of times, has always given us the same re-
sult." Voelsh, who has studied the same question, reports as the
result of his experiments that the tubercle bacillus in sputum was
not destroyed by heating to 100° C. Further experiments will be re-
quired to reconcile these contradictory results.

While the spores of the pathogenic bacteria mentioned are de-
stroyed by the boiling point of water within a few minutes, certain
non-pathogenic species resist this temperature for hours. Thus
Globig obtained a bacillus from the soil the spores of which required
five and one-half to six hours' exposure to streaming steam for their
destruction. These spores survived exposure for three-quarters of an
hour in steam under pressure at from 109° to 113° C. They were de-
stroyed, however, by exposure for twenty-five minutes in steam at
113° to 116°, and in two minutes at 127°.

In the practical application of steam for disinfecting purposes it
must be remembered that, while steam under pressure is more effec-
tive than streaming steam, it is scarcely necessary to give it the pre-

INFLUENCE OF PHYSICAL AGENTS. 159

ference, in view of the fact that all known pathogenic bacteria and
their spores are quickly destroyed by the temperature of boiling
water ; and also that superheated steam is less effective than moist
steam. When confined steam in pipes is '* superheated " it has about
the same germicidal power as hot dry air at the same temperature.
This is shown by the experiments of Esmarch, who found that an-
thrax spores were killed in streaming steam in four minutes, but
were not killed in the same time by superheated steam at a tempera-
ture of 141° C.

Desiccation. — Cultures of bacteria kept in a moist condition re-
tain their vitality for a considerable time, which varies greatly with
different species. The writer has found that a culture of the typhoid
bacillus preserved in a hermetically sealed glass tube retained its
vitality for eighteen months, as did also Bacillus prodigiosus, Bacil-
lus cavicida, and some others. According to Kitasato, the cholera
spirillum may be preserved in a moist state for seven months ; other
bacteria die out in a month or two, but, as a rule, vitality is preserved
for several months at least.

Spores in a desiccated condition preserve their vitality for a
great length of time. But desiccation is quickly fatal to some of the
pathogenic bacteria, and especially so to the cholera spirillum. Koch,
in his earlier experiments, found that his "comma bacillus" did not
grow after being dried upon a cover glass for three hours. Kitasato,
in experiments made since, found that a bouillon culture dried upon
a thin glass cover was incapable of development after three hours'
time, but that cultures in nutrient agar or gelatin survived for two
days, probably on account of the thicker layer formed and the longer
time required for complete desiccation. Pfuhl has found that the
typhoid bacillus dried upon a cover glass retains its vitality for
eight to ten weeks, and Loffler states that the diphtheria bacillus re-
sists desiccation for four or five months. Cadeac and Malet pro-
duced tuberculosis in guinea-pigs by injecting material from the
lung of a tuberculous cow which had been kept in the form of a dried
powder for nearly five months ; at a later date the virulence was
lost.

Light. — Downes and Blunt, in a communication made to the
Royal Society of London in 1877, first called attention to the fact that
light has an injurious effect upon bacteria, and that cultures may be
sterilized by exposure to direct sunlight.

Tyndall, in experiments made in the clear sunlight of the Alps,
verified the fact that the development of bacteria was restrained in
cultures during their exposure, but failed to obtain evidence that
vitality was destroyed.

Jn 1885 Duclaux took up the subject with pure cultures of various

1GO INFLUENCE OF PHYSICAL AGENTS.

bacteria, and showed that by prolonged exposure to direct sunlight the
spores of various bacilli lose their capacity to germinate. About the
same time Arloing published his researches upon the influence of
light upon the development of anthrax spores. He found that the
anthrax bacillus was not restrained in its growth by diffused lamp-
light, but its growth was retarded by an intense gaslight. Spore
formation was more abundant in darkness than in red light, and more
abundant in red than in white light. When a screen was interposed
between the culture and the source of light, consisting of an aqueous
solution of hsematoglobin, the growth of the bacilli and of spores was
much more luxuriant than in white light. In yellow light it was less
abundant than in red. The blue and violet rays were still less favor-
able for the growth of the bacillus and the development of spores.
The pathogenic power of cultures was not especially influenced by
exposure to white gaslight. In subsequent experiments with sun-
light Arloing found that two hours of exposure to the July sun suf-
ficed to destroy the vitality of anthrax spores, but that a considerably
longer exposure (twenty-six to thirty hours) was necessary when the
spores had bean allowed to germinate in a suitable culture medium.
Cultures which were not exposed long enough to destroy the vitality
of the bacilli were retarded in their growth, and subsequent exposure
for a shorter time (nine to ten hours) completely sterilized them.
Cultures which were weakened in their reproductive energy by ex-
posure to sunlight were also ' ' attenuated " as to their pathogenic
power and could be used as a vaccine in protective inoculations. Ac-
cording to Arloing, the effect produced results from the action of the
full sunlight and cannot be obtained by the use of monochromatic
light.

The experiments of Strauss seemed to give support to the view
advanced by Nocard that in Arloing's experiments spores did not
really exhibit a less degree of resisting power than the vegetating
bacilli, but that in fact they commenced to vegetate before they were
killed. Strauss placed anthrax spores in sterilized distilled water and
in bouillon, and found that, under the same conditions of exposure,
the bouillon cultures were sterilized in direct sunlight in nine
hours, while the spores suspended in distilled water grew when trans-
ferred to a suitable medium. This was accounted for on the suppo-
sition that the bouillon furnishes the necessary pabulum for the de-
velopment of the spores and that distilled water does not.

Arloing combats this view and has published additional experi-
ments which seem to disprove it. He placed small flasks containing
anthrax spores in bouillon in the direct rays of the sun in February.
Some of the flasks were placed upon a block of ice which reduced the
temperature to 4° C. ; the others were not so placed, and the tempe-

INFLUENCE OF PHYSICAL A(,KNTs. 161

rature. in the open air where all were exposed, was 11° C. All of the
spores failed to grow after an exposure of four hours. When exposed
in water the time of exposure was longer.

Roux has shown that the light also has an effect upon the culture
medium, and that sterilized bouillon which has been exposed to direct
sunlight for some hours restrains the development of anthrax spores
subsequently introduced into it, but not of the growing bacilli. His
experiments show that access of oxygen is a necessary factor in the
sterilization of cultures by sunlight.

In the experiments of Moment (ISO'-?) dry anthrax spores were
found to resist the action of light for a long time, but moist spores,
freely exposed to the air, failed to grow after forty-four hours' ex-
posure to sunlight. In the absence of spores, anthrax bacilli in a
moist condition, when freely exposed to the air, failed to grow after
exposure to sunlight for half an hour to two hours ; but in the ab-
sence of air the same bacilli were not destroyed at the end of fifty
hours' exposure.

Geisler (1892), in experiments made upon the typhoid bacillus,
found that all portions of the solar spectrum except the red rays ex-
ercised a restraining influence upon the development of this bacillus.
The electric light gave a similar result. The most decided effect was
produced by rays from the violet end of the spectrum. The restrain-
ing influence appears, from the researches of Geisler, not to be due
solely to the direct action of light upon the development of the
bacilli, but also to changes induced in the gelatin culture medium
employed in his experiments.

In his address before the International Medical Congress of Berlin,
1800, Koch states that the tubercle bacillus is killed by the action of
direct sunlight in a time varying from a few minutes to several hours,
depending upon the thickness of the layer exposed. Diffused day-
light also has the same effect, although a considerably longer time of
exposure is required — when placed close to a window, from five to
seven days.

Dieudonne (1804), in experiments upon Bacillus prod igiosus and
Bacillus fluorescens putidus, found that direct sunlight in March,
July, and August killed these bacilli in one and one-half hours, in
November in two and one-half hours. Diffuse daylight in March
and July restrained development after three and one-half hours' ex-
posure (in November four and one-half hours), and completely de-
stroyed vitality in from five to six hours.

Ward's experiments (180-2-1804) show that the blue and violet
rays have decided germicidal power, while the rays at the red end of
the spectrum are comparatively inert. This corresponds with results
previously reported by Arloing.
11

162 INFLUENCE OF PHYSICAL AGENTS.

In the writer's experiments on the cholera spirillum (1802) test
tubes, containing sterile bouillon inoculated with one or two ose of a
pure culture, were sterilized by two hours' exposure to direct sunlight
(in December).

Dieudonne (1894) found that the electric arc light destroyed his
test organisms (Bacillus prod igiosu sand Bacillus fluorescens putidus)
in eight hours. The same result was accomplished by the incandes-
cent light in eleven hours.

In view of these facts we may conclude, with Duclaux, that sun-
light is one of the most potent and one of the cheapest agents for the
destruction of pathogenic bacteria, and that its use for this purpose is
to be remembered in making practical hygienic recommendations.
The popular idea that the exposure of infected articles of clothing
and bedding in the sun is a useful sanitary precaution is fully sus-
tained by the experimental data relating to the action of heat, desic-
cation, and sunlight.

Electricity. — Cohn and Mendelssohn, in 1879, attempted to de-
termine the effect of the galvanic current upon bacteria. Cultures
were placed in U -tubes through which a constant current was passed.
A feeble current was found to be without effect. A strong current
from two elements, maintained for twenty-four hours, restrained de-
velopment in the vicinity of the positive pole, but this was probably
due to the highly acid reaction which the culture liquid acquired.
When a current from five elements was used for twenty-four hours
the liquid was sterilized, but this may have been due to the decided
changes produced in the chemical composition of the culture liquid
rather than to the direct action of the galvanic current.

The same may be said of the similar results obtained in later ex-
periments by Apostoli and Laquerriere, and by Prochownick and
Spaeth. The last-mentioned investigators found that the positive pole
had a more decided effect than the negative, and that the effect de-
pended upon the intensity and duration of the current. A current of
fifty milliamperes passed for a quarter of an hour did not kill Staphy-
lococcus pyogenes aureus, but a current of sixty milliamperes main-
tained for the same time did. The spores of Bacillus anthracis
required a current of two hundred to two hundred and thirty milli-
amperes during an hour or two. In these experiments the cultures
in gelatin were attached to the strips of platinum serving as the two
poles, and these were immersed in a solution of sodium chloride. As
chlorine was disengaged at the positive pole, the germicidal action is
attributed to this gas rather than to the direct action of the current
upon the living microorganisms.

The more recent researches of Spilker and Gottstein, made with
an induction current from a dynamo machine, are more valuable in

INFLUENCE OF PHYSICAL AGENTS. 103

estimating the power of this agent to destroy the vitality of bacteria.
The current was passed through a spiral wire which was wrapped
around a test tube of glass, containing the microorganism to be tested,
suspended in distilled water. In a first experiment Bacillus prodigi-
osus, suspended in sterilized distilled water and contained in test
tubes having a capacity of two hundred and fifty cubic centimetres,
was subjected to a current having an energy of 2.5 amperes X 1.25
volts for twenty-four hours. The temperature did not go above
30° C. No development occurred when the microorganism tested
was subsequently planted in nutrient gelatin. Further experiments
gave a similar result. It was found that stronger currents were
effective in shorter time ; but in no case was sterilization effected in
less than an hour.

Pressure. — D'Arsonval and Charrin (1894) submitted a culture
of Bacillus pyocyaneus tc a pressure of fifty atmospheres, under car-
bon dioxide. At the end of four hours cultures could still be ob-
tained, but the bacillus had lost its power of pigment production. A
few colonies were developed after six hours' exposure to this pressure;
but after twenty-four hours no development occurred.

Agitation. — Meltzer (1894) has shown that the vitality of bacteria
is destroyed by protracted and violent shaking, which causes a molec-
ular disintegration of the cells.

VII.
ANTISEPTICS AND DISINFECTANTS.

GENERAL ACCOUNT OF THE ACTION OF.

THE term antiseptic is used by some authors to designate an
agent which destroys the vitality of the microorganisms which pro-
duce septic decomposition, and others of the same class. We prefer
to restrict the use of the term to those agents which restrain the de-
velopment of such microorganisms without destroying their vitality.
The complete destruction of vitality is effected by germicides or dis-
infectants. Material containing the germs of infectious diseases is
infectious material, and we disinfect it by the use of agents which
destroy the living disease germs or pathogenic bacteria which give
it its infecting power. Such an agent is a disinfectant. But we ex-
tend the use of this term to germicides in general — that is, to those
agents which kill non-pathogenic bacteria as well as to those which
destroy disease germs. All disinfectants are also antiseptics, for
agents which destroy the 'vitality of the bacteria of putrefaction ar-
rest the putrefactive process ; and these agents, in less amount than
is required to completely destroy vitality, arrest growth and thus
act as antiseptics. But all antiseptics are not germicides. Thus a
concentrated solution of salt or of sugar will prevent the putrefac-
tive decomposition of organic material, animal or vegetable ; but these
agents do not destroy the vitality of the germs of putrefaction. In
a certain degree of concentration they are antiseptics and are largely
used for the preservation of meats and vegetables. In the same way
many mineral salts in solutions of various strengths act as antisep-
tics, and some of these in still stronger solutions are disinfectants.
Thus mercuric chloride, when introduced into a culture solution in
the proportion of 1 : 300,000, will restrain the development of anthrax
spores, but to insure the destruction of these spores a solution of
1 : 1,000 must be used. As a rule, the difference between restraining
action — antiseptic — and germicidal power — disinfectant — is not so
great as this. We give below some recent determinations by Boer
which illustrate this point, the test organism being the bacillus of
typhoid fever in a culture in bouillon twenty-four hours old :

ANTISEPTICS AXD DISINFECTANTS. 165

Restrains.

Kills.

Hydrochloric acid

1 :2100

1 -300

Sulphuric acid

1 : 1550

1 : 500

Silver nitrate

1 : 50000

1 : 4000

Sodium arscniate

1 : 6000

1 :250

Carbolic acid

1 :400

1 :200

Method of Determining Antiseptic rahte. — To determine the
restraining or antiseptic power of an agent for a particular micro-
organism, the agent is dissolved in a definite proportion in a suitable
culture medium, which is then inoculated with a pure culture of the
test organism and placed in favorable circumstances — as to tempera-
ture— for its growth. At the same time a control experiment is
made by placing another portion of the same culture medium, inocu-
lated with the same microorganism, in the same conditions, but with-
out the addition of the antiseptic agent. If development occurs in
the control experiment and not in the culture medium containing
the antiseptic, the failure to grow must be attributed to the presence
of this agent. Having made a preliminary experiment, we are
guided by the result in further experiments to determine the exact
amount required to restrain development under the same conditions.
Or we may make a series of experiments in the first instance. The
problem being, for example, to determine the antiseptic value of
carbolic acid for the typhoid bacillus, we may add this agent to a
definite amount of bouillon in test tubes in the proportion of 1 : 100,
1 : 200, 1 : 300, 1 : 400, 1 : 500. In experiments with volatile agents
the bouillon, in test tubes or small flasks, must be sterilized in ad-
vance, and the antiseptic agent introduced by means of a sterilized
pipette with great care to prevent the accidental contamination of
the nutrient medium. In experiments with non-volatile agents it will
be best to sterilize the culture medium after the antiseptic has been
added. Next we inoculate the liquid in each flask with a pure cul-
ture of the test organism. The flasks are then placed in an incubat-
ing oven at 35° to 37° C. At the same time a control, not containing
any carbolic acid, is placed in the oven. At the end of twenty-four
hours the control will be found to be clouded, showing an abundant
multiplication of the bacillus. Taking the result of Boer above given,
we would expect to find all of the solutions clear except that contain-
ing 1 : 500. This too might remain clear for some days and finally
" break down," for experience shows that when we pass the point at
which a permanent restraining influence is exerted there may be a
temporary restraint or retardation of development. For this reason
we must continue the experiment for a considerable time — not less

1GG ANTISEPTICS AND DISINFECTANTS.

than two weeks. Having found that 1 : 400 and below prevents
development, and 1 ; 500 does not, we may make further experiments
to determine the antiseptic power within narrower limits ; but this
is hardly necessary from a practical point of view.

In these experiments the result will be influenced by several cir-
cumstances, as follows :

(a) By the composition of the nutrient medium. This is a
very important factor, especially in determining the antiseptic value
of certain metallic salts. The presence of a considerable quantity
of albumin, for example, reduces greatly the antiseptic power of
mercuric chloride, silver nitrate, creolin, etc. The presence of a sub-
stance chemically incompatible, as, for example, sodium chloride in
testing nitrate of silver, will of course neutralize antiseptic action.

(b) The nature of the test organism. Within certain limits an
antiseptic for one microorganism of this class restrains the devel-
opment of all, but there are wide differences in the ability of differ-
ent species to grow in the presence of different chemical agents.
Some grow readily in the presence of a considerable amount of free
acid, others are restrained by a slightly acid reaction of the medium
in which they are placed. The Bacillus acidi lactici, for example,
can thrive in the presence of a considerable amount of the acid
which is a product of its growth, but there is a limit to its power of
developing in the presence of this and other acids. So, too, Mi-
crococcus urese, which causes the alkaline fermentation of urine,
grows in the presence of a considerable amount of carbonate of am-
monia, but is finally restrained in its growth by this alkaline salt.
The following determinations by Boer show the difference in the
antiseptic power of hydrochloric acid for certain pathogenic bacte-
ria : Bacillus of anthrax (without spores), 1 : 3,400 ; diphtheria bacil-
lus, 1 :3,400 ; glanders bacillus, 1 : 700 ; typhoid bacillus, 1 : 2,100 ;
cholera spirillum, 1 :5,500. It will be noted that the cholera spiril-
lum is restrained in its growth by about one-eighth the amount of
hydrochloric acid which is required to prevent the development of
the bacillus of glanders. The typhoid bacillus has a special tole-
rance for carbolic acid, etc.

(c) The temperature at which the experiment is made. At
the temperature most favorable for growth a greater proportion of
the antiseptic agent is required than at unfavorable temperatures-
lower or higher.

(d) The restraining influence for spores is much greater than
for the vegetative form of bacteria.

Methods of Determining Germicide Value. — The disinfecting
power of a chemical agent is determined by allowing it to act for a
given time, in a definite proportion, on a pure culture of a given

ANTISEPTICS AND DISINFECTANTS. 167

microorganism, and then testing the question of loss of vitality by
culture experiments or by inoculations of infectious disease germs
into susceptible animals.

The test by cultivation is the most reliable, but in making it
several points must be kept in view. Naturally the conditions must
be such as are favorable for the growth of the particular microor-
ganism which serves as the test ; and we must allow a considerable
time for the development of the test organism, for it often happens
that its vital activity has been weakened without being completely
destroyed, and that growth will occur after an interval of several
days, while in the control experiment it has perhaps been seen at
the end of twenty-four hours. Another most important point is the
fact that some of the disinfecting agent is necessarily carried over
with the test organisms when these are transferred to a nutrient
medium to ascertain whether they will grow, and this may be in ,
sufficient amount to restrain their development and lead to the mis-
taken inference that they have been killed. This is especially true
of mercuric chloride, which restrains the development of spores in
very minute amounts. Spores which have been subjected to its ac-
tion in comparatively strong solutions, when transferred to a culture
medium may fail to grow because of the restraining influence of
the mercuric chloride carried over at the same time. For this rea-
son liquid cultures are to be preferred in experiments of this kind.
When the test organisms are planted in a solid culture medium the
chemical agent is left associated with them ; in a liquid culture, on
the other hand, it is diluted, and the microorganisms, being distri-
buted through the nutrient medium, have the disinfecting agent
washed from their surface. In the case of mercuric chloride, how-
ever, the experiments of Geppert show that the agent is so attached
to spores which have been subjected to its action that ordinary
washing does not suffice. Moreover, spores which have been ex-
posed to the action of mercuric chloride without being killed are re-
strained in their growth by a much smaller proportion of the corro-
sive sublimate than is required for spores not so exposed — according
to Geppert, by 1 part in 2,000,000. Geppert therefore proposes, in
experiments with this agent, to neutralize the mercuric chloride
which remains attached to the test organisms by washing these in
a solution of ammonium sulphide, by which the sublimate is preci-
pitated as an inert sulphide.

With most agents simple dilution will serve the purpose of pre-
venting an erroneous inference from the restraining influence of the
chemical agent being tested. If we carry, by means of a platinum
loop, one or two ose into five to ten cubic centimetres of bouillon,
the dilution will usually be beyond the restraining influence of the

108 ANTISEPTICS AND DISINFECTANTS.

germicidal agent ; but we may carry the dilution still further, to be
on the side of safety, by inoculating a second tube containing the
same amount of sterile bouillon from the first, carrying over in the
same way one or two ose. We will still be very sure to have a
considerable number of the microorganisms to test the question of
the destruction of vitality. Instead of bouillon we may use liquefied
flesh-peptone-gelatin, which gives us the same advantage as to dilu-
tion of the disinfecting agent ; and after inoculating two tubes as
above indicated, we may make Esmarch roll tubes by turning them
upon a block of ice. The development of colonies will show that
there was a failure to disinfect ; their absence, after a proper inter-
val, will be evidence of the germicidal action of the agent employed.

Koch's Method. — In 1881 Koch published his extended experi-
ments made to determine the germicidal power of various chemical
agents as tested upon anthrax spores. His method consisted in ex-
posing silk threads, to which the dried spores were attached, in a
solution of the disinfecting agent, and at intervals transferring one
of these threads to a solid culture medium. The precaution was
taken to wash the thread in distilled water when the agent tested was
supposed to be likely to restrain development. In these experiments
a standard solution of the disinfecting agent was used, and the time
of exposure was varied from a few hours to many days.

The Writer's Method. — In the writer's experiments, made in
1880 and subsequently, a different method has been adopted. The
time has been constant — usually two hours — and the object has been
to find the minimum amount of various chemical agents which
would destroy the test organisms in this time ; and instead of sub-
jecting a few of the test organisms attached to a silk thread to the
action of the disinfecting agent, a certain quantity of a recent cul-
ture— usually five cubic centimetres — has been mixed with an equal
quantity of a standard solution of the germicidal agent. Thus five
cubic centimetres of a 1 : 200 solution of carbolic acid would be
added to five cubic centimetres of a recent culture of the typhoid
bacillus, for example, and after two hours' contact one or two ose
would be introduced into a suitable nutrient medium to test the
question of disinfection. In the case given the result obtained
would be set down as the action of a solution of carbolic acid in the
proportion of 1 : 400, for the 1 : 200 solution was diluted by the addi-
tion of an equal quantity of the culture.

Other experimenters have adopted still a different method. In-
stead of using a considerable and definite quantity of a culture con-
taining the test organism, they introduce one or two ose from such
a culture into a solution containing a given proportion of the disin-
fectant ; then after exposure for a given time the nutrient medium is
inoculated.

ANTISEPTICS AND DISINFECTANTS.

169

These different methods give results which cannot be directly
compared one with another, for to obtain corresponding results we
must have identical conditions.

Test by Inoculation into Susceptible Animals. — In testing the
action of disinfectants upon anthrax spores and other infectious dis-
ease germs, we may inoculate the microorganisms, after exposure to
the disinfectant, into a susceptible animal. This method was adopted
by the writer in a series of experiments in 1881, but he has not since
employed it, for reasons set forth in his paper giving an account of
these experiments.

"First. The test organism maybe modified as regards repro-
ductive activity without being killed ; and in this case a modified form
of disease may result from the inoculation, of so mild a character as
to escape observation. Second. An animal which has suffered this
modified form of the disease enjoys protection, more or less perfect,
from future attacks, and if used for a subsequent experiment may,
by its immunity from the effects of the pathogenic test organism,
give rise to the mistaken assumption that this had been destroyed
by the action of the germicidal agent to which it had been sub-
jected."1

In experiments to determine the value of an agent as a disinfec-
tant, no matter by what method, the following conditions, which in-
fluence the result, should be kept in view :

(a) The difference in vital resisting power of different species
of bacteria. As a rule, the pathogenic species have rather less re-
sisting power than the common saprophytes, and the micrococci
have greater resisting power than many of the bacilli. The differ-
ence in the vital resisting power of some of the best known patho-
genic species is shown in the following table, which we have made
up from determinations made by Boer — cultures in bouillon twenty-
four hours old ; time of exposure, two hours.

Hydrochloric
Acid.

Caustic
Soda.

Chloride of
Gold and
Sodium.

Nitrate
of
Silver.

Carbolic
Acid.

Anthrax bacillus

1 : 1100

1 : 450

1 :8000

1 : 20000

1:300

Diphtheria bacillus
Glanders bacillus

1:700
1 :200

1:300
1 :150

1 : 1000
1 :400

1 :2oOO
1 : 4000

1:300
1 :300

Typhoid bacillus

1 :300

1:190

1 :500

1 : 4000

1 :200

Cholera spirillum

1 : 1350

1 :150

1 : 1000

1 :4000

1 :400

(b) The presence or absence of spores. The reproductive ele-
ments known as spores have a far greater resisting power to chemi-
cal agents, as well as to heat, than have the vegetative cells. In

1 Quoted from article on " Germicides and Disinfectants/' in " Bacteria," p. 212.

170 ANTISEPTICS AND DISINFECTANTS.

practical disinfection, therefore, it is important to know what disease
germs form spores and what do not. The following are known to
form spores : The bacillus of anthrax, the bacillus of tetanus, the
bacillus of malignant oedema, the bacillus of symptomatic anthrax,
the bacillus of foul brood (infectious disease of bees). The following,
so far as is known, do not form spores : The pus cocci (Staphylo-
coccus pyogenes albus, aureus, and citreus, and Streptococcus pyo-
genes), the micrococcus of pneumonia, the bacillus of typhoid fever,
the bacillus of glanders, the bacillus of diphtheria, the spirillum of
cholera, the spirillum of relapsing fever.

Many agents which kill the growing bacteria are incapable of
destroying the vitality of spores, and others only do so in much
stronger solutions or after a long exposure to their action.

(c) The number of bacteria to be destroyed. This is an essen-
tial factor which has often been overlooked by those making experi-
ments. To destroy the bacteria carried over to five cubic centimetres
of distilled water by means of a platinum loop, is a very different
matter from destroying the immensely greater number in five cubic
centimetres of a recent bouillon culture.

(d) The nature and quantity of associated material. The
oxidizing disinfectants, like permanganate of potash and chloride of
lime, not only act upon the bacteria, destroying them by oxidation,
but upon all organic matter with which they come in contact, and at
the same time the disinfecting agent is destroyed in the chemical
reaction, which is a quantitative one. The presence, therefore, of
organic material in association with the bacteria is an important
factor, and if this is in excess the disinfectant may be neutralized
before the living bacteria are destroyed. Other substances which
precipitate the disinfecting agent in an insoluble form, or decompose
it, must of course have the same effect. Thus the presence of sodium
chloride in a culture medium would be an important circumstance if
nitrate of silver was the agent being tested, as the insoluble chloride
would be precipitated. And in the case of mercuric chloride and
certain other metallic salts the presence of albumin very materially
influences the result. Van Ermengem states that the cholera spiril-
lum in bouillon is destroyed in half an hour by mercuric chloride in
the proportion of 1: 60,000, while in blood serum 1: 800 was required
to destroy it in the same time.

(e) The time of exposure is also an important factor. Some
agents act very promptly, while others require a considerable time to
effect the destruction of bacteria exposed to their action. Thus a
solution of chloride of lime containing 0.12 per cent destroys the
typhoid bacillus and the cholera spirillum in five minutes, and
the anthrax bacillus in one minute (Mssen). On the other hand,

ANTISEPTICS AND DISINFECTANTS. 171

quicklime (milk of lime) requires a contact of several hours to in-
sure the destruction of pathogenic bacteria.

(/) The temperature at ivhich the exposure is made has a
material influence upon the result. This is shown by the experi-
ments of Henle and of Nocht. As a general rule germicidal activ-
ity increases in direct proportion to the increase in temperature from
20° 0. upward.

(g} The degree of dilution of the disinfecting agent is also a
matter of importance . This is especially true of solutions of acids
and alkalies. When a silk thread to which bacteria are attached is
suspended in an acid solution the essential point is the degree of
acidity, and not the quantity of acid in the entire solution. But if a
solution of permanganate of potash, or any other active oxidizing
agent, is used, the principal question is not the degree of dilution, but
the amount of the disinfecting agent present in the solution used. A
grain of potassium permanganate dissolved in two fluidounces of
distilled water would probably kill just as many bacteria as if it
were dissolved in half a fluidounce, although the time required for
disinfection might be longer.

From what has been said it is evident that the simple statement
that a certain agent is a germicide in a certain proportion has but
little scientific value, unless we are made acquainted with the condi-
tions under which its germicidal action has been tested.

VIII.

ACTION OF GASES AND OF THE HALOID ELEMENTS

UPON BACTERIA.

Oxygen. — Free oxygen is essential for the development of a large
number of species of bacteria — aerobics ; and it completely prevents
the growth of others — anaerobics. Many bacteria, even when freely
exposed in a desiccated condition to the action of atmospheric oxygen,
retain their vitality for a long time. The gradual loss of pathogenic
power which Pasteur has shown occurs in cultures of the anthrax
bacillus and the micrococcus of fowl cholera, is ascribed by him to
exposure to oxygen, and as proof of this he states that cultures kept
in hermetically sealed tubes do not lose their virulence in the same
degree. But other circumstances may influence the result. Thus
some of the products of growth which accumulate in culture fluids
have an injurious effect upon the vitality of the bacteria which pro-
duced them, and in time may cause a complete destruction of vitality.
In cultures exposed to the air these products would be in a more
concentrated solution from the gradual evaporation of the culture
liquid. It must also be remembered that light in the presence of
oxygen is a germicidal agent.

The experiments of Frankel show that the aerobic bacteria grow
abundantly in the presence of pure oxygen, and some species even
more so than in ordinary air. Micrococcus prodigiosus, however,
appeared to be unfavorably affected by pure oxygen, inasmuch as it
did not produce pigment so readily as when cultivated in ordinary air.

Nascent oxygen is a very potent germicidal agent, as will be seen
in our account of such oxidizing disinfectants as potassium perman-
ganate and the hypochlorite of lime.

Ozone. — It was formerly supposed that ozone would prove to be
a most valuable agent for disinfecting purposes ; but recent experi-
ments show that it is not so active a germicide as was anticipated,
and that from a practical point of view it has comparatively little
value.

Lukaschewitsch found that one gramme in the space of a cubic
metre failed to kill anthrax spores in twenty-four hours. The cholera
spirillum in a moist state was killed in this time by the same amount,
but fifteen hours' exposure failed to destroy it. Ozone for these ex-
periments was developed by means of electricity.

ACTION OF GASES AND HALOID ELEMENTS UPON BACTERIA. 173

Wyssokowicz found that the presence of ozone in a culture me-
dium restrained the development of the anthrax bacillus, the bacillus
of typhoid fever, and others tested, but concludes that this is rather
due to the oxidation of bases contained in the nutrient medium than
to a direct action upon the pathogenic bacteria.

Sonntag, in his carefully conducted experiments, in which a cur-
rent of ozonized air was made to pass over silk threads to which were
attached anthrax spores, had an entirely negative result. The an-
thrax bacillus from the spleen of a mouse, and free from spores, was
then tested, also with a negative result, even after exposure to the
ozonized air for twenty minutes at a time on four successive days. In
another experiment several test organisms (Bacillus anthracis, Bacil-
lus pneumonise of Friedlander, Staphylococcus pyogenes aureus,
Staphylococcus pyogenes albus, Bacillus murisepticus, Bacillus
crassus sputigenus) were exposed on silk threads for twenty-four
hours in an atmosphere containing 4. 1 milligrammes of ozone to the
litre of air (0. 19 volumes per cent). The result was entirely negative.
When the amount was increased to 13.53 milligrammes per litre the
anthrax bacillus and Staphylococcus pyogenes albus failed to grow
after twenty-four hours' exposure. The conclusion reached by Nis-
sen, from his own experiments and a careful consideration of those
previously made by others, is that ozone is of no practical value as a
germicide in therapeutics or disinfection.

Hydrogen. — This gas has no injurious effect upon bacteria, as is
shown by the fact that the anaerobic and facultative anaerobic species
grow readily in an atmosphere of pure hydrogen.

Hydrogen peroxide in solution in water is a valuable antiseptic
and deodorant, but its value as a germicide has been very much
overestimated. Miquel, in his experiments to determine the anti-
septic value of various agents, places H2O2 third in the list of " sub-
stances eminently antiseptic," and states that it prevents the develop-
ment of the bacteria of putrefaction in the proportion of 1: 20,000.

In the writer's experiments (1885) a solution was used which
contained at first 4. 8 per cent of H2O2, and five per cent of sulphuric
acid which was added by the chemist who prepared the solution, to
prevent loss of the hydrogen peroxide. At the end of a month the
amount of H2O2 was again estimated, and found to be 3.98 per cent.
Five weeks later the proportion was 2.4 per cent. Tested upon
^broken-down" beef tea, this solution was found to destroy the
vitality of the bacteria of putrefaction contained in it, in two hours7
time, in the proportion of thirty per cent (about 1.2 per cent of H202).
Anthrax spores were killed in the same time by a twenty-per-cent
solution (0.8 per cent H2O2). Tested upon a pure culture of pus
cocci, it was active in the proportion of ten per cent (0.4 per cent of

174 ACTION OF GASES AND OF THE

H2O2); a solution containing 0.24 per cent of H2O2 failed to kill pus
cocci. But the solution used in these experiments contained also five
per cent of sulphuric acid, which by itself kills niicrococci in the pro-
portion of 1 : 200. My conclusion was that, unless the chemists can
furnish more concentrated solutions which will keep better than that
with which I experimented, we are not likely to derive any practical
benefit from the use of hydrogen peroxide as a disinfectant.

Altehof er more recently has experimented with a solution contain-
ing 9.7 per cent of H2O2, and reports the following results: He added
to ninety-eight cubic centimetres of hydrant water two cubic centi-
metres of a bouillon culture of the typhoid bacillus, and to this was
added sufficient of his aqueous solution of H2O2 to make the propor-
tion present 1: 1,000. At the end of twenty-four hours the bacillus
was proved by culture experiments to be killed. Water containing
the cholera spirillum, treated in the same way, was not entirely steril-
ized, as a few colonies developed in Esmarch roll tubes ; but the gen-
eral result of his experiments was that the ordinary water bacteria,
and the pathogenic bacteria named (cholera, typhoid) when sus-
pended in water, required for their destruction exposure for twenty-
four hours in a solution containing one part of H2O2 in one thousand
of water.

Carbon Dioxide. — The experiments of Frankelshow that certain
bacteria grow in an atmosphere of C03 as well as in the air ; among
these are the bacillus of typhoid fever and the pneumonia bacillus
of Friedlander. Other species are slightly restricted in their growth,
e.g. Bacillus prodigiosus, Proteus vulgaris. Still others grow only
when the temperature is elevated, including the pus cocci and the
bacillus of swine pest. Most of the saprophytic bacteria failed to
grow in an atmosphere of CO2, although their vitality was not de-
stroyed by it. Certain pathogenic species were, however, killed by
the action of this gas, among others the cholera spirillum, Bacillus
anthracis, and Staphylococcus pyogenes aureus.

Leone and Hochstetter had previously reported that certain bac-
teria are injuriously affected by CO2. Frankel also found that the
growth of strictly anaerobic species was restricted in an atmosphere
of carbon dioxide. The aerobic species which failed to grow in pure
CO2 grew abundantly when a little atmospheric oxygen was ad-
mitted. In the experiments of Frankla id the cholera spirillum and
the Finkler-Prior spirillum failed to develop in an atmosphere of
CO2, and at the end of eight days were no longer capable of growth
when the carbon dioxide was replaced with atmospheric air.

Carbonic Oxide. — Frankland's experiments show that an atmo-
sphere of this gas is not favorable to the growth of the cholera spiril-
lum or of the Finkler-Prior spirillum, although it did not entirely

HALOID ELEMENTS UPON BACTERIA. 175

prevent development, and after seven days' exposure the spirilla were
not all killed, although a comparatively small number of colonies de-
veloped. Bacillus pyocyaneus failed to grow in an atmosphere of
CO, but when air was admitted, at the end of seven or eight days,
abundant development occurred.

Methane, CH4. — We have no exact experiments to determine
the action of marsh gas in a pure state on bacteria, but the experi-
ments of Kladakis upon illuminating gas maybe taken as repre-
senting approximately what might be expected from exposure in
pure CH4. An analysis of the gas used in his experiments showed
it to contain 37.97 per cent of hydrogen, 39.37 per cent of methane
(CH4), 9.99 per cent of nitrogen, 4.29 per cent of ethene (C2H4), 3.97
per cent of carbonic oxide (CO), O.G1 per cent of oxygen, and 0.41 per
cent of carbon dioxide. As hydrogen and nitrogen are neutral, and
carbonic oxide is shown by the experiments of Frankland not to act
as a germicide after several days' exposure to its action, the positive
results obtained in the experiments of Kladakis may be ascribed to
the presence of CH4 (39.37 per cent) or of C2H4 (4.29 per cent), or of
both together.

A large number of microorganisms were tested, and among these
Proteus vulgaris alone grew in an atmosphere of illuminating gas.
The others not only failed to grow in such an atmosphere, but were
destroyed by it. Cultures of Bacillus anthracis, Staphylococcus pyo-
genes aureus, and Spirillum cholerse Asiatics were sterilized in half
an hour by the action of this gas. The gas was also found to be un-
suitable for anaerobic cultures.

Xitrous Oxide, N2O. — The experiments of Frankland, made
upon the cholera spirillum, the spirillum of Finkler-Prior, and the
bacillus of green pus, gave results similar to those obtained with CO,
viz. , seven days' exposure in an atmosphere of this gas failed to de-
stroy the test organisms, but completely restrained the growth of
Bacillus pyocyaneus and interfered materially with the development
of the two species of spirillum without entirely preventing it.

Sitrogen Dioxide, NO. — Frankland found that his test organ-
isms were quickly killed by this gas (Bacillus pyocyaneus, Spirillum
cholerse Asiatics, Spirillum Finkler-Prior).

Hydrosulphuric Acid, H2S. — In the experiments of Frankland
'this gas proved to be quickly fatal to the bacteria tested (Bacillus
pyocyaneus, Spirillum cholera? Asiatics, Spirillum Finkler-Prior).
On the other hand, Grauer found that this gas did not exercise any
injurious influence upon the tubercle bacillus, the bacillus of anthrax,
the typhoid bacillus, or the cholera spirillum, after the exposure of
these microorganisms in a current of the gas for an hour.

It has been shown by the experiments of Holschewnikoff and

176 ACTION OF GASES AND OF THE

others that certain species of bacteria cause an abundant evolution
of H2S as a result of their development in an albuminous medium
(Bacillus sulfureus and Proteus sulfureus).

Sulphur Dioxide, SO2. — Very numerous experiments have been
made with this gas, owing to the fact that it has been extensively
used in various parts of the world for the disinfection of hospitals,
ships, apartments, clothing, etc.

In the writer's experiments, made in 1880, dry vaccine virus on
ivory points was disinfected by exposure for twelve hours in an at-
mosphere containing one volume per cent of this gas, and liquid
virus, exposed in a watch glass, by one-third of this amount. Sub-
sequent experiments (1885) showed that pus micrococci were killed
by exposure for eighteen hours in a dry atmosphere containing twenty
volumes per cent of SO2, but that four volumes per cent failed. In
the presence of moisture this gas has considerably greater germicidal
power than this, owing, no doubt, to the formation of the more ac-
tive agent, sulphurous acid (H2SO3). But in a pure state anhydrous
sulphur dioxide does not destroy spores. The writer has shown that
the spores of Bacillus anthracis and Bacillus subtilis are not killed by
contact for some time with liquid SO2 (liquefied by pressure). Koch
exposed various species of spore-bearing bacilli in a disinfection cham-
ber for ninety-six hours, the amount of SO2 at the outset of the ex-
periment being 6.13 volumes per cent, and at the end 3.3 per cent.
The result was entirely negative.

But in the absence of spores the anthrax bacillus, in a moist con-
dition, attached to silk threads, was destroyed in thirty minutes in
an atmosphere containing one volume per cent.

In another of Koch's experiments the amount of SO2 in the disin-
fection chamber was at the outset 0.84 per cent, and at the end of
twenty-four hours 0.55 per cent. An exposure of one hour in this at-
mosphere killed anthrax bacilli attached to silk threads, in a moist
.condition; but four hours' exposure failed to kill Bacillus prouigiosus
growing on potato, while twenty-four hours' exposure was successful.
A similar result was obtained with Bacillus pyocyaneus.

Thinot, as a result of experiments made in 1890, arrives at the
conclusion that the specific germs of tuberculosis, glanders, farcy of
cattle, typhoid fever, cholera, and diphtheria are destroyed by twenty-
four hours' exposure in an atmosphere containing SO2 developed by
the combustion of sixty grains of sulphur per cubic metre. This
amount corresponds closely with that fixed by the Committee on Dis-
infectants of the American Public Health Association on the experi-
mental evidence obtained by the writer in 1885. But the committee
insisted upon the presence of moisture and made the time of exposure
twelve hours — "exposure for twelve hours to an atmosphere con-

HALOID ELEMENTS UPON BACTERIA. 177

taining at least four volumes per cent of this gas in the presence of
moisture. "

Chlorine. — The haloid elements are active germicidal agents,
especially chlorine on account of its affinity for hydrogen, and the
consequent release of nascent oxygen when it comes in contact with
microorganisms in a moist condition. And for the same reason this
agent is a much more active germicide in the presence of moisture
than in a dry condition. The experiments of Fischer and Proskauer
showed that when dried anthrax spores were exposed for an hour in
an atmosphere containing 44. 7 per cent of dry chlorine they were not
destroyed ; but if the spores were previously moistened and were ex-
posed in a moist atmosphere for the same time, four per cent was
effective, and when the time was extended to three hours one per
cent destroyed their vitality. The anthrax bacillus, in the absence of
spores, was killed by exposure in a moist atmosphere containing 1
part to 2,500, the time of exposure being twenty-four hours, and the
same amount was effective for Micrococcus tetragenus ; the strepto-
coccus of erysipelas and the micrococcus of fowl cholera were killed in
three hours by 1 : 2,500, and in twenty-four hours by 1: 25,000. The
bacillus of mouse septica3mia and the tubercle bacillus were killed in
one hour by 1 : 200.

In the writer's experiments (1880) four children were vaccinated
with virus from ivory points which had been exposed for six hours in
an atmosphere containing one-half per cent of chlorine ; also with
four points, from the same lot, not disinfected. Vaccination was un-
successful in every case with the disinfected points, and successful
with those not disinfected. Koch found that anthrax spores failed
to grow after twenty-four hours' exposure in chlorine water. In
the experiments of De la Croix to determine the antiseptic power of
this agent, it was found that when present in unboiled beef infusion
in the proportion of 1 : 15,600 no development of bacteria occurred.
Miquel gives the antiseptic value of chlorine as 1 : 4,000.

Chloroform. — Immersion for one hundred days in chloroform
does not destroy the vitality of anthrax spores (Koch). This agent
is without effect on the virus of symptomatic anthrax" (Arloing,
Cornevin, and Thomas). Salkowski found that the anthrax bacillus
in the absence of spores, and the cholera spirillum, were killed by
being immersed in chloroform water for half an hour. Kirchner
reports still more favorable results. In his experiments a one-per-
cent solution killed the cholera spirillum in less than a minute, and
a one-quarter-per-cent solution in an hour. But the typhoid bacillus
required at least one-half per cent acting for an hour.

Iodine. — In the writer's experiments (1880) iodine in aqueous
solution with potassium iodide was found to be fatal to Micrococcus
12

178 ACTION OF GASES AND OF THE

pneumonise crouposscin the proportion of 1 : 1,000, and to the staphy-
lococci of pus in 1 : 500 — time of exposure two hours. Iodine water
was found by Koch to destroy the vitality of anthrax spores in
twenty-four hours, but a two-per-cent solution in alcohol failed to
destroy anthrax spores in forty-eight hours. In the experiments of
Schill and Fischer twenty hours' contact with a solution of the
strength of 1 : 500 failed to destroy the virulence of tuberculous spu-
tum, as tested by inoculation experiments. The antiseptic value of
iodine is given by Miquel as 1 : 4,000.

Bromine. — Fischer and Proskauer have studied the action of
bromine vapor upon various microorganisms. They found that ex-
posure for three hours in a dry atmosphere to three per cent does
not destroy the tubercle bacillus in sputum or the spores of an-
thrax. But when the atmosphere is saturated with moisture 1 : 500
is effective • and when the time of exposure was extended to twenty-
four hours, 1 : 3,500. A two-per-cent solution destroys the vitality
of anthrax spores in twenty-four hours (Koch). Bromine vapor is
an active agent for the destruction of the virus of symptomatic an-
thrax (Arloing, Cornevin, and Thomas). Miquel gives the antisep-
tic value of bromine as 1 : 1,666, which is considerably below that of
chlorine and iodine.

Iodine Trichloride. — According to Behririg, we possess in this
agent a disinfectant which possesses the potency of free chlorine and
iodine without having their disadvantages. As prepared by O. Rie-
del it is a yellowish-red powder of penetrating odor. It remains un-
changed for weeks in concentrated aqueous solution (five per cent).
A one-per-cent solution destroys anthrax spores suspended in water
almost instantly, and a 0.2-per- cent solution within a few minutes.
Anthrax spores in blood serum are killed by a one-per-cent solution
in forty minutes (Behring). Langenbuch found that a solution of
1 : 1,000 kills spores in a short time, and that when added to nutri-
ent gelatin in the proportion of 1 : 1,200 it restrains the develop-
ment of bacteria.

lodoform. — Numerous experiments have been made with this
agent, which show that it has little, if any, germicidal power ; but
it acts to some extent as an antiseptic. Tilanus reports that the tu-
bercle bacillus will not grow in glycerin-agar cultures to which a
small quantity of iodof orm has been added, and that a pure culture
of the tubercle bacillus was not killed in six days by exposure to
iodof orm vapor, but that after six weeks' exposure it failed to grow.
The experiments of Neisser and of Buchner show that while most
bacteria are not injuriously affected by exposure to iodof orm vapor,
the cholera spirillum and the Finkler-Prior spirillum are restrained in
their growth by such exposure. When plate cultures of the cholera

HALOID ELEMENTS UPON BACTERIA. 179

spirillum were placed under a bell jar beside iodoform powder
no development occurred, but when they were removed colonies de-
veloped, showing that the spirilla were not killed.

Iodoform Ether, according to Yersin, is fatal to the tubercle ba-
cillus in one-per-cent solution in five minutes. Cadeac and Meunier
found that a saturated solution required thirty-six hours to kill the
bacillus of typhoid fever.

loclol. — In experiments made by the writer (1885) this agent was
found to be without germicidal power. Riedlin found it without any
action, even upon the cholera spirillum.

Hydrofluoric Acid, HF1. — From a series of experiments made
with this gas, Grancher and Chautard arrive at the conclusion that
" the direct and prolonged action of hydrofluoric acid upon the tuber-
cle bacillus diminishes its virulence but does not kill it."

Sozoiodol Acid, according to Draer,is a phenol, in which two atoms
of hydrogen are replaced by two of iodine arid one atom by the group
HSO3. This acid and its salts with soda, potash, zinc, and mercury
have been tested by the author named. The acid and its salt with
mercury were found to destroy the cholera spirillum in two hours' time
in two-per-cent solution. A two-per-cent solution of phenol would
have accomplished the same result and in less time. Tribromphenol,
according to Draer, is less active than sozoiodol acid ; and it appears
from the experimental evidence on record that combinations of
iodine, chlorine, or bromine with phenol are less active that the
haloid elements alone. According to Karpow (1893) monochlor-
phenol, tested upon anthrax spores attached to silk threads, proved
to be decidedly more active than phenol.

Nosophen (tetraiodphenolphthalein), according to Lieveii (1895)
contains sixty-one per cent of iodine. It is entirely insoluble in
water. When added to nutrient gelatin in the proportion of one-
quarter per cent it prevented the development of the anthrax bacillus
and of Staphylococcus aureus, but failed to prevent the development
of Bacillus pyocyaneus (Lieven).

IX.

ACTION OF ACIDS AND ALKALIES.

Sulphuric Acid, H2SO4. — The experiments of Koch (1881)
showed that anthrax spores were still capable of growing after ex-
posure in a one-per-cent solution of sulphuric acid for twenty days.
In the writer's experiments (1885) a four-per-cent solution failed to
destroy the spores of Bacillus subtilis in four hours, and an eight-
per-cent solution was found to be required for the sterilization of
culture fluids containing spores ; but the multiplication of the bacte-
ria of putrefaction was prevented by the presence of this acid in a
culture solution in the proportion of 1 : 800. Pus micrococci were
destroyed by exposure for two hours in a solution containing 1 : 200.

The experiments of Boer show that there is a considerable differ-
ence in the resisting power of different pathogenic bacteria. The
time of exposure being two hours, cultures in bouillon twenty-four
hours old gave the following results :

Restrains
development.

Destroys
vitality.

1 : 2550

1 : 1300

Diphtheria bacillus

1 : 2050

1 :500

Glanders bacillus

1 :750

1 :200

Typhoid bacillus

1 : 1550

1 :500

Cholera spirillum

1 : 7000

1 : 1300

Leitz, in his studies relating to the bacillus of typhoid fever,
reports the following results : The dejections of typhoid patients,
mixed with an equal proportion of the disinfecting solution, were
sterilized by a five-per-cent solution of sulphuric acid in three days.
A pure culture was sterilized in fifteen minutes by two per cent, and
in five minutes by five per cent.

Sulphurous Acid, H2SO3. — In the writer's experiments (1885)
micrococci were destroyed in two hours by 1 : 2,000 by weight of SO,
added to water. Kitasato found that solutions of sulphurous acid
in the proportion of 0.28 per cent killed the typhoid bacillus, and
0. 148 per cent the cholera spirillum. De la Croix found that one

ACTION OF ACIDS AND ALKALIES. 181

gramme of SO2 added to two thousand of bouillon prevents the de-
velopment of putrefactive bacteria and after a time destroys the
vitality of these bacteria. The writer found that pus cocci failed to
grow in a culture solution containing one part of SO2 in five thousand
of water.

Nitric Acid, HNO3. — In the writer's experiments an eight-per-
cent solution which contained 0.819 gramme of HNO3 in each cubic
centimetre sterilized broken-down beef tea containing spores, and
five per cent failed to do so. Kitasato, in experiments upon the chol-
era spirillum and typhoid bacillus, obtained results corresponding
with those obtained with hydrochloric acid — 0.2 per cent destroyed
vitality at the end of four or five hours. In these experiments the
acid used contained 0.35 gramme HNO3 in one cubic centimetre.

Nitrous Acid. — In the writer's experiments on vaccine virus (1880)
exposure for six hours in an atmosphere containing one per cent of
nitrous acid destroyed the virulence of dried virus upon ivory points.

Hydrochloric Acid, HC1. — Anthrax spores are destroyed in ten
days by a two-per-cent solution, but not in five days (Koch). Tested
upon broken-down beef tea containing spores of Bacillus subtilis, it
was effective in two hours in the proportion of fifteen per cent, but
failed in ten per cent (Sternberg). In the experiments of Kitasato this
acid destroyed the typhoid bacillus in five hours in the proportion of
0.2 per cent, and the cholera spirillum in 0.132 per cent — the acid
used contained 0. 26 gramme HC1 in one cubic centimetre. We give
the more recent determinations of Boer in tabular form. Its germi-
cidal power was tested upon bouillon cultures which had been kept
for twenty-four hours in an incubating oven ; time of exposure to
the action of the acid solution, two hours.

Restrains
development.

Destroys
vitality.

Anthrax bacillus

1 :3400

1 : 1100

Diphtheria bacillus

1 : 3400

1 :700

Glanders bacillus *

1 : 700 '

1 -200

Typhoid bacillus

1 : 2100

1 :300

Cholera spirillum

1 : 5500

1 • 1350

Chromic Acid. — In Koch's experiments a one-per-cent solution
destroyed anthrax spores in from one to two days. In the propor-
tion of 1 : 5,000 it prevents the development of putrefactive bacteria
(Miquel).

Osmic Acid. — A solution of one per cent kills anthrax spores in
twenty-four hours (Koch). It is an antiseptic in the proportion of
1 : 6,666 (Miquel).

Phosphoric Acid. — Exposure for four or five hours to a solution

182 ACTION OF ACIDS AND ALKALIES.

containing 0.3 per cent destroys the typhoid bacillus, and 0.183 per
cent the cholera spirillum (Kitasato). The acid used contained 0.152
gramme H3PO4 in one cubic centimetre.

Acetic Acid.— A five-per-cent solution failed to kill anthrax
spores after five days' exposure (Koch). In Abbott's experiments
glacial acetic acid in fifty-per-cent solution failed in two hours to kill
anthrax spores, but micrococci were killed by two hours7 exposure to
a one-per-cent solution. A solution of 1 : 300 of glacial acetic acid
destroys the cholera spirillum in half an hour (Van Ermengem). In
the proportion of 0. 25 per cent it restrains the growth of the typhoid
bacillus, and 0. 3 per cent destroys its vitality after five hours' expo-
sure ; the cholera spirillum fails to grow in presence of 0.132 per cent
and. is destroyed by 0.2 per cent (Kitasato).

Lactic Acid. — The bacillus of typhoid fever is killed in five hours
by a solution containing 0. 4 per cent, the cholera spirillum by 0. 3 per
cent (Kitasato).

Citric Acid. — The bacillus of typhoid fever is killed in five hours
by 0.43 per cent, the cholera spirillum by 0.3 per cent (Kitasato).
The cholera spirillum is killed in half an hour by 1 : 200 (Van Er-
mengem).

Oxalic Acid. — The typhoid bacillus requires a solution of 0.36
per cent, the cholera spirillum one of 0. 28 per cent, to destroy vitality
in five hours (Kitasato).

Boracic Acid. — In the writer's experiments (1883) a saturated
solution failed to kill pus cocci in two hours. A five-per-cent solu-
tion failed to destroy anthrax spores in five days (Koch). The
typhoid bacillus is killed in five hours by 2.7 percent, the cholera
spirillum by 1.5 per cent (Kitasato). According to Arloing, Corne-
vin, and Thomas, the fresh virus of symptomatic anthrax requires
exposure to a twenty-per-cent solution for forty-eight hours for the
destruction of vitality. Boracic acid acts as an antiseptic in the pro-
portion of 1 : 143 (Miquel).

Salicylic Acid. — In the writer's experiments this agent was dis-
solved by the addition of sodium biborate, which by itself has no
germicidal power. A two-per-cent solution was found to destroy pus
cocci in two hours. Dissolved in oil or in alcohol a five-per-cent so-
lution does not destroy anthrax spores (Koch). Micrococci are de-
stroyed by solutions containing 1 : 400 (Abbott). The typhoid bacillus
is killed in five hours by 1.6 per cent, the cholera spirillum by 1.3 per
cent (Kitasato). A one-per-cent solution destroys Micrococcus Pas-
teuri in half an hour (Sternberg). It is an antiseptic in the propor-
tion of 1 : 1,000 (Miquel). A solution of 2.5 percent kills the tubercle
bacillus in six hours (Yersin). In the proportion of 1 : 300 it destroys
the cholera spirillum in half an hour (Van Ermengem).

ACTION OF ACIDS AXD ALKALIES.

Benzole Acid. — According to Miquel, this acid restrains the de-
velopment of putrefactive bacteria when present in bouillon in the
proportion of 1: 909. In the proportion of 1 : 2,000 it retards the de-
velopment of anthrax spores (Koch).

Formic Acid. — The typhoid bacillus is restrained in its growth by
0.25 per cent, and is killed in five hours by 0.35 per cent, the cholera
spirillum by 0.22 per cent (Kitasato).

Tannic Acid. — A solution of one per cent kills Micrococcus Pas-
teuri in the blood of a rabbit in half an hour (Sternberg). A five-
per-cent solution failed in ten days to destroy anthrax spores (Koch).
A twenty-per-cent solution failed in two hours to destroy the vitality
of spores of the anthrax bacillus or of Bacillus subtilis (Abbott).
Micrococci are destroyed by 1 : 400, and 1 : 800 failed (Abbott). A
twenty-per-cent solution has no effect upon the virus of symptomatic
anthrax (Arloing, Cornevin, and Thomas). A solution of 1.66 per
cent kills the typhoid bacillus in five hours, and 1.5 per cent the
cholera bacillus in the same time (Kitasato). It restrains the devel-
opment of putrefactive bacteria in the proportion of 1 : 207 (Miquel).

Tartaric Acid. — A twenty-per-cent solution of this acid fails,
after two hours' exposure, to destroy the spores of Bacillus anthracis
or Bacillus subtilis. Micrococci are killed by two hours' exposure in
a solution containing 1 : 400 (Abbott).

Malic Acid. — This was found by Kitasato to correspond with
citric acid in its germicidal power.

Valerianic Acid. — A five-per-cent solution in ether failed in five
days to destroy anthrax spores (Koch).

Oleic Acid. — A solution of five percent in ether does not destroy
anthrax spores in five days (Koch).

Thymic Acid. — In the proportion of 1 : 500 this acid prevents the
putrefactive decomposition of beef tea (Miquel).

Butyric Acid. — Five days' immersion in this acid failed to de-
stroy anthrax spores (Koch).

Arsenious Acid. — A one-per-cent solution destroys the vitality
of anthrax spores in ten days, but failed to do so in six days (Koch).
In the proportion of 1 : 166 it prevents putrefactive changes in bouillon
(Miquel).

Gallic Acid. — Abbott found this acid to destroy the bacteria in
broken-down beef tea in the proportion of 2.37 per cent, but it failed
to destroy anthrax spores in two hours in the same proportion. Mi-
crococci were killed in two hours by 1 : 142, while 1 : 250 failed.

ALKALIES.

Potassium Hydroxide, KHO. — In the writer's experiments aten-
per-cent solution of caustic potash was fatal to pus cocci, and an

184 ACTION OF ACIDS AND ALKALIES.

eight-per-cent solution failed — two hours' exposure. Exposure for
twenty-four hours to a ten-per-cent solution failed to kill the tubercle
bacillus (Schill and Fischer). A solution of one per cent kills the
anthrax bacillus, the bacillus of rothlauf, and several others (Jager).
The addition of 0. 14 per cent restrains the development of the typhoid
bacillus, and 0.18 per cent kills this bacillus in four or five hours; the
cholera spirillum failed to grow in cultures containing 0.18 per cent
and was killed by 0.237 per cent in the same time (Kitasato).

Sodium Hydroxide, NaHO. — The experiments of Jager and of
Kitasato show that soda has about the same germicidal power as
caustic potash. Boer obtained the following results with bouillon
cultures after two hours' exposure : Anthrax bacillus, 1 : 450 ; diph-
theria bacillus, 1 : 300 ; glanders bacillus, 1 : 150 ; typhoid bacillus,
1 : 190 ; cholera spirillum, 1 : 150. In about one-half the amount
required to destroy vitality the development of the above-named bac-
teria was prevented. In the proportion of 1 : 56 it acts as an anti-
septic (Miquel).

Ammonia, NH3. — In Kitasato's experiments the typhoid bacillus
was destroyed in five hours by 0.3 per cent of NH3, and the cholera
spirillum by about the same amount. Boer obtained the following
results, the time of exposure being two hours : Anthrax bacillus,
1: 300 ; diphtheria bacillus, 1 : 250 ; glanders bacillus, 1 : 250 ; typhoid
bacillus, 1 : 200 ; cholera spirillum, 1 : 350. The growth of the an-
thrax bacillus and of the diphtheria bacillus in culture solutions was
prevented by 1 : 650.

Calcium Hydroxide, Ca2HO. — According to Kitasato, the ty-
phoid bacillus and the cholera spirillum, in bouillon cultures, are
killed in four or five hours by the addition of 0. 1 per cent of calcium
oxide. Liborius had previously reported still more favorable results,
but his bouillon cultures were largely diluted with distilled water.
From a practical point of view the experiments of Pfuhl are more
valuable. Calcium hydrate was added to the dejections of typhoid
patients. When added in the proportion of three per cent steriliza-
tion was effected in six hours, and by six per cent in two hours.
When milk of lime containing twenty per cent of calcium hydrate
was used the results were still more favorable, the typhoid bacillus
and cholera spirillum being killed in one hour by the addition of
two per cent of the disinfectant. The practical value of lime-wash
applied to walls has been determined by Jager. Silk threads soaked
in cultures of various pathogenic bacteria were attached to boards
and the lime- wash applied with a cameFs-hair brush. Anthrax ba-
cilli (without spores), the glanders bacillus, Staphylococcus pyogene?
aureus, and several other pathogenic bacteria were killed by a single
application after twenty-four hours, but the tubercle bacillus was not

ACTION OF ACIDS AND ALKALIES. 185

killed by three successive applications. In the writer's experiments
(1885) the typhoid bacillus and Staphylococcus pyogenes aureus were
killed in two hours by a solution containing 1 : 40 of calcium oxide,
and 1 : 80 failed. Spores of the anthrax bacillus and of several other
spore-forming species were not killed by two hours' exposure to a
milk of lime containing twenty per cent of calcium oxide.

Potash Soap has been shown by Jolles (1895) to have considerable
germicidal value. In experiments with a soap containing 67.44
per cent of fat acids, 10.4 per cent of combined alkali, and 0.041
per cent of free alkali, the following results were obtained : The
typhoid bacillus was destroyed at 18° C. by a one-per-cent solution
in twenty -four hours; by a six-per-cent solution in thirty minutes.
The Bacillus coli communis required somewhat stronger solutions or
longer exposure — eight-per-cent solution required thirty minutes.
These experiments show that scrubbing with soap and water is a
reliable method of disinfecting surfaces. Solutions of potash — com-
mon lye — or of soda also are useful for certain purposes in domes-
tic disinfection, and scientific researches justify the continued use of
the cleansing methods which have heretofore been in use by careful
housewives.

X.
ACTION OF SALTS.

WHILE some of the metallic salts, and especially those of mer-
cury, silver, and gold, have remarkable germicidal power, others,
even in concentrated solutions, do not destroy the vitality of bacteria
exposed to their action. For convenience of reference we shall con-
sider the agents in this group in alphabetical order, but first we give
Miquel's tables of antiseptic value. This author recognizes the im-
portance of experiments to determine the restraining power of chem-
ical agents for various species of pathogenic bacteria, but says : " As
to me, faithful to a plan I adopted at the outset, I will treat the sub-
ject in a more general manner by making known simply the mini-
mum weight of the substances capable of preventing the evolution of
any bacteria or germs. The method adopted is very simple. To a
liquid always comparable to itself it is sufficient at first to add a
known weight of the antiseptic and some atmospheric germs or adult
bacteria, and to vary the quantity of the antiseptic until the amount
is ascertained which will preserve indefinitely the liquid from putre-
faction. In order to obtain germs of all kinds in a dry state it suf-
fices to take them, where they are most abundant, in the dust col-
lected in the interior of houses or of hospitals; and to procure a
variety of adult bacteria we may take the water of sewers. "

SUBSTANCES EMINENTLY ANTISEPTIC.

Efficient in the
proportion of—

Mercuric iodide, . . . . . 1 : 40000

Silver iodide, ...... 1:33000

Hydrogen peroxide, . . . . . 1 : 20000

Mercuric chloride, . . . . . 1 : 14300

Silver nitrate, . . . . . 1 : 12500

SUBSTANCES VERY STRONGLY ANTISEPTIC.

Osmicacid, ...... 1:6666

Chromic acid, . . . . . . 1 : 5000

Chlorine, ...... 1:4000

Iodine, ....... 1:4000

Chloride of gold, . ..... 1:4000

Bichloride of platinum, . . . . . 1 : 3333

Hydrocyanic acid, . . . . . 1 : 2500

ACTION OF SALTS. 187

Bromine, ....... 1:1666

Cupric chloride, . . . . . . 1 : 1428

Thymol, ....... 1:1340

Cupric sulphate, . . . . . . 1 : 1111

Salicylic acid, . . . . . 1 : 1000

SUBSTANCES STRONGLY ANTISEPTIC.

Benzoic acid, . . . . . . 1 : 909

Potassium bichromate, . . . . 1 : 909

Potassium cyanide, . . . . . 1 : 909

Aluminum chloride, ..... 1:714

Ammonia, . . . . . . 1 : 714

Zinc chloride, . . . . . .1:526

Mineral acids, . ... 1:500 to 1:333

Thymicacid, . . . . . . .1:500

Lead chloride, . . . . . . 1 : 500

Nitrate of cobalt, . . . . . .1:476

Sulphate of nickel, . . . . . 1:400

Nitrate of uranium, . . . . . . 1 : 356

Carbolic acid, . . . . . . 1:333

Potassium permanganate, . . . . 1 : 285

Lead nitrate, . . . . . . 1 : 277

Alum,. . . . . . . .1:222

Tannin, 1:207

SUBSTANCES MODERATELY ANTISEPTIC.

Bromhydrate of quinine, ..... 1:182

Arsenious acid, . . . . . . 1 : 166

Boracic acid, . . . . . . 1 : 143

Sulphate of strychnia, . . . . . 1 : 143

Arsenite of soda, ...... 1:111

Hydrate of chloral, . . . . „ 1:107

Salicylate of soda, . . . . . . 1 : 100

Ferrous sulphate, . . . . . 1 : 90

Caustic soda, . . . . . . 1 : 56

SUBSTANCES FREELY ANTISEPTIC.

Perchloride of manganese, . . . . 1 : 40

Calcium chloride, . . . . . . 1 : 25

Sodium borate, . . . . . . 1 : 14

Muriate of morphia," . . . . . 1 : 13

Strontium chloride, . . . . . 1 : 12

Lithium chloride, . . . . . -1 : 11

Barium chloride, . . . . . . 1 : 10

Alcohol, . 1:10

SUBSTANCES VERY FEEBLY ANTISEPTIC.

Ammonium chloride, . . . . . 1:9

Potassium arsenite, . . . . . .1:8

Potassium iodide, . . . . . 1:7

Sodium chloride, . . . . . .1:6

Glycerin (sp. gr. 1.25), . . . . . 1:4

Ammonium sulphate, . . . . .1:4

Sodium hyposulphite, . . . . . 1:3

188 ACTION OF SALTS.

ANTISEPTIC AND GERMICIDAL VALUE OF VARIOUS SALTS,
ARRANGED ALPHABETICALLY.

Alum. — : Antiseptic in the proportion of 1 : 222 (Miquel).

Aluminum Acetate. — According to De la Croix, this salt is an
antiseptic in the proportion of 1 : 6,310. Kuhn found it to be anti-
septic in 1 : 5,250.

Aluminum Chloride. — Antiseptic in the proportion of 1 : 714
(Miquel).

Ammonium Carbonate. — When present in the proportion of
1 : 125 it restrains the development of typhoid bacilli, and in five
hours' time it kills these bacilli in the proportion of 1 : 100 ; the
cholera spirillum is killed in the same time by 1 : 77 (Kitasato).

Ammonium Chloride. — Antiseptic in the proportion of 1:0
(Miquel). A five-per-cent solution does not kill anthrax spores in
twenty-five days (Koch).

Ammonium Fluosilicate. — The bacillus of anthrax and of ty-
phoid fever fail to grow in nutrient gelatin containing 1 : 1,000, and
a two-per-cent solution kills anthrax spores in one-quarter to three-
quarters of an hour (Faktor).

Ammonium Sulphate. — Antiseptic in the proportion of 1:4
(Miquel). A five-per-cent solution failed in two days to kill an-
thrax spores, but was effective in five days (Koch).

Barium Chloride is an antiseptic in the proportion of 1 : 10
(Miquel).

Calcium Chloride is an antiseptic in the proportion of 1 : 25
(Miquel). A saturated solution does not destroy anthrax spores
(Koch).

Calcium Hypochlorite. — This is a powerful germicidal agent
and has great value as a practical disinfectant. Good chloride of
lime contains from twenty-five to thirty per cent of available chlo-
rine as hypochlorite. The experiments made by the Committee on
Disinfectants of the American Public Health Association in 1885
showed that a solution containing 0.25 per cent of chlorine as hypo-
chlorite is an effective germicide, even when allowed to act only
for one or two minutes. In Bolton's experiments a solution of chlo-
ride of lime of 1 :2,000 (available chlorine 0.015) destroyed the ty-
phoid bacillus and the cholera spirillum in two hours. For the de-
struction of anthrax spores a one-per-cent solution was required
(available chlorine 0.3 per cent). Nissen found that the typhoid
bacillus and the cholera spirillum are destroyed with certainty in
five minutes by a solution containing 0.12 percent, anthrax bacilli
in one minute by 0. 1 per cent, Staphylococcus pyogenes aureus in
one minute by 0.2 per cent, anthrax spores in thirty minutes by a

ACTION OF SALTS. 180

five-per-cent solution and in seventy minutes by a one-per-cent solu-
tion. Experiments made by the same author upon the sterilization
of f aeces showed that 0. 5 per cent to one per cent could be relied upon
to destroy the typhoid bacillus or the cholera spirillum in faeces in
ten minutes.

Chloral Hydrate. — Antiseptic in the proportion of 1 : 107 (Mi-
quel). A twenty-per*cent solution destroys pus cocci in two hours
(Sternberg).

Cupric Chloride. — Antiseptic in the proportion of 1 • 1,428
(Miquel).

Cupric Sulphate. — Antiseptic in the proportion of 1 : 111 (Mi-
quel). Kills the cholera spirillum in the proportion of 1 : 3,000 in
ten minutes (Nicati and Rietsch). Destroys the cholera spirillum in
bouillon cultures in less than half an hour in 1 : 600, and in four
hours in 1 : 1,000 ; cultures in blood serum require 1 : 200 (Van Er-
mengem). A solution of 1 : 20 kills the typhoid bacillus in ten min-
utes (Leitz). This salt failed, in the writer's experiments, to kill the
spores of Bacillus anthracis and Bacillus subtilis in two hours' time
in a twenty-per-cent solution. In Koch's experiments a five-per-cent
solution failed to kill anthrax spores in ten days. Kills pus micro-
cocci in two hours in the proportion of 1 : 200 (Sternberg). In Bol-
ton's experiments made for the Gommittee on Disinfectants of the
American Public Health Association the following results were ob-
tained: Recent cultures in bouillon, time of exposure two hours : Ba-
cillus of typhoid fever, 1 : 200; cholera spirillum, 1 : 500; Bacillus pyo-
cyanus, 1 :200; Brieger's bacillus, 1 :200; Emmerich's bacillus, 1 : 200;
Staphylococcus pyogenes aureus, 1 : 100 ; Staphylococcus pyogenes
citreus, 1 : 100; Staphylococcus pyogenes albus, 1 : 200; Streptococcus
pyogenes, 1 : 500. When ten per cent of dried egg albumin was
added to a recent culture in bouillon of the typhoid bacillus the
amount required to insure sterilization was 1 : 10.

In the report of the Committee on Disinfectants of the American
Public Health Association this agent is recommended in " a solu-
tion of two to five per cent for the destruction of infectious material
not containing spores." The experimental data above given show
that this is a liberal allowance for material which does not contain
an excessive amount of albumin. In the experiments of Leitz the
typhoid bacillus in cultures was destroyed in ten minutes by a five-
per-cent solution.

Ferric Chloride. — A five-per-cent solution failed in two days to
destroy anthrax spores, but was effective in five days (Koch). •

Ferrous Sulphate. — In the writer's experiments (1883) a solution
of twenty per cent failed to destroy micrococci and putrefactive bac-
teria. In a more recent experiment ten per cent failed to kill pus

1'JU ACTION OF SALTS.

cocci, but was fatal to Micrococcus tetragenus — two hours' exposure.
Koch found that a five-per-cent solution failed to destroy anthrax
spores in six days. Exposure to a twenty-per-cent solution for forty-
eight hours does not destroy the virus of symptomatic anthrax (Ar-
loing, Cornevin, and Thomas). In the experiments of Jager immer-
sion in a solution of 1 : 3 destroyed the infective virulence of certain
pathogenic bacteria (fowl cholera, rothlauf, glanders), as tested by
injection into mice, but failed to kill anthrax spores and tubercle ba-
cilli. The antiseptic power of ferrous sulphate is placed by Miquel
at 1 : 90. In the writer's experiments 1 : 200 prevented the develop-
ment of micrococci and of putrefactive bacteria in bouillon placed
in the incubating oven for forty-eight hours. Leitz found that a
five-per-cent solution required three days' exposure for the destruc-
tion of the typhoid bacillus.

Gold Chloride. — Antiseptic in the proportion of 1 : 4,000 (Miquel).
Boer has made extended experiments with the chloride of gold and
sodium. We give his results below. In his disinfection experi-
ments a bouillon culture which had been in the incubating oven for
twenty-four hours was used, and the time of exposure was two hours.

Restrains
development.

Destroys
vitality.

Anthrax bacillus . . ,

1 : 40000

1 : 8000

Diphtheria bacillus

1 : 40000

1 : 1 000

Glanders bacillus

1 : 1 5000

1 • 400

Typhoid bacillus .

1 : 20000

1 :500

Cholera spirillum

1 : 25000

1 : 1000

Lead Chloride. — Antiseptic in the proportion of 1 : 500 (Miquel).

Lead Nitrate. — Antiseptic in the proportion of 1 : 277 (Miquel).

Lithium Chloride. — Antiseptic in the proportion of 1 : 11 (Mi-
quel).

Manganese Protochloride. — Antiseptic in the proportion of 1:40
(Miquel).

Mercuric Chloride. — Koch's experiments (1881) gave the follow-
ing results : A solution of 1 : 1,000 destroys anthrax spores in a few
minutes, and 1 : 10,000 is effective after a more prolonged exposure.
The writer (1884) obtained similar results — 1 : 10,000 destroyed the
spores of Bacillus anthracis and of Bacillus subtilis in two hours.
More recent experiments indicate that failure to grow in culture so-
lutions cannot be accepted as evidence of the destruction of vitality
in the case of spores exposed to the action of this agent, unless due
precautions are taken to exclude the restraining influence of the small
amount of mercuric chloride which remains attached to the spores.
Koch had ascertained that the development of spores is restrained by

ACTION OF SALTS. 1U1

the presence of 1 : 300,000 in a culture medium, and Geppert has re-
cently shown that even so small an amount as 1 : 2,000,000 will pre-
vent the development of spores the vitality of which has been reduced
by the action of a strong solution (1 : 1,000). When this restraining
action is entirely neutralized by washing the spores in a solution con-
taining ammonium sulphide it requires, according to Geppert, a solu-
tion of 1:1,000 acting for one hour to completely destroy the vitality
of anthrax spores. Frankel found that a solution of 1 : 1,000 was
effective in half an hour. The typhoid bacillus, the bacillus of mouse
septica3mia, and the cholera spirillum, in bouillon cultures and in
cultures in flesh-peptone-gelatin, are destroyed in two hours by
1 : 10,000 ; but in a bouillon culture to which ten per cent of dried
egg albumin was added a one-per-cent solution was required to de-
stroy the typhoid bacillus in the same time (Bolton). According to
Van Ermengem, cultures of the cholera spirillum in bouillon are steril-
ized in half an hour by 1 : 60,000, but cultures in blood serum require
1 : 800 to 1 : 1,000. In experiments upon tuberculous sputum Schill
and Fischer found that exposure of fresh sputum to an equal amount
of a 1 : 2,000 solution for twenty-four hours failed to disinfect it, as
shown by inoculation experiments in guinea-pigs. The antiseptic
power of mercuric chloride is given by Miquel as 1 : 14,300. In the
writer's experiments 1 : 33,000 was found to prevent the development
of putrefactive bacteria in bouillon, but a minute bacillus contained in
broken-down beef infusion multiplied, after several days, in 1 : 20,000.
The pus cocci were restrained in their development by 1 : 30,000.

In Behring's experiments the anthrax bacillus and cholera spiril-
lum were killed in one hour by 1 : 100,000 when the temperature
was 36° C., but at a temperature of 3° C. the proportion required
was 1 : 25,000. The same author states that at 22° C. Staphylo-
coccus aureus in bouillon is not always killed in twenty-five minutes
by 1 : 1,000.

Abbott (1891) has shown that a 1 : 1,000 solution does not always
destroy Staphylococcus pyogenes aureus in five minutes. He says :
" Frequently all the organisms would be destroyed after five minutes'
exposure, but almost as often a certain few would resist for that
length of time, and even longer, going in some cases to ten, twenty,
and even thirty minutes."

According to Yersin, a solution of 1 : 1,000 kills the tubercle bacil-
lus in one minute.

We might add considerably to the experimental data given, but
the results already recorded are sufficient to show the value of this
agent as an antiseptic and germicide, and justify its use for general
purposes of disinfection in the proportion of 1 : 500 or 1 : lrOOO for
material containing spores, and in the proportion of 1 : 2,000 to

192 ACTION OF SALTS.

1 : 5,000 for pathogenic bacteria in the absence of spores; due regard
being had to the fact that the presence of albumin very materially
reduces its germicidal potency, and that it may be decomposed and
neutralized by alkalies and their carbonates, by hydrosulphuric acid,
and by many other substances.

The albuminate of mercury, as has been shown by Lister, is solu-
ble in an excess of albumin, and, according to Behring, is just as
effective as an aqueous solution containing the same amount of sub-
limate when dissolved in an albuminous liquid like blood serum (?).

In practice the addition of a mineral acid to sublimate solutions,
or of sodium, potassium, or ammonium chloride, is to be recom-
mended, to prevent the precipitation of the mercuric chloride by al-
bumin in fluids containing it. Behring recommends the addition
of five parts of sodium or potassium chloride to one of the subli-
mate. Such a solution is more stable than a simple solution of sub-
limate, and no precipitate is formed by the addition of alkalies or by
albumin.

The same result is obtained, according to La Place, by the addi-
tion of five parts of hydrochloric or tartaric acid to one part of sub-
limate in aqueous solution.

Mercuric Cyanide, Hg(CN)2, and the Oxy cyanide of mercury
have been tested, with the following results : Staphylococcus aureus
is destroyed in five minutes by 1 : 100, in one hour by 1 : 1,000, in
two hours by 1 : 1,500 (Chibret). The development of Bacillus an-
thracis in culture solutions is prevented by the presence of cyanide
of mercury in the proportion of 1 : 25,000, and by the oxy cyanide by
1 : 16,000 (Behring).

Boer obtained the following results with the oxycyanide — cul-
tures in bouillon, twenty-four hours in incubating oven, time of
exposure two hours :

Restrained
development.

Destroyed
vitality.

Anthrax bacillus

1 : 80000

1 : 40000

Diphtheria bacillus

1 : 80000

1 • 40000

Glanders bacillus

1 : 60000

1 : 30000

Typhoid bacillus • ,

1 : 60000

1 : 30000

Cholera spirillum

1 : 90000

1 : 60000

Mercuric Iodide. — The antiseptic value of this salt is placed by
Miquel at 1 : 40,000, which is more than double that given by the
same author to the bichloride. In the writer's experiments upon the
antiseptic value of salts and oxides of mercury the following results
were obtained :

ACTIOX OF SALTS. 193

Active.

Failed.

Biniodide of mercury

1 : 20000

1 : 40000

Bichloride

1 : 15000

1 : 20000

Protiodide

1 . 10000

1 : 20000

Yellow oxide

1 : 1000

1 : 2000

Black oxide

1 :500

1 1000

Morphia Hydrochlorate. — Antiseptic in the proportion of 1 : 13
(Miquel).

Nickel Sulphate. — Antiseptic in the proportion of 1 : 400 (Mi-
quel).

Platinum Bichloride. — Antiseptic in the proportion of 1 : 3,333
(Miquel).

Potassium Acetate. — A saturated solution of this salt failed to
kill anthrax spores in ten days (Koch).

Potassium Arsenite. — In the writer's experiments Fowler's solu-
tion failed to kill micrococci in two hours in the proportion of four
per cent. Miquel places the antiseptic value of potassium arsenite
at 1 : 8.

Potassium Bichromate. — A five-per-cent solution failed in two
days to destroy anthrax spores (Koch). Efficient as an antiseptic in
the proportion of 1 : 909 (Miquel).

Potassium Bromide. — The bacillus of typhoid fever and the
cholera spirillum fail to grow in culture solutions containing 9 to
10.6 per cent, and are killed in four or five hours by ten to twelve
per cent (Kitasato).

Potassium Carbonate. — The development of the typhoid bacil-
lus and of the cholera spirillum is prevented by 0.74 to 0.81 per
cent, and these bacteria are killed in five hours by 1 per cent (Kita-
sato).

Potassium Chlorate. — In the writer's experiments a four-per-
cent solution failed in two hours to kill Micrococcus Pasteuri. A
five-per-cent solution failed in six days to destroy anthrax spores
(Koch).

Potassium Chromate. — A five-per-cent solution failed to kill
anthrax spores in five days (Koch).

Potassium Cyanide. — Antiseptic in the proportion of 1 : 909
(Miquel).

Potassium Iodide. — A solution of five per cent does not destroy
anthrax spores in eighty days. (Koch). Putrefactive bacteria in
broken-down beef infusion are not destroyed by two hours' exposure
in a twenty-per-cent solution (Sternberg). The typhoid bacillus and
the cholera spirillum do not grow in culture solutions containing
13

194 ACTION OF SALTS.

eight per cent, and are destroyed by five hours' exposure to 9.23 per
cent (Kitasato). Antiseptic in the proportion of 1 : 7 (Miquel).

Potassium Permanganate. — In the writer's experiments (1881)
a two-per-cent solution was required to destroy Micrococcus Pasteuri
in the blood of a rabbit. In later experiments pus cocci in bouillon
were killed by 1 : 833 — time of exposure two hours. One per cent
was found by Koch not to destroy anthrax spores in two days, but
five per cent was effective in one day. The glanders bacillus is de-
stroyed in two minutes by a one-per-cent solution (Lofner). The
experiments of Jager show that a one-per-cent solution is not reli-
able for the destruction of anthrax bacilli and other pathogenic bac-
teria tested, but a five-per-cent solution was effective. The tubercle
bacillus was not, however, killed by exposure in a five-per-cent solu-
tion. According to Miquel, permanganate of potash is an antiseptic
in the proportion of 1 : 285.

Quinine Hydrobromate. — Antiseptic in the proportion of 1 : 182
(Miquel).

Quinine Hydrochlorate. — Antiseptic in the proportion of 1 : 900
(Ceri). Quinine dissolved with hydrochloric acid destroys anthrax
spores in ten days in one-per-cent solution (Koch).

Quinine Sulphate. — The writer found that in the proportion of
1 : 800 quinine prevents the development of various micrococci and
bacilli. A ten-per-cent solution does not destroy the bacilli of symp-
tomatic anthrax (Arloing, Cornevin, and Thomas).

Silver Nitrate. — Miquel places nitrate of silver next to mercuric
chloride as an antiseptic, effective in the proportion of 1 : 12,500.
Behring also places it next to bichloride as an antiseptic and germi-
cide, and says that it is even superior to this salt in albuminous
fluids. He reports that it prevents the development of anthrax
spores when present in a culture liquid in the proportion of 1 : 80,000,
and in the proportion of 1 : 10,000 destroys these spores in forty-
eight hours. We give below the result of recent experiments by
Boer, in which the time of exposure was two hours :

Restrains
development.

Destroys
vitality.

Anthrax bacillus

1 : 60000

1 : 20000

Diphtheria bacillus

1 : 60000

1 : 2500

Glanders bacillus

1 : 75000

1 : 4000

Typhoid bacillus

1 : 50UOO

1 : 4000

Cholera spirillum

1 : 50000

1 : 4000

Silver Chloride. — A solution of chloride of silver in hyposulphite
of soda is much less effective as an antiseptic than nitrate of silver.

ACTION OF SALTS. 105

Behring found that to prevent the development of anthrax spores a
solution of 1 : 8,000 was required.

Sodium Borate. — In the writer's experiments a saturated solu-
tion of borax was found to be without germicidal power. A twenty -
per-cent solution does not destroy the virus of symptomatic anthrax
(Arloing, Cornevin, and Thomas). A five-per-cent solution failed
to destroy anthrax spores in fifteen days (Koch). Antiseptic in the
proportion of 1 : 14 (Miquel).

Sodium Carbonate. — A solution of 2.2 per cent restrains the
growth of the typhoid bacillus, and of 2.47 per cent of the cholera
spirillum. The first-named bacillus is killed by four or five hours'
exposure in a 2.47-per-cent solution, and the cholera spirillum by
3.45 per cent (Kitasato).

Sodium Chloride. — A saturated solution failed in forty-eight
hours to destroy the virus of symptomatic anthrax (Arloing, Corne-
vin, and Thomas). A saturated solution failed in forty days to de-
stroy anthrax spores (Koch). A saturated solution failed in twenty
hours to destroy the tubercle bacillus in fresh sputum (Schill and
Fischer). In the writer's experiments a five-per-cent solution failed
to kill Micrococcus Pasteuri in blood. Antiseptic in the proportion
of 1 : 6 (Miquel). According to Forster, the bacillus of typhoid
fever, the bacillus of rouget, and the streptococcus of pus are not
killed by several weeks' exposure in strong solutions of sodium chlo-
ride, but the cholera spirillum is destroyed in a few hours. Cultures
of the tubercle bacillus are not sterilized in two months by a satu-
rated solution ; and tuberculous organs from an ox, preserved in a
solution of salt, did not lose their power of infecting susceptible ani-
mals inoculated with material from the diseased tissue. The flesh
of swine which died of rothlauf was found by Petri to still contain
the bacillus in a living condition after having been preserved in
brine for a month.

Sodium Hyposulphite. — In the writer's experiments a saturated
solution failed in two hours to kill micrococci and bacilli. Exposure
for forty-eight hours to a fifty-per-cent solution does not destroy the
virus of symptomatic anthrax (Arloing, Cornevin, and Thomas).
Antiseptic in the proportion of 1 : 3 (Miquel).

Sodium Sulphite. — The results with a saturated solution of this
salt were, in the writer's experiments, entirely negative.

Tin Chloride. — A one-per-cent solution acting for two hours de-
stroyed the bacteria in putrefying bouillon, while 0. 8 per cent failed
(Abbott).

Zinc Chloride. — In the writer's experiments 1:200 destroyed
Micrococcus Pasteuri in two hours, but a two-per-cent solution was re-
quired to kill pus cocci in the same time ; spores of Bacillus anthracis

19G ACTION OF SALTS.

were not destroyed by two hours' exposure in a ten-per-cent solution,
but a solution of five per cent killed the spores of Bacillus subtilis in
the same time. Koch found that anthrax spores germinated after
being immersed in a five-per-cent solution for thirty days. The de-
velopment of Bacillus prodigiosus is only slightly retarded by expo-
sure for sixteen hours in a one-per-cent solution. Antiseptic in the
proportion of 1 : 520 (Miquel).

Zinc Sulphate. — In the writer's first experiments a twenty-per-
cent solution failed to destroy in two hours micrococci obtained from
the pus of an acute abscess. In later experiments a micrococcus from
the same source resisted two hours' exposure to a ten-per-cent solu-
tion, but Micrococcus tetragenus was destroyed by this amount.
Broken-down beef infusion mixed with an equal quantity of a forty -
per-cent solution was not sterilized after two hours' contact. In
Koch's experiments anthrax spores were found to germinate after
having been immersed for ten days in a five-per-cent solution.

ACTION OF COAL-TAR PRODUCTS, ESSENTIAL

OILS, ETC.

IN the present section we shall consider the action upon bacteria
of a variety of organic products, and for convenience will arrange
them alphabetically.

Acetone. — Anthrax spores grow freely after two days7 exposure
to the action of this agent; at the end of five days their development
is feeble (Koch).

Alcohol. — In the writer's experiments ninety-five-per-cent alco-
hol did not destroy the bacteria (spores) in broken-down beef tea in
forty-eight hours. Micrococcus Pasteuri was destroyed by two hours'
exposure in a twenty-four-per-cent solution ; pus cocci required a
forty-per-cent solution. Koch found that absolute alcohol had no
effect upon anthrax spores exposed to its action for one hundred and
ten days. Schill and Fischer found that when tuberculous sputum
was mixed with an equal amount of absolute alcohol its infecting
power was not destroyed in twenty-four hours, but that in the pro-
portion of five parts to one of sputum it was effective in destroying
the tubercle bacillus, as proved by inoculation experiments. Yersin
found that in pure cultures the tubercle bacillus is killed by five
minutes' exposure to the action of absolute alcohol.

Aniline Dyes. — Recent researches have shown that some of the
aniline colors possess very decided germicidal power. Stilling found
that solutions of methyl violet containing 1 : 30,000 exercise a re-
straining influence upon the development of putrefactive bacteria
and pus cocci, and that these^ microorganisms are destroyed by solu-
tions containing 1 : 2,000 to 1 : 1,000. Methyl violet has been placed
in the market by Merck under the name of pyoktanin. Janicke re-
ports the following results with pyoktanin : Staphylococcus pyogenes
aureus was restrained in its development by solutions containing
1 : 2,000,000, Bacillus anthracis by 1 : 1,000,000, Staphylococcus pyo-
genes by 1 : 333,300, Spirillum cholerae Asiaticse by 1 : 62,500, Bacil-
lus typhi abdominalis by 1 : 5,000. In blood serum stronger solutions
were required (1 : 500,000 for Staphylococcus pyogenes aureus). Sta-
phylococcus pyogenes aureus, Streptococcus pyogenes, and Bacillus
anthracis were killed in thirty seconds by 1 : 1,000, the typhoid bacil-

198

ACTION OF COAL-TAR PRODUCTS,

lus by the same amount in thirty minutes. Boer found malachite
green to be still more effective than methyl violet. In his experi-
ments upon bouillon cultures twenty-four hours old, with two hours'
exposure to the action of the disinfectant, he obtained the following
results :

MALACHITE GREEN.

Restrains
development.

Destroys
vitality.

1 : 120000

1 • 40000

Diphtheria bacillus

1 : 40000

1 :8000

Glanders bacillus

1 : 5000

1 :300

Typhoid bacillus .

1 : 5000

1 :300

Cholera spirillum

1 : 100000

1 : 5000

METHYL VIOLET (PYOKTANIN).

Restrains
development.

Destroys
vitality.

1 : 70003

1 -5000

Diphtheria bacillus.

1 : 10000

1 • 2000

1 : 2500

1 :150

Typhoid bacillus. •

1 : 2500

1 -150

1 : 30000

1 : 1000

Aniline Oil. — According to Riedlin, the addition of 1:5 of ani-
line water prevents the development of all bacteria in nutrient gelatin.

Aromatic Products of Decomposition. — Klein has tested the
germicidal power of phenylpropionic and phenylacetic acids. He
finds that anthrax spores resist both of these acids, in the proportion
of 1 : 400, for two days, but in the absence of spores anthrax bacilli
are quickly killed by a solution of this strength. Certain non-patho-
genic micrococci were not killed by exposure for twenty-five minutes
to 1 : 200. The caseous matter of pulmonary tuberculosis infected
guinea-pigs after exposure for ninety-six hours to 1 : 200.

Aseptol. — A ten-per-cent aqueous solution kills anthrax spores in
ten minutes, and a three- to five-per-cent solution is a reliable disin-
fectant in the absence of spores (Hueppe).

Benzene, C6H6. — Exposure in benzol for twenty days failed to
destroy the vitality of anthrax spores (Koch).

Camphor. — Alcohol saturated with camphor has no effect upon
the virus of symptomatic anthrax (Arloing, Cornevin, and Thomas)

The experiments of Cadeac and Meunier show that camphor (oil
of, or tincture?) has but little germicidal power. The typhoid ba-

ESSENTIAL OILS, ETC. 199

cillus and cholera spirillum were only destroyed after eight to ten
days7 exposure to the action of camphor ("essence").

Carbolic Acid. — Tested upon anthrax spores, Koch found a one-
per-cent solution to be without effect after fifteen days7 exposure ; a
two-per-cent solution retarded development but did not completely
destroy vitality in seven days ; a three-per-cent solution was effec-
tive in two days. In the absence of spores Koch found that a one-
per-cent solution quickly destroys the vitality of anthrax bacilli.
He recommends a five-per-cent solution for the destruction of the
"comma bacillus" in the discharges of cholera patients, and a two-
per-cent solution for the disinfection of surfaces soiled with such dis-
charges. In the writer's experiments 1 : 200 destroyed Micrococcus
Pasteuri in two hours ; and pus cocci were destroyed by 1 : 125, while
1 : 200 failed. Davaine showed by inoculation experiments that an-
thrax bacilli in fresh blood are destroyed by being exposed to the
action of a one-per-cent solution for one hour. A two-per-cent solu-
tion destroys the dried virus of symptomatic anthrax in forty-eight
hours (Arloing, Corneviii, and Thomas). Solutions in oil or in alco-
hol have been shown by Koch to be less effective than aqueous solu-
tions. Thus a five-per-cent solution in oil failed to destroy anthrax
spores in one hundred and ten days, and the same solution failed to
kill the bacilli, in the absence of spores, in less than six days. A
five-per-cent solution in alcohol did not destroy anthrax spores in
seventy days. Schill and Fischer found that a three-per-cent solu-
tion destroyed the infecting power of tuberculous sputum, as shown
by inoculation into guinea-pigs, in twenty-four hours, while solutions
of one and two per cent failed. Bolton's experiments gave the fol-
lowing results, the test organisms being in fresh bouillon cultures
and the time of exposure two hours : The cholera spirillum, the
bacillus of typhoid fever, the bacillus of schweinerothlauf, Brieger's
bacillus, the bacillus of green pus, and the pus cocci (Staphylococcus
pyogenes aureus, albus, and citreus, and Streptococcus pyogenes)
were all killed by a solution of one per cent, while in a majority of
the experiments a one-half -per-cent (1 : 200) solution failed. Cul-
tures of the typhoid bacillus in flesh-peptone-gelatin gave the same
result (1 : 100 with two hours7 exposure), and the addition of ten per
cent of dried egg albumin to bouillon cultures did not influence the
result.

The experiments of La Place show that the addition of hydro-
chloric acid to a disinfecting solution containing carbolic acid greatly
increases its germicidal power for spores. Thus it is stated that
" two per cent of crude carbolic acid with one per cent of pure hydro-
chloric acid destroyed anthrax spores in seven days, while two per
cent of carbolic acid or one per cent of hydrochloric acid alone did

200 ACTION OF COAL-TAR PRODUCTS,

not destroy these spores in thirty days. A f our-per-cent solution of
crude carbolic acid with two per cent of hydrochloric acid destroyed
spores in less than an hour ; four per cent of carbolic acid alone did
not destroy them in twelve days. Van Ermengem reports that in
his experiments the cholera spirillum in chicken bouillon was killed
in less than half an hour by 1 : 600, and that in blood serum 1 : 400
was effective. Nicati and Rietsch fix the germicidal power for the
cholera spirillum as 1 : 200, the time of exposure being ten minutes ;
Ramon and Cajal, 1 : 50. Boer gives the following results, the time
of exposure being two hours, cultures in bouillon twenty-four hours
old :

Restrains
development.

Destroys
vitality.

1 -750

1 -300

1 -500

1 -300

1 :500

1 :300

Typhoid bacillus

1 :400

1 :200

Cholera spirillum

1 :600

1 :400

Leitz reports the following results : The dejections of patients
suffering from typhoid fever, mixed in equal quantity with the disin-
fecting solution, were sterilized by a five-per-cent solution of car-
bolic acid in three days. Pure cultures of the typhoid bacillus were
sterilized in fifteen minutes by a five-per-cent solution.

In the experiments of Nocht upon anthrax spores it was found
that while at the room temperature these spores were not destroyed
by several days' exposure in a five-per-cent solution, they were de-
stroyed in three hours by the same solution at a temperature of 37.5°.

Carbolic acid prevents putrefactive changes in bouillon when pre-
sent in the proportion of 1 : 333 (Miquel). The tubercle bacillus is
killed in thirty seconds by a five-per-cent solution, and in one minute
by a one-per-cent solution (Yersin).

Coffee Infusion. — Experiments have been made by Heim and by
Liideritz on the antiseptic power of an infusion of coffee. The first-
named author found that anthrax bacilli no longer developed after
three hours' exposure in a ten-per-cent solution, but spores were not
killed at the end of a week. Streptococci in a bouillon culture re-
quired twenty-four hours' exposure, and the staphylococci of pus were
not destroyed in this time. Ltideritz found that a three-per-cent in-
fusion restrained the growth in nutrient gelatin of the typhoid ba-
cillus, and a five-per-cent infusion killed the bacillus in two days ;
the cholera spirillum failed to grow in presence of one per cent, and
a solution of this strength killed it in seven hours ; Staphylococcus

ESSENTIAL, OILS, ETC. . 201

pyogenes aureus was prevented from developing by two per cent,
and was killed in six days by a five-per-cent solution ; Streptococcus
pyogenes was prevented from growing by one per cent, and killed by
a ten-per-cent solution in one day ; Proteus vulgaris did not grow in
presence of 2. 5 per cent, and was killed in two days by ten per cent.
The question as to what constituent of the infusion of roasted coffee
was the active germicidal agent was not determined, but the authors
referred to agree that it was not caffeine.

Creolin. — This is a coal-tar product which resembles crude carbolic
acid in appearance, but smells rather like tar than like phenol. It
makes a milky emulsion with water, which has been proved by nu-
merous experiments to possess very decided germicidal power, being
superior to carbolic acid. The first careful test of the germicidal
power of this agent was made by Esmarch, who found that a solu-
tion of 1 : 200 killed the cholera spirillum in a minute, the typhoid
bacillus at the end of several days. Anthrax spores were not de-
stroyed in twenty days by a five-per-cent solution, but this solution
killed the tubercle bacillus attached to silk threads which were im-
mersed in it for a short time, and also disinfected tuberculous sputum.
Behring has shown that in albuminous liquids creolin is less effective
than carbolic acid. In blood serum 1 : 175 was required to restrain
the development of staphylococci, and 1 : 100 to destroy the same in
ten minutes. Van Ermengem, as a result of numerous experiments,
arrived at the conclusion that creolin is a cheap and useful disinfect-
ing agent, in a five-per-cent solution, for various pathogenic organ-
isms. Kaupe reports that in his experiments a ten-per-cent solution
killed anthrax spores in twenty-four hours. According to Boer, a
solution of 1 : 5,000 destroys anthrax bacilli in bouillon cultures in
two hours, 1 : 2,000 diphtheria bacilli, 1 : 300 the glanders bacillus,
1 : 250 the typhoid bacillus, and 1 : 3,000 the cholera spirillum.

Creosote. — This agent was found by the writer to be fatal to
micrococci in the proportion of 1 : 200. In the proportion of one per
cent it failed, after twenty hours5 exposure, to destroy tubercle ba-
cilli in sputum (Schill and Fischer). A saturated aqueous solution
does not destroy the tubercle bacillus in cultures in twelve hours
(Yersin). Guttman, in extended experiments upon various patho-
genic organisms, found that development was prevented by 1 : 3,000
to 1 : 4,000. A solution containing 1 : 300 killed Bacillus pyocyanus
and Bacillus anthracis in one minute, Bacillus prodigiosus in two
minutes, and the Finkler-Prior spirillum in one minute in the pro-
portion of 1 : 600.

Cresol. — This is a dark, reddish-brown, transparent fluid, some-
what thinner than creolin, and, like it, having an odor of tar. It
forms an emulsion with water, which is not so stable as that formed

202 ACTION OF COAL-TAR PRODUCTS,

by creolin. Of the three cresols, ortho-, meta-, and paracresol, the
second was found by Frankel to be most active. This author states
that the addition of sulphuric acid adds greatly to its germicidal
power. A four-per-cent solution, containing equal parts of cresol
and H2S04, killed anthrax spores in less than twenty-four hours. In
Behring's experiments a solution containing ten per cent of each killed
anthrax spores in eighty minutes, and five per cent of each in one
hundred minutes, while an eighteen-per-cent solution of sulphuric
acid alone did not kill them in twenty four hours. In the experi-
ments of Jager a two-per-cent solution destroyed the tubercle bacillus
in cultures and in sputum. As a result of his experiments Behring
concludes that cresol has no advantage over carbolic acid as a ger-
micide for the destruction of spores. Tested upon Staphylococcus
aureus, Streptococcus erysipelatos, and Bacillus pyocyanus, Frankel
found that a solution of 0. 3 per cent destroyed these microorganisms
in five minutes, while a two-per-cent solution of carbolic acid re-
quired fifteen minutes' contact to accomplish the same result.

Trikresol (Schering) has been tested, with favorable results, by
several bacteriologists. According to Hammerl it is about twice as
active a germicide as carbolic acid.

Diaphtherin (oxychinaseptol) has considerable antiseptic power,
as shown by the experiments of Rohrer and others. Two to four
drops of a one-per-cent solution was found to prevent the develop-
ment of test organisms (Staphylococcus pyogenes aureus and Bacillus
anthracis) in twelve cubic centimetres of bouillon. Stable (1893)
also finds that as an antiseptic it is far superior to carbolic acid or lysol,
and that it has the advantage of being non-toxic. Tested upon an-
thrax spores it was found to be comparatively inactive as a germicide.
A fifteen-per-cent solution destroyed anthrax spores in three days.

Disinfektol. — This is a coal-tar product similar to creolin which
has been recommended in Germany for disinfecting purposes. It is
an oily, dark-brown fluid having a specific gravity of 1.086. It forms
an emulsion with water, which has a slightly alkaline reaction. It
has been tested upon typhoid stools by Uffelmann and by Beselin.
The last-named author gives the following summary of the results
obtained : An emulsion of five per cent of disinf ektol equals in value,
for the disinfection of the liquid discharges of typhoid patients, 12.5
per cent of creolin, thirty-three per Cent of hydrochloric acid, five per
cent of carbolic acid, 1 : 500 of mercuric chloride.

Ether. — Anthrax spores may germinate after being immersed in
sulphuric ether for eight days (Koch). The tubercle bacillus is de-
stroyed by ten minutes' exposure to the action of ether (Yersin).

Essential Oils. — Chamberlain has made an extended series of
experiments to determine the antiseptic power of the vapor of vola-

ESSENTIAL OILS, ETC. 203

tile oils. A large number of essential oils tested were found to pre-
vent the development of the anthrax bacillus, while a few did not.
At the end of six days the tubes were opened and the oil absorbed by
the culture liquid allowed to evaporate. Cultures were now obtained
from all except the following, which, it was inferred, had destroyed
the vitality of the spores : Angelica, cinnamon of China, cinnamon
of Ceylon, geranium of France, geranium of Algeria, origanum.

Cadeac and Meunier have also made extended experiments upon
the typhoid bacillus and the bacillus of glanders, for the purpose of
determining the germicidal power of agents of this class. Their
method consisted in the introduction of a sterilized platinum needle
into a pure culture of the test organism, in immersing it in the
essential oil for a certain time, and then making with it a puncture
in a suitable solid culture medium. Their results are given belovr
for the typhoid bacillus.

Essences which kill the bacillus after a contact of less than
twenty-four hours :

At the end of—

Cinnamon of Ceylon, . . . . .12 minutes.

Cloves, . . . . .25

Eug-enol, ....... 30

Thyme, ...... 35

Wild thyme 35

Verbena of India, ..... 45

Geranium of France, . . . . .50

Origanum, ...... 75

Patchouly, ...... 80

Zedoary, ...... 2 hours.

Absinthe, . . . . . . 4 "

Sandal wood, . . . . . . 12 "

The following were effective in from twenty-four to forty-eight
hours: Cumin, caraway, juniper, matico, galbanum, valerian, citron,
angelica, celery, savin, copaiba, pepper, turpentine, opopanax, rose,
chamomile ; the following required from two to four days : Illicium,
sassafras, tuberose, coriander; the following from four to eight days:
Calamus, sage, fennel, mace, cascarilla, orange of Portugal; the fol-
lowing in eight to ten days : Mint, nutmeg, rosemary, carrot, mus-
tard, anise, onion, marjoram, bitter almonds, cherry laurel, myrtle,
lavender, eucalyptus, cedar, cajuput, winter green, camphor.

Biedlin reports as the result of his experiments that the essential
oils which have the greatest antiseptic value are oil of lavender, eu-
calyptus, rosemary, and cloves.

Eucalyptol. — Chabaunes and Ferret found that a five-per-cent
solution of eucalyptol is without effect upon tubercle bacilli in spu-
tum. According to Behring, eucalyptol is about four times less ac-
tive as a disinfectant than carbolic acid.

204 ACTION OF COAL-TAR PRODUCTS,

Euphorin (Phenylurethan) has been tested by Colasanti (1894),
who finds that it has rather feeble germicidal activity.

Formaldehyde (formol, formalin) has very decided germicidal
power. According to Pottevin (1894) in the absence of spores a solu-
tion of 1 : 1,000 kills bacteria, in comparatively small numbers, in from
fifteen minutes to several hours. For the destruction of spores a
much stronger solution is required — a fifteeii-per-cent solution at
15° C. killed anthrax spores in one and one-half hours, and spores
of Bacillus subtilis in twenty hours. At higher temperatures the
germicidal action is more energetic, and microorganisms exposed to
the vapor of formol are very quickly destroyed. Vanderlinden and
de Buck (1895) find that solutions of formalin are decidedly inferior
to corresponding solutions of carbolic acid, creolin, or solveol, and are
too irritating to be used in surgical practice. They report that a
solution of five per cent failed to destroy their test organisms-
Bacillus coli communis, Bacillus typhi abdominalis, Staphylococcus
pyogenes aureus. Experiments made by Reed, at the Army Medical
Museum in Washington, show that the diphtheria bacillus and other
test organisms are quickly killed by formalin vapor.

Glycerin has no action upon the virus of symptomatic anthrax
(Arloing, Cornevin, and Thomas), and is inert as regards the spores
of anthrax (Koch). Glycerin prevents putrefactive decomposition in
bouillon when present in the proportion of 1 :-4 (Miquel). Roux has
shown that the addition of five per cent of glycerin to a culture
medium is favorable to the growth of the tubercle bacillus; it is also
appropriated as pabulum by various other species.

Guaiacol. — Kuprianow, as a result of extended experiments with
this agent (1894), reports that it ranks below cresol and carbolic acid
as a germicide. In the proportion of 1 : 500 it restrains the develop-
ment of the cholera spirillum, and the author named suggests its in-
ternal administration in this disease on account of its non-toxic and
non-irritant properties.

Hydroxylamin. — Heinisch found that the development of the
anthrax bacillus is prevented by 1 : 77 of hydroxylamin hydro-
chlorate, and of the diphtheria bacillus by 1 : 75. In these experiments
a solution of soda was added to release the hydroxylamin. Marp-
mann found that 1 : 100 preserved milk without change for four
to six weeks, and that alkaline fermentation of urine was prevented
by 1:1,000.

IchthyoL — Latteux (1892) reports that the various pathogenic
bacteria used by him as test organisms were killed by a five-per-cent
solution (time •?) with exception of Streptococcus pyogenes, which
required a six to seven-per-cent solution. The more recent experi-
ments of Abel (1893) gave less favorable result, but the agent was

ESSENTIAL OILS, ETC. 205

shown to have considerable antiseptic value — 1 : 2,000 restrained the
development of streptococci; 1 : 500 of the diphtheria bacillus; 1 : 20
of Staphylococeus pyogenes aureus; 1:33 the bacillus of typhoid
fever. Streptococci and diphtheria bacilli were destroyed in twenty-
four hours by a solution of 1 : 200; Staphylococcus aureus, subjected
to the action of pure ichthyol, was destroyed in five hours — in a five-
per-cent solution it survived for four days. Cultures of the typhoid
bacillus mixed with a fifty-per-cent solution were not completely
•sterilized in thirty hours; a small number of bacilli in bouillon were,
however, destroyed by a three-per-cent solution in forty-eight hours.
Anthrax spores on silk threads were not destroyed by a fifty-per-cent
solution at the end of one hundred and forty days.

Indol. — When added in excess to water this agent failed to de-
stroy anthrax spores in eighty days (Koch).

Izal is a coal-tar product which has recently been introduced as
a disinfectant. Klein (1892) reports that in the strength of ten per
cent it kills anthrax spores in fifteen minutes. In the absence of
spores various pathogenic bacteria were killed in five minutes by a
solution containing 1 : 200.

Lanolin. — According to Gottstein, various microorganisms tested
by him failed to grow in cultures after having been in contact with
pure lanolin for five to seven days.

Loretin. — Korff (1895) claims for this agent that a two-per-cent
solution is superior to corresponding solutions of lysol, metakresol,
or phenol, and that it has the advantage of being non-toxic, odorless,
and non-irritating.

Lysol. — Weiss (1895) has tested this product and reports that a
solution of three-fourths per cent destroyed his test organisms (pus
cocci, typhoid bacillus, Bacillus coli communis, etc.) in five minutes.
Anthrax spores were destroyed by the same solution in one hour.

Naphthol . — In the proportion of 1 : 10,000 naphthol prevents the
development of the glanders bacillus, the anthrax bacillus, the typhoid
bacillus, the micrococcus of fowl cholera, of Staphylococcus aureus
and albus, and of several other microorganisms tested by Maximo-
vitch. The same author states that although insoluble in cold water,
water at 70° C. dissolves 0.44 in one thousand parts. When urine is
shaken up with naphthol in powder it does not undergo fermenta-
tion.

In the experiments of Foote hydronaphthol was found to show
some germicidal power in the proportion of 1 : 2,300, but the conclu-
sion is reached that a saturated aqueous solution (1 : 1,150) does not
equal a one-per-cent solution of carbolic acid or of creolin.

The writer, in 1892, obtained the following results in experiments
with naphthols upon the cholera spirillum.

206 ACTION OF COAL-TAR PRODUCTS,

Alpha-naphthol and beta-naplitliol have about the same antiseptic and
germicidal value. In the proportion of 1 : 16,000 both prevent the develop-
ment of the cholera spirillum in peptonized beef -tea, while 1 : 24, 000 fails to
prevent development. In the proportion of 1 : 3,000 both destroy the vital-
ity of the cholera spirillum in bouillon cultures, twenty-four hours old,
after two hours' contact, while 1 : 4,000 fails to destroy this microorganism
in the time mentioned — two hours.

In experiments made with a solution of 1:1,000, added to an equal
quantity of a twenty-four hours old bouillon culture — making- 1 : 2,000 after
mixture — and in which the time of contact varied from five to thirty minutes,
alpha-, beta-, and hydronaphthol were found to destroy the cholera germ by
fifteen minutes' exposure, but to fail after ten minutes' contact, so that the
germicidal value of each of these is similar, or nearly so.

In all these experiments the line was sharply drawn between success and
failure. No development occurred and the bouillon remained transparent
in those experiments in which the germicidal action was complete, and a
characteristic development occurred within twenty -four hours in those ex-
periments in which there was a failure to destroy the spirillum.

Benzo-naphthol has no germicidal power, probably because it is insoluble
in water. At least this is my inference from the experiments made. One
gamme was added to one thousand cubic centimetres of distilled water, and
after vigorous shaking was placed in the steam sterilizer for half an hour.
At the end of this time the greater portion, at least, of the benzo-naphthol re-
mained undissolved at the bottom of the flask. The saturated solution (?)
was then filtered and added to recent bouillon cultures of the cholera spiril-
lum in the proportion of 1 : 1, 1:2, 1 : 4, and 2:1. At the end of two hours
sterile bouillon in test tubes was inoculated from each of these and placed in
the incubating oven. At the end of forty-eight hours a characteristic devel-
opment of the cholera spirillum had occurred in all of the tubes.

Olive Oil. — Anthrax spores germinate after having been im-
mersed for ninety days in pure olive oil (Koch).

Oil of Mustard. — Koch found that the development of anthrax
spores is prevented by 1 : 33,000.

Oil of Peppermint. — A five-per-cent solution in alcohol failed in
twelve days to destroy anthrax spores, but the development of these
spores is restrained by 1 : 33,000 (Koch).

Oil of Turpentine destroys anthrax spores in five days, but failed
to do so in one day (Koch). The development of anthrax spores is
prevented by 1: 75,000 (Koch). The addition of 1 : 200 to nutrient
gelatin prevents the development of bacteria (Riedlin) . An excess
of oil of turpentine added to a liquefied gelatin culture of Staphylo-
coccus aureus does not destroy this micrococcus in five hours (v.
Christmas-Dirckinck-Holmfeld).

Saprol. — Laser (189*2) recommends tbis agent for the disinfection
of the excreta of cholera and typhoid patients. He reports that in the
proportion of 1 : 100 it sterilizes liquid fa3ces in twenty-four hours.

Skatol in excess in water has no germicidal power, as tested upon
anthrax spores (Koch).

Smoke. — The researches of Beu show that meats which have been
preserved by smoking commonly contain living bacteria capable of
growing in culture media; and Petri has shown that pork which has

ESSENTIAL OILS, ETC. 207

been salted for a month and then smoked for fourteen days may still
contain the bacillus of rothlauf in a living condition, as shown by in-
oculation experiments. It was not until about six months after smok-
ing that the bacillus failed to give evidence of vitality.

Thymol. — A five-per-cent solution in alcohol does not destroy
anthrax spores in fifteen days, but the development of these spores
is retarded by a solution of 1 : 80, 000 (Koch). The anthrax bacillus
and staphylococci fail to grow in culture media containing 1 : 3,000
(Samter). The tubercle bacillus is destroyed by contact with thy-
mol for three hours (Yersin). Thymol has about four times less
germicidal power than carbolic acid (Behring). Antiseptic in the
proportion of 1 : 1,340 (Miquel).

Tobacco Smoke. — Tassinari found that tobacco smoke restrains
the development of bacteria, and that certain species failed to de-
velop after exposure for half an hour in an atmosphere of tobacco
smoke — spirillum of cholera and Friedlander's bacillus.

XII.

ACTION OF BLOOD SERUM AND OTHER ORGANIC

LIQUIDS.

Blood Serum. — Bacteriologists have long been aware of the fact
that many species of bacteria, when injected into the circulation of a,
living animal, soon disappear from the blood, and that the blood of
such an animal a few hours after an injection of putrefactive bacte-
ria, for example, does not contain living bacteria capable of develop-
ing in -a suitable nutrient medium. Wyssokowitsch, in an extended
series of experiments, has shown that non-pathogenic bacteria in-
jected into the circulation may be obtained in cultures from the liver,
spleen, kidneys, and bone marrow after they have disappeared from
the blood, but that, as a rule, those present in these organs have lost
their vitality, as shown by culture experiments, in a period varying
from a few hours to two or three days. According to the theory of
Metschnikoff , this destruction of bacteria in the blood and tissues of a
living animal is effected by the cellular elements, and especially by
the leucocytes, which pick up and digest these vegetable cells very
much as an amoeba disposes of similar microorganisms which serve
it as food. Some such theory seemed necessary to account for the
disappearance of bacteria from the blood before the demonstration
was made that the serum of the circulating fluid, quite indepen-
dently of its cellular elements, possesses very decided germicidal
power.

Yon Fodor first (1887) called attention to the fact that anthrax ba-
cilli maybe destroyed by freshly drawn blood ; and Nuttall (1888),
in an extended series of experiments, showed that various bacteria
are destroyed within a short time by the fresh blood of warm-
blooded animals. Thus the anthrax bacillus in rabbit's blood was
usually killed in from two to four hours when the temperature was
maintained at 37°-38° C., and the same result was obtained with
pigeon's blood at 41° C. But when the blood was allowed to stand
for a considerable time, or was heated for forty-five minutes to
45° C., it served as a culture fluid, and an abundant development of
anthrax bacilli occurred in it. Bacillus subtilis and Bacillus mega-

ACTION OF BLOOD SERUM AND OTHER ORGANIC LIQUIDS. 209

therium were also destroyed in two hours by fresh rabbit's blood,
but it was without action on Staphylococcus pyogenes aureus, which
at a temperature of 37.5° C. was found to have increased in num-
bers at the end of two hours. Further researches by Nissen and
Behring show that there is a wide difference in the blood of dif-
ferent animals as to germicidal power, and that certain bacteria
are promptly destroyed, while other species are simply restrained for
a time in their development or are not affected. Thus Nissen found
that the cholera spirillum, the bacillus of anthrax, the bacillus of
typhoid fever, and Friedlander's pneumococcus were killed, while
Staphylococcus pyogenes aureus and albus, the streptococcus of ery-
sipelas, the bacillus of fowl cholera, the bacillus of rothlauf, and
Proteus hominis were able to multiply in rabbit's blood after having
been restrained for a short time in their development. In the case
of the cholera spirillum a period of ten to forty minutes sufficed for
the complete destruction of a limited number, but when the number
exceeded 1,200,000 per cubic centimetre they were no longer de-
stroyed with certainty, and after five hours an increase occurred.
The anthrax bacillus was commonly destroyed within twenty minutes
and the typhoid bacillus at the end of two hours. In the experi-
ments of Bearing and Mssen it was found that the most pronounced
germicidal effect upon the anthrax bacillus was obtained from the
blood of the rat, an animal which has a natural immunity against
anthrax ; while the blood of the guinea-pig, a very susceptible ani-
mal, had no restraining effect and served as a favorable culture
medium for the anthrax bacillus. And the remarkable fact was de-
monstrated that when the blood of a rat was added to the blood of
the guinea-pig in the proportion of 1:8, it exercised a decided re-
straining influence upon the growth of the anthrax bacillus. Later
researches have shown that cultivation in the blood of an immune
animal causes an attenuation of the virulence of an anthrax cul-
ture (Ogata and Jasuhara) ; and also that the injection of the blood
of a frog or rat — naturally immune — into a susceptible animal which
has been inoculated with a virulent culture of the anthrax bacillus,
will prevent the death of the inoculated animal.

Buchner has shown that the germicidal power of the blood of
dogs and rabbits does not depend upon the presence of the cellular
elements, but is present in clear serum which has been allowed to
separate from the clot in a cool place. Exposure for an hour to a
temperature of 55° C. destroys the germicidal action of serum as
well as of blood ; the same effect is produced by heating to 52° C. for
six hours or to 45. 6° C. for twenty hours. The germicidal power
of blood serum is not destroyed by freezing and thawing, but is
lost after it has been kept for some time. Buchner's experiments led
14

ACTION OF BLOOD SERUM AND OTHER ORGANIC LIQUIDS.

him to the conclusion that the germicidal power of fresh blood
serum depends upon the presence of some albuminous body present
in it. This view is sustained by the researches of Ogata, who has
obtained from the blood of dogs and other animals a glycerin ex-
tract of a "ferment" which is insoluble in alcohol or in ether and
which has germicidal properties.

According to Emmerich and Tsuboi (1893), when the serum-
albumin is precipitated by alcohol, dried in a vacuum at 40° C., and
dissolved in water it has no longer any germicidal activity. But if
the precipitated and dried albumin is dissolved at 3(J° C. in a weak
solution (0.05-0.08 per cent) of soda or potash it recovers its original
germicidal value.

It has been demonstrated by several experimenters that other
albuminous fluids possess a similar germicidal power. Thus Nuttall
found that a pleuritic exudation from man destroyed the anthrax
bacillus in an hour, the aqueous humor of a rabbit in two hours.
Wurz has experimented with fresh egg albumin, and found that the
anthrax bacillus failed to grow after having been exposed for an hour
to the action of albumin from a hen's egg ; other bacteria tested
were not killed so promptly, but a decided germicidal action was
manifested. Prudden has shown that the albuminous fluid obtained
from a hydrocele, or from the abdominal cavity in ascites, possesses
similar germicidal power ; and Fokker has demonstrated that fresh
milk destroys the vitality of certain bacteria which induce an acid
fermentation of this fluid.

The results heretofore referred to induced Hankin to experiment
with cell globulin obtained from the spleen or lymphatic glands of a
dog or cat. This is extracted by means of a solution of chloride of
sodium, the solution is filtered, and the globulin precipitated by the
addition of alcohol. The precipitate is washed and again dissolved
in salt solution. The result showed that this cell globulin possesses
germicidal power similar to that of blood serum.

Mucus. — The experiments of Wurtz and Lermoyez (1893) show
that nasal mucus has germicidal properties, especially for the anthrax
bacillus. Walthard (1893), in experiments with mucus from the cer-
vix uteri, was not able to demonstrate any germicidal action, but
arrived at the conclusion that it prevents the development of bacteria
simply because it is an unfavorable medium. Various bacteria were
planted upon the surface of cervical mucus in Petri dishes, and placed
in the incubating oven, but all failed to grow.

Nucleins from animal and vegetable cells have been shown by
Professor Vaughan and his associates (1893) to possess considerable
germicidal power. The nucleins of animal origin were obtained from
the testes of dogs and rats. Dissolved in a 0.5-per-cent solution of

ACTION OF BLOOD SEBUM AND 'OTHEK OKGANIC LIQUIDS. 211

caustic potash and then diluted with four volumes of physiologic salt
solution the germicidal activity was shown by the facts that Staphylo-
coccus pyogenes aureus, and the anthrax bacillus without spores,
failed to grow after twenty minutes' exposure. Kossel (1893) has
obtained similar results with nucleins from the thymus gland of the
calf.

Urine. — The experiments of Lehmann show that fresh urine has
a decided germicidal power for the cholera spirillum and the anthrax
bacillus, and no doubt for other bacteria as well. To what constitu-
ent of the urine this is due has not been determined, but it may be
due to the uric acid present.

XIII.

PRACTICAL DIRECTIONS FOR DISINFECTION.

THE Committee on Disinfectants of the American Public Health
Association (appointed in 1884), after an extended investigation with
reference to the germicidal value of various agents, in a final report
submitted in 1887 submits the following " Conclusions":

The experimental evidence recorded in this report seems to justify the
following conclusions :

The most useful agents for the destruction of spore-containing infectious
material are —

1. Fire. Complete destruction by burning.

2. Steam underpressure. 105° C. (221° F.) for ten minutes.

3. Soiling in water for half an hour.

4. Chloride of lime.1 A four-per-cent solution.

5. Mercuric chloride. A solution of 1 : 500.

For the destruction of infectious material which owes its infecting power
to the presence of microorganisms not containing spores, the committee rec-
ommends—

1. Fire. Complete destruction by burning.

2. Boiling in water for ten minutes.

3. Dry heat. 110° C. (230° F ) for two hours.

4. Chloride of lime. A two-per-cent solution.

5. Solution of chlorinated soda.* A ten-per-cent solution.

6. Mercuric chloride. A solution of 1 : 2,000.

7. Carbolic acid. A nve-per-cent solution.

8. Sulphate of copper. A ft ve-per-ceiit solution.

9. Chloride of zinc. A ten-per-cent solution.

10. Sulphur dioxide.* Exposure for twelve hours to an atmosphere con-
taining at least four volumes per cent of this gas in presence of
moisture.

The committee would make the following recommendations with refe-
rence to the practical application of these agents for disinfecting purposes :

FOR EXCRETA.

(a) In the sick-room :

1. Chloride of lime in solution, four per cent.
In the absence of spores :

2. Carbolic acid in solution, five per cent.

3. Sulphate of copper in solution, five per cent.

1 Should contain at least twenty-five per cent of available chlorine.
8 Should contain at least three per cent of available chlorine.
3 This will require the combustion of between three and four pounds of sulphur
for every thousand cubic feet of air space.

PRACTICAL DIRECTIONS FOR DISINFECTION.

(b) In privy vaults:

1. Mercuric chloride in solution, 1: 500. l

2. Carbolic acid in solution, five per cent.

Chloride of lime in powder.

FOR CLOTHING, BEDDING, ETC.

(a) Soiled underclothing1, bed linen, etc. :

1. Destruction by fire, if of little value.

2. Boiling for at least half an hour.

3. Immersion in a solution of mercuric chloride of the strength of

1 : 2,000 for four hours.

4. Immersion in a two-per-cent solution of carbolic acid for four hours.
(6) Outer garments of wool or silk, and similar articles, which would be

injured by immersion in boiling water or in a disinfecting solution:

1. Exposure in a suitable apparatus to a current of steam for ten min-

utes.

2. Exposure to dry heat at a temperature of 110° C. (230° F.) for two

hours.
(3) Mattresses and blankets soiled by the discharges of the sick:

1. Destruction by fire.

2. Exposure to superheated steam, 105° C. (221° F.), for ten minutes.

(Mattresses to have the cover removed or freely opened.)

3. Immersion in boiling water for half an hour.

FURNITURE AND ARTICLES OF WOOD, LEATHER, AND PORCELAIN.

Washing, several times repeated, with—
1. Solution of carbolic acid, two per cent.

FOR THE PERSON.

The hands and general surface of the body of attendants of the sick, and
of convalescents, should be washed with —

1. Solution of chlorinated soda diluted with nine parts of water, 1 : 10.

2. Carbolic acid, two-per-cent solution.

3. Mercuric chloride, 1 : 1,000.

FOR THE DEAD.

Envelop the body in a sheet thoroughly saturated with —

1. Chloride of lime in solution, four per cent.

2. Mercuric chloride in solution, 1 : 500.

3. Carbolic acid in solution, five per cent.

FOR THE SICK-ROOM AND HOSPITAL WARDS.

(a) While occupied, wa«h all surfaces with —

1. Mercuric chloride in solution, 1: 1,000.

2. Carbolic acid in solution, two per cent.

(b) When vacated, fumigate with sulphur dioxide for twelve hours, burn-
ing at least three pounds of sulphur for every thousand cubic feet of air
space in the room ; then wash all surfaces with one of the above-mentioned
disinfecting solutions, and afterward with soap and hot water; finally throw
open doors and windows, and ventilate freely.

1 The addition of an equal quantity of potassium permanganate as a deodorant,
and to give color to the solution, is to be recommended. [The writer no longer in-
dorses this recommendation. See his paper on " The Disinfection of Excreta," ap-
pended.]

214 PRACTICAL DIRECTIONS FOR DISINFECTION.

FOR MERCHANDISE AND THE MAILS.

The disinfection of merchandise and of the mails will only be required
under exceptional circumstances; free aeration will usually be sufficient. If
disinfection seems necessary, fumigation with sulphur dioxide will be the
only practicable method of accomplishing- it without injury.

RAGS.

(a) Rag-s which have been usea for wiping away infectious discharges
should at once be burned.

(b) Rags collected for the paper-makers during1 the prevalence of an epi-
demic should be disinfected, before they are compressed in bales, by —

1. Exposure to superheated steam of 105° C. (221° F.) for ten minutes.

2. Immersion in boiling water for half an hour.

SHIPS.

(a) Infected ships at sea should be washed in every accessible place, and
especially the localities occupied by the sick, with —

1. Solution of mercuric chloride, 1 : 1,000.

2. Solution of carbolic acid, two per cent.

The bilge should be disinfected by the liberal use of a strong solution of
mercuric chloride.

(b) Upon arrival at a quarantine station, an infected ship should at
once be fumigated with sulphurous acid gas, using three pounds of sulphur
for every thousand cubic feet of air space ; the cargo should then be dis-
charged on lighters; a liberal supply of the concentrated solution of mercuric
chloride (four ounces to the gallon) should be thrown into the bilge, and at
the end of twenty- four hours the bilge watei should be pumped out and re-
placed with pure sea water ; this should be repeated. A second fumigation,
after the removal of the cargo, is recommended; all accessible surf aces should
be washed with one of the disinfecting solutions heretofore recommended,
and subsequently with soap and hot water.

FOR RAILWAY CARS.

The directions given for the disinfection of dwellings, hospital wards, and
ships apply as well to infected railway cars. The treatment of excreta with
ja disinfectant, before they are scattered along the tracks, seems desirable at
all times in view of the fact that they may contain infectious germs. Dur-
ing the prevalence of an epidemic of cholera this is imperative. For this
purpose the standard solution of chloride of lime is recommended.

DISINFECTION BY STEAM.

The Committee on Disinfectants, in tne above-quoted "Conclu-
sions," recommends the use of " steam under pressure, 105° C. (221°
F.), for ten minutes" for the destruction of spore-containing infec-
tious material. The spores of all known pathogenic bacteria are de-
stroyed by a temperature of 100° C. maintained for five minutes, and
in view of this fact the temperature fixed by the committee is ample,
and to exact a higher temperature or longer exposure would be un-
reasonable. But in practical disinfection the temperature required
to destroy infectious material is not the only question to be considered.
Economy in the construction and operation of the steam disinfecting
apparatus must have due attention, and an important point relates

PRACTICAL DIRECTIONS FOR DISINFECTION. 215

to the penetration of porous, non-conducting articles, such as rolls of
blankets, clothing, etc. These points have been the subject of nu-
merous experimental investigations, and the principles involved
have been elucidated, especially by the investigations of Esmarch
(1887), of Budde (1889), and of Teuschner (1890).

It has been shown that streaming steam is more effective than
confined steam at the samq temperature, because it penetrates porous
objects more quickly. Also that superheated, " dry " steam is not as
effective as flowing steam at 100° C. ; on the other hand, it corre-
sponds in effectiveness with dry air, and the temperature must be
raised to 1-40° to 150° C. in order to quickly destroy the spores of
bacilli.

Esmarch's investigations show that streaming steam penetrates
porous objects, like rolled blankets, more readily than confined
steam ; but the later researches of Budde and of Teuschner show
that a temperature of 100° C. is more rapidly reached in the interior
of such rolls when the flowing steam is under pressure. With the
same pressure (fifteen pounds) a temperature of 100° C. was reached
in two and one-half minutes when the steam was flowing, and in
eleven minutes by steam at rest (Budde). Intermittent pressure
was not found by Budde to present any advantages over continuously
flowing steam ; on the contrary, the time of penetration was longer.

Teuschner, whose investigations are the most recent, arrives at
the following conclusions :

1. Strongly superheated steam is not to be recommended for practical
disinfection. On the contrary, a slight superheating- of the steam, such as
occurs in the apparatus of Schimmel, is not objectionable.

2. Those forms of apparatus in which the steam enters from above are
much safer and quicker in their disinfecting action than those in which this
is not the case. In the construction of such apparatus care must be taken,
in order to secure penetration of the objects, that the air and steam have a
free escape below.

3.. Disinfection is hastened by previously warming the apparatus.

4. The most rapid disinfecting action is secured by the use of streaming
steam in a state of tension (under pressure).

5. Objects which have been .in contact with fatty or oily substances
require a longer time for disinfection than those which have not.

6. To accomplish disinfection it is necessary to expel, as completely as
possible, all air from the objects to be disinfected, and also to secure a suffi-
cient condensation of the steam.

7. The condensation of the steam advances in a sharply defined line
from the periphery to the centre of porous objects.

8. The temperature necessary for disinfection is only found in the zone
where condensation has already taken place.

9. Only a few centimetres from the zone in which the temperature is
100° C. — when disinfection is incomplete— there may be places in which
the temperature is 40° C, or more below the boiling point.

216 PRACTICAL DIRECTIONS FOR DISINFECTION.

DISINFECTION BY FORMALDEHYDE GAS.

Recent experiments have demonstrated the valuable germicidal
properties of formaldehyde gas. Owing to its superior germicidal
value and non-toxic properties, it has to a considerable extent taken
the place of sulphur dioxide as a gaseous disinfectant. In making
practical use of this agent a suitable apparatus will be required. For
the disinfection of a room with its contents, freely exposed for sur-
face disinfection, one pound of formalin should be volatilized for
each thousand cubic feet of air space — the time of exposure to the
disinfecting action of the gas being not less than twelve hours.
When paraform is used the amount required will be sixty grammes
to one thousand cubic feet (Novy). In the absence of any appa-
ratus satisfactory results have been obtained by the Department of
Health of the city of Chicago, as follows:

"Ordinary bed sheets were employed to secure an adequate eyaporatory
surface, and these, suspended in the room, were simply sprayed with a forty-
per-cent solution of formalin through a common watering-pot rose-head. A
sheet of the usual size and quality will carry from one hundred and fifty to
one hundred and eighty cubic centimetres of the solution without dripping,
and this quaatity has been found sufficient for the disinfection of one thousand
cubic feet of space. Of course, the sheets may be modified to any necessary
number. . . . Surface disinfection was thorough, while a much greater de-
gree of penetration was shown than that secured by any other method."

Formalin may also be used in the disinfection of rooms and their
contents by spraying all exposed surfaces.

Experiments made by Kinyoun and others show that formalde-
hyde gas does not injure the color or textile strength of fabrics of
wool, silk, cotton, or linen, and that it has no injurious action upon
furs, leather, copper, brass, nickel, zinc, polished steel or gilt work.
Iron and unpolished steel are attacked by the gas.

DISINFECTION OF THE HANDS.

The importance of a reliable method of disinfecting the hands of
surgeons, obstetricians, and nurses after they have been in contact
with infectious material from wounds, puerperal discharges, etc. , is
now fully recognized, and some surgeons consider it necessary to
completely sterilize the hands before undertaking any surgical opera-
tion which will bring them in contact with the freshly-cut tissues.
The numerous experiments which have been made with a view to
ascertaining the best method of accomplishing such sterilization of
the hands show that it is by no means a simple matter to effect it,
and especially to insure the destruction of microorganisms con-
cealed beneath the finger nails. Furbringer, in an extended series
of experiments (1888), found that a preliminary cleansing with soap

PRACTICAL DIRECTIONS FOR DISINFECTION. 217

and a brush was even more important than the degree of potency of
the disinfecting wash subsequently applied. He recommends the
following procedure :

1. Remove all visible dirt from beneath and around the nails.

2. Brush the spaces beneath the nails with soap and hot water
for a minute.

3. Wash for a minute in alcohol (not below eighty per cent), and,
before this evaporates, in the following solution :

4. Wash thoroughly for a minute in a solution containing 1 : 500
of mercuric chloride or three per cent of carbolic acid.

Roux and Reynes tested the above method of Fiirbringer, and
found that it gave better results than others previously proposed, al-
though not always entirely successful in securing complete steriliza-
tion.

Boll has recently (1890) reported favorable results from the fol-
lowing method :

1. Cleanse the finger nails from, visible dirt with knife or nail scissors.

2. Brush the hands for three minutes with hot water and potash soap.

3. Wash for half a minute in a three-per-cent solution of carbolic acid,
and subsequently in a 1 : 2,000 solution of mercuric chloride.

4. Rub the spaces beneath the nails and around their margins with iodo-
form gauze wet in a five-per-ceiit solution of carbolic acid.

Welch, as a result of extended experiments made at the Johns
Hopkins Hospital, recommends the following procedure :

1. The nails are kept short and clean.

2. The hands are washed thoroughly for several minutes with soap and
water, the water being as warm as can be comfortably borne, and being fre-
quently changed. A brush sterilized by steam is used. The excess of soap
is washed off with water.

3. The hands are immersed for one or two minutes in a warm saturated
solution of permanganate of Dotash and are rubbed over thoroughly with a
sterilized swab.

4. They are then placed in a warm saturated solution of oxalic acid,
where they remain until complete decolorization of the permanganate
occurs.

5. They are then washed off with sterilized salt solution or water.

6. They are immersed for two minutes in sublimate solution, 1 : 500.
The bacteriological examination of the skin thus treated yields almost

uniformly negative results, the material for the cultures being taken from
underneath and around the nails. This is the procedure now employed in
the gynecological and surgical wards of the hospital.

THE DISINFECTION OF EXCRETA.

The contents of privy vaults and cesspools should never be allowed
to accumulate unduly or to become offensive. By frequent removal,
and by the liberal use of antiseptics, such necessary receptacles of
filth should be kept in a sanitary condition. The absorbent deodo-

218 PRACTICAL DIRECTIONS FOR DISINFECTION.

rants, such as dry earth or pounded charcoal, or the chemical de-
odorants and antiseptics, such as chloride of zinc, sulphate of iron,
etc., will, under ordinar}7 circumstances, prevent such places from
becoming offensive. Disinfection will be required only when it is
known or suspected that infectious material, such as the dejections
of patients with cholora, yellow fever, or typhoid fever, has been
thrown into the receptacles.

In the Manual for the Medical Department of the United States
Army the following directions are given :

92. When accumulations of organic material undergoing- decomposition
cannot be removed or buried, they may be treated with an antiseptic solu-
tion, or with freshly burned quicklime. Quicklime is also a valuable disin-
fectant, and may be substituted for the more expensive chloride of lime for
disinfection of typhoid and cholera excreta, etc. For this purpose freshly
prepared milk of lime should be used, containing about one part, by weight,
of hydrate of lime, to eight of water.

93. During the prevalence of an epidemic, or when there is reason to
believe that infectious material has been introduced from any source, latrines
and cesspools may be treated with milk of lime, in the proportion of five
parts to one hundred parts of the contents of the vault, and the daily addi-
tion of ten parts for one hundred parts of daily increment of faeces.

According to Behring, lime has about the same germicidal value
as the other caustic alkalies, and destroys the cholera spirillum and
the bacillus of typhoid fever, of diphtheria, and of glanders after
several hours' exposure, in the proportion of fifty cubic centimetres
normal-lauge per litre. Wood ashes or lye of the same alkaline
strength may therefore be substituted for quicklime.

Finally, it must not be forgotten that we have a ready means of
disinfecting excreta in the sick-room or its vicinity by the application
of heat. Exact experiments, made by the writer and others, show
that the thermal death-point of the following pathogenic bacteria,
and of the kinds of virus mentioned, is below 60° C. (140° F.) :
Spirillum of cholera, bacillus of anthrax, bacillus of typhoid fever,
bacillus of diphtheria, bacillus of glanders, diplococcus of pneu-
monia (Micrococcus Pasteuri), streptococcus of erysipelas, staphylo-
cocci of pus, micrococcus of gonorrhoea, vaccine virus, sheep-pox
virus, hydrophobia virus. Ten minutes' exposure to the tempera-
ture mentioned may be relied upon for the disinfection of material
containing any of these pathogenic organisms, except the anthrax
bacillus when in the stage of spore formation. The use, therefore,
of boiling water in the proportion of three or four parts to one
part of the material to be disinfected may be safely recommended
for such material. Or, better still, a ten-per-cent solution of sulphate
of iron or of chloride of zinc at the boiling-point may be used in the
same way (three parts to one).

PART THIRD.

PATHOGENIC BACTERIA.

I. MODES OF ACTION. II. CHANNELS OF INFECTION. III. SUSCEPTIBILITY AND

IMMUNITY. IV. PROTECTIVE INOCULATIONS. V. PYOGENIC BACTERIA. VI.

BACTERIA IN CROUPOUS PNEUMONIA. VII. PATHOGENIC MICROCOCCI NOT

DESCRIBED IN SECTIONS V. AND VI. VIII. TlIE BACILLUS OF AN-
THRAX. IX. THE BACILLUS OF TYPHOID FEVER. X. BAC-
TERIA IN DIPHTHERIA. XI. BACILLUS OF INFLUENZA. XII.
BACILLI IN CHRONIC INFECTIOUS DISEASES. XIII. BACILLI

WHICH PRODUCE SEPTICAEMIA IN SUSCEPTIBLE ANI-
MALS. XIV. PATHOGENIC AEROBIC BACILLI NOT

DESCRIBED IN PREVIOUS SECTIONS. XV.

BACTERIA OF PLANT DISEASES. XVI.

PATHOGENIC ANAEROBIC BACILLI.

XVII. PATHOGENIC SPIRILLA.

PAET THIED.
PATHOGENIC BACTERIA.

MODES OF ACTION.

MANY of the saprophytic bacteria are pathogenic for man, or for
one or more species of the lower animals, when by accident or ex-
perimental inoculation they obtain access to the body ; these may be
designated facultative parasites. Other species which, for a time
at least, are able to lead a saprophytic mode of life have their nor-
mal habitat in the bodies of infected animals, in which they produce
specific infectious diseases. To this class belong the cholera spirillum,
the anthrax bacillus, the bacillus of typhoid fever, and various other
microorganisms which are the cause of specific infectious diseases in
some of the lower animals. These we may speak of as parasites
and facultative saprophytes. Still others are strict parasites and
do not find the conditions for their development outside of the bodies
of the animals which they infest, except under the special conditions
in which bacteriologists have succeeded in cultivating some of them.
The best known strict parasites are the tubercle bacillus, the bacillus
of leprosy, the spirillum of relapsing fever, and the micrococcus of
gonorrhoea.

There can be but little doubt that even the strict parasites, at some
time in the past, were also saprophytes, and that the adaptation to a
parasitic mode of life was gradually effected under the laws of natural
selection. In a previous chapter (Section III., Part Second) we have
referred to the modifications in biological characters which may
occur as a result of special conditions of environment. Thus we may
obtain non-chromogenic varieties of species which usually produce
pigment, or non-pathogenic varieties of bacteria which are usually
pathogenic. There is also evidence that the tubercle bacillus, a strict

222 MODES OF ACTION.

parasite, may be so modified, by cultivation for successive genera-
tions in a culture medium containing glycerin, that it will finally
grow in ordinary beef infusion, thus showing a tendency to adapt
itself to a saprophytic mode of life.

Some of the saprophytic bacteria are indirectly pathogenic by
reason of their power to multiply in articles of food, such as milk,
cheese, fish, sausage, etc., and there produce poisonous ptomaines
which, when these articles are ingested, give rise to various morbid
symptoms, such as vomiting, gastric and intestinal irritation, fever,
etc. Or similar symptoms may result from the multiplication of
bacteria producing toxic ptomaines in the alimentary canal. No
doubt gastric and intestinal disorders are largely due to this cause,
and may be induced by a variety of saprophytic bacteria when these
establish themselves in undue numbers in any portion of the ali-
mentary tract. In Asiatic cholera the same thing occurs, but with
more fatal results from the introduction of the East Indian cholera
germ discovered by Koch. This is pathogenic for man, because it is
able to multiply rapidly in the human intestine, and there produces a
toxic substance which, being absorbed, gives rise to the morbid pheno-
mena of the disease. The spirillum itself does not enter the blood or
invade the tissues, except to a limited extent in the mucous coat of
the intestine, and the true explanation of its pathogenic power is no
doubt that which has been given.

Other microorganisms invade the tissues and multiply in cer-
tain favorable localities, but have not the power of developing in the
blood, in which they are only found occasionally and in very small
numbers or not at all. Thus the typhoid bacillus locates itself in the
intestinal glands, in the spleen, and in the liver, forming colonies of
limited extent, and evidently not finding the conditions extremely
favorable for its growth, inasmuch as it does not take complete pos-
session of these organs. The symptoms which result from its pre-
sence are doubtless partly due to local irritation, disturbance of func-
tion, and, in the case of the intestinal glands, necrotic changes
induced by it. But in addition to this its pathogenic action depends
upon the production of a poisonous ptomaine which has been isolated
and studied by the German chemist Brieger (typhotoxin).

Certain saprophytic bacteria, when injected beneath the skin of a
susceptible animal, multiply at the point of inoculation and invade
the surrounding tissues, giving rise in some instances to the forma-
tion of a local abscess, in others to an infiltration of the tissues with
bloody serum, and in others to extensive necrotic changes. These
local changes are due not simply to the mechanical presence of the
microorganisms which induce them, but to chemical products evolved
during the growth of these pathogenic bacteria. Indeed, their patho-

MODES OF ACTION. 223

genie power evidently depends, in some instances at least, upon these
toxic products of their growth, by which the vital resisting power of
the tissues is overcome.

Among the bacteria which in this way produce extensive local
inflammatory and necrotic changes are certain anaerobic species
found in the soil and in putrefying material, such as the bacillus of
malignant oedema and the writer's Bacillus cadaveris. The bacillus
of symptomatic anthrax, an infectious disease of cattle, acts in the
same way. All of these produce toxic substances which have a very
pronounced local action upon the tissues invaded by them. Other
bacteria, while they develop chiefly in the vicinity of the point of
entrance — by accident or by inoculation — produce a potent toxic sub-
stance which gives rise to general symptoms of a serious character,
such as tetanic convulsions (bacillus of tetanus) or intense fever and
nervous phenomena (micrococcus of erysipelas). Again, the local
irritation resulting from the presence of parasitic bacteria may pri-
marily give rise to the formation of new growths having a low grade
of vitality, which later may undergo necrotic changes, as in tubercu-
losis, glanders, and leprosy. In this case constitutional symptoms
are not present, or are of a mild character during the development
of these new formations, which apparently result from the local ac-
tion of substances eliminated during the growth of the parasite,
rather than from its simple presence. This is an inference based
upon the fact that non-living particles, or even living parasites, as in
trichinosis, do not produce similar new growths composed of cells,
but become encysted in a fibrous capsule.

In pneumonia we have a local process in which one or more lobes
of the lung are invaded by a pathogenic micrococcus (Micrococcus
pneumonise crouposoe) which induces a fibrinous exudation that com-
pletely fills the air cells. How far the symptoms of the disease are
due to the local inflammation and disturbance of function, and to
what extent they may be due to the absorption of a soluble toxic
substance evolved as a result of the growth of the micrococcus, has
not been determined. But the mild character of the general symp-
toms when a limited area of lung tissue is involved leads to the in-
ference that the pathogenic power of this particular pathogenic
microorganism is chiefly exercised locally.

The pus cocci and various other saprophytic bacteria, when intro-
duced beneath the skin, give rise to the formation of abscesses, un-
attended by any very considerable general disturbance ; and also to
secondary purulent accumulations — metastatic abscesses.

That this is not due simply to their mechanical presence is shown
by the fact that powdered glass and other inert substances, when
thoroughly sterilized, do not give rise to pus formation when intro-

224 MODES OF ACTION.

duced beneath the skin or injected into the cavity of the abdomen.
On the other hand, it has been demonstrated by the experiments of
Grawitz, De Bary, and others that certain chemical substances
which act as local irritants when brought in contact with the tissues
may induce pus formation quite independently of microorganisms :
nitrate of silver, oil of turpentine, and strong liquor ammonise have
been shown to possess this power. And it has been demonstrated by
the recent experiments of Buchner that sterilized cultures of a long
list of different bacteria — seventeen species tested — give rise to sup-
puration when introduced into the subcutaneous tissues.

Buchner has further shown that this property of inducing pus for-
mation resides in the dead bacterial cells and not in soluble products
present in the cultures. For the clear fluid obtained by passing
these sterilized cultures through a porcelain filter gave a negative re-
sult, while the bacteria retained by the filter, although no longer
capable of development, having been killed by heat, invariably
caused suppuration.

Individuals suffering from malnutrition are more susceptible to
invasion by specific disease germs or by the common pus cocci
than are those in vigorous health. Thus the sufferers from starva-
tion, from crowd poisoning, sewer-gas poisoning, etc., are not only
liable to be early victims during the prevalence of an epidemic dis-
ease, but are very subject to abscesses, boils, ulcers, etc. A slight
abrasion in such an individual, inoculated by the ever-present pus
cocci, may give rise to an obstinate ulcer or a phlegmonous inflam-
mation.

In the same way some of the ordinary saprophytes, which usually
have no pathogenic power, may be pathogenic for an animal whose
strength is reduced by disease or injury. Thus necrotic changes
may occur in injured tissues, or in those which have a deficient blood
supply — from occlusion of an artery, for example — due to the presence
of putrefactive bacteria which are incapable of development in the
circulation of a healthy animal or in healthy tissues. We may also
have a progressive gangrene, due to infection of wounds by bacteria
which are able to invade healthy tissues. This is seen in the so-
called hospital gangrene, which is undoubtedly due to microorgan-
isms, although the species concerned in its production has not been
determined, owing to the fact that modern bacteriologists have had
few, if any, opportunities for studying it. The history of the disease,
its rapid extension in infected surgical wards, the extensive slough-
ing which occurs within a few hours in previously healthy wounds,
and the effect of deep cauterization by the hot iron, nitric acid, or
bromine in arresting the progress of the disease, all support this view
of its etiology. Whether it is due to a specific pathogenic micro-

MODES OF ACTION. 225

organism, or to exceptional pathogenic power acquired by some one
of the common bacteria which infest suppurating wounds, cannot be
determined in the absence of exact experiments by modern methods.
But the latter view has seemed to the writer the most probably cor-
rect. There are many facts which go to show that pathogenic viru-
lence may be increased by cultivation in animal fluids, and where
wounded men are brought together under unfavorable sanitary con-
ditions, as has been the case where hospital gangrene has made its
appearance, it may be that some common saprophyte acquires the
power of invading the exposed tissues instead of simply feeding upon
the secretions which bathe its surface.

Koch has described a progressive tissue necrosis in mice, due to a
streptococcus, which he first obtained by inoculating a mouse in the
ear with putrid material. The morbid process is entirely local and
rapidly progressive, causing a fatal termination in about three days,
without invasion of the blood.

In diphtheritic inflammations of mucous membranes we have
a local invasion of the tissues and a characteristic plastic exudation.
In true diphtheria the local inflammation and necrotic changes in
the invaded tissues are not sufficient to account for the serious gen-
eral symptoms, and we now have experimental evidence that the
diphtheria bacillus produces a very potent toxic substance to which
these symptoms are no doubt largely due. The diphtheria bacillus
of Loffler appears to be the cause of the fatal malady which goes
by this name, but undoubtedly other microorganisms may be con-
cerned in the formation of diphtheritic false membranes. In cer-
tain forms of diphtheria, and especially when it occurs as a com-
plication of scarlet fever, measles, and other diseases, the Klebs-
Loffler bacillus is absent, and a streptococcus, which appears to be
identical with Streptococcus pyogenes, is found in considerable num-
bers and is probably the cause of the diphtheritic inflammation.
An epidemic of diphtheria occurring among calves was studied by
Loffler, and is ascribed by him to his Bacillus diphtherise vitulo-
rum. The same bacteriologist has shown that the diphtheria of
chickens and of pigeons is due to a specific bacillus which differs
from that found in human diphtheria, and which he calls Bacillus
diphtheria columbrarum.

Prof. Welch has studied the histological lesions produced by
filtered cultures of the diphtheria bacillus. Cultures in glycerin-
bouillon, several weeks old, were filtered through porcelain, and the
sterile filtrate was injected beneath the skin of guinea-pigs. One
cubic centimetre of this filtrate was injected into a guinea-pig on
the 10th of December, and two cubic centimetres more on the
14th of the same month. The animal succumbed at the end of
15

226 MODES OF ACTION.

three weeks and five days after the first inoculation. At the autopsy
" the lymphatic glands of the inguinal and axillary regions were
found to be enlarged and reddened; the cervical glands were swollen
and the thyroid gland was greatly congested. There was a consider-
able excess of clear fluid in the peritoneal cavity. Both layers of the
peritoneum were reddened, the vessels of the visceral layer being es-
pecially injected. The spleen was enlarged to double the average
size; it was mottled, and the white follicles were distinctly outlined
against the red ground. The liver was dark in color and contained
much blood. . . . The kidneys were congested and the cut surface
was cloudy. . . . The pericardial sac was distended with clear se-
rum. Under the epicardium were many ecchymotic spots. The
lungs exhibited areas of intense congestion or actual haemorrhage
into the tissues. . . . The histological lesions in this case are identi-
cal with those observed by us in connection with the inoculation of
the living organisms."

To what extent non-specific catarrhal inflammations of mucous
membranes are caused by the local action of microorganisms has
not been determined, but in gonorrhoea the proof is now considered
satisfactory that the " gonococcus " of Neisser is the cause of the
intense local inflammation and purulent discharge. In this disease
the action of the pathogenic microorganism seems to be limited to
the tissues invaded by it, as there is no general systemic disturbance
indicating the absorption of a toxic ptomaine.

Chronic catarrhal inflammations appear, in some cases at least,
to be kept up by the presence of microorganisms, which are always
found in the discharges from inflamed mucous surfaces.

The influence of microorganisms, and especially of the pus cocci,
in preventing the prompt healing of wounds, is now well established.
An extensive suppurating wound or collection of pus, especially if
putrefactive bacteria are present, causes fever and nervous symp
toms, due to the absorption of toxic products. More intense general
symptoms result from the presence of the streptococcus of pus than
from the less pathogenic staphylococci ; this is seen in erysipelatous
inflammations and in puerperal metritis due to the presence of this
micrococcus. Like the other pus cocci, the Streptococcus pyogenes
does not usually invade the blood, but when introduced into the sub-
cutaneous tissues it induces a local inflammatory process, with a ten-
dency to pus formation, and it invades the neighboring lymph chan-
nels, in which the conditions appear to be especially favorable for its
multiplication.

Finally, certain pathogenic bacteria, when introduced into the
bodies of susceptible animals, quickly invade the blood and multiply
in it. In so doing they necessarily interfere with its physiological

MODES OF ACTION. 227

functions by appropriating for their own use material required for
the nutrition of the tissues ; and at the same time toxic substances
are formed which play an important part in the production of the
morbid phenomena, which in this class of diseases very commonly
lead to a fatal result. The pathogenic bacteria which invade the
blood may also, in certain cases, give rise to local necrosis and dis-
turbance of function in various organs in a mechanical way by
blocking up the capillaries.

The invasion of the blood which occurs in anthrax and in vari-
ous forms of septicaemia in the lower animals, induced by subcuta-
neous inoculation with pure cultures of certain pathogenic bacteria,
does not generally immediately follow the inoculation. Usually a
considerable local development first occurs, which gives rise to more
or less inflammation of the invaded tissues, and very commonly to
an effusion of bloody serum in which the pathogenic microorganism
is found in great numbers. Even in susceptible animals the blood
seems to offer a certain resistance to invasion, which is overcome
after a time by the vast number of the parasitic host located in the
vicinity of the point of inoculation, aided probably by the toxic sub-
stances developed as a result of their vital activity.

The experiments of Cheyne (1886) seem to show that in the case
of very pathogenic species, like the anthrax bacillus or Koch's bacil-
lus of mouse septicaemia, a single bacillus introduced subcutaneously
may produce a fatal result in the most susceptible animals, while
greater numbers are required in those which are less susceptible.
Thus a guinea-pig succumbed to general infection after being inocu-
lated subcutaneously with anthrax blood diluted to such an extent
that, by estimation, only one bacillus was present in the fluid in-
jected ; and a similar result in mice was obtained with Bacillus
murisepticus. In the case of the microbe of fowl cholera (Bacillus
septicaemias haemorrhagicae) Cheyne found that for rabbits the fatal
dose is 300,000 or more, that from 10,000 to 300,000 cause a local
abscess, and that less than 10,000 produce no appreciable effect.
The common saprophyte Proteus vulgaris was found to be patho-
genic for rabbits when injected into the dorsal muscles hi sufficient
numbers. But, according to the estimates made, 225,000,000 were
required to cause death, while with doses of from 9,000,000 to 112,-
000,000 a local abscess was produced, and less than 9,000,000 gave
an entirely negative result.

Secondary infections occurring in the course of specific infec-
tious diseases are of common occurrence. Thus a pneumonia may
be developed in the course of an attack of measles or of typhoid
fever ; or infection by the common pus cocci in the course of scarlet
fever, typhoid fever, mumps, etc., may give rise to local abscesses,

228 MODES OF ACTION.

to endocarditis, etc. Again, mixed infection ma}' be induced by
injecting simultaneously into susceptible animals two species of path-
ogenic bacteria.

Bumm, Bockhart, and others have reported cases of mixed gonor-
rhceal infection in which the pyogenic micrococci gave rise to ab-
scesses in the glands of Bartholin, to cystitis, parametritis, or to
" gonorrhceal inflammation " of the knee joint. Babes gives numer-
ous examples of mixed infection in scarlet fever and in other diseases
of childhood. Anton and Fiitterer have studied the question of
secondary infection in typhoid fever. Karlinski has reported a case
of secondary infection with anthrax in a case of typhoid fever, infec-
tion occurring by way of the intestine. Many other examples of
secondary or mixed infection are recorded in the recent literature of
bacteriology and clinical medicine, but enough has been said to call
attention to the importance of the subject.

The researches of Romer, Kanthack (1892), and others show that
the injection of the filtered products of certain bacteria (Bacillus
pyocyaneus, Vibrio Metchnikovi, etc.) produces a decided leucocy-
tosis in the animals experimented upon. And a similar result, prob-
ably from a like cause, has been shown by recent experiments to
occur in pneumonia (Billings) and other infectious diseases.

Certain bacterial products have been shown by experiment to pro-
duce fever when injected into the circulation or beneath the skin of
lower animals ; others produce rapid respiration, dilatation of pupils,
diarrhoea, and paralysis or convulsions (typhotoxin of Brieger,
methyl-guanidin, etc.) ; the toxic effects of some are immediate and
of others more or less remote (toxalbumin of diphtheria) ; others have
a primary toxic effect which is followed after a time by toxic symp-
toms of a different order (Pneumobacillus liquefaciens bovis).

II.

CHANNELS OF INFECTION.

WE have abundant evidence that susceptible animals may be in-
fected by the injection of various pathogenic bacteria beneath the
skin, and accidental infection through an open tvound or abrasion
of the skin is the common mode of infection in tetanus, erysipelas,
hospital gangrene, and the " traumatic infectious diseases" generally.
Other infectious diseases, like anthrax and glanders, are frequently
transmitted in the same way. We have also satisfactory evidence
that tuberculosis may be transmitted to man by the accidental inocu-
lation of an open wound ; and in view of the fact that susceptible
animals are readily infected in this way, it would be strange if it
were otherwise.

The question whether infection may occur through the unbroken
skin has been studied by several bacteriologists and an affirmative
result obtained. Thus Schimmelbusch produced pustules upon the
thigh in two young persons suffering from pyaemia by rubbing upon
the surface a pure culture of Staphylococcus pyogenes aureus which
he had obtained from the pus of a furuncle. The same author also
succeeded in infecting rabbits and guinea-pigs with anthrax, and
rabbits with rabbit septicaemia, by rubbing pure cultures upon
the uninjured skin. Similar results had previously been reported
by Roth, who also showed that infection might occur through
the uninjured mucous membrane of the nose. Machnoff also suc-
ceeded in infecting guinea-pigs with anthrax through the unin-
jured skin of the back, and, as a result of subsequent microscop
ical examination of stained sections, arrived at the conclusion that
the principal channel through which infection was accomplished was
the hair follicles. Braunschweig, in a series of experiments in which
he introduced various pathogenic bacteria into the conjunctival sac
of mice, rabbits, and guinea-pigs, obtained a negative result with the
anthrax bacillus, the bacillus of mouse septicaemia, the bacillus of
chicken cholera, and Micrococcus tetragenus; but the bacillus ob-
tained by Bibbert from the intestinal diphtheria of rabbits gave a
positive result in five mice, two guinea-pigs, and a rabbit.

230 CHANNELS OF INFECTION.

Infection through the mucous membrane of the intestine no
doubt occurs in certain diseases. This is believed to be a common
mode of the infection of sheep and cattle with anthrax, and probably
also in the infectious disease of swine known as hog cholera. The
anthrax bacillus would be destroyed by the acid secretions of the
stomach, but if spores are present in food ingested they will reach
the intestine. The experiments of Korkunoff do not, however, sup-
port the view that infection is likely to occur in this way. In a series
of experiments upon white mice fed with bread containing a quantity
of anthrax spores the result was uniformly negative, but exception-
ally infection occurred in rabbits. The same author obtained posi-
tive results in rabbits fed with food to which a pure culture of the
bacillus of chicken cholera had been added.

Buchner, in experiments upon mice and guinea-pigs fed with
material containing anthrax spores, obtained a positive result in four
out of thirty-three animals. This is no doubt the usual mode of in-
fection in typhoid fever in man.

Infection may also occur through the mucous membrane of the
respiratory organs. This has been demonstrated by several bac-
teriologists, and especially by the experiments of Buchner, who
mixed dried anthrax spores with ly cop odium powder or pulverized
charcoal, and caused mice and guinea-pigs to respire an atmosphere
containing this powder in suspension. In a series of sixty- six experi-
ments fifty animals died of anthrax, nine of pneumonia, and seven
survived. That infection did not occur through the mucous mem-
brane of the alimentary canal was proved by comparative experi-
ments in which animals were fed with double the quantity of spores
used in the inhalation experiments. Out of thirty- three animals fed
in this way but four contracted anthrax. That infection occurred
through the lungs was also demonstrated by the microscopical ex-
amination of sections and by culture experiments, which showed that
the lungs were extensively invaded, while in many cases the spleen
contained no bacilli. Positive results were also obtained with cul-
tures of the anthrax bacillus not containing spores, which the ani-
mals were made to inhale in the form of spray. But in this case a
considerable quantity was required, and a sero-fibrinous pneumonia
was usually produced as well as general infection; the inhalation of
small quantities gave no result. Positive results in rabbits were also
obtained by causing them to inhale considerable quantities of a spray
containing the bacillus of chicken cholera.

The fact that large quantities of a liquid culture of these virulent
bacilli were required to infect very susceptible animals by way of
the pulmonary mucous membrane, and that Buchner failed to cause
the infection of these animals with small quantities of a pure culture

CHANNELS OF INFECTION. 231

inhaled in the form of spray, indicates that this is not a common
mode of infection in the absence of spores. This view receives
further support from the experiments of Hildebrandt, who made
tracheal fistulse in three rabbits, and, after the wound had entirely
healed, injected into the trachea of each a pure culture of the anthrax
bacillus, which was proved to be virulent by inoculation in mice or
guinea-pigs. All of the animals remained in good health. On the
other hand, three rabbits which received in the same way a pure cul-
ture of the bacillus of rabbit septicaemia died as a result of general
infection.

That man may be infected with anthrax by way of the respira-
tory organs seems to be well established. In England the disease
known as "wool-sorter's disease" results from infection in this way
among workmen engaged in sorting wool, which is liable to contain
the spores of the anthrax bacillus when obtained from the skin of an
animal which has fallen a victim to this disease. That infection
occurs through the lungs is shown by the fact that these organs are
first involved, the disease being, in fact, a pulmonic anthrax.

While these experiments prove the possibility of infection through
the respiratory mucous membrane, other experiments made by Hil-
debrandt show that under ordinary circumstances bacteria suspended
in the air do not reach the trachea in rabbits, but are deposited upon
the mucous membrane of the mouth, nares, and fauces. In healthy
rabbits the tracheal mucus was, as a rule, found to be free from bac-
teria, while they were very numerous in mucus obtained from the
mouth or nares. But when a rabbit was made to inhale for half an
hour an atmosphere charged with the spores of Aspergillus f umigatus
their presence in the lungs was demonstrated by cultivation, the ani-
mal being killed for the purpose half an hour after the inhalation
experiment.

The rapidity with which infection may occur is shown by the
experiments of Nissen, Pfuhl, and others. In mice inoculated with
anthrax bacilli at the tip of the tail fatal anthrax has resulted,
although the tail was amputated ten minutes after the inoculation.
Schimmelbusch inoculated fresh wounds with anthrax cultures (in
mice) and immediately after treated the wounds with strong anti-
septic solutions, but the animals succumbed to infection. Cultures
of the anthrax bacillus have been obtained from the liver, spleen, and
kidneys half an hour after the infection of an open wound on the
surface of the body (Schimmelbnsch and Ricker). The experiments
of Sherrington and others show that pathogenic bacteria may escape
by way of the kidneys into the bladder, or through the liver into the
gall bladder. But his experiments indicate that such escape does not
occur through healthy organs. Non-pathogenic bacteria injected

232 CHANNELS OF INFECTION.

into the circulation were not found in the urine, and when a consid-
erable quantity of a pathogenic species was injected into a vein there
was no immediate appearance of bacteria in the urine, but they were
found later, probably as a result of lesions in the secreting organ due
to their local action or to that of their toxic products. In man the
presence of pathogenic bacteria in the urine has been frequently veri-
fied, especially in typhoid fever, pneumonia, and streptococcus in-
fection. When, as a result of the establishment of foci of infection
in the liver, localized necrosis of tissue 'occurs, the pathogenic bac-
teria to which the infection is due escape with the bile and enter
the intestine. It is probable that escape through the walls of the
intestine does not occur unless there is a local lesion of some kind, as
in typhoid fever.

The presence of tubercle bacilli in the milk of cows has been
repeatedly demonstrated, and in a certain proportion of the cases
they have been found in the milk of cows whose udders gave
no evidence of being the seat of a tubercular process. Usually, how-
ever, when tubercle bacilli are found in the milk the cow's udder
is already involved in the disease. The milk of women with puer-
peral fever has been found to contain streptococci ; and in mastitis
from a localized infection by pyogenic cocci these are found in the
milk. It must be remembered, however, that both Staphylococcus
albus and aureus have been found in the milk of healthy women.
The micrococcus of pneumonia has been found in the milk of women
suffering from croupous pneumonia (Foa, and Bordoni-Uffreduzzi) .
Various observers (Brunner, Tizzoni, von Eiselsberg) have reported
the presence of pus cocci in the sweat of patients suffering from sep-
tica3mia, and the experiments of Brunner indicate that they may have
escaped through the sweat glands. This, however, does not appear
to be definitely established.

III.

SUSCEPTIBILITY AND IMMUNITY.

No questions in general biology are more interesting, or more
important from a practical point of view, than those which relate to
the susceptibility of certain animals to the pathogenic action of cer-
tain species of bacteria, and the immunity, natural or acquired, from
such pathogenic action which is possessed by other animals. It has
long been known that certain infectious diseases, now demonstrated
to be of bacterial origin, prevail only or principally among animals
of a single species. Thus typhoid fever, cholera, and relapsing
fever are diseases of man, and the lower animals do not suffer from
them when they are prevailing as an epidemic. On the other hand,
man has a natural immunity from many of the infectious diseases of
the lower animals, and diseases of this class which prevail among
animals are frequently limited to a single species. Again, several
species, including man, may be susceptible to a disease, while other
animals have a natural immunity from it. Thus tuberculosis is
common to man, to cattle, to apes, and to the small herbivorous ani-
mals, while the carnivora are, as a rule, immune ; anthrax may be
communicated by inoculation to man, to cattle, to sheep, to guinea-
pigs, rabbits, and mice, but the rat, the dog, carnivorous animals, and
birds are generally immune ; glanders, which is essentially a disease
of the equine genus, may be communicated to man, to the guinea-
pig, and to field mice, while house mice, rabbits, cattle, and swine
are to a great extent immune.

In addition to this general race immunity or susceptibility we
have individual differences in susceptibility or resistance to the ac-
tion of pathogenic bacteria, which may be either natural or acquired.
As a rule, young animals are more susceptible than older ones.
Thus in man the young are especially susceptible to scarlet fever,
whooping cough, and other "children's diseases," and after forty
years of age the susceptibility to tubercular infection is very much
diminished. Among the lower animals it is a matter of common
laboratory experience that the very young of a susceptible species
may be infected when inoculated with an "attenuated culture"
which older animals of the same species are able to resist.

234 SUSCEPTIBILITY AMD IMMUNITY.

Considerable differences as to susceptibility may also exist among
adults of the same species. In man these differences in individual
susceptibility to infectious diseases are frequently manifested. Of a
number of persons exposed to infection in the same way, some may
escape entirely while others have attacks differing in severity and
duration. In our experiments upon the lower animals we constantly
meet with similar results, some individuals proving to be exception-
ally resistant. Exceptional susceptibility or immunity may be to
some extent a family characteristic or one of race. Thus the negro
race is decidedly less subject to yellow fever than the white race,
and this disease is more fatal among the fair-skinned races of the
north of Europe than among the Latin races living in tropical or sub-
tropical regions. On the other hand, small-pox appears to be excep-
tionally fatal among negroes and dark-skinned races generally.

A very remarkable instance of race immunity is that of Algerian
sheep against anthrax, a disease which is very fatal to other sheep.

In the instances mentioned race immunity is probably an ac-
quired tolerance due to natural selection and inheritance. If, for
example, a susceptible population is exposed to the ravages of small-
pox, the least susceptible individuals will survive and may be the pa-
rents of children who will be likely to inherit the special bodily char-
acters upon which this comparative immunity depends. The ten-
dency of continuous or repeated exposure to the same pathogenic
agent will evidently be to establish a race tolerance ; and there is
reason to believe that such has been the effect in the case of some
of the more common infectious diseases of man, which have been
noticed to prevail with especial severity when first introduced among
a virgin population, as in the islands of the Pacific, etc.

In the same way we may explain the immunity which carnivor-
ous animals have for anthrax and various forms of septicaemia to
which the herbivora are very susceptible when the pathogenic germ
is introduced into their bodies by inoculation. From time immemo-
rial the carnivora have been in the habit of fighting over the dead
bodies of herbivorous animals, some of which may have fallen a prey
to these infectious germ diseases, and in their fighting they receive
wounds, inoculated with the infectious material from these bodies,
which would be fatal to a susceptible animal. If at any time in the
past a similar susceptibility existed among the carnivora, with indi-
vidual differences as to resisting power, it is evident that there would
be a constant tendency for the most susceptible individuals to perish
and for the least susceptible to survive.

But if we admit this to be a probable explanation of the immu-
nity of carnivorous animals from septic infection, we have not yet
explained the precise reason for the immunity enjoyed by the

SUSCEPTIBILITY AXD IMMUNITY. 235

selected individuals and their progeny. The essential difference be-
tween a susceptible and immune animal depends upon the fact that
in one the pathogenic germ, when introduced by accident or ex-
perimental inoculation, multiplies and invades the tissues or the
blood, where, by reason of its nutritive requirements and toxic pro-
ducts, it produces changes in the tissues arid fluids of the body incon-
sistent with the vital requirements of the infected animal ; while in
the immune animal multiplication does not occur or is restricted to a
local invasion of limited extent, and in which after a time the re-
sources of nature suffice to destroy the parasitic invader.

Now the question is, upon what does this essential difference de-
pend ? Evidently upon conditions favorable or unfavorable to the
development of the pathogenic germ ; or upon its destruction by
some active agent present in the tissues or fluids of the body of the
immune animal; or upon a neutralization of its toxic products by some
substance present in the body of the animal which survives infec-
tion.

What, then, are the unfavorable conditions which may be supposed
to prevent development in immune animals ? In the first place, the
temperature of the body may not be favorable. Certain pathogenic
bacteria are only able to develop within very narrow temperature lim-
its, and, if all other conditions were favorable, could not be expected
to multiply in the bodies of cold-blooded animals. Or the temperature
of warm-blooded animals, and especially of fowls, may be above the
point favorable for their development. This is the explanation
offered by Pasteur of the immunity of fowls, which are usually re-
fractory against anthrax ; and in support of this view he showed by
experiment that when chickens are refrigerated after inoculation, by
being partly immersed in cold water, they are liable to become in-
fected and to perish. But, as pointed out by Koch, the sparrow,
which has a temperature as high as that of the chicken, may con-
tract anthrax without being refrigerated. We must not, therefore,
too hastily conclude that the success in Pasteur's experiment de-
pended alone upon a reduction of the body heat. Gibier has shown
that the anthrax bacillus may multiply in the bodies of frogs or
fish, if these are kept in water having a temperature of 35° C.
But the anthrax bacillus grows within comparatively wide tempera-
ture limits, while other pathogenic bacteria are known to have a
more restricted temperature range and would be more decidedly
influenced by this factor — e.g., the tubercle bacillus.

The composition of the body fluids, and especially their reaction,
is probably a determining factor in some instances. Thus Behring
has ascribed the failure of the anthrax bacillus to develop in the
white rat, which possesses a remarkable immunity against anthrax,

236 SUSCEPTIBILITY AND IMMUNITY.

to the highly alkaline reaction of the blood and tissue juices of this
animal. Behring claims to have obtained experimental proof of the
truth of this explanation by feeding white rats on an exclusively
vegetable diet or by adding acid phosphate of lime to their food, by
which means this excessive alkalinity of the blood is diminished.
Rats so treated are said to lose their natural immunity, and to die as
a result of inoculation with virulent cultures of the anthrax bacillus.

The experiments of Nuttall, Behring, Buchner, and others have
established the fact that recently dratvn blood of various animals
possesses decided germicidal power, and Buchner has shown that
this property belongs to the fluid part of the blood and not to its
cellular elements. It has also been shown that aqueous humor, the
fluid of ascites, and lymph from the dorsal lymph sac of a frog
possess the same power. This power to kill bacteria is destroyed by
heat, and is lost when the blood has been kept for a considerable
time, but it is not neutralized by freezing. Further, this power to
destroy bacteria differs greatly for different species, being very de-
cided in the case of certain pathogenic bacteria, less so for others,
and absent in the case of certain common saprophytes. Behring
has also shown that the blood of different animals differs consider-
ably in this regard, and that the blood of the rat and of the frog,
which animals have a natural immunity against anthrax, is espe-
cially fatal to the anthrax bacillus. The experiments made show
that this germicidal power is very prompt in its action, but that it is
limited as to the number of bacteria which can be destroyed by a
given quantity of blood serum. When the number is excessive, de-
velopment occurs after an interval during which a limited destruc-
tion has taken place. It would appear that the element in the blood
to which this germicidal action is due is neutralized in exercising
this power ; and as, independently of this, blood serum is an excel-
lent culture medium for bacteria, an abundant development takes
place when the destruction has been incomplete.

Buchner (1889) first proved by experiment that the germicidal
power of the blood of dogs and rabbits does not depend upon the
presence of the cellular elements, but is present in clear serum which
has been allowed to separate from the clot in a cool place. Exposure
for an hour to a temperature of 55° C. destroys the germicidal action
of serum as well as of blood.

The researches of Buchner, of Hankin, and others, show that this
germicidal power of fresh blood serum depends upon the presence of
proteids, to which the first-named bacteriologist has given the name
of "alexins." Hankin, in his paper upon the origin of these "defen-
sive proteids" in the animal body (1892), arrives at the conclusion

SUSCEPTIBILITY AND IMMUNITY. 237

that while they are present in the cell-free serum they are the prod-
uct of certain leucocytes — Ehrlich's eosinophile cells. He believes
that the eosinophile granules become dissolved in the serum and con-
stitute the germicidal proteid which is shown to be present by ex-
periments upon bacteria. According to Hankin the separation of
these granules can be witnessed under the microscope. They first
accumulate upon one side of the cell and then gradually disappear,
and as this occurs a considerable increase in the bactericidal power
of the serum can be demonstrated. The germicidal power of the
blood serum is also said to be increased when the number of leuco-
cytes is considerably augmented, as occurs when a sterilized culture
of Vibrio Metschnikovi is injected subcutaneously. Also by treat-
ment which favors a separation of the alexin from the leucocytes,
i.e., a solution of the eosinophile granules. This may be accom-
plished by the injection of an extract of the thymus gland of the
calf, or by simply allowing the drawn blood to stand for several hours
at a temperature of 38° to 40° C.

Buchner's latest communication upon the subject shows that he
also attributes the origin of the germicidal proteid in fresh blood
serum to the leucocytes. In his paper on "Immunity," read at the
Eighth International Congress on Hygiene and Demography (Buda-
pest, 1894), he calls attention in the first place to the fact that a
clearly marked distinction must be made between natural immunity
and acquired immunity, inasmuch as the " alexins " and " antitoxins "
have very different properties. The first-mentioned proteid s are de-
stroyed by a comparatively low temperature (55° to 60° C.), while the
antitoxins resist a considerably higher temperature, and, unlike the
alexins, have no bactericidal or globulicidal action. A very remark-
able fact developed in Buchner's experiments is that the blood serum
from the dog and from the rabbit, when mixed, neutralize each other
so far as their germicidal power is concerned.

By injecting sterilized emulsions of wheat-flour paste in the
pleural cavity of rabbits and dogs Buchner succeeded in obtaining an
exudate which had more decided germicidal power than the blood
or serum of the same animal. This was evidently due to the large
number of leucocytes present, but not to their phagocytic action, as
was shown by experiment. By freezing the exudate the leucocytes
were killed, but the germicidal action of the fluid was rather in-
creased than diminished by freezing. While freezing had no effect
upon the germicidal action of the pleural exudate, this was always
neutralized by exposure to a temperature of 55° C.

Emmerich, Tsuboi, Steinmetz, and Low (1892), as a result of ex-
tended experiments, arrived at the conclusion that the germicidal
action of blood serum " depends upon a specific property of the alkali

238 SUSCEPTIBILITY AND IMMUNITY.

serumalbumin, and that it is a purely chemical process." They
state that when the germicidal power is neutralized by heat it may
be restored by the addition of an alkali. Buclmer repeated the ex-
periments of Emmerich and his associates and obtained similar re-
sults, but interprets them differently. According to him the serum
does not regain its germicidal power, but after the addition of an
alkali and subsequent dialyzing the nutritive value of the serum is so
diminished that the bacteria do not develop in it.

Pane (1892) has made experiments which give additional weight
to the assumption that the alkalinity of the blood is an important
factor in accounting for immunity. He states that carbonate of
soda, dissolved in water, in the proportion of 1:3,000, has a de-
cided germicidal action upon the anthrax bacillus, equal to that of
the blood serum of the rabbit. And that when rabbit serum is com-
pletely neutralized it no longer has any injurious action on anthrax
bacilli.

Zagari and Innocente (1892) also arrived at the conclusion that
the diminished resistance to anthrax infection resulting from curare
poisoning in frogs, and from chloral or alcohol in dogs (Platania), in
fowls as a result of starvation (Canalis and Morpurgo), in white
mice as a result of fatigue (Charin and Roger) , is, in fact, due to
diminished alkalinity of the blood, which they found to correspond
with the increased susceptibility resulting from the causes men-
tioned.

Buchner (1892) states that several of the ammonium salts, and
especially ammonium sulphate, cause an increase in the germicidal
action of blood serum, and also increase its resistance to the neutral-
izing effects of heat. The experiments of Pansini and Calabrese
(1894) show, on the contrary, that the addition of uric acid to blood
serum diminishes its bactericidal activity, as does also the presence
of glucose. That certain infectious diseases are especially virulent
in persons suffering from diabetes is a frequently repeated clinical
observation.

Van Fodor has shown by experiment that the injection of an
alkali into the circulation of a rabbit increases its resistance to
anthrax infection and the germicidal activity of its blood serum.
The same bacteriologist has found that when a rabbit is infected
with anthrax, the alkalinity of its blood is notably increased during
the first twenty-four hours, when we may suppose that the powers
of nature are brought to bear to resist the invading parasite, and that
after this time it rapidly diminishes. Ten hours after infection (by
subcutaneous inoculation?) the alkalinity of the blood had increased
21.5 per cent. Shortly before the death of the animal a diminution
of 26.3 per cent was noted. This diminution was observed in thirty-

SUSCEPTIBILITY AND IMMUNITY. 239

four" out of thirty-nine animals experimented upon, and these ani-
mals succumbed to the anthrax infection in a shorter time than did
the other five in which there was no such diminution.

It seems probable that the germicidal property of freshly drawn
blood serum is not due to its alkalinity, per se, but to the fact that
the germicidal constituent is only soluble in an alkaline fluid. The
researches of Vaughn, McClintock, and Novy indicate that this ger-
micidal constituent is a nuclein. Dr. Vaughn, in his last published
paper upon "Nucleins and Nuclein Therapy," says: "Kossel, of
Berlin, has confirmed our statements concerning the germicidal
action of the nucleins. Dr. McClintock and I have also demon-
strated that the germicidal constituent of blood serum is a nuclein.
This nuclein is undoubtedly furnished by the polynuclear white
corpuscles." Denys has (1894) reported the results of experi-
ments made in his laboratory by Van der Velde, which give sup-
port to the conclusion reached by Vaughn. In these experiments a
sterilized culture of staphylococci was injected into the pleural cavity
of rabbits in order to obtain an exudate. At intervals of two hours
this exudate was obtained by killing one of the animals in the series
experimented upon, and at the same time blood from the animal was
secured. Both the exudate and the blood were placed in a centrifugal
machine, in order to obtain a serum free from corpuscular elements.
The germicidal activity of the serum was then tested. The general
result of the experiments was to show that the longer the interval
after the injection into the pleural cavity the more potent the ger-
micidal activity of the exudate became, and that there was no corre-
sponding increase in the activity of the blood serum obtained from
the circulation. At the end of ten or twelve hours, the serum from
the exndate killed all of the staphylococci in a bouillon culture twenty
times as great in quantity as the germicidal serum used in the ex-
periment. The absence of any increase in germicidal power in the
blood serum taken from the general circulation shows that the nota-
ble increase manifested by the exudate was due to local causes ; and
as a matter of fact it corresponded with an increase in the number of
leucocytes as found in the pleural exudate.

Thus it will be seen that the independent researches of Hankin,
of Buchner, of Vaughn, and of other competent bacteriologists, have
led them to the same ultimate result so far as the origin of the ger-
micidal constituent of the blood is concerned, and that the leucocytes
appear to play an important role in the protection of the animal body
from invasion by bacteria (natural immunity).

It has been shown by several investigators that the number of
leucocytes increases in certain infectious diseases, and this increase,
together with an increased alkalinity of the blood, which has here-

240 SUSCEPTIBILITY AND IMMUNITY.

tofore been referred to, appears to be a provision of nature for over-
coming the infection which has already occurred.

It has been demonstrated by experiment that naturally immune
animals may be infected by the addition of certain substances to cul-
tures of pathogenic bacteria. Thus Arloing was able to induce symp-
tomatic anthrax in animals naturally immune for this disease by
mixing with his cultures various chemical substances, such as car-
bolic acid, pyrogallic acid, and especially lactic acid (twenty per
cent). Leo has shown that white mice, which are not subject to
the pathogenic action of the glanders bacillus, may be rendered sus-
ceptible by feeding them for some time upon phloridzin, which gives
rise to an artificial diabetes, and causes the tissues to become im-
pregnated with sugar.

Bouchard has found that very small doses of a pure culture of
Bacillus pyocyaneus are fatal to rabbits when at the same time a
considerable quantity of a filtered culture of the same bacillus is in-
jected into a vein. The animal could have withstood the filtered
culture alone, or the bacillus injected beneath its skin ; but its resist-
ing power — natural immunity — is overcome by the combined action
of the living bacilli and the toxic substances contained in the filtered
culture. The same result may be obtained by injecting sterilized
cultures of a different microorganism. Thus Roger has shown that
the rabbit, which has a natural immunity against symptomatic
anthrax, succumbs to infection when inoculated with a culture of the
bacillus of this disease, if at the same time it receives an injection of
a sterilized or non-sterilized culture of Bacillus prodigiosus. Monti
has succeeded in killing animals with old and attenuated cultures of
Streptococcus pyogenes, or of Staphylococcus pyogenes aureus, by in-
jecting at the same time a culture of Proteus vulgaris. In a similar
way, it seems probable, the normal resistance of man to infection by
certain pathogenic bacteria may be overcome. Thus when water
contaminated by the presence of the typhoid bacillus is used for
drinking by the residents of a certain town or district, not all of
those who in this way are exposed to infection contract typhoid
fever; and among those who do, there is good reason to believe that,
in certain cases at least, the result depends upon an additional factor
of the kind suggested by the above-mentioned experiments — e.g., the
consumption of food containing putrefactive products, or the respi-
ration of an atmosphere containing volatile products of putrefaction.

The natural immunity of healthy animals may also be neutralized
by other agencies which have a depressing effect upon the vital re-
sisting power. Thus Nocard and Roux found by experiment that an
attenuated culture of the anthrax bacillus, which was not fatal to
guinea-pigs, killed these animals when injected into the muscles of

SUSCEPTIBILITY AND IMMUNITY. 241

the thigh after they had been bruised by mechanical violence.
Abarrin and Roger found that white rats, which are not susceptible
to anthrax, became infected and frequently died if they were ex-
hausted, previous to inoculation, by being compelled to turn a revolv-
ing wheel for a considerable time. Pasteur found that fowls, which
have a natural immunity against anthrax, become infected and
perish if they are subjected to artificial refrigeration after inocula-
tion. This has been, confirmed by the more recent experiments of
Wagner (1891). According to Canalis and Morpurgo, pigeons
which are enfeebled by inanition eaily contract anthrax as a result
of inoculation. Arloing states that sheep which have been freely
bled contract anthrax more easily than others; and Serafini found
that when dogs were freely bled the bacillus of Friedlander, injected
into the trachea or the pleural cavity, entered, and apparently mul-
tiplied to some extent in the blood, whereas without such previous
bleeding they were not to be found in the circulating fluid. Certain
anesthetic agents have been shown also to produce a similar result.
Platariia communicated anthrax to immune animals — dogs, frogs,
pigeons — by bringing them under the influence of curare, chloral, or
alcohol; and Wagner obtained similar results in his experiments
upon pigeons to which he had administered chloral. In man, clini-
cal experience shows that those who are addicted to the excessive use
of alcohol are especially liable to contract certain infectious diseases
— pneumonia, eiysipelas, yellow fever, etc.

The micrococcus of pneumonia is habitually present in the sali-
vary secretions of many healthy individuals, and it is evident that
an attack of pneumonia does not depend alone upon the presence of
this micrococcus, which has, nevertheless, been conclusively shown
to be the usual infectious agent in cases of croupous pneumonia. No
doubt the introduction of the pathogenic micrococcus to the vulner-
able point — the lungs — is an essential factor in the development of a
case of pneumonia, but there is reason to believe that there are other
factors equally essential. Thus it is well known that an attack of
pneumonia often results from exposure to cold, which may act as an
exciting cause; and, also, that a recent attack of an acute febrile
disease — especially measles — constitutes a predisposing cause. It is
generally recognized that malnutrition, want of exercise,, insanitary
surroundings, and continued respiration of an atmosphere loaded
with dust, as in cotton mills, or a recent attack of pneumonia, con-
stitute predisposing causes to tubercular infection by way of the
lungs.

While natural immunity may be overcome by the various depress-
ing agencies referred to, it is also true that it has only a relative
value in the absence of these predisposing causes, and may be over-
16

242 SUSCEPTIBILITY AND IMMUNITY.

come by unusual virulence of the pathogenic infectious agent, or by
the introduction into the body of an excessive amount of a pure cul-
ture of the same.

The pathogenic potency of known disease germs varies as widely
as does the susceptibility of individuals to their specific action. In
general it may be said that the more recently the germ comes from
a developed case of the disease to which it gives rise the more viru-
lent it is, and the longer it has been cultivated outside of the animal
body the more attenuated is its pathogenic power. Thus when the
discharges of a typhoid fever patient find their way directly to a
water-supply of limited amount a large proportion of those who
drink the water are likely to be attacked ; but when a considerable
interval of time has elapsed since the contamination occurred,
although the germs may still be present, the liability to attack is
much less on account of diminished pathogenic virulence.

The development of an attack also depends, to some extent, upon
the number of germs introduced into a susceptible individual at one
time. The resources of nature may be sufficient to dispose of a few
bacilli, while a large number may overwhelm the resisting power of
the individual.

The experiments of Cheyne (1886) show that in the case of very
pathogenic species a single bacillus, or at least a very small number,
introduced beneath the skin, may produce fatal infection in a very
susceptible animal, while greater numbers are required in those less
susceptible. Thus a guinea-pig succumbed to general infection after
being inoculated subcutaneously with anthrax blood diluted to such
an extent that, by estimation, only one bacillus was present in the
fluid injected ; and a similar result was obtained in mice with Bacillus
murisepticus. In the case of the microbe of fowl cholera (Bacillus
septicaemia hemorrhagica}) , Cheyne found that for rabbits the fatal
dose was 300,000 or more, that from 100,000 to 30,000 cause a local
abscess, and that less than 10,000 produce no appreciable effect. The
common saprophyte, Proteus vulgaris, was found to be pathogenic
for rabbits when injected into the dorsal muscles in sufficient num-
bers. But, according to the estimates made, 225,000,000 were re-
quired to cause death, while doses of from 9,000,000 to 112,000,000
produced a local abscess, and less than 9,000,000 gave an entirely
negative result.

ACQUIRED IMMUNITY.

It has long been known that, in a considerable number of infec-
tious diseases, a single attack, however mild, affords protection
against subsequent attacks of the same disease; that in some cases
this protection appears to be permanent, lasting during the life of the

SUSCEPTIBILITY AND IMMUNITY. 243

individual; that in others it is more or less temporary, as shown by
the occurrence of a subsequent attack.

The protection afforded by a single attack not only differs in dif-
ferent diseases, but in the same disease varies greatly in different
individuals. Thus certain individuals have been known to suffer
several attacks of small-pox or of scarlet fever, although, as a rule, a
single attack is protective. Exceptional susceptibility or insuscepti-
bility may be not only an individual but a family characteristic, or
it may belong to a particular race.

In those diseases in which second attacks are not infrequent, as,
for example, in pneumonia, in influenza, or in Asiatic cholera, it is
difficult to judge from clinical experience whether a first attack exerts
any protective influence. But from experiments upon the lower ani-
mals we are led to believe that a certain degree of immunity, lasting
for a longer or shorter time, is afforded by an attack of pneumonia
or of cholera, and probably of all infectious diseases due to bacterial
parasites. In the malarial fevers, which are due to a parasite of a
different class, one attack affords no protection, but rather predis-
poses to a subsequent attack.

In those diseases in which a single attack is generally recognized
as being protective, exceptional cases occur in which subsequent
attacks are developed as a result of unusual susceptibility or expo-
sure under circumstances especially favorable to infection. Maiselis
(1894) has gone through the literature accessible to him for the
purpose of determining the frequency with which second attacks
occur in the various diseases below mentioned. The result is as
follows :

Second Third Fourth „, . ,

Attacks. Attacks. Attacks. M-

Small-pox 505 9 0 514

Scarlet fever 29 4 0 33

Measles 36 1 0 37

Typhoid fever 202 5 1 208

Cholera 29 3 2 34

These figures support the view generally entertained by physi-
cians that second attacks of scarlet fever and of measles are compar-
atively rare, while second attacks of small-pox are not infrequently
observed. Considering the very large number of cases of typhoid
fever which occur annually in all parts of Europe and America, the
number of second attacks collected does not bear a very large propor-
tion to the total number taken sick, although the recorded cases, of
course, fall far short of the total number of second attacks of this
and the other diseases mentioned.

The second attacks of cholera recorded are not numerous, and, no
doubt, a carefullly conducted investigation made in the areas of en-

244 SUSCEPTIBILITY AND IMMUNITY.

demic prevalence of this disease would show that second attacks are
more common than is indicated by these figures.

That immunity may result from a comparatively mild attack as
well as from a severe one is a matter of common observation in the
case of small-pox, scarlet fever, yellow fever, etc. ; and since the dis-
covery of Jenner we have in vaccination a simple method of produc-
ing immunity in the first-mentioned disease. The acquired immunity
resulting from vaccination is not, however, as complete or as per-
manent as that which results from an attack of the disease.

These general facts relating to acquired immunity from infectious
diseases constituted the principal portion of our knowledge with re-
ference to this important matter up to the time that Pasteur (1880)
demonstrated that in the disease of fowls known as chicken cholera,
which he had proved to be due to a specific microorganism, a mild
attack followed by immunity may be induced by inoculation with an
" attenuated virus" — i.e., by inoculation with a culture of the patho-
genic microorganism the virulence of which had been so modified
that it gave rise to a comparatively mild attack of the disease in
question. Pasteur's original method of obtaining an attenuated virus
consisted in exposing his cultures for a considerable time to the ac-
tion of atmospheric oxygen. It has since been ascertained that the
same result is obtained with greater certainty by exposing cultures
for a given time to a temperature slightly below that which would
destroy the vitality of the pathogenic microorganism, and also by ex-
posure to the action of certain chemical agents.

Pasteur at once comprehended the importance of his discovery,
and inferred that what was true, of one infectious germ disease was
likely to be true of others. Subsequent researches, by this savant
and by other bacteriologists, have justified this anticipation, and the
demonstration has already been made for a considerable number of
similar diseases — anthrax, symptomatic anthrax, rouget.

A virus which has been attenuated artificially — by heat, for ex-
ample— may be cultivated through successive generations without re-
gaining its original virulence. As this virulence depends, to a con-
siderable extent at least, upon the formation of toxic products during
the development of the pathogenic microorganism, we naturally infer
that diminished virulence is due to a diminished production of these
toxic substances.

There is reason to believe that a natural attenuation of virulence
may occur in pathogenic bacteria which are able to lead a sapro-
phytic existence during their multiplication external to the bodies of
living animals, and the comparatively mild character of some epi-
demics is probably due to this fact.

SUSCEPTIBILITY AND IMMUNITY. 245

Again, cultivation within the body of a living animal may, in
certain cases, cause a diminution in the virulence of a pathogenic
microorganism. Thus Pasteur and Thuiller have shown that the
microbe of rouget when inoculated into a rabbit kills the animal, but
that its pathogenic virulence is nevertheless so modified that a cul-
ture made from the blood of a rabbit killed by it is a suitable " vac-
cine " for the pig.

On the other hand, we have experimental evidence that the viru-
lence of attenuated cultures may be reestablished by passing them
through the bodies of susceptible animals. Thus a culture of the
bacillus of rouget, attenuated by having been passed through the
body of a rabbit, is restored to its original virulence by passing it
through the bodies of pigeons. And a culture of the anthrax bacillus
which will not kill an adult guinea-pig may be fatal to a very young
animal of the same species or to a mouse, and the bacillus cultivated
from the blood of such an animal will be found to have greatly in-
creased virulence.

In Pasteur's inoculations against anthrax "attenuated" cultures
are employed which contain the living pathogenic germ as well as
the toxic products developed during its growth. Usually two inocu-
lations are made with cultures of different degrees of attenuation—
that is to say, with cultures in which the toxic products are formed
in less amount than in virus of full power. The most attenuated
virus is first injected, and after some time the second vaccine, which
if injected first might have caused a considerable mortality. The
animal is thus protected from the pathogenic action of the most
virulent cultures.

Now, it has been shown by recent experiments that a similar im-
munity may result from the injection into a susceptible animal of the
toxic products contained in a virulent culture, independently of the
living bacteria to which they owe their origin. Chauveau, in 1880,
ascertained that if pregnant ewes are protected against anthrax by
inoculation with an attenuated virus, their lambs, when born, also
give evidence of having acquired an immunity from the disease. As
the investigations of Davaine seemed to show that the anthrax
bacillus cannot pass through the placenta from the mother to the
foetus, the inference seemed justified that the acquired immunity of
the latter was due to some soluble substance which could pass the
placental barrier. More recent researches by Strauss and Chamber-
lain, Malvoz and Jacquet, and others, show that the placenta is not
such an impassable barrier for bacteria as was generally believed at
the time of Chauveau's experiments, so that these cannot be accepted
as establishing the inference referred to. But, as stated, we have
more recent experimental evidence which shows that immunity may

246 SUSCEPTIBILITY AND IMMUNITY.

result from the introduction into the bodies of susceptible animals of
the toxic substances produced by certain pathogenic bacteria. The
first satisfactory experimental evidence of this important fact was
obtained by Salmon and Smith in 1880, who succeeded in making
pigeons immune from the pathogenic effects of cultures of the bacil-
lus of hog cholera by inoculating them with sterilized cultures of
this bacillus. In 1888 Roux reported similar results obtained by in-
jecting into susceptible animals sterilized cultures of the anthrax
bacillus. Behring and Kitasato, in 1890, reported their success in
establishing immunity against virulent cultures of the bacillus of
tetanus and the diphtheria bacillus by inoculating susceptible ani-
mals with filtered, germ-free cultures of these pathogenic bacteria.

In 1892 Behring, Kitasato, and Wassermann published the re-
sults of interesting experiments with a bouillon made from the
thymus gland of the calf. They found that the tetanus bacillus cul-
tivated in this bouillon did not form spores and had comparatively
little virulence. Mice or rabbits inoculated with it in small doses — •
0.001 to 0.2 cubic centimetre for a mouse — proved to be subsequently
immune. And the blood serum of an immune rabbit injected into
the peritoneal cavity of a mouse — 0.1 to 0.5 cubic centimetre — was
found to give it immunity from the pathogenic action of a virulent
culture of the tetanus bacillus. Similar results were obtained with
several other pathogenic bacteria cultivated in the thymus bouillon —
spirillum of cholera, bacillus of diphtheria, typhoid bacillus. We
give here the directions for preparing the thymus bouillon as used by
the authors named :

Two or three thymus glands are chopped into small pieces immediately
after they are taken from the animal. An equal part ot distilled wat^r is
added to the mass and stirred for some time ; it is then placed in an ice chest
for twelve hours. The juices are now expressed through gauze by means of
a flesh press. A clouded, slimy fluid is obtained, which constitutes a stock
solution. This is diluted with water, and a certain quantity of carbonate of
soda is added to the solution before sterilization. By this means coagulation
and precipitation of the active substance from the thymus gland are avoided.
The exact amount of water and of sodium carbonate required to prevent pre-
cipitation must be determined by experiment, as it differs for different glands.
Usually an equal portion of water and sufficient soda solution to turn litmus
paper feebly blue will give the desired result. The liquid is now heated in
a large flask, which is left for fifteen minutes in the steam sterilizer. The
liquid is allowed to cool and then filtered through fine linen to remove any
suspended coagula ; the filtrate has a milky opalescence. It is now placed
in test tubes and again sterilized. The active principle is precipitated by the
addition of a few drops of acetic acid .

In Pasteur's inoculations against hydrophobia, made subsequently
to infection by the bite of a rabid animal, an attenuated virus is in-

SUSCEPTIBILITY AND IMMUNITY. 247

troduced upon the surface of the brain, and immunity is established
during the interval — so-called period of incubation — which usually
occurs between the date of infection and the development of the
disease. That the immunity in this case also depends upon the
introduction of a chemical substance present in the desiccated spinal
cord of rabbits which have succumbed to rabies, which is used in
these inoculations, is extremely probable. But, as the germ of rabies
has not been isolated or cultivated artificially, this has not yet been
demonstrated. Wooldridge claims to have made susceptible animals
immune against anthrax by inoculating them with an aqueous ex-
tract of the testicle or of the thymus gland of healthy animals.

We may mention also the interesting results obtained by Em-
merich, Freudenreich, and others, who have shown that an anthrax
infection in a susceptible animal inoculated with a virulent culture
may be made to take a modified and non-fatal course by the simul-
taneous or subsequent inoculation of certain other non-pathogenic
bacteria — streptococcus of erysipelas, Bacillus pyocyaneus.

In a series of experiments made by the writer some years ago
evidence was obtained that, under certain circumstances, immunity
from the effects of one pathogenic bacillus may be obtained by the
previous injection of a pure culture of a different species. In the
experiments referred to injections into the cavity of the abdomen of
a culture of Bacillus pyocyaneus protected rabbits from the lethal
effects of Bacillus cuniculicida Havaniensis, when subsequently in-
jected into the cavity of the abdomen in such amount (one cubic
centimetre of a bouillon culture) as invariably proved fatal in rabbits
not protected by such injections.

Before considering the theories which have been offered in expla-
nation of acquired immunity it is desirable to call attention to certain
observations which have been made during the past few years relat-
ing to " chemiotaxis. "

The term chemiotaxis was first used by Pfeffer to designate the
property, observed by himself and others, which certain living cells
exhibit with reference to non-living organic material, and by virtue
of which they approach or recede from certain substances. The
chemiotaxis is said to be positive when the living cell approaches, and
negative when it recedes from, a chemical substance. As examples
of this we may mention the approach of motile bacteria to nutrient
material or to the surface of a liquid medium where they find the
oxygen required for their vital activities ; and of leucocytes to cer-
tain substances when these are introduced beneath the skin of warm-
er cold-blooded animals. This subject has recently received much

248 SUSCEPTIBILITY AND IMMUNITY.

attention and has been studied especially by Ali-Cohen, Massart and
Bordet, Gabritchevski, and others.

According to Gabritchevski, the following substances have a neg-
ative chemiotaxis for the leucocytes : Sodium chloride in ten-per-cent
solution, alcohol in ten-per-cent solution, quinine, lactic acid, gly-
cerin, chloroform, bile. On the other hand, a positive chemiotaxis
is excited by sterilized or non-sterilized cultures of various bacteria.
This is shown by the fact that when a small capillary tube, closed at
one end, which contains the substance to be tested, is introduced be-
neath the skin of an animal, the leucocytes are repelled from the tube
by certain substances, while those which incite positive chemiotaxis
cause them to enter the tube in great numbers. The experiments of
Buchner seem to show that the positive chemiotaxis induced by
sterilized cultures of bacteria introduced beneath the skin of an
animal, is due to the proteid contents of the cells rather than to the
chemical products elaborated as a result of their vital activity. But
that such chemical products may, in some instances at least, produce
a positive chemiotaxis independently of the bacteria is shown by
the experiments of Gabritchevski with filtered cultures of Bacillus
pyocyaneus — confirmed by Massart and Bordet.

An important observation made by Bouchard, and confirmed by
Massart and Bordet, is the following : When a tube containing a cul-
ture of Bacillus pyocyaneus is introduced beneath the skin of a rabbit
it is found, at the end of a few hours, to contain a great number of
leucocytes. But if immediately after its introduction ten cubic centi-
metres of a sterilized culture of the same bacillus are injected into the
circulation through a vein, very few leucocytes enter the tube intro-
duced beneath the skin — that is, the chemiotaxis of the leucocytes
for the bacilli contained in the tube has been neutralized by injecting
a considerable quantity of the soluble products of the same bacillus
into the circulation.

Buchner, having shown that the bacterial cells contain a proteid
substance which attracts the leucocytes, experimented with various
other proteids and found that gluten, casein from wheat, and legumin
from peas had a similar effect. Starch has no effect, but a mass of
flour, made from wheat or from peas, introduced beneath the skin of
a rabbit or of a guinea-pig, with antiseptic precautions, in the course
of a day or two is enveloped and penetrated by immense numbers of
leucocytes. If, instead of introducing these substances which induce
positive chemiotaxis beneath the skin, they are injected into the cir-
culation, Buchner has shown that a great increase in the number of
leucocytes occurs.

SUSCEPTIBILITY AND IMMUNITY.
THEORIES OF IMMUNITY.

Exhaustion Theory. — For a time Pasteur supported the view
that during an attack of an infectious disease the pathogenic micro-
organism, in its multiplication in the body of a susceptible animal,
exhausts the supply of some substance necessary for its development,
that this substance is not subsequently reproduced, and that conse-
quently the same pathogenic germ cannot again multiply in the body
of the protected animal. This view is sustained in a memoir pub-
lished in the Comptes Rendus of the French Academy in 1880, in
which Pasteur says :

" It is the life of a parasite in the interior of the body which produces the
malady commonly called ' cholera des ponies,' and which causes death.
From the moment when this culture (i.e., the multiplication of the parasite)
is 110 longer possible in the fowl the sickness cannot appear. The fowls are
then in the constitutional state of fowls not subject to be attacked by the
disease. These last are as if vaccinated from birth for this malady, because
the foetal evolution has not introduced into their bodies the material neces-
sary to support the life of the microbe, or these nutritive materials have
disappeared at an early age.

"Certainly one should not be surprised that there may be constitutions
sometimes susceptible and sometimes rebellious to inoculation — that is to
say, to the cultivation of a certain virus — when, as I have announced in my
first note, one sees a preparation of beer yeast made, exactly like one from
the muscles of fowls (bouillon), to show itself absolutely unsuited for the cul-
tivation of the parasite of fowl cholera, while it is admirably adapted to the
cultivation of a multitude of microscopic species, notably to the bacteride
charbonneuse (Bacillus aiithracis).

"The explanation to which these facts conduct us, as well of the consti-
tutional resistance of some individuals as of the immunity produced by
protective inoculations, is only natural when we consider that every culture,
in general, modifies the medium in which it is effected — a modification of
the soil when it relates to ordinary plants ; a modification of plants and ani-
mals when it relates to their parasites ; a modification of our culture liquids
when it relates to mucedines, vibrioniens, or ferments.

"These modifications are manifested and characterized by the circum-
stance that new cultivations of the same species in these media become
promptly difficult or impossible. If we sow chicken bouillon with the mi-
crobe of fowl cholera, and, after three or four days, filter the liquid in order
to remove all trace of the microbe, and subsequently sow anew in the fil-
tered liquid this parasite, it will be found quite powerless to resume th^ most
feeble development. The liquid, which is perfectly limpid after being fil-
tered, retains its limpidity indefinitely.

"How can we fail to believe that by cultivation in the fowl of the atten-
uated virus we place its body in the state of this filtered liquid which can
110 longer cultivate the microbe ? The comparison can be pushed still
further; for if we filter the bouillon containing the microbe in full develop-
ment, not on the fourth day of culture, but on the second, the filtered liquid
will still be able to support the development of the microbe, although with
less energy than at the outset. We comprehend, then, that after a cultiva-
tion of the modified (attenue) microbe in the body of the fowl we may not
have removed from all parts of its body the aliment of the microbe. That
which remains will permit, then, a new culture, but in a more restricted
measure.

"This is the effect of a first inoculation ; subsequent inoculations will

250 SUSCEPTIBILITY AND IMMUNITY.

remove progressively all the material necessary for the development of the
parasite."

In discussing this theory, in a paper published in the American
Journal of the Medical Sciences (April, 1881), the writer says:

"Let us see where this hypothesis leads us. In the first place, we must
have a material of small-pox, and a material of measles, and a material of
scarlet fever, etc., etc. Then we must admit that each of these different
materials has been formed in the system and stored up for these emergencies
—attacks of the diseases in question — for we can scarcely conceive that they
were all packed away in the germ cell of the mother and the sperm cell of
the father of each susceptible individual. If, then, these peculiar materials
have been formed and stored up during the development of the individual,
how are we to account for the fact that no new production takes place after
an attack of any one of the diseases in question ?

" Again, how shall we account for the fact that the amount of material
which would nourish the small-pox germ, to the extent of producing a case
of confluent small-pox, may be exhausted by the action of the attenuated
virus (germ) introduced by vaccination ? Pasteur's comparison of a fowl
protected by inoculation with the microbe of fowl cholera, with a culture
fluid in which the growth of a particular organism has exhausted the pabu-
lum necessary for the development of additional organisms of the same kind,
does not seem to me to be a just one, as in the latter case we have a limited
supply of nutriment, while in the former we have new supplies constantly
provided of the material — food — from which the whole body, including the
hypothetical substance essential to the development of the disease germ, was
built up prior to the attack. Besides this we have a constant provision for
the elimination of effete and useless products.

" This hypothesis, then, requires the formation in the human body, and
the retention up to a certain time, of a variety of materials which, so far as
we can see, serve no purpose except to nourish the germs of various specific
diseases, and which, having served this purpose, are not again formed in the
same system, subjected to similar external conditions, and supplied with the
same kind of nutriment."

It is unnecessary to discuss this hypothesis any further, inasmuch
as it is no longer sustained by Pasteur or his pupils, and is evidently
untenable.

The Eetention Theory, proposed by Chauveau (1880), is subject to
similar objections. According to this view, certain products formed
during the development of a pathogenic microorganism in the body
of a susceptible animal accumulate during the attack and are subse-
quently retained, and, being prejudicial to the growth of the particu-
lar microorganism which produced them, a second infection cannot
occur. Support for this theory has been found by its advocates in
the fact that various processes of fermentation are arrested after a
time by the formation of substances which restrain the development
of the microorganisms to which they are due. But in the case of a
living animal the conditions are very different, and it is hard to con-
ceive that adventitious products of this kind could be retained for
years, when in the normal processes of nutrition and excretion the
tissues and fluids of the body are constantly undergoing change.
Certainly the substances which arrest ordinary processes of fermen-

SUSCEPTIBILITY AXD IMMUNITY. 251

tation by their accumulation in the fermenting liquid, such as alco-
hol, lactic acid, phenol, etc., would not be so retained. But we can-
not speak so positively with reference to the toxic albuminous
substances which recent researches have demonstrated to be present
in cultures of some of the best known pathogenic bacteria. It is
difficult, however, to believe that an individual who has passed
through attacks of half a dozen different infectious diseases carries
about Avith him a store of as many different chemical substances pro-
duced during these attacks, and sufficient in quantity to prevent the
development of the several germs of these diseases. Nor does the
experimental evidence relating to the action of germicide and germ-
restraining agents justify the view that a substance capable of
preventing the development of one microorganism should be with-
out effect upon others of the same class ; but if we accept the re-
tention hypothesis we must admit that the inhibiting substance
produced by each particular pathogenic germ is effective only in
restraining the development of the microbe which produced it in the
first instance.

Pasteur discusses this hypothesis in his paper from which we
have already quoted, as follows :

4 ' We may admit the possibility that the development of the microbe, in
place of removing1 or destroying- certain matters in the bodies of the fowls,
adds, on the contrary, something which is an obstacle to the future develop-
ment of this microbe. The history of the life of inferior beings authorizes
such a supposition. The excretions resulting from vital processes may arrest
vital processes of the same nature. In certain fermentations we see anti-
septic products make their appearance during, and as a result of, the fer-
mentation, which put an end to the active life of the ferments and arrest
the fermentations long before they are completed. In the cultivation of our
microbe, products may have been formed the presence of which, possibly,
may explain the protection following inoculation.

"Our artificial cultures permit us to test the truth of this hypothesis.
Let us prepare an artificial culture of the microbe, and after having evapo-
rated it, in vacuo, without heat, let us bring it back to its original volume
by means of fresh chicken bouillon. If the extract contains a poison for
the life of the microbe, and if this is the cause of its failure to multiply in the
fi Itered liquid, the new liquid should remain sterile. Now, this is not the case.
We cannot, then, believe that during the life of the parasite certain substances
are produced which are capable of arresting its ulterior development."

This experiment of Pasteur appears to be conclusive so far as the
particular pathogenic microorganism referred to is concerned ; and
we may say, in brief, that more recent investigations do not sustain
the view that acquired immunity is due to the retention of products
such as are formed by pathogenic bacteria in artificial culture media,
and which act by destroying these bacteria or restraining their devel-
opment when they are introduced into the bodies of immune animals.

Moreover, if we suppose that the toxic substances which give
pathogenic power to a particular microorganism are retained in the

252 SUSCEPTIBILITY AND IMMUNITY.

body of an immune animal, we must admit that the animal has ac-
quired a tolerance to the pathogenic action of these toxic substances,
for their presence no longer gives rise to any morbid phenomena.
And this being the case, we are not restricted to the explanation
that immunity depends upon a restraining influence exercised upon
the microbe when subsequently introduced.

The Vital Resistance Theory. — Another explanation offers itself,
viz., that immunity depends upon an acquired tolerance to the
toxic products of pathogenic bacteria. This is a view which the
writer has advocated in various published papers since 1881. In a
paper contributed to the American Journal of the Medical Sci-
ences in April, 1881, it is presented in the following language:

"The view that I am endeavoring- to elucidate is that, during a non-
fatal attack of one of the specific diseases, the cellular elements implicated
which do not succumb to the destructive influence of the poison acquire a
tolerance to this poison which is transmissible to their progeny, and which
is the reason of the exemption which the individual enjoys from future
attacks of the same disease." l

In my chapter on "Bacteria in Infectious Diseases," in "Bac-
teria," published in the spring of 1884, but placed in the hands of the
publishers in 1883, I say:

" It may be that the true explanation of the immunity afforded by a mild
attack of an infectious germ disease is to be found in an acquired tolerance to
the action of a chemical poison produced by the microorganism, and conse-
quent ability to bring the resources of nature to bear to restrict invasion by
the parasite. "

The "resources of nature" are referred to in the same chapter as
follows :

' ' The hypothesis of Pasteur would account for the fact that one individual
suffers a severe attack and another a mild attack of an infectious disease,
after being subjected to the influence of the poison under identical circum-
stances, by the supposition that the pabulum required for the development
of this particular poison is more abundant in the body of one individual
than in the other. The explanation which seems to us more satisfactory is
that the vital resistance offered by the cellular elements in the bodies of
these two individuals was not the same for this poison. It is well known
that in conditions of lowered vitality resulting from starvation, profuse
discharges, or any other cause, the power to resist disease poisons is greatly
diminished, and, consequently, that the susceptibility of the same individual
differs at different times.

"From our point of view, the blood, as it is found within the vessels of a
living animal, is not simply a culture fluid maintained at a fixed tempera-
ture, but under these circumstances is a tissue, the histological elements of
which present a certain vital resistance to pathogenic organisms which may
be introduced into the circulation.

" If we add a small quantity of a culture fluid containing the bacteria of
putrefaction to the blood of an animal, withdrawn from the circulation into
a proper receptacle and maintained in a culture oven at blood heat, we will
find that these bacteria multiply abundantly, and evidence of putrefactive

1 " "What is the Explanation of the Protection from Subsequent Attacks, result-
ing from an Attack of Certain Diseases, etc ? '' American Journal of the Medical
Sciences, April, 1881, p. 37«.

SUSCEPTIBILITY AXD IMMUNITY. 263

decomposition will soon be perceived. But if we inject a like quantity of
the culture fluid with its contained bacteria into the circulation of a living
animal, not only does no increase and 110 putrefactive change occur, but the
bacteria introduced quickly disappear, and at the end of an hour or two the
most careful microscopical examination will not reveal the presence of a
single bacterium. This difference we ascribe to the vital properties of the
fluid as contained in the vessels of a living animal; and it seems probable
that the little masses of protoplasm known as white blood corpuscles are the
essential histological elements of the blood, so far as any manifestation of
vitality is concerned. The writer has elsewhere (1881) suggested that the
disappearance of the bacteria from the circulation, in the experiment
referred to, may be effected by the white corpuscles, wrhich, it is well known,
pick up, after the manner of amoebae, any particles, organic or inorganic,
which come in their way. And it requires no great stretch of credulity to
believe that they may, like an amoeba, digest and assimilate the protoplasm
of the captured bacterium, thus putting an end to the possibility of its do-
ing any harm.

" In the case of a pathogenic organism we may imagine that, when cap-
tured in this way, it may share a like fate if the captor is not paralyzed by
some potent poison evolved by it, or overwhelmed by its superior vigor and
rapid multiplication. In the latter event the active career of our conserva-
tive white corpuscle would be quickly terminated and its protoplasm would
serve as food for the enemy. It is evident that in a contest of this kind the
balance of power would depend upon circumstances relating to the inherited
vital characteristics of the invading parasite and of the invaded leucocyte."

In the same chapter the writer quotes from his paper on acquired
immunity, published in 1881, as follows :

' ' The difficulties into which this hypothesis [the exhaustion theory of Pas-
teur] leads us certainly justify us in looking further for an explanation of the
phenomena in question. This explanation is, I believe, to be found in the
peculiar properties of the protoplasm, which is the essential framework of
every living organism. The properties referred to are the tolerance which
living protoplasm may acquire to certain agents which, in the first instance,
have an injurious or even fatal influence upon its vital activity ; and the
property which it possesses of transmitting its peculiar qualities, inherent or
acquired, through numerous generations, to its offshoots or progeny.

tk Protoplasm is the essential living portion of the cellular elements of ani-
mal and vegetable tissues ; but as our microscopical analysis of the tissues has
not gone beyond the cells of which they are composed, and is not likely to
reveal to us the complicated molecular structure of the protoplasm, upon
which, possibly, the properties under consideration depend, it will be best,
for the present, to limit ourselves to a consideration of the living cells of the
body. These cells are the direct descendants of the pre-existent cells, and
may all be traced back to the sperm cell and the germ cell of the parents.
Now, the view which I am endeavoring to elucidate is that, during a non-
fatal attack of one of the specific diseases, the cellular elements implicated,
which do not succumb to the destructive influence of the poison, acquire a
tolerance to this poison which is transmissible to their progeny, and which
is the reason of the exemption which the individual enjoys from future
attacks of the same disease.

" The known facts in regard to the hereditary transmission by cells of ac-
quired properties' make it easy to believe in the transmission of such a
tolerance as we imagine to be acquired during the attack ; and if it is shown
by analogy that there is nothing improbable in the hypothesis that such a
tolerance is acquired, we shall have a rational explanation, not of heredity
and of the mysterious properties of protoplasm, but of the particular result
under consideration. The transmission of acquired properties is shown in
the budding and grafting of choice fruits and flowers, produced by cultiva-

254 SUSCEPTIBILITY AND IMMUNITY,

tion, upon the wild stock from which they originated. The acquired proper-
ties are transmitted indefinitely; and the same sap which on one twig- nour-
ishes a sour crab apple, on another one of the same branch is elaborated into
a delicious pippin.

' The tolerance to narcotics— opium and tobacco — and to corrosive poisons
— arsenic — which results from a gradual increase of dose, may be cited as an
example of acquired tolerance by living protoplasm to poisons which at the
outset would have been fatal in much smaller doses.

'The immunity which an individual enjoys from any particular disease
must be looked upon as a power of resistance" possessed by the cellular ele-
ments of those tissues of his body which would yield to the poison in the
case of an unprotected person."

This theory of immunity, advanced by the author in 1881, has
received considerable support from investigations made since that
date, and especially from the experimental demonstration by Sal-
mon, Roux, and others that, as suggested in the paper from which I
have quoted, immunity may result from the introduction into the
body of a susceptible animal of the soluble products of bacterial
growth — filtered cultures.

The theory of vital resistance to the toxic products evolved by
pathogenic bacteria is also supported by numerous experiments
which show that natural or acquired immunity may be overcome
when these toxic products are introduced in excess, or when the vital
resisting power of the animal has been reduced by various agencies.

More direct experimental evidence in favor of the view under con-
sideration is that obtained by Beumer in his experiments with steril-
ized cultures of the typhoid bacillus. He found that after the re-
peated injection of non-lethal doses mice were able to resist an
amount of this toxine which was fatal to animals of the same spe-
cies not so treated. But, on the other hand, Gamaleia found, in his
experiments upon guinea-pigs which had been made immune against
the pathogenic action of a spirillum, called by him Vibrio Metschni-
kovi, that these animals have no increased tolerance for the toxic
products of this microorganism. Although immune against infec-
tion by the living microbe, they were killed by the same quantity of
a sterilized culture as was fatal to guinea-pigs which had not been
rendered immune.

Charrin has obtained similar results in experiments with filtered
cultures of Bacillus pyocyaneus. Rabbits which had an artificial im-
munity against the pathogenic action of the bacillus were killed by
doses of a sterilized culture such as were fatal to other rahbits of the
same size not immune. In subsequent experiments by Charrin and
Gameleia " vaccinated " rabbits were found to be even more suscepti-
ble to the toxic action of filtered cultures than were those not vacci-
nated. Metschnikoff (1891) has followed up this line of experiment,
and has shown that when considerable amounts of filtered cultures
of Bacillus pyocyaneus are injected subcutaneously in rabbits a cer-

SUSCEPTIBILITY AND IMMUNITY. 255

tain tolerance to the toxic action of the same cultures is established
in some instances. But his results do not give any substantial sup-
port to the view that immunity depends upon an acquired tolerance
to the toxic action of the chemical products contained in cultures of
the pathogenic bacteria with which he experimented— Bacillus pyo-
cyaneus and Vibrio Metschnikovi.

In view of the results of experimental researches above recorded,
and of other recent experiments which show that, in certain cases at
least, acquired immunity depends upon the formation of an anti-
toxine in the body of the immune animal, we are convinced that the
theory of immunity under discussion, first proposed by the writer in
1881, cannot be accepted as a sufficient explanation of the facts in
general. At the same time we are inclined to attribute considerable
importance to acquired tolerance to the toxic products of pathogenic
bacteria as one of the factors by which recovery from an infectious
disease is made possible and subsequent immunity established.

The " vital-resistance theory" of the present writer, as set forth,
in the above-quoted extracts from his published papers, is essentially
the same as that advocated by Buchner at a later date (1883). Buch-
ner supposes that during the primary infection, when an animal re-
covers, a " reactive change " has been produced in the cells of the
body which enables it to protect itself from the pathogenic action
of the same microorganism when subsequently introduced.

Of course when we ascribe immunity to the " vital resistance" of
the cellular elements of the body, we have not explained the
modus operandi of this vital resistance or " reactive change," but
have simply affirmed that the phenomenon in question depends upon
some acquired property residing in the living cellular elements of
the body. We have suggested that that which has been acquired
is a tolerance to the action of the toxic products produced by patho-
genic bacteria. But, as already stated, in the light of recent experi-
ments this theory now appears to us to be untenable as a general
explanation of acquired immunity.

The Theory of Phagocytosis. — The fact that in certain infectious
diseases due to bacteria the parasitic invaders, at the point of inocu-
lation or in the general blood current, are picked up by the leuco-
cytes and in properly stained preparations may be seen in their in-
terior, has been known for some years. In mouse septica3mia — an
infectious disease described by Koch in his work on "Traumatic
Infectious Diseases," published in 1878 — the slender bacilli which are
the cause of the disease are found in large numbers in the interior of
the leucocytes. Koch says, in the work referred to : " Their rela-
tion to the white blood corpuscles is peculiar ; they penetrate these
and multiply in their interior. One often finds that there is

256 SUSCEPTIBILITY AND IMMUNITY.

hardly a single white corpuscle in the interior of which bacilli can-
not be seen. Many corpuscles contain isolated bacilli only; others
have thick masses in their interior, the nucleus being still recog-
nizable ; while in others the nucleus can be no longer distinguished ;
and, finally, the corpuscle may become a cluster of bacilli, breaking
up at the margin — the origin of which one could not have explained
had there been no opportunity of seeing all the intermediate steps
between the intact white corpuscle and these masses" (Fig. 78). It
will be noted that in the above quotation Koch affirms that the
bacilli penetrate the leucocytes and multiply in their interior. Now,
the theory of phagocytosis assumes that the bacilli are picked up by
the leucocytes and destroyed in their interior, and that immunity de-
pends largely upon the power of these "phagocytes" to capture and
destroy living pathogenic bacilli.

The writer suggested this as an hypothesis as long ago as 1881,
in a paper read before the American Association for the Advance-
ment of Science, in the following language:

" It has occurred to me that possibly the white corpuscles may
have the office of picking up and digesting bacterial organisms which

FIG. 78.— Bacillus of mouse septicaemia in leucocytes from blood of mouse (Koch).

by any means find their way into the blood. The propensity exhib-
ited by the leucocytes for picking up inorganic granules is well
known, and that they may be able not only to pick up but to assimi-
late, and so dispose of, the bacteria which come in their way, does
not seern to me very improbable, in view of the fact that amcebaB,
which resemble them so closely, feed upon bacteria and similar or-
ganisms."

At a later date (1884) Metschnikoff offered experimental evi-
dence in favor of this view, and the explanation suggested in the
above quotation is commonly spoken of as the Metschnikoff theory.

1 " A Contribution to the Study of Bacterial Organisms commonly found upon
Exposed Mucous Surf aces and in the Alimentary Canal of Healthy Individuals." Il-
lustrated by photomicrographs. Proceedings of the American Association for Ad-
vancement of Science, 1881, Salem, 1882, xxx., 83-94. Also in Studies from the
Biological Laboratory, Johns Hopkins University, Baltimore, vol. ii., No. 2, 1882.

SUSCEPTIBILITY AND IMMUNITY. 257

The observations which first led Metschnikoff to adopt this view
were made upon a species of daphnia which is subject to fatal infec-
tion by a torula resembling the yeast fungus. Entering with the
food, this fungus -penetrates the walls of the intestine and invades the
tissues. In certain cases the infection does not prove fatal, owing, as
Metschnikoff asserts, to the fact that the fungus cells are seized upon
by the leucocytes, which appear to accumulate around the invading
parasite (chemiotaxis) for this special purpose. If they are success-
ful in overpowering and destroying the parasite the animal recovers ;
if not, it succumbs to the general infection which results. In a simi-
lar manner, Metschnikoff supposes, pathogenic bacteria are destroyed
when introduced into the body of an immune animal. The colorless
blood corpuscles, which he designates phagocytes, accumulate at the
point of invasion and pick up the living bacteria, as they are known
to pick up inorganic particles injected into the circulation. So far
there can be no doubt that Metschnikoff is right. The presence of
bacteria in the leucocytes in considerable numbers, 'both at the point
of inoculation and in the general circulation, has been repeatedly
demonstrated in animals inoculated with various pathogenic bacteria.
The writer observed this in his experiments, made in 1881, in which
rabbits were inoculated with cultures of his Micrococcus Pasteuri ;
and it was this observation which led him to suggest the theory
which has since been so vigorously supported by Metschnikoff. But
the presence of a certain number of bacteria within the leucocytes
does not prove the destructive power of these cells for living patho-
genic organisms. As urged by Weigert, Baumgarten, and others,
, it may be that the bacteria were already dead when they were picked
up, having been destroyed by some agency outside of the blood cells.
As heretofore stated, we have now experimental evidence that blood
serum, quite independently of the cellular elements contained in it
in the circulation, has decided germicidal power for certain patho-
genic bacteria, and that the blood serum of the rat and other animals
which have a natural immunity against anthrax is especially fatal
to the anthrax bacillus.

Numerous experiments have been made with a view to deter-
mining whether pathogenic bacteria are, in fact, destroyed within
the leucocytes after being picked up, and different experimenters
have arrived at different conclusions. In the case of mouse septi-
csemia, already alluded to, and in gonorrhoea, one would be disposed
to decide, from the appearance and arrangement of the pathogenic
bacteria in the leucocytes, that they are not destroyed, but that,
on the other hand, they multiply in the interior of these cells, which
in the end succumb to this parasitic invasion. In both of the dis-
eases mentioned we find leucocytes so completely filled with the
17

258 SUSCEPTIBILITY AND IMMUNITY.

pathogenic microorganisms that it is difficult to believe that they
have all been picked up by . a voracious phagocyte, which has
stuffed itself to repletion, while numerous other leucocytes from
the same source and in the same microscopic field of view have
failed to capture a single bacillus or micrococcus. Moreover, the
staining of the parasitic invaders, and the characteristic arrange-
ment of the "gonococcus" in stained preparations of gonorrhceal
pus, indicate that their vitality has not been destroyed in the interior
of the leucocytes or pus cells, and we can scarcely doubt that the
large number found in certain cells is due to multiplication in situ
rather than to an unusual activity of these particular cells. But in
certain infectious diseases, and especially in anthrax, the bacilli in-
cluded within the leucocytes often give evidence of degenerative
changes, which would support the view that they are destroyed by
the leucocytes, unless these changes occurred before they were picked
up, as is maintained by Nuttall and others. We cannot consider
this question as definitely settled.

Going back to the demonstrated fact that susceptible animals may
be made immune by inoculating them with the toxic products pro-
duced during the growth of certain pathogenic bacteria, we may
suppose either that immunity results from the continued presence of
these toxic products in the body of the inoculated animal, or from a
tolerance acquired at the time of the inoculation and subsequently
retained — by transmission from cell to cell, as heretofore suggested.
Under the first hypothesis — retention theory — immunity may be ex-
plained as due to a continued tolerance on the part of the cellular ele-
ments of the body to the toxic substances introduced and retained ;
or to the effect of these retained toxic products in destroying the
pathogenic bacteria, or in neutralizing their products when these are
subsequently introduced into the body of the immune animal. We
cannot understand how toxic substances 'introduced in the first in-
stance can neutralize substances of the same kind introduced at a
later date. There is something in the blood of the rat which, accord-
ing to Behring, neutralizes the toxic substances present in a filtered
culture of the tetanus bacillus ; but whatever this substance may be,
it is evidently different from the toxic substance which it destroys,
and there is nothing in chemistry to justify the supposition last
made. Is it, then, by destroying the pathogenic microorganism
that these inoculated and retained toxic products preserve the animal
from future infection ? Opposed to this supposition is the fact that
the blood of an animal made immune in this v/ay, when removed
from the body, does not prove to have increased germicidal power as
compared with that of a susceptible animal of the same species.
Again, these same toxic substances in cultures of the anthrax bacillus,

SUSCEPTIBILITY AND IMMUNITY. 259

the tetanus bacillus, the diphtheria bacillus, etc., do not destroy the
pathogenic germ after weeks or months of exposure. And when we
inoculate a susceptible animal with a viruleift culture of one of these
microorganisms, the toxic substances present do not prevent the rapid
development of the bacillus ; indeed, instead of proving a germicide,
they favor its development, which is more abundant and rapid than
when attenuated cultures containing less of the toxic material are
used for the inoculation. In view of these facts we are unable to
adopt the view that acquired immunity results from the direct action
of the products of bacterial growth, introduced and retained in the
body of the immune animal, upon the pathogenic microorganism
when subsequently introduced or upon its toxic products.

But there is another explanation which, although it may appear
a priori to be quite improbable, has the support of recent experimen-
tal evidence. This is the supposition that some substance is formed
in the body of the immune animal which neutralizes the toxic
products of the pathogenic microorganism. How the presence of
these toxic products in the first instance brings about the formation
of an "antitoxin " by which they are neutralized is still a mystery;
but that such a substance is formed appears to be proved by the ex-
periments of Ogata, Behring and Kitasato, Tizzoni and Cattani, G.
and F. Klemperer, and others.

Ogata and Jasuhara, in a series of experiments made in the Hy-
gienic Institute at Tokio (1890), discovered the important fact that
the blood of an animal immune against anthrax contains some sub-
stance which neutralizes the toxic products of the anthrax bacillus.
When cultures were made in the blood of dogs, frogs, or of white
rats, which animals have a natural immunity against anthrax, they
were found not to kill mice inoculated with them. Further experi-
ments showed that mice inoculated with virulent anthrax cultures
did not succumb to anthrax septicaBmia if they received at the same
time a subcutaneous injection of a small quantity of the blood of an
immune animal. So small a dose as one drop of frog's blood or one-
half drop of dog's blood proved to be sufficient to protect a mouse
from the fatal effect of an anthrax inoculation. And the protective
inoculation was effective when made as long as seventy-two hours
before or five hours after infection with an anthrax culture. Fur-
ther, it was found that mice which had survived anthrax infection as
a result of this treatment were immune at a later date (after several
weeks) when inoculated with a virulent culture of the anthrax
bacillus.

Behring and Kitasato have obtained similar results in their ex-
periments upon tetanus and diphtheria, and have shown that the
blood of an immune animal, added to virulent cultures before in-

2GO SUSCEPTIBILITY AND IMMUNITY.

oculation into susceptible animals, neutralizes the pathogenic power
of these cultures.

They have shown by experiment that the blood of a rabbit which
has an acquired immunity against tetanus, mixed with the virulent
filtrate from a culture of the tetanus bacillus, neutralizes its toxic
power. One cubic centimetre of this filtrate was mixed with five
cubic centimetres of serum from the blood of an immune rabbit and
allowed to stand for twenty-four hours ; 0. 2 cubic centimetre of this
injected into a mouse was without effect, while 0.0001 cubic centi-
metre of the filtrate without such admixture was infallibly fatal to
mice. The mice inoculated with this mixture remained immune for
forty to fifty days, after which they gradually lost their immunity.
The blood or serum from an immune rabbit, when preserved in a
dark, cool place, retained its power of neutralizing the tetanus tox-
albumin for about a week, after which time it gradually lost this
power. The blood of chickens, which have a natural immunity
against tetanus, was found not to have a similar power. Behring
and Kitasato have also shown that the serum of a diphtheria-immune
rabbit destroys the potent toxalbumin in diphtheria cultures. It
does not, however, possess any germicidal power against the diph-
theria bacillus.

Ogata, in 1891, reported that he had succeeded in isolating from the
blood of dogs and of chickens a substance to which he ascribes the nat-
ural immunity of these animals from certain infectious diseases, and
the power of their blood to protect susceptible animals from the same
diseases. This substance is soluble in water and in glycerin, but in-
soluble in alcohol or ether, by which it is precipitated without being
destroyed. Its activity is neutralized by acids, but not by weak
alkaline solutions. Ogata supposes the substance isolated by him to
be the active agent in blood serum by which certain pathogenic bac-
teria are destroyed, as shown by the experiments of Nuttall, Buchner,
and others. Hankin had previously isolated an albuminoid sub-
stance from the spleen and blood of the rat, to which he ascribed the
immunity of this animal from anthrax. This substance, according
to the author named, is a globulin; it is insoluble in alcohol and in
distilled water, and does not dialyze.

Tizzoni and Cattani ascribe the protection of animals which have
acquired, an immunity against tetanus to the presence of an albumi-
nous substance which they call the tetanus-antitoxin. This they
have isolated from the blood of immune animals. They arrive at
the conclusion that it is a globulin, or a substance which is carried
down with the globulin precipitate, and that it is different from the
globulin, above referred to, obtained by Hankin from animals im-
mune against anthrax.

SUSCEPTIBILITY AND IMMUNITY. 261

G. and F. Klemperer, in 1891, published an important memoir in
which they gave an account of their researches relating to the ques-
tion of immunity, etc., in animals subject to the form of septicaBmia
produced by the Micrococcus pneumonia crouposaB. They were able
to produce immunity in susceptible animals by introducing into their
bodies filtered cultures of this micrococcus, and proved by experiment
that this immunity had a duration of at least six months. They
arrived at the conclusion that the immunity induced by injecting fil-
tered cultures is not directly due to the toxic substances present in
these cultures, but that they cause the production in the tissues of an
antitoxin which has the power of neutralizing their pathogenic
action. The toxic substance present in cultures of the "diplococcus
of pneumonia" they call " pneumotoxin" ; the substance produced in
the body of an artificially immune animal, by which this pneumo-
toxin is destroyed if subsequently introduced, they call " anti -pneumo-
toxin."

Emmerich, in a communication made at the meeting of the In-
ternational Congress for Hygiene and Demography, in London, re-
ported results which correspond with those of G. and F. Klemperer
so far as the production of immunity is concerned, and also gave an
account of experiments made by Donissen in which the injection of
twenty to twenty-five cubic centimetres of blood or expressed tissue
juices, filtered through porcelain, from an immune rabbit into an
unprotected rabbit, subsequently to infection with a bouillon culture
of "diplococcus pneumonise," prevented the development of fatal
septicremia. Even when the injection was made twelve to fifteen
hours after infection, by inhalation, the animal recovered.

Emmerich and Mastraum had previously reported similar results
in experiments made upon mice with the Bacillus erysipelatos suis
(rothlauf bacillus). White mice are very susceptible to the patho-
genic action of this bacillus. But mice which, subsequently to in-
fection, were injected with the expressed and filtered tissue juices of
an immune rabbit, recovered, while the control animals succumbed.
According to Emmerich, the result in these experiments was due to
a destruction of the pathogenic bacilli in the bodies of the infected
animals ; and the statement is made that at the end of eight hours
after the injection of the expressed tissue juices all bacilli in the body
of the infected animal were dead. The same liquid did not, however,
kill the bacilli when added to cultures external to the body of an
animal. The inference, therefore, seems justified that the result de-
pends, not upon a substance present in the expressed juices of an
immune animal, but upon a substance formed in the body of the
animal into which these juices are injected.

We have, however, an example of induced immunity in which

262 SUSCEPTIBILITY AND IMMUNITY.

the result appears to depend directly upon the destruction of the
pathogenic microorganism in the body of the immune animal. In
guinea-pigs which have an acquired immunity against Vibrio Metsch-
nikovi the blood serum has been proved to possess decided germicidal
power for this "vibrio," whereas it multiplies readily in the blood
serum of non-immune guinea-pigs (Behring and Msseii).

There is experimental evidence that animals may acquire an arti-
ficial immunity against the toxic action of certain toxalbumins from
other sources than bacterial cultures. Thus Sewell (1887) has shown
that a certain degree of tolerance to the action of rattlesnake venom
may be established by inoculating susceptible animals with small
doses of the " hemialbumose " to which it owes its toxic potency.
These results have been confirmed by the more recent experiments of
Calmette (1894) and of Fraser (1895). In his paper detailing the
results of his experiments the first-named author says :

"Animals may be immunized against the venom of serpents either by
means of repeated injections of doses at first feeble and progressively stronger,
or by means of successive injections of venom mixed with certain chemical
substances, among which I mention especially chloride of gold and the hypo-
chlorites of lime or of soda.

"The serum of animals thus treated is at the same time preventive, anti-
toxic, and therapeutic, exactly as is that of animals immunized against
diphtheria or tetanus.

"If we inoculate a certain number of rabbits, under the skin of the
thigh, with the same dose, one milligramme of cobra venom for example,
and if we treat all of these animals with the exception of some for control,
by subcutaneous or intraperitoneal injections of the serum of rabbits im-
munized against four milligrammes of the same venom, all of the control
animals not treated will die within three or four hours, while all of the
animals will recover which receive five cubic centimetres of the therapeutic
serum within an hour after receiving the venom."

In this connection we may remark that there is some evidence to
show that persons who are repeatedly stung by certain poisonous in-
sects— mosquitoes, bees — acquire a greater or less degree of immu-
nit}r from the distressing local effects of their stings.

Ehrlich, of Berlin, in 1891, reported his success in establishing
immunity in guinea-pigs against two toxalbumins of vegetable
origin: one — ricin — from the castor-oil bean (Ricinus communis),
the other — abrin — from the jequirity bean. The toxic potency
of ricin is somewhat greater than that of abrin, and it is esti-
mated by Ehrlich that one gramme of this substance would suffice
to kill one and a half millions of guinea-pigs. When injected be-
neath the skin, in dilute solution, it produces intense local inflamma-
tion, resulting in necrosis of the tissues. Mice are less susceptible
tfyan guinea-pigs and are more easily made immune. This is most
readily effected by giving them small and gradually increasing doses
with their food. As a result of this treatment the animal resists

SUSCEPTIBILITY AND IMMUNITY. 263

subcutaneous injections of two hundred to four hundred times the
fatal dose for animals not having this artificial immunity. The fatal
dose of abrin is about double that of ricin. When injected into mice
in the proportion of one cubic centimetre to twenty grammes of body
weight a solution of one part in one hundred thousand of water
proved to be a fatal dose. The local effects are also less pronounced
when solutions of abrin are used ; they consist principally in an ex-
tensive induration of the tissues around the point of injection and a
subsequent falling off of the hair over this indurated area. When
introduced into the conjunctival sac, however, abrin produces a
local inflammation in smaller amounts than ricin, a solution of 1 : 800
being sufficient to cause a decided but temporary conjunctivitis.
Solutions of 1 : 50 or 1 : 100 of either of these toxalbumins, introduced
into the eye of a mouse, give rise to a panophthalmitis which com-
monly results in destruction of the eye. But in mice which have
been rendered immune by feeding them for several weeks with food
containing one of these toxalbumins, no reaction follows the intro-
duction into the eye of the strongest possible solution, or of a paste
made by adding abrin to a little ten-per-cent salt solution. Ehrlich
gives the following explanation of the remarkable degree of im-
munity established in his experiments by the method mentioned:

" All of these phenomena depend, as may be easily shown, upon
the fact that the blood contains a body — antiabrin — which completely
neutralizes the action of the abrin, probably by destroying this body."

In a more recent paper Ehrlich has given an account of subse-
quent experiments which show that the young of mice which have
an acquired immunity for these vegetable toxalbumins may acquire
immunity from the ingestion of the mother's milk ; and also that
immunity against tetanus may be acquired in a very brief time by
young mice through their mother's milk. In his letanus experi-
ments Ehrlich used blood serum from an immune horse to give im-
munity to the mother mouse when her young were .already seven-
teen days old. Of this blood serum two cubic centimetres were
injected at a time on two successive days. The day after the first
injection one of the sucklings received a tetanus inoculation by
means of a splinter of wood to which spores were attached. The
animal remained in good health, while a much larger control mouse
inoculated in the same way died of tetanus at the end of twenty-six
hours. Other sucklings, inoculated at the end of forty-eight and of
seventy-two hours after the mother had received the injection of
blood serum, likewise remained in good health, while other control
mice died.

The possibility of conferring immunity by means of the milk of
an immune animal is further shown by the experiments of Brieger

264 SUSCEPTIBILITY AND IMMUNITY.

and Ehrlich (1892). A female goat was immunized against tetanus
by the daily injection of " thymus-tetanus bouillon." The dose was
gradually increased from 0.2 cubic centimetre to 10 cubic centimetres.
At the end of thirty-seven days a mouse, which received 0.1 cubic
centimetre of the milk of this goat in the cavity of the abdomen,
proved to be immune against tetanus. Further experiments gave a
similar result, even when the milk of the goat was not injected into
the peritoneal cavity of the mouse until several hours after inocu-
lation with a virulent culture of the tetanus bacillus.

When the casein was separated the milk retained its full im-
munizing activity, and by concentration in vacua a thick milk
was obtained which had a very high immunization value — 0.2 cubic
centimetre of this milk protected a mouse against forty-eight times
the lethal dose of a tetanus culture.

In a subsequent communication (1893) Brieger and Ehrlich de-
scribe their method of obtaining the antitoxin of tetanus from milk
in a more concentrated form. They found by experiment that it was
precipitated by ammonium sulphate and magnesium sulphate. From
twenty-seven to thirty per cent of ammonium sulphate added to milk
caused a precipitation of the greater part of the antitoxin. This pre-
cipitate was dissolved in water, dialyzed in running water, then
filtered and evaporated in shallow dishes at 35° C. in a vacuum.
One litre of milk from an immune goat gave about one gramme of a
transparent, yellowish-white precipitate, which contained fourteen
per cent of ammonium sulphate. This precipitate had from four
hundred to six hundred times the potency of the milk from which
it was obtained in neutralizing the tetanus toxin.

In a still later communication (1893) Brieger and Cohn give an
improved method of separating the antitoxin from the precipitate
thrown down with ammonium sulphate. The finely pulverized pre-
cipitate is shaken up with pure chloroform, and when this is allowed
to stand the antitoxin rises to the surface while the ammonium salt
sinks to the bottom. By filling the vessel to the margin with chloro-
form, the antitoxin floating on the surface can be skimmed off, after
which it quickly dries. By this method the considerable loss which
occurred in the dialyzer, used in the previously described method, is
avoided.

A most interesting question presents itself in connection with the
discovery of the antitoxins. Does the animal which is immune
from the toxic action of any particular toxalbumin also have an im-
munity for other toxic proteids of the same class? The experimental
evidence on record indicates that it does not. In Ehrlich 's experi-
ments with ricin and abrin he ascertained that an animal which had
been made immune against one of these subtances was quite as sus-

SUSCEPTIBILITY AND "IMMUNITY. 265

ceptible to the toxic action of the other as if it did not possess this
immunity, i.e., the antitoxin of ricin does not destroy abrin, and
vice versa. As an illustration of the fact, he states that in one ex-
periment a rabbit was made immune for ricin to such an extent that
the introduction into its eye of this substance in powder produced no
inflammakny reaction ; but the subsequent introduction of a solution
of abrin, of 1 to 10,000, caused a violent inflammation.

Evidently these facts are of the same order as those relating to
immunity from infectious diseases, and, taken in connection with the
experimental data previously referred to, give strong support to the
view that the morbid phenomena in all diseases of this class are due
to the specific toxic action of substances resembling the toxalbumins
already discovered ; and that acquired immunity from any one of
these diseases results from the formation of an antitoxin in the body
of the immune animal.

Hankin calls these substances produced in the bodies of immune
animals "defensive proteids," and proposes to classify them as fol-
lows : First, those occurring naturally in normal animals, which he
calls sozins ; second, those occurring in animals that have acquired
an artificial immunity — these he calls phylaxins. Each of these
classes of defensive proteids is further subdivided into those which
act upon the pathogenic microorganism itself and those which act
upon its toxic products. These subclasses are distinguished by the
prefixes myco and toxo attached to the class name.

In accordance with this classification a mycosozin is a defensive
proteid, found in the body of a normal animal, which has the power
of destroying bacteria.

A toxosozin is a defensive proteid, found in the body of a normal
animal, which has the power of destroying the toxic products of bac-
terial growth.

A inijcophylaxin is a defensive proteid produced in the body of
an animal which has an acquired immunity for a given infectious
disease, which has the power of destroying the pathogenic bacteria
to which the disease is due.

A toxophylaxin is a defensive proteid produced in the body of
an animal which has an acquired immunity for a given infectious
disease, which has the power of destroying the toxic products of the
pathogenic bacteria to which the disease is due.

Buchner had previously proposed the name " alexins " for these
defensive proteids.

The importance of the experimental evidence above referred to in
explaining the phenomena of natural and acquired immunity is ap^
parent. The facts stated also suggest a rational explanation of re*

266 SUSCEPTIBILITY AND IMMUNITY.

covery from an attack of an acute infectious disease. But the idea
that during such an attack an antidote to the disease poison is de-
veloped in the tissues is yet so novel, and the experimental evidence
in support of this view is of such recent date, that it would be pre-
mature to accept this explanation as applying to immunity in gene-
ral. It seems difficult to believe that a'n individual who has passed
through attacks of measles, mumps, whooping cough, scarlet fever,
small-pox, etc., has in his blood or tissues a store of the antitoxin of
each of these diseases, formed during the attack and retained during
the remainder of his life, or continuously produced so long as the
immunity lasts. Moreover, in those diseases to which the experi-
mental evidence above recorded relates — diphtheria, tetanus, pneu-
monia— as they occur in man, no lasting immunity has been shown
to result from a single attack, and in this regard they do not come
into the same class with the eruptive fevers and other diseases in
which a single attack usually protects during the lifetime of the in-
dividual.

In those instances in which acquired immunity has been shown
to be due to the production in the body of the immune animal of an
antitoxin, it is still uncertain whether there is a continuous produc-
tion of the protective proteid, or whether that formed during the
attack remains in the body during the subsequent immunity. The
latter supposition appears at first thought improbable ; but when we
remember that the protective proteids which have been isolated by
Hankin from the blood and spleen of rats, and by Tizzoni and Cat-
tani from the blood of animals made immune against tetanus^ do
not dialyze, it does not seem impossible that these substances might
be retained indefimtely'within the blood-vessels. On the other hand,
the passage of the tetanus antitoxin into the mother's milk, as
shown by Ehrlich's experiments upon mice, indicates a continuous
supply, otherwise the immunity of the mother would soon be lost.

The writer has obtained (May, 1892) experimental evidence that
the blood of vaccinated, and consequently immune, calves contains
something which neutralizes the specific virulence of vaccine virus,
both bovine and humanized. Four drops of blood serum from a calf
which had been vaccinated two weeks previously, mixed with one
drop of liquid lymph recently collected in a capillary tube, after con-
tact for one hour was used to vaccinate a calf ; the same animal was
also vaccinated with lymph, preserved on three quills, which was
mixed with four drops of serum from the immune calf and left for
one hour. The result of these vaccinations was entirely negative,
while vaccinations upon the same calf made with virus from the
same source, and mixed with the same amount of blood serum from
a non-immune calf, gave a completely successful and typical result.

SUSCEPTIBILITY AND IMMUNITY. 267

The experimental evidence detailed shows that in certain dis-
eases acquired immunity depends upon the formation of anti-
toxins in the bodies of immune animals. As secondary fac-
tors it is probable that tolerance to the toxic products of pathogenic
bacteria and phagocytosis have considerable importance, but it is
evident that the principal role cannot be assigned to these agencies.

As a rule the antitoxins have no bactericidal action; but it has
been shown by the experiments of Gamaleia, Pfeiffer, and others,
that in animals which have an acquired immunity against the spiril-
lum of Asiatic cholera and against spirillum Metschnikovi, there is a
decided increase in the bactericidal power of the blood serum, and
that immunity probably depends upon this fact. 9

The researches of Metschnikoff upon hog cholera, of Issaef upon
pneumonia, and of Sanarelli upon typhoid fever indicate that the
immunity conferred upon susceptible animals by protective inocula-
tions is not due to an antitoxin but to a substance present in the
blood of immune individuals which acts directly upon the pathogenic
microorganism, as is the case in cholera-immune animals. The ani-
mals immunized are said to be quite as sensitive to the action of the
bacterial poisons as are those which have not received protective
inoculations. "Their serum does not protect against the toxin, but
against the microbe" (Roux) .

According to Buchner (1894) the antitoxins are to be regarded not
as reactive products developed in the body of the immune animal,
but as modified, changed, and " entgiftete " products of the specific
bacterial cells. He insists that they do not neutralize the toxins by
direct contact, but only through the medium of the living organism.
This explanation scarcely appears tenable in view of the experimental
evidence, and the fact that the antitoxin of tetanus escapes in con-
siderable quantity with the milk of an immune goat without, ap-
parently, diminishing the immunity of the animal. In the immunity
against the toxic action of the vegetable toxalbumins — ricin and
abrin — as shown by Ehrlich's experiments, there are no " products of
bacterial cells " introduced with the pure toxalbumin from the castor
bean or the jequirity bean ; and we have sufficiently numerous ex-
periments to show that immunity, with the presence of antitoxins in
the blood, may be induced by precipitated and purified toxalbumins
from filtered cultures. Several of the experimenters, also, have re-
ported that the toxins from bacterial cultures are neutralized in vitro
by blood-serum from an immune animal, or by the precipitated anti-
toxin from such serum after contact for a certain number of hours.
If they are correct in the statement that a certain time is required
after the antitoxin has been brought in contact with the toxin, in
order that the latter may be neutralized, as shown by injection of the

268 SUSCEPTIBILITY AND IMMUNITY.

mixture into a susceptible animal, then we must admit that this
neutralizing effect occurs outside of the body of the animal, as has
been generally assumed.

The experiments of Vaillard are also opposed to*Buchner's view.
He reports that in a rabbit immunized against tetanus, " a vohfme of
blood equal to the total, amount which circulates in its body may be
withdrawn without diminishing, in an -appreciable manner, the anti-
toxic power of its serum. Therefore the antitoxin must be repro-
duced as fast as it is withdrawn." The author from whom we have
just quoted (Roux) also reports the resujts of experiments which
show that the antitoxic value of the serum of a rabbit immunized
against tetanus does not bear a direct relation to the quantity of the
tetanus toxin- introduced, but depends also upon the method adopted.
W^ien a few large doses are given the result is far lesg favorable than
that obtained by giving the same amount in repeated small doses.
The serum of an animal immunized by thirty-three small doses was
found to neutralize, in vitro, 150 parts*of toxin, while that of an
animal which received the same amount in nine* doses only neutral-
ized 25 parts of the same toxin. On the other hand we have experi-
ments which indicate that the supposed neutralization of a toxin by
an antitoxin in vitro is not really a chemical neutralization. Thus
Buchner found in his experiments with the tetanus toxin and Anti-
toxin, in a dry powder, that when mixed in a certain proportion and
injected into white mice no tetanic symptoms were induced. But the
same mixture gave rise to distinct tetanic symptoms in guinea-pigs,
showing'that the inference that the toxin had been neutralized in
vitro, based upon the experiment on mice, would have been a mis-
take. And certain observations made by Roux and Vaillard seem
to give support to the view that neutralization does not occur in
vitro, but that the result depends upon some physiological reaction
induced by the antitoxin within the body of the living animal. These
bacteriologists found that when the antitoxin was apparently in ex-
cess, tetanic symptoms could* be induced in susceptible animals if
they had been in any way exhausted prior to the injection of the
mixture of toxin -and antitoxin; and that the same result followed
when their resisting power had been reduced by injecting into them
at the same time filtered cultures of other bacteria.

In this connection the results reported by Calmette, Phisalix,
and Bertrand are of interest. These investigators found that when
the antitoxin of snake-poison was mixed with this venom in a pro-
portion which neutralized its toxic properties, as shown by experi-
mental inoculations, and the mixture then heated to 70° C., by which

SUSCEPTIBILITY AND IMMUNITY. 269

temperature the antitoxin is destroyed, subsequent inoculations
showed that the toxin was still active.

The experiments of Stern (1894) show that the typhoid bacil-
lus not only grows in blood-serum from a typhoid convalescent, which
has been proved to neutralize its pathogenic effects when injected
into a susceptible animal, but also that its toxic products are de-
veloped in this culture medium. From this Stern concludes that the
serum must in some way act upon the infected animal, causing
changes which enable it to resist infection, rather than upon the
bacillus or upon its toxic products directly. It has also been shown
by Behring (1890) for the diphtheria bacillus, by Vaillard for the
tetanus bacillus (1892), and by Issaeff (1893) for the micrococcus of
pneumonia, that these several pathogenic microorganisms may be
cultivated in the blood-serum of animals immunized for the diseases
which they produce.

In a paper published in 1897, Ehrlich advanced his "side-chain"
(seitenkette) theory. He considers the individual cells of the body to
be analogous, in a certain sense, to complex organic substances, and
that they consist essentially of a central nucleus to which secondary
atom-groups having distinct physiological functions are attached by
" side chains " —such as chemists represent in their attempts to illus-
trate the reactions which occur in the building up or pulling down of
complex organic compounds. The cell-equilibrium is supposed to be
disturbed by injury to any of its physiological atom-groups — as by a
toxin — and this disturbance results in an effort at compensatory repair
during which plastic material in excess of the amount required is
generated and finds its way into the blood. This Ehrlich regards as
the antitoxin, which is capable of neutralizing the particular toxin
to which it owes its origin, if this is subsequently introduced into the
blood. In this theory a specific combining relation is assumed to
exist between various toxic substances and the secondary atom-
groups of certain cellular elements of the body. The atom-groups
which, in accordance with this theory, combine with the toxin of any
particular disease germ, Ehrlich calls the "toxophoric side chain."
Immunity, according to Ehrlich, is either "active" or "passive."
Passive immunity results from the introduction of the immunizing
substance from an immunized animal into the circulation of a non-
immune animal, e.g., the use of diphtheria antitoxin as a prophy-
lactic. This passive immunity is more transient than the active
immunity which results from an attack of an infectious disease, from
inoculations with living vaccines, or from repeated injections of in-
creasing doses of the toxins of pathogenic bacteria. Ehrlich's ex-

270 SUSCEPTIBILITY AND IMMUNITY.

planation of immunity, however probable it may appear, can hardly
be said to rest upon a substantial experimental foundation, and we
must admit that the exact source and method of production of the
antitoxins in the animal body, and their mode of action, are still
undetermined ; and, for the present, we must be satisfied with the
knowledge that in some way these so-called antitoxins, which have
been proved, to be present in the blood-serum of immune animals,
protect these animals from infection by pathogenic bacteria. And
that when transferred to susceptible animals they confer upon them
a temporary immunity ; or if introduced after infection, may neutral-
ize the pathogenic action of the toxins produced by specific "disease
germs."

Finally, there is experimental evidence to show that immunity
from the pathogenic action of certain bacteria may be produced by
previous injections of cultures of other bacteria (sterilized or other-
wise), and even by the injection of the blood-serum of normal indi-
viduals or of other substances.

Pasteur' in 1880, communicated to the French Academy of Sciences
the results of experiments which led him to the conclusion that fowls
which had an acquired immunity against chicken cholera also had
an immunity against anthrax. Roux has reported that the blood-
serum of a horse which has been immunized against tetanus neutral-
izes the toxic power of cobra poison. But the contrary effect is not
produced — i.e., the blood-serum of an animal immunized against the
cobra poison does not neutralize the tetanus toxalbumin. The state-
ment is also made that the blood-serum of a rabbit which has been
made immune against hydrophobia will protect a susceptible animal
against the cobra venom in doses. four or five times as large as the
usually lethal dose. Also that rabbits which have been immunized
against snake-poison are less susceptible to the toxic effects of abrin,
and the reverse — i.e., antiabrin neutralizes, to some extent at least,
the toxic action of snake-poison.

The writer, in his " Report on the Etiology and Prevention of
Yellow Fever" (1890),- gives, on pp. 196 and 197, experimental evi-
dence which shows that the injection into the peritoneal cavity of
rabbits of cultures of Bacillus pyocyaneus or of Bacillus gracilis pro-
tected the animals from the fatal results of subsequent injections of
my bacillus X, which was extremely fatal to rabbits when injected
into the cavity of the abdomen in doses of 1 or 2 c.c. In referring
to these experiments I say : " The evidence favors the view that death
results from peritonitis (and toxemia?) induced by intr'a-peritoneal
injections, and that a tolerance on the part of the peritoneum may

PLATE IV.

FIGS. 1, 2, and 3. — Leucocytes from the spleen of an inoculated
monkey, containing Spirillum Obermeieri. (Soudakewitch.)

FIGS. 4 and 5. —Leucocytes (" niacrophages ") from a preparation of
muscle from a pigeon which succumbed to an anthrax inoculation. In
Fig. 4 the bacilli are deeply stained; in Fig. 5 they are pale. (Metsch-
nikoff.)

FIG. 6. — Leucocyte from a frog seventy-two hours after the injection
of anthrax spores. (Trapeznikoff . )

FIGS. 7 and 8. — Leucocytes from a chicken four hours after the in-
jection of anthrax spores. (Trapeznikoff.)

ERNBERG'S BACTERIOLOGY.

Plate F

Fig.l.

Fig. 2.

Fig 3.

Fig. 5.

Fig. 7.

PHAGOCYTES.

SUSCEPTIBILITY AND IMMUNITY. 271

be established by the injection of certain other bacilli, or of ster-
ilized cultures of bacillus X."

This corresponds with facts subsequently developed by Issaeff
(1894) in his* experiments with reference to immunity in guinea-
pigs against cholera cultures injected into the cavity of the abdomen.
He found that a certain degree of immunity was established by the
previous injection of blood-serum from normal individuals', and 'also
of various acids, alkalies, and neutral liquids. The immunity pro-
duced in this way was, . however, feeble and temporary, and could
not properly be considered as identical with that produced by inocula-
tions with attenuated cultures which give rise to a mild attack of a
specific disease.

Cesaris-Demel and Orlandi have reported (1894) their success in
immunizing animals against infection by the typhoid bacillus by
means of sterilized cultures of Bacillus coli communis, and the
reverse.

While this chapter relates especially to acquired immunity from
infectious diseases, and this immunity has been shown to depend, in
a number of these diseases at least, upon the development of anti-
toxins in the body of the immune animal, it may be worth while to
refer briefly, before closing, to some examples of acquired immunity
of a different order, We refer to the tolerance to extremes of heat
and cold which may be established by habitual exposure, and, more
especially, to the tolerance to narcotics and irritant poisons, which is
very remarkable and has never been explained in a satisfactory
manner. Samuel (1*92) has presented experimental evidence which
shows that the local inflammation which results from the application
of croton-oil to the ear of a rabbit does not occur when a second ap-
plication is made to the same ear after recovery from the effects of
the first. That a tolerance may be acquired to comparatively large
doses of arsenic is well known, and the tolerance which the victims
of drug habits acquire to enormous doses of narcotics is a matter of
daily observation. In the writer's paper on acquired immunity, pub-
lished in 1881, an attempt was made to account for acquired im-
munity in infectious diseases as analogous to the immunity to drugs »
just referred to ; but the experimental evidence presented in the pres-
ent chapter shows that the analogy has no scientific foundation in
the absence of any evidence that there is an antitoxin of morphia, of
cocaine, of narcotin, etc., in the blood of the habitues of. these drugs.

IV.
PKOTECTIVE INOCULATIONS.

ANTHRAX.

THE discovery of the anthrax bacillus by Davaine (1863), and the
demonstration of its etiological relation to the disease with which it is
associated, by the researches of Pasteur, Toussaint, Koch (1878-1881)
and other pioneers in this field of investigation, constitute the foun-
dation of our present knowledge of bacteriology and of the practical
results attained in protective inoculations and serum-therapy. And a
review of the literature relating to the anthrax bacillus would show,
in a most interesting manner, the successive steps by which we have
arrived at the important results which have gone so far toward estab-
lishing medicine upon a scientific basis. In the present volume,
however, we must confine our attention to those investigations which
relate directly to the subject in hand.

Toussaint, a pioneer in researches relating to protective inocula-
tions, has a short paper in the Comptes-Rendm of the French Academy
'of Sciences of July 12th, 1880, entitled "Immunity from Anthrax
("charbon") Acquired as a Result of Protective Inoculations."

In this paper he announces his discovery of the important fact that
the anthrax bacillus does not form spores in the tissues or liquids of
the body of an infected animal, but multiplies alone by binary divi-
sion— " sa multiplication sefait toujaurspar une division du mycelium."

In the same communication he reports his success in conferring
immunity upon five sheep by means of protective inoculations, and
also upon four young dogs. We must therefore accord him the prior-
ity in the publication of experimental data demonstrating the practi-
cability of accomplishing this result.

Toussaint does not give his method in the communication above
referred to, but the following quotation from a communication made
to the Academy of Sciences on March 19th, 1881, by Pasteur, shows
the method, and at the same time demonstrates the fact that Tous-
saint was the first to produce immunity by the use of sterilized cul-
tures. Pasteur says :

" By inoculating sheep either with defibrinated blood from an animal
dead of anthrax, after nitration through several thicknesses of- paper, or

PROTECTIVE INOCULATIONS. 273

with the same blood defibrinated and subjected to 55 C. for ten minutes,
according- to Toussaiiit, these sheep subsequently resist inoculations with
anthrax blood. . . . The bacillus, according- to Toussaint, deposits in the
blood of animals in which it multiplies a substance which may become its
own vaccine. By filtration while cold in one case, by a temperature of 55 c C.
in the other, the bacillus is said to be removed or killed ; so that the inocula-
tion of filtered or heated blood introduces into the animal inoculated vac-
cinal matter deprived of bacteria."

After thus stating Toussaiut's method and explanation Pasteur
proceeds to raise objections against this method, the principal of
which are that the anthrax bacillus is not killed by exposure to a tem-
perature of 55° C. for ten minutes, and that inoculation with a virus
prepared in this way would result in a considerable mortality among
the animals inoculated, although those surviving the inoculation would
be protected.

In a communication made to the French Academy of Sciences,
September 27th, 1880, Pasteur gave an account of an experiment made
July 14th, 1879, upon two cows, which in connection with a subsequent
experiment, made August 6th, 1880, upon four cows, led him to the
conclusion that a single attack of anthrax protects from subsequent
attacks. He says in the paper referred to :

"On the 15th of September, 1880, two cows, A and C, which had been
very ill as a result of the first inoculation, made August 6th, were reinoculated
on the left side, that is to say, on the side opposite the first inoculation. We
used five drops of culture of the bacillus of anthrax (' bacte-ridies du char-
bon"1). The following- days there was no perceptible cedema and no elevation
of temperature in either cow. The question is then resolved : a single attack
protects ('le charbon ne r&cidive pas'1)."

The next important steps in the line of experimental research
leading to protective inoculations in the disease under consideration
were reported by Pasteur in his communication to the French Acad-
emy made at the seance of February 28th, 1881 (with the collaboration
of Chamberland and Roux), entitled "De 1' attenuation des virus et
de leur retour a la virulence. " In this connection Pasteur announces
his discovery of the fact that when cultivated at a temperature of 42°
to 43° C., the anthrax bacillus no longer forms spores and rapidly
loses its virulence. He says : ^

' 'As regards its virulence, the extraordinary fact has been ascertained that
the bacillus is no longer virulent after it has been kept for eight days at a
temperature of 42 to 43" C. ; at least its cultures are inoffensive for the
guinea-pig, the rabbit, and the sheep, three species of animals which are
very susceptible to anthrax. We are able, then, not only to attenuate viru-
lence, but to effect its complete extinction, by a simple method of cultivation.

"Before the extinction of its virulence the microbe of charbon passes
through the intermediate degrees of attenuation, and, on the other hand, as
happens also with the microbe of fowl cholera, each of these degrees of vir-
18

274 PROTECTIVE INOCULATIONS.

ulence may be reproduced by cultivation. Finally, as shown in one of our
recent communications, since one attack of anthrax protects, each one of our
attenuated microbes of charbon constitutes a vaccine for the microbe of
superior virulence ; that is to say, a virus suitable to produce a more benign
malady. What, then, is more easy than to find among these a virus suitable
to give anthrax to sheep, cows, or horses, without causing them to perish,
and capable of preserving them from a subsequent fatal attack ? We have
already practised this operation upon sheep with great success."

At the end of this important communication Pasteur says :

"I concluded my communication of October 26th by remarking that the
attenuation of virus by the influence of the air is probably one of the factors
in the extinction of great epidemics. The facts just recorded, in their turn,
may serve to explain the so-called spontaneous appearance of these scourges.
An epidemic which has been terminated by the attenuation of its virus may
be relighted by the reinforcement of this virus under certain influences. The
accounts which I have read of the spontaneous appearance of the plague ap-
pear to me to offer examples of this. The plague is a virulent malady which
prevails in certain countries. In all of these countries its attenuated virus
probably exists, ready to take its active form when the necessary conditions
as to climate, famine, and distress again prevail. There are other virulent
maladies which appear spontaneously in all countries, such as camp typhus.
Without doubt the germs of the microbes which cause these diseases are
everywhere distributed. Man carries them about him, or in his intestine,
without great damage, but ready, nevertheless, to become dangerous when,
as a result of certain conditions or of successive development upon the sur-
face of wounds, in bodies enfeebled or otherwise, their virulence is progres-
sively reinforced. And from this point of view virulence appears to us
under a new light which is somewhat disquieting for humanity, unless na-
ture, in the evolution which has occurred during the past centuries, has al-
ready encountered all possible occasions for the production of virulent or
contagious diseases, an assumption which seems very improbable.

" What is an inoffensive microscopic organism for man or for a given
animal ? It is an organism which cannot develop in our body or in that of
the animal ; but nothing proves that if this microscopic organism should
penetrate into some other of the thousands of species of the creation, it could
not invade it and cause it to become sick. Its virulence, then, reinforced by
passing through a series of individuals of this species, might become such
that it could invade man or one of the domestic animals. By this means new
contagions may be created. I am disposed to believe that it is in this way
that, in the course of ages, have appeared small-pox, syphilis, the plague,
yellow fever, etc."

This broad induction has received considerable support from more
recent researches, which show that the typhoid bacillus, the cholera
spirillum, and other important pathogenic bacteria become attenuated
when they lead a saprophytic existence for some time, and regain their
virulence when they are propagated within the bodies of susceptible
animals.

In a later communication (March 21st, 1881) Pasteur says that he
has found by experiment that when attenuated varieties of the anthrax
bacillus form spores, these again reproduce the same pathogenic va-
riety, so that cultures of each degree of attenuation can be maintained
indefinitely.

PROTECTIVE INOCULATIONS. 275

On June 13th. 1881. Pasteur communicated the results of his famous
experiment at Pouilly-le-Fort, near Melun. He says :

4 'On the 5th of May. 1881, we inoculated, by means of a Pravez syringe,
twenty-four sheep, one goat, and six cows, each animal with five drops of
an attenuated culture of the anthrax bacillus. On the 17th of May we rein-
oculated these animals with a second virus, also attenuated, but more virulent
than the first.

"On the 31st of May we proceeded to make a very virulent inoculation in
order to test the efficacy of the preventive inoculations made 011 the 5th and
17th of May. For this experiment we inoculated the vaccinated animals,
and also twenty-four sheep, one goat, and four cows which had not received
any previous treatment.

"The very virulent virus used on the 31st of May was obtained from
spores preserved in my laboratory since the 21st of March, 1877.

"In order to make the experiments more comparable we inoculated alter-
nately a vaccinated and a non-vaccinated animal. When the operation was
finished all of those present were invited to reassemble 011 June 2d, i.e.,
forty-eight hours after the virulent inoculation \vas made.

" Upon the arrival of the visitors on June 2d, all were astonished at the
result. The twenty-four sheep, the goat, and the six cows which had received
the attenuated virus, all presented the appearance of health. On the con-
trary, twenty of the sheep, and the goat, which had not been vaccinated, were
already dead of anthrax; two more of the 11011- vaccinated sheep died before
the eyes of the spectators, and the last of the series expired before the end of
the day. The non -vaccinated cows were not dead. We had previously
proved that cows are less subject than sheep to die of anthrax. But all had
an extensive oedema at the point of inoculation, behind the shoulder. Cer-
tain of these oedematous swellings increased during the following days to such
dimensions that they contained several litres of liquid, deforming the animal.
One of them even nearly touched the earth. The temperature of these cows
was elevated 3 C. The vaccinated cows did not experience any elevation of
temperature, or tumefaction, or the slightest loss of appetite. The success,
therefore, was as complete for the cows as for the sheep.""

The facts that infection depends to some extent upon the number
of bacilli introduced, and that animals which have a certain degree of
immunity, like the Algerian race of sheep, may succumb when they
are inoculated with a certain quantity of virus, although they resist
a smaller amount, were announced by Chauveau in his communication
to the French Academy at the seance of June 28th, 1880. He says :

"The facts which I have just presented show that the anthrax bacillus
behaves in the organism of Algerian sheep, not as if it were deprived of the
principles necessary for its development, but rather as if it were in a medium
rendered unsuitable for its growth by the presence of substances injurious to
it. In a very small number the bacilli are arrested in their development by
the inhibitory influence of these substances. When they are very numerous,
on the contrary, they surmount more easily this obstacle to their prolifera-
tion."

This quotation shows that Chauveau had at this early date arrived
at an explanation of immunity very nearly in accord with that which
is now generally accepted.

The fact that infection is influenced by the quantity of the infec-

276 PROTECTIVE INOCULATIONS.

tious material introduced had previously been insisted upon by Da-
vaine in his paper entitled " Eeclierclies sur quelques lines des conditions
qui favorisent ou qui empechent le development de la septicemie," pub-
lished in the Bulletin of the Academy of Medicine, seance of February
18th, 1879.

Davaine says :

"A third condition relates to the quantity of bacteria introduced into the
tissues. This question of quantity has been made manifest in our experi-
ments. Not only does it differ in different species of animals, the rabbit and
the dog, for example, but it varies in different animals of the same species."

In his communication to the Academy of Sciences, made on April
4th, 1881, Chauveau gives the results of his experiments in producing
immunity by inoculations with very small quantities of virus. After
some preliminary experiments with a larger number, five sheep were
inoculated with diluted anthrax blood estimated to contain two hun-
dred and fifty bacilli for each. All of the animals survived the inocu-
lation after having manifested some slight febrile reaction. Six weeks
later all were reinoculated with a dose which should have been fatal
to an unprotected animal. One of the animals died of anthrax, the
other four resisted perfectly.

On June 26th, 1882, Chauveau reported to the Academy of Sciences
the results of his experiments relating to the protection of animals
from anthrax infection by the method of Toussaint. By carefully
conducted experiments Chauveau found that nine or ten minutes' ex-
posure to a temperature of 54° C. killed all of the bacilli in anthrax
blood, and the same result was obtained by sixteen minutes' exposure
to 52° C., while at 50° C. the time required is twenty minutes. An
attenuated virus suitable for protective inoculations is obtained by
exposure for a somewhat shorter time, and as a result of his experi-
ments Chauveau was led to the conclusion that for a first inoculation
anthrax blood heated to 50° C. for fifteen minutes afforded a good at-
tenuated virus. This was to be followed after an interval of ten to
fifteen days by a second inoculation with a stronger virus, obtained
by exposing anthrax blood to the same temperature (50° C.) for nine
or ten minutes. These inoculations sufficed to protect the animals
when they were subsequently inoculated with virus of full strength —
blood from an animal which had recently succumbed to the disease.
Chauveau says with reference to this method :

" In one hour, with a single guinea-pig [dead of anthrax], it is easy to
prepare the quantity of vaccine required to inoculate more than five hundred
sheep. The inoculation is made with the point of a lancet, charged, by the
method in use in my laboratory, with a very small quantity of virus. Two

PROTECTIVE INOCULATIONS. 277

or three large punctures under the skin, upon the internal surface of the ear,
suffice for a successful inoculation.

' ' The vaccine prepared in this way should be used at once, or at least
not later than the day after it has been prepared. Experience has shown me
that it is then quite as harmless and quite as efficacious as Pasteur's vaccine."

In the preparation of an attenuated virus by this method Chauveau
insists upon attention to the following points :

' ' The first rule to follow, and the principal one, is to practise the heating
in such a manner that all parts of the anthrax blood are raised to the re-
quired temperature and withdrawn from it at the same instant. When the
quantity of blood to be transformed to a vaccine is too great, all parts are
not uniformly acted upon by the very short exposure to heat ; the virulent
agents in the deeper layers may, in that case, preserve all of their activity,
and cause a fatal infection. To avoid this it is best to enclose the blood in
little cylindrical pipettes, 1 mm. in diameter. The extremity of these
pipettes is sealed, and the portion which contains the blood is immersed in a
considerable quantity of water maintained at the proper temperature. At
the end of the proper time they are taken from the hot bath and plunged into
cold water.

' ' Another rule should be rigorously observed if one wishes to be sure of
success. The blood should be collected under conditions which make it sure
that the virulent agents introduced into the tubes all have the same vitality,
the same activity, and that they are impressed in the same degree by the
heating. This is the case when we take the blood from a guinea-pig just
dead, after having survived from thirty-six to forty-eight hours an inocula-
tion with very active virus. Before introducing the blood into the pipettes
it should be allowed to coagulate, and the coagula should be broken and
crushed in order to obtain a defibrinated blood, which is always very rich in
virulent bacilli."

In a subsequent communication (February 26th, 1883), Chauveau
admits that the application of this method is somewhat difficult and
delicate when blood is employed, and states that it is far more satis-
factory to use pure cultures, which may be attenuated in the same
way. He prefers to cultivate the bacillus in a bouillon made from
the flesh of a chicken, and to start his culture by adding to this bouil-
lon a drop of blood from an animal just dead from anthrax. The cul-
ture is left for twenty hours in an incubating oven at a temperature of
43° 0. During this time there is an abundant development of the
bacillus, and the culture is ready to be subjected to the attenuating
action of a higher temperature. This is accomplished by exposure
to a temperature of 47° C. for a period of one, two, three, or four
hours, according to the degree of attenuation desired. After three
hours' exposure the attenuated culture no longer kills guinea-pigs.
In a later communication (March 5th, 1883) Chauveau states that he
has ascertained by experiment that the degree of attenuation produced
by this method is maintained in subsequuent cultures made at 43° C.,
from the attenuated culture thus obtained.

Another method of attenuating the virulence of anthrax cultures is

278 PROTECTIVE INOCULATIONS.

that described by Cliauveau, in 1885. Tliis consists in cultivating the
bacillus at a temperature of 38° to 39° C., under a pressure of eight
atmospheres. Cultures treated in this way killed guinea-pigs, but
did not kill sheep, cattle, or horses, and constituted a suitable atten-
uated virus for protective inoculations in these animals. One drop
was used for a sheep, and two drops for a cow or a horse, and the
immunity was proved to last for a year.

Kitt, in experiments made in 1884 and 1885, found that an atten-
uation of the virulence of anthrax bacilli may be effected by passing
them through birds, which have but little susceptibility to anthrax
infection; but the results obtained were not uniform, and the method
was not thought to have any great practical value. In the same paper
Kitt gives an account of his experiments with Pasteur's vaccine, No.
1 and No. 2, which he obtained from the agent in Paris. These ex-
periments led him to the conclusion that the attenuated cultures used
by Pasteur are too weak. But by passing them through guinea-pigs
their virulence was increased so that they served to protect cattle and
sheep, although not without danger for the last-mentioned animals.

During the year 1882 Pasteur's method was extensively practised
in the department of Eure-et-Loir, where anthrax was very prevalent
and had been the cause of extensive losses. The results of these pro-
tective inoculations were reported to the Academy of Sciences (seance
of December 18th, 1882) by Pasteur, who submitted, with some re-
marks, a report prepared by M. Boutet, from which we quote as
follows :

"The number of sheep vaccinated during1 the year has been 79,392;
among1 these flocks the average annual loss during1 the past ten years was
7,237 — 9.01 percent. Since the vaccinations but 518 animals have died — 0.65
per cent. We must observe that this year, probably 011 account of the great
humidity, the mortality in Eure-et-Loir has only been three per cent. The
losses should therefore have been 2,382, instead of 518, without the vaccina-
tions. In the flocks which were only partly vaccinated we had 2,308 vacci-
nated and 1,659 not vaccinated ; the loss among1 the first was 8, or 0.4 per
cent.; among1 the second the loss was 60, or 3.9 per cent. We call attention
to the fact that in these flocks, in different cantons of the department, the
sheep vaccinated and not vaccinated were subjected to the same conditions
of soil, of lodging, of food, of temperature, and that consequently they were
exposed to identical influences.

"The veterinary surgeons in Eure-et-Loir have vaccinated 4,562 animals
of the bovine species. Out of this number the annual loss had been 322.
Since the vaccinations only 11 cows have died. That is, the annual mortality
has been reduced from 7.03 per cent, to 0.24 per cent.

" Some engorgements, generally not serious, having occurred after vacci-
nating1 horses, and the mortality not being1 great in this species, the veterina-
rians have thought it prudent not to vaccinate horses 011 a large scale. Only
524 were vaccinated ; three of these died after the first vaccination."

Notwithstanding this favorable report some bacteriologists, and

PROTECTIVE INOCULATIONS. 279

notably Koch, were not disposed to admit the practical value of Pas-
teur's anthrax inoculations. At the conclusion of an elaborate me-
moir published in the second volume of the " Mittheilungen " of the
Imperial Board of Health of Germany (1884), Koch and his collab-
orators (Gaff k v and Loffler) say :

"As now a certain immunity against inoculated anthrax cannot be ob-
tained by the method of Pasteur, as we have seen, without considerable
losses, and as the immunity secured at the expense of considerable loss is
only an imperfect protection against contracting anthrax in the ordinary
way, we must consider the protective inoculations heretofore practised as of
doubtful utility, especially when we remember that the second inoculation
with a yet stronger virus causes the death of more animals which may serve
to further spread the disease."

The attenuating influence of light on the anthrax bacillus and the
fact that cultures attenuated in this way may be used for protective
inoculations was first ascertained by Arloing (1886). Eoux subse-
quently (1887) showed that the presence of oxygen is a necessary fac-
tor in the sterilization of cultures by exposure to sunlight. Behring,
wJio has since been so active in the field of research to which the
present volume relates, published an article in the Centralbkitt fiir
klinische Medicin in 1888 (September 22d) in which he attempted to
explain the natural immunity of white rats against anthrax infection.
His conclusions are given as follows :

"1. The blood-serum of white rats is not a favorable medium for the
anthrax bacillus."

"2. The blood-serum of rats differs from that of animals susceptible to
infection by its gi'eater alkalinity."

"3. By the addition of an acid to the blood -serum of rats this becomes a
favorable medium for the growth of the anthrax bacillus."

"4. The blood-serum of rats which are treated, during life, in such a way
as to reduce the alkalinity of the blood becomes a suitable medium for the
development of the anthrax bacillus."

As we have pointed out in the chapter on Natural Immunity, the
true explanation of the facts ascertained in Behring's experiments is
probably to be found, not in the germiciclal power of the compar-
atively small amount of alkali present in the rat's serum, but in the
fact that the germicidal proteid produced by the leucocytes is only
soluble in an alkaline medium. In a paper published in the Annals
of the Pasteur Institute (August, 1888), Roux and Chamberland have
given an account of experiments made by them which establish the
fact that immunity against anthrax maj" be established by inoculating
susceptible animals with blood from an animal dead from anthrax, in
which the anthrax bacilli had been killed by heat or removed by fil-
tration (Sur 1'immunite centre le charbon conferee par des substances

280 PROTECTIVE INOCULATIONS.

chimiques) . These experiments were commenced in 1881. The
authors named say :

" In repeating the experiments of Toussaint upon anthrax blood which
had been heated, we made several observations which convinced us that it is
possible to confer immunity against anthrax upon sheep by injecting under
their skin anthrax blood which does not contain any living bacilli."

While immunity was produced in this way, Roux and Chamber-
land remark that the sheep which had received a comparatively large
dose were quite sick when subsequently inoculated with a virulent
culture, and the immunity acquired was less reliable than that ob-
tained by Pasteur's method with two vaccines of different degrees of
attenuation.

In an investigation made by Hankin, in the laboratory of Professor
Koch at the Hygienic Institute of Berlin, the results of which are given
in a preliminary account published in the British Medical Journal
(October 12th, 1889), the important fact was ascertained that immunity
may be produced in susceptible animals by inoculating them with an
" albumose " isolated from anthrax cultures. Hankin gives the fol-
lowing account of his method of obtaining this immunizing proteid
from anthrax cultures :

" In the course of my process of preparation it is precipitated from its so-
lution by the addition of a large bulk of absolute alcohol, and well .washed
in this liquid to free it from ptomaines ; it is well known that all such sub-
stances are soluble in alcohol. It is then filtered off and dried ; then it is re-
dissolved and filtered through a .Chamberland filter. A rough estimate of
the percentage of albumose present in the clear solution thus obtained is
made colorimetrically by means of the biuret reaction and a peptone solution
of known strength."

"In one experiment four rabbits (Nos. 23 to 26) were inoculated subcuta-
neously with virulent anthrax spores. No. 26 served as a control and died
in about forty hours. The other three rabbits had the albumose solution in-
jected into the ear-vein at the same time. Nos. 24 and 25 each had about
the five-millionth of their body-weight, while No. 23 had only the ten- mil-
lionth of its body-weight of albumose. No. 25 died in less than forty-eight
hours, but Nos. 23 and 24 survived. Ten days later Professor Koch kindly
reinoculated these two rabbits for me with very virulent anthrax from an
agar-agar culture. Their temperature has remained normal since then, and
they are now alive and well a fortnight after this operation. I have also suc-
ceeded in producing immunity in mice against attenuated anthrax."

In a paper published in the Proceedings of the Koyal Society in
1890, Dr. Sidney Martin has given an account of his researches relat-
ing to " The Chemical Products of the Growth of Bacillus Anthracis,
and their Physiological Action.7' In his experiments the cultures
were maintained for from ten to fifteen days, and the bacilli were then
removed by filtering through a Chamberland filter. The filtrate was
found to contain :

PROTECTIVE INOCULATIONS.

" 1. Proto-albumose, deutero-albumose, and a trace of peptone, all with
the same chemical reactions as the similar bodies formed in peptic digestion.
2. An alkaloid. 3. Small quantities of leuciii or tyrosin. The chief char-
acteristic of the proto- and deutero-albumose obtained from anthrax cultures
was found to be their strong alkalinity in solution. This was not removed
by prolonged dialysis or by washing in alcohol, chloroform, benzene, or ether.
These proteids are precipitated in an alkaline condition by saturation with
NaCl (proto-albumose) or (NH4)2SC>4."

The alkaloid found was soluble in water or in absolute alcohol,
was strongly alkaline in solution, and readily formed salts with acids.
It was slightly volatile and lost its poisonous properties to a great ex-
tent when exposed to the air for some time. A mixture of the two
albumoses was toxic, and when injected into mice in small amounts
caused a local subcutaneous oedema ending in recovery. Larger
doses caused more extensive oedema and death. A fatal dose for a
mouse weighing twenty-two grammes was 0.3 gramme. Boiling for a
short time diminished the toxicity of these proteids without com-
pletely destroying it. The alkaloid produced similar symptoms
when injected into mice, but more promptly and in a smaller dose — 0.1
to 0.15 gramme killed a mouse weighing twenty-two grammes in two
or three hours. Hankin and Westbrook have more recently (1892)
made researches with reference to the proteids present in anthrax
cultures. To obtain an immunizing albumose they cultivated the
bacillus at 20° C. in flesh-extract solution (1:1,000) to which fibrin
was added. At the end of eight days a considerable precipitate was
obtained by means of ammonium sulphate. This was placed in a
dialyzer in running water at 42° to 45° C. ; then precipitated by alco-
hol and dissolved in a small quantity of water (thirty cubic centime-
tres)— five hundred cubic centimetres of flesh extract treated in this
way gave only 0.44 gramme of albumose. Experiments on mice
gave some evidence of the immunizing action of this albumose, but
the results were apparently not so definite as those previously re-
ported by Hankin. Nor are the experiments of Petermann, who
followed Hankin 's method (1892), more satisfactory. Arloing ob-
tained more favorable results by using culture liquids from which
the bacilli had been removed by sedimentation. A considerable
precipitate was obtained when alcohol was added to the culture
liquid, but it was found that this precipitate had no immunizing effect.
On the contrary, there remained in solution an immunizing sub-
stance. This was obtained in a concentrated form by evaporating at
50° C. in a partial vacuum. Experiments upon lambs showed the
protective power of this extract, and of the culture liquids before
treatment when injected in considerable quantity.

PROTECTIVE INOCULATIONS.

In a paper published in the Fortschritte der Medicin, Wysokowicz
gives a resume of the results obtained in Russia in protective inocula-
tions made up to date of publication (January, 1889). According to
the author named, Professor Cenkowski, who had made himself
familiar with Pasteur's method while on a visit to Paris, was the first
to employ it in Russia (1883). But he found its application to be
attended with some difficulties. The cultures attenuated as directed
by Pasteur at 42° to 43° C. "showed a very different degree of viru-
lence in different experiments, and their virulence was also changed
by keeping." Experiments were therefore made with a view to secur-
ing a more satisfactory vaccine. In an experiment made in 1885,
1,333 sheep were inoculated; of these 21 died from the first inocula-
tion and 4 from the second (1.86 per cent). Subsequently better
results were obtained, and up to the end of 1888, 20,310 sheep had
been inoculated, with an average mortality of 0.87 per cent as a re-
sult of the inoculations.

Professor Cenkowski found that greater losses occurred when the
inoculations were made in midsummer or midwinter than when they
were made in the spring or autumn. The losses from anthrax dimin-
ished among the flocks in which the protective inoculations were prac-
tised in proportion to the number of sheep inoculated, falling from
8.3 per cent in 1884, the year before the inoculations were com-
menced, to 0.13 per cent in 1888. The author of the paper states
that in some parts of Russia the annual loss among the sheep from
anthrax is as high as 33 per cent.

The reliability of the protective inoculations was tested by a com-
mission, to which Wysokowicz belonged. Fifty sheep which had been
inoculated from two to four months previously were infected with viru-
lent anthrax material. Of these only one died. Later, twenty sheep
which had been inoculated thirteen months before were inoculated with
virulent material. Of these two died. These favorable results are
ascribed by Wysokowicz to the improved method of attenuating
anthrax virus adopted by Professor Cenkowski. As a first vaccine
he employed a culture which was stronger than that of Pasteur, and
which killed mice and caused the death of one-third of the Ziesel-
mause (Spermophilus citillus) inoculated. He used as a vaccine an
attenuated culture which had been carried through a series of the
animals last mentioned. His vaccine, consisting of a bouillon cul-
ture from a drop of blood of the animal, was preserved by the addi-
tion of two parts of a thirty -per-cent solution of pure glycerin to
one part of the culture.

For inoculating a sheep of average size he used 0.1 to 0.2 cubic

PROTECTIVE INOCULATIONS.

centimetre of this first vaccine; for a larger animal, from 0.3 to 0.5
cubic centimetre. The second inoculation was made twelve days
after the first, with a virus which killed three-fourths of the Ziesel-
mause and from one-third to one-half of the rabbits inoculated with it.
Numerous experiments convinced Cenkowski that no change occurred
in the virulence of his different vaccines when they were carried
through a series of mice or of earless marmots (Zieselmause) .

Hess reports that the anthrax inoculations made by Chauveau's
method in the Canton Bern, during the years 1886, 1887, and 1888,
were not attended with any losses either from the inoculations or from
subsequent attacks of anthrax among the inoculated animals (cattle?).
In all, two hundred and fifty-three animals were inoculated during the
three years specified.

Hutyra (1890) has reported upon anthrax inoculations by Pasteur's
method, as carried out under the regulations of the Government in
1889. The number of horses inoculated was 130, 2 of which died of
anthrax at a later date — not as a result of the inoculation. This gives
a percentage of loss of 1.35, which is much below the usual rate with-
out protective inoculations. Three thousand two hundred and sev-
enty-nine cattle, belonging to 32 different estates, were inoculated.
Of these 11 died from anthrax, and 2 of these as a result of the first
inoculation. Deducting these 2 the loss was 0.27 per cent, whereas
in former years the losses in the same herds had been from 6 to
12 per cent. Twenty -two thousand seven hundred and sixty-
seven sheep were inoculated on 23 different estates. One hundred
and sixty-two of these died from the first inoculation and 59 within
twelve days after the second inoculation. In the course of the year
432 of the inoculated animals died from anthrax — in all a loss of
2.18 per cent. In the absence of protective inoculations the annual
loss in these flocks had been about 10 per cent. It was found that
lambs four months old could be inoculated with the same dose as the
older sheep, and without any greater loss as a result of the operation.

The result of anthrax inoculations made in France by Pasteur's
method during the twelve years ending in 1894 have been sum-
marized by Chamberland. The veterinarians who made the inocula-
tions were each year called upon to answer the following questions :
1. Number of animals inoculated. 2. Number of deaths from first
inoculation. 3. Number of animals dying within twelve days after
second inoculation. 4. Number of animals dying of anthrax within
a year after protective inoculations. 5. The yearly average loss
before inoculations were practised. The total number of animals
inoculated during the period to which this report refers was 1,788,-

284 PROTECTIVE INOCULATIONS.

677 sheep and 200,962 cattle. Tlie average annual loss before
these protective inoculations were practised is said to have been
about ten per cent for sheep and five per cent for cattle. The total
mortality from this disease among inoculated animals, including that
resulting from the inoculations, was 0.94 per cent for sheep and 0.34
per cent for cattle. Chamberland estimates that the total saving as a
result of the inoculations practised has been 5,000,000 francs for
sheep and 2,000,000 francs for cattle.

Podmolinoff gives the following summary of results obtained in
1892 and 1893 in the " government of Cherson " (Austria) : Number
of sheep inoculated, 67,176; loss, 294 = 0.43 per cent. Number of
horses inoculated, 1,452; loss, 8. Number of cattle inoculated,
3,652; loss, 2. The conclusion is reached that Pasteur's method of
inoculation affords an immunity against infection with virulent an-
thrax bacilli in greater amounts than could ever occur under natu-
ral conditions.

BUBONIC PLAGUE.

A number of prominent bacteriologists have been engaged in re-
searches relating to the prevention and cure of bubonic plague by
means of an antitoxic serum, obtained by the same method and in
accordance with the same fundamental scientific principle as in the
case of the antitoxic serum which is now so successfully employed in
the treatment of diphtheria. The experiments thus far made have
apparently been attended with a considerable degree of success. Pro-
fessor Calmette reports that the serum of Yersin prepared at the Pas-
teur Institute in Paris proved to be curative in a considerable propor-
tion of the cases treated during the recent outbreak at Oporto, and that
protective inoculation conferred a temporary immunity, which, how-
ever, did not last longer than twenty days. The mortality in cases
not treated by Yersin 's serum was 70 per cent, in those treated with
it 13 per cent.

The inoculations made by Haffkine in Bombay appear to have been
quite successful. In his first experiment 8,142 persons were inocu-
lated. Of these 18 subsequently contracted the disease and 2 died.
Among 4,926 persons inoculated a single time at Dharwan, 45 were
subsequently attacked and 15 died; while among 3,387 persons in
whom a second inoculation was made, only 2 were attacked. Haff-
kine uses in his inoculations a sterilized culture of the plague bacil-
lus. The inoculation is followed by slight fever and enlargement of
the nearest lymphatic glands. All symptoms disappear at the end
of two or three da vs.

PROTECTIVE INOCULATIONS. 285

The duration of the immunity resulting from these inoculations
has not been definitely determined, although in a majority of those
inoculated it appears to have afforded protection for at least five or
six months. Haffkine's method of preparing his material for protec-
tive inoculations is as follows: A kilogramme of finely chopped goat's
flesh is macerated in diluted hydrochloric acid, and then placed in an
autoclave and heated for six hours under a pressure of three atmos-
pheres. This is filtered, neutralized with KOH, and diluted up to
three litres. The plague bacillus is grown in this medium. Accord-
ing to Haffkine, when the bacillus is planted upon the surface of this
medium, a characteristic growth results. If undisturbed for five or
six days delicate thread-like processes are seen hanging in the culture
medium resembling stalactites suspended from the roof of a cavern.
This growth is said to be peculiar to the plague bacillus. To make
the prophylactic the bacillus is grown in a darkened room in large
flasks. In India it is unnecessary to use a thermostat. Five or six
crops of the stalactites are growrn and shaken to the bottom of the
flasks. This takes about six weeks. The culture is then sterilized
in a water bath at 70° C., the time required beiug about three hours.
A little carbolic acid or thymol is then added, and the material, after
shaking to distribute the bacteria, is decanted into small bottles. It
is now ready for use, and is usually injected into the subcutaneous
connective tissue in doses of two cubic centimetres. A second inocu-
lation in from fourteen to twenty days is recommended by Leumaun,
and after this the blood of the inoculated individual usually gives the
Widal reaction.

CHICKEN CHOLERA.

Pasteur's researches with reference to the etiology of the disease
known in France as cholera des ponies first led him to the discovery
that a virulent culture of a pathogenic bacterium may become " atten-
uated " by certain agencies, and that immunity may be conferred upon
susceptible animals by inoculating them with such attenuated culture.
We now know that his microbe of fowl cholera is a widely distributed
bacillus, which is frequently encountered in putrefying material, and
that it is also extremely fatal to pigeons, pheasants, sparrows, rabbits,
and mice. Also that the same or nearly allied species may produce
an infectious disease of swine (Schweineseuche), of cattle (Hinder-
seuche), and of deer ( Wildseuche).

Subcutaneous injection of a minute quantity of a virulent culture
usually kills chickens within forty-eight hours. Some time before
death the fowl falls into a somnolent condition, and, with drooping

286 PROTECTIVE INOCULATIONS.

wings and ruffled feathers, remains standing in one place until it dies.
Infection may also occur from the ingestion of food moistened with a
culture of the bacillus or soiled with the discharges from the bowels
of other infected fowls. At the autopsy the mucous membrane of the
small intestine is found to be inflamed and studded with small hem-
orrhagic foci, as are also the serous membranes ; the spleen is notably
enlarged. The bacilli are found in great numbers in the blood, in the
various organs, and in the contents of the intestine. In rabbits death
commonly occurs in from sixteen to twenty hours, and is often pre-
ceded by convulsions. The temperature is elevated at first, but
shortly before death it is reduced below the normal. The post-mor-
tem appearances are : swelling of the spleen and lymphatic glands ;
ecchymoses or diffuse hemorrhagic infiltrations of the mucous mem-
branes of the digestive and respiratory passages, and in the
muscles ; and at the point of inoculation a slight amount of inflamma-
tory oedema. The bacilli are found in considerable numbers in the
blood within the vessels, or in that which has escaped into the tissues
by the rupture of small veins. They are not, however, so numerous
as in some other forms of septicaemia— e.g., anthrax, mouse septi-
caemia— when an examination is made immediately after death ; later,
the number may be greatly increased as a result of post-mortem mul-
tiplication within the vessels. The rabbit is so extremely susceptible
to infection by this bacillus that inoculation in the cornea by a slight
superficial wound usually gives rise to general infection and death.
This animal may also be infected by the ingestion of food contami-
nated with a culture of the bacillus. It is by this means that Pasteur
proposed to destroy the rabbits in Australia, which have multiplied
in that county to such an extent as to constitute a veritable pest.
Both in fowls and in rabbits the disease may, under certain circum-
stances, run a more protracted course — e.</., when they are inoculated
with a small quantity of an attenuated culture. In less susceptible
animals — guinea-pigs, sheep, dogs, horses — a local abscess, without
general infection, may result from the subcutaneous injection of the
bacillus ; but these animals are not entirely immune. In the infec-
tious maladies of swine, cattle, deer, and other large animals, to
which reference has been made, and which are believed to be due to
the same bacillus, the symptoms and pathological appearances do not
entirely correspond with those in the rabbit or the fowl ; but the ba-
cillus as obtained from the blood of such animals corresponds in its
morphological and biological characters with Pasteur's microbe of
fowl cholera, and Koch's bacillus of rabbit septicaemia, and pure
cultures from the various sources mentioned are equally fatal to rab-

PROTECTIVE INOCULATIONS. 287

bits and to fowls. lu the larger animals pulmonary and intestinal
lesions are developed, and in swine a diffused red color of the skin,
similar to that observed in the disease known in Germany as Schicein-
erothlauf (Fr. rouget) is sometimes seen.

According to Baumgarten, bacilli from JVildseiiche or from Rinder-
seaclie inoculated into swine give rise to fatal Schweineseuche, and
bacilli from any of these forms of disease, when inoculated into pig-
eons, produce characteristic fowl cholera ; but the bacillus as obtained
from Schweinesucheoi W&dseucke is not fatal to chickens, and the ba-
cillus from Schweineseuche is fatal to guinea-pigs, which have but
slight susceptibility to the bacillus of rabbit septicaemia. Notwith-
standing these differences, he agrees with Hueppe in the view that the
bacilli from the various sources mentioned are specifically identical;
although evidently, if this view is adopted, we must admit that varie-
ties exist which differ somewhat in their pathogenic power.

In this volume this bacillus is described under the name Bac'Jlus
septiccemice hcemorrhagicce, first proposed for it by Hueppe. In the
present chapter we shall give an account of the experimental evidence
relating to protective inoculations in various animals, with the differ-
ent varieties of the bacillus in question which have been encountered.

It seems probable that the same bacillus was the cause of the fatal
form of septicaemia studied by Davaine, which resulted from the in-
oculation of susceptible animals with putrefying blood. These ex-
periments by the distinguished French physician constitute an im-
portant part of the pioneer work in this field of research. They were
commenced in 1868, and are published in the Bulletin of the Academy
of Medicine (seance of Feburary 18th, 1879).

Davaine, in the paper referred to, calls attention to the fact, devel-
oped by his experiments, that there is a great difference in the resist-
ing power of different animals to the form of septicaemia which had
been the subject of his investigations. Thus the rabbit succumbed
when inoculated with a millionth part of a drop of blood, while guinea-
pigs and dogs remained unaffected by such small doses. With refer-
ence to the specific cause of the form of septicaemia studied by him,
Davaine says :

"The virus is one of the bacteria of putrefaction. I say 'one of the bac-
teria,' because there is reason to believe that there are among these minute
organisms numerous species which do not all develop at the same time when
they are present in various media."

Davaine also discovered the fact that infection depends, within
certain limits, upon the quantity of bacteria introduced into the tis-
sues. He says :

288 PROTECTIVE INOCULATIONS.

"This question of quantity was manifest in our experiments. Not only
did it vary in different species, the rabbit and the dog, for example, but it
may vary in the same species/'

The identity of " Davaine's septicaemia " with Pasteur's cholera des
ponies is made still more probable by the experimental evidence
offered by Toussaint in a communication to the French Academy of
Sciences, made by M. Bouley at the seance of July 25th, 1881. In
this communication Toussaint says :

"Three years ago, July 8th, 1878, I had the honor to present to the
Academy an account of a malady due to microbes, which I identified with
that studied by Davaine in 1864 and 1865, and which he differentiated from
anthrax, for which it had been mistaken by Leplat and Jaillard.

"In the month of December, 1878, I made acquaintance with fowl
cholera, and already, in my thoughts, I identified this disease with that
which I had observed in my experiments made early in the year. The mi-
crobes of the two diseases resembled each other perfectly and behaved the
same when inoculated in rabbits. I had, even in 1879, sent to M. Bouley
two notes, in which I called attention to the analogies which exist between
the parasites of the two diseases and the lesions which they determine, not
only in the rabbit but also in pigeons and fowls.

"The experiments of the same kind made at the end of 1879 and in 1880
caused me to insert the note published on page 301, vol. xci., of the Comptes-
rendus, under the title of ' Identity of Acute Experimental Septicaemia
and Fowl Cholera.' I gave a resume in this note of five series of experi-
ments which had demonstrated to me that inoculations of the microbe of sep-
ticaemia give rise to the manifestations of fowl cholera. These results have
recently been confirmed by additional facts."

Toussaint closes his paper by some remarks upon the origin of
epidemics of fowl cholera, which we quote because we believe that the
additions made to our knowledge of the microbe which causes this
disease give support to the views advanced by him in 1881 :

" The causes which determine epidemics of fowl cholera are yet unknown.
It has been supposed that putrefactive substances may give rise to them, and
this has led to the recommendation of cleanliness and disinfection for their
prevention. The microbe which kills the first fowl in an epidemic certainly
came from some anterior generation which had killed others. But how was
it perpetuated ? Do not the facts which demonstrate the development of sep-
ticaemia from material undergoing putrefaction throw some light on the ques-
tion of etiology ? Is it not probable that the fowls find the conditions of
infection with cholera in the presence of organic matter undergoing putrefac-
tion, which may serve as a culture medium for the germs of septicaemia
which are in suspension in the air together with the ordinary germs of putre-
faction?"

Pasteur's first communication relating to the etiology of fowl
cholera was made to the French Academy at the seance of February
9th, 1880. In this communication he calls attention to the fact that
when fowls are fed with bread or meat soiled with a small quantity
of a culture of the microbe of fowl cholera they become infected and
their discharges contain the bacillus in large numbers, a fact which

PROTECTIVE INOCULATIONS. 289

readily accounts for the spread of the disease in a poultry yard when
a case occurs.

In the same communication Pasteur records his observation that
" by a certain change in the method of cultivation the infectious mi-
crobe may be caused to have a diminished virulence." Also the fact
that fowls inoculated with this " attenuated " virus recover and are
subsequently immune against infection by the most virulent microbes.
In concluding this communication Pasteur says :

' ' It appears to be superfluous to point out the principal result of the facts
which I have had the honor to present to the Academy. There are two, how-
ever, which it may be useful to mention. These are, first, the hope of ob-
taining artificial cultures of all kinds of virus ; second, the idea of seeking
for virus vaccines of the virulent maladies which have devastated so often,
and still devastate, the human race, and are such a scourge to J;hat branch of
agriculture which relates to the breeding of domestic animals."

In his communication of October 26th, 1880, Pasteur gives his rea-
sons for concluding that attenuation of virulence is due to the action
upon the microbe of atmospheric oxygen. He infers this from the
fact, demonstrated by experiment, that when cultures are placed in
hermetically sealed tubes, from which the oxygen present is soon ex-
hausted by the growth of the microbe, they do not become attenuated
in virulence; whereas cultures which are freely exposed to the air
gradually become attenuated. Pasteur sees in this an important fact
bearing upon the explanation of the natural extinction of epidemics.
He says :

' ' May we not suppose, then, that it is to this influence that we must at-
tribute, in the present as in the past, the limitation of great epidemics ? "

In his communication to the French Academy, made on February
28th, 1881, Pasteur treats of the attenuation of virulence by the method
above referred to and by the method of Toussaint, and also of the re-
establishment of the virulence of attenuated cultures. He says :

' ' The secret of the return to virulence rests solely, at present, upon suc-
cessive cultures in the bodies of certain animals/'

Thus he had found by experiment that the anthrax bacillus might
be so attenuated that it was harmless for grown guinea-p?gs, or even
for guinea-pigs a month or a week old, but it would still kill guinea-
pigs just born — a day old. By inoculating an older pig with the blood
of this one, and so on, the virulence was gradually augmented, until
finally a virus might be obtained which would kill adult animals, and
even sheep. In the same way the attenuated microbe of fowl cholera
could be restored to virulence by first inoculating small birds, such as

sparrows or canaries.
19

290 PROTECTIVE INOCULATIONS.

Applying these facts, demonstrated by his experiments, to the ex-
planation of the origin of epidemics, Pasteur says :

" I finished my communication on October 26th by calling attention to the
attenuation of viruses by exposure to the air as being probably one of the fac-
tors in the extinction of great epidemics. The facts presented in this paper,
in their turn, may serve to explain the so-called 'spontaneous development'
of these scourges.

' ' An epidemic which has been extinguished by the attenuation of its virus
may be reborn by the reinforcement of this virus under certain influences.
The accounts which I have read of the spontaneous appearance of the plague
appear to me to offer examples of this ; for example, the plague at Benghazi,
in 1856-58, the outbreak of which could not be traced. The plague is a viru-
lent malady which belongs to certain countries. In all of these countries its
attenuated virus ought to exist, ready to resume its active form when condi-
tions as to climate, famine, and distress again occur. There are other viru-
lent maladies which appear ' spontaneously ' in all countries ; such as camp
typhoid. Without doubt the germs of the microbes which cause these last-
mentioned maladies are everywhere distributed. Man carries them upon him
or in his intestinal canal without great damage, but ready to become danger-
ous, when, owing to constipation or to successive development upon the sur-
face of wounds, in bodies enfeebled or otherwise, their viruleiicy is pro-
gressively reinforced. M

We believe that the more complete our knowledge relating to the
origin and extinction of epidemics, of the kind referred to by Pasteur,
becomes, the more apparent will be the value of his inductions and
the clearness of his scientific foresight.

Toussaint, on July 25th, 1881, reported the results of his experi-
ments upon protecting fowls by a " new method of vaccination." This
consisted in inoculating them with the blood of a rabbit which had re-
cently died from septicaemia produced by the same microbe. As a
result of such inoculations the fowls had slight local lesions at the
point of inoculation, and soon recovered. They were subsequently
found to be immune. Cultures from the blood of a septicsemic rabbit
were found to act in the same way. When the culture had been passed
through a pigeon, and had then killed a fowl, according to Toussaint,
it preserved its virulence whsn subsequently passed through the
rabbit.

Salmon, in the " Report of the Commissioner of Agriculture " for
1881 and 1882, gives an account of his experiments in producing im-
munity by the use of a diluted virus. He says :

"The experiments of Chauveau, taken with my own, indicate that this
method is capable of generalization to the same extent as that discovered by
Pasteur : while the ease and quickness with which the vaccine is prepared,
the certainty of effects, the economy of material, and the more perfect pro-
tection are points which would appear to make it decidedly superior.
Wherever the cholera of fowls is raging a standard cultivation may be made
and the vaccine obtained within twenty-four hours ; a single drop of such a
cultivation will vaccinate ten, twenty, or even forty thousand fowls, and
within three weeks of the commencement of work the most susceptible of our

PROTECTIVE INOCULATIONS. 291

fowls are insusceptible to inoculation with the strongest virus. And this,
without any sickness, or even local necroses, which Pasteur describes as fol-
lowing vaccinations with his attenuated virus."

In discussing the practical value of this method Salmon estimates
the cost as trifling— " not more than half a day's time of one man for
one hundred fowls, even if three inoculations were made."

In a paper on protective inoculations against fowl cholera, by Kitt,
in the Deutsche Zeitsclirlft fur Tltiermedicm (December 20th, 1886), the
conclusion is reached that these inoculations undoubtedly protect the
fowls from infection either in the natural way or by inoculations with
virulent material. But Kitt doubts the practical utility of the method
for the arrest of epidemics of this disease in the poultry yard; and,
as we think with justice, prefers to depend upon cleanliness, disin-
fection, and prompt removal of infected fowls. As he points out, a
considerable time is required to produce complete immunity, and two
inoculations are often insufficient. Pasteur had previously reported
that a third inoculation is usually required. But the infection
spreads so rapidly when an epidemic is developed in a poultry yard
that a large proportion of the fowls would be likely to perish before
the protective inoculations could be carried out. Another objection
is that when inoculated in the breast muscle the value of the fowl for
the table is reduced, and when inoculated in the wing an unpleasant-
looking scab is left at the point of inoculation. The cost in material
and time required to carry out the three successive inoculations is
also an objection to the practical application of the method. More-
over, the excreta of the inoculated fowls contain the pathogenic mi-
crobe, and it would evidently be unwise to practise inoculations in
poultry yards not already infected. Kitt states also that he has
always succeeded in stamping out the disease very promptly by the
other measures referred to — disinfection, cleanliness, separation of
all fowls which show any indications of being infected.

In a more recent paper (1893) Kitt reports his success in confer-
ring immunity upon fowls by a new method, which is, however,
rather of scientific interest than of practical value. He first experi-
mented to see whether the blood serum or tissue juices of immune
fowls would give immunity against cholera to other fowls, and ob-
tained a successful result. He was not, however, able to produce im-
munity in pigeons or in rabbits by the same method. He next under-
took to determine whether the immunizing substance was present in
the eggs of fowls which had an immunity as a result of protective in-
oculations. The albumen and yolk of the egg, in doses of five to ten
cubic centimetres, was injected into the breast of fowls, and at the end

292 PROTECTIVE INOCULATIONS.

of ten days a second inoculation of the same kind was made. Six days
after the second inoculation the fowls (five) and a control hen were
inoculated with virulent blood from a pigeon, and at the same time fed
with the chopped-up flesh and liver of a pigeon just dead from fowl
cholera. The control hen died on the following day from typical
cholera, the others remained in perfect health.

CHOLERA.

The spirillum discovered by Koch in 1884 is now generally recog-
nized as the specific cause of Asiatic cholera. But recent researches
indicate that there are numerous pathogenic varieties of this spirillum,
and show that either an attenuated cholera spirillum or a closely allied
saprophyte is not infrequently found in the water of rivers in various
parts of Europe. As this spirillum is found in the intestine of cholera
patients, and not in the blood, it is evident that its pathogenic action
depends upon the chemical products developed during its growth,
and this inference is fully justified by the results of experiments upon
the lower animals. These chemical products have been studied by
Brieger, Pfeiffer, Scholl, Gamaleia, Westbrook, and others.

Brieger (1887) succeeded in isolating several toxic ptomaines from
cultures of the cholera spirillum, some of which had previously been
obtained from other sources — cadaverin, putrescin, creatinin, methyl-
guanidin. In addition to these he obtained two toxic substances not
previously known. One of these is a diamine, resembling trimethyl-
diamine ; it gave rise to cramps and muscular tremor in inoculated
animals. The other poison reduced the frequency of the heart's
action and the temperature of the body in the animals subjected to
experiment. In more recent researches made by Brieger and Frankel
(1890) , a toxalbumin was obtained from cholera cultures which, when
injected subcutaneously into guinea-pigs, caused their death in two or
three days, but had no effect upon rabbits.

Pfeiffer has more recently (1892) published his extended researches
relating to the cholera poison. He finds that recent aerobic cultures
of the cholera spirillum contain a specific toxic substance which is
fatal to guinea-pigs in extremely small doses. This substance stands
in close relation to the bacterial cells, and is perhaps an integral part
of the same. The spirilla may be killed by chloroform, thymol, or
by desiccation, without apparent injury to the toxic potency of this
substance. It is destroyed, however, by absolute alcohol, by concen-
trated solutions of neutral salts, and by the boiling temperature, and
secondary toxic products are formed which have a similar pathogenic

PROTECTIVE INOCULATIONS. 293

action but are from ten to twenty times less potent. Similar toxic
products were obtained by Pfeiffer from cultures of the Finkler-Prior
spirillum and from Spirillum Metchnikovi.

Sclioll (1890) took advantage of the fact, previously demonstrated
by Hueppe, that cultures of the cholera spirillum in egg albumen, in
the absence of oxygen, are more toxic than ordinary bouillon cultures.
Cultures were made by Hueppe's method in hen's eggs. No poison-
ous ptomaines were found, but two toxic albuminous substances were
obtained. The albuminous liquid from the egg cultures was dropped
into ten times its volume of absolute alcohol, which caused a white
precipitate, a portion of which sank to the bottom while another por-
tion floated on the surface. The portion which floated was easily
dissolved in a very dilute solution of potash and could be precipitated
from this solution by the careful addition of acetic acid, but dissolved
in an excess of this acid. It dissolved also in a seven-per-cent salt
solution, but was precipitated by a saturated solution. It gave the
biuret and xanthoprotein reaction. This substance proved to be very
poisonous. It killed guinea-pigs within twenty minutes when a few
cubic centimetres of the alkaline solution — potash — were injected into
the cavity of the abdomen. Scholl calls this substance cholera-toxo-
globulin. The precipitate which fell to the bottom of the receptacle
wras washed with alcohol, then digested with water for twenty minutes
at 40° C. Very little was apparently dissolved out by this procedure,
but this little proved to be very toxic. In from one to three minutes
after the injection of a few cubic centimetres of the solution into the
peritoneal cavity of a guinea-pig the animal died. This aqueous
solution gave the biuret and xanthoprotein reaction ; it was precipi-
tated by mercuric chlorid, nitrate of mercury, and tannin, but not by
a saturated solution of ammonium sulphate or acetic acid. This sub-
stance Scholl calls cholera-toxo-pepton. The toxic action of these
substances is destroyed by a temperature of 100° C., maintained for
half an hour, or by 40° to 45° C., maintained for twenty -four hours.
But at ordinary temperatures they retain their toxic action for several
weeks.

Gruber (1892) has also obtained a toxic albuminous precipitate by
allowing egg cultures to fall into alcohol, drying the precipitate, and
then extracting it with water.

Gamaleia (1893) has obtained a toxin which produces the typical
phenomena of cholera, which, according to him, is closely associated
with the bacteria cells, but can be extracted by a soda solution or
by heating to 55° to 60° C. The conclusion is reached that it is a
nucleo-alburnm analogous to the toxalbumins of tetanus and of

204 PROTECTIVE INOCULATIONS.

diphtheria. It is precipitated by alcohol, acids, and by magnesium
sulphate.

Finally, Westbrook, in a still more recent research (1894), arrives
at the conclusion that the cholera spirillum produces various toxio
proteids which in small amounts produce immunity in susceptible
animals, and the production of which depends to a certain extent
upon the culture medium ; or that its toxin is a substance of constant
chemical composition which is mixed with various albuminous sub-
stances, either contained in the culture medium or developed in the
culture. Duclaux is of the opinion that the last supposition is cor-
rect, and that the so-called toxalbumins are not bodies of definite
chemical composition, but mixtures of toxins and albuminous sub-
stances.

Experiments made upon the lower animals show that the intro-
duction of these cholera toxins into the body of a susceptible animal,
either with or without the living cholera spirillum, results in the
establishing oi: a certain degree of immunity against the toxic action
of cholera cultures . And there is good reason to believe that a non-
fatal attack of cholera in man gives the individual a relative immunity
from subsequent attacks, for some time at least. This has led to ex-
tended experiments with reference to the possibility of producing a
similar immunity in man by means of protective inoculations. The
experiments bearing upon this point which have been made upon the
lower animals will first engage our attention.

Hueppe (1887) first demonstrated the fact that injection of a small
amount of a cholera culture into the peritoneal cavity of a' guinea-pig
is fatal to these animals.

In the following year (1888) Gamaleia reported his success in in-
fecting guinea-pigs by subcutaneous injections of blood from an in-
fected pigeon. He found that by successive inoculations in pigeons
a considerable increase in virulence is established; and that while
guinea-pigs were not fatally infected by subcutaneous inoculations
with ordinary cultures, they invariably died when inoculated with the
more virulent culture in the blood of an infected pigeon. Also, that
when guinea-pigs were inoculated with ordinary cultures, or with
cultures sterilized by heat, they were subsequently immune, and re-
sisted inoculations with the most virulent material. In the same
year the author referred to announced the discovery of a spirillum
which closely resembles the cholera spirillum — his "Yibrio Metch-
nikovi." This was obtained from the intestinal contents of fowls
suffering from a fatal infectious malady (in Odessa). According to
Gamaleia, chickens and pigeons which have survived an inoculation

PROTECTIVE INOCULATIONS. 295

with a culture of this spirillum are subsequently immune against the
pathogenic action of the cholera spirillum, and vice versa. In subse-
quent communications Gamaleia reported that sterilized cultures of
his "Vibrio Metchnikovi " (sterilized by heat at 120° C.) were very
pathogenic for rabbits, fowls, pigeons, and even for dogs and sheep.
The rabbit proved to be the most susceptible animal, and succumbed
to doses of four cubic centimetres in from twelve to twenty hours.
Doses of one cubic centimetre per one hundred grammes of body
weight caused a temporary indisposition followed by immunity.
Pigeons were made immune by larger doses.

The researches of Pfeiffer (1889) confirmed those of Gamaleia as
to the fact that pigecns and guinea-] >igs could be made immune
against Vibrio Metchnikovi by the injection of sterilized cultures.
But guinea-pigs which had been immunized against this pathogenic
spirillum succumbed to cholera infection; and, on the other hand,
animals which had been treated in various ways with a cholera cul-
ture died without exception when infected with Vibrio Metchnikovi.
The conclusion is therefore reached that the two pathogenic spirilla
are distinct species, although very similar in many respects.

Brieger and \Vassermann (1892) have reported the results of ex-
periments with the cholera spirillum cultivated in thymus bouillon.
After twenty -four hours' development in this medium the cultures
were sterilized by heat (55° C. for fifteen minutes) and placed in an
ice-chest for twenty-four hours. Four cubic centimetres of this fluid
injected daily for four clays into the peritoneal cavity of a guinea-pig
made it immune to the cholera spirillum in doses three times as large
as were required to kill an animal not so treated. This immunity
lasted for two months. Fedoroff (1892) obtained similar results by
the subcutaneous injection of sterilized cultures in doses of one cubic
centimetre, in guinea-pigs. His cultures in thymus bouillon were
kept for from seven to ten days at 37° C., then sterilized by heating
for fifteen minutes at 65° C., then allowed to stand in a dark room for
twenty-four hours, and finally mixed with an equal volume of glycerin.

Ketscher (1892) has obtained evidence that the immunizing sub-
stance in animals which have received protective inoculations is con-
tained in the milk of females thus treated. Three goats received
subcutaneous inoculations of virulent cholera cultures, and also injec-
tions into a vein and into the peritoneal cavity. The milk of these
goats was injected into the peritoneal cavity of rabbits ; these proved
to be immune when subsequently lethal doses of a virulent cholera
culture were injected into the peritoneal cavity.

According to Gamaleia (1892), dogs are very susceptible to infec-

296 PROTECTIVE INOCULATIONS.

tion with cholera spirilla, and present symptoms closely resembling
those of cholera in man. They may also be easily immunized against
the pathogenic action of cholera cultures.

Gruber and Wiener (1892) have also found that susceptible ani-
mals are easily immunized against cholera infection either by inocu-
lation with small doses, with attenuated cultures, or with larger quan-
tities of sterilized cultures. Haffkine (1892) also reports his success
in immunizing guinea-pigs and pigeons.

Pawlowsky (1893) claims to have obtained from the blood of ani-
mals having an acquired immunity against cholera an antitoxin in the
form of an amorphous powder; and Lazarus (1892) reports that the
blood of man, after recovery from an attack of cholera, has the prop-
erty of protecting guinea-pigs from fatal infection when injected, in
very small amount, into the peritoneal cavity. Issaeff (1894) in an
extended series of experiments was not able entirely to confirm the
results reported by Lazarus. In a summary of results obtained in
his own experiments he says :

"1. The intraperitoneal or subcutaneous injection of blood serum from
normal individuals [that is, persons who have not suffered an attack of
cholera], and also of various acids, alkalies, and neutral liquids, gives to
guinea-pigs a certain resistance against intraperitoneal cholera infection.
This resistance, however, is feeble and temporary, aud cannot be considered
as identical with the true immunity which results from vaccination with the
products of the cholera bacteria.

"2. Guinea-pigs vaccinated against cholera have no immunity against
the toxins of the cholera vibrio, notwithstanding their high degree of insus-
ceptibility to infection with cultures containing the living vibrio. The blood
of immunized guinea-pigs does not possess antitoxic properties. The maxi-
mum dose of cholera toxins which immune guinea-pigs can withstand is not
greater than that which control animals withstand.

"3. The blood of guinea-pigs carefully immunized against cholera pos-
sesses specific and very pronounced immunizing, and, in a certain sense,
curative powers.

"4. The blood of cholera convalescents possesses similar specific and
curative powers. This property is first developed about the end of the third
week after the attack, and disappears completely at the end of two or three
months."

In a series of experiments made by Pfeiffer and Issaeff the results
obtained, as stated by Pfeiffer in a subsequent communication, were
as follows :

"In my research with Issaeff 'upon the explanation of cholera immu-
nity ' I proved that the serum of animals which have an active acquired im-
munity against cholera only has a specific action upon this particular species
of vibrio, and as regards other species of bacteria does not differ in its action
from the blood serum of normal animals. We also showed that this specific
influence in respect to the intraperitoneal cholera infection of guinea-pigs
was due exclusively to bactericidal processes which in some way were in-
duced by the serum of immune animals."

PROTECTIVE INOCULATIONS. 297

The view of Pfeiffer, founded upon his experimental results, is
that the destruction of the living cholera spirilla, which quickly takes
place in the peritoneal cavity of the guinea-pig, when at the same
time a minute quantity of serum from an immune animal is intro-
duced, is not directly due to the bactericidal action of this serum, but
that in some way it gives rise to a specific bactericidal action in the
exudate which is found in the peritoneal cavity as a result of such in-
jections. His experiments also lead him to the conclusion that this
is accomplished quite independently of phagocytosis.

The brief review of experimental researches relating to cholera
immunity which we have made shows that, while there is a general
agreement as to the possibility of producing immunity in susceptible
animals, there is considerable difference of opinion as to the true ex-
planation of this immunity. The supposition that it is due to an
antitoxin which has the power of neutralizing the toxic products of
the cholera spirillum does not receive any support from the most re-
cent investigations — those of Pfeiffer and Issaeff — which, on the con-
trary, seem to establish the fact that this immunity depends upon an
increased bactericidal activity of the blood serum of immune animals.
A very curious fact developed by the researches of the bacteriologists
last named is that —

"The cholera serum which in the peritoneal cavity of guinea-pigs acted
only upon the cholera bacteria, and behaved toward other vibrios exactly like
the serum of normal animals, in a test tube killed all four species of vibrios
with equal rapidity."

Unfortunately the evidence relating to the value of protective in-
oculations in man, although supported by the evidence already re-
ferred to as regards the lower animals, is, to a considerable extent,
unsatisfactory, owing to the difficulty of applying scientific methods
to experiments of this kind. The evidence, however, is in favor of
the view that a certain degree of protection is afforded by the subcu-
taneous injection of cholera cultures. Such protective inoculations
could not be expected to confer an absolute immunity, inasmuch as
the immunity resulting from a single attack has only a relative value,
and is probably not of long duration.

We quote from Shakespeare's "Eeport on Cholera in Europe and
India, 1890," the following paragraphs relating to immunity as a
result of an attack of cholera :

"IMMUNITY AFTER AN ATTACK OF CHOLERA — EXPERIENCES IN

FRANCE, 1884.

"The Academy of Medicine of Paris directed a circular letter of questions
concerning cholera to the physicians of the localities infected by that disease

298 PROTECTIVE INOCULATIONS.

iii 1884, and in group L of general observations in that questonario is found
the following : ' Have there been observed recurrences among the people
attacked, either in a former epidemic or in the present one ? Give the results
of this recurrence.' In response to their questions the Academy received 184
communications, but the committee appointed to analyze them eliminated
79 ; for various .reasons given only 104 were used for analysis. Of this num-
ber only 8 bore upon the particular question above mentioned, and it is
reasonable to assume that the other 96 observers said nothing concerning this
point because they had observed nothing bearing upon it. The results of
this analysis may be stated as follows :

"From Castelnaudary, with a population of 10,000, we learn that there
were 54 cases and 18 deaths from cholera, among which there was 1 recur-
rence ; from Aix, with 20,257, number of cases unknown, deaths, 117, among
these 2 recurrences were observed, at intervals of ten and forty days ; from
Beseges, with 11,400 inhabitants, we learn of 124 cases and 40 deaths, among
which were 2 recurrences ; from Cette, with 35,000, the number of cases is
not mentioned, but we learn that there were 92 deaths and 1 recurrence ;
from Nantes, with 124,300 inhabitants we learn of 251 cases and 112 deaths,
with 1 recurrence ; from Perpignaii, with 25,000 inhabitants, we hear of 325
cases and 225 deaths, and receive the indefinite statement that there were
some fatal recurrences ; from Pigiiaiis, population not stated, we learn of 22
attacks and 12 deaths, with 1 recurrence ; from Cadenet, with a population
of 26,000, we are not informed of the number of cases, but learned that there
were 20 deaths and 2 recurrences."

"IMMUNITY AFTER AN ATTACK OF CHOLERA — EXPERIENCE IN

SPAIN, 1885.

" While examining cholera in Spain, the writer prepared a circular con-
taining a series of twenty-five questions relating especially to the nature, eti-
ology, and prophylaxis of cholera, one of which requested the physician to
state whether or not, in his own personal experience, he had observed a sec-
ond or a third attack of cholera during the same epidemic, and in case of a
positive reply to detail the symptoms and all the circumstances surrounding
it. This circular-letter was addressed to some twenty-five hundred Spanish
physicians, located in the various cities, towns, and villages in that kingdom
which had suffered from the epidemic. Among the large number of replies
there were only eight in which a second attack was reported, and from an ex-
amination of the details of these there was 110 doubt left in our mind that six
were not genuine second attacks after a complete recovery, but were in
reality relapses due to imprudences of diet or otherwise before convalescence
and complete recovery had been established. Two of the eight cases, from the
details of the reports given, may have been genuine recurrent attacks of
Asiatic cholera, or may have been simply seizures of cholera morbus (cholera
nostras). It is well knowrii that after an attack of Asiatic cholera the di-
gestive apparatus is left in a damaged condition, and disorders of the in-
testines continue for a long time. The habits of life and the imprudences so
common to the class of people most frequently suffering from Asiatic cholera
in that country are such as to render them more than usually liable to suffer
attacks of cholera nostras. As having an important bearing upon this sug-
gestion, the writer made an analysis of the vital statistics of Spain, covering
the five years previous to 1885, for the purpose of learning the extent of
prevalence of cholera nostras among that population, and the result of the in-
quiry shows that the number of deaths attributed to that disease averaged
per year sixteen per every million inhabitants."

Dr. Ferran, who practised inoculations on an extensive scale dur-
ing the epidemic of 1885, in Spain, gives the following account of his
method of performing these inoculations :

PROTECTIVE INOCULATIONS. 299

" 1. The cholera vaccine is nothing more than a pure culture, in bouillon,
of the comma bacillus. Its easy and long preservation (four to five days)
allows of its transportability to great distances, taking care always to keep
the flask which contains the material upright.

" 2. Heat and cold do not interfere with its preservation if the vaccine is
to be used in a short time. It should not, however, be kept out of doors dur-
ing the warm season.

"3. The vaccine should be kept in flasks of the model of Ferraii, with a
flat bottom and a short neck. The stopper, which is of rubber, fits perfectly,
and is penetrated by two glass tubes. One, straight and short, which does not
extend below the inferior surface of the stopper, and which does not project
above more than some two centimetres, is plugged with a small quantity of
sterilized cotton and a superficial covering of wax. The other glass tube is
longer, and extends on the lower side as far as the bottom of the flask, while
its superior end is curved, and terminates in a capillary extremit3r, the tip of
which is closed with wax.

"4. When the vaccine is to be used it is necessary to make two principal
preparations for the operation. A small syringe for the hypodermic injection,
and a small vessel into which it is necessary to empty the fluid from the flask,
are required. The syringe should have metallic pistons and mountings,
without mastic of any kind and without rubber. Its capacity should be one
cubic centimetre, its needle thicker and shorter than that of ordinary use.
Before beginning the vaccination the syringe must be filled two or three
times with boiling water, which is aspirated and expelled through the needle.
This is called sterilizing the instrument, and by this means the extraneous
-••mis are destroyed which might be contained in it, in order to avoid the
production of phlegmons and abscesses. The trouble in taking this precaution
will be little. Acting thus, one may perform thousands of injections without
fear of any accident. It is suggested that it is a bad custom to pass the nee-
dle through a flame in order to sterilize it, because this mode of procedure
draws the temper. Another precaution that must be taken relates to the
examination of the syringe before using it, in order to be well assured that the
piston acts perfectly and that not a single drop of the liquid escapes by a leak
in the cannula. This latter defect is sufficient to reject the instrument. If the
syringe aspires air because the leather washer, which is placed at the end of
the glass tube in order to facilitate its adaptation, is dry, or the piston is in the
same condition, it is necessary to delay a little while in order to take the
syringe apart and soak it in warm water. It is convenient to keep several
syringes for use, with a sufficient number of needles, when many inocula-
tions are to be performed.

"5. The small receptacle into which the vaccine is poured in order that
the syringe may be filled readily is a capsule, a cup, or some similar vessel.
Before use, it should be washed and dried with extreme care, and imme-
diately before using passed through an alcohol or Bunsen flame, in order to
sterilize it.

"6. All these preparations having been made, the drop of wax which
closes the capillary extremity of the long tube of the flask is removed, and at
the same time also the wax covering of the cotton stopper of the short tube,
but by no means must this cotton stopper be removed ; a rubber tube, or the
extremity of a small Richardson spray apparatus, is adjusted to the short
tube. The capillary extremity of the long tube is now slightly warmed in
order to soften somewhat the wax which may have been drawn into its
lumen by capillarity, and air is forced into the flask, either by blowing into
the rubber tube or by working the Richardson atomizer ; the air injected by
pressure upon the vaccine fluid forces the latter out through the long tube
with the capillary extremity, and it is collected in the cup or small sterilized
vessel. This latter is then covered with white paper, which has been
scorched in the flame, or with a sterilized glass plate ; as often as the syringe
is filled this cover will be removed and again immediately afterward replaced.

300 PROTECTIVE INOCULATIONS.

"7. Never should the rubber stopper which closes the flask, or the cotton
which plugs the short straight tube, be removed, because otherwise the
germs of the external air might enter and contaminate the culture, and in
this way give place to local and general accidents among the inoculated.
Whenever, through the movements of transportation, the cotton plug in the
short glass tube has become so wet as to impede the passage of the air which
is to be forced into the flask in the act of expelling the vaccine from it, it
may be removed writh the point of a needle and rapidly substituted by an-
other plug of surgical cotton which has been carbonized or salicylized. If
this proceeds with cleanness and promptness, there is no danger in doing it.
When the cotton, although wet, does not impede the injection of the air, it is
better not to change it.

"8. After terminating the vaccination, again the capillary extremity of
the curved tube is passed through the flame until the small quantity of liquid
remaining in it is evaporated ; it is then stopped a second time with a small
drop of wax ; and from the other glass tube the rubber tube which has been
employed for forcing in the air is removed and another thin layer of wax is
placed over the cotton plug.

"9. If in the smaller vessel or cup any of the vaccine fluid remains after
the vaccination of all persons present, it is boiled, and in this manner the
culture is killed, for it should not be used in another operation, because at-
mospheric germs might become mixed with it.

' ' 10. The technique for the practice of the inoculation is the same as for all
hypodermic injections. The most convenient region is that of the brachial
triceps.

" 11. The dose is one cubic centimetre — or the contents of a syringe — into
each arm, for individuals of all ages and conditions. /

" 12. Five days having elapsed, re vaccinations may be performed by fol-
lowing the same instructions."

Shakespeare, who was sent by the United States Government to
Spain to investigate the results of these inoculations, reports as fol-
lows:

" And now with respect to the human inoculations: The most of these
inoculations were performed in villages in the province of Valencia. The
number of persons inoculated considerably exceeds thirty thousand. Much
has been both said and written in Spain, France, and England concerning
the results of these inoculations. The results which have been published
have appeared to very strongly back up the claim of Dr. Ferrari that chol-
eraic inoculation has the power of protecting the individual against an at-
tack of cholera, and that the extensive practice of this inoculation among
villages already invaded by the epidemic is a powerful and at the same time
harmless means of bringing the epidemic to an end. This being the case,
for those who were unwilling to accept the deductions to be made from the
published statistics the only way of escaping their force seemed to be by
an attack upon their validity.

"The statistics of the anti-choleraic inoculations have been widely at-
tacked. The first public onslaught upon these statistics of which the world,
outside of Spain, had much knowledge was made in the report of the French
Commission, with Dr. Brouardel at its head, which was presented to the
Minister of Commerce after the return of that Commission from Spain in the
summer of 1885. It is charged in that report that the results of the statistics
therein reproduced are assailable on account of having been collected by
physicians who were partisan supporters of Dr. Ferran, and that they neither
possessed any adequate official character nor did they possess sufficient de-
tails. As far as I can learn, the general impression entertained throughout
the world of the value of inoculation statistics is based, in the main, upon
this report of the French Commission.

PROTECTIVE INOCULATIONS. 301

"The statement of that Commission that the statistics which they had
been able to obtain of the preventive inoculations of Ferrari were to a con-
siderable degree void of any official character may be true, and perhaps it is
also true that they emanated from the partisan friends of Ferra.ii ; but it must
be distinctly remembered that at that day there were practically no official
statistics of this kind in the hands of any one. The official statistics collected
under the orders of the Spanish Government were gotten together at a far
later date.

' ' Upon the appointment of the Government at Madrid of the second offi-
cial Spanish Commission to investigate the Ferran question in the provinces
where the inoculations were being practised, it was ordered that official sta-
tistics of inoculation should be collected in the usual manner ; that is to
say, by the customary statistical officers of the Government. This second
medical Commission was also accompanied by an independent statistical
commission who were charged with the duty of forming statistics of those
inoculations which were expected to be witnessed by the Medical Commission
in their tour of investigation, and the report to the Spanish Government of
this statistical commission is based exclusively upon the official statistics
which they themselves collected.

' ' In estimating the value of the official character and the authority of the
official statistics, which have since the visit of the French Commission to
Spain been collected and published, the following circumstances should be
taken into account : The provincial governments of Spain are somewhat
peculiar, in that the civil governors change with the changes which take
place in the Government at Madrid, so that the political constitution of the
provincial governments is always a reflex of that of the central government
at Madrid. Moreover, the political sentiment of the provincial government
is also more or less perfectly reflected by the local governments of the towns
of the province.

" The hostility of the Minister of the Interior at Madrid to Dr. Ferr'dn, and
his attempts at the prevention of cholera by inoculation, is a well-known fact
now generally admitted ; and the hostility which Dr. Ferran met with from
the civil governor of the province of Valencia was even greater than that
manifested by the Minister of the Interior himself.

"The official statistics of the Ferrari inoculations are in the first place
signed by the physicians of the locality ; and in the next place by the judge
of the municipal court, and sometimes also by the president judge of the
judicial district, by the parochial priest, and by the mayor of the municipality,
whose signatures and seals are attested by an authorized notary public.

"It must, therefore, be obvious that the charge made by the French Com-
mission, which has been so constantly reiterated everywhere, that the public
statistics of the anti-choleraic inoculations are void of official character and
are to be regarded as ex-parte testimony of the partisans of Ferran, cannot
apply to official statistics which were collected under the supervision of the
municipal authorities of the villages wherein the inoculations were per-
formed, and attested not only by the local judicial officers and the parochial
priests, but also by the political officers — that is to say, the secretaries and
the mayors of the municipalities ; for it must be admitted that neither the po-
litical officers of the municipalities nor of the provincial governments, any
more than the parochial priest, can reasonably be charged with being the
partisans or friends of Ferran — the Minister of the Interior continuing dur-
ing the time of collection of these official statistics to be hostile to the claims
of Ferran. It therefore follows that the attack upon the statistics of the in-
oculations made by the French Commission, and so widely accepted by the
medical world as conclusive, does not apply to the official statistics of which
we are speaking. And, in view of this fact, the evidence as to the efficiency
and harmlessness of the anti-choleraic inoculations should be re-examined.
As I have already said, the results of the preventive inoculations of Ferran
as set forth in the official statistics appear to very strongly support his claim

302 PROTECTIVE INOCULATIONS.

of the protective value of the inoculations. Iii view of the great importance
of this whole subject, I have determined to place these statistics in this report
for the benefit of the readers of the English language, in order that they mav
judge for themselves of the facts as they appear to be recorded.

"From the Government statistics of cholera throughout the province of
Valencia, it appears that among the villages invaded there were 62 attacks
per one thousand of the population, and 31 deaths per thousand, which gives
a mortality of 50 per cent of those attacked. It appears from analysis of
the published official statistics of cholera in 22 towns where inocula-
tion was performed the inhabitants were divided as follows: 104,561 not
inoculated ; 30,491 inoculated. Of the latter there were 387 attacks of cholera,
or 12 per thousand, and 104 deaths, or 3 per thousand ; the mortality of those
attacked being 25 per cent. Of the former there were 8,406 attacks,
or 77 per thousand, and 3,512 deaths, or 33 per thousand, being a mortality
of those attacked of 43 per cent. It appears, therefore, that among
the population of villages wherein anti-choleraic inoculations had been more
or less extensively performed the liability of the inoculated to attacks of
cholera was 6.06 times less than that of the non-inoculated, whilst the liability
of the inoculated to death by cholera was 9.87 times less than that of the 11011-
inoculated. These figures are based exclusively upon the data furnished by
inoculations, the reiiioculatioiis being left out of consideration, because they
are much less numerous, although from the records of the inoculations it
would seem that the liability of attack, and especially of death by cholera, is
many times less among them than among those inoculated a single time.

•'The charge has also been made with respect to the published records of
the inoculations that the hygienic and physical condition of the subjects of
inoculation have not been sufficiently indicated in the records, and that the
vast majority of those profiting by the opportunity to receive the aiiti-
choleraic inoculations were of the middle and upper classes, and therefore
not of that class of inhabitants who are notoriously most liable to attack and
death from cholera. This criticism may have some justness as respects some,
perhaps many, of the villages where inoculations were performed ; but there
are certainly many of the villages wherein the results of the inoculation
seemed to be most positively in favor of the claim of Ferran where this
criticism cannot hold. I refer to villages wherein three-fourths or four-
fifths of the inhabitants were inoculated, leaving only the fraction of the
population non-inoculated. Even in the absence of any special notes indi-
cating the social conditions and hygienic surroundings of the inoculated in
these villages, it is ridiculous to assume that the vast majority of these were
people of the middle and upper classes, and were therefore but little liable to
attack and death by cholera. Any one acquainted with the character of the
Spanish population as it exists in the rural villages will admit at once that
the vast majority of this population consists of the wretched and the poor,
who live under the most unhygienic and unsalubrious conditions, and there-
fore are of that class most liable to suffer from cholera.

"There is still another result of the preventive inoculations of Ferran
apparently shown by these statistics. I refer to the apparent marked short-
ening of the course of the epidemic after a large percentage of the inhabitants
had become inoculated. It would seem, therefore, from analysis of the
official statistics, that the practice of the an ti -choleraic inoculation after the
method of Ferran, besides giving the subject inoculated a considerable im-
munity from attack and death by cholera, furnishes a means of bringing an
epidemic rapidly to an end."

With reference to Haffkine's method of inoculation we cannot do
better than to quote from a lecture which lie gave in London, in 1893 :

"In the research that I have done at the Pasteur Institute on vaccination
against Asiatic cholera I have chosen for my starting-point the inoculation

PROTECTIVE INOCULATIONS. 303

of the animal into the peritoneal cavity. Starting from this point I have
worked out a method which permits the culture of the microbe in the animal
organism in a state of purity during indefinite generations, the exaltation of
it to a well-determined maximum of strength, and keeping it at the same
degree of virulence for an unlimited period of time.

"This method is illustrated by three series of experiments which were the
subject of our publications in the Comptes rendus de la Societ£ de Biologie
of Paris, and which are :

" 1. Giving the first animal a dose larger than the fatal dose, and killing
this animal in a sufficiently short space of time to be able to find the more
resisting microbes.

"2. To expose the exudation taken from the peritoneal cavity to the air
for several hours.

"3. Then to transfer this exudation to the next animal, of large or small
size, according to the concentration of the exudation.

' ' In the hands of a number of other experimenters this method has given
the same results and showed a perlect consistency.

"The properties of the virus which is obtained in this manner of cultiva-
tion are as follows : Upon intraperitoneal inoculation it kills guinea-pigs
regularly in the space of about eight hours, and the fatal dose for this animal
is reduced to about twenty times less than that which it would have been
necessary to take for the microbe with which I started. The same inocula-
tion kills rabbits and pigeons with a dose which would have been perfectly
harmless at the beginning of the experiments. It kills guinea-pigs by intra-
muscular inoculation.

"The subcutaneous inoculation brings about the formation of a large
oedema, which tends toward sequestration of a whole part of the cutaneous
tissues and to the formation of a wide open wound, which is cured in from
two to three weeks.

"The basis of anticholeraic vaccination is founded on the virus obtained
in the manner we have just described.

"This virus, injected under the skin of a healthy animal, gives it, after
several days, immunity from all choleraic contamination, in whatever man-
ner this may arise ; that is to say, if an animal that has been thus treated be
taken, and an attempt made to infect it either by the digestive canal, by
neutralization of the gastric juice and the injection of opium into the peri-
toneum, or by the introduction of the microbe into the intestines by the
method of Nicati and Rietsch, or by intramuscular inoculation, or finally,
by intraperitoneal injection, the most terrible of all, it resists, whilst the con-
trol animals succumb.

"Anticholeraic vaccination of animals in this manner is then definitely
established. But the operation described cannot be, such as it is, applied to
man. The wound following on the subcutaneous inoculation is terrible to
look at, and, in all probability, extremely painful. Besides, although it
does not in itself present any danger to the health of the individual, it exposes
him to all the complications inseparable from an open wound.

"This power of producing necrosis of the cutaneous tissues has been
removed from the exalted vaccine by cultivating it at a temperature of 39°
C., and in an atmosphere constantly aerated. Under these conditions the
first generations of the cholera microbe would die rapidly, in an interval of
two to three days, and therefore care must be taken to sow them again in
new media immediately before death, and after a series of generations of this
kind a culture is obtained which, if injected under the skin of animals, even
in exaggerated doses, produces only a passing oedema, and prepares the
organism in such a manner that the injection of exalted virus, the definite
vaccine, only produces a local reaction of the slightest description.

304 PROTECTIVE INOCULATIONS.

"VACCINATION BY FIXED VACCINE.

"The method of vaccination thus worked out comprises, then, two vac-
cines— a mild vaccine, obtained by weakening" the fixed virus ; and a
strengthened vaccine, which is presented by the virus itself. It is easy to
understand why, to obtain the weakened vaccine, we do not use an ordinary
virus, but a virus the nature of which has been previously fixed in the labor-
atory. It is because the virus, such as is found in the natural state, especially
when it has a saprophytic phase of development, presents such pathogenic
differences that there is no certainty in its application. Respecting this we
need only recall the story of variolization, and the great danger that an indi-
vidual incurred when the infectious substance from a slightly attacked sub-
ject was transferred to him. The mildness or the gravity of an infection does
not depend only on the veritable strength of the contagious substance, but
upon the resistance of the individual from whom it is taken. Thus it hap-
pened that in taking vaccine lymph from a subject lightly affected, a very
weak substance was sometimes produced, which was incapable of producing
a protective action ; and sometimes a lymph of such strength that it killed
less resistant individuals. The great benefit of Jenhers discovery lay in
that it precisely indicated a substance fixed by passages through animals, and
of a virulence below that which is fatal to the human organism. Another
example is given in the method of Toussaint of vaccination against anthrax,
the first of its kind, which has been obliged to make way for the method of
M. Pasteur, for the sole reason that the latter, based upon virus of a fixed
nature, presented an absolute certainty in its results which was wanting in
the other. Finally, in the history of cholera itself I may recall the attempt
made in 1885 by Dr. Ferraii, of Barcelona, who, with the object of preserv-
ing the population of the Peninsula from the epidemic of cholera, made
injections in his patients of the ordinary virus taken from dead bodies and
cultivated in the laboratory. The statistics of the results obtained by this
means showed such uncertainty that 110 one dared to recommend this opera-
tion to his country in spite of the very numerous trials made in Spain.

44 The possibility of treating the animal organism by vaccines of an abso-
lutely fixed nature, prepared by means of special operations, constitutes, on
the contrary, the basis of the Pasteurian method, and here lies the whole
secret and the sole guarantee of the success of its application.

"APPLICATION OF THE METHOD TO MAN.

"The method of anticholeraic vaccination, worked out by experiments
on guinea-pigs, was tried upon rabbits and pigeons before it was applied to
man. These animals were chosen in order to have subjects very differently
organized, and in order to be able to generalize the conclusions, and to be
able to extend them to the human organism.

" The result obtained 011 all these animals being absolutely the same, it
was decided to apply the operation to man.

' ' The symptoms produced by this operation have been described in several
scientific magazines. The method has been tried at Paris, Cherbourg, and
at Moscow, on about fifty persons of both sexes, between the ages of nineteen
and sixty-eight, of French, Swiss, Russian, English, and American nation-
ality.

' ' In every case the method has shown itself absolutely harmless to health,
and the symptoms that it evoked were a rise in temperature, a local sensitive-
ness at the place of inoculation, and the formation of a transitory oedema at
the same place. The first sensations are felt about two or three hours after
inoculation ; fever and general indisposition disappear after twenty-four to
thirty-six hours ; the sensitiveness and oedema last, gradually dying away in

PROTECTIVE INOCULATIONS. 305

from three to four days. The symptoms following- the second inoculation
were generally rather more marked, but of shorter duration. The whole
recalls the sensation of a bad cold in the head, lasting about one or two days.

"The microbes introduced under the skin do not propagate, but after a
certain time they die and disappear. It is the substances which they contain,
and which are set free when they die, that act upon the animal organism and
confer immunity upon it. It is found that the same result can be obtained if
the microbe be killed before inoculation, and if their dead bodies only be in-
jected. Thus I have been enabled to prepare vaccines preserved in weak
solutions of carbolic acid. In this the microbes die at the end of several
hours, and the vaccine so prepared has been found still efficacious six
months after its preparation. It is evident that there is much advantage in
this state of preservation of the microbes. They can be used by persons
having 110 bacteriological training, and the absence of every living organism
makes them perfectly safe. The carbolic acid that they contain preserves
them against any invasion of other microbes. Finally, as they can be kept
for several months, their preparation can be entrusted to a central laboratory,
whence the vaccine ampoules can be sent out to operators. But it may be
presumed that immunity given by these preserved vaccines will not equal in
persistency that produced by living ones, and as the method is not yet backed
up by established statistics, it is better that vaccinations should be done as
much as possible with living virus, so as to obtain the most conclusive
results.

"As to the length of time that immunity produced by living vaccine
lasts, we have not yet at the laboratory animals that have been inoculated at
a very distant date ; those upon which we experimented dated from, at most,
four months and a half. At the end of this time their immunity was found
to be still perfect, and we do not despair of its lasting much longer yet.

" HARMLESSNESS OF THE METHOD.

"The inoculations upon man, added to the hundreds of experiments that
we have made upon animals, testify to the perfect harmlessness of these
operations, and there is no difficulty in proving their efficacy by experiment,
no matter on what species of animal. We have taken twelve guinea-pigs,
and vaccinated six of them with vaccines preserved in carbolic acid since
September 8th last. Yesterday, at five o'clock, six days after the first vacci-
nation, we injected into the peritoneal cavity of all the non- vaccinated ani-
mals a fatal dose of virus, and into the vaccinated animals we injected a
double dose. The six vaccinated animals are perfectly well, while of the
others two have already died of choleraic poisoning, two are very ill, and the
others will certainly soon become so. But it is evident that I cannot perform
a like experiment on man (but, however, this would be the only means of
being able to give a definite experimental demonstration)/'

Further details as to the method are given by Woodhead in the
"Edinburgh Hospital Keports," as follows:

"In order to be absolutely certain that the virus is pure, M. Haffkine
makes cultivations before each inoculation of the human subject, by Roux
and Yersin's method, one devised for the separation of the diphtheria bacillus.
A small drop of the virus exalte is taken on a spatula-shaped needle, and
streak after streak is made with the flat of this needle on the surface of the
agar in the tubes, a couple of tubes being used, so that twelve streaks per-
haps, in all, are made without the needle being recharged ; in the earlier
streaks, of course, the seed bacilli are so close together that a continuous line
of colonies makes its appearance ; but along the course of the later streaks,
colonies, with distinct intervals between them, are developed ; part of one of
these is examined under the microscope, in order to determine that it is made
20

306 PROTECTIVE INOCULATIONS.

up only of comma bacilli, and then the other part is used for seed material
for a tube culture preparatory to inoculation.

"The inoculation itself is an exceedingly simple process ; the needle and
the syringe are boiled ; the tube containing the material to be used for inocu-
lation receives a syringef ul or pipetteful of sterilized beef broth, then with a
platinum needle the culture is thoroughly mixed with this broth, so that a
kind of emulsion is prepared; this emulsion is drawn up in a sterilized
pipette, and is then passed into a sterilized conical glass covered with steril-
ized paper. If a sixth of the culture is to be introduced, two more syringe-
fuls or pipettefuls of broth are to be added, so that we now have three in all ;
if an eighth, three are added, and so on ; the whole is mixed, and then half a
syringef ul is taken for use for each patient. In inoculating, the skin, just
above the crest of the ilium, is thoroughly cleansed with flve-per-ceiit solu-
tion of carbolic acid, the attenuated virus is inoculated 011 the left side, and
then after an interval of four or five days the second vaccine, or the more
virulent form, is inoculated on the right side. After inoculation everything
that has been used is thoroughly boiled, the skin of the patient is again
washed with five-per-cent carbolic acid, and the table is washed down with
the same solution."

Haffkine commenced his experiments on man by inoculating him-
self, and has repeated the inoculation three times. He next inoculated
about fifty individuals in Paris, Cherbourg, and Moscow, and demon-
strated in a satisfactory way that the inoculations are without danger.

A first inoculation in an unprotected person is said to give rise to
some malaise and febrile reaction, to pain and tumefaction at the
point of inoculation, and swelling of the neighboring glands. The
second inoculation with a strong virus, made after an interval of six
days, causes also some elevation of temperature, but no swelling at
the point of inoculation. This slight reaction from a strong virus is
supposed to be satisfactory evidence of a certain degree of immunity
as a result of the first inoculation.

The results of the protective inoculations by Haffkine's method,
which have been practised in India, indicate that these inoculations
have a real value, but that immunity is not immediately established,
and consequently that during an epidemic a certain number of fatal
cases may be expected among the inoculated as well as among the
non-inoculated. This is illustrated by the results of inoculations
made among the prisoners in Gay a jail (1894), reported by Surgeon-
Major Macrae, I.M.S., from whose report we quote as follows:

" Cholera broke out in Gaya jail on the 9th of July, and from that date
until 2d August 34 cases occurred, with 20 deaths, there being on date of first
attack 422 prisoners in jail. The disease was clearly traceable to importa-
tion, but its diffusion among the prisoners was a question of much greater
difficulty. The sanitary condition of the jail is excellent ; it was built quite
recently, on the latest plans, and is generally considered a model jail. The
water supply, which is from a well, is of excellent quality and protected
from pollution, and it is believed that the spread of the disease was largely
due to the agency of flies finding access to food and milk after being in con-
tact with cholera poison, and contaminating them. From the 9th to the 17th
July six cases occurred, with five deaths.

PROTECTIVE INOCULATIONS.

307

"Many of the prisoners on being told about preventive inoculation wished
to be inoculated, and M. Haffkine, who had previously been communicated
with, and whose zeal and enthusiasm in the cause that he so well advocates
are beyond praise, arrived here on the 18th July, and in the presence of Sur-
geon-Colonel Harvey, who kindly assisted, and myself, inoculated 147
prisoners, and on the 19th 68, making a total of 215 out of 433 present in the
jail on that date.

"Being- purely voluntary, no selection of prisoners was possible ; but all
classes in the jail were represented, male and female, old and young, habit-
uals and less frequent offenders, strong and weakly, convalescent and even
hospital patients sent their representatives. No difference of any kind wTas
made between inoculated and non-inoculated prisoners ; they were under
absolutely identical conditions as regards food, water, accommodation, etc.,
in short, in every possible respect.

" As, owing to the progress of the epidemic, a large number of prisoners
were removed from the jail into camp, it will be found convenient to con-
sider the effect produced by the anticholera inoculation under three head-
ing^ :

"(a) TJie first will include the period from the 18th July, the date of first
inoculations, to the 24th July, the date on which final reinoc illations were
made, and refers to all the prisoners.

" (b) Tlie second concerns the prisoners who remained in jail after the
majority were removed into camp, and comprises the period from 25th July
to 2d August, on which date the final case occurred among this body of
prisoners.

' ' (c) The third refers to the body of prisoners who were moved into camp
on 25th July, and includes the period between that date and 1st August,
when the final case occurred among this body.

Average
present.

Chol-
era.

Percentage
of average
strength.

Deaths.

Percentage
of aveniirc
strength.

Percentage
of deaths to
cases.

Inoculated

211.2

No. 1.
2.37

1.89

80.0

Not inoculated

209.0

3.34

2.39

71.42

Inoculated

32.5

No. 11.
3.07

Nil.

Not inoculated

48.").-)

14.42

6.18

42.86

Inoculated

171.42

No. III.
1.16

0.58

50.0

Not inoculated

146.5

4.09

1.36

33.33

" The conclusions to be drawn from the results above recorded appear to
me to be that for the first few days the inoculations have scarcely any protec-
tive influence : then their effect seems to gradually increase. M. Haffkine in
nis publications has laid stress on the fact that he anticipates a period of ten
-days would elapse from date of first inoculations before the full effect would
be obtained.

Inoculated

Not inoculated. .

DURING THE FIRST FIVE
DAYS AFTER FIRST INOC-
ULATION.

FIRST THREE DAYS AFTER
SECOND INOCULATION.

LAST six DAYS.

Cases.

Deaths.

Cases.

Deaths.

Cases.

Deaths.

4
5

3
5

1
3

Nil.

308

PROTECTIVE INOCULATIONS.

" Further observations are necessary to prove whether the inoculations as
now practised will prove of lasting1 benefit ; the results obtained in Gaya jail
seem to me to justify the conclusion that their temporary beneficial effect is
undoubted.

"I have been informed by M. Haffkine that he proposes to introduce a
certain modification of his method, with the object of affording- protection to
patients during- the ten days necessary for the action of his vaccines. I think
there is every reason to believe that better results would have been obtained
here had the inoculations been performed at an earlier period instead of dur-
ing- the epidemic."

In a paper published in the British Medical Journal (January 26th,
1895), Haffkine gives the following summary of his inoculations in
India :

" TABLE SHOWING THE TOTAL NUMBER OF PERSONS ON WHOM OB-
SERVATIONS HAVE BEEN MADE IN CALCUTTA, GAYA, CAWNPORE,
AND LUCKNOW.

Number.

Cases.

Percentage
of Cases
to Strength.

Deaths.

Percentage
of Deaths
to Strength.

Non -inoculated

1 735

174

10.63

113

6.51

Inoculated

500

4.20

3.80

Total

2,235

195

132

Other methods of producing immunity in man have been proposed,
and experiments indicate that this may be accomplished through the
digestive tract by the ingestion of considerable quantities of steril-
ized cultures. Thus Klemperer (1892) has obtained results which
seem to show that immunity in man may be induced, not only by the
subcutaneous injection of virulent cultures, but also by the subcuta-
neous injection of the milk of immunized goats and by the ingestion of
cultures sterilized by heat. The degree of immunity, as determined
by the activity of the blood serum of the immune individual for the
protection of guinea-pigs, is considerably less, however, than when
repeated injections of virulent cultures have been made. The blood
serum of individuals made immune by the last-mentioned method is
said by Klernperer to protect guinea-pigs when injected into the cav-
ity of the abdomen in the dose of 0.005 cubic centimetre. And the
injection of five cubic centimetres of milk from an immunized goat is
said to confer such an immunity that 0.25 cubic centimetre of blood
serum from the immune individual is sufficient to protect a guinea-pig
from cholera cultures.

Sawtschenko and Sabolotny (1893), as a result of a series of ex-
periments made upon themselves and laboratory assistants, arrive at
the following conclusions :

PROTECTIVE INOCULATIONS. 309

" 1. After the ingestion of sterilized (by heat) and subsequently carbolized
agar cultures of cholera bacteria the serum of man acquires an immunizing
property as regards the cholera vibrio.

4k2. As a result of the ingestion of sterilized agar cultures the individual
is protected from infection with virulent cultures of the cholera vibrio by way
of the intestine.

" 3. The discharges of individuals immune against cholera, and to all out-
ward appearance in perfect health, may contain a great number of cholera
vibrios (in case they are in any way introduced into the intestine) and may
thus serve to propagate the malady."

DIPHTHERIA.

According to Roux and Yersin, " attenuated varieties " of the diph-
theria bacillus may be obtained by cultivating it at a temperature of
39.5° to 40° C. in a current of air; and these authors suggest that a
similar attenuation of pathogenic power may occur in the fauces of
convalescents from the disease, and that possibly the similar non-
pathogenic bacilli which have been described by various investigators
have originated in this way from the true diphtheria bacillus. These
authors further state, in favor of this view, that -from diphtheritic
false membrane, preserved by them in a desiccated condition for five
months, they obtained numerous colonies of the bacillus in question,
but that the cultures were destitute of pathogenic virulence. They
say :

" It is then possible, by commencing with a virulent bacillus of diphtheria,
to obtain artificially a bacillus without virulence, quite similar to the attenu-
ated bacilli which may be obtained from a benign diphtheritic angina, or even
from the mouth of certain persons in good health. This microbe, obtained
artificially, resembles completely the pseudo-diphtheritic bacillus ; like it, it
grows more abundantly at a low temperature ; it renders bouillon more rap-
idly alkaline ; it grows with difficulty in the absence of oxygen."

Subcutaneous inoculations in guinea-pigs of a small quantity of a
pure culture of the bacillus (0.1 to 0.5 cubic centimetre of a bouillon
culture) cause death in from one to four or five days. The usual
changes observed at the autopsy are —

" An extensive local oedema^ with more or less hyperamiia and ecchymo-
wis at the site of inoculation, frequently swollen and reddened lymphatic
glands, increased serous fluid in the peritoneum, pleura, and pericardium,
enlarged and hemorrhagic suprarenal capsules, occasionally slightly swollen
spleen, sometimes fattA degenerations in the liver, kidney, and myocardium.
We have always found the Loftier bacilli at the seat of inoculation most
abundant in a grayish- white, fibrino-purulent exudate present at the point of
inoculation, and becoming fewer at a distance from this, so that the more re-
mote parts of the oedematous fluid do not contain any bacilli " (Welch and
Abbott).

The authors quoted agree with Loffler and others in stating that
the bacillus is found only at the point of inoculation. In all cases

310 PROTECTIVE INOCULATIONS.

their cultures from the blood and from the various organs gave a
negative result.

Rabbits are not so susceptible, and may recover after the subcu-
taneous inoculation of very small doses, but usually die in from four
to twenty days when two to four cubic centimetres of a bouillon cul-
ture have been introduced beneath the skin. In these animals, also,
there is an extensive local oedema, an enlargement of the neighboring
lymphatic glands, and a fatty degeneration of the liver. Eoux and
Yersiu have shown that in rabbits, when death does not ensue too
quickly, paralysis of the posterior extremities frequently occurs, thus
completing the experimental proof of the specific pathogenic power
of pure cultures of this bacillus.

Similar symptoms are produced in pigeons by the subcutaneous
inoculation of 0.5 cubic centimetre or more, but they commonly re-
cover when the quantity is reduced to 0.2 cubic centimetre (Roux and
Yersin) .

The rat and the mouse have a remarkable immunity from the effects
of this poison. Thus, according to Roux and Yersin, a dose of two
cubic centimetres, which would kill in sixty hours a rabbit weighing
three kilogrammes, is without effect upon a mouse which weighs only
ten grammes.

Old cultures are somewhat less virulent than fresh ones, but when
replanted in a fresh culture medium they manifest their original viru-
lence. Thus a culture upon blood serum which was five months old
was found by Roux and Yrersin to kill a guinea-pig in five days, but
when replanted it killed a second animal of the same species in
twenty -four hours.

Evidently a microorganism which destroys the life of a susceptible
animal when injected beneath its skin in small quantity, and which
nevertheless is only found in the vicinity of the point of inoculation,
must owe its pathogenic power to the formation of some potent toxic
substance, which, being absorbed, gives rise to toxaemia and death.
This inference in the case of the diphtheria bacillus is fully sustained
by the results of experimental investigations. Roux and Yersiu
(1888) first demonstrated the pathogenic power of cultures which
had been filtered through porous porcelain. Old cultures were found
by these experimenters to contain more of the toxic substance than
recent ones, and to cause the death of a guinea-pig in a dose of two
cubic centimetres in less than twenty -four hours. The filtered cul-
tures produced in these animals the same effects as those containing
the bacilli — local oedema, hemorrhagic congestion of the organs,
effusion into the pleural cavity. Somewhat larger doses were fatal

PROTECTIVE INOCULATIONS.

to rabbits, and a few drops injected subcutaneously sufficed to kill a
small bird within a few hours. In their second paper (1889) the
authors mentioned state that so long as the reaction of a culture in
bouillon is acid its toxic power is comparatively slight, but that in
old cultures the reaction is alkaline, and in these the toxic potency is
greatly augmented. With such a culture, filtered after having been
kept for thirty days, a dose of one-eighth of a cubic centimetre in-
jected subcutaneously, sufficed to kill a guinea-pig; and in larger
amounts it proved to be fatal to dogs when injected directly into the
circulation through a vein.

The same authors, in discussing the nature of the poison in their
filtered cultures, infer that it is related to the diastases, and state that
its toxic potency is very much reduced by exposure to a comparatively
low temperature— 58° C., for two hours — and completely destroyed by
the boiling temperature — 100C C., for twenty minutes. It was found
to be insoluble in alcohol, and the precipitate obtained by adding al-
cohol to an old culture proved to contain the toxic substance. Loffler
also has obtained, by adding five volumes of alcohol to one of a pure
culture, a white precipitate, soluble in water, which killed rabbits in
the dose of 0.1 to 0.2 gramme when injected beneath the skin of these
animals. It gave rise to a local oedema and necrosis of the skin in
the vicinity of the point of inoculation, and to hyperaemia of the in-
ternal organs. This deadly toxin appears to be an albuminoid sub-
stance, but its exact chemical composition has not yet been deter-
mined.

Brieger and Frankel (1891) obtained results corresponding with
those previously reported by Roux and Yersin. Their researches
showed that the toxic substance contained in diphtheria cultures is
destroyed by a temperature of 60° C. ; that it is soluble in water, and
insoluble in alcohol; that it does not pass through a dialyzing mem-
brane, and has not the chemical characters of the ptomaines or toxins,
but is an albuminous body — a toxalbumin. It wras obtained by the
authors named by precipitation with slightly acidified (acetic acid)
alcohol; the precipitate, after being washed in a dialyzer and dried
in a vacuum at a temperature of 40° C., was a snow-white, amorphous,
crumbling mass.

Wassermann and Proskauer (1892) found that the alcoholic pre-
cipitate from diphtheria cultures contains two different substances,
which are distinguished by their different degrees of solubility in
dilute and absolute alcohol ; both, however, give the usual reactions
of albuminous bodies, and pass very slowly through a dialyzing mem-
brane. Only one of these substances possesses toxic properties.

312 PROTECTIVE INOCULATIONS.

After the removal of peptone and globulin from the filtered cultures,
these were evaporated and a precipitate obtained of one of the
albuminous substances by means of sixty to seventy per cent al-
cohol. The other substance remained in solution, and was sub-
sequently obtained by precipitation with absolute alcohol. The
substance first obtained by this method is toxic, and the other pre-
cipitate is not. The authors named succeeded in killing rabbits with
the toxalbumin obtained in this way, but were not able to produce
immunity in these animals by the injection of non-fatal doses. Friin-
kel (1891) had previously reported his failure to immunize guinea-pigs
by the injection of the dry precipitate, obtained in his experiments
from diphtheria cultures; but when filtered cultures, or cultures
sterilized by heat (55° C. for one hour), were injected into these ani-
mals, they showed an increased resistance to the pathogenic action of
virulent cultures. Still better results were obtained when ten cubic
centimetres of a bouillon culture, heated to 100° C., were injected
subcutaneously, but still this method was not entirely reliable. But
true immunity was established by injecting into the peritoneal cavity
ten to twenty cubic centimetres of a bouillon culture heated to from 65°
to 70° C. for one hour. The immunity was not fully established until
about fourteen days after the protective inoculation. Friinkel arrives
at the conclusion that the cultures must contain an immunizing sub-
stance as well as a toxic proteid, as the diphtheria toxalbumin is de-
stroyed by the temperature (65° to 70° C.) used in the preparation of
his cultures for producing immunity.

Behriug, in the same year (1891), commenced his experiments
upon diphtheria immunity. Guinea-pigs were made immune by the
use of sterilized cultures, and by inoculations with virulent cultures,
four weeks old, to which iodine terchloride had been added in the
proportion of 1 : 500— the mixture was allowed to stand for sixteen
hours. Animals were also immunized by injecting beneath the skin
a virulent culture of the bacillus, and then treating them with sub-
cutaneous injections of iodine terchloride (two cubic centimetres),
which was thrown under the skin for three days in succession in the
vicinity of the point of inoculation. The guinea-pigs treated in this
way remained sick for some time, but finally recovered and were
subsequently immune. Still better results were obtained when rab-
bits were subjected to the same treatment. The animals were im-
mune against the toxic action of sterilized cultures, as well as against
infection by virulent diphtheria bacilli.

In subsequent experiments (1892) Behring and Wernicke used
cultures which had been attenuated bv contact with iodine terchloride

PROTECTIVE INOCULATIONS. 313

for from thirty-six to forty-eight hours, and proved that the method
could be successfully employed in immunizing sheep ; and the fact
was ascertained that blood serum from an immune animal could be
used with success in arresting diphtheritic infection in susceptible
animals. To preserve the serum, which they obtained from immun-
ized sheep, rabbits, and guinea-pigs, they added to it 0.5 per cent of
pure carbolic acid. For producing immunity they found that a
smaller amount of serum was required than wras necessary for the
cure of an animal already infected. If the injection was made imme-
diately after infection, from one and a half to two times the amount
was required; eight hours after infection the amount was three times
as great, and twenty-four to thirty-six hours after infection the dose
required was eight times the immunizing dose.

The immunizing value of blood serum from different animals was
estimated by finding the smallest dose which would protect an animal
from fatal infection by the minimum lethal dose of a culture the toxic
potency of which had been carefully determined. The value is ex-
pressed in figures which give the proportion required compared with
the body weight of the animal. Thus an immunizing value of one
hundred would mean that one gramme of the serum is sufficient to
protect an animal weighing one hundred grammes from the fatal
effect which would be produced in a control animal of the same weight
by infection with a virulent culture of the diphtheria bacillus in the
minimum doses required to produce this result. The cultures em-
ployed are made in bouillon containing one per cent of peptone ; they
are inoculated from agar cultures and are kept in the incubating oven
for two days. Cultures prepared in this way were found to be quite
uniform in their pathogenic virulence as tested upon guinea-pigs. But
when cultures are kept for some time there is an increase in virulence.
Thus a culture obtained from a fatal case of diphtheria which in 1890
killed guinea-pigs in three to four days, when injected subcutaneously
in the dose of 0.1 cubic centimetre (two-days-old bouillon culture),
at the end of a year was fatal to these animals in the dose of 0.025
cubic centimetre. This increase in virulence is ascribed to the fact
that the cultures were renewed at long intervals.

More recently (1894) Behring has fixed a standard for what he
calls a normal therapeutic serum. This is a serum which when in-
jected into guinea-pigs in the proportion of 1 : 5,000 of bod}' weight
saves the animal from the fatal effects of ten times the minimum dose
of a culture in bouillon, two days old, which would kill a control ani-
mal not treated.

In a subsequent communication (November, 1894) Behring states

314 PROTECTIVE INOCULATIONS.

his conclusion that for producing immunity in man, one hundred and
fifty normal antitoxin units should be given, instead of sixty as he
had previously recommended.

The serum manufactured under his direction is said (September,
1894) to be of two kinds — one, obtained from the horse, has a value
of sixty normal antitoxin units ; the other has a value of one hundred
and forty units. Of the weaker serum Behring says experience has
demonstrated that for children under ten years of age ten cubic cen-
timetres is sufficient to arrest the progress of the disease and effect a
cure if given within two or three days from the outset of the attack.
For producing immunity in children subject to infection, one-tenth
of this amount (one cubic centimetre) is said to be sufficient. Of
the stronger serum one cubic centimetre is sufficient to arrest the dis-
ease during the incubation period ; and, according to Behring, out of
one hundred cases treated during the first forty-eight hours with a
single therapeutic dose (ten cubic centimetres of serum having a value
of sixty, equals six hundred normal units), not five will die. The
later the treatment is commenced the larger will be the dose required.
Behring further states that the diphtheria antitoxin has no injurious
effect upon animals in the largest doses that have been employed,
and that aside from its antitoxic power its properties are entirely
negative so far as living animals are concerned.

Aronson (1893), in experiments on dogs, succeeded in producing
immunity by the use of attenuated cultures, or of cultures to which
formaldehyde had been added ; also by feeding the animal large quan-
tities of diphtheria bouillon; and, finally, by injection of the blood of
naturally immune animals (white rats) into which large quantities (ten
cubic centimetres) of a virulent culture had been injected. Two months
after receiving several such injections it was found that 0.2 gramme
of blood-serum from the rat sufficed to save a guinea-pig from fatal
infection. In experiments on dogs an immunity was established in
six weeks by the injection of a large amount of a virulent culture. Its
serum had a value of 1 : 30,000, i.e., 0.01 cubic centimetre of this serum
sufficed to protect a guinea-pig weighing three hundred grammes.
From one hundred grammes of this serum Aronson claims to have ob-
tained 0.8 gramme of a substance which had a value of 1 : 500,000, as
tested in the treatment of an animal which had received ten times
the minimum fatal dose of a two-days' bouillon culture. A ten-per-
cent solution of this substance had, therefore, ten times the value of
Behring.' s " normal serum. " The precipitated antitoxin was soluble in
water, and more readily so in a slightly alkaline solution, and gave
all the reactions of an albuminous body. When dried in vacuo at

PROTECTIVE INOCULATIONS. 315

40° C., and then heated to 102° C., it still retained its antitoxic
potency.

Ehrlich, Kossel, and Wasserniann (1894) have made experiments
upon goats, which they found very susceptible to the action of the
diphtheria poison. Sterilized cultures were first injected in gradually
increasing amounts, and later virulent cultures. In this way they ob-
tained a serum which has a value sixty times that of Behriug's "nor-
mal serum." In a subsequent communication (1894) Wassermann
gives an account of his experiments with the milk of immunized
goats, which contains the antitoxin in considerable quantity, and
from which it was obtained in a concentrated form by the following
method : The milk is obtained in sterilized vessels and twenty cubic
centimetres of normal hydrochloric acid are added to each litre; a,t
sufficient quantity of rennet is then added to coagulate the casein,
and this is separated from the liquid, which is then shaken up with
chloroform for some time. The liquid is now allowed to stand in
order that the butter, which has been dissolved by the chloroform,
may sink to the bottom. The clear liquid is then decanted and the
antitoxin precipitated from it by means of ammonium sulphate (thirty
to thirty-three per cent). The precipitate is rapidly dried upon
porous porcelain plates, in vacuo, and then dissolved in water in the
proportion of ten parts for one hundred of milk first employed — a
concentration to one-tenth. Of this solution 0.125 cubic centimetre
was found to neutralize 0.9 cubic centimetre of a toxin which killed
guinea-pigs weighing five hundred grammes in the dose of 0.1 cubic
centimetre. This toxin was an old bouillon culture of the diphtheria
bacillus to which 0.5 per cent of carbolic acid had been added to
preserve it. In a communication of the same date Ehrlich and Was-
sermann report that they have for some time had a cow immunized
to such a degree that one cubic centimetre of its milk protects guinea-
pigs from the fatal effects of 0.9 cubic centimetre of the above-men-
tioned toxin. The antitoxic value of the milk of an immunized cow
or goat, as compared with that of its blood, is estimated by Ehrlich
and Wassermann as from 1 : 15 to 1 : 30 — usually about 1 : 20.

Aronson, in testing his antitoxin, uses a bouillon culture of the
diphtheria bacillus two and one-half months old, which he preserves
by the addition of 0.3 per cent of trikresol. He finds that the im-
munity which results from injections of the antitoxin is established
at once; that it is not accompanied by any reaction or symptom of
sickness ; and that it is of comparatively short duration.

As a result of extended experiments made at the Pasteur Institute
in Paris, Koux has perfected the following method for the production

316 PROTECTIVE INOCULATIONS.

of an antitoxin suitable for use in the treatment of diphtheria in man.
The horse has been found the most suitable animal for this purpose,
on account of his slight susceptibility and the ease with which a high
degree of immunity can be established; and because of the large
amount of blood that may be drawn without injury to the animal.
Roux prepares his toxin by cultivating the diphtheria bacillus in a
slightly alkaline bouillon made from beef and containing two per
cent of peptone and 0.5 per cent of sodium chloride. This medium
is placed in flat-bottomed flasks, and should not be more than half an
inch in depth. Two glass tubes pass into the flask, which serve for
inlet and outlet tubes to be used in passing a current of air over the
cultures. This is commenced when the growth is fairly started, at
the end of twenty-four hours, and the air should be moist to prevent
the evaporation of the culture. In Roux's laboratory a flask is used
which has a tube attached to one side, about an inch from the bottom,
and which is known as a Fernback flask. A flocculent deposit falls
to the bottom and gradually accumulates for about a month. This
consists of bacilli which have for the most part lost their vitality and
are undergoing , degeneration. At the end of thirty days, during
which time they are kept in an incubating oven at a temperature of
37° C., the cultures are passed through a Pasteur-Chamberland filter,
and 0.5 per cent of carbolic acid may be added in order to preserve
them. This filtrate is so toxic that a dose of 0.1 cubic centimetre
will kill a guinea-pig weighing five hundred grammes in less than
forty-eight hours. A healthy horse is selected and receives at first a
dose of 0.5 cubic centimetre of the filtered culture (or of the clear
fluid obtained from a culture by decantation, and containing 0.5 per
cent of carbolic acid). The dose is gradually increased at intervals
of a few days, and is followed each time by some febrile reaction and
tumefaction at the point of inoculation. When the reaction is exces-
sive, a little Gram's solution is added to the following dose. The
usual plan of treatment is stated by Kinyoun as follows :

" First day, 1 to 2 c.c. of pure toxins, of which 1 to 10 c.c. fatal to a 500-
gm. guinea-pig ; eighth day, 1 c.c. : fourteenth day, 1.5 c.c. : twentieth day,
2 c.c. ; twenty-eighth day, 3 c.c. ; thirty-third day, 5 c.c. ; thirty-eighth day,
8 c.c. ; forty- third day, 10 c.c. ; forty-seventh day, 20 c.c. ; fifty-first day, 30
c.c. ; fifty-sixth day, 50 c.c. ; sixty-second day, 50 c.c. ; sixty-eighth day, 60
c.c. ; seventy-fourth day, 100 c.c. ; eightieth day, 250 c.c. ; eighty-eighth clay,
250 c.c.

"When the first injections are given there is quite a marked local and gen-
eral reaction to the poison ; there is an oedema at the point of the injection,
which is followed by a distinct inflammatory process— hard in the centre and
soft and cedematous at its periphery. The general reaction is manifested by
a rise in the temperature, 1° to 2° C., loss of appetite, and occasionally cramps.
The reaction must be taken as the guide in the future dosage, and a sufficient

PROTECTIVE INOCULATIONS. 317

time must be allowed to elapse between the injections for the complete recov-
ery from the general and local effects. As the quantity of the toxins is in-
creased the general effects generally decrease, perhaps a rise of a degree for
twenty-four hours. The local effect partakes more of an cedenia, and has the
character of an inflammation.

" At a certain stage, usually after two months' treatment, when fifty to
sixty cubic centimetres can be injected without harm, there is no general re-
action, but a large oedema at the site of the injection, which disappears
within from twenty -four to forty-eight hours. Toward the last, even when
two hundred to three hundred cubic centimetres are given, there is only an
enormous oedema, which disappears within from twelve to eighteen hours.
When these inordinately large quantities can be given with only a local re-
action being manifest, the horse has come well under the influence, and the
blood will be found to be rich in the antitoxin.

"There is a curious fact well worth noting : At the end of the second
month of treatment, when the horse can bear as much as fifty to sixty cubic
centimetres of the toxins without discomfort, the blood will be found to con-
tain but little of the antitoxin. The antitoxin only appears after repeated
stimulation of the cells (?) by the large and frequent doses of the toxins."

The subcutaneous injections do not yield a serum as rich in the
antitoxins as when the toxins are injected directly into the blood cur-
rent. When it is desired to do this, toward the last of the treatment
the toxins are injected directly into the jugular vein. The process is
tedious and requires a longer time, and for practical purposes has not
been found so satisfactory as the simple subcutaneous injection. The
strength of the serum is tested by using young guinea-pigs of five
hundred grammes weight. One gramme of the serum usually will
protect fifty thousand grammes of guinea-pig against a fresh virulent
culture of the Bacillus diphtheria. This is the strength that is used
in the hospitals. By the intravenous injections a serum of the pro-
tective strength of 1 : 100,000 can be obtained.

When fully immunized from six to eight litres of blood may be
taken from a horse at one time, but as a rule it is better not to take
more than three. The blood is drawn from the jugular vein, by means
of a small trocar and cannula, into wide-mouthed bottles having a ca-
pacity of 2.5 litres; these are placed in an ice chest for twenty-four
hours to give time for the separation of the serum, which is then
transferred to smaller receptacles for preservation.

The dose of serum prepared in this way, when used to protect from
diphtheria infection, is five cubic centimetres for a child under ten
years of age, and ten cubic centimetres for older children. This does
not afford an absolute protection, but is believed to be generally
effective, and in case of failure the attack is said to be of a mild
character. The curative dose of Roux's serum is twenty cubic centi-
metres for children, and thirty to forty cubic centimetres for patients
over fifteen years of age. The larger dose is divided and given, at
the same time, by subcutaneous injection in two places. Antiseptic

318 PROTECTIVE INOCULATIONS.

precautions are taken in giving these injections, and a little absorbent
cotton is placed over the puncture.

FOOT-AND-MOUTH DISEASE.

This is an infectious disease of cattle, sheep, goats, and swine, the
etiology of which, so far as the specific infectious agent is concerned,
has not been determined.

The extent to which the disease in question prevails in some parts
of Europe is shown by the statistics for 1891 of the prevalence of this
disease in Germany. According to the Eeichsseuchenbericht it pre-
vailed most extensively in the southern portion of Germany. The
total number of infected farms was 47,865; the total number of in-
fected cattle was 394,640; of sheep, 240,904; of goats, 3,378; of swine,
182,208. Behla (1892) has made inoculation experiments with the
filtered saliva of infected cattle to which he added one to two per cent
of carbolic acid, and claims to have produced immunity in young pigs
and lambs. The duration is not, however, very long even in animals
which have recovered from an attack of the disease — said to be from
six months to three years — and a practical method of restricting the
disease by means of protective inoculations has not as yet been intro-
duced.

GLANDERS.

The toxic substances produced in cultures of the glanders bacillus
when concentrated in the form of a glycerin extract constitute the so-
called mallein, which has been extensively used in the diagnosis of
glanders in horses. As is the case when animals infected with tuber-
culosis are inoculated with tuberculin, animals infected with glanders
have a decided rise of temperature after receiving a sufficient dose of
mallein beneath the skin.

Babes (1892) reports that the toxic substance in cultures of the
glanders bacillus may be obtained by precipitation with alcohol; and
that mallein obtained from filtered cultures to which glycerin has
been added, or the alcoholic precipitate, may be successfully used
for protecting susceptible animals against glanders infection or for
curing the disease after infection. He has demonstrated the thera-
peutic value upon guinea-pigs and upon two horses which are said to
have been cured of chronic glanders. When large and repeated doses
are injected into healthy animals they produce nephritis and general
marasmus. The action upon horses infected with glanders is very
marked and small doses may even cause death.

Kresling (1892) recommends potato cultures as preferable to bouil-

PROTECTIVE INOCULATIONS. 319

Ion cultures for the preparation of mallein. The potatoes are to be
washed, before sterilization, in a five-per-cent bicarbonate of soda
solution, "until the wash-water remains clear." They are then
cooked for an hour and twenty minutes. After planting upon the
surface glanders bacilli from a previous culture they are placed in an
incubator at 36° to 36.5° C., with provision to prevent them from be-
coming dry. At the end of two weeks the growth is removed with a
platinum spatula and added to nine parts of water, in which it is well
mixed by rubbing. It is then allowed to stand for twenty-four hours,
after which it is sterilized for fifteen minutes at 110° 0. (a lower tem-
perature would no doubt answer quite as well). After cooling it is
passed through a Chamberlain filter by means of a pressure of six
atmospheres. The filtrate is then carefully evaporated over a water-
bath to one-fourth its volume, and to this concentrated extract gly-
cerin is added in the proportion of one part to two. The mixture is
again sterilized in the autoclave at 110° C. When injected into
healthy horses in the dose of two cubic centimetres this mallein does
not cause an elevation of temperature exceeding 0.5° to 0.8°. But
one cubic centimetre injected into a horse having glanders causes its
temperature to mount to 40° C., and at the point of inoculation a con-
siderable swelling is developed which lasts from four to six days —
in healthy horses a swelling the size of a man's fist is developed at
the point of inoculation, which disappears within twenty -four hours.
In Pasteur's laboratory, according to Nocard (1892), mallein is
prepared as follows : The glanders bacillus is first made so virulent
by successive inoculations in susceptible animals that it will kill a
rabbit or a white mouse in a few hours. This virulent bacillus is cul-
tivated in glycerin-peptone-flesh-infusion (five per cent of glycerin and
five per cent of peptone). The cultures are kept in the incubating
oven for four weeks at a temperature of 31° C., and then sterilized in
the autoclave at 110° C. They are then filtered through paper and
evaporated, in vacuo, over sulphuric acid, at a low temperature, to one-
tenth of the original volume. The result is a syrup-like, dark-brown,
strong-smelling liquid, which is about one-half glycerin. This can
be preserved in a cool and dark place for a long time. When it is to
be used nine parts of a 0.5-per-cent solution of carbolic acid are
added to one part of the glycerin extract. The concentrated extract,
when injected into a healthy horse in the dose of one-half to one cubic
centimetre, causes a local swelling which disappears after two or three
days. The temperature of the body is elevated from 1.5° to 2° C. as
a result of the injection, and there are chilliness, loss of appetite, and
debility. When the diluted mallein is injected in healthy animals in

320 PROTECTIVE INOCULATIONS.

the dose of 2.5 cubic centimetres no reaction occurs. On the other
hand, this dose causes an intense febrile reaction in horses with glan-
ders. There is a chill followed by an elevation of temperature amount-
ing to 2° to 3° C., accompanied by dyspnoea and great debility; in
some cases the animal dies as a result of the inoculation.

For the preparation of the active substance in a dry condition,
Foth gives the following directions : The cultures are evaporated at
a temperature not exceeding 80° C. to one-tenth of their volume, and
filtered. The clear and thick, 'dark-brown liquid is then slowly
dropped into absolute alcohol (twenty-five to thirty parts) with con-
stant stirring. A flaky, white precipitate is thrown down, and ac-
cumulates as a pale yellow mass upon the sides and bottom of the
vessel. After standing for twenty-four hours the alcohol is carefully
drawn off and the precipitate washed with absolute alcohol. This
is to be carefully done, and to avoid loss will require several days.
The precipitate is then placed upon a thick paper filter and thor-
oughly washed by drawing alcohol through it by means of an exhaus-
tion apparatus, after which the purified precipitate is collected and
dried with care at a low temperature — best in a vacuum over sul-
phuric acid. A spongy, crumbling mass is thus obtained, which is
easily crushed to form an extremely light white powder. This is
readily soluble in water. It is not at all hygroscopic, and can be
preserved in a dry condition without difficulty . The dose for a horse
is 0.1 gramme.

De Schweinitz and Kilborne, in a paper published in November,
1892, state that in December, 1890, they

"extracted from culture liquids of the Bacillus malleus an albumose which
appeared to be the active principle in these cultures. At that time a pre-
liminary experiment was conducted to see if this substance could be used
to make guinea-pigs immune to the disease — glanders. The result was that
out of a set of five, three vaccinated and two checks, only one, a vaccinated
animal, recovered from an inoculation of a glanders culture. This experi-
ment has since been repeated with sets of ten and twelve guinea-pigs each,
with, at present writing, only negative results. A note of this work was
published in the ' Annual Report of the Department of Agriculture for 1891.'
The albumose was best obtained from the cultures, after the removal of the
germ, by means of a Pasteur filter, by precipitation with absolute alcohol,
resolution in water, and reprecipitation."

Babes (1892) claims to have succeeded in immunizing guinea-pigs
against glanders by means of the toxic substances contained in cul-
tures of the bacillus.

Foth (1894) has reported the results of extended experiments
which have been made with his " Malleinum siccum " in Austro-Hun-
gary. These results are stated as follows :

PROTECTIVE INOCULATIONS. 321

The experiments were for the most part made by Professor Schin-
delka, of Vienna. The tests were made with doses ranging from 0.1
gramme to 0.2 gramme. The number of horses treated, for diagnos-
tic purposes, was four hundred and fifty-five; of these one hundred
and forty-seven were examined post mortem. In general the infected
horses reacted and the others did not. A reaction of 2C C. and up-
ward, running a typical course, was evidence that the animal was in-
fected, and such animals were killed and carefully examined by au-
topsy.

A reaction of 1.3° to 1.9° C., running a typical course, was taken
as evidence that the animal was probably infected, and called for its
isolation and a subsequent inoculation after an interval of four weeks.

A reaction of less than 1.2° C., or an atypical course of the febrile
reaction, was taken as evidence of non-infection.

The typical febrile reaction consisted in a rapid or gradual eleva-
tion, according to the dose, then a fall of some tenths of a degree, a
subsequent elevation to the highest previously reached point or above,
and a gradual fall to the normal. The atypical reaction, which some-
times occurs in healthy animals, consists in an early and rapid eleva-
tion followed by an equally rapid fall to the normal. To properly
distinguish the typical temperature curve, upon which the diagnosis
depends, hourly observations are considered necessary.

Schiitz (1894), as a result of his experiments on fifty-four horses,
arrives at the conclusion that malleiu may give rise to the so-called
" typical reaction " in horses which are not infected with glanders.

Hutyra and Preiz (1894), as a result of their extended researches,
arrive at the conclusion that the use of malleiu constitutes the most
important means for the early diagnosis of glanders in horses. They
conclude that a temperature of 39.4° C. may be accepted as a safe
positive mallein reaction. According to them the reaction commences
from four to six hours after the injection, and reaches its maximum
in from eight to fourteen hours — rarely in sixteen to twenty hours.
The return to the normal occurs in from twenty -four to thirty-six
hours. The authors last named give the following directions for the
preparation of mallein : The virulence of the glanders bacillus is first
increased by passing it through a series of guinea-pigs. Cultures
are then made upon sterilized potato. When the culture and potato
have become quite dry and dark colored they are collected in a glass
dish and covered with a liquid consisting of equal parts of glycerin
and distilled water, containing three to five parts per thousand of
mercuric chloride. After standing for from ten to fourteen days in
an incubating oven at 37.5° C., the liquid. is filtered through paper
21

322 PROTECTIVE INOCULATIONS.

and sterilized for an hour in a steam sterilizer. This liquid remains
sterile on account of the presence of mercuric chloride, and may be
preserved a long time without losing its activity. The dose is from
0.3 to 0.5 cubic centimetre, which is diluted to three cubic centimetres
with carbolic acid water (0.5-per-cent solution). The diluted solu-
tion may also be kept a long time without losing its activity.

Bonome and Vivaldi (1892) have tested the action of mallein ob-
tained by precipitation with alcohol upon various animals. Guinea-
pigs were found to resist comparatively large doses (ten to fifteen
milligrammes), while rabbits and cats were more sensitive to the
toxic action. In guinea-pigs and rabbits infected with glanders ba-
cilli very small doses had a favorable influence upon the progress of
the infection, and in healthy guinea-pigs a certain degree of immunity
was induced by the repeated injection of small doses.

In a subsequent paper (1894) Bonome reports that he has had
favorable results in the treatment of chronic glanders in man by doses
of yV to ^¥ cubic centimetre. The first dose is said to have caused
an elevation of temperature, headache, polyuria, etc., but upon re-
peating the dose after two or three days a decided improvement of
the general symptoms followed.

Chenot and Picq (1892) claim to have cured glanders in guinea-pigs
by injections of blood serum from the ox, which animal has an im-
munity from the disease. They also state that the blood serum of
the ox is germicidal for the glanders bacillus. Guinea-pigs treated
with ox serum, either before or after infection, recovered in seven
cases out of ten. When inoculated with very virulent cultures, which
usually killed these animals in five days, the animals are said to have
survived from twenty-one to forty-two days.

Bonome (1894) reports his success in curing infected guinea-pigs
by means of filtered cultures made in the blood serum of the ox. He
was not, however, successful in accomplishing this result with mallein
made in the usual way.

HOG CHOLERA.

The experiments thus far made with reference to protective inocu-
lations against hog cholera have not given very satisfactory results.
Selander and Metchnikoff have reported success in immunizing rab-
bits, but according to Smith their experiments were made with the
bacillus of swine plague, and not with that of hog cholera as they
supposed. The following conclusions have been formulated by Smith
as a result of his extended experiments :

"1. It is possible to produce immunity toward hog-cholera and swine-

PROTECTIVE INOCULATIONS. 323

plague bacteria in the very susceptible rabbit and the less susceptible guinea-
pig. In the rabbit the only promising- method of immunization toward hog
cholera is the use of gradually augmented doses of attenuated cultures.

"2. Immunization toward swine plague is produced artificially with
much greater ease than toward hog-cholera bacteria.

"3. The blood serum of animals protected against hog cholera and swine
plague is almost as efficacious in producing immunity soon after treatment
as the bacterial products obtained from cultures.

"4. Different degrees of culture in both hog cholera and swine plague
lead to different forms of the inoculation disease. The greater the immunity
short of complete protection the more prolonged and chronic the disease in-
duced subsequently by inoculation.

"5. Pathogenic bacteria may remain in the organs of inoculated animals
some time after apparently full recovery. Their presence may or may not
be associated with lesions recognizable by the naked eye.

" 6. The toxicity of sterilized cultures appears to be directly proportional
to the number of bacteria in the injected fluid."

The experiments of Moore, reported in Bulletin No. 6 of the
Bureau of Animal Industry, show that the bacillus of hog cholera
does not become attenuated by being passed through rabbits, and that
in the experiments of Metclmikoff, which led him to conclude that
this is the case, the bacillus of swine plague, and not that of cholera,
was used.

De Schweinitz studied the chemical products of the hog-cholera
bacillus in 1890, and obtained from the cultures cadaverin, methyl-
amine, a ptomaine ("sucholotoxin"), and an albumose ("sucholoal-
bumin ").

Novy (1890) has also obtained, by Brieger's method, a basic toxic
substance ("susotoxin") which kills rats in the dose of 0.125 to 0.25
cubic centimetre. He also obtained from concentrated cultures, by
precipitation with absolute alcohol, a toxalbumin which, when dried,
killed rats in three or four hours in the dose of 0.05 to 0.01 gramme.

De Schweinitz in a later publication (1899) reports that he has
obtained, by the method of Brieger and Boer for the isolation of the
diphtheria antitoxin, an ash-free white powder, which possesses the
antitoxic properties of serum from an immune animal; ninety cubic
centimetres of serum gave him 0.152 gramme of this powder. The
method referred to consists in precipitation by the use of zinc sul-
phate, repeated solution in sodium hydrate and precipitation by
CO,. In preparing serum for his experiments, cattle, horses, mules,
and monkeys were employed. " The animals received injections
of the filtered, sterile or live, cultures of the hog-cholera germ and
swine-plague germ, respectively, or the solutions of their products,
including cell contents, extracts, and secretions. These injections
were made either subcutaneously, intravenously, or intra-abdomi-
nally, or a combination of two or more of these methods, depending

324 PROTECTIVE INOCULATIONS.

upon the results obtained. The quantities given at first were small,
but increased gradually until large amounts of the material used could
be injected without bad results. This treatment of the animals must
be carried out very carefully, and requires six to eight months' time
before the serum is sufficiently potent to be of any practical use. As
the treatment continues, the power of the serum to check the motility
of the hog-cholera germ increases with rapidity. The length of im-
munity produced by the injection of serum is short, and more perma-
nent immunity can apparently be secured by using in addition to
serum the products of the germs."

The results of extensive inoculations (thirty-five thousand animals)
which have been made by the Agricultural Department during the past
two years have not yet been published, but it is understood that as a
rule these results have been quite satisfactory.

HOG ERYSIPELAS.

Pasteur's first studies relating to the etiology of "rouget" were
made, in collaboration with Chamberland, Roux, and Thuillier, in
1882. Pasteur found that the virulence of his cultures was increased
by passing them through pigeons and diminished by passing them
through rabbits. By a series of inoculations in rabbits he obtained
an attenuated virus suitable for protective inoculations in swine. In
practice he recommended the use of a mild virus first, and after an
interval of twelve days of a stronger virus. These inoculations have
been extensively practised in France, and the fact that immunity may
be established in this way is well demonstrated. There has been some
doubt, however, as to the practical value of the method, as its appli-
cation has been attended with some loss, and there appears to be
danger that the disease may be spread by the alvine discharges of
inoculated animals. In a region where the annual losses from the
disease are considerable, and where the soil is, perhaps, thoroughly
infected with the bacilli, protective inoculations probably afford the
best security against loss. But when it is practicable to stamp out
the disease by quarantine of infected animals, disinfection of localities
in which cases have occurred, and strict attention to cleanliness, this
will probably be found the best method of combating the malady.

Chamberland (1894) states that in the preceding seven years, dur-
ing which time protective inoculations were practised in France on a
large scale, the mortality from rouget has been reduced to 1.45 per
cent, whereas before these inoculations were practised the mortality
from this disease was about twenty per cent. Losses amounting in

PROTECTIVE INOCULATIONS. 325

some instances to as much as ten per cent have resulted from the in-
oculations. These are ascribed by Chambeiiand to secondary infec-
tion, through the inoculation wound, with other pathogenic bacteria.

Jakobi (1888) reports the results of inoculations made in 1887 and
1888 with "vaccines" obtained from Pasteur's agent in Paris. His
results agree with those previously reported by Lydtin in showing a
smaller loss, as & result of the inoculations, among young pigs than
among older ones — over sixteen weeks. The loss among young pigs
was only 1.3 per cent. The animals which survived subsequently es-
caped infection, while others not inoculated, associated with them,
succumbed to the disease.

Hutyra has given the following statistics of inoculations made in
Hungary during the year 1889, with " vaccines " obtained from the
Pasteur laboratory in Vienna: 48,637 pigs were inoculated on 117
different farms. Of these 143 (0.29 per cent) died between the first
and second inoculation. After the second inoculation 59 animals died
(0.1 per cent). During the year following the inoculations, 1,082 in-
oculated pigs died of Rothlauf. Before the inoculations the annual
loss in the same localities is said to have been from 10 to 30 per
cent. Upon one farm 220 pigs which had been inoculated were as-
sociated with 1,500 not inoculated. The loss among the latter was
50 per cent; among the former 2.27 per cent.

In a later communication (1894) Jakobi gives the following results
of inoculations made since by the same method: 1889, inoculated
133, loss 5; 1890, inoculated 151, loss 2; 1891, inoculated 158, loss
0; 1893, inoculated 223, loss 0; 1894, inoculated 145, loss 4. Total
inoculated, 1,036; total loss, 14. These inoculations were made upon
19 different farms, and principally upon pigs less than four months
old. The inoculated pigs were isolated to prevent the communication
of the disease to other unprotected pigs.

Inoculations with Blood Serum of Immune Animals. — The experi-
ments of Lorenz, commenced in 1891, seem to establish the fact that
there is an antitoxin in the blood of animals which have an acquired
immunity against this disease which may be used for producing im-
munity in other animals, or for the cure of the disease in animals
already infected. In his latest communication (1894) Lorenz says :

% ' When I read in the journals of the discovery of Behring and Kitasato
that the blood of animals immunized against tetanus, when injected beneath
the skin of other animals, gave them an immunity against tetanus, I had in
my possession rabbits which were immunized against Rothlauf. I took from
one of these some blood from the ear vein, injected it under the skin of a
mouse,, inoculated this latter with a Rothlauf culture, and made the dis-
covery, in this and a series of subsequent experiments, that the blood of an

326 PROTECTIVE INOCULATIONS.

animal immune against Rothlauf contains an immunizing substance. I
further ascertained that this substance is found only in the blood serum, and
not in the solid portions of the body organs, etc., and with the exception of
the blood was found only in the secretions of serous membranes. I also
found that the immunizing substance is only to be found for a certain time
after renewed infection in the immune animals, and that it gradually disap-
pears, without the loss of immunity in the animal, however. Finally, I dis-
covered that the animals into which one injects blood serum from immune
animals do not acquire a lasting immunity, but are only immune for a rela-
tively short time/'

In experiments made in 1893 and 1894, with a view to producing
immunizing serum for protective inoculations on a large scale, Lorenz
met with some disappointments ; but he proposes to renew his attempts
and hopes to avoid the difficulties which have been brought to light
by experience, one of which he states as follows :

"When an animal already immunized against Rothlauf receives an in-
jection of a considerable quantity of a culture of the bacillus, in order to
cause the production in its blood of a serum of high therapeutic value, the
animal bears these injections without any notable reaction. But its blood
serum contains during the following days, besides the immunizing substance,
also poisonous substances, and blood which is taken too soon (twenty-four
hours) after the injection has a toxic action upon animals which are already
infected. If this poisonous serum is injected into a mouse which has been
infected two days before with Rothlauf bacilli, in the dose of about 0.05 cubic
centimetre, death occurs in a few hours, even when scarcely any evidence of
sickness had been observed before the injection.''

The fact that mice infected with this bacillus may be cured by in-
jecting into them blood serum from an immunized rabbit has also
been demonstrated by F. Klemperer (1892) . In his experiments with
the bacillus of mouse septicaemia, and with Friedlander's bacillus, he
found that serum from an immune rabbit may be used to immunize
mice and also to cure them after infection, while serum from a non-
immune rabbit has no such action. The immunity produced in this
way was found to be specific. That is, animals immunized against
the pathogenic action of one of these bacilli were not protected against
infection by the other. The " Heilsemm " when added to cultures in
vitro did not prove to have any special bactericidal action.

HYDROPHOBIA.

Notwithstanding the extended researches made, especially in Pas-
teur's laboratory, the etiology of hydrophobia still remains unsettled.
It has been demonstrated by experiment that the virus of the disease
is located in the brain, spinal marrow, and nerves of animals which
have succumbed to the disease, as well as in the salivary secretions
of rabid animals ; and that the disease may be transmitted by intra-
venous inoculation, or by introducing a small quantity of virus beneath

PROTECTIVE INOCULATIONS.

the dura mater, with greater certainty than by subcutaneous inocula-
tions. But the exact nature of this virus has not been determined.
The fact that a considerable interval elapses after inoculation before
the first symptoms are developed indicates that there is a multiplica-
tion of the virus in the body of the infected animal ; and this is further
shown by the fact that after death the entire brain and spinal marrow
of the animal have a virulence equal to that of the material with which
it was inoculated in the first instance. The writer's experiments
(1887) show that this virulence is neutralized by a temperature of 60°
C., maintained for ten minutes— a temperature which is fatal to all
known pathogenic bacteria in the absence of spores. But recent ex-
periments show that certain toxic products of bacterial growth are
destroyed by the same temperature. We are, therefore, not justified
in assuming that the morbid phenomena are directly due to the pres-
ence of a living micro-organism ; and, indeed, it seems probable, from
what we already know, that the symptoms developed and the death of
the animal are due to the action of a potent chemical poison of the
class known as toxalbumins. But, if this is true, we have still to ac-
count for the production of the toxic albuminoid substance, and, in
the present state of knowledge, have no other way to explain its in-
crease in the body of the infected animal than the supposition that a
specific, living germ is present in the virulent material, the introduc-
tion of which into the body of a susceptible animal gives rise to the
morbid phenomena characterizing an attack of rabies.

Pasteur and his associates have thus far failed to demonstrate the
presence of microorganisms in the virulent tissues of animals which
have succumbed to an attack of rabies. Babes has obtained micro-
cocci in cultures from the brain and spinal cord of rabid animals, and
states in his article on hydrophobia in "Les Bacteries " (second edi-
tion, p. 791) that pure cultures of the second and third generation in-
duced rabies in susceptible animals ; but his own later researches do
not appear to have established the etiological relation of this micro-
coccus.

Gibier (1884) has reported the presence of spherical refractive
granules, resembling micrococci, in the brain of rabid animals, which
he demonstrated by rubbing up a little of the cerebral substance with
distilled water. As these supposed micrococci did not stain with
the usual aniline colors and were not cultivated, it appears very doubt-
ful whether the refractive granules seen were really microorganisms.

Fol (18S5) claims to have demonstrated the presence of minute
cocci, 0.2 fj. in diameter, in sections of spinal cord from rabid ani-
mals, by Weigert's method of staining. The cords were hardened in

328 PROTECTIVE INOCULATIONS.

a solution of bichromate of potash and sulphate of copper, colored
with a solution of hrematoxylon, and decolorized in a solution of fer-
rocyanide of potash and borax.

The writer (1887) has made similar preparations, carefully follow-
ing the method as described by Fol, but was not able to demonstrate
the presence of microorganisms in the numerous sections made. Nor
have the observations of Fol been confirmed by the researches of other
bacteriologists who have given their attention to the subject since the
publication of his paper.

Pasteur first announced his success in reproducing rabies in sus-
ceptible animals by inoculations of material " from the medulla oblon-
gata, the frontal lobes of the cerebral hemispheres, and the cerebro-
spinal fluid " in a communication to the Academy of Sciences made on
May 30th, 1881. At the same time he reported his success in the
discovery of " a method for considerably shortening the period of in-
cubation in rabies, and also of reproducing the disease with certainty."
This was by inoculations made after trephining, upon the surface of
the brain with material obtained from the brain of a rabid animal.
Dogs inoculated in this way developed rabies in the course of two
weeks, and died before the end of the third week — sometimes of furi-
ous rabies and sometimes of the paralytic form of the disease. In a
second communication (December llth, 1882) Pasteur reports his
success in communicating the disease by the intravenous injection of
virus from the central nervous system ; also the experimental demon-
stration of the fact that all forms of rabies may be produced by the
same virus ; also that all portions of the spinal cord of rabid animals
are virulent, as well as all parts of the brain ; also that an animal
(dog) which had recovered from a mild attack after inoculation proved
to be subsequently immune, and that " this observation constitutes a
first step toward the discovery of the prophylaxis of rabies." On
February 25th, 1884, many important facts are stated which had been
developed during the continuous study of the disease, and among
others the fact that by passing the virus through a series of animals
of the same species a fixed degree of virulence is established for each
susceptible species, as shown by a definite and uniform period of in-
cubation. By this method a virus had been obtained which produced
rabies in rabbits in seven or eight days, and another which caused
the development of the disease in guinea-pigs in five or six days after
inoculation. In a subsequent communication (May 19th, 1884) evi-
dence is given to show that by successive inoculations in monkeys the
period of incubation is prolonged, and that the attenuated virus ob-
tained from a monkey, after several successive inoculations in this

PROTECTIVE INOCULATIONS.

animal, when inoculated into the dog, no longer produces fatal rabies ;
and that dogs so treated are subsequently immune.

In his address before the International Medical Congress at Copen-
hagen (August llth, 1884), after a review of the facts developed during
his experimental researches made during the preceding four years,
Pasteur gives an account of the test made by a commission, appointed
by the Minister of Public Instruction, to determine the efficacy of his
method as applied to the protection of dogs. He says that he gave to
the commission nineteen dogs which had been rendered refractory
against rabies by preventive inoculations. These nineteen dogs and
nineteen control animals, obtained from the pound without any selec-
tion, were tested at the same time. The test was made upon some of
the animals of both series by inoculation with virulent material upon
the surface of the brain, and upon others by allowing them to be bit-
ten by rabid dogs, and upon still others by intravenous inoculations.

Not one of the protected animals developed rabies ; on the other
hand, three of the control dogs out of six bitten by a mad dog devel-
oped the disease, five out of seven which received intravenous inocu-
lations died of rabies, and five which were trephined and inoculated
on the surface of the brain died of the same disease. In a subsequent
report the commission, of which M. Boulley was president, stated that
twenty-three protected dogs which were bitten by ordinary mad dogs
all remained in perfect health, while sixty-six per cent of the control
animals, bitten in the same way, developed rabies within two months.

In his communication of October 26th, 1885, Pasteur reports his
discovery of the fact that the virulence of the spinal cord of a rabbit
is gradually attenuated by hanging it in a dry atmosphere, and is
finally entirely lost ; also that he had been able to make a practical
application of this discovery in the protection of dogs by means of
successive inoculations beneath the skin of an emulsion of spinal mar-
row attenuated in this way. The first inoculation was to be made
with a portion of spinal cord which had been kept long enough to de-
prive it of all virulence, and this was followed by daily inoculations
with more virulent material, until finally material was used from a
cord only a day or two old.

With reference to his first inoculations in man, Pasteur says :

" Making- use of this method, I have already made fifty dogs of various
races and ages immune to rabies, and -had not met with a single failure,
when, on the 6th of July, quite unexpectedly, three persons, residents of
Alsace, presented themselves at my laboratory."

These persons were Theodore Yone, who had been bitten on the
arm on July 4th ; Joseph Meister, aged nine, bitten on the same day

330 PROTECTIVE INOCULATIONS.

by the same rabid dog ; and the mother of Meister, who had not been
bitten. The child had been thrown down by the dog and bitten upon
the hand, the legs, and the thighs, in all in fourteen different places.
Pasteur commenced the treatment on July 6th, by injecting beneath
the skin of this child an emulsion of cord which had been kept for
fourteen days; this was followed by twelve more inoculations made
on successive days with cord of increasing degrees of virulence — the
last with cord a day old. On March 1st, 1886, Pasteur reported to
the Academy of Sciences the fact that the boy Meister remained in
good health and gave detailed information with reference to a number
of cases which had since been treated by the same method.

With reference to the duration of the immunity resulting from
these inoculations Pasteur says (1886) that out of fourteen dogs in-
oculated with "ordinary street virus," by trephining, at the expiration
of a year after the protective inoculations had been practised, eleven
resisted ; out of six tested in the same way at the end of two years
two proved to be immune.

In November, 1886, Pasteur communicated to the Academy of
Sciences the results of his experiments with reference to a modification
of his method as at first employed — the so-called intensive method.
This modification consisted in making the inoculations with cords of
increasing virulence in more rapid succession.

The method followed at Odessa, as reported by Gamaleia (1887),
is shown below, the day being given above and age of the cord
below.

1 2 _3^ 45 6 7 8910

14-13 12-11 10-9 8-7 6-5 4-3 2-10 8-642

Since the adoption of this method and the use of larger quantities
of virus, according to Gamaleia, there have been no deaths among
those inoculated, numbering more than two hundred at the time the
report was made. The author last referred to concludes from his ex-
perience that " the mortality diminishes in direct relation to the quan-
tity of the vaccine injected."

Bujwid (1889) reports a total of 670 inoculations, with 9 deaths,
made at Varsovie during the years 1886, 1887, and 1888. His method
is shown below.

1 2 34567

12-10 8-643643'

The results of inoculations made at the Pasteur Institute in Paris
during the years 1886 to 1890 are given in the following table :

PROTECTIVE INOCULATIONS.

331

Year.

Number Treated.

Died.

Mortality.

1886

2,671

0 94

1887

1,770

0.73

1888

1,622

0.55

1889

1,830

0.33

1890

1.540

0.32

Total

9,433

0.61

In the following table, A includes all persons treated who had been
bitten by an animal proved to be rabid ; B, persons bitten by animals
examined by veterinary surgeons and pronounced rabid; C, persons
bitten by animals suspected of being rabid. The figures relate to the
year 1890:

Number Treated.

Died.

Mortality.

416

909

0.44

215

0.46

Bordoni-Uffreduzzi gives the following statistics with reference to
the inoculations practised at the Pasteur Institute in Turin during
the years 1886 to 1891 : 81 persons were inoculated by the method
first proposed by Pasteur, with a mortality of 2.46 per cent; 925
persons were subsequently inoculated by the same method, but with
larger doses of virus, with a mortality of 1.72 per cent. Finally, 338
persons were inoculated with still larger doses, with a mortality of
0.29 per cent.

At the Pasteur Institute in Palermo the number of persons inocu-
lated in the four years prior to 1891 was 662, with a mortality among
the inoculated of 0.6 per cent. In Bologna (1890) 210 persons bitten
by dogs undoubtedly mad were inoculated, with a mortality of 0.47
per cent.

In the Pasteur Institute at Naples 810 persons were treated during
the years 1886 to 1892, with a mortality of 0.86 per cent.

During the year 1891, 1,564 persons were inoculated at the Pas-
teur Institute in Paris, with a total mortality of 0.57 per cent. In
324 of these cases the animal which inflicted the bite was proved to
be rabid by experimental inoculations.

Horsely (1889) has made a comparison of the results obtained by
the " intensive treatment " as compared with these by the treatment
first employed, and says :

PROTECTIVE INOCULATIONS.

" It is evident that the intensive treatment is very successful in coping
with the worst cases, and that, instead of being- itself a source of death, as
asserted by those who gain notoriety and subsistence by villifying and mis-
representing scientific progress, it is a powerful agent in saving life."

The following table is given by Horsely " as showing the contrast
between the old or simple treatment and the intensive treatment " :

Simple Treatment, 1886. Intensive Treatment, 1888.

Odessa 3.39 per cent. 0.64 per cent.

Warsaw 4.1 0.0* "

Moscow 8.2 f " 1.6

* The figures include sixteen months' work, and thirty individuals bitten in the
face — four by wolves.

f This unusually high rate was found to be due to imperfections in the manner
of preparing the cords for the inoculation material.

Perdrix (1890), in an analysis of the results obtained at the Pasteur
Institute in Paris, calls attention to the fact that the mortality among
those treated has diminished each year and ascribes this to improve-
ment in the method. He says :

' ' At the outset it was difficult to know what formula to adopt for the
treatment of each particular case. Upon consulting the accounts of the bites
in persons who have died of hydrophobia notwithstanding the inoculations,
we have arrived at a more precise determination as to the treatment suitable
for each case, according to the gravity of the lesions. In the cases with seri-
ous wounds we inject larger quantities of the emulsion of cord and repeat the
inoculations with the most virulent material. For the bites upon the head,
which are especially dangerous, however slight their apparent gravity may
be, the treatment is more rapid, and, above all, more intensive — that is to
say, the virulent cord is injected several times."

The statistics arranged with reference to the location of the bite
are given by Perdrix as follows :

Bitten upon the head, 684; died, 12 = 1.75 per cent.
". hands, 4,396; " 9 = 0.2
" limbs, 2,839; " 5 = 0.17

Other methods of making susceptible animals immune against
hydrophobia have been proposed and proved by experiment to be
successful. Thus Galtier in 1880-1881 claimed that the sheep and
the goat could be protected by intravenous injections of the virus of
rabies, and more recent experiments fully confirm this. Protopopoff
(1888) by injecting an emulsion of cord from a rabid animal into the
circulation of dogs succeeded in protecting them from hydrophobia
as a result of subsequent inoculation with virulent material upon the
surface of the brain. He injected into a vein, at intervals of three
days, one cubic centimetre of an emulsion of cord — first of six days,
second of three days, third of one day. Roux had previously accom-
plished the same result by a single intravenous injection of a larger

PROTECTIVE INOCULATIONS. 333

quantity (thirty -five cubic centimetres) of cord which had been kept
for five or six days. In discussing his results Roux calls attention to
the fact, which had been developed during his experiments, that the
virulence of the spinal cord of rabid animals does not depend entirely
upon the length of time it has been kept, but that large doses of cord
kept as long as twelve days will sometimes produce hydrophobia when
injected into the circulation of dogs, when smaller doses of cord kept
five or six days prove to be inoffensive. He supposes that during
desiccation the virus may not be equally acted upon throughout the
cord, but that certain " islands " in the central portion may remain
living and virulent when all the rest has been modified. A practical
point with reference to the preservation of virulent material is referred
to by Roux in a, note published in the Annals of the Pasteur Insti-
tute. This is the fact that when preserved in glycerin, portions of
the central nervous system retain their virulence for considerable
time. Other forms of virus, e.g., vaccine, may also be preserved in
the same way.

Centanni (1892) has succeeded in making rabbits immune by in-
oculating them with an attenuated virus obtained by subjecting viru-
lent material to the action of an artificial gastric juice. After digestion
for less than twelve hours the virus still kills rabbits, when inoculated
beneath the dura mater, but the period of incubation is considerably
prolonged. After from twelve to twenty hours' digestion it no longer
kills rabbits, but causes an infection, from which they recover, and
after which they are immune.

Serum-therapy. — Tizzoni and Centanni (1892) have reported suc-
cess in the treatment of infected rabbits by the use of blood serum
from immune animals of the same species — immunized by the " Ital-
ian method " above described. The animals experimented upon were
inoculated with a "street virus" which produced paralytic rabies in
rabbits and caused their death in from fourteen to eighteen days.
The blood serum was obtained from rabbits which had been proved
to be immune by resisting inoculations of virus of full strength on
the surface of the brain. The blood serum, in doses of three to five
cubic centimetres, was injected subcutaneously, or into the peritoneal
cavity, or into the circulation. Injections were made into each animal
(in all from eleven to twenty-six cubic centimetres) after the first symp-
toms of paralytic rabies had appeared (on the seventh, the tenth, the
eleventh, and the fourteenth day after infection) . Four rabbits treated
in this way fully recovered. In a subsequent experiment the bacteri-
ologists named treated three rabbits with a dry antitoxin obtained by
precipitation from the blood serum of immune rabbits. The precipi-

334 PROTECTIVE INOCULATIONS.

tate was obtained by adding one part of serum to ten parts of alcohol,
and was dried in vacuo. This dried precipitate, in doses of 0.18 to
0.25 gramme, was dissolved in sterilized water and injected as in the
previous experiment. Commencing on the eighth day after infection
five or six doses were given— in all 0.9 to 1.3 gramme. All of the ani-
mals treated recovered, while all of the control animals died. Babes
had previously (1889) reported successful results in conferring im-
munity upon susceptible animals by injections of blood serum from
immune animals.

Tizzoni and Schwartz, in pursuing this line of investigation (1892),
report that while the blood serum of immune rabbits neutralizes the
"fixed virus" of rabies in vitro, after short contact (five hours), the
blood serum of immune dogs has but slight antitoxic potency. The
immunizing substance in the rabbit serum does not dialyze, is soluble
in glycerin, is precipitated by alcohol, and in general behaves like a
globulin. In subsequent experiments Tizzoni and Schwartz used
blood serum from dogs and rabbits immunized by Pasteur's method.
The blood was drawn from the carotid of the immune animals, and
the serum from the same, mixed with virulent spinal marrow in the
form of a homogeneous emulsion, obtained by crushing and pressing
through linen. These experiments corresponded with those pre-
viously made as to the superior antitoxic power of rabbit serum,
which, after five hours' contact, neutralized the virulence of the emul-
sion of cord. By the injection of serum from an immune rabbit, in
doses of five cubic centimetres, into the circulation of other rabbits,
they were, as a rule, made immune. The immunizing substance
(antitoxin) was shown by other experiments to be present only in the
blood. Extracts from the liver, spleen, kidney, or muscles gave a
negative result.

In a later communication (1894) Tizzoni and Centanni give an
account of further experiments made principally upon sheep and dogs.
By repeated inoculations they succeeded in obtaining from these ani-
mals a serum having an immunizing value of 1 : 25, 000 or more, and
from this a precipitate was obtained estimated to have a value of
1:300,000, and which in doses of 0.23 gramme (of the dried precipi-
tate), dissolved in five times its weight of water, ought to be a sufficient
dose to protect a man from the development of hydrophobia after
being bitten by a rabid animal.

The authors named believe that inoculations with this antitoxin
would be reliable for man, and that they would possess decided ad-
vantages over Pasteur's method of inoculation. These advantages are
specified as follows :

PROTECTIVE INOCULATIONS. 335

" Applicability at any time during1 the period of incubation up to the
moment of the appearance of symptoms of rabies ; absolute absence of viru-
lence and of any injurious action : very rapid treatment by the injection of
one or several small doses of material : complete solubility and consequently
prompt absorption of the material injected and its easy preservation in a dry
condition.*'

INFLUENZA.

The bacillus discovered by Pfeiffer, in 1892, is now well estab-
lished as the specific cause of this disease. Bruschettini has recently
(1893) reported the details of his experiments upon rabbits, for which
animals this bacillus is pathogenic. As a result of these experiments
he has reached the following conclusions :

" 1. Eabbits may be vaccinated against the pathogenic action of cultures
of the influenza bacillus without great difficulty.

"2. The best material for producing a high grade of immunity is blood
cultures which have been filtered through the Berkenfeld filter.

"3. The blood serum of immunized animals has strong antitoxic proper-
ties, but has no germicidal power.

"4. The serum of vaccinated animals has the power of conferring im-
munity upon other animals, in comparatively small amounts — in the pro-
portion of 1 : 42,000 of body weight, and perhaps still less.

"5. This serum has also a decided curative action, and rescues rabbits
from death even as late as forty-eight hours after infection by injection of a
culture of the bacillus into the trachea."

These results lead the author to hope that serum-therapy may
afford a method of curing this disease in man. For this purpose the
blood of an immune rabbit would appear to be the most promising
source from which to procure an antitoxic serum.

INFLUENZA IN HORSES.

SCHUTZ (1887) has described a minute oval bacillus, usually asso-
ciated in pairs, which appears to be the specific infectious agent in
the disease known in Germany as Brusteeuche. This bacillus is path-
ogenic for mice, rabbits, pigeons, and guinea-pigs, but not for swine
or chickens. By injection of cultures into the parenchyma of the
lungs Schiitz reproduced the disease — confirmed in 1888 by Hell.

Horses which have suffered an attack of infectious influenza are
subsequently immune, and the experiments of Hell have shown that
an immunity also follows the disease which results from inoculations
with pure cultures of the Schiitz bacillus.

The extended experiments made by the War Department of the
German Government show that the disease is not produced by intra-
venous injections or by the ingestion of the bacillus with the food.
Infection occurs, however, when cultures are injected into the re-

336 PROTECTIVE INOCULATIONS.

spiratory passages. Subcutaneous injections cause a painful local
tumefaction, often followed by an abscess, but without the general
symptoms of influenza.

Experiments have been made in Germany by Hell, Siedamgrotzki,
and others, which indicate that the subcutaneous injection of blood
serum from immune horses may confer immunity on other horses.
Hell usually injected forty cubic centimetres at a time, and repeated
this at intervals until two hundred to two hundred and forty cubic
centimetres had been injected in the course of two or three weeks.
He also reports the results of treatment by injections of blood serum
into the trachea in horses already infected, and thinks these injections
had a favorable influence on the course of the disease. Experiments
made subsequently by Toepper have given a similar result, but others
have not been so fortunate, and the immunizing value of blood-serum
injections, as practised by the authors referred to, seems to be still a
matter of some doubt. Toepper (1893) gives full directions for col-
lecting the serum and a detailed account of results of experimental
inoculations made by himself and others. He prefers to inject the
serum into the breast over the ensiform cartilage. No reaction oc-
curs after the injection.

PLEURO-PNEUMONIA OF CATTLE.

Protective inoculations against this disease have long been success-
fully practised. For this purpose serum obtained from the lungs of
an animal recently dead has been employed, this having been proved
by experiment to be infectious material, although the exact nature of
the infectious agent present in it was not determined.

Willems, who wras one of the first to advocate the use of protective
inoculations in pleuro-pneumonia (1852), gave a lecture in 1894 in
which he reviewed the evidence in favor of these inoculations in
the disease under consideration. Various methods had been em-
ployed. Thus Willems states that the natives of the banks of the
Zambeze cause animals to swallow a certain quantity of the liquid
from the pleural cavity of an animal recently dead, and thus give
them immunity. The virus has been injected into the circulation by
some experimenters, and others have )roposed to attenuate it by heat.
But the method which has been most extensively employed is that
discovered by the Dutch settlers at the Cape of Good Hope (the
Boers), and consists in inoculating animals in the tail with serum
from the lungs of an animal recently dead ; or with a virus obtained
from the tumefaction produced by such an inoculation in the tail.

PROTECTIVE INOCULATIONS. 337

This secondary virus was very extensively used by Lenglen, a veter-
inarian at Arras, who communicated his results to the Academy of
Science at Paris, in April, 1863, and Willems says, in his last pub-
lished communication, that this is the method which he prefers. It
is also the method most extensively employed in Australia, into which
country infectious pleuro-pneumonia was introduced in 1858. It
(juickly spread and has caused enormous losses. The killing of all
animals, sick or suspected of being infected, was tried for several
years; but this proved to be ineffectual for stamping out the disease,
and the sacrifice was so great that this measure of prophylaxis was
abandoned.

According to Loir, attention in Australia was called to Willems'
method of protective inoculations, in 1861, by a letter from Cape
Colony published in the journals of Sydney and in Melbourne. The
method was at once applied both in Victoria and in New South Wales,
and since that date many thousands of cattle have been inoculated.
In order to obtain a sufficient supply of virus the method recom-
mended by Pasteur in 1882 has been followed. This is described by
Pasteur himself in the following words :

"With a single lung we may procure sufficient virus to serve for numer-
ous series of animals. And without having recourse to other lungs this pro-
vision may be maintained in the following manner : It is sufficient before the
supply of virus is exhausted to inoculate a young calf in the dewlap or in
the shoulder. The animal dies very promptly, and all its tissues near the
point of inoculation are infiltrated with serum, which is virulent, and may be
collected and preserved in a state of purity."

Loir prefers to obtain the virus in this way from a calf six to twelve
months old, during the second week after inoculation, when the tem-
perature of the animal has gone up to 40° to 42° C., as the virus is
then said to possess the maximum degree of intensity. This vaccine
seems to become attenuated in passing through a series of animals by
inoculation, so that when it has been passed through a series of five
animals it no longer produces death even when inoculated in the most
dangerous localities. Loir testifies to the protective value of inocula-
tions with this virus made in the tail of the animal, and gives the fol-
lowing example : A few months prior to the publication of his paper
(1893), about two thousand cows were inoculated with a virus which
had been passed through a series of five calves. At the moment of
being driven away they were joined by nineteen other cows not vac-
cinated. After being on the road for a distance of two thousand kilo-
metres, the animals arrived at their destination. The two thousand
vaccinated were in good condition, while eight of the non-vaccinated

had died of pleuro-pneumouia.
22

338 PROTECTIVE INOCULATIONS.

In the Bulletin of the Central Society of Veterinary Medicine of May
24th, 1894, M. Kobcis reports the results of inoculations made with
cultures of Arloing's Pneumobacillus liquefaciens bovis, and with in-
jections of pulmonary serum. His statistics with reference to the
last-mentioned "legal" inoculations he has obtained from official
documents relating to the Department of the Seine.

The total number of infected localities in this department during
the years 1885 to 1891 was 1,253; total number of contaminated ani-
mals, 18,356 ; total number inoculated, 18,359 ; total number of deaths
prior to inoculation, 1,753; total number of deaths after inoculation,
2,741; total number of deaths due to the inoculation, 94; total per-
centage of mortality, 22.8 per cent. After discussing these and other
statistics Robcis arrives at the conclusion that Arloing's method of
preventive inoculations with cultures of the Pneumobacillus liquefaciens
bovis gives better results than the legal method with serum from an
infected animal, the total loss among animals exposed to contagion
not being over twelve to fourteen per cent.

Nocard (1892) says that serum from the lungs of an animal dead
from pleuro-pneumonia preserves its virulence and usefulness as a
vaccine, when mixed with half a volume of pure neutral glycerin and
half a volume of a five-per-cent solution of carbolic acid. At the end
of two and a half months this mixture preserved its full virulence.

PNEUMONIA.

The micrococQus of croupous pneumonia was discovered by the
present writer in the blood of rabbits inoculated subcutaneously with
his own saliva in September, 1880. In 1885 this micrococcus, which
I had repeatedly obtained in pure cultures from the blood of rabbits
inoculated, as in the first instance, with my own saliva,' was identified
with the micrococcus of the same form present in the rusty sputum
of patients with pneumonia. In a paper read before the Pathological
Society of Philadelphia, in April, 1885, and published in the Ameri-
can Journal of Medical Sciences on July 1st of the same year, I say :

"It seems probable that this micrococcus is concerned in the etiology of
croupous pneumonia, and that the infectious nature of the disease is due to
its presence in the fibrinous exudate into the pulmonary alveoli."

This has since been fully established by the researches of Frankel,
Weichselbaum, Netter, Gameleia, and many others. Frankel first
discovered this micrococcus in his own salivary secretions in 1883,
and his first paper relating to its presence in the exudate of croupous
pneumonia was published on July 13th, 1885, i.e., thirteen days after

PROTECTIVE INOCULATIONS. 339

the publication of tlie paper from which the above quotation is made.
Under these circumstances the writer feels justified in again calling
attention to his priority in the discovery of this important pathogenic
niicrococcus, and in objecting to its beiug described as "Frankel's
pneumococcus, " the " diplococcus of Frankel," etc.

In my paper above referred to (July, 1885) I described this micro-
coccus under the name of Micrococcus Pasteuri, but in my " Manual
of Bacteriology " (1892) it is described under the name of Micrococcus
pneumonias crouposce.

This micrococcus is very pathogenic for mice and for rabbits, less
so for guinea-pigs and for dogs. Like other pathogenic microorgan-
isms of the same class, it varies greatly in virulence when obtained
from different sources. In the saliva of healthy persons, which
seems to be its normal habitat, it sometimes has comparatively little
virulence. On the other hand, when contained in the blood or in an
exudate from a serous cavity of an infected rabbit or mouse, it is very
virulent. In one instance (1881) the writer has seen a fatal result in
a dog from the subcutaneous injection of one cubic centimetre of bloody
serum from the subcutaneous connective tissue of a rabbit recently
dead.

Pneumonia never results from subcutaneous injections into sus-
ceptible animals, but injections through the thoracic walls into the
lung may induce a typical fibrinous pneumonia. This was first de-
monstrate,d by Talamon (1883), who injected the fibrinous exudate of
croupous pneumonia, obtained after death, or drawn during life by
means of a Pravaz syringe, from the hepatized portion of the lung,
into the lungs of rabbits. Gameleia has also induced pneumonia in
a large number of rabbits, and also in dogs and sheep, by injections
directly into the pulmonary tissue. Sheep were found to survive sub-
cutaneous inoculations, unless very large doses (five cubic centimetres)
of a virulent culture were injected. But intrapulinonarj' inoculations
are said to have invariably produced a typical fibrinous pneumonia
which usually proved fatal. In dogs similar injections gave rise to
a "frank, fibrinous pneumonia which rarely proved fatal, recovery
usually occurring in from ten to fifteen days, after the animal had
passed through the stages of red and gray hepatization characteristic
of this affection in man."

Without doubt an attack of pneumonia is followed by a certain
degree of immunity of longer or shorter duration. According to
Huge, who has made a careful study of the subject, relapses are
very infrequent — indicating a temporary immunity — but subsequent
attacks are more likely to occur in those who have once suffered an

340 PROTECTIVE INOCULATIONS.

attack of the disease, and as many as four or five attacks Lave been
known to occur in the same individual.

In 1,100 cases collected by Wagner but 2 relapses occurred
(=0.18 per cent). Huge reports that in 440 cases treated at the
Oharite in Berlin there were but 2 relapses. The liability to sub-
sec juent attacks at a later period is shown by the following figures,
which we copy from Euge's paper: In 280 cases reported by Stortz,
26.4 per cent had previously suffered an attack of the disease; in
133 cases reported by Morhart the proportion of previous attacks was
41.3 per cent; in 157 cases by Pohlmann, 34.4 per cent; in 166 cases
by Schapira, 31.3 per cent; in 128 cases by Keller, 36.9 per cent; in
175 cases by Grisolle, 30.9 per cent.

The writer, in a series of experiments made during the winter of
1880-81, obtained experimental evidence which showed that suscep-
tible animals (rabbits) acquire immunity from the pathogenic action
of this micrococcus as a result of inoculations with an attenuated
virus. The experiments referred to had as their object the determi-
nation of the comparative value of various germicidal agents, as tested
upon this micrococcus ; incidentally it was found " that a protective
influence has been shown to result from the injection " (into rabbits)
" of virus, the virulence of which has been modified, without being
entirely destroyed, by the agent used as a disinfectant." (Quoted
from the writer's report of the experiments referred to, "Studies
from Biological Laboratory," Johns Hopkins University, Balti-
more, 1882.)

In 1891 G. and F. Klemperer published an important memoir re-
lating to the pathogenic action of this micrococcus and the production
of immunity in susceptible animals by means of filtered cultures. In
some cases this immunity was found to last as long as six months. A
curious fact developed in their researches was that the potency of the
substance contained in the filtered cultures was increased by subject-
ing these to a temperature of 41° to 42° C. for three or four days, or
to a higher temperature (60° C. ) for an hour or two. When injected
into a vein after being subjected to such a temperature, immunity was
complete at the end of three or four days ; but the same material, not
so heated, required larger doses and a considerably longer time (four-
teen days) to confer immunity upon a susceptible animal. The un-
warmed material caused a considerable elevation of temperature, last-
ing for some days. The authors mentioned conclude from their
investigations that the toxic substance present in cultures of Micro-
coccus pneumoniae crouposse is a proteid substance, which they propose
to call pneumotoxin. The substance produced in the body of an im-

PROTECTIVE INOCULATIONS. 341

mune animal, as a result of protective inoculations, upon which the
immunity of these animals depends, is also a proteid, which they call
antipneurnotoxin. This they isolated from the blood serum of im-
mune animals. By experiment they were able to demonstrate that
the blood serum containing this protective proteid, when injected
into other animals, rendered them immune; and also that it arrested
the progress of the infectious malady induced by inoculating suscep-
tible animals with virulent cultures of the micrococcus. When in-
jected into the circulation of an infected animal, its curative action
was manifested by a considerable reduction of the body temperature.
The toxalbumin was obtained from filtered bouillon cultures of a viru-
lent variety of the micrococcus of pneumonia, in the form of an amor-
phous, yellowish-white powder. This was thrown down from the
filtered cultures by means of alcohol, and again dissolved in water
and reprecipitated in order to purify it.

Issaeff (1893) as a result of his experiments has found that the
virulence of this micrococcus can be greatly increased by successive
inoculations in the peritoneal cavity of rabbits, and that after a series
of ten or twelve such inoculations the blood of the infected animal does
not coagulate and becomes extremely toxic. In order to obtain the
toxins from this blood, Issaeff collects the blood of three or four ani-
mals just dead in a sterilized vessel, and adds to this an equal volume
of sterilized water containing one per cent of glycerin, made alkaline
by the addition of a few drops of a concentrated solution of bicarbo-
nate of soda. The mixture is sterilized by passing it through a Cham-
berland filter. This liquid sometimes kills rabbits when injected into
the circulation in the proportion of one per cent of the weight of the
animal. When heated to 70° C. its toxic power is considerably di-
minished, and a temperature of 100° C. neutralizes it completely.

Emmerich (1891) has succeeded in immunizing rabbits and mice
by the intravenous injection of a very much diluted but virulent cul-
ture of the micrococcus. Other rabbits and mice were rendered im-
mune by injecting into them material obtained from rabbits immu-
nized with diluted cultures. The flesh of these animals was rubbed up
into a pulp, and the juices were obtained by pressure through a piece
of sterilized cloth. The bloody juice, after standing for twelve hours
at a temperature of 10° C., was passed through a Pasteur filter and
then served to immunize the animals referred to.

Belfanti (1892) has succeeded in immunizing rabbits against the
pathogenic action of this micrococcus by injecting into the circulation
a filtrate obtained from the sputa of pneumonia cases. The viscid
sputa mixed with an equal part of distilled water was kept on ice for

PROTECTIVE INOCULATIONS.

twenty -four Lours and then passed through a Chamber land filter.
Ten cubic centimetres of this filtrate was injected into the ear vein of
rabbits. Some of the animals so treated proved to be immune against
general infection when inoculated with a virulent culture of the micro-
coccus, but they had a localized inflammation and oedema about the
point of inoculation. After recovering from this they proved to be
entirely refractory against subsequent inoculations.

Foa and Scabia (1892) have reported success in producing immu-
nity with filtered cultures, and also with a glycerin extract from the
blood of an infected rabbit. This, after filtration, was injected sub-
cutaneously in doses of two cubic centimetres at intervals of five days.
The authors named have also produced immunity in rabbits by the
use of " pneumo-protein. " This is an extract from the bacterial cells
obtained by first collecting these from the surface of a Chamberland
filter through which the cultures have been passed; then digesting
them for three hours at 55° C. in a five-per-cent solution of glycerin.
According to Foa and Scabia immunity produced in this way is more
decided and of longer duration than that resulting from the other
methods tested by them.

Mosny (1892) has also made numerous experiments which show
that rabbits may be immunized by means of filtered cultures, or by
the juices from the tissues of an immune animal obtained by macer-
ation and filtration. When sterilized cultures were employed the best
results were obtained by first heating very virulent cultures for three
hours at 60° C. The dose employed was ten cubic centimetres, and
immunity was not established immediately but required a period of
at least four days for its development.

The blood serum of immune rabbits was not found to have any
bactericidal power, and the micrococcus of pneumonia preserved its
vitality longer in the blood serum of immune rabbits than in that of
other animals of the same species.

G. and F. Klemperer had previously reported that the blood of
immune rabbits does not destroy the micrococcus of pneumonia or re-
strict its development.

Issaeff (1893) also reports his success in immunizing rabbits by
means of sterilized cultures or filtered blood from infected animals
recently dead. A single intravenous injection of ten cubic centi-
metres of filtered blood, prepared as heretofore indicated (p. 340),
sufficed to confer immunity. To test immunity the animals were
subsequently inoculated with two to four drops of virulent blood ; and
to maintain it the inoculations (0.5 cubic centimetre) were repeated
every four weeks. Although immune against infection these animals

PROTECTIVE INOCULATIONS. o43

are said not to have acquired any immunity against the toxins of the
micrococcus of pneumonia. Contrary to the conclusion reached by
G. and F. Klemperer, Issaeff concludes from his experiments that
"rabbits, although completely refractory against pneumonic infec-
tion, remain highly sensitive to the toxins of this microbe. Even
small doses of the toxins are not neutralized in the blood of vacci-
nated animals. We are therefore brought to the conclusion that the
existence of an antitoxic property of the blood of vaccinated animals
cannot be admitted."

The serum of immunized rabbits was not found by Issaeff to pos-
sess any bactericidal power for the micrococcus of pneumonia, and
no attenuation of virulence occurred as a result of cultivation in this
serum. But when introduced beneath the skin of an immune rabbit,
the micrococcus quickly loses its virulence. At the end of eighteen
hours it has completely lost its pathogenic power, and cultures made
in bouillon no longer have any injurious effect upon rabbits. This
attenuating effect produced in the body of an immune animal is
ascribed by Issaeff to the action of phagocytes, which are said to be
very numerous, and in the course of five or six hours to pick up all of
the cocci in the vicinity of the point of inoculation. These are not,
however, immediately destroyed in the interior of the phagocytes, but
preserve their vitality for nearly forty -eight hours, and when intro-
duced into bouillon give a culture which has no longer any patho-
genic virulence.

• RINDERPEST.

The disease of cattle known in Germany as rinderpest is due to a
bacillus closely resembling the bacillus of fowl cholera and of swine
plague (BaciUus septiccemice hcemarrhagicce).

Professor Semmer, of St. Petersburg, has reported (1892) his suc-
cess in immunizing cattle against this disease. The virulence of cul-
tures was attenuated by passing them through guinea-pigs, or by
exposure to heat, and this attenuated virus was used in protective
inoculations. Semmer says :

"By the subcutaneous injection of blood serum from immune animals
their susceptibility to rinderpest was diminished, and such blood serum de-
stroyed the 'rinderpest contagium' in one to twenty-four hours/'

SWINE PLAGUE.

As stated in the chapter on cholera in fowls, the bacillus of swine
plague (ScJnveineseuche, Loftier and Schiitz) very closely resembles
Pasteur's microbe of fowl cholera and Koch's bacillus of rabbit sep-

344 PROTECTIVE INOCULATIONS.

ticsemia, and if not identical with these at least varies from them so
slightly in its morphological and biological characters that recent
authors do not feel justified in considering it a distinct species.
Koch first obtained his bacillus of rabbit septicaemia by inoculating
rabbits with putrefying flesh infusion. Gaffky produced the same in-
fectious disease in rabbits by inoculating them with impure river water.
Davaine had previously obtained similar results by inoculating rabbits
with putrefying blood. The writer in 1887 produced the same disease
in rabbits, while in Cuba, by inoculating them with putrefying liver
from a yellow-fever cadaver. A similar, and possibly identical, ba-
cillus has been found in the blood of deer (Hueppe), of cattle (Kitt,
and of buffalo (Oreste-Armanni) suffering from a fatal infectious dis-
ease. And all of these allied species or varieties are included by
Hueppe and by the present writer under the single specific name
Bacillus septicwmice hcemorrhagicw. The bacillus of the disease known
in this country as swine plague, according to Smith, agrees in all par-
ticulars with that of the German swine plague (Sckweineseuche) de-
scribed by Loftier and Schiitz, except that the latter is more patho-
genic for swine and for rabbits.

In a publication by Smith and Moore (United States Department
of Agriculture, Bureau of Animal Industry, Bulletin No. 6, 1894) they
have given an account of their experiments relating to immunizing
animals against the pathogenic action of this bacillus. The bacilli
used in these experiments were sufficiently virulent to kill rabbits in
twenty hours when injected beneath the skin of these animals in doses
of 0.001 cubic centimetre of a fresh bouillon culture. The experiments
were made upon young adult rabbits by various methods, viz. : with
sterilized bouillon cultures ; with sterilized suspensions of agar cul-
tures; with the filtrate of agar suspensions; with defibrinated, steril-
ized blood of infected rabbits ; "with blood serum from immune animals.

"A greater or less degree of immunity was produced in rabbits by steril-
ized bouillon cultures, sterilized agar suspensions, sterilized blood from
infected rabbits, and blood serum from immunized rabbits. The sterilized
blood of diseased rabbits was capable of producing1 immunity, while the
blood serum of immune rabbits produced rather equivocal results."1

The different degrees of immunity which may be acquired by
rabbits, as shown by a subsequent inoculation with virulent material,
are classified by Moore as follows :

"1. No resistance — acute septicaemia.
"2. Slight resistance — peritonitis.

"3. Increased resistance — pleuritis and pericarditis with or without sec-
ondary pneumonia.

"4. Higher degree of resistance — pleuritis and peritonitis.

PROTECTIVE INOCULATIONS. 345

"5. Still greater resistance — irregular lesions in the form of abscesses,
subcutaneous and subperitoneal.

4k6. Nearly complete immunity — very slight reaction at the point of in-
oculation."

Up to the year 1894 the bacteriological experts of the Depart-
ment of Agriculture had not proposed to make a practical application
of the facts developed in their experimental work in the way of pro-
tecting herds of swine by means of inoculations with an attenuated
virus, or with sterilized cultures. In the report on swine plague,
made by the Bureau of Animal Industry published in 1891, the fol-
lowing measures for arresting an epidemic are recommended :

"When the disease has actually appeared in a herd the question generally
arises whether it is worth while to make any attempt to save a portion of the
herd or to leave them to their fate. As a rule it may be stated that it is best
to slaughter both healthy and diseased at once, and give the surroundings
sufficient time to rid themselves of the infection before fresh animals are
brought into them. If this be not desirable, we should recommend the fol-
lowing measures to be vigorously carried out :

"a. Removal of still healthy animals to uninfected grounds or pens as
soon as possible.

" b. Destruction of all diseased animals.

" c. Careful burial or burning of carcasses.

"d. Repeated thorough disinfection of the infected premises.

"e. Great cleanliness both as to surroundings and as regards food."

In the same report (1891) the following reference is made to pro-
tective inoculations :

" As regards swine plague the experiments which have thus far been car-
ried out indicate that this disease may prove amenable to preventive inocula-
tion. We have been able, by the injection of both living cultures and those
sterilized at a low temperature (58° C.), to make the most susceptible animals
— rabbits — insusceptible to the most virulent swine plague bacteria. By two
subcutaneous injections of cultures of swine-plague bacteria, swine have
been made insusceptible to doses injected into the circulation which proved
fatal to control pigs within twenty-four hours."

According to Smith the experiments of Metchnikoff (1892), re-
ported as made with the bacillus of hog cholera, were in fact made
with the bacillus of swine plague ; we therefore refer to them here.
These experiments showed that rabbits could be easily immunized
against the pathogenic action of virulent cultures by means of blood,
from an infected animal, sterilized by heat. Doses of 1.5 cubic centi-
metres, or more,* were fatal to rabbits; but smaller doses, repeated
several times, given either subcutaneously or by injection into the
circulation, caused the animal to become immune.

STREPTOCOCCUS INFECTION.

It is now generally recognized by pathologists that erysipelas,
puerperal fever, certain forms of diphtheritic inflammation of the

346 PROTECTIVE INOCULATIONS.

fauces, and certain acute abscesses are due to infection by a strepto-
coccus described by recent authors under the name of Streptococcus
2)yogenes. This streptococcus, like other pathogenic microorganisms
of the same class, varies greatly in its pathogenic power as a result of
conditions relating to the source of the particular variety under culti-
vation. As obtained from a case of erysipelas or puerperal fever it is
extremely virulent, but when it has led a saprophytic existence for
some time, or has been cultivated for a considerable time in the
usual artificial culture media, its pathogenic potency is greatly
diminished.

Mironoff (1893) has made a series of experiments with a view to
determining whether rabbits can be immunized against the pathogenic
action of this streptococcus, and has obtained successful results by the
following method :

Vigorous rabbits, weighing two kilogrammes, were inoculated
subcutaneousl}' with from three to six cubic centimetres of a sterilized
bouillon culture of the streptococcus. Cultures three days old were
employed, and these were sterilized for twenty minutes at 120° C..—
the reason for using so high a temperature is not apparent, inasmuch
as this streptococcus is destroyed in a few minutes by a temperature
of 60° C. At the end of ten to fifteen days, " when the animal has
fully recovered, " a second dose of from six to twelve cubic centimetres
of a culture, sterilized in the same way, is injected beneath the skin.
After another interval of ten to fifteen days two cubic centimetres of a
virulent non-sterilized culture are injected subcutaneously, and this is
repeated with gradually increasing doses (one to two cubic centime-
tres more) at intervals of the same period. Finally the animals
" support without reaction " a dose five times as great as would be re-
quired to kill an animal of the same weight not immunized. But the
author adds that more than half the animals thus treated died before
the completion of the immunizing process. These deaths resulted
from local infectious processes, such as peritonitis, pericarditis, men-
ingitis, or abscesses formed at the point of inoculation.

Further experiments showed that the blood serum of animals im-
munized in this way when injected into susceptible animals (rabbits)
in the dose of 1.5 cubic centimetres per kilogramme of body weight
conferred upon them a certain degree of immunity against streptococ-
cus infection, and with twice this amount (three cubic centimetres) a
very decided immunity was produced. The blood serum of immune
rabbits in doses of three to four cubic centimetres per kilogramme of
body weight was found to exercise a curative power, and completely to
arrest the acute septicaemia resulting from inoculations with a virulent

PROTECTIVE INOCULATIONS. 347

culture of this streptococcus, or to cause the disease to run a chronic
course, with formation of abscesses and final recovery.

In this connection we may call attention to the experiments of
Emmerich (1886), which show that the fatal course of anthrax infec-
tion, in rabbits, may be arrested by the subcutaneous or intravenous
injection of this streptococcus. Subsequent experiments by Emmerich
and de Mattei (1887) showed that eleven hours after such an injection
the anthrax bacilli were all dead and were already undergoing degen-
erative changes.

Emmerich and his associates (1894) have reported numerous addi-
tional experiments which show that the blood serum of a rabbit which
is suffering from streptococcus septicaemia (third day), when filtered
through a Pasteur-Chamberland filter to remove all living cocci, may
be used with success in arresting anthrax infection in rabbits. The
filtered serum was given four hours after the anthrax infection in the
dose of twenty-five cubic centimetres in the peritoneal cavity and fif-
teen cubic centimetres subcutaneously. This was repeated the fol-
lowing da3T at nine o'clock in the morning and five o'clock in the
evening, and again on the third day in the morning. Favorable re-
sults were also obtained by using in the same way blood serum from
a sheep infected with the streptococcus.

Cobbett (1894) reports success in immunizing rabbits by means of
attenuated varieties of the streptococcus or by filtered cultures. Also
that cutaneous erysipelas, produced by inoculation, after recovery
leaves the patient immune from a repetition of the local inflammatory
process as a result of a subsequent inoculation, and also confers a gen-
eral immunity against streptococcus infection. But this immunity is
of short duration, not lasting longer than a few weeks. Inoculation
in the ear of a rabbit, protected by a previous inoculation in the same
locality, is followed by an inflammatory reaction ; but this is of brief
duration and has disappeared before the erysipelatous inflammation
produced in a control is well under way.

SYMPTOMATIC ANTHRAX.

This disease of cattle is popularly known as " black leg, " or " quar-
ter evil," and is described by German authors under the name of
Raitschbrand — French, " cltarboii syiiiptomatiqm." The disease pre-
vails during the summer months in various parts of Europe, and to
some extent in the United States. It is characterized by the appear-
ance of irregular, emphysematous swellings of the subcutaneous tis-
sues and muscles, especially over the quarters. The muscles in the

348 PROTECTIVE INOCULATIONS.

affected areas Lave a dark color and contain a bloody serum in which
the bacillus is found to which the disease is due. This is an an-
aerobic bacillus which forms large oval spores.

The etiology of the disease was first clearly established by the re-
searches of Arloing, Cornevin, and Thomas (1880 to 1883), and sub-
sequent researches have shown that immunity may be produced in
susceptible animals by protective inoculations.

The disease causes considerable losses among cattle in certain sec-
tions. Horses do not contract it spontaneously, and when inoculated
with a culture of the bacillus present only a limited local reaction.
Swine, dogs, rabbits, fowls, and pigeons have but slight susceptibility.
The researches of the authors above mentioned have shown that the
virulence of a culture is greatly increased by adding to it twenty per
cent of lactic acid. The guinea-pig is the most susceptible animal,
and succumbs in from twenty-four to thirty -six hours when inoculated
subcutaneously with a small quantity of a pure culture. According
to Kitasato cultures in a bouillon made from the flesh of the guinea-
pig soon lose their virulence, while cultures in solid media preserve
their virulence for a long time. Cultures are readily attenuated by
heat, according to the method of Toussaint and Chauveau — exposure
to a temperature of 42° to 43° C. in the absence of spores. The
spores are attenuated by exposure for several hours to a temperature
of 80° to 100° C. Arloing, Cornevin, and Thomas recommend for the
production of immunity in cattle inoculation with a dried powder of
the muscles of animals recently dead from the disease. This is at-
tenuated by heat. According to Kitt the muscles should first be
dried at 32° to 35° C. and then powdered. Two " vaccines " are pre-
pared from this powder — a strong vaccine by exposure to a temper-
ature of 85° to 90° C. for six hours, and a weaker vaccine by exposure
for the same time to a temperature of 100° to 104° C. (dry heat). An
inoculation is first made with the weaker vaccine which gives rise to
a local reaction of moderate intensity. Later a second inoculation is
made with the stronger vaccine, after which the animal is immune
from the pathogenic action of the most virulent material. Immunity
may also be secured by intravenous injections ; or, in guinea-pigs, by
inoculations with cultures which have become attenuated by being kept
a few days, or by exposure to a temperature of 42° to 43° C. ; or by
inoculation with a very small quantity of a pure culture ; or by inocu-
lations with filtered cultures (Eoux and Chamberland) ; or with cultures
sterilized by heat (Kitasato). A non-fatal and protectivejocal infec-
tion may also be produced in cattle by inoculations with virulent ma-
terial made into the extremity of the tail. Eoux has claimed that

PROTECTIVE INOCULATIONS. 349

animals which have an acquired immunity against symptomatic an-
thrax are also immune against the pathogenic action of the bacillus of
malignant oedema; but Kitasato was unable to confirm this.

Strebel, in 1885, published the results of protective inoculations
made in Switzerland in 1884. The inoculations were made in the end
of the tail with two "vaccines," with an interval between the two of
from nine to fourteen days. The vaccines were prepared by exposure
to heat, as above recommended by Arloing, Cornevin, and Thomas.
The most favorable season for inoculations was found to be the spring,
and the most favorable age of cattle for inoculation from five months
to two years.

In seven Swiss cantons 2,199 cattle were inoculated; 1,810 inocu-
lations were made among animals which were exposed in dangerously
infected pastures. Of these but 2 died, one two months and the
other four months after the protective inoculations. Among 908 in-
oculated cattle, which were pastured with 1,650 others not inoculated,
the mortality was 0.22 per cent, while the loss among the latter was
6.1 per cent. The following year (1885), according to Strebel, the
number of inoculations, exclusive of those made in the canton of Bern,
w;ts 35,000. The losses among inoculated animals are reported as hav-
ing been about five times less than among those not protected in this
way. In the canton of Bern, in the same year, according to Hess,
15,137 cattle were inoculated by thirt}'-eight veterinarians — 12,190 of
these were pastured in dangerously infected pastures. The results
are said to have been favorable to the method, but the abstract at
hand does not give the precise figures.

In 1887 Kitt reported the results of his investigations, which were
confirmatory of those previously published by Arloing, Cornevin,
and Thomas, and also of a new method of inoculation, which pre-
sented the advantage that a single inoculation was sufficient to confer
immunity. This was made in the region of the shoulder with a vac-
cine somewhat stronger than that employed by the French bacteriol-
ogists, but which was found to be without danger for cattle. It
produced only a slight local effect. His vaccine was prepared by
heating the moistened flesh of an animal just dead from the disease
to 85° to 90° C. for six hours. This did not kill the spores present,
but caused a sufficient attenuation in their virulence.

In a later communication (1888) Kitt recommends that the flesh of
the diseased animal be first dried and pulverized, and then subjected
to a temperature of 100° C. in streaming steam for six hours, after
which it is to be again dried and used for subcutaneous inoculations.
The dose is from five to fifteen centigrammes.

350 PROTECTIVE INOCULATIONS.

Roux (1888) lias shown by experiment that sterilized cultures of
the bacillus, which have been exposed to a temperature of 115° C.,
when injected in doses of forty cubic centimetres, three times repeated,
into the cavity of the abdomen of guinea-pigs, cause these animals to
be completely immune against the most virulent material. Cultures
from which the bacilli have been separated by filtration are still more
active. And immunity could easily be conferred by the subcutaneous
inoculatioD, in guinea-pigs, of one cubic centimetre of the. filtrate from
the serum obtained from the cedematous tissues of a diseased animal.

Schuhanka (1888) has reported the results of inoculations made
in the dukedom of Salzburg during the year 1887. In all 2,596 cattle
were inoculated once, and 2,472 twice, with an attenuated virus, in
forty -seven different parishes. Most of these were from six months
to a year old. No losses occurred as a result of the inoculations.
During the summer of 1887 the 2,472 cattle which had been twice in-
oculated were associated in infected pastures with 3,561 unprotected
cattle. The loss among the former was 8, = 0.32 per cent; among the
latter it was 235, = 6.31 per cent.

Strebel reports similar results, in 1887, in the canton Freiburg,
where 1,725 cattle which had been inoculated suffered a loss of 0.23
per cent, and 1,945 associated cattle a loss of 5.28 per cent.

Lydtin (1892) reports the results of inoculations made in five dis-
tricts (Amtsbezirken) in Baden during the years 1886-91: 2,797 cattle
were inoculated with a loss of 3 only as a result of the inoculation.
None of the inoculated cattle subsequently contracted the disease.

In the Bulletin of the Central Society of Veterinary Medicine of
France (1892), Guillod and Simon give the results of 3,500 inocula-
tions made since 1884. The mortality among cattle in the region
where these inoculations were practised had been from 10 to 20 per
cent, but fell to 0.5 per cent among the inoculated animals.

The authors last named prefer inoculations in the region of the
shoulder to the plan first practised of inoculating in the end of the
tail. Strebel also (1892 ) advocates this method, which is quickly car-
ried out and attended with but little loss. According to Strebel the
loss among 13,022 inoculated in this way only amounted to 5, while
the loss among animals inoculated by the old method was twice as
great.

TETANUS.

The experiments of Kitasato (1889) show that pure cultures of the
tetanus bacillus injected into mice, rabbits, or guinea-pigs produce
typical tetanic symptoms and death. As the presence of this bacillus

PROTECTIVE INOCULATIONS. 351

at the seat of injury, in cases of tetanus in man, lias now been
demonstrated by numerous observers, there is no longer any ques-
tion that tetanus must be included among the traumatic infectious
diseases, and that the bacillus of Nicolaier and of Kitasato is the
specific infectious agent. Kitasato 's experiments (1890) show that
cultures of the tetanus bacillus which have been sterilized by fil-
tration through porcelain produce the same symptoms, and death, in
the animals mentioned, as result from inoculation with cultures con-
taining the bacillus. It is evident, therefore, that death results from
the action of a toxic substance produced by the bacillus. This is
further shown by the fact that the bacillus itself cannot be obtained in
cultures from the blood or organs of an animal which has succumbed
to an experimental inoculation with an unfiltered culture ; but the
blood of an animal killed by such an inoculation contains the tetanus
poison, and when injected into a mouse causes its death with tetanic
symptoms.

When a platinum needle is dipped into a pure culture of the teta-
nus bacillus, and a mouse is inoculated with it subcutaneously, the
animal invariably falls sick within twenty-four hours and dies of
typical tetanus in two or three days. Rats, guinea-pigs, and rabbits
are killed in the same way by somewhat larger quantities — 0.3 to 0.5
cubic centimetre (Kitasato). Pigeons are very slightly susceptible.
The tetanic symptoms are first developed in the vicinity of the point
of inoculation : if the animal is inoculated in the posterior portion of
the body, the hind legs first show tetanic contraction; if in the fore-
part of the body, the muscles of the neck are first affected. At the
autopsy there is a certain amount of hypersemia at the point of inocu-
lation, but no pus is formed; in inoculations with garden earth, or
accidental inoculations in man, pus is commonly found in the vicinity
of the inoculation wound. The various organs are normal in appear-
ance. Kitasato says that Jie has not been able to demonstrate the
presence of the bacillus or of spores in the spinal marrow, .the nerves,
muscles, spleen, liver, lungs, kidneys, or blood from the heart; nor
has he been able to obtain cultures from the various organs. In mice
which were inoculated at the root of the tail Kitasato was able to de-
monstrate the presence of the bacilli at the point of inoculation by the
microscopical examination of an excised piece of the tissues for eight
to ten hours after the inoculation ; later than this they were not found.
In pus from the inoculation wounds of men and animals accidentally
infected the bacilli are present, but the formation of spores does not
always occur. According to Kitasato, the sooner death has occurred
after accidental inoculation the less likely are spores to be found in

352 PROTECTIVE INOCULATIONS.

the rods, but from pus in which no spores are seen cultures of the
bacillus may be obtained in which spores will develop in the usual
manner.

Guinea-pigs are even more susceptible to the tetanus poison than
mice, and rabbits less so. The amount of filtrate from a slightly
alkaline bouillon culture required to kill a mouse is extremely minute
— 0.00001 cubic centimetre (Kitasato). The tetanic symptoms are
developed within three days; if the animal is not affected within four
days it escapes entirely. The tetanus poison is destroyed by a tem-
perature of 65° C. maintained for five minutes, or 60° for twenty
minutes, or 55° for an hour and a half; in the incubating oven at 37°
C. it gradually loses its toxic potency ; in diffuse daylight, also, its
toxic power is gradually lost ; in a cool, dark place it retains its origi-
nal potency indefinitely ; in direct sunlight it is completely destroyed
in from fifteen to eighteen hours ; it is not injured by being largely
diluted with distilled water; it is destroyed in an hour by hydro-
chloric acid in the proportion of 0.55 per cent; terchloride of iodine
destroys it in the proportion of 0.5 per cent; cresol in one per cent —
one hour's exposure. In general it is destroyed by acids and by
alkalies. Blood serum from cattle, horses, sheep, rabbits, rats, or
guinea-pigs does not modify its toxic properties.

Brieger (1886) first succeeded in obtaining from impure cultures
of the tetanus bacillus a crystallizable toxic substance, called by him
tetanin, which was found to kill small animals in very minute doses
and with the characteristic symptoms of tetanus. More recently Kit-
asato and Weyl have obtained the same substance, by following
Brieger's method, from a pure culture of this bacillus. From a bouil-
lon made from one and one-fourth kilogrammes of lean beef, with the
addition of twenty -five grammes of peptone, they obtained 1.7118
grammes of hydrochlorate of tetauin. This proved fatal to white mice
in six hours in the dose of 0.05 gramme, and a dose of 0.105 gramme
caused characteristic tetanic convulsions and death within an hour.
The bacteriologists last named also obtained from their cultures the
tetanotoxin of Brieger. Two mice were inoculated subcutaneous!}'
with 0.003 gramme of this substance; one died at the end of five
hours without the development of tetanic symptoms ; the other sur-
vived. In addition to these substances, indol, phenol, and buty-
ric acid were demonstrated to be present in cultures of the tetanus
bacillus.

The more recent researches of Brieger and Frankel, and of Kita-
sato, show that the toxic ptomain discovered by Brieger in 1886 is
not the substance to which cultures of the tetanus bacillus owe their

PROTECTIVE INOCULATIONS. 353

great and peculiar pathogenic power. The distinguished German
chemist and his associate have succeeded in isolating from tetanus
cultures a toxalbumin which is far more deadly than tetanin.

Brieger and Cohn in more recent investigations (1893) relating to
the toxic products of the tetanus bacillus have arrived at the following
results : The cultures were made in veal bouillon containing one per
cent of peptone and one-fifth per cent of chloride of sodium. Large
quantities of the cultures in this medium were filtered through porce-
lain filters. The active substance was precipitated from the filtrate
by means of a saturated solution of ammonium sulphate. By adding
this salt in excess the precipitate is made to rise to the surface and is
skimmed off with a platinum spatula. The liquid is removed by plac-
ing this upon porous porcelain plates and the crude toxin is dried in
a vacuum. It still contains 6.5 per cent of ammonium sulphate. The
tetanus bouillon after filtration is said to be fatal to mice in the dose
of 0.00005 cubic centimetre. A litre of this bouillon gave about one
gramme of the dried precipitate, which produced characteristic te-
tanic symptoms and death when injected into mice in the dose of
0.0000001 gramme. Kitasato in his experiments had previously ob-
tained a tetanus bouillon which was five times as toxic as that used by
Brieger and Cohn in their experiments, and which killed mice in the
dose of 0.00001 cubic centimetre. The dried precipitate obtained by
Brieger and Cohn contained various impurities, including a certain
amount of ammonium sulphate, but was found to kill susceptible ani-
mals in the proportion of 0.0000066 gramme per kilogramme of body
weight.

It was purified without loss of toxic power by placing it in a dialyzer
in running water for from twenty -four to f orb* -eight hours, after which
it was dried in vacua at 20° to 22° C. The purified toxin thus ob-
tained had a slightly yellowish color, and was in the form of trans-
parent scales, which were odorless, tasted like gum acacia, and were
easily soluble in water. The chemical reactions of this purified toxin,
according to Brieger and Cohn, show that it is not a true albuminous
body. When injected beneath the skin of a mouse weighing fifteen
grammes, in the dose of 0.00000005 gramme, it causes its death, and
one-fifth of this amount gave rise to tetanic symptoms from which the
animal recovered after a time. The lethal dose for a man weighing
seventy kilogrammes is estimated by the authors named to be 0.00023
gramme (0.23 milligramme). Comparing this with the most deadly
vegetable alkaloids known it is nearly six hundred times as potent as
atropine and one hundred and fifty times as potent as strychnine.

Fermi and Pernossi (1894), as a result of an elaborate research,
23

354 PROTECTIVE INOCULATIONS.

have determined many of the chemical characters of the tetanus toxin.
When in solution it is destroyed by a comparatively low temperature
(55° C. for one hour) and by exposure to direct sunlight, but the dry
powder resists a temperature of 120° C. It has not the properties of
an alkaloid, as it is not dissolved by any of the usual solvents of these
bodies — the only solvent thus far discovered is said to be water. It
resembles the albumins and peptones in its failure to pass through
a dialyzing membrane. The authors last referred to conclude their
summary of results as follows :

"The appended table shows that the tetanus poison, like that of diph-
theria, in its behavior as regards the action of light, heat, chemical agents,
and dialysis, as also its solvents, the agents which precipitate it, and its action
upon living animals, closely resembles the poisons of serpents (Naja tripu-
dians, Crotalus, etc.). As to the chemical nature of this group of substances,
we can at present only say that they rather have the characters of collodial
substances than otherwise, and more nearly resemble the albuminoid bodies
ihan the bases. We do not, however, reject the very probable hypothesis
that these toxins are acids or bases, or other very unstable, peculiar substances,
which are closely united with colloidal substances, as is the case, for example,
with the alkali and acid albumins and so many other albuminous bodies."

While the exact nature of the toxic substance contained in tetanus
cultures has not been determined, we probably cannot, at present, do
better than to continue to speak of it as a "toxalbumin."

Kitasato (1891) was not able to produce immunity in mice by in-
oculations with minute doses of the poison, or with a filtrate which
had been exposed to various degrees of temperature by which its
activity was diminished or destroyed. But immunity lasting for
about two months was produced in rabbits by inoculating them with
the filtrate from a culture of the tetanus bacillus, and subsequently,
in the same locality, with three cubic centimetres of a one-per-cent
solution of terchloride of iodine; this last solution was injected sub-
cutaneously in the same dose at intervals of twenty -four hours for five
days. Of fifteen rabbits treated in this way six proved to be immune
against large doses of a virulent culture of the tetanus bacillus. The
same treatment was not successful in producing immunity in mice or
guinea-pigs, but the important discovery was made that a small quan-
tity of blood (0.2 cubic centimetre) from an immune rabbit, when in-
jected into the abdominal cavity of a mouse, gave it immunity from
the effects of inoculations with the tetanus bacillus. Moreover, mice
which were first inoculated with a virulent culture of the bacillus, and,
after tetanic symptoms had appeared, received in the cavity of the
abdomen an injection of blood serum from an immune mouse, were
preserved from death. The power of the blood of an immune animal
to neutralize the tetanus poison was further shown by mixing the fil-

PROTECTIVE INOCULATIONS. 355

trate from a virulent culture with blood serum from an immune animal
and allowing it to stand for twenty -four hours; a dose three hundred
times greater than would have sufficed to kill a mouse proved to be
without effect after such admixtures with blood serum; as before
stated, the blood serum of animals which are not immune has no
effect upon the poison. The duration of immunity induced in this
way was from forty to fifty days. Blood serum from an immune rab-
bit, preserved in a cool, dark room, retains its power of neutralizing
the tetanus poison for about a week, after which time it gradually
loses it. Having found that chickens have a natural immunity against
tetanus, Kitasato made experiments to ascertain whether their blood
serum would also neutralize the tetanus poison ; the result was nega-
tive.

That the tetanus poison is present in the blood of individuals who
die from tetanus has been proved by Kitasato by injecting a small
quantity (0.2 to 0.3 cubic centimetre) of blood from the heart of a
fresh cadaver into mice; the animals develop typical tetanic symp-
toms and die in from twenty hours to three days.

Tizzoni and Cattani have (1891) reported results similar to those
obtain by Kitasato. By repeated inoculations with gradually in-
creasing doses of the tetanus poison they succeeded in making a dog
and two pigeons immune, and found that blood serum from this im-
mune dog, in very small amount, completely destroyed the toxic
power of a filtrate from cultures of the tetanus bacillus — one to two
drops of serum neutralized 0.5 cubic centimetre of filtrate after fifteen
to twenty minutes' contact. They also ascertained that small amounts
of blood serum from this immune dog injected into other dogs or
white mice produced immunity in these animals; but they were not
able to produce immunity in guinea-pigs or rabbits by the same
method.

In a later communication (May, 1891) Tizzoni and Cattani give
an account of their experiments made with a view to determining
the nature of the substance in the blood serum of an immune animal
which has the power of destroying the toxalbumin of tetanus — " tet-
anus antitoxin." They found, in the first place, that this antitoxin
in blood serum is destroyed in half an hour by a temperature of 68°
C. ; further, that it does not pass through a dialyzing membrane; that
it is destroyed by acids and alkalies. As a result of their researches
they conclude that it is an albuminous substance having the nature of
an enzyme.

Yaillard has succeeded in producing immunity in rabbits by re-
peated injections into the circulation of filtered cultures — in all twenty

350 PROTECTIVE INOCULATIONS.

cubic centimetres — which had been exposed for one hour to a temper-
ature of 60° C. At a temperature of 65° C. both the toxic and the
immunizing action is destroyed.

Behring (1892) gives the following account of a method which he
has successfully employed for producing immunity in large animals —
especially in horses: A culture of the tetanus bacillus is made, in
bouillon, of such toxic potency that 0.75 cubic centimetre will kill a
rabbit in three or four days. To two hundred cubic centimetres of
this culture he adds carbolic acid in the proportion of 0.5 per cent for
the purpose of preserving it. The horse first receives a subcutaneous
injection of ten cubic centimetres of this culture fluid to which ter-
chloride of iodine (IC13) has been added in the proportion of 0.25
per cent; at the end of eight days twenty cubic centimetres of the
same mixture are given; again in eight days the dose is repeated;
then, after an interval of three days, thirty cubic centimetres of the
same mixture. Following this, at an interval of eight days, he gives
two injections of thirty cubic centimetres each of a mixture containing
one-half the quantity of IC13 (0.175 per cent). The proportion of
the iodine terchloride is then reduced to 0.125 per cent, and two
doses of twenty cubic centimetres each are given. Finally the culture
fluid is administered in the dose of 0.5 cubic centimetre, and this dose
is doubled every five days. Before giving the first dose of culture
fluid without the addition of IC13, the immunizing value of the blood
serum of the horse is tested on mice, and if it falls below 1 : 100 a
dose of 0.25 cubic centimetre is given instead of the larger dose (0.5
cubic centimetre) above mentioned.

Schutz (1892) has applied Behring 's method to a considerable
number of horses and sheep, and arrives at the conclusion that it is a
reliable method of protecting these animals against infection with liv-
ing tetanus bacilli and against the toxic action of filtered cultures ;
that the degree of immunity" and the antitoxic power of the blood
serum increase as larger doses are gradually given. According to
Behring the immunizing value of blood serum from a horse treated in
this way is very high. As tested on mice it may be 1 : 200,000, or even
more. According to his calculations a serum having a value of 1 : 100,-
000, as tested on mice, should be given to a man weighing fifty kilo-
grammes in the quantity of fifty cubic centimetres, given in the course
of two days, in order to insure immunity.

The same author in a subsequent paper (1892) gives details as to
the method of estimating the therapeutic value of serum from an im-
mune animal. He first calls attention to the fact that the only re-
agent by which the antitoxic potency of this serum can be tested is

PROTECTIVE INOCULATIONS. 357

the body of a living animal. The test animal selected is the white
mouse. When the statement is made that a serum has the value of
1 : 1,000,000, he means that by an experimental test, made upon white
mice, it has been ascertained that these animals are protected from
fatal infection with the minimal lethal dose of a tetanus culture by the
use of 0.00002 gramme of the serum for a mouse weighing twenty
grammes. For the cure of tetanus in the mouse, after the first symp-
toms of the disease have appeared, a dose at least one thousand times
as great as the immunizing dose is required, and the more advanced
the progress of the case the greater the dose must be. A serum of
the strength above indicated, if used for the treatment of a case of
tetanus in man, should, according to Behring, be employed in doses
amounting altogether to at least one hundred cubic centimetres — given
inside of twenty -four hours in doses of twenty cubic centimetres each.
For persons sixteen years old he would give doses of ten cubic centi-
metres, and for children under six, five cubic centimetres at a dose.
The serum of this strength which he had prepared for testing its
curative value on man was preserved by the addition of 0.5 per cent
of carbolic acid.

Rotter (1892) reports a case successfully treated by Behring 's
serum. In all two hundred and fifty cubic centimetres was adminis-
tered subcutaneously. The case was not, however, one of the most
severe forms of the disease.

Brieger and Ehrlich (1892) have succeeded in immunizing goats
by means of gradually increasing doses of a culture of the tetanus
bacillus in thymus bouillon. The amount given at first was 0.2 cubic
centimetre, and this was gradually increased to ten cubic centimetres.
At the end of thirty -seven days the animal was found to be immune
against virulent cultures, and the important fact was demonstrated
that the immunizing substance (antitoxin) was present in its milk.
A mouse which received 0.1 cubic centimetre of the milk of this goat
in the peritoneal cavity proved to be immune against infection as a
result of inoculation with a tetanus culture. The immunizing value
of the milk from this goat was found to be 1,600. That is, a dose of
0.2 cubic centimetre, which was equal to 1 : 100 of the body weight of
the animal, protected a mouse from sixteen times the fatal dose of a
tetanus culture. After precipitation of the casein the milk still pre-
served its antitoxic power unimpaired, and by concentrating it in vacao
a fluid was obtained which proved to have an immunizing value of
5,000.

In a later communication (1893) Brieger and Colin give the results
of additional experiments with the milk of immunized goats. Ani-

358 PROTECTIVE INOCULATIONS.

mals were chosen which were two or three years old and had given
birth to young a few weeks before the inoculations were commenced.
It having been previously shown by Ehrlich that the precipitated
tetanus toxin from cultures could be successfully used to immunize
guinea-pigs, the same substance was employed in these experiments.
The treatment was commenced with a dose of 0.00001 gramme, which
was carefully increased to 0.00007 gramme, the injections being made
at intervals of four days. But this proved to be too much, and the
animal died of typical tetanus after the last dose. In a subsequent
experiment Brieger and Colin succeeded in immunizing a goat in a
month and a half so that the animal finally withstood a dose of 0.06
gramme, but this animal ceased to give milk, became anaemic, and
finally died.

The authors therefore resorted to a different method which had
previously been successfully employed by Ehrlich, Behring, and
others. Cultures of the tetanus bacillus in bouillon were heated to
65° C. for half an hour, and then used for immunizing two goats.
After five weeks' treatment the animals resisted doses of the precipi-
tated toxin, which were gradually increased to ten grammes, at which
time the treatment had been carried on for nearly six months and the
antitoxic value of the milk was found to be 90,000 immunization units.

The method of determining antitoxic values adopted by Brieger
and Cohn is the following : They had found by carefully conducted
experiments that their precipitated toxin (Eohgifte) killed a mouse
weighing twenty grammes in the dose of 0.0000003 gramme, but
failed to kill when injected in the dose of 0.0000002 gramme. The
first-mentioned dose was therefore accepted as the minimum fatal dose
for an animal weighing eighteen to twenty grammes, and the object
in view was to find the minimum amount of milk required to prevent
the toxic action of such a dose.

The antitoxin was obtained from the goat's milk by precipitation
with ammonium sulphate, thirty -two per cent; the precipitate was
again dissolved and treated with a solution of basic acetate of lead;
this salt does not precipitate the antitoxin when the solution is slightly
alkaline; the voluminous precipitate produced by the lead acetate is
filtered out and repeatedly washed with water; the filtered fluid and
wash water are again treated with ammonium sulphate, added to
saturation, and the resulting precipitate is dissolved in a small quan-
tity of water; a precipitate is again obtained by saturation with am-
monium sulphate, and this is dried upon porcelain plates in a vac-
uum. The ammonium sulphate remaining could not be removed by
dialysis, as experiment showed that a considerable loss of the antitoxin

PROTECTIVE INOCULATIONS. 359

occurred in a dialyzer placed in running water. But by shaking up
the dry powder in chloroform the heavy salt sank to the bottom and
the purified antitoxin floated on the surface and could be recovered by
skimming it off. The powder thus obtained consisted of a mixture of
various substances, including the antitoxin, and when obtained from
milk having an antitoxic value of 90,000 it was found to have a value
of 25,000,000 immunization units. By further purification a still
higher value was obtained (55,000,000). In experiments on mice a
dose ten thousand times as great as was necessary to produce immu-
nity proved to exercise a curative power — i.e., a dose of 0.02 gramme
for a mouse weighing twenty grammes saved it from being killed by
double the minimum fatal dose of the tetanus toxin, after tetanic
symptoms had been developed.

Reference has been made to the production of immunity by the
use of cultures made in thymus bouillon. This was made known
through the experiments of Brieger, Kitasato, and Wassermann
(1892). The thymus bouillon is made from the thymus glands of
calves, which are chopped fine in a hash machine and covered with
an equal volume of distilled water. The mixture is stirred for some
time and then placed in an ice chest for twelve hours; the liquid is
then obtained by filtration through gauze with pressure — by means of
a flesh-press machine. A turbid, slimy fluid is thus obtained, which
is diluted with an equal volume of water and made slightly alkaline
by the addition of soda solution. It is then sterilized at 100° C. for
fifteen minutes. As a result of this the liquid has a grayish-brown
color, and some large flocculi in suspension, which are removed by
passing it through fine linen. The fluid is then of a milky opal-
escence. It is next placed in test tubes and again sterilized. The
tetanus bacillus when cultivated in this medium does not form spores,
and the toxic potency of the culture is very much reduced — 1 : 5,000 to
1 : 3,000 of the toxic potency manifested by cultures of the same bacil-
lus in ordinary media. Inoculations with cultures in thymus bouillon
were found to kill mice in the dose of 0.5 cubic centimetre, while
smaller amounts failed to kill and caused the animals to be immune.
A culture in ordinary bouillon was fatal to mice in the dose of 0.001
cubic centimetre.

Experiments on rabbits (thirty -five) gave a uniformly successful
result in immunizing these animals. Immunity was established in
the course of two weeks, and the blood serum of these animals tested
on mice showed an antitoxic value of 1,000.

Reference has already been made to the earlier researches of the
Italian investigators, Tizzoni and Cattani. These have been followed

360 PROTECTIVE INOCULATIONS.

by additional investigations, the results of which have been reported
in numerous published papers. The authors named have ascertained
that when kept in a cool place (15° to 25° C.) the blood serum of
immune rabbits retains its antitoxic power for several months, and
the antitoxin, obtained by precipitation with alcohol, kept in a dry
condition for more than ten months, was found to preserve its original
activity.

Having succeeded in their earlier experiments in immunizing rab-
bits and dogs, Tizzoni and Cattani (in 1893) proceeded to experiment
upon horses, and were equally successful with these animals. As a
result of numerous injections with an attenuated virus, continued for
a period of ninety-seven days, they established an immunity which
was tested by inoculating the animal with ten cubic centimetres of a
gelatin culture, of which one two-hundredth part of a drop killed a
white mouse. The antitoxic value of the blood serum of this horse
was 1 : 5, 000, 000 — i.e., one gramme of this serum would immunize five
million grammes of mice, or two hundred and fifty thousand mice
weighing twenty grammes each. In a later communication (1894) the
authors named report that after freely bleeding immunized horses, and
allowing them to rest for one or two months, and then again treating
them with small doses of tetanus cultures, the blood serum soon be-
comes as active as before the bleeding. The greatest antitoxic power
was manifested from twenty to twenty-three days after the completion
of the protective inoculations, and a serum was obtained possessing a
value of 1 : 10,000,000. According to the authors named the precipi-
tated (by alcohol) and purified antitoxin from such a serum, judging
from their experiments on lower animals, should cure a case of teta-
nus in man in the dose of from forty to fifty centigrammes.

The authors last mentioned have reported (1892) that the young of
immune parents have a certain degree of inherited immunity. And
the more recent experiments of Ehrlich and Hubener have confirmed
this so far as the inheritance of immunity from the mother (in mice)
is concerned; but their results did not show any immunity in the
young when only the father had been rendered immune ; and the im-
munity inherited from the mother only lasted for two or three months
after birth.

TUBERCULOSIS.

Metchnikoff states that when kept at a temperature of 42° C. for
some time the tubercle bacillus undergoes a notable diminution in its
pathogenic power, and that when kept at a temperature of 43° to 44°
C. it after a time only induces a local abscess when injected subcu-

PROTECTIVE INOCULATIONS. 361

taneously into guinea-pigs. The experiments of Lote also indicate
that an "attenuation of virulence" has occurred in the cultures pre-
served in Koch's laboratory, originating in 1882 from the lungs of a
tuberculous ape. The author named made experiments with cultures
from this source (ninetieth to ninety -fifth successive cultures), and at
the same time with a culture obtained from Roux, of Pasteur's labor-
atory. Rabbits inoculated with cultures from the last-mentioned
source developed a hectic fever at the end of two weeks, and died tu-
berculous at the end of twenty -one to thirty-nine days. Twelve rab-
bits were inoculated with the cultures from Koch's laboratory ; the
injections were made either subcutaneously, or into a vein, or into the
pleural cavity, or into the cavity of the abdomen. No elevation of
temperature occurred in any of the animals, and they were found at
the end of a month to have increased in weight. At the end of six
weeks one of them was killed and tubercular nodules were found in
various organs. The remaining animals were killed at the end of
one hundred and forty -four to. one hundred and forty -eight days.
The two inoculated subcutaneously presented no sign of general tu-
berculosis, but a small yellow nodule containing bacilli was found at
the point of inoculation. Those inoculated by injection into a vein
showed one or two nodules in the lungs containing a few bacilli. In
Koch's original experiments rabbits were killed by intravenous inocu-
lation of his cultures in from thirteen to thirty-one days. That this
attenuation of virulence depends upon a diminished production of
toxic product to which the bacillus owes its pathogenic power appears
to be very certain, in view of the fact that the late cultures in a series
have a more vigorous and abundant development than the more patho-
genic cultures obtained directly from the animal body.

The discovery by Koch of a toxin in cultures of this bacillus,
which is soluble in glycerin, and which in very minute doses pro-
duces febrile reaction and other decided symptoms when injected
subcutaneously into tuberculous animals, must rank as one of the first
importance in scientific medicine, whatever the final verdict may be
as to its therapeutic value in tuberculous diseases in man.

The toxic substance contained in Koch's glycerin extract from cul-
tures of the tubercle bacillus, now generally known under the name of
tuberculin, is soluble in water, insoluble in alcohol, and passes readily
through dialyzing membranes. It is not destroyed by the boiling
temperature. According to the chemical examination of Jolles, the
" lymph " contains fifty per cent of water and does not contain alka-
loids or cyanogen compounds. It contains albuminates, which are
thrown down as a voluminous white precipitate by tannic acid, and

362 PROTECTIVE INOCULATIONS.

are redissolved by hot water containing sodium chloride and very
dilute potash solution. The elementary analysis gave N 5.90 per
cent, C 35.19 per cent, and H 7.02 per cent. The results obtained
are believed to show that the active substance present in the lymph
is a toxalbumin. In experiments made with Koch's lymph in Pas-
teur's laboratory by Bardach, a very decided elevation of temperature
was produced in tuberculous guinea-pigs by the subcutaneous injection
of 0.1 gramme, and a fatal result by the injection of 0.2 to 0.5 gramme.
In man a decided febrile reaction is produced in tuberculous patients
by very much smaller doses — 0.001 cubic centimetre.

Hammerschlag, in his chemical researches, found that the tubercle
bacillus yields a larger proportion of substances soluble in alcohol and
ether than any other bacilli tested (twenty-seven per cent). The alco-
holic extract contains fat, lecithin, and a toxic substance which pro-
duces convulsions in rabbits and guinea-pigs. The portion insoluble
in alcohol and ether contains cellulose and an albuminoid substance.
No ptomains were found, but a toxalbumin was isolated, which
caused an elevation of temperature in rabbits of 1° to 2° C., lasting
for a day or two.

Koch (1891) has given a full account of his method of prepari-ng
crude tuberculin, and also the process by which he obtains from this
a tuberculin which appears to be pure, or nearly so. To obtain con-
siderable quantities of the crude product the tubercle bacillus is culti-
vated in an infusion of calves' flesh, or of beef extract to which one
per cent of peptone and four or five per cent of glycerin have been
added. This culture liquid must be made slightly alkaline, and it
is placed in flasks with a flat bottom, which should not be more than
half filled — thirty to fifty cubic centimetres. The inoculation is made
upon the surface with small masses from a culture upon blood serum
or glycerin agar. By accident Koch discovered that these masses
floating upon the surface give rise to an abundant development, and
to the formation of a tolerably thick and dry white layer, which finally
covers the entire surface. At the end of six to eight weeks develop-
ment ceases, and the layer after a time sinks to the bottom, breaking
up meanwhile into fragments. These cultures, after their purity has
been tested by a microscopical examination, are poured into a suitable
vessel and evaporated to one- tenth the original volume over a water
bath. The liquid is then filtered through procelain. The crude tu-
berculin obtained by this process contains from forty to fifty per cent
of glycerin, and consequently is not a suitable medium for the develop-
ment of saprophy tic bacteria, if they should by accident be introduced
into it. It keeps well and preserves its activity indefinitely.

PROTECTIVE INOCULATIONS. 3G3

From this crude tuberculin Koch has obtained a white precipitate
with sixty-per-cent alcohol which has the active properties of the
crude tuberculin as originally prepared. This is fatal to tuberculous
guinea-pigs in doses of two to ten milligrammes. It is soluble in
water and in glycerin, and has the chemical reactions of an albuminous
body. In preparing it one volume and a half of absolute alcohol is
added to one volume of the crude tuberculin, and, after stirring it to
secure uniform admixture, this is put aside for twenty -four hours.
At the end of this time a flocculent deposit will be seen at the bot-
tom of the vessel. The fluid above this is carefully poured off; and
an equal quantity of sixty-per-cent alcohol is poured into the vessel
for the purpose of washing the precipitate. This is again allowed to
settle, and the procedure is repeated three or four times, after which
the precipitate is washed with absolute alcohol. It is then placed
upon a filter and dried in a vacuum exsiccator.

The " tuberculocidin " of Klebs is a purified tuberculin obtained by
precipitation with alcohol. The precipitate is washed in chloroform
and then dissolved in a mixture of carbolic acid and glycerin.

Bujwid (1894) prepares tuberculin as follows : He uses cultures on
glycerin agar or in glycerin bouillon which have been kept at a suit-
able temperature for five to eight weeks. The glycerin-agar cultures
are treated with distilled water by which the tuberculin is extracted.
After adding the water the test tubes are kept in a cool place for
twenty -four hours, and this is repeated two or three times. The ex-
tract from the agar cultures or the bouillon cultures is then sterilized
by exposure for from five to ten minutes to a temperature of 100° C. ;
then filtered through a Chamberland filter ; then evaporated at a low
temperature to a syrup-like consistence. When this crude tuberculin
is dropped into ten times its volume of strong alcohol a brown pre-
cipitate is thrown down which contains the active principle. From
the tubercle bacilli obtained by filtering his cultures Bujwid also
obtained an active substance which in doses of two milligrammes
caused an elevation of 2° C. in the temperature of an infected guinea-
pig. This substance was obtained by digesting the bacilli for two
months in glycerin and water (three per cent of glycerin), filtering
and evaporating the extract, and precipitation in six volumes of
ninety-five-per-cent alcohol. The precipitate when dried was in the
form of a white powder.

Helm an (1894) obtains tuberculin from potato cultures. The sec-
tions of potato are neutralized by leaving them for half an hour in a
solution of one-half to one per cent of bicarbonate of soda, after which
they are sterilized for twenty minutes in the autoclave at 120° C. The

364 PROTECTIVE INOCULATIONS.

best results were obtained when the potatoes were wet with a five-per-
cent solution of glycerin. The sections of potato were placed in Petri's
dishes upon blotting paper wet with a sublimate solution, and the
dishes containing the cultures were surrounded with cotton wet with
the same solution. The cultures were subsequently treated with dis-
tilled water, to extract the active principle, which was also obtained
from the bacilli by mixing them with glycerin in the proportion of
1:10.

Numerous experiments have been made with dead tubercle bacilli,
as well as with the toxic products developed in cultures. Hericourt
and Bichet (1890) found by experiment that old cultures heated to 80°
C., several days in succession, when injected into a vein in rabbits, in
the dose of ten to twenty cubic centimetres, caused the death of these
animals. Smaller doses from which the animals recovered seemed to
make them less susceptible to infection than control animals, but the
number of experiments was too limited to establish this as a fact. In
a subsequent (1891) communication the authors named claim to have
succeeded in immunizing rabbits by injecting filtered and sterilized
cultures of the tubercle bacillus, either subcutaneously (five to fifteen
cubic centimetres) or into a vein (twenty to forty drops) . The injections
were repeated every second or third day for a period of fifteen days,
after which the test inoculation was made with a culture, obtained
from a tuberculous cow in one series, and from tuberculous fowls in
another. Four vaccinated rabbits in the first series escaped general
tuberculosis, while four out of eight control animals died tuberculous.
In the second series five vaccinated animals resisted infection and
three out of four control animals died tuberculous.

De Schweinitz (1894) has reported the results of experiments with
attenuated cultures of the tubercle bacillus, and has, apparently, suc-
ceeded in conferring immunity upon guinea-pigs by inoculations
with such cultures.

Klebs (1891) , in experiments on guinea-pigs and rabbits, convinced
himself that the fatal result of an inoculation with tubercle bacilli (in
the cavity of the abdomen or subcutaneously in guinea-pigs, and in
the eye in rabbits) was greatly delayed by injections of Koch's tuber-
culin (0.3 to 0.5 cubic centimetre) either before or after infection.

Baumgarten (1891), in experiments upon rabbits inoculated with
tubercle bacilli in the anterior chamber of the eye, failed to obtain
favorable results from treatment with Koch's tuberculin given in con-
siderable doses (0.5 to one gramme) either before or after infection.

The results reported in the same year by Gramatschikoff, by
Popoff, by Alexander, and by Gasparini and Mercanti, were also un-

PROTECTIVE INOCULATIONS. 365

favorable as regards au immunizing or curative effect from inocula-
tions of tuberculin in rabbits. Donitz, on the contrary, arrives at the
conclusion that when early treatment is instituted iris tuberculosis
may be arrested and cured, and the more recent experiments of Tru-
cleau (1893) give support to this conclusion. Baumgarten, however,
insists that the tuberculin treatment does not prevent metastasis to
the lungs after inoculations in the anterior chamber of the eye.

Pfuhl (1891) treated forty-seven infected guinea-pigs, and at the
date of his report forty-four had died tuberculous, but the date of
death was somewhat postponed by the treatment. The animals not
treated succumbed at the end of eight weeks (average of all controls),
and those treated with small doses of tuberculin lived, on the average,
ten weeks. With larger doses still more favorable results were ob-
tained— four lived on an average twelve weeks, and three were still
living, eleven, fifteen, and sixteen weeks after infection, at the date of
publication.

Kitasato (1892) also obtained favorable results in the treatment
of infected guinea-pigs, and arrives at the conclusion that guinea-pigs
which have been cured by the treatment are not susceptible to a sec-
ond infection, for a certain time at least.

Bujwid (1892), in experiments upon guinea-pigs, found that in-
fected animals which received from 0.05 to 0.1 gramme of tuberculin
within three hours showed an elevation of temperature of 1.5° to 2° C.
Thirteen infected guinea-pigs treated with tuberculin lived from two
and a half to eight months, while all of the control animals (eighteen)
died in from six to nine weeks. The animal which survived eight
months was found not to be tuberculous, but presented evidence of re-
covery from a former tuberculous process. In two rabbits inoculated
in the anterior chamber the iris tuberculosis was favorably influenced
by the tuberculin treatment, but general infection occurred, and the
animals died about the same time as the controls. Three apes were
treated without any apparent result; they all died within two months
after infection.

The experiments of Gramatschikoff, Czaplewski, and Roloff, and
of Yamagiva, published in 1892, show that the tuberculin treatment
does not cure tuberculous infection in inoculated guinea-pigs and rab-
bits, and that the bacilli retain their vitality in such animals in spite
of the most persistent treatment.

Hericourt and Eichet (1892), in experiments made for the purpose
of immunizing animals against tuberculous infection, failed to obtain
positive results in the most susceptible species — guinea-pigs, rabbits,
and apes — but claim to have succeeded in immunizing dogs by intra-

366 PROTECTIVE INOCULATIONS.

venous injections of cultures of the bacillus of tuberculosis in fowls.
Animals which had been so treated after an interval of two to six
months received an intravenous injection of one cubic centimetre of a
culture of the bacillus tuberculosis from man. This was fatal to
" non-vaccinated " dogs, as a rule, in about three weeks, but the " vac-
cinated " animals survived the injection.

The results obtained by Trudeau (1893) are of such interest that
we shall quote in extenso what he says with reference to preventive
inoculations :

" Antitubercular inoculation was first tried by Falk in 1883, and all
attempts in this direction have resulted until recently in but uu unbroken
record of failures. In 1890 I added my name to the list of those who found
it impossible to produce immunity in animals by this method. In 1S1K),
Martin and Grancher, and Courmont and Dor, claimed to have produced in
rabbits a certain degree of immunity by previous inoculation, after Pasteur's
hydrophobia method, of aviaii tubercle bacilli of graded and increasing viru-
lence. These vaccinations were, however, frequently fatal to the animals,
and the immunity obtained was but slight. Richet and He'ricourt have since
claimed to produce complete immunity in dogs by intravenous inoculations
of bird tubercle bacilli. These experimenters found that though harmless to
the dog when first derived from the chicken, bird bacilli, by long cultivation
in liquid media, become pathogenic for this animal, and by thus grading the
virulence of the injections complete immunity against any form of tubercu-
lar infection was produced in the dog. As yet these striking results have not
been confirmed. The animals which I now present to you illustrate an at-
tempt I have made along the same line to produce immunity in the rabbit.
Cultures grown directly from the chicken's lesions in bouillon for, first, five
weeks, then six months, were twice injected subcutaneously at intervals of
twenty-one days in doses of 0.025 and 005, and a third injection of a still
older culture was occasionally given. About one in four of the rabbits died
within three months, profoundly emaciated, but without any visible tubercu-
lar lesions. The remaining animals recovered aud were apparently in good
health, when, together with an equal number of controls, they were inocu-
lated in the anterior chamber of the eye with cultures of Koch's bacillus
derived from the tuberculous lesions of the rabbit, and cultivated about three
months on glycerin- agar. The results of these inoculations present many
points of interest. In the controls, as is usually the case, if the operation
has been done carefully and aseptically, and with a moderate amount of
dilute virus, two days after the introduction of the virulent material in the
eye little or no irritation is observed, and little is to be noticed for two weeks,
when a steadily increasing vascularity manifests itself, small tubercles ap-
pear on the iris, which gradually coalesce and become cheesy, intense iritis
and general inflammation of the structures of the eye develop, the inocula-
tion wound becomes cheesy, and in six to eight weeks the eye is more or less
completely destroyed and the inflammation begins to subside. The disease,
however, remains generally localized in the eye for many months, and even
permanently. In the vaccinated animals, on the contrary, the introduction
of the virulent bacilli at once gives rise to a marked degree of irritation. On
the second day the vessels of the conjunctiva are tortuous and enlarged,
whitish specks of fibrinous-looking exudation appear in the iris and in the
anterior chamber, and more or less intense iritis supervenes ; but at the end
of the second to the third week, when the eyes of the controls begin to show
progressive and steadily increasing evidence of inflammatory reaction, the
irritation in those of the vaccinated animals begins slowly to subside and the
eyes to mend. The vascularity is less, the whitish spots of fibriiious material

PROTECTIVE INOCULATIONS. 367

appear smaller, the structures of the eye become clearer, the inoculation
wound is but a bluish fibrous scar, until in from six to twelve weeks, in suc-
cessful cases, all irritation has disappeared and the eyes present, as in the
animals I now show you, but fibrous evidence of the traumatism and the in-
flammatory processes which have been set up by the inoculation. In all the
controls, as you see, the inoculation wound is cheesy and the cornea and iris
are more or less destroyed by tubercle and cheesy areas.

"Some of the protected animals slowly relapse, and the one I now show
you has small tubercles growing- on the iris; but even in such eyes the
entire absence of caseation is noticeable, and the disease progresses almost
imperceptibly. I have repeated this experiment on three sets of rabbits with
about the same results each time. The vaccinations as practised are of them-
selves, in some instances, fatal : but the fact remains that where recovery
takes place a marked degree of immunity has been acquired. I do not lay
any claim, therefore, to have produced a complete or permanent immunity
by a safe method, but it seems to me that these eyes constitute a scientific
demonstration of the fact that in rabbits preventive inoculation of bird-
tubercle bacilli can retard, and even abort, an otherwise progressive localized
tubercular process so completely as to prevent destruction of the tissues
threatened, and that the future study of anti-tubercular inoculation may not
be as entirely hopeless as it has until recently appeared."

TYPHOID FEVER.

Brieger (1885) found in cultures of the typhoid bacillus small
amounts of volatile fat acids, and when grape sugar has been added
to the culture medium lactic acid. He also obtained a highly alka-
line basic substance possessing toxic properties which he named
typhotoxin (C7H17NO2). This he supposes to be the specific product
to which the pathogenic action of the bacillus is due. It produces in
mice and guinea-pigs salivation, paralysis, dilated pupils, diarrhoea,
and death.

More recent experiments by Pfeiffer (1894) lead him to conclude
that the specific poison of the typhoid bacillus is not present in fil-
tered cultures, but is closely associated with the bacterial cells. Ac-
cording to Pfeiffer the bacillus may be killed by a temperature of 54° C.
without injury to this toxic substance. The fatal dose of the dead
bacilli is from three to four milligrammes per one hundred grammes
of body weight for guinea-pigs. Susceptible animals may be im-
munized by means of this toxic substance, and their blood is found to
contain an antitoxin which has a specific bactericidal action upon the
typhoid bacillus. But, according to Pfeiffer, the blood serum of ani-
mals immunized in this way does not differ from normal serum in
its action on bacillus coli communis and other species of bacteria.
These results are believed, by the author referred to, to settle the
question of the specific character of the typhoid bacillus, and to dif-
ferentiate it from nearly allied species. The presence of a typhoid
antitoxin in the blood serum of individuals who have recently suffered
an attack of typhoid fever has also been demonstrated by Pfeiffer.

368 PROTECTIVE INOCULATIONS.

Chantemesse and Widal (1888) first showed by experiment thai
susceptible animals could be made immune against the pathogenic
action of this bacillus by the subcutaneous injection of sterilized cul-
tures. Having found that four drops of a bouillon culture, three
days old, injected into the peritoneal cavity of white mice caused the
death of these animals within thirty-six hours, they proceeded to in-
ject small quantities (one-half cubic centimetre) of a culture which
had been sterilized by heat, and found that after several such protec-
tive inoculations the mice no longer succumbed to infection by an
unsterilized culture.

In experiments made upon rabbits, Bitter (1892) arrived at the
conclusion that the immunity which he produced in these animals by
the intravenous injection of concentrated sterilized (by filtration) cul-
tures was due to the presence of an antitoxin in the blood of the
immune animals. Having found that control animals were killed by
intravenous injections of one cubic centimetre of his concentrated
solution of the products of the typhoid bacillus, he added to twice
this amount of the toxic solution a certain quantity (?) of blood
serum from an immune rabbit, and injected the mixture into the
circulation of rabbits with a negative result. Control experiments
in which the toxic solution was mixed with blood serum from non-
immune animals showed that this had no antitoxic effect, and the ani-
mals died. Bruschettini obtained (1892) similar results in his ex-
periments upon rabbits with cultures sterilized by heat (60° C.). He
concludes from his experiments that the blood serum of rabbits im-
munized in this way not only possesses antitoxic properties, but that
it has greater germicidal potency for the typhoid bacillus than the
blood of normal rabbits.

Stern (1892) has made experiments to determine whether the blood
of recent convalescents from typhoid has greater germicidal power
for the typhoid bacillus than that of other individuals. The result
showed that the blood serum from persons who had recently recovered
from typhoid fever had no increased germicidal power, but rather
showed diminished potency for the destruction of typhoid bacilli.
But blood from a man who had suffered an attack seventeen and a
half years previously was found to have unusual bactericidal power,
although it did not protect white mice from typhoid infection. On
the other hand, blood from recent convalescents served to immunize
white mice, thus indicating the presence of an antitoxin. This is
also shown by the experiments of Chantemesse and Widal (1892),
who report their success in immunizing susceptible animals by in-
jecting the blood serum of other animals previously made immune by

PROTECTIVE INOCULATIONS. 369

repeated injections of sterilized (by heat) cultures. The authors last
named have also tested the blood serum of typhoid-fever patients, of
recent convalescents from the disease, and of persons who had suf-
fered an attack some years before the experiment was made. The ex-
periments were made upon guinea-pigs. The authors conclude that
" in general the guinea-pig is immunized against the action of virulent
typhoid cultures by the subcutaneous injection of a small quantity
of serum of persons who have suffered an attack of the disease, no
matter how remote." But this immunity was shown to be of short
duration, and quite different from that induced by the injection of
sterilized cultures, which does not immediately follow the introduc-
tion of the toxic substances, but requires a certain number of days for
its development. The degree of immunity is said by the authors last
named to depend to a considerable extent upon the dose given, and
the animals treated in this way still resisted virulent cultures at the
end of two months. On the other hand, injections of blood serum
from immune individuals were effective in doses of a single cubic cen-
timetre, within a few hours, and the immunity conferred had a com-
paratively brief duration.

Protective inoculations in man have been practised on quite a large
scale by surgeons of the English army in India and in South Africa.
The method of Wright has been followed in preparing sterile cultures
for inoculation. Cultures in bouillon are made and kept in the incu-
bator at 37° C. for two or three weeks. The cultures are then drawn
into small glass tubes, which are sealed by heat. The tubes are
placed in a vessel containing cold water, and the temperature is grad-
ually raised to 60° C., where it is maintained for five minutes. Plant-
ings in a culture medium are made from these tubes to make sure that
sterilization is complete. As a further protection against the intro-
duction of living bacteria, one-half per cent of lysol may be added to
the sterilized culture. The amount used for protective inoculations
in man has been fixed at two-fifths of the minimum amount, which
would be fatal to a guinea-pig weighing two hundred and fifty
grammes. The inoculation gives rise to a well-marked local reaction,
which does not result in suppuration, and to more or less pronounced
general disturbance. Usually this is slight, but sometimes rigors,
nausea, and a tendency to syncope occur. That these inoculations
are not without effect is shown by the fact that the blood serum of
an inoculated individual exercises a marked agglutinating action
upon the typhoid bacillus in a recent culture (Widal reaction). This
is said to be equal to that resulting from an attack of typhoid fever.

Cameron, after an inoculation practised upon himself, found that at
24

370 PROTECTIVE INOCULATIONS.

the end of twenty days his blood serum exhibited an agglutinating
power forty times greater than that of normal blood. In practice it
has been found advisable to repeat the inoculation at the end of a
week. Wright reports that among 11,295 British soldiers inoculated
in India, the percentage of those who subsequently contracted typhoid
fever was 0.95, while 2.5 per cent of those not inoculated suffered an
attack of this disease. According to Foulerton the soldiers in South
Africa, during the Boer war, who have been inoculated have contracted
typhoid fever in the proportion of six per thousand, while those not
inoculated have suffered to the extent of nine per thousand. How
much value should be attached to these statistics it is difficult to say,
on account of the numerous factors which are likely to influence the
result. Thus a command on the march in a sparsely inhabited coun-
try would be much less liable to suffer from typhoid fever than another
located in a town and remaining for a considerable time on the same
camping ground. In a recent report (February, 1901) Professor
Wright states that of 539 officers, men, and women connected with the
Fifteenth Hussars at Meerut, India, 360 received protective inocula-
tion in England against typhoid fever and 179 did not. Of the former
2 (0.55 per cent) were admitted to the hospital, suffering from typhoid
fever, with 1 death (0.27 per cent) ; while of the latter 11 (6. 14 per cent
were attacked by the fever, with 6 deaths (3.35 per cent).

It is evident that, while the results reported are encouraging, this
method should not be relied upon as a substitute for those sanitary
measures which must be our main reliance for the prevention of epi-
demics of this disease, viz., sterilization of drinking-water, disinfec-
tion of excreta, sanitary police of camps, etc.

PYOGENIC BACTERIA.

THE demonstration made by Ogston, Rosenbach, Passet, and
others that micrococci are constantly present in the pus of acute
abscesses, led to the inference that there can be no pus formation in
the absence of microorganisms of this class. But it is now well
established, by the experiments of Grawitz, De Bary, Steinhaus,
Scheurlen, Kaufmann, and others, that this inference was a mis-
taken one, and that certain chemical substances introduced beneath
the skin give rise to pus formation quite independently of bacteria.
Among the substances tested which have given a positive result are
nitrate of silver, oil of turpentine, strong liquor ammoniae, cada-
verin, etc. The demonstration has also been made by numerous in-
vestigators that cultures of pus cocci, when sterilized by heat, still
give rise to pus formation when injected subcutaneously. This was
first established by Pasteur in 1878, who found that sterilized cul-
tures of his " microbe generateur du pus " induced suppuration as
well as cultures containing the living microbe. This fact lias since
been confirmed, as regards the pus staphylococci and various bacilli,
by a number of bacteriologists. Wyssokowitsch produced abscesses
containing sterile pus by injecting subcutaneously agar cultures of
the anthrax bacillus sterilized by heat. Buchner obtained similar
results in a series of forty experiments from the injection of steril-
ized cultures of Friedlander's bacillus (" pneumococcus "), and has
shown that the pus-forming property belongs to the bacterial cells
and not to a soluble chemical substance produced by them. When
cultures were filtered by means of a Chamberlain filter the clear
fluid which passed through the porous porcelain was without effect,
while the dead bacteria retained by the filter produced aseptic pus
infiltration in the subcutaneous tissues within forty-eight hours
after having been injected. Subsequent experiments gave similar
results with seventeen different species tested, including Staphylo-
coccus pyogenes aureus, Staphylococcus cereus flavus, Sarcina auran-
tiaca, Bacillus prodigiosus, Bacillus Fitzianus, Bacillus subtilis,
Bacillus coli communis, Bacillus acidi lactici, etc. From the experi-

372 PYOGENIC BACTERIA.

ments made to determine the exact cause of pus formation following
the injection of sterilized cultures Buchner arrives at the conclusion
that it is due to the albuminous contents of the bacterial cells.

While it is demonstrated that a large number of microorganisms,
either living or in sterilized cultures, may give rise to the formation
of pus, the extended researches of Rosenbach, Passet, and other
bacteriologists show that few species are usually concerned in the
formation of acute abscesses, furuncles, etc., in man. Of these the
two most important, by reason of their frequent occurrence and path-
ogenic power, are Staphylococcus pyogenes aureus and Strepto-
coccus pyogenes ; next to these comes Staphylococcus pyogenes
albus, and the following species are occasionally found : Staphylo-
coccus pyogenes citreus, Staphylococcus cereus flavus, Staphylococcus
cereus albus, Micrococcus tenuis, Bacillus pyogenes foetidus, Micro-
coccus tetragenus, Micrococcus pneumonias crouposae. Two or more
species are often found in the same abscess ; thus Passet, in thirty-
three cases of acute abscess, found Staphylococcus aureus and albus
associated in eleven, albus alone in four, albus and citreus in two,
Streptococcus pyogenes alone in eight, albus and streptococcus in
one, and albus, citreus, and streptococcus in one. Hoffa found, in
twenty-two cases of inguinal bubo, aureus in ten, albus in nine, and
citreus in three. Bumm, in ten cases of puerperal mastitis, found
aureus in seven and Streptococcus pyogenes in three. Rosenbach
found staphylococci alone sixteen times, Streptococcus pyogenes alone
fifteen times, staphylococci and streptococci associated five times,
and Micrococcus tenuis three times in thirty-nine acute abscesses and
phlegmons examined by him.

Robb and Ghrisky have shown that under the most rigid antisep-
tic treatment microorganisms are constantly found attached to su-
tures when these are removed from wounds made by the surgeon,
and that a skin abscess frequently results from the presence of the
most common of these microorganisms — Staphylococcus epidermidis
albus.

The authors named state their conclusions as follows :

"A wound, at some time of its existence, always contains organisms.
They occur either on the stitches or in the secretions.

" The number of bacteria is influenced by the constricting action of the
ligatures or drainage tube, or anything interfering with the circulation of
the tissues.

' ' The virulence of the organisms present will influence the progress of
the wound.

"The body temperature is invariably elevated if the bacteria are viru-
lent; and, indeed, in cases where many of the less virulent organisms are
found, almost without exception there is some rise of temperature. "

The organism most frequently found — Staphylococcus epidermi-

PYOGENIC BACTERIA. 373

dis albus — has but slight virulence. Out of forty-five cases in which
a bacteriological examination was made this micrococcus was ob-
tained in pure cultures in thirty-three ; in five cases it was associated
with Staphylococcus pyogenes aureus, in one case with Streptococ-
cus pyogenes, in three cases Streptococcus pyogenes was obtained
alone.

In abscesses resulting from inflammation of the middle ear the
micrococcus commonly known under the name of " diplococcus
pneumonise " —Micrococcus pneumonias crouposae — has been obtained
in pure cultures in a considerable number of cases when the pus has
been examined immediately after paracentesis of the tympanic mem-
brane. We shall not, however, describe this among the pyogenic
bacteria, but will give an account of it in the following section (Bac-
teria in Croupous Pneumonia, etc.). Bacillus pyocyaneus, which is
described by some authors among the pyogenic bacteria, is found
only in the pus of open wounds, where its presence is evidently acci-
dental. "We shall describe it among the chromogenic saprophytes.

STAPHYLOCOCCUS PYOGENES AUREUS.

Synonym. — Micrococcus of infectious osteomyelitis (Becker).

Observed by Ogston (1881) in the pus of acute abscesses, but not
differentiated from the associated staphylococci and the streptococ-
cus of pus. Obtained by Becker from the pus of osteomyelitis (1883).
Isolated from the pus of acute abscesses and accurately described by
Rosenbach (1884) and by Passet (1885).

The Staphylococcus pyogenes aureus is a facultative parasite, and
is the most common pyogenic micrococcus found in suppurative pro-
cesses generally. But it is also a common and widely distributed
saprophyte, which finds the conditions necessary for its existence on
the external surface of the human body and of moist mucous mem-
branes. This is shown by the researches of numerous bacteriolo-
gists. Thus Ullmann found it upon the skin and in the secretions of
the mouth of healthy persons, and also in the dust of occupied apart-
ments, in water, etc. ; Bockhart obtained it in cultures from the
surface of the body and from the dirt beneath the finger nails of
healthy persons ; Biondi, Yignal, and others in the salivary secre-
tions ; B. Frankel in mucus from the pharynx ; Von Besser and
Wright in nasal mucus ; Escherich in the alvine discharges of
healthy infants ; C. Frankel in the air ; and Lubbert in the soil. Its
presence in the air, in water, or in the soil is, however, quite excep-
tional, and is probably to be considered the result of accident, its
normal habitat as a saprophyte appearing to be rather, .upon the sur-
face of the body and of mucous membranes.

374 PYOGENIC BACTERIA.

Morphology.— Spherical cells having a diameter of 0.7 ^ (Hade*
lich) to 0.9 /* (0.87 /* Passet), solitary, in pairs, or in irregular
groups, occasionally in chains of three or four elements or in groups
of four. The dimensions vary somewhat in dif-
ferent culture media, being larger in a favorable
than in an unfavorable medium. The individual
cells, as pointed out by Hadelich, consist of two

hemispherical portions separated from each other
FIG. 79.-staphyiococ- by a very narrow cleft, which is not visible when

cus pyogenes aureus, ,-• •>-, j ^ j i_

from a drawing by tne cells are deeply stained, but may be demon-
Rosenbach. strated, with a high power, by staining for a short

time (two minutes or less) in a solution of f uchsin in aniline water.

This micrococcus stains quickly in aqueous solutions of the basic
aniline colors, and may also be stained with acid carmine and hsema-
toxylin. It is not decolorized by iodine solution when stained with
methyl violet — Gram's method.

Biological Characters. — Staphylococcus pyogenes aureus grows
either in the presence or absence of oxygen, and is consequently a
facultative anaerobic. It multiplies rapidly at a temperature of 18°
to 20° C. in milk, flesh infusions, and various other liquid media,
and in nutrient gelatin or agar. It liquefies gelatin, and in stab
cultures liquefaction occurs all along the line of puncture, forming a
pouch which is largest above and at the end of three or four days has
extended to the full capacity of the test tube at the surface. The
liquefied gelatin in this pouch is at first opaque from the presence of
little agglomerations of micrococci in suspension, but after a time
these are deposited and the gelatin becomes transparent. During
the period of active growth the cocci accumulate near the surface of
the gelatin, and, in contact with the air, the characteristic golden-yel-
low pigment is produced. By the subsidence of the colored masses
of cocci from this superficial stratum a yellow deposit is gradually
formed at the bottom of the pouch of liquefied gelatin (Fig. 80). This
pigment, which is the principal character distinguishing the micro-
coccus under consideration from certain other liquefying staphylo-
cocci, is only formed in the presence of oxygen. Upon the surface
of nutrient agar development occurs in the form of a moist, shining
layer, with more or less wavy outlines, having at first a pale-yellow
color, which soon deepens to an orange- or golden-yellow. The col-
onies which develop upon agar plates are spherical and opaque, and
usually acquire the golden-yellow color within a few days. Colonies
on gelatin plates or in Esmarch roll tubes first appear as small white
dots, which later are more or less granular in appearance and present
the yellow color, especially towards the centre ; but, owing to the
extensive liquefaction of the gelatin caused by them, their develop-

PYOGENIC BACTERIA.

375

ment can only be followed for two or three days. Upon potato, at a
temperature of 35° to 37° C., a rather thick, moist layer of consider-
able extent forms at the end of twenty-four to forty-eight hours ;
this is also at first of a pale-yellow, and later
of an orange-yellow color. The temperature
mentioned is most favorable for the rapid
development of this micrococcus, although
multiplication may occur at a comparatively
low temperature and is tolerably abundant at
the ordinary room temperature.

Cultures of the "golden Staphylococcus,"
and especially those upon potato, give off a
peculiar odor which resembles that of sour
paste. When cultivated in milk it gives rise
to the formation of lactic and butyric acids
and to coagulation of the casein. No poison-
ous ptomaines or toxalbumins have been iso-
lated from cultures of this micrococcus, but,
like other liquefying bacteria, it forms a sol-
uble peptonizing ferment, by which gelatin
may be liquefied independently of the living
microorganism. While the Staphylococcus
aureus gives rise to the production of acids—
principally lactic acid — in media containing Staphylococcus pyogenes aureus
glucose or lactose, it has also been shown by (

Brieger that ammonia is one of the products of its vital activity.
Unlike some other pathogenic bacteria, it is able to grow in a medium
having a distinctly acid reaction. A non-poisonous basic substance
has been isolated by Brieger from old cultures in meat infusion which
differs from any of the ptomaines obtained by him from other sources.

The thermal death-point of this micrococcus, in recent cultures in
flesh-peptone-gelatin, as determined by the writer, is between 56° and
58° C. , the time of exposure being ten minutes. When "in a desic-
cated condition a much higher temperature is required — 90° to 100° C.
— for its destruction ; and it retains its vitality for more than ten
days when dried upon a cover glass (Passet). It retains its vitality
for a long time in cultures in nutrient gelatin or agar, and may grow
when transplanted from such cultures even at the end of a year.

Very numerous experiments have been made to determine the
proportion of various chemical agents required to destroy the vitality
or to restrain the growth of this important pyogenic micrococcus.
The extended researches of Lubbert (1886) with reference to the
antiseptic power of agents added to a suitable culture medium — nu-
trient gelatin — gave the following results : Development was pre-

FIG. 80.— Gelatin culture of

376 PYOGENIC BACTERIA.

vented by the agents named in the proportion given : Nitric acid,
1 : 797 ; phosphoric acid, 1 : 750 ; boracic acid, 1 : 327 ; oxalic acid,
1 : 433 ; acetic acid, 1 : 720 ; citric acid, 1 : 433 ; lactic acid, 1 : 350 ;
benzoic acid, 1 : 400 ; salicylic acid, 1 : G55 ; iodine dissolved with
potassium iodide, 1:1,100; arsenite of potash, 1:733; mercuric
chloride, 1 : 81,400 ; chloral hydrate, 1 : 133 ; carbolic acid, 1 : 814 ;
thymol, 1 : 11,000 ; resorcin, 1 : 122 ; hydrochinon, 1 : 353 ; kairin,
1 : 407 ; antipyrin, 1 : 26 ; muriate of quinine, 1 : 550 ; muriate of
morphia, 1 : 60. For the destruction of vitality very much larger
amounts are required. In Bolton's experiments (1887) a one-per-cent
solution of carbolic acid was successful after two hours' exposure,
but two per cent failed to completely destroy vitality in the same
time ; one per cent of sulphate of copper was also successful, and but
a single colony developed after exposure to a solution of 1 : 200. In
the experiments of Gartner and Plagge the Staphylococcus aureus in
bouillon cultures is said to have been killed in a few seconds (eight)
by a solution of mercuric chloride of the proportion of 1 : 1,000 ; Behr-
ing found it was killed by the acid sublimate solution of La Place,
in the proportion of 1 : 1,000, in ten minutes ; Tarnier and Vignal
found that a solution of 1 : 1,000 was successful in two minutes.
Abbott (1891) has shown that in the same culture there may be a
considerable difference in the resisting power of the cocci, and that
while frequently all are destroyed in five minutes by a 1 : 1,000 solu-
tion, it occurs quite as frequently that some may survive after an ex-
posure of ten, twenty, and even thirty minutes.

Pathogenesis. — Subcutaneous inoculation with a small quantity
of a culture of Staphylococcus pyogenes aureus is without result in
rabbits, guinea-pigs, or mice, but when a considerable quantity is
injected beneath the skin of a rabbit or a guinea-pig an abscess is
produced, which usually results in recovery, but may give rise to
general infection and the death of the animal. Injection into a
vein or into the cavity of the abdomen in the animals mentioned
usually induces a fatal result within a few days. The most charac-
teristic pathological changes are found in the kidneys, which con-
tain numerous small collections of pus and under the microscope
present the appearances resulting from embolic nephritis. Many of
the capillaries and some of the smaller arteries of the cortex are
plugged up with thrombi consisting of micrococci. Metastatic ab-
scesses may also be found in the joints and muscles. The micro-
cocci may be recovered in pure cultures from the blood and the
various organs ; but they are not numerous in the blood, and a sim-
ple microscopical examination will often fail to demonstrate their
presence.

Animals frequently survive the injection of a small quantity of

PYOGENIC BACTERIA. 377

a pure culture made directly into the circulation, and there is evi-
dence that the pathogenic potency of this micrococcus may vary
considerably as a result of conditions relating to its origin and culti-
vation in the animal body or in artificial media. When injected in
considerable quantities it may be obtained in cultures from the
urine, but not sooner than six or eight hours after the injection, and
not until the formation of purulent foci in the kidneys has already
occurred ( Wy ssoko wit sch) .

The pyogenic properties of this micrococcus have been demon-
strated upon man by the experiments of Garre, of Bockhart, and of
Bumm. The first-named observer inoculated a small wound at the
edge of one of his finger nails with a minute quantity of a pure cul-
ture, and a subepidermal, purulent inflammation extending around

;£)> oZ^'dCT,'.»T-( N-^-»rijr,i - f-

FIG. 81.— Vertical section through a subcutaneous abscess caused by inoculation with staphylo-
cocci, in the rabbit, forty eight hours after infection; margin towards the normal tissue. X 950.
(Baumgarten.)

the margin of the nail resulted from the inoculation. Staphylococ-
cus aureus was recovered in cultures from the pus thus formed. A
more extensive and extremely satisfactory experiment was subse-
quently made by Garre, who applied a considerable quantity of a
pure culture obtained from the above-mentioned source — third gene'
ration — to the uninjured skin of his left forearm. At the end of
four days a large carbuncle, surrounded by isolated furuncles, de-
veloped at the point where the culture had been applied. This ran
the usual course, and it was several weeks before it had completely
healed. No less than seventeen scars remained to give evidence of
the success of the experiment.

In Bockhart's experiments a similar but milder result was ob-
tained, the conditions having been somewhat different. A small

378 PYOGENIC BACTERIA.

quantity of an agar culture was suspended in 0.5-per-cent salt solu-
tion, and this was rubbed upon the uninjured skin of the left fore-
arm. By gentle scratching with a disinfected finger nail the epithe-
lium was removed in places over the area to which the micrococcus
had been applied. As a result of this procedure numerous impe-
tigo pustules and occasionally a genuine furuncle developed. Por-
tions of the skin containing the smaller pustules were excised and
examined microscopically. As a result of this examination Bock-
hart concluded that the cocci penetrate by way of the hair follicles,
the sebaceous and sudoriparous glands, or, where the epidermis had
been removed by scratching, directly to the deeper layers of the skin.

In Bumm's experiments, made upon himself and several other
persons, Staphylococcus aureus suspended in sterilized salt solution
was injected beneath the skin. An abscess resulted in every case.

The very extended researches made by bacteriologists during the
past five or six years show that the golden Staphylococcus is the
most common pyogenic microorganism. Its presence has been de-
monstrated not only in furuncles and carbuncles, but also in various
pustular affections of the skin and mucous membranes — impetigo,
sycosis, phlyctenular conjunctivitis ; in purulent conjunctivitis and
inflammation of the lacrymal sac ; in acute abscesses formed in the
lymphatic glands, the parotid gland, the tonsils, the mammae, etc. ;
in metastatic abscesses and purulent collections in the joints ; in em-
pyema ; in infectious osteomyelitis ; and in ulcerative endocarditis.
The evidence relating to its presence and etiological import in the
last-mentioned affections demands special consideration.

Infectious osteomyelitis appears from the researches of Becker,
Rosenbach, Krause, Passet, and others, to be usually due to the pre-
sence of Staphylococcus aureus, although Kraske has shown that in
certain cases this is associated with other microorganisms. Becker,
who obtained this micrococcus from the pus of osteomyelitis in 1883,
was the first to show by experiment that the same affection might be
induced in rabbits by injecting cultures of the micrococcus into the
circulation, after having crushed or fractured a bone in one of its
legs. The animal usually died in from twelve to fourteen days and
presented the usual appearances of osteomyelitis at the fractured
point. The abundant yellowish-white pus contained the golden
Staphylococcus which was described by Becker, and subsequently
known in the bacteriological laboratories of Germany as the " mi-
crococcus of infectious osteomyelitis." Becker's experimental re-
sults have been confirmed by Krause and Rosenbach; and Rodet, by
injecting smaller quantities of a culture into the circulation, has suc-
ceeded in producing an osteomyelitis without previous injury to the
bone.

PYOGENIC BACTERIA. 379

Ulcerative endocarditis has been shown by the researches of
numerous bacteriologists to be occasionally accompanied by a mycotic
invasion of the affected tissues by the golden staphylococcus ; in
other cases Streptococcus pyogenes is present. The researches of
Weichselbaum, and of E. Frankel and Sanger, also show that it is
present in a certain proportion of the cases, at least, of endocarditis
verrucosa, although in smaller numbers. That the diseased condi-
tion of the cardiac valves in ulcerative endocarditis is due to mycotic
invasion is now generally admitted and is supported by experimental
evidence. Rosenbach first (1873) produced an endocarditis in lower
animals by mechanical injury to the cardiac valves, effected by in-
troducing a sound through the aorta. Following his method, Wys-
sokowitsch (1885), after injuring the cardiac valves in rabbits, in-
jected into the circulation pure cultures of various bacteria. He
obtained positive results with Staphylococcus aureus and Strepto-
coccus pyogenes only. When these micrococci were injected into
the trachea or subcutaneously the result was negative, as was the
case when very few cocci were injected into a vein, or when two
days or more were allowed to elapse after injury to the cardiac
valves. Subsequently Weichselbaum, Prudden, and Frankel and
Sanger obtained confirmatory results, thus establishing the fact that
when the valves are first injured mechanically (or chemically—
Prudden) the injection into a vein of a pure culture of Staphylococcus
aureus gives rise to a genuine ulcerative endocarditis. It has been
further shown by Ribbert that the same result may be obtained with-
out previous injury to the valves by injecting into a vein the staphy-
lococcus from a potato culture suspended in water. In his experi-
ments not only the micrococci from the surface but the superficial
layer of the potato was scraped off with a sterilized knife and mixed
with distilled water ; and the successful result is ascribed to the fact
that the little agglomerations of micrococci and infected fragments
of potato attach themselves to the margins of the valves more readily
than isolated cocci would do. In these experiments the mitral and
tricuspid valves were affected, while the semilunar valves remained
intact. In ulcerative endocarditis it is evident that cocci detached
from the diseased valves must find their way into the circula-
tion. As a matter of fact, masses of micrococci are carried away by
the blood stream and form emboli in various parts of the body, which
become secondary foci of infection and give rise to local necrotic
changes and accumulations of pus. While this undoubtedly occurs,
it is generally admitted that the mycotic infection of the cardiac
valves is usually a secondary affection, resulting from the transpor-
tation of micrococci in the blood current from some other infected
focus. But there is no general development of micrococci in the cu>

380 PYOGENIC BACTERIA.

dilating fluid, and in man, as in animals infected experimentally, a
microscopic examination of the blood for microorganisms usually
gives a negative result. Culture experiments may, however, demon-
strate their presence. Thus recent investigations by Netter, Eisel-
berg, and others show that the pus cocci are usually present in the
blood in small numbers, as demonstrated by culture experiments, in
septic infection from wounds.

STAPHYLOCOCCUS PYOGENES ALBUS.

Isolated by Rosenbach (1884) from the pus of acute abscesses, in
which it is sometimes the only microorganism present, and some-
times associated with other pus cocci. In thirty-three acute abscesses
examined by Passet (1885) it was associated with Staphylococcus
aureus in eleven, with Staphylococcus citreus in two, with Strepto-
coccus pyogenes in one, with both Staphylococcus citreus and Strep-
tococcus pyogenes in one, and was obtained alone from four.

In its morphology this micrococcus is identical with the preced-
ing, but it is distinguished from it by the absence of pigment and
by being somewhat less pathogenic. Surface cultures upon nutrient
agar or potato have a milk-white color. It liquefies gelatin in the
same way as does the golden Staphylococcus, but the deposit at the
bottom of the liquefied gelatin is without color. In the temperature
conditions favorable to its growth, and in its biological characters
generally, with the exceptions noted, it is not to be distinguished
from the species previously described. According to Fliigge, it is
more common than aureus among many of the lower animals.

Pathogenesis. — Fortunati has tested the comparative pathogenic
power of Staphylococcus aureus and Staphylococcus albus by inocu-
lations into the cornea of rabbits. A purulent infiltration of the
cornea and panophthalmitis resulted when Staphylococcus aureus
was inoculated upon the surface of the cornea by scratching with an
infected needle, but inoculations made in the same way with Staphy-
lococcus albus healed spontaneously or gave rise to a perforating
ulcer. After paracentesis of the cornea with an instrument infected
with Staphylococcus aureus panophthalmitis developed in thirty hours; v
the same result occurred at the end of sixty to seventy-two hours
when the instrument was infected with Staphylococcus albus. When
a sterilized instrument was used the result was negative. In bacteri-
ological researches made by Gallenga, in cases of panophthalmitis in
man, Staphylococcus albus was found in ten cultures and Staphy-
lococcus aureus in nine.

Staphylococcus Epidermidis Albus (Welch).
The researches of Welch show that a white Staphylococcus, prob-
ably identical with Staphylococcus pyogenes albus of Rosenbach, is

PYOGENIC BACTERIA. 3$1

the most common microorganism upon the surface of the body, and
that " it is very often present in parts of the epidermis deeper than
can be reached by any known means of cutaneous disinfection save
the application of heat." With reference to this coccus Welch
says :

"So far as our observations extend — and already they amount to a
large number — this coccus may be regarded as a nearly, if not quite, con-
stant inhabitant of the epidermis. It is now clear why I have proposed to
call it the Staphylpcoccus epidermidis albus. It possesses such feeble pyo-
genic capacity, as is shown by its behavior in wounds as well as by experi-
ments oil rabbits, that the designation Staphylococcus pyogenes albus does
not seem appropriate. Still, I am not inclined to insist too much upon this
point, as very probably this coccus, which has hitherto been unquestionably
identified by Bossowski and others with the ordinary Staphylococcus pyo-
genes albus of Rosenbach, is an attenuated or modified form of the latter
organism, although, as already mentioned, it presents some points of differ-
ence from the classical description of the white pyogenic coccus."

According to Welch, this coccus differs from Staphylococcus pyo-
genes aureus not only in color, but also in the fact that it liquefies
gelatin more slowly, does not so quickly cause coagulation of milk,
and is far less virulent when injected into the circulation of rabbits.
It has been shown by the researches of Bossowski and of Welch
that this coccus is very frequently present in aseptic wounds, and
that usually it does not materially interfere with the healing of
wounds, although sometimes it appears to cause suppuration along
the drainage tube, and it is the usual cause of " stitch abscess."
Bossowski, in fifty cases of wounds treated antiseptically, obtained
bacteria from the discharges in forty, and in twenty-six of these
cases he found Staphylococcus pyogenes albus ; Staphylococcus au-
reus was found nine times, Streptococcus pyogenes in two, and vari-
ous non-pathogenic bacteria in eight. In forty-five laparotomy
wounds examined by Ghrisky and Robb, in which strict antiseptic
precautions had been observed, bacteria were found in thirty-one, and
in nineteen of this number Staphylococcus albus was present,
Staphylococcus aureus in five, Bacillus coli communis in six, and
Streptococcus pyogenes in three.

STAPHYLOCOCCUS PYOGENES CITREUS.

Isolated by Passet (1885) from the pus of acute abscesses. In thirty -
three cases examined it was found associated with Staphylococcus albus in
two and with Staphylococcus albus and Streptococcus pyogenes in one.

In its morphology this coccus is identical with the two preceding species,
from which it is distinguished by the formation of a lemon-yellow pigment]
instead of a golden or orange- yellow as in Staphylococcus aureus. The
pigment is only formed in the presence of oxygen. This coccus is said by
Frankel to liquefy gelatin more slowly than the previously described species
— Staphylococcus aureus and Staphylococcus albus.

As to its pathogenic properties we have no definite information. It is
included among the pyogenic bacteria because of its occasional presence in

382 PYOGENIC BACTERIA.

the pus of acute abscesses, although it has heretofore only been found in as-
sociation with other microorganisms.

MICROCOCCUS PYOGENES TENUIS.

Obtained by Rosenbach (1884) from pus in three cases out of thirty- nine
examined.

Morphology. — Micrococci, somewhat irregular in size, but larger than
Staphylococcus albus, and seldom associated in masses. Frequently the in-
dividual cocci present the appearance of consisting of two deeply stained
masses separated from each other by a paler interspace. Cultures upon the
surface of nutrient agar form a very thin, transparent layer of about one
millimetre in breadth along the line of inoculation ; this resembles a thin
layer of varnish.

Pathogenesis undetermined. (Micrococcus pneumoiiiae crouposae ?)

STREPTOCOCCUS PYOGENES.

Synonyms. — Micrococcus of erysipelas (Fehleisen) ; Streptococcus
erysipelatos ; Streptococcus of pus ; Streptococcus longus (Yon Lin-
gelsheim).

Obtained by Fehleisen from the skin involved in cases of erysipe-
las (1883), and by Rosenbach (1884) and Passet (1885) from the pus
of acute abscesses. The characters of the " streptococcus of erysipe-
las" of Fehleisen and the " Streptococcus pyogenes" of Rosenbach
and Passet are generally admitted to be identical, although some
bacteriologists still describe them separately and cultures from the
two sources are still retained in bacteriological laboratories under the
names originally given them.

Rosenbach found Streptococcus pyogenes alone in fifteen cases,
and associated with staphylococci in five cases, out of thirty-nine
cases examined of acute pus formation. Passet, in thirty-three
similar cases, obtained the streptococcus alone in eight and associated
with staphylococci in two. Subsequent researches show that this
micrococcus is frequently, if not constantly, present in puerperal
metritis ; that it is the most frequent microorganism associated with
ulcerative endocarditis ; that it is frequently present in diphtheritic
false membranes, and especially in those cases of diphtheritic inflam-
mation which are secondary to scarlet fever and measles (Prudden).
Numerous investigations made by bacteriologists during the past few
years indicate that this is a very important and widely distributed
pathogenic microorganism. It has also been frequently found upon
exposed mucous surfaces — mouth, nose, vagina — of healthy in-
dividuals.

According to the researches (1891) of Von Lingelsheim, the Strep-
tococcus pyogenes differs from Streptococcus erysipelatos in be-
ing pathogenic both for mice and rabbits, while the latter is patho-

PYOGENIC BACTERIA. 383

genie for rabbits only. The author named, as a result of extended and
carefully conducted comparative studies, arrives at the following
conclusions:

" According to my observations, there are two great groups among the
streptococci. These cannot be distinguished one from the other in cultures
in highly albuminous media (pus, blood serum), but present constant dif-
ferences when cultivated in bouillon. The decisive characteristics in this
medium are : macroscopic, the cloudiness of the medium ; microscopic, the
length of the chains. The two groups are with difficulty distinguished in
agar cultures ; more easily in gelatin, in which the streptococcus which
forms short chains causes a slight liquefaction, while the Streptococcus
longus does not. Upon potato Streptococcus brevis alone shows a visible
growth. . . . We see here a group of streptococci which we separate from
the others, because of their microscopic and cultural differences, under the
name of Streptococcus brevis, which is also distinguished by having no
pathogenic action upon the animals usually experimented upon. We
recognize, on the other hand, the streptococci which we have grouped to-
gether as Streptococcus longus as all pathogenic and about in equal degree
for a certain species of animal (rabbits) ; but by experiments upon other
species (mice) we arrive at the conclusion that there must also be differences
between these streptococci. It appears that the streptococci which are dis-
tinguished by their high degree of pathogenic power upon mice are also
those which are distinguished in bouillon cultures by the formation of con-
glomerate masses. We find among these also one which is distinguished
by especial virulence for mice, and that this one is distinguished in cultures
by its scanty growth upon ox serum."

The more recent researches of Knorr (1893), and of Waldvogel
(1894), indicate that the classification of the streptococci proposed by
von Lingelsheim has no great value, and show that marked changes
in biological characters and in pathogenic power may result from
cultivation in special media, or from successive inoculations into
animals.

Morphology. — Spherical cocci, from 0.4 // to 1> in diameter, but
varying considerably in dimensions in different cultures, and even
in a single chain. Multiply by binary division,
in one direction only, forming chains, in which
the elements are commonly associated in pairs.
Under certain circumstances, instead of form-
ing chains, a culture may contain only, or

chiefly, diplococci : but usually chains contain-

*
ing from four to twenty or more elements are

formed, and these are frequently associated

in tangled masses. Occasionally one or more

cells in a chain greatly exceed their fellows in

size, and some bacteriologists suppose that FIG. 82.— PUS containing

these cells serve as reproductive spores — arthro- (jS^00

spores — but this has not been definitely proven.

Stains readily with the aniline colors and by Gram's method.

384

PYOGENIC BACTERIA.

Biological Characters. — Grows readily in various liquid and
solid culture media, including all of those usually employed in bac-
teriological researches. The most favorable temperature for its de-
velopment is from 30° to 37° C., but it multiplies freely at the ordi-
nary room temperature — 16° to 18° C.

Streptococcus pyogenes is a facultative anaerobic, growing
both in the presence and absence of oxygen. It
does not liquefy gelatin, and in gelatin stab
cultures it grows along the line of puncture,
forming numerous small, spherical, translu-
cent, whitish colonies, which are closely crowd-
ed together at the upper portion of the line of
growth, and often distinctly separated from
each other below ; upon the surface there is
often no growth, or a scanty development may
occur about the point of entrance of the inocu-
lating needle. The minute colonies along the
line of puncture are already visible at the end
of twenty -four hours in cultures kept in the
incubating oven at 30° to 35° C., and at the end
of three or four days they have reached their
full development, forming a semi-opaque, white,
granular column, upon the margins of which
the separate colonies are seen projecting into the
gelatin. On gelatin plates very small, translu-
cent colonies are developed, which upon the sur-
face spread out to form a flat, transparent disc
of about one-half millimetre. Under a low mag-
nifying power these colonies are seen to be slight-
ly granular and have a yellowish color. At a
later date they become darker and less trans-
parent, and the margin may show irregular projections made up of
tangled masses of cocci in chains. The characters of growth in
nutrient agar and in jellified blood serum are similar to those in gela-
tin, and on agar plates colonies are formed similar to those above
described, except that they are somewhat smaller and more trans-
parent. Fehleisen and De Simone state that the erysipelas coccus'
may develop upon the surface of cooked potato, but most authorities
-Flugge, C. Frankel, Passet, Baumgarten — agree that no growth
occurs upon potato. Milk is a favorable medium for the growth of
this micrococcus, and the casein is coagulated by it. A slightly acid
reaction of the culture medium does not prevent its development.
The thermal death-point, as determined by the writer, is between
52° and 54° C., the time of exposure being ten minutes. According

FIG. 83.— Streptococcus
of erysipelas iii nutrient
gelatin; stick culture at
end of four days at 16°-
18° C. (Baumgarten).

PYOGENIC BACTERIA.

385

to De Simone, a temperature of 39.5° to 41° C. maintained for two
days is fatal to this micrococcus.

Manfredi and Traversa have injected filtered cultures into frogs,
guinea-pigs, and rabbits for the purpose of ascertaining if any solu-
ble toxic substance is produced during the growth of Streptococcus
pyogenes. They report that in some cases convulsions and in others
paralysis resulted from these injections.

Von Lingelsheim has (1891) reported the following results
obtained in an extended series of experiments made to determine
the germicidal power of various chemical agents as tested upon
this microorganism — time of exposure two hours : Hydrochloric acid
1 : 250, sulphuric acid 1 : 250, caustic soda 1 : 130, ammonia 1 : 25,
mercuric chloride 1 : 2,500, sulphate of copper 1 : 200, chloride of
iron 1 : 500, terchloride of iodine 1 : 750, peroxide of hydrogen 1 : 50,
carbolic acid 1 : 300, cresol 1 : 250, lysol 1 : 300, creolin 1 : 130, naph-
thylamin 1 : 125, malachite green 1 : 3,000, pyoktanin 1 : 700.

FIG. 84.— Section from margin of an erysipelatous inflammation, showing streptococci in
lymph spaces. From a photograph by Koch, x 900.

Pathogenesis. — When inoculated into the cornea of rabbits
Streptococcus pyogenes gives rise to keratitis. Inoculations into the
ear of the same animal usually give rise to a localized erysipelatous
inflammation accompanied by an elevation of temperature in the in-
oculated ear ; at the end of thirty-six to forty-eight hours the in-
flamed area, which has well-defined margins and a bright-red color,
extends from the point of inoculation along the course of the veins to
the root of the ear. This appearance passes away in the course of a
few days and the animal recovers. Subcutaneous injections into mice
or rabbits are usually without result, and the last-named animal also
withstands injections of considerable quantities into the general cir-
culation through a vein. When, however, the animal has previously
been weakened by the injection of toxic substances the streptococcus
may multiply in its body and cause its death (Fliigge).

Fehleisen has inoculated cultures, obtained in the first instance
from the skin of patients with erysipelas, into patients in hospital
suffering from lupus and carcinoma, and has obtained positive re-
sults, a typical erysipelatous inflammation having developed
25

386 PYOGENIC BACTERIA.

around the point of inoculation after a period of incubation of from
fifteen to sixty hours. This was attended with chilly sensations and
an elevation of temperature. Persons who had recently recovered
from an attack of erysipelas proved to be immune.

Sections made from the ear of an inoculated rabbit, or of skin taken
from the affected area in erysipelas in man, show the streptococci in
considerable numbers in the lymph channels, but not in the blood
vessels. They are more numerous, according to Koch and to Fehl-
eisen, upon the margins of the erysipelatous area, and may even be
seen in the lymph channels a little beyond the red margin which
marks the line of progress of the infection.

The researches of Weichselbaum and others show that Strepto-
coccus pyogenes is the infecting microorganism in a certain propor-
tion of the cases of ulcerative endocarditis. The author named
found it in four cases out of fifteen examined, and in two cases of
endocarditis verrucosa out of thirteen. In a previously reported series
of sixteen cases (fourteen of ulcerative endocarditis and two of ver-
rucosa) the streptococcus was found in six.

In diphtheritic false membranes this streptococcus is very com-
monly present, and in certain cases attended with a diphtheritic exu-
dation, in which the Bacillus diphtherias has not been found by com-
petent bacteriologists, it seems probable that Streptococcus pyogenes
is the pathogenic microorganism responsible for the local inflamma-
tion and its results. Thus in a series of twenty-four cases studied by
Prudden in 1889 the bacillus of Loffler was not found, "but a strep-
tococcus apparently identical with Streptococcus pyogenes was found
in twenty-two." Chantemesse and Widal have also reported cases
in which a fibrinous exudate resembling that of diphtheria was as-
sociated with a streptococcus. " These forms of so-called diphtheria
are most commonly associated with scarlatina and measles, erysipe-
las, and phlegmonous inflammation, or occur in individuals exposed
to these diseases ; but whether exclusively under these conditions is
not yet established" (Prudden).

Loffler has described under the name of Streptococcus articu-
lorum a micrococcus obtained by him from the affected mucous
membrane in cases of diphtheria, and which he believes to be acci-
dentally present and without any etiological import in this disease.
In its characters it closely resembles Streptococcus pyogenes and is
perhaps a variety of this widely distributed species.- Its characters
are described by Fliigge as follows :

" Cultivated in nutrient gelatin, it forms at the end of three days small,
transparent, light-gray drops, upon the margin of which, under the micro-
scope, the cocci in twisted chains may be observed. As many as one hun-

PYOGENIC BACTERIA. 387

dred elements may be found in a single chain, and some of these are distin-
guished by their size ; occasionally whole chains are made up of these large
cocci, and when closely observed some of these may present indications of
division transversely to the axis of the chain. Subcutaneous inoculation of
cultures into mice results in the death of a considerable number of these ani-
mals — more than half ; and the streptococci are found in the spleen and other
organs. Inoculation into the ear of rabbits causes an erysipelatous inflam-
mation. When injected into the circulation of these animals through a vein
joint affections are developed in from four to six days, and a purulent ac-
cumulation occurs in which the streptococci are found. In two rabbits in-
oculated in the same way with a culture of the streptococcus of erysipelas,
Loffler has observed a similar result."

Numerous researches indicate that infection by Streptococcus
pyogenes through the endometrium is the usual cause of puerperal
fever. Thus Clivio and Monti demonstrated its presence in five
cases of puerperal peritonitis. Czerniewski found it in the lochia of
a large number (thirty-five out of eighty-one) of women suffering
from puerperal fever, but in the lochia of fifty-seven healthy puer-
peral women he was only able to find it once. In ten fatal cases he
found it in every instance, both in the lochial discharge during life
and in the organs after death. Widal carefully studied a series of
sixteen cases and arrived at the conclusion that this was the infect-
ing microorganism in all. Bumm and other observers have given
similar evidence. Eiselsberg and Emmerich have succeeded in de-
monstrating the presence of the streptococcus in hospital wards con-
taining cases of erysipelas. That puerperal fever may result from
infection through the finger of the accoucheur, when he has previ-
ously been in contact with cases of erysipelas, has long been taught,
and, in view of the facts above recorded, is not difficult to under-
stand. But in view of the fact that the streptococcus of pus has been
found in vaginal mucus and in the buccal and nasal secretions of
healthy persons, it may appear strange that cases of puerperal fever
not traceable to infection from erysipelas or from preceding cases
do not occur more frequently. This is probably largely due to an
attenuation of the pathogenic power of the streptococcus when it
leads a saprophytic existence. Widal asserts that, when cultivated
in artificial media for a few weeks, the cultures no longer have their
original virulence, and Bumm has made the same observation. On
the other hand, in " streptococcus-peritonitis " occurring as a result
of puerperal infection Bumm states that the thin, bright-yellow,
odorless fluid contained in the cavity of the abdomen is extremely
virulent ; a very slight trace, a fragment of a drop, injected into the
abdominal cavity of a rabbit, is sufficient within twenty-four hours
to cause a general septic inflammation with a bloody serous exuda-
tion, quickly terminating in the death of the animal ; injected sub-
cutaneously it gives rise to an enormous phlegmon which also

388 PYOGENIC BACTERIA.

quickly proves fatal. But cultures of Streptococcus pyogenes, after
it has been carried through successive generations in artificial media,
injected beneath the skin of a rabbit, usually produce no result, or
at most an abscess of moderate dimensions.

It seems probable that the micrococcus isolated by Fliigge from
necrotic foci in the spleen of a case of leucocythsemia, and described
by him under the name of Streptococcus pyogenes malignus, was
simply a very pathogenic variety of the streptococcus of pus. He
was not able to differentiate it from Streptococcus pyogenes by its
morphology or growth in culture media, but it proved far more
pathogenic when tested upon animals. Mice inoculated subcutane-
ously with a minute quantity of a pure culture died, without excep-
tion, in three to five days. A large abscess was formed at the point
of inoculation, and the blood of the animal contained numerous cocci
in pairs and chains. Rabbits inoculated in the ear showed at first
the same local appearances as result from inoculations with strepto-
coccus of pus and of erysipelas, but after two or three days symp •
toms of general infection were developed, and death occurred at the
end of three or four days. At the autopsy the cocci were found in
the blood, and frequently there were purulent collections in the
joints containing the same microorganism. Krause has also de-
scribed a streptococcus which only differs from Streptococcus pyo-
genes of Rosenbach and Passet by the greater virulence manifested
by its cultures.

The fact that pathogenic bacteria may attain an intensified de-
gree of virulence by cultivation in the bodies of susceptible animals
was demonstrated by Davaine many years ago, and is fully estab-
lished by the experiments of Pasteur and others. It is true of the
anthrax bacillus, of the writer's Micrococcus Pasteuri, and of other
well-known pathogenic microorganisms. The reverse of this — at-
tenuation of virulence as a result of cultivation in artificial media —
is also well established for several pathogenic species. Now it
appears that the attenuated streptococcus is far less likely to give
rise to erysipelas or to puerperal infection than is the same micro-
organism as obtained from a case of one or the other of these infec-
tious diseases. The same is probably true also of Staphylococcus
aureus and other facultative parasites which are found as sapro-
phytes upon the surface of the body and upon exposed mucous mem-
branes in healthy persons. And it is not improbable that attenuated
varieties of these micrococci which find their way into open wounds,
or into the uterine cavity shortly after parturition, if they escape
destruction by the sanguineous discharge, acquire increased patho-
genic power from their multiplication in it, as a result of which they
are able to invade the living tissues. But it appears probable that

PYOGENIC BACTERIA. 389

•

infection through open wounds does not depend alone upon the
potency of the pathogenic micrococci present in them, but also upon
the absorption of chemical poisons produced by septic (putrefactive)
bacteria, which weaken the vital resisting power of the tissues.
Gottstein, as a result of experiments made by him, is of the opinion
that the resorption of broken-down red blood corpuscles favors infec-
tion by pathogenic bacteria present in wounds ; and he has shown
that the injection into animals of certain toxic substances which de-
stroy the red corpuscles in the circulation makes them susceptible to
the pathogenic action of certain bacteria which are harmless for
them under ordinary circumstances. Thus a guinea-pig, an animal
which is immune against the bacillus of fowl cholera, succumbed to an
inoculation made after first injecting subcutaneously 0.06 gramme of
hydracetin dissolved in alcohol. At the autopsy hsemorrhagic exu-
dations were found in the serous cavities, hsemorrhagic infarctions
in the lungs, and quantities of the bacillus injected were found in
the blood and in fluid from the cavity of the abdomen.

In man the ever-present pus cocci are more likely to invade the
tissues, forming furuncles, carbuncles, and pustular skin eruptions,
or erysipelatous and phlegmonous inflammations, when the standard
of health is reduced from any cause, and especially when by absorp-
tion or retention various toxic organic products are present in the
body in excess. It is thus that we would explain the liability to these
local infections, as complications or sequelae of various specific infec-
tious diseases, in the victims of chronic alcoholism, in those exposed
to septic emanations from sewers, etc. , and probably in many cases
from the absorption of toxic products formed in the alimentary canal
as a result of the ingestion of improper food, or of abnormal fermen-
tative changes in the contents of the intestine, or from constipation.

The Pus Cocci in Inflammations of Mucous Membranes. —
To what extent the pus cocci are responsible for inducing and main-
taining non-specific inflammations of mucous membranes has not
been determined ; but having demonstrated the pyogenic properties
of these cocci, their presence in the purulent discharges from inflamed
mucous membranes can scarcely be considered as unimportant, not-
withstanding the fact that they are also frequently found in secre-
tions from healthy mucous surfaces. They are likewise found upon
the skin of healthy persons, and yet we have unimpeachable experi-
mental evidence that they may produce a local inflammation, at-
tended with pus formation, when injected subcutaneously, or even
when freely applied to the uninjured surface.

In otitis media Levy and Schrader obtained Staphylococcus
albus in pure cultures in three cases out of ten in which paracentesis
was performed, and in two others it was present in association with

390 PYOGENIC BACTERIA.

•

other microorganisms. In eighteen cases of otitis media in young
children Netter found Staphylococcus aureus six times and Strepto-
coccus pyogenes thirteen times. Scheibe, in eleven cases in which
perforation had not yet taken place, found Staphylococcus albus in
two and various other microorganisms in the remaining cases ; Sta-
phylococcus aureus was not present in any. Habermann obtained
aureus associated with other bacteria in a single case of purulent
otitis media. In a series of eight cases occurring as a sequela of
influenza Scheibe obtained Streptococcus pyogenes in two, " diplo-
coccus pneumonias" in two, Staphylococcus aureus in one, Strepto-
coccus pyogenes and Staphylococcus albus together in two, and Strep-
tococcus pyogenes in association with an undescribed micrococcus in
one. In all of these cases a slender bacillus was also present, as
shown by microscopical examination, which did not grow in any of
the culture media employed. Bordoni-Uffreduzzi and Gradenigo
have tabulated the results obtained by various bacteriologists who
have examined pus obtained through the previously intact tympanic
membrane. In thirty-two cases of this character the microorganism
most frequently found was diplococcus pneumoniae (Micrococcus
pneumonise crouposse of the present writer), which was present in a
pure culture in thirteen and associated with Staphylococcus aureus
in one, with Staphylococcus albus in one, and with Streptococcus
pyogenes in one. In the other sixteen cases the pyogenic cocci were
present in all but two, in which bacilli were found — Bacillus tenuis
in one, a non-liquefying bacillus in one. In twenty-seven cases in
which the pus was withdrawn from one to thirty days after paracen-
tesis or spontaneous rupture of the membrane, the pyogenic cocci
were present in twenty and diplococcus pneumoniae in seven.

In acute nasal catarrh Paulsen found Staphylococcus aureus in
seven cases out of twenty-four examined, and E. Frankel in two out of
four ; but it must be remembered that Von Besser has shown that this
micrococcus is frequently present in the secretions from the healthy
nasal mucous membrane, and we have experimental evidence that
the pus organisms, when introduced into the conjunctival sac of
rabbits (Widmark), do not give rise to catarrhal inflammation. On
the other hand, Widmark found that when inoculated into the cornea
of rabbits an intense conjunctivitis resulted, together with keratitis
and perforation of the cornea in fifteen per cent of the cases. The
same author in his bacteriological researches obtained the pyogenic
staphylococci from the circumscribed abscesses of blepharadenitis,
while in inflammation of the lacrymal sac Streptococcus pyogenes
was usually present.

Shougolowicz,in the bacteriological examination of twenty-six cases
of trachoma, found Staphylococcus albus in twelve, Staphylococcus

PYOGENIC BACTERIA. 391

aureus in nine, Staphylococcus citreus in three, and Staphylococcus
cereus albus in three. These pus organisms were in a number of
the cases associated with other well-known saprophytes, and in seven
cases a short bacillus not previously described was found. That
various bacilli are found in the conjunctival sac of healthy eyes
and in different forms of conjunctivitis has been shown by Fick,
whose results do not correspond in this respect with those of Gif-
ford, who found almost exclusively micrococci. Whatever may be
the final conclusion as to the role of the pus cocci heretofore de-
scribed in the etiology of acute or chronic conjunctivitis, there can be
no doubt of the power of the " gonococcus" to induce a virulent in-
flammation of the conjunctive when introduced into healthy eyes.

MICROCOCCUS GONORRHCE^E.

Synonym. — Gonococcus (Neisser).

Discovered by Neisser (1870) in gonorrhoea! pus and described by
him under the name of " Gonococcus/' Cultivated by Bumm (1885),
and infective virulence proved by inocula-
tion into man. Constantly present in viru-
lent gonorrhoea! discharges, for the most
part in the interior of the pus cells or at-
tached to the surface of epithelial cells.

Morphology. — Micrococci, usually join- ft
ed in pairs or in groups of four, in which
the elements are flattened - - " biscuit-
shaped." The flattened surf aces face each

other and are separated, in stained pre- ?§ &) ^ %$ €1
parations, by an unstained interspace. c

* . A Fia. 85. — a, gonococci from a

Ihe diameter of an associated pair ot cells pure culture, x about 1,000 ; &,gono-
varies from 0.8 to 1.6 /i in the long dia- cocci in PUS cells and epithelial CPU

, ,, from case of gonorrhoeal ophthal-

meter— average about 1.2o /*— and from mia; c, form and mode of division

0.6 to 0.8 yw in the line of the interspace of gonococci-schematic.
between the biscuit-shaped elements, which

sometimes present a slight concavity of the flattened surfaces. Mul-
tiplication occurs alternately in two planes, and as a result of this
groups of four are frequently observed. But diplococci are more
numerous and are considered as the characteristic mode of grouping.
Single, spherical, undivided cells are rarely seen.

It must be remembered that the morphology of this micrococcus
as above described does not suffice to distinguish it, for Bumm has
shown that " the biscuit form is not at all specific for the gonococcus,
but is shared with it by a number of microorganisms, which consist
of two hemispherical elements with the flattened surfaces facing each

392

PYOGENIC BACTERIA.

other and separated by a cleft, and some of these correspond in their
morphology, in every detail, with the gonococcus."

Stains quickly with the basic aniline colors, especially with
methyl violet, gentian violet, and fuchsin ; not so quickly with
methylene blue, which is, however, one of the most satisfactory
staining agents for demonstrating its presence in pus. Beautiful
double-stained preparations may be made from gonorrhoeal pus,
spread upon a cover glass and " fixed/' secundum art em, by the use
of methylene blue and eosin. Does not stain by Gram's method —
i.e., the cocci are decolorized, after having been stained with an ani-
line color, by being immersed in the iodine solution employed in
Gram's method of staining. But this character cannot be depended
upon alone for establishing the diagnosis, for Bumm has shown that

Fia. 86.—" Gonococcus " in gonorrhoeal pus. From a photomicrograph by Frankel and Pfeiffer.
X 1,000.

other diplococci are occasionally found in gonorrhoeal pus which do
not stain by this method. It serves to distinguish them, however, from
the common pus cocci heretofore described — Staphylococcus aureus,
Staphylococcus albus, Staphylococcus citreus — which retain their
color when treated in the same way. A more trustworthy diagnostic
character is that these biscuit-shaped diplococci are found within the
pus cells, sometimes one or two pairs only, but more frequently in
considerable numbers, and occasionally in such numbers as to com-
pletely fill the cell. No similar picture is presented by pus from any
other source, with the exception of that from a form of " puerperal
cystitis " which Bumm has described. But in this the diplococci
contained in the pus cells were to be distinguished by the fact that
they retained their color when treated by Gram's method. Owing

PYOGENIC BACTERIA. 393

to the difficulty of cultivating this micrococcus, and the importance,
under certain circumstances, of not making a mistake in its diag-
nosis, these characters are of exceptional value.

Biological Characters. — Bumm (1885) first succeeded in culti-
vating the " gonococcus " upon human blood serum, obtained from
the placenta of a recently delivered woman. He found that the cul-
tures thrive best in a moist atmosphere at 30° to 34° C. The growth
under the most favorable conditions is slow, and frequently no devel-
opment occurs when pus containing numerous gonococci is placed
upon blood serum in an incubating oven ; or after a slight multi-
plication development ceases and the cocci undergo degenerative
changes and quickly disappear.

Cultures upon the surface of blood serum form a very thin, often
scarcely visible layer, with a smooth, moist, shining surface, and
by reflected light a grayish-yellow color. The growth at the end of
twenty-four hours may extend for a distance of a millimetre along
the line of inoculation, but at the end of two or three days no fur-
ther development occurs and the cocci soon lose their vitality. This
micrococcus, then, is aerobic. Whether it may also be a facultative
anaerobic has not been definitely determined, but it does not grow
along the line of puncture when stick cultures are made in blood se-
rum. Its rapid and abundant multiplication in gonorrhoeal infection
of mucous membranes, and the difficulties attending its cultivation
in artificial media, show that the gonococcus is a strict parasite.

Lestikow and Loffler, prior to the publication of Bumm's impor-
tant monograph, had reported successful results in cultivating the
gonococcus upon a mixture of blood serum and gelatin. Bockhart
has since recommended a mixture of nutrient agar (two parts), lique-
fied at a temperature of 50° C., with blood serum (two to three parts)
at 20° C. By quickly mixing with this a little pus containing the
gonococcus he was able to obtain colonies upon plate cultures, made
by pouring the liquid medium upon sterile glass plates in the usual
manner.

Ghon and Schlagenhaufer in 1893 reported that they obtained
good results by adding phosphate of soda to blood-serum agar, made
according to the method of Wertheim — one part of human blood
serum from the placenta to two or three parts of nutrient agar. Also
that they were successful in cultivating the gonococcus in an acid
medium made by adding one part of urine to two of nutrient agar
(two per cent). Turro (1894) has since published the results of his
experiments relating to the cultivation of this micrococcus in acid
media. According to him it grows in normal urine, either with or
without the addition of peptone (one per cent) ; also in acid gelatin,
prepared in the usual way but without neutralization (?).

394

PYOGENIC BACTERIA.

Turro also claims to have produced specific urethritis in dogs by
inoculation with his cultures. Heiman (1895) as a result of an ex-
tended experimental research, arrives at the conclusion that "the
diplococcus described by Turro in connection with his acid media is
not the gonococcus." His inoculation experiments in dogs, made
with pure cultures of the gonococcus, gave an entirely negative result.
For the cultivation of the gonococcus, Heiman recommends a medium
made from "chest serum" obtained from a patient suffering with
hydrothorax or acute pleurisy. This was found to be superior to
placenta serum, sheep-blood serum, or peritoneum serum, because of
the great amount of serum albumin which it contains. Two per
cent of agar, one per cent of peptone, and one-half per cent of sodium
chloride were added to the chest serum, and the medium was sterilized
by "fractional sterilization."

FIG. 87.— Gonorrhoeal conjunctivitis, second day of sickness; section through the mucous mem-
brane of upper eyelid; invasion of the epithelial layer by gonococci. (Bumm.)

Schrotter and Winkler (1890) report their success in cultivating
the gonococcus upon albumin from the egg of the pewit — " Kibitz"
In the culture oven at 38° C. a thin, transparent, whitish layer was
already visible at the end of six hours and rapidly extended ; the
growth was less abundant at the end of three days, and had entirely
ceased by the fifth day. Attempts to cultivate the same microor-
ganism in albumin from hens' eggs gave a negative result.

Aufuso (1891) has cultivated the gonococcus in fluid obtained
from the knee joint in a case of chronic synovitis, but failed to culti-
vate it in the fluid of ascites. A culture of the twelfth generation
made upon the culture medium mentioned, solidified by heat, was
introduced into the urethra of a healthy man and gave rise to a
characteristic attack of gonorrhoea.

Development does not occur below 25° or above 38° C. The
writer has shown that a temperature of 60° C. maintained for ten
minutes destroys the infective virulence of gonorrhoeal pus.

Pathogenesis. — That the gonococcus is the cause of the specific
inflammation and purulent discharge characteristic of gonorrhoea is
now generally admitted upon the experimental evidence obtained by

PYOGENIC BACTERIA. 395

Bumm. Having succeeded in obtaining it in pure cultures from
gonorrhoeal pus, he made successful inoculations in the healthy ure-
thra in two cases — once with a third culture and once with one
which had been transferred through twenty successive generations.
In both cases a typical gonorrhoea developed as a result of the inocu-
lation.

The mucous membranes in man which are subject to gonorrhceal
infection are those of the urethra, the conjunctiva, the cervix uteri,
and the vagina in children — the vagina in adults is not involved.
Inoculations of gonorrhceal pus into the vagina or conjunctival sac of
the lower animals — dogs, rabbits, horses, apes — are without result.

The very numerous researches which have been made by compe-
tent bacteriologists show that the gonococcus is constantly present in
gonorrhoeal discharges, and in view of the facts above stated its etio-
logical import appears to be fully established. Bumm has studied
the development of blennorrhoea neonatorum, and has shown that
soon after infection the presence of gonococci may be demonstrated
in the superficial epithelial cells of the mucous membrane and be-
tween them ; that they soon penetrate to the deeper layers, and that
by the end of forty-eight hours the entire epithelial layer is invaded
by the diplococci, which penetrate by way of the connecting mate-
rial— " Kittsubstance " —between the cells. They also multiply in
the superficial layers of connective tissue and give rise to an inflam-
matory reaction, which is shown by an abundant escape of leuco-
cytes from the dilated capillary network. The penetration of the
gonococci to the deeper layers of the mucous membrane of the ure-
thra, and even to the corpus cavernosum, was observed by Bockhart
in a case studied by him in which death occurred during an acute
attack of gonorrhoea. But Bumm concludes from his researches
that this is not usual, and that the invasion is commonly limited to
the superficial layers of the mucous membrane.

Staphylococcus pyogenes aureus is not infrequently associated
with the gonococcus in late gonorrhoeal discharges, and the abscesses
which occasionally develop as a complication of gonorrhoea, in the
prostate, the inguinal glands, or around the urethra, are probably
due to its presence, which has been demonstrated in the pus from
such abscesses in a number of cases. The same is true of the joint
affections and endocarditis which sometimes occur in the course of
an attack of gonorrhoea. Although some authors have claimed to
find the gonococcus in these so-called metastatic gonorrhceal inflam-
mations, the evidence is not satisfactory, and it seems probable that
the Staphylococcus aureus is the usual microorganism concerned in
these affections.

VI.
BACTERIA IN CROUPOUS PNEUMONIA.

BACILLUS OF FRIEDLANDER.

Synonyms. — Pneumococcus (Friedlander); Bacillus pneumonia
(Fliigge).

Obtained by Friedlander and Frobenius in pure cultures (1883)
from the exudate into the pulmonary alveoli in cases of croup-
ous pneumonia. Subsequent researches show that it is only present
in a small proportion of the cases — nine times in one hundred and
twenty-nine cases examined by Weichselbaum, three times in seventy
cases examined by Wolf.

Morphology. — Short rods with rounded ends, often so short as

to resemble micrococci, especially in very
recent cultures ; commonly united in pairs
or chains of four, and under certain cir-
cumstances surrounded by a transparent
capsule. The gelatinous envelope — so-
FIG. 88.— Bacillus of Friedlander; called capsule — is not seen in preparations
a, from a culture; 6, from blood of ma(je f rom cuitures in artificial media, but

mouse, showing capsule. (Flugge.) . . .

is very prominent in properly stained prepa-
rations from the blood of an inoculated animal. It often has a diame-
ter equal to or greater than that of the enclosed cell, and appears to
consist of a substance resembling mucin, which is soluble in water or
dilute alcohol. Where several cells are united in a chain they may
all be enclosed in a common envelope, or each may have its own cap-
sule. This capsule is not peculiar to Friedlander's bacillus, as he
at first supposed, but is found in other bacilli and also in the writer's
Micrococcus Pasteuri.

Friedlander's bacillus stains readily with the aniline colors, but
is decolorized by the iodine solution used in Gram's method. In
preparations from the blood of an inoculated animal, stained by an
aniline color, the capsule appears as an unstained envelope surround-
ing the stained cell, but by special treatment the capsule may also be
stained. Friedlander's method is as follows : The section or cover-

BACTERIA IN CROUPOUS PNEUMONIA.

397

glass preparation is placed for twenty-four hours in a solution of
gentian violet and acetic acid, containing fifty parts of a concentrated
alcoholic solution of gentian violet, one hundred parts of distilled
water, and ten parts of acetic acid. The stained preparation is
washed for a minute or two in a one-per-cent solution of acetic acid,
dehydrated with alcohol, cleared up with oil of cloves or cedar, and
mounted in balsam. The bacillus is quickly stained in dried cover-
glass preparations by immersion in aniline- water-gentian-violet solu-
tion (two or three minutes). The stained preparation should be de-
colorized by placing it in absolute alcohol for half a minute, and then
washed in distilled water.

Biological Characters. — This bacillus does not, so far as is
known, form reproductive spores ; it is non-motile and does not
liquefy gelatin. It is aerobic and a facultative anaerobic. In
gelatin stab cultures it presents the "nail-shaped" growth first
described by Friedlander, which is not, however, peculiar to this
bacillus. The head of the nail is formed by the
development around the point of entrance of the
inoculating needle of a rounded, white mass hav-
ing a smooth, shining surface, and its stem by the
growth along the line of puncture. This consists
of closely crowded, opaque, white, spherical colo-
nies. Gas bubbles sometimes develop in gelatin
cultures, and in old cultures the gelatin about the
line of growth acquires a yellowish-brown color.
The growth in nutrient agar resembles that in
gelatin. Upon the surface of blood serum abun-
dant grayish- white, viscid masses are developed.
Upon potato the growth is abundant, quickly cov-
ering the entire surface with a thick, yellowish-
white, glistening layer which often contains gas
bubbles when the temperature is favorable. Col-
onies in gelatin plates appear at the end of twenty-
four hours as small, white spheres, which increase
rapidly in size, and upon the surface form round-
ed, smooth, glistening, white masses of consider-
able size. Under the microscope the colonies pre-
sent a somewhat irregular outline and a slightly Fia* 89 ~Friedl5nder1s

0 y bacillus; stab culture in

granular appearance. Growth occurs at compara- gelatin; end of four days
tively low temperatures— 16° to 20° C.— but is more at 16°-18° °- (Baumgar-
rapid in the incubating oven. The thermal death-
point, as determined by the writer, is about 56° C. In the ordinary
culture media it retains its vitality for a long time, and may grow
when transplanted to fresh culture material after having been pre-

398 BACTERIA IN CROUPOUS PNEUMONIA.

served for a year or more. At a temperature of 40° C. development
ceases.

Pathogenesis. — In Friedlander's experiments the bacillus from
pure cultures, suspended in water, was injected through the thoracic
wall rinto the right lung of dogs, rabbits, guinea-pigs, and mice.
Rabbits proved to be immune ; one dog out of five, six guinea-pigs
out of eleven, and all of the mice (thirty-two) succumbed to the
inoculation. At the autopsy the pleural cavities were found to con-
tain a sero-purulent fluid ; the lungs were intensely congested, con-
tained but little air, and in places showed limited areas of red infil-
tration ; the spleen was considerably enlarged ; the bacillus was
found in great numbers in the lungs, the fluid in the pleural cavi-
ties, and in the blood obtained from the general circulation or from
the various organs of the body. Similar appearances presented them-
selves in the case of the guinea-pigs which succumbed to the inocu-
lation.

These results show that the bacillus under consideration is path-
ogenic for mice and for guinea-pigs, but they are by no means
sufficient to prove that it is capable of producing a genuine croupous
pneumonia in man, and it is still uncertain whether its occasional
presence in the exudate into the pulmonary alveoli in cases of this
disease has any etiological importance.

MICROCOCCUS PNEUMONIA CROUPOS^Q.

Synonyms. — Micrococcus Pasteuri (Sternberg) ; Micrococcus of
sputum septicaemia (Frankel) ; Diplococcus pneumonise (Weichsel-
baum) ; Bacillus septicus sputigenus (Fliigge) ; Bacillus salivarius
septicus (Biondi) ; Lancet-shaped micrococcus (Talamon) ; Strepto-
coccus lanceolatus Pasteuri (Gameleia).

Discovered by the present writer in the blood of rabbits inocu-
lated subcutaneously with his own saliva in September, 1880 ; by
Pasteur in the blood of rabbits inoculated with the saliva of a child
which died of hydrophobia in one of the hospitals of Paris in De-
cember, 1880 ; identified with the micrococcus in the rusty sputum of
pneumonia, by comparative inoculation and culture experiments, by
the writer in 1885 (paper published in the American Journal of the
Medical Sciences, July 1st, 1885). Proved to be the cause of croup-
ous pneumonia in man by the researches of Talamon, Salvioli, Stern-
berg, Frankel, Weichselbaum, Netter, Gameleia, and others.

The Presence of Micrococcus Pasteuri in the Salivary Secre-
tions of Healthy Individuals. — In September, 1880, while engaged
in investigations relating to the etiology of the malarial fevers, I in-
jected a little of my own saliva beneath the skin of two rabbits as a
control experiment. To my surprise the animals died, and I fomnd

BACTERIA IN CROUPOUS PNEUMONIA.

in their blood a multitude of oval microorganisms, united for the
most part in pairs, or in chains of three or four elements. These
experiments are recorded in my paper entitled " Experimental Inves-
tigations Relating to the Etiology of the Malarial Fevers/' published
in the Report of the National Board of Health for 1881, pp. 74, 75.

Following up my experiments made in New Orleans (in Septem-
ber, 1880), in Philadelphia (January, 1881), and in Baltimore (March,
1881), I obtained the following results :

" The saliva of four students, residents of Baltimore (in March),
gave negative results ; eleven rabbits injected with the saliva of six
individuals in Philadelphia (in January) gave eight deaths and three
negative results; but in the fatal cases a less degree of virulence was
shown in six by a more prolonged period between the date of injec-
tion and the date of death. This was three days in one, four days
in four, and seven days in one. "

In a paper published in the Journal of the Royal Microscopical
Society (June, 1886) I say :

" My own earlier experiments showed that there is a difference in
the pathogenic potency of the saliva of different individuals, and I
have since learned that the saliva of the same individual may differ
in this respect at different times. Thus during the past three years
injections of my own saliva have not infrequently failed to cause a
fatal result, and in fatal cases death is apt to occur after a some-
what longer interval, seventy-two hours or more ; whereas in my
earlier experiments the animals infallibly died within forty-eight
hours."

The presence of my Micrococcus Pasteuri was demonstrated in
the blood of the rabbits which succumbed to the inoculations.

Claxton, in a series of experiments made in Philadelphia in 1882,
injected the saliva of seven individuals into eighteen rabbits. Five
of these died within five days, and nine at a later period.

Frankel, whose first publication was made in 1885, discovered
the presence of this micrococcus in his own salivary secretions in 1883,
and has since made extended and important researches with refe-
rence to it. The saliva of five healthy individuals and the sputa
of patients suffering from other diseases than pneumonia, injected
into eighteen rabbits, induced fatal " sputum septicaemia " in three
only. When he commenced his experiments his saliva was uni-
formly fatal to rabbits, but a year later it was without effect.

Wolf injected the saliva of twelve healthy individuals, and of
three patients with catarrhal bronchitis, into rabbits, and induced
" sputum septicaemia " in three.

Netter examined the saliva of one hundred and sixty-five healthy
persons, by inoculation experiments in rabbits, and demonstrated
the presence of this micrococcus in fifteen per cent of the number.

400 BACTERIA IN CROUPOUS PNEUMONIA.

Vignal, in his recent elaborate paper upon the microorganisms
of the mouth, says :

" Last year I encountered this microbe continually in my mouth
during a period of two months, then it disappeared, and I did not
find it again until April of this year, and then only for fifteen days,
when it again disappeared without appreciable cause. "

The Presence of Micrococcus Pneumonice Crouposce in Pneu-
monic Sputum. — Talamon, in 1883, demonstrated the presence of this
micrococcus in pneumonic sputum, described its morphological char-
acters, and produced typical croupous pneumonia in rabbits by in-
jecting material containing it into the lungs through the thoracic
walls.

Salvioli, in 1884, demonstrated its presence in pneumonic sputum
by injections into rabbits.

In 1885 the writer made a similar demonstration, and by compara-
tive experiments showed that the micrococcus present in the blood
of rabbits inoculated with the rusty sputum of pneumonia was iden-
tical with that which he had discovered in 1880 in rabbits inoculated
with his own saliva.

The same year (1885) A. Frankel made a similar demonstration,
and published a paper containing valuable additions to our knowl-
edge relating to the biological characters of this microorganism (first
publication appeared July 13th, 1885).

In 1886 Weichselbaum published the results of his extended re-
searches relating to the presence of this micrococcus in the fibrinous
exudate of croupous pneumonia. He obtained it in ninety-four cases
(fifty-four times in cultures) out of one hundred and twenty-nine cases
examined.

Wolf (1887) found it in sixty-six cases out of seventy examined.

Netter (1887) in seventy-five per cent of his cases, and in the sputum
of convalescents from pneumonia in sixty per cent of the cases ex-
amined, by inoculations into rabbits.

Gameleia (1887) in twelve fatal cases of pneumonia in which he
collected material from the lungs at the post-mortem examination.

Goldenberg, whose researches were made in Gameleia's labora-
tory, found it in pneumonic sputum in forty consecutive cases, by
inoculations into rabbits and mice.

The Presence of Micrococcus Pneumonice Crouposce in Menin-
gitis.— Numerous bacteriologists have reported finding diplococci in
the pus of meningitis, and frequently the microorganisms have been
fully identified as " diplococcuspneumonise." Thus Netter (1889), in
a resume of the results of researches made by him in twenty-five
cases of purulent meningitis, reports as follows :

BACTERIA IN CROUPOUS PNEUMONIA. 401

Thirteen cases were examined microscopically, by cultures, and
by inoculations into susceptible animals ; six cases by microscopical
examination and experiments on animals; and the remainder only by
microscopical examination. Four of the cases were complicated
with purulent otitis, six with pneumonia, three with ulcerative endo-
carditis. The " pneumococcus " was found in sixteen of the twenty-
five cases ; in four Streptococcus pyogenes was present ; in two
Diplococcus intracellularis meningitidis of Weichselbaum ; in one
Friedlander's bacillus ; in one Newmann and Schaffer's motile ba-
cillus ; in one a small curved bacillus.

In forty-five cases collected from the literature of the subject by

FIG. 90. FIG. 91. FIG. 98.

FIG. 90.— Micrococcus pneumonise crouposae from blood of rabbit inoculated with normal human
saliva (Dr. S.). X 1,000.

FIG. 91. — Micrococcus pneumonias crouposae from blood of rabbit inoculated subcutaneously
with fresh pneumonic sputum from a patient in the seventh day of the disease. X 1,000.

FIG. 92.— Surface culture of Micrococcus pneumonias crouposse, on nutrient agar, showing the
development of long chains. X 1,000.1

better this micrococcus was present in twenty-seven, Streptococcus
pyogenes in six, and the Diplococcus intracellularis meningitidis of
Weichselbaum in ten.

Monti (1889), in four cases of cerebro-spinal meningitis, demon-
strated the presence of the same micrococcus. In three of his cases
pneumonia was also present. In two Staphylococcus pyogenes aureus
was associated with the " diplococcus pneumonia."

Micrococcus Pneumonias Crouposce in Ulcerative Endocar-
ditis.— Weichselbaum, in a series of twenty-nine cases examined
(1888), found " diplococcus pneumonise" in seven.

Micrococcus Pneumonice Crouposce in Acute Abscesses. — In a
case of parotitis occurring as a complication of croupous pneumonia
this micrococcus was obtained from the pus in pure cultures by Testi
(1889); and in another case in which, as a complication of pneumonia,
there developed a purulent pleuritis, abscess of the parotid on both
sides, and multiple subcutaneous abscesses, the pus from all of the
sources named contained the ' ' diplococcus " in great numbers, as
shown not only by microscopical examination but by inoculation into
rabbits.

1 The above figures are from Dr. Sternberg's paper published in the American
Journal of the Medical Sciences for July and October, 1885.
26

402 BACTERIA IN CROUPOUS PNEUMONIA.

In a case of tonsillitis resulting in the formation of an abscess
Gabbi (1889) obtained the same coccus in pure cultures.

In otitis media this micrococcus has been found in a consider-
able number of cases in the pus obtained by paracentesis of the
tympanic membrane, and quite frequently in pure cultures — by Zau-
fal (1889) in six cases; Levy and Schrader (1889) in three out of ten
cases in which paracentesis was performed; by Netter (1889) in five
out of eighteen cases occurring in children.

Monti (1889) and Belfanti (1889) report cases of arthritis of the
wrist joint, occurring as a complication of pneumonia, in which this
micrococcus was obtained in pure cultures. Ortmann and Samter
(1889), in a case of purulent inflammation of the shoulder joint fol-
lowing pneumonia and pleurisy, obtained the "diplococcus pneu-
monise " in pure cultures.

Morphology. — Spherical or oval cocci, usually united in pairs, or
in chains consisting of three or four elements. Longer chains, con-
taining ten or more elements, are frequently formed, especially in
cultures upon the surface of nutrient agar, and in liquid media; it
may therefore be regarded as a streptococcus. As observed in the
blood of inoculated animals it is usually in pairs consisting of oval
or lance-oval elements, which are surrounded by a transparent cap-
sule. Owing to the elongated form of the cocci when in active
growth, it has been regarded by some authors as a bacillus; but in
cultures in liquid media, when development by binary division has
ceased, the cells are spherical, or nearly so, and in cultures on the
surface of nutrient agar the individual cells more nearly approach a
spherical form than in the blood of an inoculated animal. The " lan-
ceolate " form was first referred to by Tala-
mon, who described it as having the form of
a grain of wheat, or even still more elongated
like a grain of barley, as seen in the fibrin-
ous exudate of croupous pneumonia. The
transparent material surrounding the cells-
so-called capsule — is best seen in stained
preparations from the fibrinous exudate of
FIG. 93.-Micrococcus pneu- croupous pneumonia or from the blood of an

monise crouposae, showing cap-
sule, attached to pus cells from inoculated animal. It appears as an un-

st«ined marginal band surrounding the ellip-
tical cells, and varies greatly as to its extent
in different preparations. This capsule probably consists of a sub-
stance resembling mucin, and, being soluble in water, its extent de-
pends partly upon the methods employed in preparing specimens for
microscopical examination. It is occasionally seen in stained prep-
arations from the surface of cultures on blood serum ; and in drop

BACTERIA IN CROUPOUS PNEUMONIA. 403

cultures examined under the microscope, by using a small diaphragm
it may be seen to surround the cocci as a scarcely visible halo.

This micrococcus stains readily with the aniline colors ; and also
by Gram's method, which constitutes an important character for dis-
tinguishing it from Friedlander's bacillus.

Biological Characters. — Grows in the presence of oxygen —
aerobic — but is also a facultative anaerobic. Like other micro-
cocci, it has no spontaneous movements. It grows in a variety of
culture media when they have a slightly alkaline reaction, but will
not develop in a medium which contains the slightest trace of free
acid. Nor will it grow at the ordinary room temperature. Scanty
development may occur at a temperature of 22° to 24° C., but a
temperature of 35° to 37° C. is most favorable for its growth, which
is very rapid in a suitable liquid medium. In an infusion made from
the flesh of a chicken or a rabbit it multiplies, in the incubating
oven, with remarkable rapidity ; at the end of six to twelve hours
after inoculation the previously transparent fluid will be found to
present a slight cloudiness and to be filled throughout with the cocci
in pairs and short chains. It does not produce a milky opacity in
liquid media, like the pus cocci, for example, but the fluid becomes
slightly clouded ; multiplication ceases at the end of about forty-
eight hours or less, and the liquid medium again becomes transpa-
rent as a result of the subsidence of the cocci to the bottom of the
receptacle.

It may be cultivated in flesh-peptone-gelatin, containing fifteen
per cent of gelatin, at a temperature of 24° C., or in liquefied gela-
tin (ten per cent) in the incubating oven.
In gelatin (fifteen per cent) stab cultures
small white colonies develop all along the
line of puncture, and in gelatin plates
small, spherical, slightly granular, whitish
colonies are formed : the gelatin is not
liquefied. In agar plates extremely mi-
nute colonies are developed in the course
of forty-eight hours, which resemble little,
transparent drops of fluid, and under the
microscope some of these are observed to FIG. 94

have a Compact, finely granular Central coccus pneumonias crouposae upon

portion surrounded by a paler, transparent, XFSSSSSSSiS* °'

finely granular marginal zone. Upon the

surface of nutrient agar or coagulated blood serum development
occurs in the form of minute, transparent, jelly-like drops, which
form a thin layer along the line of inoculation in "streak cultures" ;
and in agar stick cultures the growth along the line of puncture is

icro-

404 BACTERIA IN CROUPOUS PNEUMONIA.

rather scanty, almost homogeneous, and semi-transparent. Upon
potato no development occurs, even in the incubating oven. Milk is
a favorable culture medium, and the casein is coagulated as a result
of its presence.

It ceases to grow on solid media at about 40° C., and in favorable
liquid media at 42° C. Its thermal death-point, as determined by
the writer, is 52° C. , the time of exposure being ten minutes. It
loses its vitality in cultures in a comparatively short time — four or
five days on agar — and is very sensitive to the action of germicidal
agents. Its pathogenic power also undergoes attenuation very
quickly when it is cultivated in artificial media, but may be restored
by passing it through the bodies of susceptible animals. Attenua-
tion of virulence may also be effected by exposing bouillon cultures
to a temperature of 42° C. for twenty-four hours, or by five days'
exposure to a temperature of 41° C.

Emmerich reported in 1891 to the Congress of Hygiene and
Demography in London the results of experiments made by him
relating to immunity in rabbits and mice. Rabbits were rendered
immune by the intravenous injection of a very much diluted but
virulent culture of the micrococcus. The flesh of these immune
rabbits was rubbed up into a fine paste, and the juices obtained by
compressing it in a clean, sterilized cloth. This bloody juice was kept
for twelve hours at a temperature of 10° C., and then sterilized by
passing it through a Pasteur filter. Some of this juice was injected
into a rabbit, which with twenty-five others was then made to re-
spire an atmosphere charged with a spray of a bouillon culture of
the micrococcus. As a result of this all of the rabbits died except the
one which had previously been injected with the immunizing juice.
In a similar experiment upon mice six of these animals, which had
previously been injected with the immunizing juice, survived the in-
jection of a full dose of a virulent culture, while a control mouse,
not previously injected with the juice, promptly died after receiving
the same quantity of the virulent culture.

The writer in 1881, in experiments made to determine the value
of various disinfectants, as tested upon this micrococcus, obtained
experimental evidence that its virulence is attenuated by the action
of certain antiseptic agents. Commenting upon the results of these
experiments in my chapter on " Attenuation of Virus," in " Bacte-
ria " (1884), I say :

' ' Sodium hyposulphite and alcohol were the chemical reagents which
produced the result noted in these experiments ; but it seems probable that
a variety of antiseptic substances will be found to be equally effective when
used in the proper proportion. Subsequent experiments have shown that
neither of these agents is capable of destroying the vitality of this septic
micrococcus in the proportion used (one per cent of sodium hyposulphite or

BACTERIA IN CROUPOUS PNEUMONIA. 405

one part of ninety-five-per-ceiit alcohol to three parts of virus), and that
both have a restraining- influence upon the development of this microorgan-
ism in culture fluids."

The following results were obtained by the writer in his experi-
ments (1881 and 1883) to determine the germicidal and antiseptic
value of the agents named, as tested upon this micrococcus.

Alcohol. — A twenty-four-per-cent solution was effective upon
bouillon cultures in two hours.

Boric Acid. — A saturated solution failed to destroy vitality after
two hours' exposure, but 1 : 400 restrained development.

Carbolic Acid. — A one-per-cent solution destroys vitality in two
hours, and 1 : 500 restrains development.

Cupric Sulphate destroys the virulence of the coccus in the
blood of a rabbit in the proportion of 1 : 400 in half an hour.

Ferric Sulphate failed to destroy vitality in a saturated solution,
but restrained development in the proportion of 1 : 200.

Hydrochloric Acid destroys the virulence of the blood of a rab-
bit containing this micrococcus in the proportion of 1 : 200.

Iodine, in aqueous solution with potassium iodide, destroys vital-
ity in the proportion of 1: 1,000 and prevents development in 1: 4,000.

Mercuric Chloride. — One part in forty thousand prevents the
development of this micrococcus, and 1 : 20,000 was found to destroy
vitality in two hours.

Nitric Acid. — One part in four hundred destroyed the virulence
of rabbit's blood containing this micrococcus.

Caustic Potash. — A two-per-cent solution destroyed vitality in
two hours.

Potassium Permanganate. — A two-per-cent solution destroyed
the virulence of rabbit's blood containing this coccus.

Salicylic Acid, dissolved by the addition of sodium biborate. —
A solution of 1 : 400 prevented development.

Sulphuric Acid. — One part in two hundred destroys vitality, and
1 : 800 prevents development.

In a paper by Bordoni-Uffreduzzi relating to the resisting power
of pneumonic virus for desiccation and light, the following results are
given : Pneumonic sputum attached to cloths, when dried in the air
and exposed to diffuse daylight, retained its virulence, as shown by
injection in rabbits, for a period of nineteen days in one series of ex-
periments and for fifty-five days in another. Exposed to direct sun-
light the same material retained its virulence after twelve hours'
exposure. Cultures have far less resistance, and the protection
afforded by the dried albuminous material in which the micrococci
were embedded, in the experiments referred to, probably accounts
for the virulence being retained so long a time.

406 BACTERIA IN CROUPOUS PNEUMONIA.

Kruse and Pansini (1892) have published an elaborate paper giv-
ing an account of their researches relating to "diplococcus pneumo-
nia " and allied streptococci. We give below a summary statement
of their results :

Many varieties were obtained by the observers named in their cultures
from various sources — from the lungs of individuals dead from pneumonia,
from pleuritic exudate, from pneumonic sputa, from bronchitic sputa, from
the saliva of healthy persons, from the secretion in a case of subacute nasal
catarrh, from the urine of a patient with nephritis.

Pure cultures were obtained by the use of agar plates or by inoculations
into rabbits. In all about thirty varieties were obtained and cultivated
through many successive generations. As a rule, the different varieties,
which at first were seen to nave the form of diplococci, when cultivated for
a length of time in artificial media presented the form of streptococci ; and
the elements which at first were lancet-shaped showed a tendency to become
spherical.

The more virulent varieties usually presented the form of diplococci
with lancet-shaped elements, or of short chains. A variety which formed
long chains could be pronounced, in advance of the experiments on animals,
to possess comparatively little virulence. When by inoculations in animals
the virulence of such a variety was restored, the tendency to form chains
was less pronounced.

Although, as a rule, no development occurs at 20° C., certain varieties
were obtained which, after long cultivation in artificial media, showed a de-
cided growth at 18° C.

Decided differences were shown by the cultures from various sources as
regards their growth in milk. Out of eighty-four cultures from various
sources eleven did not produce coagulation. As a rule, cultures which caused
coagulation of milk were virulent for rabbits, and when such cultures lost
their virulence they usually lost at the same time the power of coagulating
milk. Virulent cultures die out sooner than those which have become at-
tenuated by continuous cultivation in artificial media; the first, on the sur-
face of agar, usually fail to grow at the end of a week, while the attenuated
cultures may survive for three weeks or more.

Pathogenesis. — This micrococcus is very pathogenic for mice and
for rabbits, less so for guinea-pigs. The injection of a minute quan-
tity— 0. 2 cubic centimetre or less — of a virulent culture beneath the
skin of a rabbit or a mouse usually results in the death of the animal
in from twenty-four to forty-eight hours. The following is from the
writer's first published paper (1881), and refers to the pathological
appearances in rabbits :

" The course of the disease and the post-mortem appearances indicate that
it is a form of septicaemia. Immediately after the injection there is a rise of
temperature, which in a few hours may reach 2° to 3° C. (3.6° to 5.4° F.) ;
the temperature subsequently falls, and shortly before death is often several
degrees below the normal. There is loss of appetite and marked debility
after twenty -four hours, and the animal commonly dies during the second
night or early in the morning of the second day after the injection. Death
occurs still more quickly when the blood from a rabbit recently dead is in-
jected. Not infrequently convulsions immediately precede death.

"The most marked pathological appearance is a diffuse inflammatory
cedema or cellulitis, extending in all directions from the point of injection,

BACTERIA IN CROUPOUS PNEUMONIA.

407

but especially to the dependent portions of the body. Occasionally there is
a little pus near the puncture, but usually death occurs before the'cellulitis
reaches the point of producing pus. The subcutaneous connective tissue
contains a quantity of bloody serum, which possesses virulent properties and
which contains a multitude of micrococci. There is usuc lly more or less in-
flammatory adhesion of the integument to the subjacent tissues. The liver
is sometimes dark-colored and gorged with blood, but more frequently it is
of a lighter color than normal and contains much fat. The spleen is either
normal in appearance or enlarged and dark-colored. Changes in this organ
are more marked in those cases which are of the longest duration.

' ' The blood commonly contains an immense number of micrococci, usually
joined in pairs and having a diameter of about 0.5 /*. These are found in
blood drawn from superficial veins, from arteries, and from the cavities of
the heart immediately after death, and in a few cases their presence has been

FIG. 93. — Micrococcus pneumonias crouposae in blood of rabbit inoculated with pneumonic spu-
tum. X 1,000.

verified during life. Observations thus far made, however, indicate that it
is only during the last hours of life that these parasites multiply in the cir-
culating fluid, and in a certain proportion of the cases a careful search has
failed to reveal their presence in the blood in post-mortem examinations
made immediately after the death of the animal."

In animals which are not examined until some hours after death
a considerable increase in the number of micrococci occurs post mor-
tem. The fact that this micrococcus varies very much as to its
pathogenic power, as a result of conditions relating to the medium in
which it develops, was insisted upon in my first published paper, and
has been fully established by later researches (Frankel, Gameleia).
Susceptible animals inoculated with attenuated cultures acquire an
immunity against virulent cultures.

In dogs subcutaneous injections usually give a negative result,
or at most a small abscess forms at the point of inoculation. In a

408 BACTERIA IN CROUPOUS PNEUMONIA.

single experiment, however, the writer has seen a fatal result in a
dog from the injection of one cubic centimetre of bloody serum from
the subcutaneous connective tissue of a rabbit recently dead. This
shows the intense virulence of the micrococcus when cultivated in
the body of this animal. Pneumonia never results from subcutane-
ous injections into susceptible animals, but injections made through
the thoracic walls into the substance of the lung may induce a typi-
cal fibrinous pneumonia. This was first demonstrated by Talamon
(1883), who injected the fibrinous exudate of croupous pneumonia,
obtained after death, or drawn during life by means of a Pravaz
syringe from the hepatized portions of the lung, into the lungs of
rabbits. According to See, eight out of twenty animals experi-
mented upon exhibited "a veritable lobar, fibrinous pneumonia,
with pleurisy and pericarditis of the same nature." Gameleia has
also induced pneumonia in a large number of rabbits, and also in the
dog and the sheep, by injections directly into the pulmonary tissue.
Sheep were found to survive subcutaneous inoculations, unless very
large doses (five cubic centimetres) of the most potent virus were in-
jected. But intrapulmonary inoculations invariably induced a typi-
cal fibrinous pneumonia which usually proved fatal. In dogs simi-
lar injections gave rise to a " frank, fibrinous pneumonia which
rarely proved fatal, recovery usually occurring in from ten to fifteen
days, after the animal had passed through the stages of red and
gray hepatization characteristic of this affection in man."

Monti claims to have produced typical pneumonia in rabbits by
injecting cultures of this micrococcus into the trachea.

From the evidence obtained in these experimental inoculations,
and that recorded relating to the presence of this micrococcus in the
fibrinous exudate of croupous pneumonia, we are justified in con-
cluding that it is the usual cause of this disease, and consequently
have described it under the name Micrococcous pneumonias crou-
posae. We prefer this to the name commonly employed by German
authors — " diplococcus pneumonias " —because this micrococcus, al-
though commonly seen in pairs, forms numerous short chains of
three or four elements in cultures in liquid media, and upon the sur-
face of nutrient agar may grow out into long chains. It would,
therefore, more properly be called a streptococcus than a diplococcus.

While the micrococcus of pneumonia is not usually seen in the
blood in cases of pneumonia it is probably present in small numbers,
and secondary infection of the kidneys appears to be a common occur-
rence. Thus Frankel and Reiche (1894) report that in twenty-two
cases out of twenty-four in which they had an opportunity to exam-
ine the kidneys, this micrococcus was present. It was found espe-

BACTERIA IN CROUPOUS PNEUMONIA. 409

cially in the larger branches of the veins and arteries, but also in the
intertubular vessels and the glomeruli. The kidneys gave evidence
of degenerative changes, and it is probable that the " pneumococcus "
would have been found in the urine of some of these cases if a bac-
teriological examination had been made during life.

VII.

PATHOGENIC MICROCOCCI NOT DESCRIBED IN
SECTIONS V. AND VI.

DIPLOCOCCUS INTRACELLULARIS MENINGITIDIS.

DISCOVERED by Weichselbaum (1887) in the exudate of cerebro
spinal meningitis (six cases), for the most part within the cells.

Morphology. — Micrococci, usually united in pairs, in groups of
four, or in little masses ; sometimes solitary and larger (probably
being upon the point of dividing). Distinguished by their presence
in the interior of pus cells in the exudate, in this respect resembling
the gonococcus.

Stain best with Loffler's alkaline solution of methylene blue.
Do not retain their color when treated with iodine solution (Gram's
method).

Biological Characters. — This micrococcus does not grow at the
room temperature, but upon nutrient agar an abundant development
occurs in the incubating oven. Upon the surface of agar a tolerably
luxuriant, viscid growth, which by reflected light is gray and by
transmitted light grayish- white ; along the line of puncture growth
occurs only near the surface, indicating that this micrococcus will
not grow in the absence of oxygen. Upon plates made from agar-
agar (one per cent) and gelatin (two per cent) very small colonies are
formed in the interior of the mass, and larger ones, of a grayish
color, on the surface. The former, under the microscope, are seen to
be round or slightly irregular, finely granular, and of a yellowish-
brown color. The superficial colonies have a yellowish-brown nu-
cleus, surrounded by a more transparent zone. The growth upon
coagulated blood serum is very scanty, as is that in bouillon ; no
growth occurs upon potato. This micrococcus quickly loses its power
of reproduction in artificial cultures — within six days — and should
be transplanted to fresh material at short intervals — two days.

Pathogenesis. — Mice are especially susceptible, and usually die
within forty-eight hours after inoculation. Also pathogenic for
guinea-pigs, rabbits, and dogs.

NOT DESCRIBED IN SECTIONS V. AND VI. 411

MICROCOCCUS TETRAGENUS.

First described by Gaffky (Fliigge). Obtained by Koch and
Gaffky (1881) from a cavity in the lung in a case of pulmonary
phthisis. Since found occasionally in normal saliva (three times in
fifty persons examined by Biondi), and in the pus of acute abscesses
(Steinhaus, Park, Yangel). Rather common in the sputum of phthi-
sical cases.

Morphology. — Micrococci, having a diameter of about one /*,
which divide in two directions, forming tetrads, which are enclosed
in a transparent, jelly-like envelope — especially well developed as
seen in the blood and tissues of inoculated animals. In cultures the
cocci are seen in the various stages of division, as large single cells,

syvas

Fia. 96.— Micrococcus tetragenus; section of lung of mouse, x 800. (Flugge.)

pairs of oval elements, or groups of four resulting from the trans-
verse division of these latter.

Stains quickly with aniline colors, and in preparations from the
blood of an inoculated animal the transparent envelope may also be
feebly stained. Stains also by Gram's method.

Biological Characters. — This micrococcus grows, rather slowly,
in nutrient gelatin at the ordinary room temperature, without lique-
faction of the gelatin. Upon gelatin plates small white colonies are
developed in from twenty-four to forty-eight hours, which under the
microscope, with a low power, are seen to be spherical or lemon-
shaped, finely granular, and with a mulberry -like surface. When
they come to the surface they form white, elevated, and rather thick
masses having a diameter of one to two millimetres. In gelatin
stab cultures a broad and thick white mass forms upon the surface,

412 PATHOGENIC MICROCOCCI

and along the line of puncture a series of round, milk-white or yel-
lowish masses form, which usually remain distinct, but may become
confluent. Upon the surface of agar the growth is similar to that
upon gelatin, or in streak inoculations may consist of a series of
spherical, white colonies. Upon cooked potato a thick, viscous layer
is formed of milk-white color ; the growth upon blood serum is also
abundant, especially in the incubating oven. This micrococcus is a
facultative anaerobic.

Pathogenesis. — Subcutaneous inoculation of a culture of this
micrococcus in minute quantity is fatal to white mice in from two to
six days. The animals remain apparently well for the first day or
two, then remain quiet and somnolent until death occurs. The cocci
are found in comparatively small numbers in the blood of the heart,
but are more numerous in the spleen, lungs, liver, and kidneys, from
which organs beautiful stained preparations may be made show-
ing the tetrads surrounded by their transparent capsule. Common
house mice and field mice are, for the most part, immune, as are the
rabbit and the dog. Guinea-pigs sometimes die from general infec-
tion, and sometimes a local abscess is the only result of a subcutane-
ous inoculation.

MICROCOCCUS BOTRYOGENUS (Rabe).

Synonyms. — Micrococcus of " myko-desmoids " of the horse; Mi-
crococcus askoformans (Johne) ; Ascococcus Johnei (Cohn).

First described by Bollinger (1870) ; morphological characters and
location in the diseased tissues described by Johne (1884) ; biological
characters determined by Rabe (1886).

Is found in certain diffused or circumscribed growths in the con-
nective tissue of horses — " myko-desmoids."

Morphology. — Micrococci, having a diameter of 1 to 1.5 /*, usu-
ally united in pairs.

In the tissues the cocci are united in colonies of fifty to one hun-
dred yw in diameter, and these are associated in mulberry-like masses
visible to the naked eye. The separate colonies are enclosed in a
homogeneous, transparent envelope — as in Ascococcus Billrothii.
This is not the case, however, in cultures in artificial media.

Stains with the aniline colors.

Biological Characters. — In gelatin plate cultures spherical,
sharply defined, silver-gray colonies are developed ; later these have
a yellowish color and a metallic lustre, and the plate presents the ap-
pearance of being powdered with grains of pollen. It gives off a
peculiar fruit-like odor, reminding one of the odor of strawberries.
In gelatin stab cultures growth occurs along the line of puncture as
a pale grayish- white line, which later becomes milk-white ; an air

NOT DESCRIBED IN SECTIONS V. AND VI. 413

bubble forms near the surface of the gelatin ; very slight liquefac-
tion occurs in the immediate vicinity of the line of growth, and after
a time the grayish-white thread sinks into an irregular mass, lying
at the bottom of the puncture. Upon nutrient agar scarcely any de-
velopment occurs. Upon potato the growth is abundant, in the form
of a pale-yellow, circular layer, and the culture gives off the peculiar
odor above described.

Pathogenesis. — When inoculated into guinea-pigs general infec-
tion and death result. In sheep and goats it produces a local in-
flammatory oedema and sometimes necrosis of the tissues. In horses
inoculated subcutaneously an inflammatory oedema first occurs, fol-
lowed at the end of from four to six weeks by the development of new
growths in the connective tissue, resembling the tumors found in
cases of the disease in the animal from which the micrococcus in
question was first cultivated. These tumors contain characteristic
mulberry-like conglomerations of colonies made up of the coccus.

MICROCOCCUS OF MANFREDI.

Synonym. — Micrococcus of progressive granuloma formation.

Obtained by Manfredi (1886) from the sputum of two cases of
croupous pneumonia following measles.

^[orphology. — Oval micrococci, having a diameter of 0.6 to 1.0 /*
and from 1.0 to 1.5 /I in length ; usually associated in pairs, and oc-
casionally in short chains containing three or four elements.

Stains with the aniline colors and by Gram's method.

Biological Characters. — Aerobic ; does not liquefy gelatin.
Upon gelatin plates forms small, spherical colonies, at first grayish-
white, which spread out upon the surface as thin, transparent plates,
which by transmitted light have a bluish, by reflected light a pearl-
gray color. Later these become thicker and have a pearly lustre.
Under the microscope (forty to fifty diameters) the colonies are seen
to be slightly granular and the margins have an irregular outline.
In gelatin stab cultures a scanty growth occurs along the line of
puncture, and a rather thin and limited growth about the point of
inoculation. Upon blood serum a thin, greenish-yellow layer, which
has irregular margins and a slightly granular, shining surface, is
developed. The growth upon potato, at 37° C., is scanty, and con-
sists of a very thin, moist layer, which has a yellowish color and io
slightly granular. Growth occurs in favorable media — bouillon,
gelatin — at temperatures of 18° to 48° C., but ceases at a temperature
of 48° to 50° C.

Pathogenesis. — Pathogenic for dogs, rabbits, guinea-pigs, mice,
and birds. In mammals the principal pathological appearance re-
sulting from infection consists in the formation of " granulation tu-

414 PATHOGENIC MICROCOCCI

mors " in the parenchymatous organs. These vary in size from that
of a millet seed to that of a pea, and undergo caseation. They con-
tain the micrococcus and are infectious. Mammals die in from nine
to fifteen days ; birds in from one to three or four, and without the
formation of the characteristic granuloma, but with general infec-
tion of the blood. Cultures which have been kept for several months
retain their pathogenic power.

MICROCOCCUS OF BOVINE MASTITIS (Kitt).

Obtained by Kitt (1885) from the udder of cows suffering from mastitis
and giving- milk mixed with pus.

Morphology. — Micrococci, having a diameter of 0.2 to 0.5 >u, solitary,
united in pairs, in irregular groups, and occasionally in chains.

Stains with the aniline colors.

Biological Characters. — Does not liquefy gelatin. Upon gelatin plates
forms spherical, translucent, glistening colonies, the size of a hemp seed to
that of a pin's head ; in gelatin stab cultures a nail-shaped growth occurs,
the mass at the point of puncture being opaque and of a white color. Upon
potato, colonies are quickly developed which have a grayish -white or dirty
yellow color, and after a few days have a shining, wax-like appearance.
G-rows rapidly in milk, causing an acid reaction ; in six hours in the incu-
bating oven the milk is pervaded by the micrococcus, or in twelve hours at
20° C.

Pathogenesis. — Injection of pure cultures, suspended in distilled water,
into the mammary glands of cows, produces typical, acute, purulent mas-
titis (Kitt). The micrococcus produced the same result after having been
cultivated in artificial media for a year. Subcutaneous inoculations in cows,
pigs, guinea-pigs, rabbits, and mice were without result. Injections into
the mammary gland of goats were also without effect.

MICROCOCCUS OF BOVINE PNEUMONIA (?).

Isolated by Poels and Nolen (1886) from the lungs of cattle suffering
from ' ' Lungenseuche " (infectious pleuro-pneumonia of cattle).

Morphology. — Micrococci, varying considerably in size — average dia-
meter 0.9 #; solitary, in pairs, or in chains containing several elements; sur-
rounded by a transparent capsule, which stains with difficulty.

Stains with all the aniline colors, and with difficulty by Gram's method.

Biological Characters. — Does not liquefy gelatin, and grows like the ba-
cillus of Friedlander in gelatin stab cultures (nail-shaped growth). In gela-
tin plates the colonies are spherical, white, and have a very faint yellowish
tinge. Grows more rapidly on agar in the incubating oven, and upon po-
tato in the form of a very pale-yellowish layer. Is destroyed by a tempera-
ture of 66° C. maintained for fifteen minutes.

Pathogenesis — Pure cultures injected into the lungs of dogs, rabbits,
and guinea-pigs are said to give rise to pneumonic inflammation, and simi-
lar results were obtained by injection into the trachea of dogs and by in-
halation experiments. Injection of a pure culture into the lungs of a cow
caused extensive pneumonic changes ; but these did not entirely correspond
with the appearances found in the lungs of cattle suffering from infectious
pneumonia. Cattle inoculated with a pure culture, by means of a sterilized
lancet, did not fall sick, but are believed by Poels and Nolen to have been
protected from the disease by such inoculations.

The specific relation of the micrococcus above described to the disease
with which it was associated, in the researches of the authors mentioned, has
not been established by subsequent investigations.

NOT DESCRIBED IN SECTIONS V. AND VI. 415

STREPTOCOCCUS SEPTICUS (Flugge).

Found by Nicolaier and by Guameri in unclean soil during researches
made in Fliigge?s laboratory in Gottingen.

Morphology. — Cannot be distinguished from Streptococcus pyogenes, but
does not so constantly form chains, being found in the tissues of inoculated
animals, for the most part in pairs.

Biological Characters. — Grows more slowly than Streptococcus pyogenes ;
in gelatin plates very minute colonies first appear at the end of three or four
days, or along the line of puncture in gelatin stick cultures after five or six
days. Does not liquefy gelatin.

Pathogenesis. — Is very pathogenic for mice and for rabbits, causing death
from general infection in two or three days.

STREPTOCOCCUS BOMBYCIS.

Synonym. — Microzyma bombycis (Bechamp).

Found in the bodies of infected silkworms suffering from la flacherie
(maladie des morts-plats). Etiological relation established by Pasteur.

Morphology. — Oval cells, not exceeding 1.5 /* in diameter, in pairs or in
chains.

Biological Characters. — Not determined with precision.

Pathogenesis. — The infected silkworm ceases to eat, becomes weak, and
dies. Its body is soft and diffluent, and at the end of twenty-four to forty-
eight hours is filled with a dark-brown fluid and with gas.

NOSEMA BOMBYCIS.

Synonyms. — Micrococcus ovatus; Panhistophyton ovatum.

Found in the blood and all of the organs of silkworms infected with
pebriiie (Fleckenkrankheit).

First observed by Cornalia. Etiological relation established by Pasteur.

Morphology. — Shining, oval cells, three to four # long and twoy" broad;
solitary, in pairs, or in irregular groups.

Biological Characters. — Not determined with precision.

Pathogenesis. — Dark spots appear upon the skin of infected silkworms,
which lose their appetite, become slender and feeble, and soon die. The
oval corpuscles are found in all of the organs, and also in the eggs of
butterflies hatched from infected larvae. Some authors are of the opinion
that the oval corpuscles found in this disease do not belong to the bacte-
ria, but to an entirely different class of microorganisms — the Psorospermia
(Metschnikoff).

MICROCOCCUS OF HEYDENREICH.

Synonyms. — Micrococcus of Biskra button — Fr. " clou de Biskra "; Ger.
" Pendesche Geschwur."

Found by Heydenreich (1888) in pus and serous fluid obtained from the
tumors and ulcers in the Oriental skin affection known as Biskra button.

Morphology. — Diplococci, from 0.86 to 1 /* in length, surrounded by a
capsule ; sometimes associated to form tetrads.

Stains with the usual aniline colors.

Biological Characters. — An aerobic, liquefying micrococcus. Grows in
the usual culture media at the room temperature. In gelatin stick cultures,
at 20° C., at the end of forty-eight hours growth occurs along the line of
puncture in the form of small, crowded colonies, which produce a grayish-
white line ; upon the surface a thin, circular layer of a yellowish-white
color is developed. At the end of three to four days liquefaction commences
near the surface, where a funnel is formed which extends until about the
fourteenth day, when the gelatin is completely liquefied. Upon the surface

416 PATHOGENIC MICROCOCCI

of agar, at 37° C., a grayish- white or yellowish layer is formed at the end of
twenty-four hours, which has a varnish-like lustre. Upon potato, at 30° to
35° C., at the end of forty-eight hours a white or yellow layer has de-
veloped.

Pathogenesis. — According to Heydenreich, inoculations in rabbits, dogs,
chickens, horses, and sheep cause a skin affection which is identical with
that which characterizes Biskra button in man. When rubbed into the
healthy skin of man it also produces the development of abscesses.

MICROCOCCUS ENDOCARDITIDIS RUGATUS (Weichselbaum).

Obtained by Weichselbaum (1890) from the affected cardiac valves in a
fatal case of ulcerative endocarditis.

Morphology. — Micrococci, resembling the staphylococci of pus in dimen-
sions and mode of grouping; solitary, in pairs, in groups of four, or in ir-
regular masses.

Biological Characters.- — An aerobic micrococcus. Does not grow at the
room temperature. Upon agar plates, at 37° C. , at the end of three or four
days the superficial colonies consist of a small, brown, central mass sur-
rounded by a granular, semi- transparent, grayish marginal zone; gradually
they attain a characteristic wrinkled appearance ; the deep colonies, under a
low power, are irregular, finely granular, and contain a large central, yel-
lowish-brown nucleus surrounded by a narrow, grayish-brown peripheral
zone. In agar stab cultures small, spherical colonies are formed upon the
surface, which become confluent, forming a grayish-white, wrinkled layer
which has a stearin-like lustre and is very viscid ; a scanty growth occurs
along the line of puncture. Upon potato, at 373 C , a scanty development
occurs in the form of a small, dry, pale-brown mass. Upon blood serum
isolated or confluent, colorless colonies are formed the size of a poppy seed ;
these are closely adherent lo the surface of the culture medium.

Pathogenesis.- — Wben injected subcutaneously into the ear of a rabbit it
produces tumefaction and redness; in guinea-pigs, formation of pus. When
injected into the circulation of dogs, after injury to the aortic valves, an en-
docarditis is developed.

MICROCOCCUS OF GANGRENOUS MASTITIS IN SHEEP.

Obtained by Nocard (1887) from the milk of sheep suffering from gan-
grenous mastitis (rnal de pis or d'araignee), a fatal disease which attacks
especially sheep which are being milked for the manufacture of cheese, at
Roquefort and elsewhere in France.

Morphology. — Micrococci, solitary, in pairs, or in irregular groups, resem-
bling the staphylococci of pus in dimensions and arrangement.

Stains with the usual aniline colors and also by Gram's method.

Biological Characters. — An aerobic and facultative anaerobic, liquefy-
ing micrococcus. Grows at the room temperature in the usual culture me-
dia. Upon gelatin plates, at the end of forty-eight hours, the colonies are
spherical and white in color; under a low power the superficial colonies are
circular in outline, homogeneous, and brown in color ; they are surrounded
by a semi-transparent aureole ; liquefaction around the superficial colonies
occurs sooner than around those beneath the surface of the gelatin. In i
gelatin stick cultures, at 18° to 20° C., on the second day liquefaction of the!
gelatin commences near the surface ; by the fifth day a pouch of liquefied '
gelatin has formed, which has the shape of an inverted cone; at the bottom
of this an abundant deposit of micrococci is seen, while the liquefied gela-
tin above is clouded throughout. In agar stick cultures development oc-
curs upon the surface as a thick white layer, which gradually extends
over the entire surface, and after a time acquires a yellowish tint ; develop-
ment also occurs along the line of puncture. Upon potato a thin, viscid,
grayish layer is slowly developed ; the outline is irregular and the edges
thicker than the central portion ; the central portion of this layer gradually

NOT DESCRIBED IN SECTIONS V. AND VI.

417

acquires a yellow color, while the periphery remains of a dirty-white or
grayish color. Blood serum is liquefied by this micrococcus.

Pathogenesis. — A few drops of a pure culture injected subcutaneously or
into the mammary giand of sheep cause an extensive inflammatory oedema
and the death of the animal in from twenty-four to forty-eight hours. A
cubic centimetre injected into the mammary gland of a goat produced 110 re-
sult ; the horse, the calf, the pig, the cat, chickens, and guinea-pigs also proved
to be immune. Subcutaneous injections in rabbits produce an extensive ab-
scess at the point of inoculation.

STREPTOCOCCUS OF MASTITIS IN COWS.

Obtained by Nocard and Mollereau (1887) from the milk of cows suffering
from a form of chronic mastitis (mammite coiitagieuse) .

Morphology. — Spherical or oval cocci, a little less than one // in diameter,
usually united in long chains.

Stains with the usual aniline colors and also by Gram's method.

Biological Characters. — An aerobic and facultative anaerobic, non-
liquefying streptococcus. Grows in the usual culture media at the room
temperature. Develops rapidly in milk or in bouillon at a temperature of
16° to 30" C. The milk of a cow suffering from the form of mastitis produced
by this micrococcus, when drawn with proper precautions in sterilized test
tubes, at the end of twenty four hours is acid in reaction; the lower two-
thirds of the tube is filled with an opaque, dirty- white, homogeneous deposit,
and above this is a,n opalescent, serous fluid of a bluish or dirty-yellow or
slightly reddish color, according to the age of the lesion. A drop of this
milk examined under the microscope shows the presence of the streptococcus
in great numbers. The addition of two to five per cent of glucose or of gly-

Fio. 97.— Streptococcus of mastitis in cows (Nocard).

eerin to bouillon makes it a more favorable culture medium ; the reaction
should be neutral or slightly alkaline, as this streptococcus does not grow
readily in an acid medium, although it produces an acid reaction in media
containing sugar, the acid formed being lactic. In gelatin stab cultures the
growth upon the surface is scanty, in the form of a thin pellicle around the
point of puncture ; along the line of inoculation minute, opaque, granular
colonies are developed, which, being closely crowded, form a thick line with
jagged margins.

In agar stab cultures the growth is similar but more abundant. Upon
the surface of nutrient gelatin, agar, or blood serum a large number of mi-
27

418 PATHOGENIC MICROCOCCI

nute, spherical, semi-transparent colonies are developed among- the impfstrich ;
these have a bluish tint by reflected light ; they may become confluent, form-
ing a thin layer with well-defined margins. Upon gelatin plates, at 16° to
18° C., colonies are first visible at the end of two or three days; they are
spherical and slightly granular, at first transparent and later of a pale-yellow
color by transmitted light, which gradually becomes brown. At the end of
five or six weeks the colonies are still quite small, well defined, and opaque.
Pathogenesis. — Pure cultures injected into the mammary gland of cows
and goats gave rise to a mastitis resembling in its development that from
which the streptococcus was obtained in the first instance. Injections into
the cavity of the abdomen or into a vein, of one cubic centimetre of a pure
culture, gave a negative result in dogs, cats, rabbits, and guinea-pigs.

DIPLOCOCCUS OF PNEUMONIA IN HORSES.

Obtained by Schiitz (1887) from the lungs of horses affected with pneu-
monia.

Morphology. — Oval cocci, usually in pairs, surrounded by a homogene-
ous, transparent capsule.

Does not stain by Gram's method.

Biological Characters. — An aerobic, non-liquefying micrococcus. Grows
at the room temperature. Upon gelatin plates forms small, spherical, white
colonies.

In gelatin stick cultures grows along the line of puncture as small, white,
separate colonies, which grow larger without becoming confluent. Upon
the surface of agar small transparent drops are developed along the impf-
strich.

Pathogenesis. — The injection of a pure culture into the lung of a horse
produces pneumonia and causes its death in eight or nine days. Pathogenic
for rabbits, guinea-pigs, and mice.

STREPTOCOCCUS CORYZ^E CONTAGIOS.E EQUORUM.

Obtained by Schiitz (1888) from pus from the lymphatic glands involved
in horses suffering from the disease known in. Germany as Druse des
Pferdes.

Morphology. — Oval cocci, in pairs, in chains containing three or four
elements, or in long chaplets.

Stains with the usual aniline colors — very intensely with Weigert's or
Ehrlich's solution.

Biological Characters. — An aerobic and facultative anaerobic micrococ-
cus. Grows slowly at the room temperature, more rapidly at 37' C. Upon
gelatin plates at the end of three to five days minute colonies become visible ;
these never exceed the size of a pin's head. In gelatin stab cultures growth
upon the surface is scanty or absent; along the line of puncture minute
colonies are developed in rows. Upon agar plates, at 37° C., at the end of
twenty four hours lentil-shaped colonies are developed the size of a pin's
head ; under a low power the superficial colonies are seen to have a well-de-
fined, opaque nucleus surrounded by a grayish, transparent marginal zone,
which represents a half -fluid, slimy growth which does not extend after the
third day and later disappears entirely; the deep colonies are at first well-
defined, and later surrounded by wing-like outgrowths. Upon blood serum,
at 37° C., yellowish, transparent drops are first developed; these become con-
fluent and form a viscid and tolerably thick layer; this later becomes dry
and iridescent.

Pathogenesis. — Pathogenic for horses and for mice, producing in these
animals an abscess at the point of inoculation, and metastatic abscesses in
the neighboring lymphatic glands. Not pathogenic for rabbits, guinea-pigs,
or pigeons.

NOT DESCRIBED IN SECTIONS V. AND VL 419

HJEMATOCOCCUS sovis (Babes).

Obtained by Babes (1889) from the blood and various organs of cattle
which had died of an epidemic malady (in Roumaiiia) characterized by heemo-
globinuria. The cocci are found in the blood in great numbers, for the most
part enclosed in the red corpuscles.

Morphology. — Biscuit-shaped cocci united in pairs; sometimes oblong in
form, isolated or united in groups ; the free cocci are surrounded by a pale-
yellowish, shining aureole of 0.5 to 1 ja in diameter.

Stains best with Loffler's solution of methylene blue ; does not stain by
Gram's method.

Biological Characters. — An aerobic and facultative anaerobic, non-
liquefying micrococcus. Grows very slowly at the room temperature — not
below 20 J C. In the incubating oven grows in the usual culture media. In
gelatin stab cultures a scanty development of small, white colonies occurs
along the line of puncture. Upon the surface of agar small, transparent
drops are developed along the impfstrich. Upon potato, at 37° C.. a thin,
broad, yellowish, shining layer is developed in the course of a few days —
scarcely visible. Upon blood serum small, moist, transparent colonies are
developed.

Pathogenesis. — Pathogenic for rabbits and rats, which die in from six to
ten days after inoculation with a pure culture; the spleen is found to be en-
larged, the lungs hyperaemic, and a bloody serum is found in the cavity of
the abdomen; the cocci are present in the blood inconsiderable numbers,
but are rarely seen in the red corpuscles. Inoculations in oxen, horses,
goats, sheep, guinea-pigs, and birds were without effect.

STREPTOCOCCUS PERNICIOSUS PSITTACORUM.

Micrococcus of gray pirrot disease. Eberth and Wolff have described
an infectious disease of gray parrots, which is said to be extremely fatal
among the imported birds. The disease is characterized by the formation of
nodules upon the surface and in the interior of various organs, and especially
in the liver. Micrococci of medium size are found in these nodules and in
blood from the heart; these are sometimes in chains. Microscopic examina-
tion of stained sections shows that these cocci are directly related to the tis-
sue necrosis which characterizes the disease. But the micrococcus has not
been cultivated and its biological characters are undetermined.

STREPTOCOCCUS AGALACTI^ CONTAGIOS^E.

Obtained by Adametz (1894) from the milk of cows suffering from mas-
titis (Gelben Gait). According to Adametz all of the streptococci which
have been described by different investigators (Kitt, Nocard and Mollereau,
Guillebeau, and others) are probably varieties of a single species.

Morphology. — Spherical cocci in short chains — \ p in diameter.

Biological Characters. — An aerobic and facultative anaerobic, non-
liquefying streptococcus.

Upon gelatin plates forms flat, transparent, white or bluish-white,
slimy colonies, having a slight pearly lustre and an irregular outline. In
nutrient gelatin containing five per cent of milk sugar the colonies, at the
end of eight days, have a diameter of 0.85 to 1 millimetre; they are milk-
white and of a semi-fluid, slimy consistence.

Upon agar plates the deep colonies are jmnctiform and white in color-
under a low power they are seen to have an irregular dentate contour and a
brownish color; the superficial colonies gradually assume the appearance
of transparent, flat drops having a diameter of 0.5 to 0.7 millimetre. In
sterilized milk fermentation occurs, at 37 D C., in from twenty to twenty-four

420 PATHOGENIC MICROCOCCI

hours ; some hours later the casein is precipitated, fine gas bubbles are seen
in the lower part of the fluid and a foam upon the surface; the reaction is
acid and the casein is not peptonized. The power of producing acid and gas
is diminished or lost after a few successive cultures have been made.

Streptococcus mastitis sporadic^ (Guillebeau) is said by Adametz to be
distinguished from the streptococcus above described (No. 44A) by being
smaller — 0.5 // in diameter — and by the fact that the cultures do not lose the
power of producing fermentation in milk.

MICROCOCCUS MELITENSIS.

Surgeon-Major Bruce, of the British army, in 1887 demonstrated
the etiological relation of a micrococcus, now known as Micrococcus
melitensis, to the infectious disease known as Malta fever (syn-
onyms: Mediterranean fever; Neapolitan fever; Rock fever of Gib-
raltar, etc.). Subsequent researches show that this fever is not re-
stricted to the Mediterranean region, and it will probably be found
to have an extensive area of prevalence on both continents. Cases
have been recognized in America and by medical officers of the army
stationed in the Philippine Islands. Curry (Captain and Assistant
Surgeon United States Volunteers), in a recent report to the Surgeon-
General of the army, says :

"I had the honor to report to the Surgeon-General of the Army on Jan-
uary 2d, 1900, four cases of Mediterranean or Malta fever, which came under
my observation, while on duty as pathologist to the 1st Reserve Hospital in
Manila, P. I., cases occurring among our troops and originating on the
Island of Luzon.

"Later, in a report on the ' Diseases of the Philippine Islands,' I reported
twelve additional cases. In all these cases a positive serum reaction with
the Micrococcus melitensis was obtained, and the clinical history of the cases
corresponds with the descriptions of Malta fever as given by the English
army surgeons Bruce, Hughes, Wright, Semple, and others, and that de-
scribed by Manson. Included in these sixteen cases is one autopsy.

"In my report on the ' Diseases of the Philippine Islands,' under the head-
ing of 'Fevers of the Philippines,' I expressed the belief that 'Malta fever
is not an uncommon disease in the Philippine Islands,' and that it appeared
that 'Malta fever is by no means as limited geographically as has been
thought heretofore.'

"Our experience here in the Army and Navy General Hospital, Hot
Springs, Ark. , has convinced me that Malta fever is widespread in tropical
and sub-tropical regions. We are but having a repetition of the experience
of the English army surgeons at the Royal Victoria Hospital, Netley.

"Among the soldiers and sailors, here in our wards, who have been
returned from tropical stations, we have found already four to have Malta
fever. These four cases came from widely separated stations. Two cases
are in soldiers, one from the Philippines, and one from Cuba, and two are
among sailors of the United States navy who were recently returned from
South Atlantic stations.

"All four cases entered this hospital with a diagnosis of rheumatism."

Morphology. — Micrococci, about 0.5 i*. in diameter, usually soli-
tary or in pairs ; occasionally short chains are seen in cultures. In

NOT DESCRIBED IN SECTIONS V. AND VI. 421

old cultures kept at the room temperature the cells may be oval or
elongated.

Biological Characters. — An aerobic, non-liquefying micro-
coccus. Does not stain by Gram's method. Grows best in nutrient
agar. In stab cultures no growth is seen for several days. "At
length the growth appears as pearly-white spots scattered around the
point of puncture and minute, round, white colonies are also seen
along the course of the needle track"; these increase in size, and
after some weeks a rosette-shaped growth is seen upon the surface,
and the growth along the line of puncture has a yellowish-brown
color. At the end of nine or ten days, at 37° C., some of the colonies
on the surface of nutrient agar are as large as No. 4 shot; by trans-
mitted light they have a yellowish color at the centre, and the per-
iphery is bluish-white; by reflected light they have a milky- white
color. At 25° C. colonies first* become visible at the end of about
seven days, at 37° C. in three to four days. Does not grow upon
potato. Very scanty growth upon nutrient gelatin at 22° C. at the
end of a month.

This micrococcus has usually been described as non-motile, but
Gordon has demonstrated that it has from one to four flagella, which
are difficult to demonstrate by the usual staining methods.

Pathogenesis. — Pathogenic for monkeys, which suffer from fever
as a result of subcutaneous inoculations and usually die in from thir-
teen to twenty-one days. The spleen is found to be enlarged and
contains the micrococcus. Not pathogenic for mice, guinea-pigs,
or rabbits.

In man the micrococcus is found in large numbers in the spleen,
which is greatly enlarged.

Widal Reaction. — The blood serum of patients suffering from
Malta fever and of individuals who have recently recovered from the
disease causes the agglutination of Micrococcus melitensis in recent
cultures. According to Wright and Smith this reaction may be
manifested a year after recovery. Dilution of 1 : 1000 will in ex-
ceptional cases give a distinct agglutinating effect.

VIII.

THE BACILLUS OF ANTHRAX.
[Fr., CHARBON ; Ger., MILZBRAND.]

ANTHRAX is a fatal infectious disease which prevails extensively
among sheep and cattle in various parts of the world, causing heavy
losses. In Siberia it constitutes a veritable scourge and is known
there as the Siberian plague ; it also prevails to a considerable extent
in portions of France, Hungary, Germany, Persia, and India, and
local epidemics have occasionally occurred in England, where it is
known under the name of splenic fever. It does not prevail in the
United States. In infected districts the greatest losses are incurred
during the summer season.

In man accidental inoculation may occur among those who come
in contact with infected animals, and especially during the removal of
the skin and cutting up of dead animals, when there is any cut or
abrasion upon the hands. A malignant pustule is developed as the
result of such inoculation, but, as a rule, general infection does
not occur, as is the case when inoculations are made into the more
susceptible lower animals — rabbit, guinea-pig, mouse. Those who
handle the hair, hides, or wool of infected animals are also liable to
contract the disease by inoculation through open wounds, or by the
inhalation of dust containing spores of the anthrax bacillus. Cases
of pulmonic anthrax, known formerly in England as "wool-sorters'
•disease," have been occasionally observed in England and in Ger-
many, and are now recognized as being due to infection through the
lungs in the manner indicated.

The French physician Davaine, who had observed the anthrax
bacillus in the blood of infected animals in 1850, communicated to
the French Academy of Sciences the results of his inoculation experi-
ments in 1863 and 1864, and asserted the etiological relation of the
bacillus to the disease with which his investigations showed it to be
constantly associated. This conclusion was vigorously contested by
conservative opponents, but has been fully established by subsequent
investigations, which show that the bacillus, in pure cultures, induces

THE BACILLUS OF ANTHRAX. 423

anthrax in susceptible animals as certainly as does the blood of an
animal recently dead from the disease.

Owing to the fact that this was the first pathogenic bacillus cul-
tivated in artificial media, and to the facility with which it grows in
various media, it has served more than any other microorganism for
researches relating to a variety of questions in pathology, general
biology, and public hygiene, some of which are discussed in other
sections of this volume.

BACILLUS ANTHRACIS.

Synonyms. — Milzbrandbacillus, Ger.; Bacteridie du charbon, Fr.

First observed in the blood of infected animals by Pollender (1849)

and by Davaine (1850). Etiological relation affirmed by Davaine

FIG. 98.— Bacillus anthracis, from a culture, showing development of long threads in convo-
luted bundles, x 300. (Klein.)

(1863), and established by the inoculation of pure cultures by Pasteur
(1879) and by many other investigators.

Morphology. — Rod-shaped bacteria having a breadth of 1 to
1.25 /*, and 5 to 20 ^ in length; or, in suitable culture media, growing-
out into long, flexible filaments, which are frequently united in
twisted, cord-like bundles. These filaments in hanging-drop cul-
tures, before the development of spores, appear to be homogeneous ;
or the protoplasm is clouded and granular, but without distinct seg-
mentation. But in stained preparations the filaments are seen to be
made up of a series of rectangular, deeply stained segments. In
hanging-drop cultures the ends of the rods appear rounded, but in
stained preparations from the blood of an infected animal they are
seen to present a slight concavity, and a lenticular interspace is
formed where two rods come together. The diameter of the rods

424

THE BACILLUS OF ANTHRAX.

varies considerably in different culture media; and in old cultures
irregular forms are frequently seen — "involution forms."

Under favorable conditions endogenous spores are developed in
the long filaments which grow out in artificial culture media.
These first appear as refractive granules distributed at regular inter-
vals in the segments of the protoplasm, which gradually disappear
as the spores are developed ; and these are left as oval, highly re-
fractive bodies, held together in a linear series by the cellular enve-
lope, and subsequently set free by its dissolution. The germination
of these reproductive bodies results in the formation of rods and
spore-bearing filaments like those heretofore described. In this pro-
cess the spore is first observed to
lose its brilliancy, from the ab-
sorption of moisture, a promi-
nence occurs at one end of the
oval body, and soon the external
envelope — exosporium — is rup-
tured, permitting the softened
protoplasmic contents enclosed
in the internal spore membrane
— endosporium — to escape as a
short rod, to which the empty
exosporium sometimes remains
attached.

The anthrax bacillus stains
readily with the aniline colors
and also by Gram's method,
when not left too long in the
decolorizing iodine solution.
Loffler's solution of methylene
blue is an especially good stain-
ing fluid for this as well as for many other bacilli. Bismarck brown
is well adapted for specimens which are to be photographed, and also
for permanent preparations, as it is less liable to fade than the blue
and some other aniline colors.

Biological Characters. — The anthrax bacillus is aerobic, but
not strictly so, as is shown by the fact that it grows to the bottom of
the line of puncture in stab cultures in solid media. It is non-mo-
tile, and is distinguished by this character from certain common
bacilli resembling it in morphology — Bacillus subtilis — which were
frequently confounded with it in the earlier days of bacteriological
investigation.

The anthrax bacillus grows in a variety of nutrient media at a

FIG. 99.— Bacillus anthracis, from a culture, show-
ing formation of spores. X 1,000. (Klein.)

THE BACILLUS OF AXTHRAX.

425

temperature of 20° to 38° C. Development ceases at temperatures
below 12° C. or above 45° C.

This bacillus grows best in neutral or slightly alkaline media, and
its development is arrested by a decidedly acid reaction of the cul-
ture medium. It may be cultivated in infusions of flesh or of vari-
ous vegetables, in diluted urine, in milk, etc.

In gelatin plate cultures small, white, opaque colonies are devel-
oped in from twenty-four to thirty-six hours, which under the micro-
scope are seen to be somewhat irregular in outline and of a greenish
tint ; later the colonies spread out upon the surface of the gelatin,
and the darker central portion is surrounded by a brownish mass of
wavy filaments, which are associated in tangled bundles. Mycelial-
like outgrowths from the periphery of
the colony may often be seen extending
into the surrounding gelatin. At the
end of two or three days liquefaction of
the gelatin commences, and the colony
is soon surrounded by the liquefied me-
dium, upon the surface of which it floats
as an irregular white pellicle. In gela-
tin stab cultures growth occurs all
along the line of puncture as a white cen-
tral thread, from which lateral thread-
like ramifications extend into the culture
medium. At the end of two or three
days liquefaction of the culture medium
commences near the surface, where the
development has been most abundant.
At first a pasty, white mass is formed,
but as liquefaction progresses the upper
part of the liquefied gelatin becomes
transparent from the subsidence of the
motionless bacilli, and these are seen
upon the surface of the non-liquefied
portion of the medium in the form of
cloudy, white masses, while below the line of liquefaction the charac-
teristic branching growth may still be seen along the line of puncture.

In agar plate cultures, in the incubating oven at 35° to 37° C.,
colonies are developed within twenty-four hours, which under the
microscope are seen to be made up of interlaced filaments and are
very characteristic and beautiful. Upon the surface of nutrient agar
a grayish-white layer is formed, which may be removed in ribbon-like
strips ; and in stick cultures in this medium a branching growth is
seen, like that in gelatin, but without liquefaction. The addition of

FIG. 100.— Culture of Bacillus an-
thracis in nutrient gelatin : a, end
of four days ; 6, end of eight days.
( Baumgarten.)

426

THE BACILLUS OF ANTHRAX.

a small quantity of agar to a gelatin medium prevents liquefaction
of the gelatin (Fliigge).

Upon blood serum a rather thick, white layer is formed and
liquefaction slowly occurs.

Upon potato the growth is abundant as a rather dry, grayish-
white layer, of limited extent, having a somewhat rough surface and
irregular margins.

Spores are formed only in the free presence of oxygen, as in sur-
face cultures upon potato or nutrient agar, or in shallow cultures in
liquid media, and at a temperature of 20° to 35° C. They are not
formed during the development of the bacilli in the bodies of living

FIG. 101.— Colonies of Bacillus anthracis upon gelatin plates : a, at end of twenty-four hours;
6, at end of forty-eight hours, x 80. (Flugge.)

animals, but after the death of the animal the bacillus continues to
multiply for a time, and spores may be formed where the fluids
containing it come in contact with the air — as, for example, in
bloody discharges from the nostrils or from the bowels of the dead
animal.

Varieties incapable of spore production have been produced arti-
ficially, by several bacteriologists, by cultivating the bacillus under
unfavorable conditions. Roux was able to produce a sporeless va-
riety by successive cultivation in media containing a small quantity
of carbolic acid — 1 : 1,000.

Varieties differing in their pathogenic power may also be pro-
duced by cultivation under unfavorable conditions. Thus Pasteur

THE BACILLUS OF ANTHRAX. 427

produced an " attenuated virus" by keeping his cultures for a con-
siderable time before replanting them upon fresh soil, and supposed
the effect was due to the action of atmospheric oxygen. It seems
probable that it was rather due to the deleterious action of its own
products of growth present in the culture media. It has been
shown by Chamberlain and Roux that cultivation in the presence
of certain chemical substances added to the culture medium — e.g.,
bichromate of potassium 0.01 per cent — causes an attenuation of
virulence. The same result occurs when cultures are subjected to a
temperature a little below that which is fatal to the bacillus — 50° C.
for eighteen minutes (Chauveau); 42.5° C. for two or three weeks
(Koch). Attenuation of pathogenic virulence is also effected by cul-
tivation in the body of a non-susceptible animal, like the frog (Lu-
barsch, Petruschky); or in the blood of a rat (Behring); by exposure
to sunlight (Arloing); and by compressed air (Chauveau).

Anthrax spores may be preserved in a desiccated condition for
years without losing their vitality or pathogenic virulence when in-
oculated into susceptible animals. They also resist a comparatively
high temperature. Thus Koch and Wolffhiigel found that dry spores
exposed in dry air required a temperature of 140° C. , maintained for
three hours, to insure their destruction. But spores suspended in a
liquid are destroyed in four minutes by the boiling temperature,
100° C. (writer's determination).

The bacilli, in the absence of spores, according to Chauveau, are
destroyed in ten minutes by a temperature of 54° C.

For the action of various antiseptic and germicidal agents upon
this bacillus we must refer to the sections especially devoted to this
subject (Part Second).

Toussaint, by injecting filtered anthrax blood into animals, obtained
evidence that it contained some toxic substance which in his experi-
ments gave rise to local inflammation without any noticeable general
symptoms. More recent investigations show that a poisonous sub-
stance is formed during the growth of the anthrax bacillus, and that
cultures containing this toxin, from which the bacilli have been re-
moved by filtration through porcelain, produce immunity when in-
jected into susceptible animals, similar to that resulting from inocu-
lations with an attenuated virus. It is probable that the pathogenic
power of the anthrax bacillus depends largely upon the presence of
this toxin, and that the essential difference between virulent and
attenuated varieties depends upon the more abundant production of
this toxic substance by the former. It has also been shown that
virulent cultures produce a larger quantity of acid than those which
have been attenuated by any of the agencies above mentioned
(Behring).

TIIK. BACILLUS OF \\TIM;\X.

r<ll.ho<J<>iw»ix. — The anthrax hacillus is pat hogenic for cattle,
sheep, horses, rahhits, guinea-pigs, ami mice. White rats, dogs, and
frogs are immune, as is also the Algerian race of sheep. The spar-
row is suseepfihle to general infection, hut chickens, under normal
conditions, aw not. Young animals a. re, as a rule, more susceptihle
tliaii adults of the same species. Man does not hclong among the
most, susceptihle animals, hut is suhjecf to local infection as a, result
of accidental inoculation— malignant pustule — and to pulmonic an-
thrax from hrcathing air, containing spores of the anthrax hacillus,
(I iii-ing the sorting of wool or hair from infected animals. In animals
which have a, partial immunity, natural or acquired, as a result, of
inoculations with attenuated virus, the suhcutaneous introduction of
virulent cultures may give rise to a limited local inflammatory pro-
cess, with effusion of hloody serum in which the hacillus is found in
considerable numbers ; but the blood is not invaded, ami the animal,
after some slight symptoms of indisposition, recovers. In susceptihle

I i<;. Ktt. -HacilluH unthracitf In liver of mouse. X 700. (Fl&KKtJ.)

animaJs injections henea.tli the skin or into a vein tfive risc^ to general
infection, and the bacilli multiply rapidly in the circulating fluid.
Death occurs in mice within twenty-four hours, and in rabbits, as a
rule, in less than forty-eight hours. The blood of the heart and
large vessels maybe found, in an autopsy made immediately after
death, to contain mmparatively few ha.cilli ; hut in the capillaries of

the various organs, and especially in the great I y enlarged spleen, in
the liver, the kidneys, and the lungs, they will he found in great
numbers, and well t;iined sections of these organs will give an as-
tonishing picture under the microscope, which the student should not
fail to see in preparations made by himself. The capillaries in many
places will he found stulTed full of ha.cilli ; or they may even he nip-

mi: n ACILI.I s OK \\ INK \ \ 1\".»

lured MS a result of the distent inn, and tin* haeilli, tovvl her \\illi
escaped hlood corpuscles, will ho seen ill the surround in:-, ti n- In

llio kidneys tho i;-|oincmli, especially, appear MS if iujoctod \\illt «-o|
«'ivd threads, and hy rupture lliese may lind Iheir \\ a \ ml.. I he inini
ferous tuhulos.

These appearances Mild the tfoiioml symptoms indicate- Mi.,1 llio
disease produced hy (he introduction of this haeillu in!<> (lie hndie-i <>l
suseeplihle animals is a . ^ouuino H(»pl ioa'MUM. AM in olhor I'onn-* of
HO])li<gM'inia; 1 he spleen is ft mi id l« > he ;•, i • .il I \ « nl n ••. •<! ; M has a dark
e«.|oi- and is sofl and I'riahle. \\'ilh tin exception theorem pie
KOI 1 1 no not;ihlo olia 1 1 •< all IK >uj;'li I h< h \ er is a pi I < > he sol now lull
enlarged. In llio guinea piv; an exteir i\e i nllanillUil< n \ <edi m.i. e\
lei id in:'; In >m 1 he point of inocilhtt ion l< » I I n m. . I d(>pendoiil pa i I « •!
the hodv, is developed ; (he snhenlanei.iis e« .niieel i \'< ti ne i mlil
Iraled will) hloi idy serum an<l IIMM u }vla 1 1 IIOIIM upptMi ranee Tin
animal comes IM-.X! hi Hie IIH.IISC ill HUHCopI ihihl \ . and culliires \vhhh

IK', liwlllir ink Mir,.

atlennated t ex lent t halt hey will not, kill a rahhit oj a

-heep may still kill ag'in n< a pi# ; or, if not. may I-. ill ;i moilHO. PaHfenr
haB nhown that the pathogenic power of the h;willuH may h« «< iah
linh^d hy inoculations into n < -ephhle animals, and that an allenn
at<-(| cult un; whicli will not kill an adult guinea pi;' ma;, h< fatal lo
ery yf^UJJl/ UnJJIia,! of thlhi Kpeeje:^ a nd that culture'-' I roll) the hlood
of tlliH will li;i . in'Tea-e*| palh'/rM-uir- vii ulef)r , .

\''-ry ruiuutf' fjUantiti< <>\ . « nt ' -uh n • nfalhhl •, lalal to

r/jo ^ 'i '-f;ptihl<} aniinalH, hut for ral>hitH and other |< :
cr-j/tihhj BIiirnalH th(i <juantity injeeier) inlluefjee;, t),( r-e:-ull, and M

430 THE BACILLUS OF ANTHRAX.

covery may occur after subcutaneous or intravenous injection of a
very small number of bacilli.

Infection in cattle and sheep commonly results from the ingestion
of spores while grazing in infected pastures. The bacillus itself, in
the absence of spores, is destroyed in the stomach. While spores are
not formed in the bodies of living animals, their discharges contain
the bacillus, and this is able to multiply in them and to form spores
upon the surface of the ground when temperature conditions are
favorable. It is probable that this is the usual way in which pastures
become infected, and that the bloody discharges from the bladder
and bowels of animals suffering from the disease furnish a nidus for
the external development of these reproductive elements ; as also do
the fluids escaping from the bodies of dead animals. And possibly,
under specially favorable conditions, the bacillus may lead a sapro-
phytic existence for a considerable time in the superficial layers of the
soil.

Buchner has shown by experiment that infection in animals may
result from respiring air in which anthrax spores are in suspension
in the form of dust ; and in man this mode of infection occurs in the
so-called wool-sorters' disease.

The question of the passage of the anthrax bacillus from the
mother to the foetus in pregnant females has received considerable
attention. That this may occur is now generally admitted, and ap-
pears to be established by the investigations of Strauss and Chamber-
lain, Morisani, and others. That it does not always occur is shown,
however, by the researches of other bacteriologists, and especially by
those of Wolff.

Sirena and Scagliosi (1894) report, as the result of extended experi •
ments made by them, that anthrax spores may survive in distilled
water for twenty months; in moist or dry earth for two years and
nine months; in sea-water for one year and seven months; in sewage
nearly sixteen months.

Marmier (1895) has made an extended experimental research to
determine the nature of the specific toxin of the anthrax bacillus.
This he obtains from cultures, at a low temperature, in media con-
taining peptone and glycerin. It has not the reactions of an albu-
minoid body and is not destroyed by a temperature of 100° C. In
comparatively large doses it kills animals susceptible to anthrax, and
by the administration of smaller doses immunity may be established
in such animals. This toxin is contained in the bacterial cells, and
is obtained by subjecting these to the action of alcohol, or from the
filtrate when cultures are made at a low temperature in a medium
containing peptone. It has not, however, been obtained in a pure
form, and its exact nature has not been determined.

IX.
THE BACILLUS OF TYPHOID FEVER.

NUMEROUS researches support the view that the bacillus described
by Eberth in 1880 bears an etiological relation to typhoid fever —
typhus abdominalis of German authors; and pathologists have ac-
cepted this bacillus as the veritable "germ" of typhoid fever, not-
withstanding the fact that the final proof that such is the case is still
wanting.

This final proof would consist in the production in man or in one
of the lower animals of the specific morbid phenomena which char-
acterize the disease in question, by the introduction of pure cultures
of the bacillus into the body of a healthy individual. Evidently it is
impracticable to make the test upon man, and thus far we have no
satisfactory evidence that any one of the lower animals is subject to
the disease as it manifests itself in man. The experiments of
Frankel and Simmonds show, however, that this bacillus is patho-
genic for the mouse and the rabbit. We shall refer to the experi-
ments of these authors later.

Before the publication of Eberth's first paper Koch had observed
this bacillus in sections made from the spleen and liver of typhoid
cases, and had made photomicrographs from these sections. His
name is, therefore, frequently associated with that of Eberth as one
of the discoverers of the typhoid bacillus. Other investigators had no
doubt previously observed the same organism, but some of them had
improperly described it as a micrococcus. Such a mistake is easily
made when the examination is made with a low power ; even with a
moderately high power the closely crowded colonies look like masses
of micrococci, and it is only by focussing carefully upon the scattered
organisms on the outer margin of a colony that the oval or rod-like
form can be recognized.

Several observers had noted the presence of microorganisms in
the lesions of typhoid fever prior to the publication of Eberth's pa-
per, and Browicz in 1875, and Fischel in 1878, had recognized the
presence of oval organisms in the spleen which were probably identi-
cal with the bacillus of Eberth.

The researches of Gaffky (1884) strongly support the view that

432 THE BACILLUS OF TYPHOID FEVER.

the bacillus under consideration bears a causal relation to typhoid
fever. Eberth was only successful in finding the bacillus in the
lymphatic glands or in the spleen in eighteen cases out of forty in
which he searched for it. On the other hand, he failed to find it in
eleven cases of various nature — partly infectious processes — and in
thirteen cases of tuberculosis in which the lymphatic glands were
involved, and in several of which there was ulceration of the mucous
membrane of the intestine.

Koch, independently of Eberth and before the publication of his
first paper, had found the same bacillus in about half of the cases
examined by him, and had pointed out the fact that they were lo-
cated in the deeper parts of the intestinal mucous membrane, beyond
the limits of necrotic changes, and also in the spleen, whereas the
long, slender bacillus of Klebs was found only in the necrosed por-
tions of the intestinal mucous membrane.

The researches of W. Meyer (1881) gave a larger proportion of
successful results. This author confined his attention chiefly to the
swollen plaques of Peyer and follicles of the intestine which had not
yet undergone ulceration. The short bacillus which had been de-
scribed by Eberth and Koch was found in sixteen out of twenty cases
examined. The observations of this author are in accord with those
of Eberth as to the presence of the bacillus in greater abundance in
cases of typhoid which had proved fatal at an early date.

The fact that in these earlier researches the bacilli were not found
in a considerable proportion of the cases examined is by no means
fatal to the view that they bear an etiological relation to the disease.
As Gaffky says in his paper referred to :

" This circumstance admits of two explanations. Either in those
cases in which the bacillus has been sought with negative results
they may have perished collectively, before the disease process which
thev had induced had run its course ; or the proof of the presence of
bacilli was wanting only on account of the technical difficulties which
attend the finding of isolated colonies."

Gaffky's own researches indicate that the latter explanation is the
correct one.

In twenty-eight cases examined by this author characteristic
colonies of the bacillus were found in all but two. In one of these,
one hundred and forty-six sections from the spleen, liver, and kid-
neys were examined without finding a single colony, and in the other
a like result attended the examination of sixty-two sections from the
spleen and twenty-one sections from the liver. In the first of these
cases, however, numerous colonies were found in recent ulcers of the
intestinal mucous membrane, deeply located in that portion of the
tissue which was still intact. These recent ulcers were in the neigh-

THE BACILLUS OF TYPHOID FEVER. 433

borhood of old ulcers and are supposed to have indicated a relapse
of the specific process. In the second case the negative result is
thought by Gaffky to have been not at all surprising, as the patient
died at the end of the fourth week of sickness, not directly from the
typhoid process, but as a result of perforation of the intestine.

Gaffky has further shown that in those cases in which colonies
are not found in the spleen, or in which they are extremely rare, the
presence of the bacillus may be demonstrated by cultivation ; and
that, when proper precautions are taken, pure cultures of the bacil-
lus may always be obtained from the spleen of a typhoid case.
Hein has been able to demonstrate the presence of the bacillus and
to start pure cultures from material drawn from the spleen of a living
patient by means of a hypodermatic syringe. Philopowicz has re-
ported his success in obtaining cultures of the bacillus by the same
method.

The fact that a failure to demonstrate the presence of microor-
ganisms by a microscopic examination cannot be taken as proof of
their absence from an organ, is well illustrated by a case (No. 18) in
which the bacillus was obtained by Gaffky from the spleen and also
from the liver, in pure cultures ; whereas in cover-glass preparations
made from the same spleen he failed to find a single rod, and more
than one hundred sections of the spleen were examined before he
found a colony.

To obtain pure cultures from the spleen Gaffky first carefully
washes the organ with a solution of mercuric chloride, 1 : 1,000. A
long incision is then made through the capsule with a knife sterilized
by heat. A second incision is made in this with a second sterilized
knife, and a third knife is used to make a still deeper incision in the
same track. By this means the danger of conveying organisms from
the surface to the interior of the organ is avoided. From the bottom
of this incision a little of the soft splenic tissue is taken up on a ster-
ilized platinum needle, and this is plunged into the solid culture
medium, or drawn along the surface of the same, or added to lique-
fied gelatin and poured upon a glass plate. The colonies develop, in
an incubating oven, in the course of twenty-four to forty-eight hours.

Gaffky has also shown that the bacillus is present in the liver, in
the mesenteric glands, and, in a certain proportion of cases at least,
in the kidneys, in which it was found in three cases out of seven.

The appearance of the colonies in stained sections of the spleen
is shown in Figs. 104 and 105. Two colonies are seen in Fig. 104
(at a, a) as they appear under a low power — about sixty diameters.
In Fig. 105 one of the colonies is seen more highly magnified — about
five hundred diameters.

Frankel and Simmonds have demonstrated that the bacilli multi-

434

THE BACILLUS OF TYPHOID FEVER.

ply in the spleen after death, and that numerous colonies may be
found in portions of the organ which have been kept for twenty-
four to forty-eight hours before they were placed in alcohol, when
other pieces from the same spleen placed in alcohol soon after the
death of the patient show but few colonies or none at all.

This observation does not in any way weaken the evidence as to
the etiological role of the bacillus, but simply shows that dead ani-
mal matter is a suitable nidus for the typhoid germ — a fact which
has been repeatedly demonstrated by epidemiologists and insisted
upon by sanitarians.

The authors last referred to confirm Gaffky as regards the con-
stant presence of the bacillus in the spleen. In twenty-nine cases
they obtained it by plate cultures twenty-five times, and remark
that in the four cases attended with a negative result this result is

FIG. 104.

FIG. 105.

not at all surprising, inasmuch as the typhoid process had termi-
nated and death resulted from complications.

Gaffky did not succeed in obtaining cultures from the blood of
typhoid-fever patients, and concludes from his researches that if the
bacilli are present in the circulating fluid it must be in very small
numbers. He remarks that possibly the result would be different if
the blood were drawn directly from a vein instead of from the capil-
laries of the skin. Friinkel and Simmonds also report that gelatin,
to which blood drawn from the forefinger of typical cases had been
added, remained sterile when poured upon plates in the usual man-
ner— Koch's method. The blood was obtained from six different in-
dividuals, all in an early stage of the disease — the second to the
third week. A similar experiment made with blood obtained, post
mortem, from the large veins or from the heart, also gave a negative
result in every instance save one. In the exceptional case a single

THE BACILLUS OF TYPHOID FEVER. 435

colony developed upon the plate. In view of these results we are
inclined to attribute the successful attempts reported by some of the
earlier experimenters (Letzerich, Almquist, Maragliano) to accidental
contamination and imperfect methods of research. The more recent
work of Tayon does not inspire any greater confidence. This author
obtained cultures in bouillon by inoculating it with blood drawn
from a typhoid patient, and found that these were fatal, in a few
hours, to guinea-pigs, when injected into the peritoneal cavity. The
lesions observed are said to have resembled those of typhoid fever —
congestion and tumefaction of Peyer's plaques and of the mesenteric
glands, congestion of the liver, the kidneys, etc.

The presence of the bacillus of Eberth in the alvine evacuations of
typhoid patients has been demonstrated by Pfeiffer and by Frankel
and Simmonds. This demonstration is evidently not an easy mat-
ter, for while the bacilli are probably always present in some portion
of the intestine during the progress of the disease, it does not follow
that they are present in every portion of the intestinal contents. As
only a very small amount of material is used in making plate cul-
tures, and as there are at all times a multitude of bacteria of various
species in the smallest portion of faecal matter, it is not to be ex-
pected that the typhoid bacillus will be found upon every plate.
Frankel and Simmonds made eleven attempts to obtain the bacillus
by the plate method, using three plates each time, as is customary
with those who adhere strictly to the directions of the master, and
were successful in obtaining the bacillus in three instances — in two
in great numbers and in the third in a very limited number of colo-
nies.

The numerous attempts which have been made to communicate
typhoid fever to the lower animals have given a negative result in
every instance. Murchison, in 1867, fed typhoid-fever discharges to
swine, and Klein has made numerous experiments of the same kind
upon apes, dogs, cats, guinea-pigs, rabbits, and white mice, without
result. Birch-Hirschfeld, in 1874, by feeding large quantities of
typhoid stools to rabbits, produced in some of them symptoms which
in some respects resembled those of typhoid ; but these experiments
were repeated by Bahrdt upon ten rabbits with an entirely negative
result. Von Motschukoffsky met with no better success in his at-
tempts to induce the disease by injecting blood from typhoid patients
into apes, rabbits, dogs, and cats. Walder also experimented with
fresh and with putrid discharges from typhoid patients, and with
blood taken from the body after death, feeding this material to
calves, dogs, cats, rabbits, and fowls, without obtaining any posi-
tive results. Klebs has also made numerous experiments of a simi-
lar nature, and in a single instance found in a rabbit, which died

430 THE BACILLUS OF TYPHOID FEVER.

forty-seven hours after receiving a subcutaneous injection of a cul-
ture fluid containing his " typhoid bacillus," pathological lesions re-
sembling those of typhoid.

Eberth and Gaffky very properly decline to attach any import-
ance to this solitary case, in which, as the first-named writer re-
marks, a different explanation is possible, and the possibility of an
intestinal mycosis not typhoid in its nature must be considered.

Gaffky has also made numerous attempts to induce typhoid
symptoms in animals by means of pure cultures of Eberth's bacillus,
given with their food or injected into the peritoneal cavity or subcu-
taneously. The first experiments were made upon five Java apes.
For a considerable time these animals were fed daily with pure cul-
tures containing spores. The temperature of the animals was taken
twice daily. The result was entirely negative. No better success
attended the experiments upon rabbits (16), guinea-pigs (13), white
rats (7), house mice (11), field mice (4), pigeons (2), one hen and a calf.

Cornil and Babes report a similar negative result from pure cul-
tures of the typhoid bacillus injected into the peritoneal cavity and
into the duodenum in rabbits and guinea-pigs.

Frankel and Simmonds have made an extended series of experi-
ments upon guinea-pigs, rabbits, and mice, and have shown that
pure cultures of the bacillus of Eberth injected into the last-men-
tioned animals — mice and rabbits — may induce death, and that the
bacillus may again be obtained in pure cultures from their organs.
It is not claimed that the animals suffer an attack of typhoid fever
as the result of these injections, but that their death is due to the
introduction into their bodies of the typhoid bacillus, and that this
bacillus is thereby proved to be pathogenic.

BACILLUS TYPHI ABDOMINALIS.

Synonyms. — Bacillus typhosus ; Typhus bacillus.

Eberth (1880 and 1881) demonstrated the presence of this bacillus
in the spleen and diseased glands of the intestine in typhoid cada-
vers. Gaffky (1884) first obtained it in pure cultures from the same
source and determined its principal biological characters.

It is found, in the form of small, scattered colonies, in the spleen,
the liver, the glands of the mesentery, the diseased intestinal glands,
and in smaller numbers in the kidneys, in fatal cases of typhoid fever;
it has also been obtained, by puncture, from the spleen during life,
from the alvine discharges of the sick, and rarely from the urine.
It is not found in the blood of the general circulation, unless, pos-
sibly, in rare cases and in small numbers.

Morphology. — Bacilli, usually one to three //in length and about

THE BACILLUS OF TYPHOID FEVER.

437

0.5 to 0.8 [j. broad, with rounded ends; may also grow out into long
threads, especially upon the surface of cooked potato. The dimen-
sions of the rods differ considerably in different media. Spherical or
oval refractive granules are often seen at the extremities of the rods,
especially in potato cultures kept in the incubating oven ; these are
not reproductive spores, as was at first supposed. The bacilli have
numerous flagella arranged around the periphery of the cells — usually
from five to twenty, but many short rods have but a single

FIG. 106. FIG. 107.

FIG. 106.— Bacillus typhi abdominalis, from single gelatin colony. X 1,000. From a photo-
micrograph. (Frankel and Pfeiffer. >

FIG. 107.— Bacillus typhi abdominalis, from single gelafn colony. X 1,000. From a photo-
micrograph. (Sternberg.)

terminal flagellum. These flagella are spiral in form, about 0. 1 /* in
thickness, and from three to five times as long as the rods (Babes).

In stained preparations unstained " vacuoles" may often be seen
at the margins of the rods, either along the sides or at the ends ;
these appear to be due to a retraction of the protoplasm from the cell
membrane.

The typhoid bacillus stains with the aniline colors, but more
slowly than many other bacteria, and easily parts with its color when
treated with decolorizing agents — e.g., iodine solution as employed in
Gram's method. Loffler's solution of methylene blue is an excellent
staining agent for this bacillus, but permanent preparations fade out
after a time ; f uchsin, gentian violet, or Bismarck brown, in aqueous
solution, may also be used. The flagella may be demonstrated by
Loffler's method of staining (p. 32).

To stain the bacillus in sections of the spleen, etc. , it is best to
leave these in Loffler's methylene blue solution or in the carbol-
fuchsin solution of Ziehl for twelve hours or more ; or the aniline-

438

THE BACILLUS OF TYPHOID FEVER.

fuchsin solution may be used. The sections should be washed in
distilled water only, when Ziehl's solution is used, or with a very di-
lute solution of acetic acid when Ehiiich's tubercle stain is employed
(Baumgarten).

FIG. 108.— Bacillus typhi abdominal! s, stained by Loffler's method, showing flagella. x 1,000.
From a photomicrograph by Frankel and Pfeiffer.

Biological Characters. — The typhoid bacillus is a motile, aero-
bic, non-liquefying bacillus, which grows readily in a variety of
culture media at the " room temperature/' Although it grows most
abundantly in the presence of free oxygen, it may also develop in its
absence, and is consequently a facultative anaerobic.

In gelatin plate cultures small, white colonies are developed at
the end of thirty-six to forty-eight hours, which under the microscope

are seen to be somewhat irregular in
outline and of a spherical, oval, or long-
oval form ; these have by transmitted
light a slightly granular appearance and
a yellowish-brown color. At the end of
three or four days the colonies upon the
surface of the gelatin form a grayish-
white layer of one to two millimetres in
diameter, with more or less irregular
margins, and, when developed from deep
colonies, with an opaque central nucleus.

FIG. 109.— Single colony of Bacillus rrn i • ~u -j.i j T T-J.

typhi abdominalis.in nutrient gela- These Colonies, by transmitted light,

tin. (x?) From a photograph by have a yellowish-brown color towards

the centre, where they are thickest,
while the margins are colorless and transparent ; the surface is com-

THE BACILLUS OF TYPHOID FEVER.

439

feu

monly marked with a network of lines and furrows. Stab cultures in
ten-per-cent gelatin, at 18° to 20° C., at the end of three days show
upon the surface a whitish, semi-transparent layer, with sharply
defined margins and irregular outline, which has a shining, pearly
lustre ; and along the line of puncture a gray-
ish-white growth, made up of crowded colo-
nies, which are larger and more distinct at the
bottom of the line of growth. Upon nutrient
cigar, at a temperature of 35° to 37° C., the
growth is more rapid and forms a whitish,
semi-transparent layer. The cultures give off
a faint putrefactive odor. The growth upon
blood serum is rather scanty, in the form of
transparent, shining patches along the line
of inoculation.

The typhoid bacillus develops abundantly
in milky in which fluid it produces an acid
reaction ; it also grows in various vegetable in-
fusions and in bouillon.

None of the above characters of growth are
distinctive, as certain common bacilli found
in normal fasces present a very similar appear-
ance when cultivated in the same media.

The growth of this bacillus upon potato is
an important character, as was first pointed
out by Gaffky. In the incubating oven at the
end of forty-eight hours, or at the room tem-
perature in three or four days, the surface of
the potato has a moist, shining appearance,
but there is no visible growth such as is produced by many other bac-
teria upon this medium. A simple inspection would lead to the belief
that no growth had occurred ; but if with a platinum needle a little ma-
terial is scraped from any portion of the shining surface and a stained
preparation is made from it, numerous bacilli will be seen, some of
which are likely to be in the form of quite long threads, while others
are short and have rounded extremities. This " invisible growth "
has been shown by the researches of Buchner and others to be most
characteristic upon potatoes having a decidedly acid reaction, as is
usually the case. When cultivated upon potatoes having an alkaline
reaction a thin, visible film of a yellowish-brown color and of limited
extent may be developed. Inasmuch as several common and widely
distributed bacteria closely resemble the typhoid bacillus in form and
in their growth in nutrient gelatin, this character of invisible growth

FIG. 110.— Bacillus typhi
abdominalis ; stick culture
in nutrient gelatin, eighth
day at 16°-20° C. (Baum-
garten.)

440 THE BACILLUS OF TYPHOID FEVER.

upon potato is very important for its differentiation, especially as the
common bacilli referred to — Bacillus coli communis, bacillus of Em-
merich— produce a very distinct and rather thick, yellowish- white
mass upon the surface of potato. But recent researches show that
this invisible growth, although not a common character, does not
"belong exclusively to the typhoid bacillus (Babes).

This bacillus in its development in culture media produces acids — •
according to Brieger small quantities of volatile fat acids, and, in
presence of grape sugar, lactic acid. It also grows readily in a de-
cidedly acid medium, and this character has been employed as a test
for differentiating it from other similar bacilli ; but some of these
also grow in a decidedly acid medium, and too much reliance cannot
be placed upon this test.

Brieger has shown that indol is not produced in cultures of the
typhoid bacillus, and Kitasato has proposed to use the indol test for
differentiating this from other similar bacilli which are said, as a
rule, to give the indol reaction. This test consists in the addition to
ten cubic centimetres of a bouillon culture which has been in the in-
cubating oven for twenty-four hours, of one cubic centimetre of a
solution of sodium nitrite (0.02 gramme to one hundred cubic centi-
metres of distilled water), together with a few drops of concentrated
sulphuric acid. If indol is present a red color is developed.

None of the above-mentioned tests are entirely reliable, but, taken
together with the morphological and biological characters above de-
scribed, they may enable the bacteriological expert to give a tolerably
confident opinion as to the presence of this bacillus in a water supply
suspected of contamination, etc. And when a bacillus having these
characters is obtained in a pure culture from the spleen of a typhoid
cadaver the student may be very sure that he has the typhoid bacillus.
But in the presence of various similar bacilli, as in faeces, very careful
comparative researches will be required to determine in a definite
manner that a non-liquefying bacillus obtained in pure cultures by
the plate method is really the one now under consideration — espe-
cially so as the cultures of the typhoid bacillus in the same medium
may differ considerably at different times, and a number of bacilli
are known which resemble it so closely that it is still uncertain
whether they are to be considered as varieties of the typhoid bacillus
or as distinct species. Thus Babes, in an extended research, found in
the organs of typhoid cases, associated with the true typhoid bacillus,
other bacilli or varieties very closely resembling it. He has also
described three varieties (?), obtained by him from other sources,
which could only be differentiated from the true typhoid bacillus by
very careful comparison of cultures made side by side in various
media.

THE BACILLUS OF TYPHOID FEVER. 441

Cassedebat, also, in an extended examination of the river water
at Marseilles with reference to the presence of the typhoid bacillus,
found three species which very closely resembled it, but which by
careful comparison were shown to present slight but constant dif-
ferences in their biological characters. He was not able to find the
true typhoid bacillus, and his researches, together with those of Babes
and other recent investigators, make it appear probable that numerous
mistakes have been made by bacteriologists who have reported the
finding of the typhoid bacillus in river and well water, in fa3ces, etc.,
and who have depended mainly upon the character of invisible
growth upon potato in making their diagnosis. Cassedebat states
that all three of his pseudo-typhoid bacilli corresponded in their
growth upon potato with the bacillus of Eberth. They also corre-
sponded in their growth on gelatin, agar-agar, and blood serum,
which, as heretofore remarked, has no characteristic features. They
all gave a negative indol reaction. Like the typhoid bacillus, they
grew in milk without causing coagulation of the casein, but two of
them produced an alkaline reaction in this fluid, while the third cor-
responded with the typhoid bacillus in producing a decided acid re-
action. Differences were also observed in bouillon cultures, and in
bouillon and milk to which various aniline colors had been added, as
recommended by Holz.

Whether the typhoid bacillus, as obtained from the spleen of a
typhoid cadaver, is in truth specifically distinct from these similar
bacilli, or whether they are all varieties of the same species, result-
ing from modifications in their biological characters acquired during
their continuous development under different conditions, is an un-
settled question. But, in view of the experimental evidence now
available, there is nothing improbable in the supposition that they are
simply varieties, and that, as the result of a saprophytic mode of
life, this bacillus may undergo more or less permanent modifications.

In the writer's experiments (1887) the thermal death-point of the
typhoid bacillus was found to be 56° C., the time of exposure being
ten minutes ; and potato cultures containing the refractive granules
described by Gaffky as spores were found to be infallibly destroyed
by a temperature of 60° C. This result has been confirmed by Buch-
ner (1888) and by Janowsky (1890), and the inference seems justified
that these granules are not reproductive bodies, as was at first be-
lieved ; for spores are distinguished by their great resistance to heat
and other destructive agencies. According to Buchner, the bacilli
containing these refractive granules are even less resistant than fresh
cultures in which they are not present, and he is disposed to look
upon them as representing a degeneration of the protoplasm of the
cells. They do not stain by the methods which are successful in

442 THE BACILLUS OF TYPHOID FEVER.

staining the spores of other bacilli, and, in short, present none of the
characters which distinguish spores, except the form and high re-
fractive power.

The typhoid bacillus retains its vitality for many months in cul-
tures; the writer has preserved bouillon cultures for more than a year
in hermetically sealed tubes, and has found that development
promptly occurred in nutrient gelatin inoculated from these. Dried
upon a cover glass, it may grow in a suitable medium after having
been preserved for eight to ten weeks (Pfuht). When added to
sterilized distilled water it may retain its vitality for more than four
weeks (Bolton), (forty days Cassedebat), and in sterilized sea- water
for ten days (De Giaxa). Added to putrefying faeces it may preserve
its vitality for several months (Ufflemann), in typhoid stools for three
months (Karlinski), and in earth upon which bouillon cultures had
been poured for five and one-half months (Grancher and Deschamps).

In hanging-drop cultures this bacillus may be seen to exhibit very
active movements, the shorter rods rapidly crossing the field with a
darting or to-and-fro, progressive motion, while longer filaments
move in a serpentine manner.

In addition to the volatile fat acids which, according to Brieger,
are formed in small amounts in cultures of the typhoid bacillus, and
to lactic acid formed in solutions containing grape sugar, a basic
substance possessing toxic properties has been isolated by the chemist
named — his typhotoxine (C,H17!N"03). Brieger supposes that other
basic substances are likewise formed, but believes this to be the speci-
fic product to which the pathogenic action of the bacillus is due. It
is a strongly alkaline base, which produces in mice and guinea-pigs
salivation, paralysis, dilated pupils, diarrhoea, and death.

Numerous experiments have been made to determine the amounts
of various germicidal agents required to destroy the vitality of this
bacillus, and the action of antiseptics in restraining its development.
For the results of these experiments the reader is referred to the
sections in Part Second relating to the action of antiseptics and disin-
fectants.

Pathogenesis. — The very numerous experiments which have been
made on the lower animals have not been successful in producing in
any one of them a typical typhoid process. Nor is this surprising,
in view of the fact that, so far as is known, no one of them is liable to
contract the disease, as man does, by the use of infected food or
water.

The experiments of Frankel and Simmonds show that when con-
siderable quantities of a pure culture of this bacillus are injected into
the circulation of rabbits through the ear vein, or into the peritoneal
cavity of mice, a certain proportion of the inoculated animals die,

THE BACILLUS OF TYPHOID FEVER.

443

usually within forty-eight hours, and that the bacillus may be re-
covered from the various organs, although it is not present in the
blood. But death does not always occur from intravenous injections,
and subcutaneous or intraperitoneal injections in rabbits are usually
without result. Subcutaneous injections in mice proved to be fatal in
ten cases out of sixteen inoculated by A. Frankel. Seitz, by following
Koch's method — i. e. , by rendering the contents of the stomach alka-
line, and arresting intestinal peristalsis by the administration of
opium — obtained a fatal result, in a majority of the guinea-pigs experi-
mented upon, from the introduction of ten cubic centimetres of a
bouillon culture into the stomach through a pharyngeal catheter.
We may remark, with reference to these results, that while they show
that cultures of the typhoid bacillus have a certain pathogenic power,

FIG. 111.— Section through wall of intestine, showing invasion by typhoid bacilli. X 950.
(Baumgarten.)

they also show that the animals experimented upon frequently re-
covered after comparatively large doses, and that the typhoid bacil-
lus is not pathogenic in the same sense as are those microorganisms
which, when introduced into the body of a susceptible animal in very
minute amount, give rise to general infection and death. On the
other hand, a fatal result depends upon the quantity of the culture
introduced in the first instance, rather than upon the multiplication
of the bacillus in the body of the inoculated animal. This view is
confirmed by the experiments of Sirotinin, which show not only that
a fatal result depends upon the quantity injected, but also that a
similar result follows the injection of cultures which have been ster-
ilized by heat or filtration. The pathogenic action, then, depends
upon the presence of toxic substances produced during the growth of
the bacillus in artificial culture media. The researches of Brieger,
heretofore referred to, show the presence in such cultures of a toxic
ptomaine — his typhotoxine — to which the pathogenic potency of these

444 THE BACILLUS OF TYPHOID FEVER.

cultures appear to be due. White mice and guinea-pigs usually die
in from twenty-four to forty-eight hours when inoculated in the
cavity of the abdomen with a virulent culture of the typhoid bacillus
—0.1 cubic centimetre to 0.5 cubic centimetre of a bouillon culture
three days old. According to Kitasato, the virulence of cultures
from different cases of typhoid fever varies considerably.

Detection of the Typhoid Bacillus in Water. — The generally
recognized fact that typhoid fever is usually contracted by drink-
ing water contaminated by the typhoid bacillus has led to numer-
ous researches having for their object the discovery of a reliable
method of detecting this bacillus when present in water in compara-
tively small numbers in association with the ordinary water bacilli.
The use of Koch's plate method, as commonly employed, will
not suffice, because the water bacilli present grow more rapidly
and cause liquefaction of the gelatin before visible colonies of the
typhoid bacillus are formed ; and, owing to the relatively small
number of typhoid bacilli, these are likely to escape detection. The
aim of bacteriologists has, therefore, been to restrain the growth of
these common water bacilli by some agent which does not at the
same time prevent the development of the typhoid bacillus. Chan-
temesse and Widal were the first to propose the use of carbolic acid
for this purpose. They recommended the addition of 0. 25 per cent
of this agent to nutrient gelatin ; but, according to Kitasato, the de
velopment of the typhoid bacillus is restrained by an amount exceed-
ing 0.20 per cent.

Holz prepares an acid medium by adding gelatin (ten per cent) to
the juice of raw potatoes, and asserts that while the typhoid bacillus
grows luxuriantly in this medium, many other bacilli fail to develop
in it. The test is said to be still more reliable if 0.05 per cent of car-
bolic acid is added to the " potato-gelatin." According to Holz, the
addition of more than 0. 1 per cent of carbolic acid to nutrient gelatin
prevents the free development of the typhoid bacillus.

Thoinot has claimed to be able to obtain the typhoid bacillus from
mixed cultures — as, for example, from f^ces — by suspending a small
amount of material containing it for several hours in a solution con-
taining 0.25 per cent of carbolic acid. While other bacilli are
destroyed, the typhoid bacillus is said to survive such exposure.

The method of Parietti has been tested in a practical way by
Kamen, and proved to be satisfactory for the detection of the typhoid
bacillus in water which was supposed to be the source of a local
epidemic of the disease. The following solution is used :

Carbolic acid, ........ 5 grammes.

Hydrochloric acid (pure), 4

Distilled water, 100

THE BACILLUS OF TYPHOID FEVER. 445

Several test tubes, each of which contains ten cubic centimetres
of neutral, sterilized bouillon, are used in the experiment. From
three to nine drops of the acid solution are added to each of these,
and the tubes are then placed in an incubating oven for twenty-four
hours to ascertain whether they are still sterile after this addition.
If the bouillon remains clear, from one to ten drops of the suspected
water are added to each tube and they are returned to the incubating
oven. If at the end of twenty-four hours the bouillon becomes
clouded, this is due, according to Parietti, to the presence of the
typhoid bacillus, which is then to be obtained in pure cultures by the
plate method.

The following method, suggested by Hazen and White, has been
tested with favorable results by Foote. This method depends upon
the fact that most of the common water bacilli do not grow at a tem-
perature of 40° C., whereas this is a favorable temperature for the
development of the typhoid bacillus. A small quantity of the sus-
pected water is added to liquefied nutrient agar in test tubes, and
plates are made. These are placed in an incubating oven at 40° C.,
and the typhoid bacillus, if present, will develop colonies within two
or three days. At the ordinary room temperature the more numerous
water bacilli would develop upon the same plates so abundantly that
it would be difficult to recognize colonies of the typhoid bacillus.

Theobald Smith (Centralb. f. Bakteriol., Bd. xii., page 367),
has shown that the typhoid bacillus may be differentiated from other
similar bacilli (Bacillus coli communis, bacillus of hog cholera, etc.)
by the fact that it does not produce gas in culture media containing
sugar — grape sugar, cane sugar, or milk sugar. The medium recom-
mended by Smith for making this test is a peptone-bouillon contain-
ing two per cent of grape sugar and made slightly alkaline with
carbonate of soda. The liquid becomes clouded throughout at the
end of twenty-four hours, but not a trace of gas is developed even
after several days. On the other hand, the colon bacillus and other
bacilli which closely resemble the typhoid bacillus cause an abundant
development of gas in this medium.

The method of Wurtz will be found useful in the detection of
colonies of the typhoid bacillus in plate cultures from contaminated
water, etc. This consists in the addition to the nutrient medium of
lactose (two per cent) and a solution of litmus. When the colonies
develop in plates made from this medium the typhoid colonies re-
main blue, while colonies of the " colon bacillus " have a red color, on
account of the development of lactic acid.

Schild (1894) uses a bouillon containing formalin (1:7,000) and
claims that the typhoid bacillus fails to grow in this medium, while

446 THE BACILLUS OF TYPHOID FEVER.

the bacilli of the colon group multiply in it and cause the me-
dium to become clouded within twenty-four hours. Abel (1894), as a
result of extended experiments, arrives at the conclusion that the
formalin test cannot be relied upon for distinguishing the typhoid
bacillus from certain similar bacilli, which also fail to grow in for-
malin solution. • But, on the other hand, a bacillus which grows in
bouillon containing 1 : 7,000 of formalin can be definitely pronounced
to be not the typhoid bacillus.

Eisner (1895) recommends the following method for the detection
of the typhoid bacillus in water or in faeces : To potato gelatin, pre-
pared by the method of Holz, he added one per cent of potassium
iodide. But few species of bacteria will grow in this medium, but
Bacillus coli communis grows in it luxuriantly, forming fully de-
veloped colonies at the end of twenty-four hours. The typhoid col-
onies, on the contrary, are only just visible under a low power at the
end of twenty-four hours, and at the end of forty-eight hours are
seen as small, shining, drop-like, very finely granular colonies. At
the same time the colonies of the colon bacillus are much larger,
coarsely granular, and of a brownish color. By this method Eisner
succeeded in obtaining pure cultures of the typhoid bacillus from
the fa3ces in fifteen out of seventeen cases of typhoid fever, in various
stages of the disease. Lazarus (1895) has tested this method and re-
ports that he succeeded without any difficulty in obtaining pure cul-
tures of the typhoid bacillus from the alvine discharges of typhoid
patients.

When the typhoid bacillus and the colon bacillus are planted to-
gether, in the same liquid medium, the first-mentioned bacillus, even
when in excess at the outset of the experiment, soon disappears and
the Bacillus coli communis remains in full possession. According
to Wathelet (1895) the colon bacillus will grow in bouillon which
has served as a culture medium for the typhoid bacillus, or on the
surface of an agar plate from which a typhoid culture has been re-
moved; but the typhoid bacillus fails to develop in culture media
which have served for the development of the colon bacillus.

The various diagnostic tests which have been proposed, and the
extensive literature of the subject, show that the recognition of the
typhoid bacillus in water, fasces, etc., is attended with serious diffi-
culties. This is chiefly due to the fact that bacilli have been ob-
tained from various sources which resemble more or less closely the
typical typhoid bacillus as obtained from the spleen of a typhoid
patient (or cadaver) and the " colon bacillus " as found in the alimen-
tary canal of healthy men and animals; and also from the fact that
the bacillus, as obtained from typhoid cases, varies to some extent in
its biological characters, and that varieties may be produced in the

THE BACILLUS OF TYPHOID FEVER. 44?

bacillus as obtained, from a single colony, by special modes of culti-
vation. From a consideration of these facts certain authors have
been led to the conclusion that Bacillus typhi abdominalis and Bacillus
coli communis are simply varieties of the same species. This view,
however, is not generally accepted, and the characters which serve to
differentiate the two bacilli are sufficiently well defined when typical
cultures are compared. These characters, briefly stated, are: The
invisible growth of the typhoid bacillus on potato; its failure to give
the indol reaction; its failure to coagulate milk, or to produce a
change of color in litmus milk ; its failure to produce gas in culture
media containing glucose or lactose; its failure to grow in formalin
bouillon (1 : 7,000) ; and its active motility. Whether the closely re-
lated bacilli which present some of the characters above indicated,
without corresponding in all particulars with typical cultures of the
typhoid bacillus, are varieties of this bacillus, which under favorable
circumstances could give rise to typhoid infection, has not been defi-
nitely determined, but appears to be quite probable. It may be that
such varieties are developed when the typhoid bacillus in faeces finds
its way into surface waters, under conditions which are favorable for
its continued development as a saprophyte. On the other hand, it
may be that one or more of the saprophytic bacilli, which are found
in water and which closely resemble the typhoid bacillus, may give rise
to the infectious disease which we know as typhoid fever when in-
troduced into the alimentary canal of a particularly susceptible indi-
vidual, and that the special conditions attending its development as
a parasite give rise to certain modifications in its biological charac-
ters of a more or less permanent kind.

Frankland (1895), as a result of extended experiments, has arrived
at the conclusion that when the typhoid bacillus is cultivated for a
long time in media which are more and more largely diluted with
water, it acquires an increased ability to survive in river water.

A predisposition to typhoid infection is established -by various
depressing agencies, such as inanition, overwork, mental worry, in-
sanitary surroundings, etc. And there is considerable evidence in
support of the supposition that exposure to the offensive gases
given off from ill- ventilated sewers constitutes a predisposition to
the disease.

Experiments made by Alessi (1894), in the Hygienic Institute
of the University of Rome, give support to this view. The ex-
periments were made upon rats, guinea-pigs, and rabbits. The
rats were confined in a close cage with perforated bottom, which was
placed over the opening of a privy; the guinea-pigs and rabbits in
similar cages having a receptacle below in which their own excreta
was allowed to accumulate. The animals which breathed an atmo-

448 THE BACILLUS OF TYPHOID FEVER.

sphere vitiated in this way lost, after a time, their usual activity and
became emaciated, although they continued to eat greedily. When
these animals were inoculated with a small quantity of a culture of
the typhoid bacillus (0.25 to 0.5 cubic centimetre) they died within
from twelve to thirty-six hours. The same amount of the typhoid
culture injected into control animals produced no injurious effect. In
the animals which succumbed to typhoid infection there was found a
hemorrhagic enteritis, incfease in volume of Peyer's glands and of the
spleen, and typhoid bacilli in the blood, liver, and spleen. The char-
acteristic appearances of typhoid infection were more pronounced in
the rabbits and guinea-pigs than in rats. Similar experiments with
Bacillus coli communis gave similar results. The time required to
induce this predisposition for typhoid infection was from five to
seventy-two days for the rats, seven to fifty-eight for the guinea-
pigs, and three to eighteen for the rabbits. Alessi found that the
susceptibility to infection diminished after a certain time, and sug-
gests that in a similar way man may become habituated to breathing
an atmosphere containing sewer gases.

Pus- Production by Typhoid Bacilli. — The literature relat-
ing to the typhoid bacillus includes many observations as to its
presence in accumulations of pus in various parts of the body — often
in a pure culture. It has been found in a considerable number of
cases of periostitis secondary to typhoid fever, in purulent syno-
vitis, and in abscesses in various parts of the body.

Dmochowski and Janowski (1895), as the result of a review of the
literature and a painstaking experimental research, arrive at the con-
clusion that even in abscesses, occurring in typhoid fever cases, in
which only the pus cocci are found, it is probable that the typhoid
bacillus originated the process resulting in abscess formation. They
assert that the typhoid bacillus dies out in a comparatively short
time in abscesses which are directly due to its presence, and that
often it may be found in the abscess walls when its presence can no
longer be demonstrated in the purulent contents of the abscess cavity.

PLATE V.

PATHOGENIC BACTERIA.

FIG. 1. — Bacillus anthracis from cellular tissue of inoculated mouse.
Stained with gentian violet. X 1,000. Photomicrograph by Frankel
and Pfeiffer.

FIG. 2. — Bacillus anthracis in section of liver of inoculated rabbit.
Stained with Bismarck brown, x 250. Photomicrograph by Sternberg.

FIG. 3. — Micrococcus gonorrhoeas in gonorrhceal pus. Stained with
gentian violet, x 1,000. Photomicrograph by gaslight. (Sternberg.)

FIG. 4. — Anthrax spores from a bouillon culture. Double-stained
preparation — with carbol-f uchsin and rnethylene blue, x 1,000. Photo-
micrograph by Frankel and Pfeiffer.

FIG. 5. — Spirillum cholerse Asiaticee from a culture upon starched
linen at end of twenty-four hours. Stained with f uchsin. x 1,000.
Photomicrograph by Frankel and Pfeiffer.

FIG. 6. — Bacillus diphtherise from colony upon an agar plate, twenty-
four hours old. Stained with Loffler's solution of methylene blue, x
1,000. Photomicrograph by Frankel and Pfeiffer.

PLATE V.

STERNBERG'S BACTERIOLOGY.

igf. 2.

•*"^" *-<*-

**?v.- •

• •«»*

f; >

PigT. 4.

Pig. 6.

X.
BACTERIA IN DIPHTHERIA.

DIPHTHERIA is generally recognized by physicians as a specific
infectious disease, and, owing to its wide prevalence and fatal char-
acter, a precise knowledge of its etiology is of the greatest import-
ance. Until, as a result of recent researches, this was determined,
pathologist s were in doubt as to whether diphtheria should be con-
sidered as primarily a local infection, or whether the local manifesta-
tions were secondary to a general systemic infection. But this question
appears now to be definitely settled in favor of the former view. We
have to-day a very precise knowledge of the specific infecting agent,
and have evidence that it produces during its growth a very potent
toxic substance, the absorption of which from the seat of local infec-
tion accounts in a satisfactory manner for the general symptoms of
the disease, which are due to toxaemia and not to an invasion of the
blood and tissues by the pathogenic microorganism producing it.

Numerous researches by competent bacteriologists have failed to
demonstrate the presence of bacteria in the blood of patients suffer-
ing from diphtheria, but a variety of microorganisms have been ob-
tained in cultures from diphtheritic pseudo-membranes, and may be
demonstrated by the microscopical examination of stained prepara-
tions. Among these are the well-known pus organisms, and espe-
cially the Streptococcus pyogenes, which appears to be very commonly
present, and is perhaps the active agent in the production of certain
forms of pseudo-diphtheria. But the malignant, specific diphtheria,
so well known in this country and in Europe, has been demonstrated
by the recent researches of bacteriologists to be due to a bacillus first
recognized by Klebs in stained preparations of diphtheritic false
membranes (1883), and cultivated and described by Lofner in 1884.
In his first publication Lofner did not claim to have fully demon-
strated the etiological relation of this bacillus, but this appears to be
fully established by subsequent researches.

In his first research Lofner studied twenty-five cases, and in the
greater number of them found in stained preparations the bacil-
lus previously described by Klebs. From six of these cases he
29

450 BACTERIA IX DIPHTHERIA.

obtained it in pure cultures, and by inoculations in pigeons, chickens,
rabbits, and guinea-pigs proved that it gave rise to a diphtheritic
inflammation when inoculated into the mucous membrane of the
trachea, conjunctiva, pharynx, or vagina. In a second communica-
tion Loftier reported his success in finding the same bacillus in ten
additional cases, and also that he had isolated from the same source
a non-pathogenic bacillus which resembled it very closely. This
pseudo-diphtheria bacillus has since been found by other bacteri-
ologists (Von Hoffmann, Roux and Yersin), and it is uncertain
whether it is to be considered a distinct species, or a non-pathogenic
variety of the diphtheria bacillus as maintained by Roux and Yersin.
But its occasional presence does not invalidate the very positive ex-
perimental evidence relating to the specific pathogenic power of the
true diphtheria bacillus.

Loffler, in 1890, reviewed the evidence upon which this bacillus is
now generally conceded by bacteriologists to be the specific infectious
agent in true diphtheria. The following are the principal points in
the demonstration :

FIRST. — It is found in all undoubted cases of diphtheria. In
support of this we have the results of researches made by Loffler,
Wyssokowitsch, D'Espine, Von Hoffmann, Ortmann, Roux and
Yersin, Kolisko and Paltauf, Zarinko and Sorensen, who in nearly
every case have demonstrated without difficulty the presence of this
bacillus. On the other hand, Prudden failed to find it in a series of
twenty-four cases studied by him ; but his own account of these
cases indicates that they were not cases of true diphtheria. He says
in a subsequent communication :

4 'In view of the doubt existing among practitioners as to whether all
forms of pseudo-membranous inflammation should be called diphtheria or
not, and with the purpose of making a wholly objective study, the writer
distinctly stated at the outset of that paper that all the fatal cases of exten-
sive pseudo-membranous laryngitis, as well as pharyngitis, should in his
study be considered as cases of diphtheria. This left the question as to the
propriety of establishing separate groups of pseudo-membranous inflamma-
tion open and free from bias. It was distinctly stated, however, that six-
teen out of the twenty-four cases occurred in a large asylum, in which
measles and scarlet fever were prevalent during the period in which these
studies were under way. Five other cases in another asylum were ex-
posed to similar conditions."

In a subsequent series of " twelve cases of fatal pseudo-mem-
branous inflammation occurring in two children's asylums, in which
for many months there had been no scarlatina and no measles, and
in which there was no complicating suppurative inflammation and
no erysipelas," Prudden (1890) obtained Loffler's bacillus in cultures
from eleven, and he says :

"We are now, it would seem, justified, as it did not appear to the writer

BACTERIA IN DIPHTHERIA. 451

that we were two years ago, owing to the large number of important re-
searches which have been made in the interim, in saying that the name
diphtheria, or at least primary diphtheria, should be applied, and exclusively
applied, to that acute infectious disease, usually associated with a pseudo-
membranous inflammation of the mucous membranes, which is primarily
caused by the bacillus called Bacillus diphtherias of Loffler.''

With reference to the question as to how long after convalescence
is established the diphtheria bacillus may be present in the throat
of an infected person, Loffler has made the following research (1890).
In a typical case a bacteriological examination was made daily from
the commencement until fourteen days after its termination. Fever
disappeared on the fifth day, and the exudation had all disappeared
on the sixteenth day. Up to this time the bacillus was daily ob-
tained in cultures, and subsequently nearly every day up to the
twenty-fifth — that is, for three weeks after the febrile symptoms had
disappeared. Roux and Yersin have also obtained the bacillus in
cultures from mucus scraped from the throats of convalescents sev-
eral days after the disappearance of all evidence of the disease.

SECOND. The Klebs-Loffler bacillus is found only in diph-
theria.— In his earlier researches Loffler obtained the bacillus in a
single instance from the mouth of a healthy child, and this fact led
him to hesitate in announcing it as his conviction that it was the
true cause of diphtheria. But in extended researches made subse-
quently he has not again succeeded in finding it, except in associa-
tion with diphtheria, and admits now that he may have been mis-
taken as to the identity of the bacillus found. This seems not
improbable in view of the fact that very similar bacilli have been
found by various bacteriologists. Thus Von Hoffmann obtained a
very similar but non-pathogenic bacillus from the mucus of chronic
nasal catarrh and from healthy mucous membranes ; Babes from
cases of trachoma, Neisser from ulcers, Zarinko from the surface of
various mucous membranes. But all of these were shown to present
certain differences in their biological characters by which they could
be differentiated from the true diphtheria bacillus.

Welch and Abbott in their comparative studies did not find the
Loffler bacillus, "or any bacillus that an experienced bacteriologist
would be likely to confound with it." They examined mucus from
the throats of healthy children, from those suffering from simple in-
flammation of the tonsils and pharynx, and from four cases of so-
called follicular tonsillitis. As a result of their investigations they
agree with Loffler, and with Roux and Yersin, as to " the great prac-
tical value, for diagnostic purposes, of a bacteriological examination
of cover-glass specimens and by cultures " of cases in which there is
any doubt of the true character of the disease. They say further :

452 BACTERIA IN DIPHTHERIA.

i i

The only species of bacteria which we have found constantly in the
cases of diphtheria has been the Loffler bacillus. Two other species have
been present in many cases, viz., the well-known streptococcus, which grows
in much smaller colonies and less rapidly than the Loftier bacillus, and a
short, oval, often slightly pointed bacillus, growing in long chains running
parallel to each other. There are often marked irregularities in shape and
especially in size of this bacillus, even of individuals in the same chain.
The colonies of this bacillus are grayish-white, moist, larger than those of
the streptococcus, but smaller than those of the Loffler bacillus."

THIRD. As shown by Loffler's earlier researches, pure cultures
of this bacillus induce characteristic diphtheritic inflammation
when inoculated into the mucous membranes of certain lower ani-
mals. Roux and Yersiii have also shown that local paralysis is
likely to occur in inoculated animals, as is the case in diphtheria in
man. In speaking of their inoculations into the trachea in rabbits
these investigators say :

"The affection which is thus induced in the rabbit resembles croup in
man. The difficulty which the animal experiences in breathing; the noise
made by the air in passing through the obstructed trachea- the aspect of the
trachea, which is congested and covered with false membranes ; the oederna-
tous swelling of the tissues and glands of the neck, make the resemblance
absolutely remarkable."

Welch and Abbott give the following account of the results of
inoculations into the trachea in kittens :

" A half -grown kitten is inoculated into the trachea with one platinum
loop from a pure culture of the Loffler bacillus on glycerin-agar, eleven days
old, derived from Case IV. For the inoculation a small median incision was
made over the trachea, in which a hole just large enough to admit the plati-
num loop was made. The culture was rubbed over the mucosa of the trachea
for an extent about three centimetres in length, and in this process sufficient
force was used to abrade the mucous membrane. On the day following the
inoculation no special alteration in the animal was observed, but on the
morning of the second day it was found very weak. In the course of this
day it became so weak as to lie completely motionless, apparently uncon-
scious, with very feeble, shallow respiration ; several times it was thought to
be dead, but on careful examination proved still to be breathing feebly. It
was found dead on the morning of the third day. At the autopsy the wound
was found gaping and covered with a grayish, adherent, necrotic, distinctly
diphtheritic layer. For a considerable distance around the wound the sub-
cutaneous tissues were very cedematous, the oedema extending from the
lower jaw down over the sternum, and to the sides of the neck, and along
the anterior extremities. The lymphatic glands at the angle of the jaw were
markedly swollen and reddened. The mucous membrane of the trachea,
beginningat the larynx and extending down for six centimetres, wascovered
with a tolerably firm, grayish-white, loosely attached pseudo-membrane, in
all respects identical with the croupous membranes observed in the same
situation in cases of human diphtheria. "

BACTERIA IX DIPHTHERIA. 453

BACILLUS DIPHTHERIA.

First observed by Klebs (1883) in diphtheritic false membranes.
Isolated in pure cultures and pathogenic power demonstrated by
Loffler (1884).

Found in diphtheritic pseudo-membranes, and especially in the
deeper portions, intermingled with numerous cellular elements; while
the superficial layers of the membrane commonly contain but few
cells or bacilli, or are invaded by other species, especially by Strep-
tococcus pyogenes. The bacilli are not found in the affected mucous
membrane, or in sections from the internal organs in fatal cases of
this disease.

llor/tfiology. — Rods, straight or slightly curved, with rounded
ends, having a diameter of 0.5 to 0.8
ii. and from I to 3 /i in length. Ir-
regular forms are very common, and,
indeed, are characteristic of this bacil-
lus. In the same culture, and especially
in an unfavorable culture medium, very
great differences in form and dimen-
sions may be observed ; one or both ends
may appear swollen, or the central por-
tion may be notably thicker than the
extremities, or the rod may be made up
of irregular spherical or oval segments. FlG. 113> _ Bacillus diphtheria,
Multiplication occurs by fission only, from a culture upon blood serum.

I ,T_ -I -IT i , • m From a photomicrograph, x 1,000.

aild the bacilli dO not grow Out into fila- (Frankel and Pfeiffer.)

ments.

In unstained preparations certain portions of the rod, and espe-
cially the extremities, are observed to be more highly refractive than
the remaining portion ; and in stained preparations these portions
are seen to be most deeply colored. The diphtheria bacillus may be
stained by the use of Loffler 's alkaline solution of methylene blue,
but is not so readily stained with some of the other aniline colors
commonly employed. It stains also by Gram's method. For the
demonstration of the bacillus in sections of diphtheritic membrane
" nothing can surpass in brilliancy and sharp differentiation sections
stained doubly by the modified Weigert's fibrin stain and. picro-car-
mine" (Welch and Abbott).

Biological Characters. — The diphtheria bacillus is aerobic, non-
motile', and non-liquefying; it does not form spores. It grows most
freely in the presence of oxygen, but is also a facultative anaerobic.

Development occurs in various culture media at a temperature of
from 20° to 42° C., the most favorable temperature being about 359 C.

454 BACTERIA IX DIPHTHERIA.

It grows readily in nutrient gelatin having a slightly alkaline reac-
tion, in nutrient agar, glycerin-agar, or in alkaline bouillon, but the
most favorable medium appears to be that first recommended by

Loffler — viz., a mixture of three
parts of blood serum with one part
; of bouillon, containing one per cent
;f,t of peptone, one per cent of grape
sugar, and 0. 5 per cent of sodium
chloride. This mixture is steril-
ized and solidified at a low tem-
perature, as is usual with blood
serum. Upon this the develop-
Fni. us.— Colonies of Bacillus diphtherias meiit is so rapid in the incubating

in nutrient agar, end of twenty-four hours. ,, , , ,, -, - f -

X 10. (FrankelandPfeiffer.) lt> at

four hours, the large, round, ele-
vated colonies, of a grayish-white color and moist appearance, may
be easily recognized, while other associated bacteria will, as a rule,
not yet have developed colonies large enough to interfere with the
recognition of these.

Upon nutrient agar plates the deep-lying colonies, when magni-
fied about eighty diameters, appear as round or oval, coarsely granu-
lar discs, with rather ill-defined margins, or, when several colonies
are in juxtaposition, as figures of irregular form. The superficial col-
onies are grayish-yellow in color, have an irregular, not well-defined
outline and a rough, almost reticulated surface. The growth upon
glycerin-agar is very similar. The first inoculations in a plain nu-
trient agar tube often give a comparatively feeble growth, which be-
comes more abundant in subsequent inoculations in the same medium.
In stick cultures in glycerin — or plain — agar, growth occurs to the
bottom of the line of inoculation, and also upon the surface, but is
not at all characteristic. The same may be said with reference to
cultures in nutrient gelatin. Plate cultures in this medium contain-
ing fifteen per cent of gelatin, at 24° C., give rather small colonies,
which are white by reflected light and under the microscope are seen
as yellowish-brown, opaque discs, having a more or less irregular
outline and a granular structure. In alkaline bouillon the growth is
sometimes in the form of small, whitish masses along the sides and
bottom of the tube, but at others a diffusely clouded growth occurs
in this medium ; after standing for some time in the incubating oven
a thin, white pellicle may form upon the surface of the bouillon.
The reaction of the bouillon becomes at first acid, but later it has an
alkaline reaction (Welch). With reference to the growth onpotato,
authors have differed, probably because the growth is scarcely vis-
ible ; upon this point we quote from Welch and Abbott :

BACTERIA IN DIPHTHERIA.

" Our experience has been that the Bacillus diphtherias grows on ordinary
steamed potato without any preliminary treatment, but that the growth is
usually entirely invisible or is indicated by a dry, thin glaze after several
days. Doubtless the invisible character of the growth has led most observers
into the error of supposing that no growth existed, whereas the microscopi-
cal examination reveals a tolerably abundant growth, which on the first po-
tato is often feebler than on succeeding ones. Irregular forms are par-
ticularly numerous in potato cultures, and in general the rods are thicker
than on other media. In twenty-four hours, at a temperature of 35° C.,
microscopical examination shows distinct growth. We have cultivated the
bacillus for many generations on potato."

Milk is a favorable medium for the growth, of this bacillus, and,
as it grows at a comparatively low temperature (20° C.), it is evi-
dent that this fluid may become a medium for conveying the bacillus
from an infected source to the throats of previously healthy children.

Cultures of the diphtheria bacillus may retain their vitality for
several months, and when dried upon silk threads for several weeks
colonies are still developed in a suitable medium — in the room from
three to four weeks, in an exsiccator five to ten, and in one instance
fourteen weeks. In dried diphtheritic membrane, preserved in small
fragments, the bacillus retained its vitality for nine weeks, and in
larger fragments for twelve to fourteen weeks.

The thermal death-point, as determined by Welch and Abbott, is
58° 0., the time of exposure being ten minutes. Loffler had previ-
ously found that it did not survive exposure for half an hour to 60°
C. With reference to the action of germicidal and antiseptic agents,
we refer to the sections in Part Second relating to this subject.

Pathogenesis. — In view of the evidence heretofore recorded, it
may be considered as demonstrated that this bacillus gives rise to
the morbid phenomena which characterize the fatal disease in man
known as diphtheria.

We have already referred to the effects of inoculations into the
trachea in rabbits and cats, which give rise to a characteristic diph-
theritic inflammation, with general toxaemia and death from the
absorption of soluble toxic products formed at the seat of local in-
fection. This inference as to the cause of death seems justified by
the fact that the pathogenic bacillus does not invade the blood and
tissues, and is supported by additional experimental evidence (see
pages 309-317).

PSEUDO-DIPHTHERITIC BACILLUS.

Loffler, Von Hoffmann, and others have reported finding bacilli
which closely resemble the Bacillus diphtheria?, but which differ
from it chiefly in being non-pathogenic. The following account we

456 BACTERIA IN DIPHTHERIA.

take from a paper upon the subject by Roux and Yersiii (troisieme
memoire, 1890).

Found by Roux and Yersin in mucus from the pharynx and ton-
sils of children — from forty-five children in Paris hospitals, suffering
from various affections, not diphtheritic, fifteen times; from fifty-
nine healthy children in a village school on the seaboard, twenty-six
times. Of six children with a simple angina but two furnished cul-
tures of this bacillus, while it was obtained in five out of seven ca.ses
of measles.

Its characters are given as follows :

'The colonies of the pse ml o -diphtheritic bacillus, cultivated upon blood
serum, are identical with the true diphtheria bacillus At a temperature of
33° to 35 multiplication is rapid, and it continues at the ordinary tempera-
ture, although slowly. Under the microscope the appearance of the bacillus
which forms these colonies is the same as that of Bacillus diphtherige. It
stains readily with Loffler's solution of methylene blue, and intensely by
Gram's method. Sometimes it colors uniformly, at others it appears granu-
lar. It grows in alkaline bouillon, giving a deposit upon the walls of the
vessel containing the culture, and in this medium often presents the inflated
forms, pear-shaped, or club-shaped. It is destroyed in a liquid medium by a
temperature of 58° C. maintained for ten minutes. All of these characters
are common to the pseudo-diphtheritic bacillus and the true Bacillus diphthe-
riae. As a difference between them we may note that the pseudo diphtheritic
bacillusds often shorter in colonies grown upon blood serum; that its cultures
in bouillon are more abundant ; that they continue at a temperature of 20° to
22°, at which the true bacillus grows very slowly. When we make a com-
parison of cultures in bouillon they become acid and then alkaline, but the
change occurs much sooner in the case of the pseudo-diphtheritic bacillus.
Like the true bacillus, the pseudo diphtheritic grows in a vacuum, but less
abundantly than the other.

" Inoculations into animals of cultures of this bacillus have never caused
their death ; but we may remark that in some experiments a notable oedema
has been produced in guinea-pigs at the point of inoculation, while in others
there has been no local lesion. The most marked oedema resulted from cul-
tures obtained from cases of measles.

" Do the facts which we have reported explain the question which occupies
us ? Can we conclude that there is a relation between the two bacilli ? On
the one side, the presence of the pseudo diphtheritic bacillus in the mouths of
healthy persons, and of those who have anginas manifestly not diphtheritic,
seems to be opposed to the idea of a relationship between them. On the
other hand, when we consider that the non- virulent bacillus is very rare in
fatal diphtheria, that it is more abundant in benign diphtheria, that it be-
comes more common in severe cases as they progress towards recovery, and.
finally, that they are more numerous in persons who have recently had
diphtheria than in healthy persons, it is difficult to accept the idea that the
two microbes are entirely distinct. The morphological differences which
have been referred to are so slight that they prove nothing. The two micro-
organisms can only be distinguished by their action upon animals, but the
difference of virulence does not at all correspond with the difference of ori-

fin. As regards the form and the aspect of cultures, the true and false
iphtheria bacilli differ less than virulent anthrax differs from a very attenu-
ated anthrax bacillus, which, however, originate from the same source.
Besides, the sharp distinction which we make between the virulent and 11011-
virulent bacilli is arbitrary ; it depends upon the susceptibility of guinea-
pigs. If we inoculate animals still more susceptible, there are pseudo diph-
theritic bacilli which we must class as virulent; and if, on the contrary, we
substitute rabbits for guinea pigs in our experiments, there are diphtheritic
bacilli which we must call pseudo-diphtheritic. In our experiments \vo do

BACTERIA IN DIPHTHERIA. 457

not simply encounter bacilli which are very virulent and bacilli which are
non- virulent; between these two extremes there are bacilli of every degree
of virulence."

Abbott, in 1891, published the result of his researches with
reference to the presence of the pseudo-diphtheritic bacillus in
benign throat affections. He made a bacteriological study of fifty-
three patients, nine of whom were suffering from acute phaiyngitis,
fourteen from acute follicular tonsillitis, eight from ordinary post-
nasal catarrh, two from simple enlarged tonsils, fifteen from chronic
pharyngitis, one from subacute laryngitis, one from chronic laryngi-
tis, one from rhinitis, and two from an affection of the tonsils and
pharynx. In forty-nine cases nothing of particular interest was ob-
served. A variety of microorganisms were isolated, and of these
the pyogenic micrococci were the most common.

In four cases microorganisms were found which resembled the
Bacillus diphtheria of Loffler in their morphology and growth in cul-
ture media, but which proved not to be pathogenic. Abbott says :
" The single point of distinction that can be made out between the
organisms obtained from Cases L, III., and IV. and the true bacil-
lus of diphtheria is in the absence of pathogenic properties from the
former, whereas in addition to this point of distinction the organism
from Case II. gives, as has been stated, a decided and distinct
growth upon the surface of sterilized potato. "

Recent authors are generally inclined to the opinion that bacilli
which resemble the diphtheria bacilli in every respect except that
they are non-pathogenic should be regarded as attenuated varieties
of the diphtheria bacillus rather than as belonging to a distinct
species — the so-called "pseudo-diphtheria" bacillus. However, there
are bacilli which closely resemble the bacillus of diphtheria and yei
may be differentiated from it otherwise than by the test upon sus-
ceptible animals. Neisser has given us a staining method which is
especially useful in making this differential diagnosis. The culture
of the bacillus to be tested is grown upon Loifler's blood-serum mix-
ture. This is solidified at a temperature of 100° C., and grown in
an incubator at a temperature between 34° and 36° C. The staining
of a cover-glass preparation from such a culture is effected by the
following method : Methylene blue, one gramme; alcohol (96°), two
cubic centimetres ; dissolve and add distilled water, nine hundred and
fifty cubic centimetres, and acetic acid, fifty cubic centimetres. From
one to three seconds only will be required to stain the cover-glass
preparation with this solution; it should then be carefully washed in
water and stained in a solution made by adding two grammes of
vesuvin to one litre of boiling water. This solution is allowed to
cool before using, and from three to five seconds will be sufficient

458 BACTERIA IN DIPHTHERIA.

time for the action of the stain, after which the cover glass is again
washed and is then ready for examination. The diphtheria bacillus
appears in such a preparation as faintly stained brown rods, in the
interior of which one to three dark-blue granules may be seen. These
are oval in form and are found at the extremities of the bacterial
cells. Neisser and others who have made use of this method agree
that bacilli which do not stain in this way are not diphtheria bacilli.

BACILLUS DIPHTHERIA COLUMBARUM.

Described by Loffler (1884), who obtained it from diphtheritic pseudo-mem-
branes in the mouths of pig-eons dead from an infectious form of diphtheria
which prevails in some parts of Germany among these birds and among
chickens.

Reddened patches first appear upon the mucous membrane of the mouth
and fauces, and these are covered later with a rather thick, yellowish layer
of fibrinous exudate. In pigeons the back part of the tongue, the fauces,
and the corners of the mouth are especially affected ; in chickens the tongue,
the gums, the nares, the larynx, and the conjunctival mucous membrane.
The disease is especially fatal among chickens, the young fowls and those of
choice varieties being most susceptible. It is attended at the outset by fever,
and usually proves fatal within two or three weeks, but may last for several
months.

Morphology. — Short bacilli with rounded ends, usually associated in ir-
regular masses, and resembling the bacilli of rabbit septicaBmia (fowl
cholera), but a little longer and not quite so broad. In sections from the
liver they are seen in irregular groups in the interior of the vessels.

Biological Characters — An aerobic, non-motile, non-liquefying bacillus.

Grows in nutrient gelatin in the form of spherical, white colonies along
the line of puncture, and upon the surface as a whitish layer. Under the
microscope the colonies in gelatin plates have a yellowish-brown color and
a slightly granular surface. Upon blood serum the growth consists of a
semi-transparent, grayish- white layer. Upon potato SL thin layer is formed
having a grayish tint.

Pathogenesis. — Pigeons inoculated with a pure culture in the mucous
membrane of the mouth are affected exactly as are those which acquire the
disease naturally. Subcutaneous inoculations in pigeons give rise to an in-
flammation resulting in local necrotic changes. Pathogenic for rabbits and
for mice. Subcutaneous injections in mice give rise to a fatal result in about
five days. The bacillus is found in the blood and in the various organs, in
the interior of the vessels, and sometimes in the interior of the leucocytes ;
they are especially numerous in the liver. The lungs are dotted with red
spots, the spleen is greatly enlarged, and the liver has a marbled appearance
from the presence of numerous irregular white masses scattered through the
pale-red parenchyma of the organ. These white masses are seen, in sec-
tions, to consist of necrotic liver tissue, in the centre of which the bacilli
are found in great numbers, in the interior of the vessels. This appearance
is so characteristic that Loffler considers inoculations in mice to be the most
reliable method of establishing the identity of the bacillus. Not pathogenic
for chickens, guinea-pigs, rats, or dogs.

There seems to be some doubt whether the form of diphtheria which pre-
vails among pigeons, and which Loffler has shown to be due to the bacillus
above described, is identical with the diphtheria of chickens. Diphtheria in
man has been supposed by some authors to be identical with that which
prevails among fowls, and possibly this may be the case under certain cir-
cumstances. But the evidence seems to be convincing that there is an

BACTERIA IN DIPHTHERIA. 459

infectious diphtheria of fowls which is peculiar to them, and which, under
ordinary circumstances, is not communicated to man.

BACILLUS DIPHTHERIAS VITULORUM.

Described by Loffler (1884) and obtained by him from the pseudo-mem-
branous exudation in the mouths of calves suffering- from an infectious form
of diphtheria. The disease is characterized by the appearance of yellow
patches upon the mucous membrane of the cheeks, the gums, the tongue,
and sometimes of the larynx and nares of infected animals. There is a yel-
lowish discharge from the nose, an abundant flow of saliva, occasional at-
tacks of coughing, and diarrhoea. Death may occur at the end of four or
five days, but usually the animal survives for several weeks. Diphtheritic
patches similar to those in the mouth are also found in the large intestine,
and scattered abscesses in the lungs.

Loffler, in a series of seven cases examined, obtained from the deeper por-
tions of the pseudo-membranous deposit a long bacillus which appears to be
the cause of the disease.

Morphology. — Bacilli, five to six times as long as broad, usually united in
long filaments. The diameter of the rods is about half that of the bacillus
of malignant oedema.

Biological Characters.— Attempts to cultivate this bacillus in nutrient
gelatin, blood serum from sheep, and various other media were unsuccessful.
But when fragments of tissue containing the bacillus were placed in blood
serum from the calf a whitish border, consisting of the long bacilli, was de-
veloped. These could not, however, be made to grow when transferred to
fresh blood serum.

Pathogenesis. — Mice inoculated subcutaneously with the fresh diph-
theritic exudation died in from seven to thirty days. The autopsy disclosed
an extensive infiltration of the entire walls of the abdomen, which often pene-
trated the peritoneal cavity and enveloped the liver, the kidneys, and the
intestine in a yellowish exudate. The bacillus was found in this exudate,
and by inoculating a little of it into another animal of the same species a
similar result was obtained. Not pathogenic for rabbits or guinea-pigs.

BACILLUS OF INTESTINAL DIPHTHERIA IN RABBITS.

Described by Ribbert (1887) and obtained \>y him from the organs of rab-
bits which, succumbed to an affection characterized by a diphtheritic inflam-
mation of the mucous membrane of the intestine. The autopsy revealed also
swelling of the mesenteric glands and minute iiecrotic foci in the liver and
spleen.

Morphology. — Bacilli with slightly rounded ends, from three to four /*
long and 1 to 1.4 u. in diameter; often united in pairs or in filaments con-
taining several elements.

Stains with the aniline colors, but not so readily in sections as some
other microorganisms. Ribbert recommends staining with aniline-water-
fuchsin solution, washing in water, then placing the sections in methylene
blue solution, and decolorizing in alcohol. Does not stain by Gram's
method.

Biological Characters. — An aerobic, non- liquefy ing (non -motile ?) ba-
cillus. Upon gelatin plates semi-transparent, grayish colonies are formed,
which later have a brownish color; the surface of these is finely granular
and of a pearly lustre. In stick cultures in nutrient gelatin the growth
along the line of puncture is very scanty. On potato a flat, whitish layer is
formed, which extends slowly over the surface. Grows best at a temperature
of 30 to 35° C.

Pathogenesis. — Pure cultures injected into the peritoneal cavity or sub-
cutaneously in rabbits caused the death of these animals in from three to
fourteen days, according to the quantity injected. At the autopsy necrotic

460 BACTERIA IX DIPHTHERIA.

foci are found in the liver and spleen, and the niesenteric glands are en-
larged, but the intestine presents a healthy appearance. But when cultures
are introduced into the alimentary canal the characteristic diphtheritic in-
flammation of the mucous membrane of the intestine is induced. This re-
sult was obtained both by direct injection into the lumen of the intestine
and by injecting cultures into the mouth.

Additional Notes upon Diphtheria and the Diphtheria Bacil-
lus.— C. Frankel (1895) reports that he has repeatedly observed
branching forms of the diphtheria bacillus in cultures upon LCf-
fler's blood-serum medium, and that these branching forms are se<jn
more constantly and in greater numbers in cultures made upon the
surface of hard-cooked albumen from hen's eggs.

The continued presence of virulent diphtheria bacilli in the fauces
of patients who have recovered from the disease, either after the u^e
of the antitoxin or under other treatment, has been demonstrated by
several bacteriologists. Silverschmidt (1895), in forty-five cases
treated by Behring's antitoxic serum, found that the number of ba-
cilli usually diminished some days after the treatment was com-
menced, but that in cases in which complete recovery had taken
place not infrequently virulent bacilli could be obtained many days
(in one case thirty-one days) after convalescence was established.

Escherich (1893) opposes the view that the pseudo-diphtheria bacil-
lus is simply a non- virulent variety of the diphtheria bacillus. He
found this pseudo-diphtheria bacillus in the throats of thirteen out
of three hundred and twenty individuals examined. According to
him there is no evidence that this completely non-virulent pseudo-
diphtheria bacillus ever acquires pathogenic virulence, while attenu-
ated varieties of the true diphtheria bacillus readily recover their
power to produce the toxic products upon which virulence depends.

Sevestre (1895), as a result of researches made by himself and
several other bacteriologists who have made similar investigations,
arrives at the conclusion that :

" First. In a certain number of cases the bacillus of Loftier disap-
pears about the same time as the false membranes; or it may persist
for some time, but ceases to be virulent — in this case it seems to have
undergone modifications and presents the form of short bacilli. . . .

" Second. In another series of cases, less numerous but neverthe-
less considerable, the bacillus persists in a virulent condition for a
longer or shorter time after the apparent cure of the malady. . . .

" Third. The observations collected up to the present time do not
enable us to fix precisely the limits of persistence, but it is not far
out of the way if we place it at several weeks to a month for the
throat. In the nasal fossaa the bacillus often persists for a still
longer time, and its presence commonly coincides with a more or less
abundant discharge from the nose."

BACTERIA IN DIPHTHERIA. 461

Park and Beebe (1894), in an extended research made for the pur-
pose of determining the persistence of the diphtheria bacillus in the
throats of convalescents ('2,500 cultures made), found that in 304 out
of 005 consecutive cases the bacillus disappeared within 3 days after
the disappearance of the exudate; in 170 cases it persisted for 7 days;
in 64 cases for 12 days; in 36 cases for 15 days; in 13 cases for 3
weeks; in 4 cases for 4 weeks; in 2 cases for 9 weeks. Park and
Beebe arrive at the following conclusion with reference to pseudo-
diphtheria bacilli :

u The name pseudo-diphtheria bacillus should be regarded as ap-
plying to those bacilli found in the throat which, though resembling
the diphtheria bacilli in many respects, yet differ in others equally im-
portant. These bacilli are rather short, and more uniform in size
and shape than the typical Loffler bacillus. They stain equally
throughout with the alkaline methyl-blue solution, and produce
alkali in their growths in bouillon. They are found in about one
per cent of the healthy throats in New York City, and seem to have
no connection with diphtheria. They are never virulent."

Park (1894) has shown that virulent diphtheria bacilli are fre-
quently found in the throats of persons who have been associated
with diphtheria patients, although no manifestations of the disease
were visible. It is therefore apparent that infection requires not
only the presence of virulent bacilli, but also of a predisposition to
the disease. This corresponds with the facts relating to other in-
fectious diseases — e.g., tuberculosis, typhoid fever — and among the
probable predisposing causes we may mention " sewer-gas poisoning,"
catarrhal inflammations of the mucous membranes most commonly
involved, inanition, "crowd poisoning," and depressing agencies
generally.

Bacteriologists have given much attention to the question of mixed
infection in diphtheria. Funck (1894) accepts the generally received
view that mixed infections with the diphtheria bacillus and Strepto-
coccus pyogenes are more serious than an uncomplicated diphtheria,
and in an experimental research has attempted to determine whether
this is due to an increased production of the diphtheria bacillus or to
the presence of the streptococcus. His experiments on guinea-pigs
showed that when infected with streptococci these animals did not
prove to be more sensitive to the action of the diphtheria poison
(without living bacilli), and he concludes that the unfavorable influ-
ence of the streptococcus in mixed infections is due to increased patho-
genic activity on the part of the diphtheria bacillus. Bernheim
(1894) found, in his experiments on guinea-pigs, that they suc-
cumbed more rapidly to diphtheria infection when they previously

402 BACTERIA IN DIPHTHERIA.

or simultaneously received an injection of a streptococcus culture —
filtered or unfiltered.

Eesults of Treatment ivith the Antitoxin. — While questions re-
lating to therapeutics are not considered in this manual, a brief note
upon the results of treatment by the serum of immunized animals
may not be out of place. A collective investigation (1895) un-
dertaken by the Deutsche medicinische Wochenschrift gave the
following results: The number of cases collected was 10,312; all of
these occurred between the 1st of October, 1894, and the 1st of April,
1895; 5;883 of these cases were treated with the antitoxin and 4,479
without it. In the first group the mortality was 9.6 per cent, and in
the second group 14.7 per cent. Two thousand five hundred and fifty
six children treated with the antitoxin were between two and ten
years of age; among these the mortality was 4 per cent, while
among children of the same age not treated with the antitoxin the
mortality was 15.2 per cent. Six hundred and ninety-six patients
above ten years of age were treated with a mortality of 1 per cent.

Monod (1895), at a meeting of the Paris Academy of Medicine,
presented the following statistics demonstrating the influence upon
the mortality from diphtheria in France exerted by the antitoxin
since its employment from November, 1894. The following figures
represent the number of deaths from diphtheria during the first six
months in eight years in 108 French cities having a population of
more than 20,000:

1888-94. 1895.

Average. Average.

January 469 205

February 466 187

March 499 155

April 442 160

May.. 417 113

June 333 84

2,656 904

It will be seen from the above statement that during the first six
months in the year 1895 after the introduction of the antitoxin treat-
ment, the number of deaths from diphtheria in the 108 French cities
referred to was 1,552 less than the average for the preceding ten
years, and we are justified in concluding that a considerable propor-
tion of this saving at least is due to this method of treatment.

XI.

BACILLUS OF INFLUENZA.

DISCOVERED by Pfeiffer (1892) in the purulent bronchial secretion,
and by Canon in the blood of patients suffering from epidemic in-
fluenza. Pfeiffer found the bacillus in thirty-one cases examined by
him, and in uncomplicated cases it was present in the purulent bron-
chial secretion in immense numbers and in a pure culture. Canon,
whose independent observations were published at the same time,
examined the blood of twenty influenza patients in stained prepara-
tions, and found the same bacillus in nearly all of them. His method
of demonstrating it is as follows :

The blood is spread upon clean glass covers in the usual way.
After the preparations are thoroughly dry they are placed in abso-
lute alcohol for five minutes. They are then transferred to the fol-
lowing staining solution (Czenzynke's) : concentrated aqueous solu-
tion of methylene blue, forty grammes ; one-half -per-cent solution of
eosin (dissolved in seventy-per-cent alcohol), twenty grammes ; dis-
tilled water, forty grammes. The cover glasses immersed in this
staining solution are placed in an incubating oven at 37° C. for from
three to six hours, after which they are washed with water, dried,
and mounted in balsam. In. successful preparations the red blood
corpuscles are stained red by the eosin, and the leucocytes blue. The
bacillus is seen in these as a short rod, often resembling a diplococcus.
It is sometimes seen in large numbers, but usually only a few rods
are seen after a long search — four to twenty in a single preparation.
In six cases it was found in numerous aggregations containing from
five to fifty bacilli each. In these cases the blood was drawn during
a fall of temperature or shortly after.

^Morphology. — Very small bacilli, having about the same diameter
as the bacillus of mouse septicaemia, but only half as long. Solitary
or united in chains of three or four elements.

Stains with difficulty with the basic aniline dyes — best with
dilute Ziehl's solution, or Loffler's methylene blue solution, with heat.
The two ends of the bacilli are most deeply stained, causing them to
resemble diplococci. Pfeiffer says: "I am inclined to believe that
some of the earlier observers also saw the bacilli described by me,
but that, misled by their peculiar behavior with regard to staining
agents, they described them as diplococci or streptococci/' Do not
stain by Gram's method.

4G4 BACILLUS OF INFLUENZA.

Biological Characters. — An aerobic, non-motile bacillus. Does
not grow in nutrient gelatin at the room temperature. Spore forma-
tion not observed. Upon the surface of glycerin-agar in the incubat-
ing oven very small, transparent, drop-like colonies are developed at
the end of twenty-four hours. These can only be recognized by the
aid of a lens. " A remarkable point about them is that the colonies
always remain separate from each other, and do not, as all other
species known to me do, join together and form a continuous row.
This feature is so characteristic that the influenza bacilli can be
thereby with certainty distinguished from other bacteria" (Kitasato).
On 1.5 per cent sugar-agar the colonies appear as extremely small
droplets, clear as water, often only recognizable with a lens
(Pfeiffer).

In bouillon a scanty development occurs, and at the end of twen-
ty-four hours small, white particles are seen upon the surface, which
subsequently sink to the bottom, forming a white, woolly deposit,
while the bouillon above remains transparent. This bacillus does
not grow at temperatures below 28° 0.

Canon has obtained colonies, resembling those described by Kita-
sato, in cultures from the blood of influenza patients. His cultures
were made upon glycerin-agar in Petri's dishes. Ten or twelve drops
of blood from a puncture made in the finger of the patient, after
sterilization of the surface, were allowed to fall upon the agar medium,
and this was placed in the incubating oven. As the number of ba-
cilli in the blood is small, a considerable quantity is used. The
colonies are visible at the end of twenty-four to forty-eight hours.

The influenza bacillus is quickly destroyed by desiccation ; a
pure culture diluted with water and dried is destroyed with cer-
tainty in twenty hours ; in dried sputum the vitality is retained
somewhat longer, but no growth occurs after forty hours. The
thermal death-point is 60° C. with five minutes' exposure (Pfeiffer
and Beck).

Pathogenesis. — Pfeiffer infers that this is the specific cause of
influenza in man for the following reasons :

1. They were found in all uncomplicated cases of influenza ex-
amined, in the characteristic purulent bronchial secretion, often in
absolutely pure cultures. They were frequently situated in the pro-
toplasm of the pus corpuscles ; in fatal cases they were found to
have penetrated from the bronchial tubes into the peribronchitic tis-
sue, and even to the surface of the pleura, where in two cases they
were found in pure cultures in the purulent exudation.

2. They were only found in cases of influenza. Numerous con-
trol experiments proved their absence in ordinary bronchial ca
tarrh, etc.

PLATE VI.

PATHOGENIC BACTERIA.

FIG. 1. — Bacillus of influenza in bronchial niucus. x 1,000. Photo-
micrograph by Frankel,

FIG. 2. — Bacillus of influenza in bronchial mucus, after the termina-
tion of the febrile period. The bacilli are for the most part in pus cells.
X 1,000. Photomicrograph by Frankel.

FIG. 3. — Bacillus tetani from an agar culture. x 1,000. Photo-
micrograph by Frankel and Pfeiffer.

FIG. 4. — Micrococcus pneumonias crouposse in sputum of a patient
with pneumonia, x 1,000. Stained by Gram's method. Photomicro-
graph by Frankel and Pfeiffer.

FIG. 5. — Micrococcus pneumonise crouposse in blood of rabbit, x
1,000. Photomicrograph made at the Army Medical Museum, Washing-
ton, by Gray.

FIG. 6. — Bacillus of hog cholera, showing flagella. Stained by
Loffler's method. X 1,000. Photomicrograph made at the Army Medi-
cal Museum, Washington, by Gray.

BACILLUS OF INFLUENZA. 465

3. The presence of the bacilli corresponded with the course of the
disease, and they disappeared with the cessation of the purulent
bronchial secretion.

In his preliminary report of his investigations Pfeiffer says :

" Numerous inoculation experiments were made on apes, rabbits,
guinea-pigs, rats, pigeons, and mice. Only in apes and rabbits
could positive results be obtained. The other species of animals
showed themselves refractory to influenza/'

Kruse (1894) reports that he found the bacillus of Pfeiffer in
eighteen influenza patients examined by him in the hospital at Bonn.
On the other hand, he failed to find it in a considerable number of pa-
tients suffering from other diseases of the respiratory passages. His
evidence is the more valuable as he had previously (1890) reported
his failure to find the bacillus in typical cases of influenza. He now
ascribes his failure at that time to imperfect technique.

Huber (1893), Richter (1894), Borchardt (1894), and other com-
petent bacteriologists, have also confirmed the results reported by
Pfeiffer as regards the presence of this bacillus in the bronchial
secretions of persons suffering from epidemic influenza, and as to
its biological characters. Bujwid (1893) recognizes the bacillus of
Pfeiffer as identical with a bacillus which he cultivated from the
spleen of an influenza patient in 1890.

The researches of Weichselbaum, Kowalski, Friedrich, Kruse,
Bouchard, and others have given a negative result as regards the
presence of the influenza bacillus in the blood. They were not able
to demonstrate its presence either in stained preparations or by cul-
ture methods. Pfeiffer, also, during the last epidemic, has made
special researches upon this point and has never succeeded in finding
the bacillus. Day after day, both in mild and severe cases, he placed
from ten to twenty drops of blood from influenza patients on blood-
agar — a most favorable medium — but his cultures always remained
sterile.

In his experiments upon rabbits, Pfeiffer (1893) found that the
intravenous injection of a small quantity of culture on blood-agar,
twenty-four hours old, suspended in one cubic centimetre of bouillon,
caused a characteristic pathogenic effect. The first symptoms were
developed within one and a half to two hours after the injection.
The animals became extremely feeble, lying flat upon the floor with
their limbs extended, and suffered from extreme dyspnoea. The tem-
perature mounted to 41° C. or above. At the end of five or six hours
thejr were able to sit upon their haunches again, and in twenty-four
hours had nearly recovered from all indications of ill-health. Larger
doses caused the death of the inoculated animals. These results are
due to toxic products present in the cultures, and Pfeiffer has never
30

466 BACILLUS OF INFLUENZA.

observed a septicsemic infection as a result of his inoculation ex-
periments.

Pfeiffer has found in three cases of bronchopneumonia a pseudo-
influenza bacillus which closely resembles the bacillus previously de-
scribed by him as peculiar to that disease. This pseudo-influenza
bacillus resembles the genuine one in its growth in culture media,
but is larger and shows a decided inclination to grow out into long
threads. By these morphological characters, which are said to be
constant, it may, according to Pteiffer, be readily distinguished.

XII.
BACILLI IN CHRONIC INFECTIOUS DISEASES.

IN tuberculosis, leprosy, glanders, and syphilis we have a group
of infectious diseases which present many points of resemblance.
All run a chronic course ; all may be communicated to susceptible
animals by inoculation ; in all, the lymphatic glands in the vicinity
of the point of inoculation become enlarged, and new growths, con-
sisting of various cellular elements of a low grade of vitality, are de-
veloped in the tissues which are the point of predilection for each ;
in all, these new growths show a tendency to degenerative changes,
as a result of which abscesses, caseous masses, or open ulcers are
formed.

In two of the diseases in this group — tuberculosis and glan-
ders— the infectious agent has been obtained in pure cultures and its
specific pathogenic power demonstrated by inoculations in susceptible
animals; in one — leprosy — there is but little doubt that the bacillus con-
stantly found in the new growths characteristic of the disease bears
an etiological relation to it, although this has not been demonstrated,
the bacillus not having as yet been cultivated in artificial media.
The evidence with reference to the parasitic nature of the fourth dis-
ease mentioned as belonging to this group — syphilis — is still unsatis-
factory, but there is every reason to believe that it will also eventu-
ally be proved to be due to a parasitic microorganism.

The announcement of the discovery of the tubercle bacillus was
made by Koch, in March, 1882, at a meeting of the Physiological
Society of Berlin. At the same time satisfactory experimental evi-
dence was presented as to its etiological relation to tuberculosis in
man and in the susceptible lower animals, and its principal biologi-
cal characters were given.

Baumgarten independently demonstrated the presence of the tu-
bercle bacillus in tuberculous tissues and published the fact soon
after the appearance of Koch's first paper. The previous demonstra-
tion by Villemin (1865) — confirmed by Cohnheim (1877) and others —
that tuberculosis might be induced in healthy animals by inocula-
tions of tuberculous material, had paved the way for his discovery,

468 BACILLI IN CHRONIC INFECTIOUS DISEASES.

and advanced pathologists were quite prepared to accept it. The
more conservative have since been obliged to yield to the experi-
mental evidence, which has received confirmation in all parts of the
world. To-day it is generally recognized that tuberculosis is a spe-
cific infectious disease due to the tubercle bacillus.

As evidence of the thorough nature of Koch's personal researches
in advance of his first public announcement, we give the following
resume of his investigations :

In nineteen cases of miliary tuberculosis the bacilli were found in
the tubercular nodules in every instance ; also in twenty-nine cases
of pulmonary phthisis, in the sputum, in fresh cheesy masses, and in
the interior of recently formed cavities ; in tuberculous ulcers of the
tongue, tuberculosis of the uterus, testicles, etc. ; in twenty-one cases
of tuberculous — scrofulous — lymphatic glands ; in thirteen cases of
tuberculous joints ; in ten cases of tubercular bone affections ; in four
cases of lupus ; in seventeen cases of Perlsucht in cattle. His ex-
perimental inoculations were made upon two hundred and seventy-
three guinea-pigs, one hundred and five rabbits, forty-four field
mice, twenty-eight white mice, nineteen rats, thirteen cats, and upon
dogs, pigeons, chickens, etc. Very extensive comparative researches
were also made, which convinced him that the bacillus which he had
been able to demonstrate in tuberculous sputum and tissues by a spe-
cial mode of staining was not to be found in the sputa of healthy
persons, or of those suffering from non-tubercular pulmonary affec-
tions, or in organs and tissues involved in morbid processes of a
different nature.

BACILLUS TUBERCULOSIS.

Discovered by Koch (first public announcement of discovery
March 24th, 1882). The bacilli are found in the sputum of persons
suffering from pulmonary or laryngeal tuberculosis, either free or in
the interior of pus cells ; in miliary tubercles and fresh caseous
masses, in the lungs or elsewhere ; in recent tuberculous cavities in
the lungs ; in tuberculous glands, joints, bones, and skin affections
(lupus) ; in the lungs of cattle suffering from pulmonary tubercu-
losis— Perlsucht ; and in tubercular nodules generally in animals
which are infected naturally or by experimental inoculations.

In the giant cells of tubercular growths they have a peculiar and
characteristic position, being found, as a rule, upon the side of the
cell opposite to the nuclei, which are crowded together in a crescentic
arrangement at the opposite pole of the cell. Sometimes a single
bacillus will be found in this position, or there may be several.
Again, numerous bacilli may be found in giant cells in which the
nuclei are distributed around the periphery. They are more numer-

BACILLI IN CHRONIC INFECTIOUS DISEASES. 469

ous in tuberculous growths of recent origin, and often cannot be
demonstrated, by microscopical examination, in caseous material
from the centre of older nodules. But such material, when inocu-
lated into susceptible animals, gives rise to tuberculosis, and the
usual inference is that it contains spores of the tubercle bacillus.

Morphology. — The tubercle bacilli are rods with rounded ends,
of from 1.5 to 3.5 ^ in length, and are commonly slightly curved or
bent at an angle ; the diameter is
about 0.2 //. In stained preparations
unstained portions are frequently
seen, which are generally believed to
be spores, but this is by no means
certain. From two to six of these
unstained spaces may often be seen
in a single rod, and owing to this al-
ternation of stained and unstained
portions the bacilli may, under a low
power, be mistaken for chains of mi-
crococci The rods are usually soli-
tary, but may be united in pairs, or

. V i • • • * Fj°- m- — Bacillus tuberculosis.

m short chains containing three or four x 1,000. From a photomicrograph,
elements. In old cultures irregular

forms may be observed, the rods being sometimes swollen at one
extremity, or presenting the appearance of having a lateral bud-like
projection — involution forms.

The staining characters of this bacillus are extremely important
for its differentiation and recognition in preparations of sputum, etc.
Unlike most microorganisms of the same class, it does not readily
take up the aniline colors, and when stained it is not easily decolorized,
even by the use of strong acids. The failure to observe it in tuber-
culous material, prior to Koch's discovery, was no doubt due to the
fact that it does not stain in the usual aqueous solutions of the aniline
dyes. Koch first recognized it in preparations placed in a staining
fluid to which an alkali had been added — solution of methylene blue
with caustic potash ; but this method was not very satisfactory, and
he promptly adopted the method devised by Ehrlich, which consists
essentially in the use of a solution of an aniline color — fuchsin or
methyl violet — in a saturated aqueous solution of aniline oil, and de-
colorization with a solution of a mineral acid — nitric acid one part to
three parts of water.

The original method of Ehrlich gives very satisfactory results,
but various modifications have since been proposed, some of which
are advantageous. The carbol-fuchsin solution of Ziehl is now
largely employed ; it has the advantage of prompt action and of

470 BACILLI IN CHRONIC INFECTIOUS DISEASES.

keeping well. The staining is effected more quickly if heat is ap-
plied. The tubercle bacilli stain by Gram's method, but this is not
to be recommended for general use, owing to the fact that the pro-
toplasm of the rods is frequently contracted into a series of spheri-
cal, stained bodies, which might easily be mistaken for micrococci.

The examination of sputum for the presence of the tubercle ba-
cillus is recognized as a most important procedure for the early diag-
nosis of pulmonary tuberculosis. It is at-
tended with no special difficulties, and every
physician should be acquainted with the
technique.

The patient should be directed to expec-
torate into a clean, wide-mouthed bottle or
glass-covered jar the material coughed up
from the lungs, and especially, in recent
cases, that which is coughed up upon first
rising in the morning. This should be
FIQ. us.— Bacillus tubercuio- placed in the physician's hands as promptly
s^n sputum, x i.ooo. (Baum- as possibie ; although a delay of some days

does not vitiate the result, and the tubercle

bacilli may still be demonstrated after the sputum has undergone pu-
trefaction. It is well to pour the specimen into a clean, shallow vessel
having a blackened bottom — a Petri's dish placed upon a piece of dead-
black paper will answer very well. In tuberculous sputum small, len-
ticular masses of a yellowish color may usually be observed, and one
of these should be selected for microscopical examination, by picking
it up with a platinum needle and freeing it as far as possible from
the tenacious mucus in which it is embedded. If such masses are
not recognized take any purulent-looking material present in the
specimen, whether it be in small specks distributed through the mu-
cus, or in larger masses. A little of the selected material should be
placed in the centre of a clean cover glass and another thin glass
cover placed over it. By pressure and a to-and-fro motion the mate-
rial is crushed and distributed as evenly as possible ; the glasses are
then separated by a sliding motion. The film is permitted to dry by
exposure in the air. When dry the cover glass, held in forceps, is
passed three times through the flame of an alcohol lamp or Bun sen
burner to fix the albuminous coating. Too much heat causes the film
to turn brown and ruins the preparation. The staining fluid (ZiehFs
carbol-f uchsin) may then be poured upon the cover glass, or this may
be floated upon the surface of the fluid contained in a shallow watch
glass. Heat is now applied by bringing the cover glass over a
flame and holding it there until steam begins to be given off from
the surface of the staining fluid ; it is then withdrawn and again

BACILLI IN CHRONIC INFECTIOUS DISEASES. 4-71.

gently heated at intervals for a minute or two. The cover glass is
then washed in water, and the film will be seen to have a uniform
deep-red color. The next step consists in decolorization in the acid
solution (twenty-five-per-cent solution of nitric or of sulphuric acid).
The cover glass is gently moved about in this solution for a few
seconds, and the color will be seen to quickly fade to a greenish
tint. The object is to remove all color from the cells and the al-
buminous background, so that the bacilli, which retain their color in
presence of the acid, may be clearly seen. The preparation is next
washed in dilute alcohol (sixty per cent) to remove the fuchsin
which has been set free by the acid. If decolorization was not car-
ried far enough the film will be seen to still have a red color, espe-
cially in places where it is thickest, when it is removed from the
dilute alcohol and washed out in water. In this case it will be
necessary to return it to the acid solution and again wash it in the
dilute alcohol and in water. It may now be placed in a solution
of methylene blue or of vesuvin for a contrast stain. The tubercle
bacilli are distinguished by the fact that they retain the red color
imparted to them in the fuchsin solution, while other bacteria pre-
sent, having been decolorized in the acid solution, take the contrast
stain and appear blue or brown, according to the color used. The
double-stained preparation, after a final washing in water, may be
examined at once, or dried and mounted in balsam for permanent
preservation.

Of the various other methods which have been proposed, that of
Frankel, as modified by Gabbett, appears to be the most useful. This
consists in staining as above directed with ZiehPs carbol-f uchsin solu-
tion, and in then placing the cover glass directly in a second solution
which contains both the acid for decolorizing and the contrast stain.
This second solution contains twenty parts of nitric acid, thirty parts
of alcohol, fifty parts of water, and sufficient methylene blue to make
a saturated solution (one to two parts in one hundred). After re-
maining in this solution for a minute or two the cover glas& is washed
in water, and upon microscopical examination the tubercle bacilli, if
present, will be seen as red rods which strongly contrast with the
blue background.

The methods recommended for cover-glass preparations may also
be used for staining the tubercle bacillus in thin sections of tuber-
culous tissues, except that it is best not to employ heat. The sec-
tions may be left for an hour in the carbol-f uchsin solution, or for
twelve hours in the Ehrlich-Weigert tubercle stain — eleven cubic
centimetres of saturated alcoholic solution of methyl violet, ten cubic
centimetres of absolute alcohol, one hundred cubic centimetres of ani-
line water. They should then be decolorized by placing them for

472

BACILLI IN CHRONIC INFECTIOUS DISEASES.

about half a minute in dilute nitric acid (ten per cent); then wash
out color in sixty-per-cent alcohol ; counter-stain for two or three
minutes in a saturated aqueous solution of methylene blue ; dehydrate
with absolute alcohol or with aniline oil ; clear up in oil of cedar,
and mount in xylol balsam. If the aniline-water-methyl-violet solu-
tion has been used for staining the bacilli a saturated solution of
vesuvin may be used as a contrast stain.

Biological Characters. — A parasitic, aerobic, non-motile ba-
cillus, which grows only at a temperature of about 37° C. Is also a
facultative anaerobic (Frankel).

The question as to spore formation has not been definitely deter-
mined. It has been generally assumed that the unstained spaces
which are frequently seen in the bacilli are spores ; and the fact that

FIG. 116.— Section through a tuberculous nodule in the lung of a cow, showing two giant cells
containing tubercle bacilli. X 950. (Baumgarten.)

caseous material in which a microscopical examination has failed to
demonstrate the presence of bacilli may produce tuberculosis, with
bacilli, when inoculated into guinea-pigs, has been explained upon the
supposition that this material contained spores. But a few bacilli
present in such caseous material might easily escape detection. As
pointed out by Frankel, the oval spaces in stained specimens have
not the sharply defined outlines of spores. Moreover, the bacilli, when
examined in unstained preparations, do not contain corresponding re-
fractive bodies, recognizable as spores. And when the bacilli are
stained by Gram's method the protoplasm is often contracted in the
form of little, spherical stained masses, while the unstained spaces
are larger and no longer have the oval form presented in rods stained
by Ehrlich's method. The great resisting power of the bacillus to
heat and to desiccation has been supposed to be due to the presence

BACILLI IX CHRONIC INFECTIOUS DISEASES. 473

of spores. But, so far as resistance to heat is concerned, this is not
so great as was at one time believed. Schill and Fischer (1884), as-
suming that the tubercle bacillus forms spores, made quite a number
of experiments to determine its thermal death-point. They sub-
jected sputum containing the bacillus to a temperature of 100° C., and
tested the destruction of vitality by inoculations into guinea-pigs.
Exposure to steam at a temperature of 100° C. for two to five min-
utes was effective in every experiment, with one exception. One
guinea-pig died tuberculous after having been inoculated with
sputum exposed to this tamperature for two minutes. This result
was assumed to show that the bacillus would survive lower tempera-
tures, but it is evident that additional experiments were required to
establish this fact. In 1887 the writer made a few similar experi-
ments at a lower temperature, and guinea-pigs inoculated with tuber-
culous sputum exposed for ten minutes to a temperature of 90°, 80°,
and 60° C. failed to become tuberculous, while another guinea-pig,
inoculated with the same material after exposure to a temperature of
50° C. for ten minutes, died tuberculous. These results correspond
with those subsequently (1888) reported by Yersin, who tested the
thermal death-point of this bacillus by the culture method. This
author assumes that the bacilli form spores, but states as a result of
his experiments that "at the end of ten days bacilli heated for ten
minutes at 55° C. gave a culture in glycerin-bouillon ; those heated
to 00°, at the end of twenty-two days; while those heated to 70° and
above failed to grow in every instance. This experiment, repeated a
great number of times, always gave the same result. The tubercle
bacilli then resist a temperature of 60° C. for ten minutes, and it is
to be remarked that the resistance of spores to heat appears to be no
greater than that of the bacilli themselves." Yersin remarks in a
footnote that "the spores which served for these experiments did
not appear as more or less irregular granules taking the coloring
matter strongly, but as veritable spores with sharply defined outlines,
to the number of one or two in a bacillus, or three at the outside.
These spores are particularly clear in cultures upon glycerin-agar
several weeks old."

It may be that bacteriologists have been mistaken in the infer-
ence that all spores possess a greater resisting power for heat than
that exhibited by bacilli in the absence of spores. That this is true
as regards anthrax spores and many others, the thermal death-point
of which has been determined by exact experiments, does not prove
that it is true for all. And it is known that there are wide differ-
ences in the resisting power both of the spores of different species
and in the vegetating cells. To admit that the tubercle bacillus or
the typhoid bacillus, etc., may form spores which have no greater

474 BACILLI IN CHRONIC INFECTIOUS DISEASES.

resisting power against heat than the bacilli themselves, would there-
fore simply be an admission that soma bacteriologists had made a
mistaken inference based upon incomplete data. In view of the
facts stated we can simply repeat what was said at the outset, viz. ,
the question as to spore formation has not been definitely deter-
mined.

The tubercle bacillus is a strict parasite, and its biological char-
acters are such that it could scarcely find natural conditions, outside
of the bodies of living animals, favorable for its multiplication. It
therefore does not grow as a saprophyte under ordinary circum-
stances. But it has been noted by Roux and Nocard that when it
has been cultivated for a time in artificial media containing glycerin
it may grow in a plain bouillon of veal or chicken, in which media it
fails to develop when introduced directly from a culture originating
from the body of an infected animal. This would indicate the pos-
sibility of its acquiring the ability to grow as a saprophyte ; and we
can scarcely doubt that at some time in the past it was a true sapro-
phyte. The experiments of Nuttall indicate that the bacillus may
multiply, under favorable temperature conditions, in tuberculous
sputum outside of the body. And it is extremely probable that mul-
tiplication occurs in the muco-purulent secretion which accumulates
in pulmonary cavities in phthisical patients. In these cavities its de-
velopment may, in a certain sense, be regarded as saprophytic, as it
feeds upon non-living organic material.

Koch first succeeded in cultivating this bacillus upon coagulated
blood serum, prepared as directed in Section VIII., Part First, of the
present volume. Roux and Nocard have since shown (1888) that it
grows very well on nutrient agar to which glycerin has been added
(six to eight per cent), and also in veal broth containing five per cent
of glycerin. It is difficult to obtain pure cultures from tuberculous
sputum, on account of the presence of other bacteria which grow
much more rapidly and take full possession of the medium before the
tubercle bacillus has had time to form visible colonies. For this rea-
son it is best to first inoculate a guinea-pig with the tuberculous spu-
tum and to obtain cultures from it after tuberculous infection has
fully developed. The inoculated animals usually die at the end of
three or four weeks. It is best to kill one which gives evidence of
being tuberculous, and to remove one or more nodules from the
lungs through an opening made in the chest walls. The greatest
care will be required to prevent contamination by other common
microorganisms. The instruments used must be sterilized by heat,
and the skin over the anterior thoracic wall carefully turned back ;
then, after again sterilizing knives and scissors, cut an opening into
tha chest cavity, draw out the root of the lung, and take up with

BACILLI IX CHRONIC INFECTIOUS DISEASES.

slender sterilized forceps, or with a strong platinum loop, one or
more well-defined tubercular nodules. These may be conveyed di-
rectly to the surface of the solid culture medium and then broken
up and rubbed over the surface as thoroughly as possible ; or they
may first be crushed between two sterilized glass slides, and then
transferred with the platinum loop and thoroughly rubbed into the
surface of the culture medium.

This breaking-up of the tuberculous nodules and distribution of
the bacilli upon the surface of the culture medium is essential for
the success of the experiment. Instead of using the tubercular
nodules in the lungs, an enlarged lymphatic gland from the axilla or
elsewhere may be used, as first recommended by Koch. This is to
be crushed in the same way ; and it will be best to inoculate a num-
ber of tubes at the same time, as accidental contamination or failure
to develop is very liable to occur in a certain number. Owing to the
liability of the blood serum to become too dry for the development of
the bacillus, it is best to keep the cultures in a moist atmosphere, or
to prevent evaporation by applying a rubber cap over the open end
of the test tube. This should be sterilized in a solution of mercuric
chloride (1 : 1,000) ; and the end of the cotton plug should be burned
off just before applying it, for the purpose of destroying the spores
of mould fungi, which in a dry atmosphere would be harmless, but
under the rubber cap are likely to sprout and to send their mycelium
through the cotton plug to the interior of the tube, thus destroying
the culture.

Upon coagulated blood serum the growth first becomes visible at
the end of ten to fourteen days (at 37° C.), and at the end of three
weeks a very distinct and characteristic develop-
ment has occurred. The first appearance is that of
dry-looking, grayish- white points and scales, which
are without lustre, and are sometimes united to
form a thin, irregular, membranous-looking layer.
Under the microscope, with an amplification of
eighty diameters, the early, thin surface growth
upon blood serum presents a characteristic appear-
ance. The bacilli, arranged in parallel rows, form
variously curved figures, of which we may obtain
impressions by carefully applying a dry cover glass Fl(r: f117'~ Tufbe ^
to the surface. Upon staining the preparation in culture upon blood se-
the usual way the same arrangement of the bacilli rum- x 500- (Koch-)
which adhered to the thin glass cover will be pre-
served. The growth is more abundant in subsequent cultures,
which have been kept up in Koch's laboratory from his original
pure cultures up to the present time ; in these the bacillus still pre-

476 BACILLI IN CHRONIC INFECTIOUS DISEASES.

serves its characters of form and growth, and its specific pathogenic
power.

Pastor (1892) has succeeded in obtaining pure cultures of the
tubercle bacillus from sputum by the following ingenious method :
After proving by microscopic examination that the sputum of a
tuberculous individual contains numerous bacilli, he has the patient
cleanse his mouth as thoroughly as possible with sterilized water,
and then expectorate some material, coughed up from the lungs, into
a sterilized test tube. By shaking with sterilized water a fine emul-
sion is made, and this is filtered through fine gauze. The filtrate,
which is nearly transparent, contains numerous tubercle bacilli. A
few drops of the emulsion are now added to liquefied gelatin in a test
tube, and a plate is made in the usual way. This is kept for three
or four days at the room temperature, during which time the com-
mon mouth bacteria capable of growth form visible colonies. By
means of a hand lens a place is now selected in which no colonies are
seen, and a bit of gelatin is excised with a sterilized knife. This
piece is transferred to the surface of blood serum or glycerin- agar,
and placed in the incubating oven, where in due time colonies of
the tubercle bacillus will usually be found to develop.

Another method of accomplishing the same result has been
described by Kitasato. This is a method devised by Koch some time
since and successfully employed in his laboratory. The morning
expectoration of a tuberculous patient, raised from the lungs by
coughing, is received in a Petri's dish. A bit of sputum, such as
comes from the tuberculous cavity in the lungs of such a patient, is
now isolated with sterilized instruments and carefully washed in at
least ten successive portions of sterilized water. By this procedure
the bacteria accidentally attached to the viscid mass of sputum dur-
ing its passage through the mouth are washed away. In the last
bath the mass is torn apart and a small portion from the interior is
used to make a microscopic preparation, the examination of which
shows whether only tubercle bacilli are present. If this be the case
cultures upon glycerin-agar are started from material obtained from
the interior of the same mass. The colonies obtained in this way
appear in about two weeks as round, white, opaque, moist, and shin-
ing masses. Kitasato's researches show that the greater portion of
the tubercle bacilli in sputum obtained in this way, and in the con-
tents of lung cavities, are incapable of development, although this
fact cannot be recognized by a microscopic examination of stained
specimens.

On account of the greater facility of preparing and sterilizing
glycerin-agar, and the more rapid and abundant development upon
this medium, it is now usually employed in preference to blood

BACILLI IN CHRONIC INFECTIOUS DISEASES.

47:

Serum. The growth at the end of fourteen days is more abundant than
upon blood serum at the end of several weeks. When numerous
bacilli have been distributed over the surface of the culture medium
a rather uniform, thick, white layer, which subsequently acquires a
yellowish tint, is developed ; when the bacilli
are few in number or are associated in scattered
groups separate colonies are developed, which
acquire considerable thickness and have more
or less irregular outlines ; they are white at
first, then yellowish-white. Frankel describes
the tubercle bacillus as a facultative anaerobic,
and it would appear that it must be able to grow
in situations where it can obtain very little oxy-
gen from its development in the interior of tu-
berculous nodules, lymphatic glands, etc. But
in stick cultures in glycerin-agar development
only occurs near the surface, and not at all in
the deeper portion of the medium. In view of
its abundant growth on the surface it is diffi-
cult to understand this failure- to grow along
the line of puncture, if it is in truth a faculta-
tive anaerobic.

In peptonized veal broth containing five per
cent of glycerin the bacillus develops at first in
the form of little flocculi, which accumulate at
the bottom of the flask and which by agitation
are easily broken up. At the end of two or
three weeks the bottom of the flask is covered
with similar flocculi, which form an abundant
deposit.

Pawlowski and others report success in cul-
tivating the tubercle bacillus upon the Surface
of cooked potato enclosed in a test tube after
the method of Bolton andRoux. The open end
of the tube is hermetically sealed in a flame
after the bacilli have been planted upon the
obliquely-cut surface of the potato ; this prevents drying. Ac-
cording to Pawlowski, better results are obtained if the surface of
the potato is moistened with a five-per-cent solution of glycerin. The
growth is said to be seen at the end of about twelve days as grayish,
dry-looking flakes ; at the end of three or four weeks it forms a dry,
smooth, whitish layer, and no further development occurs.

The range of temperature at which this bacillus will grow is
very restricted ; 37° C. is usually given as the most favorable point,

FIG. 118.— Culture of tu-
bercle bacillus upon glyce-
rin-agar. Photograph by
Roux.

478 BACILLI IN CHRONIC INFECTIOUS DISEASES.

but Roux and RTocard say that the most favorable temperature ap-
pears to be 39°, and that development is slower at 37°.

The experiments of Koch, Schill and Fischer, and others show
that the bacilli retain their vitality in desiccated sputum for several
months (nine to ten months — De Toma) ; but they are said to undergo
a gradual diminution in pathogenic virulence,' which is more rapid
when the desiccated material is kept at a temperature of 30° to 40° C.
In the experiments of Cadeac and Malet portions of the lung from
a tuberculous cow, dried and pulverized, produced tuberculosis in
guinea-pigs at the end of one hundred and two days. They retain
their vitality for a considerable time in putrefying material (forty-
three days — Schill and Fischer ; one hundred and twenty days — Ca-
deac and Malet). The resisting power of this bacillus against ger-
micidal agents is also greater than that of certain other pathogenic
microorganisms, but not so great as to justify the inference that it
forms spores. It is not destroyed by the gastric juice in the sto-
mach, as is shown by successful infection experiments in suscep-
tible animals, by mixing cultures of the bacillus with their food
(Baumgarten, Fischer), and also by experiments with an artificially
prepared gastric juice (Falk). They are destroyed, in sputum, in
twenty hours by a three-per-cent solution of carbolic acid, even
when they present the appearance usually ascribed to the presence
of spores (Cavagnis) ; also by absolute alcohol, a saturated aqueous
solution of salicylic acid, saturated aniline water, etc. (Schill and
Fischer). The more recent experiments of Yersin upon pure cul-
tures of the bacillus gave the following results : " Tubercle bacilli,
containing spores, were killed by a five-per-cent solution of carbolic
acid in thirty seconds, by one-per-cent in one minute ; absolute alco-
hol, five minutes ; iodoform-ether, one per cent, five minutes ; ether,
ten minutes ; mercuric chloride, 1 : 1,000 solution, ten minutes ;
thymol, three hours ; salicylic acid, 2. 5 per cent, six hours.

The tubercle bacillus appears to be especially susceptible to the
action of light. In his address before the Tenth International Medi-
cal Congress (Berlin, 1890) Koch says that when exposed to direct
sunlight the tubercle bacillus is killed in from a few minutes to sev-
eral hours, according to the thickness of the layer ; it is also de-
stroyed by diffuse daylight in from five to seven days when placed
near a window. This fact has an important hygienic bearing, espe-
cially in view of the fact that the tubercle bacillus is not readily
killed by desiccation, putrefaction of the material containing it, etc.
Tuberculous sputum expectorated upon sidewalks, etc., being ex-
posed to the action of direct sunlight, will in many cases be disin-
fected by this agent by the time complete desiccation has occurred —
i.e., before it is in a condition to be carried into the air as dust.

BACILLI IN CHRONIC INFECTIOUS DISEASES.

479

Sawizky in 1891 made a series of experiments to determine
the length of time during which dried tuberculous sputum retains
its virulence. He arrived at the conclusion that virulence is not sud-
denly but gradually lost, and that in an ordinary dwelling room
dried sputum retains its specific infectious power for two and one-
half months.

Tizzoni and Cattani (1892) have presented some experimental evi-
dence which indicates that injections of Koch's tuberculin into
guinea-pigs may produce in these animals a certain degree of im-
munity against tuberculosis; and that this immunity depends upon
the presence of an anti-tuberculin formed in the body of the partially
immune animal.

Numerous experiments made by veterinary surgeons upon tuber-
culous cows show that the injection of Koch's tuberculin in these
animals, in doses of thirty to forty centigrammes, produces a rise of
temperature of from 1° to 3° C. The febrile reaction usually occurs
in from twelve to fifteen hours after the injection. Its duration and
intensity do not depend upon the extent of the tuberculous lesions,
but is even more marked when these are slight than in advanced
cases. In non-tuberculous animals no reaction occurs, and the ex-
periments made justify the suspicion that tuberculosis exists if an
elevation in temperature of a degree or more occurs as a result of
the subcutaneous injection of the dose mentioned.

When the number of tubercle bacilli in sputum is comparatively
small they may easily escape observation. Methods have therefore
been suggested for finding them under these circumstances. Ribbert
(1886) proposed the addition to the sputum of a two-per-cent solution
of caustic potash, and boiling the mixture. The tenacious mucus is
dissolved, and when the mixture is placed in a conical glass vessel
the bacilli are deposited at the bottom and may easily be found in
the sediment after removing the supernatant fluid. The same object
is accomplished by Stroschein (1889) by the addition to sputum of
three times its volume of a saturated solution of borax and boracic
acid in water.

A method of estimating the number of bacilli in sputum has
been proposed by Nuttall, which appears to give sufficiently ac-
curate results and to be useful in judging of the progress of a
case or of the results of treatment. For the details of this method
we must refer to the author's paper (Johns Hopkins Hospital Bulle-
tin, vol. xi., No. 13, 1891). It consists essentially in first making
the sputum fluid by the addition of a solution of caustic potash ; in
then shaking it thoroughly in a bottle containing sterilized gravel
or pounded glass ; in carefully measuring the total quantity of fluid,
and in dropping upon glass slides uniform drops by means of a grad-

480

BACILLI IN CHRONIC INFECTIOUS DISEASES.

uated pipette ; in spreading these uniformly by means of a platinum
needle and a turn table ; in covering the dried film with a film of
blood serum, and coagulating this by heat ; and, finally, in staining
and counting the bacilli in a series of slides from the same specimen,
and from the average number found in a single drop estimating the
total number in the sputum for twenty -four hours.

Pathogenesis. — Man, cattle, and monkeys are most subject to
contract the disease naturally, and it may be communicated by in-
oculation to many of the lower animals — guinea-pigs, field mice, rab-

FIG. 119.— Limited epithelioid celled tubercle of the iris. X 950. (Bautn^arten >

bits, and cats are among the most susceptible animals ; and in larger
doses dogs, rats, white mice, and fowls may also be infected.

When tuberculous sputum is introduced beneath the skin of a
guinea-pig the nearest lymphatic glands are found to be swollen at
the end of two or three weeks, at the same time there is a thickening
of the tissues about the point of inoculation ; later a dry crust forms
over the local tuberculous tumefaction, and beneath this is a flattened
ulcer covered with cheesy material. The animals become emaciated
and show difficulty in breathing, and usually succumb to general
tuberculosis, especially involving the lungs, within four to eight
weeks. Injections of tuberculous sputum, or of pure cultures of the
bacillus, into the peritoneal cavity give rise to extensive tuberculo-
sis of the liver, spleen, and lungs, and to death, as a rule, within
three or four weeks. Rabbits are less susceptible to subcutaneous

BACILLI IX CHRONIC INFECTIOUS DISEASES. 481

injections, but die within seventeen to twenty clays when virulent-
recent — cultures are injected into the circulation. As a result of
such an inoculation the animal : apidly loses flesh and has a decided
elevation of temperature, commencing at the end of the first week
and increasing considerably during the last days of life. At the
autopsy the spleen and liver are found to be greatly enlarged, but
they do not contain any tubercles that can be recognized by the naked
eye (Yersin). They contain, however, great numbers of tubercle
bacilli, both free and in the cells. Injections of a small quantity of
a pure culture into the anterior chamber of the rabbit's eye cause
first iris-tuberculosis, f ollowed by swelling and caseation of the near-
est lymph glands, and finally general infection and death ; when
larger quantities are injected general tuberculosis is quickly devel-
oped. The influence of quantity — number of bacilli — is also shown
in subcutaneous, intravenous, or intraperitoneal injections into guinea-
pigs and rabbits (Hirschberger, G-ebhardt, Wyssokowitsch). Thus
rabbits which received less than one hundred and fifty bacilli, in
sputum, in the experiments of Wyssokowitsch, did not develop tuber-
culosis ; and in guinea-pigs the smaller the number injected the more
protracted the course of the disease was found to be.

Tuberculosis in man no doubt results, in a large proportion of the
cases, from the respiration, by a susceptible individual, of air con-
taining the tubercle bacillus in suspension in a desiccated condition.
As already stated, it has been demonstrated by experiment that the
bacillus retains its vitality in desiccated sputum for several months.
The experiments of Cornet have demonstrated that in the dust of
apartments occupied by tuberculous patients tubercle bacilli are very
commonly present in sufficient numbers to induce tuberculosis in
guinea-pigs inoculated in the peritoneal cavity with such dust, while
negative results were obtained from inoculations with dust from
other localities. In view of these facts the usual mode of infection
is apparent. Infection may also occur through an open wound or
abrasion of the skin, as in the small, circumscribed tumors which
sometimes develop upon the hands of pathologists as a result of
handling tuberculous tissues. A few instances of accidental inocu-
lation through wounds made by glass or earthen vessels containing
tuberculous sputum have also been recorded. A more common mode
of infection, especially in children, is probably by way of the intesti-
nal glands, from the ingestion of the milk of tuberculous cows. That
infection may occur by way of the intestine has been proved by ex-
periments upon rabbits, which develop tuberculosis when fed upon
tuberculous sputum. And that the tubercle bacillus is frequently, if
not usually, present in the milk of tuberculous cows has been proved
by the experiments of Bellinger, Hirschberger, Ernst, and others.
31

482 BACILLI IN CHRONIC INFECTIOUS DISEASES.

In Hirschberger's investigations milk from tuberculous cows in-
duced tuberculosis in guinea-pigs, when .injected subcutaneously or
into the peritoneal cavity, in fifty-five per cent of the cases studied
(twenty). The conclusion is reached that the milk may contain tu-
bercle bacilli even when the udder of the cow is not involved. Ernst
also, from an examination of the milk from thirty-six tuberculous
cows in which the udder was apparently not involved, found the
tubercle bacillus by microscopical examination in five per cent of the
samples examined (one hundred and fourteen).

The prevalence of tuberculosis among cattle is shown by numer-
ous investigations, and especially by the official inspections of
slaughtered animals made in Germany. Thus in Saxony, in the
year 1889, of 611,511 cattle examined 6,135 were found to be tubercu-
lous (about one per cent) ; in Berlin, 1887-1888, out of 130,733 ani-
mals slaughtered 4,300 were found to be tuberculous (3.2 per cent).
In view of the facts stated the great mortality from tubercular dis-
eases among children, many of whom are removed from other prob-
able sources of infection, is not difficult to understand, and the
practical and simple method of preventing infection in this way, af-
forded by the sterilization (by heat) of milk used as food for infants,
must commend itself to all.

BACILLUS TUBERCULOSIS GALLINARUM.

The researches of Maffucci (1889) and of Cadiot, Gilbert, and
Roger (1890) show that the bacillus obtained from spontaneous tu-
berculosis in chickens, although closely resembling the bacillus of
human tuberculosis, is not identical with it, varying especially in its
pathogenic power. This view is sustained by the observations of
Koch, who says in his address before the Tenth International Medi-
cal Congress (Berlin, 1890) :

"The care which it is necessary to exercise in judging of the characters
which serve to differentiate bacteria, even those which are well known, I
have learned in the case of the tubercle bacillus. This species is so definitely
characterized by its staining reactions, its growth in pure cultures, and its
pathogenic qualities, and indeed by each of these characters, that it seems
impossible to confound it with other species. Nevertheless in this case also
one should not rely upon a single one of the characters mentioned for de-
termining the species, but should follow the safe rule that all available
characters should be considered, and the identity of a certain bacterium
should only be regarded as demonstrated when it has been shown to corre-
spond in all of these particulars When I made my first researches with
reference to the tubercle bacillus I was controlled by this rule, and tested
tubercle bacilli from various sources, not only with reference to their stain-
ing reactions, but also with reference to their growth in culture media and
pathogenic characters. Only in the tuberculosis of chickens I was not able
to apply this rule, as at that time it was not possible for me to obtain fresh
material from which to make pure cultures. As, however, all other forms

BACILLI IN CHRONIC INFECTIOUS DISEASES. 483

of tuberculosis had given identical bacilli, and the bacilli of chicken tuber-
culosis in their appearance and behavior towards the aniline colors entirely
corresponded with these, I believed myself justified in assuming their iden-
tity, notwithstanding the incompleteness of the research. Later I received
pure cultures from various sources, which apparently originated from tuber-
cle bacilli, but in several regards differed from these ; especially in the fact
that inoculation experiments, made by experienced and reliable investigators,
led to dissimilar results, which it was necessary to regard as unexplained con-
tradictions. At first I believed that these differences depended upon changes
such as are frequently observed in pathogenic bacteria, when these are culti-
vated in pure cultures outside of the body for a long time under more or less
unfavorable conditions. In order to solve the riddle I attempted by various
influences to change the common tubercle bacilli into the presumed variety
referred to. They were cultivated for several months at so high a tempera-
ture that only a scanty growth was obtained; in other experiments still
higher temperatures were allowed to act repeatedly for so long a time that
the cultures were brought as nearly as possible to the point of killing the
bacilli. In a similar way I subjected the cultures to the action of chemical
agents, of light, or absence of moisture ; they were cultivated for many gen-
erations in association with other bacteria ; inoculated successively in ani-
mals having but a slight susceptibility. But, in spite of all these attempts,
only slight variations were obtained in their characters — far less than other
pathogenic bacteria undergo under similar circumstances. Itappears, there-
fore, that the tubercle bacilli retain their characters with special obstinacy ;
this is in accord with the fact that pure cultures which have now been cul-
tivated by me in test tubes for more than nine years, without in the mean-
time having been in a living body, are still entirely unchanged with the ex-
ception of a slight diminution of virulence. ... It happened about a year
ago that I received a living chicken which was suffering from tuberculosis,
and I used this opportunity to make cultures directly from the diseased or-
gans of this animal, which previously I had not been able to do. When the
cultures grew I saw to my surprise that they had precisely the appearance
and all of the characters possessed by the enigmatical cultures resembling
those of the genuine tubercle bacillus. Later I learned that these also ori-
ginated from tuberculosis in fowls, but, upon the assumption that all forms
of tuberculosis are identical, had been considered genuine tubercle bacilli.
A verification of my observations I find in the recently published researches
of Prof. Maffucci with reference to tuberculosis of fowls."

According to Maffucci, adult chickens are refractory against the
action of the Bacillus tuberculosis from man, and there are slight
morphological and biological differences in the bacilli from the two
sources.

Cadiot, Gilbert, and Roger (1891) have made a series of experi-
ments with the bacillus of tuberculosis in fowls. They found
the bacilli to be very numerous in the livers of chickens suffering
from spontaneous tuberculosis, and inoculated with material from
this source six chickens, five rabbits, and twelve guinea-pigs. The
chickens, when inoculated in the cavity of the abdomen or by injec-
tion into a vein, died in from forty-one to ninety-three days from
general tuberculosis. Four of the rabbits died of general tuberculosis,
presenting the same appearance as that following inoculation with
bacilli from human tuberculosis. Of the guinea-pigs, which were
inoculated in the cavity of the abdomen, eleven remained in good

484 BACILLI IN CHRONIC INFECTIOUS DISEASES.

health and one only died of general tuberculosis. These experi-
ments show a decided difference in the pathogenic properties of
tubercle bacilli from the two sources, for the guinea-pig is especially
susceptible to tuberculosis as a result of similar inoculations with
bacilli from human tuberculosis. We must therefore conclude that
the bacillus found in spontaneous tuberculosis in fowls is a distinct
variety of Bacillus tuberculosis. Whether this variety would cause
tuberculosis in man, if introduced into susceptible subjects, has not
been determined ; and, as pointed out by Koch, this question can
only be answered in the affirmative if it should be obtained in pure
cultures from cases of human tuberculosis.

Since the above was written Maffucci has published (1892) an
elaborate memoir upon tuberculosis of fowls. His conclusions are
stated as follows :

" The bacillus of tuberculosis in fowls is distinguished from that of tuber-
culosis in mammals by the following points of difference:

"1. It does not induce tuberculosis in guinea-pig's, and seldom causes
general tuberculosis in rabbits.

' ' 2. The cultures in various media have a different appearance from those
of the Bacillus tuberculosis of mammals.

" 3. The temperature at which it develops varies between 35° and 45° C.,
and the thermal death-point is 70° C.

"4. At 45° to 50° C. the cultures show long, thick, and branched forms.

"5. The bacillus retains its vegetative and pathogenic power at the end
of two years.

" 6. This bacillus produces a substance which is toxic for guinea-pigs and
is but slightly toxic for grown fowls.

" 7. The tuberculosis produced in fowls by this bacillus is without giant
cells."

Additional Notes upon the Tubercle Bacillus (1895).— Several
authors (Metschnikoff, Czaplewski, Fischel) have described branch-
ing forms of the tubercle bacillus, and Lubinsky (1895) reports that
in certain media it grows out into long threads, which, however, he
has never observed to be branched. The media used by him are said
to give a more abundant growth than occurs upon glycerin-agar ;
the most favorable being made of flesh-peptone agar, or flesh-peptone
bouillon, containing four per cent of glycerin and mashed potato,
one kilo of finely chopped and washed potato to fifteen hundred cubic
centimetres of water; this is cooked for three or four hours and filtered
— to the filtrate is added four per cent of glycerin ; one and a half
per cent of agar is now added and the mixture is again cooked and
filtered.

Jones (1895) has observed the branching forms previously de-
scribed by several authors, and states that they are only found upon
the surface of culture media where there is free access of oxygen.
He concludes that the tubercle bacillus does not form endogenous

BACILLI IN CHRONIC INFECTIOUS DISEASES. 485

spores, such as are found in various other bacilli, but that in the rods
and branched filaments certain objects are seen which are probably
reproductive elements, and which closely resemble similar bodies
(" Kolben ") seen in the actinomyces fungus, to which Jones believes
the tubercle bacillus is closely related.

Prudden and Hodenpyl (1891) have shown that the injection of
dead tubercle bacilli in rabbits gives rise to the development of nod-
ules in the lung containing epithelioid and giant cells, but that these
never undergo caseation. This fact is supposed to justify the infer-
ence that caseation is due to the products elaborated during the
growth of living tubercle bacilli. The results reported by Vissmann
(1892) correspond with those reported by Prudden and Hodenpyl.
Gamaleia (1892) has also obtained nodules with epithelioid and
giant cells from the injection of dead tubercle bacilli, but in his ex-
periments he also found caseation of the nodules. Baumgarten sug-
gests that this was probably due to the fact that there were some liv-
ing tubercle bacilli remaining in the cultures which he injected.

Loomis (1890) and Pizzini (1892) have shown that living tubercle
bacilli are not infrequently found in the bronchial glands of individ-
uals who present no evidence of tubercular disease of the lungs or else-
where. The author last mentioned inoculated thirty guinea-pigs
with the bronchial, mesenteric, and cervical glands of thirty in-
dividuals in whom death was due to accident or acute disease, and,
who were free from tuberculosis. Twelve of these thirty guinea-
pigs developed tuberculosis as a result of the inoculation.

Straus (1894) has found tubercle bacilli in the nasal cavities of
healthy individuals.

Ernst (1895), as the result of extended researches made under the
auspices of the Massachusetts Society for Promoting Agriculture,
has arrived at the following conclusions with reference to the pres-
ence of the tubercle bacillus in the milk of tuberculous cows :

" The possibility of milk from tuberculous udders containing the
infectious element is undeniable.

" With the evidence here presented, it is equally undeniable that
milk from diseased cows with no appreciable lesion of the udder may,
and not infrequently does, contain the bacillus of the disease."

De Schweinitz (1894) has found that by continued cultivation in
an artificial medium the tubercle bacillus becomes attenuated, so that
when inoculated into guinea-pigs these animals give no evidence of
tubercular infection for six months or more. And his experiments
indicate that animals which have survived an inoculation with the
attenuated tubercle bacillus acquire an immunity against the patho-
genic action of virulent cultures.

486 BACILLI IN CHRONIC INFECTIOUS DISEASES.

Amann (1895) has given in the Centralblatt fur Bakteriologie
(Bd. xvii., page 513) a detailed account of his method for demon-
strating the presence of tubercle bacilli in sputum by sedimentation.
He mixes the sputum with two to four volumes of cold distilled
water, in a glass cylinder which should not be more than half full.
He adds one cubic centimetre of chloroform and a small quantity
of shot; the glass cylinder is then closed with a rubber cork and vio-
lently shaken for some minutes. From four to six volumes of dis-

^^i^ '"••: I ^

FIG. 120. —Section of a recent lepra nodule of the skin, x 950. (Baumgarten.)

tilled water are then added and the mixture is placed in a V-formed
glass tube for sedimentation ; two cubic centimetres of carbol-fuchsin
solution are added and distributed by gentle agitation of the tube.
At the end of two days the sedimentation is complete and the stained
bacilli, cells, connective-tissue fibres, etc., are taken up with a pipette
for examination under the microscope.

BACILLUS LEPRJE.

Discovered by Hansen (1879), chiefly in the interior of the peculiar
round or oval cells found in leprous tubercles. Discovery confirmed
by Neisser (1879) and by many subsequent observers.

While found chiefly in the leprous tubercles of the skin and mucous
membranes, the bacilli have also been found in the lymphatic glands,
the liver, the spleen, the testicles, and, in the anaesthetic form of the
disease, in the thickened portions of nerves involved in the leprous
process. Some observers have also reported finding them in the
blood, but this appears to be quite exceptional. In the leprous cells
they are commonly found in great numbers, and they may also be
seen in the lymph spaces outside of these cells. They are not found
in the epidermal layer of the skin, but, according to Babes, they may
penetrate the hair follicles.

Morphology. — The bacillus of leprosy resembles the tubercle ba-
cillus in form, but is of more uniform length and not so frequently

BACILLI IN CHRONIC INFECTIOUS DISEASES. 487

bent or curved. The rods have pointed ends ; and in stained pre-
parations unstained spaces, similar to those observed in the tubercle
bacillus and generally assumed to be spores, are to be seen, although
not quite so distinctly as in the latter. The bacilli are said by Fliigge
to be from four to six ^ in length and less than one /< in width —
probably considerably less, for the same author states that the tubercle
bacillus has about the diameter of the bacillus of mouse septicaemia,
and this is given as 0.2 />..

This bacillus stains readily with the aniline colors and also
by Gram's method. Although it differs from the tubercle bacillus
in the ease with which it takes up the ordinary aniline colors, it re-
sembles it in retaining its color when subsequently treated with
strong solutions of the mineral acids. Double-stained prepara-
tions are therefore easily made by first staining sections or cover-
glass preparations in Ziehl's carbol-f uchsiii solution or in an aqueous
solution of methyl violet, decolorizing in acid, washing in alcohol,
and counter-staining with methylene blue — or, if methyl violet was
used in the first instance, with vesuvin.

Biological Characters. — The earlier attempts to cultivate this
bacillus were without success, but recently Bordoni-Uffreduzzi has
obtained from the marrow of the bones of a leper a bacillus which
he believes to be the leprosy bacillus, and which he was able to culti-
vate upon blood serum to which a certain amount of peptone and of
glycerin had been added. At first this bacillus only grew with diffi-
culty and in the incubating oven ; but after it had been cultivated
artificially through a number of generations it is said to have grown
upon ordinary nutrient gelatin at the room temperature. The bacillus
obtained in this way is said to have retained its color when treated
with acids, after having been stained with aniline-f uchsin, correspond-
ing in this respect with the bacillus of leprosy and the tubercle ba-
cillus. But it differed considerably in its morphology from the Ba-
cillus leprse as seen in the tissues of lepers, being considerably thicker,
and it was not so promptly stained by the aniline colors as is the
bacillus found in the tissues. Moreover, attempts to cultivate the
same bacillus from leprous tubercles of the skin were unsuccessful,
as were also inoculation experiments into the anterior chamber of the
eye in rabbits. It is therefore a matter of doubt as to whether the
bacillus obtained by Bordoni-Uffreduzzi is identical with that present
in such numbers in the cells of the leprous tubercles, to which the
name Bacillus lepra? has been given.

Some of the earlier observers described the bacillus of leprosy as
motile, but this assertion seems to have been based upon some error
of observation, and it is now generally agreed that, like the tubercle
bacillus, it is without proper movements. The question of spore for-

488 BACILLI IN CHRONIC INFECTIOUS DISEASES.

mation has not been definitely settled. As before remarked, un-
stained portions, occurring at regular intervals, are seen in the rods in
stained preparations ; but no satisfactory evidence has been presented
to show that these are truly reproductive spores.

Pathogenesis. — The inference that the bacillus above described
bears an etiological relation to the disease with which it is associated
is based upon the demonstration of its constant presence in leprous
tissues — which has now been repeatedly made in various and distant
parts of the world — and of its absence from the same tissues involved
in different morbid processes. As it has not been obtained in pure
cultures, the final proof of such etiological relation is still wanting.
We have, however, experimental evidence to show that leprous tis-
sues containing this bacillus are infectious and may reproduce the
disease. The experiment has been made upon man by Arning, who
inoculated a condemned criminal subcutaneously with fresh leprous
tubercles. The experiment was made in the Sandwich Islands, and
the man was under observation until his death occurred from leprosy
at the end of about five years. The first manifestations of the disease
became visible in the vicinity of the point of inoculation several
months after the experimental introduction of the infectious material.

Positive results have also been reported in the lower animals by
Damsch, by Vossius, and by Melcher and Ortmann. The last-named
investigators inoculated rabbits in the anterior chamber of the eye
with portions of leprous tubercles excised for the purpose from a
leper. The animals died from general infection at the end of several
months, and the characteristic tubercles containing the bacillus were
distributed through the various organs.

Wolters (1893) who has made numerous inoculation experiments
and has made a critical review of all the recorded experimental evi-
dence, arrives at the conclusion that the comparatively small number
of successful results reported cannot be accepted as evidence that
leprosy can be transmitted to the lower animals by inoculation. He
believes that in some cases the tubercle bacillus has been present in
the material inoculated and that the infectious process following the
inoculation was tuberculous and not leprous. In inoculations into
the anterior chamber, in the eyes of rabbits, the considerable number
of bacilli introduced with the leprous tissue remain and retain their
staining properties, so that the bacilli originally introduced are found
in the leucocytes of the inflammatory exudate or granulation tissue
formed as a result of the introduction of foreign material. Wolters
also doubts whether the few successful results reported in the culti-
vation of the lepra bacillus are trustworthy. He has never succeeded
in his efforts to cultivate the bacillus.

BACILLI IN CHRONIC INFECTIOUS DISEASES.

489

BACILLUS MALLEI.

Synonyms. — The bacillus of glanders; Der Rotzbacillus, Ger\;
Bacille de la morve, Fr.

Discovered by Loffler and Schiitz (1882), and proved to be the
cause of glanders by the successful inoculation of pure cultures.
Found especially in the recent nodules in animals infected with
glanders ; also in the same after ulceration, and in the discharge
from the nostrils, pus from the specific ulcers, etc. ; sometimes in the
blood of infected animals (Weichselbaum).

Morphology. — Bacilli with rounded ends, straight or slightly
curved, rather shorter and decidedly thicker than the tubercle bacil-
lus ; usually solitary, but occasionally united in
pairs, or in filaments containing several elements
(in potato cultures). In stained preparations
unstained or feebly stained spaces are seen in
the rods, alternating with the deeply stained
protoplasm of the cell. As in the tubercle bacil-
lus, which presents a similar appearance, these
spaces- have been supposed by some bacteriolo-
gists to represent spores ; but Loffler believes
them to represent rather a degeneration of the
protoplasm. Baumgarten and Rosenthal claim
to have demonstrated the presence of spores by the use of Weisser's
method of staining, but they do not consider it established that the
unstained spaces in the rods referred to are of this nature.

The glanders bacillus may be stained with aqueous solutions of
the aniline colors, but the staining is more intense when the solution

FIG. 121.— Bacillus mal-
lei, x 1,000. From a pho-
tomicrograph. CFninkel
and Pfeiffer.)

ptre

-3<j*y-!fl5
FIG. 122.— Section of a glanders nodule, x 700. (Fliigge.)

is made feebly alkaline. Add to three cubic centimetres of a 1: 10,000
solution of caustic potash, in a watch glass, one cubic centimetre of
a saturated alcoholic solution of an aniline color (methylene blue,

490 BACILLI IN CHRONIC INFECTIOUS DISEASES.

gentian violet or fuchsin) ; or the aniline- water-fuchsin, or methyl
violet solution of Ehrlich may be used, with the addition just be-
fore use of an equal quantity of 1 : 10,000 solution of caustic potash.
Loffler recommends that cover-glass preparations be placed in Ehr-
lich's solution and heated for five minutes; then decolorized in a one-
per-cent solution of acetic acid to which sufficient tropseolin has
been added to give it the yellow color of Rhine wine ; then quickly
washed in distilled water. This bacillus presents the peculiarity of
losing very quickly in decolorizing solutions the color imparted to it
by the aniline staining solutions. For this reason the staining of the
bacillus in sections is attended with some difficulty. Loffler recom-
mends his alkaline methylene-blue solution for staining sections ; and
for decolorizing, a mixture containing ten cubic centimetres of distilled
water, two drops of strong sulphuric acid, and one drop of a five-
per-cent solution of oxalic acid. Thin sections should be left in this
acid solution about five seconds. The method more recently recom-
mended by Kuhne also gives good results in skilful hands (see p. 35).
Biological Characters. — An aerobic, non-motile, parasitic
bacillus, which may be cultivated in various artificial media at a
temperature of 37° C. The lowest temperature at which develop-
ment occurs (22° C. — Loffler) is a little above that at which nutrient
gelatin is liquefied ; the highest limit is 43° C. According to Frankel,
the glanders bacillus is a facultative anaerobic. Baumgarten and
Rosenthal claim to have demonstrated the presence of spores by
Neisser's method of staining. Loffler was led to doubt the forma-
tion of spores from the results of his experiments upon the thermal
death-point of this bacillus, and its comparatively slight resistance
to desiccation and destructive chemical agents. He found that ex-
posure for ten minutes to a temperature of 55° C., or for five minutes
to a three- to five-per-cent solution of carbolic acid, or for two min-
utes to a 1 : 5,000 solution of mercuric chloride, was effectual in de-
stroying its vitality. As a rule, the bacilli do not grow after having
been preserved in a desiccated condition for a few weeks ; and in a
moist condition the cultures cannot be preserved longer than three
or four months — usually not so long as this (Loffler). The bacillus
does not grow in infusions of hay, straw, or horse manure, and it is
doubtful whether it finds conditions in nature favorable for its sap-
rophytic existence. It grows, in the incubating oven, in neutral
bouillon, in nutrient gelatin, or in nutrient agar, and still better in
glycerin-agar. Upon the last-mentioned medium it grows, even at
the room temperature (Kranzfeld), but better still in the incubating
oven, as a pale- white, transparent streak along the line of inocula-
tion, which at the end of six or seven days may have a width of
seven to eight millimetres. According to Raskina, nutrient agar

BACILLI IN CHRONIC INFECTIOUS DISEASES. 491

made with milk forms an extremely favorable medium, upon which
a thick, pale-white layer develops in two or three days, which on the
third or fourth day acquires an amber-yellow color, and the deeper
layers acquire a brownish-red tint.

The growth upon solidified blood serum, in the course of three or
four days at 37° C., consists of yellowish, transparent drops, which
later coalesce into a viscid layer, which has a milky appearance from
the presence of numerous small crystals (Baumgarten). The growth
upon cooked potato is especially characteristic. In the incubating
oven, at the end of two or three days, a rather thin, yellowish, trans-
parent layer develops, which resembles a thin layer of honey. Later
this ceases to be transparent, and the amber color changes, at the
end of six to eight days, to a reddish-brown color ; and outside of
the reddish-brown layer, with more or less irregular outlines, the
potato for a short distance acquires a greenish-yellow tint.

Pathogenesis. — Glanders occurs principally among horses and
asses, but may be contracted by man from contact with infected
animals ; it has also been communicated, in one instance with a fatal
result, by subcutaneous inoculation, resulting accidentally from the
use of an imperfectly sterilized hypodermic syringe which had pre-
viously been used for injecting cultures of the bacillus into guinea-
pigs. The field mouse and the guinea-pig are especially susceptible
to infection by experimental inoculations ; the cat and the goat may
be infected in the same way. Lions and tigers in menageries are
said to have contracted glanders from being fed upon the flesh of in-
fected animals (Baumgarten). Rabbits have but slight susceptibility,
and the same is true of sheep and dogs ; swine, cattle, white mice,
and common house mice are immune.

The etiological relation of the bacillus is fully established by the
experiments of Loffler and Schiitz, confirmed by other bacteriologists,
which show that pure cultures injected into horses, asses, and other
susceptible animals, produce genuine glanders. The disease is char-
acterized in the equine genus by the formation of ulcers upon the
nasal mucous membrane, which have irregular, thickened margins
and secrete a thin, virulent mucus ; the submaxillary lymphatic
glands become enlarged and form a tumor which is often lobulated ;
other lymphatic glands become inflamed, and some of them suppurate
and open externally, leaving deep, open ulcers ; the lungs are also
involved and the breathing becomes hurried and irregular. In farcy,
which is a more chronic form of the same disease, circumscribed
swellings, varying in size from a pea to a hazelnut, appear on differ-
ent parts of the body, especially where the skin is thinnest ; these
suppurate and leave angry-looking ulcers with ragged edges, from
which there is an abundant purulent discharge. The specific bacillus

492

BACILLI IN CHRONIC INFECTIOUS DISEASES.

can easily be obtained in pure cultures from the interior of suppurat-
ing nodules and glands which have not yet opened to the surface,
and the same material will give successful results when inoculated
into susceptible animals. But the discharge from the nostrils or from
an open ulcer contains comparatively few bacilli ; and as these are
associated with various other bacteria which grow more readily in
oar culture media, it is not easy to obtain pure cultures, by the plate
method, from such material.

In the guinea-pig subcutaneous inoculation is followed in four or
five days by tumefaction at the point of inoculation, and after a time
a prominent tumor with caseous contents is developed ; ulceratioii of
the skin follows, and a chronic, purulent ulcer with irregular, indu-
rated margins results ; after a time this may cicatrize. Meanwhile
the lymphatic glands become involved, and the symptoms of general

FIG. 123.— Section through a glanders nodule in liver of field mouse. Tissue X 250. Bacilli
X 500. (Baumgarten.)

infection are developed at the end of four or five weeks ; the glands
suppurate, and in males the testicles are also involved ; finally a dif-
fuse inflammation of the joints occurs, and death results from ex-
haustion. In the guinea-pig the specific ulcers upon the nasal mu-

BACILLI IX CHRONIC INFECTIOUS DISEASES. 493

cous membrane, which characterize the disease in the horse, are rarely
developed to any great extent.

In field mice general infection occurs at once as a result of the
subcutaneous injection of a small quantity of a pure culture, and the
animal dies at the end of three or four days. Upon post-mortem
examination the principal changes are found in the liver and in the
greatly enlarged spleen. Scattered through these organs are minute
gray points which are scarcely visible to the naked eye. In the
guinea-pig, which succumbs at a later date, these nodules are larger
and* closely resemble miliary tubercles, both macroscopically and
under the microscope, in stained sections of the tissues. Similar
nodules are also found in the kidneys and in the lungs ; they have a
decided tendency to undergo purulent degeneration. The bacilli are
found principally in these nodules, of recent formation, and are com-
monly associated in groups, as if they had been enclosed in the inte-
rior of a cell the membranous envelope of which had undergone
degeneration and disappeared.

As before remarked, it is not an easy matter to demonstrate the
bacillus in sections of the tissues containing these nodules, owing to
the facility with which they lose their color in alcohol and other de-
colorizing agents. For this reason it will be best to dehydrate sec-
tions by the use of aniline oil (Weigert's method) or to resort to
Kuhne's method of staining.

It is also difficult to demonstrate the presence of the bacillus in
nodules which have undergone purulent degeneration, in the secre-
tions from the nostrils of horses suffering from glanders, or in the
pus from the specific ulcers and suppurating glands ; for they are
present in comparatively small numbers. But the virulent nature of
these discharges is shown by inoculations into guinea-pigs or mice,
and it is easier to obtain a pure culture from such virulent material
by first inoculating a susceptible animal than directly by the plate
method; for the small number of bacilli present, and their associa-
tion with other bacteria which develop more rapidly in our culture
media, make this a very uncertain procedure. For establishing the
diagnosis of glanders, therefore, Loffler recommends the inoculation
of guinea-pigs with pus from a suppurating gland or ulcer, or the
nasal discharge from a suspected animal, rather than a direct attempt
to demonstrate the presence of the bacillus by staining and culture
methods.

The method proposed by Strauss gives more prompt results.
This consists in the intraperitoneal. injection of cultures or of the
suspected products into the cavity of the abdomen of male guinea-
pigs. If the glanders bacillus is present tho diagnosis may be made
within three or four days from, the infectious process established in

494 BACILLI IN CHRONIC INFECTIOUS DISEASES.

the testicles. At the end of this time the scrotum is red and shining,
the epidermis desquamates, and suppuration occurs, the pus some-
times perforating the integument. This pus is found to contain the
glanders bacillus. The animal usually dies in the course of twelve
to fifteen days. When the animals are killed three or four days
after the inoculation, the two layers of the tunica vaginalis testis
are found to be covered with a purulent exudate containing the
glanders bacillus and to be more or less adherent. Even as early
as the second day the tunica vaginalis is seen to be covered with
granulations.

An attenuation of virulence occurs in cultures whicn have been
kept for some time, and inoculations with such cultures may give a
negative result ; or, when considerable quantities are injected, may
produce a fatal result at a later date than is usual when small
amounts of a recent culture are injected into susceptible animals.

Kalning, Preusse, and Pearson have obtained from cultures of
the glanders bacillus a glycarin extract similar to the crude tubercu-
lin of Koch — mallein. This, when injected into animals suffering
from glanders, gives rise to a considerable elevation of temperature,
and it is used as a means of diagnosis in cases of suspected infection in
animals in which the usual symptoms have not yet manifested them-
selves. The value of the test has been demonstrated by numerous
experiments.

Bonome (1894), as a result of extended researches, arrives at the
following conclusions :

" 1. The bacillus is found not only in the diseased tissues and
purulent discharges, but also in the urine and milk of infected ani-
mals.

" 2. The bacillus is found in the foetus of infected animals even
when the placenta is free from any pathological change.

" 3. The glanders bacillus is very sensitive to desiccation and will
not grow after being preserved for ten days at 25° C.

" 4. In distilled water the bacillus dies out in six days.

" 5. On the contrary, when protected from desiccation it resists
a comparatively high temperature — 70° C. for six hours; a temper-
ature of 90° to 100° C. destroys it in three minutes."

BACILLUS OF LUSTGARTEN.

Synonym. — Syphilis bacillus.

Found by Lustgarten (1884) in syphilitic lesions and secretions of syphi-
litic ulcers, and believed by him to be the specific infectious agent in this
disease. No satisfactory experimental evidence that this is the case has yet
been obtained.

Morphology.— Straight or curved bacilli, which bear considerable resem-
blance to tubercle bacilli, but differ from them in the staining reactions.
They are usually more or less curved, or bent at a sharp angle, or S-shaped ;

BACILLI IN CHRONIC INFECTIOUS DISEASES. 495

the ends often present slight knob -like swellings ; the length is from three
and one-half /" to four and one-half /*, and the diameter is from 0.25 to 0.3 /*.
With a high power the contour is seen to be not quite regular, but wavy in
outline, and bright shining spaces in the deeply stained rods may be ob-
served ; these, from two to four in a single rod, are believed by Lustgarten
to be spores. The bacilli are not found free in the tissues, but are enclosed
in cells of a round-oval or polygonal form, which are said to be about double
the size of a white blood corpuscle. The bacilli are riot numerous, and very
commonly only one or two are found in a single cell, but groups of six or
eight may sometimes be seen, especially upon the margins of a s}rphilitic
lesion, and in the tissues in the immediate vicinity of the infiltration, which
show out little change or are apparently healthy (Lustgarten).

The presence of these bacilli in syphilitic lesions was demonstrated by
Lustgarten by the following staining method : The thin sections are placed
in the Ehrlich-Weigert gentian- violet solution (one hundred parts aniline
water, eleven parts saturated alcoholic solution of gentian violet) for from
twelve to twenty-four hours at the room temperature, and two hours in the
incubating oven at 40° C. The sections are then thoroughly washed in alco-
hol and placed for ten seconds in a 1.5-per-cent solution of potassium per-
manganate; in this solution a precipitate of peroxide of manganese is

FIG. 134. FIG. 125.

FIG. 124.— Migrating cell containing syphilis bacilli. (Lustgarten. )

Fia. 125.— Pus from hard chancre containing syphilis bacilli (Lustgarten.')

formed, which adheres to the section ; this is dissolved and washed off in a
dilute aqueous solution of pure sulphuric acid; the sections are then washed
in water, and, if not completely decolorized, are returned for a few seconds to
the permanganate solution and again washed off in the acid; it may be
necessary to repeat this operation three or four times. Finally the sections
are dehydrated and mounted in balsam in the usual manner. Cover-glass
preparations are made in the same way, except that, after being taken from
the staining solution, they are washed off in water instead of in alcohol.

Another method of staining, recommended by De Giacoma, consists in
placing the sections for twenty -four hours in aniline-water-fuchsin solution
(cover-glass preparations may be stained in the same solution, hot, in a few
minutes), then washing them in water, and decolorizing in a solution of per-
chloride of iron — first in a dilute and then in a saturated solution.

The method of staining employed by Lustgarten serves to differentiate
his bacillus from many other microorganisms, but not from, the tubercle ba-
cillus and the bacillus of leprosy, which, as he pointed out, may be stained
in the same way. And it has since been shown by Alvarez and Tavel, and
by Matterstock, that in smegma from the prepuce or the vulva, bacilli are
found which have the same staining reaction and are similar in their mor-
phology to the bacillus of Lustgarten. This by no means proves that the

496

BACILLI IN CHRONIC INFECTIOUS DISEASES.

smegma bacilli found under the prepuce of healthy persons are identical
•with the bacilli found by Lustgarten and others in sections of tissues involved
'in syphilomata. In the absence of pure cultures and inoculation experiments
it is impossible to establish identity, however similar may be the characters
referred to. Several well-known pathogenic bacilli resemble quite as closely
in these particulars other bacilli which have, nevertheless, been differentiated
from them by culture and inoculation experiments. We may mention
especially in this connection the bacillus of diphtheria, as obtained from the
pseudo-membranous exudation in a genuine case of this disease, and the
pseudo diphtheria bacilli found by Roux and Yersinin the fauces of healthy
children. On the other hand, since it has been shown that similar bacilli
are common in preputial srnegma, we cannot attach great importance to the
finding of Lustgarten's bacillus in primary syphilitic sores ; and it has not
been found in sufficient numbers, or with sufficient constancy, by those who
have searched for it subsequently to the publication of Lustgarten's inves-
tigations, to give strong support to the view that it is the specific infectious
agent in syphilis. Baumgarten, who has searched in vain for Lustgarten's
bacillus in uncomplicated visceral syphilomata, suggests that the bacilli
found occasionally in such lesions were perhaps tubercle bacilli and repre-
sented a mixed infection. As the bacillus under consideration has not been
obtained in cultures, we have no information as to its biological characters
and pathogenesis.

BACILLUS OF RHINOSCLEROMA.

First observed by Von Frisch (1882) in the newly formed tubercles of
rhinoscleroma. Cultivated by Paltauf a'nd Von Eiselberg (1880).

Rhinoscleroma is a chronic affection of the skin, and especially of the
mucous membrane of the nares, which is characterized by the formation of
tubercular thickenings of the skin and tumefaction of the nasal mucous
membrane, followed sometimes by ulceratiori It prevails in Italy, Austria,
and to a slight extent in some parts of Germany. Pathologists generally
regard it as an infectious process, although this has not been proved.

The bacilli, first described by Von Frisch, appear to be constantly present
in the newly formed tubercles. They are commonly found in certain large

FIG. 126.— Bacillus of rhinoscleroma iii lymphatic vessels of the superficial part of tumor.
x 1,200. (Cornil and Babes )

hyaline cells peculiar to the disease, and may also be observed in the lym-
phatic vessels or scattered about in the involved tissues.

Morphology. — Short bacilli with rounded ends, usually united in pairs,
and surrounded by a gelatinous capsule resembling that of Friedlander's

BACILLI IN CHRONIC INFECTIOUS DISEASES. 497

bacillus. According to Eiseiiberg, the bacilli are two to three times as long
as broad, and may grow out into filaments.

These bacilli stain readily with the aniline colors and by Gram's method.
The capsule may be demonstrated by the methods usually employed in stain-
ing Friedlander's bacillus, or by the following method which is especially
recommended by Alvarez : The excised portions of tissue involved in the dis-
ease are placed for twenty-four hours in a one-per-cent solution of osmic
acid and then in absolute alcohol. When properly hardened thin sections
are made; these are stained in a hot solution of aniline-water-methyl-violet
for a few minutes, and then decolorized, by Gram's method, in iodine so-
lution.

Biological Characters. — An aerobic, non-motile, non-liquefying bacillus
(facultative anaerobic ?).

In gelatin stab cultures the growth resembles that of Friedlander's ba-
cillus— i.e., a nail-like growth, consisting of densely crowded, opaque colonies
along the line of puncture, and a heaped-up, white, glistening mass upon the
surface, hemispherical in form and viscous in consistence. Upon gelatin
plates yellowish-white, spherical colonies are developed within two or three
days, which under the microscope are seen to be granular. Upon potato a
cream-like growth occurs along the line of inoculation, which is white or
yellowish-white in color, and in which gas bubbles may be developed. De-
velopment is most rapid at a temperature of 35° to 38°, but also occurs at the
room temperature. •

Pathogenesis. — The etiological relation of this bacillus to the disease with
which it is associated has not been established. It is pathogenic for mice
and for guinea-pigs, less so for rabbits; in this regard, as in its morphology
and growth in various culture media, it bears a close resemblance to Fried-
lander's bacillus, which is also found not infrequently in the nasal secretions
of healthy persons and in those suffering from chronic nasal catarrh or ozaena.

The principal points of difference, as pointed out by Baumgarten, are as
follows : The bacillus of rhinoscleroma is usually more decidedly rod-shaped
than Friedlander's bacillus, although both may be of so short an oval as to
resemble micrococci. The first-mentioned bacillus constantly presents the
appearance of being surrounded by a transparent capsule, even in the cul-
tures in artificial media, .while Friedlander's bacillus in such media does not
usually present this appearance, unless as a result of special treatment.
Finally, the bacillus of rhinoscleroma may retain its color, in part at least,
when treated by Gram's method, while Friedlander's bacillus is completely
decolorized when placed in the iodine solution employed in this method.

Notwithstanding these points of difference, Baumgarten is not entirely
satisfied that this bacillus is a distinct species, and several bacteriologists
have maintained that it is identical with the bacillus of Friedlander.

XIII.

BACILLI WHICH PRODUCE SEPTIC^MIA
SUSCEPTIBLE ANIMALS.

WHEN, as a result of accidental (natural) or experimental inocula-
tion, a microorganism is introduced into the body of a susceptible
animal which is able to multiply in its blood, producing a general in-
fection, we speak of this general blood infection as a septicaemia.
When pathogenic microorganisms which are unable to multiply in
the blood establish themselves in some particular locality in the ani-
mal body which is favorable for their growth, and by the formation
of toxic products, which are absorbed, give rise to general symptoms
of poisoning, we designate the affection toxcemia. As examples of
this mode of pathogenic action we may mention diphtheria and
tetanus. As a rule, the various forms of septicaemia are quickly
fatal, and, as the microorganisms to which they are due multiply in
the blood of the infected animal, this fluid possesses infectious pro-
perties, and, when inoculated in the smallest quantity into another
susceptible animal, reproduces the same morbid phenomena. A typi-
cal example of this class of diseases is found in anthrax, to which
disease a special section has already been devoted (VIII.). But in
this and other forms of septicaemia subcutaneous inoculations do not,
as a rule, result in the immediate invasion of the blood by the para-
sitic microorganism. Often a local inflammatory process of consider-
able extent is first induced ; and in some cases general infection only
occurs a short time before the death of the animal, depending, per-
haps, upon a previous toxaemia from the absorption of toxic products
developed at the seat of local infection. The pathogenic action, then,
in acute forms of septicaemia appears to result, not alone from the
presence and multiplication of the pathogenic microorganism in the
blood, but also from the toxic action of products evolved during its
growth.

Some of the pathogenic bacilli of this class now known to bac-
teriologists have been discovered by studying the infectious diseases
induced by them in lower animals among which these diseases pre-
vail naturally — i.e., independently of human interference. Many

BACILLI WHICH PRODUCE SEPTICAEMIA. 4(J'J

more are known to us from experiments made in pathological labora-
tories, in testing by inoculations into animals bacteria obtained from
various sources, with reference to their pathogenic power. We in-
clude in this group only those bacilli which induce fatal septicaemia
in susceptible animals when injected into the circulation or sub-
cutaneously in a comparatively small quantity — e.g., less than half
a cubic centimetre of a bouillon culture.

BACILLUS SEPTICAEMIA H^EMORRHAGIC^E.

Synonyms. — Bacillus of fowl cholera ; Microbe du cholera des
poules (Pasteur) ; Bacillus choleras gallinarum (Fliigge) ; Bacillus der
Hiihnercholera ; Bacillus of rabbit septicaemia ; Bacillus cuniculi-
cida (Fliigge) ; Bacillus der Kaninchenseptikamie (Koch) ; Bacillus
der Rinderseuche (Kitt) ; Bacillus der Schweineseuche (Loffler and
Schiitz) ; Bacillus der Wildseuche (Hueppe) ; Bacillus der Biiffel-
seuche (Oreste-Armanni) ; (Bacterium of Davaine's septicaemia ?)

It is now generally admitted by bacteriologists that Koch's ba-
cillus of rabbit septicaemia (1881) is identical with the bacillus
("micrococcus") of fowl cholera previously described by Pasteur
(1880). The similar bacilli found in the blood of animals dead from
the infectious diseases known in Germany as Wildseuche (Hueppe),
Rinderseuche (Kitt), Schweineseuche (Schiitz), and Biiffelseuche
(Oreste-Armanni) appear also to be identical with the bacillus of
rabbit septicaemia and fowl cholera. This view is sustained by
Hueppe and by Baumgarten, and by the comparative researches of
Caneva (1891) and of Bunzl-Federn (1891).

This is evidently a widely distributed pathogenic bacillus ; it was
obtained by Koch from rabbits inoculated with pu-
trefying flesh infusion, by Gaffky from impure river

water, andbv Pasteur from the blood of fowls suffer-

. . .

ing from the infectious disease known in France as ~y~\

cholera des poules. It is not infrequently found in „ ^Q V*O '
putrefying blood, and its presence in the salivary '{j *'%
secretions of man has occasionally been demonstrated F 197 _Bacillua

(Baumgarten). septicaemia) hsemor-

With reference to the American swine plague rhagica? mthet

* & of a rabbit, x 950.

described by Salmon and Smith, we are informed by (Baumgarten.)
Smith, in his most recent publication upon the subject
(Zeitschrift fur Hygiene, Band x., page 493), that cultures of the
German Schweineseuche bacillus, received from the Berlin Hygienic
Institute, compared with his cultures from infected swine in this
country, agreed in all particulars, except that the former were de-
cidedly more pathogenic for swine and for rabbits.

It appears extremely probable that the form of septicaemia studied

500 BACILLI WHICH PRODUCE SEPTIC^MIA

by Davaine (1872), which he induced in the first instance by inject-
ing putrid ox blood into rabbits, was due to the same pathogenic ba-
cillus. The writer obtained this bacillus (1887) in Cuba from the
blood of rabbits inoculated with liver tissue taken from a yellow-
fever cadaver and kept for forty-eight hours in an antiseptic wrap-
ping. The name which we have adopted is that proposed by Hueppe
for the form of septicaemia to which it gives rise — "Septikamia
hamorrhagica. "

Morphology. — Short bacilli with rounded ends, from 0.6 to 0.7
/* in diameter and about 1.4 ^ long; sometimes united in pairs, or
in chains of three or four elements. In stained preparations the ex-
tremities are usually stained, while the central portion of the rod
remains unstained. This " end staining" causes the rods to present
the appearance of diplococci when examined with a comparatively
low power, and some of the earlier observers described the microor-
ganism under consideration as a micrococcus. It is quickly stained
by the aniline colors usually employed, but loses its color when
treated by Gram's method.

Biological Characters. — A non-motile, aerobic, non-liquefy-
ing bacillus. Does not form spores. Grows in various culture media
at the room temperature, but more rapidly at 35° to 37° C. — the
lowest temperature at which development occurs is about 13° C.
Although this is an aerobic bacillus and a certain amount of oxygen
is necessary for its development, it appears to grow better when the
amount is somewhat restricted than it does on the surface of nutrient
media.

Upon gelatin plates, at the end of two or three days, small,
white colonies are developed upon or near the surface ; these are
finely granular and spherical, with a more or less irregular outline,
and by transmitted light have a yellowish color ; later the central
portion of the colonies is of a yellowish-brown color and is sur-
rounded by a transparent peripheral zone. The superficial colonies
are commonly smaller than those which develop a little below the
surface of the gelatin. In stab cultures in nutrient gelatin the
growth upon the surface consists of a thin, whitish layer in the
vicinity of the point of puncture, having an irregular, jagged out-
line— sometimes there is no development upon the surface ; along
the line of puncture the growth consists of rather transparent, dis-
crete or confluent colonies. In streak cultures upon nutrient agar,
or gelatin, or blood serum the growth is limited to the immediate
vicinity of the line of inoculation, and consists of finely granular,
semi-transparent colonies, which form a thin, grayish-white layer
with irregular, somewhat thickened margins. Upon potato no de-
velopment occurs, as a rule, at the room temperature, but in the in-

IN SUSCEPTIBLE ANIMALS.

501

cubating oven a rather thin, transparent, grayish-white or yellowish,
waxy layer is developed in the course of a few days. According to
Bunzl-Federn, the bacillus of fowl cholera and that
of rabbit septica3mia grow upon potato, while the
bacillus of Wildseuche, Schweineseuche, and Biif-
felseuche do not. According to
Caneva, none of the bacilli of this
group grow upon potato. The
same author states that the growth
in milk is scanty and does not
produce coagulation, while Bunzl-
Federn finds that the bacillus of
fowl cholera and of rabbit septi-
caemia produce coagulation and
the others do not. These differ-
ences are not, however, consid-
ered by the author last named as
sufficient to establish the specific
difference of the bacilli from these
different sources. He looks upon
them rather as varieties of the
same species. Bunzl-Federn has
also ascertained that when cul-
tivated in a peptone solution all
of the bacilli of this group, with
the exception of that obtained
from the so-called Buffelseuche,
give the reaction for phenol and
for indol — the bacillus of Buffel-
seuche gives the indol reaction only. Development in bouillon is rapid
and causes a uniform turbidity of the fluid. Cultures of this bacillus
may retain their vitality for three months or
more when kept in a moist condition ; but
the bacillus usually fails to grow after having
been kept for a few days in a desiccated con-
dition ; according to Hueppe, it may resist
desiccation for fourteen days. The thermal
death-point, as determined by Salmon for
the bacillus of fowl cholera, is 56° C. , the time
of exposure being ten minutes (55° O. with
fifteen minutes' exposure — Baumgarten). It
is not readily destroyed by putrefaction (Kitt) .
A solution of mercuric chloride of 1 : 5,000
destroys it in one minute, and a three-per-cent solution of -carbolic

FIG. 128. — Bacillus
septicaemias haemor-
rhagicae; stick culture
in nutrient gelatin,
end of four days at 16°-
18° C. (Baumgarten )

FIG. 129.— Bacillus
of Schweineseuche ;
old stick culture
in nutrient gela-
tin. (Schutz.)

FIG. 130. — Bacillus of swine
plague; colonies on gelatin
plate, fciid of sov^a days.
X 60. (Smith.)

502 BACILLI WHICH PRODUCE SEPTICAEMIA

acid in six hours (Hueppe). Pasteur (1880) has shown that when
cultures of this bacillus (microbe of fowl cholera) in bouillon are
kept for some time they gradually lose their pathogenic virulence,
and he has ascribed this "attenuation of virulence" to the action of
atmospheric oxygen. He also ascertained that the particular degree
of virulence manifested by the mother culture after a certain interval
could be maintained in successive cultures made at short intervals.
He was thus able to cultivate different pathogenic varieties, and to
use these in making protective inoculations, by which susceptible ani-

f: ^y.> -. '.;:

V • • "" •'*. i " • -•- * »« , ""X.

^v... .. • „*. ..-. ».,..-..' .•• * . rt j,

«*>' **.'•'.,-•, "\ •• • '» . *»« .- *-* ':"\

*^ ** ^ : s . -•' , . «•„ •• M

^ /f" • ;\ : ** ••;- "•;;-•,-'--'.- -"••'v^:: f

. «*.,;'. .,•'• & -—':' h^

< -> *••••:_;.--•'•

FIG. 131.— Bacillus of Scliweineseuche, in blood of rabbit. (Schiitz.)

mals were preserved from the effects of virulent cultures injected
subsequently.

Attenuated cultures recover their virulence when inoculated into
very susceptible animals. Thus a culture which would produce a
non-fatal and protective attack in a chicken may, according to Pas-
teur, kill a small bird, like a sparrow; and by successive inoculations
from one sparrow to another the original degree of virulence may be
restored, so that a minute quantity of a pure culture would be f aid
to a chicken.

Pathogenesis. — Pathogenic for chickens, pigeons, pheasants,
sparrows, and other small birds, for rabbits and mice, also for swine
(Sehweineseuche), for cattle (Rinderseuche), and for deer (Will-
seuche). (See supra, pp. 285-287.)

The researches of Smith and of Moore show that " an attenuated
variety of bacteria, belonging to the group of swine-plague bacteria
and not distinguishable from them, inhabit the mouth and upper air

IN SUSCEPTIBLE ANIMALS. 503

passages of such domesticated animals as cattle, dogs, and cats"
(Smith).

BACILLUS OF CHOLERA IN DUCKS.

Obtained by Cornil and Toupet (1888) from the blood of ducks, in the
Jardin d'Acclhnation at Paris, which had died of an epidemic disease charac-
terized by diarrhoaa, feebleness, and muscular tremors, and which resulted
fatally in two or three days.

Morphology. — Does not differ in its morphology from the bacillus of
fowl cholera (Bacillus septicaemias hsemorrhagicae) ; short rods with rounded
ends, from 1 to 1.5 11 in length and 0.5 ju broad.

Stains with the usual aniline colors, but not by Gram's method ; the ends
stain more deeply than the central portion.

Biological Characters. — An aerobic, non-liquefying, non-motilebacillus.
Does not form spores. Grows in the usual culture media at the room tem-
perature. In its growth in various media, as well as in its morphology, Cornil
and Toupet found this bacillus to correspond with the bacillus of fowl
cholera. In gelatin stab cultures the growth upon the surface consists of a
thin, grayish layer, and along the line of puncture as small, semi transpa-
rent, slightly yellowish, spherical colonies. Upon agar, in the incubating
oven, at the end of twelve hours small, lentil shaped, waxy colonies are
formed, which later may have a diameter of three to four millimetres.
Upon potato circular, yellowish colonies are formed, which become con-
fluent and form a somewhat depressed, pale-yellow layer.

Pathogenesis. — According to Cornil and Toupet, this bacillus is patho-
genic for ducks, but not for chickens or pigeons, and only kills rabbits when
injected in considerable quantity. Ducks die in from one to three days
from subcutaneous injections, or by the ingestion of food to which the bacil-
lus has been added.

BACILLUS OF HOG CHOLERA (Salmon and Smith).

Synonyms. — Bacillus of swine plague (Billings) ; Bacillus of swine-
pest (Selaiider).

According to Smith, this bacillus was first described by Klein
(1884) ; it was first obtained in pure cultures and its principal char-
acters determined by Salmon and Smith (1885), and has since been
studied in cultures and by experimental inoculations by Selander,
Billings, Frosch, Welch, Caneva, Bunzl-Federii, and others.

The bacillus is found in the blood and various organs of hogs
which have succumbed to the infectious disease known in this country
as hog cholera ; and also in the contents of the intestine, from which
it may be obtained by inoculations into rabbits, but is not easily iso-
lated by the plate method owing to the large number of other bac-
teria present (Smith).

Morphology. — Short bacilli with rounded ends, 1.2 to 1.5 /* in
length and 0. 6 to 0. 7 /* in breadth ; usually united in pairs.

This bacillus is easily stained by the aniline colors usually em-
ployed, but does not retain its color when treated by Gram's method.
When the staining agent is allowed to act for a very short time the
ends of the rods may be stained while the central portion remains
unstained.

504

BACILLI WHICH PRODUCE SEPTICAEMIA

Biological Characters. — An aerobic (facultative anaerobic), non-
liquefying, actively motile bacillus. In many of its characters this
bacillus closely resembles the one last described (Bacillus septicaBmise
hsemorrhagicse), but it is distinguished from it by its active move-
ments, which, according to Smith, may be still observed in cultures
which have been kept for weeks or months. Does not form spores.
Grows readily in various culture media at the room temperature —
more rapidly in the incubating oven. Upon gelatin plates colonies
are developed in from twenty-four to forty-eight hours. The deep colo-
nies are spherical and homogeneous, and have a brownish color by
transmitted light; they seldom exceed one-half millimetre in diameter.

FIG. 132.— Bacillus of hog cholera; stained by Loffler's method to show flagella. x 1,000. From
a photomicrograph made at the Army Medical Museum. (Gray.)

The superficial colonies may attain a diameter of two millimetres ;
they present no distinctive characters. Upon agar plates the colonies
may have a diameter of four millimetres ; they have a grayish, trans-
parent appearance and a shining surface. In gelatin stab cultures
small, yellowish-white colonies are developed along the line of in-
oculation, which may become confluent ; upon the surface a thin,
pearly layer is developed about the point of inoculation, which may
have a diameter of six millimetres or more. Upon potato a straw-
yellow layer is developed, which later acquires a darker color. In
slightly alkaline bouillon a slight cloudiness may be observed at the
end of twenty-four hours, and at the end of one or two weeks, if
not disturbed, a deposit is seen at the bottom of the tube and a thin,
broken film may form upon the surface. The development of this
bacillus in milk produces a direct solution of the casein without pre-
vious coagulation ; when a solution of litmus has been added to milk

IN SUSCEPTIBLE ANIMALS. 505

it retains its blue color in presence of this bacillus, while the bacillus
previously described causes it to change to red. Neither phenol
nor indol is produced in solutions containing peptone (Bunzl-Federn)
—another distinguishing character from the Bacillus septicsemise
haemorrhagiccE. This bacillus may be cultivated in slightly acid
media, which after a time acquire an alkaline reaction.

In Smith's experiments this bacillus was found to resist desicca-
tion from nine days to several months, according to the thickness of
the layer dried upon the cover glass ; bacilli from an agar culture in
some experiments failed to grow after seventeen days, and in others
still gave cultures after four months. Bouillon cultures are steril-
ized in four minutes by a temperature of 70° C., in fifteen minutes
by 58° C., and in one hour by 54° C. (Smith). Novy has isolated
from cultures of the hog-cholera bacillus a toxic basic substance
which he calls susotoxin. This was obtained by Brieger's method ;
it is a yellowish-brown, syrup-like liquid, which, when injected into
rats in doses of 0.125 to 0.25 cubic centimetre, causes their death in
less than thirty-six hours. He also obtained by precipitation with
absolute alcohol, from cultures concentrated in a vacuum at 36° C.,
a toxalbumin which when dried was in the form of a white powder
easily soluble in water. Rats died in three or four hours after re-
ceiving subcutaneously a dose of 0. 1 to 0. 5 gramme.

Pathogenesis. — Pathogenic for swine, rabbits, guinea-pigs, mice,
and pigeons.

In certain parts of the United States the disease known as " hog
cholera " frequently prevails among swine as a fatal epidemic. It
may occur as an acute and quickly fatal septicaemia, or in a more
chronic form lasting from two to four weeks or even longer. In
the acute form death may occur within twenty-four hours, and hsem-
orrhagic extravasations are found upon the mucous and serous
membranes and in the parenchyma of the lungs, kidneys, and lym-
phatic glands. The spleen is greatly enlarged, soft, and dark in
color. In the chronic form of the disease the most notable changes
are found in the alimentary canal. These are most constant and
characteristic in the caecum and colon, which may be studded with
spherical, hard, necrotic masses or extensive diphtheritic patches.
According to Smith, the hsemorrhagic and necrotic form of the dis-
ease may exist at the same time in different animals of the same
herd. The bacilli are found in all of the organs, and especially in
the spleen, where they are associated in irregular colonies similar
to those of the typhoid bacillus. Smith has demonstrated their pre-
sence in urine taken from the bladder immediately after the death
of the animal, and states that the kidneys are almost always in-

BACILLI WHICH PRODUCE SEPTICAEMIA

volved, as shown by the presence of albumin and tube casts in the
urine.

An extremely minute quantity of a bouillon culture injected be-
neath the skin of a rabbit causes its death in from seven to twelve
days ; a larger quantity may produce a fatal result in five days ; in-
travenous injections of very small amounts may be fatal within
forty-eight hours. After a subcutaneous injection the animal re-
mains in apparent good health for three or four days, after which it
loses its appetite and is indisposed to move ; several days before
death the temperature is suddenly elevated from 2° to 3° C., and it
remains high until the fatal termination. At the autopsy the spleen
is found to be enlarged and of a dark-red color ; the liver is studded
with small, yellowish- white, necrotic foci; the kidneys have under-
gone parenchymatous changes ; the heart is fatty ; and the intestinal
mucous membrane is more or less marked with haemorrhagic extra-
vasations. The bacilli are found in all of the organs. In house
mice the results of experimental inoculations are similar to those in
rabbits. Guinea-pigs succumb when inoculated subcutaneously with
one-tenth cubic centimetre ; pigeons require a still larger dose —
about three-quarters of a cubic centimetre. Swine are killed by the
intravenous injection of one to two cubic centimetres of a recent
bouillon culture, but, as a rule, do not succumb to subcutaneous
injections. Cultures recently obtained from diseased animals are
more virulent than those which have been propagated for a consider-
able time in artificial media.

Smith has described a variety of the hog-cholera bacillus obtained during-
an epidemic in which the disease was of longer duration — about four weeks
— than is usual, and in which, there was commonly found at the autopsy a
diphtheritic inflammation of the mucous membrane of the stomach. This
bacillus differed from the typical form by being somewhat larger and in
forming considerably larger colonies in gelatin plates — two or three times
as large. It also produced a greater opacity in peptonized bouillon, and in
general showed a more vigorous growth in various nutrient media. It dif-
fered also in its pathogenic power, as tested upon rabbits, causing death at a
later date or not at all ; and in fatal cases the swelling of the spleen and
necrotic foci in the liver, produced by the first-described species, were absent.

Bang (1892) has obtained a bacillus from infected swine in Denmark
which corresponds with the American hog-cholera bacillus. In chronic
forms of the disease pneumonia and an extensive diphtheritic process in the
intestines occurred as a complication. This was found to be due to another
bacillus, called by Bang "vacuole-bacillus." This produced a fatal pleuro-
pneumonia when injected into the lungs in pigs. According to Bang, his
"vacuole-bacillus" is without doubt identical with the swine-plague bacillus
of Salmon and Smith, and the disease of swine studied by him was a mixed
infection. The necrotic changes in the intestine, found in cases running a
chronic course, are believed by Bang to be due to still another bacillus — his
" necrosis-bacillus." Affanassieff (1892) confirms the results previously ob-
tained by several independent observers as to the identity of the swine-plague
bacillus of Salmon and Smith with the LofBer-Schiitz bacillus. The only
difference observed was a difference in pathogenic virulence — the bacillus

IN SUSCEPTIBLE ANIMALS. 507

from America corresponding- with a somewhat attenuated variety of that
from Germany.

Welch (1894), as a result of his extended researches, arrives at the follow-
ing1 conclusion :

"Our own conclusion as to the bacteria of Schweineseuche and of swine
plague is that no difference exists between them as regards morphology,
culture behavior, and pathogenic effects on rabbits, mice, and other labora-
tory animals. Cultures of each occur which are also indistinguishable by
inoculation of pigs. The only difference by laboratory experiment which
has thus far been brought out is that there occur Schweineseuche bacilli of
higher degree of virulence as tested on pigs than any swine-plague bac-
teria which have hitherto been isolated from pigs in this country. Another
point to be considered in this connection is that Schweineseuche occurs as
an independent disease in Germany without association with hog- cholera,
whereas swine plague has not been shown to prevail with the same inde-
pendence as an epizootic in this country.''

Silberschmidt (1895) arrives at a different conclusion from that reached
by Smith, Welch, Bang, and others, He believes that the diseases of swine
known as hog cholera, swine plague, and infectious pneumo-enteritis are all
due to one and the same bacillus, which, however, varies considerably both
in its morphological characters and its pathogenic power. In view of the
results previously reached by equally competent bacteriologists, and especially
by Smith and by Welch in this country, we are not disposed to accept the
view maintained by Silberschmidt.

Smith has described several varieties of the hog-cholera bacillus, and in
his account of the "hog-cholera group of bacteria " shows that the Bacillus
enteriditis of Gartner and the Bacillus typhi murium of Loffler belong to
this group. The characters of the different varieties (or species?) belonging
to the group are given by Smith in detail (United States Department of Agri-
culture, Bureau of Animal Industry, Bulletin No. 6, 1894), and the follow-
ing1 general statement is made:

" If we attempt to sum up those characters which are to circumscribe the
hog-cholera group of bacteria we are at once confronted by the scarcity of
common characters. Pathogenesis, though of great importance from the
standpoint of pathology, is probably the last character acquired and
evidently the most variable and most readily lost. If we base the unity
of this group on morphological and biological characters, we are like-
wise met by variations in size, absence of motility, variations in the ap-
pearance of the colonies. There are, however, certain underlying char-
acters, as expressed by the behavior of these bacteria in bouillon con-
taining dextrose, saccharose, and lactose, which I think will serve as a very
important group character, differentiating such groups sharply from the
colon group. I would therefore suggest that for the present all bacteria
whose size approximates that of this group, which do not liquefy gelatin, and
whose fermentative properties are the same as those described for this group,
should be ranged under it. Future investigations into the biochemical char-
acters of these varieties or sub-species may reveal other differential charac-
ters, but the time has not yet come when such laborious work will be under-
taken oi» a sufficiently extensive scale to be of any service in differentiating
varieties and sub-species."

Selander in 1890, and Metschnikoff in 1892, have reported a rapid increase
in virulence of the bacillus of hog cholera by successive inoculations in
rabbits or pigeons. Moore (1894) has shown that this is a mistake, and that
the bacteriologists named probably did not experiment with cultures of the
hog-cholera bacillus, as they supposed, but that their experiments were
made with the bacillus of swine plague — Bacillus septicaemias hemorrhagi-
cae — which when passed through a series of rabbits attains a notable increase
in pathogenic virulence.

In a recent article, Klein, of London (1895) says: " The bacillus of
English swine plague, which I described in 1884, in Virchow's Archiv, as

508

shown by Smith and Welch, is identical with the bacillus of American hog
cholera."

BACILLUS OF BELFANTI AND PASCAROLA.

Synonym. — Impftetanusbacillus.

Obtained by Belfanti and Pascarola (1888) from the pus of wounds in an
individual who succumbed to tetanus.

Morphology. — Bacilli with rounded ends, sometimes so short as to resemble
micrococci; resemble the Bacillus septicaemias haemorrhagicae (fowl cholera).

Stains with the usual aniline colors and also by Gram's method. The
ends are commonly more deeply stained than the central portion.

Biological Characters. — An aerobic and facultative anaerobic, non-
liquefying, non-motile bacillus. Spore formation not observed. Grows in
the usual culture media at the room temperature. Upon gelatin plates yel-
lowish-gray, finely granular, spherical colonies with smooth outlines are
developed. In gelatin stab cultures, at 18° to 25° C., at the end of twenty-
four hours small, spherical colonies are developed along1 the line of punc-
ture, which are isolated or closely crowded; upon the surface a rather thin,
shining, grayish- white, iridescent, circular layer is formed ; gas is given off
which has not a disagreeable odor. Upon the surface of agar elevated,
shining, gray colonies develop along the impfstrich, or a gray, shining band
is formed which increases in thickness but not in breadth — usually less than
one-half centimetre broad. Old cultures give off an acid odor. Upon blood
serum a thin, white layer is developed along the line of inoculation. Upon
potato a thin, white, varnish-like layer is formed.

Pathogenesis. — Very pathogenic for rabbits, guinea-pigs, white mice, and
sparrows. Not pathogenic for chickens, pigeons, or geese.

BACILLUS OF SWINE PLAGUE, MARSEILLES.

Synonyms. — Bacillus der Schweineseuche, Marseilles (Rietsch
and Jobert) ; Bacillus der Frettchenseuche — ferret disease (Eberth
and Schimmelbusch) ; Bacillus der Amerikanischen Kinderseuche
(Caneva) ; Bacillus of spontaneous rabbit septicaemia (Eberth).

The researches of Caneva and of Bunzl-Federn agree as to the
identity of the bacillus obtained by Rietsch and Jobert (1887) from
swine attacked with a fatal epidemic disease in Marseilles, and the
bacillus found by Eberth and Schimmelbusch (1889) in the blood of
ferrets suffering from a fatal form of septicaBmia studied by them.
The first-named bacteriologist also identifies a bacillus supposed
by Billings to be the cause of " Texas fever " in cattle (" Ameri-
kanische Rinderseuche ") and the bacillus of swine plague (Billings)
with the above. Bunzl-Federn obtained cultures of Billings7 swine-
plague bacillus at two different times. He identifies the one first re-
ceived with the bacillus now under consideration, and the other with
the bacillus of hog cholera (Salmon).1

1 The author named says: '-With reference to the bacillus of swine plague
(Billings), I obtained, as did Caneva, a decided production of acid in the cultures
first sent by Billings ; but upon testing later cultures received directly from Bil-

IX SUSCEPTIBLE ANIMALS. 509

Morphology. — Bacilli with rounded ends, about twice as long as
broad, and one-third smaller than the bacillus of typhoid fever
(Eberth and Schimmelbusch). The bacillus of hog cholera is shorter
and more slender than the Marseilles bacillus, and the bacillus of
Loffler and Schiitz is still smaller (Rietsch and Jobert).

In stained preparations the extremities of the rods are usually
deeply stained, while the central portion remains unstained— :i polar
staining. " By Loffler's method of staining the presence of flagella
may be demonstrated (Frosch).

Stains readily with the aniline dyes usually employed, but does
not retain its color When treated by Gram's method.

Biological Characters. — An aerobic (facultative anaerobic),
non-liquefying, actively motile bacillus. Grows readily at the
room temperature, and is distinguished from the bacillus of septi-
caemia hsemorrhagica by its active movements and more rapid and
abundant development in the various culture media usually em-
ployed. It is distinguished from the bacillus of hog cholera by pro-
ducing phenol and indol in solutions containing peptone, by causing
coagulation of milk, and by producing an acid reaction in this fluid.
Grows in culture media having an acid reaction.

Rietsch and Jobert give the following account of the characters
of growth in various culture media, as compared with the bacillus of
hog cholera and the bacillus of Schweineseuche (Loffler, Schutz) :

Gelatin streak cultures. At the end of twenty-four hours this
bacillus had developed considerably, while the growth of the hog-
cholera bacillus was scarcely to be discerned with the naked eye, and
the bacillus of Schweineseuche did not form a visible growth until
the end of forty-eight hours. After several days the bacillus of
swine plague (Marseilles) formed an opaque, yellowish- white streak,
which, when examined with a low-power lens, had a brown color by
transmitted light and a bluish- white color by reflected light. The
streak of the Loffler-Schiitz bacillus was not so thick and not so
opaque, and was made up of small, nearly transparent colonies ; the
hog-cholera bacillus came between the other two. Upon blood
serum, agar, and glycerin-agar the Marseilles bacillus grew more
rapidly than the other two, forming a layer which was opaque and
of a white color, with bluish and reddish reflections. Upon potato
it formed a thick, opaque, yellowish layer, while the growth of the
hog-cholera bacillus was much thinner and that of the Loffler-Schiitz
bacillus scarcely to be seen. In bouillon the Loffler-Schiitz bacillus,
at the end of three days at 37° C., had not produced any perceptible

lings and from other sources, the result was exactly the opposite — viz., a decided
production of alkali in milk and identity with the hog-cholera bacillus of Salmon."

510 BACILLI WHICH PRODUCE SEPTICAEMIA

cloudiness, while the Marseilles bacillus at the end of twenty-four
hours had caused the fluid to be clouded, a film of bacteria had
formed upon the surface and a deposit at the bottom of the tube ; the
hog-cholera bacillus produced a less degree of opacity in the bouillon.

Pathogenesis. — This bacillus is pathogenic for sparrows and
other small birds when injected beneath the skin in small amounts,
and also for pigeons in a longer time — five to fourteen days. Frosch
reports a negative result from subcutaneous injections into rabbits,
guinea-pigs, mice, and pigeons, but his cultures appear to have be-
come attenuated, as the recent cultures of Eberth and Schimmelbusch
were fatal to pigeons in four out of five experiments. Two rabbits
were inoculated subcutaneously by Rietsch and Jobert with half a
Pravaz syringef ul of a pure culture of the Marseilles bacillus ; one of
these died on the sixth day and the other survived.

In sparrows, which succumb in from twenty-four to thirty-six
hours after receiving a small amount of a pure culture in the breast
muscle, the bacillus is present in the blood in large numbers, and a
purulent pleuritis and pericarditis is found at the autopsy. In the
ferrets from which Eberth and Schimmelbusch obtained their cultures
the bacillus was not present in the blood in sufficient numbers to be
readily demonstrated by microscopical examination, but it was ob-
tained in pure cultures from the liver, spleen, and lungs. The prin-
cipal pathological appearances noted were enlargement of the spleen
and pneumonia. Caneva reports that the Marseilles bacillus injected
into white mice gives rise to an extensive abscess at the point of in-
oculation, but does not kill adult animals. In a young mouse which
succumbed to such an injection the bacilli were not generally dis-
tributed in the tissues, but were found as emboli in the smaller capil-
laries. This bacillus, then, is distinguished from the similar bacilli
previously described by its comparatively slight pathogenic power,
as well as by its more vigorous growth in culture media, and the
other characters heretofore mentioned.

BACILLUS SEPTICUS AGRIGENUS.

Obtained by Nicolaier from soil which had been manured.

Morphology. — Resembles the bacillus of fowl cholera and of rabbit sep-
ticaemia, of which it is perhaps a variety, but is usually somewhat longer.
It also sometimes shows the end-staining characteristic of Bacillus septicae-
mias haemorrhagicae, but not so constantly and not so sharply defined.

Biological Characters. — An aerobic, (non-liquefying ?), non- motile ba-
cillus. Does not form spores.

In gelatin plate cultures spherical, finely granular colonies are developed
having a yellowish-brown central portion, which is separated by a dark
ring from a grayish-brown marginal zone; later this difference in color dis-
appears and the colonies become more decidedly granular. In stick cultures
the growth consists of a thin layer which is not at all characteristic.

Pathogenesis. — Small quantities of a pure culture injected into the ear

IX SUSCEPTIBLE ANIMALS. 511

vein of a rabbit cause its death in from twenty-four to thirty-six hours;
pathogenic also for house mice and for field mice. At the autopsy no notable
pathological changes are observed. The bacilli are found in blood from the
heart and in the capillaries of the various organs, but are not so numerous
as in rabbit septicaBmia; they show a special inclination to adhere to the
margins of the red blood corpuscles.

BACILLUS ERYSIPELATOS SUIS.

Synonyms. — Bacillus of hog erysipelas; Bacillus des Schweine-
rothlauf (Loffler, Schutz) ; Bacille du rouget du pore (Pasteur) ; Ba-
cillus of mouse septicaemia; Bacillus murisepticus (Fliigge) ; Bacil-
lus des Mauseseptikamie (Koch).

The bacillus of mouse septicaemia, first described by Koch (1878),
resembles so closely in its morphology, characters of growth, and
pathogenic power the bacillus of Schweinerothlauf of Loffler and
Schutz (1885) that they can scarcely be considered as distinct spe-
cies, although, from slight differences which have been observed, they
are perhaps entitled to separate consideration as varieties of the
same species. Fliigge, Eisenberg, Frankel, and other authors, while
recognizing the fact that the bacilli from the two sources closely re-
semble each other, apparently do not consider
them identical, and describe them separately.
Baumgarten, on the other hand, describes them
under one heading and considers it highly prob-
able that they are identical, although he also
admits slight differences in the morphological
characters and growth in culture media. These
differences are, however, no greater than we
have in artificially produced varieties of other FlG 133_Baclllus of
well-known microorganisms, and we think it mouse septicaemia in leu-
best to follow Baumgarten in describing them
under a single heading.

Koch first obtained this bacillus by injecting putrefying blood or
flesh infusion, during the first days of putrefactive change, beneath
the skin of mice. A certain proportion of the animals experimented
upon contracted a fatal form of septicaemia, and the bacillus under
consideration was found in their blood. The bacillus of Schweine-
rothlauf was obtained by Loffler and by Schutz from the blood and
various organs of swine which had succumbed to the infectious
malady known in Germany as rothlauf and in France as rouget.

Morphology. — Extremely minute bacilli, about 1 v in length and
0.2 v in diameter. The Schweinerothlauf bacilli are described as
somewhat thicker and longer by Fliigge, by Frankel, and by Ei-
senberg, but Baumgarten states that they are somewhat more

512

BACILLI WHICH PRODUCE SEPTIC^MIA

slender and on the average shorter than the bacillus of mouse septi-
ca3mia. The bacilli are solitary, or in pairs the elements of which
are often united at an angle ; occasionally a chain of three or four
elements may be observed, and in old cultures the bacilli may
grow out into short threads which are straight or more or less
curved and twisted. Small refractive bodies may sometimes be dis-
tinguished in the rods, and these have been supposed by some authors
to be spores, but this has not been demonstrated.

This bacillus stains readily with the ordinary aniline staining
agents and also by Gram's method.

Biological Characters. — A. facultative anaerobic, non-liquefy-
ing bacillus. According to Schottelius, the rothlauf bacilli are some-

FIG. 134.— Bacillus of rouget, from a pure culture. X 1,000. From a photomicrograph. (Roux.)

times motile, but Fliigge states that other observers have not seen
them in active motion. Frankel says they have the power of volun-
tary motion. Eisenberg says that the bacillus of mouse septica3mia
is motionless, and Frankel says they " seem to be incapable of volun-
tary motion. " Baumgarten remarks : " Whether the bacilli exhibit
voluntary movements has not been determined." Although this
bacillus is not strictly anaerobic, it grows better in the absence of
oxygen than in its presence. Development occurs in various culture
media at the room temperature, but is more rapid in the culture
oven. In gelatin stab cultures no development occurs upon the
surface, but the growth along the line of puncture is very character-
istic; this consists of a delicate cloud-like, radiating growth, which
extends, in the course of a few days, almost to the walls of the test
tube. The rothlauf bacillus does not extend so rapidly through the

iN SUSCEPTIBLE ANIMALS.

513

gelatin, and the branching, cloud-like growth is not as delicate;

Flugge compares it to the brush of bristles used for cleansing test

tubes. In old cultures in nutrient gelatin a

slight softening of the gelatin occurs along the

line of growth, and as a result of evaporation

and desiccation a funnel-shaped cavity is formed

in the culture medium in the course of two or

three weeks. In gelatin plates colonies are

developed in the course of two or three days in

the deeper layers of the gelatin, but not upon

the surface; these are nebulous, grayish-blue,

radiating masses, which are so delicate as to be

scarcely visible without the aid of a lens or a

dark background. Under a low power they

appear as branching feathery masses, which

have been compared by Flugge to the radiating

growth of " bone corpuscles." In older cultures

they coalesce and cause a nebulous opacity of

the whole plate, which has a bluish-gray lustre.

Upon the surface of nutrient agar or blood
serum a very scanty development occurs along
the line of inoculation. No growth occurs upon
potato. In bouillon the bacilli cause a slight
cloudiness at the outset, and later a scanty gray-
ish-white deposit upon the bottom of the test
tube ; no film is formed upon the surface.

The thermal death-point of this bacillus, as determined by the
writer (1887), is 58° C., the time of exposure being ten minutes. In
the experiments of Bolton it was destroyed in two hours by mercuric
chloride solution in the proportion of 1 : 10,000; by carbolic acid and

FIG. 135. — Bacillus of
mouse septicaemia;
culture in nutrient gela-
tin, end of four days at
18° C. (Baumgarten.)

FIG. 13H.— Bacillus of mouse septicaemia; single colony in nutrient gelatin. X 80. (Flugge.)

by sulphate of copper in one-per-cent solution. These results are
opposed to the view that the minute refractive granules which may
sometimes be seen in the interior of the rods are reproductive spores,
33

514

BACILLI WHICH PRODUCE SEPTIC^MIA

for all known spores have a much greater resisting power to heat
and the chemical agents named.

Pathogenesis.— Pathogenic for swine, rabbits, white mice, house
mice, pigeons, and sparrows. Field mice, guinea-pigs, and chickens
are immune.

Swine may be infected by the ingestion of food containing the
rothlauf bacillus, as has been demonstrated by allowing them to eat
the intestine of an animal which had recently succumbed to the dis-
ease, and also by the subcutaneous injection of pure cultures. The
disease usually terminates fatally within three or four days, and
sometimes in less than twenty-four hours. It is characterized by

FIG. 137.— Section of diaphragm of a mouse dead from mouse septicaemia, showing bacilli in a
capillary bloodvessel. (Baumgarten.)

fever, debility, loss of appetite, and by the appearance upon the sur-
face of the body of red patches, which gradually extend and become
confluent, producing after a time a uniform dark-red or brown color
of the entire surface. The discharges from the bowels frequently
contain bloody mucus. At the autopsy, in acute cases, the spleen is
notably enlarged, and the liver and kidneys are likely to be more or
less swollen, as are also the lymphatic glands, especially those of
the mesentery; the gastric and intestinal mucous membranes are
usually inflamed and spotted with ha3morrhagic extravasations ; the
serous membranes also may be inflamed, and the cavities of the
pleura3, pericardium, and peritoneum usually contain more or less
fluid. The bacilli are found in the blood vessels throughout the
body and are especially numerous in the interior of the leucocytes.

PL ATE VI.

STERNBERG'S BACTERIOLOGY.

. i.

Fig. 2.

Figr. 3.

Fig. 4.

Fig. 6.

IN SUSCEPTIBLE ANIMALS. 515

Cornevin and Kitt have shown that the contents of the intestine
also contain the bacilli in large numbers, and the disease appears to
be propagated among swine principally by the contamination of their
food with the alvine discharges of diseased animals.

Pigeons are very susceptible to the pathogenic action of this ba-
cillus, and usually die within three or four days after inoculation
with a pure culture. Rabbits are not so susceptible, although a
certain proportion die from general infection after being inoculated
in the ear. The first effect of such an inoculation is to produce an
erysipelatous inflammation. When the animal recovers it is subse-
quently immune.

White mice and house mice are extremely susceptible, but field
mice are immune. This remarkable fact was first ascertained by
Koch by experiments with his bacillus of mouse septicaemia. House
mice which have been inoculated with a minute quantity of a pure
culture of the rothlauf , or mouse septicaemia, bacillus, die in from
forty to sixty hours. The animal is usually found dead in a sitting
position, with its back strongly curved, and for many hours before
death it remains quietly sitting in the same position ; the eyes are
glued together by a sticky secretion from the conjunctival mucous
membrane. At the autopsy the spleen is found to be very much en-
larged, and there may be a slight amount of oedema at the point of
inoculation.

The bacilli are found in the blood vessels generally, and are very
numerous in the interior of the leucocytes, which are sometimes com-
pletely filled with them.

BACILLUS COPROGENES PARVUS.

Synonym. — Mauseseptikamieahnlicher Bacillus (Eisenberg).

Obtained by Bienstock from human faeces.

Morphology.— A. very minute bacillus, which is but little longer than it
is broad, and might easily be mistaken for a micrococcus.

Biological Characters. — Grows very slowly on nutrient gelatin, forming
a scarcely visible film along the line of inoculation, which at the end of
several weeks is scarcely one millimetre wide. Is not motile.

Pathogenesis. — In white mice an extensive oedema is developed at «the
point of inoculation at the end of ten or twelve hours, and the animal dies
within thirty- six hours. The bacilli are found in great numbers in the
effused serum at the point of inoculation and in comparatively small num-
bers in the blood. A rabbit inoculated with a pure culture obtained from a
mouse died at the end of eight days. The inoculation, which was made in
the ear, gave rise to a local erysipelatous inflammation.

516

BACILLI WHICH PRODUCE SEPTICAEMIA

BACILLUS CAVICIDA.

Synonym. — Brieger's bacillus. Probably a pathogenic variety of Bac-
terium coli commune of Escherich.

Obtained by Brieger (1884) from human faeces.

Morphology. — Small bacilli, about twice as long as broad, which closely
resemble the colon bacillus of Escherich (Bacterium coli commune).

Biological Characters. — An aerobic (facultative anaerobic), non-liquefy-
ing bacillus.

The growth in gelatin plate cultures is said to be very characteristic, the
colonies being " in the form of very beautifully grouped, whitish, concentric
rings, which are arranged like the ccales upon the back of a turtle " (Eisen-
berg). The writer has studied cultures of this bacillus brought from the
bacteriological laboratories of Germany, side by side with cultures of the
Bacterium coli commune of Escherich, and has found no appreciable differ-
ences in the colonies in gelatin plates, or in the growth in various culture
media. Upon potato it grows rapidly in the incubating oven, forming a
dirty-yellow, moist layer.

Pathogenesis. — This bacillus, as first obtained by Brieger, was character-
ized by being very pathogenic for guinea-pigs, which were invariably killed,
within seventy- two hours, by the subcutaneous injection of a minute quan-
tity of a pure culture. The bacillus was found in great numbers in the
blood of animals which succumbed to an experimental inoculation. The
writer's experiments with this bacillus, made in 1889, indicate that its patho-
genic power had become attenuated, inasmuch as considerable quantities of
a pure culture injected into guinea-pigs did not cause the death of the ani-
mals— culture used came originally from Germany. Not pathogenic for
rabbits or for mice.

BACILLUS CAVICIDA HAVANIENSIS.

This bacillus was obtained by the writer from the contents of the intestine
of a yellow-fever cadaver, in Havana, 1889, through inoculated guinea-pigs.

Morphology. — A bacillus with rounded ends,
from two to three u long and about 0.7 p- broad,
frequently united in pairs.

Stains readily with the ordinary aniline colors.
Biological Characters. — An aerobic and fac-
ultative anaerobic, non -liquefying, actively mo-
tile bacillus.

In gelatin stab cultures the growth upon the
surface is very scanty and thin, not extending far
from the point of puncture ; along the line of
puncture are developed small, translucent, pearl-
like, spherical colonies, which later become opaque
and sometimes granular. In gelatin roll tubes,
at the end of twenty- four hours at 22° C.,
the deep colonies are very small spheres, of a pale
stra w color ; later they become opaque, light-brown
spheres, or may have a dark central mass sur-
rounded by a transparent zone. The superficial

colonies at the end of five days are small, translucent masses of a pale straw
color towards the centre, with thin and irregular margins, sometimes with
a central light-brown nucleus ; at the end of ten days the deep colonies are
still quite small, of a brown color, and opaque.

In gtycerin-agar roll tubes, at the end of twenty-four hours, the deep colo-
nies are in the form of a biconvex lens, and appear spherical when viewed
in face and biconvex when seen from the side ; they have a straw color

FIG. 138.— Bacillus cavicida
Havaniensis; from a potato
culture. X 1,000. From a pho-
tomicrograph. (Sternberg.)

IN SUSCEPTIBLE ANIMALS,

517

by transmitted light and are bluish-white by reflected light ; the superficial
colonies are translucent, with a bluish- white lustre.

On potato, at 22° C., at the end of forty eight hours there is a thin, dirty-
yellow growth of limited extent; at the end of ten days there is a thin,
gamboge yellow layer and little masses of the same color; the growth is
quite thin, with irregular outlines, and is confined to the vicinity of the
impfstrich.

Grows in nutrient agar containing 0.2 per cent of hydrochloric acid.
Thermal death point 55° C. Grows in agua coco without forming gas, and
causes this liquid and bouillon to become slightly translucent — not milky.

Pathogenesis. — Pathogenic for guinea-pigs, less so for rabbits. Guinea-
pigs inoculated subcutaneously with a few drops of a pure culture die in ten
or twelve hours from general infection. There is usually a considerable
effusion of bloody serum in the vicinity of the point of inoculation, and the
spleen is more or less enlarged.

BACILLUS CRASSUS SPUTIGENUS.

Obtained by Kreibohm (1886) from the sputum of two individuals, and
once in scrapings from the tongue.

Morphology. — Short, thick bacilli, of oblong form, with rounded corners,
often bent or twisted — "sausage-shaped." Immediately after division the
bacilli are about one-half longer than they are broad, but before dividing

^m^ymm

f£^/-: &:?%*'. : --s^^cs •"'; -Kr<^^^^p:;%^^v':'
; ._ : • ^ffiSSS^^^Jri^-

FIG. 139.— Bacillus crassus sputigenus, from blood of mouse, x 700. (Flugge.)

again they may attain a length of three to four times the breadth. Irregular
forms with swollen ends or uneven contour are frequently seen.

This bacillus is quickly stained by the ordinary aniline colors and also
by Gram's method.

Biological Characters. — An aerobic, non-liquefying (non-motile ?) ba-
cillus. Grows in various culture media at the room temperature — more
rapidly in the incubating oven. " Appears to form spores at 35° C."
(Fliigge;.

In gelatin plates, at the end of thirty-six hours, grayish- white colonies are
developed, which soon reach the surface of the gelatin and spread out as
round, viscid, grayish white drops, which project considerably above the
surface of the culture medium. Under a low magnifying power recent colo-

518 BACILLI WHICH PRODUCE SEPTICAEMIA

nies appear as spherical, grayish-brown discs, the surface of which is marked
with dark points or lines. The superficial colonies are more transparent,
have irregular outlines, and the surface, especially near the margins, is
coarsely granular. The development in stab cultures is very rapid and re-
sembles that of Friedlander's bacillus — ' ' nail-shaped " growth. Upon potato
the growth is also similar to that of Friedlander's bacillus, and consists of a
thick, grayish- white, moist, and shining layer.

Pathogenesis. — Mice inoculated with a small quantity of a pure culture
die from acute septicaemia in about forty-ei^ht hours. The bacilli are found
in blood from the heart and from the various organs — most numerous in
the liver. Eabbits are killed within forty eight hours by intravenous injec-
tion of a small quantity, and the blood contains the bacillus in great num-
bers. Larger amounts injected into the circulation of rabbits or dogs cause
death in a few hours (three to ten), preceded by diarrhoea, and in some in-
stances bloody discharges from the bowels. At the autopsy an acute gastro-
enteritis is found.

BACILLUS PYOGENES FCETIDUS.

Obtained by Passet (1885) from an abscess of the anus.

Morphology.— Short bacilli with rounded ends, 1.45 n long and 0.58 /u
broad ; usually associated in pairs or in short chains.

Biological Characters. — An aerobic, non-liquefying, motile bacillus.
Grows rapidly in the usual culture media at the room temperature. In the
interior of the rods, in stained preparations, one or two unstained, spherical
places may sometimes be seen, which have been supposed to be spores (?).
The independent motion exhibited by this bacillus is not very active. In
gelatin plates white colonies are developed at the end of twenty-four hours,
which upon the surface spread out as grayish-white plaques, having a dia-
meter sometimes of one centimetre ; these are thickest in the centre and of
a whitish color ; the colonies may become confluent. In gelatin stab cul-
tures the growth upon the surface, at the end of twenty-four hours, consists
of a thin, grayish white layer with rather thick, irregular margins ; along the
line of puncture more or less crowded colonies. Upon potato ih& bacillus
forms an abundant, shining, pale-brown layer. The cultures give off a dis-
agreeable putrefactive odor.

According to Eisenberg, mice and guinea-pigs are killed in twenty-four
hours by injections beneath the skin or into the cavity of the abdomen, and
numerous bacilli are found in the blood.

PROTEUS HOMINIS CAPSULATUS.

Obtained by Bordoni-Uffreduzzi (1887) from two cadavers presenting the
pathological appearances of the so-called " Haderiikrankheit. "

Morphology. — Bacilli, varying considerably in dimensions; somewhat
thicker than the anthrax bacillus; often swollen in the middle or at the ex-
tremities; more or less curved; isolated, united in pairs or in long filaments;
in stained preparations from agar cultures or from blood the bacilli are sur-
rounded by a "capsule."

Stains with the usual aniline colors and also by Gram's method.

Biological Characters. — An aerobic (facultative anaerobic ?), non-lique-
fying, non motile bacillus. Formation of spores not observed. Grows in
the usual culture media at the room temperature. At a temperature of 15° to
17D C. long filaments are formed, in which the bacilli are surrounded with a
capswle ; at 22° to 24° C. the bacilli are for the most part isolated, but few fila-
ments being formed ; at 32° to 37° C. the bacilli are so short as to resemble
micrococci ; development ceases at a temperature of 8° and is very slow at
15° C.

IN SUSCEPTIBLE ANIMALS.

519

This bacillus grows as well in an acid medium as in one which is slightly
alkaline. In gelatin plates, at the end of eighteen to twenty-four hours,
colonies are formed which under a low power are seen to be spherical and
to contain a quantity of shining granules ; the following day, at a tempera-
ture of 15° to 17° C., the colonies may be as large as a pin's head and still
remain spnerical or slightly oval, but
the outline is no longer so uniform,
and between the shining points in the
interior a confused network may be
seen ; as the colony becomes larger it
is raised above the surface of the gela-
tin, becomes opaque, and has a pearly
lustre like that of Friedlander's bacil-
lus. In gelatin stab cultures the
growth resembles that of Friedlan-
der's bacillus — " nail-shaped growth."
Upon the surface of nutrient agar a
rapidly extending, semi transparent
layer is formed. Upon potato, at 15° '
to 17° C., at the end of twenty-four
hours transparent drops are seen in
the vicinity of the point of inocala-
tion, and later a moist, shining, color-
less layer, of tough consistence, is
formed, which gradually extends over

the surface. The growth upon blood
serum resembles that upon nutrient
agar, and the blood serum is not liquefied.

Pia. 140.— Proteus hominis capsulatus, from
liver of mouse. X 1,000. (Bordoni-Uffre^
duzzi.)

IMSL t»i , K/IJ.VI. vi*.\j ^ n.*~r-~r-~~ -^-^.^ «. *-^. .» — — „ ~ . ^ ~. . i liquid Diood serum or in.

bouillon the bacilli are isolated — not in filaments; they cause a clouding of
the liquid, and an abundant deposit accumulates at the bottom of the tube,
while a film of bacilli forms upon the surface. The cultures never give off
a putrefactive odor.

Pathogenesis. — Pathogenic for dogs and for mice, less so for rabbits and
for guinea-pigs. Agar cultures grown in the incubating oven at 32° to 37
G. are more pathogenic than cultures in gelatin at the room temperature.
A small quantity of a recent culture injected subcutaneously in mice cause*
their death in from one to four days, according to the quantity and age of
the culture; the recent cultures are most virulent. When the animal lives
more than twenty -four hours it has a mucous diarrhoea. At the autopsy the
spleen is found to be much enlarged and dark in color ; the lymphatic
glands are also swollen and haemorrhagic, the liver and kidneys hyperaemic;
in the vicinity of the point of inoculation is a subcutaneous oadema of jelly-
like appearance and numerous punctiform haemorrhages are seen. The ba-
cillus is found in great numbers in the effused serum from the subcutaneous
tissues, in the blood, the contents of the intestine, and in the parenchyma of
the various organs. When examined at once the bacilli in the subcutaneous
oedema and in the lymphatic glands are usually quite short, and even spherical,
while in the blood they are somewhat longer and may appear as short fila-
ments with swollen ends, surrounded by a capsule. When the examination
is made some time after the death of the animal longer filaments are quite
numerous. Rabbits and guinea-pigs are killed by the intravenous injection
of comparatively small amounts of a recent culture, but quite large doses
are required to produce a fatal result when the injection is made beneath
the skin. From two to three cubic centimetres of a recent culture injected
into the circulation of a dog give rise to symptoms of toxaemia, and the ani-
mal usually dies on the second day. At the autopsy the abdominal organs
are found to be hyperaemic, the mucous membrane of the intestine swollen,
red in color, and covered with bloody mucus. The bacillus is found in the
blood and in the various organs. When smaller doses are injected into a
vein (a few drops) the animal, after a few hours, has a mucous diarrhoea and

520 BACILLI WHICH PRODUCE SEPTICAEMIA

vomiting, or efforts to vomit. Death usually occurs at the end of two or
three days. At the autopsy the spleen is found to be normal, the other or-
gans slightly hyperaamic, and the intestinal mucous membrane in a state of
catarrhal inflammation. The bacilli are found in the blood and in the vari-
ous organs in considerable numbers.

PROTEUS CAPSULATUS SEPTICUS.

Obtained by Banti (1888) from a case of " acute haemorrhagic infection."
According to Banti, this is possibly identical with the preceding species —

Proteus hominis capsulatus — but in some respects more nearly resembles

Friedlander's bacillus.

BACILLUS ENTERITIDIS.

Obtained by Gartner (1888) from the tissues of a cow which was killed in
consequence of an attack characterized by a mucous diarrhosa, and also from
the spleen of a man who died twelve hours after eating the flesh of this
animal.

Morphology. — Short bacilli, about twice as long as broad, frequently united
in pairs; chains of four to six elements are sometimes seen.

Stains with the usual aniline colors, and presents the peculiarity of
staining deeply at one end while the remainder of the rod is but slightly
stained. When two bacilli are united the deeply stained ends are in apposi-
tion.

Biological Characters. — An aerobic, non-liquefying, motile bacillus.
Spore formation not determined. Grows in the usual culture media at the
room temperature. Upon gelatin plates pale-gray, superficial colonies are
formed at the end of twenty-four hours; under a low power these are seen
to be coarsely granular and transparent ; the central portion usually pre-
sents a greenish color ; deep colonies are spherical, indistinctly granular,
and of a brownish color ; in older colonies a marginal transparent zone is
seen which appears to be made up of minute fragments of glass of a pale-
brown color. In gelatin stab cultures but slight development occurs along
the line of puncture ; upon the surface a thick, grayish- white layer is
formed, which after a time becomes very much wrinkled. Upon the surface
of agar, at 37° C., at the end of eighteen to twenty hours a grayish- yellow
layer has formed. Upon potato a moist, shining, yellowish-gray layer is
developed. The growth upon blood serum is rapid in the form of a gray
layer along the line of inoculation.

Pathogenesis. — White mice and house mice usually die in from one to
three days when fed with a pure culture of this bacillus. Babbits and gui-
nea-pigs die in from two to five days from subcutaneous injections— less
pathogenic for pigeons and canary birds. Dogs, cats, chickens, and sparrows
are immune. A. goat died in twenty hours after receiving an intravenous
injection of two cubic centimetres of a culture in blood serum. The princi-
pal pathological appearance consists in an intense inflammation of the in-
testinal mucous membrane. The bacilli are found in blood from the heart
and also in the contents of the stomach.

BACILLUS OF GROUSE DISEASE.

Obtained by Klein (1889) from the lungs and liver of grouse which had
succumbed to an epidemic disease.

Morphology. — Bacilli with rounded ends, from 0.8 to 1.6/^long; may
also be seen as spherical or oval cells 0.6 /j. long and 0.4 jn thick; solitary, in
pairs, or in chains of three to four elements.

Stains best with Weigert's solution of methylene blue in aniline water.

Biological Characters. — An aerobic, non-liquefying, non-motile bacillus.
Spore formation not observed. Grows in the usual culture media at the

IN SUSCEPTIBLE ANIMALS. 521

room temperature — better in the incubating- oven. Upon gelatin plates, at
20° C., at the end of twenty-four hours small, angular, transparent scales
may bo seen upon the surface with a low-power lens; at the end of three or
four days these form flat, more or less irregular, shining, gray colonies, with
thin and of ten dentate margins ; these colonies may become confluent and
form a dry, scaly layer which by reflected light has a peculiar, fatty lustre
In gelatin stab cultures the superficial growth is in the form of a trans-
parent, dry, grayish layer with dentate margins, not more than three to five
millimetres in diameter. Upon agar, at 36° to 37° C., a thin, whitish-gray,
dry layer is formed.

Pathogenesis. — Pathogenic for mice, for guinea-pigs, for linnets, and for
green-finches; less so for sparrows. Chickens, pigeons, and rabbits, accord-
ing to Klein, are immune. Of eight mice inoculated subcutaneously with
one or two drops of a bouillon culture, six died within forty-eight hours
and two recovered. Out of eight guinea-pigs inoculated in the same way
four died in forty-eight hours and two recovered. At the autopsy the
lungs and liver were found to be hyperaemic, the spleen not enlarged. The
bacilli were present in large numbers in blood from the heart and in the
lungs.

BACILLUS GALLINARUM.

Obtained by Klein (1889) from the blood of chickens which succumbed
to an epidemic disease resembling " fowl cholera." The bacillus is believed
by Klein not to be identical with Pasteur's bacillus of fowl cholera, and is
said not to be pathogenic for rabbits, which would seem to differentiate it
from this bacillus (Bacillus septicaemias hsemorrhagicae).

Morphology. — Bacilli with rounded ends, from 0.8 to 2 u- long and
0.3 to 0.4 it thick ; often in pairs.

Stains with the usual aniline colors.

Biological Characters. — An aerobic, non-liquefying, non-motile bacillus.
Does not form spores. Grows in the usual culture media at the room tem-
perature— better in the incubating oven. Upon gelatin plates forms grayish-
white, superficial colonies, which later present the appearance of flat, homo-
geneous, whitish discs with thin edges and irregular margins, and by
transmitted light have a brownish color. The deep colonies are small and
spherical, and have a brownish color by transmitted light. In gelatin stab
cultures a thin, gray layer with irregular margins and of limited extent
forms upon the surface, and a scanty growth occurs along the line of punc-
ture in the form of a grayish-white line. Upon the surface of agar, at
37° C., a thin, gray layer with irregular margins has developed at the end of
twenty-four hours ; later this extends over the entire surface as a thin, gray-
ish-white layer. No growth occurs upon potato at 37° C. In bouillon, at 37°
C., development occurs, with clouding of the bouillon, within twenty -four
hours; later a deposit consisting of bacilli is seen at the bottom of the tube,
but no film forms upon the surface.

Pathogenesis. — Chickens inoculated subcutaneously with a pure culture
die in from twenty-four hours to eight or nine days. Pigeons and rabbits
are immune.

BACILLUS CAPSULATUS.

Obtained by Pfeiffer (1889) from the blood of a guinea-pig which died
spontaneously.

Morphology. — Thick bacilli with rounded ends, usually two or three
times as long as broad; often united in chains of two or three elements; may
grow out into homogeneous filaments. Stained preparations show the ba-
cilli to be enveloped in an oval capsule which may be considerably broader
than the bacilli themselves — two to five times as broad ; where several ba-
cilli are united they are surrounded by a single capsular envelope.

Stains with the usual aniline colors, but not by Gram's method. In pre-
parations which are deeply stained with hot f uchsin or gentian violet solu-

522

BACILLI WHICH PRODUCE SEPTICAEMIA

tion the capsule is so deeply stained that the bacillus is hidden; by careful
treatment with a weak solution of acetic acid the capsule may be differen-
tiated as a pale- red or violet envelope surrounding the deeply stained bacilli.

Biological Characters. — An aer-
obic aud facultative anaerobic,
non liquefying, non-motile bacillus.
Spore formation not observed.
Grows in the usual culture media
at the room temperature. The cul-
tures in agar or upon potato are very
viscid and draw out into long
threads when touched with the pla-
tinum needle; the blood of an ani-
mal killed by inoculation with this
bacillus has the same viscid charac-
ter. Upon gelatin plates minute
colonies are first visible at the end
of twenty- four to thirty-six hours;
later the deep colonies are white,
oval masses the size of a pin's head ;
the superficial colonies attain the
size of a lentil, and are flattened,
hemispherical masses with a porce-
lain-white color. In gelatin stab
cultures growth occurs to the bot-
tom of the line of puncture, and 011
the surface a shining white, circular,
arched mass forms around the point of puncture, resembling the growth of
Friedlander's bacillus. Upon the surface of agar, at 37° C , at the end of
twenty- four hours a thick, soft layer of a pure white color is formed, which
is very viscid and resembles the growth of Micrococcus tetragenus upon the
same medium. Upon potato an abundant and viscid, shining, yellowish-
white layer is quickly developed.

Pathogenesis. — Pathogenic for white mice and for house mice, which die
at the end of two or three days after being inoculated at the root of the tail
with a small quantity of a pure culture. Inoculation from mouse to mouse
increases the virulence of the cultures. At the autopsy the superficial veins
are distended with blood, the inguinal glands enlarged, the spleen consid-
erably enlarged, the liver and kidneys hyperaemic, the intestine pale, the
heart distended with blood, which usually is very viscid and is drawn out
into threads when touched with the platinum needle. The bacilli are found
in the blood and in all of the organs, in the contents of the peritoneum and
pleurae, and in the exudate in the vicinity of the point of inoculation.
Pathogenic also for guinea pigs and for pigeons; guinea-pigs are infallibly
killed within thirty-six hours by the injection of a single drop of a bouillon
culture, twenty-four hours old, into the cavity of the abdomen; the* blood
contains the bacillus in enormous numbers, as does the viscid fluid found in
the peritoneal cavity. Rabbits do not succumb to intraperitoneal or subcu-
taneous inoculations, but are killed by the intravenous injection of one
cubic centimetre of a recent bouillon culture. Putrefactive changes occur
very quickly in animals killed by inoculation with this bacillus.

FIG 141.— Bacillus capsulatus, from peritoneal
exudate of an inoculated guinea-pig. X 1,000.
From a photomicrograph. (Ffeiffer.)

BACILLUS HYDROPHILUS FUSCUS.

Obtained by Sanarelli (1891) from the lymph of frogs suffering from a
fatal infectious disease.

Morphology. — Bacilli with rounded ends, usually from 1 to 3 /* in length;
often short oval ; may grow out into filaments of 12 to 20 /* in length.

Biological Characters. — An aerobic, liquefying, motile bacillus. Grows
in the usual culture media at the room temperature. In gelatin stab cul-

IN SUSCEPTIBLE ANIMALS.

523

FIG. 143.— Bacillus hydroph'lus fus-
cus, in blood of triton. (Sanarelli.)

tures, at 18° to 20' C., liquefaction has already commenced along- the line of
puncture at the end of twelve hours, and at the end of thirty-six to forty-
eight hours half of the gelatin is liquefied iu funnel shape ; on the third or
fourth day the gelatin is completely lique-
fied, and a thick, white, flocculent deposit
is seen at bottom of the tube. In glycerin-
agar, at 37° C., a slight, bluish, diffuse
fluorescence is seen upon the surface at the
end of twelve hours, and soon after a luxu-
riant growth, which soon covers the entire
surface, is developed ; at the end of twenty-
four to thirty-six hours large gas bubbles
begin to form in the agar; gradually the
fluorescence disappears, the surface growth
becomes thicker and has a dirty -gray color
which changes later to brownish. Blood
serum is a favorable medium and is rapidly
liquefied by this bacillus. Upon potato the
growth is most characteristic. At the end
of twelve hours a thin, straw-yellow layer
is developed along the impfstrich; this
gradually becomes yellow, and at the end
of four to five days has a brown color, resembling that of the glanders bacil-
lus upon potato.

Pathogenesis. — Pathogenic for frogs, toads, lizards, and oth "cold-
blooded" animals; also for guinea-pigs, rabbits, dogs,
cats, mice, chickens, and pigeons. When a few drops of
a bouillon culture are injected into the muscles of the
thigh, swelling and redness at the point of inoculation
are quickly developed, and death usually occurs in eight
to ten hours. The bacilli are found in great numbers in
the blood and in all of the organs. Guinea pigs die from
general infection within twelve hours after receiving a
subcutaneous injection of a small amount of a pure cul-
ture; the spleen is enlarged and the liver and spleen hy-
peraemic; an extensive inflammatory oedema in the vicin-
ity of the inoculation wound is frequently observed; the
bacilli are very numerous in the blood and in all the or-
gans. Rabbits die in five to six hours from an intravenous
injection. Adult dogs are immune, but new-born dogs
(tnree to four days old) die infallibly, after receiving a
subcutaneous injection of a small quantity of a pure cul-
ture, in twelve to thirty-six hours. Young, cats also suc-
cumb to similar inoculations. Chickens and pigeons die
within five to seven hours after receiving an intravenous
injection, but resist subcutaneous injections.

FIG. 143.— Bacillus
hydrophilus fuscus;
culture in nutrieut
gelatin, end of six-
teen hours. (Sana-
relli.)

Pathogenesis.
pigs or for white

BACILLUS TENTHS SPUTIGENUS.

Obtained by Pansini (1890) from sputum.

Morphology. Short bacilli, usually in pairs and sur-
rounded by a capsule.

Stains by Gram's method.

Biological Characters. — An aerobic, non-liquefying,
non motile bacillus. Grows in nutrient gelatin at the
room temperature. Develops abundantly on potato.
Coagulates milk and produces an acid reaction in this
medium.

— Pathogenic for rabbits and white rats ; not for guinea-
mice (in small doses).

524 BACILLI WHICH PRODUCE SEPTICAEMIA

BACILLUS OF LASER.

Obtained by Laser (1892) from mice which succumbed to an epidemic dis
ease in Frankel's laboratory at Konigsberg.

In its characters this bacillus closely resembles the bacillus of swine
plague, and is perhaps identical with it.

Morphology. — A small bacillus, with rounded ends, about twice as long
as broad. Has fiagella both at the extremities and sides.

Stains by the usual aniline colors and also by Gram's method.

Biological Characters. — An aerobic and facultative anaerobic, non-
liquefying, actively motile bacillus. Spore formation not observed. Grows
either in the incubating oven or at the room temperature. Thermal death -
point 65° to 70° C. — ten minutes' exposure. Upon gelatin plates, at the end
of two days, the deep colonies are spherical, finely granular, and brownish
in color; the superficial are transparent, finely granular, and leaf -like.
In gelatin stab cultures growth occurs along the entire line of puncture as
well as upon the surface. At the end of three days a considerable evolution
of gas is usually observed. In agar an abundant development is seen at the
end of twenty- four hours in the incubating oven; upon the surface a gray-
ish-white, shining layer with dentate margins is formed along the track of
the needle. In bouillon, at 37° C., development is abundant and rapid; a
thin film is formed on the surface at the end of the second day. Upon potato
a brownish layer is formed at the end of twenty-four hours. In milk an
acid reaction is produced.

Pathogenesis. — Pathogenic for field mice, guinea-pigs, rabbits, and
pigeons. The bacillus is found in the blood and various organs of infected
mice. The spleen is found to be greatly enlarged.

BACILLUS TYPHI MURIUM (Loffler).

Obtained by Loffler (1889) from mice which died in his laboratory from
an epidemic disease due to this bacillus.

Morphology. — Short bacilli, resembling the bacillus of diphtheria in
pigeons, and varying considerably in dimensions— like the bacillus of
typhoid fever; grows out in to flexible filaments.

Stains with the aniline colors— best with Loffler's solution of methylene
blue.

Biological Characters. — An aerobic and facultative anaerobic, non-
liquefying, motile bacillus. Spore formation not determined. Has flagella
around the periphery of the cells, like those of the typhoid bacillus, and ex-
hibits similar active movements. In gelatin stab cultures, at the room
temperature, growth occurs upon the surface, at the end of forty -eight hours,
in the form of a flat, grayish- white, round, semi-transparent mass the size of
a pin's head ; later the surface colony increases in extent and has more or
less irregular margins. In gelatin plate cultures the deep colonies are at
first round, slightly granular, transparent, and grayish; later they are of a
yellowish- brown color and decidedly granular. The superficial colonies are
very granular and marked by delicate lines — similar to colonies of the
typhoid bacillus. Upon agar a grayish- white layer is developed which is
not at all characteristic. Upon potato a rather thin, whitish layer is formed,
and around this the potato acquires a dirty bluish-gray color. In milk an
abundant development occurs, and a decidedly acid reaction is produced
without causing any perceptible change in the appearance of the fluid.

Pathogenesis.— Pathogenic for white mice, which die in from one to two
weeks after infection ; also to field mice, which succumb to subcutaneous in-
jections of a pure culture, and also, in from eight to twelve days, when fed
upon potato cultures or bread moistened with a small quantity of a bouillon
culture. Loffler believes that this bacillus may be used for the destruction
of field mice in grain fields, inasmuch as they invariably die after ingesting
food which has been contaminated with it, and also from eating the bodies

IN SUSCEPTIBLE ANIMALS. 525

of other mice which have died as a result of infection. House mice are also
susceptible. Rabbits, guinea pig's, pig-eons, and chickens were found by
Loffler not to be susceptible to infection by feeding.

BACILLUS OF CAZAL AND VAILLARD.

Obtained by Cazal and Vaillard (1891) from cheesy nodules upon the
peritoneum and in the pancreas of an individual who died in the hospital
at Val de Grace.

Morphology. — Bacilli with rounded ends, but little longer than they are
broad ; solitary, in pairs, or in chains of ten to fifteen or more elements.

Stains with the usual aniline colors, but not by Gram's method ; the
extremities of the rods are more deeply stained than the central portion—
44 polar staining."

Biological Characters. — An aerobic find facultative anaerobic, liquefy-
ing, motile bacillus. Does not form spores. Grows in the usual culture media
at the room temperature — more rapidly in the incubating oven at 37° C. In
gelatin stab cultures, at the end of twenty-four hours, a series of puncti-
f orm, white colonies is developed along the line of puncture ; upon the sur-
face development is more abundant, and at the end of forty-eight hours
liquefaction commences ; this progresses slowly from above downward,
and a white, flocculent deposit accumulates at the bottom of the liquefied

Statin. ^Upon the surface of agar, at the end of twenty-four hours at 37°
., a moist, transparent, opalescent layer is developed, which rapidly ex-
tends over the entire surface ; later this layer becomes somewhat thicker,
whitish, and cream- like in consistence, without losing its transparency.
Upon potato a thick, prominent, moist, and slightly viscid layer is devel-
oped, which at first has a pale-yellow and later a yellowish-brown color.
In bouillon development is abundant, producing a milky opacity of the
liquid; a thick, flocculent deposit accumulates at the bottom of the tube ;
the reaction of the culture liquid becomes very alkaline. All of the cultures
give off a peculiar odor, slightly ammoniacal and resembling1 that of putrid
urine. The cultures retain their vitality for several months — in a closed
tube for more than a year. The thermal death-point is 60° C. with fifteen
minutes' exposure.

Pathogenesis. — Pathogenic for rabbits and mice, but not for guinea-pigs.
In mice death occurs from general infection, at the end of forty-eight to
sixty hours, from the subcutaneous injection of one eighth cubic centimetre
of a recent bouillon culture. In rabbits injection of one cubic centimetre
into the circulation causes the death of the animal in thirty-six to fifty
hours. The symptoms induced are a foetid diarrhoea and paralysis of the
extremities. When smaller doses are injected (0.5 cubic centimetre) a
chronic malady is developed, characterized at the outset by diarrhoea and
emaciation, then by the development of tumors which resemble those found
in the man from whom the cultures were first obtained. These tumors are
for the most part located in the subcutaneous connective tissue ; after a time
they attain the size of a chestnut and ulcerate, allowing the escape of a
semi-fluid, purulent material. The animals usually recover. Similar tumors
are developed as a result of subcutaneous injections of one to three cubic
centimetres of a recent bouillon culture.

BACILLUS OF BABES AND OPRESCU.

Obtained by Babes and Oprescu (1891) from a case of septicaemia hsemor-
rhagica presenting some resemblance to exanthematic typhus.

Morphology.— In agar cultures the bacilli are from 0.4 to 0.5 jj. thick, and
are frequently united in pairs ; associated with these rod-shaped bacteria are
forms which are of a short oval. In gelatin cultures oval forms are more
numerous ; they have a diameter of 0.3 to 0.4 JLI, and often appear to be
surrounded by a capsule. In fresh cultures the bacilli are often in form of

BACILLI WHICH PRODUCE SEPTICAEMIA

a figure 8, and are only stained at the point of contact of the two segments.
In potato cultures they are sometimes elongated and swollen at one ex-
tremity.

Stains with the usual aniline colors and by Gram's method.

Biological Characters. — An aerobic and facultative anaerobic, non-lique-
fying, actively motile bacillus. Spore formation not observed. Grows in the
usual culture media at the room temperature — more rapidly at 37° C. In
gelatin stab cultures yellowish- white colonies are developed along the line
of puncture; at the bottom these may have a diameter of one to two millime-
tres, and they have a brown color. Upon the surface an irregular, lobulated,
whitish, translucent, paraffin like layer is developed. At the end of eight
days the surface growth consists of large, confluent, transparent plaques,
with irregular outlines and crenated, elevated margins ; along the line of
puncture large, separate, lenticular or spherical colonies are seen ; these
have a brownish-white color. At the end of two months the surface growtli
is concentric and still more transparent, while the colonies near the surface
have become almost brown. Upon the surface of agar, at 37° C., a narrow
band is developed along the line of inoculation ; above, this is composed of
transparent, shining, flat, round colonies having a diameter of one milli-
metre or more ; below, the colonies are confluent and form a transparent,
whitish layer. In glycerin-agar development is still more abundant, and
may already be perceived at the end of twelve hours. Crystals are seen
below the surface in agar cultures and about the super ficial colonies in gela-
tin. Upon potato a uniform, thin, grayish, very transparent layer is de-
veloped, which sometimes has a brownish-gray tint. At the end of a few
days the potato acquires a brownish color. In bouillon cloudiness of the
medium is apparent at the end of ten hours ; twenty-four hours later a
whitish precipitate is seen at the bottom of the tube, which is more abun-
dant when the culture medium contains glucose; later a thin pellicle is
seen upon the surface and the bouillon acquires a yellowish color.

Pathogenesis. — Recent cultures are pathogenic for rabbits, guinea-pigs,
pigeons, and mice, which die from general infection in from two to four
days. Old cultures are less virulent.

BACILLUS OF LUCET.

Obtained by Lucet (1891) from chickens and turkeys suffering from an
infectious form of septicaemia characterized by dysenteric discharges — " Dy-
seiiterie epizootique des poules et des dindes."

Resembles Bacillus gallinarum of Klein, and is perhaps identical with
this microorganism.

Morphology. — Short bacilli, from 1.2 to 1.8 fit long, usually in pairs.

Stains with the usual aniline colors, but not by Gram's method.

Biological Characters. — An aerobic and facultative anaerobic, non-lique-
fying, non-motile bacillus. Spore formation not observed. Grows slowly in
the usual culture media at the room temperature — more rapidly at 37° 0.

In gelatin plates small, shining, moist, white, circular colonies are devel-
oped, which look like little drops of wax ; later these increase in size, and
especially in thickness, forming hemispherical masses. In gelatin stab cul-
tures grayish, punctiform colonies are developed along the line of puncture,
and upon the surface a circular, prominent, whitish plaque. Streak cultures
upon the surface of gelatin are in the form of a dirty- white or grayish- white,
moist streak, with regular margins, limited to the line of inoculation, but
increasing in thickness until it breaks loose and slips down the oblique sur-
face of the culture medium. The deposit which collects in this way acquires,
as it becomes old, in the deepest portion a reddish color. Upon agar it forms
a thick, yellowish-white, mucus-like layer with straight or slightly dentate
margins. In bouillon it produces a decided clouding of the liquid, and an
abundant grayish, pulverulent sediment accumulates at the bottom of th^
tube ; the bouillon after a time becomes transparent above this sediment and

PLATE VII.

BACILLUS OF GLANDERS.

FIG. 1. — Bacillus mallei from the liver of a field mouse, cover-glass
preparation. (Loffler.)

FIG. 2. — Bacillus mallei from a recent culture upon blood serum.
(Loffler.)

FIG. 3. — Bacillus mallei in section of spleen of a field mouse dead
from glanders. (Loffler.)

FIG. 4. — Culture of glanders bacillus upon cooked potato. (Loffler.)

STERNBERG'S BACTERIOLOGY.

Fig.l.

Fig. 2.

BACILLUS OF GIANDERS (LOEFFLER)

IN SUSCEPTIBLE ANIMALS. 527

is viscid, drawing out into threads. In the absence of oxygen the characters
of growth are the same as in its presence. The cultures acquire an alkaline
reaction ; they are sterilized by exposure for ten minutes to a temperature of
60° C. Does not grow upon potato.

Pathogenesis. — Pathogenic for chickens and turkeys. Not pathogenic for
pigeons, guinea-pigs, or rabbits when injected subcutaiieously or into the
peritoneal cavity, but kills rabbits when injected into a vein. In the in-
fected fowls the bacilli are found in small numbers in the blood, more nu-
merous in the kidneys and liver, still- more numerous in the spleen, and in
enormous numbers in the intestinal mucus, where in acute cases it is found
almost in a pure culture. Fowls do not contract the disease as a result of
the ingestioii of grains soiled with cultures of the bacillus, but become in-
fected when fed with animal food to which a pure culture has been added.

XIV.

PATHOGENIC AEROBIC BACILLI NOT DESCRIBED IN

PREVIOUS SECTIONS.

A CONSIDERABLE number of saprophytic bacilli are pathogenic for
small animals when injected into the circulation, or subcutaneously,
or into a serous cavity in considerable quantity — one to five cubic
centimetres or more — but fail to produce any appreciable effect
when introduced into the bodies of these animals in minute doses,
and do not multiply in the blood to any considerable extent, al-
though in fatal cases they may usually be recovered in cultures from
the blood and tissues. These bacilli are pathogenic by reason of the
toxic ptomaines produced by them, or because of local inflammatory
processes which they induce, or for both of these reasons combined.
Some of them may also, under certain circumstances, multiply in
the blood and thus give rise to septicaemia as well as to toxaemia ;
this is the case, for example, with the " colon bacillus " of Escher-
ich. When injected in considerable quantity into the circulation
of a guinea-pig it causes the death of the animal within twenty-four
hours, and the bacillus is found in the blood in great numbers ; but
minute amounts injected into a vein, or larger amounts injected
subcutaneousiy, do not usually produce general infection. It is,
therefore, not included among the "bacilli which produce septi-
caemia in susceptible animals." There is reason to believe, however,
that under certain circumstances this bacillus may have sufficient
pathogenic potency to produce a genuine septicaemia in guinea-pigs.
Thus the original cultures of Brieger's bacillus, which appears to be
a variety of the colon bacillus, are reported to have produced fatal
septicaemia in guinea-pigs when injected subcutaneousiy in small
amounts. A strict division into pathogenic bacilli which produce
general blood infection — septicaemia — and those which produce a
fatal result owing to the production of toxic chemical substances is
not possible; for many pathogenic bacteria produce general infection
when injected in comparatively large doses, and at the same time
give rise to symptoms of toxaemia ; or general infection may occur
in animals of one species, and fatal toxaemia without septicaemia in

PATHOGENIC AEROBIC BACILLI NOT BEFORE DESCRIBED. 529

those of another species. Many of the bacilli described in the pre-
sent section are common saprophytes, which have been shown by
laboratory experiments to be pathogenic for certain animals when
introduced into their bodies in a certain amount, which differs greatly
for different bacteria and for different species of animals. The ex-
periments of Cheyne and others show how largely the pathogenic
power of saprophytic bacteria depends upon the quantity of a cul-
ture which is injected, as well as upon the age of the culture and
the seat of the inoculation — in the blood, the abdominal cavity, the
subcutaneous tissues, or the muscles. And the bacteriologist named
has also shown that pathogenic power depends, in some instances at
least, upon the combined action of the toxic substances introduced
in the first instance and of the living bacteria. Thus Cheyne found
that one-tenth of a cubic centimetre of a bouillon culture of Proteus
vulgaris injected into the dorsal muscles of a rabbit infallibly caused
its death within forty-eight hours, but when the dose was reduced
to one-fortieth cubic centimetre the animal recovered. But if to
this amount (one-fortieth cubic centimetre) he added one cubic cen-
timetre of a sterilized (by heat) culture of the same bacillus instead
of diluting with distilled water, and injected the mixture into the
dorsal muscles of a rabbit, death occurred in every experiment
within forty-eight hours. The sterilized culture injected by itself
produced no effect in this dose (one cubic centimetre), and Cheyne
believes that the fatal result in these experiments was due to the
fact that the toxic products present in the sterilized culture over-
came the natural resisting powers of the tissues and enabled the
bacillus to multiply over a larger area than would otherwise have
been the case. As a result of this, toxic substances were produced in
the body of the animal in sufficient quantity to cause general toxae-
mia and death ; whereas the bacilli alone, in the dose mentioned,
were not able to invade the tissues in the vicinity of the point of
inoculation, and gave rise to a local abscess only. The same ex-
planation is probably true for very many of the saprophytic bacteria
which have been shown to possess pathogenic power ; and it is prob-
able that many of those which are now classed by bacteriologists as
non-pathogenic would prove to be pathogenic in the same way if
thoroughly tested upon various species of animals, although it might
be necessary to use unusually large doses to accomplish the same
result.

BACILLUS COLI COMMUNIS.

Synonyms. — Bacterium coli commune (Escherich); Colon bacillus
of Escherich ; Emmerich's bacillus (Bacillus Neapolitanus). Prob<
ably identical with Bacillus cavicida (Brieger's bacillus).
34

530 PATHOGENIC AEROBIC BACILLI

Obtained by Emmerich (1885) from the blood, various organs, and
the alvine discharges of cholera patients at Naples ; by Weisser
(1886) from normal and abnormal human faeces, from the air, and
from putrefying infusions ; by Escherich (1886) from the faeces of
healthy children ; since shown to be commonly present in the alvine
discharges of healthy men, and probably of many of the lower ani-
mals. Found by the writer in the blood and various organs of yellow-
fever cadavers, in Havana (1888 and 1889).

Numerous varieties have been cultivated by different bacteriolo-
gists, which vary in pathogenic power and to some extent in their
growth in various culture media ; but the differences described are
not sufficiently characteristic or constant to justify us in considering
them as distinct species.

Morphology. — Differs considerably in its morphology as obtained

from different sources and in various culture media. The typical

form is that of short rods with rounded ends, from two to three >w in

length and 0.4 to 0.6 /^ broad ; but under certain cir-

« «^ ^ cumstances the length does not exceed the breadth—
ff WJ/ J ^ about 0.5 fj. — and it might be mistaken for a micrococ-
cus ; again the prevailing form in a culture is a short

oval ; filaments of five /* or more in length are often
FIG. 144.— Ba- observed in cultures, associated with short rods or oval
emus coii com- u The bacilli are frequently united in pairs. The

munis. X 1 000. «_

(Escherich.) presence of spores has not been demonstrated. In un-
favorable culture media the bacilli, in stained prepara-
tions, may present unstained places, which are supposed by Escherich
to be due to degenerative changes in the protoplasm. Under certain
circumstances some of the rods in a pure culture have been observed
by Escherich to present spherical, unstained portions at one or both
extremities, which closely resemble spores,but which he was not able
to stain by the methods usually employed for staining spores, and
which he is inclined to regard as " involution forms."

This bacillus stains readily with the aniline colors usually em-
ployed by bacteriologists, but quickly parts with its color when
treated with iodine solution — Gram's method — or with diluted al-
cohol.

Biological Characters. — An aerobic and facultative anaerobic,
non-liquefying bacillus. Sometimes exhibits independent move-
ments, which are not very active. One rod of a pair, in a hanging-
drop culture, may advance slowly with a to-and-fro movement,
while the other follows as if attached to it by an invisible band
(Escherich). The writer's personal observations lead him to believe
that, as a rule, this bacillus does not exhibit independent movements.
Does not form spores. Grows in various culture media at the room

NOT DESCRIBED IN PREVIOUS SECTIONS. 531

temperature — more rapidly in the incubating oven. Grows in a de-
cidedly acid medium.

In gelatin plates colonies are developed in from twenty-four to
forty-eight hours, which vary considerably in their appearance ac-
cording to their age, and in different cultures in the same medium.
The deep colonies are usually spherical and at first are transparent,
homogeneous, and of a pale-straw or amber color by transmitted
light ; later they frequently have a dark-brown, opaque central por-
tion surrounded by a more transparent peripheral zone ; or they may
be coarsely granular and opaque ; sometimes they have a long-oval
or "whetstone" form. The superficial colonies differ still more in
appearance ; very young colonies by transmitted light often resemble
little drops of water or fragments of broken glass ; when they have
sufficient space for their development they quickly increase in size,
and may attain a diameter of three to four centimetres ; the central
portion is thickest, and is often marked by a spherical nucleus of a
dark-brown color when the colony has started below the surface of
the gelatin ; the margins are thin and transparent, the thickness
gradually increasing towards the centre, as does also the color, which
by transmitted light varies from light straw color or amber to a dark
brown. The outlines of superficial colonies are more or less irregular,
and the surface may be marked by ridges, fissures, or concentric
rings, or may be granular. The writer has observed colonies re-
sembling a rosette, or a daisy with expanded petals. Escherich
speaks of colonies which present star-shaped figures surrounded by
concentric rings.

In gelatin stab cultures the growth upon the surface is rather
dry, and may be quite thin, extending over the entire surface of the
gelatin, or it may be thicker with irregular, leaf -like outlines and
with superficial incrustations or concentric annular markings. An
abundant development occurs all along the line of puncture, which
in the deeper portion of the gelatin is made up of more or less closely
crowded colonies ; these are white by reflected light, and of an am-
ber or light-brown color by transmitted light ; later they may become
granular and opaque. Frequently a diffused cloudy appearance i&
observed near the surface of the gelatin, and under certain circum-
stances branching, moss-like tufts develop at intervals along the line
of growth. One or more gas bubbles may often be seen in recent
stick cultures in gelatin.

Upon nutrient agar and blood serum, in the incubating oven, an
abundant, soft, white layer is quickly developed. Upon potato an
abundant, soft, shining layer of a brownish-yellow color is developed.
The growth upon potato differs considerably, according to the age of
the potato. According to Escherich, upon old potatoes there may

532

PATHOGENIC AEROBIC BACILLI

be no growth, or it may be scanty and of a white color. In milk, at
37° C., an acid reaction and coagulation of the casein are produced at
the end of eight or ten days. In the absence of oxygen this bacillus
is able to grow in solutions containing grape sugar (Escherich). In
bouillon it grows rapidly, producing a milky opacity of the culture
liquid. The thermal death-point of Emmerich's bacillus, and of the
colon bacillus from faeces, was found by Weisser to be 60° C., the
time of exposure being ten minutes. The writer has obtained corre-
sponding results. Weisser found that when the bacilli from a bouil-
lon culture were dried upon thin glass covers they failed to grow

Fio. 145. FIG. 146.

Fia. 145. — Bacillus coli communis in nutrient gelatin containing twenty per cent of gelatin, end
of two weeks, showing moss-like tufts along the line of growth. (Sternberg.)

FIG. 146.— A portion of the growth shown in Fig 147, at a, magnified about six diameters.
From a photograph. (Sternberg.)

after twenty-four hours. These results give confirmation to the
view that the bacillus under consideration does not form spores.
This view receives further support from the experiments of Wal-
liczek (1894), who found that when dried upon pieces of sterile filter
paper the bacillus failed to grow at the end of eighteen hours.

Pathogenesis. — Comparatively small amounts of a pure culture
of the colon bacillus injected into the circulation of a guinea-pig
usually cause the death of the animal in from one to three days, and
the bacillus is found in considerable numbers in its blood. But when

NOT DESCRIBED IN PREVIOUS SECTIONS. 533

injected subcutaneously or into the peritoneal cavity of rabbits or
guinea-pigs, a fatal termination depends largely on the quantity in-
jected ; and although the bacillus may be obtained in cultures from
the blood and the parenchyma of the various organs, it is not present
in large numbers, and death appears to be due to toxaemia rather than
to septicaemia. Mice are not susceptible to infection by subcutaneous
injections. Small quantities injected beneath the skin of guinea-pigs
usually produce a local abscess only ; larger amounts — two to five
cubic centimetres — frequently produce a fatal result, with symptoms
and pathological appearances corresponding with those resulting
from intravenous injection. These are fever, developed soon after
the injection, diarrhoea, and symptoms of collapse appearing shortly
before death. At the autopsy the liver and spleen appear normal, or
nearly so ; the kidneys are congested and may present scattered
punctiform ecchymoses (Weisser). According to Escherich, the
spleen is often somewhat enlarged. The small intestine is hyper-
aemic, especially in its upper portion, and the peritoneal layer pre-
sents a rosy color ; the mucous membrane gives evidence of more
or less intense catarrhal inflammation, and contains mucus, often
slightly mixed with blood. In rabbits death occurs at a somewhat
later date, and diarrhoea is a common symptom. In dogs the subcu-
taneous injection of a considerable quantity of a pure culture may
give rise to an extensive local abscess.

In human pathology the colon bacillus plays an important role.
It is concerned in the etiology of a considerable proportion of the
cases of cystitis and of pyelonephritis, and peritonitis resulting from
perforation. It appears to be the cause of certain affections of the
anal region (Hartmann and Lieffring). It has been obtained in pure
culture from abscesses in various parts of the body, from the valves
of the heart in endocarditis, from the pleural cavity in empyema, etc.
It has also been found in the blood, as a result of general infection
following cystitis and pyelonephritis (Sittmann and Barnow).

Varieties. — Booker, in his extended studies relating to the bac-
teria present in the faeces of infants suffering from summer diarrhoea,
has isolated seven varieties " which closely resemble Bacterium coli
commune in morphology and growth in agar, neutral gelatin, and
potato, but by means of other tests a distinction can be made between
them."

Some of the pathogenic bacteria heretofore described are also
closely allied to the " colon bacillus " and by some bacteriologists are
supposed to belong to the same group — i.e., to be varieties of the
same species rather than independent species with fixed characters.
Whatever may be the remote relationship, the typhoid group, the hog-

534 PATHOGENIC AEROBIC BACILLI

cholera group, the Bacillus typhi murium of Loffler, the bacillus of
Laser, the Bacillus enteritidis of Gartner, and other similar bacilli
appear to be differentiated from one another by characters which
justify their description under separate names. Still it is difficult to
fix upon any one of these characters to which specific value can be
attached ; and, in view of the many varieties found in nature or pro-
duced artificially in laboratory experiments, we are not justified in
asserting that our classification of these low organisms has any sub-
stantial scientific foundation. The difficulties attending an attempt
to establish specific characters are well illustrated by the extensive
literature relating to the differentiation of bacilli belonging to the
typhoid group from those belonging to the colon group. The main
points upon which the distinction must depend have been referred to
in the section devoted to the typhoid bacillus.

Fremlin (1893) has made a comparative study of the colon bacil-
lus from various sources. He finds the common characters of gas
production in media containing sugar and coagulation of milk. Cul-
tivated from different animals the morphology is the same, but there
are differences as regards motility. The most active movements are
said to be exhibited in the bacillus from man, while the variety ob-
tained from the intestines of rabbits showed scarcely any movements.
The different varieties displayed considerable differences in their
growth upon potato.

Dreyfuss (1894) finds decided differences in the pathogenic viru-
lence of the colon bacillus from healthy individuals and from those
suffering from various intestinal disorders. A culture from the dis-
charges of a fatal case of cholera nostras proved to be exceptionally
virulent — tested by intraperitoneal injections in guinea-pigs. Gilbert
(1895), as a result of his extended researches, concludes that there are
five principal types among the bacilli most nearly related to the colon
bacillus: 1st. Bacilli which differ from the colon bacillus by their
being non-motile. This type includes two varieties : one gives thick
yellowish colonies upon gelatin plates and numerous gas bubbles on
potato — this is the bacille lactique of Pasteur and the Bacillus lactis
aerogenes of Escherich ; the other gives thin, bluish-white colonies
and includes the bacille de V endocardite of Gilbert and Lion. 2d.
Bacilli which differ from the colon bacillus by the fact that cultures
do not give the indol reaction. 3d. Bacilli which do not cause the
fermentation of lactose. 4th. Bacilli which are not motile and
do not ferment lactose. 5th. Bacilli which are not motile, do not
give the indol reaction, and do not ferment lactose.

Theobald Smith (1895) gives the following account of his method
of detecting bacilli of the " colon group " in water :

NOT DESCRIBED IN PREVIOUS SECTIONS. 535

4 ' The method followed by the writer in the general bacteriological exam-
ination of water consists, first, in the preparation of gelatin plates for the
usual enumeration ; and, second, in the addition to every one of ten fermen-
tation tubes, containing a oiie-per-cent dextrose bouillon, a certain quantity
of water. This is added most easily by first diluting the water, so that one or
two cubic centimetres are equivalent to the quantity which it is desired to add
to each tube. Pipettes graduated by drops are convenient, but not so accurate.
In case of ground water it is well to prepare in addition a flask containing
fifty to one hundred cubic centimetres of the water, and an equal, or greater,
quantity of bouillon, to which sugar is not added. Plates may be prepared
from this flask after sixteen to twenty -four hours. When gas begins to ap-
pear in the fermentation tubes, the amount accumulated at the end of each
twenty-four hours should be marked with a glass pencil on the tube. From
these tubes, which contain fifty to sixty per cent of gas on the third day, and
are very strongly acid, plates may be prepared to confirm the indications of
Bacillus coli. This, however, is not essential, for the writer has found as
yet no species having these fermentative characters which is not one of the
following : Bacillus coli, Bacillus lactis aerogenes, Bacillus enteriditis, Bacil-
lus typhi murium, Bacillus cholera? suis. The three last-mentioned species
are probably as rare in water as Bacillus typhosus itself.

' ' My own experience coincides with that of Matthews when he states that
ninety-two per cent of all bacteria in ground water are suppressed in the
thermostat. While the addition of 0.5 cubic centimetre, or even more, of
such water may fail to produce cloudiness in any of the series of fermenta-
tion tubes, the same quantity, or less, of surface water never fails to infect
the tubes."

Bacillus Coli Communis in Peritonitis. — The researches of A.
Frankel show that Bacillus coli communis may be obtained in pure
cultures from the exudate into the peritoneal cavity in a considerable
proportion of the cases of peritonitis, and there is good reason for
believing that in these cases it was the cause of the inflammatory
process. Thirty-one cases were examined by Frankel, with the fol-
lowing result: Pure cultures of Bacillus coli communis were obtained
in nine cases ; of Streptococcus (pyogenes ?) in seven ; of Bacillus
lactis aerogenes in two; of " diplococcus pneumonise " in one; of
Staphylococcus pyogenes aureus in one. Of the remaining eleven
cases, seven gave mixed cultures, and in three of these Bacillus coli
communis was the most abundant species. The author referred to
has also shown that pure cultures of Bacillus coli communis injected
into the cavity of the abdomen of rabbits cause a typical peritonitis.
The present writer has frequently obtained the same result in experi-
ments made with this bacillus. It would appear, therefore, that the
peritonitis which so constantly results from wounds of the intestine
is probably due, to a considerable extent, to the introduction of this
microorganism from the lumen of the intestine, where it is con-
stantly found, into the peritoneal cavity, where the conditions are
favorable for its rapid development.

536 PATHOGENIC AEROBIC BACILLI

BACILLUS LACTIS AEROGENES.

Obtained by Escherich (1886) from the contents of the small intestine of
children and animals fed upon milk ; in smaller numbers from the fogces of
milk-fed children, and in one instance from uncooked cow's milk.

Morphology. — Short rods with rounded ends, from 1 to
\ 2 P in length and from 0.1 to 0.5 n broad; short oval and

•j^ %* spherical forms are also frequently observed, and, under
98 certain circumstances, longer rods — 3 v— may be developed :
$*° / $ usually united in pairs, and occasionally in chains contain-
ing several elements. In some of the larger cells Escherich
has observed unstained spaces, but was not able to obtain
any evidence that these represent spores.

This bacillus stains readily with the ordinary aniline
colors, but does not retain its color when treated by Gram's

ciicricii.) . i i

method.

Biological Characters. — An aerobic (facultative anaerobic), non-liquefy-
ing, non motile bacillus. Does not form spores. Grows in various culture
media at the room temperature — more rapidly in the incubating oven.
Upon gelatin plates, at the end of twenty-four hours, small white colonies
are developed. Upon the surface these form hemispherical, soft, shining
masses which, examined under the microscope, are found to be homogeneous
and opaque, with a whitish lustre by reflected light. The deep colonies are
spherical and opaque and attain a considerable size. In gelatin stab cul-
tures the growth resembles that of Friedlander's bacillus — i.e.. an abundant
growth along the line of puncture and a rounded mass upon the surface,
forming a " nail-shaped" growth. In old cultures the upper portion of the
gelatin is sometimes clouded, and numerous gas bubbles may form in the
gelatin. Upon the surface of nutrient agar an abundant, soft, white layer
is developed. Upon old potatoes, in the incubating oven, at the end of
twenty-four hours a yellowish -white layer, several millimetres thick, is
developed, which is of paste-like consistence and contains about the peri-
phery a considerable number of small gas bubbles ; this layer increases in
dimensions, has an irregular outline, and larger and more numerous gas
bubbles are developed about the periphery, some the size of a pea; later the
whole surface of the potato is covered with a creamy, semi-fluid mass filled
with gas bubbles. On young potatoes the development is different ; a rather
luxuriant, thick, white or pale-yellow layer is formed, which is tolerably
dry and has irregular margins; the surface is smooth and shining, and a
few minute gas bubbles only are formed after several days.

Pathogenesis. — Injections of a considerable quantity of a pure culture
into the circulation of rabbits and of guinea-pigs give rise to a fatal result
within forty-eight hours.

In his first publication relating to " the bacteria found in the dejecta of
infants afflicted with summer diarrhoea," Booker has described a bacillus
which he designates by the letter B, which closely resembles Bacillus laclis
aerogeiies and is probably identical with it. He says :

" Summary of Bacillus B. — Found nearly constantly in cholera infan-
tum and catarrhal enteritis, and generally the predominating form. It
appeared in larger quantities in the more serious cases. It was not found
in the dysenteric or healthy faeces. It resembles the description of the Ba-
cillus lactis aerogenes, but the resemblance does not appear sufficient to con-
stitute an identity, and, in the absence of a culture of the latter for com-
parison, it is considered a distinct variety for the following reasons : Bacillus
B is uniformly larger, its ends are not so sharply rounded, and in all culture
media long, thick filaments are see a, and many of the bacilli have the pro-
toplasm gathered in the centre, leaving the poles clear. There is some

NOT DESCRIBED IN PREVIOUS SECTIONS.

537

difference in their colony growth on gelatin, and in gelatin stab cultures
bacillus B does not show the nail- form growth with marked end swelling in
the depth. In potato cultures the Bacillus lactis aerogenes shows a differ-
ence between old and new potatoes, while bacillus B does not show any
difference.

"Bacillus B possesses decided pathogenic properties, which was shown
both by hypodermic injections and feeding with milk cultures."

BACILLUS ACIDIFORMANS.

Obtained by the writer (1888) from a fragment of yellow-fever liver pre-
served for forty-eight hours in an antiseptic wrapping; since obtained from

FIG. 148. FIG. 149.

Fro. 148.— Bacillus aciiiformans, from a potato culture, x 1,000. From a photomicrograph*
(Sternberg )

Fio. 149. — Culture of Bacillus acidiformans in nutrient gelatin, end of four days at 22° C.
From a photograph. (Sternberg.)

liver preserved in the same way from two comparative autopsies — i.e., not
cases of yellow fever.

Morphology. — A short bacillus with rounded corners, sometimes short
oval in form ; from 1| to 3 fJ- in length and about 1.2 u- in breadth ; may grow
out into filaments of 5 to 10 //, or more, in length ; in some cultures the short
oval form, predominates.

Stains readily with the aniline colors usually employed, and by Gram's
method.

Biological Characters.— An aerobic and facultative anaerobic, non-
liquefying, non-motile bacillus. Does not form spores. Grows rapidly at
the room temperature in the usual culture media. Grows in decidedly acid
media; in culture media containing glycerin or glucose it produces an abun-
dant evolution of carbon dioxide, and a volatile acid is formed.

It does not liquefy gelatin, and in stab cultures grows abundantly both
on the surface and along the line of puncture. At the end of twenty-four
hours, at 22° C., a rounded white mass is formed upon the surface, resembling
the growth of Friedlander's bacillus ; at the bottom of the line of puncture
the separate colonies are spherical, opaque, and pearl-like by reflected light.
Gas bubbles are formed in the gelatin. At the end of a week the surface is
covered with a thick, white, semi-fluid mass.

In gelatin roll tubes the superficial colonies are translucent or opaque,
and circular or somewhat irregular in outline; by reflected light they are

538

PATHOGENIC AEROBIC BACILLI

.slightly iridescent; the deep colonies are spherical, opaque, and homo-
geneous.

The growth upon the surface of nutrient agar is abundant and rapid, of
a shining milk-white color, and cream-like in consistence. An abundant
development forms alon^ the line of puncture and the culture medium is
split up by gas bubbles. In glycerin-agar the evolution of gas is very abun-
dant and the culture medium acquires an intensely acid reaction.

On potato the growth is abundant and rapid at a temperature of 20° to
30° C., forming a thick, semi-fluid mass of a milk-white color.

I have not obtained any evidence that this bacillus forms spores; the
cultures are sterilized by ten minutes' exposure to a temperature of 160° F.

When cultivated in bouillon to which five per cent of glycerin has been
added the culture medium acquires a milky opacity, and there is a copious
precipitate, of a viscid consistence, consisting of bacilli ; during the period
of active development the surface is covered with gas bubbles, as in a sac-
charine liquid undergoing alcoholic fermentation, and the liquid has a de-
cidedly acid reaction.

Pathogenesis. — Pathogenic for rabbits and for guinea pigs when injected
into the cavity of the abdomen — one to two cubic centimetres of a culture in
bouillon. The animal usually dies in less than twenty-four hours. The
bacilli are found in the blood in rather small numbers, and are frequently
seen in the interior of the leucocytes. The spleen is enlarged, the liver
normal, the intestine usually hyperaemic.

BACILLUS CUNICULICIDA HAVANIENSIS.

Obtained by the writer (1889) from the contents of the intestine of yellow-
fever cadavers, and also from fragments of yellow-fever liver preserved for

forty-eight hours in an antiseptic wrap-
ping—my bacillus x, Havana, 1889.

Morphology. — This bacillus resembles
the colon bacillus in form, but is some-
what larger, from 2 to 4 // in length and
from 0.8 to 1 JJ> in diameter ; sometimes
associated in pairs ; may grow out into
short filaments — not common. The ends
of the rods are rounded, and under cer-
tain circumstances vacuoles are seen at
the extremities, especially in potato cul-
tures.

Stains quickly with the aniline colors
usually employed, and also by Gram's
method.

Biological Characters. — An aerobic
and facultative anaerobic, non-lique-
fying bacillus. Under certain circum-
stances may exhibit active movements,
but is usually motionless.

A very curious thing with reference
to this bacillus is that it presented ac-
tive movements in my cultures made directly from yellow-fever cadavers,
but that these movements were not constant, and that since my return to
Baltimore I have not, as a rule, observed active movements in cultures from
the same stock, which, however, preserved their pathogenic power and other
characters. In Havana these movements were usually not observed in all
the bacilli in a field under observation, but one and another would start from
a quiescent condition on an active and erratic course ; sometimes spinning
actively upon its axis, and again shooting across the field as if propelled by
a nagellum.

FIG. 150.— Bacillus cuniculicida Havani-
•ensis, from a single colony in nutrient gela-
tin, x 1,000. From a photomicrograph.
(Sternberg.)

NOT DESCRIBED IN PREVIOUS SECTIONS.

539

My notes indicate that cultures passed through the guinea-pig are more
apt to be motile.

In gelatin stab cultures the growth of bacillus x resembles that of the
colon bacillus, but the colonies at the bottom of the line of puncture are
more opaque and not of a clear amber color like that of colonies of the colon
bacillus. Upon the surface the growth is thicker than that of the colon
bacillus, and forms a milk-white, soft mass.

The colonies in gelatin Esmarch roll tubes vary considerably at different
times. Deep colonies are usually spherical, homogeneous, light brown in
color, and more opaque than the similar colonies of the colon bacillus. At
the end of a few days the deep colonies become quite opaque, and may be
Jobate, like a mulberry, or coarsely granular; sometimes the aeep colonies
.have an opaque central portion surrounded by a transparent marginal zone.

In old gelatin roll tubes these deep colonies form opaque white hemi-

FIG. 151. FIG. 153.

Fig. 151.— Bacillus cuniculicida Havaniensis; colonies in gelatin roll tube, third day at 20° C.
X 6. From a photograph. (Sternberg.)

Fio. 152. — Bacillus cuniculicida Havaniensis ; colonies in gelatin roll tube, end of forty-eight
hours. X 10. From a photograph. (Sternberg.)

spheres projecting from the surface of the dried culture medium, and little
tufts of acicular crystals are sometimes observed to project from the side of
such old colonies.

The superficial colonies are circular or irregular in outline, with trans-
parent margins a.nd an opaque central portion, sometimes corrugated. They
are finely granular and iridescent by reflected light, and of a milk-white
color ; by transmitted light they have a brownish color. Young colonies
closely resemble those of the colon bacillus. This bacillus grows well at a
temperature of 20° C. (68° F.), but more rapidly and luxuriantly at a higher
temperature — 30° to 35° C.

It grows well in agar cultures, and especially in glycerin-agar, in which
it produces some gas and an acid reaction. The growth on the surface
of glycerin-agar cultures is white, cream-like in consistence, and quite abun-
dant.

It grows well in an agar or gelatin medium made acid by the addition of
'0.2 per cent (1: 500) of hydrochloric acid.

In cocoanut water it multiplies rapidly, producing a milky opacity of the
previously transparent fluid, an acid reaction, and an evolution of carbon
dioxide.

On potato it produces a thick layer, which may cover the entire surface
in three or four days, and which has a dirty-white, cream- white, or pinkish-

540 PATHOGENIC AEROBIC BACILLI

white color and cream-like consistence. The growth upon potato varies at
different times, evidently owing1 to differences in the potato.

When stained preparations are examined with the full light of the Abbe
condenser the ends of some of the rods appear to be cut away, leaving- a con-
cave extremity ; but by using- a small diaphragm to obtain definition it will
be seen that the cell wall extends beyond the stained portion of the rod and
includes what appears to be a vacuole. There is no reason to believe that
this appearance is due to the presence of an end spore, for the supposed
vacuole is not refractive, as a spore would be, and my experiments on the
thermal death-point of this bacillus indicate that it does not form spores.
Cultures are sterilized by exposure for ten minutes to a temperature of 160°
F. (71.2° C.).

Pathogenesis. — Very pathogenic for rabbits when injected into the cavity
of the abdomen. Injections of a small quantity of a pure culture into the
ear vein or subcutaneously generally give a negative result. Injections of
from one to five cubic centimetres of a culture in bouillon, blood serum, or
agua coco, into the cavity of the abdomen, frequently prove fatal to rabbits
in a few hours — two to six.

The negative results obtained in injecting cultures beneath the skin or
into the ear vein of rabbits show that this bacillus does not induce a fatal
septicaemia in these animals, and the fatal result when injections are made
into the peritoneal cavity does not appear to be due to an invasion of the
blood, but rather to the local effect upon the peritoneum, together with the
toxic action of the chemical products resulting from its growth.

It is true that I have always been able to recover the bacillus from the
liver, or from blood obtained from one of the cavities of the heart, even in
animals which succumb within a few hours to an injection made into the
cavity of the abdomen. But the direct examination of the blood shows that
the bacilli are present in very small numbers, and leads me to believe that
the bacillus does not multiply, to any considerable extent at least, in the
circulating fluid.

The spleen is not enlarged, as is the case in anthrax, rabbit septicaemia,
and other diseases in which the pathogenic microorganism multiplies abun-
dantly in the blood.

On the other hand, there is evidence of local inflammation in the peri-
toneal cavity. When death occurs within a few hours the peritoneum is
more or less hyperaemic and there is a considerable quantity of straw-colored
fluid in the cavity of the abdomen. When the animal lives for twenty
hours or more there is a decided peritonitis with a fibrinous exudation upon
the surface of the liver and intestine. Usually the liver, in animals which
die within twenty-four hours, is full of blood, rather soft, and dark in color.
In a single instance I found the liver to be of a light color and loaded with
fat.

The rapidly fatal effect in those cases in which I have injected two or
more cubic centimetres of a culture into the cavity of the abdomen has led
me to suppose that death results from the toxic effects of a ptomaine con-
tained in the culture at the time of injection. The symptoms also give sup-
port to this supposition. The animal quickly becomes feeble and indisposed
to move, and some time before death lies helpless upon its side, breathing
regularly, but is too feeble to get up on its feet when disturbed. Death some-
times occurs in convulsions, but more frequently without— apparently from
heart failure.

Pathogenic also for guinea-pigs when injected into the cavity of the-
abdomen, but death does not occur in so short a time — eighteen to twenty
hours. The comparative researches of Reed and Carroll indicate that this is-
a pathogenic variety of the colon bacillus.

NOT DESCRIBED IN PREVIOUS SECTIONS. 541

BACILLUS LEPORIS LETHALIS.

Obtained by Dr. Paul Gibier (1888) from the contents of the intestine of
yellow-fever patients; also by the writer from the same source (1888, 1889)
in exceptional cases and in comparatively small numbers. Named and de-
scribed by present writer.

Morphology. — Bacilli with rounded ends, from 1 to 3 ^ in length and
about 0.5 H in breadth. The length may vary in the same culture from a
short oval to rods which are two or three times as long as broad, or it may
grow out into flexible filaments of considerable length. In recent cultures
the bacilli are frequently united in pairs.

Stains readily with the aniline colors usually employed. In cultures
which are several days old, or in recent cultures when the stained prepara-
tion is washed in alcohol, the ends of the rods are commonly more deeply
stained than the central portion — " end staining"; and in old cultures some
of the bacilli are very faintly stained.

Biological Characters. — A.U aerobic, liquefying, actively motile bacillus.
Does not form spores.

In gelatin stab cultures, at the end of twenty-four hours at a tempe-
rature of 20° to 22° C., there is an abundant development along- the line of
puncture and commencing liquefaction at the surface. Later the liquefaction
is funnel-shaped, and there is an opaque white central core along the line
of puncture, with liquefied gelatin around it. Liquefaction progresses most
rapidly at the surface, and in the course of three or four days the upper por-
tion of the gelatin for a distance of half an inch or more is completely lique-
fied, and an opaque white mass, composed of bacilli, rests upon the surface
of the unliquefied portion.

In gelatin roll tubes the young colonies upon the surface are transparent
and resemble somewhat small fragments of broken glass ; later liquefaction
occurs rapidly. Deep colonies in gelatin roll tubes, or at the bottom of stick
cultures, are spherical, translucent, and of a pale straw color.

Upon the surface of nutrient agar it grows rapidly, forming a rather thin,
translucent, shining, white layer, which covers the entire surface at the end
of two or three days at a temperature of 20° C.

Upon potato the growth is rapid and thin, covering the entire surface,
and is of a pale-yellow color.

This bacillus grows at a comparatively low temperature, and its vitality
is not destroyed by exposure for an hour and a half in a freezing mixture at
15° C. below zero (5° F.).

Decided growth occurred in a stick culture in gelatin exposed in Balti-
more during the month of January in an attic room. During the twenty-
two days of exposure the highest temperature, taken at 9 A.M. each day,
was 11° C., and the lowest 2° C. At a temperature of 16° to 20° C. develop-
ment in a favorable culture medium is rapid.

There is no evidence that this bacillus forms spores ; cultures are sterilized
by exposure to a temperature of 60° C. for ten minutes.

Coagulated blood serum is liquefied by this bacillus. It retains its vitality
for a long time in old cultures, having grown freely when replanted at the
end of a year from a hermetically sealed tube containing a pure culture in
blood serum.

Pathogenesis. — This bacillus is very pathogenic for rabbits when injected
into the cavity of the abdomen in quantities of one cubic centimetre or more ;
it is less pathogenic for guinea-pigs, and is not pathogenic for white rats
when injected subcutaneously. Gelatin cultures seem to possess more in-
tense pathogenic power than bouillon cultures, and cultures from the blood
of an animal recently dead as the result of an inoculation are more potent
than those from my original stock which had not been passed through a
susceptible animal.

542 PATHOGENIC AEROBIC BACILLI

The mode of death in rabbits is quite characteristic. A couple of hours
after receiving in the cavity of the abdomen two or three cubic centimetres
of a liquefied gelatin culture the animal becomes quiet and indisposed to eat
or move about. Soon after it becomes somnolent, the head drooping for-
ward and after a time resting between the front legs, with the nose on the
floor of its cage. It can be roused from this condition, and raises its head in
an indifferent and stupid way when pushed or shaken, but soon drops off
again into a profound sleep. Frequently the animals die in a sitting posi-
tion, with their nose resting upon the floor of the cage between the front
legs. I have not seen this lethargic condition produced by inoculations with
any other microorganism. Convulsions sometimes occur at the moment of
death.

The time of death depends upon the potency of the culture and its quan-
tity as compared with the size of the animal. From three to four cubic
centimetres of a liquefied gelatin culture usually kill a rabbit in from three
to seven hours.

The rapidity with which death occurs when a considerable quantity of a
liquefied gelatin culture is injected into the cavity of the abdomen, and the
somnolence which precedes death, give rise to the supposition that tV'e lethal
effect is due to the presence of a toxic chemical substance rather than to a
multiplication of the bacillus in the body of the animal. And this view is
supported by the fact that animals frequently recover when the dose admin-
istered is comparatively small and especially when it is injected subcuta-
neously.

In all cases in which death occurs, even when but a few hours have
elapsed since the inoculation was made, I have recovered the bacillus in
cultures made from blood obtained from the heart or the interior of the
liver, and, as stated, these cultures appear to have a greater virulence than
those not passed through the rabbit.

In sections of the liver and kidney stained with Loffler's solution of
methylene blue the bacilli are seen, and are often in rather long- jointed fil-
aments.

BACILLUS PYOCYANEUS.

Synonyms. — Bacillus of green pus ; Microbe du pus bleu; Bacil-
len des griinblauen Eiters ; Bacterium aeruginosum.

Obtained by Gessard (1882) from pus having a green or blue
color, and since carefully studied by Gessard, Charrin, and others.

This bacillus appears to be a widely distributed
saprophyte, which is found occasionally in the
purulent discharges from open wounds, and some-
times in perspiration and serous wound secretions
(Gessard) . The writer obtained it, in one instance,
FIG. 153. — Bacillus in cultures from the liver of a yellow-fever cada-
X 7°°' ver (Havana, 1888).

Morphology. — A slender bacillus with rounded
ends, somewhat thicker than the Bacillus murisepticus and of about
the same length (Fliigge) ; frequently united in pairs, or chains of four
to six elements; occasionally grows out into filaments.

Biological Characters. — An aerobic, liquefying, motile bacil-
lus. Grows readily in various culture media at the room tempera-
ture— more rapidly in the incubating oven. Is a facultative anae-

NOT DESCRIBED IN PREVIOUS SECTIONS. 543-

robic (Frankel). Does not form spores. The thermal death-point,
as determined by the writer, is 56° C., the time of exposure being ten
minutes. In gelatin plate cultures colonies are quickly developed,
which give to the medium a fluorescent green color ; at the end of
two or three days liquefaction commences around each colony, and
usually the gelatin is completely liquefied by the fifth day. Before
liquefaction commences the deep colonies, under a low power, appear
as spherical, granular masses, with a serrated margin, and have a
yellowish-green color ; the superficial colonies are quite thin and
finely granular ; at the centre, where they are thickest, they have a
greenish color, which fades out towards the periphery.

In stab cultures in nutrient gelatin development is most abun-
dant near the surface, and causes at first liquefaction in the form
of a shallow funnel ; later the liquefied gelatin is separated from
that which is not liquefied by a horizontal plane, and a viscid, yel-
lowish-white mass, composed of bacilli, accumulates upon this sur-
face, which gradually has a lower level as liquefaction progresses ;.
the transparent, liquefied gelatin above is covered with a delicate,
yellowish-green film, and the entire medium has a fluorescent green
color. Upon nutrient agar a rather abundant, moist, greenish- white
layer is developed, and the medium acquires a bright green-color,
which subsequently changes to olive green. Upon potato a viscid
or rather dry, yellowish-green or brown layer is formed, and the
potato beneath and immediately around the growth has a green color
when freely exposed to the air or to the vapors of ammonia. In milk
the casein is first precipitated and then gradually dissolved, while at
the same time ammonia is developed. The green pigment is formed
only in the presence of oxygen; it is soluble in chloroform and may
be obtained from a pure solution in long, blue needles ; acids change
the blue color to red, and reducing substances to yellow. According
to Ledderhose, it is an aromatic compound resembling anthracene,
and is not toxic. According to Gessard's latest researches (1890), two
different pigments are produced by this bacillus, one of a fluorescent
green and the other — pyocyanin — of a blue color. Cultivated in egg
albumin the fluorescent green pigment, which changes to brown
with time, is alone produced. In bouillon and in media containing
peptone or gelatin both pigments are formed, and the pyocyanin
may be obtained separately by dissolving it in chloroform. In an
alkaline solution of peptone (two per cent) to which five per cent of
glycerin has been added the blue pigment alone is formed.

Pathogenesis. — The experiments of Ledderhose, Bouchard, and
others show that this bacillus is pathogenic for guinea-pigs and rab-
bits. Subcutaneous or intraperitoneal injections of recent cultures —

544 PATHOGENIC AEROBIC BACILLI

one cubic centimetre or more of a culture in bouillon — usually cause
the death of the animal in from twelve to thirty-six hours. An ex-
tensive inflammatory oedema and purulent infiltration of the tissues
result from subcutaneous inoculations, and a sero-fibrinous or puru-
lent peritonitis is induced by the introduction of the bacillus into the
peritoneal cavity. The bacillus is found in the serous or purulent
fluid in the subcutaneous tissues or abdominal cavity, and also in the
blood and various organs, from which it can be recovered in pure
cultures, although not present in great numbers, as is the case in
the various forms of septicaemia heretofore described. When smaller
amounts are injected subcutaneously the animal usually recovers
after the formation of a local abscess, and it is subsequently immune
when inoculated with doses which would be fatal to an unprotected
animal. Immunity may also be secured by the injection of a con-
siderable quantity of a sterilized culture. Bouchard has also pro-
duced immunity in rabbits by injecting into them the filtered urine
of other rabbits which had been inoculated with a virulent culture of
the bacillus. It has been shown by Bouchard, and by Charrin and
Guignard, that in rabbits which have been inoculated with a culture
of the anthrax bacillus a fatal result may be prevented by soon after
inoculating the same animals with a pure culture of the Bacillus
pyocyaneus. The experiments of Woodhead and Wood indicate that
the antidotal effect is due to chemical products of the growth of the
bacillus, and not to an antagonism of the living bacterial cells. They
were able to obtain similar results by the injection of sterilized cul-
tures of Bacillus pyocyaneus, made soon after infection with the
anthrax bacillus.

Schimmelbusch (1894) reports that in researches made by Muh-
sam this bacillus was found in the axilla, the anal region, or the in-
guinal fold in fifty per cent of the healthy individuals examined.
Its presence in wounds greatly delays the process of repair and may
give rise to a general depression of the vital powers from the ab-
sorption of its toxic products. Schimmelbush states that a physician
injected 0.5 cubic centimetre of sterilized (by heat) culture into his
forearm. That as a result of this injection, after a few hours he had
a slight chill, followed by fever, which at the end of twelve hours
reached 38.8° ; an erysipelatous - like swelling of the forearm oc-
curred, and the glands in the axilla were swollen and painful. Re-
covery occurred without the formation of an abscess. Buchner has
related a similar case.

Krannhals (1894) refers to seven cases in which a general pyocy-
aneus infection in man was found, and adds an eighth from his own
experience. In this the Bacillus pyocyaneus was obtained, post mor-

NOT DESCRIBED IN PREVIOUS SECTIONS. 545

tern, from green pus in the pleural cavity, from serum in the peri-
cardial sac, and from the spleen, in pure culture.

Martha, Gruber, Maggiora, Gradenigo, Kossel, and Rohrer have
reported cases in which the Bacillus pyocyaneus has been obtained in
pure cultures from pus obtained from the tympanic cavity in middle-
ear disease. Kossel (1894) relates several cases in his own experience
which led him to the conclusion that, in children, the Bacillus pyocy-
aneus, through general blood infection or indirectly through the
absorption of its toxic products, may be the cause of death.

The following varieties of this bacillus have been described by
bacteriologists:

BACILLUS PYOCYANEUS /? (P. Ernst).

Found in pus from bandages colored green.

Morphology. — Slender bacilli from 2 to 4 /-i long — occasionally 5 to 6 fj- —
and from 0.5 to 0.75 Abroad; sometimes united in pairs, or chains of three
elements.

Biological Characters. — An aerobic, liquefying, actively motile, chro-
mogenic bacillus. Produces a yellowish-green pigment; when old cul-
tures are shaken up with chloroform and this is allowed to stand, three
layers are formed — an upper, clouded, dirty -yellow layer ; below this is a
milky, pale-green layer ; and at the bottom a transparent, azure-blue layer.
Spore formation has not been demonstrated. Grows in the usual culture
media at the room temperature — more rapidly at 35° C. Upon gelatin plates
colonies are formed resembling those of the well-known Bacillus pyocyaneus,
but liquefaction is more rapid. In gelatin stick cultures funnel-shaped
liquefaction occurs at the upper part of the line of puncture by the third
day, and progresses more rapidly than is the case with Bacillus pyocyaneus
under the same circumstances ; on the fifth day a bluish-green color is de-
veloped; by the twelfth day liquefaction has obliterated the entire line of
growth and extends to the margins of the tube ; the liquefied gelatin for a
depth of about one centimetre has a dark emerald-green color, and a film
consisting of bacilli is seen upon the surface. Upon the surface of agar a
flat, greenish- white, dry layer is formed along the line of inoculation, and
the agar around, at the end of a week, acquires a bluish-green color. Upon
potato, at the end of three days, an abundant dry layer of a fawn-brown
color has developed ; this is surrounded by a pale-green coloration of the
potato, and at points where the surface is fissured, an intense dark-green
color is developed; the growth on potato has a more or less wrinkled appear-
ance ; when one of the fawn-colored colonies is touched with the platinum
needle, the point touched, at the end of two to five minutes, acquires an in-
tense dark leaf -green color, which reaches its maximum intensity in about
ten minutes, and has faded out again at the end of half an hour. Ernst con-
siders this "chameleon phenomenon" the most characteristic distinction
between the bacillus under consideration and Bacillus pyocyaneus. In milk
a green color is developed at the surface, the casein is precipitated and sub-
sequently peptonized.

Bacillus pyocyaneus pericarditidis. Found by H. C. Ernst in
fluid obtained by tapping the pericardial sac of a man aged forty-
seven years. Fluid was drawn from the pericardial sac on four dif-
ferent occasions. The man subsequently "eloped." Ernst gives the
following description of this bacillus :
35

546 PATHOGENIC AEROBIC BACILLI

ORIGIN. — Pericardial fluid, containing also bacilli of tuberculosis.

FORM AND ARRANGEMENT. — Small straight bacilli, with rounded ends,
three or four times as long as broad, and on most media slightly larger than
the Bacillus pyocyaneus of Gessard, occurring within the cells in the origi-
nal fluid, and sometimes showing two or three end to end, but never observed
in long chains.

MOTILITY. — Actively motile in hanging-drop culture. No cilia or flagel-
la have been demonstrated.

GROWTH — Gelatin : Plates. — Colonies appear at the end of thirty-six to
forty-eight hours as fine white points in the interior, and upon the surface of
the medium ; edges are sharply defined ; soon there appears a circular zone
of liquefaction, finally passing through the stratum of the medium with
the colony at the bottom. Under a low power the centre of the colony may
be of a brownish color. On the second day a greenish tinge may be seen
about the individual colonies on the surface which spreads through the
entire medium. The plates may always be distinguished from those of the
Bacillus pyocyaneus of Gessard by the bluish-green when contrasted with
the yellowish-green color of this latter.

Gelatin: Needle Cultures. — At the end of twenty-four hours a small,
saucer-shaped depression of liquefaction at upper end of needle track, which
gradually spreads and deepens until the liquefaction extends straight across
the tube, and about half-way down the needle track. A bluish-green fluores-
cence appears about the liquefied portion at the very upper part of the gela-
tin, later changing into a yellowish green. The colony is deposited as a
yellowish, heavy sediment at the bottom of the liquefied portion, the upper
part of which is clear. A small, whitish growth occurs along the remainder
of the needle track. Old cultures, in which a certain amount of evapora-
tion has occurred, assume a very dark greenish-black color.

Agar-agar. — Along the needle track appears a flat, dry colony of a dirty
grayish- white color spreading out upon each side of the needle track and
growing at first upon the surface of the water of condensation, later depos-
iting a white sediment at the bottom. From the first there may be detected,
by reflected light, a metallic lustre on the surface of the colony in places,
which metallic sheen later spreads over the whole colony and furnishes a
marked differentiating point. In addition to this, within twenty-four to
forty-eight hours at 37° C., there appears a green fluorescence throughout
the whole of the medium, which increases slowly to a marked bluish-green
color, and never assumes the nut-brown of the Bacillus pyocyaneus of
Gessard upon the same medium. The colony is not especially viscid.

Potato. — There appears a reddish-brown colony along the needle track,
elevated and moist, confined to the line of the needle. It presents no change
of color upon touching with the needle, but certain specimens (as do some of
the Bacillus pyocyaneus) develop later a heavy green color extending over
the whole surface of the potato, which later changes almost to black.

Bouillon. — Twenty-four hours at 37° C. gives a growth, especially on the
surface, which is a wrinkled scum ; no cloudiness of the bouillon, and a very
faint greenish fluorescence one centimetre below the surface. At this time
it differs from the Bacillus pyocyaneus of Gessard, in that the latter shows
cloudiness of the medium all through. Later the same cloudiness appears in
bouillon cultures of this new bacillus, together with a whitish sediment de-
posited at the bottom of the tube, and then the cultures are indistinguishable
from each other. The same changes, but slower, occur at room tempera-
ture.

Peptone. — One, 3.5, and six-per-cent solution. Twenty-four hours at 37°
C. gives a faint bluish tinge at upper edge of medium with very faint cloudi-
ness ; later (in one or two weeks) there forms a marked scum upon the sur-
face that is difficult to break up by shaking, and the whole medium assumes
a grass-green color of more or less intensity, and not seen on other similar
bacilli. The shape and size of the organism, under the microscope, differ

NOT DESCRIBED IX PREVIOUS SECTIONS. 547

very markedly in this medium from any other bacilli examined. The same
changes are to be seen at room temperature, but more slowly.

Egg- Albumin: Plain. — Twenty- four hours at 37° C., yellowish-white,
very profuse growth all along1 the needle track ; yellowish-green spreading
out from it almost to sides of tube, and in the condensation water as well.
The growth has no especial distinguishing characteristics. Irregular lique-
faction occurs, but the growth at no time differs in any marked way from
other varieties of the Bacillus pyocyaneus.

Blood Serum. — Twenty-four hours at 37° C. shows flat, moist colony
with bluish-green fluorescence in its neighborhood. Liquefaction begins
early and goes on slowly until complete in from one to two weeks, with an
increasing intensity of color which becomes markedly blue, and eventually
almost black.

Milk. — Behaves as do the other bacteria.

BEHAVIOR TO TEMPERATURE. — Grows at 15°-25° C. slowly; much more
freely at 35-38° C., when it produces the coloi more quickly.

RAPIDITY OF GROWTH. — Moderate.

SPORE-PRODUCTION. — Not observed.

NEED OP AIR. — Does not grow under mica. Facultatively anaerobic, but
does not produce color except with free access of oxygen.

GAS-PRODUCTION. — Produces faint foul odor.

BEHAVIOR TO GELATIN. — Liquefies gelatin slowly.

COLOR-PRODUCTION. — Produces a bluish-green color which in old cul-
tures changes almost to a black. Upon the addition of acids (both vegetable
and mineral) to cultures the color changes to red, and upon the addition of
alkalies a bright grass-green appears. This reaction is best seen in bouillon
and gelatin cultures, but occurs in other media as well, notably blood-serum.

BEHAVIOR TO ANILINE DYES. — Stains easily and well with any of the
aniline dyes usually employed, and by Gram's method.

MICROSCOPIC APPEARANCE IN DIFFERENT MEDIA. — Under the micro-
scope, its general appearance on various media is of a rod larger than the
Bacillus pyocyaneus. In peptone cultures this difference is verv marked.
In this case, the Bacillus pyocyaneus tested appeared as very short, oval,
bacilli, almost like micrococci, while the new bacillus showed as a long,
fine rod, from four to six times as long as broad — length about one-half the
diameter of a red-blood corpuscle — and arranged sometimes two or three
end to end. These same cultures transferred to gelatin became indistin-
guishable from each other in size.

PATHOGENESIS. — Injections of small quantities (0.5 centimetre) of a bouil-
lon culture twenty -four hours old into the abdominal cavity of rabbits and
guinea-pigs, killed fifty per cent in from twenty-four to thirty-six hours.
Autopsy showed general congestion of abdominal viscera, slight effusion into
the peritoneal cavity, and cover-glass preparations and cultures showed the
bacilli in the effusion in the abdominal cavity, as well as in the blood from
the heart and various organs.

BACILLUS OF FIOCCA.

Found by Fiocca in the saliva of cats and dogs.

Closely resembles the influenza bacillus of Pfeiffer and of Canon.

Morphology. — Resembles the bacillus of rabbit septicaemia, but is only
half as large— from 0.2 to 0.33 /* in breadth. The length is but little greater
than the breadth. Usually seen in pairs, closely resembling diplococci.
When cultivated on potato it appears to be a micrococcus, but in the blood
of infected animals and in bouillon cultures it is seen to be a short bacillus.

Stains with difficulty with the usual aniline colors, but is readily stained
by Ehrlich's method or with Ziehl's solution.

Biological Characters. — An aerobic and facultative anaerobic, non-
liquefying, non-motile bacillus. Spore formation not observed. Grows best

548

PATHOGENIC AEROBIC BACILLI

at 37° C. and does not develop at temperatures below 15° C. In agar plates,
at 37° C., small, punctiform colonies are developed at the end of twenty-four
hours; these do not increase in size later; under the microscope the deep
colonies are seen to be spherical, granular, and dark yellow in color; the
superficial colonies are more or less round, with irregular outlines, trans-
parent, slightly granular, and often have a shining nucleus at the centre.
Upon gelatin plates the colonies have a similar appearance, but are not vis-
ible in less than four or five days. In streak cultures upon the surface of
agar small, punctiform colonies are seen along the track of the needle at the
end of twenty-four hours, resembling fine dewdrops; the following day
these colonies are a little larger and less transparent ; they remain distinct,
especially along the margins of the line of growth. Upon potato a very
thin, transparent layer is developed, which does not change the appearance
of the surface of the potato, but slightly increases its resistance to the plati-
num needle. In bouillon small flocculi. suspended in the clear 'liquid, are
-developed within twenty-four hours; these subsequently sink to the bottom.

Milk is not coagulated by this bacillus, and no gas is produced in media
containing sugar.

Pathogenesis. — Pathogenic for rabbits, guinea-pigs, young rats, and mice,
in which animals it produces general infection, and death— in rabbits — at
the end of twenty-four hours. The bacillus is found in the blood in great
numbers.

PROTEUS VULGARIS.

Obtained by Hauser (1885) from putrefying animal substances,
and since shown to be one of the most common and widely distrib-
uted putrefactive bacteria. This and the other species of Proteus

Fia. 154. — Proteus vulgaris; " swarming islands " from a gelatin culture. X 285. (Hauser. j

described by the same bacteriologist (Proteus mirabilis, Proteus Zen-
keri) have no doubt frequently been encountered by previous observ-
ers, and are among the species formerly included under the name
" Bacterium termo," which was applied to any minute motile bacilli
found in putrefying infusions.

Morphology. — Bacilli with rounded ends, about 0.6 jn broad, and

NOT DESCRIBED IN PREVIOUS SECTIONS. 549

varying greatly in length, being sometimes short oval, and at others
from 1.25 to 3.75 /* in length ; also grow out into flexible filaments,
which may be more or less wavy or spiral in form. The short rods
are commonly seen in pairs ; they have terminal flagella ; involution
forms are frequently seen, the most common being spherical bodies
about 1.6 jit in diameter. In old cultures in bouillon, or in cultures
made in meat infusion in the incubating oven, the short oval forms
greatly predominate, but in recent cultures in nutrient gelatin fila-
ments of considerable length are encountered in association with
shorter rods.

Stains readily with fuchsin or gentian violet — not so well with
the brown aniline colors ; does not stain by Gram's method (Cheyne).

Biological Characters. — Anaerobic and facultative anaerobic,
liquefying, motile bacillus. Grows rapidly in the usual culture
media at the room temperature.

The growth upon gelalin plates (five per cent of gelatin) at the
room temperature is very characteristic ; at the end of six or eight
hours small depressions in the gelatin are observed, which contain
liquefied gelatin and grayish- white masses of bacilli. Under a low
power these depressions are seen to be surrounded by a marginal
zone consisting of two or three layers, outside of which is a zone of a
single layer, from which amoeba-like processes extend upon the sur-
face of the gelatin. These processes are constantly undergoing
changes in their form and position, and may become separated from
the mother colony, or remain temporarily attached to it by a narrow
thread consisting of bacilli ; after a time the entire surface of the
gelatin is covered with wandering, amoeba-like colonies ; these
rapidly cause liquefaction, which by the end of twenty-four to forty-
eight hours has reached a depth of one millimetre or more over the
entire surface. The deep colonies also are surrounded by processes
projecting into the gelatin, which may be observed to suddenly ad-
vance and again to be retracted towards the central zoogloea-like
mass. Liquefaction around the colony rapidly progresses, and
actively motile rods and spiral filaments may be seen about the peri-
phery of this liquefied gelatin, while about it is a radiating crown of
irregular processes, some of which may be screw-like or corkscrew-
formed. In ten-per-cent gelatin the migration of surface colonies,
above described, is not observed. In gelatin stab cultures liquefac-
tion occurs along the entire line of puncture, and soon the contents
of the tube are completely liquefied ; near the surface of the liquefied
gelatin the growing bacilli form a grayish-white cloudiness, and at
the bottom of the tube an abundant flocculent deposit is formed.
Upon the surface of nutrient agar a rapidly extending, moist, thin,
grayish- white layer is formed. Upon potato this bacillus produces a

550 PATHOGENIC AEROBIC BACILLI

dirty -white, moist layer. The cultures in media containing albumin
or gelatin have a putrefactive odor and acquire a strongly alkaline
reaction. A temperature of 20° to 24° C. is most favorable for the
growth of this bacillus. It is a facultative anaerobic and grows in
an atmosphere of hydrogen or of carbon dioxide, although not so
rapidly as in the presence of oxygen. The movements are often ex-
tremely active and difficult to follow under the microscope ; again
they may be quite deliberate, or the bacilli may remain motionless
for a time and again dart off in active motion. The long terminal
flagella may sometimes be discerned by means of a good objective
and careful manipulation of the light.

Pathogenesis. — Pathogenic for rabbits and for guinea-pigs when
injected into the circulation, into the cavity of the abdomen, or sub-
cutaneously in considerable quantity. Cultures in nutrient gelatin
are said by Cheyne to be more pathogenic (toxic) than those in bouil-
lon. When injected into the muscles of rabbits a much smaller
dose produces a fatal result than when injected subcutaneously.
In Cheyne's experiments, made in London (1886), one-tenth cubic
centimetre of a liquefied gelatin culture, injected into the dorsal
muscles, was invariably fatal in from twenty-four to thirty-six hours;
a dose of one-twentieth cubic centimetre, injected in the same way,
usually caused death; while one-fortieth cubic centimetre gave rise to
an extensive local abscess, and the animals died at the end of six or
eight weeks. Doses of less than one-five-hundredth cubic centimetre
produced no effect. Cheyne estimates that one cubic centimetre of a
culture in nutrient gelatin contains 4,500,000,000 bacilli, and, conse-
quently, that a smaller number than 9,000,000 produced no effect when
injected into the muscular tissue of rabbits. Injections into the sub-
cutaneous connective tissues of a dose twice as large as that which in-
variably proved fatal when injected into the muscles usually caused
an extensive abscess, but did not kill the animal ; and, after re-
covery from the effects of such an injection, the rabbit was found to
be immune against a similar dose injected into the muscles. Foa
and Bonome have succeeded in producing immunity against the
effects of virulent cultures of this bacillus by inoculating rabbits with
filtered cultures, and also by injecting beneath the skin of these ani-
mals a solution of neurin, which they believe to be the principal
toxic product present in the cultures.

Proteus Vulgar is in Cholera Infantum. — The extended re-
searches of Booker have led him to the conclusion that this bacillus
plays an important part in the production of the morbid symptoms
which characterize cholera infantum. Proteus vulgaris was found
in the alvine discharges in a considerable proportion of the cases ex-
amined, but was not found in the faeces of healthy infants. ' The

NOT DESCRIBED IN PREVIOUS SECTIONS. 551

prominent symptoms in the cases of cholera infantum in which the
proteus bacteria were found were drowsiness, stupor, emaciation
and great reduction in flesh, more or less collapse, frequent vomiting
and purging, with watery and generally offensive stools."

The researches of Krogius, Schnitzler, Schmidt and Aschoff, and
others, show that in cases of cystitis and of pyelonephritis this bacil-
lus is frequently found in pure cultures, or associated with other bac-
teria. The authors last named state that in sixty cases of cystitis
reported by various authors the colon bacillus was found in pure cul-
tures, and in thirteen cases the proteus of Hauser. Next to Bacillus
coli communis Proteus vulgaris appears to be the microorganism
most frequently concerned in the etiology of pyelonephritis.

Levy (1895) isolated from sour yeast a bacillus, which he identified
as "Proteus Hauseri," and made numerous experiments on dogs to
test its pathogenic power. From five to ten cubic centimetres of a
liquefied gelatin culture injected into the circulation, through a vein,
caused the typical symptoms of "sepsin poisoning," as formerly de-
scribed by Bergmann and Schmeideberg (1868). In two dogs which
died at the end of forty-eight hours the intestinal tract was found in
a condition of intense hemorrhagic infiltration. The spleen and
glands of the mesentery were much enlarged. But a bacteriological
examination gave an entirely negative result, showing that death
resulted from toxemia and not from septica3mia. Further experi-
ments showed that the dried precipitate obtained from liquefied gela-
tin cultures, by the addition of alcohol, had the same pathogenic
action on dogs, rabbits, and mice as cultures containing the living
bacilli. That a similar pathogenic effect is produced in man by the
products of growth of this bacillus was shown by the following facts :
While conducting his experiments Levy had an opportunity to make
a bacteriological examination in the case of a man who died after a
brief attack of cholera morbus. From the vomited material and the
stools he obtained a pure culture of proteus ; but the blood, collected
at the autopsy, was sterile. In the mean time seventeen other per-
sons who had eaten at the same restaurant were taken sick in the same
way. Upon an examination at the restaurant it was found that the
bottom of the ice chest in which the proprietor kept his meats was
covered with a slimy, brown layer, which gave off a disagreeable
odor. Cultures from this gave the proteus as the principal micro-
organism present. Levy concludes from his own investigations and
those of other bacteriologists that in so-called " flesh-poisoning " bac-
teria of this group are chiefly at fault, and that the pathogenic effects
are due to toxic products evolved during their development.

PATHOGENIC AEROBIC BACILLI

PROTEUS OF KARLINSKI.

Synonym.— Bacillus murisepticus pleomorphus (Karlinski). Probe bly
identical with Proteus vulgaris of Hauser.

Obtained by Karlinski (1889) from a fibro-purulent uterine discharge, and
from abscesses in the uterus and its appendages in a puerperal woman.

Morphology. — Eesembles Proteus vulgaris of Hauser in its morphology,
and presents various forms under different circumstances relating to the
culture medium, the temperature, age of culture, etc. — sometimes as spheri-
cal or short oval cells, at others as longer or shorter rods or spiral filaments;
usually as bacilli with round ends two and a half times as long as thick,'
often united in pairs.

Stains with the usual aniline colors, but not by Gram's method.

Biological Characters. — An aerobic and facultative anaerobic, liquefy-
ing, motile bacillus. Spore formation not observed. Grows rapidly in the
usual culture media at the room temperature. In gelatin plate cultures, at
the end of ten hours, small colonies are developed which have well-defined
outlines, are oval or whetstone-shaped, of a light-brown color by transmitted
light and white by reflected light, with a somewhat darker margin and a
smooth surface, sometimes marked by shallow clefts ; at the end of twenty
hours the colonies commence to have irregular margins, and the surface of
the gelatin above them is marked by concentric rings. At the end of thirty
hours the colonies have formed a bulb-shaped liquefaction of the gelatin,
and delicate, ray-like offshoots are seen around the margin. At the end of
two days the bulbous cavities are aboutone and a half millimetres in diameter
and contain a cloudy, grayish-white liquid; they are surrounded by a moist-
looking, gray, irregular marginal zone. In gelatin stab cultures, at the end
of twenty-four hours, a funnel-shaped liquefaction of the gelatin occurs near
the surface, and a grayish-white, cloudy mass is developed along the line of
puncture; at the end of forty- eight hours a sac-like pouch of liquefied gela-
tin has formed, and in the course of four or five days the gelatin is entirely
liquefied. Upon agar plates the colonies are at first oval in form and white
by reflected light, or pale brown by transmitted light ; at the end of thirty
hours the surface becomes wrinkled or folded and is surrounded by radiat-
ing, delicately twisted offshoots. Upon the surface of agar a white layer
is developed. Upon potato a whitish-gray, soft, homogeneous layer, which
after standing along time has a darker color. Upon blood serum a thin,
grayish-white layer is formed and the serum is rapidly liquefied. Gelatin
cultures acquire a strongly alkaline reaction and give off a disagreeable
odor resembling that of butyric acid.

Pathogenesis. — White mice inoculated at the root of the tail die in from
twenty-two to twenty-four hours ;^the spleen is greatly enlarged; the bacilli
are found in blood from the various organs — less numerous in blood from
the heart. Field mice and house mice are less susceptible. Subcutaneous
injections in rabbits may give rise to local inflammation and also to general
infection. In white rats and guinea-pigs a local abscess may result from a
subcutaneous inoculation.

PROTEUS MIRABILIS.

Obtained by Hauser (1885) from putrefying animal substances.

Morphology. — Bacilli resembling very closely the preceding species (Pro-
teus vulgaris), but presenting more numerous involution forms, which may'
be spherical, pear-shaped, or spermatozoa-like, etc. The bacilli are about
0.6 n in diameter and vary greatly^in length, being sometimes nearly spheri-
cal, or forming rods of 2 to 3.75 /* in length, or long filaments.

Biological Characters. — An aerobic and facultative anaerobic, liquefy-
ing, motile bacillus. Spore formation has not been observed. Grows in the
usual culture media at the room temperature. Does not liquefy gelatin as

NOT DESCRIBED IN PREVIOUS SECTIONS.

553

rapidly as Proteus vulgaris. Upon gelatin plates, at the end of twelve
hours, superficial colonies of two to three millimetres in diameter are formed ;
under a low power these appear finely granular and brownish in color, and
have an irregular outline; outgrowths from the margin extend in various
directions and form new colonies, which may be attached for a time by a
long and slender thread consisting of bacilli. The movement of these new
colonies is not as pronounced as in the case of the preceding species, and

FIG. 155. — " Swarming islands " of Proteus mirabilis, from a gelatin culture, x 285. (Hauser.)

they are characterized by the presence of numerous distorted bacilli — invo-
lution forms. The deep colonies form spiral zooglcea masses.

In gelatin stab cultures the whole surface is first covered with threads
and islands of bacilli, which after a time form an anastomosing network, and
finally a confluent layer which at the end of forty-eight hours is rather thick,

"FiG. 156.— Spiral zooglcea from a culture of Proteus mirabilis. X 95. (.Hauser.)

with a moist, shining surface and grayish color, and appears to be perforated
with numerous small, sieve-like openings. These thinner and transparent
places disappear after a time, and at the end of two or three days liquefac-
tion of the gelatin commences; complete liquefaction does not occur until
the fifth or sixth day, or even later. Along the line of puncture finely gran-
ular colonies are first formed, from which long threads are given off, which
form after a short time a tolerably broad zone of threads and spiral zooglcea
masses.

554 PATHOGENIC AEROBIC BACILLI

Pathogenesis. — In Hauser's experiments filtered cultures (two to six cubic
centimetres), injected into the circulation or into the cavity of the abdomen
in rabbits, caused fatal toxaemia.

PROTEUS ZENKERI.

Obtained by Hauser (1885) from putrefying animal substances.

Morphology. — Bacilli which vary greatly in length — average about 1. 65 /*,
and about 0.4 ju broad.

Biological Characters. — An aerobic and facultative anaerobic, non-
liquefying, motile bacillus. Spore formation not observed. Grows in the
usual culture media at the room temperature. Upon the surface of nutrient
gelatin a laminated mass forms about the point of puncture, from the peri-
phery of which offshoots are given off, at the extremities of which colonies
are formed, as in the case of Proteus mirabilis. Gradually a rather thick,
grayish-white, opaque layer is formed, which covers the entire surface of the
gelatin and is easily detached from it. This species is distinguished from
the two preceding by the fact that it does not liquefy gelatin or blood serum
and does not give off a decided putrefactive odor when cultivated in these
^nedia.

Pathogenesis. — Considerable quantities injected into small animals give
rise to local abscesses and to symptoms of toxaemia.

PROTEUS SEPTICUS.

Obtained by Babes (1889) from the mucous membrane of the intestine and
the various organs of a boy who died of septicaemia.

Morphology. — Bacilli about 0. 4 ju. broad and varying greatly in length;
slightly curved rods or flexible filaments, often associated in loose chains.

Stains by the usual aniline colors and by Gram's method.

Biological Characters. — An aerobic, liquefying, motile bacillus. Spore
formation not observed. Grows in the usual culture media at the room
temperature. In gelatin plates centres of liquefaction are quickly formed
and rapidly extend. The spherical, liquefied places have at first a wavy or
dentate outline, and are surrounded by a branching, transparent, granular
margin which rapidly extends in advance of the liquefaction. In stab cul-
tures in nutrient gelatin liquefaction of the entire contents of the tube may
take place within twenty-four hours, or a broad, liquefied sac is formed
along the line of puncture. Gelatin cultures give off a very disagreeable
•odor. Upon the surface of nutrient agar, at 37° C., a peculiar, thick net-
work extends over the surface in the course of a few hours. Upon potato an
^elevated, brownish-white, shining layer is formed. Blood serum is lique-
fied by this bacillus.

Pathogenesis. — Pathogenic for mice, less so for rabbits. In mice death
occurs in from one to three days after the subcutaneous injection of a small
quantity of a pure culture ; the bacilli are present in the blood in small
numbers.

PROTEUS LETHALIS.

Synonym. — Proteus bei Lungengangran des Menschen (Babes).

Obtained by Babes (1889) from the spleen and gangrenous portions of the'
lung of a man who died of septicaemia.

Morphology. — Short rods with round ends, from 0.8 to 1.5 ju thick ; often
•swollen in the middle, like a lemon or a flask ; forms short, flexible filaments
which also present similar swellings.

Stains with the usual aniline colors and also by Gram's method.

Biological Characters. — An aerobic and facultative anaerobic, non-
fying, motile bacillus. Not observed to form spores. Grows in the
usual culture media at the room temperature. In gelatin plates forms hemi-
spherical, elevated, whitish, translucent colonies, which later send out

NOT DESCRIBED IN PREVIOUS SECTIONS. 555

coarse branches which ramify over the surface of the gelatin. • A similar
growth is observed upon the surface of gelatin stab cultures, and an abun-
dant development takes place along the line of puncture. Upon nutrient
agar a thick, opaque, slightly yellowish layer is formed. Upon potato a
moist, shining, brownish layer is developed, and the potato acquires a
brownish color. Upon blood serum the growth is less abundant than on
agar; the blood serum is not liquefied. This bacillus grows rapidly at the
room temperature; it is destroyed by a temperature of 80° C., and presum-
ably does not form spores.

Pathogenesis. — Recent cultures are very pathogenic for mice and for
rabbits, less so for guinea-pigs. The subcutaneous injection of a small
quantity of a pure culture kills susceptible animals in two or three days.
More or less oedema is found at the point of inoculation. Injections into the
rectum of rabbits gave rise to haemorrhagic enteritis, peritonitis, and death
at the end of four days.

BACILLUS A OF BOOKER.

Obtained by Booker (1889) from the alvine discharges of children suffer-
ing from cholera infantum.

Morphology. — Bacilli with round ends, varying greatly in length, usually
three to four // long and 0.7 /* broad (in recent agar cultures). In older cul-
tures the bacilli are shorter and smaller.

Biological Characters. — An aerobic and facultative anaerobic, lique-
fying, motile bacillus. Grows at the room temperature in the usual culture
media. In gelatin plates colonies are visible at the end of twenty -four
hours; under the microscope these are nearly colorless, and liquefaction
•soon occurs around them. In gelatin stab cultures complete liquefaction
occurs in three or four days . Upon agar a colorless layer covering the entire
surface is developed in three or four days, and an abundant development
occurs along the line of puncture. Agar colonies have a bluish look, and
are surrounded by an indistinct halo which shades off gradually into the
surrounding agar ; under a low power the colonies are light-brown and the
borders indistinct ; the surface has a delicate, wavy appearance. Upon po-
tato the growth is luxuriant and of a dirty-brown color. Blood serum is
liquefied by this bacillus.

Milk is coagulated into a gelatinous mass having an alkaline reaction ;
later the coagulum is dissolved.

Pathogenesis. — Mice and guinea-pigs fed with cultures in milk die in from
one to eight days.

BACILLUS ENDOCARDITIDIS GRISEUS.

Obtained by Weichselbaum (1888) from the affected valves in a case of
endocarditis recurrens ulcerosa.

Morphology. — Short rods with rounded or somewhat pointed ends, about
two to three times as long as broad — of about the same dimensions as the
bacillus of typhoid fever.

Stains with the usual aniline colors and also by Gram's method; the
longer rods from old cultures are irregularly stained.

Biological Characters. — An aerobic, non-liquefying, motile bacillus.
Refractive bodies may be seen in some of the rods, which resemble spores and
are stained by the method of Ernst, but they do not show the resistance of
known spores to physical and chemical agents. Grows well in the usual
culture media at the room temperature. Upon gelatin plates colonies are
formed which resemble those of Friedlander's bacillus, but which gradually
acquire a gray or grayish- white color. The prominent, convex, superficial
colonies under a low power are finely granular and grayish -brown in color;
the deep colonies are yellowish-brown in color, have slightly notched mar-
gins, and the surface is covered with minute projections. In stab cultures

PATHOGENIC AEROBIC BACILLI.

a rather thin, circular layer forms about the point of puncture ; this has the
appearance of stearin ; later it becomes grayish- white and the margins are
marked by radiating lines. Upon the surface of nutrient agar a similar
growth occurs which has a pale-brown or reddish-gray color. Upon potato
in the incubating oven an abundant development occurs, forming a dry-
looking layer of a grayish-brown color and having irregularly notched mar-
gins. Upon blood serum an abundant, grayish-white growth of cream-like
consistence forms along the impfstrich ; later this has a reddish gray color.
This bacillus grows to the bottom of the line of puncture in stick cultures,
and is no doubt a facultative anaerobic.

Pathogenesis. — Pathogenic for white mice and for guinea-pigs.

BACILLUS ENDOCARDITIDIS CAPSULATUS.

Obtained by Weichselbaum (1888) from thrombi and embolic infarctions
in the spleen and kidneys of a man who died from endocarditis with forma-
tion of thrombi.

Morphology. — Resembles Friedlander's bacillus, and is frequently sur-
rounded by a capsule, which may be stained ; also forms long, curved fila-
ments, in the protoplasm of which vacuoles may be observed in stained pre-
parations.

Stains with the usual aniline colors, but not by Gram's method ; by
staining with fuchsin arid carefully decolorizing with diluted alcohol the
presence of a capsule may be demonstrated.

Biological Characters.— An aerobic, no n-liquefying bacillus. Grows in
the usual culture media at the room temperature.

In gelatin stab cultures development occurs along the line of puncture,
and on the surface as a rather thin, white, dry layer which resembles stearin.
In agar plates the superficial colonies are thin, about two millimetres in
diameter and gray in color ; under a low power the margins are trans-
parent and colorless, and the centre resembles the deep colonies; these are
very small and grayish- white in color ; under a low power the surface is
seen to be covered with tooth-like, projecting masses, the margin is dentate
and has a pale-yellow color, while the centre is yellowish- brown.

Pathogenesis. — Rabbits are killed by the injection of a considerable quan-
tity of a pure culture into the cavity of the abdomen or subcutaneously.

BACILLUS ALVEI.

Synonym. — Bacillus of foul brood (of bees).

Obtained by Cheshire and Cheyne (1885) from the larvae in hives infected
with " foul brood." The larvae in the interior of cells in the comb die and
become almost fluid as a result of parasitic invasion by this bacillus.

Morphology.— Bacilli with rounded ends, from 2.5 to 5 ju in length (aver-
age about 3.6 //) and 0.8 u in diameter. Grow out into filaments and form,
large oval spores which have a greater diameter than the rods in which they
are developed — 1.07 n.

Stains readily with the aniline colors usually employed, also by Gram's
method.

Biological Characters.— An aerobic and facultative anaerobic, liquefy-
ing, motile bacillus. Forms endogenous spores. Grows readily in the usual
culture media at the room temperature.

In gelatin plates small, round or oval colonies are formed, which later
become pear-shaped; a branching outgrowth occurs about the margins of the
colonies, and especially from the small end of the pear-shaped mass. In
streak cultures upon the surf ace of gelatin growth occurs first along the impf-
strich, and from this an outgrowth occurs consisting of bacilli in a single
row or in several parallel rows, and forming irregular or circular figures

NOT DESCRIBED IN PREVIOUS SECTIONS. 557

from which other similar outgrowths occur ; the branching1 outgrowths may
anastomose. The gelatin is liquefied in the vicinity of these lines of growth,
forming a network of channels. A similar growth is seen upon the surface
of gelatin stab cultures, and along the line of puncture white, irregular
masses are formed, from which rather coarse branches are given off which
often have a club-shaped extremity. In older cultures the finer branches
disappear, so that the secondary centres of growth are disconnected from the
original colonies ; complete liquefaction of the gelatin occurs in about two
weeks; the liquefied gelatin has a yellowish color and peculiar odor. Upon
the surface of nutrient agar, at 37° C., a white layer is formed. Upon potato
the development is slow and results in the formation of a dry, yellowish
layer. In milk coagulation first occurs, and the coagulum is subsequently
dissolved; a slightly acid reaction is produced. This bacillus grows best in
the incubating oven, at 37°, and does not develop at temperatures below 16°
C. The spores require for their destruction a temperature of 100° C. main-
tained for four minutes (determined by the writer, 1887).

Pathogenesis. — The introduction of pure cultures of this bacillus into
hives occupied by healthy swarms causes them to become infected with foul
brood; grown bees also become infected when given food containing the ba-
cillus (Cheshire) . Mice injected subcutaiieously with a considerable quan-
tity die within twenty-four hours, guinea-pigs in. six days (Eisenberg).
Small amounts injected beneath the skin of mice or rabbits produce no appa-
rent result.

BACILLUS OP ACNE CONTAGIOSA OF HORSES.

Obtained by Dieckerhoff and Grawitz (1885) from pus and dried scales
from the pustules of " acne contagiosa " of horses.

Morphology.— Short rods, straight or slightly bent, 0.2 /* in diameter.

Stains best with an aqueous solution of fuchsin, and also by Gram's
method ; does not stain well with Loffler's alkaline solution of methylene
blue.

Biological Characters. — Anaerobic, non-liquefying bacillus. In gelatin
stab cultures a very scanty growth occurs along the line of puncture ; upon
the surface a white mass forms about the point of puncture. Upon blood
serum and nutrient agar an abundant growth at the end of twenty -four
hours at 37° C., consisting of white colonies along the impfstrich, which
later have a yellowish-gray color. The growth is more abundant and rapid
upon blood serum than upon other media.

Pathogenesis. — Pure cultures of the bacillus described are said by Diecker-
Tioff and Grawitz to produce typical acne pustules when rubbed into the skin
of horses, calves, sheep, and dogs. When rubbed into the intact skin of
guinea-pigs a phlegmonous erysipelatous inflammation was produced, and
the animal died at the end of forty-eight hours with symptoms of toxaemia.
Subcutaneous injections in guinea-pigs caused toxaemia and death at the end
of twenty-four hours. At the autopsy a haemorrhagic infiltration of the in-
testinal mucous membrane was observed ; the bacilli were not found in the
internal organs. In rabbits pure cultures rubbed into the intact skin caused
a development of pustules and a severe inflammation of the subcutaneous
connective tissue, from which the animal usually recovered. Subcutaneous
injections in rabbits sometimes caused a fatal toxaemia. House mice, field
mice, and white mice were not affected by the application of cultures, by
rubbing, to the uninjured skin, but succumbed to subcutaneous injections in
twenty-four hours or between the fifth and tenth days. Those which died
at a late date presented the pathological appearances which characterize
pyaemia.

558 PATHOGENIC AEROBIC BACILLI

BACILLUS OF PURPURA HJEMORRHAGICA OF TIZZONI AND GIO-

VANNINI.

Obtained by Tizzoni and Giovannini (1889) from the blood of two children,
who died of purpura haemorrhagica following impetigo.

Morphology. — Bacilli with round ends, from 0.75 to 1.3 /* long and 0.2
to 0.4 yu broad; often seen in pairs or in groups like streptococci.

Stains with the usual aniline colors, but not by Gram's method.

Biological Characters. — An aerobic and facultative anaerobic, non-
liquefying, non-motile bacillus. Spore formation not observed. Grows in
the usual culture media at the room temperature. Upon gelatin plates the
colonies at first resemble those of Streptococcus pyogenes. Upon the surface
small, opaque points are seen at the end of > forty-eight hours, which at the
end of four to five days develop into spherical, yellowish-gray colonies with
irregular margins, surrounded by a growth resembling tufts of curly hair.
Upon agar the growth is similar, but more rapid and of a pale color, often
with a central nucleus surrounded by a net-like marginal zone. Upon
blood serum the growth is similar to that upon agar. Upon potato, at 37°
C., a limited development occurs about the point of inoculation, which has
a dark-yellow color. The cultures give off a very penetrating odor.

Pathogenesis. — Pathogenic for dogs, rabbits, and guinea-pigs when in-
jected subcutaiieously. Not pathogenic for white mice or pigeons. The
symptoms resulting from a subcutaneous injection are said to be fever, al-
buminuria and, in some cases, anuria, haemorrhagic spots upon the skin,
convulsions ; death occurs in from one to three days. At the autopsy there
are found oedema about the point of inoculation , haemorrhages in the skin and
muscles, and sometimes in the internal organs and in serous cavities; the
blood does not coagulate. The bacilli are found in the subcutaneous con-
nective tissue, but not in the blood or in the various organs. Sections show
coagulation necrosis of the liver cells and of the renal epithelium.

BACILLUS OF PURPURA H^EMORRHAGICA OF BABES.

Obtained by Babes (1890) from the spleen and lungs of an individual who
died from purpura haemorrhagica with symptoms of septicaemia. Resembles
the bacillus previously described by Tizzoni and Giovannini, and still more
that of Kolb ; but, according to Babes, differs in some respects from both of
these, although they all belong evidently to the same group.

Morphology. — Bacilli with rounded ends, oval or pear-shaped, about 0.3 /*
thick, surrounded by a narrow capsule.

Stains with the aniline colors, but not deeply, and still less intensely by
Gram's method.

Biological Characters. — An aerobic and facultative anaerobic, non-
liquefying, non-motile bacillus. Does not form spores. Grows in the usual
culture media at the room temperature. In gelatin stick cultures, at the
end of three days, a thin, transparent, irregular layer has developed upon
the surface, and a whitish, punctate stripe along the line of inoculation. In
agar stick cultures an abundant development occurs along the line of punc-
ture, and at the end of three days the growth upon the surface consists of
small, moist, transparent drops; later of larger, flat, shining, yellowish-
white plaques which have ill-defined margins. Upon blood serum the de-
velopment is somewhat more abundant in the form of small, white, moist
colonies one to two millimetres broad. Upon potato, at the end of three
days, moist, whitish drops with ill-defined margins.

Pathogenesis. — Inoculations in the conjunctivas of rabbits produce ecchy-
moses of the conjunctiva. At the autopsy numerous haemorrhagic extrava-
sations are found in all the organs, especially in the lungs and liver ; the
spleen is enlarged ; the bacilli can be recovered in pure cultures from the
various organs. Old cultures proved to have lost their virulence. Patho-
genic for mice, which die from general infection in the course of a few days ;

NOT DESCRIBED IN PREVIOUS SECTIONS.

the spleen is enlarged, and haemorrhages in the serous membranes are usually
seen.

BACILLUS OF PURPURA H^EMORRHAGICA OF KOLB.

Obtained by Kolb (1891) from the various organs of three individuals
who died in from two to four days from attacks characterized by suddenly
developed fever, purpura, and albuminous urine.

Morphology. — Oval bacilli, usually in pairs, 0.8 to 1.5/*long andO.8/*
broad, surrounded by a narrow capsule, which is only seen distinctly in.
preparations from the organs.

Stains with the aniline colors, but not deeply, and still more feebly by
Gram's method.

Biological Characters. — An aerobic and facultative anaerobic, non-
liquefying non-motile bacillus. Does not form spores. Grows in the usual
culture media at the room temperature. In gelatin stick cultures, at the end
of four days, a very small, thin, hyaline growth is seen about the point of
inoculation. The development is more abundant along the line of puncture.
Upon the surface of agar a thin layer is formed with smooth margins.
Upon potato, at the end of three to four days, a whitish, moist, shining stripe
is seen along the impfstrich which is about three millimetres broad.

Pathogenesis. — Injections of 0.5 to 1 cubic centimetre of a bouillon
culture into the abdominal cavity of rabbits cause symptoms of general in-

Fia. 157. FIG. 158.

Fi». 157.— Bacillus gracilis cadaveris, from a gelatin culture. X 1,000. From a photomicro-
graph. (Steinberg.)

FIG. 158. — Bacillus gracilis; colonies in gelatin roll tube, end of forty -eight hours. X 12. From
a photograph. (Steinberg )

fection in the course of a few days, and not infrequently haemorrhagic ex-
travasations are seen in the ear muscles. More than one cubic centimetre
may cause death in from one to three days. At the autopsy haemorrhagic
extravasations are found in the subcutaneous tissues and in the serous and
mucous membranes. The blood has little disposition to coagulate; the
bacillus may be recovered in pure cultures from the various organs. In
guinea-pigs local ecchymoses are sometimes produced, otherwise not patho-
genic for this animal. Pathogenic for mice, which die from general infec-
tion, after being inoculated with a small quantity of a pure culture, in from
two to three days ; spleen enlarged; lymphatic glands often haemorrhagic.
Not fatal to dogs, but animals which were inoculated with one cubic centi-
metre of a bouillon culture and subsequently killed proved to have haemor-
rhagic extravasations in the various organs.

560 PATHOGENIC AEROBIC BACILLI

BACILLUS GRACILIS CADAVERIS (Steinberg) .

Obtained (1889) from a fragment of liver, of man, kept for forty-eight
hours in an antiseptic wrapping.

Morphology. — Bacilli about 1 JLI broad and 2 jn long, associated in long
chains.

Biological Characters. — An aerobic and facultative anaerobic, non-
motile, non-liquefying bacillus. Spore formation not observed. In gelatin
roll-tubes the deep colonies are opaque and spherical ; superficial colonies
circular or slightly irregular in outline, white in color, and opaque or slightly
translucent. In gelatin stab cultures, at 22° C. , at the end of five days a
rather thick, white mass at the point of puncture, covering one-third of the
surface, and closely crowded, opaque colonies at bottom of line of puncture,
with slender, branching outgrowth above. In nutrient agar, at the end of
five days at 22° C., a milk-white growth upon the surface and opaque
growth to bottom of line of puncture. On potato, at end of five days at
22° C., rather thick, cream-white growth with irregular margins along the
impfstrich. Cultures in bouillon have a milky opacity and a very disagree-
able odor. Grows in agua coco without formation of gas.

Pathogenic for rabbits when injected into the cavity of the abdomen.

CAPSULE BACILLUS OF NICOLAIER.

Obtained by Nicolaier (1894) from pus contained in an abscess of the kid-
ney— obtained post-mortem.

Morphology. — Thick bacilli, with rounded ends, usually four times as
long as thick, and frequently presenting irregular outlines ; often united in
pairs, and sometimes growing out into filaments; cocci-like forms also occur.
Often surrounded by a capsule which remains unstained in stained prepara-
tions. Does not stain by Gram's method.

Biological Characters. — An aerobic, and facultative anaerobic, non-
liquefying, non-motile bacillus. Does not form spores. Grows at the room
temperature and more rapidly at 37° C. Upon gelatin plates at 20° C., at
the end of twenty-four to thirty-six hours punctiform colonies are devel-
oped, which under a low power appear finely granular, and grayish-yellow
spheres. At the end of forty-eight to sixty hours the superficial colonies ap-
pear as round or slightly irregular, grayish- white discs, which project but lit-
tle above the surface of the gelatin, and have thin, transparent margins.
The deep colonies have a sharply defined contour, with dark-brown centre
and a purely granular pale-brown marginal zone. In gelatin stab cultures
a slightly elevated, moist-looking, sticky layer with more or less transparent
margins is developed. In slanting cultures this growth gradually slips down
to the lowest part of the exposed surface, leaving a thin, gray, transparent
layer over the gelatin ; along the line of puncture a ribbon-like, grayish-
white growth with irregular margins is developed. In media containing
glucose some gas bubbles are developed. The growth is much more rapid
in the incubating oven at 37° C., and there is an abundant development of
gas in agar tubes. Upon potato a grayish- white, slimy mass with a shining
surface is quickly developed. In bouillon, at the end of twenty-four hours,
at 37° C., the medium is clouded throughout, and a grayish- white deposit ac-
cumulates at the bottom of the tube. Development occurs also in acid
media.

Pathogenesis. — Pathogenic for house mice, white mice, and for rats — not
for rabbits or guinea-pigs — by subcutaneous injections. As Nicolaier has
made a careful comparison of the characters of the various " capsule bacilli"
described, we quote from him as follows :

"Our bacillus in its morphology and growth in various media closely re-
sembles that of Fasching and of Abel, both of which were obtained in patho-

NOT DESCRIBED IN PREVIOUS SECTIONS. 561

logical products from man. It is distinguished from them by its pathogenic
action upon mice. White and gray mice when infected with our bacillus
die from septicaemia and show, in addition to a serous exudation at the point
of inoculation, constant pathological changes in the kidneys, which may usu-
ally be recognized by a macroscopic examination. Also by the spleen, which
is not always enlarged, and the liver, which only in a few cases showed any
microscopic changes. In mice inoculated with the bacillus of Faschmg, or
that of Abel, which died of septicaemia, there was constantly seen an en-
largement of the spleen (Fasching, Abel) and of the liver (Abel), and a
cloudy swelling of the liver and kidneys (Abel) which our mice failed to
show. The macroscopic and microscopic changes which we found in the
kidneys in mice, and also in some cases in the liver and spleen, were not ob-
served by Fasching or by Abel. Eecently Paulseii has described a capsule ba-
cillus from atrophic rhinitis, and Marchand a capsule bacillus — not further
described — which he obtained in great numbers from the exudate in a case
of lobar pneumonia. Both appear to be very similar to Fasching's bacillus.
They are pathogenic for mice, but do not cause the changes in the kidneys
which we have described. These capsule bacilli are therefore not identical
with ours. Marchaiid's bacillus is further distinguished by the fact that it is
pathogenic for guinea-pigs. . . . The bacillus of Kockel is distinguished
from ours by the following characters : It forms upon the surface of gelatin,
as well as in stick cultures, highly elevated, button like colonies, while our
bacillus grows more in flat and broad layers. It also lacks the semi-fluid
character of growth upon slanting agar, which distinguishes our bacillus,
and as a result of which the growth slips down to the lowest point on the
slanting surface ; further it forms upon potato a yellowish layer, while ours
is grayish- white ; and it does not grow in acid media. Finally, it is patho-
genic for rabbits by intravenous injection, while ours is not."

BACILLUS MUCOSUS OZ^N^.

Obtained by Abel (1893) from cases of ozi^na simplex (rhinitis atrophicans
fcetida). As this bacillus appears to correspond in its morphological and bio-
logical characters with the capsule bacillus above described we shall not
repeat this description, but quote from Abel, as follows:

"Thi.s bacillus, found in the secretion from cases of ozaena, as the de-
scription we have given shows, closely resembles Friedlander's pneumo-
bacillus. It is distinguished from it by certain constant characters. The
ozrena bacillus forms in cultures a more fluid mass than Friedlander's. As
a result of this it does not form the characteristic nail-head culture, but
spreads out over the surface of the gelatin. Upon slanting gelatin cultures
the growth slips down to the lowest point. In old cultures it never shows a
brown coloring of the culture medium. It never forms gas on potato, and
in agar and gelatin cultures but little gas is developed. Mice always suc-
cumb to subcutaneous inoculations, while Friedlander's bacillus does not
kill mice. Intraperitoneal infection of guinea-pigs with the ozseiia bacillus
always causes their death. Friedlander's bacillus only killed about half the
guinea-pigs inoculated in the cavity of the abdomen. Finally, Friedlander's
bacillus has a greater tendency to cocci-like forms. The resemblance to
Pfeiffers capsule bacillus is closer. But the tenacious layer described by
Pfeiffer as found upon the intestinal coils and the lungs in mice, and the
sticky condition of the blood and tissue juices (fadenziehende) are want-
ing. The reaction at the point of inoculation in mice is also much more
pronounced with my bacillus."

It seems extremely probable that this bacillus, the Bacillus capsulatus mu-
cosus of Fasching, and the above-described capsule bacillus of Nicolaier
are simply pathogenic varieties of one and the same bacillus.

562 PATHOGENIC AEROBIC BACILLI

CAPSULE BACILLUS OF VON DUNGERN.

Obtained by von Dungern (1893), post mortem, from a new-born child
which died of hemorrhagic septicaemia — infection through umbilicus.

Morphology. — A short, thick bacillus, from 1 to 2 p. long and half as
broad, surrounded by a capsule which is slightly stained by gentian violet —
best seen in the body of infected mice ; sometimes seen in pairs or in chains
of four elements ; also grows out into filaments, especially in bouillon.
Upon potato usually only small spherical elements, resembling micrococci,
are seen. Does not stain by Gram's method.

Biological Characters. — An aerobic and facultative anaerobic, non-
motile, non-liquefying bacillus. Does not form spores. Coagulates milk,
in which it causes an abundant development of gas at 38° C. Has feeble
indol reaction. Grows well at room temperature, more rapidly in incubator.
Upon gelatin plates the deep colonies at end of twelve hours are the size of a
pin's head, finely granular, spherical, and sharply defined. Upon the sur-
face, porcelain-like, elevated, white colonies are developed, which in two or
three days attain the size of lentils. In gelatin stab cultures development
occurs all along the line of puncture, frequently with formation of gas bub-
bles. Upon agar a thick, soft layer of a white color is developed. In bouil-
lon, at 38° C., there is considerable development of gas. Upon potato the
growth is very abundant, of a pale yellowish-white color, thick, soft, some-
what sticky, and filled with gas bubbles. A great portion of the surface is
covered by this growth at the end of twenty -four hours, even at the room
temperature. These cultures give off a peculiar odor, sometimes aromatic-
foetid and sometimes recalling that of fresh bread. Some of the cultures on
potato soon become cream-like in consistence. At first they have an alkaline
and later an acid reaction, when they have the odor of acetic acid.

Pathogenesis. — Very pathogenic for white mice. The bacilli are found
in the blood and in all the organs in enormo'us numbers. At the point of
inoculation there is frequently a hemorrhagic oedema. The spleen is greatly
enlarged. Also pathogenic for guinea-pigs when injected into the cavity of
the abdomen — less pathogenic for rabbits.

According to von Dungern, this bacillus can not be distinguished by its
morphological and biological characters from Fried lander's bacillus, Bacil-
lus capsulatus of Pfeiffer, or Bacillus canalis capsulatus of Mori. But it is
distinguished from these by greater virulence, especially for rabbits, and by
the fact that it frequently gives rise to hernorrliagic extravasations in inocu-
lated animals. In our opinion the characters given do not justify the view
that this bacillus is a distinct species from the bacilli above mentioned.

BACILLUS PESTIS (Kitasato and Yersin).

Discovered by Kitasato (1894) in the blood of living patients, and
in the buboes, blood, and organs of those who had recently died from
the infectious malady known as bubonic plague. Kitasato was sent
to Hong-Kong by the Japanese Government for the purpose of inves-
tigating this disease. According to Lowson the bacilli are found in
the faeces, in the contents of the buboes, and in the blood.

Morphology. — In his preliminary note, Kitasato described the
plague bacilli as "rods with rounded ends," which are readily
stained by the ordinary aniline dj-es, the poles being stained darker
than the middle part, especially in blood preparations, and present-
ing a capsule sometimes well marked, sometimes indistinct.

NOT DESCRIBED IN PREVIOUS SECTIONS. 563

Yersin, who was sent by the French Government to study the
bubonic plague at Hong-Kong, arrived in that city on the loth of
June, 1894. He describes the bacillus found in the contents of the
buboes as being short and thick, with rounded ends, staining easily
with the aniline colors, but not by Gram's method. " The extremities
stain more intensely than the centre, so that they often present a
clear space in the middle. Sometimes the bacilli appear to be sur-
rounded by a capsule. ... In bouillon the bacillus has a very char-
acteristic appearance, resembling the cultures of the streptococcus of
erysipelas — a clear liquid with grumous deposits on the walls and at
the bottom of the tube. These cultures examined under the micro-
scope show veritable chains of short bacilli, presenting in places a
considerable spherical enlargement."

This bacillus is sometimes seen to be motile, and it has flagella,
which, however, are difficult to stain (Gordon).

In agar cultures, in the incubator at 37° C., involution forms soon
appear. These may be spherical, oval, pyriform, etc., and are often
many times larger than the typical bacillus.

Biological Characters. — We quote from Kitasato's preliminary
report as follows :

The bacilli show very little movement, and those grown in the incubator,
in beef -tea, make the medium somewhat cloudy. The growth of the bacilli is
strongest 011 blood serum at the normal temperature of the human body
(34° C.) ; under these conditions they develop luxuriantly and form a col-
ony moist in consistence and of a yellowish-gray color ; they do not liquefy
the serum. On agar-agar jelly (the best is good glycerin agar) they also
grow freely. The different colonies are of a whitish-gray color and by re-
flected light have a bluish appearance ; under the microscope they appear
moist aiicl in rounded patches with uneven edges ; at first they appear every-
where as if piled up with "glass-wool," later as if having dense, large cen-
tres. If a cover-glass preparation is made from a cultivation on agar-agar,
and, after having been stained, is observed under the microscope, long
threads of bacilli are seen, which might, by careless inspection, be mistaken
for a coccus chain, but are recognized with certainty as " threads of bacilli "
under closer observation. The growth on agar-gelatin is similar to that 011
agar-agar ; in a puncture cultivation at the ordinary temperature after a few
days they are found growing as a fine dust in little points alongside the
puncture, but with very little growth on the surface. Whether these ba-
cilli are able to liquefy ordinary gelatin or not I am at present unable to de-
cide, as the temperature of Hong-Kong ranges so high that the employment
of simple nutritive gelatin is out of the question. I shall give further infor-
mation on this question later. On potatoes at a temperature of from 28°
to 30° C., there was no growth after ten days' observation, but at a tempera-
ture of 37° C. the bacilli developed sparingly after a few days ; the growth
was whitish-gray in color and exsiccated. As mentioned before, the bacilli
grow best at a temperature of from 38° to 39° C. ; at how low a temperature
growth is possible I am unable at present to state. So far I have been un-
able to observe the formation of spores.

Experiments on Animals. — Mice, rats, guinea-pigs, and rabbits are sus-
ceptible to inoculation. If these animals are inoculated with pure culti-
vations, or with the blood of a plague patient in which the bacilli have been

504 PATHOGENIC AEROBIC BACILLI

observed, or with the contents of a bubo, or with pieces of internal organs,
or even with the contents of the intestine, they begin to become ill in from
one to two days, according to the size of the animal. Their eyes become wa-
tery, they begin to show disinclination for any effort, later they avoid their
food, and hide quietly in a corner of the cage. The temperature rises to
41.5° C., and with convulsive symptoms they die in from two to five days. I
must observe that in Hong-Kong I could only obtain small guinea-pigs
(weight from one hundred to one hundred and fifty grammes) and small
rabbits (from two hundred to two hundred and fifty grammes). If I could
have experimented upon larger animals it is possible that life would have
been prolonged somewhat beyond the periods mentioned above. The parts
around the point of inoculation are infiltrated with a reddish gelatinous
exudation, the spleen is enlarged, sometimes there is a swelling of the lym-
phatic glands, and in all the orsrans the bacilli are found. The results found
after death in animals are very similar to those found in anthrax and in
oedema malign um. Pigeons do not appear to be susceptible to the influence
of the bacilli. I made experiments by feeding some mice and guinea-pigs
with pure cultivations of the bacillus and with small pieces of the internal
organs : the result was, such animals perished in a few days under the same
symptoms as those which had been inoculated. In all the internal organs
of animals so destroyed I found the bacilli. With the dust of dwelling-
houses from which the plague-stricken had been removed, I made sev-
eral experiments upon animals. Some of the animals died from tetanus.
In one case only a guinea-pig died with plague symptoms, and in this ani-
mal the same bacilli were found in the internal organs as in those of
plague patients who had succumbed. These experiments with the dust from
infected houses I shall certainly continue. Many rats and mice at present
die spontaneously in Hong-Kong. I examined some of them. In the inter-
nal organs of a mouse I discovered the same bacilli.

Experiments with Desiccation, — The contents of a bubo in which the
bacilli were present in great numbers were wiped over cover glasses (per-
fectly cleansed by heat and alcohol), 'and some of these cover-glasses were
dried in the air of a room at a temperature ranging from 28° to 30 C. Oth-
ers I exposed directly to the sun's rays, and from among them, after an expo-
sure of from one, two, and three hours up to six days, I removed some parts,
putting such portions in beef-tea and placing them in the incubator. Those
which had been standing in the room from one to thirty-six hours showed a
pretty good growth in the incubator, but those which had been in the room
for more than four days were unable to show any growth even after one
week's incubation. Those exposed directly to the sun were all destroyed after
from three to four hours. Further cultivations 011 serum were treated
exactly like the contents of the bubo with very similar results.

Experiment* tn'th Heat. — Beef -tea cultivations which had been heated
for thirty minutes in a water bath up to 80° C. were destroyed: at 100° C., in
the vapor apparatus they were destroyed in a few minutes.

Yersin reports that when fragments of the spleen or liver of
animals which have died of the plague are fed to rats and mice they
usually become infected and die, and the bacillus is found in their
organs, lymphatic glands, and blood. He also demonstrated the pres-
ence of the bacilli in dead rats found in the houses or streets of
Hong-Kong.

Without doubt rats play an important part in the propagation of
the disease. Monkeys are also very susceptible to infection, and it is
said that the disease has been known to occur as an epidemic among
these animals. There is also good reason to believe that fleas have
some influence in the propagation of the disease, by transferring the

NOT DESCRIBED IN PREVIOUS SECTIONS. 565

bacillus from infected rats to man, or from one individual to another.
Infection in man occurs by inoculation through lesions of the skin
and also by the respiratory passages (pulmonic form).

BACILLUS PISCICIDUS AGILIS (Sieber) .

Discovered by Sieber (1895) in infected fish, which died of an epidemic
disease in the laboratory of Professor Nencki, at St. Petersburg.

Morphology. — Short bacilli, often united in pairs.

Biological Characters. — An aerobic and facultative anaerobic, motile,
liquefying bacillus. In old cultures in bouillon spores are developed.
Grows at temperatures of from 12° to 37.5° C. Thermal death point, 60 to
65° C. On gelatin and agar plates forms granular, grayish, or yellowish
colonies, which appear to be made up of three concentric rings — the outer one
having a jagged outline. Gas is developed during the growth of the bacillus
—carbon dioxide and methyl merecaptan in small amount. Upon potato it
forms yellowish-brown, pearl -like colonies. Causes coagulation of milk.
Ketains its vitality and virulence for months in well or river water.

Pathogenesis. — Pathogenic for fish, frogs, guinea-pigs, rabbits, mice,
'and dogs (not for birds). Old cultures are more pathogenic than recent
ones, and gelatin cultures are the most active. Frogs are killed in half an
hour by 0.1 cubic centimetre of a bouillon culture six days old. Filtered
cultures are as toxic as those containing the living bacillus ; they give with
iron chloride a characteristic color reaction — an intense red color. Sieber
has obtained from his cultures an extremely toxic alkaloid in the form of a
hydrochlorate. Two litres of filtered culture gave 0.1 gramme of this salt.
An aqueous solution of this killed a frog in. fifteen minutes in the dose of
0.0035 gramme.

BACILLUS OF MERES HKOWSKY.

Obtained by Mereshkowsky (1894) from infected animals (Sperniophilus
musicus) which died from an epidemic malady developed in his laboratory.

Morphology. — Closely resembles Loffler's Bacillus typhi murium.

Biological Characters. — An aerobic, motile, non-liquefying bacillus.
Spore formation not observed. Grows in the usual culture media at the
room temperature — best at 37.5° C. In. bouillon, at the end of twenty -four
hours, the medium is clouded and a white pellicle is seen upon the surface,
which breaks up into small flocculi and falls to the bottom when the tube
is slightly shaken. On gelatin plates minute, slightly granular, pale-brown
colonies may be seen, under a low power at the end of twenty-four hours;
on the second day these are visible as white spheres, which under the micro-
scope have a pale-brown color and a more or less transparent, peripheral
zone. In media containing glucose no gas is developed. The growth upon
agar and potato presents nothing characteristic.

Pathogenesis. — Pathogenic for Zieselmausen (Spermophilus musicus),
for Spermophilus guttatus, for squirrels (Sciurus vulgaris) for house mice,
for field mice (Arvicola arvalis). Not pathogenic for man or for the domes-
tic animals tested, horse, swine, sheep, fowls. Mereshkowsky proposes to
use cultures of this bacillus for the extermination of field mice, which die in
from one to ten days after being fed upon biscuit wet with a bouillon cul-
ture.

BACILLUS OF EMMERICH AND WEIBEL.

Obtained by Emmerich and Weibel (1894) from infected trout in ponds
belonging to an establishment for raising these fish. The disease appeared
as a superficial " f urunculosis with secondary development of abscesses con-
taining bloody pus." Death occurred in from twelve to twenty days. The
pustules and secondary abscesses and blood from the heart and various or-
gans contained bacilli, which proved to be the cause of the infectious malady.

5G6 PATHOGENIC AEROBIC BACILLI

Morphology. — Bacilli about as long1 as the tvplioid bacillus, but not so
thick, very frequently united in pairs ; occasionally grows out into filaments.

Biological Characters.— An aerobic and facultative anaerobic, lique-
fying, non-motile bacillus. Does not form spores. Thermal death point, 60 D
C. Stains with the usual aniline colors but not by Gram's method. Grows
best at 10° to 15° C. The growth in gelatin is quite characteristic. At the
end of two or three days, in gelatin plates, at the room temperature, small
white colonies are developed ; in four or five days small gas bubbles or ex-
cavations are seen, at the bottom of which lie the scale-like or rosetta-formed
colonies. The margin of the colonies is irregular and later jagged. At
first the colonies are grayish- white or yellowish, later brownish. The
superficial colonies have a peculiar lustre. In gelatin stab cultures, colo-
nies develop along the line of puncture, which at first resemble the growtli
of Streptococcus pyogenes, and no development is seen on the surface. At
the end of five to seven days in place of the line of colonies is seen a channel
filled with air, or gas developed by the separate colonies, the bubbles from
which coalesce. The funnel formed in this way is somewhat larger above,
and at the bottom contains a whitish sediment consisting of bacteria con-
tained in a few drops of liquefied gelatin. Along the sides of the funnel
bubble-like cavities may frequently be seen, at the bottom of which the bac-
teria have accumulated. In bouillon a slight cloudiness is seen near the
surface, on the walls of the test tube; when slightly shaken this falls to the
bottom, leaving the bouillon entirely clear. In agar -agar tubes, a veil-
like stripe develops along the line of puncture, and a grayish-yellow, moist
layer, with irregular outlines upon the surface. After some weeks this
acquires a brown color. No growth occurs upon potato. No development
occurs in the incubating oven at 37" C.

Pathogenesis. — Trout became infected and died through direct infection,
subcutaneous or intramuscular inoculations, or through the addition of cul-
tures to the water in which they were kept, or by placing infected fish in the
same tank with healthy ones.

BACILLUS OF BECK.

Synonym. — Der Bacillus der Brustseuche beim Kaninchen.

Obtained by Beck (1892) from rabbits which died of an infectious malady
in the Institut fur Infectionskrankheiten, in Berlin,

Morphology. — Very small and slender bacilli, about twice as long and
twice as thick as the influenza bacillus ; some what pointed at the extremities ;
show a tendency to grow out into filaments.

Biological Characters. — An aerobic (strict) non-liquefying, non-motile
bacillus. Spore formation not observed. Grows at the room-temperature
and more vigorously at 38J C. Does not stain by Gram's method. Thermal
death point, 50° C. (five minutes). Resists desiccation, at the room tempera-
ture, for seventeen days, at 37° C. for three days.

On gelatin plates, at the end of forty-eight hours, small, finely granular,
glass-like colonies are developed ; older colonies have a pale-brown appear-
ance. In gelatin stab cultures a granular growth of a white color is seen
along the line of puncture. Upon agar, at 37° C., an abundant development
occurs in twenty -four hours. The line of puncture seen from above is gray-
ish-white, by transmitted light bluish and porcelain-like with a brownish
tint. On agar plates the colonies have a yellowish-gray appearance ; the
margin of the finely granular colonies is sharply defined. In agar cultures
several days old the colonies are sticky and may be picked up as a compact
mass, or drawn out into threads. In bouillon, at 37° C., there is a slight
cloudiness at the end of twenty-four hours ; later the bouillon is clear and a
white sediment is seen at the bottom of the tube. In bouillon cultures
especially, the bacillus grows out into long filaments.

NOT DESCRIBED IN PREVIOUS SECTIONS. 567

Pathogenesis. — From 0.25 to 1 cubic centimetre of a bouillon culture
injected into the pleural cavity of a rabbit caused a development of all of
the symptoms of influenza (Brustseuche) — viz., elevation of temperature at
the end of five or six hours, cough, nasal discharge, dyspnoea, and death—
usually in from three to five days. The autopsy showed a distinct pleuro-
pneumoiiia and a general blood infection by the bacillus in question.
Injections into the circulation also give rise to the symptoms of influenza,
including pneumonia, and to death at the end of from ten to fourteen days.
Subcutaneous injections resulted in the development of an abscess and of ex-
tensive necrosis of the tissues, but did not cause a general blood infection.
Guinea-pigs were somewhat less susceptible than rabbits, but injections into
tlie pleural cavity produced similar symptoms and death at a later date.
White mice and house mice, as a result of intraperitoneal injections, died
within two or three days from general blood infection.

BACILLUS PISCICIDUS (Fischel and Enoch).

Obtained by Fischel and Enoch (1892) from an infected carp.

Morphology. — Bacilli solitary or in chains of four to five elements, 1.2
to 3 /i long and 0.25 ^ thick. Stains by the usual aniline colors and by
Gram's method.

Biological Characters. — An aerobic and facultative anaerobic, non-
motile, liquefying bacillus. Forms spores. In gelatin plates forms round
colonies of a pale yellowish-brown color, having a slightly toothed border
and a granular surface. At the end of twenty-four hours a narrow zone of
liquefaction can be discerned around the colonies, and at the end of about
ten days the gelatin is entirely liquefied. In gelatin stab cultures a scanty
growth is seen along the line of inoculation at the end of twelve hours ; the
growth upon the surface is rapid, and liquefaction commences at the end of
twenty-four hours. Upon agar, at 37° C. at the end of eighteen hours, a thin
granular layer is seen, which consists of small, pale-gray colonies. In agar
stick cultures a scanty growth occurs along the line of puncture, which does
not increase after thirty-six hours. Upon the surface the growth is abundant,
forming, at the end of five days a tolerably thick grayish-white layer. No

frowth occurs upon potato at the room temperature, but at 37° C. a tolera-
ly thick, sticky layer of a grayish- white color is developed in three or four
days. In bouillon, at 37° C., the medium is clouded at the end of twelve
hours, and a thin pellicle is seen upon the surface at the end of thirty-six
hours ; this falls to the bottom when the tube is slightly agitated. At the
end of four days development has ceased, and the bouillon is again transpar-
ent, while a flocculent deposit is seen at the bottom of the tube. The bouillon
gives off a penetrating odor, like that of burnt milk. The same odor is given
off from cultures in milk, which is peptonized by the action of the bacillus.
At the end of twenty days, at 37° C. , the entire contents of the tube have be-
come transparent.

Pathogenesis. — Produces a fatal infectious disease in fish ("gold carp")
when inoculated beneath the skin ; also pathogenic for mice and for guinea-
pigs.

BACILLUS PYOGENES FILIFORMIS (Flexner).

Obtained by Flexner (1895) from the interior of the uterus and from an
exudate in the pericardial and pleural cavities, of a rabbit which died on the
fifth day after parturition.

Morphology. — Pleomorphous cocci-like forms, short or long bacilli, and
long threads are seen in cover slips prepared from the exudate. "Very few
of the bacilli stain regularly ; for the most part brightly stained spots appear
between stained areas. An outer membrane always stains, enclosing the

568 PATHOGENIC AEROBIC BACILLI

stained dots in a colorless ground. The threads, as a rule, present delicate,
sinuous, and wavy outlines ; the short forms are straight with rounded ends."

Biological Characters. — All attempts to cultivate this bacillus in the
usual media, either in the presence of oxygen or in an atmosphere of hydro-
gen, proved unsuccessful. But successive cultures were made by inoculations
in the pleural cavity of rabbits — a bit of pleural exudate suspended in bouil-
lon was used for this purpose. The bacillus was also propagated upon the
lungs, heart, uterus, and kidney of healthy rabbits. The organs were re-
moved with great care to prevent contamination and placed in sterilized test
tubes. Transplantations from these cultures were only successful for one or
two generations. Better results were obtained ty cultivating the bacillus
upon the one-third to one-half grown foetuses of rabbits.

Pathogenesis. — "Considerable variations were observed according as the
inoculations were made into the pleural cavity, the peritoneal cavity, the sub-
cutaneous tissue, beneath the dura mater, or directly into the circulation.
The inoculations gave positive results in all cases except a few, in which they
were made subcutaneously. The death of the animal occurred soonest when
inoculation was made beneath the dura mater. A small portion of the skull
was trephined, care being taken to exclude extraneous microorganisms, and
a drop of the pleural fluid or a speck of the fibrinous exudate was introduced
beneath the membranes, care being taken not to injure the brain. These
animals, which quickly recovered from the effects of the operation, died on
an average about twelve hours after the inoculation. . . .

" The pleural inoculations were followed by death, as before stated, in ev-
ery instance, the death of the animal occurring upon the third or fourth day.
The appearances presented at the autopsy were for the most part an exact
reproduction of those observed in the animal which had succumbed to the
natural disease. Upon the side of inoculation a thick, grayish-yellow, shaggy
membrane covered the pleural surfaces, being at times four or five millime-
tres in thickness. The pleural cavity contained several cubic centimetres of
a clear haemoglobin-colored fluid, the lung for the most part being com-
pressed. At times smaller or larger areas of lobular pneumonia would be
present ; and, as a rule, the inflammation was not limited to the serous mem-
brane of the side of inoculation, but extended into the opposite pleural cavity
and into the pericardial sac. However, in these situations the process was,
as a rule, less intense, the solid exudate being less considerable, and in the
case of the opposite pleural cavity sometimes entirely wanting. The super-
ficial vessels, however, were injected and the serous surface of the affected
membrana covered with a slimy, clear fluid. In addition to this the oppo-
site pleural cavity always contained a similar pink serum to that described
upon the side of inoculation.

' ' The study of the exudate upon the side of inoculation as well as the
fluid contained in the opposite pleural cavity and in the pericardium showed
the same organisms as had been introduced."

BACILLUS DYSENTERIC.

The researches of Shiga, of Flexner, and of the board of medical
officers of the army engaged in the study of tropical diseases in the
Philippine Islands (1890) give support to the belief that there is a
form of acute dysentery which is due to infection by the bacillus of
Shiga, which Flexner describes as follows : '

"Bacillus of the average size of B. coli communis. There is variation in
length : almost none in thickness. The individuals are usually separate ;

Johns Hopkins Hospital Bulletin, vol. xi., No. 115.

NOT DESCRIBED IX PREVIOUS SECTIONS. 569

sometimes they are united in pairs, but only very rarely do they occur as
filaments. The ends are slightly rounded. The bacillus shows moderate
motility : Gram's stain is negative. .

" Growth takes place upon all culture media at the room temperature,
but better in the thermostat. Gelatin is not liquefied. The colonies resemble
those of B. typhosus, being more nearly like them when first isolated from
the dejecta than after a period of cultivation outside the body. After many
months of such saprophytic growth the colonies become thicker, exhibit a
moist surface, and are less translucent. The strokes upon agar slants show a
similar alteration. At first the growth extends but little laterally, but later
on it becomes two to three millimetres in width, and generally shows distinct
indentations at the edges. Upon gelatin the colonies are more delicate ; the
stab extends along the line of puncture only, spreading very little at the sur-
face of the medium.

"On potato, growth takes place along the line of inoculation and spreads
beyond. After some days it is a little elevated and of a pale-brown tint. On
unfavorable potatoes the growth is slight, moist, and membranous, resem-
bling, except for the greater amount of moisture, that of B. typhosus when
typical.

" Sugars — glucose, lactose, and saccharose — are not fermented gaseously.
In glucose media a moderate acid production takes place.

" Bouillon is clouded diffusely and a sediment forms. There is no pro-
duction of a pellicle.

" Litmus milk assumes, after twenty-four to seventy-two hours, a faint
lilac tinge. After the lapse of from six to eight days alkali begins to be pro-
duced, which increases in amount until the litmus is rendered deep blue in
color. No coagulation of the milk ensues.

"Indol is not always formed. Even in sugar-free bouillon it may fail to
appear, or it may be produced in small quantities only.

"Suitable cultures of this organism, when tested for the agglutination re-
action with the blood serum of persons suffering from dysentery — the host of
another individual — give, in many cases, a positive result.

"The bacillus is pathogenic for the ordinary laboratory animals. It is
abundant in the acute cases in which it may be the predominating organism ;
it becomes more difficult to find as the cases progress toward recovery or
chronicity. In the ordinary chronic dysentery of Manila, in which amoebae
are commonly encountered, it was not found. It can be cultivated from the
dejecta during life, and the intestinal contents, mucous membrane, and
mesenteric glands in fatal cases.

"Since the publication of Shiga's studies, Escherich and Celli have both
attempted to show that the organisms obtained from their respective epidemics
of dysentery are identical with the B. dysenteriae. In both cases they have
proceeded upon the false assumption that Shiga's microorganism was a
variety of B. coli communis, whereas, in point of fact, it is much more nearly
related in its cultural and physiological properties to B. typhosus.

"The question naturally arises, In what ways does it differ from B.
typhosus? Comparison of the Eberth-Gaffky and Shiga bacilli show the
criteria of difference to be by no means numerous. The main features, how-
ever, are as follows : The latter shows less marked motility when first iso-
lated and a tendency to lose motility rapidly in artificial cultivations ; it
displays a more uniform generation of indol ; after a brief preliminary acid
production in milk it gives rise to a gradually increasing alkalinization ; it is
inactive to blood serum from typhoid cases ; but reacts with serum from
dysenteric cases to which B. typhosus does not respond. . . .

' ' Bearing directly upon these considerations are the results of Lieutenant
Strong's studies, continued after our departure from Manila. He writes :
' After you left we had a large number of acute cases of dysentery. It seems
certain that this form, which we have begun to speak of as acute infectious
dysentery, is independent of amcebae. I have now records of fourteen cases

570

(not all were fatal) which I studied bacteriologically. From the stools in all
of these, there has been obtained a bacillus which agrees with the organism
obtained by you. I have also obtained the organisms from the mesenteric
glands in three fatal cases. In one case of acute dysentery with secondary
acute fibrinous peritonitis I obtained it from the exudate. The agglutina-
tion reaction is not invariable. Amoebae were never demonstrable in any of
these fourteen cases. On the other hand, in every case with certain anatomi-
cal lesions we always find the amoebae. In some cases of dysentery in which
the amoebae were absent and the bacilli present, that have lasted four to five
weeks (one case lasted nearly two months) and then resulted fatally, we see
a continuation of the same process that is observed in the acute fatal cases.
The lesions are those of necroses of the mucous membrane and induration of
the gut.' "

XV.

BACTERIA OF PLANT DISEASES.

I SHALL not attempt to give a full account of the bacteria which
have been described as bearing an etiological relation to various in-
fectious diseases of plants, but a " text- book of bacteriology " would
be incomplete without some reference to the best known of these
bacteria. In the following descriptions of species I have preferred
to quote largely from the published papers of Dr. Erwin F. Smith,
of the Department of Agriculture, United States, a recognized au-
thority in the investigation of plant diseases, rather than to rewrite
his descriptions.

BACILLUS SOLANACEARUM (Smith).

Causes a bacterial disease of the tomato, egg plant, and Irish
potato.

"Morphology. — A medium-sized bacillus, with rounded ends; often in
pairs, with a plain constriction ; elliptical, but of variable size, depending- on
age of culture or the length of time the tissues of the plant have been occu-
pied ; usually one and one-half to three times as long as broad. On cover-
glass preparations made from peptone beef bouillon cultures forty-eight
hours old and stained with a watery solution of methyl violet, many are 1.5
by 0.5 //, but these measurements must not be taken too literally, since the size
depends not only on the age of the culture but also on the kind of stain em-
ployed, i.e., on whether or not the cell wall stains. Organism motile, often
only sluggishly so, especially when taken from the plant, but sometimes very
actively motile, especially in young cultures. Flagella much longer than
the rod ; several — exact number and place of attachment not made out
clearly, owing to imperfect preparations (Van Ermengem's method), but ap-
parently arising from any part of the rod. An attempt to stain them by
L6 filer's method was unsuccessful. No spores observed either in the plant
or in culture media, but the search has not been continued long enough to
warrant any opinion as to their existence. Zooglcea are formed almost from
the start in fluid culture media.

"Symptoms Produced in the Plant. — The first indication of this disease,
or at least the first one to attract the farmer's attention, is the sudden wilting
of the foliage. This may occur first on a single shoot, but finally it affects
the whole plant. Subsequently, and especially if the plant is young or not
very woody, the stem shrivels, first changing to a yellowish-green or to a
muddy green, and finally to brown or black. The vascular bundles become
brown long before the shrivelling takes place, and in the potato often show
through the outer green parts of the stem as long, dark streaks, or the bac-
teria run out on the petioles, after the manner of pear blight, forming nar-
row, black lines. The vessels of such bundles are filled with the bacilli,

572 BACTERIA OF PLANT DISEASES.

which ooze out when the stein is cut across. The foliage may wilt with or
without a preliminary yellowing1. If the bacteria are abundant in the ves-
sels of the stem, the wilt is often very sudden and the foliage has no time to
become yellow. The progress of the disease seems to be more rapid in young
than in old plants and in hot than in cold weather.

"In the case of the potato the tubers are also finally attacked and de-
stroyed, the organism reaching them by way of the vascular bundles of the
stem. A brown or black rot ensues, beginning in the stem end of the tuber
in the vascular ring and extending in all directions therefrom. All stages
of this rot of the tubers (both in 1895 and in 1896) were obtained repeatedly
from pure cultures of the bacillus pricked into the stem several feet above
ground.

" Bouillon and Peptone Cultures. — This organism grows well at room
temperatures of 20° to 30° C., in beef broth peptoiiized (Witte's peptonum
siccum). It seemed to make little difference whether the bouillon was left
acid or rendered slightly alkaline with carbonate of soda. The gathering of
the zooglcea in the upper layers of the fluid is very distinct, especially if the
tubes are left undisturbed in an upright position for some days. On shaking
the turbidity becomes uniform. The organism produces a copious, dirty
white precipitate (much more precipitate than B. tracheiphilus).

"The inoculated tubes of litmus milk developed no acid — i.e., showed
no trace of reddening. After two or three days the litmus became perceptibly
bluer than in the control tubes, and this bluing increased from day to day,
indicating a progressing alkalinity. This change took place at room tem-
peratures of 20° to 30 C., and also in the thermostat at 37° C. The casein
was not precipitated.

" Gelatin. — In plate cultures of nutrient gelatin the buried colonies are
circular in outline (globose), yellowish or brownish, granular (under Zeiss
sixteen millimetres objective and 12 compensating ocular), and with well-
defined margins. No oblong or spindle-shaped colonies could be found.
The circular outline and regularity and distinctness of the margin of the
colony were especially noteworthy. Whether these features will be found
constant with all gelatins is a question yet to be determined. Occasionally,
after a few days, a narrow, clear zone appeared around the margin of many of
these colonies as if liquefaction had set in. This, however, did not progress,
or increased but very slowly, and was clearly visible only under the compound
microscope. The buried colonies remained small, as if requiring more
oxygen than they were able to get. The surface colonies were circular, thin,
thin -edged, smooth, white, and wet-shining. They did not spread over the
plate rapidly or cause any liquefaction (fifteen per cent gelatin, temperature
20° to 27° C.).

"The organism grew best in a gelatin of the following composition:
Lean minced beef, five hundred cubic centimetres ; distilled water, one thou-
sand cubic centimetres ; mixed and set twenty-four hours in a cool place ;
filtered and added ten grammes of Witte's peptonum siccum and one hundred
and fifty grammes of L. and F. gelatin. This gelatin was clarified with egg
and rendered alkaline with sodium hydrate, titrating with phenolphthalein.
The degree of alkalinity was between twelve and fourteen of Mr. Fuller's
scale.

"Agar. — In poured plates of nutrient agar the buried colonies differed
considerably from those in gelatin. Instead of being circular with a very
smooth margin, they were irregularly round or even oblong, with a decidedly
irregular granular margin. These colonies were brown or yellowish-brown
under sixteen millimetres objective and 12 ocular. After some weeks the
whole body of the agar became decidedly brown. No spindle-shaped colonies
were to be seen The surface colonies grew rather slowly. They were dirty-
white, smooth, wet-shining, and did not spread widely over the agar.

' * The behavior on potato is very characteristic. In twenty-four to forty-
eight hours (temperature 27° to 32° C.) the fluid became turbid and the pro-

BACTERIA OF PLANT DISEASES. 573

jecting part of the cylinder was covered with a copious, wet-shining growth.
At first this growth was white or dirty white, but after some days (three to
ten) it became brown, and finally, in places, nearly or quite black (smoke
brown is perhaps the proper term). The growth on potato was not wrinkled.
The substratum and the fluid in the bottom of the tube also became brown.
The rapidity and the degree of pigmentation seem to depend on the slightly
varying composition of the potato, apparently on the amount of glucose
present. No gas was formed in any of the many potato cultures. No acid
was detected in any stage of the growth of the cultures, not even when tested
at the end of the first twenty-four hours. The potato cultures, which were
slightly acid on the start (normal acidity of the tuber), soon became strongly
alkaline to litmus paper. With Nessler's solution the alkaline potato cultures
gave an immediate,* copious, orange-yellow reaction, indicating ammonia.
These cultures developed a peculiar odor, often noticed in rotting potatoes,
but not specially disagreeable. This odor was likened by one person to the
smell of sour bran. Its chemical nature has not been determined. The
cylinders did not fall into pieces, but retained their shape for several
weeks.

44 Gas Production. — No gas appeared in any of the many cultures. The
organism is not a gas producer.

"Relation to Oxygen. — This bacillus appears to be strictly aerobic. If
ever facultative anaerobic, it is not so with any of the carbohydrates yet
tested.

"Acids — No acid reaction could be detected in any stage of any of the
cultures. Potato cultures only twenty-four hours old and which were acid
on the start (normal acidity of the tuber) gave a decided alkaline reaction to
litmus paper. If any acid whatever is formed it is masked by the presence
of alkali and is not butyric acid.

"Alkalies. — This organism is a very vigorous alkali producer. On
warming the cultures over a gas flame or on placing the blued strips of
litmus paper 011 a warm glass plate the alkaline reaction quickly disappears.
On adding a few drops of Nessler's reagent, as already stated, a copious
orange-yellow precipitate is at once developed. This would indicate that at
least a part of the alkali is clue to ammonia. Probably amiiie bases are also
present.

kk The bacillus grows well in the thermostat at 37° C. — possibly a trifle
better than outside at summer temperatures ranging from 25° to 32° C.
Under either condition it grows rapidly. It still grew readily from bouillon
cultures after several weeks' exposure to 37 D C. (three weeks' exposure in
one case, seven weeks' exposure in another).

"Pigments. — A brown pigment is formed in course of a few days in the
host plants (potato, tomato, etc.), and in culture media containing grape,
fruit, or cane sugar (nutrient agar, steamed potato, fermentation tubes).
This pigment is soluble in water and glycerin. It is insoluble in ethyl alco-
hol, ether, chloroform, xylol, and carbon bisulphide."

"BACILLUS HYACINTHI (Wakker).

" Pseudomonas hyacinth! (Wakker). — A yellow, rod-shaped organism,
multiplying by fission ; ends rounded ; single, in pairs, or fours, more rarely
in the form of chains or filaments ; motile by means of one polar flagellum.
In the host plant, when the bundles are crowded full of the yellow slime and
broken down, it is generally 0.8 to 1.2 by 0.4 to 0.6 /<. In alkaline beef
broth or on agar it usually "measures 1 to 2 by 0.4 to 0.6 /^. In old cultures
rich in sugar it often grows out into long, slender chains, or into filaments
(50 to 100 p long) in which there are no distinct septa. Non-sporiferous. Color
distinctly yellow, but somewhat variable. Chrome yellow to pale cadmium
in the host plant, i.e., bright yellow (Ridgway's nomenclature of colors).
On culture media, when not interfered with by the brown pigment, generally

574 BACTERIA OF PLANT DISEASES.

gamboge, chrome yellow, or canary yellow, but sometimes paler. Old
cultures on some media darken from the production of a soluble, pale-brown
pigment. This feeble brown stain is best developed in hyacinth broth, in
potato broth with peptone, on turnips, on radishes, and on banana rinds. It
was not observed in acid or alkaline beef broth, on cocoanut flesh, on sugar
beets, in nutrient starch jelly, in agar, or in gelatin., with or without sugar.
This organism grows readily on potato cylinders standing in distilled water,
but it never becomes copious or fills the water with a solid yellow slime,
owing to its feeble diastatic action. Potatoes on which it has grown, even for
several months, always give a strong starch reaction with iodine. It behaves
the same on nutrient sta.rch jelly free from assimilable sugars. It liquefies
nutrient gelatin and Loffler's blood serum, but does so slowly, and will not
liquefy gelatin at all if ten per cent cane sugar is added. Growth on
nutrient agar or nutrient starch jelly is inhibited (unless the inoculation be
from a solid culture and very copious) by the addition of ten per cent
glycerol, and is greatly retarded by five-per-cent glycerol; even two and a
half per cent of glycerol retarded growth. Growth in beef broth was much
retarded by the addition of 1.5-per-cent sodium chloride. Organisms ex-
tremely sensitive to plant acids, including those of the hyacinth. Aerobic ;
doubtfully, if ever, facultative anaerobic; not a gas producer. Does not
redden litmus milk, but makes it bluer, and slowly separates the casein from
the whey by means of a lab ferment. Produces under some circumstances,
and slowly, a small amount of non-volatile acid (slime acid ?) with various
sugars (grape, cane, etc.), which acid is frequently obscured by the moderate
production of alkali. In the presence of air produces an organic acid
(probably acetic) from ethyl alcohol dissolved in milk or bouillon. Inverts
cane sugar, but apparently without the intervention of any enzyme. Will
not grow on thirty-per-cent grape-sugar agar. Resists dry air very well, i.e.,
more than forty -eight days when spread on cover glasses in thin layers.

" In Dunham's solution with methylene blue the color is reduced in a
few days, but re-oxidizes quickly on shaking ; final color (fifty- six days) bright
blue. In Dunham's solution with indigo carmine the color changes to a
bright blue, which persists for a long time ; final color yellowish. In Dun-
ham's solution with rosolic acid and enough HC1 to render the fluid yellow-
ish, Ps. hyacinthi did not redden the fluid, but made it colorless, the bac-
terial precipitate becoming rosy or salmon-colored. Produces indol slowly
in peptonized beef broth and in peptonized Uschinsky's solution ; does not
produce nitrites in these solutions. Does not reduce potassium nitrate to
nitrite in peptonized beef bouillon. Not a strong-smelling germ. Not
readily destroyed by its own decomposition products except in media contain-
ing alcohol.

' ' Will not grow in the thermostat at 37° C. , and grows very feebly 011
some media and not at all on others at 34° to 35 C. Optimum temperature
28° to 30° C., or thereabouts. Minimum temperature approximately 4° C.
Thermal death point (ten minutes' exposure), 47.50 C. ; nearly all the rods
are killed at 47° and a great many at 46.50° C. Did not grow at room tem-
perature after six days' exposure in alkaline beef broth in the thermostat at
35° to 36.35°. Does not grow well in Uschinsky's solution. Grows much
better in Uschinsky's solution when peptone is added to it. Grows well
with a bright yellow color on cylinders of steamed cocoanut flesh, standing
with one end in distilled water.

"Pathogenic to hyacinths. Enters the plant through wounds, through
the blossoms, etc., and multiplies in the vascular system, filling the vessels,
especially those of the bulb, with a bright yellow slime consisting of bacteria.
The walls of the vessels are destroyed and extensive cavities are formed in
the bundles. The parenchyma around the bundles is also involved, but only
very slowly, the organism being a feeble destroyer of cell walls. The host
plant is not rapidly destroyed, a year or more being necessary. The cells
are first separated by solution of the middle lamella, but the wall itself seems

BACTERIA OF PLANT DISEASES. 575

finally to disappear. The cavities contain innumerable bacteria mingled
with fragments of the dissolved bundles and of the surrounding parenchyma.
"First described by Dr. J. H. Wakker from the Netherlands, where it
often causes serious losses in the hyacinth gardens. Not known to occur in
any other part of the world " (E. F. Smith).

BACILLUS CAMPESTRIS (Pammel) .

The cause of brown rot in Cruciferous plants.

" Pseudomonas campestris (Pammel). — Yellow, rod-shaped, motile
micro-organism. Size and color varying according to substratum, food
supply, etc. Generally 0.7 to 3.0 by 0.4 to 0.5 ,«. Coior dull wax yellow or
canary yellow. Occasionally as bright as light cadmium or as pale as prim-
rose yellow (Ridgway's color scale). One polar flagellum. Non-sporiferous,
so far as known. Pathogenic for various Cruciferous plants, entering and
dwarfing or destroying the host plant through the vascular system, which
becomes decidedly brown. Aerobic but, so far as known, not a gas or acid
producer, i.e., not facultative anaerobic. Forms cavities around the bundles
but seems to be only feebly destructive to cellulose. Produces a brown pig-
ment in the host plant and on steamed Cruciferous substrata, especially the
turnip. Grows very rapidly on steamed potato cylinders at room tempera-
tures, but without odor or the formation of any brown pigment. Liquefies
gelatin. Grows feebly at 7° C., better at 10a C., but still feebly ; grows well
at 17° to 19° C. ; grows luxuriantly at 21C to 26' C. ; grows very feebly at 37D
to 38° C. ; will not grow at 40° C. ; and is killed by ten minutes' exposure to
51° C. Organism closely related to Wakker's Bacterium hyacinthi, from
which it differs, so far as I have been able to observe, chiefly in its patho-
genic properties, its duller yellow color and its higher thermal death point'*
(E. F. Smith).

PSEUDOMONAS STEWARTI (Smith).

"A medium-sized rod rounded at the end and motile by means of one
polar flagellum, size 0.5 to 0.9 by 1 to 2 //, no spores observed ; found in enor-
mous numbers in the vascular bundles of corn (Zea mays) associated with a
destructive disease of which it is probably the cause ; color in the host plant
and in culture media yellow (buff to chrome or ochre, occasionally a pale,
dirty yellow) ; aerobic and facultative anaerobic ; grows in all ordinary cul-
ture media ; bears alkali well (soda) and plant acids extremely well ; grows
luxuriantly in Uschinsky's solution ; growth enormously stimulated by cane
sugar, grape sugar, and galactose ; growth not favored by five-per-cent doses
of lactose, maltose, dextrin, mannite, or glycerin in nutrient starch jelly ;
diastatic action feeble, i.e., able to obtain food from starch only with much
difficulty ; produces alkalies in all sorts of media and acids in the "presence of
grape and cane sugar; reduces litmus slowly; does not liquefy gelatin
(Stewart) ; does not liquefy Loftier1 s blood serum ; grows well at summer
temperatures of 25° to 30 C. ; does not die out quickly in culture media ; does
not produce gas; sensitive to light (Stewart); occurs in New York and
Michigan and maybe looked for in all parts of the United States" (E. F.
Smith).

BACILLUS AMYLOVORUS (Burrill).

Described by Burrill (1880) as the cause of pear blight. Etiologi-
cal relation to this disease confirmed by Arthur (1884 to 1887) and
by Waite (1891 to 1895).

' ' Beginning in the spring the germs on the new growth of the season first
appear on the negative discs of the blossoms. The bacilli live and multiply
in the nectar and are able to enter the nectar glands without a puncture or

576 BACTERIA OF PLANT DISEASES.

injury, and thus normally get inside their hosts. The distribution from
flower to flower and tree to tree is through the agency of insects, mainly
flower- visiting source. Infection also occurs on the young shoots and less
frequently on the fleshy bark through injuries. Insects and birds are agents
of distribution and inoculation in these cases. No evidence could be found
that the germs are carried by the wind. The blight germs usually die out in
the twigs which are blighted and dead, but in certain cases the germs manage
to keep alive through the summer by making slow progress in the fleshy
bark. Such cases may succeed in living over winter. Winter weather is
favorable to the longevity on account of the moisture and low temperature.
The cases of "hold over" blights start oft' again in spring and exude quanti-
ties of gummy matter full of the bacilli. This is visited by insects, especially
flies and wasps, and carried on to the newly opened flowers, thus completing
the life cycle.

' ' An oval rod-like bacillus 0.6-0.8 n by 1 to 6 // long. Constant in diameter
but varying greatly in length. Occurs singly or in young cultures in pairs,
chains, or masses. Stains readily with the ordinary aniline dies either watery
or alcoholic solutions. Has 110 capsule, but is supplied with several flagella
scattered over the surface. It is actively motile. Does not produce spores.
On nutrient beef and potato broth produces first a strong turbidity and a
slight granular pellicle on the surface, which breaks up and settles to the
bottom. The color of the mass is milky white on all solid media.

"On agar plates the outside colonies at ordinary temperature (18° to 20° C.)
reach a diameter of about one millimetre in forty-eight hours, and at the end
of a week become five to six millimetres across. A temperature of 36° to
37° C. starts the growth more promptly, but results in a feebler ultimate de-
velopment.

"The addition of malic or citric acid in small amounts so as to acidify
the agar, increases the vigor of growth, while an excess of alkali diminishes
it. On gelatin made from the commercial brands the opposite effect is pro-
duced. Gelatin should be neutral to phenolphthalein. to insure vigorous
development. There is moderate liquefaction in good gelatin culture. A
moderate growth is made on sterile potato cylinders.

"In the fermentation tube it decomposes sugar without the formation of
gas. It is most vigorous on maltose, the cultures becoming strongly acid,
and is slightly less so on cane sugar, dextrose, and levulose. It is aerobic and
facultative anaerobic. It produces no pigment or coloring matter of any sort,
and no odor. It does not decompose starch. Its principal food consists of
nitrogenous matter, sugars, and probably, to some extent, certain organic
acids, the very substances which occur in vigorous, young, growing tissues
of the host. Certain statements formerly made are now known to be erro-
neous.

"The germ mass is said to be yellowish -white on potato. This could
only come from an impure culture, as the true pear-blight germ is always
white. Gas, in some places COs, is said to be formed. This never oc-
curs. Butyric acid is said to be one of the products of its decomposition.
The germ produces acid but never butyric. Starch is said to be decomposed
and used as a food, but so far we have never been able to demonstrate this.
The germ is said to live over winter in the soil. In our search we have
failed to find it in such places, and its life cycle is complete without it" (Waite).

BACILLUS TRACHEIPHILUS (Smith).

The cause of "wilt " in various species of Cucurbitacece — cucum-
bers and melons.

"Bacilli, often two or three times as long as broad, of medium size; soli-
tary or in pairs, occasionally in chains of four. The dimensions vary greatly
in the infected plant ; many rods are 1.2 to 2.5 // long by 0.5 to 0.7 p broad

BACTERIA OF PLANT DISEASES. 577

In cultures the dimensions vary still more. In recent cultures the bacilli
exhibit active movements, which are soon lost. The bacilli are often asso-
ciated in viscous masses, forming- milk-white drops, which when touched
with a platinum needle may be drawn out into long threads. This viscosity
appears to be due to a swollen and partially liquefied capsule, which may be
demonstrated under the microscope in stained or unstained preparations.
Does not form spores. Grows in bouillon, Dunham's solution, etc. Does
not form a surface film or a deposit at the bottom of the test tube, but the
culture medium is slightly clouded. Grows very slowly or not at all in gela-
tin and does not liquefy. Upon agar-agar it grows as a thin, smooth, milk-
white, sticky layer, which extends only a short distance from the point of
inoculation. In stab cultures it grows all along the line of puncture, form-
ing, after a time, finger-like projections, which under a lens are seen to be
finely granular. Upon potato it forms a thin, smooth, white, moist-looking
layer, which only extends a short distance from the line of inoculation.
The color of the growth resembles that of the potato, and is much whiter
than that of most bacteria. It produces no pigment and causes no change
in the color of the potato. In culture solutions containing dextrose, saccha-
rose, lactose, or maltose no gas is developed. It does not cause coagulation
of milk. It grows best in alkaline media. It is destroyed by a temperature
of 43° C. maintained for ten minutes. Cultures in liquid media or on potato
usually die out within three weeks. It stains best with carbol-fuchsin solu-
tion. In properly stained preparations it is seen to have a capsule and
flagella — in some bacilli one flagellum at each extremity of the rod, while in
others there are more " (Smith).

XYI.
PATHOGENIC ANAEROBIC BACILLI.

STRICTLY anaerobic bacilli are not able to multiply in the blood
of living animals ; but some of them may multiply in the subcuta-
neous connective tissue or in the muscles, when introduced by in-
oculation, and are pathogenic because of the local inflammatory or
necrotic processes to which they give rise, or because they produce
soluble toxic substances which are absorbed and cause death by
their special action upon the nervous system or by general toxaemia.

BACILLUS TETANI.

Synonyms. — The bacillus of tetanus ; Tetanusbacillus, Ger.

Nicolaier (1884) produced tetanus in mice and rabbits by intro-
ducing garden earth beneath their skin, and showed that the disease
might be transmitted to other animals by inoculations with pus or
cultures in blood serum containing the tetanus bacillus, which, how-
ever, he did not succeed in obtaining in pure cultures. Carle and
Rattone (1884) showed that tetanus is an infectious disease, which
may be transmitted by inoculation from man to lower animals — a
fact which has since been verified by the experiments of Rosenbach
and others. Obtained in pure cultures by Kitasato (1889).

The writer produced tetanus in a rabbit in 1880 by injecting be-
neath its skin a little mud from the street gutters in New Orleans.
The tetanus bacillus appears to be a widely distributed microorgan-
ism in the superficial layers of the soil in temperate and especially in
tropical regions. In Nicolaier's experiments it was not found in soil
from forests or in the deeper layers of garden earth.

Morphology. — Slender, straight bacilli, with rounded ends,
which may grow out into long filaments. Spores are developed at
one extremity of the bacilli, which are spherical in form and consid-
erably greater in diameter than the rods themselves, giving the
spore-bearing bacilli the shape of a pin.

Stains with the usual aniline colors and also by Gram's method.
The method of Ziehl may be employed for double-staining bacilli and
spores.

PATHOGENIC ANAEROBIC BACILLI.

579

Biological Characters. — An anaerobic, liquefying, motile
bacillus. Forms spores. Grows at the room temperature, in the
absence of oxygen, in the usual culture media. Grows best at a
temperature of 36° to 38° C.; in nutrient gelatin, at 20° to 25° C.,
development is first seen at the end of three or four days ; does not
grow at a temperature below 14° C. Spores are formed in cultures
kept in the incubating oven at 36° C., at the end of thirty hours ;
in gelatin cultures at 20° to 25° C., at the end of a week (Kitasato).
The bacilli exhibit voluntary movements which are not very active ;
those containing spores are not motile. It may be cultivated in an
atmosphere of hydrogen, but does not grow in the presence of oxy-
gen— strictly anaerobic — or in an atmosphere of carbon dioxide.
The addition of one and one-half to two per cent of grape sugar to
nutrient agar or gelatin causes the development to be more rapid

FIG. 159.

FIG. 160.

FIG. 159.— Tetanus bacillus, from a gelatin culture, x 1,000. From a photomicrograph by
Pfeiffer.

FIG. 160.— Tetanusbacillus, from an agar culture ; spore-beating rods. X 1,000. From a photo-
micrograph by Pfeiffer.

and abundant. The culture medium should have a feebly alkaline-
reaction.

Colonies in gelatin plates, in an atmosphere of hydrogen, re-
semble somewhat colonies of Bacillus subtilis, the opaque central
portion being surrounded by a circle of diverging rays ; liquefaction
is, however, much slower, and the resemblance is lost after a short
time. Older colonies resemble the colonies of certain microscopic
fungi, being made up of diverging rays. In long gelatin stab cul-
tures development occurs along the line of puncture, at a consid-
erable distance below the surface, in the form of a radiate out-
growth ; the gelatin is slowly liquefied, and a small amount of gas is
at the same time formed. In peptonized bouillon having a slightly
alkaline reaction, under hydrogen gas, the development is abundant

580

PATHOGENIC ANAEROBIC BACILLI.

and the cultures give off a characteristic odor — " brenzlichen Ge-
ruch " (Kitasato).

According to Kitasato, blood serum is not a very favorable me-
dium for the growth of the tetanus bacillus, and — contrary to the

statement of Kitt, Tizzoni, and others-
it does not cause liquefaction of this
medium.

The spores of the tetanus bacillus re-
tain their vitality for months in a desic-
cated condition, and are not destroyed in
two and one-half months when present
in putrefying material (Turco). They
withstand a temperature of 80° C. main-
tained for an hour, but are killed by
five minutes' exposure to steam at 100° C.
They are not destroyed in ten hours by
a five-per-cent solution of carbolic acid,
but did not grow after fifteen hours' ex-
posure in the same solution. A five-
per-cent solution of carbolic acid, to
which 0.5 per cent of hydrochloric acid
has been added, destroys them in two
hours ; in sublimate solution containing
1 : 1,000 of mercuric chloride they are
destroyed at the end of three hours, or
in thirty minutes when 0.5 per cent of
hydrochloric acid is added to the solu-
tion. Kitasato succeeded in obtaining
pure cultures from the pus formed in
the vicinity of inoculation wounds, by
destroying the associated bacilli after
the tetanus bacilli had formed spores.

This was effected by heating cultures from this source for about an
hour at a temperature of 80° C. The spores of the tetanus bacillus
survived this exposure, and colonies were obtained from them in flat
flasks especially devised for anaerobic cultures ; from these colonies
pure cultures in nutrient agar or gelatin — long stick cultures — or in
peptonized bouillon were easily obtained.-

BACILLUS CEDEMATIS MALIGNI.

Synonyms.— Bacillus of malignant oedema; Yibrion septique
(Pasteur).

Discovered by Pasteur (1877); carefully studied by Koch (1881).

FIG. 161.— Culture of Bacillus tetani
in nutrient gelatin. (Kitasato.)

PATHOGENIC ANAEROBIC BACILLI.

581

This bacillus is widely distributed, being found in the superficial
layers of the soil, in dust, in putrefying substances, in the blood of
animals which have been suffocated (by invasion from the intestine),
in foul water, etc.

It may usually be obtained by introducing beneath the skin of a
rabbit or a guinea-pig a small quantity of garden earth. The animal
dies within a day or two, and this bacillus is found in the bloody
serum effused in the subcutaneous connective tissue for a consider-
able distance about the point of inoculation.

Morphology. — Bacilli from 3 to 3.5 p long and 1 to 1.1 /^ broad;

FIG. 162.— Bacillus oedematis maligni, from subcutaneous connective tissue of inoculated
guinea-pig. X 950. (Baumgarten.)

frequently united in pairs, or chains of three elements ; may grow
out into long filaments 15 to 40 /* long — these are straight, or bent
at an angle, or more or less curved. They resemble the bacillus of

anthrax, but are not quite as broad, have
rounded ends,, and in stained preparations
the long filaments are not segmented as is
the case with the anthrax bacillus. By
Loffler's method of staining they are seen to
have flagella arranged around the periphery
of the cells. Large, oval spores may be de-
veloped in the bacilli (not in the long fila-
ments), which are of greater diameter than
the rods, and produce a terminal or central
swelling of the same, according to the loca-
tion of the spore.

Stains readily by the aniline colors usu-
ally employed, but is decolorized when treated by Gram's method.

FIG. 163. -Bacillus osdema-
tis maligni, from an agar cul-
ture, showing spores. X 1,000
From a photomicrograph.
(Frankel and Pfeiffer.)

582

PATHOGENIC ANAEROBIC BACILLI.

In stained preparations the long filaments may present a somewhat
granular appearance from unequal action of the staining agent.

Biological Characters. — A strictly anaerobic, liquefying, mo-
tile bacillus. Forms spores. Grows in the usual culture media
when oxygen is excluded — in an atmosphere of hydrogen. Grows
at the room temperature — better in the incubating oven at 37° C.
The spores are formed most abundantly in cultures kept in the in-
cubating oven, but may also be formed at a temperature of 20° C.
In the bodies of animals which succumb to an experimental inocula-
tion no spores are found immediately
after death, but the bacilli multiply rap-
idly in the cadaver, and form spores
when the temperature is favorable.

The malignant- oedema bacillus may
be cultivated in ordinary nutrient gela-
tin, but its development is more abun-
dant when one to two per cent of grape
sugar has been added to the culture
medium. In deep stab cultures in this
medium development occurs at first only
near the bottom of the line of puncture ;
the gelatin is liquefied and has a grayish-
white, clouded appearance ; an abundant
development of gas occurs, and as this
accumulates the growth and liquefaction
of the gelatin extend upward. A very
characteristic appearance is obtained
when the bacilli are mixed in a test
tube with gelatin which has been liquefied by heat, and which is then
allowed to solidify. Spherical colonies are developed, in the course
of two or three days, in the lower portion of the gelatin ; these are
filled with liquefied gelatin of a grayish- white color, and when ex-
amined with a low power are seen to be permeated with a network
of filaments, while the periphery presents a radiate appearance. In
nutrient agar growth also occurs at the bottom of a deep punc-
ture ; it has an irregular, jagged outline and a granular appearance;
the considerable development at the deepest portion and gradual
thinning out above give the growth a club shape ; in the incubating
oven there is an abundant development of gas, which often splits up
the agar medium and forces the upper portion against the cotton
stopper. An abundant development of gas also occurs in cultures
in blood serum, and the medium is rapidly liquefied ; at a tempera-
ture of 37° it is changed in a few days to a yellowish fluid, at the
bottom of which some irregular, corroded fragments of the solidified

FIG. 164.— Bacillus oedematis ma-
ligni, cultures in nutrient gelatin; a,
long stab culture : fr, colonies at bot-
tom of gelatin tube. (Flugge.)

PATHOGENIC ANAEROBIC BACILLI. 583

serum may be seen. In agar plates, placed in a close receptacle
from which oxygen is excluded, cloudy, dull-white colonies are
formed which have irregular outlines and under the microscope
are seen to be made up of branching and interlaced filaments radi-
ating from the centre. Cultures of the malignant-oedema bacillus
give off a peculiar, disagreeable odor, which cannot, however, be
designated as " putrefactive."

Pathogenesis. — Pathogenic for mice, guinea-pigs, rabbits, and,
according to Kitt, for horses, dogs, goats, sheep, calves, pigs, chick-
ens, and pigeons. According to Arloing and to Chauveau, cattle are
immune. The disease is rarely developed except as a result of ex-
perimental inoculations, but horses occasionally have malignant
oedema from accidental inoculation, and cases have been reported
in man — "gangrene gazeuse." A small quantity of a pure cul-
ture injected beneath the skin of a susceptible animal gives rise to
an extensive inflammatory oedema of the subcutaneous connective
tissue and of the superficial muscles, which extends from the point
of inoculation, especially towards the more dependent portions of
the body. The bloody serum effused is without odor and contains
little if any gas. But when malignant oedema results from the in-
troduction of a little garden earth beneath the skin of a guinea-pig or
other susceptible animal, the effused serum is frothy and has a pu-
trefactive odor, no doubt from the presence of associated bacteria.
Injections into the circulation do not give rise to malignant oedema,
unless at the same time some bacilli are thrown into the connective
tissue. While small animals usually die from an experimental in-
oculation with a moderately small quantity of a pure culture, larger
ones (dogs, sheep) frequently recover. At the autopsy, if made at
once, the bacilli are found in great numbers in the effused serum,
but not in blood from the heart or in preparations made from the
parenchyma of the various organs ; later they may be found in all
parts of the body as a result of post-mortem multiplication. This
applies to rabbits and to guinea-pigs, but not to mice ; in these little
animals the bacilli may find their way into the blood during the last
hours of life, and their presence may be demonstrated in smear prepa-
rations of blood from the heart or from the parenchyma of the spleen
or liver. In mice the spleen is considerably enlarged, dark in color,
and softened ; in rabbits and guinea-pigs less so. With this excep-
tion the internal organs present no very notable pathological changes.

Animals which recover from malignant cedema are said to be
subsequently immune (Arloing and Chauveau). Roux and Cham-
berlain have shown that immunity may be induced in guinea-pigs by
injecting filtered cultures of the malignant-oedema bacillus (about
one hundred cubic centimetres of a bouillon culture in three doses)

584

PATHOGENIC ANAEROBIC BACILLI.

into the abdominal cavity ; or, better still, by the injection of fil-
tered serum from animals which have recently succumbed to an ex-
perimental inoculation (one cubic centimetre repeated daily for seven
or eight days).

BACILLUS CADAVERIS.

Obtained by the writer (1889) from pieces of liver and kidney, from yel-
low-fever cadavers, which had been preserved for forty-eight hours in an
antiseptic wrapping, at the summer temperature of Havana; also in two

Fio. 165.— Bacillus cadaveris; smear preparation from liver of yellow-fever cadaver, kept
twenty-four hours in an antiseptic wrapping. X 1,000. From a photomicrograph. (Sternberg.)

cases from pieces of yellow-fever liver immediately after the autopsy ; also
from liver preserved in an antiseptic wrapping from comparative autopsies
made in Baltimore.

Morphology. — Large bacilli with
square or slightly rounded corners,
from 1.5 to 4 ^ in length and about
1.2 // broad; frequently associated in
pairs; may grow out into straight or
slightly curved filaments of from 5
to 15 fj. in length.

Biological Characters. — An an-
aerobic, non-motile bacillus ; not
cultivated in nutrient gelatin ; not
observed to form spores.

Bacillus cadaveris is a strict anae-
robic and is difficult to cultivate. I
have succeeded best with nutrient
agar containing five per cent of
glycerin, removing the oxygen
thoroughly by passing a stream of
hydrogen through the liquefied me-
dium. The colonies in a glycerin-
agar roll tube (containing hydrogen

FIG. 166.— Bacillus cadaveris, from an anae-
robic culture in glycerin-agar. X 1,000. From
a photomicrograph. (Sternberg.)

and hermetically sealed) are opaque,
irregular in outline, granular, and of

PATHOGENIC ANAEROBIC BACILLI.

585

a white color by reflected light. The culture medium acquires an acid re-
action as a result of the development of the bacillus.

Liver tissue containing this bacillus, after having been kept in an anti-
septic wrapping for forty-eight hours, has a fresh appearance, a very acid re-
action, and is without any putrefactive odor.

Pathogenesis. — Liver tissue containing this bacillus is rery pathogenic
for guinea-pigs when injected subcutaneously, and causes an extensive in-
flammatory cedema extending from the point of inoculation. Pure cul-
tures of the bacillus are less pathogenic, and the few experiments which I
made in Havana gave a somewhat contradictory result, recovery having
occurred in one guinea-pig which received a subcutaneous injection of ten
minims of liquid from an anaerobic culture in glycerin-agar, while another
died at the end. of twenty hours from a subcutaneous injection of three
minims, with extensive inflammatory cedema in the vicinity of the point of
inoculation.

BACILLUS OF SYMPTOMATIC ANTHRAX.

Synonyms. — Rauschbrandbacillus, Ger. ; Bacille du charbon
symptomatique, Fr.

First described by Bellinger and Feser (1878); carefully studied
and its principal characters determined by Arloing, Cornevin, and
Thomas (1880-83).

FIG. 167.

FIG. 168.

FIG. 167.— Bacillus of symptomatic anthrax, from an agar culture, x 1,000. From a photomi-
crograph- (Frankel and Pfeiffer.)

FIG. 168.— Bacillus of symptomatic anthrax, from muscles of inoculated guinea-pig. From a
photomicrograph. (Roux.)

Found in the affected tissues of animals — principally cattle — suf-
fering from " black leg," " quarter evil," or symptomatic anthrax (Fr. ?
" charbon symptomatique"; Ger., " Rauschbrand "). The disease

586

PATHOGENIC ANAEROBIC BACILLI.

prevails during the summer months in various parts of Europe, and
is characterized by the appearance of irregular, emphysematous
swellings of the subcutaneous tissue and muscles, especially over the
quarters, hence the name "quarter evil." The muscles in the
affected areas have a dark color and contain a bloody serum in
which the bacillus is found.

Morphology. — Bacilli with rounded ends, from three to five /*
long and 0.5 to 0.6 /* broad ; sometimes united in pairs, but do not
grow out into filaments. The spores are oval, somewhat flattened on
one side, thicker than the bacilli, and lie near the middle of the rods,
but a little nearer to one extremity. The bacilli containing spores
.are somewhat spindle-formed (Kitasato). "Involution forms " are
quite common in old cultures or in unfavorable
media ; in such cultures variously distorted and
often greatly enlarged bacilli may be seen, some
being greatly swollen in the middle — spindle-
shaped. When properly stained, by Loffler's
method, a number of flagella are seen around the
periphery of the cells.

Stains with the aniline colors usually em-
ployed, but not by Gram's method. Spore-bear-
ing bacilli may be double-stained by first stain-
ing the spores by ZiehFs method, and then the
bacilli with a solution of methylene blue.

Biological Characters. — An anaerobic, liq-
uefying, motile bacillus. Forms spores. Grows
at the room temperature in the usual culture media,
in the absence of oxygen, in an atmosphere of hy-
drogen, but not in carbon dioxide. This bacillus
grows more rapidly and abundantly in nutrient
agar or gelatin to which 1.5 to 2 per cent of
grape sugar or five per cent of glycerin has been
added. Colonies in gelatin, in an atmosphere of
hydrogen, are at first spherical, with irregular out-
lines and a wart-like surface ; later the gelatin is
liquefied around them, and radiating filaments
grow out into the gelatin, so that by transmitted
light they present the appearance of an opaque
central mass with an irregular surface surrounded
by rays. In stab cultures in nutrient gelatin, at
20° to 25° C., at the end of two or three days
•development occurs at the bottom of the line of puncture to within
about two fingers' breadth of the surface ; the gelatin is slowly
liquefied and considerable gas is formed. In old cultures the

fto

FIG. 169.— Bacillus
of symptomatic an-
thrax; long stab cul-
ture in nutrient gela-
tin, ten days at 18°-
20° C. (Kitasato.)

PATHOGENIC ANAEROBIC BACILLI. 587

growth and liquefaction of the gelatin extend nearly to the sur-
face. In agar stab cultures, in the incubating oven, develop-
ment begins within a day or two and extends to within one
finger's breadth of the surface ; considerable gas is evolved, and
the cultures have a peculiar, acid, penetrating odor. Development
is most rapid at 36° to 38° C., but may occur at a temperature of 16°
to 18° C. — not lower than 14°. Spores are quickly formed in cul-
tures kept in the incubating oven — not so quickly at the room tem-
perature. These withstand a temperature of 80° C. maintained for
an hour, but are killed in five minutes by a temperature of 100° C.
(in steam). In the bodies of infected animals spores are not formed
until after the death of the animal, at the end of twenty-four to forty-
eight hours (Kitasato).

The spores are destroyed by a five-per-cent solution of carbolic
acid in ten hours, and the bacilli, in the absence of spores, in five
minutes ; a 1 : 1,000 solution of mercuric chloride destroys the spores
in two hours (Kitasato). According to Kitasato, certain shining
bodies of irregular form, which stain readily with the aniline colors,
are to be seen in the rods as they are found in the bloody serum from
an animal recently dead ; but these are not spores, as some bacterio-
logists have supposed.

Pathogenesis. — Cattle, which are immune against malignant
oedema, are most subject to infection by the bacillus of symptomatic
anthrax, and the disease produced by this anaerobic bacillus prevails
almost entirely among them ; horses are not attacked spontaneously
— i. e. , by accidental infection — and when inoculated with a culture of
this bacillus present only a limited local reaction. Swine, dogs, rab-
bits, fowls, and pigeons have but slight susceptibility, but the re-
searches of Aiioing, Cornevin, and Thomas, and of Roger show that
by the addition of a twenty-per-cent solution of lactic acid to a cul-
ture its virulence is greatly increased, and animals which have but
little susceptibility, like the rabbit or the mouse, succumb to such in-
jections ; similar results were obtained by Roger by the simultaneous
injection of sterilized or non-sterilized cultures of Bacillus prodigiosus
or of Proteus vulgaris.

Klein (1894) has obtained from the spleen of sheep a bacillus
which corresponds with the bacillus of malignant oedema in every
respect, except that it proved to be without pathogenic power — "a
non-virulent variety of the Rauschbrand bacillus" (Klein).

BACILLUS CEDEMATIS MALIGNI NO. II (Novy).

Obtained by Novy (1894) from the subcutaneous oedema in guinea-pigs
which were inoculated with a solution of milk-nuclein, which had been pre-
pared from fresh casein.

Morphology. — Bacilli with rounded ends, usually solitary, from 2.5 to

588 PATHOGENIC ANAEROBIC BACILLI.

5 // long and from 0.8 to 0.9 // broad. Occasionally short and straight fila-
ments, 8 to 14 ," long, are seen — very rarely these reach a length of 22 to 35,".
Long and slender spiral filaments are found in pure cultures which are be-
lieved to be gigantic flagella. These are seen in preparations stained with
gentian violet as unstained spiral filaments, usually from 17 to 25 j* long ;
some are of uniform thickness and others spindle-formed, having a thickness
of 1.7 to 2.6 p in the middle, and tapering to a scarcely visible line at the ex-
tremities. These flagella are readily stained by Loffler's method. They are
attached to the periphery of the rods, as in the typhoid bacillus. In artifi-
cial cultures they are usually from 40 to 50 p long. With reference to the
peculiar spindle-formed bodies found in the cultures Novy says : " As to the
character of these gigantic flagella little can be said. Loftier, who, so far as
I know, was the first to observe these singular forms, regarded them as bun-
dles or collections of flagella."

Although at first inclined to doubt this, Novy says, in a postscript to his
paper, that an examination of photo-micrographs, which had been made to
accompany it, convinces him that Loffler's explanation is probably correct.

Biological Characters. — An anaerobic, motile bacillus. The motions are
not active, but consist in a very moderate to-and-fro swinging motion. Does
not form spores. Does not grow at the room temperature. Grows at tem-
peratures of 24° to 38° C. The best media for its development are slightly
alkaline bouillon, gelatin, or agar, containing two per cent of glucose. May
be cultivated in a vacuum or in an atmosphere of hydrogen, carbon dioxide,
or illuminating gas. Also in long stick cultures in agar. In glucose-agar
plates colonies develop in fifteen hours at 38° C. These appear as small,
white masses the size of a pin's head, which, under the microscope, appear
to be made up of thickly felted threads. The smaller colonies appear as a
network of branching lires, very similar to the colonies of the tetanus ba-
cillus ; larger colonies have a dark centre, with an irregular, fringed margin,
and are surrounded by delicate filaments. In glucose-agar stab cultures
growth occurs along the line of puncture to within one cubic centimetre of
the surface, but is not as abundant as the growth of the bacillus of malig-
nant oedema or of symptomatic anthrax. At 38° C. development occurs within
twelve to sixteen hours, and has reached its maximum at the end of twenty-
four hours. An abundant development of gas occurs, which splits up the
agar and forces the upper portion towards the top of the tube. The develop-
ment of gas is most abundant in alkaline media, being almost absent in media
having a neutral or acid reaction. The most favorable medium is a fresh al-
kaline bouillon containing two per cent of gelatin, of glucose, and of pep-
tone.

Pathogenesis. — Pathogenic for rabbits, guinea-pigs, white mice, white rats,
pigeons, and cats. Death usually results in from twelve to thirty-six hours
after the subcutaneous injection of one-tenth to one-fourth cubic centimetre
of a pure culture. At the autopsy an extensive subcutaneous oedema is found
extending from the point of inoculation. The fluid in the brawny connective
tissue is usually colorless, sometimes of a pale-red color. A small amount of
gas is commonly present. The pleural cavities contain an enormous amount
of serous exudate, which at first is fluid, but when the autopsy is delayed be-
comes gelatinous. In rabbits and guinea-pigs the amount of this serum ob-
tained from the pleural cavities may be from fifty to sixty cubic centimetres.
The bacilli are usually not very numerous in this serum from the subcuta-
neous tissues and pleural cavity.

Kerry (1894) has described a " new pathogenic anaerobic bacillus " which
resembles that of Novy in several particulars. It does not grow at the room
temperature, does not form spores, and is pathogenic for mice, rats, rabbits,
and guinea-pigs ; it forms ' ' very long and thick flagella, which may be spir-
alig gechlangelt." This bacillus was obtained from a guinea-pig inoculated
with dried blood (suspended in water containing lactic acid and glucose)
which had been obtained from a cow that was supposed to have died of
Rauschbrand.

PATHOGENIC ANAEROBIC BACILLI. 589

BACILLUS AEROGENES CAPSULATUS.

Found by Welch in the blood vessels of a patient with thoracic aneurism,
opening- externally ; autopsy made in cool weather eight hours after death —
the vessels found full of gas bubbles.

Morphology. — Straight or slightly curved bacilli with slightly rounded
or sometimes square-cut ends ; a little thicker than Bacillus aiithracis, and
varying in length — average length 3 to 6 // ; long threads and chains are oc-
casionally seen. The bacilli, both from cultures and in the animal body, are
enclosed in a transparent capsule.

Biological Cliaracters. — An anaerobic, non-motile, non-liquefying ba-
cillus. Does noc form spores. Grows in the usual culture media, in the ab-
sence of oxygen, at the room temperature, and produces an abundant de-
velopment of gas in all. In nutrient gelatin there is no marked liquefaction,
but the gelatin is slightly peptonized. In agar, colonies are developed which
are usually one to two millimetres in diameter, but may attain a diameter of
one centimetre ; they are grayish- white in color and in the form of flattened
spheres, ovals, or irregular masses, beset with little projections or hair-like
processes. Bouillon is rendered diffusely cloudy, with an abundant white
sediment. Milk is coagulated in one or two days. The cultures in agar and
bouillon have a faint odor, comparable to that of stale glue. Upon potato a
pale grayish-white layer is developed; growth occurs at 18° to 20° C., but is
much more rapid at 30° to 37° C. Bouillon cultures are sterilized by ex-
posure to a temperature of 58° C. for ten minutes.

Pathogenesis. — "Quantities up to 2.5 cubic centimetres of fresh bouillon
cultures were injected into the circulation of rabbits without any apparent
effect, except in one instance in which a pregnant rabbit was killed, by the
injection of one cubic centimetre, in twenty-one hours. If the animal is
killed shortly after the injection the bacilli develop rapidly after death, with
an abundant formation of gas in the blood vessels and organs, especially the
liver. At temperatures of 18° to 20° C. the vessels, organs, and serous cavi-
ties may be full of gas in eighteen to twenty-four hours, and at tempera-
tures of 30° to 32° C. in four to six hours, when one cubic centimetre of a
bouillon culture has been injected into the circulation shortly before death."

It is suggested by Welch and Nuttall that in some of the cases in
which death has been attributed to the entrance of air into the veins, the gas
found at the autopsy may not have been atmospheric air, but may have been
produced by this or some similar microorganism entering the circulation and
developing after death.

In a paper published in the Bulletin of the Johns Hopkins Hos-
pital (September, 1900) Professor Welch says: " Our further studies
of the gas bacillus obtained from different sources have shown a
moderate range of variation in some of its properties. This is true
especially of spore formation, rapidity of liquefaction of gelatin,
presence of capsules, and virulence."

This bacillus has been shown by recent researches to be widely
distributed in nature, its natural habitat being the intestinal canal of
man and lower animals and the soil. It has considerable importance
in human pathology, having been found in various localized infec-
tious processes in the subcutaneous tissues, the uterus, the urinary
tract, the liver, the lungs, and the pleural cavities.

XVII.
PATHOGENIC SPIRILLA.

SPIRILLUM OBERMEIERI.

Synonyms. — Spirochaete Obermeieri ; Spirillum of relapsing fe-
ver ; Die Recurrensspirochate.

Discovered by Obermeier (1873) in the blood of persons suffering
from relapsing fever.

This spirillum is present, in very great numbers, in the blood of
relapsing-fever patients during the febrile paroxysms. It has not
been found under any other circumstances, and its etiological rela-
tion to the disease with which it is associated is generally admitted.

Morphology. — Very slender, flexible, spiral or wavy filaments,
with pointed ends ; from sixteen to forty /* in length and consider-
ably thinner than the cholera spirillum — about 0.1 /*. Koch has
demonstrated the presence of flagella (Eisenberg).

Stains readily with the aniline colors, especially with fuchsin,
Bismarck brown, and in Loffler's solution of methylene blue.

Biological Characters. — An aerobic, motile spirillum which
has not been cultivated in artificial media. This spirillum appears to
be a strict parasite, whose habitat is the blood of man. The disap-
pearance of the parasite from the blood soon after the termination
of a febrile paroxysm, and its reappearance during subsequent par-
oxysms, have led to the inference that it must form spores, but this
has not been demonstrated. In fresh preparations from the blood
the spirillum exhibits active progressive movements, accompanied
by very rapid rotation in the long axis of the spiral filaments, or by
undulatory movements. The movements are so vigorous that the
comparatively large red blood corpuscles are seen, under the micro-
scope, to be thrown about by the slender spiral filaments, which it is
difficult to see in unstained preparations. When preserved in a one-
half -per-cent salt solution they continue to exhibit active movements
for a considerable time. Efforts to cultivate this spirillum in artificial
media have thus far been unsuccessful, although Koch has observed
an increase in the length of the spirilla and the formation of a
tangled mass of filaments.

ERNBERO/S BACTERIOLOGY

Plate- VII

Spirillurti Obernieieri ia blood of two monkeys,
inoculated after removal of spleen..
( Soudakewil clij.

PATHOGENIC SPIRILLA.

591

In experiments made by Heydenreich the spirillum was found to
preserve its vitality (motility) for fourteen days at a temperature of

Fio. 170.— Spirillum Obermeieri in blood of man. x 1,009. From a photomicrograph..
(Frankel and Pfeiffer. )

16° to 22° C., for twenty hours at 37°, and at 42.5° for two or three
hours only.

Pathogenesis. — Causes in man the disease known as relapsing
fever. Munch and Moczutkowsky have produced typical relapsing.

FIG. in.—Spirillum Obermeieri in blood of an inoculated ape. x 700. (Koch.

fever in healthy persons by inoculating them with blood containing
the spirillum of Obermeier. The spirilla are found in the blood dur-
ing the febrile paroxysm, and for a day or two, at the outside, after

592 PATHOGENIC SPIRILLA.

its termination ; sometimes they are present in great numbers, and
at others can only be found by searching several microscopic fields;
they are not present in the various secretions — urine, sweat, saliva,
etc. In fatal cases the principal pathological changes are found in
the spleen, which is greatly enlarged, and in the liver and marrow
of the bones, which contain inflammatory and necrotic foci. Koch
and Carter have succeeded in transmitting the disease to monkeys
by subcutaneous inoculations with small amounts of defibrinated
blood containing the spirillum. After an incubation period of seve-
ral days typical febrile paroxysms were developed, during which
the actively motile spirilla were found in the blood in large numbers.
Blood from one animal, taken during the attack, induced a similar
febrile paroxysm when inoculated into another of the same species-
relapses, such as characterize the disease in man, were not observed.
One attack did not preserve the animals experimented upon from a
similar attack when they were again inoculated after an interval of
a few days. Soudakewitch (1891) has made successful inoculation
experiments in monkeys, and has shown that in monkeys from which
the spleen has previously been removed the spirilla continue to
multiply very abundantly in the blood and the disease has a fatal
termination, whereas in monkeys from which the spleen has not been
removed the spirilla disappear from the blood within a few days
after the access of the febrile paroxysm and the animal recovers.

SPIRILLUM ANSERUM.

Synonym. — Spirochseta anserina (Sakharoff).

Obtained by Sakharoff (1890) from the blood of geese affected by a fatal
form of septicaemia due to this spirillum. This disease prevails among geese
in Caucasia, especially in swampy regions, appearing annually and destroy-
ing a large number of the domestic geese.

Morphology. — Resembles the spirillum of relapsing fever. The long and
flexible spiral filaments, when the disease is at its height, are often seen in
interlaced masses, around the margins of which radiate single filaments
which by their movements cause the whole mass to change its place, as if it
were a single organism. These masses are sometimes so large that a single
one occupies the entire field of the microscope.

Stains with the usual aniline colors.

Biological Characters. — An aerobic, motile spirillum. Not cultivated
in artificial media. The movements are very active, resembling those of
Spirillum Obermeieri, but cease in an hour or two in preparations made from
the blood of geese containing it.

Pathogenesis. — A small quantity of blood from an infected goose inocu-
lated into a healthy animal of the same species induces the disease after a
period of incubation of four to five days. The infected goose ceases to eat,
becomes apathetic, remaining in one place, and usually dies at the end of a
week ; the temperature is increased, and in some cases there is diarrhoea.
The spirilla are found in the blood at the outset of the malady, but after
death they are not seen either in the blood or in the various organs. The
heart and the liver are found to have undergone a fatty degeneration, and
yellowish, cheesy granules the size of a millet seed are seen upon the surface
of these organs. The spleen is soft and easily broken up by the fingers.

PATHOGENIC SPIRILLA. 593

Inoculations into chickens and pigeons were without result ; in one
chicken the spirilla were found in the blood 011 the fourth day after inocula-
tion, but the fowl recovered.

SPIRILLUM CHOLER^E ASIATICS.

Synonyms. — Spirillum ("bacillus") of cholera; Comma bacillus
of Koch ; Kommabacillus der Cholera Asiatica ; Bacille-virgule
cholerigene.

Discovered by Koch (1884) in the excreta of cholera patients and
in the contents of the intestine of recent cadavers.

The researches of Koch, made in Egypt and in India (1884), and
subsequent researches by bacteriologists in various parts of the
world, show that this spirillum — so-called " comma bacillus "• —is con-
stantly present in the contents of the intestine of cholera patients
during the height of the disease, and that it is not found in the con-
tents of the intestine of healthy persons or of those suffering from

FIG. 172. FIG. 173.

FIG. 172.— Spirillum cholerae Asiaticae. x 1,000. From a photomicrograph. (Koch.)
FIG. 173.— Spirillum cholerae Asiaticae, involution forms. X 700. (Van Ermengem.)

other diseases than cholera. The etiological relation of this spiril-
lum to Asiatic cholera is now generally admitted by bacteriologists.
Morphology. — Slightly curved rods with rounded ends, from 0.8
to 2 n in length and about 0.3 to 0.4 /*iii breadth. The rods are
usually but slightly curved, like a comma, but are occasionally in
the form of a half-circle, or two united rods curved in opposite
directions may form an S-shaped figure. Under certain circum-
stances the curved rods grow out into long, spiral filaments, which
may consist of numerous spiral turns, and in hanging-drop cultures
the S-shaped figures may also be seen to form the commencement
of a spiral ; in stained preparations the spiral character of the long
filaments is often obliterated, or nearly so. When development is
very rapid the short, curved rods or S-shaped spirals only are seen ;
but in hanging-drop cultures, or in media in which the develop,
38

594

PATHOGENIC SPIRILLA.

ment is retarded by an unfavorable temperature, the presence of a
little alcohol, etc. , the long, spiral filaments are quite numerous, and
bacteriologists generally agree that the so-called " comma bacillus "
is really only a fragment of a true spirillum. By LofHer's method
of staining the rods may be seen to have a single terminal flagel-
lum. In old cultures the bacilli frequently lose their characteristic
form and become variously swollen and distorted — involution forms.
Hueppe has described the appearance of spherical bodies in the
course of the spiral filaments, which he believes to be reproductive
elements — so-called arthrospores.

Stains with the aniline colors usually employed, but not as quick-
ly as many other bacteria ; an aqueous solution of fuchsin is the

FIG. 174. FIG. IT.").

Fio. 174 —Spirillum choleree Asiaticae; colonies upon gelatin plate, end of thirty hours. X 100.
Photograph by Frankel and Pfeiffer.

Fio. 175.— Spirillum cholerae Asiaticse, from a gelatin culture, x 1,000. From a photomicro-
graph. (Frankel and Pfeiffer.)

most reliable staining agent; is decolorized by iodine solution-
Grants method. Sections may be stained with Loffler's solution.

Biological Characters. — An aerobic (facultative anaerobic),
liquefying, motile spirillum. Grows in the usual culture media at
the room temperature — more rapidly in the incubating oven. Does
not grow at a temperature above 42° or below 14° C. Does not form
endogenous spores (forms arthrospores, according to Hueppe ?).

In gelatin plate cultures, at 22° C., at the end of twenty-four
hours small, white colonies may be perceived in the depths of the
gelatin ; these grow towards the surface and cause liquefaction of
the gelatin in the form of a funnel which gradually increases in

PATHOGENIC SPIRILLA.

595

depth, and at the bottom of which is seen the colony in the form of
a small, white mass ; as a result of this the plates on the second or
third day appear to be perforated with numerous small holes ; later

the gelatin is entirely liquefied. Under
a low power the young colonies, before
liquefaction has commenced, present a
rather characteristic appearance ; they
are of a white or pale-yellow color, and
have a more or less irregular outline,
the margins being rough and uneven;
the texture is coarsely granular, and the
surface looks as if it were covered with
little fragments of broken glass, while

the colony has a shining appearance ; when liquefaction commences an
ill-defined halo is first seen to surround the granular colony, which
by transmitted light has a peculiar roseate hue. In stab cultures in
nutrient gelatin development occurs all along the line of inoculation,

FIG. 176.— Colonies of the cholera
spirillum; a, end of twenty hours ; 6,
end of thirty hours; c, end of forty-
eight hours; d, after liquefaction of
the gelatin. (Flugge.)

FIG. 177.— Spirillum choleras Asiaticse; a, one day old; b. three days old; c, four days old; d, five
days old; e, seven days old; /, 10 days old. From photographs by Koch.

but liquefaction of the gelatin first occurs only near the surface ; on
the second day, at 22° C., a short funnel is formed which has a
comparatively narrow mouth, and the upper portion of which con-
tains air, while just below this is a' whitish, viscid mass ; later the
funnel increases in depth and diameter, and at the end of from four
to six days may reach the edge of the test tube ; in from eight to
fourteen days the upper two-thirds of the gelatin is completely lique-
fied. Owing to the slight liquefaction which occurs along the line of
growth during the first three or four days, the central mass which

596

PATHOGENIC SPIRILLA.

had formed along the line of inoculation settles down as a curled
or irregularly bent, yellowish-white thread in the lower part of a
slender tube filled with liquefied gelatin, the upper part of which
widens out and is continuous with the funnel above. Upon the sur-
face of nutrient agar a moist, shining, white layer is formed along
the line of inoculation — impfstrich. Blood serum is slowly liquefied
by this spirillum. Upon the surface of cooked potato, in the incu-
bating oven, a rather thin and semi-transparent brown or grayish-
brown layer is developed. In bouillon the development is rapid and
abundant, especially in the incubating oven ; the fluid is only slightly

FIQ. 178.— Cultures in nutrient gelatin, at the room temperature (16° to 18° C.), at the com-
mencement of the fourth day; a, Spirillum cholerae Asiaticse; &, Spirillum tyrogenum ; c, Spirillum
of Finkler and Prior. (Baurngarten.)

clouded, but the spirilla accumulate at the surface, forming a wrin-
kled membranous layer. Sterilized milk is also a favorable culture
medium. In general this spirillum grows in any liquid containing a
small quantity of organic pabulum and having a slightly alkaline
reaction. An acid reaction of the culture medium prevents its de-
velopment, as a rule, but it has the power of gradually accommo-
dating itself to the presence of vegetable acids, and grows upon
potatoes — in the incubator only — which have a slightly acid reaction.
Abundant development occurs in bouillon which has been diluted
with eight or ten parts of water, and the experiments of Wolffhugel

PATHOGENIC SPIRILLA. 597

and Riedel show that it also multiplies to some extent in sterilized
river or well water, and that it preserves its vitality in such water
for several months. But in milk or water which contains other bac-
teria it dies out in a few days. Gruber and Schottelius have shown,
however, that in bouillon which is greatly diluted the cholera spiril-
lum may take the precedence of the common saprophytic bacteria,
and that they form upon the surface of such a medium the charac-
teristic wrinkled film. Koch found in his early investigations that
rapid multiplication may occur upon the surface of moist linen, and
also demonstrated the presence of this spirillum in the foul water of
a " tank " in India which was used by the natives for drinking
purposes. In the experiments of Bolton (1886) the cholera spirillum
was found to multiply abundantly in distilled water to which
bouillon was added in the proportion of fifteen to twenty-five parts
in one thousand.

The thermal death-point of the cholera spirillum in recent cul-
tures in flesh-peptone-gelatin, as determined by the writer (1887), is
52° C., the time of exposure being four minutes ; a few colonies only
developed after exposure to a temperature of 50° for ten minutes.
In Kitasato's experiments (1889) ten or even fifteen minutes' expo-
sure to a temperature of 55° C. was not always successful in destroy-
ing the vitality of the spirillum, although in certain cultures exposure
to 50° for fifteen minutes was successful. He was not, however,
able to find any difference between old and recent cultures as regards
resistance to heat or to desiccation. In a moist condition this spiril-
lum retains its vitality for months — as much as nine months in agar
and about two months in liquefied gelatin. It is quickly destroyed
by desiccation, as first determined by Koch, who found that it did
not grow after two or three hours when dried in a thin film on a
glass cover. In Kitasato's experiments (1889) the duration of vital-
ity was found to vary from a few hours to thirteen days, the differ-
ence depending largely upon the thickness of the film. When dried
upon silk threads they may retain their vitality for a considerably
longer time (Kitasato). Very numerous experiments have been
made to determine the amount of various disinfecting agents re-
quired to destroy the vitality of this microorganism. We give be-
low the results recently reported by Boer (1890), whose experiments
were made in Koch's laboratory. Experiments upon a culture in
bouillon kept for twenty-four hours in the incubating oven, time of
exposure two hours : hydrochloric acid, 1 : 1,350 ; sulphuric acid,
1 : 1,300 ; caustic soda, 1 : 150 ; ammonia, 1 : 350 ; mercuric cyanide,
1 : 60,000 ; gold and sodium chloride, 1 : 1,000 ; silver nitrate, 1: 4,000;
arsenite of soda, 1 : 400 ; malachite green, 1 : 5,000 ; methyl violet,
1:1,000; carbolic acid, 1 : 400 ; creolin, 1:3,000; lysol, 1:500. In

598 PATHOGENIC SPIRILLA.

Bolton's experiments (1887) mercuric chloride was effective in two
hours in the proportion of 1 : 10,000 ; sulphate of copper, 1 : 500.

The low thermal death-point and comparatively slight resisting
power for desiccation and chemical agents indicate that this spiril-
lum does not form spores, and most bacteriologists agree that this
is the case. Hueppe, however, has described a mode of spore for-
mation which is different from that which occurs among the bacilli,
viz., the formation of so-called arthrospores ; these are said to be
developed in the course of the spiral threads, not as endogenous re-
fractive spores, but as spherical bodies which have a somewhat
greater diameter than the filament and are somewhat more refrac-
tive. This mode of spore formation has not been observed by Kita-
sato and other bacteriologists who have given attention to the ques-
tion, and cannot be considered as established. In competition with
the ordinary putrefactive bacteria the cholera spirillum soon disap-
pears, and, as determined by Neffelman and by Kitasato, they only
survive for a few days when mixed with normal faeces.

A test for the presence of the cholera spirillum has been found
by Bujwid and by Dunham in the reddish- violet color produced in
bouillon cultures containing peptone, or in cultures in nutrient gela-
tin, when a small quantity of sulphuric acid is added to the culture.
According to Frankel, this test serves to distinguish it from the ordi-
nary bacteria of the intestine and from the Finkler-Prior spirillum,
but not from Metschiiikoff's spirillum (" vibrio"). The reaction is
shown by bouillon cultures which have been in the incubating oven
for ten or twelve hours, and by gelatin cultures in which liquefac-
tion has occurred. The sulphuric acid used should be quite pure ;
the color quickly appears and is reddish- violet or purplish-red. Ac-
cording to Salkowski, the red color is due to the well-known indol
reaction, which in cultures of the cholera spirillum is exceptionally
intense and rapid in its development. A test which is said to dis-
tinguish cultures of the cholera spirillum from the spirillum of De-
neke and that of Finkler-Prior, has been proposed by Cahen. This
consists in adding a solution of litmus to the bouillon and in making
the culture at 37° C. The cholera cultures show on the following
day a decoloration which does not occur at this temperature with the
other spirilla named.

For determining as promptly as possible whether certain suspected
excreta contain cholera spirilla, a little of the material may be used
to inoculate greatly diluted bouillon, gelatin plates being made at
the same time. At the end of ten or twelve hours the cholera spiril-
lum, if present, will already have formed a characteristic wrinkled
film upon the surface ; a little of this should be used to start a new
culture in diluted bouillon, and a series of gelatin plates made from

PATHOGENIC SPIRILLA.

599

it, after which the color test may be applied. The result of this, in
connection with the morphology of the microorganisms forming the
film and the character of growth in the gelatin plates, will estab-
lish the diagnosis if the cholera spirillum is present in considerable
numbers. If but few are present in the original material it may be
necessary to make two or more series of plates and bouillon cultures
before a pure culture can be obtained and a positive diagnosis made.

/•

c •*

Fia. 179.— Section through mucous membrane of intestine from cholera cadaver; a tubular
gland (a) is cut obliquely; in the interior of this (6), and between the epithelial and basement
membrane, are numerous spirilla. X 600. (Fliigge.)

The spirillum is not found in the blood or in the various organs of
individuals who have succumbed to an attack of cholera, but it is
constantly found in the alvine discharges during life and in the con-
tents of the intestine examined immediately after death ; frequently in
almost a pure culture in the colorless " rice-water" discharges. It is
evident, therefore, that if we accept it as the etiological agent in this
disease, the morbid phenomena must be ascribed to the absorption of
toxic substances formed during its multiplication in the intestine. In
cases which terminated fatally after a very brief sickness Koch found
but slight changes in the mucous membrane of the intestine, which
was slightly swollen and reddened ; but in more protracted cases the
follicles and Peyer's patches were reddened around their margins, and
an invasion of the mucous membrane by the " comma bacilli " was
observed in properly stained sections ; they penetrated especially
the follicles of Lieberkiihn, and in some cases were seen between the
epithelium and basement membrane. As a rule, the spirillum is not

600 PATHOGENIC SPIRILLA.

present in vomited matters, but Koch found it in small numbers in
two cases and Nicati and Rietsch in three. In about one hundred
cases in which Koch examined the excreta, or the contents of the in-
testine of recent cadavers, during his stay in Egypt, in India, and in
Toulon, his " comma bacillus" was constantly found, and other ob-
servers have fully confirmed him in this particular — Mcati and
Rietsch in thirty-one cases examined at Marseilles ; Pf eiff er, twelve
cases in Paris ; Schottelius in cases examined in Turin ; Ceci in
Genoa, etc. On the other hand, very numerous control experiments
made by Koch and others show that it is not present in the alvine
discharges of healthy persons or in the contents of the intestine of
those who die from other diseases. In the writer's extended bacte-
riological studies of the excreta, and contents of the intestine of ca-
davers, in yellow fever, he has not once encountered any microor-
ganism resembling the cholera spirillum.

As none of the lower animals are liable to contract cholera during
the prevalence of an epidemic, or as a result of the ingestion of food
contaminated with choleraic excreta, we have no reason to expect
that pure cultures of the spirillum introduced by subcutaneous inocu-
lation or by the mouth will give rise in them to a typical attack of
cholera. Moreover, it has been shown by experiment that this spi-
rillum is very sensitive to the action of acids, and is quickly de-
stroyed by the acid secretions of the stomach, of man or the lower
animals, when the functions of this organ are normally performed.
By a special method of procedure, however, Nicati and Rietsch, and
Koch, have succeeded in producing in guinea-pigs choleraic symp-
toms and death. The first-named investigators injected cultures of
the spirillum into the duodenum, after first ligating the biliary duct;
the animals experimented upon died, and the intestinal contents con-
tained the spirillum in large numbers. The fact that this procedure
involves a serious operation which alone might be fatal, detracts
from the value of the results obtained. Koch's experiments on
guinea-pigs are more satisfactory, and, having been fully controlled
by comparative experiments, show that the " comma bacillus " is
pathogenic for these animals when introduced in a living condition
into the intestine. This was accomplished by first neutralizing the
contents of the stomach with a solution of carbonate of soda — five
cubic centimetres of a five-per-cent solution, injected into the stomach
through a pharyngeal catheter. For the purpose of restraining in-
testinal peristalsis the animal also receives, in the cavity of the abdo-
men, a tolerably large dose of laudanum — one gramme tincture of
opium to two hundred grammes of body weight. The animals are
completely narcotized by this dose for about half an hour, but re'
cover from it without showing any ill effects. Soon after the ad'

PATHOGENIC SPIRILLA. 601

ministration of the opium a bouillon culture of the cholera spirillum
is injected into the stomach through a pharyngeal catheter. As a
result of this procedure the animal shows an indisposition to eat and
other signs of sickness, its posterior extremities become weak and
apparently paralyzed, and, as a rule, death occurs within forty-eight
hours. At the autopsy the small intestine is found to be congested
and is filled with a watery fluid containing the spirillum in great
numbers. Comparatively large quantities of a pure culture injected
into the abdominal cavity of rabbits or of mice often produce a fatal
result within two or three hours ; and Nicati and Rietsch have ob-
tained experimental evidence of the pathogenic power of filtered cul-
tures not less than eight days old. The most satisfactory evidence
that this spirillum is able to produce cholera in man is afforded by an
accidental infection which occurred in Berlin (1884), in the case of a
young man who was one of the attendants at the Imperial Board of
Health when cholera cultures were being made for the instruction of
students. Through some neglect the spirillum appears to have been
introduced into his intestine, for he suffered a typical attack of
cholera, attended by thirst, frequent watery discharges, cramps in
the extremities, and partial suppression of urine. Fortunately he
recovered ; but the genuine nature of the attack was shown by the
symptoms and by the abundant presence of the " comma bacillus"
in the colorless, watery discharges from his bowels. Mcati and
Riet3eli observed a certain degree of attenuation in the pathogenic
power of the spirillum after it had been cultivated for a considerable
time at 20° to 25° C. ; and the observation has since been made that
cultures which have been kept up from Koch's original stock have
no longer the primitive pathogenic potency.

Cunningham, as a result of researches made in Calcutta (1891),
arrives at the conclusion that Koch's "comma bacillus" cannot
be accepted as the specific etiological agent in this disease. This
conclusion is based upon the results of his own bacteriological
studies, which may be summed up as follows : First, in many un-
doubted cases of cholera he has failed to find comma bacilli. Sec-
ond, in one case he found three different species. Third, in one case
the reaction with acids could not be obtained. From sixteen cases
in which Cunningham made cultures he obtained ten different vari-
eties of comma bacilli, the characters of which he gives in his pub-
lished report. It may be that in India, which appears to be the
permanent habitat of the cholera spirillum, many varieties of this
microorganism exist ; but extended researches made in the laborato-
ries of Europe show that Cunningham is mistaken in supposing that
spirilla resembling Koch's " comma bacillus " are commonly present
in the intestine of healthy persons. The view advocated is that

G02

PATHOGENIC SPIRILLA.

during the attack these spirilla are found in increased numbers be-
cause conditions are more favorable for their development, but that
they have no etiological import. The writer would remark that, in
very extended researches made in the United States and in Cuba, he
has never found any microorganism resembling Koch's cholera spi-
rillum in the faeces of patients with yellow fever or of healthy indi-
viduals, or in the intestinal contents of yellow-fever cadavers.

SPIRILLUM OF FINKLER AND PRIOR.

Synonym. — Vibrio proteus. -

Obtained by Finkler and Prior (1884) from the faeces of patients with
cholera nostras, after allowing the dejecta to stand for some days. Subse-

Fio. 180.

FIG. 181.

FIG. 182.

X 1,000. From a photomicro-

FIG. 180.— Spirillum of Finkler and Prior, from a gelatin culture,
graph. (Frankel and Pfeiffer.)

Fia. 181.— Spirillum of Finkler an! Prior; colonies upon gelatin plate; a, end of sixteen hours;
6, end of twenty-four hours; c, end of thirty-six hours, x 80. (Fliigge )

FIG. 182.— Spirillum of Finkler and Prior; culture in nutrient gelatin; c, two days old; d, four
days old. (Flugge.)

quent researches have not sustained the view that this spirillum is the speci-
fic cause of cholera morbus.

Morphology. — Resembles the spirillum of Asiatic cholera, but the curved
segments (" bacilli") are somewhat longer and thicker and not so uniform
in diameter, the central portion being usually thicker than the somewhat
pointed ends; forms spiral filaments, which are not as numerous, and are
usually shorter than those formed by the cholera spirillum. In unfavorable
media involution forms are common — large oval, spherical, or spindle-
shaped cells, etc. Has a single flagellum at one end of the curved segments,
which is from one to one and one-half times as long as these.

Stains with the usual aniline colors — best with an aqueous solution of
fucbsin.

PATHOGENIC SPIRILLA.

Biological Characters. — An aerobic and facultative anaerobic, liquefy-
ing, motile spirillum. Spore formation not demonstrated. Grows in the
usual culture media at the room temperature. Upon gelatin plates small,
white, punctiform colonies are developed at the end of twenty four hours,
which under the microscope are seen to be finely granular and yellowish or
yellowish-brown in color ; liquefaction of the gelatin around these colonies
progresses rapidly, and at the end of forty-eight hours is usually complete in
plates where they are numerous. Isolated colonies on the second day form
saucer-shaped depressions in the gelatin the size of lentils, having a sharply
denned border. In gelatin stab cultures liquefaction progresses much more
rapidly than in similar cultures of the cholera spirillum, and a stocking-
shaped pouch of liquefied gelatin is already seen on the second day, which
rapidly increases in dimensions, so that by the end of a week the gelatin is
usually completely liquefied ; upon the surface of the liquefied medium a
whitish film is seen. Upon agar a moist, slimy layer, covering the entire
surface, is quickly developed. The growth in blood serum is rapid and
causes liquefaction of the medium. \Jponpotato this spirillum grows at the
room temperature and produces a slimy, grayish-yellow, glistening layer,
which soon extends over the entire surface. The cholera spirillum does not
grow upon potato at the room temperature. The cultures of the Finkler-
. Prior spirillum give off a tolerably strong putrefactive odor, and, according
to Buchner, in media containing sujrar an acid reaction is produced as a re-
sult of their development. Th«y have a greater resistance to desiccation than
the cholera spirillum.

Pathogenesis. — Pathogenic for guinea-pigs when injected into the
stomach by Koch's method, after previous injection of a solution of car-
bonate of soda, but a smaller proportion of the animals die from such injec-
tions (Koch). At the autopsy the intestine is pale, and its watery contents,
which contain the spirilla in great numbers, have a penetrating, putrefactive
odor.

SPIRILLUM TYROGENUM.

Synonyms — Spirillum of Deneke; Kasespirillen.

Obtained by Deneke (1885) from old cheese.

Morphology. — Curved rods and long, spiral filaments resembling the
spirilla of Asiatic cholera. The diameter of the curved segments is some-
what less than that of the cholera spirillum, and the turns in the spiral fila-
ments are lower and closer together. The diame-
ter of the "commas" is uniform throughout, so
that this spirillum more closely resembles the
cholera spirillum than does that of Finkler and
Prior.

Stains with the usual aniline colors — best
with an aqueous solution of fuchsin.

Biological Characters. — An aerobic and fac-
ultative anaerobic, liquefying, motile spirillum. ^^
Spore formation not demonstrated. Grows in

the usual culture media at the room temperature FIG. 183.— Spirillum tyroge-
— more rapidly than the cholera spirillum and num. x TOO. (Flugge.)
less so than that of Finkler and Prior. Upon

gelatin plates small, punctiform colonies are developed, which on the second
day are about the size of a pin's head and have a yellowish color; under
the microscope they are seen to be coarsely granular, of a yellowish-green
color in the centre and paler towards the margins. The outlines of the colo-
nies are sharply defined at first, but later, when liquefaction has commenced,
the sharp contour is no longer seen. At first liquefaction of the gelatin
causes funnel-shaped cavities resembling those formed by the cholera spiril-
lum, but liquefaction is more rapid. In gelatin stab cultures liquefaction
occurs all along the line of puncture, and the spirilla sink to the bottom of

004

PATHOGENIC SPIRILLA.

the liquefied gelatin in the form of a coiled mass, while a thin, yeflowish
layer forms upon the surface ; complete liquefaction usually occurs in
about two weeks. Upon the surface of agar a thin, yellowish layer forms

FIG. 184.— Spirillum tyrogenum; colonies in gelatin plate; a, end of sixteen hours; 7>, end of
twenty-four hours ; c, end of thirty-six hours. X 80. (Fliigge.)

along the impfstrich. Upon potato, at a temperature of 37° C., a thin, yel-
low layer is usually developed (not always — Eisenberg) ; this contains, as a
rule, beautifully formed, long1, spiral filaments.

Pathogenesis. — Pathogenic for guinea-pigs when introduced into the
stomach by Koch's method ; three out of fifteen animals treated in this way
succumbed.

SPIRILLUM METSCHNIKOVI.

Synonym. — Vibrio Metschnikovi (Gameleia).

Obtained by Gamele'ia (1888) from the intestinal contents of chickens
dying of an infectious disease which prevails in certain parts of Russia dur-
ing the summer months, and which in some respects re-
sembles fowl cholera. The experiments of Gamele'ia show
that the spirillum under consideration is the cause of the
disease referred to, which he calls gastro-enteritischol erica.
Morphology.— Curved rods with rounded ends, and spi-
ral filaments ; the curved segments are usually somewhat
shorter, thicker, and more decidedly curved than the
" comma bacillus" of Koch. The size differs very consid-
erably in the blood of inoculated pigeons, the diametei*-
being sometimes twice as great as that of the cholera spiril-
lum, and at others about the same. A single, long, undu-
lating flagellum may be seen at one extremity of the spiral
filaments or curved rods in properly stained preparations.

Stains with the usual aniline colors, but not by Gram's
method.

Biological Characters. — An aerobic (facultative an-
aerobic ?), liquefying, ni(/tile spirillum. According to
Gamale'ia, endogenous spores are formed by this spirillum ;
but Pfeiffer does not confirm this observation, and it must
be considered extremely doubtful in view of the slight
resistance to heat — killed in five minutes by a temperature
of 50° C. Grows in the usual culture media at the room
temperature. Upon gelatin plates small, white, puncti-
form colonies are developed at the end of twelve to six-
teen hours ; these rapidly increase in size and cause lique-
faction of the gelatin, which is, however, much more rapid
with some than with others. At the end of three days
large, saucer-like areas of liquefaction may be seen resem-
bling that produced by the Finkler-Prior spirillum and the
contents of which are turbid, while other colonies have
produced small, funnel-shaped cavities filled with transparent, liquefied gel-
atin and resembling colonies of the cholera spirillum of th? *snne age. Under

FIG. 185.-Spiril-
lum Metschnikovi ;
culture in nutrient
gelatin, end of forty-
eight hours. From a
photograph. (Fran-
kel and Pfeiffer.)

PATHOGENIC SPIRILLA. 605

the microscope the larger liquefied areas are seen to contain yellowish-brown
granular masses which are in active movement, and the margins are sur-
rounded by a border of radiating filaments. In gelatin stab cultures the
growth resembles that of the cholera spirillum, but the development is more
rapid. Upon agar, at 37° C., a yellowish layer resembling that formed by
the cholera spirillum is quickly developed. Upon potato no growth occurs
at the room temperature, but at 37° C. a yellowish-brown or chocolate-col-
ored layer is formed, which closely resembles that produced by the cholera
spirillum under the same circumstances. In bouillon, at 37° C., develop-
ment is extremely rapid, and the liquid becomes clouded and opaque, having
a grayish-white color, while a thin, wrinkled film forms upon the surface.
When muriatic or sulphuric acid is added to a culture in peptomzed bouillon
a red color is produced similar to that produced in cultures of the cholera
spirillum, and even more pronounced. In milk, at 35° C., rapid development
occurs, and the milk is coagulated at the end of a week ; the precipitated
casein accumulates at the bottom of the tube in irregular masses and is not
redissolved. The milk acquires a strongly acid reaction and the spirilla
quickly perish.

Pathogenesis. — Pathogenic for chickens, pigeons, and guinea-pigs; rab-
bits and mice are refractory except for very large doses. Chickens suffering
from the infectious disease caused by this spirillum remain quiet and somno-
lent, with ruffled feathers ; they have diarrhoea ; the temperature is not ele-
vated above the normal, as is the case in chicken cholera. At the autopsy
the most constant appearance is hypersemia of the entire alimentary canal.
A grayish-yellow liquid, more or less mixed with blood, is found in con-
siderable quantity in the small intestine ; the spleen is not enlarged and the
organs generally are normal in appearance. In adult chickens the spirillum
is not found in the blood, but in young ones its presence may be verified by
the culture method and by inoculation into pigeons, which die in from
twelve to twenty hours after being inoculated with two to four cubic cen-
timetres. The pathological appearances in pigeons correspond with those
found in chickens, but usually the spirillum is found in great numbers in
blood taken from the heart. A few drops of a pure culture inoculated sub-
cutaneously in pigeons or injected into the muscles cause their death in
eight to twelve hours. Gameleia claims that the virulence of cultures is
greatly increased by successive inoculations in pigeons, but Pfeiffer has
shown that very minute doses are fatal to pigeons and that no decided in-
crease of virulence occurs as a result of successive inoculations. According
to Gameleia, chickens may be infected by giving them food contaminated
with the cultures of the spirillum, but pigeons resist infection in this way.
Guinea-pigs usually die in from twenty to twenty-four hours after receiving
a subcutaneous inoculation ; at the autopsy an extensive subcutaneous
oedema is found in the vicinity of the point of inoculation, and a superficial
necrosis may be observed ; the blood and the organs generally contain the
" vibrio " in great numbers, showing that the animals die from general in-
fection— acute septicaemia. When infection occurs in these animals by way
of the stomach the intestine will be found highly inflamed and its liquid con-
tents will contain numerous spirilla.

Gameleia has shown that pigeons and guinea-pigs may be made immune
by inoculating them with sterilized cultures of the spirillum— sterilized by
heat at 100° C. Old cultures contain more of the toxic substance than those
of recent date. Thus two to three cubic centimetres of a culture twenty days
old will kill a guinea-pig when injected subcutaneously, while five cubic
centimetres of a culture five days old usually fail to do so. According to
Pfeiffer, old cultures have a decidedly alkaline reaction, and their toxic power
is neutralized by the addition of sulphuric acid.

Gameleia has claimed that by passing the cholera spirillum of Koch
through a series of pigeons, by successive inoculation, its pathogenic power

606 PATHOGENIC SPIRILLA.

is greatly increased, and that when sterilized cultures of this virulent vari-
ety of the " comma bacillus " are injected into pigeons they become immune
against the pathogenic action of the " vibrio Metschnikoff , " and the reverse.
Pfeiffer (1889), in an extended and carefully conducted research, was not
able to obtain any evidence in support of this claim.

NOTES RELATING TO THE PATHOGENIC SPIRILLA.

Quite a number of spirilla have been obtained from various sources
which resemble more or less closely the spirillum of Asiatic cholera.
It appears probable that some of these are in fact varieties of Koch's
" comma bacillus " which have undergone various modifications as a
result of the conditions under which they have maintained their ex-
istence as saprophytes. Others are evidently essentially different,
and have no very near relationship to the cholera spirillum. The
principal points of difference between these recently described spirilla
and Spirillum cholerse Asiatics are given in the following resume,
for which we are indebted to Dieudonne (1804).

' ' Since the outbreak of cholera in 1892, various vibrios have been de-
scribed which resemble more or less closely the cholera vibrio. When these
are tested as to their morphological characters, growth in peptone solutions,
in gelatin and agar plates, cholera-red reaction, and pathogenic power, they
may be divided, at the outset, into two groups : viz., such vibrios as show
only a remote resemblance to the cholera vibrio, and therefore are easily dif-
ferentiated from it, and such as present only minor differences or none at
all that have been demonstrated.

"To the first group belongs the spirillum isolated by Russell from sea
water — Spirillum marinum — which rapidly liquefies gelatin and does not
grow at the body temperature. Reiion isolated from water, obtained at Bil-
lancourt, a vibrio which likewise quickly liquefies gelatin, but is not patho-
genic for guinea-pigs, either by subcutaneous or intraperitoneal inoculation.
Grunther, in examining the Spree water, found a vibrio which, upon gelatin
plates, formed circular colonies with smooth margins, very finely granular
and of a brown color. This vibrio did not give the indol reaction, and all
infection experiments gave a negative result. Giinther named this sapro-
phyte Vibrio aquatilis. About the same time (1892) Kiessling obtained from
water, from Blankenese, a vibrio which presented similar characters and
probably is identical with that of Giinther. Weibel obtained from well-water
a vibrio which liquefies gelatin more rapidly than the cholera vibrio ; its
pathogenic action was not tested. Bujwid (1893) isolated from Weichsel
water a vibrio which at low temperatures (12° C.) grew almost the same as
the cholera vibrio, but at higher temperatures was easily distinguished from
it. Bujwid's assistant, Orlowski, found in a well at Lubin a very similar
vibrio. Loffler (1893) obtained from the Peeiie water a vibrio which at 37°
C. grows rapidly and liquefies gelatin very rapidly, like the Finkler-Prior
spirillum. Fokker (1893), from water of the harbor at Groiiingeii, obtained
a vibrio which rapidly liquefied gelatin and occasionally gave the indol re-
action. Injections into the peritoneal cavity of mice and guinea-pigs gave
a negative result. Fokker supposes that this is an attenuated cholera bacil-
lus, because it forms the same ensyme as cholera bacteria, and when culti-
vated for three months its characters, especially its peptonizing power, had
changed. Fischer (1893) found in the stools of a woman suffering from diar-
rhoea a vibrio which in gelatin cultures resembled that of Finkler and
Prior. In bouillon and peptone solution it caused clouding and formation of

PATHOGENIC SPIRILLA. 607

a pellicle, but only gave a slight indol reaction. A portion of the mice in-
oculated subcutaiieously had after a time abscesses, from the contents of
which Fischer was able to cultivate his vibrio, which he named Vibrio helco-
genes. Vogler (1893), in an extended series of examinations of faeces, found
a vibrio which showed manv points of resemblance to the cholera vibrio in
its growth in gelatin. But it constantly gave a negative indol reaction, and
was not pathogenic for guinea-pigs when injected into the peritoneal cavity.
Bleisch obtained from the dejecta of a man who died with choleraic symptoms
a bacterium which upon gelatin plates grew at first like the cholera bacillus, but
was distinguished from it by many points of difference in other respects :
short rods, sometimes bent, but never showing spiral forms. It gave the
cholera-red reaction. Wolf (1883) obtained from cervical secretion, from a
woman suffering from chronic eiidometritis, a comma-formed bacillus, which
in its growth 011 gelatin plates resembled the cholera vibrio. The liquefac-
tion was, however, much more rapid, a culture a day old being as far ad-
vanced as a cholera culture of three to four days. The addition of sulphuric
acid to a bouillon culture caused a faint rose-red color, which upon standing
changed to brown. The addition of sulphuric acid and potassium iodide paste
did not cause a blue color, so there was 110 formation of nitrites. Bonhoff
(1893), in water from Stolpe, in Pommerania, discovered two vibrios, one of
which in the first twenty-four hours grevsr like the cholera vibrio, but did not
give the cholera-red reaction. Out of four guinea-pigs inoculated one only
died with cholera-like symptoms. The other vibrio gave the cholera-red reac-
tion, but did not liquefy gelatin and was very inconstant as regards its patho-
genic power. Zorkendorfer (1893) isolated a vibrio from the stools of a
woman who died with choleraic symptoms, which at first grew upon gelatin
plates like the cholera vibrio, but after the second day liquefied the gelatin
very rapidly, so that it could no longer be taken for the same. The indol
reaction was constantly absent, and it was not pathogenic for guinea-pigs,
rabbits, or pigeons. Blackstein (1893) obtained from the water of the Seine
a comma bacillus which resembled the cholera vibrio in many particulars, but
was distinguished by the finer granulation and more opaque appearance of
its colonies. Sanarelli (1893), by the use of special media, isolated from the
water of the Seine and of the Marne 110 less than thirty-two vibrios, four of
which resembled the cholera vibrio in giving the indol reaction. Three
others gave the indol reaction after eight days ; the remainder did not give it
at ail, or only very faintly. The vibrios which upon a first inoculation gave
110 results or only very slight evidence of pathogenic power, when carried
through a series of animals caused a fatal infection. When a sterilized cul-
ture of the colon bacillus was injected at the same time death always oc-
curred. Sanarelli believes that these vibrios must have had a common ori-
gin— from the dejecta of cholera patients. Fischer (1894) has described a
number of vibrios from sea-water which are distinguished from the cholera
vibrio especially by a preference for media containing sea-water. Finally,
the vibrios found in water, referred to by Koch (' Ueber den augenblicklicheii
Stand der Cholera-diagnose,' Zeitschr. fur Hygiene, Bd. xiv., page 319),
belong here.

"Quite different from these is a second group of vibrios which in their in-
vestigation offered great and often almost insuperable difficulties for the
differential diagnosis. Here, first of all, is the Vibrio Berolinensis, found by
Neisser in August, 1893, and described by Rubner, Neisser, and Giinther.
This was isolated from water which had previously contained cholera vibrios,
for which reason Dun bar considers it not impossible that this is a genuine
cholera vibrio, somewhat changed perhaps by long-continued development
in water. Neither in its morphology nor in its behavior in gelatin stick cul-
tures, in milk and other media, could it be distinguished from the genuine
comma bacillus ; the indol reaction and pathogenic action upon guinea-pigs
were the same ; on the contrary, a differentiation was easily made in gelatin
plate cultures. At the end of twenty -four hours it formed small, spherical,

G08 PATHOGENIC SPIRILLA.

finely granular colonies, which at the end of forty-eight hours were not yet
visible to the naked eye. Heider (1893) isolated from the water of the Donau
canal a vibrio which he called Vibrio Danubicus. This resembles the chol-
era vibrio fully in its morphology. As a distinguishing character it was
found that this vibrio, in thinly planted plates, forms flat, superficial colo-
nies having irregularly rounded margins and other slight differences ; also
the pathogenic action upon mice inoculated subcutaneously, and the ease with
which guinea-pigs are infected by way of the respiratory passages. It is
worthy of note that the day after the sample was taken a man was taken sick
with cholera who had worked on the Donau the day before — on the principal
stream at a place far below the junction of the canal. Dunbar (1893) found
vibrios in the Elbe, in the Rhine, in the Pegnitz, and in the Amstel at Amster-
dam. These presented no decided characters by which he was able to differ-
entiate them from the cholera vibrio. The most careful comparative investi-
gations did not lead to the discovery of any points of difference which had
not already been observed in genuine cholera cultures. Everything, there-
fore, indicated that these were genuine cholera bacilli, especially as these
vibrios disappeared from the rivers when cholera ceased to prevail. It was
first possible through an observation of Kutscher's to differentiate a portion
of these water bacteria, and certain vibrios isolated from the discharges of
persons suspected of having cholera from cultures of the cholera spirillum.
In the presence of oxygen, at a suitable temperature, they give off a greenish-
white phosphorescence.

"As phosphorescence has never been observed in undoubted cholera cul-
tures, we can assert with tolerable certainty that such phosphorescent vibrios
are not genuine cholera bacteria. But as this phosphorescent property was
inconstant in thirty-eight out of sixty-eight cultures, Dunbar believes that
some reserve must be exercised in accepting this as evidence that these are
not genuine cholera vibrios. Maasseii (1894) gives as a further distinguishing
character of these phosphorescent vibrios the fact that they form a strong,
usually wrinkled pellicle in bouillon, of proper alkalinity, containing gly-
cerin or carbohydrates (cane sugar, lactose) ; also that in such media the
formation of indol and a subsequent return to an alkaline reaction may be
observed.

"As already stated, Sanarelli isolated from Seine water a considerable
number of vibrios, and among them four — viz. : one from St. Cloud, Point-
du-Jour, Gennevilliers No. 5, and Versailles (Seine), which after twenty-four
hours gave a distinct indol reaction and were more or less pathogenic for
guinea-pigs (the one from St. Cloud was also pathogenic for pigeons}. I van-
off (1893) describes a vibrio which he isolated from the faeces of a patient with
typhoid fever. But as the discharges had been mixed with Berlin hydrant
water, Ivanoff admits the possibility that his vibrio came from this water.
It closely resembles the cholera vibrio, but is distinguished by its colonies in
gelatin plates, which, at the end of twenty -four to thirty-six hours, in place
of the usual coarse granulation of cholera colonies shows a distinct formation
of filaments. Morphologically the vibrio is distinguished by a decided ten-
dency to preserve the spiral form, and especially by its size. Celli and Saii-
tori (1893) describe a Vibrio romanus, which they isolated from twelve
undoubted cases of cholera. This does not give the indol reaction, is not
pathogenic for animals, and does not grow in bouillon or agar at 37° C.
This is considered by the authors named an atypical variety of the cholera
vibrio, especially as the distinguishing characters did not prove to be perma-
nent. After eight months' cultivation the cultures gave the indol reaction, but
the pathogenic power was still almost absent. Recently Chaiitemesse (1894)
has described a vibrio which he found in the spring of 1894 during the chol-
era epidemic at Lisbon. This differed in many particulars from the genuine
cholera vibrio, resembling more closely the vibrio of Finkler-Prior. As in
the Lisbon epidemic, with a large number taken sick, only one death occurred,
and in view of the results of the bacteriological examination, Chantemesse

PATHOGENIC SPIRILLA. 609

supposes this to have been an epidemic of cholera nostras. Finally, Pfuhl
(1894) found a vibrio in the north harbor of Berlin which from its growth in
gelatin and pathogenesis for pigeons he believes to be identical with Vibrio
Metschnikovi."

To the list of vibrios above referred to as resembling more or less
closely the cholera spirillum we must add those described by Cun-
ningham (1894) and obtained by him from the discharges of cholera
patients. He has described "thirteen distinct forms obtained from
cases of cholera and one of non-choleraic origin."

Pfeiffer and Issaeff (1894) report that they have found a sen-
sitive test for the differentiation of these vibrios in the specific
character of cholera immunity. They found that guinea-pigs which
were immunized against cholera infection have a lasting immunity,
and that the serum of such immunized animals has a specific ac-
tion in protecting against infection by genuine cholera vibrios
only, while for other species it has no action different from that
of the blood serum of normal animals. In all cases where the cholera
serum acted specifically the vibrios were promptly destroyed, while
in cases where this specific action was absent the injected vibrios
multiplied rapidly and caused the death of the animal. By means of
this method the vibrios isolated from water — the phosphorescent
vibrios of D unbar, Vibrio Danubicus, Cholera Massanah — are shtfwn
to be distinct species, while the vibrio of Ivanoff behaves like the
genuine cholera vibrio. In a subsequent paper Pfeiffer reports the
interesting fact that a trace of highly active cholera serum, added to
a culture of the cholera spirillum, when injected into the peritoneal
cavity of a guinea-pig, within a surprisingly brief time causes the
destruction of the cholera vibrios ; whereas no such effect is produced
upon other species. A similar destruction occurs when cholera vib-
rios are injected into the abdominal cavity of immunized guinea-
pigs. The researches of Dunbar (1894) indicate that Pfeiffer 's test
is not so reliable as he supposed ; and also that phosphorescence can-
not be relied upon for distinguishing similar water bacteria from
genuine cholera vibrios. Rumpel has reported the fact that two un-
doubted cultures of the cholera spirillum, from different sources, after
being passed through pigeons and cultivated for some time in arti-
ficial media, showed phosphorescence. One of these cultures was ob-
tained originally from the discharges of Dr. Oergel, who was a vic-
tim to cholera from laboratory infection (case reported by Reincke, in
the Deutsche medicinisclie Wochenschrift, No. 41, 1894). Another
case of supposed laboratory infection, in which recovery occurred, is
reported by Lazarus, in the Berliner medicinisclie Wochenschrift9
1893, page 1,241.

That cholera vibrios may be present in the alimentary canal of
39

610 PATHOGENIC SPIRILLA.

healthy individuals without giving rise to any symptoms of ill-health
appears to be demonstrated. In support of this conclusion we quote
as follows from a recent paper by Abel and Claussen:

" In Wehlau (East Prussia), in the autumn of 1894, seven cases of
cholera occurred about the same time. The members of the family
were at once isolated and their fa3ces examined almost daily. Of
especial interest were seventeen individuals who belonged to families
in which three fatal cases occurred. Of these seventeen persons, who
were not sick at all or only had for a brief time a diarrhoea, thirteen
had cholera vibrios in their discharges for a considerable time. As
the table shows, many of these comma bacilli were not found in dis-
charges every day, but were obtained again after being absent" (in
the cultures) " for a day or two."

Abel and Claussen (1895), as a result of very extended experi-
ments, arrive at the conclusion that cholera vibrios in faeces as a rule
do not survive longer than twenty days, and often cannot be ob-
tained after two or three days ; exceptionally they were obtained in
cultures at the end of thirty days — Karlinsky and Dunbar have re-
ported finding them at the end of fifty-two days and four months.
Karlinsky (1895) has also reported that upon woollen and linen goods,
cotton batting and wool, which were soaked in the discharges of
cholera patients and preserved from drying by being wrapped in
waxed paper, the cholera vibrio retained its vitality for from twelve
to two hundred and seventeen days.

The researches of Kasansky (1895) show that the cholera spiril-
lum is not destroyed by alow temperature (—30 C.) and that it
even resists repeated freezing and thawing — three or four times.

Behring and Ransom (1895) as a result of an extended experi-
mental research, arrive at the conclusion that cholera cultures from
which the bacteria have been removed have specific toxic properties,
and cause symptoms similar to those which result from the intro-
duction into guinea-pigs of the living bacteria ; that from these fil-
tered cultures a solid substance can be obtained having the same
toxic properties, and that from susceptible animals which have been
treated with this toxic substance a serum can be obtained which is
active not only against the cholera poison, but against the cholera
vibrio. These results support those previously reached by other
bacteriologists and lead to the hope that a specific treatment of the
disease may be successfully employed. The results obtained by
Haffkine in India are favorable to the view that his method of prophy-
laxis, by the subcutaneous injection of virulent cholera cultures, has
a real value.

PLATE IX.

FIG. 1. — Bacillus diphtherias (Klebs-Loffler) from culture on blood
serum. Stained with Loftier' s solution of mechylene blue. x 1,000.
Photomicrograph by oil lamp. (Borden.)

FIG. 2. — Micrococcus gonorrhoeas in urethral pus. Stained with
Loffler's solution of methylene blue, x 1,000. Photomicrograph by oil
lamp. (Borden.)

FIG. 3. — Bacillus tuberculosis in sputum. x 1,000. Photomicro-
graph by oil lamp. (Borden.)

FIG. 4. — Bacillus typhi abdominalis, from agar culture, x 1,000.
Photomicrograph by oil lamp. (Borden.)

FIG. 5. — Streptococcus pyogenes (longus). x 1,000. Photomicro-
graph made at the Army Medical Museum by sunlight. (Gray.)

FIG. 6. — Bacillus mallei, x 1,000. Photomicrograph made at the
Army Medical Museum by sunlight. (Gray.)

PLATEIX.

STERNBERG'S BACTERIOLOGY.

Fig. 2.

v».

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i r

••«£#,- H *'l
' -' rf IT'', »^"* .'.

^^^:, JH#

Pig. 3.

"«••!•

. 5.

Pigr. 4.

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. e.

PATHOGENIC BACTERIA.

PART FOURTH.

SAPROPHYTES.

I. BACTERIA IN THE AIR. II. BACTERIA IN WATER. III. BACTERIA IN
THE SOIL. IV. BACTERIA ON THE SURFACE OF THE BODY AND OF EX-
POSED Mucous MEMBRANES. V. BACTERIA OF THE STOMACH AND
INTESTINE. VI. BACTERIA OF CADAVERS AND OF PUTREFYING
MATERIAL FROM VARIOUS SOURCES. VII. BACTEBIA
itf ARTICLES or FOOD.

BACTERIA IX THE AIR.

THE saprophytic bacteria are found wherever the organic material
which serves as their pabulum is exposed to the air under conditions
favorable to their growth. The essential conditions are presence of
moisture and a suitable temperature. The organic material may be
in solution in water or in the form of moist masses of animal or
vegetable origin, and the temperature may vary within considerable
limits — 0° to 70° C. But the species which takes the precedence will
depend largely upon special conditions. Thus certain species multi-
ply abundantly in water which contains comparatively little organic
pabulum, and others require a culture medium rich in albuminous
material or in carbohydrates ; some grow at a comparatively low or
high temperature, while others thrive only at a temperature of 20° to
40° C. or have a still more limited range ; some require an abun-
dant supply of oxygen, and others will not grow in the presence of
this gas. Our statement that saprophytic bacteria are found wherever
the organic material which serves as their pabulum is exposed to the
air — under suitable conditions — relates to the fact that it is through
the air that these bacteria are distributed and brought in contact
with exposed material. It is a matter of common laboratory experi-
ence that sterilized organic liquids quickly undergo putrefactive de-
composition when freely exposed to the air, and may be preserved in-
definitely when protected from the germs suspended in the air by
means of a cotton air filter. But the organic pabulum required for
the nourishment of these bacteria is not found in the air in any con-
siderable amount, and if they ever multiply in the atmosphere it
must be under very exceptional conditions. Their presence is due to
the fact that they are wafted from surfaces where they exist in a
desiccated condition, and, owing to their levity, are carried by the
wind to distant localities. But, under the law of gravitation, when
not exposed to the action of currents of air they constantly fall
again upon exposed surfaces, which, if moist, retain them, or from
which, if dry, they are again wafted by the next current of air.
Under these circumstances it is easy to understand why, as deter-

614

BACTERIA IX THE AIR.

mined by investigation more bacteria are found near the surface of
the earth than at some distance above the surface, more over the
land than over the ocean, more in cities with their dust-covered
streets than in the country with its grass-covered fields.

Careful experiments have shown that bacteria do rot find their
way into the atmosphere from the surface of liquids, unless portions
of the liquid containing them are projected into the air by some
mechanical means, such as the bursting of bubbles of gas. Cultures
of pathogenic bacteria freely exposed to the air in laboratories do not
endanger the health of those who work over them ; but if such a cul-
ture is spilled upon the floor and allowed to remain without disin-
fection, when it is desiccated the bacteria
contained in it will form part of the dust of
the room and might be dangerous to its
occupants. Bacteria do not escape into the
air from the surface of the fluid contents of
sewers and cesspools, but changes of level
may cause a deposit upon surfaces, which
is rich in bacteria, and when dried this ma-
terial is easily carried into the atmosphere
by currents of air.

Tyndall's experiments (1869) show that
in a closed receptacle in which the air is
perfectly still all suspended particles are af-
ter a time deposited on the floor of the closed
air chamber. And common experience de-
monstrates the fact that the dust of the at-
mosphere is carried by the wind from ex-
posed surfaces and again deposited when the
air is at rest. This dust as deposited, for
example, in our dwellings contains innu-
merable bacteria in a desiccated condition,
and the smallest quantity of it introduced
into a sterile organic liquid will cause it to
undergo putrefactive decomposition, and
by bacteriological methods it will be found
to contain various species of bacteria. Such
dust also contains the spores of various
mould fungi which are present in the atmo-
sphere, usually in greater numbers than the
bacteria. The mould fungi are air plants

which vegetate upon the surface of moist organic material and form
innumerable spores, which are easily wafted into the air, both on
account of their low specific gravity and minute size, and because they

FIG. 186. — Penicillium glau-
cum; m, mycelium, from which
is given off a branching pedicle
bearing spores. X 150.

BACTERIA IX THE AIR.

Gio

are borne upon projecting pedicles by which they are removed from
the moist material upon which and in which the mycelium develops
(Fig. 186), and, being dry, are easily carried away by currents of air.

Bacteriologists have given much attention to the study of the mi-
croorganisms suspended in the atmosphere, with especial reference to
hygienic questions. The methods and results of these investigations
will be considered in the present section.

Pasteur (1860) demonstrated the presence of living bacteria in the
atmosphere by aspirating a considerable quantity of air through a
filter of gun-cotton or of asbestos contained in a glass tube. By dis-
solving the gun-cotton in alcohol and ether he was able to demon-
strate the presence of various microorganisms by a microscopical ex-
amination of the sediment, and by placing the asbestos filters in
sterilized culture media he proved that living ge.rms had been filtered
out of the air passed through them.

FIG. 187.

A method employed by several of the earlier investigators con-
sisted in the collection of atmospheric moisture precipitated as dew
upon a surface cooled by a freezing- mixture. This was found to con-
tain living bacteria of various forms. The examination of rain water,
which in falling washes the suspended particles from the atmosphere,
gave similar results.

The first systematic attempts to study the microorganisms of the
air were made by Maddox (1870) and by Cunningham (1873), who
used an aeroscope which was a modification of one previously de-
scribed by Pouchet. In the earlier researches of Miquel a similar
aeroscope was used. This is shown in Fig. 187. The opening to the
cylindrical tube A is kept facing the wind by means of a wind vane,
and when the wind is blowing a current passes through a small aper-
ture in a funnel-shaped partition which is properly placed in the
cylindrical tube. A glass slide, upon the lower surface of which a

616 BACTERIA IN THE AIR.

mixture of glycerin and glucose has been placed, is adjusted near the
opening of the funnel, at a distance of about three millimetres, so
that the air escaping through the small orifice is projected against it.
By this arrangement a considerable number of the microorganisms
present in the air, as well as suspended particles of all kinds, are ar-
rested upon the surface of the slide and can be examined under the
microscope or studied by bacteriological methods. But an aeroscope
of this kind gives no precise information as to the number of living
germs contained in a definite quantity of air. The microscopical ex-
amination also fails to differentiate the bacteria from particles of
various kinds which resemble them in shape, and the microorgan-
isms seen are for the most part spores of various fungi mingled with
pollen grains, vegetable fibres, plant hairs, starch granules, and
amorphous granular material.

Another method, which has been employed by Cohn, Pasteur,
Miquel, and others, consists in the aspiration of a definite quantity of
air through a culture liquid, which is then placed in an incubating
oven for the development of microorganisms washed out of the air
which has been passed through it. This method shows that bacteria
of different species are present, but gives no information as to their
relative number, and requires further researches by the plate method
to determine the characters of the several species in pure cultures.

A far simpler method consists in the exposure of a solid culture
medium, which has been carefully sterilized and allowed to cool on a
glass plate or in a Petri's dish, for a short time in the air to be ex-
amined. Bacteria and mould fungi deposited from the air adhere to
the surface of the moist culture medium, and form colonies when the
plate, enclosed in a covered glass dish, is placed in the incubating oven.
The number of these colonies which develop after exposure in the
air for a given time enables us to estimate in a rough way the num-
ber of microorganisms present in the air of the locality where the
exposure was made ; and the variety of species is determined by ex-
amining the separate colonies, each of which is, as a rule, developed
from a single germ. By exposing a number of plates at different
times this method enables us to determine what species are most
abundant in a given locality and the comparative number in dif-
ferent localities, as determined by counting the colonies after ex-
posure for a definite time — e.g., ten minutes. Of course we will only
obtain evidence of the presence of such aerobic bacteria as will
grow in our culture medium. The anaerobic bacteria may be studied
by placing plates exposed in a similar way in an atmosphere of hydro-
gen. Bacteria which grow slowly and only under special conditions,
like the tubercle bacillus, would be likely to escape observation, as
the mould fungi and common saprophytes would take complete pos-

BACTERIA IN THE AIR.

617

session of the surface of the culture medium before the others had
formed visible colonies. Students will do well to employ this simple
and satisfactory method for the purpose of making themselves familiar
with the more common atmospheric organisms, and they will find
the shallow glass dishes with a cover, known as Petri's dishes, very
convenient for the purpose. These dishes should be sterilized in the
hot-air oven and sufficient sterile nutrient gelatin or agar poured
into them to cover the bottom. After the culture medium has be-
come solid by cooling, the exposure may be made by simply remov-
ing the cover and replacing it at the end of the time fixed upon.

FIG. 188.

To determine in a more exact way the number of microorganisms
contained in a given quantity of air will require other methods. But
we may say, en passant, that such a determination is usually not of
great scientific importance. The number is subject to constant fluc-
tuations in the same locality, depending upon the force and direction
of the wind. If we have on one side of our laboratory a dusty
street and on the other a green field, more bacteria will naturally be
found when the wind blows from the direction of the street than
when it comes from the opposite direction ; or, if the air is filled with
dust from recently sweeping the room, we may expect to find very

CIS BAfTKKIA IX THK AIR.

many more than, when the room has been undisturbed for some time.
The painstaking researches which have already been made have es-
tablished in a general way the most important facts relating to the
distribution of atmospheric bacteria, but have failed to show any de-
finite relation between the number of atmospheric bacteria and the
prevalence of epidemic di .In the apparatus of Hesse, Fig.

188, a glass tube, having a diameter of four to five centimetres and a
length of half a metre to a metre, is employed. In use this is sup-
ported upon a tripod, as shown in the figure, and air is drawn
through it by a wat'-r aspirator consisting of two flasks, also shown.
The upper flask being filled with water, this flows into the lower
flask by siphon action, and upon reversing the position of the flasks
number one is again filled. By repeating this operation as many
times as desired a quantity of air corresponding with the amount of
water passed from the upper to the lower flask is slowly aspirated
through the horizontal glass tube. The microorganisms present are
d< -pi^ited upon nutrient gelatin previously allowed to cool upon the
lower portion of the large glass tube. The air enters through a small
opening in a piece of sheet rubber which is tied over the extremity
of the horizontal tube, and before the aspiration is commenced this
opening is covered by another piece of sheet rubber tied over the
first. Experience shows that when the air is slowly aspirated most
of the germs contained in it are < i t ed near the end of the tube
through \vhich it enters. The colonies which develop upon the nu-
trient gelatin show the number and chara<-t;-r of living microorgan-
< -o 1 1 1 ; i i 1 1 ed in the measured quantity of air aspirated through the

appacaf us. Tin- method with a soluble filter of ptil v.-ri/rd sugar, to

be described hereafter, is preferable when exact results are desired;
and for the purpose of determining the relative abundance and the
variety of microorganisms present in the atmosphere of a given lo-
cality the exposure of nutrient gelatin in Potri's dishes is far simpler,
and, as a nil.-, will furnish all the information that is of real
value.

In his extended researches made at the laboratory of Montsouri,
in Paris, Miquel has used various forms of apparatus and has ob-
tained interesting results ; but his method of ensemencements frac-

//o// //As requires a great expenditure of time and patience, and the
more recent method with soluble filters is to be preferred.

In his latest modification of the m-; hod rrfn-rod to Miquel used a
flask like that shown in Fig. 189. From twenty to forty cubic cen-
1 i metres of distilled water are introduced into this flask. The cap A
contains a cotton air filter and is fitted to the neck of the flask by a

-•n.iind John. This is removed during t'x> r\ prrimml. The tube ('
is ronnertrd with a- irator. It Contains two Cotton Or asbestos

K.U TKR1A IX THK A IK.

619

FIG.

filters, c and b. The cap being- removed and the aspirator attached,
the air is drawn through the water, by which suspended germs are
arrested : or if not they are caught by the inner cotton ping b. The
led point of the tube B is now broken oft*, and the contents of the
tlask equally divided in thirty to forty tubes containing bouillon,
which are placed in the incubating oven.
Twenty-five cubic centimetres of bouillon
are also introduced into the tlask. and the
cotton plug /> is pushed into it so that any
bacteria arrested by it may develop. If
one-fourth or one-tifth of the bouillon tubes
show a development of bacteria it is in-
ferred that each culture originated from
a single germ, a*ul the number present in
the amount of air drawn through the tlask
i- estimated from the number of tubes in
which development occurs.

The method adopted by Straus and \Viirt /. is more convenient and
more reliable in its results. This consists in passing the air by means
of an aspirator through liquefied nutrient gelatin or agar. The ap-
paratus shown in Fig. 1W is used for this purpose. Two cotton
plugs are placed in the tube B, to which the aspirator is attached,
and after the determined |iiantity of air has been passed through the
liquefied medium the inner plug is pushed down with a sterili/.ed

platinum needle so as to wash out in the culture
medium any germs arrested by it. Finally the
gelatin or agar is solidified upon the walls of
the tube A by rotating it upon a block of ice or
under a stream of cold water. It is now put
aside for the development of colonies, which are
counted to determine the number of germs pre-
sent in the quantity of air passed through the
liquefied culture medium. The main difficulty
with this apparatus is found in the fact that the
nutrient gelatin foams when air is bubbled
through it ; for this reason an agar medium is
to be preferred. In using this it will be neces-
sary to place the liquefied agar in a. bath main-
tained at W C. Foaming of the gelatin is pre-
vented by adding a drop of olive oil before ster-
ili/.ation in the steam sterili/.or. But this inter-
feres with the transparency of the medium.
In the earlier experiments upon atmospheric organisms Pasteur
used a filter of asbestos, which was subsequently washed out in a

FlO. ISHI.

620

BACTERIA IN THE AIR.

culture liquid. A filter of this kind washed out in liquefied gelatin
or nutrient agar would give more satisfactory results, as the culture
medium could be poured upon plates or spread upon the walls of a
test tube and the colonies counted in the usual way. Petri prefers
to use a filter of sand, which he finds by experiment arrests the mi-
croorganisms suspended in the atmosphere, and which is subsequently
distributed through the culture medium. The sand used is such as

has been passed through a wire sieve having
openings of 0. 5 millimetre in diameter. This is
sterilized by heat, and is supported in a cylin-
drical glass tube by small wire-net baskets. The
complete arrangement is shown in Fig. 191.
Two sand filters, c, and C2, are used, the lower
one of which serves as a control to prove that
all microorganisms present in the air have been
arrested by the upper one. The upper filter is
protected, until the aspirator attached to the
tube h is put in operation, by a sterile cotton
plug, not shown in the figure which represents
the filter in use. Petri uses a hand air pump as
an aspirator, and passes one hundred litres of
air through the sand in from ten to twenty
minutes. The sand from the two filters is then
distributed in shallow glass dishes and liquefied
gelatin is poured over it ; this is allowed to sol-
idify and is put aside for the development of
colonies. The principal objection to this method
is the presence of the opaque particles of sand
in the culture medium. This objection has been
overcome by the use of soluble filters, a method
first employed by Pasteur and since perfected
by Sedgwick and by Miquel. The most useful
material for the purpose appears to be cane
sugar, which can be sterilized in the hot-air oven
at 150° C. without undergoing any change in
its physical characters. Loaf sugar is pulver-
ized in a mortar and passed through two sieves
in order to remove the coarser grains and the
very fine powder, leaving for use a powder having grains of about
one-half millimetre in diameter. This powdered sugar is placed
in a glass tube provided with a cap having a ground joint and a cot-
ton plug to serve as an air filter (A, Fig. 192), or in a tube such as is
shown at B, having the end drawn out and hermetically sealed. Two
cotton plugs are placed at the lower portion of the tube, at a and at b.

FIG. 191.

BACTERIA IN THE AIR.

621

Glass tubing having a diameter of about five millimetres is used in
making these tubes, and from one to two grammes of powdered sugar
is a suitable quantity to use as a filter. The whole apparatus is steril-
ized for an hour at 150° C. in a hot-air oven after the pulverized
sugar has been introduced. Before using it will be necessary to
pack the sugar against the supporting plug a by gently striking the
lower end of the tube, held in a vertical position, upon some horizon-
tal surface ; and during aspiration
the tube must remain in a vertical
position, or nearly so, in order that
the sugar may properly fill its entire
calibre. The aspirator is attached to
the lower end of the tube by a piece
of rubber tubing. When the tube B
is used the sealed extremity is broken
off at the moment that the aspirator
is set in action, and it is again sealed
in a flame after the desired amount
of air has been passed through the
filter. The next step consists in dis-
solving the sugar in distilled water
or in liquefied gelatin. To insure
the removal of all the sugar the cot-
ton plug a may be pushed out with a
sterilized glass rod, after removing b
with forceps. From fifty to five hun-
dred cubic centimetres of distilled
water, contained in an Erlenmeyer
flask and carefully sterilized, may be
used, the amount required depending
upon circumstances relating to the
conditions of the experiment. By
adding five or ten cubic centimetres
of this water, containing the sugar
and microorganisms arrested by it,
to nutrient gelatin or agar liquefied by heat, and then making Es-
march roll tubes, the number of germs in the entire quantity is easily
estimated by counting the colonies which develop in the roll tubes.

Sedgwick and Tucker, in a communication made to the Boston
Society of Arts, January 12th, 1888, were the first to propose the use
of a soluble filter of granulated sugar for collecting atmospheric
germs. Their complete apparatus consists of an exhausted receiver,
from which a given quantity of air is withdrawn by means of an air
pump. A vacuum gauge is attached to the receiver, which is coupled

FIG. 192.

FIG. 193.

G22 BACTERIA IX THE AIR.

with the glass tube containing the granulated -sugar filter by a piece
of rubber tubing. Instead of transferring the soluble filter to gela-
tin in test tubes, they use a large glass cylinder having a slender
stem, in which the sugar is placed (Fig. 193). After the aspiration
liquefied gelatin is introduced into the large glass cylinder, which is
held in a horizontal position ; the sterilized cotton plug is then re-
placed in the mouth of the cylinder, the sugar is pushed into the
liquefied gelatin and dissolved, and by rotating the cylinder upon a
block of ice the gelatin is spread upon its walls as in an Esmarch roll
tube. For convenience in counting the colonies lines are drawn upon
the surface of the cylinder, dividing it into squares of uniform di-
mensions.

GENERAL RESULTS OF RESEARCHES MADE.

As already stated, the presence of bacteria in the atmosphere de-
pends upon their being wafted by currents of air from surfaces where
they are present in a desiccated condition. That they are not carried
away from moist surfaces is shown by the fact that expired air from
the human lungs does not contain microorganisms, although the in-
spired air may have contained considerable numbers, and there are
always a vast number present in the salivary secretions. The moist
mucous membrane of the respiratory passages constitutes a germ
trap which is much more efficient than the glass slide smeared with
glycerin used in some of the aeroscopes heretofore described, for it
is a far more extended surface. As a matter of fact, most of the sus-
pended particles in inspired air are deposited before the current of
air passes through the larynx.

Air which passes over large bodies of water is also purified of its
germs and other suspended particles. The researches of Fischer
show that at a considerable distance from the land no germs are
found in the atmosphere over the ocean, and that it is only upon ap-
proaching land that their presence is manifested by the development
of colonies upon properly exposed gelatin plates.

Uffelmann found, in his researches, that in the open fields the
number of living germs in a cubic metre of air averaged two hundred
and fifty, on the sea coast the average was one hundred, in the court-
yard of the University of Rostock four hundred and fifty. The num-
ber was materiallv reduced after a rainfall and increased when a
dry land wind prevailed.

Frankland fonnd that fewer germs were present in the air in
winter than in summer, and that when the earth was covered with
snow the number was greatly reduced, as also during a light fall of
snow ; the air of towns was found to be more rich in germs than the

BACTERIA IX THE AIR.

air of the country ; the lower strata of the atmosphere contained
more than the air of elevated localities.

Von Freudenreich also found that the air of the country contained
fewer germs than that of the city. Thus in the city of Berne a cubic
metre of air often contained as many as two thousand four hundred
germs, while the maximum in country air was three hundred. His re-
sults corresponded with those of Miquel in showing that the number
of atmospheric organisms is greater in the morning and the evening,
between the hours of 6 and 8, than during the rest of the day. Neu-
mann, whose researches were made in the Moabite Hospital, found
the greatest number of bacteria in the air in the morning after the
patients able to sit up had left their beds and the wards had been
swept. The number of germs was then from eighty to one hundred
and forty in ten litres of air, while in the evening the number fell to
four to ten germs in ten litres.

Miquel has given the following summary of results obtained in
his extended experiments, made in Paris during the years 1881, 1882,
and 1883 :

Average for
« «

« «

1880

Number of Germs in a Cubic Metre of Air.

Air of Laboratory,
Montsouri.

Air of Park, Mont
souri.

215
348
550

71
62
51

1881

1882..

Rue de Rivoli, average for one year, 750 ; summit of Pantheon, 28 ;
Hotel-Dieu, 1880, average for four months, male ward 6,300, female
ward 5,120 ; La Piete Hospital, average of fifteen months, 11,100.

It must be remembered that the figures given relate both to bac-
teria and to the spores of mould fungi, and that the latter are com-
monly the most numerous when the experiment is made in the open
air. Petri has shown that when gelatin plates are exposed in the air
the relative number of spores of mould fungi deposited upon them is
less than is obtained in aspiration experiments.

The number of colonies which develop on exposed plates does not
represent the full number of bacteria deposited, for these colonies
very frequently have their origin in a dust particle to which several
bacteria are attached, or in a little mass of organic material contain-
ing a considerable number.

It is generally conceded that sea air and country air are more
wholesome than the air of cities, and especially of crowded apart-
ments, in which the number of bacteria has been shown to be very
much greater. But it would be a mistake to ascribe the sanitary
value of sea, country, and mountain air to the relatively small num-

624 BACTERIA IN THE AIR.

her of bacteria present in such air. There are other important fac-
tors to be considered, and we have no satisfactory evidence that the
number of saprophytic bacteria present in the air has an important
bearing upon the health of those who respire it. We do know that
the confined air of crowded apartments, and especially of factories
in which a large quantity of dust is suspended in the air, predisposes
those breathing such air to pulmonary diseases and lowers the gen-
eral standard of health. But it has not been proved that this is due
to the presence of bacteria. Infectious diseases may, under certain
circumstances, be communicated by way of the respiratory passages
as a result of breathing air containing in suspension pathogenic bac-
teria ; but there is reason to believe that this occurs less frequently
than is generally supposed.

Kruger has shown that the dust of a hospital ward in which pa-
tients with pulmonary consumption expectorated occasionally upon
the floor contained tubercle bacilli. This was proved by wiping up
the dust on a sterilized sponge, washing this out in bouillon, and in-
jecting this into the cavity of the abdomen of guinea-pigs. Two
animals out of sixteen injected became tuberculous. In pulmonic
anthrax, which occasionally occurs in persons engaged in sorting
wool — "wool-sorters' disease" — infection occurs as a result of the
respiration of air containing the spores of the anthrax bacillus.

Among the non-pathogenic saprophytes iound in the air certain
aerobic micrococci appear to be the most abundant, and, as a rule,
bacilli are not found in great numbers or variety. In some localities
various species of sarcinse are especially abundant. The following
is a partial list of the species which have been shown by the researches
of various bacteriologists to be occasionally present in the air. But,
as heretofore remarked, their presence is to be regarded as acci-
dental, and so far as we know there is no bacterial flora properly be-
longing to the atmosphere :

Micrococcus urese (Pasteur), Diplococcus rosens (Bumm), Diplococcus
citreus conglomeratus (Bumm), Micrococcus radiatus (Fliigge), Micrococcus
flavus desidens (Fliigge), Micrococcus flavus liquefaciens (Fliigge), Micro-
coccus tetragenus versatilis (Sternberg), Micrococcus pyogenes aureus (Rosen-
bach), Micrococcus pyogenes citreus (Passet), Micrococcus cinnabareus
(Fliigge), Micrococcus flavus tardigradus (Fliigge), Micrococcus versicolor
(Fliigge), Micrococcus viticulosus (Katz), Micrococcus candidans (Fliigge),
Pediococcus cerevisias (Balcke), Sarcina lutea (Schroter), Sarcina rosea
(Schroter), Sarcina aurantiaca, Sarcina alba, Sarcina Candida (Reinke),
Bacillus tumescens (Zopf), Bacillus subtilis (Ehrenberg), Bacillus multipedi-
culosus (Fliigge), Bacillus mesentericus fuscus (Fliigge), Bacillus mesenteri-
cus ruber (Globig), Bacillus inflatus (A. Koch), Bacillus mesentericus vul-
gatus, Bacillus prodigiosus, Bacillus aerophilus (Liborius), Bacillus pestifer
(Frankland), Spirillum aureum (Weibel), Spirillum flavescens (Weibel), Spi-
rillum flavum (Weibel), Bacillus Havaniensis (Sternberg).

In the researches of Welz, made in the vicinity of Freiburg, twenty-
three different micrococci and twenty-two bacilli were obtained from the
air.

BACTERIA IX THE AIR. 625

ADDITIONAL NOTES UPON BACTERIA IN THE AIR.

Ruete and Enoch (1895) have examined the air of closed schoolrooms
with the following results. Eighteen different species were obtained, only
one of which proved to be pathogenic for mice, guinea-pigs, and rabbits. The
number of bacteria per cubic metre varied from 1,500 to 3,000,000, the aver-
age being about 268,000. The observations were made during the winter
months.

Marpmaiin (1893), in his examination of dust collected in the streets of Leip-
zig for tubercle bacilli, obtained positive results from a considerable pro-
portion of the specimens examined. Evidently these bacilli in dust from the
streets are liable to be blown into the air and deposited upon the mucous
membrane of the respiratory passages of those breathing this air. Christian!
(1893) has shown that, as a rule, no bacteria are present in the air at an alti-
tude of one thousand metres or more above the soil (air collected during
balloon ascensions).

Dyar (1895) has made a careful study of the microorganisms found in. the
air in the city of New York. He has described numerous species of micro-
cocci and bacilli found chiefly in the air of the hallway of the College of
Physicians and Surgeons. Some of these are new and some have been
identified as previously described species.

II.

BACTERIA IN WATER.

THE water of the ocean, of lakes, ponds, and running streams
necessarily contains bacteria, as they are constantly being carried
into it by currents of air passing over the neighboring land surfaces,
and by rain water which washes suspended microorganisms from
the atmosphere ; and, as such water contains more or less organic
material in solution, many of the saprophytic bacteria multiply in it
abundantly. It is only in the water of springs and wells which
comes from the deeper strata of the soil that they are absent. The
number and variety of species present in water from any given
source will depend upon conditions relating to the amount of organic
pabulum, the temperature, the depth of .the water, the fact of its
being in motion or at rest, its pollution from various sources, etc.
The comparatively pure water of lakes and running streams contains
a considerable number of bacteria which find their normal habitat
in such waters and which multiply abundantly in them, notwith-
standing the small quantity of organic matter and salts which they
contain. The water of stagnant, shallow pools, and of sluggish
streams into which sewage is discharged, contains a far greater
number and a greater variety of species.

The study of these bacteria in water has received much attention
on account of the sanitary questions involved, relating to the use of
water from various sources for drinking purposes. In the present
section we shall first give an account of the methods of bacteriologi-
cal water analysis, and then a condensed statement of results ob-
tained in the very numerous investigations which have been made.

A very important point to be kept in view is the fact that a great
increase in the number of bacteria present, in samples of water col-
lected for investigation, is likely to occur if these samples are kept
for some time. A water which, for example, contains only two
hundred to three hundred bacteria per cubic centimetre when the ex-
amination is made at once, may contain several thousand at the end
of twenty-four hours, and at the end of the second or third day
twenty thousand or more may be present in the same quantity.

BACTERIA IX WATER. 627

Later, on account of the exhaustion of organic pabulum, the num-
ber is again reduced as the bacteria present gradually lose their
vitality. Under these circumstances it is evident that an estimate of
the number of bacteria present in water from a given source can
have no value, unless a sample is tested by bacteriological methods
within a short time after it has been collected. Not more than an
hour or two should be allowed to elapse, especially in warm weather.
By placing the water upon ice the time may be extended somewhat,
but Wolffhiigel has shown that the number of germs is gradually
diminished when water is preserved in this way, and it will be safest
to make an immediate examination when this is practicable.

The collection may be made in a sterilized Erlenmeyer flask pro-
vided with a cotton air filter, or in a bottle having a ground-glass
stopper which has been wrapped in tissue paper and sterilized for an
hour or more at 150° C. in the hot-air oven. Or the small flasks with
a long neck may be used, as first recommended by Pasteur. These
are prepared as follows : The bulb is first gently heated, and the ex-
tremity of the tube dipped into distilled water, which mounts into

FIG. 1W.

the bulb as it cools ; the water is then made to boil, and when all
but a drop or two has escaped and the bulb is filled with steam the
extremity of the tube is hermetically sealed. When the steam has
condensed by the cooling of the bulb a partial vacuum is formed,
and the tube is ready for use at any time. It is filled with water by
breaking off the sealed extremity under the surface of the water of
which a sample is desired. This is done with sterilized forceps, and
care must be taken that the exterior of the tube is properly sterilized
before the collection is made. The end is immediately sealed in the
flame of a lamp. A difficulty with these vacuum tubes is that they
are so completely filled with water that this cannot be readily drawn
from them again in small quantities. The writer therefore prefers
to make the collection in a tube shaped as shown in Fig. 194, in which
a partial vacuum is formed just before the collection by heating tho
air in the bulb. The water mounts into the tube as the air in tho
bulb cools, and is readily forced out again for making cultures by
applying gentle heat to the bulb. As a lamp is needed to seal the end
of the tube in either case, there is no special advantage in having a
vacuum formed in advance, and, as stated, the vacuum tubes are so

628

BACTERIA IX WATER.

nearly filled with water that it is not so simple a matter to obtain the
contents for our culture experiments without undue exposure to at-
mospheric germs. In practice small glass bottles with ground-glass
stoppers will be found most convenient, and, when properly steril-
ized, are unobjectionable. They should be filled at a little distance
below the surface, as there is often a deposit of dust upon the surface

of standing water, and sometimes a
delicate film made up of aerobic bac-
teria. When water is to be obtained
from a pump or a hydrant it should
be allowed to flow for some time before
the collection is. made. To collect
water at various depths the apparatus
shown in Fig. 195 is recommended by
Lepsius. An iron frame supports an
inverted flask, A, filled with sterilized
mercury and containing about three
hundred cubic centimetres. The flask
B is intended to receive the mercury
when, at the desired depth, it is al-
lowed to flow through the capillary
tube b. This is sealed at the extremity
and bent as shown in the figure. By
pulling upon the cord c this tube is
broken, and as the mercury flows from
the flask this is filled with water
through the tube a. The extremity
of the broken tube b is closed by the
mercury in the flask B when A is full
of water, and the apparatus can be
brought to the surface with only such
water as was collected at the depth
from which a sample was desired.

The bacteriological analysis is
made by adding a definite quantity
of the water under investigation to
liquefied gelatin or agar-gelatin, and
making a plate or Esmarch roll tube, which is put aside for the devel-
opment of colonies. Miquel and others have preferred to use liquid
cultures and the method of fractional cultivation described in the
previous section. The use of a solid culture medium has, however,
such obvious advantages that we do not consider it necessary to do
more than refer to the other method as one which, when applied
with skill and patience, may give sufficiently accurate results.

FK,. 1 1

BACTERIA IX WATER. 629

The amount of water which should be added to the usual quan-
tity of liquefied flesh-peptone-gelatin in a test tube, in order that the
colonies which develop may be well separated from each other and
easily counted, can only be determined by experiment. If the water
is from an impure source a single drop may be too much, and it will
be necessary to dilute it with distilled water recently sterilized. But
for ordinary potable water it will usually be best, in a first experi-
ment, to make two trials, one with one cubic centimetre and one
with one-half cubic centimetre added to the liquefied nutrient gelatin.
The water in the collecting bottle should be shaken, to distribute the
bacteria which may have settled to the bottom, before drawing off by
means of a sterilized pipette the amount used for the experiment, and
the germs present in it are to be distributed through the liquefied
gelatin by gently moving the tube to and fro.

Koch's method of preparing a gelatin plate is illustrated in Fig.
1'JO. A glass dish, containing ice water and covered with a large

. lit*;.

plate of glass, is supported upon a levelling tripod. By means of a
spirit level this is adjusted to a horizontal position, so that when the
liquefied gelatin is poured upon the smaller sterilized glass plate, seen
in the centre of the large plate of glass, it will not flow, but may be
evenly distributed over the surface by means of a sterilized glass rod.
The glass cover resting against the side of the apparatus is placed
over the gelatin plate while it is cooling, to protect it from atmo-
spheric germs, and when the gelatin is hard the plate is transferred
to a shallow glass dish, which is kept at a temperature of about
20° C. for several days for the development of colonies. It is difficult
to count colonies when more than five thousand develop upon a plate
of the usual size, and for this reason it will be best to repeat the ex-
periment with a smaller quantity of water from the same source, if
this is at hand, rather than to attempt to count an overcrowded
plate. Before pouring the gelatin upon the plate the lip of the test
tube containing it should be sterilized by passing it through a flame.
The liquefied gelatin should ba carefully distributed to cover a rect-

630 BACTERIA IN WATER.

angular surface and leaving a margin of about one centimetre around
the edge of the plate. The Koch's dish in which the gelatin plate is
placed for the development of colonies should be carefully sterilized
by heat or by washing it out with a sublimate solution. A circular
piece of filtering paper, saturated with sublimate solution or distilled
water, is placed at the bottom of the lower dish to keep the air in a
moist condition and prevent drying of the gelatin. Usually two or
three plates made at the same time are placed one above the other on
glass supports made for this purpose. If many liquefying organisms
are present it will be necessary to count the colonies before these run
together — usually on the second day ; but in the absence of liquefy-
ing colonies it is best to wait until the third, or even the fifth day, as
the number of visible colonies and the ease of counting them will be
greater than at an earlier date. The development of a few scattered
liquefying colonies which threaten to spoil the plate may be arrested
by taking up the liquefied gelatin from each with a bit of filtering
paper, and then, by means of a camers-hair brush, applying a solu-
tion of potassium permanganate to the margin of the colony. The
growth of colonies of mould fungi, which have developed from spores,
from the atmosphere falling upon the plate while it is exposed, can
be checked by the application of collodion containing bichloride of
mercury.

Counting of the colonies is a simple matter when they are few
in number ; when they are numerous it is customary to place the
plate over a dark background, and to place above it a glass plate
divided into square centimetres by lines ruled with a diamond. By
means of a lens of low power the colonies in a certain number of
squares are counted and the average taken. This multiplied by the
number of square centimetres in the gelatin-covered surface gives
approximately the entire number of colonies which have developed
from the amount of water used in the experiment.

Instead of using Koch's original plate method, as above described,
the shallow, covered glass dishes recommended by Petri may bo
employed. These are from one to one and one-half centimetres high
and from ten to fifteen centimetres in diameter. The liquefied gel-
atin is poured into the lower dish and the cover at once placed over
it. The gelatin does not dry out very soon, but, if necessary, several
of these Petri's dishes may be placed in a larger jar, which serves as
a moist chamber.

The roll tubes of Esmarch may also be used, and have the ad-
vantage that accidental colonies from air-borne germs are excluded.
The counting of colonies is not quite as easy, but by the use of a
mounted lens especially designed for the purpose it is attended with
no great difficulty. The surface of the tube is divided into squares

BACTERIA IX WATER. 631

by colored lines, and the number of colonies in several squares is
counted in order to obtain an average and estimate the entire
number.

Water which contains numerous liquefying bacteria had better
be examined by the use of nutrient agar instead of gelatin; and in
very warm weather it will be necessary to use an agar medium, as
ten-per-cent gelatin is likely to melt if the temperature goes above
22° C. A difficulty in the use of agar for plates consists in the lia-
bility of the film to slip from the glass. This may be remedied to
some extent by adding a few drops of a concentrated solution of gum
acacia to the liquefied agar medium. Petri's dishes are well adapted
for the use of the agar medium, as the objection referred to does not
apply to them. The gelatin-agar medium, containing 5 per cent
of gelatin and 0. 75 per cent of agar, may also be used with advan-
tage in the bacteriological analysis of water. Much stress was at
one time laid upon the enumeration of liquefying colonies, upon
the supposition that the liquefying bacteria were especially harmful
as compared with the non-liquefying, and that a water containing
many liquefying colonies was to be looked upon with suspicion. We
now know, however, that there are many common and harmless
saprophytes which cause the liquefaction of gelatin, and that some
of the most dangerous pathogenic bacteria do not liquefy gelatin.
This distinction has therefore no special value, and the question for
bacteriologists to-day is not how large is the comparative number of
liquefying colonies, but what species are represented by the colonies
present, liquafying and non-liquefying, and what are the special
pathogenic properties of each. The answer to these questions, in
the case of any particular water supply, calls for special knowledge
and great patience and care in the isolation in pure cultures, and
careful study of the various species present.

It is now generally recognized that a mere enumeration of the
number of colonies which develop from a water under investigation
is not a sufficient indication upon which to found an opinion as to its
potability. An excessive number of bacteria is an indication that
the water contains a large amount of the organic material which
serves as pabulum for these microorganisms. But the chemists are
able to determine the amount of organic matter present in water
with greater precision ; and, as we have seen, the number of bacteria
may increase many-fold in water which is kept standing in the labo-
ratory for two or three days in a well-corked bottle. As a matter of
fact, the enumeration of bacteria in water, although it has given us
results of scientific interest, has not materially added to the methods
previously applied for estimating the sanitary value of water ob-
tained from various sources for drinking purposes. But the bacte-

632 BACTERIA IX AVATER.

riological examination may prove to be of great value if it succeeds
in demonstrating the presence of certain pathogenic bacteria and in
thus preventing the use of a dangerous water. We do not mean to
say, however, that an enumeration of the bacteria present in drink-
ing water has no practical value. An excessive number indicates an
excessive amount of organic pabulum, which may have come from
a dangerous source; and the dangerous pathogenic bacteria are not
only more likely to be present in such water, but they can more
readily multiply in it, while in a pure water they would fail to in-
crease in number, and, as has been shown by experiment, would die
out within a short time.

The number of bacteria present in rain water, or in snow which
has recently fallen, varies greatly at different times. Naturally the
number is greater when the surface of the earth is dry and the at-
mosphere loaded with dust by currents of wind passing over it, and
less when the surface is moist and the atmosphere has been purified
by recent rains.

In snow from the surface of a glacier in Norway, Schmelck found
two bacteria and two spores of mould fungi per cubic centimetre of
water from the melted snow. Ganowski, in experiments made with
freshly fallen snow collected in the vicinity of Kiew, obtained the fol-
lowing results : February 2d, 1888 : temperature of the air, 7.2° C. ;
snowfall, 0. 1 millimetre ; number of bacteria in 1 cubic centimetre
of water from melted snow, 34 in one sample and 38 in another.
February 20th, 1888: temperature, 11.1° C. ; snowfall, 1.1 milli-
metres ; number of bacteria in one sample, 203, in another 384.

Miquel obtained from rain water collected at Montsouri during a
rainy season 4. 3 germs per cubic centimetre ; in rain water collected
in the centre of the city of Paris, 19 per cubic centimetre.

.HcuZhas also been shown to contain bacteria inconsiderable num-
bers. Bujwid found in hailstones which fell at Warsaw 21,000
bacteria in 1 cubic centimetre ; but this is exceptional, and is supposed
to be due to the fact that surface water had been carried into the air
by the storm and frozen. Fontin examined hail which fell in St.
Petersburg, and obtained an average of 729 bacteria per cubic centi-
metre of water from the melted hail.

River water has been carefully examined by numerous bacterio-
logists in various localities and at different seasons of the year. We
give below some of the results reported :

Water of the Seine at Choisy, before reaching Paris, 300 ; at
Bercy, 1,200 ; at Saint-Denis, after receiving the sewer water from
the city, 200,000 germs per cubic centimetre (Miquel).

Water of the Spree beyond Kopenick, 82,000 ; two hundred steps
below the mouth of the Wuhle, 118,000 ; in Berlin above the mouth

BACTERIA IX WATER. 633

of the Panke, 040.000; below the mouth of the Panke, 1,800,000
(Koch).

Water of the Main above the city of Wiirzburg, in the month of
February, 530 ; below the city, 15,500 (Rosenberg).

Water of the Potomac, at Washington, in 1886 : January, 3,774 ;
February, 2,536; March, 1,210; April, 1,521; May, 1,004; June,
348 ; July, 255 ; August, 254 ; September, 178 ; October, 75 ; No-
vember, 116 ; December, 967 (Theobald Smith).

The Thames, in the autumn of 1885, in the vicinity of London
Bridge two hours after high water, contained 45,000 germs per cubic
centimetre ; the water of the Lea at Lea Bridge, 4,200,000 (Bisch-
off).

The Neva inside the city of St. Petersburg, in September, 1883,
contained 1,500 in one sample and 1,040 in another ; in November
(20th), 6,500 (Poehl).

The water of the Oder, collected within the limits of the city of
Stettin, was found by Link to contain from 5,240 to 15,000 bacteria
per cubic centimetre ; that of the Limmat, at Zurich, 346 in one
specimen and 508 in another (Cramer).

Lake water, as a rule, contains fewer bacteria than river water.

Wolff htigel, in researches extending from July, 1884, to July,
1885, obtained from the water of the Tegeler Lake an average of 396
bacteria per cubic centimetre. Cramer obtained an. average of 168
per cubic centimetre during the months of October, December, and
January, 1884, from the water of Lake Zurich ; in June of the same
year the average of 42 examinations gave 71 per cubic centimetre.
In 'Lake Geneva, Fol and Dunant obtained from water collected some
distance from the shore an average of 38 bacteria per cubic centi-
metre.

Ice which is usually collected from lakes and rivers contains a
greater or less number of bacteria, according to the depth and purity
of the water. The ice used in Berlin, collected from the surface of
lakes and rivers in the vicinity of the city, contains from a few hun-
dred to 25,000 bacteria to the cubic centimetre (Friinkel). In the ex-
periments of Heyroth samples of ice from the same source gave less
than 100 per cubic centimetre in three, from 100 to 500 in eight, from
500 to 1,000 in six, from 1,000 to 5,000 in seven, and 14,400 in one.

Prudden obtained from Hudson River ice, put up six miles below
the city of Albany, an average of 398 bacteria per cubic centimetre
from transparent ice, and in the superficial "snow ice" 9,187. Ice
collected lower down the river contained an average of 189 in the
transparent and 3,693 in the snow ice.

Ice from the Dora at Turin was found by Bordoni-Uffreduzzi to
contain from 120 to 3,546 bacteria per cubic centimetre.

634 BACTERIA IX WATER.

Hydrant ivater, as supplied to cities, has received the attention
of numerous investigators. The water supply of Berlin was ex-
amined by Plagge and Proskauer at intervals of a week from June,
1885, to April, 1886. Their tabulated results show considerable
variations. We give the figures for a single day, June 30th, 1885 :
Stralauer works, water of the Spree, unfiltered 4,400, filtered 53 ;
Tegeler works, water of the lake, unfiltered 880, filtered 44 ; high re-
servoir at Charlottenberg, 71 ; 75 W. Wilhelmstrasse, 121 ; Fried-
richstrasse, 41-42 S. W., 160 ; Schmidstrasse, 165 E., 51 ; Friedrich-
strasse, 126 N., 151 ; Weinmeisterstrasse, 15 C., 63.

Wells which are supplied by water from deep strata contain few
bacteria, unless contaminated by surface water in which they are
usually very abundant. Roth examined the water of sixteen surface
wells in Belgard, which has a very porous subsoil, and found from
4,500 to 5,000 bacteria in three, from 7,800 to 15,000 in six, from
18,000 to 35,000 in six, and 130,000 per cubic centimetre in one.

Forty-seven wells in Stettin, the water of which was examined by
Link, gave the following results : Less than 100 in six, 100 to 500 in
twenty-one, and in the remainder (sixteen) from 1,000 to 18,000.

Sixty-four wells in Mainz examined by Egger, and 53 in Gotha
by Becker, gave more favorable results ; the number of wells in the
former city, in which less than 100 colonies developed from 1 cubic
centimetre, was 34, and in the latter the same (34). Bolton examined
the water of 13 wells in Gottingen, and found but 1 in which the
number of colonies from 1 cubic centimetre was less than 100 ; in 12
the number varied from 180 to 4,940.

The water of deep wells and springs may be entirely free from
bacteria, or nearly so. Egger found in the water of an artesian well
at Mainz 4 bacteria per cubic centimetre, and the same number was
found by Hueppe in the deep well at the Wiesbaden slaughter-house.
The artesian well at the gasworks of Kiel was found by Brennig to
contain from 6 to 30 bacteria per cubic centimetre. In a spring at
Batiolettes, Fol and Dunant found 57 bacteria per cubic centimetre.
Fiirbringer obtained from springs at Jena 156 from one, 51 from
another, 32 from another, and 109 from another. The water supplied
to Danzig from the Prangenaur Spring was found in several experi-
ments to be free from bacteria (Freimuth).

In a summary of results obtained in various German cities Tie •
mann and Gartner find that sixty-nine per cent of the wells from
which samples of water were examined contained less than 500 bac-
teria per cubic centimetre.

The water of seivers is naturally rich in bacteria. Miquel found
that at Clichy the sewer water contained 6,000,000 bacteria per cubic
centimetre. Bischoff found in water from London sewers 7,500,000,

BACTERIA IX WATER. 635

and numerous observations show that tho number of bacteria in river
water is greatly increased in tlia vicinity of and below the mouths
of city sewers.

We conclude from the experimental data recorded that water
containing less than 100 bacteria to the cubic centimetre is presum-
ably from a deep source and uncontaminated by surface drainage,
and that it will usually be safe to recommend such water for drink-
ing purposes, unless it contains injurious mineral substances.
Water that contains more than 500 bacteria to the cubic centimetre,
although it may in many cases be harmless, is to be looked upon
with some suspicion, and water containing 1,000 or more bacteria is
presumably contaminated by sewage or surface drainage and should
be rejected or filtered before it is used for drinking purposes. But,
as heretofore .stated, the danger does not depend directly upon the
number of bacteria present, but upon contamination with pathogenic
species which are liable to be present in surface water and sewage.
In swallowing a glassful of pure spring water a number of bacteria
from the buccal cavity are washed away and carried into the stomach,
which, if enumerated, would doubtless far exceed in numbers those
found in the most impure river water.

The number of bacteria does not depend alone upon the amount
of organic pabulum contained in a water, and cannot be depended
upon in forming an estimate of this ; for, as has been shown by
Bolton, certain water bacteria multiply abundantly in water con-
taining comparatively little organic matter, while other species fail
to grow unless the quantity is greater. In a water containing con-
siderable nutrient material the water bacteria may be restrained in
their development by other species present until the amount of pabu-
lum is reduced so that these no longer thrive, when the common
water bacteria will take the precedence, and an enumeration may
show a greater number of colonies than at first. But, in general,
water rich in organic material contains a greater number of bacteria
and a greater variety of species than that which is comparatively
pure.

That certain bacteria may multiply in water which has been
carefully distilled has been shown by Bolton and others. Two com-
mon water bacteria — Micrococcus aquatilis and Bacillus erythrospo-
rus — multiplied abundantly in doubly distilled water, and when
this water was again sterilized and re-inoculated with one of these
species the same abundant increase occurred. This was repeated six
times with the same result (Bolton). Computing the number of
these water bacteria in ten cubic centimetres of distilled water at
twenty millions, and estimating their specific gravity at one, and the
diameter of the individual cells at one yw, the total weight of the entire

BACTERIA IN WATER.

number, according to Bolton, would be less than one-hundredth
of a milligramme, and at least three-fourths of this must consist of
water. The organic material represented by this number of bacteria
would therefore be so minute that it might be supplied by dust par-
ticles accidentally falling into the distilled water.

Rosenberg has shown that while many of the species which he
obtained in pure cultures from the water of the river Main multiplied
in sterilized distilled water, other species quickly died out in such
water. The growth of certain bacteria depends not only upon the
quantity of nutritive material present, but upon its quality, the con-
ditions in this regard being widely different for different species. .

In view of the facts heretofore stated bacteriologists are now giv-
ing more attention to a careful study of the kinds of bacteria pre-
sent in their examinations of water. Rosenberg, in his examinations
of the water of the Main in the vicinity of Wiirzburg (1886), found
that before the river reached the city the water contained more
micrococci than bacilli, but that after receiving the sewage of the
city the number of bacilli was greatly in excess.

Adametz (1888) has described eighty-seven species obtained by
him from water in the vicinity of Vienna ; Maschek found fifty-five
different species in the drinking water used at Laitmeritz; and Tils
(1890) has described fifty-nine species obtained by him from the city
water supply at Freiburg.

Among the pathogenic bacteria which are liable to find their
way into water used for drinking purposes, the most important, from
a sanitary point of view, are the bacillus of typhoid fever and the
spirillum of Asiatic cholera. Both of these microorganisms are pre-
sent in great numbers in the excreta of persons suffering from the
specific forms of disease to which they give rise, and are consequently
liable to contaminate wells and streams which receive surface water,
when such excreta are thrown upon the surface or into sewers, etc.
Epidemics of these diseases have frequently been traced to the use
of such contaminated water, and in a few instances the presence of
these specific disease germs in water has been demonstrated by bac-
teriological methods. Laboratory experiments indicate, however,
that an increase of these pathogenic bacteria in drinking water is not
likely to occur, except under special conditions, and that they die
out after a time, being at a disadvantage in the struggle for exist-
ence constantly going on among the numerous species which have
their normal habitat in water.

Bolton, Frankland, and others have shown that the anthrax ba-
cillus, not containing spores, dies out in hydrant water within five or
six days. In the experiments of Kraus the anthrax bacillus added
to well water, not sterilized, at a temperature of 10.5° C., was still

BACTERIA IN WATER. 637

present in a living condition on the second day, but 110 colonies de-
veloped aftar the third day ; the typhoid bacillus died out between
the fifth and seventh days ; the cholera spirillum was no longer found
on the second day. In the meantime the common water bacteria
had increased in numbers enormously. Similar results have been
reported by Hochstetter and others. Hueppe, in ten experiments in
which the typhoid bacillus was added to well water of a bad quality,
found that in two no development of this bacillus occurred after the
fifth day, while a few colonies developed in the other experiments as
late as the tenth day. In these experiments the temperature was
comparatively low (10.5° C.). At a higher temperature the experi-
ments of Wolffhugel and Riedel show that an increase may take
place. At the room temperature (about 20° C.) the typhoid bacillus
added to distilled water, to well water, and to Berlin hydrant water
was still present, in some instances, at the end of thirty-two days.
And it was found that in some cases a decrease in the number
occurred, then a notable increase, and finally a second diminution.

Koch found the cholera spirillum in a water tank at Calcutta
during a period of fourteen days, and in his experiments showed that
it preserved its vitality in well water for thirty days, in Berlin sewer
water for six to seven days, and in the same mixed with faeces for
twenty-seven hours only. In the experiments of Nicati and Rietsch
the cholera spirillum preserved its vitality in distilled water for
twenty days, in sewer water (of Marseilles) thirty-eight days, in
water of the harbor for eighty-one days. The numerous experiments
recorded by the observers named, and by Bolton, Hueppe, Hoch-
stetter, Maschek, Kraus, and others, show that while the cholera
spirillum may sometimes quickly die out in distilled water, in other
experiments it preserves its vitality for several weeks (Maschek), and
that it lives still longer in water of bad quality, such as is found in
sewers, harbors, etc. Bolton found that for its multiplication a
water should contain at least 40 parts in 100,000 of organic material,
while the typhoid bacillus grew when the proportion was considerably
less than this— 6.7 parts in 100,000.

Russell (1891) has studied the bacterial flora of the Gulf of
Naples, and of the mud at the bottom of this gulf, collected at
various depths up to eleven hundred metres. His investigations
show that sea water does not contain as many bacteria as an
equal volume of fresh water; that bacteria are found in about
equal numbers in water from the surface and in that from various
depths ; that the mud at the bottom constantly contains large num-
bers of bacteria ; that some of the species isolated grow best in a
Culture medium containing sea water.

At a depth of 50 metres the water contained 121 bacteria per cubic

BACTERIA IX WATER.

centimetre, and the mud from the bottom 245,000 ; at 100 metres the
water contained 10 and the mud 200,000 per cubic centimetre ; at
500 metres the water contained 22 and the mud 12,500 per cubic
centimetre ; at 1,100 metres the mud contained 24,000.

The following new species were obtained by Russell from the
source mentioned : Bacillus thalassophilus, Cladothrix intricata,
Bacillus granulosus, Bacillus limosus, Spirillum marinum, Bacillus
litoralis, 'Bacillus halophilus.

The bacterial flora of fresh and sea water is very extensive, as
will be seen by the following list of species which have been described
by various bacteriologists who have given their attention to its
study :

XOX-PATHOGEXIC MICROCOCCI.

Micrococcus aurantiacus (Cohii), Micrococcus luteus (Cohn), Micrococcus
violaceus (Cohn), Micrococcus flavus liquefaciens (Fliigge), Micrococcus fla-
vus desideiis (Fliigge), Micrococcus radiatus (Fliigge), Micrococcus ciimaba-
reus (Fliigge), Micrococcus flavus tardigradus (Fliigge), Micrococcus versi-
color (Fliigge), Micrococcus agilis (Ali-Cohen), Micrococcus fuscus (Maschek),
Diplococcus luteus (Adametz), Pediococcus albus (Lindner), Micrococcus
cerasiiius siccus (List), Micrococcus citreus (List), Micrococcus aquatilis
(Bolton), Micrococcus fervidosus (Adametz), Micrococcus plumosus (Brauti-
gam), Micrococcus viticulosus (Katz), Micrococcus cremoides(Zimmermann),
Micrococcus carneus (Zimmerman.n), Micrococcus concentricus (Zimmer-
mann), Micrococcus rosettaceus (Zimmermann), Micrococcus urese (Pasteur),
Weisser Streptococcus (Maschek), Wurmformiger Streptococcus (Maschek),
Micrococcus aerogenes (Miller), Sarcina alba, Sarcina Candida (Reinke),
Sarcina lutea.

PATHOGENIC MICROCOCCI.

Staphylococcus pyogenes aureus (Rosenbach), Micrococcus of Heyden-
reich — " Micrococcus Biskra/'

XOX-PATHOGEXIC BACILLI.

Bacillus arborescens (Fraiikland), Bacillus viscosus (Frankland), Bacil-
lus aquatilis (Frankland), Bacillus liquidus (Frankland), Bacillus nubilis
(Frankland), Bacillus vermicularis (Frankland), Bacillus aurantiacus
(Frankland), Bacillus coeruleus (Smith), Bacillus glaucus (Maschek), Bacil-
lus albus putidus (Maschek), Bacillus fluoresceiis liquefaciens. Bacillus ftuo-
rescens mvalis (Schmolck), Bacillus lividus (Plagge and Prpskauer), Bacil-
lus rubidus (Eisenberg), Bacillus sulfureum (Holschewriikoff), Bacillus
violaceus, Bacillus gasoformans (Eisenberg), Bacillus liquefaciens (Eisen-
berg), Bacillus phosphorescens indicus (Fischer), Bacillus phosphoresces s
indigenus (Fischer), Bacillus phosphorescens gelidus (Katz), Bacillus sma-
ragdino-phosphoresceiis (Katz), Bacillus argenteo-pbosphorescens Nos. I.,
II., and III. (Katz), Bacillus cyaneo-phosphoresceiis (Katz), Bacillus ar-
genteo-phosphorescens liquefaciens (Katz), Bacillus ramosus, Bacillus sub-
tilis (Ehrenberg), Proteus sulfureus (Lindenborn), Bacillus aureus (Ada-
metz), Bacillus brunneus (Adametz), Bacillus flavocoriaceus (Adametz),
Bacillus fluorescensnon-liquefaciens, Bacillus latericeus (Adametz), Bacillus
stolonatus (Adametz), Bacillus berolinensis indicus (Classen), Bacillus ery-
throsporus (Eidam), Bacillus luteus (List), Bacillus aquatilis sulcatus Nos.
1, 2, 3, 4, ar>d 5 (Weichselbaum), Bacillus albus (Eisenberg), Bacillus multi-
pediculosus( Fliigge), Bacillus Ziirnianum (List), Bacillus fulvus (Zimmer-
mann), Bacillus helvolus (Zimmermann), Bacillus ochraceus (Zimmer-

BACTERIA IN WATER. 639

maim), Bacillus plicatus, Bacillus devorans (Zimmermann), Bacillus gracilis
(Zimmermann), Bacillus guttatus (Zimmermann), Bacillus implexus (Zim-
mermann), Bacillus punctatus (Zimmermann), Bacillus radiatus aquatilis
(Zimmermann), Bacillus vermiculosus (Zimmermann), Bacillus constrictus
(Zimmermann), Bacillus fluorescens aureus (Zimmermann), Bacillus fluo-
rescens longus (Zimmermann), Bacillus fluorescens tenuis (Zimmermann),
Bacillus fuscus (Zimmermann), Bacillus rubefaciens (Zimmermann), Bacil-
lus subflavus (Zimmermann), Bacillus janthinus (Zopf), Bacillus mycoides
(Fliigge), Bacillus tremelloides (Tils), Bacillus cuticularis (Tils), Bacillus
filiformis (Tils), Bacillus ubiquitus (Jordan), Bacillus circulans (Jordan),
Bacillus superficialis (Jordan), Bacillus reticularis (Jordan), Bacillus ru-
bescens (Jordan), Bacillus hyalinus (Jordan), Bacillus cloacae (Jordan),
Bacillus delicatulus (Jordan), Bacillus violaceus laurentius (Jordan).

PATHOGENIC BACILLI.

Bacillus typhi abdominalis (Eberth, Gaffky), Bacillus erysipelatos suis
(" Bacillus murisepticus, " Koch), Bacillus septicaemias haemorrhagicae
("Bacillus cuniculicida," Koch), Proteus vulgaris (Hauser), Proteus mira-
bilis (Hauser), Bacillus canalis capsulatus (Mori), Bacillus canal is parvus
(Mori), Spirillum choleroe Asiaticse (''Comma bacillus," Koch), Bacillus coli
communis (Escherich), Bacillus hydrophilus fuscus (Sanarelli), Bacillus
venenosus (Vaughan), Bacillus yenenosus brevis (Vaughan), Bacillus vene-
nosus invisibilis (Vaughan), Bacillus venenosus liquefaciens (Vaughan).

The following additional species are described by Zimmermann (1894) in
his second publication ("Die Bakterien unserer Trink- und Nutzwasser ").
Micrococcus candidus, Micrococcus coralloides, Streptococcus cinereus, Mi-
crococcus sulphureus, Micrococcus galbaiiatus, Micrococcus erythromyxa,
Sarcina flavea, Sarcina aurantiaca, Sarcina rosea. Bacillus ruber, Bacillus
miniaceus, Bacillus mesentericus roseus, Bacillus carnosus, Bacillus chryso-
gloia, Bacillus multipediculus flavus, Bacillus villosus, Bacillus radiatus,
Bacillus fluorescens albus, Bacillus viridans, Bacillus turcosa, Bacillus halans,
Bacillus nacreaceus, Bacillus mirabilis, Bacillus umbilicatus, Bacillus lactis
viscosus, Bacillus synxaiithus, Bacillus sericeus, Bacillus minutus, Bacillus
stellatus, Bacillus radicosus, Bacillus vernicosus, Bacillus mucosus, Bacillus
centralis, Bacillus spumosus, Bacillus aimulatus, Bacillus liquefaciens, Bacil-
lus disciformans.

The following spirilla and " vibrios" have also been found in water —
chiefly in river water :

Spirillum volutans, Spirillum sanguineum, Spirillum serpens, Vibrio ru-
gula, Spirillum plicatile, Spirillum marinum (Russell). Spirillum cholerae
Asiaticae, Spirillum of Renon, Vibrio aquatilis (Gunther), Vibrio of Weibel,
Vibrios of Bujwid (Bacillus choleroides a and 6), Vibrio of Loftier, Vibrios
of Bonhoff, Vibrio of Blackstein, Vibrios of Sanarelli, Vibrios of Fischer,
Vibrio Berolinensis, Vibrio Danubicus, Vibrio of Pfuhl (v. Metchnikovi ?).
Several of the " vibrios" in this list which have recently been obtained from
river water in various parts of Europe are probably varieties of the cholera
spirillum.

ADDITIONAL NOTES UPON BACTERIA IN WATER.

It is now generally recognized by bacteriologists that the potability of
water is to be determined by an investigation relating to the presence or ab-
sence of known pathogenic bacteria, rather than by an estimate of the num-
ber of bacteria present in each cubic centimetre of the water under exami-
nation. From a sanitary point of view the most important of these pathogenic
bacteria are the cholera spirillum and allied " vibrios," the bacilli of the " ty-
phoid group " (Bacillus typhi abdominalis and allied forms), the bacilli of
the "colon group" (Bacillus coli communis with its varieties and similar
bacilli of faecal origin). When one of these pathogenic bacilli is present in a

G40 BACTERIA IN WATER.

water-supply hi small numbers as compared with the number of saprophytic
bacteria, it is not an easy matter to demonstrate the fact by the ordinary
plate method, especially in the case of non-liquefying species like the typhoid
bacillus. If we harve, for example, one typhoid bacillus to one thousand ba-
cilli of other species it is evident that in a series of three plates, made in the
usual way for the purpose of obtaining isolated colonies, there would be but a
small chance of obtaining a colony of the typhoid bacillus in plate No. 3,
and a plate containing one thousand colonies or more would be so crowded
that the detection of the single typhoid colony would be very difficult. For
this reason, it is necessary to resort to special methods by which the more
numerous saprophytic bacteria will be excluded, or their numbers greatly
reduced. Some of the methods which have been successfully employed for
the detection of the typhoid bacillus and of the cholera spirillum are given
in the sections devoted to these microorganisms. We give below some de-
tails relating to the methods employed by bacteriologists of recognized com-
petence in recent investigations :

Marpmaim (1895) considers all water which contains faecal bacteria as
dangerous as a supply for drinking purposes. For the detection of patho-
genic bacteria he recommends the following procedure :

The pathogenic bacteria are divided into two groups by cultivation in nu-
trient agar containing 0.2 percent of citric acid, and in the same medium con-
taining two per cent of sodium carbonate. The bacilli of the typhoid group
are said to grow in the acid medium but not in that containing two per cent
of sodium carbonate. On the other hand, cholera vibrios develop in the al-
kaline medium but not in that containing 0.2 per cent of citric acid. The ba-
cilli of the colon group also (" cloaca-bacilli") do not grow in the medium
containing citric acid. Bouillon containing the same amounts of acid and
alkali is also employed. The water to be examined is first mixed with an
equal portion of acid and of alkaline bouillon in two test tubes, and these are
kept at a temperature of 30° C. for twenty -four hours, during which time
the pathogenic bacteria, if present, will multiply and cause a clouding of the
culture media. Inoculations are now made into the acid arid alkaline agar
and gelatin. Growth in alkaline gelatin at the room temperature (10° to 18°
C.) is due to "cloaca-bacteria" ; growth in acid gelatin at 20° to 23° C. is due
to bacilli of the typhoid group. Plates should also be made from the clouded
bouillon, acid and alkaline ; and the colonies resembling those of the typhoid
or of the colon group should be tested in nutrient gelatin containing sugar
to ascertain whether there is development of gas, in which case the bacilli are
of the colon group.

When typhoid and colon bacilli are associated in water the last-mentioned
bacillus takes the precedence, and the typhoid bacillus has a tendency to dis-
appear. This is shown by the experiments of Girnbert (1894), who introduced,
at the same time, colon bacilli and typhoid bacilli into water, and found that
at the end of forty-eight hours he was no longer able to isolate the typhoid
bacillus from plates. In view of this fact failure to find the typhoid bacillus
does not relieve the water from the suspicion of being dangerous if the colon
bacillus is present. But, on the other hand, this bacillus is so common that
it is perhaps the exception when it is not present in surface waters. As
pointed out by von Freudenreich (1895) it may, however, escape detection
unless a considerable quantity of water is used in making the test. When
the quantity is from one hundred to five hundred cubic centimetres, in-
stead of from one to five cubic centimetres, as was formerly the usual amount
employed, it is found not infrequently even in spring water (von Freuden-
reich).

The author last mentioned says that when present in small numbers it
may be demonstrated by the method of Vincent, as follows : Mix of the water
ninety cubic centimetres with ten cubic centimetres of a twenty-per-cent
solution of peptone, and one cubic centimetre of a seven-per-cent solution of
carbolic acid; place in the incubating oven at 42 C. If development «K>

BACTERIA IN WATER. 641

curs it will probably be due to the colon bacillus, but it will be necessary to
make plates and pure cultures from single colonies in order to determine
this with certainty. The demonstration may be made more quickly, accord-
ing to von Freudeiireich, by using a medium containing milk sugar (five per
cent) and cultivating at 35° C. If the colon bacillus is present there will be
an abundant development of gas in from twelve to twenty -four hours, and
the bacillus may then be readily isolated by the plate method. The colon
bacillus has been found byMoissan and Gimbert in mineral waters bottled in
France. Poncet (1895) has made a careful study of the bacteria found in the
various springs at Vichy. The species described are all harmless water bac-
teria and have little interest from a sanitary point of view.

Kruse (1894), as a result of his extended researches and of a critical con-
sideration of the experimental data available, arrives at the conclusion that a
sanitary inspection of the sources of supply is more important, in determin-
ing the safety of the supply from a sanitary point of view, than a chemical
or bacteriological examination. The writer has for some years past enter-
tained the same opinion. Kruse says, however, that for the control of fil-
tering plants bacteriological "counting-methods" are indispensable. He
also ascribes a "high scientific value" to investigations relating to the pres-
ence of the more important pathogenic bacteria; but says that, notwith-
standing the improvements in methods of research, we cannot wait for a
demonstration of the presence of the cholera or typhoid bacteria before con-
demning a water as probably unsafe, if sources of contamination are dis-
covered— or, we would add, if cases of cholera or typhoid fever can be traced
with a fair degree of certainty to the use of water from a given source.

Fischer (1894), in his account of the researches made during the Plankton
expedition, has given a summary of the experimental evidence relating to the
presence of bacteria in the waters of the ocean. The species found were for
the most part different from those found in lakes and rivers, and at some
distance from the shore none of the previously known species of micrococci
and bacilli were encountered. The number of bacteria in samples from the
surface at a distance from the shore was comparatively small (usually less
than five hundred per cubic centimetre), but in the vicinity of land very
large numbers were sometimes found. At a distance of ten metres below the
surface the number found was greatly in excess of the number at the surface
— the difference being probably due to the germicidal action of sunlight. At
depths of four hundred metres bacteria were constantly found in great num-
bers, and water from a depth of eleven hundred metres was still found to
contain them.

III.

BACTERIA IN THE SOIL.

SURFACE soil, and especially that which is rich in organic matter,
contains very numerous bacteria of many different species. Some of
these are of special interest on account of their pathogenic power.
Thus the bacillus of malignant oedema and the bacillus of tetanus
have been shown to be widely distributed species, which have been
obtained by investigators in various parts of the world by inoculating
susceptible animals — guinea-pigs or mice — with a little rich surface
soil. Other species are interesting because of their action in nitrifi-
cation and in the destructive decomposition of organic material by
which it is fitted for assimilation by the higher plants. Many of the
bacteria present in the soil are strictly anaerobic, and in attempts to
estimate the number and kind of microorganisms present in a given
sample this fact must be kept in view.

The simplest method of studying the bacteria in the soil consists
in introducing a small quantity into liquefied gelatin in test tubes,
and, after carefully crushing it with a sterilized glass rod and thor-
oughly mixing it with the gelatin, making roll tubes in the usual
way. Some of these should be put up for anaerobic cultures — i.e.,
the tube should be filled with an atmosphere of hydrogen according
to Frankel's method. If the object in view is to estimate the num-
ber of bacteria in a given sample of soil the difficulty is encountered
that, however finely crushed, the little masses of earth are likely to
contain numerous bacteria, and we cannot safely assume that each
colony originates from a single germ. Thoroughly washing a small
quantity of soil, by agitation, in a considerable quantity of distilled
water, and then adding a definite quantity of the water to nutrient
gelatin and making roll tubes or plates, as in water analysis, sug-
gests itself as a simple method ; but Frankel has shown that it is far
from being reliable when the object is to estimate the number of
bacteria. He obtained more uniform and accurate results by intro-
ducing the earth at once into liquefied gelatin and crushing it as
thoroughly as possible with a strong platinum wire, after which as
thorough a mixture as possible was effected by tilting the tube up

BACTERIA IN THE SOIL. 643

and down. But for the purpose of obtaining pure cultures from sin-
gle colonies of the various species present, we should prefer to wash
the earth in distilled water and to allow the sediment to settle before
taking a portion of the water to add to the nutrient medium.

In some experiments made in 1881 Koch ascertained that in soil
which had not been disturbed but few bacteria were to be found at
the depth of a metre; and this fact has since been established by the
extended researches of Frankel, who devised a special boring instru-
ment for obtaining samples of earth from different depths. Miquel,
in 1879, estimated the number of bacteria in one gramme of earth
collected in the park of Montsouri, Paris, at a depth of twenty centi-
metres, at 700,000; and in a cultivated field which had been treated
with manure, at 900,000. The following results were obtained by
Adametz : One gramme of earth from a sandy soil contained at the
surface 380,000, at a depth of twenty to twenty-five centimetres
400,000 ; the same quantity of clayey soil contained at the surface
500,000, at a depth of twenty to twenty-five centimetres 460,000.

In experiments made by Beumer (1886) and by Maggiora (1887)
considerably greater numbers were found, but the last-named ob-
server, in some instances at least, kept the earth for some time after
collecting it, which may have materially influenced the result.
Beumer obtained from a specimen of sandy humus taken from a
depth of three metres 45,000,000 to the gramme ; at four metres,
10,000,000; at five metres, 8,000,000; at six metres, 5,000,000.
These specimens were obtained from the vicinity of hospitals at
Greifswald. In a churchyard, at a depth of four metres, the num-
ber in one experiment was 1,152,000, and in another 1,278,000.

Frankel has given special attention to the examination of undis-
turbed soil not in the immediate vicinity of dwellings. In samples
from a fruit orchard near Potsdam he found that the superficial
layers contained from 50,000 to 350,000 germs per cubic centimetre.
The greatest number was not immediately upon the surface, but at
from one-quarter to one-half metre below the surface. The num-
ber was found to be greater in summer than in winter, the maximum
being in July and August. At a depth of three-quarters of a metre
to a metre and a half there was a very great and abrupt diminution in
the number of germs. From 200, 000 at one-half metre the number fell
to 2,000 at a depth of a metre, from 250,000 at three-quarters of a
metre to 200 at one metre, etc. , and at a depth of one and one-half
metres, in some instances, no more living germs were obtained. In
other experiments a few colonies developed from earth obtained at a
depth of three or four metres, but these were slow in making their
appearanc'e, and often several days, or even weeks, elapsed before
they became visible in Esmarch roll tubes. In experiments with sur-

G44 BACTERIA IN THE SOIL.

face soil, on the contrary, a multitude of colonies developed within
twenty-four to forty-eight hours, and, as many liquefying bacteria
were present, it was necessary to make the enumeration on the first
or second day, at which time, no doubt, many of the bacteria present
had not yet formed visible colonies. The results obtained have,
therefore, only a relative value.

The most important fact developed by FrankePs researches is that
in virgin soil there is a dividing line at a depth of from three-quarters
to one and one-half metres, below which very few bacteria are found,
and that, consequently, the " ground- water region " is free from micro-
organisms, or nearly so, notwithstanding the immense numbers pre-
sent in the superficial layers.

The extended researches of Maggiora, made in the vicinity of
Turin, led him to the following conclusions :

1. The number of germs in desert and forest soils is much smaller, other
conditions being- equal, than in cultivated lands, and in these it is less than
in inhabited localities.

2. In desert soils the number of germs bears a relation (a) to the geologi-
cal epoch to which the lands belong, and, within certain limits, to the height
above the level of the sea — the older the soil and the greater the altitude,
other things being equal, the fewer the germs ; (&) to the compactness and
aeration of the soil — the more compact and impermeable to air the smaller
the number of germs capable of developing in gelatin ; (c) to the nature of
the soil — sandy soils contain fewer germs than soils rich in clay and in
humus.

3. In cultivated lands the number of germs augments with the activity
of cultivation and the strength of the fertilizers used.

4. In inhabited localities the number of germs in the superficial layers is
very great. In the deep layers it usually diminishes rapidly, as is the case
in all other soils.

As to the kinds of bacteria present, and their biological characters
and functions in preparing organic material for assimilation by the
plants whose roots penetrate the soil, we have yet much to learn.
Frankel remarks that the species most frequently encountered in the
deeper strata of the soil were three bacilli which also abound in the
superficial layers — viz., the " hay bacillus," the "wurzel bacillus,"
and the "hirnbacillus." In all eleven bacilli were isolated and cul-
tivated. Micrococci were only found four times, and spirilla not at
all. Mould fungi were more abundant, and especially one previously
obtained from the air by Hesse and called by him "brauner Schim-
melpilz." Anaerobic bacilli, contrary to expectation, were not ob-
tained in Frankel's researches, and no pathogenic species were found
in the deeper layers of the soil. We have already referred to the
fact that the bacillus of malignant oedema and the bacillus of tetanus,
two pathogenic, anaerobic species, are common in rich surface soil in
various parts of the world.

BACTERIA IX THE SOIL. 645

The results obtained in the researches referred to, in which nutri-
ent gelatin was used as a culture medium, are no doubt very in-
complete, not only on account of the liquefaction of the gelatin by
common liquefying bacilli before other species present have formed
visible colonies, but also because this is not a favorable culture me-
dium for some of the species present in the soil. Thus Frankland has
succeeded in isolating a nitrifying ferment which he calls " Bacillo-
coccus," which grows abundantly in bouillon, but fails to grow in
nutrient gelatin. Winogradski has also obtained in pure cultures a
nitrifying ferment from the soil in the vicinity of Zurich, which he
has called " Nitromonas."

Comparatively few micrococci are found in the soil, while in the
air they are usually found to be more abundant than bacilli. This
is perhaps due to the fact that the bacilli are more promptly destroyed
by desiccation and the action of sunlight.

Several bacteriologists have made investigations relating to the
duration of vitality of pathogenic bacteria in the soil. Frankel found
that in Berlin the bacillus of anthrax, in Esmarch roll tubes, when
buried in the soil at a depth of two metres, only occasionally gave
evidence of growth, and at three metres no development occurred.
The comparatively low temperature at this depth was no doubt an
important factor in influencing the result. The cholera spirillum in
the months of August, September, and October grew at a depth of
three metres, but in the remaining months of the year failed to grow
at two, while growth occurred at one and one-half metres. The
bacillus of typhoid fever grew at three metres during the greater
portion of the year.

Giaxa has made extended and interesting experiments with the
cholera spirillum, cultures of which he added to different kinds of
soil (garden earth, clay, sand) and placed at different depths below
the surface — one-quarter, one -half, and one metre. Some of the earth
was sterilized and some was not. In the unsterilized earth he found
the cholera spirillum in considerable numbers at the end of twenty-
four hours at the greatest depth tested (one metre), but at the end of
forty eight hours it had disappeared in five experiments out of seven
— the lowest temperature at this depth was 20° C. In the sterilized
soil the result was different ; the cholera spirillum was present in
enormous numbers at the end of four days at a depth of a metre,
and was still found in smaller numbers at the end of twelve days, but
had disappeared at the end of twenty-one days. These results indicate
that the presence of common saprophytes in the soil is prejudicial to
the development of the cholera spirillum, and that under ordinary
circumstances it succumbs in the struggle for existence with these
more hardy microorganisms.

C46 BACTERIA IN THE SOIL.

The researches of Proskauer (1891) confirm those of Frankel and
others as to the rapid diminution in the number of bacteria in the
deeper layers of the soil. They also agree with those of Gartner in
showing that in the soil of churchyards the number of bacteria
diminishes greatly in the soil beneath the layer containing coffins.
In general the influence of dead bodies upon the bacteria in the soil
in the vicinity of coffins was very slight; in the subsoil of the grave-
yard there were not many more bacteria than in similar soil outside
of this. Reimers had previously shown that samples of earth from
two graves, in one of which the body had been buried for thirty-five
years and in the other for one and one-half years, gave similar re-
sults when examined by bacteriological methods.

Manfredi in 1892 published the results of his extended investiga-
tions relating to the dust in the streets of Naples, The number of
bacteria varied greatly in different parts of the city. In streets
where the traffic was least and hygienic conditions the best the
average number was 10,000,000 per gramme. In dirty and busy
thoroughfares the average was 1,000,000,000, and in certain locali-
ties the number was even five times as great as this. Injections into
guinea-pigs gave a positive result in seventy-three per cent of the
animals experimented upon. Among the known pathogenic bacte-
ria obtained in this way were the pus cocci (in eight), Bacillus tuber-
culosis (in three), the bacillus of malignant oedema, and the tetanus
bacillus.

In the memoir of Fulles (1891) the following species are described
as having been found by him in the soil at Freiburg, Germany:

MICROCOCCI.

(a) Non-liquefying. — Micrococcus aurantiacus (Colin), Micrococcus can-
didus (Cohn), Micrococcus luteus (Cohn), Mjcrococcus candicans (Fliigge),
Micrococcus versicolor (Fliigge), Micrococcus cinnabareus (Fliigge), Micro-
coccus cereus albus (Passet), Micrococcus fervitosus (Adametz), Bother coc-
cus (Maschek*.

(b) Liquefying. — Micrococcus flavus liquefaciens (Fliigge), Micrococcus
flavus desidens (Fliigge), Diplococcus luteus (Adametz), Sarcina lutea.

NON-PATHOGENIC BACILLI.

(a) Non-liquefying. — Bacillus fluorescens putidus (Fliigge), Bacillus mus-
coides (Liborius), Bacillus scissus (Frankland), Bacillus candicans, Bacillus
diffusus (Frankland), Bacillus filiformis (Tils), Bacillus luteus (Fliigge),
Fluorescent water bacillus (Eisenberg), Bacillus viridis pallescens (Frick),
Bluish-green fluorescent bacillus (Adametz), Bacillus stolonatus (Adametz),
Bacillus Ziirnianum (List), Bacillus aerogenes (Miller), Bacillus No. 1 and
Bacillus No. 2 (Fulles).

(6) Liquefying.— Bacillus ramosus liquefaciens (Flugge\ Bacillus liqui-
dus (Frankland), Bacillus ramosus— "wurzel bacillus," Bacillus subtilis

BACTERIA IN THE SOIL. G47

(Ehrenberg), Bacillus mesentericus fuscus (Fliigge), Bacillus mesentericus
vulgatus (Fliigge), Bacillus fluorescens liquefaciens (Fliigge), Lemon-yellow
bacillus (Maschek), Green yellow bacillus (Eisenberg), Gas-forming bacillus
(Eisenberg), Gray bacillus (Maschek), Bacillus prodigiosus (Ehrenberg),
Proteus mirabilis (Hauser), Proteus vulgaris (Hauser), Bacillus mesentericus
vulgatus, Bacillus cuticularis (Tils), " Weisser bacillus" (Eisenberg).

In addition to the above the following species have been described by
other authors: Bacillus liquefaciens magnus (Liideritz), Bacillus radiatus
(Liideritz), Bacillus solidus (Liideritz), Bacillus mycoides roseus (Scholl),
Bacillus viscpsus (Frankland), Bacillus candicans (Frankland), Bacillus
poliformis (Liborius), Clostridium fcetidum (Liborius).

Pathogenic species. — Staphylococcus pyogenes aureus (Rosenbach), Ba-
cillus tetani(Nicolaier), Streptococcus septicus (Nicolaier), Pseudo-cedema ba-
cillus (Liborius), Bacillus septicus agrigenus (Nicolaier), Bacillus of Utpadel.

IV.

BACTERIA OF THE SURFACE OF THE BODY AND OF
EXPOSED MUCOUS MEMBRANES.

GREAT numbers of bacteria of various species multiply upon the
surface of the human body, where they find the necessary pabulum
in the excretions from the skin and the exfoliated epithelium. Evi-
dently the number will be largely influenced by the clothing worn,
the atmospheric conditions as to heat and moisture, personal habits,
etc. The writer has frequently inoculated culture media with a drop
of sterilized fluid which had been placed upon the surface of the body
of patients in hospitals and of healthy persons. By friction with a
platinum needle at the point where the drop of fluid is applied the
surface is washed and a little epithelium detached. Cultures may
always be obtained by inoculating nutrient media from a drop of fluid
applied in this way. Micrococci of various species, including the pus
cocci, are very commonly encountered ; sarcinse and various bacilli
are also frequently met with. Even the hands, which by reason of
their exposure and frequent ablutions are freer from exfoliated epi-
thelium than portions of the body covered with clothing, have con-
stantly attached to their surface a considerable number of bacteria.
This is shown by the experiments of Kiimmel and Forster, of Fiir-
bringer and others, with reference to the disinfection of the hands.
Forster found that after the most careful cleaning of the hands with
soap, water, and a brush, contact of the fingers with nutrient gelatin
always resulted in the development of a greater or less number of
colonies.

Bordoni-Uffreduzzi, in his researches relating to the bacteria of
the skin, obtained in pure cultures five different species of micrococci
and two bacilli. Pure cultures of his Bacterium graveolens, which
was usually found between the toes, gave off a disagreeable odor like
that observed from this locality in certain individuals. In his re-
searches made in Havana the writer frequently encountered in cul-
tures from the surface, associated with various micrococci, his Micro-
coccus tetragenus versatilis.

Fiirbringer found quite frequently in the spaces beneath the fin-

BACTERIA OF THE SURFACE OF THE BODY. 649

ger nails Staphylococcus pyogenes aureus associated with various
other microorganisms. A similar result had previously been reported
by Bockhart.

In his examinations of water from various sources Miquel found
that "wash-water" from the floating laundries on the Seine con-
tained more bacteria than water from any other source, even than
the water of the Paris sewers. His enumeration gave twenty-six
million germs per cubic centimetre.

Hohein has enumerated the colonies developing from undercloth-
ing worn for various lengths of time and made of different kinds of
material. A piece of the goods to be tested was sewed fast to the
underclothing, so as to come in immediate contact with the body ; at
the end of a given time a fragment one-quarter of a centimetre square
was cut up as fine as possible and distributed in nutrient gelatin.
Plates were made and the colonies counted at the end of five or six
days.

In an experiment in which sterilized woven goods were worn next
to the skin of the upper arm the following results were obtained :
Linen goods, at the end of one day 28, two days 4,180 colonies ; cot-
ton goods, end of one day 105, end of two days 1,870 ; woollen goods,
end of one day 606, end of two days 6,799. When the material had
been in contact with the skin for four days the colonies which devel-
oped were so numerous that they could not be counted.

Maggiora isolated twenty-two species of bacteria from his cultures
inoculated with epidermis from the foot. None of these proved to
be pathogenic for mice, rabbits, or guinea-pigs. Several gave off a
strong odor of trimethylamin, similar to that of sweating feet.

The following species have been found upon the surface of the
body :

Non-pathogenic. — Diplococcus albicans tardus (Unna and Tommasoli),
Diplococcus citreus liquefacieiis (Unna and Tommasoli), Diplococcus flavus
liquefaciens tardus (Unna and Tommasoli), Staphylococcus viridis flaves-
cens (Guttmann), Bacillus graveolens (Bordoni-Uffreduzzi), Bacillus epider-
midis (Bordoni), Ascobacillus citreus (Unna and Tommasoli), Bacillus fluo-
rescens liquefaciens minutissimus (Unna and Tommasoli), Bacillus aureus
(Unna and Tommasoli), Bacillus ovatus minutissimus (Unna and Tomma-
soli), Bacillus albicans pateriformis (Unna and Tommasoli), Bacillus spini-
ferus (Unna and Tommasoli), Bacillus of Scheurlen, Micrococcus tetragenus
versatilis (Sternberg), Bacillus Havaniensis liquefaciens (Sternberg).

Pathogenic. —Staphylococcus pyogenes albus, Staphylococcus pyogenes
aureus, Streptococcus pyogenes, Diplococcus of Demme, Bacillus of L emme,
Bacillus of Schimmelbusch, Bacillus of Tommasoli, Bacillus saprogenes II.
(Rosenbach) , Bacillus parvus ovatus (Loftier).

SURFACE OF MUCOUS MEMBRANES.

Cultures made from the conjunctivas of healthy persons usually
show the presence of various micrococci, and sometimes of bacilli.

650 BACTERIA OF THE SURFACE OF THE BODY

McFarland (1895) says that in his researches the microorganisms
found were for the most part "those already described by others and
of common occurrence in the air." He encountered, however, sev-
eral bacilli not previously described (" Bacillus hirsutus, Bacillus
ccerulefaciens, Bacillus circurnscriptus, Bacillus succinacius, Bacillus
violaceus flavus"). Lachowicz (1895) failed to obtain any bacteria
in his cultures from the con junctival sac in sixty-nine per cent of the
healthy eyes examined by him (sixty-three eyes in all). He con-
cludes that the microorganisms, which at times are found in the
healthy conjunctival sac, come principally from the air; that they
are present in small numbers and probably remain only for a short
time. His experiments show that most species when artificially
introduced rapidly diminish in numbers and soon disappear entirely.
Cultures of Streptococcus pyogenes and of Bacillus xerosis conjunc-
tive introduced into healthy eyes did not cause the slightest irrita-
tion. In this connection we may remark that the same is true as
regards pathogenic bacteria introduced into the bladder, but that
when there is some cause of local irritation or injury a chronic
cystitis is likely to be developed. In like manner, we believe, chronic
conjunctivitis may be developed as the result of local irritation in
connection with the presence of pathogenic bacteria and especially of
the pyogenic micrococci.

The extended researches of Bach (1894) gave results corresponding
with those of previous investigators, and not with those reported by
Lachowicz, who, as stated above, failed to obtain cultures from sixty-
nine per cent of the healthy eyes examined. Bach says : " In a large
percentage of the cases the presence of bacteria may be demonstrated,
even when the conjunctiva presents a perfectly normal appearance;
the conjunctival sac must therefore be regarded as constantly in-
fected." Bach describes twenty-seven different microorganisms ob-
tained by him in pure cultures from this source, of these eighteen
are micrococci. He recognizes the fact that most of them come from
the air, while others are introduced by the hands in rubbing the
eyes, etc. In diseased conditions these are more numerous than in
health, but the pus cocci are not infrequently found in healthy eyes.

As bacteria are constantly present in the air, they are necessarily
deposited upon the moist mucous membrane of the nose during in-
spiration. Indeed, it would appear as if an important function of
this extended mucous membrane is to purify the air from suspended
particles, and it has been shown by experiment that expired air is
practically free from bacteria. The greater number of those con-
tained in inspired air are deposited upon the mucous membrane of
the anterior nares. In culture experiments made by Von Besser,
Wright, and others the nasal mucus was found to contain a great

AND OF EXPOSED MUCOUS MEMBHANES. 651

variety of bacteria; among others the pus cocci were frequently
found by both of the observers mentioned. In eighty one cases Von
Besser found the " diplococcus pneumonias " fourteen times, Staphy-
lococcus pyogenes aureus fourteen times, Streptococcus pyogenes
seven times, and Friedlander's bacillus twice. Twenty-eight of the
cases examined were convalescents in hospital; among these the
pathogenic species mentioned were found less frequently than in other
individuals. The following non-pathogenic species were isolated:
Micrococcus liquefaciens albus in twent}*-two cases, Micrococcus al-
bus in nine cases, Micrococcus cumulatus tenuis in fourteen cases,
Micrococcus flavus liquefaciens in three cases, Bacillus striatus albus
in ten cases, etc.

Paulsen (1890) made thirty-one cultures in nutrient gelatin from
sixteen persons and thirty-three in nutrient agar from twenty-two
persons, with the following result : Eleven remained sterile, nineteen
showed not more than ten colonies, sixteen less than one hundred,
twelve more than one hundred, and in six the number was so great
that they could not be counted. Micrococci were more numerous
than bacilli ; of these a " sulphur-yellow coccus " in tetrads was found
in eight individuals. Various species of liquefying cocci, resem-
bling the pus cocci, were isolated, but the conclusion was reached
that none of these were identical with the staphylococci of pus,
which Von Besser and Wright both found in a considerable propor-
tion of the culture experiments made by them.

Thomson and Hewlett (1895) have recently reported results which
differ to some extent from those previously reported. While they
found numerous bacteria in the vestibulum naris, cultures made from
mucus obtained from the interior of the nose usually gave a negative
result — sixty-four out of seventy-six remained absolutely sterile,
while in seven there was a scanty growth only. They conclude that
while microorganisms are occasionally found upon the Schneider-
ian membrane they are not numerous and are often entirely absent;
and that the}* are rarely found upon the pituitary membrane. Straus
(1895) has examined the nasal secretions of persons associated with
tubercular patients for the purpose of ascertaining if the tubercle ba-
cillus was present. The presence of this bacillus was demonstrated,
by inoculation into guinea-pigs, in nine healthy individuals out of
twenty -nine examined ; two of these were physicians and six were
nurses.

Very extended researches have been made with reference to the
bacteria present in the human mouth, which show that numerous
species are constantly present in the buccal secretions and upon the
surface of the moist mucous membrane. Some of these are occa-
sional and accidental, while others appear to have their normal habi-

652

tat in the mouth, where the conditions as to temperature, moisture,
and presence of organic pabulum are extremely favorable for their
development. A minute drop of saliva spread upon a glass slide,
dried, and stained with one of the aniline colors, will always be
found to contain an immense number of bacteria of various forms.
Some of these are attached to epithelial cells and some scattered about
singly or in groups. Among those seen in a single specimen we will
usually find cocci in tetrads, in chains, and in irregular groups,
bacilli of various dimensions, and occasionally spirilla. According
to Prof. Miller, of Berlin, the following species almost invariably
occur in every mouth : Leptothrix innominata, Bacillus buccalis max-
imus, Leptothrix buccalis maxima, lodococcus vaginatus, Spirillum
sputigenum, Spirochaate dentium. All of these fail to grow in ordi-
nary culture media. Miller has made extended attempts to obtain
cultures by varying the medium used and attempting to imitate as
nearly as possible the natural medium in which they are found; but
his attempts have been unsuccessful, or nearly so — " only line cultures
afforded a limited growth, but the colonies never developed more
than fifteen to twenty cells, and a transference to a second plate
proved futile, no further growth taking place."

Up to the year 1885 Miller had isolated twenty-two different
species of bacteria from the human mouth. Ten of these were cocci,
five short bacilli, six long bacilli, and one a spirillum. Later the
same author cultivated eight additional species. Vignal has isolated
and described seventeen species obtained by him in pure cultures
from the healthy human mouth; most of these are bacilli, and Miller,
who found micrococci to be more numerous, supposes the difference
in results to be due to the fact that many of the cocci do not grow in
nutrient gelatin, which was the medium employed by Vignal. In
the researches of the last-named author the following species were
obtained most frequently, in the order given: 1. Bacterium termo.
2. Bacillus e (Bacillus ulna ?). 3. Potato bacillus. 4. Coccus a. 5.
Bacillus b. 6. Bacillus d. 7. Bacillus c (Bacillus alvei ?). 8. Bacil-
lus subtilis. 9. Staphylococcus pyogenes albus. 10. Staphylococcus
pyogenes aureus.

Among the species above enumerated we find two of the most
common pus cocci, Staphylococcus albus and aureus. but no mention
is made of another important pathogenic micrococcus which is fre-
quently found in the healthy human mouth, viz., the micrococcus of
sputum septicaemia, first named by the writer Micrococcus Pasteuri.
This does not grow at ordinary temperatures, and consequently
would not be obtained in gelatin plate cultures. Very different re-
sults have been reported by different observers as to the frequency
with which the pathogenic cocci are found in the buccal cavity.

AND OF EXPOSED MUCOUS MEMBRANES. 053

Black found in the saliva of ten healthy individuals the Staphy-
lococcus pyogenes aureus seven times, Staphylococcus pyogenes al-
bus four times, and Streptococcus pyogenes three times. On the
other hand, Netter found Staphylococcus aureus only seven times in
one hundred and twenty-seven individuals examined. Miller also
has rarely found the pus cocci in the mouths of healthy persons.
Streptococcus pyogenes was not found by Vignal in his extended
researches. The experiments of the writer, of Vulpian, Frankel,
letter, Claxton, and others show that the micrococcus which in
1885 I named Micrococcus Pasteuri, and which is identical with the
" diplococcus pneumonia " of German authors, is frequently present
in the healthy human mouth— now called Micrococcus pneumonia
crouposaB. Ketter examined the saliva of one hundred and sixty-five
healthy individuals and obtained it in fifteen per cent of the number
examined.

Another pathogenic micrococcus which is frequently present in
the mouths of healthy persons is the Micrococcus tetragenus of Koch.
The following pathogenic bacteria have also been isolated and de-
scribed : Bacillus crassus sputigenus (Kreibohm), Bacillus salivarius
septicus (Biondi). The Streptococcus septo-pyaBmicus of Biondi is
described as having characters identical with those of the Strepto-
coccus pyogenes of Rosenbach. Two other pathogenic species de-
scribed by Biondi were each found in a single case only. Miller
has described the following pathogenic species isolated and studied
by him : Micrococcus gingiva3 pyogenes, Bacterium gingivse pyo-
genes, Bacillus dentalis viridans, Bacillus pulpa3 pyogenes.

Rosenthal (1893) examined the secretions from the mouths of
fourteen individuals and obtained twenty-eight different bacteria; of
these twenty-one had been previously described. Five species be-
lieved to be new are described in detail by Rosenthal, viz. : Sarcina
viridis flavesceris, Micrococcus Reessii, Micrococcus ochraceus, Dip-
lococcus Hauseri, Bacterium cerasinum.

Vignal has tested a considerable number of microorganisms,
obtained by him in his cultures from the healthy human mouth, with
reference to their peptonizing action upon various kinds of food, with
the idea that some of them may have an important physiological
function of this kind. Out of nineteen species he found ten which,
after a longer or shorter time, dissolved fibrin, nine which dissolved
gluten, ten which dissolved casein, and five which dissolved albumin ;
nine changed lactose into lactic acid, seven inverted cane sugar, seven
caused the fermentation of glucose, and seven coagulated milk.

Sanarelli (1891) has shown that normal saliva has the power
of destroying the vitality of a limited number of certain patho-
genic bacteria, including the following species: Staphylococcus
pyogenes aureus, Streptococcus pyogenes, Micrococcus tetragenus

654 BACTERIA OF THE SURFACE OF THE BODY

Bacillus typhi abdominalis, Spirillum cholerse Asiaticse. When to
ten cubic centimetres of saliva, sterilized by filtration through porce-
lain, the above-mentioned pathogenic bacteria were added in small
numbers by means of a platinum needle carried over from a pure
culture, no development occurred, and at the end of twenty-four
hours the bacteria introduced were incapable of growth in a suitable
medium. But when this amount of filtered saliva was inoculated
with a large platinum loop — an ose — a certain number of the bacteria
survived, and at the end of three or four days an abundant develop-
opment occurred. At first, however, the number of living cells was
considerably diminished. In saliva to which one ose of a culture of
Staphylococcus aureus was added thirteen thousand eight hundred
and forty colonies developed in a plate made immediately after inocu-
lation, while a plate made at the end of twenty-four hours contained
but one hundred and thirty-two colonies, and one at the end of forty-
eight hours had but eight colonies. Subsequently multiplication
occurred, and a plate made on the ninth day after inoculation con-
tained so many colonies that they could not be counted.

The diphtheria bacillus was not destroyed in filtered saliva, but
did not multiply in it. On the other hand, it proved to be a very
favorable medium for the development of Micrococcus pneumonise
crouposaB.

Mucus from the surface of the meatus urinarius of man and
woman, or from the vagina, will always be found to contain various
bacteria ; but the bladder, the uterus, and Fallopian tubes in healthy
individuals are free from microorganisms.

Winter has isolated twenty-seven different species from vaginal
and cervical mucus, and reports that he found Staphylococcus pyo-
genes albus in one-half of the cases examined. A streptococcus was
also encountered which resembled Streptococcus pyogenes, although
not positively identified with it. Samschin, on the other hand, failed
to obtain the pus cocci in vaginal mucus from healthy women.

Donderlein, Yon Ott, and others have carefully examined the
lochial discharge with reference to the presence of bacteria. The
first-named author found that in healthy women the lochial discharge
obtained from the uterus was free from germs, but when collected
from the vagina various microorganisms were obtained. In one case
in which some fever existed Staphylococcus pyogenes aureus was
found in the vagina, while the discharge from the uterus was free
from germs. In five cases of puerperal fever Streptococcus pyogenes
was obtained in the lochial discharge from the uterus. The results
of Yon Ott correspond with those of Donderlein. Czerniewski, in
the lochia of fifty-seven healthy women, found the Streptococcus
pyogenes but once, while in the lochial discharge of fatal cases of
puerperal fever it was always present.

AND OF EXPOSED MUCOUS MEMBRANES.

Steffeck (1892) has examined the vaginal secretion of twenty-nine
pregnant females who had not been subjected to digital examina-
tion, and found Staphylococcus pyogenes albus in nine, Staphylo-
coccus pyogenes aureus in three, and Streptococcus pyogenes in one.
These results indicate that puerperal septicaemia from self-infection
may occur in exceptional cases. In seventeen of the twenty-nine
cases examined none of these pyogenic micrococci were found.

Hofmeister (1894) has shown that bacteria are found not only
upon the mucous membrane of the meatus urinarius in man, but
that they may usually be obtained from the urethral canal at a depth
of eight centimetres or more, although the number rapidly diminishes
in the deeper portion of the urethra.

Walthard (1895) arrives at the conclusion that while in pregnant
females bacteria are constantly found in the vagina and the lower
portion of the cervical canal, they are absent from the upper part of
the cervical canal, the uterus, and the tubes; and that during the
puerperal condition the uterine cavity is preserved from spontaneous
infection per vias naturalis by the plug of mucus in the cervical
canal. In the vaginal secretions of one hundred pregnant women,
who had not been subjected to a digital examination, streptococci
were obtained twenty-seven times in cultures. These were not viru-
lent, but, according to Walthard, these saprophytic streptococci be-
come virulent when, owing to a diminished resisting power, they are
enabled to invade the tissues as parasites.

Kronig (1894) concludes from his investigations that the vaginal
secretions of pregnant women are usually so acid that Streptococcus
pyogenes could not multiply in them; also that when the secretion is
normal it is almost always sterile.

Doclerlein (1894) insists that the failure of Kronig to obtain micro-
organisms in his cultures was due to the fact that suitable media
were not used ; also that certain bacilli are constantly found in nor-
mal, acid vaginal secretions, and that in the pathological secretions
which are feebly acid, neutral, or in some cases slightly alkaline a
great variety of bacteria are found, including Streptococcus pyo-
genes, as demonstrated by himself and other investigators. In a
later paper (1894) Kronig reports his success in obtaining cultures
from normal, acid vaginal secretions by using acid media and by
cultivating under anaerobic conditions. He reports also that patho-
genic bacteria (streptococci, staphylococci, and Bacillus pyocyaneus)
introduced into the vaginaB of pregnant women lose their power of
reproduction in from six to forty-eight hours (streptococci did not
grow after six hours). In a still later communication (1894) Kronig
reports that the bacteria present in the vaginal secretions of pregnant

656 BACTERIA OF THE SURFACE OF THE BODY

women are for the most part strictly anaerobic species, and that
among these he found two non-pathogenic streptococci.

Menge (189-4) has examined the vaginal secretions in fifty non-
pregnant women who had been in bed for at least fourteen days —
after laparotomy. Microscopical examination showed the presence
of bacteria in all cases, but in only six cases was a development of
colonies obtained — upon agar plates; in one case Streptococcus pyo-
genes was present. Menge concludes from his investigations that
spontaneous infection during childbirth cannot occur, and that with
the exception of the gonococcus the known pathogenic bacteria can-
not multiply in the cervical canal.

Gawronsky (1894) has examined the secretions from the healthy
urethra in sixty-two women, most of whom were under treatment
for uterine disease or displacement. The material for his cultures
was obtained b}* means of a platinum loop, introduced through a
glass cylinder, at a distance of one or one and. one-half centimetres
from the external orifice of the urethra. In fifteen out of the sixty-
two cases examined a positive result was obtained, as follows: In
three cases Streptococcus pj^ogenes, in eight Staphylococcus pyogenes
aureus, in one Staphylococcus pyogenes albus, in two Bacillus coli
communis, in one Bacterium tholoideum of Gessner.

The following species have been obtained from the nasal and
buccal secretions :

FROM THE NOSE.

Non-pathogenic. — Micrococcus nasalis (Hajek), Diplococcus coryzoe
(Hajek), Micrococcus albus liquefaciens (Von Besser), Micrococcus cumu-
latus tenuis (Von Besser), Micrococcus tetragenus subflavus (Von Besser),
Diplococcus fluorescens f retidus (Klamann), Micrococcus foetidus (Klamann),
Vibrio nasalis (Weibel), Bacillus striatus flavus (Von. Besser), Bacillus
striatus albus (Von Besser).

Pathogenic. — Staphylococcus pyogenes aureus, Staphylococcus pyogenes
albus, Streptococcus pyogenes. Bacillus of Friedlander, Bacillus of rhino-
scleroma (?), Bacillus foetidus ozaeiise (Hajek), Bacillus mallei (Loffler), Ba-
cillus smaragdmus foetidus (Reimann).

FROM THE MOUTH.

Non-pathogenic. — Micrococcus roseus (Eisenberg), Micrococcus A, B, C,
D, E of Podbielskij, Sarcina pulmonum (Hauser), Sarcina lutea, Micrococcus
candicans (Fliigge), Bacillus of Miller, Bacillus virescens (Frick), Vibrio
rugula, Vibrio lingualis (Weibel), Pseudo-diphtheria bacillus (Von Hoff-
mann), Bacillus mesentericus vulgatus, Bacillus subtiHs, Bacillus a, b, c, d,
e, /, gr, h, i, and,/ of Vignal, Bacillus subtilis similis, Bacillus radiciformis
(Eisenberg), Bacillus luteus, Bacillus fluorescens non-liquefaciens, Bacillus
ruber, Bacillus viridiflavus, Proteus Zenkeri, Bacillus G, H, I, J, K, L, M.
N, and Vibrio O andP of Podbielskij, Vibrio viridaiis (Miller), Micrococcus
nexifer (Miller), lodococcus magnus (Miller), Ascococcus buccalis (Miller),
Bacillus fuscans (Miller).

Pathogenic. — Staphylococcus pyogenes albus, Staphylococcus pyogenes
aureus, Staphylococcus salivarius septicus (Biondi), Streptococcus pyogenes,
Micrococcus salivarius septicus (Biondi), Micrococcus tetra<jenus (Gaffky),

AXD OF EXPOSED MUCOUS MEMBRANES. 657

Micrococcus gingivaa pyogenes (Miller), Streptococcus septo-pyaeinicus (Bi-
ondi), Streptococcus articulorum (Loffler), Micrococcus of Manfredi, Micro-
coccus pneuinonise crouposae — ' ' Micrococcus Pasteuri " (Sternberg) ; Bacillus
diphtherias (Loffler), Bacillus tuberculosis (Koch), Bacillus of Friedlander,
Bacillus bronchitidae putridse (Lurnnitzer), Bacillus septicaamiae haemorrha-
gicae, Bacillus gingivae pyogenes (Miller), Bacillus pulpse pyogenes (Miller),
Bacillus dentalis viridans (Miller), Bacillus crassus sputigenus (Kreibohm),
Bacillus saprogenes No. 1 (Rosenbach), Bacillus pneumoniae agilis (Schou),
Bacillus pneumonias of Klein, Bacillus pneumosepticus (Babes).

As the secretions of the mouth contain numerous bacteria, these
must constantly find their way to the stomach, but conditions are
not favorable for their development when the stomach is in a healthy
state and its secretions normal. Under certain circumstances, how-
ever, there may be an abundant development in the stomach of spe-
cies which give rise to various fermentations, and no doubt dyspep-
tic symptoms are frequently due to this cause. In the present
section we are, however, only concerned with the bacteria of the
healthy stomach. Most of these, we think, are to be considered as
only temporarily and accidentally present in this viscus as the result
of the swallowing of the buccal secretions and of food and drink con-
taining them.

The experiments of Straus and Wiirtz and of others show that
normal gastric juice possesses decided germicidal power, which is
due to the free hydrochloric acid contained in it. Hamburger (1890)
found that gastric juice containing free acid is almost always free
from living microorganisms, and that it quickly kills the cholera
spirillum and the typhoid bacillus, but has no effect upon anthrax
spores. Straus and Wiirtz found that the cholera spirillum is killed
by two hours' exposure in gastric juice obtained from dogs, the
typhoid bacillus in two to three hours, anthrax bacilli in fifteen to
twenty minutes, and the tubercle bacillus in from eighteen to thirty-
six hours. The experiments of Kurlow and Wagner, made with
gastric juice obtained from the stomach of healthy men by means of
a stomach sound, gave the following results : Anthrax bacilli with-
out spores failed to grow after exposure to the action of human gas-
tric juice for half an hour, but spores were not destroyed in twenty-
four hours ; the typhoid bacillus was killed in one hour ; the
cholera spirillum, the bacillus of glanders, and Bacillus pyocyanus
were all destroyed at the end of half an hour ; the pus cocci showed
greater resisting power. Certain bacteria have a greater resisting
power for acids than any of those above mentioned, and some of them
may consequently pass through the healthy stomach to the intestine

BACTERIA OF THE STOMACH AXD INTESTINE. 659

in a living condition, but there is good reason to believe that t_ie
spirillum of cholera or the bacillus of anthrax would not. On tli3
other hand, the tubercle bacillus and the spores of other bacilli can,
no doubt, pass through the stomach to the intestine without losing
their vitality.

Of nineteen species isolated by Vignal in his cultures from the
healthy human mouth, the greater number resisted the action of the
gastric juice for more than an hour, and six species which did not
form spores were found to retain their vitality in gastric juice for
more than twenty-four hours.

In making a bacteriological analysis of the contents of the healthy
stomach the more resistant microorganisms and those which form
spores will. naturally be found in greater or less numbers, inasmuch
as some of them are likely to be present in food and water ingested.

Yan Puteren (1888) obtained a variety of microorganisms in very
considerable numbers from the stomachs of infants fed upon un-
sterilized cow's milk, but in healthy nursing infants the number was
much smaller, especially when the mouth was washed out with dis-
tilled water immediately before and after nursing. In 18 per cent
of the cases no microorganisms were found under these circum-
stances, and in 41 per cent the number fell below one thousand per
cubic centimetre, Among the nursing infants examined (eighty-
five) the following species were most numerous : Monilia Candida,
Bacillus lactis aerogenes, a non-liquefying coccus, Staphylococcus
pyogenes aureus, Bacillus subtilis. In infants fed upon cow's milk
(eleven) Bacillus lactis aerogenes was present in 45.4 per cent of
the cases, and Staphylococcus pyogenes aureus in 27.2 per cent, non-
liquefying cocci in 54. 4 per cent, liquefying cocci in 72. 7 per cent,
Bacillus subtilis in 36.3 percent, and Bacillus butyricus (Hueppe)
in all of the cases ; next to these Bacillus flavescens liquefaciens
was the most abundant. The author named reaches the conclusion
that no species is constant and that the presence of those found de-
pends upon accidental circumstances.

Abelous (1889) found in his own stomach, washed out while fast-
ing, a considerable number of species of bacteria, viz* : Sarcina
ventriculi, Bacillus pyocyaneus, Bacillus lactis aerogenes, Bacillus
subtilis, Bacillus mycoides, Bacillus amylobacter, Vibrio rugula,
and eight other undescribed bacilli and one coccus. All of these
microorganisms were able to resist the action of hydrochloric acid
in the proportion of 1.7 grammes in 1,000 grammes of water.
Several were found to be facultative anaerobics.

The action of the bacteria isolated by him was tested by Abelous
upon various alimentary substances. The time required to effect
changes, such as the digestion of fibrin, the changing of starch

GGO BACTERIA OF THE STOMACH AND INTESTINE.

into glucose, etc,, was found to be so long that there was no reason
to suppose that any one of the microorganisms tested was con-
cerned in ordinary stomach digestion.

In the intestine conditions are favorable for the development of
many species of saprophytie bacteria, and the smallest quantity of
excrementitious material from the bowels, spread upon a glass slide
and stained with one of the aniline colors, will be found to contain
a multitude of microorganisms of this class, of various forms.
Among these are certain species which have their normal habitat in
the intestine, and which may always be obtained in cultures from
this source, while others, having been present in food or water in-
gested, and having escaped destruction in the acid juices of the
stomach, are accidentally and temporarily present. These latter
may or may not increase in the organic pabulum which abounds in
the intestine, according as the conditions are favorable or otherwise.
The strictly aerobic bacteria could not multiply because of the ab-
sence of oxygen, and the species encountered are for the most part
anaerobics or facultative anaerobics. The Bacillus coli communis
of Escherich, which is the most constant and abundant species found
in the intestine of man and of certain of the lower animals, is a facul-
tative anaerobic, which grows readily in the ordinary culture media,
either in the presence of oxygen or in an atmosphere of hydrogen.
But certain other bacteria of the intestine are strictly anaerobic and
do not grow readily in the media commonly employed by bacteri-
ologists.

Escherich has shown that in new-born infants the meconium is
free from bacteria. At the end of twelve to eighteen hours after
birth bacteria appear in the alvine discharges, and the number is
already considerable at the expiration of the first twenty-four hours
of independent existence. The species first found are cocci and yeast
cells which no doubt come from the atmosphere, having been de-
posited upon the moist mucous membrane of the mouth and swal-
lowed with the buccal secretions. When the meconium is replaced
by " milk faeces " these contain in large numbers the Bacillus coli
communis, heretofore spoken of as the most common species found in
the intestine of adults. Another species associated with this, but
not so abundant, is the Bacillus lactis aerogenes of Escherich.
Other bacilli and cocci are found occasionally in smaller numbers.
These bacilli do pot liquefy gelatin, and, as a rule, the microor-
ganisms found in the alvine discharges of healthy persons are noii-
liquefying bacteria. Eschericlrs researches led him to the conclu-
sion that the Bacillus lactis aerogenes is constantly present in the
small intestine of milk-fed children as the most prominent species,
and that its multiplication there is favored by the presence of milk

BACTERIA OF THE STOMACH AND INTESTINE. 661

sugar, and that Bacillus coli communis finds the most favorable
conditions for its growth in the large intestine.

Brieger, in 1884, isolated from faeces and carefully studied two
bacilli, one of which has since been called by his name. This is a
non-liquefying bacillus which is very pathogenic for guinea-pigs,
and which in its morphology and characters of growth closely re-
sembles the Bacillus coli communis of Escherich. Indeed, a num-
ber of non-liquefying bacilli, differing but slightly in their morpho-
logical and biological characters, have been obtained by various
investigators from the alimentary canal of man and the lower ani-
mals, and it is still a question whether they are to be regarded as
distinct species or as varieties of the "colon bacillus " of Escherich.
The bacillus obtained by Emmerich from cholera cadavers in Na-
ples belongs to this group, and, if not identical with the colon bacil-
lus, resembles it so closely that its differentiation is extremely diffi-
cult. Brieger's bacillus forms propionic acid in solutions containing,
grape sugar. A second bacillus obtained by him from the same
source resembles the * ' pneumococcus " of Friedlander ; this causes
the fermentation of saccharine solutions, with production of ethyl
alcohol.

Bienstock (1883) isolated four species of bacilli from normal faeces,
two of which are comparatively large and resemble Bacillus sub-
tilis in their morphology and in the formation of spores. A third
species is described as an extremely slender pathogenic bacillus, re-
sembling the bacillus of mouse septicaemia. The fourth species is an
actively motile bacillus which forms end spores, causing the rods to
have the form of a drumstick. This is said to cause the decomposi-
tion of albumin, with production of ammonia and carbon dioxide.
Later researches do not sustain Bienstock's conclusion that the ba-
cilli described by him are the principal forms found in normal faeces.

Among the species encountered by Escherich, in addition to those
mentioned above (Bacillus coli communis and Bacillus lactis aero-
genes), are the following : Proteus vulgaris, found three times in
meconium, and constantly in the faeces of dogs fed upon flesh ; Strep-
tococcus coli gracilis, found in meconium, but not during the period
of nursing, is constantly present in the intestine when a flesh diet is
employed.

The intestine of carnivorous and omnivorous animals contains a
greater number of bacteria than that of the herbivora, and in the
large intestine they are far more numerous than in the small intes-
tine (De Giaxa). Sucksdorf has enumerated the colonies developing
from one milligramme of faeces from individuals on mixed diet. He
obtained an average of 380,000 from a series of observations in which
the maximum was 2,300,000 and the minimum 25,000.

CG2 BACTERIA OF THE STOMACH AND INTESTINE.

The constant presence of certain species of bacteria in the intes-
tine of man and the lower animals has led to the supposition that
they may serve a useful purpose, or perhaps even have an essential
physiological role in connection with intestinal digestion. While
this question has not been definitely settled, the experiments of
Vallin, Abelous, and others have thrown some light upon it, and a
recent experiment by Nuttall and Thierf elder (1895) has considerable
importance as bearing upon its solution. The experiment consisted
in removing a foetus from a pregnant guinea-pig by Caesarean sec-
tion, placing it under conditions which protected it from the micro-
organisms present in the atmosphere, and feeding it upon sterilized
milk. Great technical skill was shown in carrying out this experi-
ment for a period of eight days, during which time the little animal
was kept in a sterilized atmosphere and was fed every hour day and
night. At the end of this time it had consumed over three hundred
and thirty cubic centimetres of sterilized milk, and was as active and
healthy as other guinea-pigs of the same age. It was now killed, and
a careful bacteriological examination showed that the discharges
from the bowels and the contents of the intestine were entirely sterile.

ADDITIONAL NOTES UPON BACTERIA OF THE STOMACH AND

INTESTINE.

Oppler (1894) has examined material, obtained in the early morning, from
the stomach of persons suffering from indigestion, and found nearly always
numerous masses of sarcinae. Five different species were obtained from this
source, which were distinguished by the following characters : No. 1, colo-
nies sulphur yellow ; No. 2, colonies greenish yellow ; No. 3, colonies white ;
No. 4, colonies white, does not liquefy gelatin ; No. 5, colonies orange yel-
low. Nos. 1 and 3 were most frequently encountered.

Kauffmann (1895) in a carefully studied case of chronic dyspepsia obtained
from the contents of the stomach in the morning before breakfast, and after
a test meal, the following bacteria : Yellow sarcina, Micrococcusaurantiacus,
Staphylococcus cereus albus. Bacillus subtilis, Bacillus ramosus, ' ' a large
thick bacillus," " a short bacillus resembling Bacillus coli communis." The
last-mentioned bacillus was found in large numbers, and Kauffmami suggests
that it may have been the cause of the fermentation in the digestive tract
which caused the unpleasant symptoms in the case under investigation.

Macf ayden (1887) and Gillespie (1893) have also obtained a bacillus from
the stomach which appears to be identical with Bacillus coli communis. In
the researches of Gillespie it was obtained from a patient with dilatation of
the stomach who suffered from flatulence, etc. In all, twenty-four different
microorganisms were obtained by Gillespie from the contents of the stomach
of different individuals. This number includes three species of saccha-
romyces and a mucor. Among the conclusions reached by Gillespie are the
following :

"14. Although bacteria are of no aid to peptic digestion, and a hindrance
to the pancreatic ferment if in quantity in the duodenum, they still are of
great use in the small intestine, where they control putrefaction. This seems
paradoxical : microorganisms obstructing microorganisms but assisting diges-
tion. It seems, however, to be true. The organisms which most easily
pass the searching examination of the stomach are those which give rise by

BACTERIA OF THE STOMACH AND INTESTINE. 663

their growth to the fatty acids, as they are the most resistant to the action of
acids. Their products in the small intestine are sufficient to keep the con-
tents of that viscus acid, and they thereby prevent or control putrefaction.
In the large intestine the secretion is so alkaline that the putrefactive organ-
isms reassert themselves.

"15. Increased putrefaction in the intestinal canal may therefore be due,
in some cases, either to insufficient mortality among the putrefactive organ-
isms in the stomach, or to too great mortality among the acid-forming bac-
teria and yeasts.

"16. The lactic acid which appears during the first stages of digestion is
due to the action of organisms.

"17. The lactic, acetic, butyric, and succiiiic acids found in gastroectasis
are due also to organisms which luxuriate in the too stationary contents.
The marsh gas, the Brennender-gas of the Germans, is probably due to the
same cause ; in the only case of this character with which I have had the
good fortune to meet no material for examination could be obtained."

The following species have been isolated from faeces and the con-
tents of the intestine of cadavers :

Non-pathogenic. — Streptococcus coli gracilis (Escherich), Micrococcus
aerogenes (Miller), Micrococcus tetragenus versatilis (Sternberg), Micrococ-
cus ovalis (Escherich), "Yellow liquefying staphylococcus" (Escherich),
" Porzellancoccus " (Escherich), Bacillus subtilis, Bacillus aerogenes (Miller),
Bacterium aerogenes (Miller), Bacillus lactis erythrogenes (Hueppe), Clostri-
dium fcetidum (Liborius), Bacillus muscoides (Liborius) , Bacillus putrificus
coli (Bienstock), Bacillus subtilis similis I. and II. (Bienstock), Bacillus
Zopfii, Bacillus liquefaciens communis (Sternberg), Bacillus intestinus lique-
faciens (Sternberg), Bacillus intestinus motilis (Sternberg), Bacillus fluores-
cens liquefaciens (Fliig-ge), "Colorless fluorescent liquefying bacillus"
(Escherich), "Yellow liquefying bacillus" (Escherich), Bacillus mesenteri-
cus vulgatus, Bacilli of Booker, A to T, first series ; a to s, second series ;
Bacilli of Jeffries A to Z, and or, ft.

^ Pathogenic.— Staphylococcus pyogenes aureus, Bacillus typhi abdo-
minalis, Bacillus septicaemise haemorrhagicaa, Bacillus of Belfanti and Pas-
carola, Bacillus enteritidis (Gartner), Bacillus of Lesage, Bacillus pseudo-
murisepticus (Bienstock), Bacillus coli communis (Escherich), Bacillus lactis
aerogenes (Escherich), Bacillus cavicida (Brieger), Bacillus of Emmerich,
Bacillus coprogenes fcetidus (Schottelius), Bacillus of Utpadel, Bacillus leporis
lethalis (Sternberg), Bacillus acidiformans (Sternberg), Bacillus cuniculicida
Havaniensis (Sternberg), Bacillus cadaveris (Sternberg), Bacillus cavicida
Havaniensis (Sternberg), Proteus vulgaris (Hauser), Bacillus tuberculosis,
Spirillum choleras Asiaticee Spirillum of Finkler and Prior.

VI.

BACTERIA OF CADAVERS AND OF PUTREFYING
MATERIAL FROM VARIOUS SOURCES.

THE putrefactive changes which occur so promptly in cadavers,
when temperature conditions are favorable, result chiefly from post-
mortem invasion of the tissues by bacteria contained in the alimen-
tary canal. But it is probable that under certain circumstances
microorganisms from the intestine may find their way into the cir-
culation during the last hours of life, and that the very prompt putre-
factive changes in certain infectious diseases in which the intestine
is more or less involved are due to this fact. The writer has made
numerous experiments in which a portion of liver or kidney re-
moved from the cadaver at an autopsy made soon after death — one
to six hours — has been enveloped in an antiseptic wrapping and kept
for forty-eight hours at a temperature of 25° to 30° C. In every in-
stance there has been an abundant development of bacteria, although
as a rule none were obtained from the same material immediately after
the removal of the organ from the body. This shows that a few
scattered bacteria were present. The same result was obtained in
cases of sudden death from accident, as from portions of liver or
kidney removed from the bodies of persons dying of yellow fever,
tuberculosis, and other diseases.

Numerous researches show that the blood of healthy men and
animals is free from bacteria, and that saprophytic bacteria injected
into a vein soon disappear from the circulation ; and recent experi-
ments show that blood serum has decided germicidal power. But in
spite of this fact the experiments of Wyssokowitsch show that cer-
tain bacteria injected into the circulation may be deposited in the
liver, the spleen, and the marrow of the bones, and there retain their
vitality for a considerable time. The spores of Bacillus subtilis were
found by the observer named to preserve their vitality in the liver or
spleen of animals into which they had been injected, for a period of
two or three months. In the writer's experiments the microorgan-
isms which first developed in fragments of liver preserved in an an-
tiseptic wrapping were certain large anaerobic bacilli, and especially

BACTERIA OF CADAVERS AND OF PUTREFYING MATERIAL. C05

my Bacillus cadaveris, together with the Bacillus coli communis
of Escherich, my Bacillus hepaticus fortuitus, and other non-lique-
fying bacilli of the " colon group/'7

These bacteria did not give rise to a putrefactive odor, and the
fragment of liver when cut into had a fresh appearance and a very
acid reaction. Later, putrefactive changes occurred and Proteus

FIG. 197.— Smear preparation from liver of yellow-fever cadaver, kept forty-eight hours in an
antiseptic wrapping, x 1,000. From a photomicrograph. (Steinberg.)

vulgaris and other putrefactive bacteria obtained the precedence.
Evidently all of these species must have been present in the liver at
the time it was removed from the cadaver, although in such small
numbers that they were rarely seen in smear preparations or ob-
tained in cultures from the fresh liver tissue. The appearance of a
smear preparation from the interior of a fragment preserved for
forty-eight hours in an antiseptic wrapping is shown in Fig. 197.

The horribly offensive gases which are given off from dead ani-
mals in a state of putrefaction appear to be due to certain large an-
aerobic bacilli which are found in such material,
and which have not yet been thoroughly studied ^k

owing to the difficulty of cultivating them in arti-
ficial media ; among them is a large bacillus with
round ends which forms an oval spore at one ex-
tremity of the rather long rod. This the writer
has described under the name of Bacillus cada-
veris grandis, Fig. 198.

In the interior of a putrefying mass of this kind
only those bacteria are found which are able to grow in the absence
of oxygen, but aerobic saprophytes may multiply upon the surface of

FIG

666 BACTERIA OF CADAVERS AND OF PUTREFYING MATERIAL.

such a mass, or in organic liquids to which the air has free access.
Among the most common putrefactive bacteria are the Proteus vul-
garis, Proteus mirabilis, and Proteus Zenkeri of Hauser. Formerly
the minute motile bacteria found in putrefying animal infusions, etc.,
were commonly spoken of as belonging to the species " Bacterium
termo," but recent researches show that several different species were
included under this name by those whose researches were made be-
fore the introduction of Koch's method for isolating and differentiat-
ing microorganisms of this class by the use of solid culture media.
The different species of Proteus are all facultative anaerobics. They
are more or less pathogenic, and according to Hauser produce a chem-
ical poison which, when injected into small animals, causes death with
all of the symptoms of putrid intoxication. The bacillus of mouse
septicaemia, which was first obtained by Koch from a putrefying meat
infusion, is also pathogenic, as are the writer's Bacillus cadaveris
and various other anaerobic bacteria found in putrefying material.

Some account of the various products of putrefaction and the
microorganisms concerned in their production will be found in Sec-
tion IV., Part Second, of the present volume.

VII.

BACTERIA IN ARTICLES OF FOOD.

Milk always contains bacteria, unless drawn with special precau-
tions into a sterilized flask. In the healthy udder of the cow it is
sterile, but in tuberculous cows, when the milk glands are involved,
tubercle bacilli may find their way into the milk in considerable
numbers. Ag ordinarily obtained and preserved, milk is greatly ex-
posed to bacterial contamination from various sources ; desquamated
cuticle from the external surface of the udder and from the hands of
the milker, and floating particles from the air of the stable, fall into it
at the very moment it is drawn, and it is subsequently contaminated
by bacteria from the air, and from water used in washing the recep-
tacles in which it is placed or added to it by the thrifty milkman.
As it furnishes an excellent nutrient medium for many of the bacteria
which are thus introduced into it, under favorable conditions of tem-
perature it quickly undergoes changes due to the multiplication in it
of one or more of these microorganisms. The acid fermentation and
coagulation of the casein which so constantly occurs is completely
prevented by sterilizing fresh milk in flasks provided with a close-
fitting cork or cotton air filter. Numerous researches have been
made with reference to the microorganisms found in milk and the
various fermentations to which they give rise. Naturally a great
variety of species will be found in an extended research, but all are
accidentally present, and only those demand special attention which
produce the various fermentations of this fluid commonly encoun-
tered, or which have special pathogenic properties.

Several different bacteria produce an acid fermentation and con-
sequent coagulation of milk, but the usual agent in producing this
fermentation is the Bacillus acidi lactici, which is identical with the
' ' ferment lactique " of Pasteur. When a pure culture of this bacillus
is introduced into sterilized milk kept at a temperature of 25° to 30° C.,
coagulation occurs in from fifteen to twenty-four hours. A uniform,
gelatinous mass is produced which does not subsequently become
dissolved (Adametz). Various other bacteria produce a similar
change, including a number of common water bacteria, several spe-

668 15. UTERI A IN ARTICLES OF FOOD.

cies of sarcina, Staphylococcus pyogenes aureus, and other pus cocci.
Usually coagulation is due to the combined action of several bacteria,
among which Bacillus acidi lactici is apt to be the most prominent.

Other bacteria produce coagulation without the lactic acid fer-
mentation. This appears to be due to the formation of a soluble
ferment which acts like rennet, causing the coagulation of milk
which has a neutral or slightly alkaline reaction. The coagu-
lated casein in this case is subsequently redissolved. The bacteria
which produce this change for the most part form spores, while the
lactic acid ferments do not. If, therefore, milk is heated nearly to the
boiling point the acid-forming bacteria will be destroyed and the
spores of the other species surviving will give rise to coagulation
without the production of lactic acid. Among the more common
microorganisms of this group are the Bacillus butyricus (Hueppe),
Bacillus mesentericus vulgatus, Loffler's " white milk-bacillus," and
the bacilli described by Duclaux under the generic name of Tyrothrix.

Other fermentations are produced by certain chromogenic bacteria,
and these, as a rule, are not as harmless from a sanitary point of view
as those above referred to. Blue milk is produced by the presence of
Bacillus cyanogenus, yellow milk by Bacillus synxanthus (Schroter)
and by a species obtained by List from the faeces of a sheep and
another found by Adametz in cheese. The well-known Bacillus
prodigiosus produces its characteristic red pigment when present in
milk, and a bluish-red color is caused by Bacterium lactis ery throgenes
(Hueppe).

Viscous fermentation in milk is produced by several different bac-
teria, among others by a micrococcus studied by Schmidt-Muhlheim,
and a short bacillus isolated by Adametz — Bacillus lactis viscosus.
Milk which has undergone this change is unwholesome as food ; it
is recognized by the long filaments which are produced when it is
touched with any object and this is slowly withdrawn.

The Caucasian milk ferment, Bacillus Caucasicus, produces a
special fermentation, which has been referred to in Section IV. , Part
Second (page 139).

Various pathogenic bacteria have occasionally been found in milk
in addition to the tubercle bacillus already referred to. Thus Adametz
found Staphylococcus pyogenes aureus in two samples which had
been submitted to him for examination, one of which had given rise
to vomiting and diarrhoea. Wyssokowitsch cultivated from milk
which had been standing some time a pathogenic bacillus, named by
him Bacillus oxytocus perniciosus.

The special microorganism which produces the poisonous pto-
maine called by Vaughan tyrotoxicon has not yet been isolated ; nor
do we know the exact cause of scarlet fever, although there is evi-

BACTERIA IX ARTICLES OF FOOD.

669

dence that this disease lias been spread by the use of contaminated
milk, as have also diphtheria and typhoid fever, which diseases are
due to bacilli now well known. As the cholera spirillum grows
readily in milk, this disease could no doubt also be transmitted in
the same way.

Sedgwick and Batchelder (1892) have examined a large number
of specimens of milk obtained in Boston and vicinity, for the purpose
of determining the number of bacteria present. They found, as an
average of several trials, that milk obtained in a clean stable, from
a well-kept cow, by milking in the usual way into a sterilized bottle,
contained 530 bacteria per cubic centimetre. 'When, however, the
milkman used the ordinary milk pail of flaring form, seated himself
with more or less disturbance of the bedding, and vigorously shook
the udder over the pail during the usual process of milking,*' the
numbers were very much higher — on an average 30,500 per cubic
centimetre immediately after milking. The average of fifteen sam-
ples taken from the tables of persons living in the suburbs of Boston
was 09,143 per cubic centimetre. The average of fifty-seven sam-
ples of Boston milk, obtained directly from the milk wagons and
plated at once, was 2,355,500 per cubic centimetre. The average of
sixteen samples from groceries in the city of Boston was 4,577,000
per cubic centimetre.

Prof. Eenk found in the milk supply of Halle from 6,000,000 to
oU, 000, 000 bacteria per cubic centimetre — a number considerably ex-
ceeding that usually found in the sewage of American cities (Sedg-
wick).

Cohn and Neumann (1801) have shown that the milk of healthy
women frequently contains bacteria, and that Staphylococcus pyo-
genes albus is the species most frequently found. This has been
confirmed by the researches of Palleske (1892), Ringel (1893) and
others. The last-mentioned author examined the milk of 25 women
recently confined, "12 of whom were healthy and 13 sick." In 3
cases only was the milk sterile; in 17 cases Staphylococcus pyogenes
albus was found; in 2 cases Staphylococcus pyogenes aureus; in 1
case both albus and aurens ; in 2 cases Staphylococcus pyogenes albus
and Streptococcus pyogenes. The streptococci were found in a case
of mild puerperal fever and in a case of phlebitis.

The researches of Hirshberger (1889), of Ernst (1895), and of
others show that the milk of tuberculous cows may contain tubercle
bacilli even when the udder of the animal presents no evidence of a
localized tubercular infection. In 121 samples of milk examined by
Ernst from 36 different cows, 19 gave a positive result; all from the
milk of 12 cows in which no evidence of tuberculosis of the udder
was found in a carefully made post-mortem examination. Among

670 BACTERIA IN ARTICLES OP FOOD.

the bacteria which produce unwholesome chaL-g^s in milk are several
which cause it to become viscous or soapy. Among these we may
mention Micrococcus lactis viscosus of Conn, Micrococcus Freuden-
reichi of Guillebeau, Bacillus mesentericus vulgatus, and Bacillus
lactis saponacei of Weighmann and Zirn. A considerable number
of bacilli are known which give rise to the production of butyric
acid fermentation in milk and its products. Some of these are an-
aerobic and some aerobic. The list includes the following : Bacillus
butyricus of Prazmowski, Bacillus of Liborius, Bacillus of Botkin,
Bacilli of Kadrowski.

The bitter taste which milk and cheese sometimes acquire is due
to the presence of special bacterial ferments ; among these the best
known are an aerobic, liquefying micrococcus described by Conn, a
bacillus described by Weighmann, Micrococcus casei amari and Ba-
cillus liquefaciens lactis amari of De Freudenreich (1895).

In fresh butter of good quality comparatively few microorganisms
are found, but the researches of Conn show that the characteristic
and agreeable flavor of fresh butter is due to, or at least may be imi-
tated by, a bacillus which is concerned in the ripening of cream
under normal conditions. Cultures of this bacillus (Bacillus 41 of
Conn) have already been used in a practical way by butter makers
to improve the flavor of their product.

Krenger (1890) obtained from "cheesy butter," having a disa-
greeable odor, various bacteria. Among these the most numer-
ous were an oval micrococcus (Micrococcus acidi lactici, Kreuger), a
slender bacillus resembling Bacillus fluorescens, and Bacillus acidi
lactici of Hueppe.

Klecki (1894) has isolated from rancid butter several bacteria not
previously described, one or more of which are no doubt concerned
in the production of the rancid taste and odor. These are described
under the following names: Bacillus butyri, Diplococcus butyri, a
bacillus resembling lodococcus vaginatus of Miller, Tetracoccus
butyri, Bacillus butyri No. 2.

Duclaux (1887) has isolated from different kinds of cheese no less
than eleven different species of bacteria, which he believes are con-
cerned in the "ripening process." Seven of these are aerobic and
four anaerobic species. Adametz (1889) has also isolated and studied
a number of species to which he attributes the ripening of cheese.

More recently Henrici (1895) has studied the bacterial flora of
cheese, and Marchal (1895) has shown that the ripening of certain
kinds of cheese (fromages mous) is probably due to Oidinm lactis.

Meats, even when salted and smoked, may contain living patho-
genic bacteria which were present prior to the death of the animal,
and, when not properly preserved, are of course liable to be invaded
by putrefactive bacteria.

BACTERIA IN ARTICLES OF FOOD. 671

The researches of Foster (1889) show that the typhoid bacillus,
the pus cocci, the tubercle bacillus, and the bacillus of swine plague
resist the action of a saturated solution of salt for weeks and even for
months; and the same observer found that the ordinary processes of
salting and smoking did not destroy the tubercle bacillus in the flesh
of a cow which had succumbed to tuberculosis. Beu has made cul-
tures from a large number of specimens of fresh, salted, and smoked
meats and fish, with the general result that the fresh and salted meats
were found to contain a limited number of bacteria of various species,
and that smoking for several days did not insure the destruction of
these microorganisms. In specimens of sausage six days' smoking
did not destroy a liquefying bacillus which was present, but at the
end of six weeks' exposure to smoke this bacillus no longer grew,
while a non-liquefying bacillus present in the same specimen had not
been destroyed. Fourteen days' smoking sufficed to destroy all the
microorganisms in a specimen of bacon, but this was not sufficient
for the interior portions of a ham. Among the bacteria obtained by
Beu from smoked meats he mentions the following : Staphylococcus
cereus albus, Proteus vulgaris, Staphylococcus pyogenes aureus, Ba-
cillus liquefaciens viridis, etc. The number of colonies which de-
veloped from a fragment, the size of a mustard seed to that of a flax-
seed, taken from the interior of the meats examined, was usually
small; and the presence of a few scattered bacteria of these common
species has no significance from a sanitary point of view, except as
showing that pathogenic bacteria may survive in infected meats after
they have been exposed to the usual processes of salting and smoking.

Petri, in experiments upon the bacillus of swine plague (Schweine-
rothlauf), arrived at the following results :

The flesh of swine which died of this disease preserved its infec-
tious properties after having been preserved in brine for several
months, and the same flesh salted or pickled for a month and then
smoked for fourteen days contained the rothlauf bacillus in a living
and unattenuated condition. At the end of three months virulent
rothlauf bacilli were still obtained from a smoked ham, but they were
no longer found at the end of six months.

Schrank (1888) has made cultures from both the albumin and the
yolk of fresh eggs, and finds that they are free from bacteria. He
thinks that, as a rule, putrefactive bacteria obtain access to the inte-
rior through injured places in the sheh1, although exceptionally the
egg may be infected with them in the oviduct of the fowl. The usual
bacteria concerned in the putrefactive changes in eggs are, according
to the author mentioned, a variety of Proteus vulgaris and Bacillus
fluorescens putidus.

Zorkendorfer (1893) has cultivated from rotten eggs sixteen dif-

072 BACTERIA IN ARTICLES OF FOOD.

ferent bacilli, all of which are described in detail and none of which
were found to correspond with previously described species as given
in Eisenberg's Bacteriological Diagnosis.

Peters (1889) has studied the flora of the "sauerteig" used in
Germany as yeast for leavening bread. In addition to the numerous
cells of three species of Saccharomyces, he finds that bacilli are present
in great numbers, as shown by direct microscopical examination and
culture experiments. He describes five species, designated Bacillus
A, B, C, D, and E, which are commonly present, and to which the
acid fermentation of the dough is ascribed.

In Graham bread which had undergone changes making it unfit
to eat, Kratschmer and Niemilowicz have found the Bacillus mes-
entericus vulgatus, which appears to have been the cause of the
fermentation, which was produced in bread having a slightly alka-
line reaction by inoculating it with a pure culture of this bacillus.
The infected bread has a brownish color, a peculiar odor, and be-
comes sticky and viscid.

Uffelmann (1890) has also studied the bacteria in spoiled rye
bread, and obtained, in addition to common mould fungi, Bacillus
mesentericus vulgatus and Bacillus liodermus.

Waldo (1894) has shown that baking does not sterilize bread.
This was to have been expected in the case of the spores of bacilli,
but it is somewhat surprising to find that two species of Sarcina and
two micrococci survived the baking process. In all Waldo obtained
thirteen species of bacteria from the interior of sixty-two loaves
examined. Bacillus subtilis and allied spore-forming bacilli were
most frequently found, and the statement is made that a loaf " from
a low-class, dirty bakery will almost invariably contain more living
bacteria (or their spores) than .one from a good, clean bakery."

Lehmann (1894) under the name Bacillus levans has described a
microorganism which closely resembles Bacillus coli communis. This
was obtained from sour dough, and was believed to be the cause of
the acid fermentation which so often interferes with success in ob-
taining sweet and wholesome bread. When a culture of this bacil-
lus was added to flour and water, without the addition of yeast, an
active fermentation occurred and the dough became acid.

INDEX

ABBOTT'S study of pseudo-diphtheritic

bacilli, 450

Abel's capsule bacillus, 561
Abriu, 262, 265, 267
Abscesses, formation of, 223
acute, micrococci in, 14

micrococcus pneumonise crou-

posae in, 401

Absinthe, antiseptic power of, 203
Acetic acid, action of, on spores, 182

as a decolorizer in staining, 28
Aceton-celluloidm solution, 36
Acetone, gerniicidal power of, 197
Acetylene gas in photomicrography, 110
Acid media, bacterial growth in, 166
Acids, action of, on bacteria, 180
produced by bacteria, 137
in culture media, 46
prejudicial to the vitality of the

cholera spirillum, 600
diluted, as decolorizers in staining,

Acne contagiosa of horses, bacillus of, 557
Aerobic bacteria, 16, 172

in stab cultures. 70
Agar as a medium of growth for :
bacillus A of Booker, 555

aerogenes capsulatus, 589

of Babes and Oprescu, 526

Beck, 566

of Belfonti and Pascarola, 508

capsulatus, 522

capsule, of Nicolaier, 560

of Cazal and Vaillard, 525

of cholera in ducks, 503

dysenterise, 569

enteritidis, 520

of Fiocca, 548

gallinarum, 521

of grouse disease, 521

Agar as a medium of growth for:
bacillus of hog cholera. 504, 509
of Laser, 524
of Lucet, 526
piscicidus, 567
piscicidus agilis, 565
of purpura haemorrhagica, Kolb,

559
of purpura hsemorrhagica, Tiz-

zoni and Giovannini, 558
pyocyaneus ,3, 545
pyocyaneus pericarditidis, 546
cedenmtis maligni. No. II., 588
solanacearum, 572
of symptomatic anthrax, 586, 587
of swine plague, Marseilles, 509
typhi muriuni, 524
micrococcus tetragenus, 412
proteus of Karlinski, 552
spirillum of Finkler and Prior, 603
Metschnikovi, 605
tyrogenum, 604

Agar-agar as a solid culture medium, 43
characters of growth in stab cultures

in, 72
bacillus of Emmerich and Weibel in,

566

bacillus pestis in, 563
Agar-ge latin, 44

growth of bacillus pestis in, 563
bacillus acidiformans,- 588
Agar, nutrient, Abbott's method of pre-
paring, 46

in analysis of water, 631
as a culture medium for :

bacillus of acne contagiosa of

horses, 557
bacillus alvei, 557
bacillus coli communis, 531
bacillus diphtherias, 454

674

INDEX.

Agar as a culture medium for :

bacillus endocarditidis griseus,

556

bacillus erysipelatos suis, 513
bacillus gracilis cadaveris, 560
bacillus leporis lethalis, 541
bacillus cedeniatis uialigni, 582
bacillus pyocyaneus, 543
bacillus typhi abdominalis, 439
micrococcus botryogenus, 413
micrococcus melitensis, 421
proteus horninis capsulatus, 519
proteus lethalis, 555
proteus septicus, 554
proteus vulgar is, 549
nutrient, Shultz's method of prepar-
ing, 45
spirillum choleras asiaticae in,

596
staphylococcus pyogenes aureus

in, 374

streptococcus pyogenes in, 384
plates, bacillus anthracis on, 425

bacillus endocarditidis capsula-
tus on, 556
bacillus cedematis nialigni on,

583
micrococcus pneumonias croupo-

sae, 403

solutions, filtration of, 44
stick cultures, bacillus of purpura

haemorrhagica, Babes, 558
Age, as influencing susceptibility to dis-
ease, 233
Agitation, influence of, on bacterial

growth, 163

Agua coco as a culture medium, 39
analysis of, 40
bacillus cavicida havaniensis

in, 517

bacillus cuniculicida havanien-
sis in, 538
Air, demonstrations of micro-organisms

in, 615

list of saprophytes found in, 624
optically pure, 5
of city streets, bacteria in, 625
of closed schoolrooms, bacteriology

of, 625
of hospitals, pathogenic germs in, 624

Albuminous fluids, germicidal powers of,

210

Alcohol as a decolorizer in staining, 28
action of, on bacterial growth, 197
on micrococcus pneumonias crou-

posae, 405

absolute, action of, on bacillus tuber-
culosis, 478
Alessi's experiments on the pathogenic

action of typhoid bacilli, 447
Alexins (Buchner), 236, 265
Algerian sheep, insusceptibility of, to

anthrax, 2o4

Alkalies, action of, on bacteria, 180
Alkalinity of blood as affecting immu-
nity, 236

Alkaloid in anthrax cultures, 428
Almonds, bitter, antiseptic power of,

203

Alpha-naphthol, antiseptic power of, 206
Alum, antiseptic value of, 188
Aluminum acetate, antiseptic value of,

188

chloride, antiseptic value of, 188
Amann's method of separating tubercle

bacilli from sputum, 486
Ammonia, action of, on bacterial growth,

184

as a nutrient of bacteria, 126
oxidation of, by bacteria, 144
Ammonium carbonate, antiseptic value

of, 188

fluosilicate, antiseptic value of, 188
sulphate, antiseptic value of, 188
Amplification of photomicrographs, 106
Anaerobic bacteria, 16

cultivation of, 80
in stab cultures, 71
isolation of, 83
concerned in putrefaction, 143
Anaesthetics in experiments on animals,

Analysis, chemical, of Friedlander's ba-
cillus, 124
of putrefactive bacteria by

Nencki, 124

of purified'tuberculin, 362
Angelica, antiseptic power of, 203
Aniline blue solution, Loffler's, 29
colors as stains, 25

INDEX.

675

Ani Hue colors for staining the anthrax

bacillus, 424
in culture media, 46, 64
dyes, germicidal power of, 197
Aniliue-geutian-violet solution, Ehr-

lich's, 29
Aniline-uiethyl-violet solution, Ehrlich-

Weigert, 29
Aniline oil, gerinicidal power of, 198

water for staining solutions, 29
Animals, experiments upon, 96

inoculations into susceptible, 169
post-mortem examinations of inocu-
lated, 101

Anise, antiseptic power of, 203
Annales de 1'Insthut Pasteur, a reference

to, 8
de Micrographie, a reference to,

9
Anthrax, causation of, discovered by

Davaine, 6

animals immune to, 233, 234
antidotal effect of bacillus pyocya-

neus in, 544

susceptibility of herbivora to, 234
bacillus, 422

general infection by, 428

pathogenesis of, 428

specific toxin of, 430

sporeless varieties of, 4213

staining of, 424

thermal death-point of, 157

vitality of, in various waters,

637

cultures, attenuation of, 277
chemical products in, 428
toxalbumin of, 150
protein. 124

spores, formation of, 424, 426
influence of chlorine on, 177
influence of sunlight on, 161
survival of, in various media,

430
symptomatic, 347

bacillus of, 585
Antiabrin, 263
Anticholeraic inoculations, Haffkine's,

302

harmlessness of, 305
Antipneumatoxin, 261, 341

Antiseptic agents, attenuation of viru-
lence by, 131
power, 164

determination of, 165
values, Miquel's tables of, 186
Antiseptics, definition of, 164
Antitetanic blood serum^ therapeutic

value of, 356
Antitoxic values, Brieger and Colin 's

method of determining, 358
Antitoxin, diphtheria, 314
from goat's in ilk, 358
tetanus, Qurative power of, 359
typhoid, present in the blood serum
of persons who have recently suf-
fered from the fever, 367
Antitoxins, 237

bactericidal value of, 267
discovery of, 259
immunity dependent on, 267
Apparatus, sterilization of, 53

for coagulating blood serum, 58
for filtering nutrient agar-agar, 4^
for hydrogen in cultivating anae-
robic bacteria, 86
incubation, of d' Arson val. 94
for photomicrography, 106
for sterilizing by filtration, 59, 60
Aqueous humor, germicidal power of, 236
Arloing's experiments on spore forma-
tion, 160
Aromatic products of decomposition,

germicidal power of, 198
Aronson's diphtheria antitoxin, 314
Arsenious acid, action of, on bacteria,

183

Arthritis of the wrist, inicrococcus pneu-
moniae crouposse in. 402
Arthrobacterium of Hueppe, 123
Arthrospores, 16, 19, 123

of the comma bacillus, 554, 598
Artificial culture media, 40
Asbestos, filtration of air through, 619
Ascitic fluid, germicidal power of, 236
Ascococcus, general characters, 17
grouping of, 21
Johnei (Cohn), 412
Aseptol, germicidal power of, 198
Atmosphere, the, relatively free from
bacteria, 613, 622

C76

INDEX.

Attenuated virus, Pasteur's method of

obtaining, 244

restoration of virulence to, 245
of anthrax, 427

cultures of the anthrax bacillus, 429
Attenuation of virulence, 130

by cultivation in the blood of an

immune animal, 132
of the bacillus of diphtheria, 457,

458

of the bacillus of tuberculosis. 3(?0
of cultures of bacillus mallei, 4i>4
of the microbe of fowl cholera, 502

JJABES, on the pigments of bacillus pyo-

cyaneus, 134
Babes' bacillus of purpura hsemorrhagica,

558

Bacille du charbon symptomatiqne, 585
de la morve (B. mallei), 489
du rouget du pore (Pasteur), 511
virgule cholerigene, 593
Bacillen des griinblauen Eiters, 542
Bacilli, general characters, 18
morphology, 22
anaerobic (pathogenic), 578
in chronic infectious diseases, 467
movements, 120
of Baumgarten, 12
non-pathogenic, list of species found

in the soil (Fillies), 646
list of species found in water,

638
pathogenic (anaerobic), 578

list of species found in soils

(Fttlles), 646
list of species found in water,

639

pseudo-typhoid, 441
which produce septicaemia in sus-
ceptible animals, 498
Bacillus A of Booker (from cholera in-

fantuni), 555

aceticus, acid produced by, 137
acidiformans (Sternberg), 139/537

pathogenesis of, 538
acidi lactici, 667

thermal death-point of, 155
of acne contagiosa of horses, 557
aerogenes capsulatus (Welch), 587

Bacillus alvei, 556

pathogenesis, 557
arnylovorus (Burrill), 575
anthracis, 422

biological characters, 424
destroyed by gastric juice, 658
morphology of, 423
pathogenesis, 428
spore formation, 426
resistance of, to high tempera-
tures, 154

thermal death-point of, 150
vitality of, in waters of various

characters, 637
vitality of, when buried in the

soil, 645

of anthrax, symptomatic, 585
biological characters, 586
methods by which virulence is

increased, 587
morphology, 586
pathogenesis of, 587
vital resistance of its spores, 586
B of (Booker), 536
of Babes and Oprescu, 525
of Beck, from infected rabbits, 566
of Belfanti and Pascarola, 508
der Brustseuche beim Kaninchen,

566
der Bliffelseuche (Oreste-Armanni),

499

butyricus, acid produced by, 138
cadaveris (Sternberg) from yellow-
fever cadavers, 584
grandis (Sternberg) of putrefac-
tion, 665

campestris (Pammel), 575
capsulatus, 521

inucosus (Fasching), 561
cavicida, 516

liavaniensis (Sternberg), 516
thermal death-point of, 155
vitality of, in moist atmos-
pheres, 159

of Cazal and Vaillard, 525
of cholera (see Spirillum), 593
of cholera in ducks (Cornil and

Toupet), 503

cholerae gallinarum (Fliigge), 499
coli communis, 529

INDEX.

G77

Bacillus coli commimis, probably identi-
cal with bacillus cavicida, 529
commimis, discovery of, 530
commimis, biology and mor-
phology of, 530

commimis, pathogenesis of, 532
communis. varieties of, 533
coprogenes parvus. 515
crassus sputigenus. 517

sputi genus, thermal death-point

of, 155
cuniculicida (Fliigge), 499

havaniensis (Sternberg), 538
havanieusis pathogenesis, 540
cyanogenus. thermal death-point of,

155
diphtheria, 449. 453

biological and morphological

characters, 4~>'-\
co 1 u in bar u in, 458
columbarum, pathogenesis, 455
columbarum, thermal death-
point of, 455
vitulorum. 4-V.i
dysenteric (Shiga), 568

distinguished from bacillus ty-

phosus, 569
of Eberth, 431

of Emmerich and Weibel from in-
fected trout, 565
endocarditidis capsulatus, 550

griseus, 555

enteritidis (Gartner), 520
erysipelatos suis, 511

suis, biological characters, 512
suis, morphology, 511
suis, pathogenesis, 514
suis, action of germicides on, 513
of Fiocca, from saliva of cats and

dogs. 547
fluorescens, thermal death-point of,

155
pntridus, influence of light on,

161

of foul brood (of bees), 556
of fowl cholera, 499

thermal death-point of, 501
action of antiseptics on. 501
der Frettchenseuche (Eberth and
Schimmelbusch). o08

Bacillus of Friedlander. 390

gallinarum (Klein) not identical with
Pasteur's bacillus of fowl chol-
era, 521

thermal death-point of, 150
gracilis cadaveris (Sternberg) from

liver of man, 560
of green pus, 542
of grouse disease (Klein), 520
of hog cholera (Salmon and Smith),

503

pathogenesis, 505
der Hlilinercholera. 499
hyacinthi (Walker), 573
hydrophilus fuse us (Sanarelli), 522
indicus. thermal death-point of, 155
of influenza (Pfeiffer), 403

toxin of, 405

der Kanincheuseptikamie(Koch), 499
lactis aerogenes (Escherich), 536
of Laser, perhaps identical with the
bacillus of swine plague, Mar-
seilles, 524
leporis lethalis (Sternberg, Gibier),

541
leprae, 486

pathogenesis, 488

of Lucet, perhaps identical with ba-
cillus gallinarum of Klein, 526
mallei, 489

pathogeuesis, 491
thermal death-point of, 156
des Mauseseptikamie (Koch), 511
of Mereshkowski, 565
of mouse septicaemia, 511

action of chlorine on, 177
mucosus ozffinae (Abel), 561
murisepticus (Fltigge), 511

thermal death-point of, 155
pleomorphus (Karlinski), 552
neapolitauus, thermal death-point

of, 155

cedematis malign i, 580
maligni, biology. 582
malign i, morphology, 581
maligni, pathogenesis, 583
maligni, No. II. (Novy). 587
pestis (Kitasato and Yersin), 562
biological characters as given by
Kitasato. 563

678

INDEX.

Bacillus pestis, influence of desiccation

on, 564
piscicidus (Fischel and Enoch), 567

agilis (Sieber), 565
pneumonias (Fliigge) (see Micrococ-

cus), 396

thermal death-point of, 155
of preputial sniegnia similar mor-
phologically to the syphilis bacil-
lus. 405

prodipio8us,pigment produced by, 135
influence of electric currents on,

163

influence of light on, 161
influence on the virulence of

anthrax cultures, 587
thermal death-point of, 155
vitality of, in moist atmospheres,

159
of purpura hsemorrhagica of Babes,

558

hsemorrhagica of Kolb, 559
haemorrhagica of Tizzoni and

Giovanini, 558
pyocyaneus, 542

Mitidotal effect of, in inoculated

anthrax, 544

butyric acid produced by, 139
green pigment of, 134, 543
general infection by, 544
pathogenesis of, 543
thermal death-point of, 155
varieties of, 545
pyocyaneus ft (P. Ernst), 545

distinguished from bacillus

pyocyaneus, 545
pyocyaneus pericarditidis (H. C.

Ernst), 545
pyogenes liliformis (Flexner), 567

fcetidus (Passet), 518
of rabbit septicaemia, 499
of rhinoscleroma, 496

similarity of, to Friedlander's

bacillus, 497
der Rinderseuche (Kitt), 499

amerikanischen (Caneva), 508
salivarius septicus (Biondi), 398
septiceemiae haemoiThagicaB, 499

hsemorrhagicse. biology and mor-
phology of, 500

Bacillus septicsemise haemorrhagicse, va~

rieties of, 344
septicus agrigenus (Nicolaier), 510

sputigenus (Fliigge), 398
des Schweinerothlauf (Loffler and

Schutz), 511

thermal death-point of, 155
der Schweineseuche (Loffler and

Schutz), 499

Marseilles (Rietsch-Jobert), 508
solanacearum (Smith), 571
of spontaneous rabbit septicaemia

(Eberth), 508

of swine pest (Selander), 503
plague (Billings), 503
plague, Marseilles, 508
post-mortem appearances in

death from, 510

subtilis, resistance of, to high tem-
perature, 154

tenuis sputigenus (Pansini), 523
tetani, 578

tracheiphilus (Smith1), 576
tuberculosis, 468

attenuation of, 485
biological characters, 472
branching forms of, 484
chlorine, action of, on, 177
cultures, difficulties of obtaining

pure, 474
cultures, methods of obtaining,

476
gallinarum, not a variation of

bacillus tuberculosis, 482
germicides, action of, on, 478
light, action of, on, 161
morphology, 469
in nasal cavity, healthy, 485
pathogenesis, 480
reproductive elements of, 485
resistance of, to freezing, 153
not ordinarily saprophytic, 474
spore formation undetermined,

472

sputum, examination of. for, 470
sputum, estimation of number

of, in, 479

staining methods for, 30
thermal death-point of, 158
vitality of, 478

INDEX.

679

Bacillus, typhi abdominalis (see Typhoid
Bacillus), 430

murium, 524
typhosus, 431, 436
ureae, 140

der \Vildseuche (Hueppe), 499
X (Steinberg) from yellow- fever ca-
davers, 538
Bacteria, a distinct class of vegetable

organisms, 11
action of acids on, 180

of alkalies, 183

of coal-tar products, 197

of gases, 172

of the haloid elements, 172

of salts, 186

chemical composition of, 124
classification of, 10
conditions of growth of, 125
culture media for, 37
dependent on pabulum, 153
dimensions of, 20

influence of pressure on growth of,
163

of physical agents, 153

of one species on the growth of

another, 128

mistaken for infusoria, 3, 10
modes of grouping, 20
modifications of biological charac-
ters, 129

morphology of, 20
motions of, 117
the photographing of, 103
probably seen by Leeuwenhoeck, 3, 10
products of vital activity, 133

acids, 137.

butyric acid, 138

fermentation, 136

fermentation, alkaline in urine
141

phosphorescence, 145
a possible nucleus in, 117
reproduction of, 117
rapidity of reproduction, 120
structure of. 117
thermal death-point of, 155
varieties of, 117

vitality of, in moist atmospheres,
159

Bacteria in the air, G13

number of relatively unimpor-
tant, 624

in articles of food, 667
in croupous pneumonia, 396
in diphtheria, 449
in the dust of city streets, 646
in the soil, methods of studying, 642
action of, on buried pathogenic

bacteria, 645
number of, in various localities

and at various depths, 643
of graveyards, 643, 646
in water, 62* >

species very numerous, 636
distilled, 125

of cadavers and of putrefying ma-
terial from various sources, 664
of plant diseases, 571
of the skin and exposed mucous

membranes, 648
species found on, 649
of the stomach and intestine, 658
nitrifying, 144
non-pathogenic, thermal death-point

of, 157
parasitic, 127
pathogenic, fatal dose of, 242

modes of action of, 219
Bacteriaceae of Zopf, 12
Bacterial diseases, immunity afforded by

an attack, 243
Bacte'ridie du charbon, 423
Bacteriology, literature of, 8

early progress of, 6
Bacterium aeruginosum, 542

coli commune (Escherich), 516, 529
of Davaine's septicaemia, 499, 500
of Ehrenberg, 3, 10
termo, 548

Barium chloride, antiseptic value of, 188
Baurngarten, bacilli of, 12

classification of bacteria, 12
on the influence of Koch's tubercu-
lin, 364

Bedding, disinfectants for, 213
Beef extracts as culture media, 41
Beggiatoa, sulphur in, 24
Behring's discovery of the antitoxins. 8
antitoxin of diphtheria, 314

680

INDEX.

Behring's discovery of the immunization

of large animals against tetanus, 356

serum for tetanus, strength and doses

of, 357
standard of diphtheritic antitoxin,

313
Belfonti's experiments on micrococcus

pneumonia; crouposse, 341
Benzene, germicidal power of, 198
Benzoic acid, antiseptic value of, 183
Benzo-naphthol, no germicidal power,

206

Beta-naphthol, antiseptic -power of, 206
Binary division of bacteria, 120
Biological classification of bacteria, 13
Biskra button, micrococcus of, 415
Bismarck brown as a stain, 25

for staining anthrax bacilli, 424
Blackleg, 347, 585
Bladder, urinary, free from bacteria in

health, 654

Blood, alkalinity of, as affecting immu-
nity, 236

free from bacteria in health. 664
germicidal power of, 236
of typhoid patients does not yield

cultures, 434
serum as a culture medium, -'57

germicidal powers of, 208, 257
immunizing value of, 313
mixture, Loffler 's, 48
solidification of, 57
solidified, streak cultures on, 77
sterilization of, 57
Sternberg's method of collect-
ing, 38

Blood serum as a medium of growth for .
bacillus A of Booker, 555
bacillus of acne contagiosa of

horses, 557

bacillus anthracis, 426
bacillus coli communis, 531
bacillus endocarditidis griseus,

556

bacillus enteritidis, 520
bacillus erysipelatos suis, 513
bacillus of Friedlander, 397
bacillus hydrophilus fuscus, 523
bacillus mallei, 491
bacillus cedematis maligni, 582

Blood serum as a medium of growth for:
bacillus pestis, 563
bacillus of purpura hsemorrhag-

ica (Baoes), 558

bacillus of purpura hsemorrhag-
ica (Tizzoni and Giovannini),
558

bacillus of swine plague, Mar-
seilles, 509
bacillus tetani, 580
bacillus tuberculosis, 474, 475
bacillus typhi abdominalis, 439
micrococcus gonorrhoeas, 393
micrococcus Manfredi, 413
proteus hominis capsulatus, 519
proteus of Karlinski, 552
proteus lethalis, 555
proteus septicus, 554
spirillum cholerse asiaticse, 596
spirillum of Finkler and Prior,

603
Bluchers apparatus for an atmosphere of

hydrogen, 87
Blue milk, 668

rays of spectrum, germicidal power

of, 161

Bonn's thernio-regulator, 90
Boiling in water, as a disinfectant, 212,

213
Booker's bacillus B, probably identical

with bacillus lactis aerogenes, 536
Boracic acid, action of, on bacteria, 182
Borden's method of photographing by oil

light, 111
Bordoni-Uffreduzzi on the bacteria of the

human skin, 648
bacillus of, from the marrow of
the bones of a leper, probably
not identical with bacillus
leprse, 487
Boric acid, action of, on micrococcus

pneumonise crouposae, 405
Botkin's apparatus for an atmosphere of

hydrogen, 87
bacillus butyricus, 139
Bougies of the Chamberland filter, 59,

60
Bouillon as a culture medium, 41

for bacillus aerogenes capsula-
tus. 589

INDEX.

681

Bouillon as a culture medium for bacil-
lus of Babes aud Oprescu, 526
for bacillus of Beck, 566
for bacillus of Cazal and Vail-

lard, 525
for bacillus coli communis,

532

for bacillus diphtherias, 454
for bacillus dysenteriae, 569
for bacillus of Emmerich and

Weibel, 566
for bacillus erysipelatos suis,

513

for bacillus of Fiocca, 548
for bacillus galliuarum, 521
for bacillus gracilis cadaveris,

560
for bacillus of hog cholera, 504,

509

for bacillus of influenza, 464
for bacillus of Laser, 524
for bacillus of Lucet, 526
for bacillus of Mereshkowsky,

565

for bacillus of Nicolaier (cap-
sule), 560
for bacillus cedematis maligni,

No. II., 588

for bacillus piscicidus, 567
for bacillus pyocyaneus pericar-

ditidis, 546
for bacillus septicaemia haem-

orrhagicae, 501, 509
for bacillus solanacearum, 572
for bacillus of swine plague,

Marseilles, 509
for bacillus tetani, 579
for bacillus of von Dungern

(capsule), 562

for micrococcus Manfredi, 413
for proteus hominis capsulatus,

519
for spirillum cholerae asiaticee,

596
for spirillum of Metschnikovi,

605
Bovine mastitis, micrococcus of (Kitt),

414

pneumonia, micrococcus of, 414
Branching forms of bacilli, 123

Bread, not sterilized by the heat of bak-
ing, 672

paste as a culture medium, 49
Brieger's bacillus, 516

toxic ptomains from cholera cultures,

21)2

typhotoxin, 367
Bromine, influence of, on bacterial

growth, 178
Bronchial glands, living tubercle bacilli

found in, 485
Broncho-pneumonia, a pseudo-influenza

bacillus in, 466
Brownian movements, 119
Brustseuche, 335
Bubonic plague, 562

discovery of the bacillus of, 8
infection in man by inoculation
through cutaneous lesions and
by the respiratory passages,
565
rats and fleas as propagators of,

564

Buchner's method of cultivating anaero-
bic bacteria, 85
theory of immunity, 255
Bujwid's test for the presence of the

cholera spirillum, 598
experience with tuberculin, 365
method of preparing tuberculin, 363
Bulbs, Steruberg's, 38, 66
Bureau of Animal Industry, report of
measures for arresting swine plague,
345

Butter, bacteria in, 670
Butyric acid, action of, on bacteria, 183
produced 'by bacteria, 138

CADAVERIN, 146

Cajuput, antiseptic power £»f, 203
Calamus, antiseptic power of, 203
Calcium chloride, antiseptic value of, 188
hydroxide, action of, on bacterial

growth, 184
hypochlorite, antiseptic value of,

188

light in photomicrography, 107
Camphor, germicidal power of, 199, 203
Canada balsam for mounting bacterial
slides, 28

682

INDEX.

Canon's method of demonstrating the ba-
cillus of influenza, 463
Capsule bacilli, Nicolaier's comparison

of the characters of, 560
bacillus of Nicolaier, 560
of von Dungern, 562
Caraway, antiseptic value of, 203
Carbolic acid in culture media, 47
as a disinfectant, 212, 213
action of. on anthrax spores,

587
action on bacillus tuberculosis,

478
action on micrococcus pneu-

monice crouposse, 405
for restraining growth of water
bacteria, in searching for the
typhoid bacillus, 444
Carbol-fuchsin solution, Ziehl's, 29
Carbon for bacterial growth, 126

dioxide, influence of, on bacterial

growth, 174

prevents growth of the anaerobic
bacillus of symptomatic an-
thrax, 586

does not prevent growth of the
anaerobic bacillus cedernatis
maligni No. II., 588
monoxide, influence of, on bacterial

growth, 174

Carnivora immune to tuberculosis, 233
Carrot, antiseptic power of, 203
Cars, railway, disinfection of, 214
Cascarilla, antiseptic power of, 203
Cattle, prevalence of tuberculosis among,

482

Caucasian milk ferment, 139
Caustic potash, action of, on micrococcus

pneimionise crouposse, 405
Cedar, antiseptic power of, 203
Celery, antiseptic power of, 203
Cell-globulin, germicidal power of, 210
Cells, vital activity of, 133
Cellular structure of bacteria, 117
Cellulose, fermentation of, 141
" Centralblatt fur Bakteriologie," 9
Cerebrospinal meningitis, diplococcus

of, 410

Cesspools, disinfection of, 217
Chamomile, antiseptic power of, 203

Chantemesse and Widal's immunization
against the typhoid bacillus,
368

method of searching for the
typhoid bacillus in water sup-
plies, 444

Chaplets, grouping of bacteria in, 22
Charbon, bacillus of, 42U
symptomatique, 347

Chauveau's retention theory of immu-
nity, 250
Cheese, bacteria in, 670

toxic principle in, 148
Chemical agents, influence of, on cultures

of the cholera spirillum, 597
analysis* of water, advantages of, 631
Chemiotaxis, 247, 257
Cherry laurel, antiseptic power of, 203
Cheyne's experiments on the toxicity of

cultures of proteus vulgaris, 550
observations on the pathogenic power

of saprophytes, 529

Chloral hydrate, antiseptic value of, 189
Chloride of lime as a disinfectant, 212, 213
Chlorinated soda as a disinfectant, 213
Chlorine, influence of, on bacterial

growth, 177

Chloroform, action of, on bacteria, 177
Cholera, a disease of man, 233
cultures, toxalbumin of, 150
experimental evidence of its produc-
tion in man by the comma bacil-
lus, 601

immunity after an attack of, 297
infection, heat as a disinfectant for,

154

ptomains, 149
second attacks of, 243
spirillum, influence of light on, 162
vaccine (Ferran), 299

(Haffkine), 302
Cholera infantum, bacillus A of Booker

in, 555

proteus vulgaris in, 550
Cholera morbus not caused by the Fink-

ler-Prior spirillum, 602
proteus Hauseri in, 551
Cholin, 147

Chromic acid, action of, on putrefactive
bacteria, 181

INDEX.

683

Chromogenic organisms, 14, 135
Cinnamon, antiseptic power of, 203
Cities, bacteriology of the air of, 025
Citric acid, action of, or pathogenic bac-
teria, 182

Citron, antiseptic power of. 203
Cladothrix, apparent branching in, 24
Cladotrichese (Zopf), general characters,

12, 19

Classification of bacteria, 10
Clostridinm, general characters of, 18
spore formation in, 122
of bacilli, 23

Clothing, disinfection of, 213
Clou de Biskra, micrococcus of, 415
Cloves, antiseptic power of, 203
Cocci of Baumgarten, 12
Coccocese (Zopf), 12
Cocoanut water analysis of, 40
as a culture medium, 39
for bacillus cavicida havanien-

sis in, 517
for bacillus cuniculicida hava-

niensis in, 539

Coffee infusion, antiseptic power of, 200
Cohn's classification of bacteria, 11

solution, 40, 126

Cohnheim, production of tuberculosis by

inoculation of tuberculous material, 467

Cold, influence of, on bacterial vitality,

153
Colon bacilli, difficulty of distinguishing

from allied groups, 534
and typhoid bacilli, varieties of

the same species? 447
frequency of, in water supplies,

640
methods for their detection in

water supplies, 640
Smith's method for their detec-
tion, 535
bacillus of Escherich, 529

its important role in human

pathology, 533

Colonies on plates of solid media. 72
Comma bacillus of Koch, 593
Conjunctiva, bacterial flora of, 649
Contact preparations, 27
Copaiba, antiseptic power of, 203
Copper sulphate as a disinfectant, 212

Coriander, antiseptic power of, 203
Corn, Indian, bacterial diseases of, 575
Cotton, first use of, as a filter for micro-
organisms, 4
plugs for test tubes, 62

for tubes containing aerobic bac-
teria, 54

in growing tubercle bacilli, 475
Cover-glass preparations, 25
Cows, cocci of haemoglobinuria of, 419

streptococcus of mastitis of, 419
Creolin, germicidal power of, 201
Creosote, germicidal power of, 201
Cresol, germicidal power of, 201
Cruciferous plants, brown rot in, 575
Cuba, cases of Malta fever from, 420
Cucumbers, bacterial diseases of, 576
Cucurbitacese, bacterial diseases of, 576
Culture media, 37

artificial, 40
for bacillus mallei, 490
for bacillus tuberculosis, 474
liquid, 40
natural, 37
neutralization of, 49
solid, 41

sterilization of, 52
Cultures of anthrax, chemical products

in, 428

of bacillus diphtherias, vitality of, 455
of typhoid bacilli obtained from the
spleen in typhoid fever cases, 433,
436

drop, 64

in liquid media, 62, 67
in solid media, 69
stab, 69
streak, 77

Cumin, antiseptic power of, 203
Cunningham's objections to accepting
Koch's bacillus as the specific
cause of cholera, 601
vibrios resembling those of cholera.

609

Cupric chloride, antiseptic value of, 189
sulphate, antiseptic value of, 189
action of, on micrococcus pneu-

moniae crouposse, 405
Curry on Malta fever in the Philippine
Islands, 420

684

INDEX.

Cystitis, proteus vulgaris in, 551
Czenzynke's staining solution for the
bacillus of influenza, 468

D'ARSOXVAL'S incubating apparatus, 04
Davaine, causation of anthrax discovered

by, 0. 422

classification of bacteria, 10
Dead, disinfection of the. 213
Death-point, thermal, of bacteria, 155
De Bary's classification of bacteria, 12
Decolorization in staining, 28
Defensive proteids of Haffkine, 265
De Giacoma's method of staining syph-
ilis bacilli, 495
Deneke's spirillum, 603
De Schweinitz, immunity from tubercu-
losis conferred by attenuated cultures,
364
Desiccation, influence of, on bacteria,

159

resistance of anthrax spores to, 427
influence of, on bacillus pestis, 564

on the comma bacillus, 597
Developers in photomicrography, 113
Diaphtherin, germicidal power of, 202
Dieudonne on the vibrios resembling the

cholera spirillum, 606
Dimethylamine, 147
Diphtheria antitoxin, 314

results of treatment with, 462
bacillus of, found in throat for weeks
after subsidence of fever, 451,
460

attenuated varieties, 309
branching forms of (C. Frankel),

460

bacteria in, 449
cultures, toxalbumin of, 312
evidence on which the Klebs-Lb'ffler
bacillus is regarded as the cause
of, 450

heat as a disinfectant for, 154
intestinal in rabbits, bacillus of, 459
a local infection. 440
mixed infections in, 461
post-mortem appearances of, 226
toxalbumin of, 140

Diphtheritic false membranes, strepto-
coccus pyogenes in. 386

Diphtheritic inflammation, cause of,

225

Diplococcns, general characters, 17
of Frankel, 339
grouping of, 21

intracellularis ineningitidis, 410
pneumonise, discovery of, 7
pneumonise (Weichselbaum), 398
in meningitis, 400.
in otitis media, 373, 390
Discontinuous heating in sterilizing, 53,

56, 57
Disease, race immunity to, 234

susceptibility to, 233
Diseases of plants, bacteria of, 571
Disinfectant, importance of sunlight as

a, 102
Disinfectants, action of, 164

influence of, on cholera cultures, 507
Disinfection, practical directions for, 212
Disinfektol, germicidal power of, 202
Distilled water, bacteria in, 125, 635
Double staining of bacterial preparations,

of spore preparations, 31
Drop cultures, 04

Druse des Pferdes. streptococcus of, 418
Ducks, bacillus of cholera in (Cornil and

Toupet), 503

Duclaux's solution for butyric-acid fer-
ment, 138

Dunham's solution, 151
peptone solution, 41
test for the presence of the cholera

spirillum, 598

Dust, atmospheric, bacterial constitution
of, 614

EBERTH'S bacillus. 431
Egg albumen as a culture medium for
bacillus pyocyaneuspericarditidis. 547
Eggs, bacteriology of, 671

as a culture medium, 40
Ehrenberg's vibrioniens, 3, 10
Ehrlich's aniline-gentian-violet solution,

29
solution in staining bacillus mallei,

400

method of staining bacillus tubercu-
losis, 469

INDEX.

685

Ehrlich's side chain theory, 269
Ehrlich-Weigert's aniline-methyl-violet

solution, 29, 471

method of staining the tubercle ba-
cillus, 30

stain for syphilis bacilli, 495
Electric light in photomicrography, 107
Electricity, influence of, on bacterial

growth, 162

Eisner's method for detecting the ty-
phoid bacillus in water and faeces,
446
Emmerich's bacillus (B. ueapolitanus),

529

thermal death-point of, 155
experiments on rnicrococcus pneu-
monias crouposae, 341
Endocarditis, ulcerative, micrococcus

pneumonise crouposae in, 401
Endospores, 16

Enzymes produced by liquefying bac-
teria, 136
Eosin as a double stain, 28

collodion in photographing bacteria,

103

Eosinophile cells, 237
Epidemics, Pasteur on the origin of, 290
Erlenmeyer flasks, 60, 63
Escherich's view that pseudo-diphther-
itic bacilli are distinct from the true
bacillus, 460
Esmarch's roll tubes, 76.

tubes in water analysis, 628,

630

method of cultivating anaerobic bac-
teria, 84

Essential oils, antiseptic power of, 202
Ether, action of, on bacillus tuberculosis,

478

antiseptic power of, 202
in experiments on animals, 99
Eugenol, antiseptic power of, 203
Euphorin, feeble germicidal activity of,

204

Eucalyptol, antiseptic power of, 203
Eucalyptus, antiseptic power of, 203
Excreta, disinfection of, 212, 217

method of testing for the cholera

spirillum, 598
Exhaustion theory of immunity, 249

Expired air free from micro-organisms,

622
Eye, inoculations into, 99

FACULTATIVE anaerobics, 16, 18

in stab cultures, 70
parasites, 15, 127, 221
saprophytes, 221

Faeces, list of species of pathogenic bac-
teria found in, 663
Fallopian tubes free from bacteria in

health, 654

Farcy, symptoms of, 491
Fasching's capsule bacillus, 561
Feet, the, bacteria on, 649
Fennel, antiseptic power of, 203
Ferment lactique of Pasteur, 667
Fermentation by bacteria, 136

of grape sugar in water sup-
plies, 535

in liquid media, 68
putrefactive, 141
test for colon and typhoid bacilli in

water, 445
tubes, 68
of urea, 140
viscous, 141
Ferments, soluble, produced by bacteria,

143
Fermi and Pernossi on the properties of

tetanus poison, 354

Ferran's anticholeraic inoculations, 301
Ferret disease, bacillus of, 508
Ferric chloride, antiseptic value of,
189
sulphate, action of, on micrococcus

pneumonise crouposae, 40")
antiseptic power of, 189
Filters, soluble, in analysis, 620
Filtration as a means of sterilizing, 58
Finger-nails, bacteria found under, 648
Finkler and Prior, spirillum of, 602
Fiocca's bacillus, 547

method of staining spores, 32
Fire as a disinfectant, 212, 213
Fish, boiled, as a culture medium, 40
Flagella of bacteria, 24, 119
methods of staining, 32
of bacillus amylovorus, 576
campestris, 575

INDEX.

Flagella of bacillus of hog cholera

(Loffler's staining), 504
hyacinthi, 573
cedeinatis nialigni, 581
redernatis maligni, No. II., 588
solanacearum, 571
of swine plague, 509
of symptomatic anthrax, 586
typhi abdominalis, 437
typhi murium, 524
proteus vulgar is, 550
pseudomonas Stewarti, 575
spirillum of Finkler and Prior, 002
Metschnikovi, (504
Obermeieri, 590
Flasks, Erlennieyer's, for liquid media,

60, 63

Pasteur's, 63
Sternberg's, 38, 66

Fleas, propagators of bubonic plague, 504
Fleckenkrankheit, 415
Flesh-peptone solution as a culture me-
dium, 41
gelatin, preparation of, 41

Koch's, use of, 69
Flesh water as a culture medium, 40
Flexner's description of bacillus dysen-

terise, 568
Fliigge's micrococcus septicus, 415

directions for preparing fermented

milk, 139

Foa and Scabia's experiments on micro-
coccus pneumonise crouposse, 342
Foot-and-mouth disease, protective inoc-
ulations in, 318

Formaldehyde as a disinfectant, 204, 216
Formalin, disinfectant power of, 204
use of, as a test for typhoid bacilli,

446

Formic acid as a germicide, 183
Formol, disinfectant power of, 204
Foth's directions for preparing mallein,

320
Fowl cholera, causes of, 288

bacillus of, action of chlorine

on, 177
bacillus of, action of germicides

on, 501

bacillus of, thermal death-point
of, 156

Frankel's method of cultivating anae-
robic bacteria, 82
pneumococcus, :)•']'.>
researches on the bacteria of the soil,

643

Frankel-Gabbett method of staining ba-
cillus tuberculosis, 471
Fraukel and Simmonds, multiplication
of typhoid bacilli in the spleen after
death, 433

Freezing, influence of, on bacterial vi-
tality, 153
French commission, report of, on Fer-

ran's anticholeraic inoculations, 301
Freudenreich's modification of Koch's

plate method, 71)
Friedlander's bacillus, analysis of, 124

thermal death-point of, 155
pneumococcus, 396
method of staining the tubercle ba-
cillus, 30

Fuchsin as a stain, 25, 27
Fuller's method of determining the re-
action of nutrient media, 50
Furniture, disinfectants for, 213

GABBETT'S method of staining bacillus

tuberculosis, 30

Gaffky's explanation of the failure to
find Eberth's bacillus in every case of
typhoid fever, 432
Galbanum, antiseptic power of, 203
Gallic acid, action of, on bacteria, 183
Gas developed by the bacillus of Em-
merich and Weibel, 566
by bacillus oedematis nialigni,

No. II., 588

by bacillus piscicidus agilis, 565
by bacillus of von Dungern (cap-
sule), 562

bacillus (the) in its relations to hu-
man pathology, 589
Gases, action of, on bacteria, 172
Gaslight, photomicrography by, 109
Gastric juice, germicidal power of, 658
protective by its acidity against

attacks of cholera, 600
Gastro-enteritis cholerica of chickens,
symptoms and post-mortem appear-
ances, 605

INDEX.

687

Gelatin as a culture medium, 41

liquefaction of, by bacteria, 135
growth of bacillus aerogenes capsu-

latus on, 589

of bacillus dysenteriae on, 569
of bacillus cedeiiiatis rnaligni

No. II. on, 588

of symptomatic anthrax on, 086
of bacillus solanacearum on. 072
nutrient, as a culture medium, 42
for bacillus diphtherias. 454
for bacillus cedeiiiatis maligni,

582

plates, counting the colonies on, 630
plates in the cultivation of :
bacillus A of Booker, 555
bacillus acidiformans, 537
bacillus alvei, 556
bacillus anthracis, 425
bacillus of Beck, 566
bacillus of Belfonti and Pasca-

rola, 508

bacillus capsulatus, 522
bacillus cavicida, 516
bacillus coli communis, 531
bacillus crassus sputigenus, 517
bacillus of Emmerich and Wei-

bel, 566
bacillus endocarditidis griseus,

555

bacillus enteritidis, 520
bacillus erysipelatos suis, 512,

513

bacillus of Fiocca, 548
bacillus of Friedlander, 397
bacillus gallinarum, 521
bacillus of grouse disease, 521
bacillus of hog cholera, 504, 509
bacillus lactis aerogenes, 536
bacillus of Laser, 524
bacillus of Lucet, 526
bacillus of Mereshkowsky, 565
bacillus of Nicolaier (capsule),

560

bacillus piscicidus, 567
bacillus piscicidus agilis, 565
bacillus of purpura hasmorrhagi-

ca, Tizzoni and Giovannini,

558
bacillus pyocyaneus, 543

Gelatin plates in the cultivation of :
bacillus pyocyaneus /3, 545
bacillus pyocyaneus pericardi-

tidis, 546

bacillus pyogenes fcetidus, 518
bacillus septicaemias hasmor-

rhagicae, 500, 509
bacillus tetani, 579
bacillus typhi abdominalis, 438
bacillus typhi murium, 524
bacillus of von Dungern (cap-
sule, 562

micrococcus bothryogenus, 412
inicrococcus Manfredi, 413
micrococcus tetragenus, 411
proteus hominis capsulatus, 519
proteus of Karlinski, 552
proteus lethalis, 554
proteus mirabilis, 553
proteus septicus, 554
proteus vulgaris, 549
proteus Zenkeri, 554
spirillum choleras asiaticae, 594
spirillum of Finkler and Prior,

603

spirillum Metschnikovi, 604
spirillum tyrogenum, 603
staphylococcus pyogenes aureus,

374

streptococcus pyogenes, 384
in water analysis, 628
stab cultures of :

bacillus A of Booker, 555
bacillus acidiformans, 537
bacillus acne contagiosa of

horses, 557
bacillus alvei, 557
bacillus anthracis, 425
bacillus of Babes and Oprescu,

526

bacillus of Beck, 566
bacillus of Belfonti and Pasca-

rola, 508

bacillus capsulatus, 522
bacillus cavicida havaniensis,

516
bacillus of Cazal and Vaillard,

525

bacillus of cholera in ducks, 503
bacillus coli communis, 531

688

INDEX.

Gelatin, stab cultures of :

bacillus crassus sputigenus, 518
bacillus cuniculicida havanien-

sis, 539
bacillus of Emmerich and Wei-

bel, 560
bacillus endocarditidis capsula-

tus, 556
bacillus endocarditidis griseus,

555

bacillus enteritidis, 520
bacillus erysipelatos suis, 512
bacillus of Friedlander, 397
bacillus gallinaruui, 521
bacillus gracilis cadaveris, 560
bacillus of grouse disease, 521
bacillus of hog cholera, 504
bacillus hydrophilus fuscus, 522
bacillus lactis aerogenes, 536
bacillus of Laser, 524
bacillus leporis lethalis, 541
bacillus of Lucet. 526
bacillus of Nicolaier (capsule),

560

bacillus cedeinatis maligni, 582
bacillus piscicidus, 567
bacillus of purpura hEemorrha-

gica (Babes), 558
bacillus of purpura hsemorrha-

gica (Kolb), 559
bacillus pyocyaneus, 543
bacillus pyocyaneus ft, 545
bacillus pyocyaneus pericardi-

tidis, 546

bacillus pyogenes fcetidus, 518
bacillus of symptomatic anthrax,

586

bacillus tetani, 579
bacillus typhi abdominalis, 439
bacillus typhi murium, 524
bacillus of von Dungern, 562
micrococcus bothryogenes, 412
micrococcus Man f red i, 413
micrococcus tetragenus, 41 1
proteus hominis capsulatus, 519
proteus of Karlinski, 552
proteus lethalis, 555
proteus mirabilis, 553
proteus septicus, 554
proteus vulgar is, 549

Gelatin stab cultures of :

spirillum cholerae asiaticae, 595
spirillus of Finkler and Prior,

603

spirillum Metschnikovi, 605
spirillum tyrogenum, 603
staphylococcus pyogenes aureus,

374

streptococcus pyogenes, 384
roll-tubes for :

bacillus acidiformans, 537
bacillus cavicida havaniensis,

516

bacillus cuniculicida havanien-
sis, 539

bacillus grandis cadaveris, 560
bacillus leporis lethalis, 541
stick cultures, staining for, 36
streak culture of bacillus of Lucet,

526
culture of bacillus of swine

plague, Marseilles, 509
Gentian violet as a stain, 25, 27
Geranium, antiseptic power of, 203
Germicides, definition of, 164

methods of determining value of, 166
Germs in air, number of, 623
Gessard -on the pigments produced by

bacillus pyocyaneus, 134
researches on bacillus pyocyaneus,

543

Giant cells in tuberculous nodules, 468, 472
in lungs from injection of dead

tubercle bacilli, 485
Glaeogenes of Cohn, 11
Glanders, animals affected by, 233
bacillus of (B. mallei), 489
methods by which infection is com-
municated, 491

test for, by mallein, 152, 321, 494
by obtaining pure cultures by

inoculation, 493
Glucose in culture media, 46

influence of, on bacterial growth, 128
Glycerin, action of, on bacteria, 204
Glycerin-agar, preparation of, 43

bacillus acidiformans, growth of, on,

538

of Babes and Oprescu, 626
cuniculicida havaniensis, 539

INDEX.

G89

Glycerin-agar. bacillus hydrophilus fus-

cus, 523
mallei, 490
pestis, 563

of swine plague. Marseilles, 509
tuberculosis, 476
roll -tube cultures of bacillus cavi-

cida havaniensis, 517
of bacillus cadaveris in hydro-
gen atmospheres, 584
Goat's milk immunized, antitoxin of, 357
precipitation of antitoxin from,

358

Gold chloride as an antiseptic, 190
Gonococcus of Neisser, 226, 391

biological characters of, 393

discovery of, 7

multiplication of, in leucocytes,

253

Graham bread, bacteriology of, 672
Grain's method of staining, 29, 34

solution for decolorizing in staining,

Grape sugar in liquid culture media, 68
growth of bacillus coli com-

munis in solutions of, 532
Graveyards, bacteria in the soil of, 643,

(.4(5

Gray-parrot disease, micrococcus of, 419
Ground-water region of the soil, free from

bacteria, 644

Guaiacol, antiseptic power of, 204
Guinea-pigs, effects of injection of tuber-
culous matter into, 480

ILEMATOCOCCUS bovis (Babes), 419
Hsemoglobinuria of cows, cocci of, 419
Haffkine's anticholeraic inoculations,

302

Hail, number of bacteria in, 632
Haloid elements, action of, on bacteria,

172

Hands, disinfection of the, 216, 648
Hanging-drop cultures, 64

of typhoid bacilli, appearance

of, 442
Hansen's discovery of the bacillus of

leprosy, 7
Hazen and White's method of separating

typhoid bacilli in water supplies, 445
44

Heat, action of, on anthrax spores, 587
attenuation of virulence by, 131
influence of, on bacterial growth,
153

on bacillus pestis, 564
isolation of spores by, 68
as a sterilizer of culture media, 52
dry, as a disinfectant, 212, 213

influence of, on bacterial growth,

154

non-penetration of, 154
as a sterilizing agency, 53
moist, influence of, on bacterial vi-
tality, 154
Heating, discontinuous, as a sterilizing

measure, 53

Heliostat in photomicrography, 107
Helman's tuberculin from potato cul-
tures, 363

Helmholtz'S experiments opposed to spon-
taneous generation, 4
Hernialbumose, 262

Herbivora, susceptible to bacterial injec-
tion, 96
to anthrax, 234
Hericourt and Richet, experiments of, on

bacillus tuberculosis, 364, 365
Hesse's apparatus for the bacteriological

analysis of air, 618

Heterogenesis or spontaneous genera-
tion, 5

Heydenreich's micrococcus, 415
Hoffman's experiments on the preserva-
tion of putrescible liquids, 5
Hog cholera, bacillus of (Salmon and

Smith), 503

prevalence, symptoms and post-
mortem appearances of, 505,
506
protective inoculations against,

322
group of bacteria, varieties or

species '? 506, 507
erysipelas, bacillus of, 511

protective inoculations against,

324

symptoms and post-mortem ap-
pearances of, 514

Holz's method of searching for the ty-
phoid bacillus in water supplies, 444

690

INDEX.

Horse, symptoms of glanders in the, 491
immunization of, against tetanus,

360
Horses, bacillus of acne contagiosa of,

557

diplococcus of pneumonia in, 418
streptococcus coryzae contagiosae of,

418

Hot-air oven for sterilizing, 53
Hot Springs, Ark., Malta fever from the

tropics at, 420
Hueppe's apparatus for sterilizing and

coagulating blood serum, 58
classification of bacteria, 12
Hyacinth, bacterial disease of the, 574
Hydrant water, number of bacteria in,

634

Hydrocele fluid as a culture medium, 40
Hydrochloric acid, action of, on micro-
coccus pneumoniae crouposse,
405

as a decolorizer in staining, 28
influence of, on bacterial growth,

181
Hydrofluoric acid, influence of, on the

tubercle bacillus, 179
Hydrogen generator, 86

in the cultivation of anaerobic bac-
teria, 82

influence of, on bacterial growth, 173
peroxide as a disinfectant, 173
Hydronaphthol, antiseptic power of, 205
Hydrophobia, influence of temperature
on the infectious material of, 156
protective inoculations against, 332
toxalbumin of, 327

Hydrosulphuric acid, a bacterial produc-
tion, 141
influence of, on bacterial growth,

175

Hydroxylamin, antiseptic power of, 204
Hypodermic injection of cholera vaccine,

299
syringe for inoculations, 97

ICE, number of bacteria in, 633

cream, toxic principle in, 148
Ichthyol, antiseptic power of, 204
Illicium, antiseptic power of, 203
Immunity from disease, 233

Immunity, acquired, 242

alkalinity of the blood as affecting,

236

dependent on antitoxins. 267
influence of animal temperature ont

235

natural agencies by which neutral-
ized, 240
theories of, 249
to anthrax by filtered cultures of the

bacillus, 428
by anthrax toxin, 427
to cholera by a previous attack, 2!>7
to serpent venom, 262
to tetanus by cultures in thymus

bouillon, 359
inherited, 360

to tuberculosis by attenuated cul-
tures, 485
Immunization of swine against rouget,

method and disadvantages of, 515
Impf tetanus bacillus, 508
Incubating ovens, 88
Indigestion, sarcinae in stomach of per-
sons suffering* from, 662
Indol, antiseptic power of, 205
production of, 151

by the cholera spirillum, 598
by vibrios resembling the chol-
era spirillum, 607
by bacillus septicaemias hsemor-
rhagicae in peptone solution,
501
by bacillus of swine plague,

Marseilles, 509
by capsule bacillus of von Dun-

gern, 562
not produced by the bacillus of hog

cholera, 505

test for the typhoid bacillus, 440
Infection, rapidity of, 231

by inhalation, 102
Infections, mixed, 228

secondary, 227
Infectious diseases, chronic, bacilli in,

467
Influenza, protective inoculations against,

335

bacillus, discovery of, 8
morphology. 463

INDEX.

691

Influenza, bacillus, pathogenesis, 464
stains for, 4i;:J
toxin of. 405
vitality of. 4(54
in horses, protective inoculations

against. 335

Inhalation, infection of animals by, 102
Inoculation experiments with bacillus

pest is, 503
of liquid media, 03

Inoculations, protective, in cholera, 204
Insects, transmission of plant diseases

by, 570
Intestinal bacteria, not concerned in the

physiology of digestion, 002
mucousmembrane, infection through,

230
Intestine, bacterial injections into. W

of man, bacteria found in, 000, 00.--J
Involution forms of bacilli, 23

of bacillus of symptomatic an-
thrax, 580

Iodine, action of, on micrococcus pneu-
monias crouposae, 405
influence of, on bacterial growth, 178
trichloride as a disinfectant, 178
lodoform as an antiseptic, 178
lodoform-ether, action of, on bacillus

tuberculosis. 179, 478
lodol without germicidal power, 179
Issaeff 's experiments on the micrococcus

pneumonias crouposae, 342
Izal, disinfectant power of, 205

JAHRESBE RIGHT (the) of Baumgarten, 9
Jequirity solution as a culture medium, 47
"Journal of Hygiene." 9
"Journal of Pathology and Bacteri-
ology, " 9
Juniper, antiseptic power of, 203

KAESESPIRILLEX, 003
Kidneys, cultures of typhoid bacilli ob-
tained from the. 433, 430
specially affected by staphylococcus

pyogenes aureus, 370
Kinyoun's treatment of the horse for

diphtheria antitoxin, 310
Kitasato, bacteriological work of, 8

distinguishing biological characters
of the bacillus of influenza, 404

Kitasato, experiments on the bacillus of

tetanus. 351

results with tuberculin, 305
Kitt's micrococcus of bovine mastitis,

414

Klebs on the influence of Koch's tuber-
culin. 304

method of obtaining pure cultures,
^___6_Z_

Klebs-Loffler bacillus of diphtheria, 449
value of, for diagnostic purposes,

451

Klemperer's experiments on cholera im-
munization. 308
Koch's apparatus for coagulating blood

serum, 58

bacteriological researches, 7
decolorizing solution, 28
differentiation of species, 482
discovery of the tubercle bacillus,

407, 408

experiment showing that the comma
bacillus will infect when intro-
duced in a living condition into
the intestine, 000

experiments on the thermal death-
point of bacteria, 154
flesh-peptone gelatin, use of, 09
inoculation syringe, 97
method of determining the germi-
cidal power of chemical agents on
anthrax spores, 108
method of staining flagella, 32
plate method, 74

method, modifications of, 79
method, in water analysis, 029
statement of the action of sunlight

on the tubercle bacillus, 478
steam sterilizer, 55
tuberculin, 151, 301
production of, 302
experiments favorable to, 479
typhoid bacillus, 432
vibrios remotely resembling the chol-
era spirillum, 007
views of the interior structure of the

bacterial cell. 118
Kockel's capsule bacillus, 501
Kolb's bacillus of purpura haeinorrhagicai
559

692

INDEX.

Kommabacilhis der cholera asiatica, 593

Kreibohm's bacillus, 517

Kruse's modification of Koch's plate

method, 79
Kuhne's method of staining bacteria in

tissues, 35
silicate jelly, 47

staining method for bacillus mallei,
400

LACTIC acid, action of, on bacteria, 182

Lactose-litmus-agar, 47

Lactose and litmus for detecting typhoid

bacilli, 445

Lake water, number of bacteria in, 633
Lancet-shaped micrococcus (Talamon),

398

Lanolin, antiseptic power of, 205
Laundries, bacteria in wash-water from,

649

Lavender, antiseptic power of, 203
Lead chloride as an antiseptic, 190

nitrate as an antiseptic, 190
Leprosy, bacillus of, 487
Leptotrichese of Zopf, general characters,

12, 19
Leucocytes, protective against bacterial

invasion, 239

multiplication of gonococci in, 258
Leuconostoc, general characters of, 17
Liborius' method of cultivating anaero-
bic bacteria, 85
Light, attenuating influence of, 279

influence of, on bacterial growth, 159
action of, on bacillus tuberculosis,

478

for photomicrography, 107
Linen underclothing, bacteria on, 649
Liquefying bacteria, 16

in stab cultures, 71

Liquid media, cultures of anaerobic bac-
teria in, 81
Liquids, non-evolution of bacteria from

the surface of, 614

Lithium chloride as an antiseptic, 190
Litmus, in culture media, 46, 64

action of bacillus of hog cholera on,

504, 509

Liver, cultures of typhoid bacilli ob-
tained from the, 433, 436

Lochial discharge, presence of bacteria

in, 054

Loftier 's aniline-blue solution, 29
blood -serum mixture, 48
culture medium for bacillus diph-
therias, 454

method of staining bacteria in tis-
sues, 34

of staining flagella, '.}•!
solution for staining anthrax bacilli,

424

for staining the typhoid bacil-
lus, 4:;:

Loretin, disinfectant power of, 205
Liibert's researches on the action of anti-
septic agents on staphylococcus pyo-
genes aureus, 375
Lustgarten's bacillus, 494
Lydton's report on protective inocula-
tions in Baden, 350
Lymph, germicidal power of, 236
Lysol, disinfectant power of, 205

MACE, antiseptic power of, 203
Maffucci 's conclusions on the tuberculo-
sis of fowls, 484
Maggiora's researches on the bacteria of

the soil, 644
Magnesium light, in photomicrography,

107

Mail matter, disinfection of, 214
Mai d'araigne"e, micrococcus of, 416
Mai de pis, micrococcus of, 416
Malic acid as a germicide, 183
Malignant oedema, bacillus of, 580

pustule in man, 422, 428
Mallein, a glycerin extract from cultures
of the bacillus of glanders, 318, 494
as a test for glanders in horses, 321
preparation of, in Pasteur's labora-
tory, 319

Foth's method of preparing, 320
Malnutrition, influence of, on develop-
ment of pus cocci, 224
Malta fever, micrococcus of, 420

Widal reaction in, 421
Maminite contagieuse, micrococcus of,

417

Manganese protochlorite as an antiseptic,
190

INDEX.

693

Marchand's capsule bacillus, 561
Marjoram, antiseptic power of, 203
Marmier's experiments on the toxin of

anthrax, 430
Marpmann's method for the detection of

pathogenic bacteria in water, 040
Marsh gas, a bacterial product, 141
Mastitis in cows, micrococcus of. 417

streptococci of, varieties of a

single species, 419
gangrenous, in sheep, micrococcus

of, 410

Matico, antiseptic power of, 203
Mauseseptikamiealmlicher bacillus (Eis-

enberg), 515

Measles, second attacks of, 243
Meat infusions as culture media, 40
Meats, bacteria in, 070
Meatus urinarius of man, bacteria in,

654

Meconium, free from bacteria, 660
Media, liquid, cultures in, 62

characters of growth in, 64
solid, cultures in, 69
Mediterranean fever, micrococcus of, 420
Melons, bacterial diseases of, 576
Merchandise, disinfection of, 214
Mercuric chloride action of, on anthrax

spores, 587

•action of, on bacillus tubercu-
losis, 478
action of, on micrococcus pneu-

rnoniae crouposae, 405
as a disinfectant, 212, 213
as an antiseptic, 190
cyanide as a disinfectant, 192
iodide as an antiseptic, 192
Merismopedia, general characters of, 17

grouping of, in tetrads, 22
Mesenteric glands, cultures of typhoid

bacilli obtained from, 433, 436
Metachromatic granules, 118
Metacresol, germicidal power of, 202
Methane, influence of, on bacterial

growth, 175
Methylamine, 147, :323
Methylene blue as a stain, 25, 27
Methyl-guanidin, 148
Metschnikoff's theory of phagocytosis,
208, 256

Mice, subcutaneous injection of, 99

field, immune to mouse septicaemia,

515

Mereshkowsky 's suggested meth-
od of destroying, 567
Microbe du cholera des poules (Pasteur),

499

du pus bleu, 542

Micrococci, general characters, 17
infrequent in the soil, 645
list of species found in the soil

(Fulles), 646
non-pathogenic, list of species found

in water, 638
pathogenic, list of species found in

water, 638

Micrococcus askofornians (Johne), 412
of Biskra button, 415
botbryogenus (Rabe), 412
of bovine mastitis, 414

pneumonia, 414
endocarditidis rugatus (Weichsel-

baum), 416

of erysipelas (Fehleisen), 382
of fowl cholera, thermal death-point

of, 150

of gangrenpus mastitis in sheep, 416
gonorrhceal, 391

growth of, in acid media, 393
thermal death-point of, 156
of Heydenreich, 415
of Manfredi, 413
melitensis. 420

of myko-dermoids of the horse, 412
ovatus, 415

Pasteuri (Sternberg), 339, 398
discovery of, 7, 339, 398
found in the mouth. 652
thermal death-point of, 155
pneumoniae crouposae, 339, 398

crouposae, biological characters

of, 403

crouposae, capsule of, 402
crouposae, discovery of, 7
crouposae, morphology of, 402
crouposae, pathogenesis, 400
crouposae, thermal death-point

of, 404

crouposae, Belfonti's experi-
ments on, 341

694

INDEX.

Micrococcus pneumonias crouposae, Em-
merich's experiments on, 341
crouposae, Foa and Scabia's ex-
periments on, 342
crouposae, Issaeff's experiments

on, 341

crouposae, Kleniperer's experi-
ments on, 342
crouposae in endocarditis, ulcer-

ative, 401

crouposae in meningitis, 400
crouposae in otitis media. 390,

402
of progressive granuloma formation,

413

pyogenes tenuis, 382
of sputum septicaemia (Frankel), 398
tetragenus, 411

action of chlorine on, 177
thermal death-point of, 155
urese, 140

liquefaciens, 140
Micromillimetre (the), 20
Microzyma bombycis, 415
Middle-ear disease, bacillus pyocyaneus

in, 545
Milk, bacteria in, 232, 663

number of present, 669
fermented, 139
immunization by, 263
of immunized goats, antitoxic, 357
toxic principle in, 148
of healthy women, bacteria in, 669
of cows affected with chronic masti-
tis, 417
of tuberculous cows, infective, 482,

485, 669
as a culture medium, 39

for bacillus A of Booker, 555
for bacillus aerogenes capsulatus,

589

for bacillus alvei, 557
for bacillus coli communis, 532
for bacillus diphtheriae, 455
for bacillus dysenteriae, 569
for bacillus of Fiocca, 548
for bacillus of hog cholera, 504,

509

for bacillus of Laser, 524
for bacillus piscicidus, 567

Milk as a culture medium for bacillus

piscicidus agilis, 565
for bacillus pyocyaneus, 543
for bacillus pyocyaneus pericar-

ditidis, 547

for bacillus solanacearum, 572
for bacillus tenuis sputigenus,

523
for bacillus typhi abdominalis,

439

for bacillus typhi rnurium, 524
for bacillus of vonDungern (cap-
sule), 562
for micrococcus pneumonias

crouposae, 404
for spirillum cholera asiaticae,

596

for spirillum Metschnikovi, 605
Milzbrand, bacillus of, 422
Mint, antiseptic power of, 203
Miquel's aeroscope, 615

later methods of air analysis, 618
tables of antiseptic values, 186
Mironoff's experiments against strepto-
cocci c infection, 346
Mitylotoxin, 148
Mixed infections, 228
Moist chamber for drop cultures, 65
Moitessier's gas-pressure regulator, 88
Moller's method of staining spore prepa-
rations, 32
Monkeys, susceptible to the infection of

bubonic plague, 564

Monochlorophenol as a disinfectant, 179
Morphia hydrochlorate as an antiseptic,

193
Morphological classification of bacteria,

12
Morphology of bacteria, general account,

20
insufficient to differentiate species,

20
Mosny, experiments on micrococcus

pneumonias crouposae, 342
Motile bacteria, 16
Moulds, spores of, in atmospheric dust,

614

Mouse septicaemia, symptoms and post-
mortem appearances of, 515
Mouth, bacteria found in the, 651, 656

INDEX.

695

Mucorini of Nageli, 11

Mucus, gerrnicidal power of, 210

Miiiicke's thermo-regalator. 92

Muscarini, 147

Mushrooms, toxic principle of, 147

Mussels, toxic principle in, 148

Mustard, antiseptic power of, 203

oil of, antiseptic power of, 200
Mycelium of moulds, 614
Mycoderma, 64

acetic acid produced by, 137
Mycophylaxin, 265
Mykoprotein, 124
Myrtle, antiseptic value of, 203

NAOELI'S classification of bacteria, 11
Nails, finger, surgical care of, 217
IS" ail-shaped growth of stab cultures, 397
Naphthol, disinfectant power of, 205
Nasal catarrh, pus cocci in, 390
Natural culture media, 37
Neapolitan fever, micrococcus of, 420
Negro, insusceptibility of, to yellow

fever, 234
Neisser's gonococcus, 226

discovery of, 7

stain for bacillus diphtherias, 457
double staining of spore prepara-
tions, 31

Nematogenes of Colin, 11
Neuridin, 146
Neurin, 147

Nickel sulphate as an antiseptic, 193
Nicolaier's capsule bacillus, 560

discovery of the tetanus bacillus, 8
Nitrates, reduction of, 144
Nitric acid as a decolorizer in staining,

28
influence of, on bacterial growth,

181
action of, on micrococcus pneu-

monise crouposse, 405
Nitrification produced by bacteria, 144
Nitrogen necessary for bacterial growth,

126
dioxide, influence of, on bacterial

growth, 175
Nitrous acid as a test for indol, 151

influence of, on bacterial growth,
181

Nitrous oxide, influence of, on bacterial

growth, 175
Non-liquefying bacteria, 16

characters of growth in stab

cultures, 70

Non-motile bacteria, 16
Non-pathogenic bacteria from the mouth,

656

from the nose, 656
list of species from faeces, 663
Nose, bacteria found in the, 650, 656
Nosema bombycis, 415
Nosophen, 179
Nuclein, the gerrnicidal constituent of

blood serum, 239

Nucleins, germicidal power of, 210
Nutmeg, antiseptic power of, 203
Nutrient agar (see Agar, nutrient)
gelatin (see Gelatin, nutrient)
NuttalTs method of estimating bacilli in
tuberculous sputum, 479

OIL light, Borden's method of photograph-
ing by, 111
Oleic acid, influence of, on bacterial

growth, 183
Olive oil, no influence on anthrax spores,

206

Onion, antiseptic power of, 203
Opopanax, antiseptic power of, 203
Orange of Portugal, antiseptic power of,

203

Origanum, antiseptic power of, 203
Orthochromatic photographic plates, 105,

113

Orthocresol, germicidal power of, 202
Orthophenol, germicidal power of, 197
Ose, 74
Osmic acid, fatal to anthrax spores,

181
Osteomyelitis, micrococcus of, 373

thermal death-point of, 375, 378
Otitis "media, micrococcus pneumonias

crouposse in, 402
pus cocci present in, 389
Oxalic acid, action of, on bacteria, 182
Oxychinaseptol. antiseptic power of, 202
Oxycyanide of mercury as a disinfectant,

192
Oxygen, as a disinfecting agent, 172

696

INDEX.

Oxygen, essential for certain species of

bacteria, 172 ,

influence of, on pigment production,

133

on spore formation, 12o
nascent, as a germicidal agent, 172

PANHISTOPHYTON ovatum, 415
Paracresol, germicidal power of, 202
Paralysis, local, caused by diphtheritic

inoculations, 452
Parasites, strict, 221
Parasitic bacteria, 15
Parietti's method of separating typhoid

bacilli from water supplies, 444
Park and Beebe's observations on the
disappearance of the Loftier bacillus in
cases of diphtheria, 461
Parrot disease, micrococcus of, 419
Pasteur's bacteriological researches, 7
cholera des poules, 288
demonstration of living organisms in

the atmosphere, 615
discovery of the causation of pe"-

brine, 6
experiments on the attenuation of

virus, 130

on the attenuation of the mi-
crobe of fowl cholera, 502
on the etiology of rouget, 516
on the preservation of putresci-

ble liquids, 5
flasks for liquid media, 63
inoculations of rabbits with hydro-
phobic virus, 99
solution, 40, 126
tubes for the collection of water

samples, 627

views on the origin of epidemics, 290
on rabies, 328

on the influence of temperature
on the destruction of organ-
isms, 5

Pasteur-Chamberland filter, 60
Pasteur Institute, production of diph-
theria antitoxin at, 316
of Naples, statistics of, 331
of Palermo, statistics of, 331
of Paris, statistics of, 331
of Turin, statistics of, 331

Patchouly, antiseptic power of, 203
Pathogenic anaerobic bacteria, 80

bacteria, list of species found in

faeces, 6(5:;
in milk, 068
in the mouth, 652, 656
in the nose, <>.~,i;
in the soil, duration of vitality

of. (i4o
in water, 6:56

in water, detection of, 640
organisms, 14

power of saprophytes dependent on
quantity and age of cultures and
site of the inoculation, 529
spirilla, 590

Paulsen's capsule bacillus, 561
Pear blight, 575

Pe'brine, silkworms infected with, 415
causation of, discovered by Pasteur,

not a bacterial disease ? 415
Pendesche Geschwur, 415
Pepper, antiseptic power of, 203
Peptone, a product of bacterial action,

142

Peptotoxin, 148

Peritoneum, injections into, 98
Peritonitis, bacillus coli commuuis in,

535

Person, disinfection of the, 213
Petermann's experiments with filtered

anthrax cultures, 428
Petri dishes, 76

use of, in air analyses, 617

use of, in water analyses, 630,

631

Petri 's sand filter for air analyses, 620
Petroleum lamps for incubating ovens,

95
Pfeiffer's capsule bacillus, 561

evidence of his bacillus being the

specific cause of influenza, 464
researches into the toxins of cholera

cultures, 599
views on the toxin of the typhoid

bacillus, 367

Pfeiffer and Issaeff's method of distin-
guishing the cholera spirillum from
other vibrios, 609

INDEX.

097

Pfuhl's experiments with tuberculin, 365
Phagocytosis, 255, 267

Metschnikoff's theory of, 208, 256
Phenol, produced by bacillus septicse-

mise haemorrhagicse, 501
by bacillus of swine plague,

Marseilles, 509
not produced by bacillus of hog

cholera, 505

Phenolphthalein, to determine the reac-
tion of media, 49
Philippine Islands, Malta fever in the,

420
Phosphorescence by bacteria, 145

an indication that vibrios are not

the cholera spirillum, 608, 609
Phosphoric acid, influence of, on the ty-
phoid bacillus, 181
Photomicrographs of bacteria 103

amplification of, 106
Photomicrography, apparatus for, 106
Physicians, interest of, in bacteriological

work, 6
Pigment, production of, by bacteria, 133,

135
by staphylococcus pyogenes au-

reus 374
Pitfield-Muir method of staining flagella,

Plant diseases bacteria of, 571
Platinum bichloride a^an antiseptic, 193
loop for inoculations, 63
needle for inoculations, 65, 69
Plectridium, spore formation in, 122
Pleuropneumonia of cattle, protective

inoculations against. 336
Pneumonia, causation of, 241
the micrococcus of, 339
percentages of relapses in, 340
protective inoculations against, 338
Pneumotoxin, 261, 340
Polar granules, 118

Porcelain, unglazed, as a filtering me-
dium, 59
Post-mortem examination of inoculated

animals, 101
Potash soap, action of, on bacterial

growth, 185

Potassium hydroxide, influence of, on
bacteria, 183

Potassium nitrate in culture media, 46
permanganate as an antiseptic, 194
action of, on micrococcus pneu-

moniab crouposse, 405
salts as antiseptics 193
Potato as a culture medium, 40, 48, 78
for bacillus A of Booker, 555
for bacillus acidiformans, 538
for bacillus aerogenes capsulatus,

589

for bacillus alvei, 557
for bacillus anthracis 426
for bacillus of Babes and Opres-

cu, 526

for bacillus capsulatus, 522
for bacillus cavicida havanien-

sis, 516
for bacillus of Cazal and Vail-

lard, 525
for bacillus of cholera in ducks,

603

for bacillus coli communis 531
for bacillus crassus sputigenus,

518
for bacillus cuniculicida havani-

ensis, 539

for bacillus diphtherias, 454
for bacillus dysenterise. 569
for bacillus of Emmerich and

Weibel, 566
for bacillus endocarditidis gri-

seus, 556

for bacillus enteritidis, 520
for bacillus of Fiocca, 548
for bacillus of Friedlander, 397
for bacillus gallinarum, 521
for bacillus gracilis cadaveris,

554
for bacillus of hog cholera 504,

509
for bacillus hydrophilus fuscus,

523

for bacillus lactis aerogenes 536
for bacillus of Laser, 524
for bacillus leporis lethalis, 541
for bacillus of Lucet, 527
for bacillus mallei, 491
for bacillus of Nicolaier (cap-
sule), 560
for bacillus pestis, 563

698

INDEX.

Potato as a culture medium for bacillus

piscicidus, 567

for bacillus piscicidus agilis, 565
for bacillus of purpura hsemor-

rhagica (Babes), 558
for bacillus of purpura hsemor-

rhagica (Kolb). 559
for bacillus of purpura hsemor-

rhagica (Tizzoni and Giovan-

nani), 658

for bacillus pyocyaneus, 543
for bacillus pyocyaneus ft, 645
for bacillus pyocyaneus pericar-

ditidis, 546
for bacillus pyogenes fcetidus,

618
for bacillus septicsemise hsemor-

rhagicse, 500, 509
for bacillus solanacearum, 572
for bacillus of swine plague,

Marseilles, 509
for bacillus tenuis sputigenus,

523

for bacillus tuberculosis, 477
for bacillus typhi abdominalis,

439

for bacillus typhi murium, 524
for bacillus of von Dungern, 562
for micrococcus bothryogenus,

413

for micrococcus Manfredi, 413
for micrococcus pneumoniae

crouposse, 403

for micrococcus tetragenus, 412
for proteus hominis capsulatus,

518

for proteus of Karlinski, 552
for proteus lethalis, 555
for proteus septicus, 554
for proteus vulgaris, 549
for spirillum cholerae asiaticse,

596
for spirillum of Finkler and

Prior, 603
for spirillum of Metschnikovi,

605

for spirillum tyrogenum, 604
for staphylococcus pyogenes au-

reus, 375
rot, symptoms of, 571

Pregl's method of staining tubercle ba-
cilli in tissues, 36

Pressure, influence of, on growth of bac-
teria, 163

Privy vaults, disinfection of, 217
Protection (standard of) in inoculation

of an ti tetanic serum, 356
Protective inoculations in tuberculosis,

360
against typhoid fever in the

British army, 369

Proteus capsulatus septicus (Banti), 520
Hauseri (Levy) from sour yeast,

551

of Karlinski, probably identical
with proteus vulgaris (Hauser), 552
lethalis, 554
bei Lungengangran des Menschen

(Babes), 654
mirabilis, swarming islands of, 552,

553

septicus, 554
vulgaris (Hauser), 548

in cholera infantum, 550
influence of, on the virulence of

anthrax cultures, 587
instanced as illustrating varia-
tions in the pathogenic power
of saprophytes, 529
Zenkeri, 554

Prudden's failure to find the Klebs-
Loffler bacillus in pseudo-membranous
inflammations, 450
Pseudo-diphtheria bacilli, 461

non pathogenic. 450, 456
attenuations of bacillus diph-
theria, 260

Pseudo-influenza bacillus in broncho-
pneumonia, 466
Pseudomonas campestris, 575
hyacinthi (Wakker), 573
Stewarti (Smith), 575
Ptomains, 133, 146
Puerperal fever caused by streptococcus

pyogenes, 387, 654
self-infection possible, 655, 656
Pulmonary mucous membrane, infection

through, 230

Pulmonic anthrax, 422, 428
Pure cultures on gelatin plates, 72

INDEX.

699

Purpura haeinorrhagica, bacillus of Babes,

558

bacillus of Kolb, 559
bacillus of Tizzoni and Giovan-

nini, 558
Pus, formation of, 223

independent of bacteria, 371
of acute abscess micrococci of, 14
production of. by typhoid bacilli

448

cocci found in healthy eyes, 650
found in the mouth, 652
present in diphtheritic mem

branes, 449
on inflamed mucous membranes,

389
sterilized by heat give rise to

pus formation, 371
Putrefaction, 141, 665

anaerobic bacteria active in, 142

products of, 142

in small intestine restrained by

bacteria, 662
Putrescin 147

Pyelonephritis, proteus vulgaris in, 551
Pyocyanin, 134, 543
Pyogenic bacteria, 371
Pyoktanin, germicidal power of, 197
Pyrogallic acid in the cultivation of an
aerobic bacteria, 85

QUARTER evil, 347, 586

Quinine, salts of, as antiseptics, 194

RABBITS, bacillus of intestinal diphtheria

in, 459
inoculations of with the diphtheria

bacillus, 452, 455
with hydrophobic virus, 99
with tuberculous matter, 481
Race immunity to disease. 234
Rags, disinfection of, 214
Railway cars, disinfection of, 214
Rain-water, number of bacteria in, 632
Ranvier's moist chamber, 65
Rats as propagators of bubonic plague,

564

Rauschbrand, 347
Rauschbrandbacillus. 585

anon -virulent variety of (Klein), 587

Reaction of culture media, 127
Recurrens spirochaete, 590
Red rays of spectrum non germicidal, 161
Reichert's ther mo-regulator, 90
Relapsing fever, a disease of man, 233
spirillum of, 590
transmission of, by inoculation

with infected blood, 591
Retention theory of immunity, 250
Rhinoscleroma, bacillus of, 496
Rhorbeck's thermo-regulator. 88
Ricin, immunity against, 262, 265, 267
Rinderpest, influence of temperature on

the infectious material, 156
protective inoculations against, 343
Rinderseuche, 287

River water, number of bacteria in, 632
Rose, antiseptic power of, 203
Rosemary, antiseptic power of, 203
Rotzbacillus (see Bacillus mallei), 489
Rouget, 287

bacillus of, 511

Koch's experiments on the etiology

of, 515

protective inoculations against, 324
Roux's attenuated varieties of diphtheria

bacillus 309

method of producing diphtheria anti-
toxin, 316

incubator and thermo-regulator, 95
Roux and Yersin, inoculation of the ba-
cillus of diphtheria into rabbits,
452

study of the pseudo-diphtheritic ba-
cilli, 456

SACCHAROMYCETES of Nageli, 11
Sach's classification of bacteria 11
Sage, antiseptic power of, 203
Salicylic acid, action of. on bacillus tu-
berculosis, 478
germicidal action of, 182
action of. on micrococcus pneu-
monias crouposas, 405
Saliva, peptonizing action of, 653

prejudicial to the action of certain

pathogenic organisms 653
Salmon's immunizing experiments, 290
Salmonson's culture method, 79
tube, 82

700

INDEX.

Salt meat, bacteria in, 671

saturated solution of, inefficient as a

germicide, 573
Salts, antiseptic and germicidal value of,

158
Sanarelli's water vibrios resembling the

cholera spirillum, 607
Sandalwood, antiseptic power of, 203
Sand filtration in air analysis, 620
Saprin, 147

Saprol, disinfectant power of 206
Saprophytes, 611

conditions favorable to their growth,

613

in air, list of, 624

Saprophytic bacilli which are patho-
genic, 528
bacteria, 15

Sarcina, general characters of, 17
grouping of, 22
in stomach of persons suffering from

indigestion, 662
aurantiaca, thermal death-point of,

155

lutea, thermal death-point of, 155
Sassafras, antiseptic power of, 203
Savin, antiseptic power of, 203
Sawizky on the vitality of dried tuber-
culous sputum, 479
Scarlet fever, second attacks of, 243
Scheinfaden, 120
Schizomycetes of Nageli, 11
Schoolrooms, bacteriology of the air of,

625
Schroder and Van Dusch, use of cotton

as a filter by, 4
Schuhanka, protective inoculations in

Salzburg 350
Schulze's (Franz) experiments opposed to

spontaneous generation. 4
Schiitz's diplococcus of pneumonia in

horses 418

Schwann's experiments opposed to spon-
taneous generation, 4
Schweinerothlauf, 287
Schweineseuche, 287, 344

(Loffler and Schtitz), 343
Sea water, number of bacteria in, 637,

641
Secondary infections, 227

Septicaemia, definition and general char-
acters of, 498
produced by saprophytic bacteria,

528, 529

Septicsemic bacteria, attenuated varie-
ties in the mouth and air passages of
certain animals, 503
Serpent venom, immunization against,

262
Serum-albumin, germicidal power of, 210,

238
Serum of blood, germicidal action of,

236, 257

immunizing value of, 313
Sevestre's observations on the disappear-
ance of the Loffler bacillus after diph-
theria, 460

Sewer water, number of bacteria in, 634
Shakespeare's report on anticholeraic in-
oculations, 300

Sheep, micrococcus of gangrenous masti-
tis in, 416
Sheep-pox, influence of temperature on

the infectious material of, 156
Ships, disinfection of, 214
Siberian plague, 422
Silicate jelly as a culture medium, 47
Silkworms infected with "la flacherie,"

415

with pe"brine, 415
Silver chloride as an antiseptic, 194

nitrate as an antiseptic, 194
Sjobring's method of demonstrating the

structure of bacterial cells, 117
Skatol, no germicidal power, 206
Skin, abscesses caused by staphylococcus

epidermidis albus, 372
human, bacterial flora of, 648

list of species found on the, 649
infection through the, 229
Sleskin's silicate jelly, 47
Smallpox, heat as a disinfectant for, 154

second attacks of, 243
Smear preparations, 26
Smith (Erwin F.) quoted on bacterial

diseases of plants, 571
Smith (Theobald), fermentation tube of,

views on the hog ckolera group of
bacteria, 506

INDEX.

701

Smith (Theobald), detection of colon ba-
cilli in waters, 535
identification of colon and typhoid

bacilli in water supplies, 445
Smoke, antiseptic power of, 206

tobacco, antiseptic power of, 207
Snow, number of bacteria in, 632
Sodium borate as an antiseptic, 195
carbonate as an antiseptic, 195
chloride as an antiseptic, 195
in culture media, -Mi
influence of, on bacterial growth,

128
hydroxide, influence of, on bacterial

growth, i84

hyposulphite as an antiseptic, 195
sulphite as an antiseptic, 195
Soil bacceria, 642
Solid culture media, 41
Solidification of blood serum, 57
Sour dough, bacteria of, 672
Sozoiodol acid as a disinfectant, 179
Spallanzani's sterilization of putrescible

liquids, 4

Species not differentiated by morpholog-
ical characters alone, 13
Sperulina (Hueppe), general characters of,

Spirilla, general characters of, 18
of Bauingarten, 13
list of species found in water, 639
movements of, 119
pathogenic, 590
resembling the spirillum of Asiatic

cholera, 606

Spirillum anserum, from blood of af-
fected geese, 592
cholera? asiaticae, 292, 593

asiaticae, attenuation of, by con-
tinuous cultivation, 601
asiaticie, biological characters,

594

asiaticae, influence of disinfect-
ing agents on, 597
asiaticae influence of gastric

juice on, 658
asiaticae, influence of light on,

162

asiaticae, influence of tempera-
ture on. 610

Spirillum choleras asiaticae occasionally
present in healthy persons, 610
asiaticae, phosphorescence occa-
sionally found in, 609
asiaticae, thermal death-point of,

155
asiaticae, vitality of, in faeces,

610
asiaticae, vitality of, when

buried in the soil, 64">
asiaticae, vitality of, in waters

of various characters, 637
of Ehrenberg, 3, 10
of Finkler and Prior, 602
pathogenesis, 603
thermal death-point of, 155
Metschnikovi, 604

pathogenesis, 605

murinum (Russell) remotely resem-
bling the spirillum of cholera, 606
Obermeieri, in the blood during the
febrile paroxysms of relapsing
fever, 590
pathogenesis, 591
tyrogenum (Deneke), 603

thermal death-point of, 155
Spirochaete, general characters, 18
anserina (Sakharoff), 592
of Ehrenberg, 3, 10
Obermeieri, 590

discovery of, 7
Spleen, influence of, on the spirillum of

relapsing fever, 592
pure cultures of the typhoid bacillus

from the, 433
Splenic fever, 422

Spontaneous generation proved errone-
ous, 4
Spores, 16

action of acetic acid on, 182

endogenous, 120

germination of, 122

influence of oxygen and temperature

on, 123

isolation of, by heat, 68
resisting powers of, 5, 157
restraining influence of temperature

on, 166

staining of, 31
thermal death-point of, 53, 473

702

INDEX.

Spores, vitality of, 121, 122
Spores formed by :

bacillus alvei, 556
bacillus anthracis, 424, 426
bacillus anthracis, multiplica-
tion of, 429
bacillus anthracis, survival of,

in various media, 430
bacillus anthracis, exceptions

to, 426

bacillus crassus sputigenus, 517
bacillus cedematis maligni, 582
bacillus piscicidus agilis, 565
bacillus of symptomatic anthrax,

586, 587

bacillus tetani, 578
bacillus tetani, influence of heat

and germicides on, 580
Spores not formed by or not observed in :
bacillus acidi formans, 538
bacillus aerogenes capsulatus,

589

bacillus amylovorus, 576
bacillus of Babes and Oprescu,

526

bacilus of Beck, 566
bacillus coli communis, 530
bacillus cuniculicida havanien-

sis, 540
bacillus of Emmerich and Wei-

bel, 566

bacillus erysipelatos suis, 513
bacillus of Fiocca. 547
bacillus gracilis cadaver is, 560
bacillus hyacinthi, 573
bacillus lactis aerogenes, 536
bacillus of Laser, 524
bacillus leprse, 488
bacillus mallei, 489, 490
bacillus of Mereshkowsky, 565
bacillus of Nicolaier (capsule),

560
bacillus cedematis maligni No.

II., 588

bacillus pestis, 563
bacillus piscicidus, 567
bacillus of purpura hsemor-

rhagica (Babes), 558
bacillus of purpura haemor-

rhagica (Kolb), 559

Spores not formed by or not observed in :
bacillus pyocyaueus. 543
bacillus pyocyaneus pericardi-

tidis. 547

bacillus solanacearum, 571
bacillus tracheiphilus, 577
bacillus tuberculosis, 472, 478
bacillus of von Dungern (cap-
sule), 562

proteus lethalis, 554
proteus Zenkeri, 554
spirillum cholerae asiaticse, 594
spirillum of Finkler and Prior,

603

spirillum of Metschnikovi, 604
spirillum tyrogenum, 603
Spores of moulds and fungi in atmos-
pheric dust, 614

of non-pathogenic bacteria, resist-
ance of, to heat, 158

Spring water, number of bacteria in, 634
Sputum, examination of, for bacillus

tuberculosis, 470
micrococcus tetragenus in, 411
separation of bacilli from, by sedi-
mentation, 486

tuberculous, estimation of the num-
ber of bacilli in, 479
Stab cultures, 69

character of growth in, 70
of anaerobic bacteria, 81
from roll-tube colonies, 77
Stained preparations, the photographing

of, 104

Staining methods in bacteriology, 25
of a dried bacterial film, 27
sections of gelatin stab cultures,

bacteria in tissues, 34
sections of tuberculous tissues, 471
of spores, 31

Staining materials and methods for :
bacillus acidiformans, 537
bacillus acne contagiosa of

horses, 557
bacillus alvei, 556
bacillus anthracis, 424
bacillus of Babes and Oprescu,

526
bacillus of Beck, 566

INDEX.

703

Staining materials and methods for:

bacillus of Belfonti and Pasca-

rola, 508

bacillus capsulatus, 521
bacillus cavicida havaniensis,

516
bacillus of Cazal and Vaillard,

526
bacillus of cholera in ducks,

"503

bacillus coli communis, 580
bacillus crassus sputigenus, 517
bacillus cuniculicida havanien-
sis, 538, 540
bacillus diphtherias, 453
bacillus dysenterise, 569
bacillus of Emmerich and Wei-

bel, 566

bacillus endocarditidis capsula-
tus, 556
bacillus endocarditidis griseus,

555

bacillus enteritidis, 520
bacillus erysipelatos suis, 512
bacillus of Fiocca, 547
bacillus of Friedlander, 396
bacillus of grouse disease, 520
bacillus of hog cholera, 504
bacillus of influenza, 463
bacillus of intestinal diphtheria

in rabbits, Ribbert's method,

459

bacillus lactis aerogenes, 536
bacillus leporis lethalis, 541
bacillus leprse, 487
bacillus of Lucet, 526
bacillus mallei, 489
bacillus of Nicolaier (capsule),

560

bacillus cedematis inaligni, 581
bacillus cedematis maligni No.

II., 588

bacillus piscicidus, 567
bacillus of purpura hsemor-

rhagica (Babes), 558
bacillus of purpura hsemor-

rhagica (Kolb), 559
bacillus of purpura hsemor-

rhagica (Tizzoni and Giovan-

ninl), 558

Staining materials and methods for :

bacillus pyocyaneus pericardi-

tidis, 547

bacillus pyogenes filiformis, 567
bacillus cf rhinoscleroma, 497
bacillus of septicaemia hsemor-

rhagica, 500

bacillus solanacearum, 571
bacillis of swine plague, Mar-
seilles, 509

bacillus of syphilis, 495
bacillus of symptomatic anthrax,

586

bacillus of tetani, 578
bacillus tuberculosis, 30, 469
bacillus typhi abdominalis, 437
bacillus typhi niurium, 524
bacillus of von Dungern, 562
diplococcus intracellularis men-

ingitidis, 410

micrococcus bothryogenus, 412
micrococcus gonorrhoea, 392
micrococcus of Manfredi, 413
micrococcus tetragenus, 411
proteus hominis capsulatus, 518
proteus lethalis, 554
proteus septicus, 554
proteus vulgaris, 549
spirillum cholerse asiaticae, 594
spirillum of Finkler and Prior,

602

spirillum of Metschnikovi, 604
spirillum of Obermeieri, 590
spirillum tyrogenum, 603
staphylococcus pyogenes aureus,

374

streptococcus pyogenes, 383
Staphylococcus, grouping of, 21
epidermidis albus (Welch), 380
pyogenes albus, 380

albus, thermal death-point of,

155

aureus, 373

aureus, resistance of, to freez-
ing, 153
aureus, thermal death-point of,

155

aureus toxalbumins from cul-
tures of, 150
citreus, 381

704

INDEX.

Staphylococcus pyogenes citreus, thermal

death-point of, 155
Starvation, influence of, on development

of pus cocci, 224
Steam under pressure as a disinfectant,

159, 212, 213
compared with streaming steam,

215

as a sterilizer, 56
sterilizers, 55

Sterilization of media, etc., 37, 52
by filtration, 58

imperfect, errors arising from, 62
of a piston syringe, 97
of a platinum loop for inoculation,

Sternberg's bacillus X, 538
bulbs, 38, 66

discovery of bacillus coli communis
in the blood and organs of yellow-
fever cadavers, 530
experiments on attenuation of virus

by antiseptic agents, 131
on the thermal death-point of

bacteria, 154

method of collecting blood serum, 38
liquid culture method, 65
method of cultivating anaerobic bac-
teria, 83

of determining the germicidal
power of chemical agents on
anthrax spores, 168
syringe for inoculation, 97
theory of immunity, 252
thermo-regulator, 92
tubes for the collection of water

samples, 627
Stomach, bacteria of, not concerned in

digestive action, 660
of nursing infants, bacteria in, 659
Strauss' method of diagnosticating glan-
ders, 493
Streak cultures of bacillus septicaemias

haemorrhagicae, 500, 509
Strebel's protective inoculations, 348, 349
Streptococci, 17, 129
grouping of, 22
infection of, 347

Streptococcus agalactise contagiosae, 419
articulorum (Loffler), 386

Streptococcus bombycis, 415

coryzse contagiosae equorum, 418
erysipelatos, 382

action of chlorine on, 177
lanceolatus Pasteuri (Ganieleia), 398
longus (von Lingelsheim), 382

germicidal power of chemicals

on, 385

of mastitis in cows, 417
mastitis sporadiae, 420
perniciosus psittacorum, 419
pyogenes, 346, 382

in diphtheria, 449, 452, 453, 461
thermal death-point of, 155, 384
malignus (Fliigge), 388
septicus (Fliigge), 415
Strict anaerobics, 16
parasites, 15, 221
Sucholoalbumin, 323
Sucholotoxin, 323
Sugar as a filter in air analysis, 620
Sulpho-carbolic acid, germicidal power

of, 197

Sulphur dioxide, influence of, on bac-
terial growth, 176
as a disinfectant, 212, 213, 214
Sulphuretted hydrogen, a bacterial pro-
duction, 141

Sulphuric acid as a decolorizer in stain-
ing, 28

action of, on bacterial growth, 180
action of, on micrococcus pneu-

moniae crouposaa, 405
Sulphurous acid, action of, on bacterial

growth, 180

Sunlight, importance of, as a disinfect-
ant, 162

Susceptibility to disease, 233
Susotoxin, 323

of Novy from cultures of bacillus of

hog cholera, 505
Swine plague, 343

(American) bacillus of (Salmon
and Smith), identical with
bacillus septicaemias haemor-
rhagicae, 499
practical measures for arresting,

345

protective inoculations against,
322

INDEX.

705

Symptomatic anthrax, 347

bacillus of, 585
Syphilis, Lustgarten's bacillus of, 494

not obtained in cultures, 496

TANNIC acid as a disinfectant, 183
Tartaric acid as a disinfectant, 183
Temperature, influence of, on bacterial

growth, 126, 153
on spore formation, 123
on the destruction of organ-
isms, 5
resistance of anthrax spores to a

high, 427
animal, influence of. on immunity,

235
Test tubes, use of, for cultures, 62

sterilization of, 53
Tetanin, 149

Brieger's method of obtaining, 352
Tetanotoxin of Brieger, 149, 352
Tetanus, a traumatic infection, 350
antitoxin, 260

curative power of, 359
Tizzoni and Cattani 's experi-
ments on, 355
bacillus, 578

cultures, toxalbumin of, 150, 151
poison of, 352

neutralized by the blood of an

immune animal, 354
not neutralized by the blood of

chickens, 355
Tetrads, 22, 120

Tetraiodphenolphthalein, antiseptic val-
ue of, 179

Texas fever in cattle, bacillus of, 508
Thallophytes of Sachs, 11
Therapeutic value of antitetanic blood

serum, 356

Thermal death-point of bacteria, 52, 155
of bacillus of Beck, 566
of bacillus cavicida havaniensis,

517
of bacillus of Cazal and Yaillard,

525

of bacillus coli communis, 532
of bacillis of Emmerich and

\Veibel, 566

of bacillus erysipelatos suis, 513
45

Thermal death-point of bacillus of Laser,

524

of bacillus piscicidus agilis, 565
of bacillus pyocyaneus, 543
of bacillus septicaemias hsemor-

rhagicae, 501
of bacillus typhi abdoruinalis,

441
of spirillum choleras asiaticse,

597

Thermo-regulators, 88
Thoinot's method of isolating the typhoid

bacillus, 444

Thyme, antiseptic power of, 203
Thymic acid, action of, on bacteria, 183
Thymol, antiseptic power of, 207

action of, on bacillus tuberculosis,

478

Thymus bouillon, 246, 357
preparation of, 359
immunization to tetanus by cul-
tures in, 359

Tin chloride as an antiseptic, 195
Tizzoni and Cattani, on the preservation

of tetanus antitoxin, 359
experiments favorable to Koch's

tuberculin, 479
Tizzoni and Giovannini's bacillus of

purpura hsernorrhagica, 558
Tobacco smoke, antiseptic power of, 207
Torula chains, 22
Toussaint on fowl cholera, 288
Toxaemia, definition of, 498

caused by anaerobic bacteria, 578

by saprophytic bacilli, 528, 529
in diphtheria, 449

Toxalbumin of diphtheria cultures, 312
of hog cholera, 505
of hydrophobia, 327
of tetanus cultures, 353
of typhoid culture, 150
Toxalbumins, 146, 149
Toxic power of the culture products of

bacillus tetani, 353
of virulent cultures neutralized
by the blood of immune ani-
mals, 259
Toxin of anthrax, 430

immunity produced by, 427
of cholera cultures, 610

706

INDEX.

Toxin of bacillus of hog cholera, 505
of influenza, 465
piscicidus agilis, 565
tuberculosis, 152, 361
Toxins, 146, 151
Toxophylaxin, 265
Trachoma, pus cocci in, 390
Tribroniophenol as a disinfectant. 179
Trikresol, germicidal power of, 202
Trimethylamine, a non-toxic ptomain,

147

Trudeau's successful antitubercular inoc-
ulations, 366
Tryptic enzymes, 135
Tubercle bacilli, method of staining in

tissues, 36
in milk of cows, 232
bacillus (see Bacillus tuberculosis)
Tuberculin, action of, on tuberculous ani-
mals, 479

Bujwid's method of preparing, 363
Helman's method of preparing, 363
Koch's, 151, 361
use of, by various experimenters,

364, 365

Tuberculocidin of Klebs, 363
Tuberculosis, carnivora immune to, 233
among cattle, prevalence of, 482
usual methods of infection, 481
protective inoculations in, 360
Tuberose, antiseptic power of, 203
Turpentine, antiseptic power of, 203,

206
Tympanic cavity, bacillus pyocyaneus

present in inflammation of, 545
Tyndall's optically pure air, 5
Typhoid bacilli and colon bacilli, varie-
ties of the same species? 447
bacillus, 431, 436

attenuation of, 242
biological characters of, 438
culture medium for, 47
dead animal matter a suitable

nidus for, 434

destroyed by gastric juice, 658
difficulty of differentiating, 440
influence of light on the growth

of, 161

morphology. 436
pathogenesis of, 442

Typhoid bacillus, pathogenic to certain
of the lower animals, but does
not convey typhoid fever, 436
pus production by, 448
resistance of, to freezing, 153
thermal death-point of, 155, 441
vitality of, in various media,

442
vitality of, in moist atmospheres,

159
vitality of, when buried in the

soil, 645
colonies in the spleen, post-mortem

multiplication of, 434
cultures, toxalbumin of, 150
fever, a disease of man, 233

failure to communicate it to ex-
perimental animals, 435, 442
heat as a disinfectant for, 154
protective inoculations in. 3i»7
second attacks of, 243
Typhotoxin, 148, 222

of Brieger, 442
Tyrotoxicon, 148

ULCERATIVE endocarditis, 379, 386
Underclothing, worn, bacteria on, 649
Urea, fermentation of, 140
Uric acid, influence of, on the bacterici-
dal activity of blood serum, 238
Urine as a culture medium, 39

germicidal power of, 211
Uterus, free from bacteria in health, 654

VACCINE virus, influence of temperature
on, 156

Vagina, bacteria of, 654

Vaillard, immunity to tetanus produced
in rabbits, 355

Valerian, antiseptic power of, 203

Valerianic acid as a disinfectant, 183

Van Ermengem's method of staining
flagella, 33

Veal broth peptonized as a culture me-
dium for bacillus tuberculosis, 477

Vegetable infusions as culture media, 41

Veins, injections into, 98

Venom, serpent, immunization against,
262

Verbena, antiseptic power of, 203

INDEX.

707

Vesuvin as a stain, 25

as a double stain in bacterial prepa-
rations, 28
Vibrio, general characters, 18

aquatilis (Giinther), remotely resem-
bling the spirillum of cholera, <306
Berolinensis (Neisser), its differenti-
ation from the cholera spirillum,
607

Danubicus (Heider), its differentia-
tion from the cholera spirillum,
608

of Ehrenberg, 3, 10
helcogenes ^Fischer), remotely resem-
bling the cholera spirillum, 607
Metschnikovi (Gamele'ia), 604
Vibrion septique (Pasteur), 580
Vibrionieus of Ehrenberg, 3, 10
Vibrio-proteus, 602
Vibrios of Celli and Santori, 60S

resembling the spirillum of cholera,

600

list of species found in water, 639
phosphorescent, not genuine cholera

germs, 608, 609

Vincent's method of detecting colon ba-
cilli in water supplies, 640
Violet rays of spectrum, germicidal

power of, 161
Virulence, attenuation of, in cultures,

130, 388

intensification of, 132, 388
recovery of, 132
Viscous fermentation, 141
Vital resistance theory of immunity, 252
Vitality of bacillus of hog cholera, 505
of the typhoid bacillus in cultures,

442

Von Dungern's capsule bacillus distin-
guished from others of similar mor-
phology, 562

WATER, action of, on the cholera spiril-
lum, 597
bacteria in, 626 <-

list of species found in, 638
number not dependent on the
amount of organic matter pres-
ent, 635
distilled, growth of bacteria in, 635

Water, pathogenic bacteria that may be

present in, 636
bacteria, detection of, 640
bacteriological analysis of, 628
collection of. for analysis, 627
importance of immediate exam-
ination, 627
chemical analysis of, advantages of,

631
filtration, efficiency determined by

counting colonies, 641
supplies, detection of colon bacilli

in, 535
difficulty of finding typhoid

bacilli in, 441, 444
sanitary inspection of source
more important than bacterio-
logical or chemical analysis,
641
vibrios, remotely resembling the

spirillum of cholera, 606
Waters, surface, frequency of the colon

bacilli in, 640
Weichselbaum's diplococcus intracellu-

laris ineningitidis, 410
micrococcus endocarditidis rugatus,

416
Weigert's method of staining bacteria in

tissues, 35
of staining used in sections of

nodules of glanders, 493
Welch and Abbott's cultivation of the
bacillus of diphtheria on po-
tato, 455
inoculations of kittens with the

diphtheria bacillus, 452
Well water, number of bacteria in, 634
Widal reaction given by blood serum of
those subjected to antityphoid
inoculation, 369
in Malta fever, 421
Wildseuche, 287, 499
Winogradsky's silicate jelly, 47
Wintergreen, antiseptic power of, 203
Wolffhugel's experiments on the thermal

death-point of bacteria, 154
Wolter's opinion that positive results in
attempts to inoculate leprosy were due
to associated tubercle bacilli, 488
Woollen underclothing bacteria on, 649

708

INDEX.

Wool-sorter's disease, 422, 430

a pulmonary affection, 2."H
Woven underclothing, bacteria on, 649
Wurtz's method of identifying typhoid
bacilli in water supplies, 44-~>

XYLOL for solution of Canada balsam, 28

YELLOW fever, insusceptibility of negro

to, 234

cadavers, bacillus coli conmmnis
in the blood and organs of, 530
milk. 668

Yersin's attenuated varieties of diph-
theria bacillus, oOD
description of the bacillus pestis, 563

ZEA mays, bacterial diseases of, 575

Zedvary, antiseptic power of, 203
"Zeitsckrift fur Hygiene," 8
Ziehl's carbol-fuchsin solution. 2!)
for bacillus lepra, 487
for bacillus tuberculosis, 469-

471

for the typhoid bacillus, 438
Ziehl-Nelson method of staining the tu-
bercle bacillus, 30, 36
Zinc chloride as an antiseptic and disin-
fectant, 195, 212
sulphate as a germicide, 196
Zooglea, 21

formed by bacillus solanacearum, 571

proteus mirabilis, 553
Zopf, classification of, 12
Zymogenic organisms, 14

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